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Ammonium uptake by rice roots Wang, Miao Yuan 1994

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AMMONIUM UPTAKE BY RICE ROOTS by  MIA0 YUAN WANG B.Sc. Zhejiang Agricultural University, Hangzhou, 1981 M.Sc. University of Saskatchewan, Saskatoon, 1987  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (DEPARTMENT OF BOTANY)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA JUNE 1994 © M. Y. WANG, 1994  In presenting this thesis in  partial fulfilment of the requirements for an advanced  degree at the University of British Columbia, I agree that the Ubrary shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by  his  or  her  representatives,  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department  of  The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  ABSTRACT  4 uptake was studied using 3-week-old rice plants (Oryza NH 13 sativa L. cv. M202), grown hydroponically in modified Johnson’s nutrient solution containing 2, 100 or 1000 !IM NH 4 (referred to hereafter as G2, G100 or G1000 plants, respectively). At steady-state, the influx and efflux of 13 4 was increased as 4 NH NH ’- provision during growth was increased. The half-life of cytoplasmic ‘ 4 N 3 H exchange was calculated to be 8 mm while the half-life for cell wall exchange was 1 mm. Cytoplasmic 4 [NH ’-] of G2, G100 and G1000 roots was estimated to be 3.72, 20.55, and 38.08 mM respectively. However about 72% to 92% of total root NH 4 was located in the vacuole. During a 30 minute period G100 plants metabolized 19% of the newly absorbed 13 -’- and the remainder was partitioned among the 4 NH cytoplasm (41%), vacuole (20%) and efflux (20%). Of the metabolized 13 N, roughly one half was translocated to the shoots. In short-term, perturbation experiments, below 1 mM external concentration ) 0 + 4 ([NH ] 13 , ÷ influx of G2, G100 and G1000 roots was 4 NH saturable and operated by means of a high affinity transport system (HATS). The Vmax values for this transport system were negatively correlated and Km values were positively correlated with NH 4 provision during growth and root [NH +]. Between 1 and 40 mM , 4 0 + 4 [NH ] 13 4 NH influx showed a linear response to external concentration due to a low affinity transport system (LATS). The 13 -’- influxes by the HATS, and to 4 NH a lesser extent the LATS, are energy-dependent processes. Selected metabolic inhibitors reduced influx of the HATS by 50 to 80%, but of the LATS by only 31 to 51%. Estimated Q 10 values for HATS were greater than 11  2.4 at root temperatures from 5 to 10°C and constant at 1.5 between 5 to 30°C for the LATS. Influx of 4 NH by the HATS was insensitive to 1 ÷ external pH in the range from 4.5 to 9.0, but influx by the LATS declined significantly beyond pH 6.0. The transmembrane electrical potential differences (z’P) of epidermal and cortical cells of intact roots were in the range from -120 to -140 millivolts (mV) in the absence of NH -’- in bathing solution and were -116 4 mV and -89 mV for G2 and G100 plants in 2 and 100 jiM NH 4 solutions, respectively. Introducing 4 NH ’- to the bathing medium caused a rapid depolarization which exhibited a biphasic response to external 4 [NH -]. Plots of membrane depolarization versus ‘ 4 N 3 H influx were also biphasic, indicating distinct coupling processes for the two transport systems, with a reak-point between the two concentration ranges around 1 mM NH . 4 Depolarization of z’P due to NH 4 uptake was eliminated by a protonophore (carboxylcyanide-m-chlorophenylhydrazone), inhibitors of ATP synthesis (sodium cyanide plus salicylhydroxamic acid), or an ATPase inhibitor (diethyistilbestrol). 4 N 3 ‘ H influx was regulated by internal ammonium and its primary metabolites, amides and amino acids. When internal amide or amino acids concentrations were increased, the influx of 13 4 was reduced. However, NH treating rice roots with L-Methionine DL-Sulfoximine (MSX) reduced the levels of ammonium assimilates but did not increase 13 4 influx NH probably because internal 4 [NH ’-] was increased. Short-term nitrogen depletion stimulated 1 4 N H influx, but long-term N depletion caused NH 4 influx to be reduced probably due to N limitation of carrier synthesis. A cascade regulation system is proposed to explain the multi-level regulation of NH 4 influx. 111  The interaction between ammonium and potassium showed that when N is adequate, K promoted NH 4 uptake and utilization. Likewise, proper N nutrition promoted K-’- uptake but the presence of NH 4 in uptake solution strongly inhibited the K ( Rb+) 8 6 uptake at the transport step. The results indicated that NH 4 and K-I- may share the same channel but are regulated by different feedback signals.  iv  TABLE OF CONTENTS Abstract Table of Contents List of abbreviation List of Tables List of Figures Dedication Acknowledgment  ii v xii xiv xv xviii Xix  Chapter 1. RESEARCH BACKGROUND  1  1.1. General Introduction Rice 1.1.1. Essentiality of nitrogen 1.1.2. Necessity of N fertilization 1.1.3. Bio-availability of nitrogen 1.1.4.  1 1 1 2 2  1.2. Ammonium Uptake 1.2.1. Importance of transport research Transport of NH 1.2.2. 4 by lower plants Transport of NH 1.2.3. 4 by higher plants 1.2.3.1. Carrier-mediated transport 1.2.3.2. Concentration-dependent kinetics 1.2.3.3. Depolarization of membrane potential 1.2.3.4. Energy dependence  3 3 4 5 5 6 6 7  1.3. Major Factors Affecting Ammonium Uptake Effects of photosynthesis 1.3.1. 1.3.1.1. Dependence on soluble carbohydrates 1.3.1.2. Periodic variations of light and growth 1.3.1.3. Ambient environmental factors Effects of root temperature 1.3.2. 1.3.2.1. Short-term perturbation 1.3.2.2. Qio value for NH 4 uptake 1.3.2.3. Long-term low temperature effects 1.3.3. Effects of pH on NH 4 uptake 1.3.3.1. Acidification of rhizosphere by NH 4 uptake V  7 7 7 8 9 10 10 10 11 12 12  1.3.3.2. Retarded plant growth in acidic medium 4 toxicity and acidic damage 1.3.3.3. NH 4 fluxes at the plasma membrane NH 1.3.4. 1.3.4.1. Net flux 1.3.4.2. Influx 1.3.4.3. Efflux 1.3.4.4. Balance of fluxes 1.3.4.5. N cycling in the whole plant  15 16  1.3.5. Regulation of ammonium uptake 1.3.5.1. Negative feedback regulation 1.3.5.2. Enhanced NH 4 uptake Interaction between NH 1.3.6. 4 and K 1.3.6.1. Mutual beneficial effects between N and K 1.3.6.2. Inhibition of K uptake by NH 4 1.3.6.3. Inhibition of NH 4 uptake by K  17 17 17 18 18 18 19  1.4. Research Objectives  19  Chapter 2. MATERIALS AND METHODS 2.1. Plant 2.1.1. 2.1.2. 2.1.3.  13 13 14 14 14 15  Growth Seed germination Growth conditions Provision of nutrients  2.2. N Isotopes For Studying N Uptake 2.2.1. Isotopic tracer 2.2.2. Nitrogen Isotopes 2.2.3. Stable ‘ N techniques 5 Radioactive isotope, 13 2.2.4. N 2.2.4.1. Use in biological studies 2.2.4.2. Production of 1 N 2.2.4.3. Advantages of the use of 13 N in biological studies 2.2.4.4. Considerations of using ‘ N in nitrogen uptake 3 2.2.4.5. Use of 13 N in nitrogen transport studies 2.2.4.6. Use of ‘ N in nitrogen assimilation 3 2.2.4.7. Use of 1 N in denitrification 2.2.5. Protocol for 4 NH production in present study 3 ‘ vi  22 22 22 22 23 24 24 24 25 26 26 27 29 30 31 32 33 33  2.3. Measurement Of NH 4 Fluxes 2.3.1. Influx of 13 4 NH 2.3.2. N 3 Effluxof’ 4 ’H 2.3.3. Net flux of NH ÷ 4  35 35 35 36  2.4. Compartmental (Efflux) Analysis 2.4.1. Compartmentation of plant cells 2.4.2. Development of theory 2.4.3. Models for compartmental analysis 2.4.4. The general procedure of compartmental analysis 2.4.5. Procedures for compartmental analysis in the present study  36 36 37 38 42 44  2.5.  Determination Of Ammonium  46  2.6.  Preparation Of Metabolic Inhibitors  46  2.7. Electrophysiological Study 2.7.1. Transmembrane electrical potential measurement 2.7.2. Single impalement and membrane potential 2.7.3. Setup for measuring membrane potential 2.8.  Determination of amino acids in root tissue  Chapter 3. FLUXES AND DISTRIBUTION OF 13 4 IN CELLS NH 3.1.  Introduction  55 57 57  3.2. Materials And Methods Plant growth and ‘ 3.2.1. N production 3 Measurement of fluxes 3.2.2. 3.2.2.1. 4 NH influx 3 ‘ 3.2.2.2. Net NH 4 flux 3.2.2.3. Time course of 13 4 uptake NH 3.2.3. Compartmental analysis 3.2.4. Partition of absorbed 4 NH 1 3.2.4.1. Separation of ‘ N-compounds in plant tissue 3 3.2.4.2 Chemical assay of NH 4 in root tissue 3.2.5.  47 47 53 54  Calculation of flux to vacuole (Øcv) vii  59 59 59 59 59 60 60 60 60 61  61  4.  3.3. Results 3.3.1. Compartmental analysis Metabolism and translocation of ‘ 3.3.2. N 3 Time course of 4 3.3.3. NH influx in rice roots 3 ‘  62 62 71 71  3.4. Discussion 3.4.1. The half-lives of 13 ÷ exchange 4 NH Fluxes of 4 3.4.2. NH into root cells 3 ‘ The NH 4 pools in roots 3.4.3. Model of 4 3.4.4. NH uptake by rice plants 3 ‘  75 75 78 82 83  3.5.  86  SUMMARY  NH INFLUX 3 ‘ KINETICS OF 4 4.1.  Introduction  88 88  4.2. Materials And Methods Plant growth and 1 4.2.1. N production 4.2.2. Relative growth rate Influx measurement 4.2.3. Kinetic study 4.2.4. 4.2.5. Metabolic inhibitor study 4.2.6. Temperature study pH profile study 4.2.7.  90 90 90 91 91 92 93 93  4.3.  94  Results  Kinetics of 13 4.3.1. 4 influx NH 4.3.2.1. HATS 4.3.1.2. LATS 4.3.2. Effect of metabolic inhibitors on the influx of 13 4 NH 4.3.3. Effect of root temperature on 4 NH influx 3 ‘ 4.3.4. Effect of solution pH on 4 NH influx 3 ‘ 4.4. Discussion Kinetics of ammonium uptake 4.4.1. 4.4.2. Energetic of ammonium uptake 4.4.3. Effect of pH profile on ammonium uptake 4.4.4. Regulation of ammonium uptake viii  94 94 98 98 101 104 104 104 107 111 112  4.5.  Summary  114  Chapter 5. ELETROPHYSIOLOGICAL STUDY 5.1.  Introduction  115 115  5.2. Materials And Methods 5.2.1. Growth of plants Measurements of cell membrane potential 5.2.2. 5.2.3. Experimental treatments 5.2.3.1. Effect of [NH4+] 0 on A’F 5.2.3.2. Effect of accompanying anion on A’P 5.2.3.3. Effects of metabolic inhibitors on NH4-induced AP depolarization  116 116 117 118 118 118  5.3. Results 5.3.1. Transmembrane electrical potentials of rice roots 5.3.2. Contribution of the accompany anions to AW Effect of 0 5.3.3. C1] on A’P 4 [NH Effect of metabolic inhibitors on Z’{’ 5.3.4.  120 120 120 123 126  5.4. Discussion 5.4.1. Anion effect 5.4.2. Depolarization of A’P by HATS and LATS 5.4.3. Calculation of the free energy for NH -’- transport 4 5.4.4. Mechanisms of NH 4 uptake by HATS and LATS  130 130 131 135 138  5.5.  Summary  139  Chapter 6. REGULATION OF AMMONIUM UPTAKE 6.1.  119  Introduction  141 141  6.2. Materials And methods 6.2.1. Plant growth and 13 N production 6.2.2. Experimental design 6.2.2.1. Experiment I. Depletion and repletion study 6.2.2.2. Experiment II. Effects of MSX 6.2.2.3. Experiment III. Effects of exogenous amino acids 6.2.2.4. Experiment IV. Effects of selected inhibitors 6.2.3. Determination of free ammonium in root tissue 6.2.4. Determination of amino acids in root tissue  143 143 144 144 144 145 145 145 146  6.3. Results 6.3.1. Experiment I. Depletion and repletion study 6.3.2. Experiment II. Effects of MSX  146 146 156  lx  6.3.3. 6.3.4.  Experiment III. Effects of exogenous amino acids Experiment IV. Effects of selected inhibitors  6.4 Discussion Negative feedback on NH4 uptake by NH 6.4.1. 4 assimilates Effect of MSX: reduced amino acid pool 6.4.2. 6.4.3. Effect of short-term N depletion 6.4.4. 4 influx after long-term N depletion Stimulated NH Negative feedback on 4 6.4.5. NH influx from internal 1 -I4 NH 6.4.6. Cascade regulation system of nitrogen uptake Chapter 7. INTERACTION BETWEEN K AND NH 4 7.1.  Introduction  163 172 176 176 178 181 183 185 188 193 193  7.2. Materials And Methods Plant growth and 1 7.2.1. N production 7.2.2. Experimental design 7.2.1.1. Experiment I: Effects of K and NO - in pretreatment 3 and K-- and NH ÷ in uptake solutions on net K+ and 4 4 fluxes NH 7.2.1.2. Experiment II: Effects of NH 4 provision during growth and of K-- and NH4 in pretreatment and uptake solutions on 86 Rb (K+) influxes 7.2.1.3. Experiment III: Effects of NH 4 provision during growth and presence in uptake solution upon influx isotherms for 86 Rb (K+) 7.2.1.4. Experiment IV: Effects of NH 4 provision during growth and short-term pretreatment upon 86 Rb (K) influx 7.2.1.5. Experiment V: Effect of NH 4 concentrations present in uptake solution upon influx isotherms for 86 Rb (K-I-) 7.2.1.6. Experiment VI: Effects of K provision during growth and presence in uptake solutions upon influx isotherms for 4 NH 1 7.3. Results 7.3.1. Experiment I: Effects of K and N0 3 in pretreatment K-I4 NH 1and and in uptake solutions on net K+ and 4 fluxes NH x  194 194 194  195  195 196 196 196 197 197  197  7.3.2. 7.3.3. 7.3.4.  7.3.5. 7.3.6.  Experiment II. Effects of NH 4 provision during growth and of K and NH 4 in pretreatment and uptake 86Rb+ solutions on (K) influxes Experiment III: Effects of NH 4 provision during growth and presence in uptake solution upon influx Rb+ (K) 6 isotherms for 8 Experiment IV: Effects of NH 4 provision during Rb+ 6 growth and short-term pretreatment upon S (K) influx Experiment V: Effect of NH 4 concentrations present in uptake solution upon influx isotherms for 86 Rb (K) Experiment IV: Effects of K provision during growth and presence in uptake solutions upon influx isotherms for 4 NH 1  7.4. Discussion 7.4.1. Plant growth in response to provisions of NH 4 and K 7.4.2. Effect of plant N status on K Rb) 86 uptake ( 7.4.3. 4 in the uptake solution on K Rb+) Effect of NH 86 ( uptake Effect of K on NH 7.4.4. 4 uptake 7.4.5. Shared transport and different feedback signal? Chapter 8. GENERAL CONCLUSIONS  200 203  206 206 210 216 216 218 220 222 224 226  REFERENCES 228 APPENDIX A. Reported studies on using radioactive isotope ‘ 262 N 3 APPENDIX B. Reported values of half-life (t ) and ion contnt (Q.) of 2 / 1 various compartments of root cells 263  xi  Abbreviations AA AFS AOA Arg Asn Asp Azaserine CCCP CN DES DMRT DNP DON  amino acids Appearent free space amino-oxyacetate Arginine Asp arigine Aspatarte O-diazoacetyl-L-serine; carboxylcyanide-m-chlorophenyl-hydrazone; (sodium) cyanide; diethyistilbestrol; Duncan’s multiple range test. 2 ,4-dinitrophenol; 6-diazo-5-oxo-L-norleucine; transmembrane electrical potential difference; rate of assimilation of 4 NH in roots; 3 ‘ mass flux across the tonoplast into vacuole; translocation of 13 N labeled metabolites to xylem (shoots); inward, outward and net fluxes (iimol g’FW h-i) Poc, ‘P, and net across the plasmalemma, respectively; G2, G100 and G1000 plants rice seedlings grown in MJNS containing 2, 100 or 1000 jiM NH , respectively; 4 G2M, GlOOM and G1000M MJNS containing 2, 100 or 1000 jiM , respectively, as growth media; 4 NH glutamate dehydrogenase (GDH; EC 1.4.1.2) GDH Gln Glutamine Glutamate Glu GOGAT glutamate synthase; glutamine synthetase; GS HATS or LATS high affinity or low affinity NH 4 transport systems, respectively; the external ion concentration giving half of the maximum Km rate (jiM); LSD Least significant difference; MA methylamine MJNS modified Johnson’s nutrient solution; xii  MSX NiR NR pCMBS  L-Methionine DL-Sulfoximine nitrite reductase nitrate reductase p-chloromercuribenzene-sulfonate; Qj, Q, Q ., ammonium contents (jimol g’FW) of root, cytoplasm and 3 vacuole, respectively; SHAM salicyihydroxamic acid; 0 and Sradioisotopic specific activities of external media and S cytoplasmic compartments, respectively; the calculated maximum rate of ion influx (jimol g-’FW h-’); Vmax cytoplasmic ammonium concentration (iiM or mM); [NH4] 1 J 4 [NH root (internal) ammonium concentration ( i,M or mM); 1 0 ] 4 [NH external ammonium concentration (jiM or mM); vacuolar ammonium concentrations (jiM or mM); ] 4 [NH  xlii  List of Tables Table Table Table  1. Separation of 13 N-labeled compounds by cation exchange column.  64  2. Estimated half-lives of 4 NH exchange for three 1 compartments of root cells.  66  3. Comparison of 4 NH fluxes across the plasmalemma of 1 root cells.  67  Table 4. Size of ammonium pools in root cells at steady-state.  (P,) from cytoplasm into vacuole.  Table  5. Calculation of the flux  Table  6. Distribution of newly absorbed 13 N in shoot and root tissues.  Table  70 72 73  7. Kinetic parameters for 4 NH influx of G2, G100, G1000 3 ‘  plants.  96  Table  8. Reduction of 4 N 3 ‘ ’H influx by metabolic inhibitors.  102  Table  9. Qo values for 4 NH influx by the HATS or LATS. 3 ‘  103  Table 10. Effect of uptake solution pH on 13 4 influx. NH  105  Table 11. Membrane potentials of G2 and G100 plants measured in different bathing solutions.  121  Table 12. Effect of metabolic inhibitors on the depolarization of A’P.  129  Table 13. Net 86 Rb flux of rice plants grown with or without either potassium and ammonium.  198  Table 14. Net NH4 flux of rice plants grown with or without either potassium and ammonium.  199  Table 15. Michaelis-Menten kinetic parameters for 86 Rb influx of plants grown in different levels of NH -’- and K. 4 208 Table 16. Effects of NH -- and K on plant growth. 4  211  Table 17. Michaelis-Menten kinetic parameters for 4 N 3 ‘ ’H influx of plants grown in different levels of potassium and ammonium. 213 xiv  List of Figures  Figure  4 N 3 H convertion in laboratory. 1. Scheme of ‘  34  Figure  2. Diagrame of the setup for measuring cell membrane potential.  56  Figure  3. A represented pattern of 13 4 released intact roots. NH  65  Figure  4. Fluxes of G2, G100 and G1000 plants at steady-state.  69  Figure  5. Cumulative uptake of 1 4 N H by G2 and G100 roots.  74  Figure  6. Influxes of 4 NH into G2 and G100 roots at steady3 ‘ state.  76  Figure Figure  7. Proposed  model for ammonium conpartmentation in rice roots.  uptake  and 84  8. Concentration dependence of 13 4 influx at low range NH (<1 mM).  95  9. Relationship between kinetic parameters of NH 4 uptake and root ammonium concentrations of rice seedlings.  97  Figure 10. Concentration dependence of 13 4 influx at low range NH (>1mM).  99  Figure  Figure 11. Effect of metabolic inhibitors on ‘ 4 N 3 H influx.  100  Figure 12. Effects of some anions on AW depolarization.  122  Figure 13. The A’P depolarization of root cell by 4 NH C 1.  124  Figure 14. Concentration dependence of net A’P depolarization of root cells.  125  Figure 15. Effects of metabolic inhibitors on A’F depolarization of root cells.  127  Figure 16. Effects of metabolic inhibitors on NH C 1. induced by 4  128  &{-‘  depolarization  Figure 17. The relationship between 4 NH influx and zP 3 ‘ depolarization at the same [NH . 0 ] 4 xv  134  Figure 18. Free energy requirement for NH 4 uptake as a function of +J. 4 external {NH  136  Figure 19. 13 4 influx of repleted G2 plants. NH  147  NH influx of depleted G1000 plants. 1 Figure 20. 4  148  Figure 21. Internal ammonium content of repleted G2 plants.  149  Figure 22. Total amino acid concentration plants.  ) 1 ([AA]  of repleted G2 151  13NH influx (23A) and internal ammonium content + Figure 23. 4 (23B) of repleted G2 or depleted G1000 roots.  153  Figure 24. Total 1 [AA] of repleted G2 or depleted G1000 roots.  154  Figure 25. Tissue amide or amino acid contents of repleted G2 or depleted G1000 roots.  155  Figure 26. Effect of MSX on 1 4 N H influx of rice roots.  158  Figure 27. Effect of MSX on ammonium content of rice roots.  159  Figure 28. Effect of MSX on total [AA] 1 of rice roots.  160  Figure 29. Effect of MSX on root content of amide or amino acid.  161  Figure 30. Effect of exogenous glutamine on root 13 4 influx. NH  164  Figure 31. Effect of exogenous glutamine on root contents of amide and amino acid.  165  Figure 32. Effects of exogenous glutamine on ‘ 4 N 3 H influx.  166  Figure 33. Effects of exogenous amides and amino acid on root ammonium content.  168  Figure 34. Effects of exogenous amides and amino acid on total amino acid content.  169  Figure 35. Effects of exogenous amides and amino acid on amino acid content.  170  Figure 36. Effects of exogenous amides and amino acid on amino acid content.  171  Figure 37. Effects of MSX, DON and AOA on 13 4 influx. NH xvi  173  Figure 38. Effects of MSX, DON and AOA on internal ammonium and total amino acid content. Figure 39.  174  Effects of MSX, DON and AOA on major amino acids content.  175  Figure 40. Effect of NH 4 in the growth media, pretreatment and uptake solutions on 86 Rb influx.  201  Figure 41. Effects of NH ÷ and K in growth media and uptake 4 solutions on 86 Rb influx.  202  Figure 42. Relationship between estimated Vm of 86 Rb influx and roots internal {K÷].  204  Figure 43. Effect of short-term NH 4 pretreatment on 86 Rb influx.  205  Figure 44. Effects of NH 4 and K in growth media and uptake solutions on 86 Rb uptake isotherm.  207  Figure 45. Effects of NH 4 and K in growth media and uptake solutions on 86 Rb translocated to shoots.  209  Figure 46. Effect of K in uptake solution on ‘ 4 N 3 H influx isotherm.  212  Figure 47. Effect of K in uptake solution on ‘ 4 N 3 H influx by HATS  214  Figure 48. Effect of K in uptake solution on 4 NH influx By 3 ‘ HATS+LATS.  215  xvii  To my wife, Xiao Ge for her love, understanding and sacrifice  xviii  ACKNOWLEDGMENTS  My sincere gratitude to research supervisor Dr. A.D.M. Glass, for his guidance, encouragement and moral support throughout this project. His time and patience in editing this thesis is greatly appreciated. Gratitude is extended to my advisory committee members: Dr. A. A. Bomke, Dr. P. J. Harrison, and Dr. I. F. P. Taylor, for their guidance. A special thanks must be expressed to Dr. M. Y. Siddiqi, for his invaluable suggestions and dependable assistance. The financial assistance provided by the Potash & Phosphate Institute of Canada is gratefully acknowledged. Sincere thanks is due to Dr. J.E. Hill of University of California, Davis, U.S.A. for providing rice seeds as a generous gift during this research project. To perform experiment using 13 N, with a half-life of 9.98 mm, requires team-work. Special appreciation is extended to member of the 13 brigade’: Mala Fernando, Bryan J. King, Hebert Kronzucker, Jarnail ‘ N Mehroke for ‘lending a hand’ and ‘sparkling’ discussions. My thanks also go to the Botany workshop, Mr. Mel Davis and Ken Jeffries for their willingness and skillfulness to help me out in my technical problems. Many thanks is due to the team in TRIUMF, UBC, who provided 13 N for this study. I greatly appreciate willingly cooperation from Michael Adams, Tamara Hurtado, Salma Jivan and other team members during ‘ N 3 production and transportation. My gratitude also extends to the Radiological unit in University Hospital, UBC site, for allowing me to pick up ‘Red Rabbiter’ in their terminal of the underground Pipe-line. The appreciation is also extended to Drs. John Hobbs and Krystyne Piotrowska for amino acids analysis. I would like to express my sincere appreciation to the U.S. Plant Soil and Nutrition Laboratory, USDA-ARS, Cornell University, and particularly to Dr. L.V. Kochian for hosting me as a visiting scientist to carry out the electrophysiological study in his laboratory. My special gratitude extend to Mr. J. E. Shaff, who patiently taught me how to operate various items of equipment and for kindly looking after me during my stay in Ithaca. I xix  Ms. L. Armstrong and would also like to thank Drs. J.W. Huang and P. Ryan, Mr. T. Toulemonde for assistance and discussions. staff and faculty I am grateful to fellow graduate students as well as port and friendship. I members of the Botany department for their sup in Soil and Fertilizer wish to express my gratitude to former colleagues . Institute, Zheijiang Academy of Agricultural Sciences back home in A very special thanks is to all my family members families of Bill and Kris, China. I am grateful to all my friends, particular to a brother and gave me Jame and Jill, Warren and Liz, who treated me like and my family strong support in many aspects. expressed to my At last, but not least, my special gratitude must be support all through this wife, Xiao ge and my son, Li ren for their love and program.  xx  1  Chapter 1.  RESEARCH BACKGROUND  1.1. GENERAL INTRODUCTION  1.1.1. Rice Rice (Oryza sativa Linneaus) is a semi-aquatic, annual grass plant in the family Poaceae (formerly Graminae). Rice is grown in over 100 countries on every continent except Antarctica, extending from 5 3°N to 35°S latitude, from sea level to 3000 m altitude (Lu and Chang, 1980; Mikkelsen and De Datta, 1991). Rice grows either as an upland (dry) or lowland (wet) crop in the tropic, subtropics, temperate, and subtemperate zones and on plains, hilly regions, and plateaus. About 53% of total land area under rice cultivation is irrigated, producing 73% of the world’s rice (De Datta, 1988). More than 90% of the world’s rice is produced in Asia (IRRI, 1988). Rice is the staple food and the energy source for about 40% of the world’s population (De Datta, 1981, 1986b); it supplies the energy source for more than half of the world’s population and provides 75% of the caloric intake of Asia’s over two billion people (Buresh and De Datta, 1991).  1.1.2. Essentiality of nitrogen Nitrogen is required for the synthesis of amino acids, proteins, nucleic acids and many secondary plant products such as alkaloids. It is involved in the whole life cycle of plants; in enzymes for biochemical  2 processes, in chlorophyll for photosynthesis, and in nucleoproteins for the control of hereditary and developmental processes. Since N is present in so many essential compounds, it is not surprising that growth without sufficient N is slow. Nitrogen is the single most important chemical element limiting crop yield.  1.1.3. Necessity of N fertilization Proper application of N increases both yield and protein content of rice (Patrick et al., 1974; Gomez and De Datta, 1975; Allen and Terman, 1978). The intensification of rice production has involved a tremendous increase in the use of nitrogen fertilizers and the selection of high yielding varieties that are highly responsive to nitrogen. However, research on the effects of nitrogen fertilizers on rice production has focused mainly on the agronomic context, in terms of grain yield, carbohydrate metabolism, growth patterns or morphological characteristics. Information concerning physiological and biochemical aspects of nitrogen uptake by rice as well as other higher plants, is limited, which is unfortunate since these details may prove to be important for the production of new varieties with improved nitrogen utilization.  L1.4. Blo-availability of nitrogen  Ammonium is the predominant and most readily bio-available nitrogen form in paddy soil; it is the preferred nitrogen species taken up by rice plants (Sethi, 1940; Sasakawa and Yamomoto, 1978; Goyal and Huffaker, 1984). Besides NH , rice roots also absorb N0 4 3 (Malavolta,  3 1954) and organic nitrogen such as urea, Gin and Arg (Arima and Kumazawa, 1977; Mon et al., 1979; Mon and Nishizawa, 1979; Harper, 1984).  1.2. AMMONIUM UPTAKE  1.2.1. Importance of transport research Information on the ammonium transport system(s) of root cells of rice, and their regulations, is meagre. Moreover, the relationships among uptake, assimilation and other metabolic processes are not as well understood as is the case for other plant nutrients. To understand the ammonium transport system(s), generally, it is necessary to characterize their kinetics, energetic and genetic properties. In order to achieve this, fluxes should be measured in response to variation of concentration, temperature and pH, and the effects of metabolic inhibitors should be determined. Where transport mutants are available, the genetic basis of the transport system(s) can be evaluated (Kleiner, 1981, 1985; Glass, 1988). This information satisfies more than the researcher’s curiosity; it provides a better understanding of ammonium uptake for the development of better fertilization practice and improved variety selection.  4 4 by lower plants 1.2.2. Transport of NH Ammonium uptake has been well studied in bacteria, fungi and algae (Kleiner, 1975, 1981, 1985; Roon et al., 1977; Pelley and Bannister, 1979; Smith and Walker, 1978; Boussiba et al., 1984). In brief, ammonium can be accumulated against its concentration and electrochemical potential gradients, resulting in significant ammonium concentration within plant cells (Smith and Walker, 1978; Pelley and Bannister, 1979; Kleiner, 1981; Boussiba et al., 1984). NH 4 uptake is concentration dependent and its isotherm in the low range of external concentration conformed to Michaelis-Menten kinetics (Hackette et al., 1970; Dubois and Grenson, 1979; Felle, 1980; Fuggi et al., 1981; Smith, 1982; Box, 1987). NH 4 transport across the plasma membrane has been claimed to occur via an electrogenic uniporter which depolarizes membrane electrical potential differences (Barr et al., 1974; Haines and Wheeler, 1977; Slayman, 1977; Raven and Smith, 1976; Smith et al., 1978; Smith and Walker, 1978; Walker et al., 1979a, 1979b; Laane, 1980; Raven, 1980; Smith, 1980; Kleiner and Fitzke,1981; Berti et al., 1984; Ulirich et al., 1984). The Qo value for NH + uptake has been reported to be 4  2.0 (Hackette et al., 1970)  and ATP may be involved in the transport step, hence the uptake system is inhibited by anaerobiosis or several metabolic inhibitors (Stevenson and Silver, 1977; Cook and Anthony, 1978a; Felle, 1980). The responses of NH 4 uptake to pH changes is complex (Hackette et al., 1970; Roon et al., 1977; Kleiner, 1981). The optimum pH was 67 for bacteria and fungi. The existence of specific proteinaceous carriers for NH 4 uptake is supported by biochemical, kinetic and physiological evidences. Moreover, NH 4 transport mutants have been isolated and some transport genes have been identified and cloned (Arst and Page, 1973; Castorph and Kleiner, 1984; Holtel and Kleiner, 1985; Franco et al., 1987; Reglinski et al., 1989).  5 1.2.3. Transport of NH 4 by higher plants There is a limited literature available regarding NH ÷ transport in 4 higher plants (Highinbotham et al., 1964), although a number of kinetic studies were reported for NO - uptake (Deane-Drummond and Glass, 3 1982a, 1983a; Siddiqi et al., 1990; Hole et al., 1990; Wieneke, 1992). Generally the NH 4 transport systems in higher plants are very similar to those in lower plants. Ammonium transport is localized perhaps at the plasma membrane and possible other membranes (Kleiner, 1981; Churchill and Sze, 1983). Evidence from kinetic studies of ammonium uptake by plant roots indicates that NH 4 transport is a carrier-mediated process (Nissen, 1973; Joseph et al., 1975). There are several lines of evidence that support the existence of the proteinaceous carriers to be discussed in the following sections. 1.2.3.1. Carrier-mediated transport Evidence indicating that ammonium transport is a carrier-mediated process (Nissen, 1973; Joseph et al., 1975) comes from determining kinetic parameters for NH 4 accumulation in cells (Kleiner, 1985). The uptake of 4 by barley, rice, ryegrass, tomato, and wheat is concentration NH dependent and follows Michaelis-Menten kinetics, (Tromp, 1962; Lycklama, 1963; Fried et al., 1965; Cox and Reisensuer, 1973; Rao and Rains, 1976; Bloom and Chapin, 1981; Youngdahl et al., 1982; McNaughton and Presland, 1983; Bloom, 1985; Deane-Drummond and Thayer, 1986; Smart and Bloom, 1988). Presland and McNaughton (1986) examined the rates of NH4 uptake as a function of external [NH ] in corn, and reported 4 a saturable system below 1 mM 4 [NH ’-]. In a continuously flowing nutrient solution system, NH 4 uptake rates of intact rice plants were fitted to a Michaelis-Menten model (Fried et al., 1965; Youngdahl et al., 1982).  6 1.2.3.2. Concentration dependent kinetics A biphasic pattern of NH 4 uptake, with both saturable and linear phases, was first reported in Lemna, by Ulirich et al., (1984). For crops, like corn, and rice, NH 4 uptake kinetics (below 1 mM 4 [NH commonly ) 0 ] conform to Michaelis-Menten patterns with Km values ranging from 0.0 14 to 0.167 mM (Fried et al., 1965; Youngdahl et al., 1982; Presland and McNaughton, 1986; Glass, 1988). Km values of 0.075 and 0.103 mM and a Vmax of 0.061 and 0.017 mmol kg-’ s’ were obtained for 4-week-old and  9-week-old rice plants, respectively, (Youngdahl et al., 1982). The second system, above 1 mM 4 [NH ] , failed to correspond to Michaelis-Menten kinetics (Ullrich et al., 1984; Presland and McNaughton, 1986). Generally, uptake studies at high external concentration have been achieved only with some difficulty, because depletion of the external solution is so small. 1.2.3.3. Depolarization of membrane potential The inward movement of ammonium occurs as the cation 4 NH ’(Walker et al., 1979a, 1979b; MacFarlane and Smith, 1982; Kleiner, 1985; Deane-Drummond, 1986). Only one report measuring AW in rice roots has appeared in the literature: Usmanov (1979) reported AP to be -160 mV. As early as 1964, Higinbotham et al., noted the marked depolarizing effect of 0 + 4 [NH ] on coleoptile cells AP in oats. Ullrich et al., (1984) found that, in Lemna, depolarization of zSM’ by NH 4 below 0.2 mM [NH 0 was ] 4 concentration-dependent and both NH 4 uptake and z’P depolarization responded in a saturable fashion with half saturation values of 17 pM for both processes. From 0.2 to 1 mM, net uptake of NH ÷ responded linearly 4 to , 0 + 4 [NH ] with no further AP depolarization. Since 4 NH ’- is the main species taken by plant roots, it must be taken up via active transport and/or  7  facilitated diffusion. Both processes are coupled to an energy source, either directly (the former) or indirectly (the latter). 1.2.3.4. Energy dependence  Metabolic energy is important to NH 4 uptake. Macklon et al., (1990) has shown that NH 4 absorption by excised root segments of Allium cepa L. was an active process. The uptake of ammonium at high temperature (2530°C) is closely associated with metabolism (Sasakawa and Yamamoto, 1978), and the uptake process was also decreased when carbohydrate levels were reduced (see Section 1.3.1.1.) or when temperatures were lowered (see Section 1.3.2.1.).  1.3.  MAJOR FACTORS AFFECTING AMMONIUM UPTAKE Besides the mechanism and kinetics of ammonium uptake, research  on ammonium uptake has also included other related issues such as the effect of energy status, nitrogen cycling within the plant, the effects of root pH and temperature. It must be emphasized that when environmental factors are concerned, one must be aware of the root’s capacity to adapt ion uptake in response to changed conditions, especially in long-term experiments.  1.3.1. Effects of photosynthesis 1.3.1.1. Dependence on soluble carbohydrates  Of major importance in the uptake of ammonium is the energy status of the plant. The energy status of rice plants had a substantial influence on  8 the uptake of 4 NH ’- and on its conversion into high molecular weight N compounds (Mengel and Viro, 1978). The high demand for carbohydrate is in order to achieve active transport of NH 4 at low external concentration, and to supply carbon skeletons for the rapid assimilation of 4 NH ’- as it is absorbed by roots (Givan, 1979; Fentem et al., 1983a, 1983b). When the availability of carbohydrate is low, the assimilation of 4 NH ’- is also low (Breteler and Nissen, 1982), and consequently a high efflux rate of NH 4 may result. A general relationship exists between the proportion of total nitrogen absorbed as NH 4 from mixed N sources such as NH 3 N 4 O and the availability of soluble carbohydrates in roots. (Raper et al., 1992). The concentration of soluble carbohydrates in the leaves of NH4-fed plants was greater than that of 3 N0 -fed plants, but was lower in roots of NH 4 fed plants, regardless of pH (Chaillou et al., 1991). The study of NH 4 uptake isotherms in Chiorella revealed that preincubation with glucose drastically increased Vmax (5-fold), with no change of Km (Schlee and Komor, 1986). It was reported that glucose induced a glucose transport system and two specific amino acid transport systems (Cho et al., 1981). Glucose also induced the transport systems for ammonium, nitrate and urea (Schlee et al., 1985). Removal of the endosperm of rice seedling suppressed NH 4 uptake markedly (Sasakawa and Yamamoto, 1978), while the addition of 30 mM sucrose restored uptake. In higher plants, provision of carbon skeletons in the form of a-ketoglutarate increased uptake and association of NH 4 in Lemna (Monselise and Kost, 1993). 1.3.1.2. Periodic variations of light and growth  There is a great variation in NH 4 assimilation rates between day and night during the tillering stage of rice plants (Ito, 1987). This is probably  9  related to the diurnal changes in carbohydrate flux from shoot to root resulting from changes in relative source-sink activity of shoots (Rufty et al., 1989; Lim et al., 1990). This periodic variation of carbohydrate supply is also influenced by morphological variations of plant growth (Henry and Raper, 1989a; Vessey et al., 1990b). The net rate of NH 4 uptake oscillated between a maximum and a minimum with a periodicity co-ordinate with intervals of leaf emergence (Tolley and Raper, 1985; Tolley-Henry et al., 1988; Henry and Raper, 1989a; Rideout et al., 1994). Changes of both influx and efflux were responsible for the observed differences of net 4 NH ’uptake (Henry and Raper, 1989). 1.3.1.3. Ambient environmental factors  The ability of the plant root to absorb nitrogen was affected by previous growth conditions of the examined plants (Mon et al., 1979), since environmental factors will influence the carbohydrate status. Susceptibility of plants to NH 4 toxicity is also related to plant carbohydrate status (Nightingale, 1937; Prianishnikov, 1941; Givan, 1979). The soluble carbohydrate concentration in roots increased with increasing root temperature (Clarkson et al., 1975; Macduff et al., 1987a) and with nitrogen deprivation (Rufty et al., 1988; Henry and Raper, 1991), and decreasing rhizospheric pH (Chaillou et al., 1991). High ambient C02 concentration increased total plant N and total nitrate-N content and leaf area but not leaf number of soybeans.  10 1.3.2. Effects of root temperature 1.3.2.1. Short-term perturbation Ammonium transport across the plasma membrane is sensitive to temperature. Although ion accumulation at steady-state may be independent of external concentration or temperature, both of these factors influence short-term fluxes (Cram, 1973; Smith, 1973, Glass, 1983). In a 5-hour root temperature perturbation study, it was found that the uptake and assimilation of ammonium were profoundly affected in both Indica and Japonica rice plants (Ta and Ohira, 1981). This might be explained by the dependence of the NH 4 uptake system on the rate of metabolism (Raven and Smith, 1976), or effects of low temperature on enzymes of 4 NH ’- assimilation (Shen, 1972). The effect of temperature on ion uptake may also be due to physical changes in different parts of the cell membrane (e.g. membrane fluidity) instead of on the transport process (Clarkson and Warner, 1979). 1.3.2.2. Qo value for NH 4 uptake  Qio values can be used to indicate the temperature dependence of ion transport. When temperature is lowered or increased by 10°C, the ratio of the two transport rates can be calculated by equation: 1 2 {(t /10] LnQjo= ) 1 / 2 Ln(V t ) V  [1]  where t 1 and t 2 are the temperature before and after the change, and Vi. and V 2 are the transport rates at respective temperatures. When Qo is close to 1, the transport rates are the same at the different temperatures, and ion transport is insensitive to temperature. A Qo value greater than 2 is often considered as indicating the metabolic dependence of a  11 physiological process such as ion transport. In a seven hours perturbation of root temperature, Sasakawa and Yamamoto (1978) found that the uptake of ammonium by 9-days old rice seedlings was closely associated with metabolism. The Qo values between 9  24°C were> 2.5 for 15 ÷ 4 NH  absorption by rice roots estimated from Ta and Ohira’s (1981) data. Low  Qio values (1.0  1.5) were reported for net ammonium uptake of low-  temperature adapted ryegrass and oilseed rape (Clarkson and Warner, 1979; Macduff et al., 1987). 1.3.2.3. Long-term low temperature effects  The effect of root temperature on ion uptake varies with the treatment duration. Plants may adjust rates of ion transport in the longterm so that net uptake is independent of external variables such as temperature (Clarkson, 1976). As a result of plant adaptation to low root temperatures,  4 is absorbed more readily than N0 NH 3  at low  temperatures by roots of Italian and perennial ryegrass (Lycklama, 1963; Clarkson and Warner, 1979; Clarkson et al., 1986) and lettuce (Frota and Tucker, 1972). Ammonium uptake by 4 day corn roots occurred even at temperatures as low as 0°C (Yoneyama et al., 1977). In both Indica and Japonica rice plants ammonium and nitrate uptake and assimilation were strongly affected by temperature (Ta and Ohira, 1981). The uptake as well as assimilation of the two forms of nitrogen were greatly inhibited at low temperature and low light intensity. At low root temperature, uptake of NH 4 was higher than that of N0 . The 3 proportion of 4 NH ’- absorbed from mixed 4 NH ’- and N0 3 solution was increased as root temperature decreased from 13 to 3°C (Macduff and Wild, 1989). Likewise, transferring corn roots from 30°C to 0°C, reduced  12 3 N 5 ‘ 0 uptake more drastically than 1 4 N H uptake (Yoneyama et al., 1977). NH uptake to reduced temperature + The lower sensitivity of 4 (compared to NO - uptake) might be explained by a lesser dependence of 3 + uptake on the rate of metabolism and energy production (Raven and 4 NH Smith, 1976), or less effect of low temperature on enzymes of NH + 4 assimilation (GS-GOGAT) compared to those enzymes of NO - uptake and 3 reduction (NR and NiR).  1.3.3. Effects of pH on NH 4 uptake It has frequently been reported that NH + uptake is higher at 4 elevated pH while NO - uptake is stimulated at low pH (van den Honert 3 and Hooysman, 1955; Fried et al., 1965; Jungk, 1970). When plants are grown in medium containing NH ÷ as the solo source of N, the inevitable 4 acidification of the medium may cause damage to the roots and even death of plants (Loo, 1931; Raven and Smith, 1976). Moreover root growth may be restricted in NH 4 medium even when the pH of the medium is controlled between 6.0 and 6.5 (Lewis et al., 1987). 1.3.3.1. Acidification of rhizosphere by NH 4 uptake A major factor in N uptake is the change of rhizosphere pH associated with NH 4 uptake and its effect on plant growth, root morphology and capacity for ion uptake. It is well known that NH 4 uptake will cause acidification of the growth medium (Raven and Smith, 1976). At high NH 4 concentrations an enhanced 4 NH ’- uptake by ectomycorrhizal fungi caused an accelerated medium acidification that indirectly inhibited growth  13 (Jongbloed and Borst-Pauwels, 1990). NH 4 has greater detrimental effects on plant roots than on shoots (Loo, 1931; Raven and Smith, 1976). Plants supplied with moderate concentrations of NH 4 generally grow poorly compared with plants supplied with other sources of nitrogen (Rufty et al., 1982b) or mixed 4 3 N0 /NH ’- supplies. Increased proportions of NH 4 in mixed NH 4 and NO - nutrient solutions increased shoot:root ratios at all 3 levels of root-zone pH (Vessey et al., 1990). When NH 4 and NO - were 3 supplied together, cumulative uptake of total nitrogen was not affected by 4 : NO pH or solution NH - ratio (Raper et al., 1991b). 3 1.3.3.2. Retarded plant growth in acidic medium Acidic growth medium will, in turn, affect plant growth and NH ÷ 4 uptake. Root growth was restricted by increased acidity between pH 6.0 to 4.0 (Arnon and Johnson, 1942; Islam et al., 1980). As the pH of the rootzone declined, therefore, NH 4 uptake decreased and N0 3 uptake increased (Vessey et al., 1990). It was reported that the growth rate of soybean shoots and roots was reduced by increasing pH (Rufty et al., 1982b). 1.3.3.3. NH 4 toxicity and acidic damage If acidification of the root medium is controlled, plant growth with 4 as the sole N source may be equal to growth with NO NH 3 (Barker et al., 1966; Rufty et al., 1983; Tolly-Henry and Raper, 1986a, 1989; Findenegg, 1987; Vessey et al., 1990). Soybean plants can effectively utilize NH 4 as a nitrogen source as long as root-zone pH is strictly controlled and a balance is maintained between carbohydrate availability and acquisition of NH 4 (Rufty et al., 1983). It was suggested that the inhibition of plant growth at -1- uptake and a consequential limitation 4 low pH was due to a decline in NH of growth by N stress (Vessey et al., 1990).  14 1.3.4. NH 4 fluxes at the plasma membrane 1.3.4.1. Net flux NET FLUX (net) describes the ‘net’ rate of ion uptake by roots. The net  ion uptake from the medium (outside) into the cytoplasm is determined by the balance between influx and efflux. In practice, net flux Pnet  =  Poc  -  Øco  [2]  is measured by the disappearance of tested ion in the uptake solution. 1.3.4.2. Influx INFLUX  () is defined as the rate of inward movement of solute  across a particular membrane. Strictly speaking influx should refer to the unidirectional movement measured during a very short period, short enough to discount the efflux. NH ÷ influx is negatively correlated with 4 plant N status in lower plants (Silver and Perry, 1981; Hartmann and Kleiner, 1982; Wiegel and Kleiner, 1982; Boussiba et al., 1984; Mazzucco and Benson, 1984; Rai et al., 1984; Jayakumar et al., 1985), and higher plants (McCarthy and Goldman, 1979; Pelley and Bannister, 1979; Smith, 1982; Ulirich et al. 1984; Holtel and Kleiner, 1985; Clarkson, 1986; Lee and Rudge, 1986; Morgan and Jackson, 1988a, 1988b; Clarkson and Luttge, 1991). MA influxes of pea seedlings decreased after pretreatment with glutamine and NH 4 and increased after pretreatment with asparagine (Deane-Drummond, 1986). 1.3.4.3. Efflux EFFLUX  (Ø) is the rate of outward solute flow from cytoplasm across  the plasma membrane. Efflux of ions from plant roots was identified in  15 plants under stress or damaged conditions (Pitman, 1963; Hope et al., 1966; Jackson and Edwards, 1966; Hiatt and Lowe, 1967; Ayers and Thornton, 1968; Bowen, 1968). In intact plants, efflux of K, Na, 4 P 2 H , 0 Cl-, Br-, or NO - has been observed from roots (MacRobbie, 1964; Cram, 3 1968, 1973; Dodd et al., 1966; Poole, 1969, 1971a, 1971b; Pitman, 1971; Morgan et a!., 1973; Macklon, 1975 a, 1975b; Macklon and Sim, 1976, 1981; Behi and Jeschke, 1982; Jeschke, 1982; Lazof and Cheeseman, 1986; Siddiqi et al., 1991). 4 efflux may be a common feature of net NH Continuous NH 4 uptake by roots of higher plants (Morgan and Jackson, 1973). In a study using N 4 ‘ -grown 0 roots were equilibrated in a intact ryegrass, 3 3 N 5 ‘ 0 solution enriched with ‘ N (97.5 atmo %). The results suggested that there was a 5 simultaneous occurrence of the influx of 3 N 5 ‘ 0 and efflux of ‘ 3 N 4 0 (Morgan et al., 1973). Moreover, careful measurements of 14 4 efflux NH revealed that there must have been generation of NH 4 by breakdown of nitrogen compounds during the course of the experiment. There was excess quantity of 4 NH effluxes compared with the initial content in the roots (Morgan and Jackson, 1988a). There is even an ‘ NH efflux from 14 4 3 N0 grown roots (Morgan and Jackson, 1988b). 1.3.4.4. Balance of fluxes  There is thought to be an ammonium cycle across the root cell plasma membrane (Morgan and Jackson, 1988b). It was reported that endogenous NO - effluxes to the unstirred layers were recycled through 3 - influx (Morgan et al., 1973). The same could be expected for NH 3 NO 4 efflux. Substantial ammonium cycling occurred during net ammonium uptake (Jackson et aL, 1993), yet plants grown under low N conditions possess a low NH -’- efflux. Morgan and Jackson (1988a) suggested that the 4  16 regulation of NH 4 uptake by roots of higher plants may involve changes of both influx and efflux in response to plant nitrogen status. It was found that net 15 4 influx was increased and net ‘ NH NH efflux was decreased 4 in nitrogen depleted wheat and oat seedlings (Morgan and Jackson, 1988a), and net 4 NH ’- uptake of barley and maize plants previously grown with NH 4 ’- was decreased subsequently (Morgan and Jackson, 1988b). The determining factor may be the internal [NH ] of the root cell. 4 For example, enhanced NH 4 influx by MSX treatment was claimed to be due to the enlargement of cytoplasmic and vacuolar NH 4 pools of root tissue several times (Jackson et al., 1993; Lee and Ayling, 1993) which appeared enhance the influx of ‘ 4 N 3 H of (maize and barley) plants by reducing isotopic efflux (Lee et al., 1992; Lee and Ayling, 1993). However, the enlarged [NH 1 was also advanced to explain the enhanced efflux ] 4 observed in their system (Morgan and Jackson, 1988b). 1.3.4.5. N cycling in the whole plant  Within the plant, N cycling, the simultaneous movement of Ncompounds from root to shoot, and from shoot to root (Cooper and Clarkson, 1989; Larsson et al., 1991) may enable N absorption to be regulated to match the demand imposed by plant growth (Drew and Saker, 1975; Edwards and Barber, 1976). The concentrations of amides (Gln and Asn) in the roots will be the result of the balance between their synthesis from absorbed inorganic N (NH 4 or N0 ), their import via the phloem, and 3 their export via the xylem (Lee et al., 1992).  17 1.3.5. Regulation of ammonium uptake Feedback inhibition of NH 4 uptake by nitrogenous effectors has been implicated in lower plants (Kleiner, 1985; Ullrich et al., 1984; Pelley and Bannister, 1979; MacFarlane and Smith, 1982; Wiame et al., 1985; Wright and Syrett, 1983; Thomas and Harrison, 1985) and higher plants (Cook and Anthony, 1978b; Breteler and Siegerist, 1984; Wiame et al., 1985; Revilla et a!., 1986; Lee and Rudge, 1986; Morgan and Jackson, 1988a). There is, however, only limited information available concerning the possible mechanism(s) of regulating 4 NH ’- uptake by either NH 4 per se or its primary assimilates. 1.3.5.1. Negative feedback regulation  At high nitrogen status, plant 4 NH ’- uptake could be suppressed due to (i) low energy supply to the root system, (ii) accumulation in the root tissue of nitrogenous compounds that exerts negative feedback on the transport system, or (iii) high efflux of endogenous NH 4 (Morgan and Jackson, 1988b). Repression of NH 4 uptake may be due to continual generation of ammonium from degradation of organic nitrogenous sources within roots and rapid accumulation of ammonium in roots of N-depleted plants upon initial exposure to ammonium (Morgan and Jackson, 1988a, 1988b). However, Morgan and Jackson (1988b) indicated that the immediate assimilates of NH -’-, such as glutamine, are more likely negative 4 effectors on 4 NH ’- uptake. 1.3.5.2. Enhanced NH 4 uptake  Negative correlation between ammonium uptake and cell nitrogen status have commonly been observed (McCarthy and Goldman, 1979; Pelley and Bannister, 1979; Smith, 1982; Ullrich et al. 1984; Holtel and  18 Kleiner, 1985; Clarkson, 1986; Lee and Rudge, 1986; Morgan and Jackson, 1988a, 1988b; Clarkson and Luttge, 1991). It has been recognized that the capacity for nitrogen uptake is enhanced in N-depleted plants such as wheat (Tromp, 1962; Minotti et al., 1969; Jackson et al., 1976b; Morgan and Jackson, 1988a, 1988b); ryegrass (Lycklama, 1963); maize (Ivanko and Ingversenm, 1971; Lee et al., 1992); barley (Lee and Rudge, 1986); and oats (Morgan and Jackson, 1988a, 1988b).  1.3.6. Interactions between NH 4 and K 1.3.6.1. Mutual beneficial effects between N and K N and K are essential plant nutrients, required for healthy plant growth and high yield production (Ajayi et al., 1970; Dibb and Welch, 1976; Kemmler, 1983; Dibb and Thompson, 1985; Grist, 1986; Biswas et al., 1987; Dey and Rao, 1989; Ichii and Tsumura, 1989; Fageria et al., 1990; Xu et al., 1992). Mutual beneficial effects of K and N on plant growth have often been described. An adequate K supply has been shown to enhance 4 uptake and assimilation (Ajayi et al., 1970; Barker and Lachman, NH 1986; Scherer and MacKown, 1987). Sufficient N nutrition normally promotes K-’- uptake due to the biological dilution effect of better plant growth (Noguchi and Sugawara, 1966; Kirkby, 1968; Claassen and Wilcox, 1974; Faizy, 1979; Lamond, 1979; Beusichem and Neeteson, 1982). 1.3.6.2. Inhibition of K÷ uptake by NH 4 However, NH 4 has been shown to strongly inhibit the absorption of K-’- in short-term experiments in many species including wheat, barley, maize and tobacco (Breteler, 1977; Munn and Jackson, 1978; Rufty et al.,  19 1982; Rosen and Carison, 1984; Scherer et aL, 1984). There was a negative correlation between the external NH 4 concentrations and K uptake (Rosen and Carlson, 1984; Scherer et al., 1987; Jongbloed et al., 1991), and net ammonium uptake was correlated with potassium efflux (Morgan and Jackson, 1989). The inhibitory effect of NH 4 on K uptake has been claimed to be independent of K-i- provision or pretreatments; it is probably exerted on the transport processes at the plasma membrane. Insufficient evidence is available to draw a conclusion regarding the inhibition of K uptake by 4 in terms of competitive and non-competitive effects (Deane NH Drummond and Glass, 1983b; Scherer et al., 1984). K uptake was suppressed during rapid 4 NH ’- uptake by N-starved plants (Tromp, 1962), but K-starvation did not produce the same effect as N-starvation on the transport of NH 4 (Tromp, 1962; Lee and Rudge, 1986). 1.3.6.3. Inhibition of NH 4 uptake by K  On the other hand, NH4 uptake of plants was not reduced by K in the nutrient medium (Mengel et al., 197 6; Rosen and Carison, 1984; Scherer and Mackown, 1987). However, the influence of K÷ on NH 4 uptake has not been consistent. It was reported that K had inhibitory effects but did not compete with NH 4 for selective binding sites in the absorption process (Ajayi et al., 1970; Dibb and Welch, 1976; Mengel et al., 1976).  1.4. REsEARCH OBJECTIVES  The objective of this study was to investigate the mechanisms and characteristics of ammonium uptake by rice plants. In particular, the  20 studies have emphasized short-term responses of fluxes to changes in ambient conditions. This particular goal was achieved by using the shortlived radioisotope 1 N  2 / 1 (t  =  9.98 mm), addressing five different areas:  (1). By measuring NH 4 influx and efflux, the exchange of N at the plasma membrane and the relationships between these fluxes were quantified. Subcellular distribution of absorbed NH 4 was also estimated. The results of these studies are interpreted in terms of a root cell model in Chapter 3. (2). To describe the kinetics of NH 4 uptake and the pattern(s) of its concentration dependence, NH 4 influx was measured in perturbation experiments in plants grown in different levels of N. By altering ambient conditions such as medium pH, root temperature, and by treating roots with various metabolic inhibitors, the energetic of NH 4 uptake was investigated. These are described in Chapter 4. (3). By measuring electrical potential differences together with assaying cytoplasmic 4 [NH ] , the electrochemical potential gradient for NH 4 between external solution and cytosol were defined in order to explore the mechanisms of NH 4 uptake. Membrane electrical potential differences of rice roots were recorded as a function of external NH 4 concentration. This information is incorporated with data dealing with biochemical, kinetic and energetic aspects of NH -’- uptake to formulate a model for the mechanisms 4 of NH -’- uptake (Chapter 5). 4 (4). Without information on the regulation of NH 4 uptake, the uptake model is incomplete. NH 4 influx was measured as a function of root N status. Internal 4 [NH ’-l was determined as well as the concentrations of individual amino acids. In Chapter 6, the results are discussed in reference to existing reports to develop a model of the regulation of NH -’- uptake. 4  21 (5). Chapter 7 deals with the interactions between NH 4 and K-’- at the uptake level and explores the effects of prior exposure to these ions on subsequent ion uptake.  22  Chapter 2.  METHODS AND MATERIALS  In this chapter, the general methods used in this study are described. Method(s) used in a particular experiment will be addressed in the corresponding chapter.  2.1. PLANT GROWTH  2.1.1. Seed germination  Rice seeds (Oryza sativa L. cv. M202) were surface sterilized in 1% NaOC1 for 30 mm  and rinsed several times with de-ionized distilled water.  Seeds were imbibed overnight in aerated dc-ionized distilled water at 3 8°C, then placed on plastic mesh mounted on Plexiglas discs. The discs were set in a Plexiglas tray filled with dc-ionized distilled water just above the level of the seeds, and seeds were allowed to germinate in a growth chamber in the dark (at 38°C) for 4 d. During the following 2 d, the temperature was stepped down to 20°C (by 9°C per day). Then discs, with one-week-old rice seedlings, were transferred to 40-L Plexiglas tanks.  2.1.2. Growth conditions Plants were grown hydroponically in 40-L Plexiglas tanks located in a walk-in growth room, in which growth conditions were maintained as follows: temperature: 20 ± 2°C; relative humidity: 75%; and irradiance: 300  1 under fluorescent light-tubes (VITA LITE, Duro-Test) on a cycle j.tE m 2 s  23 of 16 h light and 8 h dark. Plants were 3-week-old when they were used for most experiments unless specifically indicated.  2.1.3. Provision of nutrients The growth medium was modified based on the recipe of modified Johnson’s nutrient solution (Johnson et al., 1957; Epstein, 1972) and a recipe from the International Rice Research Institute (Yoshida et al., 1972), in which ammonium 4 (NH C 1) was the only source of nitrogen (except for some specific experiments as specifically indicated) and silicon was added as 0 S 2 Na . 3 5H iO . This modified Johnson’s nutrient solution (hereafter referred to as MJNS) was also the medium used to carry out all experiments. The composition of this MJNS, in micromolar (iiM), was 200 for Ca, K and P, 100 for Mg, 300 for S, 16 for B, 5 for Si and Fe, 1 for Mn and Zn, 0.3 for Cu and Mo. The external ammonium concentration ) 0 ÷ 4 ([NH ] was varied as indicated at the appropriate places. Generally plants were grown in MJNS containing 2, 100, or 1000 jiM , 0 + 4 [NH ] referred to hereafter as G2, G100, G1000 plants, respectively. The concentrations of nutrients in growth medium were maintained by infusion of appropriate stock solutions, through peristaltic pumps (Technicon Proportioning Pump II, Technicon Inst. Corp.). Generally 2 liters per day of stock solution were supplied and stock concentrations were determined from daily chemical analyses of medium samples. Solutions were mixed continuously by circulating pumps (Circulator Model IC-2, Brinkmann Inst., Inc.), and aerated continuously. The pH of growth medium was maintained at 6.0 ± 0.5 by adding powdered CaCO 3 (13 g/tank), according to measured pH values, 12 times daily.  24 2.2. N ISOTOPES FOR STUDYING N UPTAKE  2.2.1. Isotopic tracer There is now widespread use of isotopic tracers, particular radioactive tracers, in the biological sciences (Thain, 1984). Carbon (“C, 4C), phosphorus (32P), sulfur (5S), chlorine (36C1), potassium K), 42 ( rubidium ( Rb), 8 6 calcium ( Ca) 4 5 and sodium ( Na) 2 2 have been employed to determine the kinetics of transport and transformation of these elements in living systems. Measurements of radioisotopic influx and/or efflux have been used to obtain an estimate of the unidirectional fluxes of the stable isotope of the ion at the plasmalemma and tonoplast and to estimate the separate amounts of the stable isotopes in the cytoplasm and vacuole (Cooper, 1977; Thain, 1984). The utility of radiochemical techniques is afforded by (i) their great sensitivity compared to other analytical methods. Radioisotopic tracers may offer 10 -fold increased detection sensitivity over stable isotope 8 methods (Cooper, 1977; Krohn and Mathis, 1981); (ii) the fact that they “label” the atoms of molecules without significantly altering their chemical properties (Cooper, 1977; Boyer, 1986).  2.2.2. Nitrogen Isotopes There are six isotopes of nitrogen known, ranging in mass number from 12 to 17 (Kamen, 1957). The stable isotopes of nitrogen are ‘ N and 4 N, the latter being present to the extent of 0.365 atom per cent. 15 Radioactive isotopes 12 N and ‘ N are positron emitters with half-lives of 3  25 0.0125 seconds and 9.98 minutes respectively. ‘ N and ‘ 6 N are negatron 7 emitters with half-lives of 7.35 and 4.14 seconds respectively, 17 N also emits neutrons. The longest-lived radioactive isotope of nitrogen is ‘ N 3 which is the only radioactive isotope that has been used in tracer research (Kamen, 1957; Krohn and Mathis, 1981; Bremner and Hauck, 1982). The use of 1 N (Burns and Miller, 1941) in biological studies started as early as N (Ruben et al., 1940). 3 the use of ‘  2.2.3. Stable 15 N techniques Since the first use of 1 N (Burns and Miller, 1941), this isotope has been widely used in agricultural research (Hauck, 1982; Knowles and Blackburn, 1993), and the analytical methodology has been continuously improved (Clusius and Backer, 1947; loch and Weisser, 1950; Hürzeler and Hostettler, 1955; Broida and Chapmen, 1958; Faust, 1960; Mulvaney and Liu, 1991; Hoult et al., 1992). N has been used in characterizing the N0 5 ‘ 3  and 4 NH ’- uptake  processes of plants (Fried et al., 1965; Yoneyama and Kaneko, 1989; Yoneyama et al., 1991) and tracing the metabolism of nitrogen in plant cells (Yoneyama and Kumazawa, 1975; Arima and Kumazawa, 1977). 1 N is also widely used in studying 2 -N fixation in soil-plant systems, aquatic and sediment systems (Watanabe, 1993; Warembourg,  1993) and N  transformation in soils (Azam et al., 1993). It is also employed in studying the mineralization of soil organic N (Powlson and Barraclough, 1993) and nitrification and denitrification of soil N (Mosier and Schimel, 1993). N-labeled nitrogen fertilizer has also been used in the study of fertilizer 15 use efficiency (Azam et al., 1991).  26 Stable N isotope techniques have several advantages over techniques using radionuclides. As a biochemical tracer, ‘ N offers the advantages of 5 being relatively inexpensive, widely available, free of radiation hazard and less limiting in terms of experiment duration. The advantages of using ‘ N 5 also embodies a major disadvantage in its use as a tracer: a sizable background, present in all nitrogenous materials, against which added tracer must be measured (Cooper et al., 1985). In order to measure significant enrichment of 5 N in specific metabolic compartments, investigators have to administer a large amount of 5 ‘ N -labeled nonphysiological precursors to biological systems (Cooper et al., 1985). In addition it requires tedious preparation to convert samples to N 2 gas prior to mass or emission spectrometry.  2.2.4. Radioactive isotope, 13 N 2.2.4.1. Use in biological studies N was first made in 1934 by Joliot and Curie as ‘ 3 ‘ 4 N 3 H and was one of three isotopes generated artificially by induction of radioactivity in otherwise stable elements (boron) by bombardment with particles emitted by polonium (Joliot and Curie, 1934). It was first used as a biological tracer in studying the 2 -N fixation of non-legume barley plants (Ruben et al., 1940), which was one year earlier than the first report of using 15 2 to N study N 2 fixation (Burns and Miller, 1941). Much of the early tracer work in biochemistry was carried out with positron-emitting radionuclides, such as “C, and to a lesser extent 13 N, but with the introduction of  N, their importance declined over a C and 15 4 ‘  period of two decades. Only in the past 10 years or so, have these short-  27 lived isotopes again become important as tracers particularly in the field of biochemical research. With about 70 medical cyclotrons, there are at least 12 groups, that generate ‘ N for biological studies (Cooper et al., 1985). In 3 biological studies, there are several groups using ‘ N in study nitrogen 3 nutrition of plants (Appendix A). 2.2.4.2. Production of 13 N  N can be obtained from targets containing boron, carbon, nitrogen 13 or oxygen and an appropriate accelerated particle (Cooper et al., 1985). The N; ‘ 13 B(o,n) 10 C(d,n) 13 2 N; 12 C(p,y) 13 C(p,n) 13 3 N; ‘ N; ‘ N(p,pn) 13 4 N; 14 N(n,2n) ‘ N 3 and 16 N reactions have all been used to make ‘ 3 O(p,c)’ N (Krohn and 3 Mathis, 1981; Tilbury, 1981). The method most widely used at present for the production of ‘ N-ammonia is the proton irradiation of water 3 3 Q 6 (‘ N (p,)’ ), followed by reduction of the ‘ N0 and ‘ 3 2 N 3 0 formed under typical conditions of irradiation (Park and Krohn, 1978; McElfresh et al., 1979; Lindner et al., 1979; Tiedje et al., 1979; Chasko and Thayer, 1981; Cooper et al., 1985). An example flow scheme of ‘ 2 production based on nuclear N 3 reactions of N 3 O 6 ‘ (p,c’ is as follows: (Meeks et al.,1985) 20 MeV, 20 1 A  1. Generation:  0 2 H  >  3 N0 13  2 N 3 ‘ 0  +  +  4 N 3 ‘ H  HPLC/SAX column  2. Concentration: 60 ml N0 3 ‘  up to 3 ml ‘ N0 3  >  Devarda’s Alloy (Cu/Al/ Zn)  3. Reduction:  N0 3 ‘  >  NH 1 3  65°C Saturated NaOH  4. Trapping:  3 NH 13  +  H  >  4 N 1 H  28 Na/KOBr  5. Oxidation:  4 NH 13  >  [‘3N]-N  1.5 imo1 ‘ NH4 4  N varies with the types of nuclear reaction, target 3 The yield of ‘ material, and particle energy. Bombarding 10 ml pure water with an 10 jiA proton beam of high energy (>19 MeV) could yield 36 mCi pA 4 20 miw 1 (Vaalburg et al., 1975). The ‘ N species, , 3 N0 2 3 ‘ NO and , 1 4 N 1 H are present in the radioactive sample. The relative concentrations of these species is dependent upon the irradiation dose as well as on other factors such as the previous irradiation history of the target foil (Tilbury and Dahi, 1979). The study of the effect of integrated dose showed that at low dose 4 N 3 ‘ ’H is greater than ‘ 2 N 3 0 and at high dose NH 13 is less than ‘ 4 2 N 3 0 (Tilbury and Dahi, 1979). There are also some contaminants in the radioactive product. It was found that irradiated unprocessed water contains 18 F 1 (t = 2 / 1.8 h), 150 (t = 2 / 1 2.0 mm), and 48V 1 (t = 2 / 16.2 d). Both 18 F and 48V produce no problems with 4 NH since these radloisotopes do not distil. Since l8F is 3 ‘ from the reaction of F 0(p,n)’ 8 ‘ , its contamination can be minimized by depleting 180 in water (Skokut et al., 1978). Though 15Q can be detected in N-ammonia solution, it will disappear during preparations lasting more 3 ‘ than 20 mm  (Vaalburg et al., 1975).  The 13 N isotope disintegrates by emission of a positron (E3, 1.2 MeV of maximum emission energy) giving rise to ‘ C (Meeks, 1993). In 3 annihilation reaction between a positron and an electron, two gamma photons are formed each of 0.511 MeV energy traveling in nearly opposite directions (Cooper et al., 1985; Meeks, 1993). Therefore the detection of radioactive decay in the sample is accomplished in a gamma counter. Since N decay results in Cerenkov light, it may be counted by the 3 ‘  29 photomultiplier tubes in liquid scintillation systems (Glass et al., 1985). Radioactivity is typically counted immediately in a gamma counter and all counts are decay-corrected to a common time. The admitted 13 N in plant tissues can be observed by placement of multiple Gieger-Mueller tubes along the plant axis (McNaughton and Presland, 1983; Caidwell et al., 1984), by autoradiography on X-ray film between blocks of dry ice for 20 -  30 mm  (Deane-Drummond and Thayer, 1986), or by hand-sectioning of  the tissue and scintillating counting. 2.2.4.3. Advantages of the use of 13 N in biological studies The use of 4 N 3 ‘ N H in biological studies of nitrogen nutrition has several advantages: (1) The chief advantage is that such nuclide can be prepared at a very high specific activity increasing sensitivity for detection approximately -fold, to trace rapid kinetics and metabolic pathways (Krohn and 8 10 Mathis 1985). Because of the great sensitivity of the radioactive isotope technique, 13 N has proved to be of value in elucidating biological mechanism over very short time intervals. (Hanck, 1982). (2) In order to measure the initial events in biological processes it may be necessary to determine events on a time scale of seconds to minutes. High specific activity tracers which are detected with high efficiency (e.g. 13 N) make possible such measurements. It is clear that time resolution of a tracer-influx experiment is crucial for subsequent interpretation of the fluxes. In short term experiment, by using , N0 one is able to monitor 3 ‘ net uptake and disappearance of ‘ N0 simultaneously, thus increasing 3 the experimental resolution compared with experiments where plants  30 have to be sampled and further prepared before assay (Oscarson et aL, 1987). (3) The isotope decays rapidly  2 / 1 (t  =  9.98 mm). After allowing sufficient  time for decay, repeat studies can be carried out in the same system without interference from previously administered tracer (Cooper et al., 1985). In tissue dissection or in vitro studies, the total quantity of tracer present in rather large specimens can be determined rapidly and accurately, with little sample preparation, by gamma counting techniques. (4) 13 N is inherently less hazardous to use in comparison with conventional, much longer lived tracers. The problem of radioactive waste disposal is eliminated (Cooper et al., 1985). Nevertheless, the disadvantages are also related to its short half-life. It is only available at relatively few research centers located close to the cyclotron. Its production requires a suitable accelerator and a correspondingly large capital investment (Cooper et al., 1985). Its short half-life limits the period over which it can be used to a maximum of perhaps 4 hours or so depending on the application (Meeks, 1993). Techniques of precursor synthesis, labeling, product purification, metabolic separation and analysis must be appropriately rapid (Fuhrman et al., 1988). 2.2.4.4. Considerations of using 13 N in nitrogen uptake To study nitrogen uptake, especially ammonium uptake by plant roots, several facts have to be considered: (1) Membrane fluxes of nitrogen are of utmost importance for the over-all nitrogen utilization in plant growth.  31 (2) Ammonium is rapidly metabolized to amino acids and amides within the root before transport to the shoot (Pate, 1973). Evidence showed that the NH 4 uptake rate is also regulated by the N assimilation and translocation rates of the plants (Wiame et al., 1985; Morgan and Jackson, 1988). Therefore it is necessary to identify the nitrogen compounds in the uptake, assimilation and transport processes. (3) It is difficult to measure the subcellular, i.e. cytoplasmic and vacuolar, 4 directly due to their small size and rapid turn pools of N0 3 and/or NH over. It was found that the half-time for exchange of the cytoplastic NO 3  -  pool ranged from 2 to 5 minutes in roots of Zea Mays (McNaughton and Presland, 1983). (4) Ion uptake of plant roots is able to adapt during a long-term experiments in response to changes of environmental conditions, such as temperature or pH (Macduff et al., 1987). Therefore the tracer technique can be chosen as a proper approach to study ammonium uptake by rice roots in consideration of high sensitivity, rapid measurement and short duration of experiments. Another point is that uptake by depletion is so slow from high external concentration that it can not be measured except with 3 ‘. N 2.2.4.5. Use of 1 N in nitrogen transport studies  In short-term experiments, ‘ N has been used to study nitrogen 3 uptake by plant roots (McNaughton et al., 1983; Glass et al., 1985; Lee et al., 1986; Oscarson et al., 1987). Most reported studies used ’ NO in 3 ‘ uptake experiments; few made use of . 4 N 1 H ‘ N0 has been used to 3 identify and characterize the transport systems (Thayer and Huffaker, 1982; McNaughton and Presland, 1983; Siddiqi et al., 1990; Glass et al.,  32 1990); regulation of influx (Glass et al., 1985; Oscarson et al., 1987; Siddiqi et al., 1989; Rufty et al., 1991); and cell compartmentation (Presland and McNaughton, 1984; Lee et al., 1986; Siddiqi et al., 1991). Presland et al., (1986) were able to use NH 13 to study ammonium uptake by roots of 4 hydroponically grown maize seedlings and the transport of 13 N to the shoot. It was found that the rate of uptake of ammonium, by Zea mays, was a function of external ammonium ion concentration at less than 1 mM. 2.2.4.6. Use of 13 Nin nitrogen assimilation N has also proven useful in understanding nitrogen assimilation in 13  plant cells. Gin is the first major organic product of ‘ 4 N 3 H assimilation (Skokout et al., 1978) and the GS/GOGAT pathway is the primary route of assimilating fixed ‘ N (Meeks et al., 1978a). It was found that MSX 3 inhibited the incorporation of 4 N 3 ‘ ’H into Gln more than into Glu. The opposite was true for 3 N0 In tobacco cells GDH only plays a minor role ‘ . (Skokout et al., 1978) but in non-leguminous angiosperm 2 -N fixers, GDH may play a major role in the assimilation of exogenously supplied NH 4 (Schubert et al., 1981). Since 13 4 can be produced in hundreds of millicuries, it should be NH possible to synthesize a large number of 13 N-labeled amino acids, nucleotides, amino sugars, and other metabolites via known enzymatic routes (Cooper et al., 1985). Organic N-containing compounds, such as L (1 3 N  N)-glutamine, are also synthesized from ) -glutamate and L- ( amide-’ 3  4 and used in studies of NH NH 13 4 and glutamine assimilation pathways (Suzuki et al., 1983; CalderOn et al., 1989). It was found in Neurospora crassa that ( N)-Gln 1 3 is metabolized to N)-Glu 13 by GOGAT and to (  4 N}I 13  by the glutamine transaminase-o-amidase pathway. Then released 1 4 N H is reassimilated by both GDH and GS (Calderón et al., 1989). Extracted 3 ‘ N -  33 labeled amino acids or amides can be separated by HPLC and electrophoresis (Cooper et al., 1979; Meeks, 1993). It was found that translocation of N compounds can also be traced by N 1. Barley leaves exposed to 3 NH gas for 30 mm, incorporated 1 ‘ N mainly into free Gin and Glu and 1 to 3% of these were exported to the sheaths through the phloem (Hanson et al., 1979). 2.2.4.5. Use of 13 N in denitrification In addition 1 N has also been used to study denitrification in soils (Gersberg et al., 1976; Tiedje et al., 1979; Bremner and Hauck, 1982). Use of ‘ N allows the direct quantitative measurements of denitrification rates 3 over short time intervals, without changing the concentration of N0 3 in the soil system from flooded rice fields (Gersberg et al., 1976).  2.2.5. Protocol for 13 4 production in present study NH The short-lived radioisotope 13 N  (t 1/2  =  9.98 minutes) was produced  as described by Siddiqi et al., (1989), by 20 MeV-proton irradiation of H 0 2 on an ACEL CP42 cyclotron. Contaminants in the ‘ N0 sample (mainly 3 F) were removed by passing the samples twice through a SEP-PAC 18 Alumina-N cartridge (Waters Associates). Reduction of ‘ N0 to 13 3 3 was NH achieved by using Devarda s alloy at 70°C in a water bath (Vaalburg et al., T 1975; Meeks et al., 1978); 1 3 N H was separated from remaining chemical species by distillation at alkaline pH, and trapping in acid solution as 13 4 NH ’-. The flow scheme for this conversion is shown in Figure 1.  34  1  F 8 13N0+l  LJc  SEP-PAK  Labelled Uptake Solution  Leod holder  D e v a rd a• s A110U iN NOH  0 CU urn  7 Oc  Figure 1. The flow scheme for 13 4 production. (As described in Section NH 2.2.5.)  35 2.3. MEAsuREMENT OF NH 4 FLuxEs  2.3.1. Influx of 4 NH 3 ‘ Standard procedures for 13 4 uptake were as follows: (a) loading: NH rice roots were loaded in NH 13 4 labeled MJNS (hereafter referred to as ‘loading’ solution) for designated periods; (b) pre-wash and post-wash: prior to and after loading, roots were pre-washed and post-washed in un labeled MJNS (hereafter referred to as ‘washing’ solution) for 5 mm and 3 mm, respectively. The choice of these times is rationalized in the Discussion section (section 3.4.). Experiments were conducted at steady-state with respect to , 0 4 [NH -] i.e., the [NH 0 of ‘washing’ solutions and ‘loading’ ] 4 solutions were the same as those provided during the growth period or in experiments to define influx isotherms; plants were exposed to different 0 for short (perturbation) experiments. Immediately after the post] 4 [NH wash period, plants were cut into shoots and roots and the surface liquid adhering to the roots was removed by a standard 30 sec spin in a slowspeed table centrifuge (International Chemical Equipment, Boston). Roots and shoots were introduced into separate scintillation vials and immediately counted in a gamma counter (MINAXI y, Packard). The 5000 fresh weights of roots and shoots were recorded immediately after counting.  2.3.2. Efflux of 13 4 NH  Roots of rice seedlings were immersed in the 1 4 N H labeled ‘loading’ solution for 30 mm. At the end of this time plants were transferred to an  36 elusion vessel and tracer leaving the roots in exchange for ‘ NH in the 4 un-labeled identical ‘washing’ solution. This solution was collected at prescribed interval in 20-ml scintillation vials for counting.  4 2.3.3. Net flux of NH Net NH 4 flux was measured in uptake solutions by the depletion method. Solution samples (S 1 and S ) were taken at different times (t 2 1 and t ) 2 , and the difference of assayed [NH ] was used to calculate net NH 4 4  flux. Net NH 4 flux can also be estimated by subtracting efflux from influx of the same roots.  2.4.  CoMPARTMENTAL (EFFLux) ANALYsIs  2.4.1. Compartmentation of plant cells Plant cells are highly compartmentalized. They are surrounded by the cell wall, and the plasma membrane encloses the cytoplasm, in which are found the vacuole, mitochondria, nucleus, plastids and other organelles. Up to 80% or more of cell volume is occupied by the vacuole which is enclosed by the tonoplast (Salisbury and Ross, 1985). The cytoplasm is the vital part of cell. The major functions of the vacuole are to maintain turgor which contributes to cell shape and to store solutes. The compartmentation of the cell has important consequences for nutrient uptake, unidirectional fluxes, assimilation, distribution and translocation. Because higher plant cells are too small to dissect and the size of the compartments is even  37 smaller, it seems technically impossible to obtain information on the composition of each compartment. However, through various methods, such as NMR, ion-specific electrodes, EDX, compartmental analysis, or fluorescent dyes, the ion concentration of one or more particular compartments, or fluxes between compartments can be estimated. Compartmental analysis is the only systematic method of investigating transport processes and estimating the size of compartments and to analyze the kinetics of movement of ions to or from a tissue (Cram, 1968). Therefore it has been established as a tool for characterizing the exchange properties of multicompartment systems.  2.4.2. Development of theory Compartmental analysis was first used by Fourier in 1822 to describe the relationships between heat flow and temperature gradients and, in 1855, it was adopted by the biologist, Fick, in studying diffusive flow along a concentration gradient (Zierler, 1981). Not until one century later, was it introduced by MacRobbie and Dainty (1958) to study ion transport in Nitellopsis. Soon after, Pitman (1963) was the first to use this method to investigate multicompartmental transport processes in a higher plant. Compartmental analysis has mostly been used by plant physiologists to calculate the fluxes, characterize internal ion pool sizes and membrane kinetic parameters for ion exchange. The basic assumption of this methodology is that the system is at steady state, or at equilibrium. Additional assumptions include that (1) the substance of interest flows into and from the separate compartments of the system; (2) the flux is proportional to the quantity (or concentration) of  38 the substance in the compartment from which the material flows. It is assumed that the material under study is neither destroyed nor synthesized in any compartment, and that each compartment is homogeneous, or well stirred; (3) the concentration of an ion species or its flux is described by a first-order linear differential equation with constant coefficients which are independent of elapsed time and of the conjugate (Zierler, 1981). For higher plant systems, the additional assumption is that the relevant compartments of the experimental system are functionally in series with each other (Walker and Pitman, 1976; Cheeseman, 1986). These assumptions may not always be valid (Lazof and Cheeseman, 1986). It is suggested that compartmental efflux analysis should not be used alone, but integrated with other methods such as influx measurements (Cheeseman, 1986).  2.4.3. Models for compartmental analysis The testing model or the analysis process can be varied with the research subject (excised tissue or intact plant), number of compartments (2, 3, or more), nutrition status (steady or non-steady) (Walker and Pitman, 1976). The conventional compartmental analysis is suited to determine unidirectional fluxes and compartmental contents of ions in excised root tissues, or suspension-cultured cells (Pitman, 1963; Cram, 1968; Poole, 1971; Macklon, 1975a; Pfrüner and Bentrup, 1978; Jeschke and Jambor, 1981). Since it was considered to be small in excised roots (Macklon, 1975), the xylem transport in intact plants was not included in this conventional model (Pitman, 1963; Etherton,1967; Pallaghy et al., 1970). However, the method was modified by Pitman (1971, 1972) to study Cl- uptake and transport in barley roots. Tracer efflux from the  39 cortical cell surface and the transport of tracer into the xylem were measured and analyzed separately. A three compartment model, including xylem transport, was tested in the study of unidirectional fluxes of Na in roots of intact sunflower seedlings (Jeschke and Jambor, 1981). In two compartment models, xylem transport was also considered in studies of N0 fluxes in roots of intact barley seedlings (Lee and Clarkson, 1986; 3 ‘ Siddiqi et al., 1986). The two compartments included the cell wall and cytoplasm, respectively. The short half-life of 13 N decay (9.98 mm) precluded analysis of the vacuole. The testing model for higher plants by compartmental analysis (Walker and Pitman, 1976) is based on the assumption that (1) the cytoplasm and vacuole are in series; (2) the cytoplasmic content is very much less than the vacuolar content; (3) the tissue is in a steady state (Cram, 1968). Therefore one may expect that at steady-state conditions of roots: S  =  0 (1 S  -  ekct)  [3]  when roots are exposed to a radioisotope-labeled medium with specific activity S , the radioisotope content of the cytoplasm S increases 0 exponentially with time (t) and the rate of tracer exchange of the cytoplasm (kc) is given by the relationship 2 (kc=O.693/tl/ ) . The quantity of radioactivity inside the cell Q is given by Qc*AtcpocSc  [4]  where A is a cross section constant and p is the flux from outside to cytoplasm. The fluxes in opposite directions, between cytoplasm and vacuole are considered to be equal at steady state:  40 Øcv  [5]  =  then the flux into the cytoplasm Poc  =  Øco +  -  xc;  (if 4  <<  0 it may be neglected)  [6]  therefore net uptake of an ion Joc  =  Poc  -  Pco  [7]  and the transport of ion from root to shoot through xylem would be Jox  =  -  Pxc  [8]  if roots were uniformly labeled after 16-24 hours loading: 0 S,= S= S  [9]  and the specific activity in the xylem can be estimated from the transport rate of tracer (cI(t)) and transport rate of ion 0 (J ( t)) with the assumption that the symplasm behaves like a rapidly mixed phase and has a uniform specific activity Sc =  I(t) / 0 J ( t)  [10]  Based on these relationships, one is able to estimate unidirectional fluxes and other parameters for each of the compartments. A biphasic efflux pattern suggests two phases, outside and inside the plasma membrane (Luttge and Higinbotham, 1979). Since the fastest component was found in both living tissue and chloroform-killed tissue, Cram (1965) concluded that the fastest component of efflux of tracer Cl from carrot tissue probably corresponded to the apparent free space (AFS). After treating barley roots with either sodium dodecyl sulphate  41  (SDS) or 70°C hot-water for 30 mm, the amounts of released N0 during 3 ‘ initial efflux were similar to the control plants (Siddiqi et al., 1991). Therefore this rapid efflux component probably corresponds to the AFS. Another approach has been to use different sizes of molecules to confirm the AFS phase. It was found that 3 [1,2H ] polyethylene glycol ( H-PEG) is 3 too large to diffuse into AFS, but 4 D-[1-’ C ] mannitol is able to diffuse freely in the AFS without been absorbed by root cells (Shone and Flood, 1985). After loading with a mixture of 3 H-PEG and D-[1-’ CJ mannitol, plant roots 4 were washed in unlabeled solution. Since the ratio of 3 H and ‘ C should be 4 same from the surface film of ‘loading’ solution carried over with the roots, C] mannitol must be washed out from AFS, and can be 14 the extra D-[1used to assess the volume of the AFS. It was found that there was an initial rapid release of 90% of H and ‘ C within the first 1 mm but more ‘ 4 C was 4 subsequently released (Lee and Clarkson, 1986). Therefore the rapidly released radioactivity during early efflux is probably from the AFS. A tricompartmental efflux pattern (including the apparent free space) were reported for Cl- in carrot root slides or isolated corn root cortex (Cram, 1968; 1973), and excised or intact barley roots (Pitman, 1963, 1971); and for Na and K÷ in intact barley roots (Poole, 1971a, 1971b; Jeschke, 1982). Based on the results of compartmental analysis and other studies, Cram (1965) concluded that, in addition to the fastest efflux from the AFS, the two slower components were considered to be subcellular in origin, the cytoplasm and the vacuole. Further quantitative considerations and model fitting suggested that the cytoplasm and the vacuole are arranged in series with direct connection between the external solution and the cytoplasm, but not between the external solution and the vacuole (MacRobbie, 1964; Cram, 1965).  42 Also a third small symplastic kinetic compartment may exist in addition to the bulk cytoplasm and vacuole (Luttge and Higinbotham, 1979; Lazof and Cheeseman, 1986). In a study of sodium transport in Spergularia marina, Lazof and Cheeseman (1986) found. that the rapid fluxes involved only a very small portion of the total Na in the roots but the authors were unable to identify the physical entity corresponding to the compartment identified. There were also several similar reports in other transport studies. The additional compartment could be the small portion of the bulk cytoplasm connecting to the vacuole (Pitman, 1963); or the cytoplasm can exchange with both vacuole and plastids (Walker and Pitman, 1976); or the possible involvement of vesicles moving in the cytoplasm (Dodd et al., 1960; Luttge and Osmond, 1970); or the involvement of vesicular transport of ER (Arisz, 1960; MacRobbie, 1970; Stelzer et al., 1975; Tanchak et al., 1984).  2.4.4. The general procedures of compartmental analysis The general procedure for compartmental analysis has been described in detail (Walker and Pitman, 1976; Zierler, 1981; Rygiewicz et al., 1984). Several radioisotopes have been used in compartmental analyses, 36C1, 82 Br, 42 K or 86Rb+, 22 Na, 45 Ca, and 28 Mg. One part of this technique involves the use of radioisotopic tracers to measure influx and efflux, the separate components of the net flux. The second part is a more systematic method to analyse the kinetics of movement of ions to or from different compartments (Cram, 1968). The basic assumption of this procedure is that radioisotope loaded into different compartments will be washed out with different rate constants.  43 After allowing plant tissues, cells or roots to load with radioactive tracer for a designated duration, the efflux of this radioisotope is measured for a prescribed period of time. Depending on the type of ion studied, there are two ways to count the radioactivity. For nonmetabolized ions, such as Cl-, Br-, K-’-, Na, and Mg+, the radioactivity remaining in the tissue at the end of elution can be counted. By counting the eluates at different times the counts remaining in the tissue at these times can be estimated. For metabolized ions, however, counts remaining in the tissues would be misleading because they consist of the radioactive ion under examination and the metabolic products of its assimilation. In the latter case the rate of efflux, rather than counts remaining must be estimated as a function of the duration of elusion. However, even this method requires that the identity of the effluxed ion be confirmed. Plotted as a function of time on a semi-logarithmic plot, the activity data (e.g. cpm remaining in system or efflux rate) are resolved into different linear phases which have been interpreted as corresponding to different compartments within the cells. One flaw in this method has been the subjective basis of line fitting (curve-peeling) of data which has implications for the number of exponential terms and their coefficients. To improve the method, Rygiewicz et al. (1984) proposed a microcomputer method in which maximization of r 2 for linear regression serves as the criterion to determine data points belonging to each compartment. This development greatly increased the accuracy of parameter estimation (Rygiewicz et al., 1984) and the objectivity of the estimated results (Cheeseman, 1986). Selected parameters obtained from compartmental analysis from several sources are shown in Appendix B. It was reported that the half-  44 lives of C1 exchange for apparent free space, cytoplasm and vacuole were 1.4 mm, 10 mm and 300 h, respectively for carrot root tissue (Cram, 1968). In excised barley roots, a slow, vacuolar compartment, was not visible even after 10 h of exchange (Behl and Jeschke, 1982). It must be kept in mind that compartmental analysis alone does not allow one to identify each compartment (Luttge and Higinbotham, 1979), one must interpret the results with necessary caution and verify these correlations independently. For example, several techniques are available to identify and quantify the vacuole (Clarkson and Luttge, 1984).  2.4.5. Procedures for compartmental analysis in the present study For better time control of the separation of ‘washing’ solutions from the NH 13 4 labeled roots during the efflux process and to reduce disturbance of roots, I devised a simple apparatus in which to perform the efflux study. The spout of a plastic funnel (100 mm diameter) was cut to fit into the barrel of a 25 cc plastic syringe, into which it was sealed. A length of rubber tubing replaced the needle end of the syringe and a metal spring clip on the tubing functioned as drainage control. A small hole was drilled in the wall of the syringe barrel near the bottom, and a needle introduced through this hole to provide for aeration. This technique also resulted in good mixing of the ‘washing’ solution. Roots of rice seedlings used for compartmental analysis were immersed for 30 mm  in the ‘loading’ solution. These pre-labeled roots  were carefully introduced into the syringe barrel for elution. Samples of 20 ml ‘washing’ solution were poured into the efflux-funnel and allowed to exchange with the 13 N-labeled roots. After prescribed intervals, this  45 solution was drained from the funnel directly into a 20-ml scintillation vial, by opening the drainage clip. Fresh ‘washing’ solution was poured into the efflux-funnel from the top of the funnel, immediately after closing the drainage clip. The duration of successive washes were: 1 x 5 s, 1 x 10 s, 7 x 15 s, 2 x 30 s, 5 x 1 mm and 5 x 2 mm. After the last wash, the plants were cut into shoots and roots and introduced into separate scintillation vials. The radioactivities of all samples were counted immediately. In order to be assured that the 13N species that had effluxed from the roots was  4 MI 13  rather than any metabolic products, two other sets of NH 13 4 labeled roots were effluxed for 30 mm in 750 ml ‘washing’ solution. Two 20-ml samples of the efflux solution from each beaker were taken and separated by the CEC procedure (see below) and counted for radioactivities. The radioactivities released from intact rice roots into efflux solutions during 18 mm  efflux experiments, were counted, converted to efflux rates and  plotted versus time in semi-log plots (see Fig. 2 in section 3.3.1.). This method of analysis is required because NH 4 is rapidly metabolized in rice roots (Yoneyamo and Kumazawa, 1974), and converted into amino acids and proteins. As a consequence, standard methods of compartmental analysis (Walker and Pitman, 1976), based on semi-log plots of cpm remaining in the tissue plotted against time are not appropriate. Hence the values of log of rate 13 4 released against time were plotted using the NH methods detailed by Lee and Clarkson (1986) in an automated computer analysis (Siddiqi et al., 1991).  46 2.5.  DETERMINATIoN OF AMMONIUM  Intracellular NH 4 was extracted from rice roots by use of a Cation Exchange Column (CEC) separation based on the methods of Fentem et al., (1983a) and Belton et al., (1985) and determined by the indophenol blue colorimetric method (Solorzano, 1969). The procedure was as described in Wang et al., (1993a): in brief, after desorbing in 4 NH free MJNS for 3 mm to remove NH 4 in the cell wall, the roots were cut, weighed, and ground with liquid nitrogen in a pre-cooled porcelain mortar and extracted with 10 ml of 10 mM sodium acetate buffer (pH 6.2). The resulting slurry was passed through a Whatman #1 filter paper and then washed 3 times each with 5 ml of the same buffer solution. The filtrate was passed through the CEC filled with 3 ml of resin (Dowex-50, 200-400 mesh, Na form). The 4 adsorbed on the CEC column was eluted using 250 mM KC1. The NH concentration of NH 4 in solution was determined by the indophenol blue colorimetric method (Solorzano, 1969).  2.6.  PREPARATIoN OF METABoLIC INHIBITORS  The same metabolic inhibitors were used in the 1 4 N H influx study (Chapter 5) and electrophysiological study (Chapter 6). The inhibitors used were  as follows:  (1)  CCCP  (10 iiM): carbonylcyanide m-chloro  phenyihydrazone dissolved in ethanol; (2) CN- plus SHAM (1 mM): NaCN plus salicylhydroxamic acid dissolved in water. The resulting alkaline pH was adjusted by titration with H 4 5 2 0 to pH 6; (3) DES (50 1 iM): diethylstilbestrol dissolved in ethanol; (4) DNP (0.1 mM): 2,4-dinitrophenol dissolved in ethanol; (5) Mersalyl (50 iiM): Mersalyl acid dissolved in water; (6) pCMBS (1 mM): p-chloromercuribenzene-sulfonate dissolved in  47 water. The acidic pH was adjusted by titration with Ca(OH) 2 to pH 5.8. Ethanolic solutions of CCCP, DES and DNP were added to the nutrient solutions to give a final ethanolic concentration of 1%. Control solutions were treated with ethanol at the same concentration.  2.7. ELEcm0PHYsI0L0GIcAL STUDY  2.7.1. Transmembrane electrical potential measurement Usually plant cell transmembrane potential differences are in the range of -100 to -200 mV negative inside (Higinbothan, 1973; Tester, 1990). In the early 1930’s, Umrath started to use microelectrodes to measure the membrane potential across the tonoplast (Findlay and Hope, 1976). Since then, other electrical properties of plant cells have also been studied such as membrane capacitance (Curtis and Cole, 1938), membrane conductances (Cole and Curtis, 1939), and membrane resistance (Higinbotham et al., 1964; Spanswick, 1970; Anderson et al., 1974). The contemporary climax of electrophysiology occurred when Neher and Sakmann (1976) developed of patch-clamping techniques. The combination of molecular gene cloning and patch-clamp analysis (Hedrich et al., 1987) represents a particularly powerful means of elucidating the mechanism of ion transport through cell membranes. The chemical potential of an ion  (j) is composed of all those  components that enable it to do work and can be expressed by the equation [11]:  48 iO  where  j.i,j  +RTlna g FVP+m 1 +z h  [11]  is the chemical potential of the ion fin joules mol1 and  is the’  standard state chemical potential of 1 mole of the ions f per liter at 0°C; R is the gas constant (8.314 J mol” °K’); T is absolute temperature in °K (°K =  273  +  3 is the activity of the ion; [°C] ); a  3 Z  is its valency; F is the Faraday  constant (9.65 x 1O J mol ); V is the electrical potential in volts; V V 1 1 is the volume; P is the pressure; m 1 is the mass; g is the gravitational acceleration; and h is the height above sea level. In terms of solute transport across the membrane, V 1 is very small and h is generally negligible. When the concentration (C ) of the solute is low so that the 1 activity and concentration are close, concentration C 1 (mol rn) can be used 3 in place of the activity a 1 (a 1=  yj  3 ), where C  yj  is the activity coefficient.  Simple diffusion is a non-mediated transport process whereby the solute moves along the free energy gradient. In addition to the lipid composition, the difference of ion concentration just inside and outside the plasma membrane determines the diffusion of solute across a membrane. Ion diffusion through membranes may be described by the permeability coefficient which is the flux per unit driving force (in its original conception, the concentration gradient). For the diffusion of small noncharged molecules such as 3 NH and 2 H 0 , the chemical potential =  +  RT ln C 1  [12]  can be expressed as in equation [12]. Since the driving force is only due to the concentration gradient from high to low (negative sign), the net flux J 3 (mol m 2  3 J  -1)  =  is expressed as in equation [13]: 1 J (-dji K /dx) 1  [13]  49 Differentiating in equation [6] and replacing K 1 RT (in equation [14]) by D 1 (the diffusion coefficient) (Stein,1986) gives equations [141 and [15]:  1 J  =  -  1 RT d K  / dx  [14]  /dx) 1 1 (dC 1 =-D J  [15]  Equation [15] is Fick’s First Law of diffusion, where K 1 is the proportional coefficient or the mobility of the ion j, and D 1 is the diffusion coefficient of species fin m 2 s. If P 1  (m s-i) is the permeability coefficient of the  medium or the membrane for ion j, then P 3 =-D / z\x  [16]  therefore, for the concentration gradient zC 1  1 J  =  1 zC P 3  =  1 1 (Co P  -  =  1 Co  -  C1  ) 1 C’  [17]  The permeability (P ) of a chemical species (j) is a measure of the 1 ability of the species of small non-electrolyte to pass through a membrane. The permeability coefficient for isopropanol or phenol is 10-6 m sec 1 across the plasma membrane (Nobel, 1983). The diffusion of most ions across the membrane is very low due to their low permeability compared to non-electrolytes. In addition to the concentration gradient, the electrical potential gradient must be included in the driving force. Therefore, equation [11] can be presented as: =  +  RT ln C 1  +  3 F-I’ z  [18]  For a particular ion, the electrochemical potential gradient (11*]) determines the potential for passive ion flux. At equilibrium both outside and inside electrochemical potentials are the same:  50 =  0  =  -  [19]  combining equation [18] and [19] =  (RT in C 1  +  zF’{ - (RT in C 0  +  zF%)  [20]  where : z$C 10 is the electrochemical potential difference across the membrane;  and C 1 are the electrochemical potential outside and inside  the cell membrane respectively, ‘P 1 and ‘P 0 represent the inside and outside electrical potentials, respectively, measured as V; C 1 and C 0 are the concentration (mM or mol m ) inside and outside the cell membrane, 3 respectively. Because of the selective and permeable nature of membranes and the existing concentration asymmetry, the electrical potential difference at zero net flux, when zs.i  0, is defined as the Nernst potential  =  (‘PN)  as in  equation [21]: RT =  ------  0 C ln (  zF  )  [21]  1 C  This is the Nernst equation which describes the electrochemical potential of an ion distributed at thermodynamic equilibrium between two phases separated by a cell membrane. Considering monovalant cations and assuming temperature to be 25°C equation [21] can be simplified to [22]: 0 C =  - 59 log  (  )  [22]  Ci When jC  0, equation [20] and [21] can be rearranged as: =  zF ((‘{- ‘P )  -  (RT in C )/zF) 1 0 RT ln C -  51 =  where  ‘PM  zF  -  [23]  ‘PN)  is the measured membrane electrical potential differences across  the membrane in volts (‘-PM  =  ‘P-• ‘fe), normally this potential difference  across the plasma membrane is large for plant cells (about -200 mV), negative inside (Dainty, 1962; MacRobbie, 1971; Higinbotham, 1973). The membrane potential (‘PM) can be generated from three sources (Nicholls, 1982). One is due to diffusion potentials which may contribute 30 to 40% of measured membrane potential (Pierce and Higinbotham, 1970; Higinbotham et aL, 1970). Salts (e.g. KC1 and NaC1) in solution dissolve to release cations (K-i- and Na) and an aniàn (Cl-) which may have different membrane permeability (PK÷, PNa and Pcii. Presuming that there is initially no electrical asymmetry across the membrane, when ions move along their chemical potential gradient, different mobilities of cations and anions result in charge separation which creates an electrical potential difference, known as a diffusion potential (‘PD ). It can be assessed by the Goldman voltage equation: RT  PK[K]o + PNa[Nalo + Pi[Cl]j +  ln ( F  ) PK[KJj  +  PNa[Na]j  +  Pi[C1]o  [24]  +  The second source of membrane potential is the Donnan potential, though the contribution is relatively small. Inside the plant cell, there are many large organic molecules, such as protein and other large polymers (RNA and DNA), with a large number of immobile carboxyl, phosphate and amino groups from which H can dissociate. The asymmetrical distribution of diffusible cations leads to a small negative potential across the plasma membrane (negative inside) (Nobel, 1983).  52 Thirdly, a major component is a metabolically-driven potential due to the operation of an electrogenic ion pump  -  the H pump. The H-’- pump  (H-translocating ATPase) carries a net positive charge across the membrane and contributes directly to the membrane potential (Poole, 1973; Sza, 1984). The activity of H pump depends on the hydrolysis of ATP catalyzed by a plasma membrane ATPase (Hodges, 1973; Poole, 1978; Spanswick, 1981). From equation [20], one can obtain an equation which calculates the electrochemical potential difference for proton at 25°C: AtH  =  A’P + 59 zpH  [25]  A proton concentration difference (ApH) and an electrical potential difference (AP) are two related entities that make up the electrochemical difference generated in part by the H-’--translocating ATPase (Sze, 1984). By actively pumping out H across the plasma membrane, a ‘proton motive force’ is built up which can provide the free energy necessary to transport other ions, both actively and passively into the cell (Poole, 1978). In other words, the H-’--pump generates both a potential difference (AW) to drive electrogenic uniport, and an electrochemical gradient of protons to drive transport of ions in antiport or symport with H. Since the electrochemical potential difference (AJI*jo) across a membrane is the combined chemical potential and electrical potential difference (equation [18]), it is used to describe the free energy status of a solute in a particular location. It is assumed that a difference of free energy between two points of a system represents the driving force for a passive flux of ions from one point to another. When the resultant chemical potential difference is just balanced by the resultant electrical potential difference (AI*jo  =  0), there is no net flux of solute by passive forces.  53 Alternatively it can be stated that no energy is expended in moving ions between the two locations. 2.7.2. Single impalement and membrane potential Microelectrodes are commonly prepared from a micropipette filled with electrolyte solution. It is a filament-containing or single-barreled borosilicate glass capillary tube with the fine-tip which is pulled with either a vertical or horizontal electrode puller (Purves, 1960; Findlay and Hope, 1976). The external diameter of the tip should be 0.5% or less of the diameter of the plant cell which it is to impale (Purves, 1960). For cytoplasmic insertion, a tip diameter of 1 to 2 jim is usually satisfactory (Findlay and Hope, 1976). A tip diameter of <0.5 jim has often been used (Kochian et al., 1989; Ullrich and Novacky, 1990; Glass et al., 1992). However, the smaller the tip diameter the higher the tip potential or electrical resistance (Findlay and Hope, 1976). Membrane potential difference can be easily expressed in a number of equations (refers to section 2.7.1.), such as the Nernst potential (Eq. [21]), or electrochemical potential (Eq. [24]), or the Goldman diffusion potential (Eq. [21]). When the potential difference is measured by inserted microelectrode, the value is an apparent resting potential which is the real potential difference plus the total offset potential (Purves, 1981). The total offset potential includes the liquid junction potential, the tip potential and the potential due to the possible dissimilarity between the indifferent electrode and the electrode which contacts the microelectrode’s filling solution. The latter can be compensated by the offset control of the oscilloscope amplifier. The liquid junction potential occurs between the microelectrode’s filling solution and the electrolyte outside the tip. It can  54 be decreased by use of 3 M KC1 as filling solution since the diffusion coefficients of K and of C1 are almost identical. The tip potential is due to the characteristics of glass wall, electrolyte concentration difference between inside and outside of the tip of micropipette and can be eliminated by filling the micropipette with low pH solution or other treatments (Purves, 1960). A tip potential of -5 to -30 mV was recorded for the microelectrode filled with 0.5 M KC1 plus 0.1 M Mes (pH 5) (Ulirich and Novacky, 1990). The electrolyte solution could be 3 M KC1 at pH 2.0 (Kochian et al., 1989; Glass et al., 1992) to get a high concentration of ions in the tip and a low electrical resistance. As pointed out by Purves (1960) the history of microelectrode technology can be regarded as a succession of attempts to minimize tip diameter and resistance simultaneously.  2.7.3. Setup for measuring membrane potential The fundamental setup for measuring electrical potential difference between two aqueous phases (cell ambient and cytoplasm), is an electrical circuit which should be connected by a salt-bridge, i.e. Hg C1 plus KC1 2 (Willians and Wilson, 1981). The microelectrode is such a micro-salt-bridge or miniaturized Calomel half-cell, and connected to the circuit with the silver wire or silver/silver pellet (Purves,  1960). Besides the  microelectrode which impales the cell cytoplasm, another reference microelectrode (or the indifferent electrode) is also immersed in bathing solution and connected to the ground. The electrical signals are amplified through a preamplifier (or electrometer), and are sent to the output devices such as the oscilloscope, the tape recorder, the pen recorder the  55 digital voltmeter or the audio monitor (Findlay and Hope, 1.976). Since the plant cells are tiny, vivid and fragile, the impalement the cell through the cell  wall  and  cell  membrane  is  operated  by  three-way  micromanipulators (Kochian et al., 1989; Glass et al., 1992) under the microscope on an anti-vibration table (Purves, 1960). A diagram of such a setup is shown in Figure 2.  2.8. Determination of amino acids in root tissue Free amino acids in root tissue were determined, after the method reported by Fentem et al., (1983a, 1983b), as follows: weighed root samples were ground with liquid N 2 in a porcelain mortar and extracted with 80% aqueous ethanol. After centrifugation (IEC Clinic Centrifuge), the supernatant was transferred to an evaporating flask. The extraction and centrifugation were repeated 5 times. Pooled extracts were evaporated under vacuum at 35°C on a flash evaporator (Buchler Evapomix). The crude extracts were then re-suspended into 5 ml of distilled deionized water. After mixing 5 ml of chloroform with the crude extract, the supernatant (aqueous phase) was collected into an Eppendorf tube (1.5 ml) for further centrifugation and lyophilization. The extracts were derivatized with phenylisothiocyanate (PTC) automatically on an Amino Acid Analyzer (ABI, Model 402A) equipped to derivatize and hydrolyze applied samples, and then separated by HPLC analysis (Separation system, ABI 130A). The amino acid concentrations were determined by the Amino Acid Analyzer and analyzed by means of an ABI 920A data analysis module. The chemicals used as amino acid standards were from Sigma.  56  1. Compressed air  11. Impaling electrode  2. Air-regulator  12. Reference electrode  3. Bathing solution reservoir  13. Grounding electrode  4. Needle valve for controlling flow rate  14. Electrode holder  5. Small chamber for impalement  15. Preamplifier  6. Small pins on the wall to support the root 7. Focusing plate of microscope  16. 3-dimensional manipulator (course) 17. 3-dimensional manipulator (fine)  8. Large chamber for the rest of root  18. Amplifier  9. Over-flow of the bathing solution (level) 10. Rice plant  19. Chart recorder  Figure 2. Setup for measuring cell membrane electrical potential.  57  Chapter 3. FLUXES AND DISTRIBUTION OF 13 4 IN CELLS NH  3.1.  INTRODUCTION  The short-lived radioisotope ‘ N (t 3 172  =  9.98 mm) has been used as a  tracer in studies of the fluxes of NO - and NH 3 4 into intact roots of corn and barley plants (McNaughton and Presland, 1983; Glass et al., 1985; Lee and Clarkson, 1986; Hole et al., 1990; Siddiqi et al., 1991). It provides a methodology for the measurement of unidirectional fluxes (influx or efflux) across biological membranes over extremely short times and with great sensitivity (McNaughton and Presland, 1983). Because of its strong  ‘  emission, 13 N can be determined rapidly and accurately, with little sample preparation, even in intact plants, by gamma counting techniques (McNaughton and Presland, 1983; Cooper et al., 1985; Meeks, 1992). The major emphasis in studies of N uptake has been upon N0 , 3 reflecting the widely held perception that N0 3 is the predominant form of N available to crop species. Relatively less is known about the uptake and subcellular partitioning of NH 4 in higher plants. Nevertheless in rice cultivation (Sasakawa and Yamomoto, 1978), in forest ecosystems (Lavoie et al., 1992), in Arctic tundra (Chapin et al., 1988) and even in winter varieties of cereals growing in cold soils (Bloom and Chapin, 1981), NH 4 may represent the more important form of available nitrogen. It was demonstrated that net fluxes of NH 4 into rice roots gradually acclimated between 0.1 and 1 mM external 4 [NH + ] so that net flux at steady-state varied little between plants grown in these concentrations (Wang et al., 1991). Nevertheless, there is a lack of information about  58 fluxes between subcompartments in relation to acclimation or to the mechanism(s) of NH 4 uptake. For example, Presland and McNaughton (1986) failed to observe ‘ 4 N 3 H efflux from maize roots. By contrast, a sizable net efflux of endogenous 14 4 was reported in wheat, oat, and NH barley upon transfer to ‘ 4 N 5 H solution, although there was no exact correlation between root ammonium concentration and net 14 4 efflux NH (Morgan and Jackson, 1988a, b). The internal 4 NH ’- concentration of plant roots can readily be assayed, after extraction, by methods based on colorimetry or ion-specific electrodes (Fentem et al., 1983a; Morgan and Jackson, 1988a, 1988b; Roberts and Pang, 1992). However, such analyses fail to provide information on the subcellular distribution of NH . On the basis of 4 biochemical analysis, it was concluded that more than one intracellular 4 existed in roots of rice (Yoneyama and Kumazawa, 1974, pool of NH 1975; Arima and Kumazawa, 1977). Two other methods have been employed to determine subcellular NH 4 distribution, namely, efflux analysis (Macklon et al., 1990) and the nuclear magnetic resonance spectroscopy (Lee and Ratcliffe, 1991; Roberts and Pang, 1992). These studies recognized several 4 NH ’- fractions of roots, corresponding to those of the superficial, water free space, Donnan free space, the cytoplasm and the vacuole. + 4 In this chapter, the results of compartmental analyses, using 13NH efflux, are used to estimate the half-lives of NH 4 exchange and the size of major compartments in root cells, as well as NH 4 fluxes between these compartments. Together with data obtained from chemical fractionation, it was possible to develop a detailed analysis of the initial fate of absorbed 4 N 3 ‘ . H In addition, the t 2 values for 13 / 1 4 exchange provide essential NH  59 parameters for the design of appropriate protocols for influx measurement, particularly the duration of ‘ 4 N 3 H loading and post-wash treatments. To evaluate the methodology of the compartmental analyses, influx and net flux of NH 4 were also measured by independent methods.  3.2.  MATERIALS AND METHODS  3.2.1. Plant growth and ‘ N production 3 Details of seed germination, growth conditions, provision of nutrients and production of 1 4 N H are described in Sections 2.2.. 2.3.. 2.4., and 2.5., respectively.  3.2.2. Measurement of fluxes 3.2.2.1  4 N 1 H Influx  Checks of the fluxes derived from efflux analysis: After ‘loading’ for 10, 20, and 30 mm, respectively, at steady-state conditions, influx of 4 was also determined by two independent methods: (1) the NH 13 accumulation of 13 N by seedling roots (see section 2.3.2.); (2) the rate of depletion of NH 13 4 ’- from ‘loading’ solution. 3.2.2.2. Net NH 4 flux  In addition, the net flux of NH 4 was also measured based on the rate of depletion of 14 4 (see section 2.3.4.). NH  60  3.2.2.3. Time course of 13 4 uptake NH In the time-course experiments, G2 or G100 plants were exposed to 2 iM or 100 jiM 13 -labeled loading solutions, respectively, for durations 4 NH ranging from 10 sec to 31 mm. As described in section 2.3.1., roots were subjected to a standard pre-wash, loading and post-wash procedure.  3.2.3. Compartmental Analysis The procedure for compartmental analysis was followed as described in section 2.4.5.  3.2.4. Partition of absorbed ‘NH 4 3.2.4.1. Separation of 13 N-compounds in plant tissue 13 4 NH ’- was separated from its immediate metabolic products by Cation Exchange Column (CEC) Separation described in section 2.5. After plants were loaded in 100 jiM ‘ 4 N 3 H for 30 minutes, the separated, frozen 13 4 NH labeled shoots and roots were first counted in the gamma counter and then ground in liquid nitrogen. After the filtration, the radioactivity remaining on the filter was referred to as root debris. The filtrate was passed through the CEC filled with 3 ml of resin (Dowex-50, 200-400 mesh, Na-’- form) resulting in an elute (Off-CEC) and a CEC-bound fraction (On CEC). Two sets of G100 plants, containing 100 120 plants each, were used.  61  3.2.4.2 Chemical assay of NH 4 in root tissue  4 contents Root NH  (Qj)  of G2, G100, and G1000 seedlings were  separated and determined as described in section 2.5.  3.2.5. Calculation of flux to vacuole  (4,)  The results of CEC separation quantified the un-metabolized 4 NH 1 fraction in roots following 30 mm  4 N 1 H loading. This amount  represented the combined values of cytoplasmic  (Q*c)  (Q*c+v)  and vacuolar  radioactivities that can be converted to a chemical quantity  (Q*v)  (Q+) after  dividing by the specific activity of ÷ 4 N 3 ‘ H in the external solution 0 (S ) : Qc+v=Q*c+v/So  [261  The specific activity of ‘ 4 N 3 H within the cytoplasm (Sc) during loading will increase to its steady-state value according to the rate constant for tracer exchange of the cytoplasm (kc  =  0.693 / ti,’ 2  ) as given in the following  equation (Walker and Pitman, 1976). S  =  0 (1 S  -  ekct)  [31  Thus, if S 0 and t 2 are known, S can be determined for any particular / 1 time (t). By 30 mm  of loading (equivalent to 4 cytoplasmic half-lives, see  Table 2), the specific activity of cytoplasmic ‘ 4 N 3 H (Sc) is brought to approximately 94% of S and 13 4 accumulated within the cytoplasm NH also reaches about 94% of Q (in Table 4). Therefore, the proportion of Q+ transferred to the vacuole is given by:  62 [27]  and from Equation [27], the flux to the vacuole estimated (Method I). The portion of vacuole  () can be roughly  Q*c+v that is transferred to the  (Q*) is given by: /  v—  *_(  ‘  +vI\  *  c+v  The accumulation of tracer in the root vacuole is related to the chemical flux to vacuole  (Ø) and the specific activity of the cytoplasm at each  interval: *  v (t)  C ‘I’cv •-‘c (t)  _f —  Q*Oi*ES(t)  and  [30]  The sum of tracer accumulation within the vacuole by Equation [28], and E Sc  (t)  Q* (  Q*v (t)) is given  can be calculated for each minute from  Equation [3]. Therefore, by means of Method II, it is possible to estimate  Ø, more rigorously from Equation [30].  3.3. RESULTS  3.3.1. Compartmental analysis Analysis of the ‘ N released into ‘washing’ solutions during 3 compartmental analysis revealed that 99.5% of the radioactivity was retained on the CEC (Table 1). Since positively charged amino acids (arginine, histidine and lysine) represented only 5% of total amino acids in  63 3-week-old rice roots (Yoneyama and Kumazawa, 1974), I interpreted this result to indicate that 4 NH was the predominant N species released from 3 ‘ roots and adsorbed on the cation exchange resins. The influence of [NH 0 on compartmental analyses was investigated ] 4 by using G2, G100, or G1000 plants, to represent inadequate, adequate and excess N supply, respectively, prior to efflux measurements. A representative sample of such data (18 mm  efflux) for G1000 plants is  shown in Fig. 3. Three distinct phases, having different slopes with high r 2 values were found for each of the three types of plants tested (G2, G100 and G1000). These compartments were tentatively defined as corresponding to: (I) the superficial solution adhering to roots, (II) the cell wall and (III) the cytoplasm, respectively. The half-lives for exchange ) of these compartments were calculated to be 2 / 1 (t  3 sec, 0.5 to 1 mm,  and 7 to 8.5 mm, respectively (Table 2). According to Duncan’s multiple range test, there were no significant differences for these values among plants grown under different concentrations of 4 NH except for the cell, wall fraction of G2 plants. One important part of the compartmental analysis was to calculate the fluxes of NH 4 across the plasma membrane of root cells. These calculated fluxes are in good agreement with the values obtained by more direct methods using the same root material (Table 3). Influx  (%) varied  with the NH 4 level provided during the growth period. Average NH 4 influx values for G2, G100 and G1000 plants were estimated to be 1.32 ± 0.07, 6.08 ± 0.61, and 10.16 ±0.31 j.tmol g’FW h’, respectively. Net flux (Pnet)  was estimated by subtracting the estimated values of 4 N 3 ‘ - efflux H  (derived from efflux analysis) from the influx of NH 13 or by measuring , 4  64  Table 1. Separation of 13 N-labeled compounds by cation exchange column. The loading solution, efflux solution and shoot extract were assayed. Each mean is the average of two replicates ± Se.  Radioactivity adsorbed on CEC (% of total cpm in sample)  1) in loading solution  99.7 ± 0.1 (2)  2) in efflux solution  99.5 ± 0.5 (2)  3) in shoot extract  0.7 ± 0.2 (2)  65 8 7•  —‘S  .—  C.?  E 6-  .  5. 4. ‘5-,  —  3. — ‘-,  2-  z  1  0-  0  •  -  I  I  •  5  •  10  Efflux  time  15  20  (mm)  Figure 3. A representative pattern of 13 4 released from intact roots. NH 13 (log(cpm) g’FW mm-i) released from intact rice roots 4 The rate of NH of G1000 plants during 18 mm efflux (see text for details). Three phases (I, II, and III) of 13 4 releasing were determined by correlation coefficient. NH  66  Table 2. Estimated half-lives of 13 4 exchange for three compartments of NH root cells. Means for half-lives of ‘ 4 N 3 H exchange (t ) for three 2 / 1 compartments (superficial, cell wall, and cytoplasm) were estimated from the efflux analysis. G2, G100 and G1000 plants were loaded in 13 N-labeled MJNS for 30 mm  and effluxed in un-labeled identical MJNS for 18 mm  at  steady-state conditions with regards to [NH . Each mean is the average 0 ] 4 of 4 individual efflux tests ± Se. Compartments  G2  G100  G1000  I.  Superficial (s)a  3.42 ± 1.00 a  3.83 ± 0.24 a  3.38 ± 0.37 a  II.  Cell wall (mm)  1.06 ± 0.10 b  0.57 ± 0.09 a  0.43 ± 0.06 a  III.  Cytoplasm (mm)  6.95 ± 1.14 a  7.36 ± 0.12 a  8.33 ± 0.60 a  a Duncan’s multiple range test was used to compare the means of each compartment. Only means followed by a different letter are significantly different at the 5% level of significance.  67  Table 3. Comparison of ‘ 4 N 3 H fluxes across the plasma membrane of root 4 N 3 1 H fluxes (influx, efflux, and net flux) is the average cells. Each mean + of 3 or 4 replicates with ± Se. Methods  G2  Influx (%): -- efflux analysis a 4 (1) ‘NH  G100  G1000  (jimol g’FW h’) 1.20 ± 0.07  5.97 ±0.41  10.51 ±2.04  (2) 1 4 N H accumulated in roots b 1.39 ±0.02  5.27 ±0.20  10.16 ±0.23  4 depletion of medium b 1.37 ±0.02 (3) NH  6.99 ±0.51  10.29 ±0.29  NH depletion of medium b 1.33 ± 0.01 1 (4) 4  6.11 ±0.32  9.66 ±0.63  1.06 ±0.07  4.80 ± 0.39  7.41 ± 1.55  -’- depletion of medium a 1.11 ± 0.04 4 NH (6) 14  4.32 ±0.15  6.08 ±0.27  Net flux (Pnet): 4 efflux analysis a (5) NH  Efflux (): (7) 4 NH efflux analysisa 1  0.13 ±0.02  1.17 ±0.14  3.09 ±0.56  (8) Subtracted (6) from (2)  0.27 ±0.02  0.94 ± 0.05  4.09 ±0.04  (9) Subtracted (6) from (4)  0.22 ±0.17  1.79 ±0.47  3.58 ±0.36  a Based on 30-mm uptake; b Based on 10-mm uptake.  68 net depletion of 4 NH from the uptake solution. Both methods gave ‘ similar results with average values of 1.09 ± 0.03, 4.56 ± 0.24, and 6.75 ± 4 g’FW h-i for G2, G100 and G1000 plants, respectively. The 0.67 jimol NH influx and net flux values of G100 plants were 4 fold higher than those of G2 plants (Table 3). Fluxes of G1000 plants were about 1.5 times the values of G100 plants. Efflux values, expressed as percentages of influx, were 11%, 20%, and 29% for G2, G100 and G1000 plants, respectively, (Fig. 4). Since the volumes of subcellular compartments are very different (Steer, 1981; Patel, 1990), it is necessary to distinguish between 4 NH ’content (Q) expressed as moles per unit weight of roots (pmol g’), and NH 4 ’- concentration 4 ([NH ] ) expressed as moles per unit volume of a compartment (jiM or mM). The results of estimated cytoplasmic NH 4 concentration 4 ([NH ] 4 contents ), chemically assayed total root NH  (Q) of  G2, G100 and G1000 plants, as well as calculated values of , 1 4 [NH -] 4 [NH ] , -’-] and [NH 4 [NH Q and Q, are presented together in Table 4. Values of 1 -’-J 4 were higher with higher levels of 4 NH provision. The values of [NH4-’-]c, were 5 to 6 fold higher in G100, and 10 fold higher in G1000 plants than in G2 plants. The values for the vacuolar pool were based on the differences between the total NH 4 content in the roots  (Qj) and the  cytoplasmic pool (O). Of the total NH 4 of the roots, 92% was localized within the vacuole in G2 plants and about 72% to 76 % in G100 and G1000 plants. Chemical and radioisotopic quantities for various compartments used in calculating cytoplasm (S E Q*v  (t)  (t))  are presented in Table 5. The specific activity of  was calculated for each minute from t=1 to 30 mm. Both  and E S  (t)  were used for estimating  methods I and II are given in Table 5.  cv.  The  P, estimated by  69  90  .Efflux 11%:.  Net Flux 899k  G2  Efflux 20%  iIIralIJIiIdlIl  Net Flux 8O9  Net Flux 719k  Efflux  G100  G1000  Plants Figure 4. Fluxes of G2, G100, G1000 plants. Efflux percentage of influx  (Ø)  (Ø)  and net flux  (ønet)  as  for G2, G100 and G1000 plants at steady-state  based on the data of compartmental analysis in Table 2.  70 Table 4. Size of ammonium pools in root cells. Ammonium pools in root cells of G2, G100 and G1000 plants at steady-state. The contents of un metabolized NH ÷ in root tissues (Q) and cytoplasm (Q) and vacuole (Q,) 4 and their corresponding NH 4 concentrations ([NH4], and [NH4-’-]V), as well as that of the cell wall pool 4 ([NH ’-]), are presented. 4 content NH  Plant b  ÷ concentrationa 4 NH -1-]w 4 [NH  (imol g FW root) 4  ] 4 [NH  [NH ÷ 4 ]  (mM)  G2  2.38  0.19 ( 8%)  2.19 (92%)  0.56  3.72  2.58  G100  4.31  1.03 (24%)  3.28 (76%)  2.27  20.55  3.86  G1000  6.85  1.94 (28%)  4.91 (72%)  14.41  38.08  5.78  a The values of [NH and 4 ] 4 [NH were estimated from compartment analysis with ] four replicates each and [NH 4 was estimated from Q . 3 b The values of 4 assay with three replicates each Q were obtained from chemical NH and are the same as the values of 4 [NH +]. C  The values of Q were calculated from ENH ] based on the assumption that the 4 cytoplasm only had 5% of total cell volume. d The values for Q, are based on the difference between Q and Q and the assumption that the vacuole occupies 85% of cell volume. In parenthesis, Q or Q, respectively, are presented as percentages of Q.  71 3.3.2. Metabolism and translocation of 13 N Virtually none of the ‘ 4 N 3 H absorbed by rice roots was translocated to the shoots (Table 1). It is improper to express the translocation of ‘ N 3 (to the shoot) as j.tmol NH 4 per gram fresh weight of roots because (a) ‘ N 3 is transported from the root in the form of amino acids and (b) the specific activities of these amino acid pools were unknown. Therefore the translocation was expressed as a percentage of the total radioactivity (cpm accumulated in roots plus shoots during the loading period). This total radioactivity is equivalent to net absorption of . 4 N 1 H Further fractionation of root tissues of G100 plants by the CEC separation revealed that about 8.6% of the radioactivity provided by influx during 30 mm 4 N 3 ‘ H loading was retained in a metabolized form (Table 6). By combining the 13 N translocated to shoots (10%) with ‘root debris’ (4%) and the ‘Off CEC’ fraction (5%), an estimation of the proportion (19%) of absorbed 1 4 N H that was metabolized during the 30 mm was obtained. The partitioning of radioactivity was also calculated based on the total cpm remaining in roots (Table 6).  3.3.3. Time course of ‘ 4 N 3 H influx in rice roots The results of steady-state 4 N 3 ‘ ’H uptake by G2 and G100 plants, establishing the pattern of ‘ 4 N 3 H accumulation in rice roots, are shown in Fig. 5. The accumulation of ‘ 4 N 3 H appeared to be linear for the duration of the 30 mm  uptake experiments; the coefficient of determination of these  lines (0.87 and 0.99 for G2 and G100 plants, respectively) were high. In all cases, the intercept on the ordinate differed significantly from zero (at 5% significance level). G100 plants had a higher accumulation rate than G2  72 Table 5. Calculation of the flux () from cytoplasm into vacuole. The data used in calculation were taken from the results of the compartmental analysis (Table 4) and root partitioning experiment (Table 6). The calculation procedure is in section 3.2.4.2. Parameter  value  S  164214  4 cpmimol  (t=30 mm)  3361875  cpm pmol-’  ZQ*v(t) (t=3Omin)  79666  cpmg’  238982  cpmg’  1.46  4 imolg  0.97  jimolg’  0.49  4 iimol g  Sc (t)  Q*c+v  Q__v  unit  Pcv  (Method I)  0.97  jimol g’ h’  øcv  (Method II)  1.42  jimol g’ h-’  73  Table 6. Distribution of newly absorbed ‘ N in shoot and root tissues. After 3 30 minutes loading in 13 N-labeled MJNS containing 100 jiM 4 NH ’-, Fractionation of radioactivity in shoots and roots of G100 plants were carried out according to sections 2.5. and 3.2.4.1. Radioactivities are expressed as percentages of total cpm in plants. Each analysis used 100 to 120 plants and data given are means of two replicates (±se). % cpm in plant  [A]  in shoots  [B]  in roots  [C]  9.7 ± 0.9  i.  total  ii.  % recovery after CEC  90.3 ±0.9  (a)  On-CEC  (b)  Root debris  5.1 ± 1.0  (c)  Off-CEC  3.5 ±0.6  81.7 ±2.5  Metabolized  a ‘Metabolized’ is the sum of lines [A]  18.3 ± 2.5  ,  (b) and (c) based on the total cpm in whole  plants or the sum of lines (b) and (c) based on the total cpm in root.  74 4.  ‘GlOO’ plants (R”2 = 0.99) 0  ‘G2’ plants (R”2 = 0.87)  E  1 +r  z 0 0  5  10  20  15  Uptake  time  25  35  30  (mm)  Figure 5. Cumulative uptake of 4 NH by G2 and G100 roots. Time course 3 ‘ study of 13 4 uptake by G2 and G100 roots at steady-state. G2 or G100 NH rice plants were grown and loaded in ‘ N-labeled MJNS containing 2 p.M 3  (a)  or 100 p.M (+) [NH , respectively. Uptake is expressed as the 0 ] 4 H (p.mol g’FW). Each datum point is the average of 3 ’4 N 3 ‘ accumulation of replicates with standard errors as vertical bars.  75 plants. The data for ‘ N accumulation were used to calculate the rate of ‘ 3 N 3 accumulation (influx) as a function of time (Fig. 6). Based upon very short +, the influx of G100 plants appeared 4 exposures (less than 2.5 mm) to 13NH to be about 20 to 30% higher than the steady value of influx. Beyond 5 to 10 mm, influx in both G2 and G100 plants remained essentially unchanged; 1 and 7.5 .mo1 g’FW h’ for G2 and G100 plants, respectively.  3.4.  DiscussioN  3.4.1. The half-lives of 13 4 exchange NH Three kinetically distinct phases (I, II, III) with half-lives for exchange of approximately 3 sec, 1 mm  ÷ 4 NfI 13  and 8 mm, respectively, were  identified by means of compartmental analysis (Table 2). Phase I is probably due to the surface solution on roots carried-over from the ‘loading’ solution (Fig. 3). The second phase is attributed to the cell wall fraction, or the apparent free space (AFS) which is the sum of the Water Free Space (WFS) and the Donnan Free Space (DFS) (McNaughton and Presland, 1983 and references therein). The half-life of this phase (0.5 to 1 mm) was shorter than the equivalent phase reported for corn roots (2.5 mm) by Presland and McNaughton (1986), but similar to the half-life for 3 exchange (0.5 mm) in barley roots (Siddiqi et al., 1991). By using the N0 ‘efflux-funnel’, shorter efflux intervals were achieved. This allowed for resolution of these two rapid phases (I and II) and more accurate estimation of the cell wall half-life.  76 16  A-  14  G100 Plants G2 Plants  +.  4  Uptake  time  (mm)  Figure 6. Influxes of 13 4 into G2 and G100. Steady-state influxes of NH 4 into G2 and G100 roots were measured in the time course study. NH 13 Symbols are the same as in Fig. 4. Influx is expressed as (p.mol g-’FW h’). Each datum point is the average of 3 replicates with standard error (± se) as vertical bars.  77  The third phase is believed to be the cytoplasm. The half-lives of cytoplasmic exchange for G2, G100 and G1000 plants ranged from 6.9 to 8.3 mm, but the differences were statistically insignificant, although the cytoplasmic pool sizes varied according to the provision of 4 NH - during growth (Table 2). Siddiqi et al., (1991) showed that barley roOts, treated with SDS or pretreated by immersion in water at 70°C for 30 mm, accumulated and released significantly less ‘ N0 from phase III, but 3 phase II appeared unaffected. These results were consistent with phase III being the cytoplasm. In studies of 1 4 N H efflux from spruce roots, Kronzucker, H. (personal communication) has found that elevated [Ca 0j ] 2 the loading and washing solutions reduced the extent of phase II for 4 N 3 ‘ H exchange in spruce roots, (which had similar half-lives to those observed in rice) as would be expected if this phase corresponded to the cell wall compartment. The short half-life of ‘ N decay, and long half-life 3 of exchange of the vacuole (Lee and Clarkson, 1986; Macklon et al., 1990), precludes the estimation of vacuolar parameters by efflux analysis using ‘. Using 15 N 3 , Macklon et al., (1990) estimated the half-lives for 4 NH cytoplasmic and vacuolar exchange to be 44 mm  and 8.2 to 22.8 hours,  respectively for excised onion roots. Cooper and Ford (cited in Macklon et al., 1990) observed much shorter t 172 values for cytoplasmic ‘ 4 N 3 H exchange, ranging from 4 to 10 mm in roots of wheat. The latter values are much closer to those obtained in the present studies, i.e. 6.9 to 8 mm (Table 2). The longer tl,/ 2 values reported by Macklon et al., (1990) may have arisen from species differences and/or differences of methodology. In order to select appropriate durations for the loading and washing periods employed in influx studies, it is important to estimate the half lives for NH 13 4 ’- exchange between different compartments (Cram, 1968).  78 The choice of a 10 mm  loading time, used in the present study and in  + influx studies, was arrived at from considering the 4 subsequent 13NH following: (1) the half-life of 13 N decay is short 2 ,’ 1 (t  =  9.98 mm) and  therefore the influx period should be as short as possible. As the isotope decays, the statistical uncertainty in the measurement of 13 N retained by the plant roots or transported to the stem becomes as high as ±15% after about 40 mm  (McNaughton and Presland, 1983); (2) if the loading time is  long, compared to the t 2 for cytoplasmic exchange for , / 1 4 N 3 ‘ H the specific activity of the cytoplasmic pool may approach saturation and the 4 N 3 ‘ H efflux term  c0) 4 (  will be maximized. The measured 13 4 influx under NH  these conditions would approximate the net flux  (Pnet  =  Øoc  -  ‘Pco);  (3)  although the over-estimation of influx (see below) was minimized by 20 minutes, 10 minutes loading reduced that over-estimation to less then 10% (Fig. 6). The duration of the loading period and the post-wash period is a compromise (Lee and Clarkson, 1986). Since the goal was to measure the unidirectional flux across the plasma membrane  (Ø),  N present in the 13  cell wall should be removed during the post-wash period. Based on the estimated t 2 of the cell wall fraction, a short post-wash period of 3 mm / 1 (corresponding to 3 to 6 half-lives, Table 1) was adopted in all influx experiments. In order to equilibrate the cell wall fraction to any changes of 0 4 [NH , -] rice roots were, therefore, always pretreated for 5 mm in identical un-labeled MJNS before loading in 13 N-labeled MJNS.  3.4.2. Fluxes of 13 4 into root cells NH + appeared to be 4 The results of the present study showed that 13NH accumulated at a constant rate (r 2  =  0.874 and 0.997, respectively) during  79 30 mm  loading of G2 and G100 plants under steady-state conditions (Fig.  5). Moreover, ‘ 4 N 3 H accumulation increased with increasing [NH 0 + 4 ] of the loading solution. This observation is similar to previous reports indicating that the accumulation of ‘ N (either as 3 N0 or ÷ 3 ‘ 4 N 3 ‘ H ) by plant roots increases in linear fashion during short (usually <15 mm) loading periods (Presland and McNaughton, 1984; Lee and Drew, 1986). The data for 13 4 NH ’- accumulation by G2 and GlOD plants are also presented as plots of influx versus time (Fig. 6). Influx values based upon very short exposures to 4 N 3 ‘ ’H were accompanied by large errors probably associated with the lower counts accumulated and a large multiplicative factor involved in calculating influx on a per hour basis. Nevertheless, the data indicated that initial influx values were 20 to 30% higher than those recorded after 2 to 5 mm. After loading for more than 5 mm, the influxes were 1 and 7.5 jtmol g F 1 W h 4 for G2 and G100 plants respectively, and notwithstanding some variation, remained reasonably constant for the next 25 mm. Presland and McNaughton (1984) noted a higher rate of 4 NH accumulation in maize 1 roots during the first 2 mm  that they attributed to apoplasmic filling. In  the present study, although the roots were subjected to a 3 mm post-wash, any tracer uptake by rice roots during the post-wash period would represent an over-estimate. The impact of these additional counts would be to over-estimate the calculated influx values at shorter loading intervals due to the multiplicative effect in calculating fluxes on a per hour basis. This effect, which decreases as the duration of the influx period increased, was minimized at about 20 mm  (Fig. 6). This interpretation is in  contrast to that of Lee and Ayling (1993) who argue that the lower counts recorded after 2 to 5 mm  represent an under-estimate of influx due to  release of absorbed ‘ N or ‘ 3 N as cytoplasmic specific activity reaches 5 steady-state. I question this interpretation because: (1) the t 172 for  80 exchange of 4 NH from the cytoplasmic phase was 1  8 mm for rice roots  grown at various nitrogen conditions (Table 2); (2) the absolute value of the efflux from cytoplasm to outside  () varied from 10 to 30% of influx  (Ø) according to compartmental of analyses (Table 3). Therefore I consider it unlikely that a significant reduction of measured influx would result from efflux of tracer during the short duration of these exposures. Values for influx  efflux  (Ø) and net flux  (Ønet)  of 13 4 NH  determined by efflux analyses corresponded very well with those obtained by other (more direct) methods (Table 3). This close correspondence allows us to accept the parameters derived from ‘ 4 N 3 H compartmental analysis with some degree of confidence. Influxes of 13 4 into rice roots under NH steady-state conditions increased according to the levels of [NH 0 + 4 ] in the growth media (Table 3). A similar trend was shown for net fluxes determined either by efflux analysis or by depletion methods. Net uptake (Pnet)  tended to show only a small increase as [NH 0 increased from 100 ] 4  to 1000 1 iM (Table 3). This confirms my previous report that net uptake of 4 was acclimated to [NH NH 0 + 4 ] in growth media, although the acclimation was not achieved by G2 plants (Wang et al., 1991). These results demonstrated that NH 4 fluxes are closely related to the nitrogen status of plants, which is determined by plant growth conditions. Estimated effluxes of NH 4 from rice roots were about 10, 20 and 29% of the influx values for G2, G100 and G1000 plants, respectively (Table 3 and Fig. 4). In addition, efflux was positively correlated with the [NH 4 ’-] (Table 3 and 4). This result agrees with the suggestion that continuous NH 4 efflux may be a common feature of net NH 4 uptake by roots of higher plants (Morgan and Jackson, 1988a). Nitrogen efflux (either 4 or 3 NH NO ) has been reported to be quite significant, particularly at  81 elevated concentrations of N (Morgan et al., 1973; Breteler and Nissen, 1982). Indeed, Deane-Drummond and Glass (1983a, b) suggested that nitrate efflux might regulate net uptake by means of a type of ‘pump and leak’ mechanism. By contrast, Lee and colleagues have emphasized the importance of influx in the regulation of net uptake of nitrate, although nitrate efflux was equivalent to almost 40% of nitrate influx in barley roots (Lee and Clarkson, 1986; Lee and Drew, 1986). Morgan and Jackson (1988a, b) also found a sizable net efflux of endogenous N 4 ‘ ’H occurred upon transfer to 15 4 solutions in wheat, oat, and barley adequately NH supplied with nitrate. However an exact parallel between root ammonium concentrations and net 14 4 efflux was not observed. Although plasma NH membrane influx determines the maximum rate of net uptake (Lee and Clarkson, 1986), efflux certainly makes a significant contribution to determining net uptake. Because of its short half-life, ‘ N is unsuitable for the determination 3 of vacuolar parameters by efflux analysis. Nevertheless, the combination of 4 N 3 ‘ H efflux analysis and the CEC separation of ‘ N products enabled us to 3 estimate  Ø,  using two methods. Both results give values for  in the  range from 1 to 1.5 jimol g-’FW h. Method (I) is based on the estimated 1 4 accumulation during 30 mm NH 13  loading, while method (II) involved  the use of S values estimated minute by minute from a knowledge of the half-life of cytoplasmic exchange (see section 3.2.5.). Therefore method (II) is probably more refined than the value derived from method I. These values are somewhat lower than those obtained by efflux analysis in onion (Macklon et al., 1990), however the Macklon’s study was undertaken at 2 mM , 0 + 4 [NH 1 compared to my analyses undertaken with G100 plants at 100  82 jiM NH . The differences may also reflect the methodology and plants 4 species employed.  NH pools in roots 3.4.3. The 4 In the present study, the values of Q were in the range from 2.38 to 6.85 jimol g’FW for roots grown with different levels of NH -’- (Table 4). 4 Fentem et al., (1983b) reported a value of 3.2 jimol g’FW in 9-d-old barley roots grown in 1 mM NH . For barley, wheat and oat grown in NO 4 3 or N-free conditions, the value of 1 +] was in the range of 0.4 to 2 jimol 4 [NH FW (Morgan and Jackson, 1988a,b, 1989). However, when plants grown 1 gin NH 3 were pretreated with 0.5-1.5 mM 0 4 or in N0 +] for various 4 [NH periods of time, the values of Qj were high and varied from 6 to 35 jimol g ‘FW (Lee and Ratcliffe, 1991; Morgan and Jackson, 1988a). The relatively low intracellular NH 4 content, particularly, under steady state conditions, may reflect the efficiency of NH 4 assimilation (Goyal and Huffaker, 1984). Irrespective of the 0 -’-] provided during the growth period, the 4 [NH bulk of absorbed NH 4 was localized in the vacuole (Table 4). Nevertheless, because of the large size of the vacuole, the values of [NH -’-] were 4 significantly lower than those of the [NH +] 4 [NH ÷] (Table 4). Increasing 0 4 from 2 to 1000 jiM, caused [NH4]c to increase more than 10 fold, while ÷] increased by only 4 [NH  2 fold. Cytoplasmic NH 4 concentrations of rice  roots estimated in the present study (Table 4) were in the range of reported values for wheat, maize, barley and onion (Fentem et al., 1983b; Cooper and Clarkson, 1989; Macklon et al., 1990; Lee and RatcIiffe, 1991). On the basis of NMR studies of NH 4 distribution in root tip of maize, cytoplasmic [NH +] ranging from 3 to 438 jiM were reported (Roberts and 4  83 Pang, 1992). However, in that study, lower values might be expected since root tips were excised from 2-day-old maize seedlings and maintained without an exogenous source of NH 4 during estimation of [NH ] by NMR. 4 My indirect estimation of [NH4]v provided a range from 2.6 to 5.8 mM for G2, G100 and G1000 plants (Table 4). Using 5 ‘, Macklon et al. (1990) N reported a similar range (3.9 to 10.9 mM) for [NH4] in cortical cells of onion roots. Slightly higher values (15 to 36 mM) for 4 [NH ’-] were estimated in maize roots by ‘ N-NMR spectroscopy (Lee and Ratcliffe, 4 1991).  3.4.4. Model of NH 4 uptake by rice plants Despite the widespread use of compartmental analysis to investigate compartmentation of non-metabolized ions, e.g. Cl- (Cram, 1968), Na (Jeschke and Jambor, 1981), and K (Memon et al., 1985), relatively few studies have been undertaken using metabolizable ions such as PO 4 (Lefebvre and Clarkson, 1984), NO - (Presland and McNaughton, 1984; Lee 3 2 (Cram, 1983) and NH 4 and Clarkson, 1986; Siddiqi et al., 1991), S0 4 (Macklon et al., 1990). Presland and McNaughton (1984) postulated the existence of four compartments (three in the roots and one in the shoot) based upon the distribution of ‘ N among these tissues in maize plants. 3 Using 1 4 N H efflux analysis with excised onion roots, the compartmental parameters for superficial, water free space, Donnan free space, cytoplasm and vacuole were identified (Macklon et al., 1990). The present study has characterized two intra-cellular compartments and one extra cellular compartment for 13 4 in rice roots. The biochemical NH fractionation approach was also used to identify different compartments  84  Plasmakmma  Root cell  Stele  —  ‘I. Cytoplasm [13  NH4  .  OC 1 i5.78J 0  ‘S “S ‘.S5 “S ‘S  [_[_]  Metabolites  A55 1%  I  cI1O%  cxc(?)  ‘S  Figure 7. Proposed model for ammonium uptake and compartmentation in rice G100 roots. The bold values in parentheses are estimated fluxes of absorbed 13 4 (jimol g”FW h”). The percentages represent the relative NH distributions of ‘NH 4 among the compartments as a proportion of the isotope entering the cell during the 30 mm  loading. oc, from outside  plasmalemma to cytoplasm; øco, from cytoplasm to outside plasmalemma; 4cv, from cytoplasm to vacuole; vc, from vacuole to cytoplasm; ‘Icx, metabolites translocation from root to shoot; ‘Pxc, metabolites translocation from shoot to root;  cIASS,  assimilation rate; ØDEG, degradation rate;  p  represents chemical flux and 1 represents radioisotopic flux. Fluxes accompanied by (?) indicate fluxes for which data are not available from the present study.  85 for NH 4 assimilation. By using 15 , three compartments were found 4 NH corresponding to different cell types and a storage pool in barley roots (Fentem et al., 1983a) or different organelles (Rhode et al., 1980). Spatial differences in the activities of enzymes involved in 4 NH ’- assimilation are also found along the root (Fentem et al., 1983a). In addition to this form of heterogeneity, there are distinct isozymes of glutamine synthetase, located within the cytosol and within plastids (Miflin and Lea, 1980). Much less information is available concerning the partitioning of newly absorbed ammonium between these compartments, particularly concerning the partitioning between metabolized and un-metabolized fractions in the root and translocation to the shoot. In the present experiments, nearly 90% of absorbed ‘ N remained in the roots, of which 3 80% was in the cation form ) 4 N 3 (‘ H after 30 mm  ‘loading’ (Table 6).  Among the ‘metabolized’ ‘ N pools (ass) in roots, significant quantities of 3 absorbed ‘ N (10%) were translocated to shoots (F) during the 3 experiment (Table 6), and analysis of this ‘ N by ion-exchange 3 chromatography (Table 1) revealed a virtual absence of 13 . The 4 NH remaining metabolized fractions consisted of 5.5% that failed to be held on the CEC, presumed to be amino acids and/or soluble protein, and 3.9%, which was not soluble and remained associated with the ‘Root debris’. Calculations derived from results of both efflux and chemical analyses showed that un-metabolized NH 4 in the cytoplasm (Q) constituted only 8% of Qj for G2 roots and 30% for G100 and G1000 roots, respectively, (Table 4). Taking G100 plants as an example, a model describing the spatial and biochemical compartmentation of newly absorbed 4 NH ’- uptake by rice roots is given in Fig. 7. About 24% of un-metabolized NH 4 was allocated to the cytoplasm and 76% to the vacuole. Based on the influx of 13 ÷ into 4 NH  86 roots, 21% and 40% of ‘ N remained in the cytoplasmic and vacuolar 3 compartments, respectively, along with 20% that was effluxed and 19% that was assimilated. Of the 19% assimilated, roughly half (10% of influx) was translocated to shoots. This assimilation rate was based on total 13 N transported across the plasmalemma and may underestimate the true assimilation rate because during the loading period, the cytoplasmic 4 NH 3 ‘ pool would not have reached steady state.  3.5. SUMMARY Uptake of 13 4 by roots and distribution of 13 NH 4 among plant NH parts and sub-cellular compartments was determined on rice plants grown hydroponically in MJNS containing 2, 100 or 1000 jiM NH . At steady4 state, the influx of 4 NH was determined to be 1.31, 5.78 and 10.11 jimol 3 ‘ g’FW h’, respectively, for G2, G100 and G1000 plants; efflux was 11, 20, and 29%, respectively, of influx. The NH 4 flux to the vacuole was calculated to be between 1 to 1.4 jimol g’FW h-i. By means of 4 NH 1 ÷ efflux analysis, three kinetically distinct phases (superficial, cell wall, and + exchange of 3 4 cytoplasm) were identified, with half-lives for i3NH seconds, 1 and 8 minutes, respectively. Cytoplasmic [NH ] was estimated 4 to be 3.72, 20.55, and 38.08 mM for G2, G100 and G1000 plants, respectively. These concentrations were higher than vacuolar 4 [NH ] , yet 72% to 92% of total root NH 4 was located in the vacuole. Distributions of newly absorbed 13 4 between plant parts and among the compartments NH were also examined. During a 30 minute period G100 plants metabolized 19% of the influxed 13 . The remainder (81%) was partitioned among 4 NH  87 the vacuole (20%), cytoplasm (41%) and efflux (20%). Of the metabolized N, roughly one half was translocated to the shoots. 3 ‘  88  Chapter 4.  KINETICS OF ‘3NH 4 INFLUX  4.1. INTRODUCTION Despite the potential benefits of nitrate for the growth of rice plants, especially under anaerobic conditions (Malavolta, 1954; Bertani et al., 1986), ammonium is the predominant and most readily bio-available nitrogen form in paddy soil (Yu, 1985). It is the preferred nitrogen species taken up by rice (Fried et al., 1965; Sasakawa and Yamomoto, 1978), and in terms of the efficiency of fertilizer ultilization, ammonium is superior to nitrate in paddy soil (Craswell and Vlek, 1979). Ammonium uptake systems have been well defined as concentrative, energy-dependent and carrier-mediated in algae (Smith and Walker, 1978), fungi (Kleiner, 1981), bacteria (Kleiner, 1985), and cyanobacteria (Boussiba and Gibson, 1991). However compared to the extensive investigations of N0 3 uptake, the kinetics and energetics of ammonium transport in higher plants have received relatively little attention. In both rice plants and Lemna NH 4 uptake followed a bi-phasic pattern, with a saturable carrier-mediated system operating at low external NH 4 ([NH ) 0 ] 4 and either a second saturating system (Fried et al., 1965) or a linear diffusive component at elevated [NH 0 (Ullrich et al., 1984). In N-starved ] 4 Lemna both NH 4 uptake by the saturable system and depolarization of  plasma membrane potential were found to exhibit the same concentration dependence (Km 5 for both processes were 17 jiM). At higher [NH T 0 4 ’-] the uptake by the linear system was not accompanied by further depolarization of membrane potential (Ullrich et al., 1984). The saturable  89 4 uptake was sensitive to some metabolic inhibitors component of NH (Sasakawa and Yamamoto, 1978) and to changes of root temperature (Bloom and Chapin, 1981). In addition, 4 NH ’- uptake is subject to negative feedback, supposedly from N metabolites (Lee and Rudge, 1986; Morgan and Jackson, 1989; Clarkson and Luttge, 1991). Youngdahl et al., (1982) demonstrated that NH 4 uptake in rice decreased with plant age. However, despite these studies, the mechanism(s) of 4 NH ’- uptake by roots of higher plants remain unclear. In particular, the high concentration system represents virtually unexplored territory. Ammonium is unique among inorganic cations, because following absorption by plant roots, it is rapidly assimilated into organic poois. This has made the analysis of uptake and the subsequent fate of absorbed NH 4 much more complicated than for cations such as K or Ca . The availability 2 of 13 N to this laboratory has enabled us to measure short-term 13 4 NH influx into roots of intact rice plants (Wang et al., 1991, 1993a, 1993b). This is critically important for two main reasons. Firstly, this technique allows determination of the particular flux (e.g. unidirectional plasma membrane influx or efflux), which is responding to the imposed conditions. By contrast, net uptake measurement, often obtained by means of longterm depletion experiments, actually measures the difference between influx and efflux. This is especially relevant because nitrogen (either NH 4 or 3 NO ) efflux has been reported to be significant, particularly at elevated concentrations of N (Morgan and Jackson, 1989; Breteler and Nissen, 1982; Wang et al., 1993). Secondly, by judicious choice of appropriate influx and desorption times, based upon the half-lives for exchange of the sub compartments of the root (Lee and Clarkson, 1986; Presland and McNaughton, 1986; Siddiqi et al., 1991; Wang et al., 1993), it is possible to  90 measure the plasma membrane influx as opposed to other fluxes (to vacuole or to stele) which result from long-term experiments (Cram, 1968). The objective of this study was to investigate the mechanisms and characteristics of ammonium uptake by rice plants. I have particularly emphasized short-term responses of 13 4 influxes to changes in [NH NH 0 ] 4 of uptake solutions over a wide range of external concentrations, in order to define the transport mechanisms responsible for influx across the plasma membrane. I have examined the influence of prior NH 4 provision upon the kinetic parameters for influx by both components of the bi phasic system for 4 NH transport. In addition the sensitivities of these fluxes to metabolic inhibitors, short-term variations in temperature and pH were determined with a view to clarifying the mechanisms of these fluxes.  4.2.  METHoDS AND MATERIALS  4.2.1. Plant growth and 1 N production See section 2.2. Seed germination; section 2.3. Growth conditions; section 2.4. Provision of nutrients; and section 2.5. Production of 13NH + 4  4.2.2. Relative growth rate Rice seedlings were grown in 2, 100, and 1000 iM NH 4 (designated, hereafter, as G2, G100 and G1000 plants, respectively) to represent inadequate, adequate and excess nitrogen provision. Total fresh weights of  91 plants were recorded for three treatments at ages of 14, 21 and 28 d. They were used to calculate relative growth rates (RGR).  4.2.3. Influx measurement See section 2.3.1.  4.2.4. Kinetic study Influxes of G2, G100 or G1000 plants, respectively, were measured in N-labeled MJNS varying in [NH 3 ‘ 0 4 ’-] from 2 pM to 40 mM in perturbation experiments. Perturbation experiments are defined as those in which plants are grown at one particular , 0 + 4 [NH ] and influxes are measured in a range of [NH . Measured 13 0 ] 4 ÷ influxes at various [NH 4 NH 0 + 4 ] were fitted to the Michaelis-Menten equation V  =  (Vmax  io)/(Km 4 [NH  +  0 ’ 4 [NH ) -]  [31]  and a more comprehensive equation V  ]o)/(Km 4 (Vmax • [NH  +  0 ’ 4 [NH ) -]  +  b  •  0 ’ 4 [NH -]  +  a  [32]  by means of a non-linear regression method using the computer program “Systat” (Wilkinson, 1987). In the equation, V (imol g’FW h-i) stands for the influx measured at a particular [NH . Vmax is the calculated 0 ] 4 maximum rate of influx while Km (.tM) represents 0 -’-] giving half of the 4 [NH maximum influx; b and a are constants characterizing the linear phase. At each concentration tested, influxes were determined in two to six separate  92 experiments with three or four replicates. Each replicate consisted of about 20 rice seedlings. Based on the results of the kinetics studies (see Results), measured 4 influx from NH  <  1 mM [NH 0 + 4 ] appeared to result from a saturable high  affinity transport system (hereafter referred to as HATS). Since the influx by the HATS had saturated between 0.1 and 1.0 mM , 0 4 [NH ’-] influx from 0.1 mM [NH 0 + 4 ] was selected as a concentration representative of the HATS in the following studies. Above 1 mM 0 +] measured NH 4 [NH , 4 influx appeared to result from the participation of both the HATS and a low affinity transport system (hereafter referred to as LATS). Therefore, the difference between measured influx at concentrations >1 mM [NH 0 and ] 4 the saturated values of the HATS were taken to represent fluxes due to the LATS.  4.2.5.  Metabolic inhibitor study Influxes were measured in MJNS containing representative levels of  either 0.1 mM to estimate the activities of the HATS, or 20 mM 4 NH C 1 for the HATS plus LATS, in the presence or absence of different metabolic inhibitors. The inhibitors used were as follows: (1) 10 iiM CCCP; (2) 1 mM CN plus SHAM; (3) 50 j.tM DES; (4) 0.1 mM DNP; (5) 50 iM Mersalyl; (6) 1 mM pCMBS. Details of preparation refers to Section 2.9. In this study, both 3-week-old G2 and G100 plants were used. Before labeling with radioisotope, rice roots were treated with un-labeled MJNS containing the same concentrations of CN- plus SHAM for 30 mm. There were no pretreatments for the other inhibitors. Measurements of influx  93 were undertaken as in the kinetic study. Each inhibitor experiment was repeated twice with three replicates for each treatment. Each replicate consisted of about 20 seedlings. Therefore the means for influxes and standard errors were calculated from six replicates and represented the mean for approximately 120 seedlings.  4.2.6. Temperature study Rice plants were grown under the same conditions as described previously, so that they were adapted to 20 ± 2°C. Influxes were subsequently measured in MJNS with either 0.1 mM or 20 mM 4 NH C 1 at solution temperatures of 5, 10, 20 and 30°C. During the pre-wash, uptake and post-wash, solutions were maintained at the designated temperatures. The measurements of influx were undertaken as in the kinetic study.  4.2.7. pH profile study Rice plants were grown in MJNS containing 2 jiM NH 4 under the conditions described in METHODS AND MATERIALS and adapted to growth medium at pH 6. Uptake solutions were adjusted to pH values of 3.0, 4.5, 6.0, 7.5 and 9.0 by additions of HC1 or NaOH, respectively. To examine the effects of solution pH upon ‘ 4 N 3 H influx, roots were exposed to the designated pH levels during 5 mm mm  pre-wash, 10 mm  influx as well as 3  post-wash. Influxes of ÷ 4 N 3 ‘ H were measured in either 0.1 mM or 10  mM NH 4 solution. The choice of 10 mM NH , rather than 20 mM was 4 dictated by the desire to minimize additions of HC1 or NaOH in adjusting pH levels in the uptake solutions.  94 4.3. RESuLTS  4.3.1. Kinetics of NH 4 influx Influxes of ‘ 4 N 3 H in response to external concentrations in the range from 0.002 to 40 mM [NH 0 were resolved into two distinct phases, ] 4 presumably mediated by two separate transport systems; at low [NH 0 (< ] 4 1 mM), a saturable high affinity transport system (HATS); and at high 0 (> 1 mM), the combined activities of a saturated HATS and a linear ] 4 [NH low affinity transport system (LATS). 4.3.2.1. HATS In the low concentration range (< 1 mM 4 [NH ) 0 ] , the values of NH 13 4 ’influx into roots of G2, G100 or G1000 rice plants conformed to Michaelis Menten kinetics (Fig. 8). The kinetic parameters of Vmax and Km were estimated using non-linear regression analysis (Table 7) to fit the Michaelis-Menten equation. Analysis by means of a more comprehensive equation (see equation [32] in section 4.2.4.) gave similar trends although actual values of Vm and Km were slightly different (data not shown). With increasing provision of NH 4 from 2, through 100 to 1000 jiM in the period of two weeks prior to uptake measurements, root [NH ] increased from 4 2.37, through 4.31 up to 6.85 jimoles g’FW, respectively. As shown in Fig. 9, increasing 4 [NH was associated with decreasing Vm values, from 12.8 1 ] through 8.2 down to 3.4 jimol g’FW h’, and increasing Km values, from 32 through 90 up to 188 jiM, for G2, G100 and G1000 plants, respectively.  95 15  -  G2  110. G100  5.  G1000  z  0  0  0.1  0.2  External  0.3  0.4  ammonium  0.5  0.6  0.7  concentration  0.8  0.9  1.0  (mM)  Figure 8. Concentration dependence of 4 -NH influx at low [NH 1 . Influx 0 ] 4 of 13 4 into rice roots was measured in perturbation experiments. Rice NH seedlings were grown at 2, 100 or 1000 jiM NH 4 (G2 (A), G100 (0) or G1000 (x), respectively). Each datum point is the mean of 16 replicates with standard error as a vertical bar. The solid lines are estimated from Vm and Km values (Table 7) of G2, G100 and G1000 plants, respectively.  96  Table 7. Kinetic parameters for saturable and linear ‘ 4 N 3 H influx of G2, G100 or G1000 roots as functions of [NH . The relationships between 0 J 4 +] of uptake solution were estimated from 4 [NH 4 N 3 ‘ H influx and 0 Michaelis-Menten kinetics for influx measured between 2 to 1000 tM 0 and for linearity in the range of 1 to 40 mM, where ‘a’ is the ] 4 [NH intercept and ‘b’ is the slope. G2 HATS a  HATS+LATS  LATS  G100  G1000  Vm  12.8 ± 0.2 b  Km  32.2 ± 2.1  90.2 ± 23.2  188.1 ± 34.5  a  13.21  10.14  4.59  b  0.67  0.79  1.30  2 r  0.97  0.97  0.99  8.2 ±  0.7  3.4 ±  a  0.41  1.94  1.19  b  0.67  0.79  1.30  2 r  0.98  0.96  0.98  0.2  a HATS represents the high affinity transport system, measured below 1 mM [NH4] . 0  Influx measured at concentrations above 1 mM [NH 0 is considered to be the ] 4 combined contributions of both high and low affinity transport systems (HATS+LATS). LATS represents the low affinity transport system and is estimated by subtracting HATS from HATS+LATS. regression with ± se,  b Vmax and Km were estimated by non-linear  97  250  15  -200  10 E —0—  & E  Vmax  Km  -100  5 -50 G2  G1OO  Gi000  •  0.0  Figure 9.  .  2.0 Root  4.0 ammonium  6.0 8.0 concentration (mM)  •  10.0  Relationship between kinetic parameters of NH 4 uptake and  root ammonium concentrations ([NH ) of rice seedlings. The values of 1 ] 4 Vmax (0) and Km (z) from Fig. 8, were plotted against 1 +] for G2, G100, 4 [NH  or G1000 plants, indicated by (L) on the X axis.  98 4.3.1.2. LATS  In the higher range from 1 to 40 mM, the relationship between 0 and 13 ] 4 {NH 4 influx was linear (Fig. bA). The Y intercepts of these NH lines (13.21, 10.14 and 4.59 for G2, Gb00 and G1000, respectively) decreased according to the ammonium provision during the growth and agreed well with the corresponding Vm for the HATS (Table 7). Thus it is concluded that the measured fluxes at elevated [NH 0 + 4 ] result from the combined activities of the HATS and the LATS. To evaluate the effect of prior NH 4 provision on the LATS for ‘ 4 N 3 H influx without the influence of the HATS, the Vmax values for HATS were subtracted from the measured influxes at elevated [NH 0 values. The derived LATS values were re ] 4 plotted accordingly (Fig. lOB). As shown in Fig. lOB, 13 4 influx by LATS NH is higher for G1000 than for G100 or G2. Slopes of the lines increased according to the NH 4 level during growth period (0.67 for G2, 0.79 for G100 and 1.30 for G1000 in Table 7). These linear relationships at high 0 were confirmed by means of F-tests for linearity (Zar, 1974). ] 4 [NH Statistical analyses revealed that the slope of the G1000 line was significantly different from the slopes of the G2 and G100 lines (data not shown).  4.3.2. Effect of metabolic inhibitors on the influx of ‘ 4 N 3 H In most cases NH 13 influxes of G2 plants were reduced by the 4 presence of metabolic inhibitors in the uptake solutions as shown in Fig. 11. Net reductions of influxes, listed in Table 8, were calculated by using the influx of the control as zero reduction (0%). The HATS for NH 4 influx  99 70  60  1OA  G1000  lOB  I 30•  20  :  x  Gl000  0  G100 G2  o  5  110  External  15  20  Ammonium  2’5  30  Concentration  (mM)  Figure 10. Influx of ‘ 4 N 3 H into rice roots at high [NH 0 in perturbation ] 4 experiments. 1OA: Influxes of NH 13 4 - into G2 (A), G100 (0), or G1000 (x) roots, respectively, were plotted against [NH . Each datum point is the 0 ] 4 mean of more than 6 replicates with ± se as vertical bar. lOB: The estimated LATS Fluxes after subtracting the Vrnax of the HATS of G2, G100 or G1000, respectively, from the corresponding measured influxes (in 9-A). These plotted lines of LATS have the same slopes as their corresponding lines in 9-A but with slightly different values of the intercept, 0.53, 1.96 and 0.99 for G2, G100, and G1000 plants, respectively.  100 30 HATS+LATS  EJ HATS E:L!1LATS  --  20  :i  Control  CCCP  CN+SHAM  Inhibitors  DES  in  uptake  DNP  I1Lr  Marselyl  pCMBS  solution  Figure 11. Effect of metabolic inhibitors on 13 4 influx. Rice plants were NH grown in MJNS containing 2 iM 4 NH C 1. Influxes of ‘ 4 N 3 H were measured in MJNS with either 0.1 mM or 20 mM NH 4 in the presence or absence of a specific metabolic inhibitor. Each datum point is the average of more than 6 replicates with standard error as vertical bar. Abbreviations: CCCP (10 mM): Carboxylcyanide m-chlorophenylhydrazone; CN- plus SHAM (1 mM): NaCN and salicylhydroxamic acid; DES (50 mM): diethyistilbestrol; DNP (0.1 mM): 2,4-dinitrophenol; Mersalyl (50 mM): mersalyl acid; pCMBS (1 mM): p-chloromercuri-benzenesulfonate.  101 was reduced by 81 to 87% by the protonophore (CCCP) or the un-coupler of electron-transport-chain (CN plus SHAM) and inhibitors of ATP synthesis (DNP). These three treatments reduced the LATS by only 31 to 51%. ATPase inhibitor DES reduced ‘ 4 N 3 H influx due to the HATS by 51% but had negligible effects on LATS. External protein modifiers of the membrane surface, pCMBS and Mersalyl, reduced 13 ÷ influx of HATS by 4 NH about 40% with slightly less or similar reductions of LATS (22 to 46%). These patterns of inhibition were also observed for G100 plants (data not shown).  4.3.3. Effect of root temperature on ‘ 4 N 3 H influx Short-term perturbations of root temperature significantly affected NH into rice roots that were adapted to the growth 3 1 + the influx of 4 temperature of 20°C (data not shown). Table 9 shows the calculated Qj.o values for G2 and G100 plants in the temperature range from 5°C to 30°C. In this temperature range the Qj.o values for HATS fell from> 2.4 between 5 to 10°C to 1.25 between 20 to 30°C. The results of F-tests in conjunction with Duncan’s Multiple Range Tests demonstrated that Qo values for the different temperature ranges were significantly different for the HATS (P> 0.05). In contrast, there were no significant differences between the Qio values for LATS in the same three temperature ranges for both G2 and G100 plants (P  >  0.05). Nevertheless Qjo values for the LATS were  significantly greater than 1.  102 Table 8. Reduction of ‘ 4 N 3 H influx into roots of G2 plants by various metabolic inhibitors. Treatment  Inhibitor Level  % Reduction of HATS a  Control  None  0  LATS b 0  CCCP  10 mM  84.58  30.72  CN+SHAM  1 mM  80.84  43.20  DES  50 mM  53.96  4.00  DNP  0.1mM  86.72  50.55  Mersalyl  50 mM  41.97  22.40  pCMBS  0.5mM  41.33  46.11  a The influxes of HATS were measured in the representative {NH 0 (0.1 mM). ] 4 Reduction of HATS (%) was calculated by setting the ‘Control’ value, the reduction of influx value measured in 0.1 mM NH4C1 uptake solutions without the inhibitor, as 0%. b Reduction of LATS (%) was calculated by first determining the influx due to LATS by subtracting the influx values measured at 0.1 mM NH 4 from that at 20 mM 4 NH - for control and for each inhibitor treatment, respectively. The reduction of influx value due to LATS under control conditions was then set at 0%.  103 Table 9. Calculated Qjo values for 13 4 influx by the HATS or LATS of NH rice plants grown at 20°C with 2 or 100 iM 4 NH C 1 (G2 and G100 plants). Temperature  G2 Plants a  DMRT b  G100 Plants  DMRT  Range (a)HATS:  5-10°C  2.48±0.04  a  2.59±0.21  a  20°C  1.79 ± 0.08  b  1.68 ± 0.22  b  20-30°C  1.25±0.16  c  1.44±0.16  b  10  (b) LATS  C:  5  -  10°C  1.41 ± 0.21  1.54 ± 0.27  20°C  1.49 ± 0.06  1.90 ± 0.46  20-30°C  1.56±0.06  1.33±0.12  10  -  -  a Each value (± Se) is the average of three means from duplicate experiments; each mean is derived from three replicates. b DMRT stands for Duncan’s Multiple Range Test for comparing all possible pairs of treatment means. Means having a common letter are not significantly different at the 5% significance level.  C  Both F-tests and  DMRT indicated that means for the LATS were not significantly different at the 5% level.  104 4.3.4. Effect of solution pH on ‘ 4 N 3 H influx The effect of uptake solution pH on ‘ 4 N 3 H influx was also investigated. The percentage of the control was computed on the basis of the influx value at pH 6.0 for either HATS or LATS (Table 10). In this case, a Least Significant Difference test (LSD) was used for making pairwise comparisons between the control and other treatments. In the range from 4.5  -  9.0, solution pH had only a small effect  ti  4 influx from 0.1 mM NI-J 13  NH influx by LATS decreased very significantly with 3 1 + , whereas 4 0 ] 4 [NH increasing ambient pH beyond pH 6.0. By contrast, reduction of solution pH down to 3.0 drastically reduced 13 4 influx by HATS as well as LATS. NH  4.4. DISCuSSION  4.4.1. Kinetics of ammonium uptake In Chapter 3 and in Wang et al., (1993a) it was demonstrated that the half lives for ‘ 4 N 3 H exchange of the cell wall and cytoplasmic phases of rice roots (G2, G100 or G1000 plants) were approximately 1 and 8 mm, respectively (Section 3.3.1., Table 2). By using 10 mm and 3 mm  + 4 exposures to 13NH  post-washes, therefore, estimates of plasma membrane influxes  rather than net flux or quasi-steady fluxes to vacuole were obtained (see Cram, 1968). The results of the present study revealed that 4 NH ’- influx across the plasma membrane into rice roots exhibits a bi-phasic pattern: in the low range (below 1 mM ) 0 ÷ 4 [NH , ] influx occurred via a saturable high affinity transport system (HATS); while from 1 to 40 mM [NH 0 + 4 ] a second, low affinity, non-saturable transport system (LATS) became apparent. This  105 Table 10. Effect of uptake solution pH on N 1 H4 influx into rice roots of 3-week-old G2 plants grown at pH  =  6.0 in MJNS. Influx of 13 NH4 was  measured in MJNS at various pH levels (3.0, 4.5, 6.0, 7.5, and 9.0) with 0 at either 0.1 mM for the HATS or 10 mM for the HATS+LATS. The [NH4--] value of LATS was obtained by subtracted the values of HATS from HATS+LATS of each treatment.  (a)HATS:  (b) LATS:  pH  Influx a  3.0  6.91±1.43  4.5  LSD b  (%) of Control  *  53  12.02±0.46  ns  87  6.0  13.22±0.27  control  100  7.5  14.51±0.39  ns  109  9.0  12.94 ± 0.30  ns  95  3.0  15.75 ± 0.45  *  87  4.5  18.63±2.80  ns  103  6.0  18.07 ± 0.49  control  100  7.5  11.44± 1.37  *  63  9.0  9.29 ± 1.54  *  51  C  a Each value (± Se) (i.tmol g’FW h’) is the average of four means of duplicate experiments. Each mean is derived from three replicates.  b LSD stands for Least  Significant Difference test, used for making pairwise comparisons between the control at pH 6.0 and other treatments. significant.  C  *  =  significant at 5% level and ns  =  not  The percentages of control were calculated using the NH 4 influx  measured at pH=6.O as 100%.  106 bi-phasic pattern of uptake has been reported for NH 4 uptake by Lemna (Ulirich et al., 1984), for K uptake by corn roots (Kochian and Lucas, 1982), and for NO - uptake by barley roots (Siddiqi et al., 1990). 3 Plasma membrane ‘ 4 N 3 H influx at low [NH 0 + 4 ] conformed to Michaelis-Menten kinetics (Table 7) in accord with earlier studies of net 4 uptake by rice (Youngdahl et al., 1982; Wang et al., 1991). This has NH also been found to be the case for roots of other species, including corn (Becking, 1956), rye-grass (Lycklama, 1963), and barley (Bloom and Chapin, 1981), where net NH 4 uptake rates saturated in the range from 100 to 1000 iM [NH . The significance of this HATS for NH 0 ] 4 4 in rice roots is that it allows plants to absorb sufficient nitrogen 4 (NH -) from very low levels in the rhizosphere to meet the minimum requirement for plant growth. In the present experiments, for example, by three weeks, the relative growth rates were independent of [NH 0 from 100 to 1000 pM ] 4 . The relative growth rates calculated from total fresh weight of both 4 NH G100 and G1000 plants were at —0.16 d’ for the third week of growth while for G2 the value was 0.06 d’. By the fourth week the differences in RGR had diminished to 0.05, 0.06, and 0.06 d’, respectively for G2, G100 and Gl000 plants. The reduced growth rates of G2 plants were accompanied by increased root:shoot ratios, and leaves were slightly paler than those of plants grown at higher [NH . 0 ] 4 At the higher range of [NH 0 + 4 ] (1 to 40 mM), a linear, low affinity transport system (LATS) also participated in NH 4 uptake by rice roots, as is the case for other ions and plant species (Kochian and Lucas, 1982; Ullrich et al., 1984; Pace and McClure, 1986; Siddiqi et al., 1990). The Y intercepts for lines of measured influx (due to both transport systems) against [NH 0 were in good agreement with the corresponding Vmax ] 4  107 values for the HATS (Table 7), which suggests that the two distinct transport systems (HATS and LATS) are additive. Despite the importance of NH 4 as principal source of N for many plant species and the increasing availability of techniques for the measurement of short-term NH 13 and 15 4 4 influxes, few detailed NH influx isotherms (as distinct from net uptake isotherms) have been reported for NH 4 influx into roots of higher plants. Nevertheless, Ullrich et aL, (1984) were able to demonstrate linear kinetics of NH 4 uptake by Lemna between 0.1 to 1.0 mM [NH 0 using a depletion method. The ] 4  question of the saturation of this apparently linear system at higher concentrations remained unresolved. Clearly, it is difficult to measure net fluxes by employing concentration depletion methods at high external concentrations without extending the uptake experiment for long periods of time. By using short-lived radioisotopes, such as 3 ‘, it has been N possible to measure unidirectional fluxes of N0 3 and NH ÷ at the plasma 4 membrane of intact plant roots (Glass et al., 1985; Ingemarson, 1987; Presland and McNaughton, 1986; Lee and Clarkson, 1986; Siddiqi et al., 1990; Wang et al., 1993). Even at concentrations as high as 40 mM, there was no evidence of saturation of the LATS system (Fig. 10).  4.4.2. Energetics of ammonium uptake The influx of ammonium by HATS is clearly dependent on metabolic energy. In the present study metabolic inhibitors, CCCP, DNP or CN plus SHAM, diminished ‘ 4 N 3 H influxes of HATS by more than 80% (Table 8). The effects of these inhibitors on the LATS were much smaller (31 to 51% inhibition). Further evidence from the 0110 values (Table 9) supported the  108 notion of energy dependence. A Qo value greater than 2 is considered to indicate the metabolic dependence of physiological processes such as ion transport. Short-term perturbations of temperature between 5 to 10°C, significantly increased the Qo values for HATS up to  2.5 compared to  1.5 between 20 to 30°C. In a 7 h concluded that the uptake of ammonium by 9-day old rice seedlings was closely associated with metabolism. However, such long-term studies probably measure the Qo for NH 4 assimilation rather than the transport process. The values of Qo estimated from Ta and Ohira’s data (1982) provided values larger than 2.5 for 15 -’4 NH absorption by rice roots between 9 to 24°C. Lower Qio values (1.0 to 1.6) were reported for net ammonium uptake of low-temperature adapted ryegrass (Clarkson and Warner, 1979); barley (Bloom and Chapin, 1981); and oilseed rape (Macduff et al., 1987) indicating that 4 NH ’- transport had acclimated to the low temperature growth conditions. Consistent with the results of the metabolic inhibitor studies, the present 1 Q 0 study indicated that LATS was less sensitive to changes of root temperature than the HATS (Table 9). The apparent energy-dependence of the HATS may not necessarily mean that NH 4 uptake is an active transport process, although active transport systems for ammonium have been proposed in bacteria, fungi and algae (Kleiner, 1981; Schlee and Komor, 1986, Singh et al., 1987). The accumulation of NH 4 against its concentration gradient could be achieved by active or passive uptake mechanisms: the former, by direct use of metabolic energy to carry a solute across a membrane towards a region of higher electrochemical potential; while the latter, by solute flux across a membrane along the electrochemical potential gradient, a process that may be only indirectly related to metabolic energy.  109 According to the compartmental analysis (Chapter 3 and in Wang et al., 1993a), the cytoplasmic concentration of NH ÷ in G2 roots was 4 estimated to be 3.7 mM. Using this value and -130 mV as measured plasma membrane membrane electrical potential difference for G2 plants in ‘MJNS’ minus Nitrogen solutions (Wang et al., 1992), predictions derived from the Nernst equation indicated that net ammonium uptake would be active only when 0 +] falls below 125 jiM. This is rather similar to the 4 [NH value of 67 jiM calculated for Lemna (Ulirich et al., 1984). However, this calculation only serves to predict the feasibility of the process occurring under the prescribed conditions. The precise relationship between the calculated electrochemical potential difference for an ion and the putative transport systems, predicted on the basis of concentration-dependent influx curves, are difficult to realize. In the present case, for example, there are no discontinuities in the uptake curve corresponding to the predicted concentration at which the switch between active and passive transport (-425 jiM [NH ) occurs. This issue is raised to warn against a 0 ] 4 too literal interpretation of the thermodynamic predictions. While on thermodynamic grounds influx is uphill below 125 jiM and downhill beyond this level, the kinetic data reveal no apparent change of transport mechanism. The characteristics of the two transport systems for NH 4 influx have significant features in common with those described for K-i- uptake in which (incidentally) there is yet no clear consensus regarding the mechanisms of influx into higher plant roots. Likewise, the mechanism of the apparently active transport of ammonium below 125 jiM is unknown. It might occur by means of a specific ATPase or a secondary transport system such as an NH : 4 H symport that is driven by the proton motive force (pmf). As  110 proposed for K’- uptake by Neurospora, for each K entering, one H-’- is co transported and 2H are extruded by the proton pump (Rodriquez-Navarro et al., 1986). The net result is therefore a 1:1 K/H-’- exchange. Is it possible that NH 4 influx is mediated by an analogous system? It has long been documented that 4 NH uptake is associated with strong acidification of the external medium (e.g. Becking, 1956). Likewise in the present study, when pH was not adjusted daily in the initial growth experiments, external pH dropped so low that plants failed to grow normally. So far as the passive uptake of ammonium is concerned at higher concentrations, several authors have proposed that 4 NH ’- influx may occur by an electrogenic uniport in response to the electrical gradient (Kleiner, 1981; Ullrich, 1984). When ambient concentration is beyond the predicted threshold for active uptake, the concentrative NH 4 uptake may be due to a facilitated transport system driven by the electrochemical potential difference for NH . This has two components; the difference in chemical 4 potential of NH 4 (L$LNH ÷) between cytoplasm and outside and the 4 electrical potential difference (z\’-P) generated in part by proton efflux across the transducing membrane. The actual mechanistic link, if one exists, between NH 4 influx and the pmf across the plasma membrane is unclear at present. Certainly the results of the treatments with the protonophore (CCCP) or the un-coupler of ATP formation (DNP and CN plus SHAM), which caused greater than 81% reduction of influx due to HATS, are consistent with a dependence of NH 4 influx on transmembrane pmf. Further support for this hypothesis is provided by the effect of ATPase inhibitor, DES, which reduced ‘ 4 N 3 H influx due to HATS by 54% but had negligible effects on LATS.  111 4.4.3. Effect of pH profile on ammonium uptake In the present study, influx by the HATS was strongly reduced below pH 4.5. By contrast, in the range from pH 4.5 to 9.0, 4 NH influx by the 1 HATS appeared to be relatively insensitive to pH. 13 4 influx by the NH LATS actually decreased with increasing ambient pH beyond pH 6.0. It has been reported for several species that the specific uptake rate of 4 NH - can be reduced by short-term decreases in pH below 6.0 (Munn and Jackson, 1978; Marcus-Wyner, 1983; Vessey, 1990) and even terminated alltogether at pH 4.0 (Tolly-Henry and Raper, Jr., 1986). Tanaka (1959) suggested that rice is very sensitive to pH below 4. Most probably this reflects a general detrimental effect of such acidic conditions on the transport systems. In addition, it has been observed that when plants were grown at such low pH values over extended periods of time, the roots became stunted and discolored. It has been suggested that both high pH and/or high ammonium concentration of solution may result in high rates of NH 3 uptake due to increased NH 3 concentration and the higher permeability of cell membranes to NH 3 than NH 4 (see Macfarlane and Smith, 1982). However, in many studies this expectation has not been observed, and uptake failed to increase at elevated pH (MacFarlane and Smith, 1982; Deane-Drummond, 1984; Schlee and Komor, 1986). Likewise, in the present study, influxes of 4 NH due to the LATS were reduced by 1 25  -  35% at higher pH (7.5  -  9.0), despite a predicted increase of [NH ] 3  from less then 0.1% of total [NH 4  +  ] at pH 6.0, to 36% at pH 9.0 3 NH  according to the pKa for NH 4 (9.2 5). Furthermore, membrane electrical potentials of rice roots have been shown to be depolarized by elevated ammonium concentrations (Wang et al., 1992). These observations indicate the entry of cation 4 (NH + ) rather than neutral ammonium 3 (NH ) . The  112 evidence from our electrophysiological study of rice roots indicated a linear relationship between depolarization of membrane potential and 4 from 1 to 40 mM (data not shown). Therefore, at elevated influx of NH concentration and pH, it is unlikely that simple diffusion of NH 3 could be considered as a major component of the influx of LATS. Nevertheless, in their study using Lemna, Ullrich et al., (1984) reported that depolarization of membrane potential was saturated at  0.1 mM even though net uptake  continued to 1 mM in a linear pattern. This observation is consistent with 3 entry by the LATS in Lemna. NH  4.4.4. Regulation of ammonium uptake Although the bi-phasic pattern of NH -’- influx was independent of the 4 4 exposure, the individual systems, particular the HATS, were prior NH extremely sensitive to prior NH 4 exposure (Figs. 8, 9, 10). Evidently NH -’4 influx by the HATS was subject to regulation by negative feedback: with increasing [NH 0 in the growth medium, root [NH ] 4 +J increased and NH 4 4 influx decreased (Fig. 9). It is noteworthy that in the present case, negative feedback regulation appeared to affect both Vm and Km values (Table 7, Figs. 8 and 9). It has commonly been observed that Vmax is strongly and unequivocally influenced by the level of nutrient supplied during growth. By contrast, an effect on Km has rarely been observed (Lee, 1982). Only in the case of K (Glass, 1976) was the Km strongly influenced by K status although other ions such as CF do show small changes (Lee, 1982). In the present study, the values of Km were strongly influenced by the prior level of NH -’- supply, and are positively correlated with 1 4 -’-] 4 [NH .  113 Contrary to expectation, ‘ 4 N 3 H influxes due to the LATS were higher in plants previously maintained at 1000 jiM NH 4 than in those maintained at 2 jiM 4 NH ’-. The reverse was found to be the case for N0 influx in 3 ‘ barley (Siddiqi et al., 1990). This positive correlation between provision of NH 4 ’- and 13 4 influxes at high [NH NH 0 + 4 ] may indicate that the LATS may not be subject to regulation by negative feedback. Another possible explanation is that better nitrogen nutrition may provide more building materials (protein?) for constructing transporters. However, exposures to high [NH 0 4 ’-] (>1 mM) were brief and in longer exposures NH 4 influx may be down-regulated in accord with expectation. The present study has demonstrated the strong negative downregulation of influx by the HATS in response to elevated 4 NH supply during growth. At present the mechanism(s) and signals responsible for this down-regulation of uptake are unclear. Feedback signals may result from un-metabolized ammonium of root cells or reduced nitrogen (Lee, 1982; Morgan and Jackson, 1989). Lee and Rudge (1986) have suggested that in barley the uptake of NH 4 and N0 3 are under common negative feedback control from a product of 4 NH ’- assimilation rather than NH 4 and/or N0 3 accumulation per Se. Reduced N pools which cycle in xylem and phloem from root to shoot have been implicated in the whole plant regulation of N uptake by plant roots (Cooper and Clarkson, 1989). However, Siddiqi et al. (1990) have suggested that in the case of N0 3 influx, vacuolar accumulation of N0 3 per se may also, at least indirectly, participate in flux regulation. Further support for this proposal has come from studies of nitrate reductase mutants of barley that are capable of normal induction of N0 3 uptake and appear to show diminished ‘ N0 3 influx as N0 3 accumulates (King et al., in press). In the present study, also,  114  there was a close negative correlation between NH 4 influx and 4 [NH ’-j In root tissues (Fig. 9). However, the altered NH 4 status in G2, G100, and G1000 plants was probably also associated with changes in organic N fractions. Since efflux was estimated to be 10 to 30% of influx for G2, G100 and G1000 plants, respectively (Wang et al., 1993), negative feedback acts very strongly on the influx step of the HATS, but since efflux also increased with increasing [NH , this flux will exert significant effects 0 ] 4 upon net uptake.  4.5. SUMMARY The work described provides the first detailed characterization of 4 influx across the plasma membrane of rice roots. Ammonium influx is NH bi-phasic, mediated by two discrete transport systems. Metabolic inhibitor studies and Qo determinations indicated that both systems were energydependent, although the HATS consistently showed greater sensitivity to metabolic interference than the LATS. Nevertheless, thermodynamic evaluations indicate that only at quite low [NH 0 is there a need to invoke ] 4 active transport of NH 4 against the electrochemical gradient. It is highly unlikely that the LATS is active. The HATS was found to be extremely sensitive to prior exposure to ammonium as indicated by the altered values of Km and Vmax. General insensitivity of influx to pH in the range from 4.5 to 9.0 argues strongly against significant entry of NH 3 across the plasma membrane even at high . 0 + 4 [NH ]  115  Chapter 5.  5.1.  ELECTROPHYSIOLOGICAL STUDY  INTRODUCTION  Ammonium influx by rice roots (Oryza sativa L. cv. M202) has been shown to exhibit a biphasic dependence on [NH 0 (Wang et al., 1991, ] 4 1992b; 1993b). At low , 0 + 4 [NH 1 influx is mediated by a saturable HATS which exhibits high Qio values between 10 and 30 °C and a significant sensitivity to metabolic inhibitors (Wang et al., 1993b). At elevated [NH 0 ] 4 (between 1 and 40 mM), NH 4 influx increases in a linear fashion with increasing [NH , and though still exhibiting energy-dependence, this 0 ] 4 LATS was shown to be less responsive to metabolic inhibitors (Wang et al., 1993b). A biphasic pattern of NH 4 uptake of this sort, with both saturable and linear phases, was first reported in Lemna, by Ullrich et al., (1984). In order to make a definitive evaluation of the thermodynamics of 4 influx (passive versus active transport), it is essential to determine NH the chemical potential difference for NH 4 between the cytoplasm and external media, and &P across the plasma membrane. In Chapter 3, compartmental analysis was used to estimate cytoplasmic 4 [NH ] . So far as I am aware, only one report measuring A’P in rice roots has appeared in the literature: Usmanov (1979) reported AW to be -160 mV. As early as 1964, Higinbotham et al. noted the marked depolarizing effect of [NH 0 j 4 coleoptile cell z’P in oats. Likewise, Walker et al. (1979a, b) demonstrated the transport of ammonium and methylamine across the plasma membrane of Chara, and the depolarizing effects of these cations. The most  116 detailed study of the concentration dependence of A’P depolarization by NH 4 ’- was undertaken by Ullrich et al. (1984), using Lemna. Below 0.2 mM 0 both NH ] 4 [NH 4 uptake and A’P depolarization responded in a saturable fashion with half-saturation values of 17 j.tM for both processes. From 0.2 to 1 mM, net uptake of NH 4 responded linearly to [NH , with no further 0 ] 4 P depolarization. On the basis of this observation, Ulirich et al. (1984)  concluded that the linear system might result from diffusion of NH 4 or 3 across the plasma membrane. NH The present study was initiated, therefore, to estimate M’ in intact rice roots, under conditions corresponding to those employed to estimate cytoplasmic  j in our previous study, and to determine the 4 [NH  concentration dependence of the depolarizing effect of [NH . The effects 0 ] 4 of metabolic inhibitors on Af were also examined.  5.2. MATERIALs AND METHODS  5.2.1. Growth of plants Rice (Oryza sativa L. cv. M202) seeds were surface sterilized in 1% NaOCI for 30 mm  and rinsed with deionized water. Seeds were imbibed  overnight in aerated deionized water at 38°C before planting on plastic mesh mounted on the bottoms of polyethylene cups. Four cups (3 to 4 seeds per cup) were set in the lid of a 1-L black polyethylene vessel with the solution level just above the seeds. Seeds were allowed to germinate in the dark (at 20°C) for 4 days. At day 5, rice seedlings were exposed to light and MJNS containing the designated levels of 4 NH C 1. The composition of  117 MJNS, growth conditions, nutrient supply and pH adjustment were those described in Section 2.1.2. The growth medium in the 1-litre polyethylene vessels were completely replaced on alternate days and the nutrient levels were topped up with concentrated stock solutions daily. Rice plants used in the experiments were 3-week-old G2 or G100 plants respectively.  5.2.2. Measurements of cell membrane potential Plasma membrane P of rice roots were measured as described by Kochian et al. (1989) and Glass et al. (1992). In short, rice plants were secured in the larger part of a flow-through Plexiglas impalement chamber, and one intact root was carefully placed over the platinum pins in a narrow section of the chamber. This root was held firmly during the impalement by two short lengths of Tygon tubing, from each of which a small wedge had been cut. The tubing was placed on either side of the impalement zone to clamp the root in place. All impalements were made in a region about 1 to 3 cm behind the root tip, using a hydraulically driven, three-dimensional micromanipulator (Model MO-20, Narashige, USA). Both the Plexiglas impalement chamber and micromanipulator were mounted on the microscope stage. Microelectrodes (including impaling, reference and grounding electrodes) were made from 1.0 mm single-barreled borosilicate glass tubing pulled to a tip diameter of 0.5 iM and filled with 3M KC1 (adjusted to pH 2 to reduce tip potentials). Measured membrane potentials of root cells, which are the voltage differences between the impaling and reference electrode, were amplified and recorded on a strip chart recorder. During impalement, solutions were continuously delivered from an air-pressured reservoir to the chamber through tygon tubing at controlled flow rates (7.5 ml minl).  118 5.2.3. Experimental treatments At the beginning of each experiment, the impalement was made on G2 or G100 roots bathed in their growth media (MJNS containing 2 or 100 iM 4 NH C 1, respectively) and the membrane potential was recorded or  A’I’GlOO).  (A.PG2  MJNS without NH 4 is referred to throughout as the -N solution.  Before applying each treatment, the -N solution was introduced to obtain a resting membrane potential, MN, as the point of reference. Roots were allowed to equilibrate for at least 3 to 5 mm in this -N solution to reach the resting potential before introducing subsequent treatment solutions. 5.2.3.1. Effect of 0 +] 4 [NH  Roots were exposed to {NH 0 of 2, 5, 10, 25, 50, 75, 100, 250, 500 J 4 jiM for studying the HATS, and 1, 2.5, 5, 10, 20, 30, and 40 mM 4 NH C 1 for investigating the LATS, in a background of MJNS. When roots were exposed to several different [NH 0 during a single impalement, G2 or G100 ] 4 medium was flushed through the chamber before each change of NH 4 concentration. When  ‘P  returned to its original  (A’PG2  or  A’’G1OO)  value, it  was satisfied that the physiological status of the root had returned to its original condition. 5.2.3.2. Effect of accompanying anion on 4’i’  To evaluate the contribution of the accompanying anion to the observed depolarization of AP by 4 NH salts in the low concentration range, A’P were measured in the following solutions in sequence: (a) 50 jiM , (b) 50 jiM CaSO 2 CaC1 , (c) 100 jiM NH 4 C1, (d) 50 jiM . 4 S0 Likewise in 2 ) 4 (NH the high concentration range, &P was measured in (e) 5 mM CaCl , (f) 5 2 mM CaSO , (g) 10 mM 4 4 NH C 1, and then (h) 5 mM . S0 These 2 ) 4 (NH  119  concentrations were chosen to provide equivalent anion charge in all treatments. 5.2.3.3. Effects of metabolic inhibitors on 4 NH induced Af’ depolarization  The same metabolic inhibitors used in the 13 4 influx study NH (Section 2.9.), were used to investigate effects on 4 NH induced depolarization of AP. These included 1 mM NaCN plus 1 mM SHAM, 10 iiM CCCP, 50 jiM DES, and 1 mM pCMBS. This study involved three steps: (1) the responses of z’P to additions of 0.1 or 10 mM 4 NH C 1 were determined in sequence; (2) the inhibitor to be evaluated was first introduced in -N solution. When z’P had reached a new steady-state, this solution was replaced with the  inhibitor plus 0.1 or 10 mM 4 NH C 1 in sequence; (3) the solution containing inhibitor plus 4 NH C 1 was replaced by -N solution. When a new steady value of L\M&N had been reached, 0.1 and then 10 mM 4 NH C 1 were added to the -N solution in sequence. The 4 NH C 1 concentrations, 0.1 or 10 mM in MJNS, were selected as representative levels for the operation of the HATS or the combined HATS and LATS (Wang et al., 1993a).  120 5.3. Results  5.3.1. Transmembrane electrical potentials of rice roots Plasma membrane A’P for epidermal and cortical cells of 3-week-old rice roots (Table 11) were measured in 0.2 mM CaSO 4 alone solution, or G2 and G100 media (‘‘PN,  ‘PG2  and  ‘‘PG1OO,  (A’PCaSO4),  or -N  respectively). As  presented in Table 11, &PCaSQ4 values were consistently more negative than &p measured in other solutions. Likewise the negative than the corresponding  A’PG2  or  &P.N  were more  values. The depolarizing  zPG 100  effect of 4 NH C 1 additions can be directly compared in Table 11 for a particular root type because -N and G2 or G100 media differed only by the presence of NH C1 in MJNS. Therefore, both 4  APG2  and z’+’oo represented  the membrane potentials of root cells adapted to their respective growth conditions.  5.3.2. Contribution of the accompany anions to  ‘i’  Figure 12 reveals that there was a very small depolarizing effect of Ca 2 salts compared to 4 NH salts, under conditions where the concentration of the accompanying anion was held constant. Also there was 2. 4 virtually no difference between the depolarizing effects of Cl- and S0 This was true also at the higher concentrations of Ca -salts and 4 2 NH salts (Traces e, f, g and h in Fig. 12). In the lower concentration range, no repolarization of  M’  was observed until the 2 Ca salts or 4 NH salts were  withdrawn from the chamber. By contrast, in 5 mM CaC1 , complete 2 repolarization and even hyperpolarization was evident within 10 mm  of  121 Table 11. Membrane potentials of G2 and G100 plants measured in different bathing solutions. The bathing solution for measurements were 0.2 mM CaSO4; MJNS-N; MJNS  +  2 iM 4 NH or MJNS ,  G2 plants (mV)  4 A’PCaSO  -140±3.5  ( ) 5 d  ‘‘N  -129±1.0  (n=184)  z’{’G2orLS.WG1OO  -116±2.1  (n=14)  a  +  100 jiM 4 NH ’-.  G100 plants (mV)  -135±1.8  (n=53)  -131±0.6  (n=197)  -89±2.4  (n=28)  G2 or G100 plants were impaled in 0.2 mM CaSO4 solution; b G2 or G100 plants were  impaled in -N solution;  C  G2 or G100 plants were impaled in MJNS containing either 2 iM or 100 tM NH4C1, respectively; d Average value ± standard error, n: number of observations;  122  Addition of ions U 2 a. 50 jiM CaC V -138  4 b. 50 jiM CaSO  -110  —  CI 4 100 jiM NH d. 50 jiM  S 2 ) 4 (NH r NN O -114  2 e. 5 mM CaCI  v  4 f.5mMCaSO  g.1OmMNH C 4 I omV{  S0 2 ) (NH h. 5 mM 4  -130  rnnes  Figure 12. Effects of some anions on A’P depolarization. Representative traces to demonstrate the contribution of the accompany anions to depolarization of &P elicited by exposure of roots to different salts at various concentrations. V: the salts were withdrawn from MJNS. Each treatment was repeated on three separate plants.  123 evidence of repolarization in 4 NH C 1 (Fig. 12, trace g) but this was only partial. Only after removal of the NH -salts was complete repolarization 4 observed.  C1] on z\P 4 [NH 5.3.3. Effect of 0 C1 to the -N solution induced a strong 4 The addition of NH depolarization of &P (Fig. 13). This depolarization occurred rapidly after the introduction of 4 NH C 1, even at very low concentrations (e.g. 2 jiM NH C 4 1). The time required to reach the initial maximum depolarization was from 0.5 to 2 mm, increasing with increasing . 0 C 4 [NH 1] The depolarization of A’P was positively correlated with . 0 C 4 [NH 1] A saturable pattern was evident in the range from 2 to 1000 jiM 4 NH C 1 (Fig. 14A) for both G2 and G100 plants. Estimated half-saturation values for net depolarization (analogous to a Km value) were 21.8 ± 2.7 jiM for G2 plants and 35.0 ± 8.0 jiM for G100 plants, while the maximum depolarization (analogous to a Vm value) was 50.6 ± 2.0 mV for G2 plants and 34.3 ± 1.9 mV for G100 plants. Kinetic parameters were obtained by fitting the data to the Michaelis-Menten equation by means of a nonlinear regression computer program “Systat” (Wilkison, 1987) as used in our earlier kinetic study of 13 4 influx (Wang et al., 1993b). Between 1 to 40 mM {NH NH 0 C 4 1] (Fig. 14B), the magnitude of the depolarization increased linearly with increasing concentrations of NH C1. This relationship was observed for both 4 G2 and G100 rice plants, although the extent of depolarization was smaller for the latter.  124 C 1 4 Addition of NH /  10  S minutes  I NH C 40 mM 4  C1 4 20 mM NH I NH C 10 mM 4  1 NH C 5 mM 4 I NH C 1 mM 4  I NH C 500 iiM 4  I NH C 100 iiM 4 5OjiM  I NH C 4  M lO i 1  I NH C 4  2M  1 NH C 4  1. Representative NH C Figure 13. The &P depolarization of root cell by 4 traces from G2 plants showing the depolarization of root cell zVP induced by C1. V 4 adding various concentrations of NH MJNS.  1 was withdrawn from NH C 4  125 60  -  1 4A  .—  0 •G2 0  10-  U-  •  G100  I.I.I.I.I.I.I.I.I.I.I.  0  100  200  300  External  400  500  600  Ammonium  700  800  chloride  900  1000  (jiM)  120-  14 B  100  20  •  G2  0  G100 r”2= 0.99  r’2=O.94  0 0  5  10  15  External  20  25  Ammonium  30  Chloride  35  40  45  (mM)  Figure 14. Concentration dependence of net A’P depolarization of root cells. Rice seedlings were grown in either 100 jiM (G100) or 2 jiM NH 4 (G2). The  -N media were used as basal solutions for the resting AP. Each point is the average of 3 measurements from each of 3 individual plants. The vertical bar is the standard error. 14A: Low 0 C1] range (<1 mM); 14B: High 4 [NH range (1 to 40 mM).  126 Figure 15. shows the effects of four metabolic inhibitors on &T’ recorded in -N solutions. The largest depolarization of z’P (95 mV), was induced by the protonophore, CCCP, while CN+SHAM and the ATPase inhibitor, DES, elicited depolarizations of 82 mV and 40 mV, respectively. The external protein modifier, pCMBS, caused only a small depolarization (8 mV). Representative traces depicting the effects of each of these inhibitors on NH -induced depolarization of A’P are shown in Fig. 16. In 4 Table 12, the effects of these inhibitors on the 4 NH induced depolarization of z’P are expressed as a percentage of the depolarization under the control conditions, in absence of the inhibitor. The data are presented as follows: (i) control: in absence of the inhibitor the reduction of 4 NH induced depolarization of A’P is zero; (ii) plus inhibitor: reduction of 4 -NH ’--induced depolarization of zXP varied from 0 to 91%, depending upon the inhibitor used and ; 0 + 4 [NH ] and (iii) residual effect: the residual effect after removal of the inhibitor from external solutions on 4 NH induced depolarization of A’P. The [NH 0 C 4 1J employed were 0.1 mM and 10 mM, respectively, chosen to represent the HATS and the combined HATS+LATS. In Table 12 the depolarizations of A’P caused by 0.1 mM [NH 0 C 4 1] were subtracted from those caused by 10 mM 0 CIj to represent the effect due to IATS alone. 4 [NH In the presence of the various inhibitors, the depolarization of zs’1’ induced by HATS was generally reduced by greater than 50%. By contrast, depolarization of AP due to NH 4 uptake through the LATS was only slightly affected by the presence of inhibitors. Table 12 also reveals that there was virtually no recovery from the inhibitor treatments following removal of the inhibitors from the external medium.  127  -34 10 m V  [ -44  012345/ mm  Add Inhibitor  )  /  SHAM ==  80 mm  f—j  Figure 15. Effects of metabolic inhibitors on AW depolarization of root cells. Effects of metabolic inhibitors on AP depolarization of root cells. Representative traces showed effects of metabolic inhibitors on A’P in time course. The inhibitors were: (A) 10 iM CCCP; (B) 1 mM CN+SHAM; CN- was added into -N medium alone and then SHAM was added at (J.I); (C) 50 iiM DES; (D) 1 mM pCMBS. Each treatment was repeated on at least three individual roots. The space between two bars (I I) is the omitted period as minutes.  128 -53  -68 (C) DES  (A) CCCP  6oiovE (iii  0 12 3 4 5 mm  L add 0.1 mM N1LCl I NEI C add 10 mM 4  (i) control (ii) inhibition (inhibitor presented) (iii) residual effect (inhibitor removed)  1 induced A’P NH C Figure 16. Effects of metabolic inhibitors on 4 I on P NH C depolarization. Representative traces for the effects of 4 depolarization in the presence or absence of metabolic inhibitors in -N media. Metabolic inhibitors were those shown in Figure 15..  129 Table 12. Effect of metabolic inhibitors on the depolarization of zXP due to 4 uptake via HATS or LATS in G2 plants. The inhibitors used were: (A) NH 10 jiM CCCP; (B) 1 mM CN-  Inhibitor  +  1 mM SHAM; (C) 50 jiM DES; (D) 1 mM pCMBS.  CCCP  Treatment  CN+SHAM  DES  pCMBS  Reduction of A’P depolarization (%)  1. Due to NH 4 uptake by HATS a (i)  control  (ii)  0  0  0  0  plus inhibitor  89  91  72  52  (iii) residual effect  91  68  81  90  2. Due to NH 4 uptake by LATS b (i)  control  0  0  0  0  (ii)  plus inhibitor  9  0  14  -  (iii) residual effect  34  -  3  -  a The values of zP were measured when roots were bathed in MJNS containing 0.1 mM NH 4 in the absence (i and iii) and presence (ii) of the inhibitors. The percentage reductions of P depolarization were calculated from the differences between control values for zSP induced by 4 NH and depolarization values in the presence of the inhibitor (ii) or after removal of the inhibitor (iii);  b The values of ‘P for LATS  were the differences between measured s’-P at 10 mM (for HATS+LATS) and at 0.1 mM NH C 4 1 (for HATS). Then the percentage were calculated as described above (a); calculated values were negative due to the less zSW depolarization of the control.  C  The  130 5.4. DISCuSSION  5.4.1. Anion effect A perennial problem associated with attempts to evaluate the electrical effect of a particular ion is the contribution of the accompanying counterion. This problem has rarely been acknowledged in published studies. However, indirect approaches, such as comparisons of the depolarizing effects of NO - in N03-induced and un-induced plants have 3 been employed in order to dissect out the anion effect (Glass et al., 1992). Another approach that has proven effective is to switch from one anion to another (e.g. CaC1 2 to Ca(N0 2 without changing the accompanying cation ) 3 or its concentration. As a result, the observed changes of Af are due solely to the anion effect (McClure et al., 1990; Glass et al., 1992). The results of such studies have demonstrated that N0 3 can strongly depolarize A’1’ and these observations have formed the basis of currently proposed proton/nitrate cotransport mechanisms (Ulirich and Novacky, 1981; McClure et al., 1990; Glass et a!., 1992). In the present study, low concentrations of C1 (100 jiM) provided in the form of the calcium salt elicited a very small depolarization (Fig. 12, trace a). Replacing this solution with the same concentration of CaSO 4 (Fig. 12, trace b) confirmed that C1  was responsible for most of this  depolarization. Thus when these calcium salts were replaced by their ammonium equivalents, maintaining the same anion concentration, the significant depolarization of AP could largely be attributed to NH . 4 Although the depolarizing effects of the calcium salts, presented at 5 mM were significantly higher than at 50 jiM (Fig. 12, traces e and f), the effects  131 of transfer to the equivalent ammonium salts can be seen to induce a much larger depolarization (53 mV compare to 18 mV; Fig. 12, traces g and e). Even though it was not possible to quantitatively isolate the contribution of Cl- for studies of LATS, I consider that the 4 NH ’- effect still predominated, even at high external . 0 C 4 [NH 1] In fact, the difference between traces g and e (Fig. 12) can be attributed to the difference between 4 NH ’- and Ca 2 effects, since Cl- was maintained at the same level. Thus the depolarizations referred to in the remainder of the paper were interpreted as predominantly due to the transport of NH . 4 A feature of these initial studies was the apparent repolarization of 1 following depolarization in the chloride solutions (Fig. 12, traces e and z+ g) at high [Cl1 . Although repolarization to the resting potential was not 0 complete in 10 mM NH C1, the extent of the initial repolarization was 4 comparable to that in CaCl , where repolarization was completed. A similar 2 spontaneous repolarization of z’P was noted in Lemna and in barley roots following depolarization of A’P by N0 3 (Ulirich and Novacky, 1981; Glass et al., 1992).  5.4.2. Depolarization of AP by HATS and LATS Addition of ammonium chloride into -N solutions induced a rapid depolarization of membrane potential of rice epidermal and cortical cells (Figs. 12 and 13). This was evident even at very low concentration (2 jiM NH C 4 1) (Fig. 13). Ullrich et al. (1984) reported that addition of NH 4 immediately decreased the membrane potentials of Lemna gibba. Likewise, the zP of green thallus cells of Riccia fluitans were rapidly depolarized by 4 [NH C 1] as low as 1 jiM (Felle, 1980). As can be seen from  132 Fig. 13, the time to reach initial maximum depolarization increased from 0.5 to 3 mm with increasing concentrations of NH C1. 4 The depolarization of z’P by NH 4 exhibited a biphasic concentrationdependence (Figs. 14A and 14B), similar to NH 4 influx into roots of rice (Wang et al., 1993b). In the low concentration range (<1 mM), depolarization of the membrane potential saturated in response to [NH 0 ] 4 (Fig. 14A). Both net flux and unidirectional influx of NH 4 in rice roots have been shown to respond to [NH 0 in a similar fashion (Youngdahl et al., ] 4 1982; Wang et al., 1991; 1993b). Estimated half-saturation values for NH 4 induced depolarization (analogous to a Km value) were 21.8 ± 2.7 .tM for G2 plants and 35.0 ± 8.0 jiM for G100 plants. These values were somewhat lower than the Km for 4 NH influx, 32 jiM and 90 jiM, 1 respectively (Wang et al., 1993b). Since our studies were undertaken with the same rice variety as employed for the ‘ 4 N 3 H influx experiments, these differences may represent differences in growth conditions for plants used for the two studies, or that membrane depolarization reflects the net, rather then the unidirectional, effect of ion fluxes. Another factor, already addressed above, is the possible effect of the accompanying anions. The maximum depolarizations (analogous to a Vmax value) were 50.6 ± 2.0 mV and 34.3 ± 1.9 mV for G2 and G100 plants, respectively. The larger depolarizing effects of [NH 0 4 ’-] in G2 compared to G100 plants (Figs.. 14A and 14B) correspond to the higher values of ‘ 4 N 3 H influx observed in G2 compared to G100 plants (Wang et al., 1993b). Clearly the depolarization of z’ in response to 0 -’-] (<1 mM) was due to the carrier-mediated NH 4 [NH 4  uptake that exhibited Michaelis-Menten kinetics (Wang et al., 1993b). Similar saturable patterns of A’P depolarization were associated with the uptake of either 4 NH ’- or N0 3 in Lemna (Ulirich and Novacky, 1981; Ulirich  133  et al., 1984) and the uptake of both 4 NH and CH NH in cells of Riccia 3 fluitans (Felle, 1980). Between 1 and 40 mM, the depolarization of A’{’ increased linearly with increasing [NH 0 C 4 1] (Fig. 14B) in a manner similar to that observed for 4 influx (Wang et al., 1993b). Both G2 and G100 rice plants exhibited NH 13 this linear response, but the extent of depolarization was smaller in G100 + influx was also smaller. The concentration-dependent 4 plants, where 13NH data for depolarization of zM’ by LATS was fitted by linear regression with 2 values of 0.94 and 0.99 for G2 and G100 rice plants, respectively. A r similar linear response to [NH 0 was reported for net NH ] 4 4 uptake by Lemna at [NH 0 between 0.1 to 1 mM (Ulirich et al., 1984). However, in ] 4  this concentration range, NH 4 uptake by Lemna was not associated with further depolarization of A’P. Ulirich et al., (1984) interpreted this pattern as due to a diffusive uptake of NH 4 or NH . It is clear that NH 3 3 influx would not depolarize z’P. However it is not clear how 4 NH uptake could occur without further zXi’ depolarization, unless NH 4 influx was associated with a stoichiometric anion influx or cation efflux resulting in an electroneutral transport. To better understand the relationship between NH 4 uptake and changes in z’P, the observed values of A’P depolarization were paired with the data for ‘ 4 N 3 H influx from Wang et al. (1993b) at each [NH 0 4 ’-] (Fig. 8). It is evident that the depolarization of A’P was strongly correlated with 4 N 3 ‘ H influx, and that the relationship was biphasic. By use of a computer-based procedure to determine the ‘break-points’ for the biphasic pattern objectively (Rygiewicz et al., 1984), the correlation coefficient established a break-point at 1 mM [NH . The biphasic pattern (Fig. 17) 0 ] 4  134  120  (a)  G2 plants  (b)  G100  plants  100 r”2  •  80  =  0.93 r’2  =  0.99  60  at 1 mM NH 4 Cl 40  20  r”2  =  0.98  r”2  =  10  20  0.93  0  I  0  10  20  30  40  1 3 NIT 4  50  Influx  0  30  40  50  (pmol/gFW/h)  Figure 17. The relationship between ‘ 4 N 3 H influx and z’P depolarization at the same [NH NH influx is from Figs. 8 and 10 and net 1 . 4 0 ] 4 depolarization of membrane potentials is from Figs. 14 A and 14B for G2 and G100 plants measured at the same [NH . 0 J 4  135 indicates that NH 4 influx and the depolarization of A’P are due to two distinct systems for NH 4 uptake by rice roots, i.e. a high affinity transport system (HATS) and a low affinity transport system (LATS). The larger slope of the lines for the low concentration range for G2 and G100 plants suggests that the HATS is more electrogenic than the LATS. This may be due to the increasingly electroneutral NH 4 transport at high . 0 C 4 {NH 1J In the present study, the electrophysiological evidence suggested that at high 0 C 4 [NH 1] ammonium is taken up by rice roots in the cation form 4 (NH ’-) despite the presence of a relatively high concentration of NH 3 in solution. Alternatively, it might be argued that depolarization of AP may be due to the inhibition of the H-ATPase by NH 3 at high . 0 + 4 [NH ] However, the lack of a pronounced increase of ‘ N uptake at pH values approaching the pKa 3 for 4 NH ’- does not support this interpretation (Wang et al., 1993b). In addition, the rapid repolarization of A’P following removal of external NH 4 (in Fig. 12, traces g and h) is unexpected considering that the cytoplasmic ‘ N exchange is 7 mm 3  5.4.3.  2 ti,i  for  (Wang et al., 1993a).  Calculation of the free energy for NH 4 transport The average A’P values were substantially more negative in G2 plants  impaled in 2 iM NH -’- than in G100 plants impaled in 100 iM NH 4 4 (Table 11). Furthermore, the extent of the depolarization of A’f by NH 4 was consistently greater for G2 plants than G100 plants at a particular . 0 4 [NH -] The average P value was -116 mV for G2 plants and -89 mV for G100 plants (Table 11). For both G2 and G100 plants, the resting potentials in the absence of NH -’- (AW-N) were in the range of -120 to -140 mV. In low 4 salt bathing medium (0.2 mM 4 CaSO ) , the transmembrane electrical potentials (AW 02  mM CaSO4)  were 25 mV more negative than A’YN and 45 mV  136 —  0  4000  G100 G2 2000  0  0  .  -2000 0.0  0.2  External  0.4  ammonium  0.6  concentration  0.8  (mM)  Figure 18. Free energy requirement for NH 4 uptake as a function of external 4 [NH ] . Values of cytoplasmic 4 [NH + ] were taken from our previous study (Wang et al., 1993a). Arrows indicate the [NH 0 below which 4 J 4 NH ’uptake is against the electrochemical potential gradient for G2 and G100 plants, respectively.  137 more negative than  &‘G2  and  APG100,  respectively. These differences  reflect the contributions to the membrane depolarization from the various ions present in MJNS. Since the values of APN, and  M’G2  and  A’PG100,  were  measured in the same basal medium (MJNS), the observed differences must largely be due to the [NH 0 in the bathing medium. ] 4 The measured  A’P,  together with values for cytoplasmic 4 {NH ÷ ], are  needed to estimate the electrochemical potential difference for NH 4 across the plasma membrane, which in turn allows us to determine the energy requirement for transport (Findlay and Hope, 1976). Taking 3.72 mM and 20.55 mM as cytoplasmic 4 {NH + ], and -116 mV and -89 mV as steady-state A’P  for G2 and G100 roots, respectively (Wang et al., 1993a), at a series of  given [NH 0 the Nernst potentials (EN) were estimated for G2 and G100 ] 4 roots, respectively. From these values, the free energy  (zSt)  required to  transport NH 4 across the plasma membrane can be computed from the differences between measured membrane potentials  (A’PG2  or  zS’PG100)  and  estimated Nernst potentials at specific [NH 0 (Fig. 18). The estimated free ] 4 energy differences  (Aji)  for NH 4 distribution were positive at or below 42  jiM for G2 and 655 jiM for G100 plants (Fig. 18). This means that below these concentrations, NH 4 uptake by G2 and G100 roots respectively, must be active (Fig. 18). These concentrations represent the lower limits for active transport under steady-state conditions. However, displacing [NH 0 ] 4 to values greater than 2 or 100 jiM, respectively, will elevate the limit for active transport because of further AW depolarization and increased cytoplasmic 4 [NH ] . Above these minimum levels, the uptake of NH -- may 4 occur via passive transport systems, down the electrochemical potential gradients for 4 NH -. As pointed out previously (Wang et al., 1993b), these free energy estimations only provide a prediction of the feasibility of the uptake process occurring under the prescribed conditions. For both G2 and  138 G100 plants, the predicted [NH 0 4 ’-] for the shift from active to passive uptake was quite a bit lower than the break-point determined by the kinetics analyses (42 jiM and 655 jiM versus 1 mM). Thus, one must be cautious in identifying a specific transport system based purely on thermodynamic or kinetic considerations.  5.4.4. Mechanisms of NH 4 uptake by HATS and LATS The preceding section has demonstrated that at low [NH 0 4 ’-] (<42 jiM for G2 plants and 655 jiM for G100 plants), NH 4 influx appears to be an active process in roots of rice plants. However, the details of this mechanism are unknown for rice and for any higher plants. Possible mechanisms for this active uptake via HATS include: (a). a proton : NH 4 symport; (b). a specific NH 4 ATPase. The results of the inhibitor studies, both for the electrical potentials in the present study and 4 N 3 ‘ ’H influx (Wang et al., 1993b) provide evidence for a dependence (either direct or indirect) on the proton motive force. Application of CCCP caused 89% and 85% inhibition, respectively, of membrane depolarization by 4 NH ’- and 4 influx in solution containing 100 jiM NH NH 13 . The strong inhibitory 4 effects of CN+SHAM on depolarization of zSP (9 1%) and on ‘ 4 N 3 H influx (8 1%) confirm the dependence of these processes on a source of metabolic energy without distinguishing the nature of the mechanisms. The effects of DES, an inhibitor of the H-ATPase, indicated the involvement of the proton pump, suggesting speculatively that H+-transport might be involved. The results of the present and earlier studies (Wang et al., 1993b), strongly suggest that the two systems, HATS and LATS, have different mechanisms of energy coupling. Above 42 jiM for G2 and 655 jiM for G100  139 plants, NH 4 transport was predicted to be a passive process. This prediction is borne out by the generally smaller effects of metabolic inhibitors at high external 4 [NH ’-] than at low 0 +] (present study and in 4 [NH Wang et al., 1993b), although 13 4 influx showed greater sensitivity to NH inhibitors than the AP depolarization. There is virtually no information available regarding the energy coupling for the LATS. Passive entry of NH 4 ’- might occur via an electrogenic uniport (Kleiner, 1981; Ullrich et al., 1984). This may be a specific channel for NH 4 or a shared cation channel. For example, the recently described K channel in Arabidopsis has been shown to have an NH 4 conductance that is 30% of the K conductance (Schachtman et al., 1992). Also, in the cyanobacterium Anabaena variabiis (Avery et al., 1992), the uptake of Cs (a K analog at the uptake step) and 4 was closely related. Thus low affinity 4 NH NH transport might occur via the K channel.  5.5. SUMMARY The transmembrane electrical potential differences (&P) were measured in epidermal and cortical cells of intact roots of 3-week-old rice  (Oryza sativa L. cv. M202) seedlings grown in 2 or 100 micromolar (jiM) ÷ (G2 or G100 plants, respectively). In modified Johnson’s nutrient 4 NH solution (MJNS) containing no nitrogen, A’I’ was in the range of -120 to -140 millivolts (mV). Introducing 4 NH to the bathing medium caused a rapid depolarization. At the steady-state, average &P of G2 and G100 plants were -116 mV and -89 mV, respectively. This depolarization exhibited a biphasic response to external [NH ] similar to that reported for 4 4 N 3 ‘ H influx isotherms (Wang et al., 1993b). Plots of membrane depolarization versus 13 4 influx were also biphasic, indicating distinct NH  140 coupling processes for the two transport systems, with a break-point between two concentration ranges around 1 mM NH . The extent of 4 depolarization was also influenced by nitrogen status, being larger for G2 plants than G100 plants, corresponding to the larger NH 4 influxes in G2 plants than G100 plants. Depolarization of A’P due to NH 4 uptake was eliminated by a protonophore (carboxylcyanide-m-chlorophenyl hydrazone), inhibitors of ATP synthesis (sodium cyanide plus salicyihydroxamic acid), or an ATPase inhibitor (diethyistilbestrol).  141  Chapter 6.  6.1.  REGULATION OF AMMONIUM UPTAKE  INTRODUCTION  2-, Cl-, their 4 When plants are deficient in nutrients, such as 4 PO , S0 uptake capacity is greatly enhanced (Lee, 1982). This phenomenon has been known since the works of Brezeale (1907 in Glass, 1989) that nutritional history of a plant can profoundly affect its subsequent capacity to absorb the same ion (see also Hoagland and Broyer, 1936; Broyer and Hoagland, 1943). Such relationships between the ions provided during plant growth and their subsequent uptake by roots or tissues was well defined in several species for the uptake of K (Leigh and Wyn Jones, 1973; Glass, 1975; 1976; 1978; Pettersson, 1975; Dunlop et al., 1979; Jensen and Pettersson, 1979; Pettersson and Jensen, 1979), C1 (Sanders, 1980; Smith and MacRobbie, 1981; Greenway, 1965; Pitman, 1971; Cram, 3- (Lefebvre and Glass, 1982; Lee, 4 1973; Hodges and Vaadia, 1964), p0 S0 2 - (Lee, 1982) and N0 1982) 4 3 (Jackson et al., 1974; MacKown et al., 1982; Glass et al., 1985; Siddiqi et al., 1989, 1990; Jackson and Volk, 1992; King et al., 1993). However, the quantitative basis of the correlation between the rate of N absorption and the N-status of the plant material is not precise (Lee and Rudge, 1986). It have been demonstrated that plants are able to adapt to available sources of N over a wide range of concentrations (Clement et al., 1978; Wang et al., 1991). The existence of distinct transporters with different affinities for either nitrate or ammonium (Siddiqi et al., 1989; Wang et al., 1993b) represents an important part of this capacity for adaptation. Typically, nitrogen starvation leads to elevated fluxes of nitrogen, while N  142 excess leads to down regulation of uptake. However, the underlying mechanisms responsible for these changes are largely unknown. Several hypotheses have been advanced concerning the sources of feedback regulation responsible for controlling N uptake. These include the importance of products of N assimilation (Lee and Rudge, 1986; Cooper and Clarkson, 1989; Jackson and Volk, 1992), as well as the effects of accumulated ions (NO ) on influx or efflux (Morgan and Jackson, 4 - and NH 3 1988a, 1988b; Siddiqi et al., 1989; King et al., 1993; Wang et al., 1993a). It has been suggested by Morgan and Jackson (1988b), that at high plant N status, reduction or suppression of net ammonium uptake may be due to (i) low energy supply to the root system, (ii) accumulation in the root tissue of a nitrogenous compound which exerts negative feedback on NH 1-. This accumulated the influx system, (iii) high efflux of endogenous 4 regulating effector could be ammonium ions generated by degradation of organic nitrogenous sources within roots, or rapid accumulation of ammonium in N-depleted roots upon initial exposure to ammonium, or relative ease of outward ammonium movement (Morgan and Jackson, 1988a, 1988b). The regulation of influx may therefore reflect the interplay among suppression of influx by a product of ammonium assimilation, the accumulation of root ammonium and associated ammonium efflux, and a stimulation by ammonium of its own uptake (Morgan and Jackson, 1992). It was found that 13 4 influxes into intact roots of rice were NH negatively correlated with the level of NH 4 provision during growth and the internal [NH ] in root tissues (Wang et al., 1993 a, 1993b). It has been 4 suggested that the regulation of NH 4 uptake could result from feedback effects of accumulated NH -’- or products of NH 4 -’- assimilation (Ullrich et al., 4 1984; Lee and Rudge, 1986; Morgan and Jackson, 1988; Lee et al., 1992;  143 Jackson and Volk, 1992; Wang et al., 1993a). These exert effects on both influx and efflux although the principle effect is upon influx (Wang et al., 1993a). However, the mechanism(s) of regulation are still unclear. In order to explore the basis of the negative feedback regulation of ÷ uptake, I investigated the effects of the following pretreatments on 4 NH 4 influx: (1) repleting N-depleted plants in 1 mM NH NH 13 4 in the presence or absence of MSX; (2) depleting N-repleted plants in 2 iiM NH 4 solution in the presence or absence of MSX; (3) elevating root glutamine concentrations by supplying this amino acid exogenously; (4) altering internal concentrations of NH , glutamine and other amino acids in root 4 tissue of the above treatments; (5) using selected inhibitors of ammonium assimilation to study the effect of perturbing ammonium metabolism on ammonium uptake. The results of these experiments are interpreted in terms of a cascade model for the regulation of NH 4 influx in rice roots.  6.2. MATERIALS AND METHODS  6.2.1. Plant growth and ‘ N production 3 Section 2.2. Seed germination; Section 2.3. Growth conditions; Section 2.4. Provision of nutrients; Section 2.5. Production of . 4 N 3 ‘ H  144 6.2.2. Experimental design 6.2.2.1. Experiment I. Depletion and repletion study  To investigate NH 4 uptake by roots in response to changing plant N status, 13 4 influx was measured in 4 NH NH repleted G2 plants or -NH 4 1-depleted G1000 plants as well as G2 and G1000 plants under their growth conditions. At designated times, the assigned G2 plants were transferred to the G1000 medium and G1000 plants were transferred to the G2 medium. The time periods of repletion were 1, 2, 3, 3.5, 4, 4.5, 5, 6.5, 7.5, 8, 9.5, 12, 13.5, 24, 48, 72 h. The time periods of depletion were 0, 0.33, 0.58, 0.92, 1.75, 2.75, 3.75, 12, 18, 25, 50, 60, 72, 97, 126, 145, 161, 192 h. 6.2.2.2. Experiment II. Effects of MSX  The objective of this study was to investigate the time course of 4 N 3 1 H influx. Either G2 or G1000 plants were pretreated effects of MSX on + in their respective growth media in the presence of 1 mM MSX (G2+MSX or G1000+MSX) for 1, 4, 12 and 24 h before the ‘ 4 N 3 H influx measurement. A second set of plants was used to investigate MSX effects during repletion and depletion: plants were first transferred into growth media with MSX containing the same [NH 0 as they had been grown in (i.e. in G2+MSX for ] 4 G2 plants or G1000+MSX for G1000 plants) at 24 h before measurement, and then G2 plants were transferred from G2+MSX to G1000+MSX or G1000 plants were transferred from G1000+MSX to G2+MSX at times of 1, 4, 12 and 24 h. For comparison, a third set of plants was transferred from growth medium to pretreatment medium i.e. G2 plants to G1000 medium or G1000 plants to G2 medium at times of 1, 4, 12 and 24 h. In another experiment, the pretreatment times for both G2 plants repleted in G1000  145 medium and G1000 plants depleted in G2 medium were 0, 1, 4, 12, and 24 h. The influxes were measured for 10 mm  in 100 jiM 13 -’--labeled 4 NH  solution without MSX. Each datum point is the mean of 6 replicates and the vertical bar represents the standard error (± Se). 6.2.2.3. Experiment III. Effects of exogenous amino acids (1) Effects of pretreatment with glutamine on 1 4 N H influx of rice roots: G100 plants were pretreated in G100 medium with or without 10 mM glutamine for 16 h before measuring 13 4 influx. 13 NH 4 influxes NH were then measured in 2, 10, 25 and 100 jiM NH 13 4 labeled solution without glutamine. (2) The effects of various exogenously supplied amino acids on the influx of 13 : G2 plants were pretreated in G2 medium or 4 NH G100 medium plus 10 mM glutamate, glutamine or asparagine for 16 h, NH influxes were measured in 100 mM labeled ÷ 1 respectively. 4 4 N 1 H solution in the presence of the same amino acids. Each experiment was repeated twice, with 3 replicates. 6.2.2.4. Experiment IV. Effects of selected inhibitors Inhibitors of glutamine synthesis (L-methionine DL- sulfoximine, MSX), glutamate synthesis (6-diazo-5 -oxo-L-norleucine, DON) and aminotransferases (amino-oxyacetate, AOA) were used to perturb tissue concentrations of glutamine and glutamate to investigate the effect of change of these compounds on 1 4 N H influx. All treatments of inhibitors were administered for 16 h at 100 mM. 4 NH influxes were measured in 1 either 100 mM or 10 mM labeled 1 4 N H solution. 6.2.3. Determination of free ammonium in root tissue See section 2.5.  146 6.2.4. Determination of amino acids in root tissue See section 2.8.  6.3. RESULTS  6.3.1. Experiment I. Depletion and repletion study As shown in Fig. 19, the initial ‘ 4 N 3 H influx of nitrogen-deficient rice plants (G2 plants) was 11.10 jimol g’FW h’, which is close to the Vm (12.8 jimol g’FW h-i) of G2 plants (Wang et al., 1993b). After repletion in G1000 medium, influx increased to nearly 3 times its initial value (to 31.97 FW h 4 ) during the first 5 h. Between 6 to 12 h of loading, influxes 4 iimol g declined to about 10 p.mol g’FW h’. After three days in 1 mM 4 NH solution, the ‘ 4 N 3 H influx dropped below 5 jimol g’FW h-’. When G2 plants were repleted in 10 or 100 jiM NH 4 solution, G2 roots responded with a similar pattern, but showed a delay in reaching the maximum of influx (data not shown). Nitrogen-sufficient rice seedlings were grown in G1000 medium for at least 13 days and transferred to G2 medium for periods varying from 0.3 to 192 h, respectively, before measurement of ‘ 4 N 3 H influx. As shown in Fig. 20A, initial ‘ 4 N 3 H influx of G1000 plants was quite low (1.15 jimol g F 1 W h-i) in agreement with previous reports (Wang et al., 1993b). Short term depletion in G2 medium, for periods of 0.5 to 4 h, caused N 1 H4 influxes to increase almost 10 fold. Between 4 to 24 hours, ‘ 4 N 3 H influx of these N-depleted plants was close to the Vm for 13 4 influx of G2 NH  147 40  19A  40  Repletion  in  G1000  Media  (h)  4 N 3 H influx of repleted G2 plants. After repletion in G1000 Figure 19. ‘ medium for various periods, 13 4 influx of G2 plants was measured in NH 100 jiM 13 -labeled solutions. Insert 19B shows, in expanded form, the 4 NH first 24 h of repletion. Each datum point is the mean of 3 to 6 replicates and the vertical bar represents the standard error (± Se).  148 15  20A  jlO  0  20  40  60  80  Depletion in  100  120  140  160  180  G2 media (h)  Figure 20. 4 NH influx of G1000 plants during depletion in G2 medium 1 for various periods. The influxes were measured in 100 iM NH 13 4 labe1ed solution. Insert 20B shows in expanded form the data for the first 24 h of depletion. Each datum point is the mean of 3 to 6 replicates and the vertical bar represents the standard error (± se).  200  149  6  21A  0  5  10  15  20  25  2 .—  0  E E 0•  •  •  0  I  20  •  I  40  •  I  60  •  I  80  •  I  100  •  I  120  Depletion in G2 media  I  •  140  •  I  •  160  I  180  200  (h)  Figure 21. Internal ammonium content of depleted G1000 roots. G1000 roots were depleted in G2 medium for various periods and internal ammonium content were assayed. Insert 2 lB shows in expanded form the data for the first 24 h of depletion. Each datum point is the mean of 6 replicates and the vertical bar represents the standard error (± se).  150 plants (—41 pmol g’FW h-i) (Fig. 20B). After 24 hours depletion, the 4 N 3 ‘ H influxes declined but were still higher than those of G1000 plants at steady-state. The results indicated, that depletion in G2 medium for up to 8 days, caused no further decline of influx, which remained at about 6 imol g-’FW h-i. Meanwhile, root 4 NH concentrations dropped rapidly ÷ during the first 4 h depletion of N, from 5.6 to 3.6 iimol g’FW (Fig. 21A). After 24 h depletion, internal 4 [NH + j remained at a low level (0.6 !Imol g 1 FW, in Fig. 21B). Figures 20B and 21B reveal that there was a negative correlation (r 2  =  0.74) between [NH 1 and 13 ] 4 4 influx during 24 h NH  depletion of N. Beyond 24 h of N depletion, no correlation was found. Changes of the total AA content in root tissue of G1000 plants during depletion in G2 medium, are presented in Fig. 22A. In the first 4-5 h of depletion of N, total amino acid concentration ([AA]) increased (Fig. 22B). In fact the total {AA] 1 remained above the original level through 200 h of depletion. The contents of the major amino acids and amides, [Gln] , 1 , {Glu] 1 , and [Asp] were also found to have increased in the same fashion 1 [Asn] (data not shown). The phenomenon of stimulated influx observed during the first hours following exposure of G2 plants to 1000 jiM NH 4 was not as pronounced in the second experiments (open circles in Fig. 23A) as in the first experiment (Fig. 19A). This may have been due to differences of experimental conditions. In the first experiment, the depletion/repletion was carried out in a large volume of nutrient solution (in 35-liters Plexiglas tanks) in which the NH 4 concentrations were held relatively constant. In the second experiment, the same treatments were performed in a volume of 20 ml of medium. Such a small volume may have limited the repletion process and consequently affected the extent of the influx response. For example, typical cytoplasmic and vacuolar 4 [NH + ] were 0.19 and 2.19 jimol g’FW for  151 5.  2.5____  22A  :::  0  25  50  100  125  G2  media  (h)  Total amino acid concentration  ([AA] )  Depletion  Figure  22.  75  After depletion in  G2  in  expanded form the data for  se).  6  24 h  ([AA] ) 1 .  2025  175  150  of depleted  medium for various periods,  assayed for tissue amino acid concentration  mean of  15  10  G 1000  G1000 Insert  200  roots.  roots were  22B  shows in  of repletion. Each datum point is the  replicates and the vertical bar represents the standard error  (±  152 G2 roots and 1.94 and 4.91 jimol g’FW for G1000 roots, respectively (see Table 4). This means that in order to convert G1000 plants to G2 plants there is about 4.47 jimol NH 4 4 g F W to be depleted either by metabolism or efflux to the external media. Assuming that rates of efflux and assimilation are equivalent at about 20% of the rate of influx (Chapter 3 and Wang et al., 1993a), then the release of NH 4 could elevate external ] to nearly 100 tiM. In the small volume employed for this 4 [NH experiment the released NH 4 would readily be re-absorbed, slowing down the change from G1000 to G2 statues. As shown in Fig. 23A, when G2 plants were repleted with 4 NH - in G1000 medium, the ‘ 4 N 3 H influx (closed circle) increased from 8.17 to 10.00 j.tmol g’FW h’ during the first hour, then dropped to 8.61 at 4 h and 1.95 iimol g-’FW h-i after 24 h repletion. Root [NH 1 (closed square) ] 4 increased rapidly in the first hour from 2.21 to 6.48 iimol g’FW and increased only slightly to 7.13 iimol g’FW during the next 23 h of NH 4 repletion (Fig. 6B). By contrast, depletion of G1000 plants in G2 medium increased 13 4 influx only very slightly during the first hours. Then NH influx increased rapidly from 0.72 to 7.29 iimol g’FW h’ (open circle in Fig. 23A). During the depletion in G2 medium, the [NH 1 + 4 ] of G1000 plants (open square) decreased gradually from 6.35 to a value similar to that of G2 at steady-state, 2.36 jimol g’FW by 12 h of depletion. During the next 12 h, there was only a small further decrease of 4 [NH + ] (Fig. 23B). The changes of tissue amino acids present different patterns for plants undergoing nitrogen depletion or repletion. During the repletion process, G2 plants were exposed to 1000 .tM NH 4 for up to 24 h. The total  153 12-  23A  L0,  G2IG1000 G1000IG2  —  —0—  G2IG1000 G1000IG2  —D  —  E  23B  —  8  E  :.  -.  U  —  4  z  II  .  2E E .  0 5  0  10  15  20  00  25  Pretreatment  Figure 23. repleted  4 NH 13  G2  5  10  time  15  20  (h)  influx (23A) and internal ammonium content (23B) of  or depleted  G1000  roots, after pretreatment for  roots. 23A:  1, 4, 12  respectively, were measured in  and  100 1 iM  4 N 3 ‘ H 24 h  influxes of  in  G1000  or  G2  or  G2  medium,  G1000  13 4 NH labeled solution. 23B:  Internal ammonium content of the same roots. Each datum point is the mean of  6  replicates and the vertical bar represents the standard error.  25  154  0  3000 0  E 2000  1000  —s——  G2/G1000M  —O----  G1000/G2M  U-  0  5  10  Pretreatment  15  time  20  25  (h)  Figure 24. Total [AA] 1 of repleted G2 or depleted G1000 roots. Total  1 [AAJ  of G2 or G1000 roots were assayed after pretreatment for 1, 4, 12 and 24 h in G1000 or G2 medium, respectively. Each datum point is the mean of 6 replicates and the vertical bar represents the standard error (± Se).  155 2000 -  25A G1n —0-—  1500  25B Glu  G2/G1000M G1000IG2M  —C]—  1000-  100  500  50  -  u0  0 600°  G1000/G2M  150  -  0  G2/G1000M  5  10  15  20  25  10  5  15  20  25  600-  25D Asp  25C Asn——— G2/G1000M —0— G1000/G2M  ———  —ETh—  400-  400  200  200  G2/G1000M G1000/G2M  —  E  -.  0•  0 0  5  10  15  20  25  Pretreatment  0  5  time  10  15  20  (h)  Figure 25. Tissue amide or amino acid contents of repleted G2 or depleted G1000 roots. After pretreatment for 1, 4, 12 and 24 h in G1000 or G2 media, respectively, the amino acid contents of G2 and G1000 roots were assayed. 25A for [Gin] ; 25B for [Glu] 1 ; 25C for 1 1 [Asn] , ; 25D for [Asp] . Each 1 datum point is the mean of 6 replicates and the vertical bar represents the standard error (± Se).  25  156 1 increased during 12 h of repletion and stayed at more or less the [AA] same level during the next 12 h (Fig. 24). The content of Gln (Fig. 25A, closed circles) changed in the same pattern as the total [AA] 1 but the Glu content (Fig. 25B, closed circle) decreased continuously during 4 NH repletion. Although the reduction of [Glu] was 37%, [Gln] 1 increased 372% during 24 h of repletion. In contrast, [Asn] 1 decreased by about 24% during the first hour, then it increased nearly 39% of the initial level in the next 12 h (Fig. 25C, closed circles). [Asp] 1 was reduced (49%) in G2 roots during the first 4 h (Fig. SD, closed squares), after which it increased slightly. When G1000 plants were depleted in G2 medium, total  [AA] as well as the  four major amino acids decreased rapidly for the first hour (Figs. 24, 25AD, open symbols). This is interesting because despite big changes in these [amino acid], influx changed little. After that, the [AA], [Gln] , [Glu] 1 , 1 , and [Asp] 1 [Asn] 1 increased 65%, 353%, 61%, 40% and 31%, respectively, within 23 h of commencing the depletion process.  6.3.2.  Experiment II. Effects of MSX  Short periods (< 12 h) of MSX treatment increased 4 NH influx of G2 3 ‘ roots (closed circles in Fig. 26) from 8.17 to 16.93 jimol g FW h’, but 1 longer (12  -  24 h) exposures reduced influx slightly, to 12.64 jimol g’FW  h’. During 24 h pretreatment of G2 plants in G1000+MSX, ‘ 4 N 3 H influxes (open squares) remained essentially constant at about 10 iimol g’FW h’ and were lower than those of in G2+MSX (closed circles). Likewise, G1000 plants, pretreated in G2+MSX or G1000+MSX media, exhibited very low 4 N 3 ‘ H influx values (closed and open squares) which remained essentially constant for the duration of the experiment. Fluxes of G1000 plants were  157 significantly lower than in G2 plants in G2+MSX or G1000+MSX (compare open to closed symbols in Fig. 26). For G2 plants pretreated in G2+MSX, root [NH 1 + 4 ] increased rapidly from 2.21 to 7.19 at the first hour and remained at the same level for the remainder of the experiment (closed circles in Fig. 27A), but pretreatment in G1000+MSX caused root [NH 1 to increase rapidly from 2.21 to 8.49 ] 4 imol g’FW during the first hour, reaching a value of 9.35 after 24 h repletion (closed squares in Fig. 27B). G1000 plants possessed a higher initial [NH 1 (6.35 jimol 1 ] 4 g F W) (Figs. 27A and 27B), which continuously increased to 8.57 imol 1 g F W after 24 h during treatment of G1000+MSX medium. Root 1 -’-] in G1000 plants treated in G2+MSX declined 4 [NH gradually from 7.36 at 1 h to 5.77 between 4 and 24 h (open circles in Fig. 27A). The increment of [NH 1 4 ’-] in MSX treated plants varied with prior 4 provision during growth and additional depletion or repletion NH treatments (Figs. 27A and 27B). During the first hour, the [NH 1 4 ’-] of G2 plants increased 230% in G2+MSX medium and 320% in G1000+MSX medium. The [NH 1 of G1000 plants increased 35% in G1000+MSX ] 4 medium, and 16% during the same time period in G2+MSX medium, the latter then decreased to 9% after 24 h. The total [AA] 1 of G2 or G1000 plants in the four treatments pretreated with 1 mM MSX, remained at similar levels, respectively, over the 24 h period (Figs. 28A and 28B). G1000 plants (open symbols) had a higher total  [AA] than G2 plants (closed symbols). Both plants showed a  small increase in the G1000+MSX treatment (Fig. 28B). Pretreatment in G2+MSX, caused the [Gin] 1 of G2 roots to decline at the first hour but no further changes were observed during the remainder of the experiment (Fig. 29A, closed circles). The opposite effect was observed in G1000+MSX  158 25  •  G2/G2M+MSX G2/G1000M+MSX  —0— D  G1000/G2M+MSX G1000/G1000M+MSX  20  0  210  10  Pretreatment  time  25  (h)  Figure 26. Effect of MSX pretreatment on 4 NH influx of rice roots. G2 1 (closed symbols) or G1000 plants (open symbols) were pretreated with 10 mM MSX for a maximum duration of 24 h including 0, 1, 4, 12, and 24 h in G2+MSX medium (open or closed circles) and in G1000+MSX medium (open or closed squares), respectively. The influxes were measured in 100 jiM -labe1ed solution without MSX. Each datum point is the mean of 6 4 NH 13 replicates and the vertical bar represents the standard error (± Se).  159 —•  12  in G2M+MSX  27A  in G1000M+MSX  27B  10.  G2  21  —0— •  0  I  5  •  I  10  •  I  15  G1000 0  •  20  Depletion  25  or  •  0  •  5  repeltion  I  —a—  G2  —D—  G1000  •  15  10  time  I  •  I  20  (h)  Figure 27. Effect of MSX on internal ammonium content of rice roots. The pretreatments and symbols are same as in Fig. 26. Each datum point is the mean of 6 replicates and the vertical bar represents the standard error (± se).  25  160 ‘—‘  3000  3000  28A  ——  —0— 00  28B  in G2M+MSX  in G1000M+MSX  G2 G1000  —  G2  —D—  G1000  0  5  10  15  20  Depletion  25  or  0  5  repeltion  10  time  15  20  (h)  Figure 28. Effect of MSX on total [AA] 1 of rice roots. The pretreatments and symbols are same as in Fig. 9. Figures hA and 11B are for the plants pretreated in G2+MSX medium and in G1000+MSX medium, respectively. Each datum point is the mean of 6 replicates and the vertical bar represents the standard error (± Se).  25  161  :::z ; 100•  100•  EE  ooo  0• 0 ‘  I  I  150  100  29C  5  10  15  20  25  150  in G2M+MSX  —0——  0  G2 G1000  o  5  29D  10  * —D—-  G2  15  20  G1000 25  in G1000M+MSX  —U—  100  G2 G1000  II  .  ..  25  .  Depletion  or  repeltion  time  (h)  Figure 29. Effect of MSX on amide or amino acid content of rice roots. The pretreatments and symbols are the same as in Fig. 26. Figures 29A, 29C, 29E, 29G is for [Gin] , [Glu] 1 , [Asn] 1 , and [Asp] 1 1 of plants pretreated in G2+MSX medium, respectively. Figs. 29B, 29D, 29F, 29H is for [Gin] , [G1u], 1 , and [Asp] 1 [Asn] 1 of plants pretreated in G1000+MSX medium, respectively.  162 300  •  —0—  f  300  29E in G2M+MSX G2 G1000  in G1000M÷MSX  29F  —•—  20O  200  G2  I I  E 100  0 0  5  10  15  20  25  0 0  5  300  300  15  20  25  in G1000M+MSX  29H  in G2M+MSX  29G  10  II ——  —0— —  —  G2 G1000  ——  —D—  00  L1 200  100  100  G2 G1000  E  0• 0  U-  5  10  15  20  Depletion  Figure 29. (Continued).  25  or  0  repeltion  5  time  10  (h)  15  20  25  163 (Fig. 29B, closed squares). The [Gin] 1 of G1000 roots was reduced more in G2+MSX (Fig. 29A, open circles) than in G1000+MSX (Fig. 29B, open squares). In the latter medium, Gln recovered slightly after 24 h pretreatment (Fig. 29B). The levels of [Glu] 1 in roots declined rapidly within the first 4 h of pretreatment in G2+MSX and in G1000+MSX (Figs. 29C and 29D) except in the G2 plants treated in G2+MSX, in that it took a longer time to achieve the same reduction (Fig. 29C, closed circles). The [Asn] 1 and 1 of G2 roots were also significantly reduced in all four pretreatments [Asp] (Figs. 29EH). A similar extent of reduction of [Asn] 1 was reached in a shorter time period when G1000 plants were pretreated with MSX in either repletion with or depletion of NH 4 (open circles in Fig. 29C and open squares in Fig. 29D) whereas the change of [Asp] 1 was more gradually in G2+MSX (Fig. 29G) than in G1000+MSX (Fig. 29H); in the latter treatment the reduction occurred within 4 h of pretreatment.  6.3.3. Experiment III. Effects of exogenous amino acids Pretreatment of G100 roots with 10 mM glutamine significantly reduced ‘ 4 N 3 H influx at all concentrations tested (Fig. 30). Assays of 1 revealed that glutamine pretreatment was associated with higher ] 4 [NH -1-] (6.2 ± 0.5 .tmol g’ FW) than those pretreated without glutamine 4 [NH 1 (2.3 ± 0.8 pmol g-’FW). The 18 h pretreatment in 10 mM Gin raised the contents of Gin, Glu, and Asp near 4 times and Asn 7 times (Figs. 3 1A and 31B). The interaction of exogenous amino acids and nitrogen status were also investigated. When G2 plants were treated with either 10 mM [Gin] 0 or 0 for 18 h, 1 [Glu] 4 N H influxes were significantly reduced (from 8.94  164 6 0  without glutamine  [NH  Jo (jiM)  Figure 30. Effect of exogenous glutamine on ‘ 4 N 3 H influx of roots. G100 plants were pretreated in G100 medium with or without 10 mM glutamine for 16 h before measuring ‘ 4 N 3 H influx. ‘ 4 N 3 H influxes were measured in 2, 10, 25 and 100 jiM NH 13 4 labe1ed solution without glutamine. Each datum point is the mean of 6 replicates and the vertical bar represents the standard error (± se).  165 6000  2000  -  31A Total  31B  Asp Glu  F, F?  0  F,  Asn  F— F— —F  LJGIn  —F —F  4000-  F— F— F’  0  ‘F F—  1000  F— —F —F ‘F —F  -.  FF  2000  .‘  ,  F F  I  F  0  /  F..  F— ‘F  0-  0+Gln  -Gin  F• —  +Gin  -Gin  Pretreatment  Figure 31. Effect of exogenous glutamine on the contents of amides and amino acids of root tissues. The pretreatments are same as in Fig. 30. Fig. , [Glu] 1 , {Asnj 1 , and [Asp] 1 . Each 1 1 and Fig. 31B is [Gin] 31A is total [AA] datum point is the mean of 6 replicates and the vertical bar represents the standard error (± se).  166 12  12  32A  32B  G2/G2M 10  10  E  8  —  6  —  4.  z 2  0  G2/G100M  •  0 T)  •Im•  ‘Il/I,  Control  Gin  Glu  Asn  8  T  6  4  2-  0-  -I-  Control  Gin  Glu  Asn  Pretreatment  Figure 32. Effect of exogenous amides and amino acid on ‘ 4 N 3 H influx. G2 plants were pretreated in G2 medium (Fig. 32A) or G100 medium (Fig. 32B) in the presence of 10 mM of either Gin, or Giu, or Asn for 6 h. The influxes were measured in 100 jiM 13 -labeied solution. Each datum 4 NH point is the mean of 6 replicates and the vertical bar represents the standard error (±se).  167 iimol g’ FW h-i of the control to 5.12 and 2.30, respectively, Fig. 32A). No -NH influx occurred as a result of Asn 1 significant reduction of 4 pretreatment (Figs. 32A, B). Comparisons of pretreatments in G2 medium and G 100 medium for G2 plants, revealed that the higher concentration of NH influxes of the 3 ‘ 4 in the latter medium led to a reduction of 4 NH control and the Asn-pretreated plants from 8.94 and 9.26 imol g-’ FW h-i (Fig. 32A) down to 7.04 and 5.36 j..tmol g’ FW h-i, respectively (Fig. 32B). 0 or [Glu] 0 and 10 mM [Gln] ] 4 0 (Fig. 32A) The combination of 100 iM [NH 4 influx further than the pretreatments of 2 jiM NH failed to reduce 13 0 or [Glu] 0 (Fig. 32A). Pretreatments with 0 and 10 mM [Gln] ] 4 [NH 1 from 1.1 to 3.6 ] 4 exogenous amides or amino acids increased [NH  -  5.5  4 conditions (G2 medium) (Fig. 33A). In jimol g 1 FW at low external NH +] was higher for the Glu pretreatment, 4 G100 treatment, internal [NH followed by the control, and the pretreatments with Gln and Asn (Fig. 33B). Total AA concentrations were significantly higher for plants pretreated in G100 medium than in G2 medium (Fig. 34). In both cases, the total AA was higher in the pretreatments of Glu and Asn (Fig. 34B). When exogenous amides or amino acids were provided during pretreatments, 1 was highest in the Gin pretreatment (Figs. 35A and 36A), except for [Gln] the [Glu] 0 pretreatment in G2 medium that had the highest [Gln] (Fig. 34A). Compared to the control, the concentrations of Gln were doubled in both 1 was highest in GIn pretreatments, followed by the Asn media. [Glu] pretreatment (Figs. 35B and 36B). Both [Asn] 1 and [Asp] were highest in the Asn pretreatment (Figs. 35C, 36C, 35D and 36D).  168 0 0  6  33A0 G2IG2M  T  33 B  G2/G100M  5.  0  -.S  C  T_  4.  Wf WFA  3.  Ff1  III  2-  E 1-  4  2-  C  E  0 Control  Gin  Glu  Asn  0” Control  Gin  Glu  Asn  Pretreatment  Figure 33. Effects of exogenous amides and amino acid on internal ammonium content. Details as in Fig. 32.  169 6  34A  -.  E  18  34B  4.  12  G2/G100M  -  9-  -.  C.?  -  G2IG2M  2  C  0  6-  Liii  Control  Gin  3.  Glu  Asn  Control  Gin  Glu  Asn  Pretreatment Figure 34. Effects of exogenous amides and amino acid on total amino acid content. Details as in Fig. 32.  170 1500  35A  Gin  35B  Glu  C C  ‘V  200  1000  100-  500-  C  0-  I  / 0-  Control Gin 300  35C  Giu  Asn  Control 3000  Asn  35D  Gin  Glu  Asn  Asp  -.‘  C C 1  200  2000 ???1 ?1 ‘‘‘I ‘—‘F ‘—‘F F—,—  100-  1000  ——‘F ‘F,— F,—— —F—— \\‘•  ___ \\% F,,  C  F——, F—,— F,—,  0  0  Control  Gin  Glu  Asn  FFF F,,—  IF,FFI I’ ••. •‘ ‘.1 IFtFPI  Control Gin Pretreatment  ___ \\%\ .‘..  •..  •s “  ‘.  .tFZ  Giu  Asn  Figure 35. Effect of exogenous amides and amino acid on contents of amino acids in G2 roots. Pretreatments are same as in Fig. 32. Figs. 35AD is for [G1n], [Glu] , [Asn] 1 , and {Asp] of plants pretreated in G2 medium, 1 respectively.  t71 8000  36A  8000  Gin  36B  Glu  0 0  6000  -  4000  -  VA  6000-  /  —  0  -.S  2000  4000  -I  -  0  0Control 3000  Gin  Glu  2000-  0  Asn  Control  Gin  Giu  Asn  6000  -  36C  36D  Asn  p  2000-  Asp  —cF—,— —F—— —F—— —F——  400W  ———F ———F F——— F  _F__  •‘.\ ____ ‘\\ F___  FFF  FFF ‘.\\ F__F FF  ‘%‘\  1000-  F__F ‘‘.%\ ____  2000  ____ \‘\  _,F_  FF. \‘‘•,  F__F F,,,  ‘\‘\  FF?  ‘%.\  F__F •\‘  ____ ____  0  ‘‘,,I \‘‘‘I —‘F_I ‘‘.‘.\‘I FFI  c-)  \•\ FFFF \%‘  FF \‘.•%  _,__ \‘s’.\ FFFF \\\  ‘F. \‘‘\  FFF ,\•\  F,,, ,\•\  F,,, F,,, ‘.\\‘ ‘FF ,‘\•  ?FF  ‘•\•  0-  ‘I  Control Gin  Glu  Asn  Control  Gin  Giu  Asn  Pretreatment  Figure 36. Effect of exogenous amides and amino acid on contents of amino acids in G100-pretreated roots. Pretreatments are same as in Fig. 32. Figs. 36AD is for [Gin] , [Giu] 1 , [Asnj 1 , and [Asp] of plants pretreated in G100 1 medium, respectively.  172 6.3.4. Experiment IV. Effects of selected inhibitors G100 plants were treated with inhibitors of glutamine synthesis, MSX, glutamate synthesis, DON, and aminotransferases, AOA, for 16 h, respectively. The ‘ 4 N 3 H influxes were measured in either 100 jiM or 10 NH solution without inhibitors. The largest effect of the 1 mM labeled 4 inhibitors of NH 4 assimilation was associated with AOA pretreatment (Fig. NH influx due to HATS (high affinity transport system) and 3 1 36A). The 4 LATS (low affinity transport system) were reduced by 68% and 32%, respectively (Figs. 37A and 37B). MSX reduced ÷ 4 N 3 ‘ H influx by the LATS 25% and by the HATS 19%. DON treatment produced only a slight reduction of 1 4 N H influx (16% for LATS and 4% for HATS). As can be seen in Fig. 38A, MSX significantly increased [NH 1 almost j 4 3 fold. The level of [NH 1 was 1.9 times higher as a result of AOA ] 4 pretreatment, while rice roots treated with DON actually had a lower ] than the control. The total 4 [NH  1 was doubled by the AOA treatment [AA]  (Fig. 38B). While slightly increased by MSX, the total [AA1 was greatly reduced by DON treatment. Looking at the four major amides and amino acids, (as shown in Figs. 39A, B, C, D), the pretreatment of AOA significantly increased all four, to a level which was at least double that of the control. There were no dramatic changes due to the MSX pretreatment. The four major amino acids were reduced to about half that of controls after treating plants with DON (Figs. 39A, B, C, D).  173 5  Uptake in 0.1 mM  37A  15  37B  Uptake in 10 mM  4. —  F  10 : V  /  —  — +.1.  z o  •  Control  MSX  DON  /4’  0-  .  AOA  Control  MSX  DON  AOA  Pretreatment  Figure 37. Effect of MSX, DON and AOA on 4 NH influx. G100 plants were 1 pretreated with MSX, DON, and AOA for 16 h, respectively. The influxes were measured in either 100 1 iM (Fig. 37A) or 10 mM (Fig. 37B) labeled 4 solution without inhibitors. Each datum point is the mean of 6 NH 13 replicates and the vertical bar is the standard error (± Se).  174  -.S  12  3  38A  Ammonium  38B  8  Total AA  2  —  E  :i  4. C  0  Pd/”II  Control  MSX  DON  AOA  0 Control  MSX  DON  AOA  Pretreatment  Figure 38. Effect of MSX, DON and AOA on internal ammonium and total amino acid content. Pretreatments are same as in Fig. 37. Fig. 38A is for internal ammonium and Fig. 38B is for total amino acid content.  175 1000’  ,IflhI  39A  39B  Gin  Glu  C  800 300 600 200 400 100’  200 C  C,)  I  II  Control  MSX  DON  AOA  Control 600-  “7  39C  U  Asn  MSX  39D  DON  AOA  Asp ‘\,‘ ‘I,, %‘\\ ... , , F ‘,.‘.  ,  400’  400  ,,, \\\\ ‘,‘f -  ,  ‘.‘\\ ,.,, ‘.‘‘...  ,,, ‘‘‘\ ‘/_f ‘‘\‘ f—f, %,‘ f_f ,  ‘  ‘f_f  200  ‘f_f %‘‘ ‘f_f  2 “'‘ U)  -.S  /,,, \\\ f \•\  tf  T7 ,,,,  \%\\ f_f,  ,,__  ,f,f  \‘‘.\  ,,,,  \\s\ ,,,,  C  ,,,, \\ ,,__  ‘.‘‘.\  Control  MSX  DON  AOA  0  •  f_f, \\\ ,,,, \s\% ,,,, ‘\s ‘.  —  Control  ,,,, %\‘\ ,,,, \\ .‘  MSX  f_f, ,%\‘ ,f__ \‘.•..\ ,,,_  DON  ,,f, f_f, ,,,,  AOA  Pretreatment  Figure 39. Effect of MSX, DON and AOA on major amino acid contents. Pretreatments are same as in Fig. 37. Figs. 39AD is for [Gin] , [Glu] 1 , [Asn] 1 , 1 and [Asp], respectively. Each datum point is the mean of 6 replicates and the vertical bar is the standard error (± Se).  176 6.4  DiscussioN  6.4.1. Negative feedback on NH 4 uptake by NH 4 assimilates 4 uptake is probably regulated continuously in response to the N NH status of the plant, but it is not clear how this is achieved. Increase in ammonium influx upon nitrogen limitation and decrease in influx as cell nitrogen status rises have commonly been observed (McCarthy and Goldman, 1979; Pelley and Bannister, 1979; Smith, 1982; Ulirich et al. 1984; Holtel and Kleiner, 1985; Clarkson, 1986; Lee and Rudge, 1986; Morgan and Jackson, 1988a, 1988b; Clarkson and Luttge, 1991). Feedback inhibition of NH 4 uptake by nitrogenous effectors has been implicated in organisms like Lemna, algae, yeast and higher plants (Kleiner, 1985; Ulirich et al., 1984; Pelley and Bannister, 1979; MacFarlane and Smith, 1982; Wiame et al., 1985; Wright and Syrett, 1983; Thomas and Harrison, 1985; Clarkson and Luttge, 1991). The product(s) of ammonium assimilation have been proposed to act as the negative feedback factors for the NH 4 uptake process (Cook and Anthony, 1978b; Breteler and Siegerist, 1984; Wiame et al., 1985; Revilla et al., 1986; Lee and Rudge, 1986; Morgan and Jackson, 1988a). In the review by Clarkson and Luttge (1991) a central role for glutamine in regulating the uptake of N by fungi and microalgae was presented. Glutamine or asparagine are the low molecular weight N-containing compounds stored or translocated by plants in the family of Poaceae (Gramineae) (Marschner, 1986). Lee and Rudge (1986) showed sizable increases in NH 4 uptake by barley following N-depletion, and the increased capacity for NH 4 uptake was inversely related to the reduced-N  177 status of the root tissue. In tobacco cells cultured on nitrate, urea, or ammonium, Gln is the first major organic product of assimilation of ‘ 4 N 3 H (Skokout et al., 1978). It is also true for rice, because glutamine and glutamate were the primary products of ammonium assimilation in rice roots (Arima and Kumazawa, 1977). However the studies by Lee et al., (1992) and by several other workers (summarized in Clarkson and Luttge, 1991) showed that other amino acids may participate in the regulation of N uptake. In the present study, evidence supporting a central role for glutamine or other amino acids in controlling NH 4 influx was equivocal. When plants were maintained at 2 tM or 1000 iM NH 4 respectively, 4 N 3 ‘ H influx was inversely correlated with [Glfl]i (closed symbols compared to open symbols in Figs. 29A and 29B). Likewise, when the internal concentrations of Gln and other amino acids were increased by pretreatment with Glu, ‘ 4 N 3 H influx declined (Figs. 30, 31B and 35A). The results indicated that Glu had an inhibitory effect on ‘ 4 N 3 H influx, greater than Gln or Asn (Figs. 32A and 36A). This point was supported by the results of the AOA treatment. After treating plants with AOA, under the conditions of the present study there was a significant increase of [Gln] 1 (Fig. 39A), [Glu] 1 (Fig. 39B), [Asn] 1 (Fig. 39C), and [Asp] 1 (Fig. 39D). This increment was associated with a significant reduction of 1 4 N H influx (Fig. 20A). It must be pointed out that the above mentioned reductions of 4 influx in rice also coincided with a significant increase of [NH NH 13 1 ÷ 4 ] (Figs. 33A and 38A). Pretreatment with 10 mM Gln doubled the [NH4]j from 2.30 to 6.10 iimol g FW (also in Fig. 33A) and decreased 4 4 NH 1 influx.  178 In the depletion experiment shown in Fig. 23A transfer of G1000 plants to G2 solution failed to increase NH ÷ influx until 4 h had elapsed. 4 Yet, the amino acid analysis indicated strong reduction of total  [AA] and  [Gin], [Giu] and [Asp] (Figs. 24, 25AD). Strong reductions of amino acids were not correlated with 13 4 influx. Therefore, it is not entirely clear NH which N derivative is responsible for limiting influx. Although applying organic N to the growth media has been found to increase crop yield (Mon et al., 1977; Mon and Uchino, 1977), the treatment of organic N suppresses the uptake of inorganic N. For example, maize roots pretreated with Gin or Asn exhibited reduced net uptake of 4 and N0 NH 3 (Lee et al., 1992). The uptake of ‘ 3 N 5 0 by barley roots was depressed by pretreatment with Arg and His (Mon et al., 1979). It was suggested that transport activity for ammonium was controlled by intracellular rather then extracellular metabolites (Jayakumar and Barner, 1984).  6.4.2. Effect of MSX: reduced amino acid pooi MSX inhibited the activity of glutamine synthetase in plant roots, and stopped the ‘ N labeling of free amino acids, particularly glutamine and 5 glutamate in roots of barley or rice (Arima and Kumazawa, 1977; Lewis et al., 1983). Preventing the assimilation of newly absorbed 4 NH or releasing 4 from the catabolism of internal N-containing compounds rapidly NH increased the NH 4 concentration in roots (Arima and Kumazawa, 1977; Lewis et al, 1983; Lee et al., 1992). Two major effects are expected: the amino acid pool is reduced and NH 4 pool is increased. After treating with MSX, tissue [Gin] 1 is typically decreased (Steward and Rhode, 1976; Fentem  179 et al., 1983a, 1983b) and consequently the amide donor to Asn synthesis is decreased, since the concentrations of Gin and Asn closely correlated (Lee et al., 1992). When products of ammonium assimilation were reduced by treatment of MSX, NH 4 influx was increased (Jackson et al., 1993), though - influx was not stimulated (Lee et al., 1992). 3 NO MSX increased the cytoplasmic ammonium concentration in root tissue of rice (Arima and Kumazawa, 1977), Datura (Probyn and Lewism 1979), barley (Lewis et al., 1983; Fentem et al., 1983b; Morgan and Jackson, 1988a, 1988b); wheat (Morgan and Jackson, 1988a, 1988b), maize (Lee and Ratcliffe, 1991; Lee et al., 1992). A ten fold increment of the cytoplasmic pooi was reported in maize roots compared to the control (Lee and Ratcliffe, 1991; Lee and Ayling, 1993). This increase is due to two effects: (a) the assimilation of NH 4 into amino acids is blocked, and (b) the production of NH 4 from breakdown of amino derivatives remains unaffected. It has been claimed that release of NH 4 from this degradation path occurs at a rate which is 50% higher than the rate of NH 4 influx (Jackson et al., 1993). As a result, ammonium appeared in the xylem sap (Lee and Ratcliffe, 1991) and net NH 4 efflux was increased substantially (Morgan and Jackson, 1988a). Arima and Kumazawa (1974, 1975, 1976, 1977) proposed that most of the glutamine is synthesized adjacent to the outer membrane of plasma membrane of root cells, through which ammonium with a high 15 N abundance permeates from the external solution. MSX treatment might enlarge this ammonium compartment near the membrane. Another explanation for the enhanced NH 4 influx by MSX treatment is that MSX enlarged the cytoplasmic and vacuolar NH 4 pools of root tissue several times (Jackson et aL, 1993; Lee and Ayling, 1993). The enlarged  180 4 pools in cell enhanced influx of 1 NH 4 N H in maize and barley (Lee et al., 1992; Lee and Ayling, 1993). According to Lee and Ayling (1993) this resulted in a large value of NH 4 influx because what was measured under these circumstances was a true value of influx. By contrast, under ‘normal’ circumstances (they claim) even short ‘ 4 N 3 H influx measurements are compromised by a significant efflux. The results of studies on rice and barley (Siddiqi et al., 1991; Wang et al., 1993a) repudiate this interpretation because the half-life of the cytoplasmic compartment is too long (7-8 mm) and the efflux term is too small (10%  -  30%) compared to  influx (Wang et al., 1993a). The results from this study show that, after treating plants with MSX, the concentrations of major amides and amino acids were all reduced to different extents (Figs. 29A-H), accompanied by an increased . 1 4 [NH ’-] The increment of [NH ] was varied with NH 4 4 provision and additional depletion or repletion treatments (Figs. 27A and 27B). However the + 4 treatment with MSX in this experiment failed to increase the 13NH influxes of G1000 plants treated in either G2+MSX or G1000+MSX medium (open symbols in Fig. 26) compared to the effects of depletion or repletion by the same plants in the absence of MSX (Fig. 23A). However, G2 plants treated with G2+MSX conditions revealed a significant increase of influx (Fig. 26). When the same G2 plants were treated in G1000 medium plus MSX there was no decline of influx of the sort observed in the absence of MSX (Fig. 23A). This is consistent with an important role of amino N in down-regulating influx in low-N plants. The lack of an increased influx when G1000 plants were transferred to G2 medium with MSX (Fig. 26) argues that internal 4 [NH ’-] is important in maintaining low NH 4 fluxes in high-N plants. This has also been claimed by Causin and Barneix (1993) in  181  wheat. Thus the results of these experiments indicated that both [NH ] 4 and  1 may play important role in regulating NH [AA] 4 fluxes.  6.4.3. Effect of short-term N depletion It has been recognized that the nitrogen (both ammonium and nitrate) uptake capacity of plant roots is enhanced when plants undergo nitrogen depletion (Humphries, 1951; Jackson et al., 1976; Clement et al., 1979; MacKown et al., 1981; Breteler and Nissen, 1982; Lee and Rudge, 1986; Ingemarsson et al., 1987; Oscarson et al., 1987; Teyker et al., 1988; Siddiqi et al., 1989; Jackson and Volk, 1992). 4 NH uptake shows a particularly strong response in several species, such as wheat (Tromp, 1962; Minotti et al., 1969; Jackson et al., 1976b; Morgan and Jackson, 1988a,  1988b), ryegrass (Lycklama,  1963), maize (Ivanko and  Ingversenm, 1971; Lee et al., 1992), barley (Lee and Rudge, 1986), and oats (Morgan and Jackson, 1988a, 1988b). In the present study, rice also responded to nitrogen depletion with enhanced NH 4 influx (Fig. 20). The short-term depletion of high +-grown plants (G1000) in low N medium (G2 medium) stimulated 4 NH 4 influx during the first 4 to 5 h of depletion. NH NH 13 influx remained 4 high for the next 20 h, then declined to a relatively lower rate for the next 20 h of depletion (Fig. 20B). Similar rapid initial increases of NH 4 uptake were observed when plants were depleted of N for the first 0.25 and 1 h (Lycklama, 1963; Minotti et al., 1969; Breteler, 1975; Deane-Drummond, 1986; Goyal and Huffaker, 1986; Morgan and Jackson, 1988a, 1988b, 1989).  182 A likely explanation for this enhancement is the removal of a factor which exerts negative feedback regulation on NH 4 uptake. Both 4 NH - and its primary assimilate were suggested as such factors in uptake regulation (Breteler and Siegerist, 1984; Revilla et al., 1986; Lee and Rudge, 1986; Morgan and Jackson, 1988a). Another explanation for this enhancement is due to enhanced influx and reduced efflux (Morgan and Jackson, 1988a, 1988b). Substantial ammonium cycling occurred during net ammonium uptake (Jackson et al., 1993), yet plants grown in low N possess a low NH 4 efflux. For G2, G100 and G1000 plants at steady-state with respect to 0 4 [NH , ’-] the effluxes of NH 4 were 10%, 20% and 29%, respectively, of influx (Wang et al., 1993a). However, changes of these relatively small proportions may not account for the large increases of NH 4 uptake such as were observed in the present study. In the present study, [NH 1 was negatively correlated with influx ] 4 during 4 h of depletion (Figs. 20B and 21B). It was also observed that the 4 influx was negatively correlated with internal NH 4 (Wang et Vmax for NH al., 1993b). When plants were subjected to N depletion, the tissue content of NH 4 (Fig. 2 1B) dropped rapidly to lower levels and possibly resulted in a relief of N-suppression of the uptake process. [NH4]c is a likely candidate for negative feedback regulation since the free NH 4 pools (cytoplasmic and vacuolar) will be drained in two opposite directions: efflux out of the tissue and metabolism into amino acids. In such a short time, [NH ] will 4 be the first fraction to be drained to a minimum. Therefore internal NH 4 is a likely factor to exert a negative signal on NH 4 transport across the plasma membrane (also in section 6.4.5.). It is generally believed that short periods of N depletion, less than 24 to 48 h, would not cause a decline of growth rate (Siddiqi et al., 1989;  183 Jackson and Volk, 1992). Though it was reported that enhanced uptake reached a maximum after 3 days of depletion when nitrogen stress was not severe enough to alter the RGR significantly (Lee and Rudge, 1986), longer N depletion may not sustain the maximum enhanced uptake rate due to possible adjustment of the RGR. For 8-d-old maize plants grown on 5 mM 3 NO , NH 4 uptake rates increased steadily, and within 72 h of Ndepletion, rates of NH 4 uptake initially increased followed by a decline and a subsequent increase (Jackson and Volk, 1992). This enhanced NH 4 uptake or NH 4 influx may be due to a relief of the uptake process from Nsuppression. As suggested by Morgan and Jackson (1992), this type of response reflects the interplay of suppression by a product of ammonium assimilation, the accumulation of root ammonium and associated ammonium efflux, and a stimulation by ammonium of its own uptake.  6.4.4. Stimulated 4 NH influx after long-term N depletion -l4 When N-depleted roots are first exposed to elevated levels of NH there is an initial increase of NH 4 influx for the first few hours of exposure to NH 4 (Goyal and Huffaker, 1986; Morgan and Jackson, 1988a). The above workers observed a 25-35% increase of influx in wheat during the period from 2-10 h after exposure to NH ; further exposure caused 4 influx to decline. This phenomenon was found in wheat but not oat (Morgan and Jackson, 1988a). In the present study, an even greater effect was observed when G2 plants were repleted in G1000 medium. Within the first two hours repletion with 4 NH ’-, 13 4 influx increased rapidly from NH 11.10 imol g’FW h’ to 31.97 iimol g’FW h’ (Fig. 19B). Then, influx dropped to the initial rate of about 10 p.mol g’FW h 1 after 8 h more repletion. A smaller stimulation can also be seen in Fig. 6A.  184 There are at least two possible explanations suggested for this phenomenon (Morgan and Jackson, 1988a). First, a second system for ammonium influx may be initiated (induced?) as N-depleted plants are exposed to ammonium for a short period before negative feedback become active. Another possibility is that there are two effectors (positive and negative) to regulate a single transport system. The positive effector could be NH 4 and the negative one may be a product of 4 NH assimilation (Morgan and Jackson, 1988a). Ammonium concentrations were related to the stimulation in influx whereas a product of ammonium assimilation was subsequently responsible for its reduction/inhibition (Wiame et al., 1985; Cook and Anthony, 1978a, 1978b). The initial increase of NH 4 influx may be resulted from provision of N to synthesize more transporters that are sacrificed when plants are under N stress. In this sense, NH 4 would exert an effect as a source of N for transporters and also as a transport regulator. It was observed in a separate study (Fig. 43 in Chapter 7) that rice plants grown in low N and low K doubled their 86 Rb influx after preloading in 1 mM NH 4 for 2 h. In such a situation, it may be hypothesized that immediately after exposure to NH , more transporters are synthesized. This may not necessarily 4 involve the synthesis of a different carrier for K system. Re-supplying -- provides the ‘building blocks’ to assemble more transporters to 4 NH promote uptake and meet plant demand for N and K. Subsequently, negative feedback mechanisms begin to exert their regulation.  185  6.4.5. Negative feedback on 13 4 influx from internal NH NH 4 As discussed above, the theory of amides or amino acids as N uptake regulators can not explain all the observed results on the regulation of 4 influx. The data seem to indicate that internal NH NH 13 4 may able play a role in regulating NH 4 influx. It has been reported that ammonium transport is repressed by intracellular ammonium per se but not by its assimilates or de novo protein synthesis (Rai et al., 1986; Franco et al., 1987, 1988). The active, specific transport of 1 4 N H and 4 ‘ C -MA in both wild type and mutant cells of Aspergillus nidulans is regulated by the concentrations of internal ammonium (Pateman et al., 1973, 1974). One of the major reported reasons for excluding NH 4 as a negative feedback factor was that there was not an exact parallel between root ammonium concentrations and net NH 4 influx (Lee and Rudge, 1986) or effiux (Morgan and Jackson, 1988a). Therefore endogenous 4 NH - in roots appeared to exert no effect on uptake of either NH 4 (Lee and Rudge, 1986; Morgan and Jackson, 1988) or N0 3 (Rufty et al., 1982a; Chaillou et al., 1991; Vessey et al., 1990a). Despite this claim by the above workers, there were negative correlations between NH 4 absorption and tissue concentration. It was reported that when plants were depleted of nitrate for a week, net NH 4 uptake was increased 5 to 10-fold (Morgan and Jackson, 1988a) “because of low internal NH 4 (1.—2 iimol g’)” (Morgan and -1-] is 4 Jackson, 1988a, 1988b, 1989). But this appears to agree that [NH correlated (negatively) with NH 4 uptake. While there is a positive correlation between the N provision during growth and the internal content of NH 4 in root tissue, the Vmax of 4 NH influx was negatively 3 ‘ correlated with these two conditions (Wang et al., 1993a). Nitrogen depletion rapidly altered the N-status of the plants, particular the tissue  186 concentration (Vose and Bresse, 1964; Lee and Rudge, 1986). In the present study, within 4 to 12 hours of depletion of G1000 roots in G2 medium, 4 NH influxes increased and were closely correlated with 3 ‘ decreases of internal 4 NH content (Figs. 20B, 21B, 23A, and 23B). Lee and Ratcliffe (1991) argued that at steady-state, cytoplasmic ammonium concentration would be not in the millimolar range because the activity of GS was considerable higher than the uptake rate of NH -’- (4 4 jimol g’FW h’). Glutamine synthetase from higher plants has a high affinity for ammonium (Km 20 iiM) (Steward et al., 1980; Milflin and Lea, 1976). It would seem that if NH 4 is not accumulated to a certain level in the cytosol it would not be necessary to invoke a possible regulatory role for this N form. However, most reported estimates of cytoplasmic NH -’4 concentration are in the millimolar range in roots of barley, maize, rice, onion, and wheat (Fentem et al., 1983b; Cooper and Clarkson, 1989; Macklon et al., 1990; Lee and Ratcliffe, 1991; Wang et al., 1993a). In the present study, the indirect estimation of cytoplasmic NH 4 concentrations would give 0.8 mM as the lowest value (Fig. 20A). However low concentrations in the cytoplasm may be due to its rapid movement into the vacuole. It was calculated that half of the total free NH 4 was in a ‘storage pool’ in the roots (Fentem et al., 1983a). In rice roots, it was estimated, that 4 was stored in the root vacuoles (Wang et al., 1993a). above 70% of NH The proportional distribution of newly absorbed NH 4 to N assimilation and to storage may depend on the balance between the gradient across the tonoplast and, the capacity of the GS/GOGAT system, which is probably influenced by whole plant N status. Since high external NH 4 repressed the activity of GS reversibly (Rhodes et al., 1976; Arima and Kumazawa, 1977) and NR (Siddiqi et al., 1993; King et al., 1993), NH 4 should have a role in regulating the NH 4 transport across plasma membrane but not the overall  187 N assimilation, which would include transport across the plasma membrane, metabolism, translocation and utilization (as discussed in section 6.4.6.). Second, the rapid dispersion of NH 4 may be the reason it is so difficult to reveal the contribution of NH 4 to the regulation process. At low 4 entering across the plasma membrane is rapidly external 4 [NH ] , NH metabolized by GS/GOGAT at a rate that is potentially faster than influx (Lee and Ratciiffe, 1991), or is transferred to the vacuole for storage. There may be only limited opportunity for NH 4 per se to exert any direct regulation on NH 4 influx (transport step) under these conditions. Under conditions of elevated NH 4 supply, when the GS/GOGAT system and vacuole are relatively saturated, internal NH 4 may increase to a level which enables it to exert a negative feedback on the transport step. Under such condition, there may be a good correlation between [NH 1 and ] 4 accumulated primary products such as Gin. Ideally, the treatment with MSX blocks the assimilation of NH 4 into Gin therefore leading to increased ] and decreased [amino acids] in roots. As a result one might expect 4 [NH the influx to be increased. This was observed in the present study. Pretreatment of G1000 plants with MSX resulted in a decrease of all major primary products of NH 4 assimilation (open symbols in Figs. 29AD). However these changes did not result in the enhanced 4 N 3 ‘ ’H influxes as would be expected. Internal [NH ] remained at essentially the same level 4 though there was a trend to reduce [NH 1 in G2+MSX after 24 h ] 4 pretreatment (open circles in Fig. 27A). This may be the reason 4 NH 3 ‘ influxes increased during the first hour and remained at the same level thereafter (closed symbols in Fig. 26).  188 A comparison of the G2 plants treated with MSX in 2 mM or 1000 mM solutions (Fig. 26) revealed a significantly higher influx in the G2 plant treated in G2+MSX than in the G2 plant treated in G1000+MSX at 4 and 12 h. Yet the amino concentrations in the G2 plant treated in G1000+MSX showed no significant change during this period (Fig. 29). However [NH 1 4 -] appeared to be higher in the G2 plant treated in G1000+MSX (Figs. 27A and 27B), consistent with an inhibitory effect of [NH 1 4 -] on 13 4 influx NH whenever 4 [NH ÷ ] is elevated; either by growth in high N condition or as a result of MSX treatment.  6.4.6. Cascade regulation system of nitrogen uptake The process of NH 4 uptake may be sensitive to regulation from several signals, related to N status of the plant. These may include internal N pools (NH , 3 4 NO , AA), the GS/GOGAT system, translocation (and recycling) and utilization. Clearly all these processes interact strongly. To imagine that only single cytosolic substrate (e.g. glutamine) might regulate the critical uptake step, may be naive. Therefore, there may be a cascade system with many levels of negative feedback regulation on 4 NH ’- uptake. In addition to N signals, nitrogen 4 (NH + ) uptake may be limited by the supply of carbohydrate from shoots (Kleiner, 1985). This could be considered as an important component of the regulation at the whole plant level. The ambient conditions such as light intensity and temperature will effect the production of carbohydrates. It was found, for example, that net 4 uptake rates oscillate between maximum and minimum with a NH periodicity co-ordinated with intervals of leaf emergence (Tolley and Raper, 1985; Tolley-Henry et al., 1988; Henry and Raper, 1989a; Rideout et al., 1994). At the time of emergence and early expansion of a new leaves  189 there is a requirement for large amount of nitrogen (Radin and Boyer, 1982; Steer et al., 1984), and carbohydrate (Turgeon, 1989). Therefore new leaves become the sink of photosynthate (Turgeon, 1989) and the flux of carbohydrate to roots is reduced. Nitrogen uptake depends on and competes (with other growth process) for soluble carbohydrate from the shoot (Raper et al., 1978; Lim et al., 1990; Henry and Raper, 1991), since carbohydrates provide metabolic energy for nitrogen uptake and translocation (Minotti and Jackson, 1970; Penning de Vries et al., 1974; Jackson et al., 1976). Translocation of carbohydrate from shoot to roots is responsive to concentration of carbohydrate in the shoot pool (Wann et al., 1978; Granato and Raper, 1989; Lim et al., 1990). Since NH 4 is assimilated rapidly and almost exclusively in roots as it is absorbed (Given, 1979; Chaillou et al., 1991), this source of carbon skeletons is equally important for NH 4 uptake and assimilation. It appears that regulation of both NH -’4 and N0 3  uptake at the whole-plant level is subject to common  mechanisms that influence diverse processes within the root and are differentially affected by nitrogen stress (Rideout et al., 1994). The next level of this cascade may be nitrogen assimilation and the major regulators responsible for controlling NH 4 uptake would be active inside root cells. These might include amides and some major amino acids (Pelley and Bannister, 1979; MacFarlane and Smith, 1982; Wright and Syrett, 1983; Ulirich, 1984; Kleiner, 1985; Thomas and Harrison, 1985; Wiame et al., 1985; Lee and Rudge, 1986; Morgan and Jackson, 1988b). As the primary product of NH -’- assimilation, glutamine is the primary 4 candidate for negative effector (Cook and Anthony, 1978a, 1978b; Dubois and Grenson, 1979; Wiame et al., 1985). Within the N cycling of plants, the simultaneous movement of N-compounds from root to shoot, and from shoot to root (Cooper and Clarkson, 1989; Larsson et al., 1991) may enable  190 N absorption to be regulated to match the demand imposed by plant growth (Drew and Saker, 1975; Edwards and Barber, 1976). The concentrations of amides (Gin and Asn) in the roots will be the result of the balance between their synthesis from absorbed inorganic N (NH + or 3 4 NO ), their import via the phloem, and their export via the xylem (Lee et ai., 1992). 4 has not been considered as a negative feedback Internal NH effector for NH 4 uptake (Lee and Rudge, 1986; Morgan and Jackson, 1988a, 1988b; Raper et al., 1992), because it is claimed that there is no correlation between cumulative uptake of NH 4 and endogenous 4 NH - in roots (Chaillou et ai., 1991; Vessey et ai., 1990a). One may consider NH ÷ to 4 be at the center of a vital process of uptake and metabolism. Unlike K-i-, NH 4 ’- will be rapidly consumed into amino acids within the root. Therefore, tissue 4 [NH ’-] is not an ideal indicator of N status. A second reason is that, it was observed by Morgan and Jackson (1988b) that during the first two days of N-deprivation, root NH 4 concentration and NH 4 uptake were closely correlated. After 5 d of N-deprivation, the root NH 4 concentrations were found increased slightly and the rate of NH ÷ uptake was continued 4 to increase. Based on present studies, NH 4 would be expected to be the negative effector when internal NH 4 levels increase beyond a certain level. Below this level one may assume that any free NH 4 would be immediately drawn into the metabolic process to meet the high demand for plant growth. There may be a critical nitrogen status below which the system is impaired and above which it is subject to repression and/or inhibition (Breiman and Barash, 1980). It is proposed, therefore, that internal NH 4 represents a third level of control, operating whenever internal 4 [NH + ] is elevated. The site(s) for  191 its putative effects may include the transport step at the plasma membrane, or the transcriptional level involving the genes coding for NH -’4 transport. In view of the different effects of internal NH 4 on NH 4 influx of N-repleted G2 plants and on N-depleted G1000 plants, it is possible that negative feedback regulation of NH 4 uptake may be facilitated by either 4 or its assimilates. In low N-grown roots the up-regulation of influx NH may be exerted through products of NH ÷ assimilation, while in high 4 4 may participate in the down-regulation of N-grown roots, internal NH 4 uptake systems. NH In the case of the up-regulation of 13 4 influx following transfer of NH G1000 plants to G2 medium (Fig. 20A), the [NH 1 dropped during the first ] 4 two hours of depletion (Fig. 20B) and then decreased gradually to a value similar to that of G2 plants at steady-state. I consider that cytoplasmic j may be the controlling effector here. This is based upon the 4 [NH following additional observations: first, similar negative correlations were found in the 24 h depletion experiment (Figs. 23A, 23B), however 4 NH 3 ‘ influxes were negatively correlated with the [NH +] (Figs. 23A, 23B) but 4 not the content of amides or amino acids (Figs. 24, 25A-D); Second, when the GS-GOGAT pathway was blocked by MSX, 4 NH influx remained at 3 ‘ low rate (Fig. 26) due to higher [NH ] (Figs. 27A and 27B) despite a large 4 decrease of four major amides and amino acids (Figs. 29A-H). Third, 13 4 NH influxes were different when G2 plants were pretreated with MSX for the same 24 h (Fig. 26), but transferred to either G2 or G1000 medium, which resulted in higher 1 +] for plants in G1000+MSX than in G2+MSX 4 {NH medium (Fig. 27). Since estimated half-life for cytoplasmic NH 4 exchange is <10 mm  (Wang et al., 1993a), it would be expected that this component  192 of internal [NH ] would respond more dynamically to change of external 4 [NH 4 ’-] than the vacuolar 4 [NH ’-]. In contrast, the observed declines of 1 4 N H influxes were related to [NH + high 4 ] and major amino acids (Figs. 23B, 24, and 25A-D). I interpreted this result to indicate that the decline of ÷ 4 N 3 ‘ H influx normally observed when G2 plants are loaded in G1000 medium, depends upon products of NH 4 assimilation. This conclusion was supported by the results of glutamine pretreatment (Fig. 30), which reduced 13 4 influx at NH all concentrations tested. Further proof to this effect is provided by our amino acid analyses. Figure 25A and 25C show that transfer from G2 to G1000 medium caused [Gln] 1 and [Asn] 1 to increase several times while in the presence of MSX this increase was prevented (Figs. 29A and 29C). In addition the 1 4 N H influx was strongly correlated (negatively) with increased Gln, Glu, Asn, and Asp after treatment with AOA (Figs. 37A and 39A-D).  193  Chapter 7.  INTERACTION BETWEEN K+ AND NH ÷ 4  7.1. INTRODUCTION Potassium uptake has been well studied in higher plants (Glass, 1975; 1976, 1978; Glass et al., 1981; Kochian and Lucas, 1982, 1988; Glass and Fernando, 1992). Likewise, the kinetics of ammonium transport have also been characterized (Becking, 1956; Fried et al., 1965; Ullrich et al., 1984; Wang et al., 1993a, 1993b). Despite the similarities between K and , such as charge, hydrated ion diameter and some aspects of transport 4 NH processes (Haynes and Goh, 1978), the interaction of these two cations is poorly understood. The interaction between K and NH 4 may be examined at different levels, such as the bioavailability in soils, effects on plant growth, and effect on plant roots’ uptake/transport of these ions. Mutual beneficial effects of K and N on plant growth have often been described. An adequate K supply has been shown to enhance NH 4 uptake and assimilation (Ajayi et al., 1970; Barker and Lachman, 1986; Scherer and MacKown, 1987) and is very important for nitrogen use efficiency. On the other hand, NH 4 may promote K-I- stress in rice (Noguchi and Sugamara, 1966) or reduce the K concentration of plants (Claassen and Wilcox, 1974; Faizy, 1979; Lamond, 1979). A number of studies have been carried out to investigate the interactions of K and NH 4 at the transport level. In short-term experiments, the uptake of K was significantly reduced by the presence of 4 in the uptake solution (Deane-Drummond and Glass, 1983b; Rosen NH  194 and Carison, 1984; Morgan and Jackson, 1988). However the influence of K-’on 4 NH ’- uptake has not been consistent. In most cases, the uptake of 4 NH ’by plant roots has appeared to be independent of K-’- levels in the uptake solution and the K status of the plants (Rufty et al., 1982; Rosen and Carison, 1984; Scherer and MacKown, 1987). Nevertheless, Bange et al., (1965) reported that K is capable of inhibiting NH 4 uptake in barley plants. The objective of this study was to investigate the interactions between K-’- and NH 4 at the membrane transport step, and the influences of tissue K and N status on these ion fluxes, using 86 Rb and , 4 N 3 ‘ H respectively, as tracers.  7.2.  METHoDs AND MATERIALS  7.2.1. Plant growth and ‘ N production 3 Section 2.2. Seed germination; section 2.3. Growth conditions; section 2.4. Provision of nutrients; section 2.5. Production of 13 . 4 NH  7.2.2. Experimental design Three experimental variables were employed in this study involving N and K supply. These were (i) provision during three-week-growth periods or less as designated; (ii) pretreatment for up to three days prior to flux measurement; and (iii) presence in the uptake solutions. Test materials were 3-week-old rice seedlings. Each experiment was repeated  195 twice with three replicates. Both influxes of 13 4 and 86 NH Rb were calculated based on root fresh weight and 10 mm uptake periods, except in experiment I, where the net fluxes of NH 4 and 86 Rb were calculated from 30 mm  uptake periods. Before and after transfer into or out of the  radioactive isotopic labeled uptake solution, plant roots were prewashed and postwashed in an identical unlabeled solution for 5 and 3 mm, respectively. These time periods were based on a previous study (Wang et al., 1993a, 1993b). 7.2.1.1. Experiment I: Effects of K and NO - in pretreatment, K and NH 3 4 in uptake solutions on net K and NH 4 fluxes. Plants were grown in MJNS containing 200 iM K plus 1.5 mM N0 3 for 18 days, and were transferred to pretreatment solutions for three days. The pretreatments were MJNS with or without K and N (+K+N, -K+N, +K-N, -K-N) in which +K  200 1 iM , 4 P 2 KH O -K  mM Ca(N0 ; and -N 2 ) 3  =  100 iiM 4 P0 +N Ca(H ; 2 )  =  0.75  0.75 mM CaC1 . The 86 2 Rb influxes were measured  from radioisotope-labeled MJNS (+K*+N, +K*N) containing 200 jiM K-’- with or without 200 jiM NH . Net NH 4 4 fluxes were measured from MJNS containing 200 j.tM NH 4 with or without 200 jiM K (+K+N, -K+N). 7.2.1.2. Experiment H: Effects of NH 4 provision during growth and of K and NH 4 in pretreatment and uptake solutions on 86 Rb÷ (K+) influxes. Plants were grown in MJNS containing 200 jiM K plus 10, 50 or 100 j.tM NH , hereafter referred as Gb, G50, or G100 plants, respectively. The 4 plants were transplanted for three days to MJNS with or without additions of K and N, in which +K  =  200 jiM ; 4 P 2 KH O -K  10, 50 or 100 jiM NH C1; -N 4  =  =  100 jiM ) 4 P Ca(H ; 2 0 and +N  =  5, 25 or 50 jiM CaC1 2 for G2, Gb, or G100  plants, respectively. The 86 Rb-’- influxes were measured from radioactive  196 isotopic labeled uptake solutions (MJNS containing 200 iiM K with or +). 4 without 100 iM NH 7.2.1.3. Experiment III: Effects of NH 4 provision during growth and presence in uptake solution upon influx isotherms for 86 Rb÷ (K÷).  Plants were grown in four different growth media containing 2 or 100 pM NH 4 plus either 2 or 200 .tM K, hereafter referred as G2/2, G2/200, G100/2, G100/200 plants, respectively. The 86 Rb influxes were measured in MJNS containing 2, 10, 50, 75, 100, 250 or 500 iM K, respectively, plus 2 .tM NH 4 for G2/2 and G2/200 plants, or 1.00 jiM 4 NH ’for G100/2 and G100/200 plants. 7.2.1.4. Experiment 1½ Effects of NH 4 provision during growth and shortterm pretreatment upon 86 Rb÷ (K÷) influx.  Plants were pretreated for 0, 2, 4, 8, 24 h in 1 mM NH 4 plus 2 jiM K for G2/2 and G100/2 plants, or in 1 mM NH 4 plus 200 jiM K-’- for G2/200 8 Rb+ influxes were measured during 10 mm and G100/200 plants. 6  in  uptake solution containing 100 jiM NH 4 and 200 jiM K. 7.2.1.5. Experiment V: Effect of NH 4 concentrations present in uptake solution upon influx isotherms for 86Rb+ (K).  The 86 Rb influxes of G2/2, G2/200, G100/2, G100/200 plants were measured in MJNS containing 2, 25, 50, 100, or 200 jiM K, plus 2, 25, 50, or 100 jiM NH . The translocations of 86 4 Rb into plant shoots were also estimated based on the radioactivity recorded from plant shoots.  197 7.2.1.6. Experiment VI: Effects of K provision during growth and presence in uptake solutions upon influx isotherms for . 4 N 1 H The 13 4 influxes of G2/2, G2/200, G100/2, G100/200 plants were NH measured in uptake solutions (a) containing 2, 10, 50, 100, or 200 jiM NH 4 plus either 0, or 200 jiM K; (b) containing 100 jiM or 10 mM NH 4 plus 2, 20, 200, or 2000 jiM K.  7.3. RESuLTS  7.3.1.  Experiment I: Effects of K-’- and NO - in pretreatment, K-- and NH 3 -’4  in uptake solutions on net K and NH 4 fluxes. Pretreatment with NH 4 during three days prior to the uptake measurement generally increased 86 Rb (K+) uptake. Only in -K+N, -K-N treatments was there no increase of 86 Rb uptake; all other treatments increased influx by 1.35 times (-K+N, -K-N) and 3.4 times (+K+N, +K-N) when 4 NH ’- was absent from the uptake solution and by 1.85 times (+K+N, +K-N) when 4 NH ’- was present (Table 13). Yet, the means for +K+N and -K+N were not significantly different at the 5% level of probability when NH 4 was present in the uptake solution. When NH 4 was absent, the means (3.29/0.96 for +K+N/+K-N and 8.72/6.44 for -K+N/-K-N) were statistically different at the 1% level. The removal of K during pretreatment caused a much greater effect on 86 Rb accumulation, increasing 86 Rb÷ (K+) uptake by 2.65 and 6.7 times when it was absent from uptake solution, and by 5.25 and 10.2 times  198 Table 13. Net 86Rb+ flux measured with or without ammonium. Rice plants were grown in MJNS containing 1.5 mM NO - and pretreated 3 days 3 in 4 different solutions with or without either 200 ji,M K (+K or -K) or 1.5 mM NO - (+N or -N). The net flux of 86 3 Rb was measured in the following uptake solutions: +K+N or +K-N (N  =  200 jiM NH 4 and K  200 p.M K÷)  labeled with S6RbC1. Fluxes were calculated based on 30 mm  uptake  periods. Uptake solution Pretreatment  +K+N  +K-N (jimol g’FW h-i)  *  +K+N  0.63 ±0.07  d  3.29 ±0.14  c  -K+N  3.31 ±0.22  c  8.72 ±0.53  a  +K-N  0.34 ± 0.02  d  0.96 ± 0.02  d  -K-N  3.47 ± 0.28  c  6.44 ± 0.28  b  For comparing all possible pairs of treatment means (±se), Duncan’s Multiple Range  s R b+ flux. Means having a Test were performed, separately, on the data of net 6 common letter are not significantly different at the 5% significance level for small letter.  199  Table 14.  4 flux measured with or without potassium. Rice plants Net NH  were grown in MJNS containing 1.5 mM 3 NO and pretreated 3 days in 4 different solutions with or without either 200 jiM K (+K or -K) or 1.5 mM - (+N or -N). The net NH 3 NO 4 flux was measured in the uptake solutions (+K+N, N  =  4 and K 200 jiM NH  =  200 jiM K; or -K+N) for 30 mm uptake. Uptake solution  Pretreatment  +K+N  -K+N (jimol g FW h-i) 4  *  +K+N  4.97 ± 0.45  d*  5.92 ± 0.63  cd  -K+N  6.36 ± 0.18  cd  8.19 ± 0.64  abc  +K-N  8.06 ±0.34  abc  9.76 ± 1.40  a  -K-N  8.65 ±0.91  ab  7.00 ±0.85  bcd  For comparing all possible pairs of treatment means (±se), Duncan’s Multiple Range  Test were performed, separately, on the data of net NH4 flux. Means having a common letter are not significantly different at the 5% significance level for small letters.  200 when NH 4 was present (Table 13). In these -K plants the presence or 4 during pretreatment caused only a much smaller effect absence of NH (compare fluxes for -K+N and -K-N pretreatments). Clearly, the presence of 4 in the uptake solution caused a large reduction of 86Rb+ (K-’-) uptake, NH regardless of the pretreatments. 4 fluxes were reduced by 3 days of pretreatment with The net NH 4 in all treatments (Table 14). Removing K from pretreatment NH 4 uptake as they had for 86Rb+ (K+) solutions caused small increases in NH uptake, but these differences were not significant at the 5% level of probability. The presence of K in the uptake solutions caused statistically 4 uptake in all pretreatments except -K-N non-significant reductions in NH when NH 4 uptake actually decreased. Here again, however, the difference was not statistically significant.  4 provision during growth, and of K 7.3.2. Experiment II. Effects of NH Rb (K--) influxes. and NH 4 in pretreatment and uptake solutions on 86 The effects of three factors (N provision during the growth period, -’- in 4 4 pretreatment, and the presence or absence of NH 3 d of K and NH Rb Rb (K) uptake were examined in Exp II. 86 the uptake solution) on 86 4 influx was increased in virtually all treatments by increased levels of NH provision during the growth period (see Figs. 40A and 40B). In those experiments where NH 4 was present during influx measurement the 4 pretreatment was reduced or absent at the noted positive effect of NH highest level of NH -’- (100 riM) but was still pronounced between 10 and 4 . As in Experiment I, provision of NH 4 4 during the 3 d 50 iM NH pretreatment caused the greatest increase of 86 Rb (K) influx in low K  201 10  5.  A.  —A--— —h-—  0  without  uptake -K+N -K-N  N]  B. uptake with N1 A  ::  50100  Ammonium  levels  0  for  50100  plant  growth  (jiM)  Figure 40. Effects of NH 4 in the growth media, pretreatment and uptake solutions on 86 Rb influx. Gb, G50, and G100 plants were pretreated for 3 days in four solutions including +K+N (closed squares), +K-N (open squares), Rb+ influxes were 6 -K+N (closed triangles), -K-N (open triangles). The 8 measured in MJNS containing 200 j.tM K without NH 4 (Fig. 40A) or with 100 iM NH 4 (Fig. 40B). Data points are the average of three replicates with ±se as vertical bars.  202 12  -  G10012  10-  — —  8-  I  E  :i ‘V—’  6-  G212 4  2’  G100/200 A  0-  G21200  0  100  200  [K Jo  300  400  500  (riM)  Figure 41. Effects of NH 4 and K in growth media and uptake solutions on Rb÷ influx. The 86 86 Rb influxes of G2/2, G2/200, G100/2, G100/200 plants were measured for 10 minutes in 86 Rb labeled MJNS, containing 2, 10, 50, 75, 100, 250 or 500 iiM K--, respectively, plus 2 j.tM NH 4 for G2/2 (open circle) and G2/200 (open triangle), or plus 100 iM N}l 4 for G100/2 (closed circle) and G100/200 (closed triangle). Data points are the average of three replicates with ±se as vertical bars.  203 plants when NH4 was absent from the uptake solutions (Fig. 40A) and the least effect in high K plants in the presence of 4 NH ’- during uptake (Fig. 40B). However, as in Experiment I, the presence of NH 4 during flux measurements, reduced 86 Rb (K+) influx in all treatments. Again, removing K from the pretreatment solution caused increased 86 Rb (K) influx, and this effect was more pronounced when adequate N was provided (compare squares and triangles to note the K’- effect, and closed and open triangles to note the N effect).  7.3.3.  Experiment III: Effects of NH 4 provision during growth, and  Rb+ (K-’-). 6 presence in uptake solution upon influx isotherms for 8 Figure 41 presents the 86 Rb influx isotherms for plants grown under G2/2, G2/200, G100/2 and G100/200 conditions. The data were fitted to Michaelis-Menten equations. The kinetics of 86 Rb uptake were influenced by the provision of both NH4 and K during three weeks growth. The 86 Rb influx curves for G2/200 and G100/200 plants (grown in higher external K+) revealed a low Vmax, 0.34 and 0.59 j.imol g’FW h-’, respectively. By contrast, plants grown in low K (2 riM), exhibited much higher Vmax value (3.74 and 9.58 jimol g FW h-i for G2/2 and G100/2 plants, respectively). 4 As in the previous experiments the provision of NH 4 during the growth prior to influx measurements caused a significant positive effect; Vmax for 6Rb (K-’-) influx was increased 3 fold. The estimated values of Km were 8 also higher for plants grown in higher K conditions (15.02 jiM for G2/200 and 38.59 jiM for G100/200 plants) than for those grown in low K supply (18.00 jiM for G100/2 and 3.47 jiM for G2/2). The relationship between estimated kinetic parameters and measured tissue K concentrations clearly indicated the operation of negative feedback inhibition of 86 Rb influx.  204 12  10  8  E  6  4.  E 2  0•  I  0  I  •  20  •  40 Internal  [K  +  1  I  60  (mM)  Figure 42. Relationship between estimated kinetic parameter of 86 Rb influx (Vmax) and the assayed roots internal [K+J. The vertical bars are standard errors for Vmax and the horizontal bars are standard errors for [K].  205  8’  G10012  —  6’  —A--—  G100/200  -0-  G212  —  zr  E  G21200  4, —  2’ “C  0’  I  0  •  I  5  •  I  10  I  •  I  •  15  Growth condition (NH  •  20  /K  I  25  30  )  Figure 43. Effect of short-term NH 4 pretreatment on 86 Rb influx. Plants were pretreated in 1 mM NH 4 plus either 2 jiM K for (G2/2 and G100/2) or 200 j.iM K for (G2/200 and G100/200 plants), respectively, for 0, 2, 4, 8, 24 h. 86 Rb influxes were measured for 10 minutes in 86 Rb labeled MJNS containing 200 jiM K and 100 jiM NH . Data points are the average 4 of three replicates with ± se as vertical bars.  206 Figure 42 showed a strong negative correlation between Vmax values and internal [K] values.  7.3.4. Experiment IV: Effects of NH 4 provision during growth, and shortterm pretreatment upon 86 Rb (K÷) influx. When the N status of plants was changed by short-term exposures to NH 4 ’-, 86Rb+ influxes were also altered, as shown previously for 3 days exposures to NH 4 (Table 13, Figs. 40 and 41). For plants grown in higher N (G100/2 or G100/200) the 86 Rb influxes were affected little by loading in 1 mM NH 4 for various periods (Fig. 43). For plants grown in low N, the results of pretreatment in 1 mM NH 4 varied according to the differences in the K status. The 86 Rb influxes of G2/2 plants were greatly increased during the first 4 h pretreatment in 1 mM NH . In contrast, 86 4 Rb influxes of G2/200 declined slightly after the first 4 h.  7.3.5. Experiment V: Effect of NH4 concentrations present in uptake solution upon influx isotherms for 86 Rb (Kj. To further understand the inhibitory effect of NH 4 in uptake solutions, 86 Rb influxes were measured at five [K+] 0 levels in the presence of four levels of 0 -’-] Generally, 86 4 [NH . Rb influxes for G100/2 were higher than for G2/2 and G100/200. G2/200 plants had the lowest rates of potassium uptake. Generally, S6Rb+ influx decreased with increasing 0 4 [NH ’-] in the uptake solutions, but the effect on G100/2 is not so evident (Fig. 44). Even at 2 p.M [K-’-] , the inhibitory effect of NH 0 4 was evident. Table 15 presents estimated Michaelis-Menten parameters for all 86 Rb  207 G21200  G100/200  —------  16.00  16.00  12.00  12.00  Eoo  Y  8.00 4.00 0.00 5  2 x  oioo 5  100  X  G100I2  G2/2 z  z  16.00  16.00  12.00  12.00 Y  8.00  00 1 00 50 25  4.00 0.00 2  /  25 50100  ;‘  2 X  8.00  y  4.00 100 J./50  0.00 2  25  50  ‘25 2 X  Figure 44. Effects of NH 4 and K in growth media and uptake solutions on Rb influx. The 86 86 Rb influx of G2/2, G2/200, G100/2, or G100/200 plants, respectively, were measured in MJNS containing 2, 10, 50, 100, or 200 jiM K-’- plus 2, 25, 50, or 100 jiM NH , respectively. In plots: X 4 (jiM), Y= [K] (jiM), Z  =  =  ] 4 [NH  Rb influx (j.tmol K-’- g’FW root h-i), respectively. 86  208 Table 15.  Rb+ influx for four 6 Michaelis-Menten kinetic parameters for 8  groups of plants (G2/2, G2/200, G100/2 or G100/200). Based on the data of Fig. 44, the parameters were estimated by nonlinear procedure on replicated influx data (n=2). [NH 0 i 4 (riM)  Vmax ± se (pmol g’FW h-i)  Km ± se (iiM)  2 r  G2/2 plants 2  11.07± 1.04  11.82±  5.90  0.82  25  7.83 ± 0.78  13.23 ±  7.59  0.79  50  8.27 ± 0.80  23.36 ±  8.73  0.86  100  5.83 ± 0.81  18.39 ± 11.34  0.86  G100/2 plants 2  14.97 ± 1.81  25  8.63 ±  9.05  0.89  14.02 ± 1.77  10.99 ± 7..47  0.78  50  15.79 ± 1.80  49.53 ± 16.08  0.93  100  12.34± 0.89  11.58±  4.66  0.82  1.57  0.17  G2/200 plants 2  1.02 ± 0.13  25  0.41 ± 0.06  105.93 ± 31.03  0.96  50  0.41 ± 0.04  136.18 ± 21.07  0.90  100  0.55 ± 0.12  186.75 ± 70.79  0.98  3.09 ±  G100/200 plants 2  7.36 ± 0.55  17.60 ±  5.61  0.80  25  4.06 ± 0.54  36.25 ± 15.26  0.81  50  2.18 ± 0.32  17.68 ± 10.73  0.81  100  2.31± 0.58  63.88± 41.43  0.95  209 G1 00/20Q]  G2/200  z 16.00\  16.00 12.00 4  8.00 4.00  “  0.00 2  Y  Y  4  00 00  50 25 25  0.00  X  50100  00  -r5 0 . 4 0 50  x  100  Gi00/2  G212J  z 16.00 12.00 8.00 4.00i/ 50 25  0.00 2  2  25 100  100  Figure 45. Effects of NH 4 and K in growth media and uptake solutions on Rb÷ translocated to shoots. (Details as in Fig. 44) In plots: X 86 Y= [K] (jiM), Z  =  =  [NH + 4 ] (.tM),  S6Rb+ translocated (nmol K g’FW shoot h’), respectively.  210 influxes isotherms. Generally, Vmax values decreased with increasing 0 + 4 [NH ] in the uptake solutions. In contrast, Km values tended to increase +] in the uptake solutions for G2/200 and G100/200 4 {NH with increasing 0 plants (Table 15). However, Km values remained relatively constant for G2/2 and G100/2 plants. Similar inhibitory effects were true for the 6Rb) to shoots (Fig. 45). It was evident that higher 8 translocation of K ( rates of 86Rb translocation were associated with growth on sufficient N (G100/200) or insufficient K (G2/2 and Gt00/2). In this experiment, plant biomass was recorded in order to make comparisons of the effects of growth conditions. There were statistically significant differences among total fresh and dry weights of plants (G100/200  >  G2/200  >  G100/2  >  G2/2) although the ratios of dry:fresh  weight were relatively constant (Table 16). Both fresh or dry shoot weights of plants grown in well-supplied media (G100/200) were significantly higher than for other types of plant. With inadequate supply of either K or 4 NH ’-, plants (G2/200 or G100/2) plants had smaller biomass but these were still significantly higher than that of G2/2 plants. However, the differences of root weight indicated that K played a more important role in root growth than did NH 4 (compare G100/200 to G2/200). When K was adequately supplied, plant roots grew better. Under K-’- stress, 4 NH ’- seemed to have little effect on root biomass.  7.3.6.  Experiment IV: Effects of K-’- provision during growth and presence  in uptake solutions upon influx isotherms for 13 . 4 NH The effects of K in the uptake solutions on the ‘ 4 N 3 H influx were examined using G2/2, G2/200, G100/2, and G100/200 plants (Fig. 46). The  211  4 and K on plant growth. Rice plants were grown in Table 16. Effects of NH 4 plus either 2 or 200 jiM K (G2/2, G2/200, either 2 1 iM or 100 jiM NH G100/2 or G100/200, respectively). Each value is the average of 40 sample means (mg per plant) with ± Se. Plants  G2/2  G2/200  G100/2  Total FW (mg)  179 ±  5 d  225 ±  6 c  TotalDW(mg)  21±  1 d  30±  1 c  Total D/F  0.13 ± 0.00 b  368 ±  3 a  2 b  49±  2 a  38±  0.14 ± 0.01 a 258± 10 a  114±  4c  163±  StDW(mg)  18±  ic  26±  0.16 ± 0.00 a  293 ± 14 b  0.13 ± 0.OOAb 0.14 ± 0.01 b  StFW(mg)  St D/F  G100/200  5 b  168±  8 b  b  27±  1 b  7  0.16 ± 0.00 a  0.17 ± 0.01 a  40±  2 a  0.14 ± 0.01 a  RtFW(mg)  56±  2c  62±  1 c  125±  6 a  110±  4 b  RtDW(mg)  4±  Oc  4±  0 c  11±  1 a  9±  1 b  Rt D/F  0.06 ± 0.00 b 0.06 ± 0.00 b 0.09 ± 0.01 a  FW St/Rt  2.03 ± 0.00 c  2.61 ± 0.00 a  1.36 ± 0.00 D 2.38 ± 0.02 b  DW St/Rt  5.43 ± 0.27 b  7.20 ± 0.23 a  2.88 ± 0.24 C 4.92 ± 0.38 b  *  0.09 ± 0.01 a  For comparing all possible pairs of treatment means, Duncan’s Multiple Range Test  were performed, separately, on the data of net 86 Rb flux. Means having a common letter are not significantly different at the 5% significance level for small letter.  212 16  8  G21200  G1001200  T  12  6  E  •  o  50  •  100  •  150  200  50  0  100  150  200  8  16  G212  I  G10012  -  12  6  Q  EEE “iZ 1 ‘  0  50  100  150  200  [NH  0  50  100  150  200  Jo (riM)  Figure 46. Effects of K in uptake solution on 4 NH influx isotherm. Root 3 ‘ 4 influx of G2/2, G2/200, G100/2, or G100/200 plants, respectively, NH 13 were measured in MJNS containing 100 1 iM NH 4 in the presence of either 2 (open circle) or 200 iM K-’- (closed circle). Predicted isotherms (dashed lines for 0 jiM K-’- and solid lines for 200 jiM K) were calculated from the computed Vm and Km for different plants (Table 17).  213  Table 17. Michaelis-Menten kinetic parameters for 1 4 N H influx for four plants (G2/2, G2/200, G100/2 or G100/200) derived from influx isotherms 4 with or without 200 jiM K-i- (+K or based on 2, 25, 50, 100, or 200 jiM NH -K). The parameters were estimated by nonlinear procedures on replicated influx data. 4 solution NH 13  Vm ± se  G2/2  +K  10.07 ± 0.94  17.40 ±  3.13  G2/2  -K  10.69 ± 0.91  13.62 ±  2.80  G2/200  +K  13.99± 2.77  16.36±  1.09  G2/200  -K  13.95 ± 2.93  16.22 ±  0.31  G100/2  +K  4.60± 1.09  359.36±121.31  G100/2  -K  13.07± 2.29  209.11± 57.13  G100/200  +K  3.94± 0.61  37.89± 20.17  G100/200  -K  4.62 ± 0.80  30.56 ± 11.93  Plants  Km ± se  214 12 • G2/200  G2/2  G1001200  1  9. —  E  zL  6  z  G10012  3.  0  —I—  o  —  20  [K  200 Jo  2000  (jiM)  Figure 47. Effects of K in uptake solution on 13 4 influx by HATS. Root NH 4 N 3 ‘ H influxes of G2/2, G2/200, G100/2, or G100/200 plants, respectively, were measured in MJNS containing 100 iM NH 4 in the presence of 0, 20, 200, 2000 tM K. Data points are the average of three replicates with ±se as vertical bars.  215 40 • G2/200  G2/2  • G1001200  G10012  —  zL 20  + •  10  z 00  200  2000  [K Jo (pM) 4 influx by NH Figure 48. Effects of K in the uptake solution on 13 H influxes of G2/2, G2/200, G100/2, or G100/200 4 N 3 HATS+LATS. Root ‘ 4 in plants, respectively, were measured in MJNS containing 1000 jiM NH the presence of 0, 200, 2000 jiM K. Data points are the average of three replicates with ±se as vertical bars.  216 presence of K in the ‘ 4 N 3 H uptake solutions failed to significantly reduce 4 influx except in the case of the G100/2 plants where significant NH 13 differences were apparent. The estimated influx kinetics also showed the same trends (Table 17). Nevertheless, there were slight reductions of 4 N 3 ‘ H influx which failed to satisfy statistical evaluation in G2/2 and G100/200 plants. It was noted that plants grown at low N levels had NH influxes when the K nutrition was adequate during growth 1 higher 4 (compare G2/200 and G2/2 plants). When K in the uptake solution was increased from 0, 2, 20, 200, and 2000 jiM (Fig. 47), a strong inhibitory effect of K (in the uptake solutions) on 1 4 N H influxes of G100/2 and G100/200 plants was evident. By contrast, ‘ 4 N 3 H influxes were significantly increased by growth in low N (G2/2 and G2/200) with no effects of K÷ when present in the uptake solutions. The ‘ 4 N 3 H influxes measured in 10 mM NH 4 were not changed significantly although the influxes were lower in the presence of 2000 jiM K (Fig. 48).  7.4. DISCuSSION  7.4.1. Plant growth in response to provisions of NH 4 and KBoth N and K are very important to crop growth and yield. Uptake of K and N, plant dry weight, and paddy yields of rice increased with increasing K and N application rate (Biswas et al., 1987; Ichii and Tsumura H,1989; Fageria et al., 1990). Deficiency of either N or K in the nutrient solution decreased the tissue content of either N or K, influenced  217 photosynthetic rate and translocation of carbohydrates, caused lower grain weight and therefore reduced rice yield (Grist, 1986; Dey and Rao, 1989). Reduction in photosynthetic rate may be due to impairment of stomatal diffusive conductance and decreased N content/unit leaf area (Dey and Rao, 1989). High tissue K not only promoted CO 2 assimilation, starch formation and the transport of the assimilates but also improved the nitrogen metabolism of the plant and nitrogen use efficiency (Kemmler, 1983; Dibb and Thompson, 1985). K enhanced NH 4 assimilation and reduced the toxic effects of NH 4 such as stem lesions in tomato or leaf lesion in corn (Ajay et al., 1970; Dibb and Welch, 1976). In a recent paper by Yong et al., (1993) the presence of K in Arabidopsis’ growth media was responsible for preventing toxic effects of NH 4 on root growth. Supplying high levels of K÷ to 4 NH N grown plants stimulated shoot growth and more vigorous root growth (Xu et al., 1992). In the present study the total fresh and dry weights were significantly higher in the sequence of G100/200, G2/200, G100/2 and G2/2 (Table 16). The significant difference between G2/200 and G100/200 indicates the importance of K for plant growth when the N nutrition is adequate. Comparing both fresh and dry weights of roots among four treatments in Table 16, higher K in the growth media produced significantly higher root mass (G100/200 and G2/100) than growth in low K (G2/2 and G100/2), whereas the shoot fresh and dry weights, were not significantly different between G2/200 and G100/2. A greater root mass of seedlings grown in higher K indicated that K may play an important role in facilitating root development (Beaton and Sekhon, 1985; Xu et al., 1992). There was a significant positive correlation between total root weight and K uptake (Table 16). Total root length and dry weight increased as crop growth advanced and N supply increased (Chamuah and Dey, 1988).  218 However, the root number was negatively correlated with 4 NH N uptake in lowland rice (Ichii and Tsumura,1989). As shown in Table 16, the Shoot:Root ratios for both fresh and dry weights were higher for G100/2 than G200/200 or G2/2. G2/200 plants had the lowest Shoot:Root ratio. It has been reported that N deficiency decreased S/R ratios of seedling plants (Zsoldos et al., 1990). N stress reduces plant growth, particularly shoot growth, through several mechanisms operating on different time scales. The possible signals may be related to N stress-induced changes of abscisic acid and cytokinins (Goring and Mardanov, 1976; Sattelmacher and Marschner, 1978; Chapin et al., 1988a, 1988b; Kuiper et al., 1989). This lower ratio of shoot:root may also due to higher root mass in higher K condition as discussed above.  7.4.2. Effect of plant N status on K (86Rb+) uptake The nitrogen status of plants had a significant influence on K Rb) 86 ( uptake. Typically, S6Rb+ influxes of Gb, G50 and G100 plants were increased with increasing 4 [NH j e levels in growth media (Figs. 40A and 40B). The presence of NH 4 during the pretreatment period also caused increased 86 Rb influx (Figs. 40A and 40B). 86 Rb uptake by roots exposed to +K+N and -K+N pretreatments were significantly higher than that for +K N or -K-N pretreatments (Table 13). This positive effect of N status on K uptake may be related to protein synthesis for K transport. The long term regulation of ion uptake probably involves induction or derepression of carrier synthesis. It is known that plants respond to K deprivation rapidly by synthesizing novel polypeptides in the plasma membrane (Fernando et al., 1992) which are believed to form part of the high affinity K-’- transport  219  system (Glass and Fernando, 1992). When plants were grown in low K (2 iM), with sufficient N supply (100 1 1 6Rb+) influx was 8 +), K ( 4 iM NH promoted (Figs. 41, 44). However, when the supply of nitrogen was limited during plant growth, the synthesis of K transporters in the cell membrane may be limited. In the present study, when G2/2 plants were pretreated with 1 mM NH 4 for 4 hours, more transporters could be synthesized and Rb+ influx was significantly increased and remained relative high 6 the 8 during the 24 h pretreatment (Fig. 43). This raises an important question concerning the ‘induction’ of increased NH 4 uptake observed when low N plants are first exposure to 4 NH (Goyal and Huffaker, 1986; Morgan and Jackson, 1989; Wang et al., 1993b; in Chapter 6). The observation that exposure to NH 4 also increased K uptake on a similar time scale indicates that this 4 NH ’- effect is not specific as for example the induction of N0 3 uptake by exposure. Rather, it appears that the so-called ‘induction’ may be general positive N effect associated with N-depleted plants. Another possible explanation for the positive effect of NH 4 may be due to the effect of N supply on growth rate. The influx of ions into roots may be negatively correlated with the internal concentration of a particular ion, such as Cl- (Cram, 1973); K (Young et al., 1970; Pitman and Cram, 1973; Glass, 1975; Glass and Dunlop, 1978); NO - (Siddiqi et al., 3 S0 2 - (Smith, 1975). Figure 3 showed that the Vm for 86Rb+ 1992), and 4 influx was negatively correlated with internal K levels in agreement with previous reports (Glass, 1975; Clarkson, 1983; Pettersson, 1986; Zsoldos et al., 1990). Vmax decreased and Km increased exponentially with increased tissue K concentration (Dunlop et al., 1979; Glass, 1976, 1977, 1978). In the present study, the 86 Rb influx was increased in the sequence of G100/2, G2/2, G100/200, and G2/200 (Figs. 41 and 44) and coincides with the sequence of 1 [K] of these roots. Higher 86 Rb influxes also resulted  220 from three days pretreatment in minus K solution (Figs. 40A and 40B). Therefore high N supply, resulting in increased plant growth, would cause the opposite effect on tissue [K] and K ( Rb) 8 6 influx, i.e. a biological dilution effect. This may explain why NH 4 supplement to rice plants promotes K stress (Noguchi and Sugawara, 1966), or reduced K concentration of plants (Claassen and Wilcox, 1974; Faizy, 1979; Lamond, 1979).  7.4.3. Effect of NH 4 in the uptake solution on K-’- Rb) 86 uptake ( Despite the positive effect of NH 4 provided during the growth period and the pretreatment period, NH 4 has been shown to strongly inhibit the absorption of K in short-term experiments (Bange et al., 1965; Moraghan and Porter, 1975; Breteler, 1977; Munn and Jackson, 1978; Rosen and Carison, 1984; Scherer et al., 1984). In the present study, 86 Rb influxes were inhibited by the presence of 4 NH ’- in the uptake solution (Figs. 40A, 40B and 44, Table 13 and 15). The inhibition of 86 Rb influx increased with increasing 4 [NH ’-] in the uptake solutions (Table 15). The uptake of K by excised rice roots decreased markedly with increasing concentrations of 4 in the uptake solution (Scherer et al., 1987). Greater inhibition of K NH uptake was exerted by 1000 jiM NH 4 than 100 jiM NH 4 (Rosen and Carison, 1984), and the inhibition by 1000 jiM 4 NH occurred after 90 mm treatment and the inhibition by 100 jiM NH 4 took about 240 mm (Jongbloed et al., 1991). Since this inhibitory effect of NH 4 on K ( Rb+) 8 6 influx is independent of K provision or pretreatments, it is probably exerted on the transport processes at the plasma membrane. It is suggested that certain  221 solutes are bound to, or associated with, a particular transporter. When an ion of a particular species is attached to this transporter, another similar ion (of the same or a different species) may compete for the same binding site and reducing its uptake. Mixed competitive and non-competitive inhibition between K and 4 NH ’- has been reported for tobacco (Scherer et al., 1984) and barley (Dean-Drummond and Glass, 1983). Although 4 NH ’may not always inhibit K uptake competitively, NH ÷ often has a lower 4 affinity for the carrier than K (Conway and Duggan, 1958; Jongbloed et aL, 1991). Likewise, it was found that the Km for K was increased by NH 4 supplementation in ectomycorrhizal fungi (Boxman et aL, 1986; Jongbloed et al., 1991). There was a considerable K efflux induced by NH 4 influx during 4 uptake by roots of corn, wheat or oat (Becking, 1956; Morgan and NH Jackson, 1989). NH 4 markedly inhibits K uptake in many species including wheat (Tromp, 1962), barley (Bange et al., 1965; Meijer, 1970). maize (Rufty et al., 1982) and tobacco (Scherer et al., 1984). It was also reported that exposure of seedlings of Scots pine and Douglas fir to NH 4 induced a loss of K (Boxman and Roelofs, 1986; Bledsoe and Rygiewicz, 1986). The need to maintain cation-anion balance may explain some aspects of this inhibitory effect. For example, the presence of monovalent cations 4 (NH ’-, K, Na) in the uptake solution depressed 45 2 influx due Ca to stimulated Ca 2 extrusion (Siddiqi and Glass, 1984). Generally plants supplied with NH -’--N contain lower concentrations of inorganic cations 4 such as Ca , Mg 2 , K-’- (Kirkby and Mengel, 1967; Barker and Maynard, 2 1972; Harada et al., 1968; Moraghan and Porter, 1975; Magalhaes and Wilcox,, 1983; Scherer et al., 1984; Siddiqi and Glass, 1984). It was found that K content of white mustard leaves was reduced to near half that of N0 3 -N grown by growth on NH -’--N (Kirkby, 1968). Similar competitive 4  222 effects were also found in maize and sugar beet when grown on either urea or 4 NH N (Beusichem and Neeteson, 1982). Although NH 4 may stimulate the leakage of K-’-, it may not be the main mechanism responsible for the inhibition of K-’- influx. It is well known that NH 4 uptake is associated with H-’- efflux and acidification of growth media (Pitman, 1970; Riley and Barber, 1971; Pitman et al., 1975; Revan and Smith, 1976; Haynes and Goh, 1978; Bagshaw et al., 1982; Marschner and ROmheld, 1983; Nye, 1986; Youssef and Chino,1989; Jongbloed and Borst-Pauwels, 1990; Chaillou et al., 1991). It is a common practise to add base to neutralize the H generated in growth media (Barker et al., 1966; Rufty et al., 1983; Thoresen et al., 1984; Vessey et al., 1990; Wang et al., 1993b). It was estimated that the uptake of 1 mol 4 NH ’required the excretion of 1.33 mol H-’- and 0.33 mol K entered root cells (Raven, 1985). Further, K uptake is intimately associated with active H efflux (Mitchell, 1970; Glass et al., 1981). The K:H  exchange  stoichiometries were almost consistently greater than 2:1 (Glass and Siddiqi, 1982). Last, but not least, the efflux of K was not significant in uptake regulation (Glass, 1983) compared to the importance of K influx (Johansen et al., 1970; Yong and Sims, 1972). Since the presence of NH 4 in solution inhibited K-’- uptake to a greater extent in K-loaded plants than in K-starved plants (Rosen and Carlson, 1984), the efflux of K may not affected by the addition of NH 4 (Jongbloed et al., 1991).  7.4.4. Effect of K-’- on NH 4 uptake The uptake of NH4 by young rice plants, as well as tomato and plum was not competitively affected by the K concentration of the nutrient medium (Mengel et aL, 1976, 1978; Rosen and Carlson, 1984) or by plant K  223 status (Rosen and Carison, 1984; Scherer and Mackown, 1987). However it was found that the addition of high concentrations of K caused a reduction in methylamine transport rate in Anacystis nidulans (Boussiba et al., 1984). There is a synergistic behavior between N and K in the scope of crop growth and production (Mengel, 1989). Plant 4 NH N nutrition was improved by supplying K-’- (Mengel et aL, 1976; Dibb and Thompson, 1985). For example, barley response to increasing N concentrations was dependent on levels of K in the whole plant sample (MacLeod, 1969). The much higher N and K uptake with the higher K supply rate suggested that there might be a complementary uptake effect between NH 4 and K-’- (Dibb and Thompson, 1985). Lee and Rudge (1986) found that both K-’- and NH 4 uptake were stimulated to the same extent in N-starved roots. In greenhouse tests, K application tended to increase grain N content and total N uptake by rice plants (Chakravorti, 1989). Tomato plants grown in sand culture with high NH 4 appeared to display symptoms of NH 4 toxicity related to increased ethylene synthesis that declined as K supply increased (Corey and Barker, 1989). In the present study, 4 NH influxes of G100/2, G100/200 and 1 G2/2plants were reduced by the presence of K in the uptake solution. Clearly K was most inhibitory to NH 4 influx when plants were N sufficient (Figs. 46 and 48) and K-deficient, especially at high [K-’-] 0 (Fig. 47). In the former condition, the NH 4 influx would be relative low and probably mediated by the high affinity transport system (Wang et al., 1993b). Studies on rice and tomato showed that K had inhibitory effects but did not compete with 4 NH ’- for selective binding sites in the absorption process (Ajay et al., 1970; Dibb and Welch, 1976; Mengel et al., 1976).  224 7.4.5. Shared transport and different feedback signal? It is known that at low external concentrations, both 4 NH ’- and K-’transport depend on a source of metabolic energy (Kochian and Lucas, 1982; Hong and Stutte, 1987) and conform to Michaelis-Menten kinetics (Epstein, 1972; Debnam and Levin, 1975; Polley and Hopkins, 1979; Fischer and Luttge, 1980; Kochian and Lucas, 1982; Luttge and Higinbotham, 1982; Wang et al., 1993b). The rapidity of the inhibitory effects of 4 NH ’- and K on each other observed in the present studies indicated that inhibition probably occurred at the level of membrane transport although this inhibition may not be a competitive one. Similar results were reported for maize roots (Shaff et al., 1993). This suggests that NH 4 and K may share a common transport pathway, such as an ion channel (Wang et al., 1992b, 1993b; Shaff et al., 1993) and this hypothesis is supported by molecular evidence. In a recently cloned K channel from Arabidopsis, the NH 4 conductance was determined to be 30% of the K conductance for the KAT1 K channel (Schachtman et al., 1992). Uptake of both NH 4 and K caused depolarization of plasma membrane electrical potentials (Kochian and Lucas, 1989; Ulirich et al., 1984; Wang et al., 1992b). Since the influx of both cations may be driven by the proton motive force (at high external concentration), diminishing membrane potential may lead to reduced ion uptake by influencing the proton motive force. It has been reported that the depolarization of the plasma membrane by NH 4 may increase the Km for K (Kleiner, 1981; Borst-Pauwels et al., 1971; Roomans and Borst-Pauwels et al., 1977; Jongbloed et a!., 1991). However the effect on membrane potential can not explain why 4 NH ’- inhibited K÷ uptake in all four nutrient treatments  225  (G2/2, G2/200, G100/2, G100/200) and K only inhibited 4 NH ’- influx at high N/low K plant status. 4 N 3 ‘ H influx and its kinetic parameters (Vm and Km) of N-deficient plants (G2/2) were not significantly affected by the presence of K in uptake solution except as noted above for the G100/2 plants. Also the (8 R b+) influx by NH inhibition of K 6 4 was lower when plants were K starved. The uptake of K by excised rice roots decreased markedly with increasing concentrations of NH 4 in the uptake solution, while the uptake of NH 4 was little affected by the concentration of K-’- in the uptake solution (Scherer et al., 1987). K uptake was suppressed during rapid 4 NH ’- uptake by N-starved plants (Tromp, 1962), but K-starvation did not produce the same effect as N-starvation on the transport of NH 4 (Tromp, 1962; Lee and Rudge, 1986). This biased inhibitory effect between NH 4 and K may suggest that NH 4 and K share a common transport pathway, but the regulation signal for these two ions may arise from separate sources. The superior competitive behavior of NH 4 over K is similar to the inhibitory effect of NH 4 on N0 3  uptake which has also been linked to the  depolarizing effects of NH 4 on 1 P (see Lee and Draw, 1989 for discussion). Yet it is clear that, although K causes a depolarization of AP similar to that caused by NH , it is not inhibitory to N0 4 3 uptake, nor is it as effective inhibiting 4 NH ’- uptake. Hence it is unlikely that the inhibitory effect of NH 4 ’- is due to membrane depolarization/dissociation of pmf. The basis of 4 inhibitory effect remains to be resolved. NH  226  Chapter 8.  GENERAL CONCLUSIONS  This study has identified and characterized the ammonium uptake system in rice roots in terms of cellular compartmentation (Chapter 3), kinetics (Chapter 4), energetics, electrophysiology (Chapter 5) and biochemistry (Chapter 6). The interaction between NH 4 and K on the plant growth and ion uptake was also examed (Chapter 7). Ammonium is absorbed by rice roots in the cation form even at elevated . 0 + 4 [NH ] Newly absorbed NH 4 is either stored in the root cell vacuoles or rapidly metabolized to amino acids in roots. Amino acids, but not NH , are consequently translocated to the shoots. Cytoplasmic 4 4 [NH ’-] may range from 3 to 38 mM according to the N provision during growth. The concentration dependence of NH 4 uptake demonstrated that, at least, two individual systems, HATS and LATS, operate at the plasma membrane to transport NH 4 into root cells. A saturable pattern of 4 N 1 ’H influxes is due to HATS and a linear relationship between 13 ÷ influx 4 NH and [NH 0 + 4 ] is mediated by LATS. HATS and LATS are not only kinetically different, but also different in energy dependence and stoichiometry of membrane potential depolarization. Significant efflux of NH 4 was observed even when plants were grown at lower level of , 0 4 [NH ’-] 2 i1 M. Efflux increased as [NH 0 + 4 ] increased from 2 to 100 and 1000 i1 M, correspoding to 10, 20 30% respectively of influx at these [NH . 0 ] 4 NH 4 ’- uptake is subjected to negative feedback regulation by both 4 and its metabolites.The effects of pretreatment with exogenous Gln, NH  227  Glu and Asn were found to reduce influx to differing extents. A cascade regulation system is proposed to explain the regulation of ammonium uptake in response to changes of internal NH 4 and its metabolites. This involves regulation at many levels, from the whole plant down to the molecular level. The results of NH 4 and K interaction studies at the level of plant growth and uptake gave quite different results. Both cations are essential for plant growth, and utilization of each nutrient is optimized when each is in adequate supply. At the uptake level, pretreatment with 4 NH ’- caused a strong stimulation of K uptake, but was inhibitory to K uptake when it was present in the uptake solution. By contrast, K was inhibitory to NH -’4 uptake only when plants were K starved and N (NH +) sufficient. The 4 inhibitory effect of these cations is probably not due to competition for p.m.f., but to direct effect of these ions on the individual transporters.  228  REFERENCES Ajayi 0, Maynard DN, Barker AV (1970) The effects of potassium on ammonium nutrition of tomato (Lycopersicon esculentum Mill.). Agron J 62:818-821 Au AA, Ikeda M, Yamada Y (1987) Effect of the supply of potassium, calcium, and magnesium on the absorption translocation and assimilation of ammonium and nitrate-nitrogen in wheat plants. Soil Sci Plant Nutr 33:585-594 Allen S, Terman GL (1978) Yield and protein content of rice as affected by rate source method and time of applied N. Agron J 70:238-242 Anderson WP, Hendrix DL, Higinbotham N (1974) Higher plants cell membrane resistance by a single intracellular electrode method. Plant Physiol 53:122-124 Arima Y (1974) Rapid incorporation of 15 N into amide nitrogen of rice seedling roots from (15NH4)2S04 I. Physiological significance of glutamine on nitrogen absorption and assimilation in plants. J Sci Soil Manure 45:509-512 Arima Y, Horinouchi T, Kumazawa K (1976) Physiological significance of glutamine on nitrogen absorption and assimilation in plants. IV. Variation and regulation of glutamine synthetase activity in rice seedlings fed with ammonium or nitrate. J Sci Soil Manure 47:198-203 Arima Y, Kumazawa K (1975a) Physiological significance of glutamine on nitrogen absorption and assimilation in plants. II. A kinetic study of amide and amino acid synthesis in rice seedling roots fed with ‘ N labelled ammonium. J Sci Soil 5 Manure 46:355-361 Arima Y, Kumazawa K (1975b) Physiological significance of glutamine on nitrogen absorption and assimilation in plants. III. Properties and intracellular localization of glutamine synthetase in rice seedling roots. J Sd Soil Manure 46:389-394 Arima Y, Kumazawa K (1977) Evidence of ammonium assimilation via the glutamine synthetase-glutamate synthase system in rice seedling roots. Plant Cell Physiol 18:1221-1229 Arisz WH (1958) Influence of inhibitors on the uptake and the transport of chloride ions in leaves of Vallisneria spiralis. Acta Bot Neerl 7:1-3 2 Arnon DI, Fratzke WE, Johnson CM (1942) Hydrogen ion concentration in relation to absorption of inorganic nutrients by higher plants. Plant Physiol 17:5 15-5 24 Arst HN, Page MM (1973) Mutants of Aspergillus nidulans altered in the transport of methylammonium and ammonium. Mol Gen Genet 121:239-245 Atkins GL (1969) Multicompartmental models for biological systems. Methuen, London Avery SV, Codd GA, Gadd GM (1992) Caesium transport in the cyanobacterium Anabaena variabilis: Kinetics and evidence for uptake via ammonium transport system(s). FEMS Micro Letters 95:253-256  229 Azam F, Ashraf M, Lodhi A, Sajjad MI (1991) Relative significance of soil and nitrogenous fertilizer in nitrogen nutrition and growth of wetland rice (Oryza sativa L.). Biol Fertil Soils 11:57-61 Azam F, Simmons FW, Mulvaney RL (1993) Immobilization of ammonium and nitrate and their interaction with native N in three Illinois Mollisols. Biol Fertil Soils 15:50-54 Bagshaw R, Vaidyanathan LV, Nye PH (1982) The supply of nutrient ions by diffusion to plant roots in soil. VI Effect of onion plant roots on pH and phosphate desorption characteristics in a sandy soil. Plant Soil 37:627-639 Bange GGJ, J Tromp, S Henkes (1965) Interactions in the absorption of potassium sodium, and ammonium ions in excised barley roots. Acta Botanica Neerlandica 14:116-130 Barker AV, Lachman WH (1986) Potassium and ammonium interactions in nutrition of tomato cultivars and mutants. J Plant Nutr 9:1-21 Barker AV, Maynard DN (1972) Cation and nitrate accumulation in pea and cucumber plants as influenced by nitrogen nutrition. J Amer Soc Hort Sci 97:27-30 Barker AV, Volk JA, Jackson WA (1966) Root environment acidity as a regulatory factor in ammonium assimilation by the bean plant. Plant Physiol 41:11931199 Barr CE, Koh MS, Ryan TE (1974) NH3 efflux as a means for measuring H extrusion in Nitella. In U Zimmermann, J Dainty, eds, Membrane transport in plants, Springer, Berlin, pp 180-118 Baruah BP, Saikia L (1989) Potassium nutrition in relation to stem rot incidence in rice. J Potassium Res 5 (3): 12 1-124 Bastida J, Liabrés JM, Viladomat F, Cusió RM, Codina C (1988) Free amino acids and alkaloid content in snapdragon plants grown with nutrition. J Plant Nutr 11:115 Beaton JD, Sekhon GS (1985) Potassium nutrition of wheat and other small grains. In RD Munson, ed, Potassium in agriculture, ASA-CSSA-SSSA, Madison, pp 701-752 Becking JH (1956) On the mechanism of ammonium uptake by maize roots. Acta Bot Neerl 5:2-79 Behl R, Jeshke WD (1982) Potassium fluxes in excised barley roots. J Exp Bot 33:584600 Below FE, Heberer JA (1990) Time of availability influences mixed-nitrogen-induced increases in grown and yield of wheat. J Plant Nutr 13:667-676 Belton PS, Lee RB, Ratcliffe RC (1985) A ‘ N Nuclear Magnetic resonance study of 4 inorganic nitrogen metabolism in barley maize and pea roots. J Exp Bot 3 6:190210 Bertani A, Brambilla I, Reggiani (1986) Effect of exogenous nitrate on anaerobic root metabolism. In RMM Crawford, ed, Plant life in aquatic and amphibious habitats. British Ecological Society Special Symposium, Blackwell, Oxford, pp 255-264  230 Bertl A, Felle H, Bentrup FW (1984) Amine transport in Riccia fluitans. Cytoplasmic and vacuolar pH recorded by a pH-sensitive microelectrode. Plant Physiol 76:75-78 Beusichem ML, Neeteson JJ (1982) Urea nutrition of young maize and sugar-beet plants with emphasis on ionic balance and vascular transport of nitrogenous compounds. Neth J Agri Sci 30:317-330 Bhat KKS, (1983) Nutrient in flows into apple roots. Plant Soil. 71:371-380 Biswas CR, Bhattacharya B, Bandyopadhyay BK, Bandyopadhyay AK (1987) N, P, and K uptake of rice on coastal saline soils. Inter Rice Res Newsletter 12 (2):42 Bledsoe CS, Rygiewicz PT (1986) Ectomycorrhizas affect ionic balance during ammonium uptake by Douglas fir roots. New Phytol 102:271-283 Blevins DG (1989) An overview of nitrogen metabolism in higher plants. In JE Poulton, JT Romeo, EE Conn, eds, Plant Nitrogen Metabolism. Recent advances in phytochemistry, Vol. 23, Plenum press, New York and London, pp 1-41 Bloom AJ (1985) Wild and cultivated barley show similar affinities for mineral nitrogen. Oeceologia 65:555-557 Bloom AJ, Chapin FS, III (1981) Differences in steady-state net ammonium and nitrate influx by cold and warm adapted barley varieties. Plant Physiol 68:1064-1067 Bock BR (1987) Increases in maximum yield of spring wheat by maintaining relatively high ammonium/nitrate ratios in soil. J Fert Issues 4:68-72 Borst-Pauwels GWFH, Wolters GHJ, Henricks JJG (1971) The interaction of 2,4dinitrophenol with anaerobic Rb transport across the cell membrane. Biochem Biophys Acta 225:269-276 Boussiba S, Dilling W, Gibson J (1984) Methylammonium transport in Anacystis nidulans R-2. J Bacteriology 160:204-2 10 Boussiba S, Gibson J (1991) Ammonia translocation in cyanobacteria. FEMS Mirobiol Rev 88:1-14 Bowen JE (1968) Borate absorption in excised sugar cane leaves. Plant Cell Physiol 9:467-472 Box RJ (1987) The uptake of nitrate and ammonium nitrogen in Chara hispida L.: the contribution of the rhizoid. Plant Cell Envrion 10:169-176 Boxman AW, Roelofs JGM (1986) Some effects of nitrate versus ammonium nutrition on the nutrient fluxes in Pinus sylvestris seedlinbgs. Effect of mycorrhizal infection. Can J Bot 66:1091-1097 Boxman AW, Sinke RJ, Roelofs JGM (1986) Effects of ammonium on the growth and Rb) uptake of various ectomycorrhizal fungi in pure culture. Water Air 86 K( Soil Pollut 32:517-522 Boyer RF (1986) Modern Experimental Biochemistry. Addison-Wesley Pubi Company, Reading  231 Breiman A, Barash I (1980) Methylamine and ammonia transport in Stemphylium botryosum. J Gen Microbiol 72:248-2 56 Bremner JM, Hauck RD (1982) Advances in methodology for research on nitrogen transformations in soils. In FJ Stevenson, ed, Nitrogen in Agricultural Soils. Amer Soc Agron, Madison Wis, pp 467-5 02 Breteler H (1975) Carboxylates and the uptake of ammonium by excised maize roots. Agricultural Research Reports, 837. Centre for agricultural Publishing and Documentation, Wageningen, The Netherlands Breteler H (1977) Ammonium-rubidium uptake interaction in excised maize roots. In M Thellier, ed, Transmembrnae ionic exchanges in plants. Centre National de la Recherche Scientifique, Paris, pp 185-191 Breteler H, Nissen P (1982) Effect of exogenous and endogenous nitrate concentration on nitrate utilization by dwarf beans. Plant Physiol 70:754-759 Breteler H, Siegerist M (1984) Effect of ammonium on nitrate utilization by roots of Dwarf. Plant Physiol 75:1099-1103 Broida  HP, Chapman MW (1958) Stable nitrogen isotope analysis by optical spectroscopy. Anal Chem 30:2049-2055  Broyer TC, Hoagland DR (1943) Metabolic activities of roots and their bearing on the relation of upward movement of salts and water in plants. Amer J Bot 30:261273 Buchanan JM (1973) Formylglycinarnide ribonucleotide amidotransferase. In S Prusiner, ER Stadtman, eds, The Enzymes of Glutamine Metabolism. Academic Press. New York and London, pp 387-408 Buresh RJ, De Datta SK (1991) Nitrogen dynamics and management in rice-legume cropping systems. Adv in Agron 45:1-59 Burns RH, Miller CE (1941) Application of 15 N to the study of biological nitrogen fixation. Science 93:114-115 Calderón J, Cooper AJL, Gelbard AS, More J (1989) ‘ N isotope studies of glutamine 3 assimilation pathways in Neurospora crass. J Bacteriol 171:1772-1774 Caidwell CD, Fenson DS, Bordeleau L, Thompson RG, Drouin R, Didsury R (1984) Translocation of 13 N and 11 C between nodulated roots and leaves in alfalfa seedlings. J Exp Bot 35:43 1-443 J Exp Bot 35:43 1-443 Castorph H, Kleiner D (1984) Some properties of a Kiebsiella pneumoniae ammonium transport negative mutant. Arch Microbiol 139:245-247 Causin HF, Barneix AJ (1993) Regulation of NH 4 uptake in wheat plants. Effect of root ammonium concentration and amino acids. Plant Soil 151: 211-218 Chaillou S, Vessey JK, Morot-Gaudry JF, Raper,Jr CD, Henry LT, Boutin JP (1991) Expression of characteristics of ammonium nutrition as affected by pH of the root medium. J Exp Botany 42(235):189-196  232 Chakravorti SP (1989) Effect of increasing levels of potassium supply on the content and uptake of various nutrients by rice. J Potassium-Res 5 (3): 104-114 Chamuah GS, Dey JK (1988) Root growth and potassium uptake of rice at variable supply of nitrogen. J Potassium Res 4:12-15 Chapin FS, III, Clarkson DT, Lenton JR, Walter CHS (1988a) Effect of nitrogen stress and abscisic acid on nitrate absorption and transport in barley and tomato. Planta 173:340-351 Chapin FS, III, Walter CHS, Clarkson DT (1988b) Growth response of barley and tomato to nitrogen stress and its control by abscisic acid, water relations and photosynthesis. Planta 173:352-366 Chasko JH, Thayer JR (1981) Rapid concentration and purification of 13 N-labelled anions on a High Performance Anion Exchanger, Tnt J Appi Radiat Isotops 32:645-649 Cheeseman JM (1986) Compartmental efflux analysis: an evaluation of the technique and its limitations. Plant Physiol 80:1006-1011 Cho BH, Sauer N, Komor E, Tanner W (1981) Glucose induces two amino acid transport systems in Chiorella. Proc Natl Acad Sd USA 78:3591-3594 Churchill KA, Sze H (1983) Anion-sensitive, Hf-pumping ATPase in membrane vesicles from oat roots. Plant Physiol 71:610-617 Claassen MHT, Wilcox GE (1974) Effect of nitrogen form on growth and composition of tomato and pea tissue. J Amer Soc Hort Sci 99:17 1-174 Clarkson DT (1976) The influence of temperature on the exudation of xylem sap from detached root systems of rye (Secale cereale) and barley (Hordeum vulgare). Planta 132:297-304 Clarkson DT (1986) Regulation of the absorption and release of nitrate by plant cells: A review of current ideas and methodology. In H Lambers, JJ Neetson, I Stulen, eds, Fundamental, ecological and agricultural aspects of nitrogen metabolism in higher plants. Martinus Nijhoff Pubi, Dordrecht/Bosten, Lancaster, pp 3-27 Clarkson DT, Hopper MJ, Jones LHP (1986) The effect of root temperature on the uptake of nitrogen and the relative size of the root system in Lolium perenne. I. Solutions containing both NH4 and NO -. Plant Cell Environ 9:535-545 3 Clarkson DT, Jones LHP, Purves JV (1992) Absorption of nitrate and ammonium ions by Lolium perenne from flowing solution cultures at low root temperatures. Plant Cell Environ 15:99-106 Clarkson DT, Luttge U (1984) II Mineral Nutrition: Vacuoles and Tonoplasts. Prog Bot 46:56-67 Clarkson DT, Luttge U (1991) II. Mineral nutrition: Inducible and repressible nutrient transport systems. Progress in Bot 52:61-83 Clarkson DT, Mercer ER, Johnson MC, Jones LHP (1975) The uptake of nitrogen (ammonium and nitrate) by different segments of the roots of intact barley  233 plants. Agricultural Research Council Letcombe Laboratory Annual Report for 1974, pp 10-13 Clarkson DT, Smith FW, Vanden Berg PJ (1983) Regulation of sulphate transport in a tropical legume, Macroptiliurn atropurpureurn, cv. Sirato. J Exp Bot 34:14631483 Clarkson DT, Warner A (1979) Relationships between root temperature and transport of ammonium and nitrate ions by Italian and perennial ryegrass Loliurn multiflorum and Loll urn perenne. Plant Physiol 64:557-561 Clement CR, Hopper MJ, Jones LHP (1978) The uptake of nitrate by Lolium perenne from flowing nutrient solution. I. Effect of NO - concentration. J Exp Bot 25:813 99 Clement CR, Jones LHP, Hopper MJ (1979) Uptake of nitrogen from flowing nutrient solution: effect of terminated and intermittent nitrate supplies. In EJ Hewitt, CV Cutting, eds, Nitrogen assimilation of plants, Academic Press, London, pp 123-133 COic Y, Lesaint C, LE Roux F (1962) Effects of ammonium and nitrate nutrition and a change of ammonium and nitrate supply on the metabolism of anions and cations in tomatoes. Ann Physiol Veg 4:117-125 Cole KS, Curtis HJ (1938) Electrical impedance of Nitella during activity. J Gen Physiol 22:37-64 (1939) Cole KS, Curtis HJ (1939) Electrical impedance of Nitella during activity. J Gen Physiol 22:37-64 Conway EJ, Duggan F (1958) A cation carrier in the yeast cell wall. Biochem J 69:265274 Cook RJ, Anthony C (1978a) The ammonia and methylamine active transport system of Aspergilus nidulans. J Gen Microbiol 109:265-274 Cook RJ, Anthony C (1978b) Regulation by glutamine of ammonia transport in Aspergillus nidulans. J Gen Microbiol 109:275-286 Cooper AJL, Gelbard AS, Barry RF (1985) Nitrogen-13 as a biochemical tracer. Adv in Enzyme 57:25 1-356 Cooper ATL, McDonald JM, Gelbard AS, Geldhill RF, Duffy TE (1979) The metabolic fate of nitrogen-13 labeled ammonia in rat brain. J Biol Chem 2 54:4982-4992 Cooper HD, Clarkson DT (1989) Cycling of amino-nitrogen and other nutrients between shoots and roots in cereals- A possible mechanism integrating shoot and root in the regulating of nutrient uptake, J Exp Bot 40:753-762 Cooper TG (1977) The Tools of Biochemistry. John Wiley & Sons, New York. Corey KA, Barker AV (1989) Ethylene evolution and polyamine accumulation by tomato subjected to interactive stresses of ammonium toxicity and potassium deficiency. J Amer Soc Horti Sd 114 (4): 65 1-655  234 Cox WJ, Reisenauer HM (1973) Growth and ion uptake by wheat seedlings supplied nitrogen as nitrate, or ammonium, or both. Plant and Soil 38:363-380 Cram Wi (1968) Compartmentation and exchange of chloride in carrot root tissue. Biochim Biophys Acta 163:339-353 Cram Wi (1973) Internal factors regulating nitrate and chloride influx in plant cells. J Exp Bot 34:1463-1483 Cram Wi (1983) Characteristics of sulfate transport across plasmalemma and tonoplast of carrot root cells. Plant Physiol 72: 204-211 Craswell ET, Viek PLG (1979) Fate of fertilizer nitrogen applied to wetland rice. In Nitrogen and Rice. IRRI, Los Banos, pp 174-192 Criddle RS, Ward MR, Huffaker RC (1988) Nitrogen uptake by wheat seedlings, interactive effects of four nitrogen sources: N0 , N0 3 , NH 2 4 and urea. Plant Physiol 86:166-175 Curtis Hi, Cole KS (1938) Transverse electric impedance of the squid giant axon. J Gen Physiol 2 1:757-765 Dainty 1 (1962) Ion transport and electrical potentials in plant cells. Annu Rev Plant Physiol 13:379-402 Davis DD (1973) Metabolic control in higher plants. In Miliborrow BV, ed, Biosynthesis and its control in plants. Academic Press. London, pp 1-20 De Datta SK (1981) Principles and practices of rice production. John Wiley & Sons, New York De Datta SK (1986) Improving nitrogen fertilizer efficiency in lowland rice in tropical. Asia Fert Res 9:171-186 De Datta SK (1988) Urea: Experience in lowland rice. In E Pushparajah, A Husin, AT Bachik, eds, Proc Tnt Symp Urea Technology and Utilization. Malaysia Soc Soil Sci, Kuala Lumpur, pp 23-37 Deane-Drummond CE (1984) Mechanism of nitrate uptake into Chara corallina cells: lack of evidence for obligatory coupling to proton pump and a new 3 /N0 N0 exchange model. Plant, Cell Environ 7:317-323 Deane-Drummond CE (1986) Some regulatory aspects of [‘ C]methylamine influx into 4 Pisum sativm L. cv. Feltham first seedlings. Planta 169:8-15 Deane-Drummond CE, Glass ADM (1982) Nitrate uptake into barley (Hordeum vulaare) plants A new approach using 36 3 as an analogue for N0 C10 3 Plant Physiol 70:50-54 Deane-Drummond CE, Glass ADM (1983a) Short term studies of nitrate uptake into barley plants using ion-specific electrodes and 36 . I. Control of net uptake 3 C10 by N03- efflux. Plant Physiol 73:100-104 Deane-Drummond CE, Glass ADM (1983b) Short term studies of nitrate uptake into barley plants using ion-specific electrodes and 36 . II. Regulation of N033 ClO efflux by t’fJ-J . Plant Physiol 73:105-110 4  235 Deane-Drummond CE, Jacobsen E (1986) Characteristics of 36 3 influx into nitrate C10 reductase deficient mutant El Pisum sativum seedlings: evidence for restricted ‘induction’ by nitrate compared with wild type. Plant Sci 46:169-173 Deane-Drummond CE, Thayer JR (1986) Nitrate transport characteristics in Hordeum vulgare L. seedlings using three different tracer techniques. J Exp Bot 37:423439 Debnam ES, Levin RJ (1975) An experimental method of identifying and quantifying the active transfer electrogenic component from the diffusive component during sugar absorption measured in vivo. J Physiol 246:181-196 Deigna MT, Lewis OAM (1988) The inhibition of ammonium uptake by nitrate in wheat. Mew Phytol 110: 1-3 Deuel TF, Lerner A, Albrycht D (1973) Regulation of glutamine synthetase from rat liver and rat kidney. In S Prusiner, ER Stadtman, eds, The Enzymes of Glutamine Metabolism. Academic Press. New York and London, pp 129-144 Dey SK, Rao CN (1989) Influence of nutrient deficiency on photosynthesis and productivity in early rice varieties. Oryza 26:317-319 Dibb DW, Thompson WR, Jr. (1985) Interaction of potassium with other nutrients. In RD Munson, ed, Potassium in agriculture, ASA-CSSA-SSSA, Madison, pp 515-533 Dibb DW, Welch LF (1976) Corn growth as affected by ammonium vs nitrate absorbed from soil. Agron J 68:89-94 Dodd WA, Pitman MG, West KR (1966) Sodium and potassium transport in the marine alga Chaetomorpha darwinii. Aust J Biol Sci 19:341-3 54 Drew MC, Saker LR (1975) Nutrient supply and growth of the seminal root system in barley. I. Localized compensatory increases in lateral root growth and rates of nitrate uptake when nitrate supply is restricted to only part of the root system. J Exp Bot 26: 79-90 Drew MC, Saker LR (1980) Assessment of a rapid method using soil cores for estimating the amount and distribution of crop roots in the field. Plant and Soil 55:297-305 Dubois F, Grenson M (1979) Methylamine/ammonia uptake systems in Saccharomyces cerevisae: multiplicity and regulation. Mol Gen Genet 175:67-76 Dunlop J, Glass ADM, Tomkins BD (1979) The regulation of K uptake by ryegrass and white clover roots in relation to their competition for potassium. New Phytol 83:365-370 Edwards JH, Barber SA (1976) Nitrate flux into corn roots as influenced by shoot requirements. Agron J 68:47 1-473 El-Shinnawi MM, El-Seidy M, Omran MS, Barsoom SW (1988a) Nitrogen forms in plants as affected by nitrogen source. Egyptian J Soil Sci 28:269-287 El-Shinnawi MM, Omran MS, El-Seidy M, Barsoom SW (1988b) Amino acids content in certain plant seedlings supplied with various nitrogen sources. Egyptian J Soil Sci 2 8:183-196  236 Elliot GC, Nelson PV (1983) Relationship among nitrogen accumulation, nitrogen assimilation and plant growth in chrysanthemums. Physiol Plant 57:250-259 Epstein E (1966) Dual pattern of ion absorption by plant cells and by plant. Nature 212:1324-1327 Epstein E (1972) Mineral Nutrition of Plants: Principles and perspectives. John Wiley and Sons, Inc. New York Etherton B (1967) Steady state sodium and rubidium effluxes in Pisum sativum roots. Plant Physiol 42:685-690 Etherton E (1963) The relationship of cell transmembrane electropotential to potassium and sodium accumulation ratios in oat and pea seedlings, Plant Physiol 38:581-585 Fageria NH (1974) Kinetics of phosphate absorption by intact rice plants. Aust J Agric Res 25:395-400 Fageria NK, Baligar VC, Wright RJ, Carvalho JRP (1990) Lowland rice response to potassium fertilization and its effect on N and P uptake. Ferti Res 21:157-162 Faust  H (1986) ‘ N in biological nitrogen fixation studies 5 Mitteilungen 114:3-120  -  A bibliography. Zfl  Felle H (1980) Amine transport at the plasmalemma of Riccia fluitans. Biochimica et Biophysica Acta 602:181-195 Fentem PA, Lea PJ, Stewart GR (1983a) Ammonia assimilation in the roots of nitrate and ammonia-grown Hordeum vulgare L (cv Golden Promise). Plant Physiol 71:496-501 Fentem PA, Lea PJ, Stewart GR (1983b) Action of inhibitors of ammonia assimilation on amino acid metabolism in Hordeum vulgare L (cv Golden Promise). Plant Physiol 71:502-506 Fernando M, Mehroke J, Glass ADM (1992) De Novo synthesis of plasma membrane and tonoplast polypeptides of barley roots during short-term K deprivation. Plant Physiol 100:1269-1276 Findenegg GR (1987) A comparative study of ammonium toxicity at different constant pH of the nutrient solution. Plant Soil 103:239-243 Findlay GP, Hope AB (1976) Electrical properties of plant cells: Methods and Findings. In U Luttge, MG Pitman, eds, Transport in Plants II, Part A Cells. SpringerVerlag, Berlin, pp 53-92 Fischer E, Luttge U (1980) Membrane potential changes related to active transport of glycine in Lemna gibba Gl. Plant Physiol 65:1004-1008 Franco AR, Cárdenas J, Fernández E (1987) A mutant of Chiamydomonas reinhardtii altered in the transport of ammonium and methylammonium. Mol Gen Genet  206:414-418  Franco AR, Cárdenas J, Fernández F (1988) Two different carriers transport both ammonium and methylammonium in Chiamydomonas reinhardtii. J Biol Chem  263:14039-14043  237 Fried MF, Zsoldos F, Vose PB, Shatokhin IL (1965) Characterizing the NO - and NH 3 4 uptake process of rice roots by use of 15 N labelled 3 NO Physiol Plant 4 NH . 18:313-320 Frota JNE, Tucker TC, (1972) Temperature influence on ammonium and nitrate absorption by Lettuce. Soil Sd Soc Am Proc 36:97-100 Fuggi A, DI Rigano M, Vona V, Rigano C (1981) Nitrate and ammonium assimilation in algal cell suspension and related pH variations in the external medium, monitored by electrodes. Plant Sci Lett 23:129-138 Gashaw L, Mugwira LM (1981) Ammonium-N and nitrate-N effects on growth and mineral composition of triticale, wheat and rye. Agron J 73:47-51 Gentry LE, Wang XT, Below FE (1989) Nutrient uptake by wheat seedlings that differ in response to mixed nitrogen nutrition. J Plant Nutr 12:363-373 Gersberg R, Krohn K, Peek N, Goldman CR (1976) Denitrification studies with 13 Nlabeled nitrate. Science 192:1229-1231 Gharbi A (1989) The effect of potassium fertilizer application on N, P and K uptake and on the yield of durum wheat (Triticum durum). Agricoltura-Mediterranea, 119 (3): 272-275 Ginsburg, A. and E.R. Stadtman. 1973. Regulation of glutamine synthetase in Escherichia coli. In S Prusiner, ER Stadtman, eds, The Enzymes of Glutamine Metabolism. Academic Press. New York and London, pp 9-43 Givan CV (1979) Metabolic detoxication of ammonia in tissues of higher plants. Phytochemistry 18:375-382 Glass ADM (1975) The regulation of potassium absorption in barely roots. Plant Physiol 56:337-380 Glass ADM (1976) Regulation of potassium absorption in barley roots. An allosteric model. Plant Physiol 56:337-380 Glass ADM (1977) Regulation of K influx by barley roots: Evidence for direct control by internal K. Aust J Plant Physiol 4:313-318 Glass ADM (1983) Regulation of ion transport. Ann Rev Plant Physiol 34: 3 11-326 Glass ADM (1988) Nitrogen uptake by plant roots. Animal and Plant Sd  1:15 1-6  Glass ADM (1989) Plant Nutrition. An introduction to current concepts. Jones and Bartlett Publishers, Boston Glass ADM, Dunlop J (1978) The influence of potassium content on the kinetics of potassium influx into excised ryegrass and barley roots. Planta 141:117-119 Glass ADM, Fernado M (1992) Homeostatic processes for the maintenance of the K+ content of plant cells: A model. Israel J Botany 41:145-166 Glass ADM, Shaff J, Kochian LV (1992) Studies of the uptake of nitrate in barley. 4. Electrophysiology. Plant Physiol 99:456-463  238 Glass ADM, Siddiqi MY (1982) Cation-stimulated H efflux by intact roots of barely. Plant Cell Environ 5:385-393 Glass ADM, Siddiqi MY (1984) The control of nutrient uptake rates in relation to the inorganic composition of plants. Adv of Plant Nutr 1:103-147 Glass ADM, Siddiqi MY, Giles KI (1981) Correlation between potassium uptake and hydrogen efflux in barley varieties. Plant Physiol 68:45 7-459 Glass ADM, Siddiqi MY, Ruth TJ, Rufty, Jr TW (1990) Studies of the uptake of nitrate in barley. II Energetics. Plant Physiol 93:1585-1589 Glass ADM, Thompson RG, Bordeleau L (1985) Regulation of NO - influx in barley 3 studies using 3 N0 Plant Physiol 77:379-381 ‘ . Gomez KA, De Datta SK (1975) Influence of environment on protein content of rice. Agron J 67:565-568 Goyal SS, Haffaker RC (1986) The uptake of N0 , NO 3 - and NH 2 4 by intact wheat (Triticurn aestivurn) seedlings. I Induction and kinetics of transport systems. Plant Physiol 82:1051-1056 Goyal SS, Huffaker RC (1984) Nitrogen toxicity in plants. In RD Hauck, ed, Nitrogen in Crop Production, ASA.CSSA.SSSA, Madison Goyal SS, Huffaker RC, Orens OA (1982) Inhibitory effects of ammoniacal nitrogen on growth of radish plants. II. Investigation on the possible causes of ammonium toxicity to radish plants and irreversal by nitrate. J Am Soc Hort Sci 107:130135 Granato TC and Raper CD, Jr. (1989) Proliferation of Maize (Zea mays L.) roots in response to localized supply of nitrate. J Exp Bot 40:263-275 Greenway H (1965) Plant responses to saline substrates. IV. Chloride uptake by Horde urn vulgare as affected by inhibitors, transpiration, and nutrients in the medium. Aust J Biol Sci 18:249-268 Grist DH (1986) Rice. Ed6, Longman, London and New York Hackette SL, Skye GE, Burton C, Segel In (1970) Characterization of an ammonium transport system in filamentous fungi with 14 methylammonium- as the C substrate. J Biol Chem 245 :4240-4249 Hageman RH (1984) Ammonium versus nitrate nutrition of higher plants. In RD Hauck, ed, Nitrogen in crop production, Amer Soc Agro, Madison, pp 67-85 flames KC, Wheeler PA (1977) Ammonium and nitrate uptake by the marine macrophyte Hypn ea rn usciformis (Rhodophyta) and Macrocys tis pyrifera (Phaeophyta). J Phycol 14:319-324 Hanck RD (1982) Nitrogen-Isotope-ratio analysis. In AL Page, RH Miller, DR Keeney, eds, Methods of Soil Analysis. Part 2-Chemical and microbiological properties. (2ndEd.). ASA-SSSA, Madison. Hanson AD, Tully RE (1979) Amino acids translocated from turgid and water-stressed barley leaves. II. Studies with 13N and 14C. Plant physiol 64:467-47 1  239 Harada T, Takaki H, Yamada Y (1968) Effect of nitrogen source on the chemical components in young plants. Soil Sci Plant Nutr 14:47-5 5 Harper JE (1984) Uptake of organic nitrogen forms by roots and leaves. In RD Hauck, ed, Nitrogen in crop production. Amer Soc Agron/Crop Sci Soc Amer/Soil Sci Soc Amer, Madison, pp 165-170 Hartman SC (1973) Relationships between glutamine amidotransferases and glutaminases. In S Prusiner, ER Stadtman, eds, The Enzymes of Glutamine Metabolism. Academic Press. New York and London, pp 319-330 Hartmann A, Kleiner D (1982) Ammonium (methylammonium) transport by Azospirillum spp. FEMS Micro Lett 15:65-67 Hauck RD (1982) Nitrogen-Isotope-Ratio Analysis. In AL Page, RH Miller, DR Keeney, eds, Method of Soil Analysis, part 2, Chemical and microbiological properties, Ed2 Amer Soc Agro/Soil Sci Soc Amer, Madison, pp 73 5-779 Haynes RJ (1986) Mineral nitrogen in the plant-soil system. Academic Press, Orlando Haynes RJ, Goh KM (1978) Ammonium and nitrate nutrition of plants. Biol Rev 53:465-510 Heberer JA, Below FE (1989) Mixed nitrogen nutrition and productivity of wheat grown in hydroponics. Ann Bot 63:643-649 Hedrich R, Schroeder JI, Fernandez JM (1987) Patch-clamp studies on higher plant cells: a perspective. TIBS 12:49-52 Henry LT, Raper CD, Jr. (1988) Assessment of an apparent relationship between availability of soluble carbohydrates and reduced nitrogen during floral initiation in tobacco. Bot Gaz 149:289-294 Henry LT, Raper CD, Jr. (1989a) Cyclic variations in nitrogen uptake rate of soybean plants. Plant Physiol 91:1345-1350 Henry LT, Raper CD, Jr. (1989b) Effects of root-zone acidity on utilization of nitrate and ammonium in tobacco plants. J Plant Nutr 12:811-826 Henry LT, Raper CD, Jr. (1991) Soluble carbohydrates allocation to roots, photosynthetic rate of leaves, and nitrate assimilation as affected by nitrogen stress and irradiance. Bot Gaz 152:23-33 Henry LT, Raper CD, Jr., Rideout JW (1992) Onset of and recovery from nitrogen stress during reproductive growth of soybean. mt J Plant Sd 153:178-185 Hiatt AJ (1967) The relationship of cell sap pH to organic acid change during ion uptake. Plant Physiol 42:294-298 Hiatt AJ, Lowe RH (1967) Loss of organic acids, amino acids, potassium and chloride from barley roots treated anaerobically and with metabolic inhibitors. Plant Physiol 42:1731-1736 Higinbotham N (1970) Movement of ions and electrogenesis in higher plant cells. Am Zoology 10:393-403 Higinbotham N (1973) Eletropotentials of plant cells. Ann Rev Plant Physiol 24:25-46  240 Higinbotham N, Anderson WP (1974) Electrogenic pumps in higher plant cells. Can J Bot 52:1011-102 1  Higinbotham N, Etherton B, Foster RJ (1964) Effect of external K, NH , Na, Ca, Mg, and 4 H ions on the cell transmembrane electropotential of Avena coleoptile. Plant physiol 39:196-203 Hoagland DR, Broyer TC (1936) General nature of the process of salt accumulation by roots with description of the experimental methods. Plant Physiol 11:471-507 Hodges TK (1973) Ion absorption by plant roots. Adv Agron 25:163-207 Hodges TK, Vaadia Y (1964) Uptake and transport if radiochioride and tritiated water by various zones of onion roots of different chloride status. Plant Physiol 39:104-108 Hole DJ, Emran AM, Fares Y, Drew MC (1990) Induction of nitrate transport in maize roots and kinetics of influx measured with nitrogen-13. Plant Physiol 93:642647 Holtel A, Kleiner D (1985) Regulation of methylammonium transport in Paracoccus denitrificans. Arch Microbiol 142:285-288 Hong YP, Stutte CA (1987) Rice root uptake and translocation of 32 P and 86 Rb. The Research Reports of the Rural Development Administration 29:48-59, Suweon, Rep of Korea Hope AB, Simpson A, Walker NA (1966) The efflux of chloride from cells of Nitella and Chara. Aust J Biol Sci 19:355-362 Horowitz B, Meister A (1973) Utilization of glutamine for the biosynthesis of asparagine. In S Prusiner, ER Stadtman, eds, The Enzymes of Glutamine Metabolism. Academic Press. New York and London, pp 5 73-603 Hoult DI, Preston C (1992) Inexpensive plasma discharge source for molecular emission spectroscopy with application to ‘ N analysis. Rev Sci Instrum 5 63:1927-1931 Humphries EC (1951) The absorption of ions by excised root systems. II. Observation soon roots of barley grown in solutions deficient in phosphorus, nitrogen or potassium. J Exp Bot 2:344-379 Ichii M, Tsumura H (1989) Comparison of nutrient uptake in ecospecies and ecotypes of rice seedlings. Japan J Crop Sci 5 8:7-12 Ikeda H, Osawa T (1988a) Effects of CO 2 concentration in the air, and shading, on the utilization of N03 and NH 4 by vegetable crops. J of the Japan Soc Hort Sci 57:52-61 Ikeda H, Osawa T (1988b) The effects of 3 /NO ratios and temperature of the 4 NH nutrient solution on growth, yield and blossom-end rot incidence in tomato. J of the Japan Soc Hort Sci 57:62-69 Ikeda M (1990) Nitrogen assimilating enzyme activity of tomato plant in response to the supply of ammonium or nitrate or both. J Fac Agr Kyushu Univ 34(3):255263  241 Ingemarsson B (1987) Nitrogen utilization in Lemna. II. Studies of nitrate uptake using 13N03-. Plant Physiol 85:860-864 IRRI (1988) World Rice Statistics. International Rice Research Institute, Manila, Philippines Islam AKMS, Edward DG, Asher CJ (1980) pH optima for crop growth: results of a flowing solution culture experiment with six species. Plant Soil 54:339-357 Ito A (1987) Changes of water temperature, pH, dissolved oxygen, inorganic nitrogen, and phosphorus concentrations in flowing irrigation water on paddy surface. Soil Sci Plant Nutr 33:449-459 Ivanko S, Ingversenm J (1971) Investigation on the assimilation of nitrogen by maize roots and the transport of some major nitrogen compounds by xylem sap. I. Nitrate and ammonia uptake and assimilation in the major nitrogen fractions of nitrogen-starved maize roots. Physiol Plant 24:59-65 Jackson PC, Edwards DG (1966) Cation effects on chloride fluxes and accumulation levels in barley roots. J Gen Physiol 50:225-241 Jackson WA, Chaillou S, Morot-Gaudry J, Volk RJ (1993) Endogenous ammonium generation in maize roots and its relationship to other ammonium fluxes. J Exp Bot 264:731-739 Jackson WA, Johnson RE, Volk RJ (1974) Nitrate uptake by nitrogen-depleted wheat seedlings. Physiol Plant 32:37-42 Jackson WA, Kwik KD, Volk RJ (1976) nitrate uptake during recovery from nitrogen deficiency. Physiol Plant 36:174-181 Jackson WA, Pan WL, Moll RH, Kamprath EJ (1986) Uptake, translocation, and reduction of nitrate. In CA Netra, ed, Biochemical basis of plant breeding. Vol 2 Nitrogen metabolism. CRC Press, Boca Raton, FL pp 73-98 Jackson WA, Volk RJ (1992) Nitrate and ammonium uptake by maize: adaptation during relief from nitrogen suppression. New Phytol 12 2:439-446 Jayakumar A, Barner EM, Jr. (1984) The role of glutamine in regulation of ammonium transport in Azotobacter vinelandii. Arch Biochem Biophys 231:95-101 Jayakumar A, Epstein W, Barnes Jr. EM (1985) Characterization of ammonium (methylammonium)/potassium antiport in Esclierichia coli. J Biol Chem 260:7528-7532 Jensen P, Pettersson S (1979) Allosteric regulation of potassium uptake in plant roots Physiol Plant 42:207-2 13 Jeschke WD (1982) Shoot-dependent regulation of sodium and potassium fluxes in roots of whole barley seedlings. J Exp Bot 33(135):601-618 Jeschke WD, Jambor W (1981) Determination of unidirectional sodium fluxes in roots of intact sunflower seedlings. J Exp Bot 32:1257-1272 Johansen C, Edwards DG, Loneragan JF (1970) Potassium fluxes during potassium absorption by intact barley roots of increasing potassium content. Plant Physiol 45:601-603  242 Johnson CM, Stout PR, Broyer TC, Canton AB (1957) Comparative chlorine requirements of different plant species. Plant and Soil 8:337-353 Joliot F, Curie I (1934) Artificial production of a new kind of radio-element. Nature 133:201-202 Jongbloed RH, Clement JMAM, Borst-Pauwels GWFH (1990) Effects of ammonium and pH on growth of some ectomycorrhizal fungi in vitro. Acta Bot Neerl 39:349358 Jongbloed RH, Clement JMAM, Borst-Pauwels GWFH 1991 Kinetics of NH 4 and K uptake by ectomycorrhizal fungi Effect of NH4 on K uptake Physiol Planta 83:427-43 2 Joseph RA, Hai TV, Lambert J (1975) Multiphasic uptake of ammonium by soybean roots. Physiol Plant 34:32 1-325 Jungk A(1970) Interactions between the nitrogen concentration (NH4, 3 NO and 4 NH , -) and the pH of the nutrition solution, and their effects on the growth and 3 NO ion balance of tomato plants. Gartenbauwissenschaft 35:13-26 Kamen MD (1957) Isotopic Tracers In Biology-An Introduction to Tracer Methodology. Academic Press Inc., New York. Kemmier G (1983) Modern aspects of wheat manuring. PIP-Bulletin No. 1 Revised, 2nd Ed, International Potash Institute, CH-3 048 Bern-Worblaufen, Switzerland King BJ, Siddiqi MY, Glass ADM (1992) Studies of the uptake of nitrate on barley. V. Estimation of root cytoplasmic nitrate concentration using nitrate reductase activity--Implication for nitrate influx. Plant Physiol 99:1582-1589 King BJ, Siddiqi MY, Ruth TJ, Warner RL, Glass ADM (1993) Feedback regulation of nitrate influx in barley roots by nitrate, nitrite, and ammonium. Plant Physiol 102:1279-1286 Kirkby BA (1968) Influence of ammonium and nitrate nutrition on the cation-anion balance and nitrogen carbohydrate metabolism of white mustard plants grown in dilute nutrient solution. Soil Sci 105:133-151 Kirkby EA, Hughes AD (1970) Some aspects of ammonium and nitrate nutrition in plant metabolism. In EA Kirkby Nitrogen Nutrition of the Plant. Univ. of Leeds, pp 69-77 Kirkby EA, Mengel K (1967) Ionic balance in different tissues of the tomato plant in relation to nitrate, urea or ammonium nutrition. Plant Physiol 65:6-14 Kleiner D (1975) Ammonium uptake by nitrogen fixing bacteria. I. Azotobacteria vinelandii. Arch Microbiol 104:163-169 Kleiner D (1981) The transport of NH 3 and NH 4 across biological membranes. Biochim Biophys Acta 639:41-52 Kleiner D (1985) Bacterial ammonium transport. FEMS Microbiol Rev 32:87-100  243 Kleiner D, Fitzke E (1981) Some properties of a new electrogenic transport system: the ammonium (methylammonium) carrier from Clostridium pasteurianum. Biochim Biophys Acta 641:138-147. Knowles R, Blackburn TH (1993) Nitrogen Isotope Techniques. eds, Academic Press mc, San Diego Kochian LV, J Shaff, WJ Lucas (1989) High affinity K uptake in Maize roots. Plant Physiol 91:1202-1211 Kochian LV, Lucas W (1988) Potassium transport in roots. Advances in Botanical Research 15:93-178 Kochian LV, Lucas WJ (1982) Potassium transport in corn roots. I. Resolution of kinetics into a saturable and linear component. Plant Physiol. 70:1723-1731 Kochian LV, Shaff JE, Lucas WJ (1989) High affinity K uptake in maize roots. A lack of coupling with H efflux. Plant Physiol 91:1202-1211 Krohn KA, Mathis CA (1981) The use of isotopic nitrogen as a biochemical tracer. In JW Root, KA Krohn, eds, Short-lived radionuclides in chemistry and biology, Adv in Chem Ser 197, Amer Chem Soc, Washington, DC, pp 233-249 Kuiper D, Kuiper PJ, Lambers H, Schuit J, Staal M (1989) Cytokinin concentration in relation to mineral nutrition and benzyladenine treatment in Plantago major ssp. pleiosperma. Plant Physiol 75:511-517 Laane C, Krone W, Konings W, Haaker H, Veeger C (1980) Short-term effect of ammonium chloride on nitrogen fixation by Azotobacter vinelandii and bacteroids of Rhizobium leguminoniae cells. Eur J Biochem 103:39-46 LaRoche J, Harrison WG (1989) Reversible kinetic model for the short-term regulation of methylammonium uptake in two phytoplankton species, Dunaliella tertiolecta (Chlorophyceae) and Pha eoda ctyl urn tricorn u turn (Bacillariophyceae). J Phycol 25:36-48 Larsson C-M, Larsson M, Purves JV, Clarkson DT (1991) Translocation and cycling through roots of recently absorbed nitrogen and sulphur in wheat (Triticurn aestivurn) during vegetative and generative growth. Physiol Plant 67:30-3 6 Lauchli A (1984) Salt exclusion: An adaptation of legumes for crops and pastures under saline conditions. In RC Staples, GH Toennissen, eds, Strategies for crop improvement. John Wiley and Sons, New York, pp 17 1-187 Lavoie N, Vezina L-P, Maogolis HA (1992) Absorption and assimilation of nitrate and ammonium ions by Jack pine seedlings. Tree Physiol 11:171-183 Lazof D, Cheeseman JM (1986) Sodium transport and compartmentation in Spergukaria marina. Partial characterization of a functional symplasm. Plant Physiol 8 1:742-747 Lee RB (1982) Selectivity and kinetics of ion uptake by barley plants following nutrient deficiency. Ann of Bot 50:429-449  244 Lee RB and Ayling SM (1993) The effect of methionine sulphoximine on the absorption of ammonium by maize and barley roots over short periods. J Exp Bot 258:53-63 Lee RB, Clarkson DT (1986) Nitrogen-13 studies of nitrate fluxes in barley roots. I. Compartmental analysis from measurements of ‘ N efflux. J Exp Bot 185:17533 1767 Lee RB, Drew MC (1986) Nitrogen-13 studies of nitrate fluxes in barley roots. II. Effect of plant N-status on the kinetic parameters of nitrate influx. J Exp Bot 37(185):1768-1779 Lee RB, Drew MC (1989) Rapid reversible inhibition of nitrate influx in barley by ammonium. J Exp Bot 40(216):741-752 Lee RB, Purves JV, Ratcliffe RG, Saker LR (1992) Nitrogen assimilation and the control of ammonium and nitrate absorption by maize roots. J Exp Bot 43:13851396 Lee RB, Ratcliffe RG (1991) Observation on the subcellular distribution of the ammonium ion in maize root tissue using in-vivo ‘ N-nuclear magnetic 4 resonance spectroscopy. Planta 183:359-367 Lee RB, Rudge KA (1986) Effects of nitrogen deficiency on the absorption of nitrate and ammonium by barley plants. Annals of Botany 57:47 1-486 Lefebvre DD, Clarkson DT (1984) Compartmental analysis of phosphate in roots of intact barley seedlings. Can J Bot 62:1076-1080 Lefebvre DD, Glass ADM (1982) Regulation of phosphate influx in barley roots: effects of phosphate deprivation and reduction in influx with provision of orthophosphate. Physiol Plant 54:199-206 Leigh RA, Wyn Jones RG (1973) The effect of increased internal ion concentration on the ion uptake isotherms of excised maize root segments. J Exp Bot 24:787-795 Lewis OAM, Chadwick S (1983) An 15 N investigation into nitrogen assimilation by hydroponically-grown barley (Hordeum vulgare L cv. Clipper) in response to nitrate, ammonium and mixed nitrate and ammonium nutrition. New Phytol 95:635-645 Lewis OAM, DM James and EJ Hewitt (1982) Nitrogen assimilation in barley (Hordeum vulgare L. cv. Mazurka) in response to nitrate and ammonium nutrition. Ann Bot 49:39-49 Lewis OAM, Fulton B, von Zelewski AAA (1987) Differential distribution of carbon in response to nitrate, ammonium, and nitrate + ammonium in wheat. In WR Ulirich, PJ Aparicio, PJ Syrett, F Castillo, eds, Inorganic nitrogen metabolism. Springer-Verlag, Berlin, pp 240-246 Lewis OAM, S Chadwick and J Withers (1983) The assimilation of ammonium by barley roots. Planta 159:483-486 Lewis OAM, Soares MIM, Lips SH (1986) A photosynthetic and 15 N investigation of the differential growth response of barley to nitrate, ammonium, and nitrate + ammonium nutrition. In H Lambers, JJ Neeteson, I Stulen, eds, Fundamental,  245 ecological and agricultural aspects of nitrogen metabolism in higher plants, Martinus Nijhoff Publ Dordrecht, The Netherlands, pp 285-300 Lim JT, Wilkerson GG, Raper Jr CD, Gold HJ (1990) A dynamic growth model of vegetative soybean plants: Model structure and behaviour under varying root temperature and nitrogen concentration. J Exp Bot 41:229-241 Lin W (1984) Further characterization on the transport property of plasmalemma NDAH oxidation system in isolated corn root protoplasts. Plant Physiol 74:219222 Lindner L, Helmer J, Brinkman GA (1979) Water “loop”-target for the In-cyclotron production of ‘ N by the reaction 3 3 0(pcL)’ 6 ‘ N . Interna J Appi Radia Isotop 30:506-507 Loo T-L (1931) Studies on the absorption of ammonia and nitrate by the root of Zea maysOseedlings, in relation to the concentration and the actual acidity of culture solution. J Facul Agr Hokkaido Imp Univ Vol XXX, Part I, pp 1-118 Lu JJ, Chang TT (1980) Rice in its temporal and spatial perspectives. In BS Luh, ed, Rice: Production and Utilization, Westport, CT:AVI, pp 1-74 Luttge U, Higinbotham N (1979) Transport in plants. Springer Verlag, New York Luttge U, Higinbotham N (1982) Transport in plant cells. Annu Rev Plant Physiol 22:75-96 Luttge U, Osmond CB (1970) Ion absorption in Atriplex leaf tissue. III. Site of metabolic control of light dependent chloride secretion to epidermal bladders. Aust J Biol Sci 23:17-25 Lycklama JC (1963) The absorption of ammonium and nitrate by perennial ryegrass. Acta Bot Neerl 12:36 1-423 Macduff JH, Hopper MJ, Wild A (1987) The effect of root temperature on growth and uptake of ammonium and nitrate by (Brassia napus L.) in flowing solution culture. II. Uptake from solutions containing 3 NO J Exp Bot 38(186):53-66 4 NH . Macduff JH, Wild A (1989) Interactions between root temperature and nitrogen deficiency influence preferential uptake of NH 4 and N0 3 by oilseed rape. J Exp Bot 40(211):195-206 Macfarlane JJ, Smith FA (1982) Uptake of methylamine by Ulva rigida: Transport of cations and diffusion of free base. J Exp Bot 33:195-207 Macklon AES (1975a) Cortical cell fluxes and transport to the stele in excised root segments of Allium cepa L. I. Potassium Sodium and Chloride. Planta 122:109130 Macklon AES (1975b) Cortical cell fluxes and transport to the stele in excised root segments of Allium cepa L. II. Calcium. Planta 122:131-141 Macklon AES, Ron MM, Sim A (1990) Cortical cell fluxes of ammonium and nitrate in excised root segments of Allium cepa L.: studies using 15 N. J Exp Bot 41:359-370  246 Macklon AES, Sim A (1976) Cortical cell fluxes and transport to the stele in excised root segments of Allium cepa L. III. Magnesium. Planta 12 8:5-9 Macklon AES, Sim A (1981) Cortical cell fluxes and transport to the stele in excised root segments of Allium cepa L. IV. Calcium as affected by its external concentration. Planta 152:381-387 MacKown CT, Jackson WA, Volk RJ (1982a) Restricted nitrate influx and reduction in corn seedlings exposed to ammonium. Plant Physiol 69:353-359 MacKown CT, Jackson WA, Volk RJ (1982b) Nitrate assimilation by decapitated corn root systems: effects of ammonium during induction. Plant Sci Lett 24:295-302 MacKown CT, Volk RJ, Jackson WA (1981) Nitrate accumulation, assimilation and transport by decapitated corn roots: effects of prior nitrate nutrition. Plant Physiol 68:133-138 MacLeod LB (1969) Effects of N, P, and K and their interactions on the yield and kernel weight of barley in hydroponic culture. Agron J 6 1:26-29 MacRobbie EAC (1964) Factors affecting the fluxes of potassium and chloride ions in Nitella translucens. J Gen Physiol 47:859-877 MacRobbie EAC (1970) The active transport of ions in plant cells. Quart Revs Biophy 3:251-294 MacRobbie EAC (1971) Vacuolar fluxes of chloride and bromide in Nitella tra.nslucens. J Exp Bot 22:487-502 MacRobbie EAC, Dainty J (1958) Ion transport in Nitellopsis obtusa. J Gen Physiol 42:335-353 Magalhães JR, Huber DM, Tsai CY (1992) Evidence of increased ammonium assimilation in tomato plants with exogenous c-ketoglutarate. Plant Science 85:135-141 Magalhaes JR, Wilcox GE (1983) Tomato growth and nutrient uptake patterns as influenced by nitrogen form and light intensity. J Plant Nutr 6:941-956 Magasanik BM, Prival J, Brenchley JE (1973) Glutamine synthetase, regulator of the synthesis of glutamate-forming enzymes. In S Prusiner, ER Stadtman, eds, The Enzymes of Glutamine Metabolism. Academic Press. New York and London, pp 65-76 Malavolta E (1954) Study on the nitrogenous nutrition of rice. Plant Physiol 29:98-99 Marcus-Wyner L (1983) Influence of ambient acidity on the absorption of N0 3 and 4 by tomato plants. J Plant Nutr 6:657-666 NH Marcus-Wyner L, Rains DW (1982) Simultaneous measurement of NH 4 absorption and N2 fixation by Glycine max L. Response to temperature, pH, and external nitrogen concentration. Plant Physiol 69:460-464 Marschner H, Römheld V (1983) In vivo measurement of root-induced pH changes at the soil-root interface. Effect of plant species and nitrogen source. Z Pflanzenphysiol 111: 241-251  247 Mazzucco CE, Benson DR (1984) C]-Methylammonium 14 transport by Frankia sp. [ strain Cp. II. J Bacteri 160:636-641 McCarthy JJ, Goldman JC (1979) Nitrogenous nutrition of marine phytoplankton in nutrient-depleted waters. Science 203:670-672 McClure PR, Kochian LV, Spanswick RM, Shaff J (1990) Evidence for cotransport of nitrate and protons in maize roots. I. Effects of nitrate on the membrane potential. Plant Physiol 93:281-289 McElfresh MW, Meeks JC, Parks NJ (1979) The synthesis of ‘ N-labelled nitrate of high 3 specific activity and purity. J Radioanaly Chem 53:337-344 McNaughton GS, Presland MR (1983) Whole plant studies using radioactive 13nitrogen. I. Techniques for measuring the uptake and transport of nitrate and ammonium ions in by hydroponically grown Zea mays. J Exp Bot 34:880-892 Meeks JC (1993) ‘ N Techniques. In R Knowles, TH Blackburn, eds, Nitrogen Isotope 3 Techniques, Academic Press mc, San Diego, pp 273-303 Meeks JC, Stewinberg NA, Joseph CM, Enderlin CS, Jorgensen PA, Peters GA (1985) Assimilation of exogenous and dinitrogen-derived 13 NH4 by Anabaena azollae separated from Azolla caroliniana Wild. Arch Microbiol 142:229-233 Meeks JC, Wolk CP, Lockau W, Schillimg N, Jeseph CM, Chien W-S (1978) Pathways of assimilation of 2 4 by cyanobacteria with and without NH N]N and 13 3 [‘ heterocysts. J Bacteriol 134:125-130 Meijer CLC (1970) Kinetics observations concerning the uptake of ammonium by several cereals. Thesis, University of Leiden Memon AR, Siddiqi MY, Glass ADM (1985) Efficiency of K utilization by barley varieties: activation of Pyruvate kinase. J Exp Bot 3 8:79-90 Mengel K (1989) The role of potassium in improving nitrogen uptake and nitrogen utilization by crops. Technical Bulletin- National Fertilizer Development Centre. No. 4:111-122 Mengel K, Viro M (1978) The significance of plant energy status for the uptake and incorporation of NH -nitrogen by young rice plants. Soil Sci Plant Nutr 4 24(3):407-416 Mengel K, Viro M, Hehi G (1976) Effect of potassium on uptake and incorporation of ammonium-nitrogen of rice plants. Plant Soil 44:547-558 Miflin BJ, Lea PJ (1980) Ammonium assimilation. In BJ Miflin, ed, The Biochemistry of Plants, Vol 5. Academic Press, New York, pp 169-202 Miflin BJ, Lea PT (1976) The pathway of nitrogen assimilation in plants. Phytochemistry 15:873-885 Mikkelsen DS, De Datta SK (1991) Rice culture. In BS Luh, Ed2, Rice. Vol I. Production. Van Nostrand Reinhold, New York, pp 103-186  248 Miller RE (1973) Glutamate synthase from Escherichia coli: an iron-sulfide flavoprotein. In S Prusiner, ER Stadtman, eds, The Enzymes of Glutamine Metabolism. Academic Press. New York and London, pp 183-205 Minotti PL, Craig D, Jackson WA (1969) Nitrate uptake by wheat as influenced by ammonium and other cations. Crop Sci 9:9-14 Minotti PL, Jackson WA (1970) Nitrate reduction in the roots and shoots of wheat seedlings. Planta 95:36-44 Mitchell P (1970) Membrane of cells and organelles: morphology, transport and metabolism. Symp Soc Gen Microbiol 20:121-166 Monseilse EB-I, Kost D (1993) Different ammonium-ion uptake metabolism and detoxification efficiencies in two Lemnacear. A 5 ‘ N -nuclear magnetic resonance study. Planta 189:167-173 Moraghan JT, Porter OA (1975) Maize growth as affected by root temperature and form of nitrogen. Plant Soil 43 :479-487 Morgan MA, Jackson WA (1988a) Inward and outward movement of ammonium in root systems: transient responses during recovery from nitrogen deprivation in presence of ammonium. J Exp Bot 39:179-19 1 Morgan MA, Jackson WA (1988b) Suppression of ammonium uptake by nitrogen supply and its relief during nitrogen limitation. Physiol Planta 73:38-45 Morgan MA, Jackson WA (1989) Reciprocal ammonium transport into and out of plant roots: modifications by plant nitrogen status and elevated root ammonium concentration. J Exp Bot 40:207-2 14 Morgan MA, Volk RJ, Jackson WA (1973) Simultaneous influx and efflux of nitrate during uptake by perennial ryegrass. Plant Physiol 51:267-272 Mon 5, Nishimura Y, Nishizawa N (1979) Nitrogen absorption by plant root from the culture medium where organic and inorganic nitrogen coexist. I. Effect of pretreatment nitrogen on the absorption of treatment nitrogen. Soil Sci Plant Nutr 25:39-50 Mon S, Nishizawa N (1977) Nitrogen absorption by plant root from the culture medium where organic and inorganic nitrogen coexist. II. Which nitrogen is preferentially absorbed among (U, (2,3,2 C) G1uNH 4 H) Arg and NaNO 3 ? Soil 3 Sci Plant Nutr 25:541-58 Mon 5, Nishizawa N (1979) Nitrogen absorption by plant root from the culture medium where organic and inorganic nitrogen coexist. II. Which nitrogen is preferentially absorbed among [U-( , [2,32 C]G1uNH 14 H]Arg and 3 3 N0 Soil 5 Na(’ ? Sci Plant Nutr 25:51-58 Mon 5, Uchino H (1977) Criticism to the mineral nutrition theory. V. Growth features of barley water-cultured with amino acids nitrogens, Abstracts of the 1976 Meeting, Soc Sd Soil Manure 23:6 1 Mon 5, Uchino H, Sago F, Suzuki S, Nishikawa A (1985) Alleviation effect of arginine on artificially reduced grain yield of NH - or N0 4 - fed rice. Soil Sd Plant Nutr 3 31:55-67  249 Mulvaney BL, Liu YP (1991) Refinement and evaluation of an automated mass spectrometer for nitrogen isotope analysis by the Rittenberg technique. J Automatic Chem 13:273-280 Munn DA, Jackson WA (1978) Nitrate and ammonium uptake by rooted cutting of sweet potato. Agron J 70:312-3 16 Murphy AT, Lewis OAM (1987) Effect of nitrogen feeding source on the supply of nitrogen from root to shoot and the site of nitrogen assimilation in maize (Zea mays L. cv. R201). New Phyto 107:327-333 Nicholls DG (1982) Bioenergetics. An introduction to the chemiosmotic theory. Academic Press, London Nightingale GT (1937) Ammonium and nitrate nutrition of dormant delicious apple trees at 48F. Bot Gaz 95:43 7-452 Nissen P (1973) Multiphasic uptake in plants. II. Mineral cations, chloride, and boric acid. Physiol plant 29:298-354 Nobel PS (1983) Introduction to Biophysical plant Physiology. Freeman, San Francisco Noguchi Y, Sugawara T (1966) Potassium and Japonica rice. Summary of 25 years’ research. Internationa Potash institute, Bern. Nye PH (1986) Acid-base changes in the rhizasphere. In B Tinker, A Läuchli, eds, Advances in Plant Nutrition Vol 2, Praeger Publ, New York Oertle JJ (1967) The salt absorption isotherm. Physiol Plant 20:1014-1026 Oji Y (1989) Differential preference of plant for ammonium or nitrate. Nippon Nogeikagaku Kaishi 63(8):1382-1385 Oji Y, Izama G (1971) Rapid synthesis of glutamine during the initial period of ammonia assimilation in roots of barley plants. Plant Cell Physiol 12: 817-821 Omran MS, El-Shinnawi MM, El-Seidy M, Barsoom SW (1988a) The influence of nitrogen source on plants growth. Egyptian J Soil Sci 28:167-181 Oscarson P, Ingemarsson B, af Ugglas M, Larsson C-M (1987) Short-term studies of 3 uptake in Pisum using 13 N0 . Planta 170:550-555 3 N0 Pace GM, McClure PR (1986) Comparison of nitrate uptake kinetic parameters across maize inbred lines. J Plant Nutr 9:1095-1111 Pallaghy CK, Luttge U, von Willert K (1970) Cytoplasmic compartmentation and parallel pathways of ion uptake in plant root cells. Z Pflphysiol 62:5 1-57 Park NJ, Krohn KA (1978) The synthesis of 13N labeled ammonia, dinitrogen, nitrite, and nitrate using a single cyclotron target system. Interna J Appl Radia Isotop 29:754-75 6 Pate JS (1973) Uptake, assimilation and transport of nitrogen compounds by plants Soil Biol Biochem 5:109-119  250 Patel DD, Barlow PW, Lee RB (1990) Development of vacuolar volume in the root tips of pea. Ann Bot 65:159-169 Pateman JA, Dunn E, Kinghorn JR, Forbes EC (1974) The transport of ammonium and methylammonium in wild type and mutant cells of Aspergillus nidulans. Mol Gen Genet 133:225-236 Pateman JA, Kinghorn JR, Dunn E, Forbes EC (1973) Ammonium and regulation in Aspergilus nidulans. J Bacteriol 114:943-950 Patrick WH Jr., Delaune RD, Peterson FJ (1974) Nitrogen utilization by rice using 15 Ndepleted ammonium sulfate. Agron J 66:819-820 Pearson CJ, Volk RJ, Jackson WA (1981) Daily changes in nitrate influx, efflux and metabolism in maize and pearl millet. Planta 152:319-324 Pelley  JL, Bannister TT (1979) Methylamine uptake in the green alga Chiorella pyrenoidosa. J Phycol 15:110-112  Penning de Vries FWT, Brunsting AHM, van Laar HH (1974) Products, requirements and efficiency of biosynthesis: a quantitative approach. J Theoret Biol 45:339377 Pettersson S (1975) Ion uptake efficiency of sunflower roots Physiol Plant 34:28 1-285 Pettersson S (1986) Growth, contents of K and kinetics of K Rb) 86 uptake in barley ( cultured at different low supply rates of potassium. Physiol plant ???:122-128 Pettersson 5, Jensen P (1979) Allosteric and non-allosteric regulation of rubidium influx in barley roots. Physiol Plant 44:110-114 Pfruner H and Bentrup F-W (1978) Fluxes and compartmentation of K Na and C1 and action of auxins in suspension-cultured Petroselinum cells. Planta 143:213-223 ,  Pierce WS, Higinbotham N (1970) Compartments and fluxes of K+, Na+, and Cl- in Avena coleoptile cells. Plant Physiol 46:666-673 Pitman MG (1963) The determination of the salt relations of the cytoplasmic phase in cells of beet root tissue. Aust J Biol Sci 16:647-668 Pitman MG (1971) Uptake and transport of ions in barley seedlings. I. Estimation of chloride fluxes in cells of excised roots. Aust J Biol 24:407-421 Pitman MG (1972) Uptake and transport of ions in barley seedlings. III. Correlation between transport to the shoot and relative growth rate. Aust J Biol Sci 25:905919 Pitman MG, Schaeler N, Wildes RA (1975) Relation between permeability to potassium and sodium ions and fusicuccin-stimulated hydrogen-ion efflux in barley roots. Planta 126: 61-73 Pitman MG, Cram WJ (1973) Regulation of inorganic ion transport in plants. In WP Anderson, ed, Ion transport in plants. Academic Press, London, pp 465-48 1  251 Polley LD, Hopkins JW (1979) Rubidium (potassium) uptake by Arabidopsis. A comparison of uptake by cells in suspension culture and by roots of intact seedlings. Plant Physiol 64:374-378 Poole RJ (1969) Carrier mediated potassium efflux across the cell membrane of red beet. Plant Physiol 44:48 5-490 Poole RJ (1971a) Effect of sodium on potassium fluxes at the cell membrane and vacuole membrane of red beet. Plant Physiol 47:731-734 Poole RJ (1971b) Development and characteristics of sodium-selective transport in red beet. Plant Physiol 47:735-739 Poole RJ (1973) The H+ pump in red beet. In WP Anderson, ed, Ion transport in plants. Academic Press, London, pp 129-134 Poole RJ (1978) Energy coupling for membrane transport. Annu Rev Plant Physiol 29:437-460 Poulton JE, Romeo JT, Conn EE (1989) eds, Plant Nitrogen Metabolism. Recent advances in phytochemistry, Vol. 23, Plenum press, New York and London. Presland MR, McNaughton GS (1984) Whole plant studies using radioactive 13Nitrogen. II. A Compartmental model for the uptake and transport of nitrate ions by Zea mays. J Exp Bot 35:1277-1288 Presland MR, McNaughton GS (1986) Whole plant studies using radioactive 13Nitrogen. IV. A Compartmental model for the uptake and transport of ammonium ions by Zea mays. J Exp Bot 37:1619-1632 Prianishnikov DN (1941) Nitrogen in the life of plants. [Translated from Russian] Kramer Business Service, Madison, Wisc Probyn TA, Lewis OAM (1979) The route of nitrate-nitrogen assimilation in the root of Datura stramonium L. J Exp Bot 30:299-305 Purves RD (1981) Microelectrode methods for intracellular recording and ionophoresis. Academic Press, London Purves RD (1981) Microelectrode methods for intracellular recording and ionophoresis. Academic Press, London Radin JW, Boyer JS (1982) Control of leaf expansion by nitrogen nutrition in sunflower plants: role of hydraulic conductivity and turgor. Plant Physiol 69:771-775 Rai AN, Rowell P, Stewart WD (1984) Evidence for an ammonium transport system in free living and symbiotic cyanobacteria. Arch Microbiol 13 7:241-246 Rai AN, Singh DT, Singh HN (1986) Regulation of ammonium/ methylammonium transport by ammonium in the cyanobacterium Anabaena variabilis Physiol Planta 68:320-322 .  Rao KP, Rains DW (1976) Nitrate absorption by barley. I. Kinetics and energetics. Plant Physiol 57:55-58  252 Raper CD, Jr., Parsons LR, Patterson DT, Kramer PJ (1977) Relationship between growth and nitrogen accumulation for vegetative cotton and soybean plants. Bot Gaz 138:129-137 Raper CD, Jr., Vessey JK, Henry LT, Chaillou S (1991b) Cyclic variations in nitrogen uptake rate of soybean plants: effects of pH and mixed nitrogen sources. Plant Physiol Biochem 29:205-2 12 Raper CD, Jr., Wann M, Weeks WW (1978) Interdependence of root and shoot activities in determining nitrogen uptake rate of roots. Bot Gaz 13 8:289-294 Raven J (1985) Regulation of pH and generation of osmolarity in vascular plants: a cost-benefit analysis in relation to efficiency of use of energy: nitrogen and water. New Phytol 101:25-77 Raven JA (1980) Nutrient transport in micro-algal. Adv Micro Physiol 2 1:47-226 Raven JA, Farquhar GD (1981) Methylammonium transport in Phaseolus vulgaris leaf slices. Plant Physiol 67:859-863 Raven JA, Smith FA (1976) Nitrogen assimilation and transport in vascular land plants in relation to intracellular pH regulation. New Phytol 76:205-212 Reglinski A, Rowell P, Kerby NW, Stewart WP (1989) Characterization of methylammonium/ammonium transport in mutant strains of Anabaena variabiis resistant to ammonium analogous. J Gen Microbiol 135:1441-1451 Reglinski A, Rowell P, Kerby NW, Stewart WP (1989) Characterization of methylammonium/ammonium transport in mutant strains of Anabaena variabilis resistant to ammonium analogous. J Gen Microbiol 135:1441-1451 Reisenauer HM (1978) Absorption and utilization of ammonium nitrogen by plants. In DR Nielson, JG Macdonald, eds, Nitrogen in the Environment. Vol 2. SoilPlant-Nitrogen relationship, pp 157-199 Revilla F, Llobell A, Raneque A (1986) Energy-dependence of the assimilatory nitrate uptake in Azotobactor chroococcum. J Gen Microbiol 132:917-923 Rhodes D, Brunk DG, Magalhaes JR (1989) Chapter 6. Assimilation of ammonia by glutamate dehydrogenase? In JE Poulton, JT Romeo, EE Conn, eds, Plant Nitrogen Metabolism. Recent advances in phytochemistry. Vol. 23. Plenum Press. New York and London, pp 191-226 Rhodes D, Rendon GA, Stewart GR (1976) The regulation of ammonia assimilating enzymes in Lemna minor. Planta 129:203-2 10 Rhodes D, Sims AP, Folkers BF (1980) Pathway of ammonium assimilation in illuminated Lemna minor. Phytochemistry 19:357-365 Rideout JW, Chaillou S, Raper CD, Jr., Morot-Gaudry J-F (1994) Ammonium and nitrate uptake by soybean during recovery from nitrogen deprivation. J Exp Bot 45:23-33 Riech S, Almon H, Boger P (1987) Comparing short-term effects of ammonia and methylamine on nitrogenase activity in Anabaena variabilis (ATCC 29413). Z Naturforsch 42c:902-906  253 Riley D, Barber SA (1969) Bicarbonate accumulation and pH charges at the soybean (Glycine max (L.) Merr.) root-soil interface. Soil Sci Soc Am Proc 3 3:905-908 Riley D, Barber SA (1971) Effect of ammonium fertilization on phosphorus uptake as related to root-induced pH changes at the root-soil interface. Soil Sci Soc Amer Proc 3 5:301-306 Ritchie RJ (1987) The permeability of ammonia methylamine and ethylamine in the Charophyte Chara corallina (C. australis). J Exp Bot 38:67-76 Ritchie RJ (1988) The ionic relations of Ulva lactuca. J Plant Physiol 133:183-92 Ritchie RJ and Gibson J (1987a) Permeability of ammonia and amines in Rhodobacter sphaeroides and Bacillus firmus. Arch Biochem Biophys 258:332-341 Ritchie RJ and Gibson J (1987b) Permeability of ammonia methylamine and ethylamine in the cyanobacterium, Synechoccus R-2 (Anacystis nidulans) PCC 7942. J Membrane Biol 95:3131-142 Roberts JKM, Pang MKL (1992) Estimation of ammonium ion distribution between cytoplasm and vacuole using nuclear magnetic resonance spectroscopy. Plant Physiol 100:1571-1574 Robison D (1986) Limits to nutrient influx rates in roots and root systems. Physiologia. Planta. 68:55 1-559 Rodriquez-Navarro A, Blatt MA, Slayman CL (1986) A potassium-proton symport in Neurospora crassa. J Gen Physiol 87:649-674 Roomans GM, Borst-Pauwels GWFH (1977) Interaction of phosphate with monovalent cation uptake in yeast. Biochem Biophys Acta 470:84-9 1 Roon RJ, Even HL, Dunlop P, Larimore FL (1975) Methylamine and ammonia transport in Saccharomyces cerevisiae. J Bacteriol 122:502-509 Roon RJ, Meyer GM, Larimore FS (1977) Negative interactions between amino acid and methylamine/ammonia transport systems of Saccharomyces cerevisiae. J Biol Chem 252:3599-3604 Rosen CJ, Carison RM (1984) Characterization of K and NH 4 absorption by myrobalan plum and tomato: Influence of plant potassium status and solution concentrations of K and NH4. J Amer Soc Hort Sci 109:552-559 Ruben S, Hassid WZ, Kamen MD (1940) Radioactive nitrogen in the study of N2 fixation by non-leguminous plants. Science 91:578-579 Rufty TW Jr, Mackown CT, Volk RJ (1989) Effects of altered carbohydrate availability on whole-plant assimilation of 3 N0 Plant Physiol 89:457-463 5 ‘ . Rufty TW, Jr., Jackson WA, Raper CD, Jr. (1982a) Inhibition of nitrate assimilation in roots in the presence of ammonia: The moderating influence of potassium J Exp Bot 33:1122-1137 Rufty TW, Jr., Raper CD, Jr., Jackson WA (1983) Growth and nitrogen assimilation of soybeans in response to ammonium and nitrate nutrition Bot Gaz 144(4),466470  254 Rufty TW, Jr., Raper CD, Jr., Jackson WJ (1982b) Nitrate uptake, root and shoot growth, and ion balance of soybean plants during acclimation to root-zone acidity. Bot Gaz 143:5-14 Rufty TW, Jr., Siddiqi MY, Glass ADM, Ruth TJ (1991) Altered 3 N0 influx in ‘ phosphorus limited plants Plant Science 76:43-48 Rygiewicz PT, Bledsoe CS, Glass ADM (1984) A comparison of methods for compartmental analysis of Rb efflux from barley and Douglas-fir roots. Plant Physiol 76:913-917 Sahulka J (1977) The effect of some ammonium salts on nitrate reductase level, on in vivo nitrate reduction and on nitrate content in excised Pisum sativum roots. Biol Plant 19:113-128 Salisbury FB, Ross CW (1985) Plant Physiology, Ed3,Wadsworth Pubi Comp, Belmont Salsac L, Chaillou S, Morot-Gaudry JE, Lesaint C, Jolivet E (1987) Nitrate and ammonium nutrition in plants. Plant Physiol and Biochem 25:805-8 12 Sanders D (1980) The mechanism of C1 transport at the plasma membrane of Chara corallina. I. Cotransport with H. J Membr Biol 53:129-141 Sasakawa H, Yamamoto Y (1978) Comparison of the uptake of nitrate and ammonium by rice seedlings,--influences of light, temperature, oxygen concentration, exogenous sucrose, and metabolic inhibitors. Plant Physiol 62: 665-669 Sattelmacher B, Marschner H (1978) Nitrogen nutrition and cytokinin activity in Solanum tuberosum. Physiol Plant 42:185-189 Schachtman DP, Schroeder JI, Lucas WJ, Anderson JA, Gader RF (1992) Expression of an inwardly-rectifying potassium channel by the Arabidopsis KAT1 cDNA. Science 258:1654-1658 Schenk, M. and J. Wehrman (1979). The influence of ammonia in nutrient solution on growth and metabolism of cucumber plants. Plant Soil 52:403-414 Scherer HW, Leggett JE, Sims JL, Krasaesindhu P (1987) Interactions among ammonium, potassium and calcium during their uptake by excised rice roots. J Plant Nutr 10:67-8 1 Scherer HW, MacKown CT (1987) Dry matter accumulation, N uptake and chemical composition of tobacco grown with different N sources at two levels of K. J Plant Nutr 10:1-14 ,  Scherer HW, MacKown CT, Leggett JE (1984) Potassium-ammonium uptake interactions in tobacco seedlings. J Exp Bot 35:1060-1070 Schlee J, Cho B-H, Komor E (1985) Regulation of nitrate uptake by glucose in Chiorella. Plant Sci 39:25-30 Schlee J, Komor E (1986) Ammonium uptake by Chiorefla. Planta 168:232-238 Schrader LE, Domska D, Jung PE, Jr., Peterson LA (1972) Uptake and assimilation of ammonium-N and nitrate N and their influence on growth of corn (Zea mays L.). Agron J 64:690-695  255 Schubert KR, Coker III GT, Firestone RB (1981) Ammonia assimilation in Alnus glutinosa and Glycine max. Short-term studies using N[ammonium. 13 Plant [ Physiol 67:662-665 Scott TJ, Mitchell Mi, Santos A, Destaffen P (1989) Comparison of two methods for measuring ammonia in solution samples. Commun In Soil Sci Plant Anal 20:1131-1144 Shaff JE, Lucas WJ, Kochian LV (1993) Evidence for common transport systems for K and NH4 absorption in maize roots: an investigation utilizing extracellular vibrating K and NH 4 microelectrodes. Plant Physiol 102:5 94 Shen TC (1972) Nitrate reductase of rice seedlings and its induction by organic nitro compounds. Plant Physiol 49:546-549 Shimabukuro RH, Hoffer BL (1992) Effect of diclofop on the membrane potentials of herbicide-resistant and -susceptible annual ryegrass root tips. Plant Physiol 98:1415-1522 Shone MGT, Flood AV (1985) Measurements of free space and sorption of large molecules by cereal roots. Plant, Cell Environ 8:309-315 Siddiqi MY, ADM Glass, Ruth TJ (1991) Studies of the uptake of nitrate in barley. III Compartmentation of N0 . J Exp Bot 42(244):1455-1463 3 Siddiqi MY, Bryan JK, Glass ADM (1992) Effects of nitrite, chlorate, and chlorite on nitrate uptake and nitrate reductase activity. Plant Physiol 100:644-650 Siddiqi MY, Glass ADM (1984) The influence of monovalent cations upon influx and efflux of Ca 2 in barley roots. Plant Sci Letter 33:103-114 Siddiqi MY, Glass ADM (1986) A model for the regulation of K influx and tissue potassium concentrations by negative-feedback effects upon plasmalemma influx. Plant Physiol 8 1:1-7 Siddiqi MY, Glass ADM, Ruth TJ, Fernando M (1989) Studies of the regulation of nitrate influx by barley seedlings using 3 N0 Plant Physiol 90:806-8 13 ‘ . Siddiqi MY, Glass ADM, Ruth TJ, Rufty TW (1990) Studies of the uptake of nitrate in barley I.Kinetics of 13 3 influx. Plant Physiol 93:1426-143 2 N0 Silver S, Perry RD (1981) Tracer studies with 13NH4+, 42 K, and 28 : A bugs eye 2 Mg view of the periodic table. In JW Root, KA Krohn, eds, Advances in chemistry series No. 197: Short-lived radionuclides in chemistry and biology, Amer Chem Soc, Washington, DC, pp 453-468 Singh DT, Ghesh R, Singh HN (1987) Physiological characterization of the ammonium transport system in the free-living Diazotrophic Cyanobacterium Anabaena cycadeae. J Plant Physiol 127:231-239 Skokout TA, Wolk CP, Thomas J, Meeks JC, Schatter PW, Chien WS (1978) Initial organic products of assimilation of ‘ N ammonium and 3 3 N-nitrate by tobacco cell cultures on different sources of nitrogen. Plant Physiol 62:298-304 Slayman CL (1977) Energetics and control of transport in Neurospora. In AM Jungrreis, TK Hodges, A Kleinzeller, SG Schultz, eds, Water relations in  256 membrane transport in plants and animals. Academic Press, New York, pp 6986 Smart DR, Bloom AJ (1988) Kinetics of ammonium and nitrate uptake among wild and cultivated tomatoes. Oecologia 76:336-340 Smith FA (1973) The internal control of nitrate uptake into excised barley roots with differing salt contents. New Phytol 72:769-782 Smith FA (1982) Transport of methylammonium and ammonium ions by Elodea densa. J Exp Bot 33:221-232 Smith FA, MacRobbie EAC (1981) Comparison of cytoplasmic pH and C1 influx in cells of Chara corallina following ‘Cl--starvation’. J Exp Bot 32:827-835 Smith FA, Raven JA, Jayasuriya HD (1978) Uptake of methylammonium ions by Hydrodictyon africanum. J Exp Bot 29:12 1-122 Smith FA, Walker NA (1978) Entry of methylammonium and ammonium ions into Chara internodal cells. J Exp Bot 29: 107-120 Smith FW, Thompson JF (1971) Regulation of nitrate reductase in excised barley roots. Plant Physiol 48:219-223 Smith 1K (1975) Sulfate transport in cultured tobacco cells. Plant Physiol 55:303-307 Smith 1K (1980) Regulation of sulfate assimilation in tobacco cells. Plant Physiol 66:877-883 Solorzano L (1969) Determination of ammonia in natural waters by the phenol hypochlorite method. Limnology and Oceanography 14:799-801 Spanswick RM (1970) Electrophysiological techniques and the magnitudes of the membrane potentials and resistance of Nitella translucens. J Exp Bot 21:617627 Steer BT, Hocking PJ, Kortt AA, Roxburgh CM (1984) Nitrogen nutrition of sunflower (Helianthus annuus L.) yield components, the timing of their estabolishment and seed characteristics in response to nitrogen supply. Field Crop Res 9:219236 Steer MW (1981) Understanding cell structure, Cambridge Univ Press, Cambridge. Stevenson R, Silver S (1977) Methylammonium uptake by Escherichia coli: evidence 4 transport system. Biochem Biophys Res Commun 75:1133for a bacterial NH 1139 Steward GR, Mann AF, Fentem PA (1980) Enzymes of glutamate metabolism. In BJ Miflin, ed, The biochemistry of plants, Vol 5, Academic Press, New York, pp 271-327 Steward GR, Rhodes D (1976) Evidence for the assimilation of ammonia via the glutamine pathway in nitrate-grown Lemna minor L. FEBS Letters 64:296-299 Suzuki K, Tamate K, Nakayama T, Yamazaki T, Kasida Y, Fukushi K, Maruyama Y, Maekawa H, Nakaoka H (1983) Development of an equipment for the automatic  257 procedure of 3 NH and L-( ‘ N)-glutamate. J Labelled Compounds and 13 Radiopharmaceuticals 109:1375-1377 Suzuki Y, Yoshida H, Morooka M (1988) Heterosis for rate of nitrogen uptake in F 1 rice hybrids. Soil Sci Plant Nutr 34:87-95 Swiader JM (1986) Characterization on ammonium and nitrate absorption in Pumpkin (Cucurbita moschata Poir.). J Plant Nutr 9:103-113 Sze H (1984) H-translocating ATPases of the plasma membrane and tonoplast of plant cells. Physiol Plant 61:683-691 Ta TC, Ohira K (1981) Effects of various environmental and medium conditions on the response of Indica and Japonica rice plants to ammonium and nitrate nitrogen. Soil Sci Plant Nutr 27(3):347-355 Tanaka A, Patnaik S, Abichandani CT (1959) Studies on the nutrition of rice plant. Proc md Acad Sci B49:389-396 Tanchak MA, Griffing LR, Mersey BG (1984) Endocytosis of cationized ferritin by coated vesicles of soybean protoplasts. Planta 162:481-486 Tate SS, Meister A (1973) Glutamine synthetases of mammalian liver and brain. In S Prusiner, ER Stadtman, eds, The Enzymes of Glutamine Metabolism. Academic Press. New York and London, pp 77-127 Tester M (1990) Plant ion channels: whole-cell and single-channel studies. New Phytol 114:305-340 Teyker RH, Moll RH, Jackson WA (1989) Divergent selection among maize seedlings for nitrate uptake. Crop Sci 29:879-884 Thain JF (1984) The analysis of radioisotopic tracer flux experiments in plant tissues. J Exp Bot 3 5:444-453 Thayer J.R and R.C Huffaker 1982 Kinetic evaluation using ‘ N reveals two 3 assimilatory nitrate transport systems in Klebsiella pneurnoniae. J Bacteriology 149 (1):198-202 Thomas TE, Harrison PJ (1985) Effect of nitrogen supply on nitrogen uptake, accumulation in Porphyra perforata (Rhodophyta). Mar Biol 85:269-278 Thoresen S.S J.R Clayton,Jr and S.I Ahmed 1984 The effect of short-term fluctuations in pH on N0 3 uptake and intracellular constituents in Skeletonema costa turn (Grey.) Clever. J Exp Mar Biol Ecol 83:149-157 Tiedje  JM, Firestone RB, Firestone MK, Betlach MR, Smith MS, Caskey WH (1979) Methods for the production and use of nitrogen-13 in studies of denitrification. Soil Sci Soc Am J 43:709-7 15  Tiemeier DC, Smotkin D, Milman G (1973) Regulation of glutamine synthetase in Chinese hamster cells. In S Prusiner, ER Stadtman, eds, The Enzymes of Glutamine Metabolism. Academic Press. New York and London, pp 145-166 Tilbury RS (1981) The chemical form of ‘ N produced in various nuclear reactions 3 and chemical environments: a review. Adv Chem Ser 197  258 Tilbury RS, Dahl JR (1979) 13 N species formed by proton irritation of water. Radiation Research 79:22-33 Tolley LC, Raper CD, Jr (1985) Cyclic variations in nitrogen uptake rate in soybean plants. Plant Physiol 78:320-323 Tolley-Henry L, Raper Jr CD, Granato TC (1988) Cyclic variations in nitrogen uptake rate in soybean plants: effects of external nitrate concentration. J Exp Bot 39:613-622 Tolly-Henry L, Raper CD Jr (1986) Utilization of ammonium as a nitrogen source: Effects of ambient acidity on growth and nitrogen accumulation by soybean. Plant Physiol 82:54-60 Tromp J (1962) Interactions in the absorption of ammonium, potassium, and sodium ions by wheat roots. Acta Bot Neerl 11:147-192 Turgeon R (1989) The sink-source transition in leaves. Ann Rev Plant Physiol Plant Mol Biol 40:119-138 Ullrich WR, Larsson M, Larsson C-M, Lesch S, Novacky A (1984) Ammonium uptake in Lemna gibba G 1, related membrane potential changes, and inhibition of anion uptake. Physiol Plant 61:369-376 Ullrich WR, Novacky A (1981) Nitrate-dependent membrane potential changes and their induction in Lemna gibba G1. Plant Sci Let 22:211-217 Ullrich WR, Novacky A (1990) Extra- and intracellular pH and membrane potential changes induced by K, Cl, 4 P0 and NO uptake and Fusicoccin in root hairs 2 H , of Lirnnobiurn stoloniferurn. Plant Physiol 94:1561-1567 Usmanov lY (1979) Modification of the bioelectric and redox processes in certain grasses under the action of ammoniacal and nitrate nitrogen. Soviet Agricultural Science 9:8-11 Vaalburg W, Kamphuis JAA, Beerling-van der Molen HB, Reiffers S, Rijskamp A, Woidring MG (1975) An improved method for the cyclotron production of 1 3Nlabeled ammonia. mt j Am Rad Isot 26:316-318 Van Den Honert T.H J.J.M Hooymans and W.S Volkers (1955) Experiments on the relation between water absorption and mineral uptake by plant roots Acta Botanica Neerlandica 4:139 Vessey JK, Henry LT, Chaillou S, Raper CD, Jr. (1990a) Root-zone acidity affects relative uptake of nitrate and ammonium from mixed nitrogen sources. J Plant Nutr 13(1): 95-116 Vessey JK, Tolley-Henry L, Raper CD, Jr., Henry LT (1990b) Nitrogen nutrition and temporal effects of enhanced carbon dioxide on soybean growth. Crop Sci 30:287-294 Vose PB, Bresse EL (1964) Genetic variation in the utilisation of nitrogen by ryegrass species Loll urn perenne and Lolium nultiflorum. Ann of Bot 28:25 1-270  259 Walker NA, Beily MJ, Smith FA (1979a) Amine uniport at the plasmalemma of Charophyte cells: I. Current-voltage curves, saturation kinetics, and effect of unstirred layers. J Membrane Biol 49:21-55 Walker NA, Pitman MG (1976) Measurement of fluxes across membranes. In U Luttge, MG Pitman, eds, Encyclopedia of plant physiology, Vol 2. Part A. Springer, Berlin, pp 93-126 Walker NA, Smith FA, Beily MJ (1979b) Amine uniport at the plasmalemma of Charophyte cells: II. Ratio of matter to charge transported and permeability of free base. J Membrane Biol 49:283-296 Wang MY, Glass ADM, Shaff JE, Kochian LV (1992a) Electrophysiological studies of ammonium uptake by rice roots (abstract No. 404). Plant Physiol 99:S-68 Wang MY, Siddiqi MY, Glass ADM (1991) The mechanism of ammonium uptake by rice roots. (Abstract 957). Plant Physiol 96:S-145 Wang MY, Siddiqi MY, Glass ADM (1992b) Energetics of 13 4 uptake by rice roots NH (abstract No. 405). Plant Physiol 99:S-68 Wang MY, Siddiqi MY, Ruth TJ and Glass ADM (1993a) Ammonium Uptake by Rice Roots. I. Fluxes and Subcellular distribution of ‘ NH4. Plant Physiol 103:12493 1258 Wang MY, Siddiqi MY, Ruth TJ and Glass ADM (1993b) Ammonium Uptake by Rice Roots. II. Kinetics of 13 4 influx across the plasmalemma. Plant Physiol NH 103:1259-1267 Wang X, Below FE (1992) Root growth, nitrogen uptake, and tillering of wheat induced by mixed-nitrogen source. Crop Sci 32:997-1002 Wann M, Raper CD Jr., Lucas HL Jr. (1978) A dynamic model for plant growth: a simulation of dry matter accumulation for tobacco. Photosynthetica 12:12 1-136 Warncke DD, Barber SA (1973) Ammonium and nitrate uptake by Corn (Zea mays L.) as influenced by nitrogen concentration and 3 /NO ratio. Agron J 65:9504 NH 953 Warncke DD, Barber SA (1978) Ammonium and nitrate uptake by corn (Zea mays L.) as influenced by nitrogen concentration and NH4/N0 3 ratio. Agron J 65:950953 Weissman GS (1951) Nitrogen metabolism of wheat seedlings as influenced by the ammonium:nitrate ratio and the hydrogen ion concentration. Amer J Bot 38:162-174 Wells BR (1991) Ed, Arkansas Rice Research Studies, Arkansas Agricultural Experiment Station, Fayetteville, Arkansas, pp 1-188 Wheeler PA (1979) Uptake of methylarnine (an ammonium analogue) by Ma cr0 cystis pyrifera (Phaeophyta). J Phycol 15:12-17 Wheeler PA, McCarthy JJ (1982) Methylamine uptake by Chesapeake Bay phytoplankton: Evaluation of the use of the ammonium analogue for field uptake measurements. Limnol Oceanogr 27:1129-1140  260 Whoihueter RW, Schtt H, Hoizer H (1973) Regulation of glutamine synthesis in vivo in E. coli. In S Prusiner, ER Stadtman, eds, The Enzymes of Glutamine Metabolism. Academic Press. New York and London, pp 45-7 6 Wiame JM, Grenson M, Arst HN (1985) Nitrogen catabolite: repression in yeasts and filamentous fungi. Adv in Micro Physiol 26:1-88 Wiegel J, Kleiner D (1982) Survey of ammonium (methylammonium) transport by aerobic N -fixing bacteria-the special case of Rhizobium. FEMS Microb Lett 2 15:61-63 Wieneke J (1992) Nitrate fluxes in squash seedlings measured with 13 N. J Plant Nutr 15:99-124 Wilkinson L (1987) Systat: The system for Statistics. SYSTAT, Inc., Evanston, IL Willians BL, Wilson K (1981) A Biologist’s Guide to Principles and Techniques of Practical Biochemistry, Ed2, (Contemporary biology) Edward Arnold. pp 195200 Wright SA, SyrettPJ (1983) The uptake of methylammonium and dimethylammonium by the diatom Phaeodactyhum tn corn utum. New Phytol 95:189-202 Wyngaarden JB (1973) Glutamine phosphoribosylpyrophosphate amidotransferase. In S Prusiner, ER Stadtman, eds, The Enzymes of Glutamine Metabolism. Academic Press, New York and London, pp 365-386 Xu QF, Tsai CL, Tsai CY (1992) Interaction of potassium with the form and amount of nitrogen nutrition on growth and nitrogen uptake of maize. J Plant Nutr 15:23-33 Yoneyama T and C Sano (1977) Nitrogen nutrition and growth of rice plant. I. Nitrogen circulation and protein turnover in rice seedlings. Soil Sci Plant Nutr 23:237-245 Yoneyama T Kaneko A (1989) Variations in the natural abundance of 15N in nitrogenous fractions of Komatsuma plants supplied with nitrate Plant Cell Physiol 30:957-962 Yoneyama T Omata T Nakata S Yazaki J (1991) Fractionation of nitrogen isotopes during the uptake and assimilation of ammonia by plants Plant Cell Physiol 32:1211-1217 Yoneyama T, Akiryama Y, Kumazawa K (1977) Nitrogen uptake and assimilation by corn roots. Soil Sd Plant Nutr 23:85-9 1 Yoneyama T, Kumazawa K (1974) A kinetic study of the assimilation of 15 N-labelled ammonium in rice seedling roots. Plant Cell Physiol 15: 655-661 Yoneyama T, Kumazawa K (1975) A kinetic study of the assimilation of 15 N-labelled nitrate in rice seedling. Plant Cell Physiol 16: 21-26 Yoneyama T, Sano C (1978a) Nitrogen nutrition and growth of rice plant II Consideration concerning the dynamics of nitrogen in rice seedling. Soil Sci Plant Nutri 24:191-198  261 Yoneyama T, Sano C (1978b) Nitrogen nutrition and growth of rice plant Ill. Origin of amino-acid nitrogen in the developing leaf. Soil Sci Plant Nutri 24:199-205 Yong M, Sims AP (1972) The potassium relations of Lemna minor L. I. Potassium uptake and plant growth. J Exp Bot 29:885-894 Yoshida S, Forno DA, Cock JH, Gomez KA (1972) Laboratory manual for physiological studies of rice, Ed2, The International Rice Research Institute, Los Baños, Philippines Young GM, Sims AP (1970) The potassium relations of Lemna minor L. I. Potassium uptake and plant growth. J Exp Bot 23:958-969 Youngdahl U, Pacheco R, Street JJ, Viek PLG (1982) The kinetics of ammonium and nitrate: uptake by young rice plants. Plant and Soil 69: 225-232 Youssef R.A and M Chino 1989 Root-induced changes in the rhizosphere of plants I pH changes in relation to the bulk soil Soil Sci Plant Nutr 3 5:461-468 Yu TR (1985) Chapter 10 Soil and Plants. In T-R Yu, ed, Physical Chemistry of Paddy Soils. Science Press, Beijing, pp 197-217 Zar JH (1974) Biostatistical Analysis. Prentice-Hall, Inc., Englewood, Cliffs, New Jersey, pp 228-236 Zierler K (1981) A critique of compartmental analysis. Ann Rev Biophys Bioeng 10:531-562 Zsoldos F, Haunold E, Herger P, Vashegyi A (1990) Effects of sulfate and nitrate on K uptake and growth of wheat and cucumber. Physiol Plant 80:42 5-430 Zsoldos F, Vashegyi A (1990) Effects of pH and nitrate on potassium and phosphate uptake and growth of rice seedlings Acta Univ Szeged Acta Biol 3 6:95-98  262  Appendix A. Reported studies on using radioactive isotope ‘ N 3 Year  Author  Berkeley, U.S.A. 1940 Ruben et al, England 1961 Nicholas et al, Manitoba, Canada 1967 Campbell et al, Michigan, U.S.A. 1974 & 76 Wolk et al, 1975 & 77 Thomas et al, 1977 & 78(a) Meeks et al, 1978 (b) Meeks et al, 1978 Skoukit et al, 1979 Hanson et al, 1979 & 81 Tiedje et al, 1981 Schubert et al, Davis, U.S.A. 1976 Gersberg et al, 1978 Gersberg et al, 1982 Thayer&Huffaker Meeks et al, 1985 Lower Hutt, New Zealand 1981 McCallum et al, 1983 & 85 McNaughton et al, 1984 & 86 Presland et al, Quebec, Canada 1984 Caidwell et at, Vancouver, Canada 1985 Glass et al, Siddiqi et al, 1989 Wantage, England 1986 (a,b) Lee et al, Stockholm, Sweden 1987 Oscarson et al., 1988 (a,b) Ingemarsson et al., New York, U.S.A. 1989 Calderon et al, Houston, U.S.A. 1990 Hole et at, Jülich, Germen. 1992 Wieneke  N Species  Objective  Material  2 N 13  -Fixation 2 N  Non-legume  2 N 13  -Fixation 2 N  Bacteria  2 N 13  -Fixation 2 N  Microorganism  13 2 N 2 N 13 2 N 13 2 N 13 13 , 3 NO 13 4 NH 3 NH 13 N 13 4 NH 13  -N 2 Fixation -Fixation 2 N -Fixation 2 N -Fixation 2 N N assimilation Translocation Denitrification -Fixation 2 N  Blue-green algae Blue-green algae Blue-green algae Soybean Tobacco cells Barley soils Non-legume  13 3 N0 N 13 3 N0 13 3 N0 13  Denitrification N assimilation 3 transport N0 Translocation  Flooded rice soil Phytoplankton Kiebsiella Cynobacteria  NH 1 , 3 N0 13 + 4 3 3 N0 13  Denitrification N uptake, Flux N uptake, Flux  Soils Maize Maize  -Fixation 2 N ‘ , N 3 t 4 2 N ’ ’ H 0 N  Alfalfa  13 3 N0 3 N0 13  N uptake, Flux N uptake, Flux  Barley Barley  3 N0 13  N uptake, Flux  Barley  13 3 N0 3 N0 13  N uptake, Flux N uptake, Flux  Pisum Lemna  N-glutamine 13  assimilation  Neurospora crassa  3 NO 13  3 transport NO  Maize  3 NO 13  3 transport NO  Squash  263 Appendix B Reported values of half-life (t ) and ion content (0) of 172 various compartments of root cells. Superficial  Free space  Cytoplasm t  (sec) K  onion barley barley Na onion barley barley Ca onion Mg onion onion Cl3 barley N0 barley  15  43  3.3  19  2.8  13 18 18-52  1.3  -  1/2  (mm)  (mm)  7.3  82 103 25- 75 29 30 23 18 20- 22 17- 25 54 56 74 90-104  -  -  -  18 12  -  -  3.7  -  1.5 3.2 9.5-17.9 -  Vacuole  -  -  (h) 80 108 14- 30 23 11 326 362 77-231 35-390 12 30 49- 71 68-137 -  -  -  -  Q(p,mol g-’) K-’-  onion barley Na onion barley barley Ca onion Mg onion Clonion 3 barley N0 barley  0.2-1.4  <0.1  -  0.8  0.3-1.0 <0.1  -  0.3  0.8-1.3 11.0-22.0 <0.4 0.1 0.6 3.6 0.5-2.8 1.6 2.5 0.32 <0.1 0.1 -  -  1.2 1.3 0.23 <0.1 0.4 -  -  0.4 0.6 0.05 <0.1 0.1 -  -  -  -  72.7-73.1 28.0-95.6 34.3 34.9 37.9 76.0 25.0-46.0 5.4 5.9 11.1 11.2 68 137 -  -  -  -  -  

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