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Ammonium uptake by rice roots Wang, Miao Yuan 1994

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AMMONIUM UPTAKE BY RICE ROOTSbyMIA0 YUAN WANGB.Sc. Zhejiang Agricultural University, Hangzhou, 1981M.Sc. University of Saskatchewan, Saskatoon, 1987A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(DEPARTMENT OF BOTANY)We accept this thesis asconforming to the required standardTHE UNIVERSITY OF BRITISH COLUMBIAJUNE 1994© M. Y. WANG, 1994In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Ubrary shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives, It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of__________________The University of British ColumbiaVancouver, CanadaDateDE-6 (2/88)ABSTRACT13NH4 uptake was studied using 3-week-old rice plants (Oryzasativa L. cv. M202), grown hydroponically in modified Johnson’s nutrientsolution containing 2, 100 or 1000 !IM NH4 (referred to hereafter as G2,G100 or G1000 plants, respectively). At steady-state, the influx and effluxof 13NH4was increased as NH4-’- provision during growth was increased.The half-life of cytoplasmic‘3NH4 exchange was calculated to be 8 mmwhile the half-life for cell wall exchange was 1 mm. Cytoplasmic [NH4-’-] ofG2, G100 and G1000 roots was estimated to be 3.72, 20.55, and 38.08 mMrespectively. However about 72% to 92% of total root NH4 was located inthe vacuole. During a 30 minute period G100 plants metabolized 19% of thenewly absorbed 13NH4-’- and the remainder was partitioned among thecytoplasm (41%), vacuole (20%) and efflux (20%). Of the metabolized 13N,roughly one half was translocated to the shoots.In short-term, perturbation experiments, below 1 mM externalconcentration ([NH4+]0),13NH4÷ influx of G2, G100 and G1000 roots wassaturable and operated by means of a high affinity transport system(HATS). The Vmax values for this transport system were negativelycorrelated and Km values were positively correlated with NH4 provisionduring growth and root [NH4+]. Between 1 and 40 mM [NH4+]0,13NH4influx showed a linear response to external concentration due to a lowaffinity transport system (LATS). The 13NH4-’- influxes by the HATS, and toa lesser extent the LATS, are energy-dependent processes. Selectedmetabolic inhibitors reduced influx of the HATS by 50 to 80%, but of theLATS by only 31 to 51%. Estimated Q10 values for HATS were greater than112.4 at root temperatures from 5 to 10°C and constant at 1.5 between 5 to30°C for the LATS. Influx of 1NH4÷ by the HATS was insensitive toexternal pH in the range from 4.5 to 9.0, but influx by the LATS declinedsignificantly beyond pH 6.0.The transmembrane electrical potential differences (z’P) of epidermaland cortical cells of intact roots were in the range from -120 to -140millivolts (mV) in the absence of NH4-’- in bathing solution and were -116mV and -89 mV for G2 and G100 plants in 2 and 100 jiM NH4 solutions,respectively. Introducing NH4-’- to the bathing medium caused a rapiddepolarization which exhibited a biphasic response to external [NH4--]. Plotsof membrane depolarization versus ‘3NH4 influx were also biphasic,indicating distinct coupling processes for the two transport systems, with areak-point between the two concentration ranges around 1 mM NH4.Depolarization of z’P due to NH4uptake was eliminated by a protonophore(carboxylcyanide-m-chlorophenylhydrazone), inhibitors of ATP synthesis(sodium cyanide plus salicylhydroxamic acid), or an ATPase inhibitor(diethyistilbestrol).‘3NH4 influx was regulated by internal ammonium and its primarymetabolites, amides and amino acids. When internal amide or amino acidsconcentrations were increased, the influx of 13NH4was reduced. However,treating rice roots with L-Methionine DL-Sulfoximine (MSX) reduced thelevels of ammonium assimilates but did not increase 13NH4 influxprobably because internal [NH4-’-] was increased. Short-term nitrogendepletion stimulated1NH4influx, but long-term N depletion caused NH4influx to be reduced probably due to N limitation of carrier synthesis. Acascade regulation system is proposed to explain the multi-level regulationof NH4 influx.111The interaction between ammonium and potassium showed thatwhen N is adequate, K promoted NH4 uptake and utilization. Likewise,proper N nutrition promoted K-’- uptake but the presence of NH4 in uptakesolution strongly inhibited the K(86Rb+) uptake at the transport step. Theresults indicated that NH4 and K-I- may share the same channel but areregulated by different feedback signals.ivTABLE OF CONTENTSAbstract iiTable of Contents vList of abbreviation xiiList of Tables xivList of Figures xvDedication xviiiAcknowledgment XixChapter 1. RESEARCH BACKGROUND 11.1. General Introduction 11.1.1. Rice 11.1.2. Essentiality of nitrogen 11.1.3. Necessity of N fertilization 21.1.4. Bio-availability of nitrogen 21.2. Ammonium Uptake 31.2.1. Importance of transport research 31.2.2. Transport of NH4 by lower plants 41.2.3. Transport of NH4 by higher plants 51.2.3.1. Carrier-mediated transport 51.2.3.2. Concentration-dependent kinetics 61.2.3.3. Depolarization of membrane potential 61.2.3.4. Energy dependence 71.3. Major Factors Affecting Ammonium Uptake 71.3.1. Effects of photosynthesis 71.3.1.1. Dependence on soluble carbohydrates 71.3.1.2. Periodic variations of light and growth 81.3.1.3. Ambient environmental factors 91.3.2. Effects of root temperature 101.3.2.1. Short-term perturbation 101.3.2.2. Qio value for NH4 uptake 101.3.2.3. Long-term low temperature effects 111.3.3. Effects of pH on NH4 uptake 121.3.3.1. Acidification of rhizosphere by NH4 uptake 12V1.3.3.2. Retarded plant growth in acidic medium 131.3.3.3. NH4 toxicity and acidic damage 131.3.4. NH4 fluxes at the plasma membrane 141.3.4.1. Net flux 141.3.4.2. Influx 141.3.4.3. Efflux 151.3.4.4. Balance of fluxes 151.3.4.5. N cycling in the whole plant 161.3.5. Regulation of ammonium uptake 171.3.5.1. Negative feedback regulation 171.3.5.2. Enhanced NH4 uptake 171.3.6. Interaction between NH4 and K 181.3.6.1. Mutual beneficial effects between N and K 181.3.6.2. Inhibition of K uptake by NH4 181.3.6.3. Inhibition of NH4 uptake by K 191.4. Research Objectives 19Chapter 2. MATERIALS AND METHODS 222.1. Plant Growth 222.1.1. Seed germination 222.1.2. Growth conditions 222.1.3. Provision of nutrients 232.2. N Isotopes For Studying N Uptake 242.2.1. Isotopic tracer 242.2.2. Nitrogen Isotopes 242.2.3. Stable ‘5N techniques 252.2.4. Radioactive isotope, 13N 262.2.4.1. Use in biological studies 262.2.4.2. Production of 1N 272.2.4.3. Advantages of the use of 13N in biological studies 292.2.4.4. Considerations of using ‘3N in nitrogen uptake 302.2.4.5. Use of 13N in nitrogen transport studies 312.2.4.6. Use of ‘3N in nitrogen assimilation 322.2.4.7. Use of 1N in denitrification 332.2.5. Protocol for‘3NH4production in present study 33vi2.3. Measurement Of NH4 Fluxes 352.3.1. Influx of 13NH4 352.3.2. Effluxof’3NH4-’- 352.3.3. Net flux of NH4÷ 362.4. Compartmental (Efflux) Analysis 362.4.1. Compartmentation of plant cells 362.4.2. Development of theory 372.4.3. Models for compartmental analysis 382.4.4. The general procedure of compartmental analysis 422.4.5. Procedures for compartmental analysis in the presentstudy 442.5. Determination Of Ammonium 462.6. Preparation Of Metabolic Inhibitors 462.7. Electrophysiological Study 472.7.1. Transmembrane electrical potential measurement 472.7.2. Single impalement and membrane potential 532.7.3. Setup for measuring membrane potential 542.8. Determination of amino acids in root tissue 55Chapter 3. FLUXES AND DISTRIBUTION OF 13NH4 IN CELLS 573.1. Introduction 573.2. Materials And Methods 593.2.1. Plant growth and ‘3N production 593.2.2. Measurement of fluxes 593.2.2.1. ‘3NH4influx 593.2.2.2. Net NH4 flux 593.2.2.3. Time course of 13NH4uptake 603.2.3. Compartmental analysis 603.2.4. Partition of absorbed1NH4 603.2.4.1. Separation of‘3N-compounds in plant tissue 603.2.4.2 Chemical assay of NH4 in root tissue 613.2.5. Calculation of flux to vacuole (Øcv) 61vii3.3. Results 623.3.1. Compartmental analysis 623.3.2. Metabolism and translocation of ‘3N 713.3.3. Time course of‘3NH4influx in rice roots 713.4. Discussion 753.4.1. The half-lives of 13NH4÷exchange 753.4.2. Fluxes of‘3NH4into root cells 783.4.3. The NH4 pools in roots 823.4.4. Model of‘3NH4uptake by rice plants 833.5. SUMMARY 864. KINETICS OF‘3NH4INFLUX 884.1. Introduction 884.2. Materials And Methods 904.2.1. Plant growth and 1N production 904.2.2. Relative growth rate 904.2.3. Influx measurement 914.2.4. Kinetic study 914.2.5. Metabolic inhibitor study 924.2.6. Temperature study 934.2.7. pH profile study 934.3. Results 944.3.1. Kinetics of 13NH4 influx 944.3.2.1. HATS 944.3.1.2. LATS 984.3.2. Effect of metabolic inhibitors on the influx of 13NH4 984.3.3. Effect of root temperature on‘3NH4influx 1014.3.4. Effect of solution pH on‘3NH4influx 1044.4. Discussion 1044.4.1. Kinetics of ammonium uptake 1044.4.2. Energetic of ammonium uptake 1074.4.3. Effect of pH profile on ammonium uptake 1114.4.4. Regulation of ammonium uptake 112viii4.5. Summary 114Chapter 5. ELETROPHYSIOLOGICAL STUDY 1155.1. Introduction 1155.2. Materials And Methods 1165.2.1. Growth of plants 1165.2.2. Measurements of cell membrane potential 1175.2.3. Experimental treatments 1185.2.3.1. Effect of [NH4+]0on A’F 1185.2.3.2. Effect of accompanying anion on A’P 1185.2.3.3. Effects of metabolic inhibitors on NH4-induced APdepolarization 1195.3. Results 1205.3.1. Transmembrane electrical potentials of rice roots 1205.3.2. Contribution of the accompany anions to AW 1205.3.3. Effect of [NH4C1]0on A’P 1235.3.4. Effect of metabolic inhibitors on Z’{’ 1265.4. Discussion 1305.4.1. Anion effect 1305.4.2. Depolarization of A’P by HATS and LATS 1315.4.3. Calculation of the free energy for NH4-’- transport 1355.4.4. Mechanisms of NH4 uptake by HATS and LATS 1385.5. Summary 139Chapter 6. REGULATION OF AMMONIUM UPTAKE 1416.1. Introduction 1416.2. Materials And methods 1436.2.1. Plant growth and 13N production 1436.2.2. Experimental design 1446.2.2.1. Experiment I. Depletion and repletion study 1446.2.2.2. Experiment II. Effects of MSX 1446.2.2.3. Experiment III. Effects of exogenous amino acids 1456.2.2.4. Experiment IV. Effects of selected inhibitors 1456.2.3. Determination of free ammonium in root tissue 1456.2.4. Determination of amino acids in root tissue 1466.3. Results 1466.3.1. Experiment I. Depletion and repletion study 1466.3.2. Experiment II. Effects of MSX 156lx6.3.3. Experiment III. Effects of exogenous amino acids 1636.3.4. Experiment IV. Effects of selected inhibitors 1726.4 Discussion 1766.4.1. Negative feedback on NH4 uptake by NH4assimilates 1766.4.2. Effect of MSX: reduced amino acid pool 1786.4.3. Effect of short-term N depletion 1816.4.4. Stimulated NH4 influx after long-term N depletion 1836.4.5. Negative feedback on 1NH4 influx from internalNH4-I- 1856.4.6. Cascade regulation system of nitrogen uptake 188Chapter 7. INTERACTION BETWEEN K AND NH4 1937.1. Introduction 1937.2. Materials And Methods 1947.2.1. Plant growth and 1N production 1947.2.2. Experimental design 1947.2.1.1. Experiment I: Effects of K and NO3- in pretreatmentand K-- and NH4÷ in uptake solutions on net K+ andNH4 fluxes 1957.2.1.2. Experiment II: Effects of NH4 provision duringgrowth and of K-- and NH4 in pretreatment anduptake solutions on 86Rb (K+) influxes 1957.2.1.3. Experiment III: Effects of NH4 provision duringgrowth and presence in uptake solution upon influxisotherms for 86Rb (K+) 1967.2.1.4. Experiment IV: Effects of NH4 provision duringgrowth and short-term pretreatment upon 86Rb(K) influx 1967.2.1.5. Experiment V: Effect of NH4 concentrations presentin uptake solution upon influx isotherms for 86Rb(K-I-) 1967.2.1.6. Experiment VI: Effects of K provision during growthand presence in uptake solutions upon influxisotherms for1NH4 1977.3. Results 1977.3.1. Experiment I: Effects of K and N03 in pretreatmentand K-I- and NH4-1- in uptake solutions on net K+ andNH4 fluxes 197x7.3.2. Experiment II. Effects of NH4 provision during growthand of K and NH4 in pretreatment and uptakesolutions on 86Rb+ (K) influxes 2007.3.3. Experiment III: Effects of NH4 provision duringgrowth and presence in uptake solution upon influxisotherms for86Rb+ (K) 2037.3.4. Experiment IV: Effects of NH4 provision duringgrowth and short-term pretreatment upon S6Rb+(K) influx 2067.3.5. Experiment V: Effect of NH4 concentrations present inuptake solution upon influx isotherms for 86Rb(K) 2067.3.6. Experiment IV: Effects of K provision during growthand presence in uptake solutions upon influxisotherms for1NH4 2107.4. Discussion 2167.4.1. Plant growth in response to provisions of NH4 and K 2167.4.2. Effect of plant N status on K(86Rb) uptake 2187.4.3. Effect of NH4 in the uptake solution on K(86Rb+)uptake 2207.4.4. Effect of K on NH4 uptake 2227.4.5. Shared transport and different feedback signal? 224Chapter 8. GENERAL CONCLUSIONS 226REFERENCES 228APPENDIX A. Reported studies on using radioactive isotope ‘3N 262APPENDIX B. Reported values of half-life (t1/2) and ion contnt (Q.) ofvarious compartments of root cells 263xiAbbreviationsAA amino acidsAFS Appearent free spaceAOA amino-oxyacetateArg ArginineAsn AsparigineAsp AspatarteAzaserine O-diazoacetyl-L-serine;CCCP carboxylcyanide-m-chlorophenyl-hydrazone;CN (sodium) cyanide;DES diethyistilbestrol;DMRT Duncan’s multiple range test.DNP 2 ,4-dinitrophenol;DON 6-diazo-5-oxo-L-norleucine;transmembrane electrical potential difference;mass rate of assimilation of‘3NH4in roots;flux across the tonoplast into vacuole;translocation of 13N labeled metabolites to xylem (shoots);Poc, ‘P, and net inward, outward and net fluxes (iimol g’FW h-i)across the plasmalemma, respectively;G2, G100 and G1000 plants rice seedlings grown in MJNS containing2, 100 or 1000 jiM NH4, respectively;G2M, GlOOM and G1000M MJNS containing 2, 100 or 1000 jiMNH4, respectively, as growth media;GDH glutamate dehydrogenase (GDH; EC 1.4.1.2)Gln GlutamineGlu GlutamateGOGAT glutamate synthase;GS glutamine synthetase;HATS or LATS high affinity or low affinity NH4 transport systems,respectively;Km the external ion concentration giving half of the maximumrate (jiM);LSD Least significant difference;MA methylamineMJNS modified Johnson’s nutrient solution;xiiMSX L-Methionine DL-SulfoximineNiR nitrite reductaseNR nitrate reductasepCMBS p-chloromercuribenzene-sulfonate;Qj, Q, Q3., ammonium contents (jimol g’FW) of root, cytoplasm andvacuole, respectively;SHAM salicyihydroxamic acid;S0 and Sradioisotopic specific activities of external media andcytoplasmic compartments, respectively;Vmax the calculated maximum rate of ion influx (jimol g-’FW h-’);[NH4] cytoplasmic ammonium concentration (iiM or mM);[NH4J1 root (internal) ammonium concentration (1i,M or mM);[NH4]0 external ammonium concentration (jiM or mM);[NH4] vacuolar ammonium concentrations (jiM or mM);xliiList of TablesTable 1. Separation of 13N-labeled compounds by cation exchangecolumn. 64Table 2. Estimated half-lives of 1NH4 exchange for threecompartments of root cells. 66Table 3. Comparison of1NH4 fluxes across the plasmalemma ofroot cells. 67Table 4. Size of ammonium pools in root cells at steady-state. 70Table 5. Calculation of the flux (P,) from cytoplasm into vacuole. 72Table 6. Distribution of newly absorbed 13N in shoot and roottissues. 73Table 7. Kinetic parameters for‘3NH4 influx of G2, G100, G1000plants. 96Table 8. Reduction of‘3NH4-’- influx by metabolic inhibitors. 102Table 9. Qo values for‘3NH4influx by the HATS or LATS. 103Table 10. Effect of uptake solution pH on 13NH4 influx. 105Table 11. Membrane potentials of G2 and G100 plants measured indifferent bathing solutions. 121Table 12. Effect of metabolic inhibitors on the depolarization of A’P. 129Table 13. Net 86Rb flux of rice plants grown with or without eitherpotassium and ammonium. 198Table 14. Net NH4 flux of rice plants grown with or without eitherpotassium and ammonium. 199Table 15. Michaelis-Menten kinetic parameters for 86Rb influx ofplants grown in different levels of NH4-’- and K. 208Table 16. Effects of NH4-- and K on plant growth. 211Table 17. Michaelis-Menten kinetic parameters for‘3NH4-’- influx ofplants grown in different levels of potassium andammonium. 213xivList of FiguresFigure 1. Scheme of‘3NH4convertion in laboratory. 34Figure 2. Diagrame of the setup for measuring cell membranepotential. 56Figure 3. A represented pattern of 13NH4released intact roots. 65Figure 4. Fluxes of G2, G100 and G1000 plants at steady-state. 69Figure 5. Cumulative uptake of1NH4by G2 and G100 roots. 74Figure 6. Influxes of‘3NH4 into G2 and G100 roots at steady-state. 76Figure 7. Proposed model for ammonium uptake andconpartmentation in rice roots. 84Figure 8. Concentration dependence of 13NH4 influx at low range(<1 mM). 95Figure 9. Relationship between kinetic parameters of NH4 uptakeand root ammonium concentrations of rice seedlings. 97Figure 10. Concentration dependence of 13NH4 influx at low range(>1mM). 99Figure 11. Effect of metabolic inhibitors on‘3NH4influx. 100Figure 12. Effects of some anions on AW depolarization. 122Figure 13. The A’P depolarization of root cell by NH4C1. 124Figure 14. Concentration dependence of net A’P depolarization ofroot cells. 125Figure 15. Effects of metabolic inhibitors on A’F depolarization ofroot cells. 127Figure 16. Effects of metabolic inhibitors on &{-‘ depolarizationinduced by NH4C1. 128Figure 17. The relationship between ‘3NH4 influx and zPdepolarization at the same [NH4]0. 134xvFigure 18. Free energy requirement for NH4 uptake as a function ofexternal {NH4+J. 136Figure 19. 13NH4 influx of repleted G2 plants. 147Figure 20. 1NH4influx of depleted G1000 plants. 148Figure 21. Internal ammonium content of repleted G2 plants. 149Figure 22. Total amino acid concentration ([AA]1) of repleted G2plants. 151Figure 23. 13NH4+ influx (23A) and internal ammonium content(23B) of repleted G2 or depleted G1000 roots. 153Figure 24. Total [AA]1 of repleted G2 or depleted G1000 roots. 154Figure 25. Tissue amide or amino acid contents of repleted G2 ordepleted G1000 roots. 155Figure 26. Effect of MSX on1NH4influx of rice roots. 158Figure 27. Effect of MSX on ammonium content of rice roots. 159Figure 28. Effect of MSX on total [AA]1 of rice roots. 160Figure 29. Effect of MSX on root content of amide or amino acid. 161Figure 30. Effect of exogenous glutamine on root 13NH4 influx. 164Figure 31. Effect of exogenous glutamine on root contents of amideand amino acid. 165Figure 32. Effects of exogenous glutamine on‘3NH4influx. 166Figure 33. Effects of exogenous amides and amino acid on rootammonium content. 168Figure 34. Effects of exogenous amides and amino acid on totalamino acid content. 169Figure 35. Effects of exogenous amides and amino acid on aminoacid content. 170Figure 36. Effects of exogenous amides and amino acid on aminoacid content. 171Figure 37. Effects of MSX, DON and AOA on 13NH4influx. 173xviFigure 38. Effects of MSX, DON and AOA on internal ammonium andtotal amino acid content. 174Figure 39. Effects of MSX, DON and AOA on major amino acidscontent. 175Figure 40. Effect of NH4 in the growth media, pretreatment anduptake solutions on 86Rb influx. 201Figure 41. Effects of NH4÷ and K in growth media and uptakesolutions on 86Rb influx. 202Figure 42. Relationship between estimated Vm of 86Rb influx androots internal {K÷]. 204Figure 43. Effect of short-term NH4 pretreatment on 86Rb influx. 205Figure 44. Effects of NH4 and K in growth media and uptakesolutions on 86Rb uptake isotherm. 207Figure 45. Effects of NH4 and K in growth media and uptakesolutions on 86Rb translocated to shoots. 209Figure 46. Effect of K in uptake solution on‘3NH4influx isotherm. 212Figure 47. Effect of K in uptake solution on‘3NH4influx by HATS 214Figure 48. Effect of K in uptake solution on ‘3NH4 influx ByHATS+LATS. 215xviiTo my wife, Xiao Gefor her love, understanding and sacrificexviiiACKNOWLEDGMENTSMy sincere gratitude to research supervisor Dr. A.D.M. Glass, for hisguidance, encouragement and moral support throughout this project. Histime and patience in editing this thesis is greatly appreciated. Gratitude isextended 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 mustbe expressed to Dr. M. Y. Siddiqi, for his invaluable suggestions anddependable assistance.The financial assistance provided by the Potash & PhosphateInstitute 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 agenerous gift during this research project.To perform experiment using 13N, with a half-life of 9.98 mm,requires team-work. Special appreciation is extended to member of the‘13N brigade’: Mala Fernando, Bryan J. King, Hebert Kronzucker, JarnailMehroke for ‘lending a hand’ and ‘sparkling’ discussions. My thanks also goto the Botany workshop, Mr. Mel Davis and Ken Jeffries for theirwillingness and skillfulness to help me out in my technical problems.Many thanks is due to the team in TRIUMF, UBC, who provided 13Nfor this study. I greatly appreciate willingly cooperation from MichaelAdams, Tamara Hurtado, Salma Jivan and other team members during ‘3Nproduction and transportation. My gratitude also extends to theRadiological unit in University Hospital, UBC site, for allowing me to pick up‘Red Rabbiter’ in their terminal of the underground Pipe-line. Theappreciation is also extended to Drs. John Hobbs and Krystyne Piotrowskafor amino acids analysis.I would like to express my sincere appreciation to the U.S. Plant Soiland Nutrition Laboratory, USDA-ARS, Cornell University, and particularlyto Dr. L.V. Kochian for hosting me as a visiting scientist to carry out theelectrophysiological study in his laboratory. My special gratitude extend toMr. J. E. Shaff, who patiently taught me how to operate various items ofequipment and for kindly looking after me during my stay in Ithaca. Ixixwould also like to thank Drs. J.W. Huang and P. Ryan, Ms. L. Armstrong andMr. T. Toulemonde for assistance and discussions.I am grateful to fellow graduate students as well as staff and facultymembers of the Botany department for their support and friendship.Iwish to express my gratitude to former colleagues in Soil and FertilizerInstitute, Zheijiang Academy of Agricultural Sciences.A very special thanks is to all my family members back home inChina. I am grateful to all my friends, particular to families of Bill andKris,Jame and Jill, Warren and Liz, who treated me like a brother and gavemeand my family strong support in many aspects.At last, but not least, my special gratitude must be expressed to mywife, Xiao ge and my son, Li ren for their love and support all through thisprogram.xx1Chapter 1. RESEARCH BACKGROUND1.1. GENERAL INTRODUCTION1.1.1. RiceRice (Oryza sativa Linneaus) is a semi-aquatic, annual grass plant inthe family Poaceae (formerly Graminae). Rice is grown in over 100countries on every continent except Antarctica, extending from 5 3°N to35°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) orlowland (wet) crop in the tropic, subtropics, temperate, and subtemperatezones and on plains, hilly regions, and plateaus. About 53% of total landarea 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% ofthe world’s population (De Datta, 1981, 1986b); it supplies the energysource for more than half of the world’s population and provides 75% ofthe caloric intake of Asia’s over two billion people (Buresh and De Datta,1991).1.1.2. Essentiality of nitrogenNitrogen is required for the synthesis of amino acids, proteins,nucleic acids and many secondary plant products such as alkaloids. It isinvolved in the whole life cycle of plants; in enzymes for biochemical2processes, in chlorophyll for photosynthesis, and in nucleoproteins for thecontrol of hereditary and developmental processes. Since N is present in somany essential compounds, it is not surprising that growth withoutsufficient N is slow. Nitrogen is the single most important chemical elementlimiting crop yield.1.1.3. Necessity of N fertilizationProper application of N increases both yield and protein content ofrice (Patrick et al., 1974; Gomez and De Datta, 1975; Allen and Terman,1978). The intensification of rice production has involved a tremendousincrease in the use of nitrogen fertilizers and the selection of high yieldingvarieties that are highly responsive to nitrogen. However, research on theeffects of nitrogen fertilizers on rice production has focused mainly on theagronomic context, in terms of grain yield, carbohydrate metabolism,growth patterns or morphological characteristics. Information concerningphysiological and biochemical aspects of nitrogen uptake by rice as well asother higher plants, is limited, which is unfortunate since these details mayprove to be important for the production of new varieties with improvednitrogen utilization.L1.4. Blo-availability of nitrogenAmmonium is the predominant and most readily bio-availablenitrogen form in paddy soil; it is the preferred nitrogen species taken upby rice plants (Sethi, 1940; Sasakawa and Yamomoto, 1978; Goyal andHuffaker, 1984). Besides NH4, rice roots also absorb N03 (Malavolta,31954) and organic nitrogen such as urea, Gin and Arg (Arima andKumazawa, 1977; Mon et al., 1979; Mon and Nishizawa, 1979; Harper,1984).1.2. AMMONIUM UPTAKE1.2.1. Importance of transport researchInformation on the ammonium transport system(s) of root cells ofrice, and their regulations, is meagre. Moreover, the relationships amonguptake, assimilation and other metabolic processes are not as wellunderstood as is the case for other plant nutrients. To understand theammonium transport system(s), generally, it is necessary to characterizetheir 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 bedetermined. Where transport mutants are available, the genetic basis ofthe transport system(s) can be evaluated (Kleiner, 1981, 1985; Glass,1988). This information satisfies more than the researcher’s curiosity; itprovides a better understanding of ammonium uptake for thedevelopment of better fertilization practice and improved varietyselection.41.2.2. Transport of NH4 by lower plantsAmmonium 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 beaccumulated against its concentration and electrochemical potentialgradients, resulting in significant ammonium concentration within plantcells (Smith and Walker, 1978; Pelley and Bannister, 1979; Kleiner, 1981;Boussiba et al., 1984). NH4 uptake is concentration dependent and itsisotherm in the low range of external concentration conformed toMichaelis-Menten kinetics (Hackette et al., 1970; Dubois and Grenson,1979; Felle, 1980; Fuggi et al., 1981; Smith, 1982; Box, 1987). NH4transport across the plasma membrane has been claimed to occur via anelectrogenic uniporter which depolarizes membrane electrical potentialdifferences (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 Qovalue for NH4+ uptake has been reported to be 2.0 (Hackette et al., 1970)and ATP may be involved in the transport step, hence the uptake systemis inhibited by anaerobiosis or several metabolic inhibitors (Stevenson andSilver, 1977; Cook and Anthony, 1978a; Felle, 1980). The responses of NH4uptake to pH changes is complex (Hackette et al., 1970; Roon et al., 1977;Kleiner, 1981). The optimum pH was 67 for bacteria and fungi. Theexistence of specific proteinaceous carriers for NH4 uptake is supportedby biochemical, kinetic and physiological evidences. Moreover, NH4transport mutants have been isolated and some transport genes have beenidentified and cloned (Arst and Page, 1973; Castorph and Kleiner, 1984;Holtel and Kleiner, 1985; Franco et al., 1987; Reglinski et al., 1989).51.2.3. Transport of NH4 by higher plantsThere is a limited literature available regarding NH4÷ transport inhigher plants (Highinbotham et al., 1964), although a number of kineticstudies were reported for NO3- uptake (Deane-Drummond and Glass,1982a, 1983a; Siddiqi et al., 1990; Hole et al., 1990; Wieneke, 1992).Generally the NH4 transport systems in higher plants are very similar tothose in lower plants. Ammonium transport is localized perhaps at theplasma membrane and possible other membranes (Kleiner, 1981; Churchilland Sze, 1983). Evidence from kinetic studies of ammonium uptake byplant roots indicates that NH4 transport is a carrier-mediated process(Nissen, 1973; Joseph et al., 1975). There are several lines of evidence thatsupport the existence of the proteinaceous carriers to be discussed in thefollowing sections.1.2.3.1. Carrier-mediated transportEvidence indicating that ammonium transport is a carrier-mediatedprocess (Nissen, 1973; Joseph et al., 1975) comes from determining kineticparameters for NH4 accumulation in cells (Kleiner, 1985). The uptake ofNH4 by barley, rice, ryegrass, tomato, and wheat is concentrationdependent and follows Michaelis-Menten kinetics, (Tromp, 1962;Lycklama, 1963; Fried et al., 1965; Cox and Reisensuer, 1973; Rao andRains, 1976; Bloom and Chapin, 1981; Youngdahl et al., 1982; McNaughtonand Presland, 1983; Bloom, 1985; Deane-Drummond and Thayer, 1986;Smart and Bloom, 1988). Presland and McNaughton (1986) examined therates of NH4 uptake as a function of external [NH4] in corn, and reporteda saturable system below 1 mM [NH4-’-]. In a continuously flowing nutrientsolution system, NH4 uptake rates of intact rice plants were fitted to aMichaelis-Menten model (Fried et al., 1965; Youngdahl et al., 1982).61.2.3.2. Concentration dependent kineticsA biphasic pattern of NH4 uptake, with both saturable and linearphases, was first reported in Lemna, by Ulirich et al., (1984). For crops, likecorn, and rice, NH4 uptake kinetics (below 1 mM [NH4]0) commonlyconform to Michaelis-Menten patterns with Km values ranging from 0.0 14to 0.167 mM (Fried et al., 1965; Youngdahl et al., 1982; Presland andMcNaughton, 1986; Glass, 1988). Km values of 0.075 and 0.103 mM and aVmax of 0.061 and 0.017 mmol kg-’ s’ were obtained for 4-week-old and9-week-old rice plants, respectively, (Youngdahl et al., 1982). The secondsystem, above 1 mM [NH4], failed to correspond to Michaelis-Mentenkinetics (Ullrich et al., 1984; Presland and McNaughton, 1986). Generally,uptake studies at high external concentration have been achieved onlywith some difficulty, because depletion of the external solution is so small.1.2.3.3. Depolarization ofmembrane potentialThe inward movement of ammonium occurs as the cation NH4-’-(Walker et al., 1979a, 1979b; MacFarlane and Smith, 1982; Kleiner, 1985;Deane-Drummond, 1986). Only one report measuring AW in rice roots hasappeared in the literature: Usmanov (1979) reported AP to be -160 mV. Asearly as 1964, Higinbotham et al., noted the marked depolarizing effect of[NH4+]0on coleoptile cells AP in oats. Ullrich et al., (1984) found that, inLemna, depolarization of zSM’ by NH4 below 0.2 mM [NH4]0 wasconcentration-dependent and both NH4 uptake and z’P depolarizationresponded in a saturable fashion with half saturation values of 17 pM forboth processes. From 0.2 to 1 mM, net uptake of NH4÷ responded linearlyto [NH4+]0,with no further AP depolarization. Since NH4-’- is the main speciestaken by plant roots, it must be taken up via active transport and/or7facilitated diffusion. Both processes are coupled to an energy source, eitherdirectly (the former) or indirectly (the latter).1.2.3.4. Energy dependenceMetabolic energy is important to NH4 uptake. Macklon et al., (1990)has shown that NH4 absorption by excised root segments of Allium cepa L.was an active process. The uptake of ammonium at high temperature (25-30°C) is closely associated with metabolism (Sasakawa and Yamamoto,1978), and the uptake process was also decreased when carbohydratelevels were reduced (see Section 1.3.1.1.) or when temperatures werelowered (see Section 1.3.2.1.).1.3. MAJOR FACTORS AFFECTING AMMONIUM UPTAKEBesides the mechanism and kinetics of ammonium uptake, researchon ammonium uptake has also included other related issues such as theeffect of energy status, nitrogen cycling within the plant, the effects of rootpH and temperature. It must be emphasized that when environmentalfactors are concerned, one must be aware of the root’s capacity to adaption uptake in response to changed conditions, especially in long-termexperiments.1.3.1. Effects of photosynthesis1.3.1.1. Dependence on soluble carbohydratesOf major importance in the uptake of ammonium is the energy statusof the plant. The energy status of rice plants had a substantial influence on8the uptake of NH4-’- and on its conversion into high molecular weight Ncompounds (Mengel and Viro, 1978). The high demand for carbohydrate isin order to achieve active transport of NH4 at low external concentration,and to supply carbon skeletons for the rapid assimilation of NH4-’- as it isabsorbed by roots (Givan, 1979; Fentem et al., 1983a, 1983b). When theavailability of carbohydrate is low, the assimilation of NH4-’- is also low(Breteler and Nissen, 1982), and consequently a high efflux rate of NH4may result. A general relationship exists between the proportion of totalnitrogen absorbed as NH4 from mixed N sources such as NH4O3and theavailability of soluble carbohydrates in roots. (Raper et al., 1992). Theconcentration of soluble carbohydrates in the leaves of NH4-fed plantswas greater than that of N03--fed plants, but was lower in roots of NH4-fed plants, regardless of pH (Chaillou et al., 1991).The study of NH4 uptake isotherms in Chiorella revealed thatpreincubation with glucose drastically increased Vmax (5-fold), with nochange of Km (Schlee and Komor, 1986). It was reported that glucoseinduced a glucose transport system and two specific amino acid transportsystems (Cho et al., 1981). Glucose also induced the transport systems forammonium, nitrate and urea (Schlee et al., 1985). Removal of theendosperm of rice seedling suppressed NH4 uptake markedly (Sasakawaand Yamamoto, 1978), while the addition of 30 mM sucrose restoreduptake. In higher plants, provision of carbon skeletons in the form ofa-ketoglutarate increased uptake and association of NH4 in Lemna(Monselise and Kost, 1993).1.3.1.2. Periodic variations of light and growthThere is a great variation in NH4 assimilation rates between day andnight during the tillering stage of rice plants (Ito, 1987). This is probably9related to the diurnal changes in carbohydrate flux from shoot to rootresulting from changes in relative source-sink activity of shoots (Rufty etal., 1989; Lim et al., 1990). This periodic variation of carbohydrate supplyis also influenced by morphological variations of plant growth (Henry andRaper, 1989a; Vessey et al., 1990b). The net rate of NH4 uptake oscillatedbetween a maximum and a minimum with a periodicity co-ordinate withintervals of leaf emergence (Tolley and Raper, 1985; Tolley-Henry et al.,1988; Henry and Raper, 1989a; Rideout et al., 1994). Changes of both influxand efflux were responsible for the observed differences of net NH4-’-uptake (Henry and Raper, 1989).1.3.1.3. Ambient environmental factorsThe ability of the plant root to absorb nitrogen was affected byprevious growth conditions of the examined plants (Mon et al., 1979),since environmental factors will influence the carbohydrate status.Susceptibility of plants to NH4 toxicity is also related to plantcarbohydrate status (Nightingale, 1937; Prianishnikov, 1941; Givan, 1979).The soluble carbohydrate concentration in roots increased with increasingroot temperature (Clarkson et al., 1975; Macduff et al., 1987a) and withnitrogen deprivation (Rufty et al., 1988; Henry and Raper, 1991), anddecreasing rhizospheric pH (Chaillou et al., 1991). High ambient C02concentration increased total plant N and total nitrate-N content and leafarea but not leaf number of soybeans.101.3.2. Effects of root temperature1.3.2.1. Short-term perturbationAmmonium transport across the plasma membrane is sensitive totemperature. Although ion accumulation at steady-state may beindependent of external concentration or temperature, both of thesefactors influence short-term fluxes (Cram, 1973; Smith, 1973, Glass, 1983).In a 5-hour root temperature perturbation study, it was found that theuptake and assimilation of ammonium were profoundly affected in bothIndica and Japonica rice plants (Ta and Ohira, 1981). This might beexplained by the dependence of the NH4 uptake system on the rate ofmetabolism (Raven and Smith, 1976), or effects of low temperature onenzymes of NH4-’- assimilation (Shen, 1972). The effect of temperature onion uptake may also be due to physical changes in different parts of thecell membrane (e.g. membrane fluidity) instead of on the transport process(Clarkson and Warner, 1979).1.3.2.2. Qo value for NH4 uptakeQio values can be used to indicate the temperature dependence ofion transport. When temperature is lowered or increased by 10°C, the ratioof the two transport rates can be calculated by equation:LnQjo= {(t2-t1)/10] Ln(V2/V1) [1]where t1 and t2 are the temperature before and after the change, and Vi.and V2 are the transport rates at respective temperatures. When Qo isclose to 1, the transport rates are the same at the different temperatures,and ion transport is insensitive to temperature. A Qo value greater than 2is often considered as indicating the metabolic dependence of a11physiological process such as ion transport. In a seven hours perturbationof root temperature, Sasakawa and Yamamoto (1978) found that theuptake of ammonium by 9-days old rice seedlings was closely associatedwith metabolism. The Qo values between 9 24°C were> 2.5 for 15NH4÷absorption by rice roots estimated from Ta and Ohira’s (1981) data. LowQio 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 effectsThe effect of root temperature on ion uptake varies with thetreatment duration. Plants may adjust rates of ion transport in the long-term so that net uptake is independent of external variables such astemperature (Clarkson, 1976). As a result of plant adaptation to low roottemperatures, NH4 is absorbed more readily than N03 at lowtemperatures by roots of Italian and perennial ryegrass (Lycklama, 1963;Clarkson and Warner, 1979; Clarkson et al., 1986) and lettuce (Frota andTucker, 1972). Ammonium uptake by 4 day corn roots occurred even attemperatures as low as 0°C (Yoneyama et al., 1977).In both Indica and Japonica rice plants ammonium and nitrateuptake and assimilation were strongly affected by temperature (Ta andOhira, 1981). The uptake as well as assimilation of the two forms ofnitrogen were greatly inhibited at low temperature and low light intensity.At low root temperature, uptake of NH4was higher than that of N03.Theproportion of NH4-’- absorbed from mixed NH4-’- and N03 solution wasincreased as root temperature decreased from 13 to 3°C (Macduff andWild, 1989). Likewise, transferring corn roots from 30°C to 0°C, reduced12‘5N03- uptake more drastically than 1NH4 uptake (Yoneyama et al.,1977).The lower sensitivity of NH4+ uptake to reduced temperature(compared to NO3- uptake) might be explained by a lesser dependence ofNH4+ uptake on the rate of metabolism and energy production (Raven andSmith, 1976), or less effect of low temperature on enzymes of NH4+assimilation (GS-GOGAT) compared to those enzymes of NO3- uptake andreduction (NR and NiR).1.3.3. Effects of pH on NH4 uptakeIt has frequently been reported that NH4+ uptake is higher atelevated pH while NO3- uptake is stimulated at low pH (van den Honertand Hooysman, 1955; Fried et al., 1965; Jungk, 1970). When plants aregrown in medium containing NH4÷ as the solo source of N, the inevitableacidification of the medium may cause damage to the roots and even deathof plants (Loo, 1931; Raven and Smith, 1976). Moreover root growth maybe restricted in NH4 medium even when the pH of the medium iscontrolled between 6.0 and 6.5 (Lewis et al., 1987).1.3.3.1. Acidification of rhizosphere by NH4 uptakeA major factor in N uptake is the change of rhizosphere pH associatedwith NH4 uptake and its effect on plant growth, root morphology andcapacity for ion uptake. It is well known that NH4 uptake will causeacidification of the growth medium (Raven and Smith, 1976). At high NH4concentrations an enhanced NH4-’- uptake by ectomycorrhizal fungi causedan accelerated medium acidification that indirectly inhibited growth13(Jongbloed and Borst-Pauwels, 1990). NH4 has greater detrimental effectson plant roots than on shoots (Loo, 1931; Raven and Smith, 1976). Plantssupplied with moderate concentrations of NH4 generally grow poorlycompared with plants supplied with other sources of nitrogen (Rufty et al.,1982b) or mixed N03-/NH4’- supplies. Increased proportions of NH4 inmixed NH4 and NO3- nutrient solutions increased shoot:root ratios at alllevels of root-zone pH (Vessey et al., 1990). When NH4 and NO3- weresupplied together, cumulative uptake of total nitrogen was not affected bypH or solution NH4 : NO3- ratio (Raper et al., 1991b).1.3.3.2. Retarded plant growth in acidic mediumAcidic growth medium will, in turn, affect plant growth and NH4÷uptake. Root growth was restricted by increased acidity between pH 6.0 to4.0 (Arnon and Johnson, 1942; Islam et al., 1980). As the pH of the root-zone declined, therefore, NH4 uptake decreased and N03 uptake increased(Vessey et al., 1990). It was reported that the growth rate of soybeanshoots and roots was reduced by increasing pH (Rufty et al., 1982b).1.3.3.3. NH4 toxicity and acidic damageIf acidification of the root medium is controlled, plant growth withNH4 as the sole N source may be equal to growth with NO3 (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 NH4 as anitrogen source as long as root-zone pH is strictly controlled and a balanceis maintained between carbohydrate availability and acquisition of NH4(Rufty et al., 1983). It was suggested that the inhibition of plant growth atlow pH was due to a decline in NH4-1- uptake and a consequential limitationof growth by N stress (Vessey et al., 1990).141.3.4. NH4 fluxes at the plasma membrane1.3.4.1. Net fluxNET FLUX (net) describes the ‘net’ rate of ion uptake by roots. The netion uptake from the medium (outside) into the cytoplasm is determined bythe balance between influx and efflux. In practice, net fluxPnet = Poc - Øco [2]is measured by the disappearance of tested ion in the uptake solution.1.3.4.2. InfluxINFLUX () is defined as the rate of inward movement of soluteacross a particular membrane. Strictly speaking influx should refer to theunidirectional movement measured during a very short period, shortenough to discount the efflux. NH4÷ influx is negatively correlated withplant N status in lower plants (Silver and Perry, 1981; Hartmann andKleiner, 1982; Wiegel and Kleiner, 1982; Boussiba et al., 1984; Mazzuccoand Benson, 1984; Rai et al., 1984; Jayakumar et al., 1985), and higherplants (McCarthy and Goldman, 1979; Pelley and Bannister, 1979; Smith,1982; Ulirich et al. 1984; Holtel and Kleiner, 1985; Clarkson, 1986; Lee andRudge, 1986; Morgan and Jackson, 1988a, 1988b; Clarkson and Luttge,1991). MA influxes of pea seedlings decreased after pretreatment withglutamine and NH4 and increased after pretreatment with asparagine(Deane-Drummond, 1986).1.3.4.3. EffluxEFFLUX (Ø) is the rate of outward solute flow from cytoplasm acrossthe plasma membrane. Efflux of ions from plant roots was identified in15plants under stress or damaged conditions (Pitman, 1963; Hope et al.,1966; Jackson and Edwards, 1966; Hiatt and Lowe, 1967; Ayers andThornton, 1968; Bowen, 1968). In intact plants, efflux of K, Na, H2P04-,Cl-, Br-, or NO3- has been observed from roots (MacRobbie, 1964; Cram,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; Siddiqiet al., 1991).Continuous NH4efflux may be a common feature of net NH4 uptakeby roots of higher plants (Morgan and Jackson, 1973). In a study usingintact ryegrass,‘4N03--grown roots were equilibrated in a‘5N03-solutionenriched with ‘5N (97.5 atmo %). The results suggested that there was asimultaneous occurrence of the influx of ‘5N03- and efflux of ‘4N03(Morgan et al., 1973). Moreover, careful measurements of 14NH4 effluxrevealed that there must have been generation of NH4 by breakdown ofnitrogen compounds during the course of the experiment. There was excessquantity of4NH effluxes compared with the initial content in the roots(Morgan and Jackson, 1988a). There is even an‘4NH efflux from 14N03-grown roots (Morgan and Jackson, 1988b).1.3.4.4. Balance of fluxesThere is thought to be an ammonium cycle across the root cellplasma membrane (Morgan and Jackson, 1988b). It was reported thatendogenous NO3- effluxes to the unstirred layers were recycled throughNO3- influx (Morgan et al., 1973). The same could be expected for NH4efflux. Substantial ammonium cycling occurred during net ammoniumuptake (Jackson et aL, 1993), yet plants grown under low N conditionspossess a low NH4-’- efflux. Morgan and Jackson (1988a) suggested that the16regulation of NH4 uptake by roots of higher plants may involve changes ofboth influx and efflux in response to plant nitrogen status. It was foundthat net 15NH4 influx was increased and net‘4NH efflux was decreasedin nitrogen depleted wheat and oat seedlings (Morgan and Jackson, 1988a),and net NH4-’- uptake of barley and maize plants previously grown withNH4-’- was decreased subsequently (Morgan and Jackson, 1988b).The determining factor may be the internal [NH4] of the root cell.For example, enhanced NH4 influx by MSX treatment was claimed to bedue to the enlargement of cytoplasmic and vacuolar NH4 pools of roottissue several times (Jackson et al., 1993; Lee and Ayling, 1993) whichappeared enhance the influx of‘3NH4 of (maize and barley) plants byreducing isotopic efflux (Lee et al., 1992; Lee and Ayling, 1993). However,the enlarged [NH4]1 was also advanced to explain the enhanced effluxobserved in their system (Morgan and Jackson, 1988b).1.3.4.5. N cycling in the whole plantWithin the plant, N cycling, the simultaneous movement of N-compounds from root to shoot, and from shoot to root (Cooper andClarkson, 1989; Larsson et al., 1991) may enable N absorption to beregulated to match the demand imposed by plant growth (Drew and Saker,1975; Edwards and Barber, 1976). The concentrations of amides (Gln andAsn) in the roots will be the result of the balance between their synthesisfrom absorbed inorganic N (NH4 or N03), their import via the phloem, andtheir export via the xylem (Lee et al., 1992).171.3.5. Regulation of ammonium uptakeFeedback inhibition of NH4 uptake by nitrogenous effectors hasbeen implicated in lower plants (Kleiner, 1985; Ullrich et al., 1984; Pelleyand 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 concerningthe possible mechanism(s) of regulating NH4-’- uptake by either NH4 per seor its primary assimilates.1.3.5.1. Negative feedback regulationAt high nitrogen status, plant NH4-’- uptake could be suppressed dueto (i) low energy supply to the root system, (ii) accumulation in the roottissue of nitrogenous compounds that exerts negative feedback on thetransport system, or (iii) high efflux of endogenous NH4 (Morgan andJackson, 1988b). Repression of NH4 uptake may be due to continualgeneration of ammonium from degradation of organic nitrogenous sourceswithin roots and rapid accumulation of ammonium in roots of N-depletedplants upon initial exposure to ammonium (Morgan and Jackson, 1988a,1988b). However, Morgan and Jackson (1988b) indicated that theimmediate assimilates of NH4-’-, such as glutamine, are more likely negativeeffectors on NH4-’- uptake.1.3.5.2. Enhanced NH4 uptakeNegative correlation between ammonium uptake and cell nitrogenstatus have commonly been observed (McCarthy and Goldman, 1979;Pelley and Bannister, 1979; Smith, 1982; Ullrich et al. 1984; Holtel and18Kleiner, 1985; Clarkson, 1986; Lee and Rudge, 1986; Morgan and Jackson,1988a, 1988b; Clarkson and Luttge, 1991). It has been recognized that thecapacity for nitrogen uptake is enhanced in N-depleted plants such aswheat (Tromp, 1962; Minotti et al., 1969; Jackson et al., 1976b; Morgan andJackson, 1988a, 1988b); ryegrass (Lycklama, 1963); maize (Ivanko andIngversenm, 1971; Lee et al., 1992); barley (Lee and Rudge, 1986); andoats (Morgan and Jackson, 1988a, 1988b).1.3.6. Interactions between NH4 and K1.3.6.1. Mutual beneficial effects between N and KN and K are essential plant nutrients, required for healthy plantgrowth 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; Xuet al., 1992). Mutual beneficial effects of K and N on plant growth haveoften been described. An adequate K supply has been shown to enhanceNH4 uptake and assimilation (Ajayi et al., 1970; Barker and Lachman,1986; Scherer and MacKown, 1987). Sufficient N nutrition normallypromotes K-’- uptake due to the biological dilution effect of better plantgrowth (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 NH4However, NH4 has been shown to strongly inhibit the absorption ofK-’- in short-term experiments in many species including wheat, barley,maize and tobacco (Breteler, 1977; Munn and Jackson, 1978; Rufty et al.,191982; Rosen and Carison, 1984; Scherer et aL, 1984). There was a negativecorrelation between the external NH4 concentrations and K uptake (Rosenand Carlson, 1984; Scherer et al., 1987; Jongbloed et al., 1991), and netammonium uptake was correlated with potassium efflux (Morgan andJackson, 1989).The inhibitory effect of NH4 on K uptake has been claimed to beindependent of K-i- provision or pretreatments; it is probably exerted on thetransport processes at the plasma membrane. Insufficient evidence isavailable to draw a conclusion regarding the inhibition of K uptake byNH4 in terms of competitive and non-competitive effects (DeaneDrummond and Glass, 1983b; Scherer et al., 1984). K uptake wassuppressed during rapid NH4-’- uptake by N-starved plants (Tromp, 1962),but K-starvation did not produce the same effect as N-starvation on thetransport of NH4 (Tromp, 1962; Lee and Rudge, 1986).1.3.6.3. Inhibition ofNH4 uptake by KOn the other hand, NH4 uptake of plants was not reduced by K inthe nutrient medium (Mengel et al., 197 6; Rosen and Carison, 1984;Scherer and Mackown, 1987). However, the influence of K÷ on NH4 uptakehas not been consistent. It was reported that K had inhibitory effects butdid not compete with NH4 for selective binding sites in the absorptionprocess (Ajayi et al., 1970; Dibb and Welch, 1976; Mengel et al., 1976).1.4. REsEARCH OBJECTIVESThe objective of this study was to investigate the mechanisms andcharacteristics of ammonium uptake by rice plants. In particular, the20studies have emphasized short-term responses of fluxes to changes inambient conditions. This particular goal was achieved by using the short-lived radioisotope 1N (t1/2 = 9.98 mm), addressing five different areas:(1). By measuring NH4 influx and efflux, the exchange of N at the plasmamembrane and the relationships between these fluxes were quantified.Subcellular distribution of absorbed NH4 was also estimated. The resultsof these studies are interpreted in terms of a root cell model in Chapter 3.(2). To describe the kinetics of NH4 uptake and the pattern(s) of itsconcentration dependence, NH4 influx was measured in perturbationexperiments in plants grown in different levels of N. By altering ambientconditions such as medium pH, root temperature, and by treating rootswith various metabolic inhibitors, the energetic of NH4 uptake wasinvestigated. These are described in Chapter 4.(3). By measuring electrical potential differences together with assayingcytoplasmic [NH4], the electrochemical potential gradient for NH4between external solution and cytosol were defined in order to explore themechanisms of NH4 uptake. Membrane electrical potential differences ofrice roots were recorded as a function of external NH4 concentration. Thisinformation is incorporated with data dealing with biochemical, kinetic andenergetic aspects of NH4-’- uptake to formulate a model for the mechanismsof NH4-’- uptake (Chapter 5).(4). Without information on the regulation of NH4 uptake, the uptakemodel is incomplete. NH4 influx was measured as a function of root Nstatus. Internal [NH4-’-l was determined as well as the concentrations ofindividual amino acids. In Chapter 6, the results are discussed in referenceto existing reports to develop a model of the regulation of NH4-’- uptake.21(5). Chapter 7 deals with the interactions between NH4 and K-’- at theuptake level and explores the effects of prior exposure to these ions onsubsequent ion uptake.22Chapter 2. METHODS AND MATERIALSIn this chapter, the general methods used in this study are described.Method(s) used in a particular experiment will be addressed in thecorresponding chapter.2.1. PLANT GROWTH2.1.1. Seed germinationRice 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 at3 8°C, then placed on plastic mesh mounted on Plexiglas discs. The discswere set in a Plexiglas tray filled with dc-ionized distilled water just abovethe level of the seeds, and seeds were allowed to germinate in a growthchamber in the dark (at 38°C) for 4 d. During the following 2 d, thetemperature was stepped down to 20°C (by 9°C per day). Then discs, withone-week-old rice seedlings, were transferred to 40-L Plexiglas tanks.2.1.2. Growth conditionsPlants were grown hydroponically in 40-L Plexiglas tanks located ina walk-in growth room, in which growth conditions were maintained asfollows: temperature: 20 ± 2°C; relative humidity: 75%; and irradiance: 300j.tE m2 s1 under fluorescent light-tubes (VITA LITE, Duro-Test) on a cycle23of 16 h light and 8 h dark. Plants were 3-week-old when they were usedfor most experiments unless specifically indicated.2.1.3. Provision of nutrientsThe growth medium was modified based on the recipe of modifiedJohnson’s nutrient solution (Johnson et al., 1957; Epstein, 1972) and arecipe from the International Rice Research Institute (Yoshida et al., 1972),in which ammonium (NH4C1) was the only source of nitrogen (except forsome specific experiments as specifically indicated) and silicon was addedas Na2SiO3.5H0.This modified Johnson’s nutrient solution (hereafterreferred to as MJNS) was also the medium used to carry out allexperiments. The composition of this MJNS, in micromolar (iiM), was 200for Ca, K and P, 100 for Mg, 300 for S, 16 for B, 5 for Si and Fe, 1 for Mnand Zn, 0.3 for Cu and Mo. The external ammonium concentration ([NH4÷]0)was varied as indicated at the appropriate places. Generally plants weregrown in MJNS containing 2, 100, or 1000 jiM [NH4+]0,referred to hereafteras G2, G100, G1000 plants, respectively. The concentrations of nutrients ingrowth medium were maintained by infusion of appropriate stocksolutions, through peristaltic pumps (Technicon Proportioning Pump II,Technicon Inst. Corp.). Generally 2 liters per day of stock solution weresupplied and stock concentrations were determined from daily chemicalanalyses of medium samples. Solutions were mixed continuously bycirculating pumps (Circulator Model IC-2, Brinkmann Inst., Inc.), andaerated continuously. The pH of growth medium was maintained at 6.0 ±0.5 by adding powdered CaCO3 (13 g/tank), according to measured pHvalues, 12 times daily.242.2. N ISOTOPES FOR STUDYING N UPTAKE2.2.1. Isotopic tracerThere is now widespread use of isotopic tracers, particularradioactive tracers, in the biological sciences (Thain, 1984). Carbon (“C,4C), phosphorus (32P), sulfur (5S), chlorine (36C1), potassium (42K),rubidium(86Rb), calcium(45Ca) and sodium(22Na) have been employed todetermine the kinetics of transport and transformation of these elementsin living systems. Measurements of radioisotopic influx and/or efflux havebeen used to obtain an estimate of the unidirectional fluxes of the stableisotope of the ion at the plasmalemma and tonoplast and to estimate theseparate amounts of the stable isotopes in the cytoplasm and vacuole(Cooper, 1977; Thain, 1984).The utility of radiochemical techniques is afforded by (i) their greatsensitivity compared to other analytical methods. Radioisotopic tracersmay offer 108-fold increased detection sensitivity over stable isotopemethods (Cooper, 1977; Krohn and Mathis, 1981); (ii) the fact that they“label” the atoms of molecules without significantly altering their chemicalproperties (Cooper, 1977; Boyer, 1986).2.2.2. Nitrogen IsotopesThere are six isotopes of nitrogen known, ranging in mass numberfrom 12 to 17 (Kamen, 1957). The stable isotopes of nitrogen are ‘4N and15N, the latter being present to the extent of 0.365 atom per cent.Radioactive isotopes 12N and ‘3N are positron emitters with half-lives of250.0125 seconds and 9.98 minutes respectively. ‘6N and ‘7N are negatronemitters with half-lives of 7.35 and 4.14 seconds respectively, 17N alsoemits neutrons. The longest-lived radioactive isotope of nitrogen is ‘3Nwhich is the only radioactive isotope that has been used in tracer research(Kamen, 1957; Krohn and Mathis, 1981; Bremner and Hauck, 1982). Theuse of 1N (Burns and Miller, 1941) in biological studies started as early asthe use of ‘3N (Ruben et al., 1940).2.2.3. Stable 15N techniquesSince the first use of 1N (Burns and Miller, 1941), this isotope hasbeen widely used in agricultural research (Hauck, 1982; Knowles andBlackburn, 1993), and the analytical methodology has been continuouslyimproved (Clusius and Backer, 1947; loch and Weisser, 1950; Hürzeler andHostettler, 1955; Broida and Chapmen, 1958; Faust, 1960; Mulvaney andLiu, 1991; Hoult et al., 1992).‘5N has been used in characterizing the N03 and NH4-’- uptakeprocesses of plants (Fried et al., 1965; Yoneyama and Kaneko, 1989;Yoneyama et al., 1991) and tracing the metabolism of nitrogen in plantcells (Yoneyama and Kumazawa, 1975; Arima and Kumazawa, 1977). 1N isalso widely used in studyingN2-fixation in soil-plant systems, aquatic andsediment systems (Watanabe, 1993; Warembourg, 1993) and Ntransformation in soils (Azam et al., 1993). It is also employed in studyingthe mineralization of soil organic N (Powlson and Barraclough, 1993) andnitrification and denitrification of soil N (Mosier and Schimel, 1993).15N-labeled nitrogen fertilizer has also been used in the study of fertilizeruse efficiency (Azam et al., 1991).26Stable N isotope techniques have several advantages over techniquesusing radionuclides. As a biochemical tracer, ‘5N offers the advantages ofbeing relatively inexpensive, widely available, free of radiation hazard andless limiting in terms of experiment duration. The advantages of using ‘5Nalso embodies a major disadvantage in its use as a tracer: a sizablebackground, present in all nitrogenous materials, against which addedtracer must be measured (Cooper et al., 1985). In order to measuresignificant enrichment of 5N in specific metabolic compartments,investigators have to administer a large amount of‘5N-labelednonphysiological precursors to biological systems (Cooper et al., 1985). Inaddition it requires tedious preparation to convert samples to N2 gas priorto mass or emission spectrometry.2.2.4. Radioactive isotope, 13N2.2.4.1. Use in biological studies‘3N was first made in 1934 by Joliot and Curie as‘3NH4and was oneof three isotopes generated artificially by induction of radioactivity inotherwise stable elements (boron) by bombardment with particles emittedby polonium (Joliot and Curie, 1934). It was first used as a biological tracerin studying theN2-fixation of non-legume barley plants (Ruben et al.,1940), which was one year earlier than the first report of using 15N2 tostudy N2 fixation (Burns and Miller, 1941).Much of the early tracer work in biochemistry was carried out withpositron-emitting radionuclides, such as “C, and to a lesser extent 13N, butwith the introduction of ‘4C and 15N, their importance declined over aperiod of two decades. Only in the past 10 years or so, have these short-27lived isotopes again become important as tracers particularly in the field ofbiochemical research. With about 70 medical cyclotrons, there are at least12 groups, that generate ‘3N for biological studies (Cooper et al., 1985). Inbiological studies, there are several groups using ‘3N in study nitrogennutrition of plants (Appendix A).2.2.4.2. Production of 13N13N can be obtained from targets containing boron, carbon, nitrogenor oxygen and an appropriate accelerated particle (Cooper et al., 1985). The10B(o,n)3N;‘2C(d,n)13N; 12C(p,y)13N;‘3C(p,n)13N;‘4N(p,pn)13N; 14N(n,2n)‘3Nand 16O(p,c)’3Nreactions have all been used to make ‘3N (Krohn andMathis, 1981; Tilbury, 1981). The method most widely used at present forthe production of‘3N-ammonia is the proton irradiation of water(‘6Q(p,)’3N), followed by reduction of the‘3N0 and‘3N02 formed undertypical 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 ‘3N2 production based on nuclearreactions of‘6O(p,c’3Nis as follows: (Meeks et al.,1985)20 MeV, 20 1A1. Generation: H20 > 13N03 + ‘3N02 + ‘3NH4HPLC/SAX column2. Concentration: 60 ml‘3N0- > up to 3 ml‘3N0Devarda’s Alloy (Cu/Al/ Zn)3. Reduction: ‘3N0 > 1NH365°C Saturated NaOH4. Trapping: 13NH3 + H > 1NH428Na/KOBr5. Oxidation: 13NH4 > [‘3N]-N1.5 imo1‘4NH4The yield of ‘3N varies with the types of nuclear reaction, targetmaterial, and particle energy. Bombarding 10 ml pure water with an 10 jiAproton beam of high energy (>19 MeV) could yield 36 mCi pA4 20 miw1(Vaalburg et al., 1975). The ‘3N species, ‘3N01NO2 and 1NH4,arepresent in the radioactive sample. The relative concentrations of thesespecies is dependent upon the irradiation dose as well as on other factorssuch 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‘3NH4-’- is greater than‘3N02 and at high dose 13NH4 is less than ‘3N02(Tilbury and Dahi, 1979).There are also some contaminants in the radioactive product. It wasfound that irradiated unprocessed water contains 18F (t1/2=1.8 h), 150(t1/2=2.0 mm), and 48V (t1/2=16.2 d). Both 18F and 48V produce noproblems with ‘3NH4 since these radloisotopes do not distil. Since l8F isfrom the reaction of‘80(p,n)’F, its contamination can be minimized bydepleting 180 in water (Skokut et al., 1978). Though 15Q can be detected in‘3N-ammonia solution, it will disappear during preparations lasting morethan 20 mm (Vaalburg et al., 1975).The 13N isotope disintegrates by emission of a positron (E3, 1.2 MeVof maximum emission energy) giving rise to ‘3C (Meeks, 1993). Inannihilation reaction between a positron and an electron, two gammaphotons are formed each of 0.511 MeV energy traveling in nearly oppositedirections (Cooper et al., 1985; Meeks, 1993). Therefore the detection ofradioactive decay in the sample is accomplished in a gamma counter. Since‘3N decay results in Cerenkov light, it may be counted by the29photomultiplier tubes in liquid scintillation systems (Glass et al., 1985).Radioactivity is typically counted immediately in a gamma counter and allcounts are decay-corrected to a common time. The admitted 13N in planttissues can be observed by placement of multiple Gieger-Mueller tubesalong 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 ofthe tissue and scintillating counting.2.2.4.3. Advantages of the use of 13Nin biological studiesThe use of‘3NH4-N in biological studies of nitrogen nutrition hasseveral advantages:(1) The chief advantage is that such nuclide can be prepared at a very highspecific activity increasing sensitivity for detection approximately108-fold, to trace rapid kinetics and metabolic pathways (Krohn andMathis 1985). Because of the great sensitivity of the radioactive isotopetechnique, 13N has proved to be of value in elucidating biologicalmechanism over very short time intervals. (Hanck, 1982).(2) In order to measure the initial events in biological processes it may benecessary to determine events on a time scale of seconds to minutes. Highspecific activity tracers which are detected with high efficiency (e.g. 13N)make possible such measurements. It is clear that time resolution of atracer-influx experiment is crucial for subsequent interpretation of thefluxes. In short term experiment, by using‘3N0, one is able to monitornet uptake and disappearance of ‘3N0 simultaneously, thus increasingthe experimental resolution compared with experiments where plants30have to be sampled and further prepared before assay (Oscarson et aL,1987).(3) The isotope decays rapidly (t1/2 = 9.98 mm). After allowing sufficienttime for decay, repeat studies can be carried out in the same systemwithout interference from previously administered tracer (Cooper et al.,1985). In tissue dissection or in vitro studies, the total quantity of tracerpresent in rather large specimens can be determined rapidly andaccurately, with little sample preparation, by gamma counting techniques.(4) 13N is inherently less hazardous to use in comparison withconventional, much longer lived tracers. The problem of radioactive wastedisposal 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 thecyclotron. Its production requires a suitable accelerator and acorrespondingly large capital investment (Cooper et al., 1985). Its shorthalf-life limits the period over which it can be used to a maximum ofperhaps 4 hours or so depending on the application (Meeks, 1993).Techniques of precursor synthesis, labeling, product purification, metabolicseparation and analysis must be appropriately rapid (Fuhrman et al.,1988).2.2.4.4. Considerations of using 13Nin nitrogen uptakeTo study nitrogen uptake, especially ammonium uptake by plantroots, several facts have to be considered:(1) Membrane fluxes of nitrogen are of utmost importance for the over-allnitrogen utilization in plant growth.31(2) Ammonium is rapidly metabolized to amino acids and amides withinthe root before transport to the shoot (Pate, 1973). Evidence showed thatthe NH4 uptake rate is also regulated by the N assimilation andtranslocation rates of the plants (Wiame et al., 1985; Morgan and Jackson,1988). Therefore it is necessary to identify the nitrogen compounds in theuptake, assimilation and transport processes.(3) It is difficult to measure the subcellular, i.e. cytoplasmic and vacuolar,pools of N03 and/or NH4 directly due to their small size and rapid turnover. It was found that the half-time for exchange of the cytoplastic NO3-pool ranged from 2 to 5 minutes in roots of Zea Mays (McNaughton andPresland, 1983).(4) Ion uptake of plant roots is able to adapt during a long-termexperiments in response to changes of environmental conditions, such astemperature or pH (Macduff et al., 1987). Therefore the tracer techniquecan be chosen as a proper approach to study ammonium uptake by riceroots in consideration of high sensitivity, rapid measurement and shortduration of experiments. Another point is that uptake by depletion is soslow from high external concentration that it can not be measured exceptwith ‘3N.2.2.4.5. Use of1Nin nitrogen transport studiesIn short-term experiments, ‘3N has been used to study nitrogenuptake by plant roots (McNaughton et al., 1983; Glass et al., 1985; Lee etal., 1986; Oscarson et al., 1987). Most reported studies used ‘3NO’ inuptake experiments; few made use of 1NH4.‘3N0 has been used toidentify and characterize the transport systems (Thayer and Huffaker,1982; McNaughton and Presland, 1983; Siddiqi et al., 1990; Glass et al.,321990); regulation of influx (Glass et al., 1985; Oscarson et al., 1987; Siddiqiet al., 1989; Rufty et al., 1991); and cell compartmentation (Presland andMcNaughton, 1984; Lee et al., 1986; Siddiqi et al., 1991). Presland et al.,(1986) were able to use 13NH4 to study ammonium uptake by roots ofhydroponically grown maize seedlings and the transport of 13N to theshoot. 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 of13Nin nitrogen assimilation13N has also proven useful in understanding nitrogen assimilation inplant cells. Gin is the first major organic product of‘3NH4 assimilation(Skokout et al., 1978) and the GS/GOGAT pathway is the primary route ofassimilating fixed ‘3N (Meeks et al., 1978a). It was found that MSXinhibited the incorporation of‘3NH4-’- into Gln more than into Glu. Theopposite was true for‘3N0.In tobacco cells GDH only plays a minor role(Skokout et al., 1978) but in non-leguminous angiosperm N2-fixers, GDHmay play a major role in the assimilation of exogenously supplied NH4(Schubert et al., 1981).Since 13NH4can be produced in hundreds of millicuries, it should bepossible to synthesize a large number of 13N-labeled amino acids,nucleotides, amino sugars, and other metabolites via known enzymaticroutes (Cooper et al., 1985). Organic N-containing compounds, such as L(13N ) -glutamate and L- ( amide-’3N)-glutamine, are also synthesized from13NH4 and used in studies of NH4 and glutamine assimilation pathways(Suzuki et al., 1983; CalderOn et al., 1989). It was found in Neurosporacrassa that(13N)-Gln is metabolized to(13N)-Glu by GOGAT and to 13N}I4by the glutamine transaminase-o-amidase pathway. Then released1NH4is reassimilated by both GDH and GS (Calderón et al., 1989). Extracted ‘3N-33labeled amino acids or amides can be separated by HPLC andelectrophoresis (Cooper et al., 1979; Meeks, 1993). It was found thattranslocation of N compounds can also be traced by 1N. Barley leavesexposed to‘3NH gas for 30 mm, incorporated 1N mainly into free Gin andGlu and 1 to 3% of these were exported to the sheaths through the phloem(Hanson et al., 1979).2.2.4.5. Use of 13Nin denitrificationIn addition 1N has also been used to study denitrification in soils(Gersberg et al., 1976; Tiedje et al., 1979; Bremner and Hauck, 1982). Useof ‘3N allows the direct quantitative measurements of denitrification ratesover short time intervals, without changing the concentration of N03 inthe soil system from flooded rice fields (Gersberg et al., 1976).2.2.5. Protocol for 13NH4production in present studyThe short-lived radioisotope 13N (t 1/2 = 9.98 minutes) was producedas described by Siddiqi et al., (1989), by 20 MeV-proton irradiation of H20on an ACEL CP42 cyclotron. Contaminants in the ‘3N0 sample (mainly18F) were removed by passing the samples twice through a SEP-PACAlumina-N cartridge (Waters Associates). Reduction of‘3N0 to 13NH3 wasachieved by using DevardaTsalloy at 70°C in a water bath (Vaalburg et al.,1975; Meeks et al., 1978); 1NH3 was separated from remaining chemicalspecies by distillation at alkaline pH, and trapping in acid solution as13NH4-’-. The flow scheme for this conversion is shown in Figure 1.3413N0+l8FiN NOH1Figure 1. The flow scheme for 13NH4production. (As described in SectionSEP-PAKLJcLeod holderD e v a rd a• sA110ULabelledUptake Solution7 Oc0 CU urn2.2.5.)352.3. MEAsuREMENT OF NH4 FLuxEs2.3.1. Influx of‘3NH4Standard procedures for 13NH4uptake were as follows: (a) loading:rice roots were loaded in 13NH-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 unlabeled MJNS (hereafter referred to as ‘washing’ solution) for 5 mm and 3mm, respectively. The choice of these times is rationalized in the Discussionsection (section 3.4.). Experiments were conducted at steady-state withrespect to [NH4--]0, i.e., the [NH4]0 of ‘washing’ solutions and ‘loading’solutions were the same as those provided during the growth period or inexperiments to define influx isotherms; plants were exposed to different[NH4]0for short (perturbation) experiments. Immediately after the post-wash period, plants were cut into shoots and roots and the surface liquidadhering to the roots was removed by a standard 30 sec spin in a slow-speed table centrifuge (International Chemical Equipment, Boston). Rootsand shoots were introduced into separate scintillation vials andimmediately counted in a gamma counter (MINAXI y-5000, Packard). Thefresh weights of roots and shoots were recorded immediately aftercounting.2.3.2. Efflux of 13NH4Roots of rice seedlings were immersed in the1NH4labeled ‘loading’solution for 30 mm. At the end of this time plants were transferred to an36elusion vessel and tracer leaving the roots in exchange for‘4NH in theun-labeled identical ‘washing’ solution. This solution was collected atprescribed interval in 20-ml scintillation vials for counting.2.3.3. Net flux of NH4Net NH4 flux was measured in uptake solutions by the depletionmethod. Solution samples (S1 and S2) were taken at different times (t1 andt2), and the difference of assayed [NH4] was used to calculate net NH4flux. Net NH4 flux can also be estimated by subtracting efflux from influxof the same roots.2.4. CoMPARTMENTAL (EFFLux) ANALYsIs2.4.1. Compartmentation of plant cellsPlant cells are highly compartmentalized. They are surrounded bythe cell wall, and the plasma membrane encloses the cytoplasm, in whichare found the vacuole, mitochondria, nucleus, plastids and other organelles.Up to 80% or more of cell volume is occupied by the vacuole which isenclosed by the tonoplast (Salisbury and Ross, 1985). The cytoplasm is thevital part of cell. The major functions of the vacuole are to maintain turgorwhich contributes to cell shape and to store solutes. The compartmentationof the cell has important consequences for nutrient uptake, unidirectionalfluxes, assimilation, distribution and translocation. Because higher plantcells are too small to dissect and the size of the compartments is even37smaller, it seems technically impossible to obtain information on thecomposition of each compartment. However, through various methods,such as NMR, ion-specific electrodes, EDX, compartmental analysis, orfluorescent dyes, the ion concentration of one or more particularcompartments, or fluxes between compartments can be estimated.Compartmental analysis is the only systematic method of investigatingtransport processes and estimating the size of compartments and toanalyze the kinetics of movement of ions to or from a tissue (Cram, 1968).Therefore it has been established as a tool for characterizing the exchangeproperties of multicompartment systems.2.4.2. Development of theoryCompartmental analysis was first used by Fourier in 1822 todescribe the relationships between heat flow and temperature gradientsand, in 1855, it was adopted by the biologist, Fick, in studying diffusiveflow along a concentration gradient (Zierler, 1981). Not until one centurylater, was it introduced by MacRobbie and Dainty (1958) to study iontransport in Nitellopsis. Soon after, Pitman (1963) was the first to use thismethod to investigate multicompartmental transport processes in a higherplant. Compartmental analysis has mostly been used by plant physiologiststo calculate the fluxes, characterize internal ion pool sizes and membranekinetic parameters for ion exchange.The basic assumption of this methodology is that the system is atsteady state, or at equilibrium. Additional assumptions include that (1) thesubstance of interest flows into and from the separate compartments ofthe system; (2) the flux is proportional to the quantity (or concentration) of38the substance in the compartment from which the material flows. It isassumed that the material under study is neither destroyed norsynthesized in any compartment, and that each compartment ishomogeneous, or well stirred; (3) the concentration of an ion species or itsflux is described by a first-order linear differential equation with constantcoefficients which are independent of elapsed time and of the conjugate(Zierler, 1981). For higher plant systems, the additional assumption is thatthe relevant compartments of the experimental system are functionally inseries with each other (Walker and Pitman, 1976; Cheeseman, 1986). Theseassumptions may not always be valid (Lazof and Cheeseman, 1986). It issuggested that compartmental efflux analysis should not be used alone, butintegrated with other methods such as influx measurements (Cheeseman,1986).2.4.3. Models for compartmental analysisThe testing model or the analysis process can be varied with theresearch subject (excised tissue or intact plant), number of compartments(2, 3, or more), nutrition status (steady or non-steady) (Walker andPitman, 1976). The conventional compartmental analysis is suited todetermine unidirectional fluxes and compartmental contents of ions inexcised root tissues, or suspension-cultured cells (Pitman, 1963; Cram,1968; Poole, 1971; Macklon, 1975a; Pfrüner and Bentrup, 1978; Jeschkeand Jambor, 1981). Since it was considered to be small in excised roots(Macklon, 1975), the xylem transport in intact plants was not included inthis conventional model (Pitman, 1963; Etherton,1967; Pallaghy et al.,1970). However, the method was modified by Pitman (1971, 1972) tostudy Cl- uptake and transport in barley roots. Tracer efflux from the39cortical cell surface and the transport of tracer into the xylem weremeasured and analyzed separately. A three compartment model, includingxylem transport, was tested in the study of unidirectional fluxes of Na inroots of intact sunflower seedlings (Jeschke and Jambor, 1981). In twocompartment models, xylem transport was also considered in studies of‘3N0 fluxes in roots of intact barley seedlings (Lee and Clarkson, 1986;Siddiqi et al., 1986). The two compartments included the cell wall andcytoplasm, respectively. The short half-life of 13N 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) thecytoplasm and vacuole are in series; (2) the cytoplasmic content is verymuch less than the vacuolar content; (3) the tissue is in a steady state(Cram, 1968). Therefore one may expect that at steady-state conditions ofroots:S = S0 (1 - ekct) [3]when roots are exposed to a radioisotope-labeled medium with specificactivity S0, the radioisotope content of the cytoplasm S increasesexponentially with time (t) and the rate of tracer exchange of thecytoplasm (kc) is given by the relationship (kc=O.693/tl/2) The quantity ofradioactivity inside the cell Q is given byQc*AtcpocSc [4]where A is a cross section constant and p is the flux from outside tocytoplasm. The fluxes in opposite directions, between cytoplasm andvacuole are considered to be equal at steady state:40Øcv = [5]then the flux into the cytoplasmPoc = Øco + - xc; (if 4 << 0 it may be neglected) [6]therefore net uptake of an ionJoc = Poc - Pco [7]and the transport of ion from root to shoot through xylem would beJox = - Pxc [8]if roots were uniformly labeled after 16-24 hours loading:S,= S= S0 [9]and the specific activity in the xylem can be estimated from the transportrate of tracer (cI(t)) and transport rate of ion (J0t)) with the assumptionthat the symplasm behaves like a rapidly mixed phase and has a uniformspecific activity Sc= I(t) /J0(t) [10]Based on these relationships, one is able to estimate unidirectional fluxesand other parameters for each of the compartments.A biphasic efflux pattern suggests two phases, outside and inside theplasma membrane (Luttge and Higinbotham, 1979). Since the fastestcomponent was found in both living tissue and chloroform-killed tissue,Cram (1965) concluded that the fastest component of efflux of tracer Clfrom carrot tissue probably corresponded to the apparent free space(AFS). After treating barley roots with either sodium dodecyl sulphate41(SDS) or 70°C hot-water for 30 mm, the amounts of released‘3N0-duringinitial 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 confirmthe AFS phase. It was found that [1,2-3H] polyethylene glycol(3H-PEG) istoo large to diffuse into AFS, but D-[1-’4C] mannitol is able to diffuse freelyin the AFS without been absorbed by root cells (Shone and Flood, 1985).After loading with a mixture of3H-PEG and D-[1-’4CJ mannitol, plant rootswere washed in unlabeled solution. Since the ratio of 3H and ‘4C should besame from the surface film of ‘loading’ solution carried over with the roots,the extra D-[1-14C] mannitol must be washed out from AFS, and can beused to assess the volume of the AFS. It was found that there was an initialrapid release of 90% of H and ‘4C within the first 1 mm but more ‘4C wassubsequently released (Lee and Clarkson, 1986). Therefore the rapidlyreleased radioactivity during early efflux is probably from the AFS.A tricompartmental efflux pattern (including the apparent freespace) were reported for Cl- in carrot root slides or isolated corn rootcortex (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 andother studies, Cram (1965) concluded that, in addition to the fastest effluxfrom the AFS, the two slower components were considered to besubcellular in origin, the cytoplasm and the vacuole. Further quantitativeconsiderations and model fitting suggested that the cytoplasm and thevacuole are arranged in series with direct connection between the externalsolution and the cytoplasm, but not between the external solution and thevacuole (MacRobbie, 1964; Cram, 1965).42Also a third small symplastic kinetic compartment may exist inaddition to the bulk cytoplasm and vacuole (Luttge and Higinbotham,1979; Lazof and Cheeseman, 1986). In a study of sodium transport inSpergularia marina, Lazof and Cheeseman (1986) found. that the rapidfluxes involved only a very small portion of the total Na in the roots butthe authors were unable to identify the physical entity corresponding tothe compartment identified. There were also several similar reports inother transport studies. The additional compartment could be the smallportion of the bulk cytoplasm connecting to the vacuole (Pitman, 1963); orthe cytoplasm can exchange with both vacuole and plastids (Walker andPitman, 1976); or the possible involvement of vesicles moving in thecytoplasm (Dodd et al., 1960; Luttge and Osmond, 1970); or theinvolvement 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 analysisThe general procedure for compartmental analysis has beendescribed in detail (Walker and Pitman, 1976; Zierler, 1981; Rygiewicz etal., 1984). Several radioisotopes have been used in compartmentalanalyses, 36C1, 82Br, 42K or 86Rb+, 22Na, 45Ca, and 28Mg. One part ofthis technique involves the use of radioisotopic tracers to measure influxand efflux, the separate components of the net flux. The second part is amore systematic method to analyse the kinetics of movement of ions to orfrom different compartments (Cram, 1968). The basic assumption of thisprocedure is that radioisotope loaded into different compartments will bewashed out with different rate constants.43After allowing plant tissues, cells or roots to load with radioactivetracer for a designated duration, the efflux of this radioisotope is measuredfor a prescribed period of time. Depending on the type of ion studied, thereare two ways to count the radioactivity. For nonmetabolized ions, such asCl-, Br-, K-’-, Na, and Mg+, the radioactivity remaining in the tissue atthe end of elution can be counted. By counting the eluates at differenttimes the counts remaining in the tissue at these times can be estimated.For metabolized ions, however, counts remaining in the tissues would bemisleading because they consist of the radioactive ion under examinationand the metabolic products of its assimilation. In the latter case the rate ofefflux, rather than counts remaining must be estimated as a function of theduration of elusion. However, even this method requires that the identityof the effluxed ion be confirmed.Plotted as a function of time on a semi-logarithmic plot, the activitydata (e.g. cpm remaining in system or efflux rate) are resolved intodifferent linear phases which have been interpreted as corresponding todifferent compartments within the cells. One flaw in this method has beenthe subjective basis of line fitting (curve-peeling) of data which hasimplications for the number of exponential terms and their coefficients. Toimprove the method, Rygiewicz et al. (1984) proposed a microcomputermethod in which maximization of r2 for linear regression serves as thecriterion to determine data points belonging to each compartment. Thisdevelopment 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 fromseveral sources are shown in Appendix B. It was reported that the half-44lives of C1 exchange for apparent free space, cytoplasm and vacuole were1.4 mm, 10 mm and 300 h, respectively for carrot root tissue (Cram, 1968).In excised barley roots, a slow, vacuolar compartment, was not visibleeven after 10 h of exchange (Behl and Jeschke, 1982). It must be kept inmind that compartmental analysis alone does not allow one to identifyeach compartment (Luttge and Higinbotham, 1979), one must interpret theresults with necessary caution and verify these correlations independently.For example, several techniques are available to identify and quantify thevacuole (Clarkson and Luttge, 1984).2.4.5. Procedures for compartmental analysis in the present studyFor better time control of the separation of ‘washing’ solutions fromthe 13NH4-labeled roots during the efflux process and to reducedisturbance of roots, I devised a simple apparatus in which to perform theefflux study. The spout of a plastic funnel (100 mm diameter) was cut tofit into the barrel of a 25 cc plastic syringe, into which it was sealed. Alength of rubber tubing replaced the needle end of the syringe and a metalspring clip on the tubing functioned as drainage control. A small hole wasdrilled in the wall of the syringe barrel near the bottom, and a needleintroduced through this hole to provide for aeration. This technique alsoresulted in good mixing of the ‘washing’ solution.Roots of rice seedlings used for compartmental analysis wereimmersed for 30 mm in the ‘loading’ solution. These pre-labeled rootswere carefully introduced into the syringe barrel for elution. Samples of 20ml ‘washing’ solution were poured into the efflux-funnel and allowed toexchange with the 13N-labeled roots. After prescribed intervals, this45solution was drained from the funnel directly into a 20-ml scintillationvial, by opening the drainage clip. Fresh ‘washing’ solution was poured intothe efflux-funnel from the top of the funnel, immediately after closing thedrainage clip. The duration of successive washes were: 1 x 5 s, 1 x 10 s, 7 x15 s, 2 x 30 s, 5 x 1 mm and 5 x 2 mm. After the last wash, the plants werecut into shoots and roots and introduced into separate scintillation vials.The radioactivities of all samples were counted immediately. In order to beassured that the 13N species that had effluxed from the roots was 13MI4rather than any metabolic products, two other sets of13NH4-labeled rootswere effluxed for 30 mm in 750 ml ‘washing’ solution. Two 20-ml samplesof the efflux solution from each beaker were taken and separated by theCEC procedure (see below) and counted for radioactivities. Theradioactivities released from intact rice roots into efflux solutions during18 mm efflux experiments, were counted, converted to efflux rates andplotted versus time in semi-log plots (see Fig. 2 in section 3.3.1.). Thismethod of analysis is required because NH4 is rapidly metabolized in riceroots (Yoneyamo and Kumazawa, 1974), and converted into amino acidsand proteins. As a consequence, standard methods of compartmentalanalysis (Walker and Pitman, 1976), based on semi-log plots of cpmremaining in the tissue plotted against time are not appropriate. Hence thevalues of log of rate 13NH4 released against time were plotted using themethods detailed by Lee and Clarkson (1986) in an automated computeranalysis (Siddiqi et al., 1991).462.5. DETERMINATIoN OF AMMONIUMIntracellular NH4 was extracted from rice roots by use of a CationExchange Column (CEC) separation based on the methods of Fentem et al.,(1983a) and Belton et al., (1985) and determined by the indophenol bluecolorimetric method (Solorzano, 1969). The procedure was as described inWang et al., (1993a): in brief, after desorbing in NH4-free MJNS for 3 mmto remove NH4 in the cell wall, the roots were cut, weighed, and groundwith liquid nitrogen in a pre-cooled porcelain mortar and extracted with10 ml of 10 mM sodium acetate buffer (pH 6.2). The resulting slurry waspassed through a Whatman #1 filter paper and then washed 3 times eachwith 5 ml of the same buffer solution. The filtrate was passed through theCEC filled with 3 ml of resin (Dowex-50, 200-400 mesh, Na form). TheNH4 adsorbed on the CEC column was eluted using 250 mM KC1. Theconcentration of NH4 in solution was determined by the indophenol bluecolorimetric method (Solorzano, 1969).2.6. PREPARATIoN OF METABoLIC INHIBITORSThe same metabolic inhibitors were used in the 1NH4influx study(Chapter 5) and electrophysiological study (Chapter 6). The inhibitors usedwere as follows: (1) CCCP (10 iiM): carbonylcyanide m-chlorophenyihydrazone dissolved in ethanol; (2) CN- plus SHAM (1 mM): NaCNplus salicylhydroxamic acid dissolved in water. The resulting alkaline pHwas adjusted by titration with H2504 to pH 6; (3) DES (50 1iM):diethylstilbestrol dissolved in ethanol; (4) DNP (0.1 mM): 2,4-dinitrophenoldissolved in ethanol; (5) Mersalyl (50 iiM): Mersalyl acid dissolved inwater; (6) pCMBS (1 mM): p-chloromercuribenzene-sulfonate dissolved in47water. 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 nutrientsolutions to give a final ethanolic concentration of 1%. Control solutionswere treated with ethanol at the same concentration.2.7. ELEcm0PHYsI0L0GIcAL STUDY2.7.1. Transmembrane electrical potential measurementUsually plant cell transmembrane potential differences are in therange of -100 to -200 mV negative inside (Higinbothan, 1973; Tester,1990). In the early 1930’s, Umrath started to use microelectrodes tomeasure the membrane potential across the tonoplast (Findlay and Hope,1976). Since then, other electrical properties of plant cells have also beenstudied such as membrane capacitance (Curtis and Cole, 1938), membraneconductances (Cole and Curtis, 1939), and membrane resistance(Higinbotham et al., 1964; Spanswick, 1970; Anderson et al., 1974). Thecontemporary climax of electrophysiology occurred when Neher andSakmann (1976) developed of patch-clamping techniques. The combinationof molecular gene cloning and patch-clamp analysis (Hedrich et al., 1987)represents a particularly powerful means of elucidating the mechanism ofion transport through cell membranes.The chemical potential of an ion (j) is composed of all thosecomponents that enable it to do work and can be expressed by theequation [11]:48iO +RTlna+z1FVP+mgh [11]where j.i,j is the chemical potential of the ion fin joules mol-1 and is the’standard state chemical potential of 1 mole of the ions f per liter at 0°C; Ris the gas constant (8.314 J mol” °K’); T is absolute temperature in °K (°K= 273 + [°C] ); a3 is the activity of the ion; Z3 is its valency; F is the Faradayconstant (9.65 x 1O J mol1V);V is the electrical potential in volts; V1 isthe volume; P is the pressure; m1 is the mass; g is the gravitationalacceleration; and h is the height above sea level. In terms of solutetransport across the membrane, V1 is very small and h is generallynegligible. When the concentration (C1) of the solute is low so that theactivity and concentration are close, concentration C1 (mol rn-3) can be usedin place of the activity a1 (a1= yj C3 ), where yj is the activity coefficient.Simple diffusion is a non-mediated transport process whereby thesolute moves along the free energy gradient. In addition to the lipidcomposition, the difference of ion concentration just inside and outside theplasma membrane determines the diffusion of solute across a membrane.Ion diffusion through membranes may be described by the permeabilitycoefficient which is the flux per unit driving force (in its originalconception, the concentration gradient). For the diffusion of smallnoncharged molecules such as NH3 and H20, the chemical potential= + RT ln C1 [12]can be expressed as in equation [12]. Since the driving force is only due tothe concentration gradient from high to low (negative sign), the net flux J3(mol m2 -1) is expressed as in equation [13]:J3 = K1 J (-dji1/dx) [13]49Differentiating in equation [6] and replacing K1 RT (in equation [14]) by D1(the diffusion coefficient) (Stein,1986) gives equations [141 and [15]:J1 = - K1 RT d /dx [14]J1 =-D1 (dC1/ x) [15]Equation [15] is Fick’s First Law of diffusion, where K1 is the proportionalcoefficient or the mobility of the ion j, and D1 is the diffusion coefficient ofspecies fin m2 s. If P1 (m s-i) is the permeability coefficient of themedium or the membrane for ion j, thenP =-D3/z\x [16]therefore, for the concentration gradient zC1 = Co1- C1J1 = P1 z C3 = P1 (Co1 - C’1) [17]The permeability (P1) of a chemical species (j) is a measure of theability of the species of small non-electrolyte to pass through a membrane.The permeability coefficient for isopropanol or phenol is 10-6 m sec1across the plasma membrane (Nobel, 1983).The diffusion of most ions across the membrane is very low due totheir low permeability compared to non-electrolytes. In addition to theconcentration gradient, the electrical potential gradient must be includedin the driving force. Therefore, equation [11] can be presented as:= + RT ln C1 + z3 F-I’ [18]For a particular ion, the electrochemical potential gradient (11*]) determinesthe potential for passive ion flux. At equilibrium both outside and insideelectrochemical potentials are the same:50=-= 0 [19]combining equation [18] and [19]= (RT in C1 + zF’{ - (RT in C0 + zF%) [20]where : z$C10 is the electrochemical potential difference across themembrane; and C1 are the electrochemical potential outside and insidethe cell membrane respectively, ‘P1 and ‘P0 represent the inside and outsideelectrical potentials, respectively, measured as V; C1 and C0 are theconcentration (mM or mol m3) inside and outside the cell membrane,respectively.Because of the selective and permeable nature of membranes andthe existing concentration asymmetry, the electrical potential difference atzero net flux, when zs.i = 0, is defined as the Nernst potential (‘PN) as inequation [21]:RT C0=------ ln ( ) [21]zF C1This is the Nernst equation which describes the electrochemical potentialof an ion distributed at thermodynamic equilibrium between two phasesseparated by a cell membrane. Considering monovalant cations andassuming temperature to be 25°C equation [21] can be simplified to [22]:C0=- 59 log ( ) [22]CiWhen jC 0, equation [20] and [21] can be rearranged as:= zF ((‘{- ‘P ) - (RT in C0 - RT ln C1)/zF)51= zF- ‘PN) [23]where ‘PM is the measured membrane electrical potential differences acrossthe membrane in volts (‘-PM = ‘P-• ‘fe), normally this potential differenceacross 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 30to 40% of measured membrane potential (Pierce and Higinbotham, 1970;Higinbotham et aL, 1970). Salts (e.g. KC1 and NaC1) in solution dissolve torelease cations (K-i- and Na) and an aniàn (Cl-) which may have differentmembrane permeability (PK÷, PNa and Pcii. Presuming that there isinitially no electrical asymmetry across the membrane, when ions movealong their chemical potential gradient, different mobilities of cations andanions result in charge separation which creates an electrical potentialdifference, known as a diffusion potential (‘PD ). It can be assessed by theGoldman voltage equation:RT PK[K]o + PNa[Nalo + Pi[Cl]j +ln ( ) [24]F PK[KJj + PNa[Na]j + Pi[C1]o +The second source of membrane potential is the Donnan potential,though the contribution is relatively small. Inside the plant cell, there aremany large organic molecules, such as protein and other large polymers(RNA and DNA), with a large number of immobile carboxyl, phosphate andamino groups from which H can dissociate. The asymmetrical distributionof diffusible cations leads to a small negative potential across the plasmamembrane (negative inside) (Nobel, 1983).52Thirdly, a major component is a metabolically-driven potential dueto the operation of an electrogenic ion pump - the H pump. The H-’- pump(H-translocating ATPase) carries a net positive charge across themembrane and contributes directly to the membrane potential (Poole,1973; Sza, 1984). The activity of H pump depends on the hydrolysis ofATP catalyzed by a plasma membrane ATPase (Hodges, 1973; Poole, 1978;Spanswick, 1981). From equation [20], one can obtain an equation whichcalculates the electrochemical potential difference for proton at 25°C:AtH = A’P + 59 zpH [25]A proton concentration difference (ApH) and an electrical potentialdifference (AP) are two related entities that make up the electrochemicaldifference generated in part by the H-’--translocating ATPase (Sze, 1984).By actively pumping out H across the plasma membrane, a ‘proton motiveforce’ is built up which can provide the free energy necessary to transportother ions, both actively and passively into the cell (Poole, 1978). In otherwords, the H-’--pump generates both a potential difference (AW) to driveelectrogenic uniport, and an electrochemical gradient of protons to drivetransport of ions in antiport or symport with H.Since the electrochemical potential difference (AJI*jo) across amembrane is the combined chemical potential and electrical potentialdifference (equation [18]), it is used to describe the free energy status of asolute in a particular location. It is assumed that a difference of freeenergy between two points of a system represents the driving force for apassive flux of ions from one point to another. When the resultant chemicalpotential difference is just balanced by the resultant electrical potentialdifference (AI*jo = 0), there is no net flux of solute by passive forces.53Alternatively it can be stated that no energy is expended in moving ionsbetween the two locations.2.7.2. Single impalement and membrane potentialMicroelectrodes are commonly prepared from a micropipette filledwith electrolyte solution. It is a filament-containing or single-barreledborosilicate glass capillary tube with the fine-tip which is pulled witheither a vertical or horizontal electrode puller (Purves, 1960; Findlay andHope, 1976). The external diameter of the tip should be 0.5% or less of thediameter of the plant cell which it is to impale (Purves, 1960). Forcytoplasmic 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 orelectrical resistance (Findlay and Hope, 1976).Membrane potential difference can be easily expressed in a numberof equations (refers to section 2.7.1.), such as the Nernst potential (Eq.[21]), or electrochemical potential (Eq. [24]), or the Goldman diffusionpotential (Eq. [21]). When the potential difference is measured by insertedmicroelectrode, the value is an apparent resting potential which is the realpotential difference plus the total offset potential (Purves, 1981). The totaloffset potential includes the liquid junction potential, the tip potential andthe potential due to the possible dissimilarity between the indifferentelectrode and the electrode which contacts the microelectrode’s fillingsolution. The latter can be compensated by the offset control of theoscilloscope amplifier. The liquid junction potential occurs between themicroelectrode’s filling solution and the electrolyte outside the tip. It can54be decreased by use of 3 M KC1 as filling solution since the diffusioncoefficients of K and of C1 are almost identical.The tip potential is due to the characteristics of glass wall, electrolyteconcentration difference between inside and outside of the tip ofmicropipette and can be eliminated by filling the micropipette with low pHsolution or other treatments (Purves, 1960). A tip potential of -5 to -30mV was recorded for the microelectrode filled with 0.5 M KC1 plus 0.1 MMes (pH 5) (Ulirich and Novacky, 1990). The electrolyte solution could be 3M KC1 at pH 2.0 (Kochian et al., 1989; Glass et al., 1992) to get a highconcentration of ions in the tip and a low electrical resistance. As pointedout by Purves (1960) the history of microelectrode technology can beregarded as a succession of attempts to minimize tip diameter andresistance simultaneously.2.7.3. Setup for measuring membrane potentialThe fundamental setup for measuring electrical potential differencebetween two aqueous phases (cell ambient and cytoplasm), is an electricalcircuit which should be connected by a salt-bridge, i.e. Hg2C1 plus KC1(Willians and Wilson, 1981). The microelectrode is such a micro-salt-bridgeor miniaturized Calomel half-cell, and connected to the circuit with thesilver wire or silver/silver pellet (Purves, 1960). Besides themicroelectrode which impales the cell cytoplasm, another referencemicroelectrode (or the indifferent electrode) is also immersed in bathingsolution and connected to the ground. The electrical signals are amplifiedthrough a preamplifier (or electrometer), and are sent to the outputdevices such as the oscilloscope, the tape recorder, the pen recorder the55digital voltmeter or the audio monitor (Findlay and Hope, 1.976). Since theplant cells are tiny, vivid and fragile, the impalement the cell through thecell wall and cell membrane is operated by three-waymicromanipulators (Kochian et al., 1989; Glass et al., 1992) under themicroscope on an anti-vibration table (Purves, 1960). A diagram of such asetup is shown in Figure 2.2.8. Determination of amino acids in root tissueFree amino acids in root tissue were determined, after the methodreported by Fentem et al., (1983a, 1983b), as follows: weighed rootsamples were ground with liquid N2 in a porcelain mortar and extractedwith 80% aqueous ethanol. After centrifugation (IEC Clinic Centrifuge), thesupernatant was transferred to an evaporating flask. The extraction andcentrifugation were repeated 5 times. Pooled extracts were evaporatedunder vacuum at 35°C on a flash evaporator (Buchler Evapomix). The crudeextracts 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 furthercentrifugation and lyophilization. The extracts were derivatized withphenylisothiocyanate (PTC) automatically on an Amino Acid Analyzer (ABI,Model 402A) equipped to derivatize and hydrolyze applied samples, andthen separated by HPLC analysis (Separation system, ABI 130A). Theamino acid concentrations were determined by the Amino Acid Analyzerand analyzed by means of an ABI 920A data analysis module. Thechemicals used as amino acid standards were from Sigma.561. Compressed air2. Air-regulator3. Bathing solution reservoir4. Needle valve for controlling flow rate5. Small chamber for impalement6. Small pins on the wall to support the root7. Focusing plate of microscope8. Large chamber for the rest of root9. Over-flow of the bathing solution (level)10. Rice plant11. Impaling electrode12. Reference electrode13. Grounding electrode14. Electrode holder15. Preamplifier16. 3-dimensional manipulator (course)17. 3-dimensional manipulator (fine)18. Amplifier19. Chart recorderFigure 2. Setup for measuring cell membrane electrical potential.57Chapter 3. FLUXES AND DISTRIBUTION OF 13NH4 IN CELLS3.1. INTRODUCTIONThe short-lived radioisotope ‘3N (t172 = 9.98 mm) has been used as atracer in studies of the fluxes of NO3- and NH4 into intact roots of corn andbarley plants (McNaughton and Presland, 1983; Glass et al., 1985; Lee andClarkson, 1986; Hole et al., 1990; Siddiqi et al., 1991). It provides amethodology for the measurement of unidirectional fluxes (influx orefflux) across biological membranes over extremely short times and withgreat sensitivity (McNaughton and Presland, 1983). Because of its strong ‘emission, 13N can be determined rapidly and accurately, with little samplepreparation, 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 N03,reflecting the widely held perception that N03 is the predominant form ofN available to crop species. Relatively less is known about the uptake andsubcellular partitioning of NH4 in higher plants. Nevertheless in ricecultivation (Sasakawa and Yamomoto, 1978), in forest ecosystems (Lavoieet al., 1992), in Arctic tundra (Chapin et al., 1988) and even in wintervarieties of cereals growing in cold soils (Bloom and Chapin, 1981), NH4may represent the more important form of available nitrogen.It was demonstrated that net fluxes of NH4 into rice roots graduallyacclimated between 0.1 and 1 mM external [NH4+] so that net flux atsteady-state varied little between plants grown in these concentrations(Wang et al., 1991). Nevertheless, there is a lack of information about58fluxes between subcompartments in relation to acclimation or to themechanism(s) of NH4 uptake. For example, Presland and McNaughton(1986) failed to observe ‘3NH4 efflux from maize roots. By contrast, asizable net efflux of endogenous 14NH4was reported in wheat, oat, andbarley upon transfer to ‘5NH4 solution, although there was no exactcorrelation between root ammonium concentration and net 14NH4efflux(Morgan and Jackson, 1988a, b).The internal NH4-’- concentration of plant roots can readily beassayed, after extraction, by methods based on colorimetry or ion-specificelectrodes (Fentem et al., 1983a; Morgan and Jackson, 1988a, 1988b;Roberts and Pang, 1992). However, such analyses fail to provideinformation on the subcellular distribution of NH4. On the basis ofbiochemical analysis, it was concluded that more than one intracellularpool of NH4 existed in roots of rice (Yoneyama and Kumazawa, 1974,1975; Arima and Kumazawa, 1977). Two other methods have beenemployed to determine subcellular NH4 distribution, namely, effluxanalysis (Macklon et al., 1990) and the nuclear magnetic resonancespectroscopy (Lee and Ratcliffe, 1991; Roberts and Pang, 1992). Thesestudies recognized several NH4-’- fractions of roots, corresponding to thoseof the superficial, water free space, Donnan free space, the cytoplasm andthe vacuole.In this chapter, the results of compartmental analyses, using 13NH4+efflux, are used to estimate the half-lives of NH4 exchange and the size ofmajor compartments in root cells, as well as NH4 fluxes between thesecompartments. Together with data obtained from chemical fractionation, itwas possible to develop a detailed analysis of the initial fate of absorbed‘3NH4.In addition, the t1/2 values for 13NH4 exchange provide essential59parameters for the design of appropriate protocols for influx measurement,particularly the duration of‘3NH4 loading and post-wash treatments. Toevaluate the methodology of the compartmental analyses, influx and netflux of NH4 were also measured by independent methods.3.2. MATERIALS AND METHODS3.2.1. Plant growth and ‘3N productionDetails of seed germination, growth conditions, provision of nutrientsand production of1NH4are described in Sections 2.2.. 2.3.. 2.4., and 2.5.,respectively.3.2.2. Measurement of fluxes3.2.2.1 1NH4InfluxChecks of the fluxes derived from efflux analysis: After ‘loading’ for10, 20, and 30 mm, respectively, at steady-state conditions, influx of13NH4 was also determined by two independent methods: (1) theaccumulation of 13N by seedling roots (see section 2.3.2.); (2) the rate ofdepletion of 13NH4-’- from ‘loading’ solution.3.2.2.2. Net NH4 fluxIn addition, the net flux of NH4was also measured based on the rateof depletion of 14NH4 (see section 2.3.4.).603.2.2.3. Time course of13NH4uptakeIn the time-course experiments, G2 or G100 plants were exposed to 2iM or 100 jiM13NH4-labeled loading solutions, respectively, for durationsranging from 10 sec to 31 mm. As described in section 2.3.1., roots weresubjected to a standard pre-wash, loading and post-wash procedure.3.2.3. Compartmental AnalysisThe procedure for compartmental analysis was followed asdescribed in section 2.4.5.3.2.4. Partition of absorbed ‘NH43.2.4.1. Separation of13N-compounds in plant tissue13NH4-’- was separated from its immediate metabolic products byCation Exchange Column (CEC) Separation described in section 2.5. Afterplants were loaded in 100 jiM‘3NH4for 30 minutes, the separated, frozen13NH4-labeled shoots and roots were first counted in the gamma counterand then ground in liquid nitrogen. After the filtration, the radioactivityremaining on the filter was referred to as root debris. The filtrate waspassed 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 (OnCEC). Two sets of G100 plants, containing 100 120 plants each, were used.613.2.4.2 Chemical assay ofNH4 in root tissueRoot NH4 contents (Qj) of G2, G100, and G1000 seedlings wereseparated 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 1NH4fraction in roots following 30 mm 1NH4 loading. This amount (Q*c+v)represented the combined values of cytoplasmic (Q*c) and vacuolar (Q*v)radioactivities that can be converted to a chemical quantity (Q+) afterdividing by the specific activity of‘3NH4÷in the external solution (S0):Qc+v=Q*c+v/So [261The specific activity of‘3NH4within the cytoplasm (Sc) during loading willincrease to its steady-state value according to the rate constant for tracerexchange of the cytoplasm (kc = 0.693 / ti,’2 ) as given in the followingequation (Walker and Pitman, 1976).S = S0 (1 - ekct) [31Thus, if S0 and t1/2 are known, S can be determined for any particulartime (t). By 30 mm of loading (equivalent to 4 cytoplasmic half-lives, seeTable 2), the specific activity of cytoplasmic‘3NH4 (Sc) is brought toapproximately 94% of S and 13NH4 accumulated within the cytoplasmalso 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 () can be roughlyestimated (Method I). The portion of Q*c+v that is transferred to thevacuole (Q*) is given by:*_( / \ *v— ‘ +vI c+vThe accumulation of tracer in the root vacuole is related to the chemicalflux to vacuole (Ø) and the specific activity of the cytoplasm at eachinterval:*_f Cv (t)— ‘I’cv •-‘c (t)and Q*Oi*ES(t) [30]The sum of tracer accumulation within the vacuole Q* ( Q*v (t)) is givenby Equation [28], and E Sc (t) can be calculated for each minute fromEquation [3]. Therefore, by means of Method II, it is possible to estimateØ, more rigorously from Equation [30].3.3. RESULTS3.3.1. Compartmental analysisAnalysis of the ‘3N released into ‘washing’ solutions duringcompartmental analysis revealed that 99.5% of the radioactivity wasretained on the CEC (Table 1). Since positively charged amino acids(arginine, histidine and lysine) represented only 5% of total amino acids in633-week-old rice roots (Yoneyama and Kumazawa, 1974), I interpreted thisresult to indicate that‘3NH4was the predominant N species released fromroots and adsorbed on the cation exchange resins.The influence of [NH4]0on compartmental analyses was investigatedby using G2, G100, or G1000 plants, to represent inadequate, adequate andexcess N supply, respectively, prior to efflux measurements. Arepresentative sample of such data (18 mm efflux) for G1000 plants isshown in Fig. 3. Three distinct phases, having different slopes with high r2values were found for each of the three types of plants tested (G2, G100and G1000). These compartments were tentatively defined ascorresponding to: (I) the superficial solution adhering to roots, (II) the cellwall and (III) the cytoplasm, respectively. The half-lives for exchange(t1/2) of these compartments were calculated to be 3 sec, 0.5 to 1 mm,and 7 to 8.5 mm, respectively (Table 2). According to Duncan’s multiplerange test, there were no significant differences for these values amongplants grown under different concentrations of NH4, except for the cell-wall fraction of G2 plants.One important part of the compartmental analysis was to calculatethe fluxes of NH4 across the plasma membrane of root cells. Thesecalculated fluxes are in good agreement with the values obtained by moredirect methods using the same root material (Table 3). Influx (%) variedwith the NH4 level provided during the growth period. Average NH4influx 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‘3NH4-- efflux(derived from efflux analysis) from the influx of 13NH4,or by measuring64Table 1. Separation of13N-labeled compounds by cation exchange column.The loading solution, efflux solution and shoot extract were assayed. Eachmean 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)658—‘S 7•.—E 6-C.? .5.4.‘5-,3.——‘-,2-z10- • - I • I •0 5 10 15 20Efflux time (mm)Figure 3. A representative pattern of 13NH4 released from intact roots.The rate of 13NH4 (log(cpm) g’FW mm-i) released from intact rice rootsof G1000 plants during 18 mm efflux (see text for details). Three phases (I,II, and III) of 13NH4releasing were determined by correlation coefficient.66Table 2. Estimated half-lives of 13NH4exchange for three compartments ofroot cells. Means for half-lives of ‘3NH4 exchange (t1/2) for threecompartments (superficial, cell wall, and cytoplasm) were estimated fromthe efflux analysis. G2, G100 and G1000 plants were loaded in 13N-labeledMJNS for 30 mm and effluxed in un-labeled identical MJNS for 18 mm atsteady-state conditions with regards to [NH4]0.Each mean is the averageof 4 individual efflux tests ± Se.Compartments G2 G100 G1000I. Superficial (s)a 3.42 ± 1.00 a 3.83 ± 0.24 a 3.38 ± 0.37 aII. Cell wall (mm) 1.06 ± 0.10 b 0.57 ± 0.09 a 0.43 ± 0.06 aIII. Cytoplasm (mm) 6.95 ± 1.14 a 7.36 ± 0.12 a 8.33 ± 0.60 aa 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% levelof significance.67Table 3. Comparison of‘3NH4fluxes across the plasma membrane of rootcells. Each mean13NH4+fluxes (influx, efflux, and net flux) is the averageof 3 or 4 replicates with ± Se.Methods G2 G100 G1000Net flux (Pnet):NH4efflux analysis a 1.06 ±0.0714NH4-’- depletion of medium a 1.11 ± 0.04Efflux ():(7) 1NH4efflux analysisa(8) Subtracted (6) from (2)(9) Subtracted (6) from (4)(jimol g’FW h’)5.97 ±0.41 10.51 ±2.045.27 ±0.20 10.16 ±0.236.99 ±0.51 10.29 ±0.296.11 ±0.32 9.66 ±0.63Influx (%):(1) ‘NH4-- efflux analysis a 1.20 ± 0.07(2) 1NH4accumulated in roots b 1.39 ±0.02(3) NH4depletion of medium b 1.37 ±0.02(4) 1NH4depletion of medium b 1.33 ± 0.01(5)(6)4.80 ± 0.394.32 ±0.151.17 ±0.140.94 ± 0.051.79 ±0.477.41 ± 1.556.08 ±0.273.09 ±0.564.09 ±0.043.58 ±0.360.13 ±0.020.27 ±0.020.22 ±0.17a Based on 30-mm uptake; b Based on 10-mm uptake.68net depletion of ‘4NH from the uptake solution. Both methods gavesimilar results with average values of 1.09 ± 0.03, 4.56 ± 0.24, and 6.75 ±0.67 jimol NH4 g’FW h-i for G2, G100 and G1000 plants, respectively. Theinflux and net flux values of G100 plants were 4 fold higher than those ofG2 plants (Table 3). Fluxes of G1000 plants were about 1.5 times thevalues 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 NH4-’-content (Q) expressed as moles per unit weight of roots (pmol g’), andNH4-’- concentration ([NH4]) expressed as moles per unit volume of acompartment (jiM or mM). The results of estimated cytoplasmic NH4concentration ([NH4]), chemically assayed total root NH4 contents (Q) ofG2, G100 and G1000 plants, as well as calculated values of [NH4--]1,[NH4],Q and Q, are presented together in Table 4. Values of [NH4-’-]1and [NH4-’-Jwere higher with higher levels of NH4 provision. The values of [NH4-’-]c,were 5 to 6 fold higher in G100, and 10 fold higher in G1000 plants thanin G2 plants. The values for the vacuolar pool were based on thedifferences between the total NH4 content in the roots (Qj) and thecytoplasmic pool (O). Of the total NH4 of the roots, 92% was localizedwithin the vacuole in G2 plants and about 72% to 76 % in G100 and G1000plants. Chemical and radioisotopic quantities for various compartmentsused in calculating are presented in Table 5. The specific activity ofcytoplasm (S (t)) was calculated for each minute from t=1 to 30 mm. BothE Q*v (t) and E S (t) were used for estimating cv. The P, estimated bymethods I and II are given in Table 5.69.Efflux 11%:. iIIralIJIiIdlIl90 Efflux 20%EffluxNet Flux 899k Net Flux 8O9 Net Flux 719kG2 G100 G1000PlantsFigure 4. Fluxes of G2, G100, G1000 plants. Efflux (Ø) and net flux (ønet) aspercentage of influx (Ø) for G2, G100 and G1000 plants at steady-statebased on the data of compartmental analysis in Table 2.70Table 4. Size of ammonium pools in root cells. Ammonium pools in rootcells of G2, G100 and G1000 plants at steady-state. The contents of unmetabolized NH4÷ in root tissues (Q) and cytoplasm (Q) and vacuole (Q,)and their corresponding NH4 concentrations ([NH4], and [NH4-’-]V), as wellas that of the cell wall pool ([NH4-’-]), are presented.Plant NH4 content NH4÷ concentrationab [NH4-1-]w [NH4] [NH4÷](imolg4FW root) (mM)G2 2.38 0.19 ( 8%) 2.19 (92%) 0.56 3.72 2.58G100 4.31 1.03 (24%) 3.28 (76%) 2.27 20.55 3.86G1000 6.85 1.94 (28%) 4.91 (72%) 14.41 38.08 5.78a The values of [NH4]and [NH4]were estimated from compartment analysis withfour replicates each and [NH4 was estimated from Q3.b The values of Q were obtained from chemical NH4 assay with three replicates eachand are the same as the values of [NH4 +].C The values of Q were calculated from ENH4]based on the assumption that thecytoplasm only had 5% of total cell volume.d The values for Q, are based on the difference between Q and Q and the assumptionthat the vacuole occupies 85% of cell volume. In parenthesis, Q or Q, respectively,are presented as percentages of Q.713.3.2. Metabolism and translocation of 13NVirtually none of the‘3NH4absorbed by rice roots was translocatedto the shoots (Table 1). It is improper to express the translocation of ‘3N(to the shoot) as j.tmol NH4 per gram fresh weight of roots because (a) ‘3Nis transported from the root in the form of amino acids and (b) the specificactivities of these amino acid pools were unknown. Therefore thetranslocation was expressed as a percentage of the total radioactivity (cpmaccumulated in roots plus shoots during the loading period). This totalradioactivity is equivalent to net absorption of 1NH4. Furtherfractionation of root tissues of G100 plants by the CEC separation revealedthat about 8.6% of the radioactivity provided by influx during 30 mm‘3NH4loading was retained in a metabolized form (Table 6). By combiningthe 13N translocated to shoots (10%) with ‘root debris’ (4%) and the ‘Off CEC’fraction (5%), an estimation of the proportion (19%) of absorbed 1NH4that was metabolized during the 30 mm was obtained. The partitioning ofradioactivity was also calculated based on the total cpm remaining in roots(Table 6).3.3.3. Time course of‘3NH4influx in rice rootsThe results of steady-state‘3NH4-’- uptake by G2 and G100 plants,establishing the pattern of‘3NH4accumulation in rice roots, are shown inFig. 5. The accumulation of‘3NH4appeared to be linear for the duration ofthe 30 mm uptake experiments; the coefficient of determination of theselines (0.87 and 0.99 for G2 and G100 plants, respectively) were high. In allcases, the intercept on the ordinate differed significantly from zero (at 5%significance level). G100 plants had a higher accumulation rate than G272Table 5. Calculation of the flux () from cytoplasm into vacuole. The dataused in calculation were taken from the results of the compartmentalanalysis (Table 4) and root partitioning experiment (Table 6). Thecalculation procedure is in section 3.2.4.2.Parameter value unitS 164214 cpmimol4Sc (t) (t=30 mm) 3361875 cpm pmol-’ZQ*v(t) (t=3Omin) 79666 cpmg’Q*c+v 238982 cpmg’1.46 imolg40.97 jimolg’Q__v 0.49 iimol g4Pcv (Method I) 0.97 jimol g’ h’øcv (Method II) 1.42 jimol g’ h-’73Table 6. Distribution of newly absorbed ‘3N in shoot and root tissues. After30 minutes loading in 13N-labeled MJNS containing 100 jiM NH4-’-,Fractionation of radioactivity in shoots and roots of G100 plants werecarried out according to sections 2.5. and 3.2.4.1. Radioactivities areexpressed as percentages of total cpm in plants. Each analysis used 100 to120 plants and data given are means of two replicates (±se).% cpm in plant[A] in shoots 9.7 ± 0.9[B] in rootsi. total 90.3 ±0.9ii. % recovery after CEC(a) On-CEC 81.7 ±2.5(b) Root debris 5.1 ± 1.0(c) Off-CEC 3.5 ±0.6[C] Metabolized 18.3 ± 2.5a‘Metabolized’ is the sum of lines [A] , (b) and (c) based on the total cpm in wholeplants or the sum of lines (b) and (c) based on the total cpm in root.744.‘GlOO’ plants (R”2 = 0.99)0 ‘G2’ plants (R”2 = 0.87)E+r1z00 5 10 15 20 25 30 35Uptake time (mm)Figure 5. Cumulative uptake of‘3NH4by G2 and G100 roots. Time coursestudy of 13NH4uptake by G2 and G100 roots at steady-state. G2 or G100rice plants were grown and loaded in‘3N-labeled MJNS containing 2 p.M (a)or 100 p.M (+) [NH4]0, respectively. Uptake is expressed as theaccumulation of‘3NH4-’- (p.mol g’FW). Each datum point is the average of 3replicates with standard errors as vertical bars.75plants. The data for ‘3N accumulation were used to calculate the rate of ‘3Naccumulation (influx) as a function of time (Fig. 6). Based upon very shortexposures (less than 2.5 mm) to 13NH4+, the influx of G100 plants appearedto be about 20 to 30% higher than the steady value of influx. Beyond 5 to10 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. DiscussioN3.4.1. The half-lives of 13NH4exchangeThree kinetically distinct phases (I, II, III) with half-lives for 13NfI4÷exchange of approximately 3 sec, 1 mm and 8 mm, respectively, wereidentified by means of compartmental analysis (Table 2). Phase I isprobably due to the surface solution on roots carried-over from the‘loading’ solution (Fig. 3). The second phase is attributed to the cell wallfraction, or the apparent free space (AFS) which is the sum of the WaterFree Space (WFS) and the Donnan Free Space (DFS) (McNaughton andPresland, 1983 and references therein). The half-life of this phase (0.5 to 1mm) was shorter than the equivalent phase reported for corn roots (2.5mm) by Presland and McNaughton (1986), but similar to the half-life forN03 exchange (0.5 mm) in barley roots (Siddiqi et al., 1991). By using the‘efflux-funnel’, shorter efflux intervals were achieved. This allowed forresolution of these two rapid phases (I and II) and more accurateestimation of the cell wall half-life.761614 A- G100 PlantsG2 Plants+. 4Uptake time (mm)Figure 6. Influxes of 13NH4 into G2 and G100. Steady-state influxes of13NH4 into G2 and G100 roots were measured in the time course study.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.77The third phase is believed to be the cytoplasm. The half-lives ofcytoplasmic exchange for G2, G100 and G1000 plants ranged from 6.9 to8.3 mm, but the differences were statistically insignificant, although thecytoplasmic pool sizes varied according to the provision of NH4-- duringgrowth (Table 2). Siddiqi et al., (1991) showed that barley roOts, treatedwith SDS or pretreated by immersion in water at 70°C for 30 mm,accumulated and released significantly less ‘3N0 from phase III, butphase II appeared unaffected. These results were consistent with phase IIIbeing the cytoplasm. In studies of 1NH4 efflux from spruce roots,Kronzucker, H. (personal communication) has found that elevated [Ca2]0jthe loading and washing solutions reduced the extent of phase II for‘3NH4 exchange in spruce roots, (which had similar half-lives to thoseobserved in rice) as would be expected if this phase corresponded to thecell wall compartment. The short half-life of ‘3N decay, and long half-lifeof exchange of the vacuole (Lee and Clarkson, 1986; Macklon et al., 1990),precludes the estimation of vacuolar parameters by efflux analysis using‘3N. Using 15NH4,Macklon et al., (1990) estimated the half-lives forcytoplasmic 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 etal., 1990) observed much shorter t172 values for cytoplasmic ‘3NH4exchange, ranging from 4 to 10 mm in roots of wheat. The latter values aremuch 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) mayhave arisen from species differences and/or differences of methodology.In order to select appropriate durations for the loading and washingperiods employed in influx studies, it is important to estimate the halflives for 13NH4-’- exchange between different compartments (Cram, 1968).78The choice of a 10 mm loading time, used in the present study and insubsequent 13NH4+ influx studies, was arrived at from considering thefollowing: (1) the half-life of 13N decay is short (t1,’2 = 9.98 mm) andtherefore the influx period should be as short as possible. As the isotopedecays, the statistical uncertainty in the measurement of 13N retained bythe plant roots or transported to the stem becomes as high as ±15% afterabout 40 mm (McNaughton and Presland, 1983); (2) if the loading time islong, compared to the t1/2 for cytoplasmic exchange for‘3NH4,the specificactivity of the cytoplasmic pool may approach saturation and the‘3NH4--efflux term (4c0) will be maximized. The measured 13NH4 influx underthese conditions would approximate the net flux (Pnet = Øoc- ‘Pco); (3)although the over-estimation of influx (see below) was minimized by 20minutes, 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 acompromise (Lee and Clarkson, 1986). Since the goal was to measure theunidirectional flux across the plasma membrane (Ø), 13N present in thecell wall should be removed during the post-wash period. Based on theestimated t1/2 of the cell wall fraction, a short post-wash period of 3 mm(corresponding to 3 to 6 half-lives, Table 1) was adopted in all influxexperiments. In order to equilibrate the cell wall fraction to any changes of[NH4--]0,rice roots were, therefore, always pretreated for 5 mm in identicalun-labeled MJNS before loading in 13N-labeled MJNS.3.4.2. Fluxes of 13NH4into root cellsThe results of the present study showed that 13NH4+ appeared to beaccumulated at a constant rate (r2 = 0.874 and 0.997, respectively) during7930 mm loading of G2 and G100 plants under steady-state conditions (Fig.5). Moreover,‘3NH4accumulation increased with increasing [NH4+]0of theloading solution. This observation is similar to previous reports indicatingthat the accumulation of ‘3N (either as‘3N0-or‘3NH4÷) by plant rootsincreases in linear fashion during short (usually <15 mm) loading periods(Presland and McNaughton, 1984; Lee and Drew, 1986). The data for13NH4-’- accumulation by G2 and GlOD plants are also presented as plots ofinflux versus time (Fig. 6). Influx values based upon very short exposuresto‘3NH4-’- were accompanied by large errors probably associated with thelower counts accumulated and a large multiplicative factor involved incalculating influx on a per hour basis. Nevertheless, the data indicated thatinitial influx values were 20 to 30% higher than those recorded after 2 to 5mm. After loading for more than 5 mm, the influxes were 1 and 7.5 jtmolg1FW h4 for G2 and G100 plants respectively, and notwithstanding somevariation, remained reasonably constant for the next 25 mm. Presland andMcNaughton (1984) noted a higher rate of1NH4accumulation in maizeroots during the first 2 mm that they attributed to apoplasmic filling. Inthe present study, although the roots were subjected to a 3 mm post-wash,any tracer uptake by rice roots during the post-wash period wouldrepresent an over-estimate. The impact of these additional counts wouldbe to over-estimate the calculated influx values at shorter loadingintervals due to the multiplicative effect in calculating fluxes on a per hourbasis. This effect, which decreases as the duration of the influx periodincreased, was minimized at about 20 mm (Fig. 6). This interpretation is incontrast to that of Lee and Ayling (1993) who argue that the lower countsrecorded after 2 to 5 mm represent an under-estimate of influx due torelease of absorbed ‘3N or ‘5N as cytoplasmic specific activity reachessteady-state. I question this interpretation because: (1) the t172 for80exchange of1NH4from the cytoplasmic phase was 8 mm for rice rootsgrown at various nitrogen conditions (Table 2); (2) the absolute value ofthe efflux from cytoplasm to outside () varied from 10 to 30% of influx(Ø) according to compartmental of analyses (Table 3). Therefore Iconsider it unlikely that a significant reduction of measured influx wouldresult from efflux of tracer during the short duration of these exposures.Values for influx efflux (Ø) and net flux (Ønet) of 13NH4determined by efflux analyses corresponded very well with those obtainedby other (more direct) methods (Table 3). This close correspondence allowsus to accept the parameters derived from‘3NH4compartmental analysiswith some degree of confidence. Influxes of 13NH4 into rice roots understeady-state conditions increased according to the levels of [NH4+]0in thegrowth media (Table 3). A similar trend was shown for net fluxesdetermined either by efflux analysis or by depletion methods. Net uptake(Pnet) tended to show only a small increase as [NH4]0increased from 100to 1000 1iM (Table 3). This confirms my previous report that net uptake ofNH4was acclimated to [NH4+]0in growth media, although the acclimationwas not achieved by G2 plants (Wang et al., 1991). These resultsdemonstrated that NH4 fluxes are closely related to the nitrogen status ofplants, which is determined by plant growth conditions.Estimated effluxes of NH4 from rice roots were about 10, 20 and29% of the influx values for G2, G100 and G1000 plants, respectively(Table 3 and Fig. 4). In addition, efflux was positively correlated with the[NH4-’-] (Table 3 and 4). This result agrees with the suggestion thatcontinuous NH4 efflux may be a common feature of net NH4 uptake byroots of higher plants (Morgan and Jackson, 1988a). Nitrogen efflux (eitherNH4 or NO3-) has been reported to be quite significant, particularly at81elevated concentrations of N (Morgan et al., 1973; Breteler and Nissen,1982). Indeed, Deane-Drummond and Glass (1983a, b) suggested thatnitrate efflux might regulate net uptake by means of a type of ‘pump andleak’ mechanism. By contrast, Lee and colleagues have emphasized theimportance of influx in the regulation of net uptake of nitrate, althoughnitrate 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‘4NH-’- occurredupon transfer to 15NH4solutions in wheat, oat, and barley adequatelysupplied with nitrate. However an exact parallel between root ammoniumconcentrations and net 14NH4efflux was not observed. Although plasmamembrane influx determines the maximum rate of net uptake (Lee andClarkson, 1986), efflux certainly makes a significant contribution todetermining net uptake.Because of its short half-life, ‘3N is unsuitable for the determinationof vacuolar parameters by efflux analysis. Nevertheless, the combination of‘3NH4efflux analysis and the CEC separation of ‘3N products enabled us toestimate Ø, using two methods. Both results give values for in therange from 1 to 1.5 jimol g-’FW h-1. Method (I) is based on the estimated13NH4 accumulation during 30 mm loading, while method (II) involvedthe use of S values estimated minute by minute from a knowledge of thehalf-life of cytoplasmic exchange (see section 3.2.5.). Therefore method (II)is probably more refined than the value derived from method I. Thesevalues are somewhat lower than those obtained by efflux analysis in onion(Macklon et al., 1990), however the Macklon’s study was undertaken at 2mM [NH4+10,compared to my analyses undertaken with G100 plants at 10082jiM NH4. The differences may also reflect the methodology and plantsspecies employed.3.4.3. The NH4 pools in rootsIn the present study, the values of Q were in the range from 2.38 to6.85 jimol g’FW for roots grown with different levels of NH4-’- (Table 4).Fentem et al., (1983b) reported a value of 3.2 jimol g’FW in 9-d-oldbarley roots grown in 1 mM NH4.For barley, wheat and oat grown in NO3-or N-free conditions, the value of [NH4+]1was in the range of 0.4 to 2 jimolg-1FW (Morgan and Jackson, 1988a,b, 1989). However, when plants grownin NH4 or in N03 were pretreated with 0.5-1.5 mM [NH4+]0for variousperiods 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 relativelylow intracellular NH4 content, particularly, under steady state conditions,may reflect the efficiency of NH4 assimilation (Goyal and Huffaker, 1984).Irrespective of the [NH4-’-]0 provided during the growth period, thebulk of absorbed NH4was localized in the vacuole (Table 4). Nevertheless,because of the large size of the vacuole, the values of [NH4-’-] weresignificantly lower than those of the [NH4÷] (Table 4). Increasing [NH4+]0from 2 to 1000 jiM, caused [NH4]c to increase more than 10 fold, while[NH4÷] increased by only 2 fold. Cytoplasmic NH4 concentrations of riceroots estimated in the present study (Table 4) were in the range ofreported 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 NH4 distribution in root tip of maize,cytoplasmic [NH4+] ranging from 3 to 438 jiM were reported (Roberts and83Pang, 1992). However, in that study, lower values might be expected sinceroot tips were excised from 2-day-old maize seedlings and maintainedwithout an exogenous source of NH4 during estimation of [NH4]by NMR.My indirect estimation of [NH4]v provided a range from 2.6 to 5.8 mM forG2, G100 and G1000 plants (Table 4). Using ‘5N, Macklon et al. (1990)reported a similar range (3.9 to 10.9 mM) for [NH4] in cortical cells ofonion roots. Slightly higher values (15 to 36 mM) for [NH4-’-] wereestimated in maize roots by‘4N-NMR spectroscopy (Lee and Ratcliffe,1991).3.4.4. Model of NH4uptake by rice plantsDespite the widespread use of compartmental analysis to investigatecompartmentation of non-metabolized ions, e.g. Cl- (Cram, 1968), Na(Jeschke and Jambor, 1981), and K (Memon et al., 1985), relatively fewstudies have been undertaken using metabolizable ions such as PO4(Lefebvre and Clarkson, 1984), NO3- (Presland and McNaughton, 1984; Leeand Clarkson, 1986; Siddiqi et al., 1991), S042 (Cram, 1983) and NH4(Macklon et al., 1990). Presland and McNaughton (1984) postulated theexistence of four compartments (three in the roots and one in the shoot)based upon the distribution of ‘3N among these tissues in maize plants.Using 1NH4efflux analysis with excised onion roots, the compartmentalparameters for superficial, water free space, Donnan free space,cytoplasm and vacuole were identified (Macklon et al., 1990). The presentstudy has characterized two intra-cellular compartments and one extracellular compartment for 13NH4 in rice roots. The biochemicalfractionation approach was also used to identify different compartments84Plasmakmma Root cell Stele —‘I..Cytoplasm Metabolites I[13.OC 1 i5.78J [_[_] A551% cI1O%NH4 0 cxc(?)‘S “S ‘.S5 “S ‘S ‘SFigure 7. Proposed model for ammonium uptake and compartmentation inrice G100 roots. The bold values in parentheses are estimated fluxes ofabsorbed 13NH4 (jimol g”FW h”). The percentages represent the relativedistributions of ‘NH4 among the compartments as a proportion of theisotope entering the cell during the 30 mm loading. oc, from outsideplasmalemma 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 translocationfrom shoot to root; cIASS, assimilation rate; ØDEG, degradation rate; prepresents chemical flux and 1 represents radioisotopic flux. Fluxesaccompanied by (?) indicate fluxes for which data are not available fromthe present study.85for NH4 assimilation. By using 15NH4,three compartments were foundcorresponding to different cell types and a storage pool in barley roots(Fentem et al., 1983a) or different organelles (Rhode et al., 1980). Spatialdifferences in the activities of enzymes involved in NH4-’- assimilation arealso found along the root (Fentem et al., 1983a). In addition to this form ofheterogeneity, there are distinct isozymes of glutamine synthetase, locatedwithin the cytosol and within plastids (Miflin and Lea, 1980).Much less information is available concerning the partitioning ofnewly absorbed ammonium between these compartments, particularlyconcerning the partitioning between metabolized and un-metabolizedfractions in the root and translocation to the shoot. In the presentexperiments, nearly 90% of absorbed ‘3N remained in the roots, of which80% was in the cation form (‘3NH4)after 30 mm ‘loading’ (Table 6).Among the ‘metabolized’ ‘3N pools (ass) in roots, significant quantities ofabsorbed ‘3N (10%) were translocated to shoots (F) during theexperiment (Table 6), and analysis of this ‘3N by ion-exchangechromatography (Table 1) revealed a virtual absence of 13NH4.Theremaining metabolized fractions consisted of 5.5% that failed to be held onthe 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 analysesshowed that un-metabolized NH4 in the cytoplasm (Q) constituted only8% 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 spatialand biochemical compartmentation of newly absorbed NH4-’- uptake by riceroots is given in Fig. 7. About 24% of un-metabolized NH4was allocated tothe cytoplasm and 76% to the vacuole. Based on the influx of 13NH4÷into86roots, 21% and 40% of ‘3N remained in the cytoplasmic and vacuolarcompartments, 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 13Ntransported across the plasmalemma and may underestimate the trueassimilation rate because during the loading period, the cytoplasmic‘3NH4pool would not have reached steady state.3.5. SUMMARYUptake of 13NH4by roots and distribution of 13NH4 among plantparts and sub-cellular compartments was determined on rice plants grownhydroponically in MJNS containing 2, 100 or 1000 jiM NH4. At steady-state, the influx of‘3NH4was determined to be 1.31, 5.78 and 10.11 jimolg’FW h’, respectively, for G2, G100 and G1000 plants; efflux was 11, 20,and 29%, respectively, of influx. The NH4 flux to the vacuole wascalculated to be between 1 to 1.4 jimol g’FW h-i. By means of 1NH4÷efflux analysis, three kinetically distinct phases (superficial, cell wall, andcytoplasm) were identified, with half-lives for i3NH4+ exchange of 3seconds, 1 and 8 minutes, respectively. Cytoplasmic [NH4]was estimatedto be 3.72, 20.55, and 38.08 mM for G2, G100 and G1000 plants,respectively. These concentrations were higher than vacuolar [NH4], yet72% to 92% of total root NH4 was located in the vacuole. Distributions ofnewly absorbed 13NH4between plant parts and among the compartmentswere also examined. During a 30 minute period G100 plants metabolized19% of the influxed 13NH4.The remainder (81%) was partitioned among87the vacuole (20%), cytoplasm (41%) and efflux (20%). Of the metabolized‘3N, roughly one half was translocated to the shoots.88Chapter 4. KINETICS OF ‘3NH4 INFLUX4.1. INTRODUCTIONDespite 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-availablenitrogen form in paddy soil (Yu, 1985). It is the preferred nitrogen speciestaken up by rice (Fried et al., 1965; Sasakawa and Yamomoto, 1978), andin terms of the efficiency of fertilizer ultilization, ammonium is superior tonitrate 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 extensiveinvestigations of N03 uptake, the kinetics and energetics of ammoniumtransport in higher plants have received relatively little attention. In bothrice plants and Lemna NH4 uptake followed a bi-phasic pattern, with asaturable carrier-mediated system operating at low external NH4 ([NH4]0)and either a second saturating system (Fried et al., 1965) or a lineardiffusive component at elevated [NH4]0(Ullrich et al., 1984). In N-starvedLemna both NH4 uptake by the saturable system and depolarization ofplasma membrane potential were found to exhibit the same concentrationdependence (KmT5 for both processes were 17 jiM). At higher [NH4-’-]0 theuptake by the linear system was not accompanied by furtherdepolarization of membrane potential (Ullrich et al., 1984). The saturable89component of NH4 uptake was sensitive to some metabolic inhibitors(Sasakawa and Yamamoto, 1978) and to changes of root temperature(Bloom and Chapin, 1981). In addition, NH4-’- uptake is subject to negativefeedback, supposedly from N metabolites (Lee and Rudge, 1986; Morganand Jackson, 1989; Clarkson and Luttge, 1991). Youngdahl et al., (1982)demonstrated that NH4 uptake in rice decreased with plant age. However,despite these studies, the mechanism(s) of NH4-’- uptake by roots of higherplants remain unclear. In particular, the high concentration systemrepresents virtually unexplored territory.Ammonium is unique among inorganic cations, because followingabsorption by plant roots, it is rapidly assimilated into organic poois. Thishas made the analysis of uptake and the subsequent fate of absorbed NH4much more complicated than for cations such as K or Ca2.The availabilityof 13N to this laboratory has enabled us to measure short-term 13NH4influx into roots of intact rice plants (Wang et al., 1991, 1993a, 1993b).This is critically important for two main reasons. Firstly, this techniqueallows determination of the particular flux (e.g. unidirectional plasmamembrane influx or efflux), which is responding to the imposed conditions.By contrast, net uptake measurement, often obtained by means of long-term depletion experiments, actually measures the difference betweeninflux and efflux. This is especially relevant because nitrogen (either NH4or NO3-) efflux has been reported to be significant, particularly at elevatedconcentrations of N (Morgan and Jackson, 1989; Breteler and Nissen, 1982;Wang et al., 1993). Secondly, by judicious choice of appropriate influx anddesorption times, based upon the half-lives for exchange of the subcompartments of the root (Lee and Clarkson, 1986; Presland andMcNaughton, 1986; Siddiqi et al., 1991; Wang et al., 1993), it is possible to90measure the plasma membrane influx as opposed to other fluxes (tovacuole or to stele) which result from long-term experiments (Cram, 1968).The objective of this study was to investigate the mechanisms andcharacteristics of ammonium uptake by rice plants. I have particularlyemphasized short-term responses of 13NH4influxes to changes in [NH4]0of uptake solutions over a wide range of external concentrations, in orderto define the transport mechanisms responsible for influx across theplasma membrane. I have examined the influence of prior NH4 provisionupon the kinetic parameters for influx by both components of the biphasic system for NH4 transport. In addition the sensitivities of thesefluxes to metabolic inhibitors, short-term variations in temperature and pHwere determined with a view to clarifying the mechanisms of these fluxes.4.2. METHoDS AND MATERIALS4.2.1. Plant growth and 1N productionSee section 2.2. Seed germination; section 2.3. Growth conditions;section 2.4. Provision of nutrients; and section 2.5. Production of 13NH4+4.2.2. Relative growth rateRice seedlings were grown in 2, 100, and 1000 iM NH4 (designated,hereafter, as G2, G100 and G1000 plants, respectively) to representinadequate, adequate and excess nitrogen provision. Total fresh weights of91plants were recorded for three treatments at ages of 14, 21 and 28 d. Theywere used to calculate relative growth rates (RGR).4.2.3. Influx measurementSee section 2.3.1.4.2.4. Kinetic studyInfluxes of G2, G100 or G1000 plants, respectively, were measured in‘3N-labeled MJNS varying in [NH4-’-]0 from 2 pM to 40 mM in perturbationexperiments. Perturbation experiments are defined as those in whichplants are grown at one particular [NH4+]0,and influxes are measured in arange of [NH4]0.Measured 13NH4÷influxes at various [NH4+]0were fitted tothe Michaelis-Menten equationV = (Vmax [NH4io)/(Km + [NH4’-]0) [31]and a more comprehensive equationV (Vmax • [NH4]o)/(Km + [NH4’-]0)+ b • [NH4’-]0+ 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 forthe influx measured at a particular [NH4]0.Vmax is the calculatedmaximum rate of influx while Km (.tM) represents [NH4-’-]0giving half of themaximum influx; b and a are constants characterizing the linear phase. Ateach concentration tested, influxes were determined in two to six separate92experiments with three or four replicates. Each replicate consisted of about20 rice seedlings.Based on the results of the kinetics studies (see Results), measuredNH4 influx from < 1 mM [NH4+]0appeared to result from a saturable highaffinity transport system (hereafter referred to as HATS). Since the influxby the HATS had saturated between 0.1 and 1.0 mM [NH4-’-]0,influx from0.1 mM [NH4+]0was selected as a concentration representative of the HATSin the following studies. Above 1 mM [NH4+]0, measured NH4 influxappeared to result from the participation of both the HATS and a lowaffinity transport system (hereafter referred to as LATS). Therefore, thedifference between measured influx at concentrations >1 mM [NH4]0andthe saturated values of the HATS were taken to represent fluxes due to theLATS.4.2.5. Metabolic inhibitor studyInfluxes were measured in MJNS containing representative levels ofeither 0.1 mM to estimate the activities of the HATS, or 20 mM NH4C1 forthe HATS plus LATS, in the presence or absence of different metabolicinhibitors. The inhibitors used were as follows: (1) 10 iiM CCCP; (2) 1 mMCN plus SHAM; (3) 50 j.tM DES; (4) 0.1 mM DNP; (5) 50 iM Mersalyl; (6) 1mM pCMBS. Details of preparation refers to Section 2.9.In this study, both 3-week-old G2 and G100 plants were used. Beforelabeling with radioisotope, rice roots were treated with un-labeled MJNScontaining the same concentrations of CN- plus SHAM for 30 mm. Therewere no pretreatments for the other inhibitors. Measurements of influx93were undertaken as in the kinetic study. Each inhibitor experiment wasrepeated twice with three replicates for each treatment. Each replicateconsisted of about 20 seedlings. Therefore the means for influxes andstandard errors were calculated from six replicates and represented themean for approximately 120 seedlings.4.2.6. Temperature studyRice plants were grown under the same conditions as describedpreviously, so that they were adapted to 20 ± 2°C. Influxes weresubsequently measured in MJNS with either 0.1 mM or 20 mM NH4C1 atsolution temperatures of 5, 10, 20 and 30°C. During the pre-wash, uptakeand 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 studyRice plants were grown in MJNS containing 2 jiM NH4under theconditions described in METHODS AND MATERIALS and adapted to growthmedium 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 theeffects of solution pH upon ‘3NH4 influx, roots were exposed to thedesignated pH levels during 5 mm pre-wash, 10 mm influx as well as 3mm post-wash. Influxes of‘3NH4÷were measured in either 0.1 mM or 10mM NH4 solution. The choice of 10 mM NH4, rather than 20 mM wasdictated by the desire to minimize additions of HC1 or NaOH in adjusting pHlevels in the uptake solutions.944.3. RESuLTS4.3.1. Kinetics of NH4 influxInfluxes of‘3NH4in response to external concentrations in the rangefrom 0.002 to 40 mM [NH4]0 were resolved into two distinct phases,presumably mediated by two separate transport systems; at low [NH4]0 (<1 mM), a saturable high affinity transport system (HATS); and at high[NH4]0(> 1 mM), the combined activities of a saturated HATS and a linearlow affinity transport system (LATS).4.3.2.1. HATSIn the low concentration range (< 1 mM [NH4]0),the values of 13NH4-’-influx into roots of G2, G100 or G1000 rice plants conformed to MichaelisMenten kinetics (Fig. 8). The kinetic parameters of Vmax and Km wereestimated using non-linear regression analysis (Table 7) to fit theMichaelis-Menten equation. Analysis by means of a more comprehensiveequation (see equation [32] in section 4.2.4.) gave similar trends althoughactual values of Vm and Km were slightly different (data not shown). Withincreasing provision of NH4 from 2, through 100 to 1000 jiM in the periodof two weeks prior to uptake measurements, root [NH4]increased from2.37, through 4.31 up to 6.85 jimoles g’FW, respectively. As shown in Fig.9, increasing [NH4]1was associated with decreasing Vm values, from 12.8through 8.2 down to 3.4 jimol g’FW h’, and increasing Km values, from 32through 90 up to 188 jiM, for G2, G100 and G1000 plants, respectively.9515 -G2110.G1005.G1000z_ ___00 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0External ammonium concentration (mM)Figure 8. Concentration dependence of1-NH4 influx at low [NH4]0.Influxof 13NH4 into rice roots was measured in perturbation experiments. Riceseedlings were grown at 2, 100 or 1000 jiM NH4 (G2 (A), G100 (0) orG1000 (x), respectively). Each datum point is the mean of 16 replicateswith standard error as a vertical bar. The solid lines are estimated fromVm and Km values (Table 7) of G2, G100 and G1000 plants, respectively.96Table 7. Kinetic parameters for saturable and linear‘3NH4 influx of G2,G100 or G1000 roots as functions of [NH4J0.The relationships between‘3NH4 influx and [NH4+]0 of uptake solution were estimated fromMichaelis-Menten kinetics for influx measured between 2 to 1000 tM[NH4]0 and for linearity in the range of 1 to 40 mM, where ‘a’ is theintercept and ‘b’ is the slope.G2 G100 G1000HATS a Vm 12.8 ± 0.2 b 8.2 ± 0.7 3.4 ± 0.2Km 32.2 ± 2.1 90.2 ± 23.2 188.1 ± 34.5HATS+LATS a 13.21 10.14 4.59b 0.67 0.79 1.30r2 0.97 0.97 0.99LATS a 0.41 1.94 1.19b 0.67 0.79 1.30r2 0.98 0.96 0.98a HATS represents the high affinity transport system, measured below 1 mM [NH4]0.Influx measured at concentrations above 1 mM [NH4]0 is considered to be thecombined contributions of both high and low affinity transport systems(HATS+LATS). LATS represents the low affinity transport system and is estimated bysubtracting HATS from HATS+LATS. b Vmax and Km were estimated by non-linearregression with ± se,9715 250-20010E—0— Vmax& Km-100E 5-50•G2 G1OO Gi000. •0.0 2.0 4.0 6.0 8.0 10.0Root ammonium concentration (mM)Figure 9. Relationship between kinetic parameters of NH4 uptake androot ammonium concentrations ([NH4]1)of rice seedlings. The values ofVmax (0) and Km (z) from Fig. 8, were plotted against [NH4+]1for G2, G100,or G1000 plants, indicated by (L) on the X axis.984.3.1.2. LATSIn the higher range from 1 to 40 mM, the relationship between{NH4]0and 13NH4 influx was linear (Fig. bA). The Y intercepts of theselines (13.21, 10.14 and 4.59 for G2, Gb00 and G1000, respectively)decreased according to the ammonium provision during the growth andagreed well with the corresponding Vm for the HATS (Table 7). Thus it isconcluded that the measured fluxes at elevated [NH4+]0 result from thecombined activities of the HATS and the LATS. To evaluate the effect ofprior NH4 provision on the LATS for‘3NH4influx without the influence ofthe HATS, the Vmax values for HATS were subtracted from the measuredinfluxes at elevated [NH4]0 values. The derived LATS values were replotted accordingly (Fig. lOB). As shown in Fig. lOB, 13NH4influx by LATSis higher for G1000 than for G100 or G2. Slopes of the lines increasedaccording to the NH4 level during growth period (0.67 for G2, 0.79 forG100 and 1.30 for G1000 in Table 7). These linear relationships at high[NH4]0 were confirmed by means of F-tests for linearity (Zar, 1974).Statistical analyses revealed that the slope of the G1000 line wassignificantly different from the slopes of the G2 and G100 lines (data notshown).4.3.2. Effect of metabolic inhibitors on the influx of‘3NH4In most cases 13NH4 influxes of G2 plants were reduced by thepresence of metabolic inhibitors in the uptake solutions as shown in Fig.11. Net reductions of influxes, listed in Table 8, were calculated by usingthe influx of the control as zero reduction (0%). The HATS for NH4 influx9970 1OA60 lOB G1000I30•20 x Gl0000 G100:o 5 110 15 20 2’5 30G2External Ammonium Concentration (mM)Figure 10. Influx of‘3NH4into rice roots at high [NH4]0in perturbationexperiments. 1OA: Influxes of 13NH4-- into G2 (A), G100 (0), or G1000 (x)roots, respectively, were plotted against [NH4]0.Each datum point is themean of more than 6 replicates with ± se as vertical bar. lOB: Theestimated LATS Fluxes after subtracting the Vrnax of the HATS of G2, G100or G1000, respectively, from the corresponding measured influxes (in 9-A).These plotted lines of LATS have the same slopes as their correspondinglines in 9-A but with slightly different values of the intercept, 0.53, 1.96and 0.99 for G2, G100, and G1000 plants, respectively.10030HATS+LATSEJ HATSE:L!1LATS-- 20:i____I1LrControl CCCP CN+SHAM DES DNP Marselyl pCMBSInhibitors in uptake solutionFigure 11. Effect of metabolic inhibitors on 13NH4influx. Rice plants weregrown in MJNS containing 2 iM NH4C1. Influxes of‘3NH4were measuredin MJNS with either 0.1 mM or 20 mM NH4 in the presence or absence of aspecific metabolic inhibitor. Each datum point is the average of more than6 replicates with standard error as vertical bar. Abbreviations: CCCP (10mM): Carboxylcyanide m-chlorophenylhydrazone; CN- plus SHAM (1 mM):NaCN and salicylhydroxamic acid; DES (50 mM): diethyistilbestrol; DNP (0.1mM): 2,4-dinitrophenol; Mersalyl (50 mM): mersalyl acid; pCMBS (1 mM):p-chloromercuri-benzenesulfonate.101was reduced by 81 to 87% by the protonophore (CCCP) or the un-coupler ofelectron-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‘3NH4 influx due to the HATS by 51% buthad negligible effects on LATS. External protein modifiers of themembrane surface, pCMBS and Mersalyl, reduced 13NH4÷influx of HATS byabout 40% with slightly less or similar reductions of LATS (22 to 46%).These patterns of inhibition were also observed for G100 plants (data notshown).4.3.3. Effect of root temperature on‘3NH4influxShort-term perturbations of root temperature significantly affectedthe influx of 13NH4+ into rice roots that were adapted to the growthtemperature of 20°C (data not shown). Table 9 shows the calculated Qj.ovalues 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 between5 to 10°C to 1.25 between 20 to 30°C. The results of F-tests in conjunctionwith Duncan’s Multiple Range Tests demonstrated that Qo values for thedifferent temperature ranges were significantly different for the HATS (P>0.05). In contrast, there were no significant differences between the Qiovalues for LATS in the same three temperature ranges for both G2 andG100 plants (P > 0.05). Nevertheless Qjo values for the LATS weresignificantly greater than 1.102Table 8. Reduction of‘3NH4 influx into roots of G2 plants by variousmetabolic inhibitors.Treatment Inhibitor % Reduction ofLevelHATS a LATS bControl None 0 0CCCP 10 mM 84.58 30.72CN+SHAM 1 mM 80.84 43.20DES 50 mM 53.96 4.00DNP 0.1mM 86.72 50.55Mersalyl 50 mM 41.97 22.40pCMBS 0.5mM 41.33 46.11a The influxes of HATS were measured in the representative {NH4]0 (0.1 mM).Reduction of HATS (%) was calculated by setting the ‘Control’ value, the reduction ofinflux 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 bysubtracting the influx values measured at 0.1 mM NH4 from that at 20 mM NH4-- forcontrol and for each inhibitor treatment, respectively. The reduction of influx valuedue to LATS under control conditions was then set at 0%.103Table 9. Calculated Qjo values for 13NH4 influx by the HATS or LATS ofrice plants grown at 20°C with 2 or 100 iM NH4C1 (G2 and G100 plants).Temperature G2 Plants a DMRT b G100 Plants DMRTRange(a)HATS: 5-10°C 2.48±0.04 a 2.59±0.21 a10 - 20°C 1.79 ± 0.08 b 1.68 ± 0.22 b20-30°C 1.25±0.16 c 1.44±0.16 b(b) LATS C: 5 - 10°C 1.41 ± 0.21 1.54 ± 0.2710 - 20°C 1.49 ± 0.06 1.90 ± 0.4620-30°C 1.56±0.06 1.33±0.12a Each value (± Se) is the average of three means from duplicate experiments; eachmean is derived from three replicates. b DMRT stands for Duncan’s Multiple RangeTest for comparing all possible pairs of treatment means. Means having a commonletter are not significantly different at the 5% significance level. C Both F-tests andDMRT indicated that means for the LATS were not significantly different at the 5%level.1044.3.4. Effect of solution pH on‘3NH4influxThe effect of uptake solution pH on ‘3NH4 influx was alsoinvestigated. The percentage of the control was computed on the basis ofthe 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 pairwisecomparisons between the control and other treatments. In the range from4.5- 9.0, solution pH had only a small effect ti 13NI-J4 influx from 0.1 mM[NH4]0,whereas 13NH4+ influx by LATS decreased very significantly withincreasing ambient pH beyond pH 6.0. By contrast, reduction of solution pHdown to 3.0 drastically reduced 13NH4 influx by HATS as well as LATS.4.4. DISCuSSION4.4.1. Kinetics of ammonium uptakeIn Chapter 3 and in Wang et al., (1993a) it was demonstrated thatthe half lives for‘3NH4exchange of the cell wall and cytoplasmic phasesof rice roots (G2, G100 or G1000 plants) were approximately 1 and 8 mm,respectively (Section 3.3.1., Table 2). By using 10 mm exposures to 13NH4+and 3 mm post-washes, therefore, estimates of plasma membrane influxesrather than net flux or quasi-steady fluxes to vacuole were obtained (seeCram, 1968). The results of the present study revealed that NH4-’- influxacross the plasma membrane into rice roots exhibits a bi-phasic pattern: inthe low range (below 1 mM [NH4÷]0), influx occurred via a saturable highaffinity transport system (HATS); while from 1 to 40 mM [NH4+]0a second,low affinity, non-saturable transport system (LATS) became apparent. This105Table 10. Effect of uptake solution pH on1NH4 influx into rice roots of3-week-old G2 plants grown at pH = 6.0 in MJNS. Influx of 13NH4 wasmeasured in MJNS at various pH levels (3.0, 4.5, 6.0, 7.5, and 9.0) with[NH4--]0 at either 0.1 mM for the HATS or 10 mM for the HATS+LATS. Thevalue of LATS was obtained by subtracted the values of HATS fromHATS+LATS of each treatment.pH Influx a LSD b (%) of Control C(a)HATS: 3.0 6.91±1.43 * 534.5 12.02±0.46 ns 876.0 13.22±0.27 control 1007.5 14.51±0.39 ns 1099.0 12.94 ± 0.30 ns 95(b) LATS: 3.0 15.75 ± 0.45 * 874.5 18.63±2.80 ns 1036.0 18.07 ± 0.49 control 1007.5 11.44± 1.37 * 639.0 9.29 ± 1.54 * 51a Each value (± Se) (i.tmol g’FW h’) is the average of four means of duplicateexperiments. Each mean is derived from three replicates. b LSD stands for LeastSignificant Difference test, used for making pairwise comparisons between thecontrol at pH 6.0 and other treatments. * = significant at 5% level and ns = notsignificant. C The percentages of control were calculated using the NH4 influxmeasured at pH=6.O as 100%.106bi-phasic pattern of uptake has been reported for NH4 uptake by Lemna(Ulirich et al., 1984), for K uptake by corn roots (Kochian and Lucas,1982), and for NO3-uptake by barley roots (Siddiqi et al., 1990).Plasma membrane ‘3NH4 influx at low [NH4+]0 conformed toMichaelis-Menten kinetics (Table 7) in accord with earlier studies of netNH4 uptake by rice (Youngdahl et al., 1982; Wang et al., 1991). This hasalso been found to be the case for roots of other species, including corn(Becking, 1956), rye-grass (Lycklama, 1963), and barley (Bloom andChapin, 1981), where net NH4 uptake rates saturated in the range from100 to 1000 iM [NH4]0.The significance of this HATS for NH4 in rice rootsis that it allows plants to absorb sufficient nitrogen (NH4--) from very lowlevels in the rhizosphere to meet the minimum requirement for plantgrowth. In the present experiments, for example, by three weeks, therelative growth rates were independent of [NH4]0from 100 to 1000 pMNH4.The relative growth rates calculated from total fresh weight of bothG100 and G1000 plants were at —0.16 d’ for the third week of growthwhile for G2 the value was 0.06 d’. By the fourth week the differences inRGR had diminished to 0.05, 0.06, and 0.06 d’, respectively for G2, G100and Gl000 plants. The reduced growth rates of G2 plants wereaccompanied by increased root:shoot ratios, and leaves were slightly palerthan those of plants grown at higher [NH4]0.At the higher range of [NH4+]0 (1 to 40 mM), a linear, low affinitytransport system (LATS) also participated in NH4 uptake by rice roots, asis 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 Yintercepts for lines of measured influx (due to both transport systems)against [NH4]0 were in good agreement with the corresponding Vmax107values for the HATS (Table 7), which suggests that the two distincttransport systems (HATS and LATS) are additive.Despite the importance of NH4 as principal source of N for manyplant species and the increasing availability of techniques for themeasurement of short-term 13NH4 and 15NH4 influxes, few detailedinflux isotherms (as distinct from net uptake isotherms) have beenreported for NH4 influx into roots of higher plants. Nevertheless, Ullrich etaL, (1984) were able to demonstrate linear kinetics of NH4 uptake byLemna between 0.1 to 1.0 mM [NH4]0 using a depletion method. Thequestion of the saturation of this apparently linear system at higherconcentrations remained unresolved. Clearly, it is difficult to measure netfluxes by employing concentration depletion methods at high externalconcentrations without extending the uptake experiment for long periodsof time. By using short-lived radioisotopes, such as ‘3N, it has beenpossible to measure unidirectional fluxes of N03 and NH4÷ at the plasmamembrane 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, therewas no evidence of saturation of the LATS system (Fig. 10).4.4.2. Energetics of ammonium uptakeThe influx of ammonium by HATS is clearly dependent on metabolicenergy. In the present study metabolic inhibitors, CCCP, DNP or CN plusSHAM, diminished‘3NH4influxes 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 the108notion of energy dependence. A Qo value greater than 2 is considered toindicate the metabolic dependence of physiological processes such as iontransport. Short-term perturbations of temperature between 5 to 10°C,significantly increased the Qo values for HATS up to 2.5 compared to1.5 between 20 to 30°C. In a 7 h concluded that the uptake of ammoniumby 9-day old rice seedlings was closely associated with metabolism.However, such long-term studies probably measure the Qo for NH4assimilation rather than the transport process. The values of Qo estimatedfrom Ta and Ohira’s data (1982) provided values larger than 2.5 for 15NH4-’-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 adaptedryegrass (Clarkson and Warner, 1979); barley (Bloom and Chapin, 1981);and oilseed rape (Macduff et al., 1987) indicating that NH4-’- transport hadacclimated to the low temperature growth conditions. Consistent with theresults of the metabolic inhibitor studies, the present Q10 study indicatedthat LATS was less sensitive to changes of root temperature than the HATS(Table 9).The apparent energy-dependence of the HATS may not necessarilymean that NH4 uptake is an active transport process, although activetransport systems for ammonium have been proposed in bacteria, fungiand algae (Kleiner, 1981; Schlee and Komor, 1986, Singh et al., 1987). Theaccumulation of NH4 against its concentration gradient could be achievedby active or passive uptake mechanisms: the former, by direct use ofmetabolic energy to carry a solute across a membrane towards a region ofhigher electrochemical potential; while the latter, by solute flux across amembrane along the electrochemical potential gradient, a process that maybe only indirectly related to metabolic energy.109According to the compartmental analysis (Chapter 3 and in Wang etal., 1993a), the cytoplasmic concentration of NH4÷ in G2 roots wasestimated to be 3.7 mM. Using this value and -130 mV as measuredplasma membrane membrane electrical potential difference for G2 plantsin ‘MJNS’ minus Nitrogen solutions (Wang et al., 1992), predictions derivedfrom the Nernst equation indicated that net ammonium uptake would beactive only when [NH4+]0falls below 125 jiM. This is rather similar to thevalue of 67 jiM calculated for Lemna (Ulirich et al., 1984). However, thiscalculation only serves to predict the feasibility of the process occurringunder the prescribed conditions. The precise relationship between thecalculated electrochemical potential difference for an ion and the putativetransport systems, predicted on the basis of concentration-dependentinflux curves, are difficult to realize. In the present case, for example,there are no discontinuities in the uptake curve corresponding to thepredicted concentration at which the switch between active and passivetransport (-425 jiM [NH4]0)occurs. This issue is raised to warn against atoo literal interpretation of the thermodynamic predictions. While onthermodynamic grounds influx is uphill below 125 jiM and downhillbeyond this level, the kinetic data reveal no apparent change of transportmechanism.The characteristics of the two transport systems for NH4 influx havesignificant features in common with those described for K-i- uptake in which(incidentally) there is yet no clear consensus regarding the mechanisms ofinflux into higher plant roots. Likewise, the mechanism of the apparentlyactive transport of ammonium below 125 jiM is unknown. It might occurby means of a specific ATPase or a secondary transport system such as anNH4: symport that is driven by the proton motive force (pmf). As110proposed for K’- uptake by Neurospora, for each K entering, one H-’- is cotransported and 2H are extruded by the proton pump (Rodriquez-Navarroet al., 1986). The net result is therefore a 1:1 K/H-’- exchange. Is it possiblethat NH4 influx is mediated by an analogous system? It has long beendocumented that NH4 uptake is associated with strong acidification of theexternal medium (e.g. Becking, 1956). Likewise in the present study, whenpH was not adjusted daily in the initial growth experiments, external pHdropped so low that plants failed to grow normally.So far as the passive uptake of ammonium is concerned at higherconcentrations, several authors have proposed that NH4-’- influx may occurby an electrogenic uniport in response to the electrical gradient (Kleiner,1981; Ullrich, 1984). When ambient concentration is beyond the predictedthreshold for active uptake, the concentrative NH4 uptake may be due toa facilitated transport system driven by the electrochemical potentialdifference for NH4. This has two components; the difference in chemicalpotential of NH4 (L$LNH4÷) between cytoplasm and outside and theelectrical potential difference (z\’-P) generated in part by proton effluxacross the transducing membrane. The actual mechanistic link, if oneexists, between NH4 influx and the pmf across the plasma membrane isunclear at present. Certainly the results of the treatments with theprotonophore (CCCP) or the un-coupler of ATP formation (DNP and CN plusSHAM), which caused greater than 81% reduction of influx due to HATS,are consistent with a dependence of NH4 influx on transmembrane pmf.Further support for this hypothesis is provided by the effect of ATPaseinhibitor, DES, which reduced‘3NH4 influx due to HATS by 54% but hadnegligible effects on LATS.1114.4.3. Effect of pH profile on ammonium uptakeIn the present study, influx by the HATS was strongly reduced belowpH 4.5. By contrast, in the range from pH 4.5 to 9.0, 1NH4 influx by theHATS appeared to be relatively insensitive to pH. 13NH4 influx by theLATS actually decreased with increasing ambient pH beyond pH 6.0. It hasbeen reported for several species that the specific uptake rate of NH4-- canbe reduced by short-term decreases in pH below 6.0 (Munn and Jackson,1978; Marcus-Wyner, 1983; Vessey, 1990) and even terminated all-together at pH 4.0 (Tolly-Henry and Raper, Jr., 1986). Tanaka (1959)suggested that rice is very sensitive to pH below 4. Most probably thisreflects a general detrimental effect of such acidic conditions on thetransport systems. In addition, it has been observed that when plants weregrown at such low pH values over extended periods of time, the rootsbecame stunted and discolored. It has been suggested that both high pHand/or high ammonium concentration of solution may result in high ratesof NH3 uptake due to increased NH3 concentration and the higherpermeability of cell membranes to NH3 than NH4 (see Macfarlane andSmith, 1982). However, in many studies this expectation has not beenobserved, and uptake failed to increase at elevated pH (MacFarlane andSmith, 1982; Deane-Drummond, 1984; Schlee and Komor, 1986). Likewise,in the present study, influxes of1NH4due to the LATS were reduced by25- 35% at higher pH (7.5- 9.0), despite a predicted increase of [NH3]from less then 0.1% of total [NH4 + NH3] at pH 6.0, to 36% at pH 9.0according to the pKa for NH4 (9.2 5). Furthermore, membrane electricalpotentials of rice roots have been shown to be depolarized by elevatedammonium concentrations (Wang et al., 1992). These observations indicatethe entry of cation (NH4+) rather than neutral ammonium (NH3). The112evidence from our electrophysiological study of rice roots indicated alinear relationship between depolarization of membrane potential andinflux of NH4 from 1 to 40 mM (data not shown). Therefore, at elevatedconcentration and pH, it is unlikely that simple diffusion of NH3 could beconsidered as a major component of the influx of LATS. Nevertheless, intheir study using Lemna, Ullrich et al., (1984) reported that depolarizationof membrane potential was saturated at 0.1 mM even though net uptakecontinued to 1 mM in a linear pattern. This observation is consistent withNH3 entry by the LATS in Lemna.4.4.4. Regulation of ammonium uptakeAlthough the bi-phasic pattern of NH4-’- influx was independent of theprior NH4 exposure, the individual systems, particular the HATS, wereextremely sensitive to prior NH4 exposure (Figs. 8, 9, 10). Evidently NH4-’-influx by the HATS was subject to regulation by negative feedback: withincreasing [NH4]0in the growth medium, root [NH4+J increased and NH4influx decreased (Fig. 9). It is noteworthy that in the present case, negativefeedback regulation appeared to affect both Vm and Km values (Table 7,Figs. 8 and 9). It has commonly been observed that Vmax is strongly andunequivocally influenced by the level of nutrient supplied during growth.By contrast, an effect on Km has rarely been observed (Lee, 1982). Only inthe case of K (Glass, 1976) was the Km strongly influenced by K statusalthough other ions such as CF do show small changes (Lee, 1982). In thepresent study, the values of Km were strongly influenced by the prior levelof NH4-’- supply, and are positively correlated with [NH4-’-]1.113Contrary to expectation,‘3NH4influxes due to the LATS were higherin plants previously maintained at 1000 jiM NH4 than in those maintainedat 2 jiM NH4-’-. The reverse was found to be the case for‘3N0-influx inbarley (Siddiqi et al., 1990). This positive correlation between provision ofNH4-’- and 13NH4influxes at high [NH4+]0may indicate that the LATS maynot be subject to regulation by negative feedback. Another possibleexplanation is that better nitrogen nutrition may provide more buildingmaterials (protein?) for constructing transporters. However, exposures tohigh [NH4-’-]0 (>1 mM) were brief and in longer exposures NH4 influx maybe down-regulated in accord with expectation.The present study has demonstrated the strong negative down-regulation of influx by the HATS in response to elevated NH4 supplyduring growth. At present the mechanism(s) and signals responsible forthis down-regulation of uptake are unclear. Feedback signals may resultfrom un-metabolized ammonium of root cells or reduced nitrogen (Lee,1982; Morgan and Jackson, 1989). Lee and Rudge (1986) have suggestedthat in barley the uptake of NH4 and N03 are under common negativefeedback control from a product of NH4-’- assimilation rather than NH4and/or N03 accumulation per Se. Reduced N pools which cycle in xylemand phloem from root to shoot have been implicated in the whole plantregulation of N uptake by plant roots (Cooper and Clarkson, 1989).However, Siddiqi et al. (1990) have suggested that in the case of N03influx, vacuolar accumulation of N03 per se may also, at least indirectly,participate in flux regulation. Further support for this proposal has comefrom studies of nitrate reductase mutants of barley that are capable ofnormal induction of N03 uptake and appear to show diminished‘3N0influx as N03 accumulates (King et al., in press). In the present study, also,114there was a close negative correlation between NH4 influx and [NH4-’-j Inroot tissues (Fig. 9). However, the altered NH4 status in G2, G100, andG1000 plants was probably also associated with changes in organic Nfractions. Since efflux was estimated to be 10 to 30% of influx for G2, G100and G1000 plants, respectively (Wang et al., 1993), negative feedback actsvery strongly on the influx step of the HATS, but since efflux alsoincreased with increasing [NH4]0,this flux will exert significant effectsupon net uptake.4.5. SUMMARYThe work described provides the first detailed characterization ofNH4 influx across the plasma membrane of rice roots. Ammonium influx isbi-phasic, mediated by two discrete transport systems. Metabolic inhibitorstudies and Qo determinations indicated that both systems were energy-dependent, although the HATS consistently showed greater sensitivity tometabolic interference than the LATS. Nevertheless, thermodynamicevaluations indicate that only at quite low [NH4]0is there a need to invokeactive transport of NH4 against the electrochemical gradient. It is highlyunlikely that the LATS is active. The HATS was found to be extremelysensitive to prior exposure to ammonium as indicated by the alteredvalues of Km and Vmax. General insensitivity of influx to pH in the rangefrom 4.5 to 9.0 argues strongly against significant entry of NH3 across theplasma membrane even at high [NH4+]0.115Chapter 5. ELECTROPHYSIOLOGICAL STUDY5.1. INTRODUCTIONAmmonium influx by rice roots (Oryza sativa L. cv. M202) has beenshown to exhibit a biphasic dependence on [NH4]0 (Wang et al., 1991,1992b; 1993b). At low [NH4+10,influx is mediated by a saturable HATSwhich exhibits high Qio values between 10 and 30 °C and a significantsensitivity to metabolic inhibitors (Wang et al., 1993b). At elevated [NH4]0(between 1 and 40 mM), NH4 influx increases in a linear fashion withincreasing [NH4]0,and though still exhibiting energy-dependence, thisLATS was shown to be less responsive to metabolic inhibitors (Wang et al.,1993b). A biphasic pattern of NH4 uptake of this sort, with both saturableand linear phases, was first reported in Lemna, by Ullrich et al., (1984).In order to make a definitive evaluation of the thermodynamics ofNH4 influx (passive versus active transport), it is essential to determinethe chemical potential difference for NH4 between the cytoplasm andexternal media, and &P across the plasma membrane. In Chapter 3,compartmental analysis was used to estimate cytoplasmic [NH4]. So far asI am aware, only one report measuring A’P in rice roots has appeared inthe literature: Usmanov (1979) reported AW to be -160 mV. As early as1964, Higinbotham et al. noted the marked depolarizing effect of [NH4j0coleoptile cell z’P in oats. Likewise, Walker et al. (1979a, b) demonstratedthe transport of ammonium and methylamine across the plasmamembrane of Chara, and the depolarizing effects of these cations. The most116detailed study of the concentration dependence of A’P depolarization byNH4-’- was undertaken by Ullrich et al. (1984), using Lemna. Below 0.2 mM[NH4]0both NH4 uptake and A’P depolarization responded in a saturablefashion with half-saturation values of 17 j.tM for both processes. From 0.2to 1 mM, net uptake of NH4 responded linearly to [NH4]0,with no furtherP depolarization. On the basis of this observation, Ulirich et al. (1984)concluded that the linear system might result from diffusion of NH4 orNH3 across the plasma membrane.The present study was initiated, therefore, to estimate M’ in intactrice roots, under conditions corresponding to those employed to estimatecytoplasmic [NH4j in our previous study, and to determine theconcentration dependence of the depolarizing effect of [NH4]0.The effectsof metabolic inhibitors on Af were also examined.5.2. MATERIALs AND METHODS5.2.1. Growth of plantsRice (Oryza sativa L. cv. M202) seeds were surface sterilized in 1%NaOCI for 30 mm and rinsed with deionized water. Seeds were imbibedovernight in aerated deionized water at 38°C before planting on plasticmesh mounted on the bottoms of polyethylene cups. Four cups (3 to 4seeds per cup) were set in the lid of a 1-L black polyethylene vessel withthe solution level just above the seeds. Seeds were allowed to germinate inthe dark (at 20°C) for 4 days. At day 5, rice seedlings were exposed to lightand MJNS containing the designated levels of NH4C1. The composition of117MJNS, growth conditions, nutrient supply and pH adjustment were thosedescribed in Section 2.1.2. The growth medium in the 1-litre polyethylenevessels were completely replaced on alternate days and the nutrient levelswere topped up with concentrated stock solutions daily. Rice plants used inthe experiments were 3-week-old G2 or G100 plants respectively.5.2.2. Measurements of cell membrane potentialPlasma membrane P of rice roots were measured as described byKochian et al. (1989) and Glass et al. (1992). In short, rice plants weresecured in the larger part of a flow-through Plexiglas impalementchamber, and one intact root was carefully placed over the platinum pinsin a narrow section of the chamber. This root was held firmly during theimpalement by two short lengths of Tygon tubing, from each of which asmall wedge had been cut. The tubing was placed on either side of theimpalement zone to clamp the root in place. All impalements were made ina region about 1 to 3 cm behind the root tip, using a hydraulically driven,three-dimensional micromanipulator (Model MO-20, Narashige, USA). Boththe Plexiglas impalement chamber and micromanipulator were mountedon the microscope stage. Microelectrodes (including impaling, referenceand grounding electrodes) were made from 1.0 mm single-barreledborosilicate glass tubing pulled to a tip diameter of 0.5 iM and filled with3M KC1 (adjusted to pH 2 to reduce tip potentials). Measured membranepotentials of root cells, which are the voltage differences between theimpaling and reference electrode, were amplified and recorded on a stripchart recorder. During impalement, solutions were continuously deliveredfrom an air-pressured reservoir to the chamber through tygon tubing atcontrolled flow rates (7.5 ml minl).1185.2.3. Experimental treatmentsAt the beginning of each experiment, the impalement was made onG2 or G100 roots bathed in their growth media (MJNS containing 2 or 100iM NH4C1, respectively) and the membrane potential was recorded (A.PG2or A’I’GlOO). MJNS without NH4 is referred to throughout as the -N solution.Before applying each treatment, the -N solution was introduced to obtain aresting membrane potential, MN, as the point of reference. Roots wereallowed to equilibrate for at least 3 to 5 mm in this -N solution to reach theresting potential before introducing subsequent treatment solutions.5.2.3.1. Effect of [NH4+]0Roots were exposed to {NH4J0of 2, 5, 10, 25, 50, 75, 100, 250, 500jiM for studying the HATS, and 1, 2.5, 5, 10, 20, 30, and 40 mM NH4C1 forinvestigating the LATS, in a background of MJNS. When roots were exposedto several different [NH4]0 during a single impalement, G2 or G100medium was flushed through the chamber before each change of NH4concentration. When ‘P returned to its original (A’PG2 or A’’G1OO) value, itwas satisfied that the physiological status of the root had returned to itsoriginal condition.5.2.3.2. Effect of accompanying anion on 4’i’To evaluate the contribution of the accompanying anion to theobserved depolarization of AP by NH4-salts in the low concentrationrange, A’P were measured in the following solutions in sequence: (a) 50 jiMCaC12, (b) 50 jiM CaSO4, (c) 100 jiM NH4C1, (d) 50 jiM (NH4)2S0.Likewise inthe high concentration range, &P was measured in (e) 5 mM CaCl2, (f) 5mM CaSO4, (g) 10 mM NH4C1, and then (h) 5 mM (NH4)2S0.These119concentrations were chosen to provide equivalent anion charge in alltreatments.5.2.3.3. Effects of metabolic inhibitors on NH4-induced Af’ depolarizationThe same metabolic inhibitors used in the 13NH4 influx study(Section 2.9.), were used to investigate effects on NH4-induceddepolarization of AP. These included 1 mM NaCN plus 1 mM SHAM, 10 iiMCCCP, 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 NH4C1 weredetermined in sequence;(2) the inhibitor to be evaluated was first introduced in -N solution. Whenz’P had reached a new steady-state, this solution was replaced with theinhibitor plus 0.1 or 10 mM NH4C1 in sequence;(3) the solution containing inhibitor plus NH4C1 was replaced by -Nsolution.When a new steady value of L\M&N had been reached, 0.1 and then 10mM NH4C1 were added to the -N solution in sequence. The NH4C1concentrations, 0.1 or 10 mM in MJNS, were selected as representativelevels for the operation of the HATS or the combined HATS and LATS(Wang et al., 1993a).1205.3. Results5.3.1. Transmembrane electrical potentials of rice rootsPlasma membrane A’P for epidermal and cortical cells of 3-week-oldrice roots (Table 11) were measured in 0.2 mM CaSO4 alone (A’PCaSO4), or -Nsolution, or G2 and G100 media (‘‘PN, ‘PG2 and ‘‘PG1OO, respectively). Aspresented in Table 11, &PCaSQ4 values were consistently more negativethan &p measured in other solutions. Likewise the &P.N were morenegative than the corresponding A’PG2 or zPG 100 values. The depolarizingeffect of NH4C1 additions can be directly compared in Table 11 for aparticular root type because -N and G2 or G100 media differed only by thepresence of NH4C1 in MJNS. Therefore, both APG2 and z’+’oo representedthe membrane potentials of root cells adapted to their respective growthconditions.5.3.2. Contribution of the accompany anions to ‘i’Figure 12 reveals that there was a very small depolarizing effect ofCa2-salts compared to NH4-salts, under conditions where theconcentration of the accompanying anion was held constant. Also there wasvirtually no difference between the depolarizing effects of Cl- and S042.This was true also at the higher concentrations ofCa2-salts and NH4-salts(Traces e, f, g and h in Fig. 12). In the lower concentration range, norepolarization of M’ was observed until the Ca2-salts or NH4-salts werewithdrawn from the chamber. By contrast, in 5 mM CaC12, completerepolarization and even hyperpolarization was evident within 10 mm of121Table 11. Membrane potentials of G2 and G100 plants measured indifferent bathing solutions. The bathing solution for measurements were0.2 mM CaSO4; MJNS-N; MJNS + 2 iM NH4,or MJNS + 100 jiM NH4-’-.G2 plants (mV) G100 plants (mV)A’PCaSO4 -140±3.5 (5)d-135±1.8 (n=53)‘‘N -129±1.0 (n=184) -131±0.6 (n=197)z’{’G2orLS.WG1OO -116±2.1 (n=14) -89±2.4 (n=28)a G2 or G100 plants were impaled in 0.2 mM CaSO4 solution; b G2 or G100 plants wereimpaled in -N solution; C G2 or G100 plants were impaled in MJNS containing either 2iM or 100 tM NH4C1, respectively; d Average value ± standard error, n: number ofobservations;122Addition ofionsUa. 50 jiM CaC2V-138—b. 50 jiM CaSO4-110100 jiM NH4CId. 50 jiM(NH)2SOr N-114e. 5 mM CaCI2f.5mMCaSO4 vg.1OmMNH4CIomV{-130h. 5 mM (NH4)2S0 rnnesFigure 12. Effects of some anions on A’P depolarization. Representativetraces to demonstrate the contribution of the accompany anions todepolarization of &P elicited by exposure of roots to different salts atvarious concentrations. V: the salts were withdrawn from MJNS. Eachtreatment was repeated on three separate plants.123evidence of repolarization in NH4C1 (Fig. 12, trace g) but this was onlypartial. Only after removal of the NH4-salts was complete repolarizationobserved.5.3.3. Effect of [NH4C1]0on z\PThe addition of NH4C1 to the -N solution induced a strongdepolarization of &P (Fig. 13). This depolarization occurred rapidly afterthe introduction of NH4C1, even at very low concentrations (e.g. 2 jiMNH4C1). The time required to reach the initial maximum depolarization wasfrom 0.5 to 2 mm, increasing with increasing [NH4C1]0.The depolarization of A’P was positively correlated with [NH4C1]0.Asaturable pattern was evident in the range from 2 to 1000 jiM NH4C1 (Fig.14A) for both G2 and G100 plants. Estimated half-saturation values for netdepolarization (analogous to a Km value) were 21.8 ± 2.7 jiM for G2 plantsand 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 ± 1.9mV for G100 plants. Kinetic parameters were obtained by fitting the datato the Michaelis-Menten equation by means of a nonlinear regressioncomputer program “Systat” (Wilkison, 1987) as used in our earlier kineticstudy of 13NH4influx (Wang et al., 1993b). Between 1 to 40 mM {NH4C1]0(Fig. 14B), the magnitude of the depolarization increased linearly withincreasing concentrations of NH4C1. This relationship was observed for bothG2 and G100 rice plants, although the extent of depolarization was smallerfor the latter.124Addition of NH4C 1/10S minutes40 mM NH4CI20 mM NH4C110 mM NH4CI5 mM NH4C11 mM NH4CI500 iiM NH4CI5OjiM NH4CI100 iiM NH4CIlO1iM NH4CI2M H1Figure 13. The &P depolarization of root cell by NH4C1. Representativetraces from G2 plants showing the depolarization of root cell zVP induced byadding various concentrations of NH4C1. V NH4C1 was withdrawn fromMJNS.12560 -1 4A.—0•G210- 0 G100U- • I.I.I.I.I.I.I.I.I.I.I.0 100 200 300 400 500 600 700 800 900 1000External Ammonium chloride (jiM)120-100- 14 B• G2 r’2=O.9420 0 G100 r”2= 0.9900 5 10 15 20 25 30 35 40 45External Ammonium Chloride (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 NH4 (G2). The-N media were used as basal solutions for the resting AP. Each point is theaverage of 3 measurements from each of 3 individual plants. The verticalbar is the standard error. 14A: Low [NH4C1]0 range (<1 mM); 14B: Highrange (1 to 40 mM).126Figure 15. shows the effects of four metabolic inhibitors on &T’recorded in -N solutions. The largest depolarization of z’P (95 mV), wasinduced by the protonophore, CCCP, while CN+SHAM and the ATPaseinhibitor, 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 theseinhibitors on NH4-induced depolarization of A’P are shown in Fig. 16. InTable 12, the effects of these inhibitors on theNH4-induced depolarizationof z’P are expressed as a percentage of the depolarization under the controlconditions, in absence of the inhibitor. The data are presented as follows:(i) control: in absence of the inhibitor the reduction of NH4-induceddepolarization of A’P is zero; (ii) plus inhibitor: reduction ofNH-’--induceddepolarization of zXP varied from 0 to 91%, depending upon the inhibitorused and [NH4+]0;and (iii) residual effect: the residual effect after removalof the inhibitor from external solutions on NH4-induced depolarization ofA’P. The [NH4C1J0employed were 0.1 mM and 10 mM, respectively, chosento represent the HATS and the combined HATS+LATS. In Table 12 thedepolarizations of A’P caused by 0.1 mM [NH4C1]0were subtracted fromthose caused by 10 mM [NH4CIj0to represent the effect due to IATS alone.In the presence of the various inhibitors, the depolarization of zs’1’ inducedby HATS was generally reduced by greater than 50%. By contrast,depolarization of AP due to NH4 uptake through the LATS was onlyslightly affected by the presence of inhibitors. Table 12 also reveals thatthere was virtually no recovery from the inhibitor treatments followingremoval of the inhibitors from the external medium.127-3410 m V [012345/ -44mmAdd / SHAMInhibitor == 80 mm) f—jFigure 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 timecourse. The inhibitors were: (A) 10 iM CCCP; (B) 1 mM CN+SHAM; CN- wasadded into -N medium alone and then SHAM was added at (J.I); (C) 50 iiMDES; (D) 1 mM pCMBS. Each treatment was repeated on at least threeindividual roots. The space between two bars (I I) is the omitted period asminutes.128-68 -53(A) CCCP (C) DES6oiovE(iii0 12 3 4 5mmL add 0.1 mM N1LCl (i) control(ii) inhibition (inhibitor presented)add 10 mM NEI4CI (iii) residual effect (inhibitor removed)Figure 16. Effects of metabolic inhibitors on NH4C1 induced A’Pdepolarization. Representative traces for the effects of NH4CI on Pdepolarization in the presence or absence of metabolic inhibitors in -Nmedia. Metabolic inhibitors were those shown in Figure 15..129Table 12. Effect of metabolic inhibitors on the depolarization of zXP due toNH4 uptake via HATS or LATS in G2 plants. The inhibitors used were: (A)10 jiM CCCP; (B) 1 mM CN- + 1 mM SHAM; (C) 50 jiM DES; (D) 1 mM pCMBS.Inhibitor CCCP CN+SHAM DES pCMBSTreatment Reduction of A’P depolarization (%)1. Due to NH4 uptake by HATS a(i) control 0 0 0 0(ii) plus inhibitor 89 91 72 52(iii) residual effect 91 68 81 902. Due to NH4 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 0.1mM NH4 in the absence (i and iii) and presence (ii) of the inhibitors. The percentagereductions of P depolarization were calculated from the differences betweencontrol values for zSP induced by NH4 and depolarization values in the presence ofthe inhibitor (ii) or after removal of the inhibitor (iii); b The values of ‘P for LATSwere the differences between measured s’-P at 10 mM (for HATS+LATS) and at 0.1 mMNH4C1 (for HATS). Then the percentage were calculated as described above (a); C Thecalculated values were negative due to the less zSW depolarization of the control.1305.4. DISCuSSION5.4.1. Anion effectA perennial problem associated with attempts to evaluate theelectrical effect of a particular ion is the contribution of the accompanyingcounterion. This problem has rarely been acknowledged in publishedstudies. However, indirect approaches, such as comparisons of thedepolarizing effects of NO3- in N03-induced and un-induced plants havebeen 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 toanother (e.g. CaC12 to Ca(N03)2without changing the accompanying cationor its concentration. As a result, the observed changes of Af are due solelyto the anion effect (McClure et al., 1990; Glass et al., 1992). The results ofsuch studies have demonstrated that N03 can strongly depolarize A’1’ andthese observations have formed the basis of currently proposedproton/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 inthe form of the calcium salt elicited a very small depolarization (Fig. 12,trace a). Replacing this solution with the same concentration of CaSO4 (Fig.12, trace b) confirmed that C1 was responsible for most of thisdepolarization. Thus when these calcium salts were replaced by theirammonium equivalents, maintaining the same anion concentration, thesignificant depolarization of AP could largely be attributed to NH4.Although the depolarizing effects of the calcium salts, presented at 5 mMwere significantly higher than at 50 jiM (Fig. 12, traces e and f), the effects131of transfer to the equivalent ammonium salts can be seen to induce a muchlarger depolarization (53 mV compare to 18 mV; Fig. 12, traces g and e).Even though it was not possible to quantitatively isolate the contribution ofCl- for studies of LATS, I consider that the NH4-’- effect still predominated,even at high external [NH4C1]0.In fact, the difference between traces g ande (Fig. 12) can be attributed to the difference between NH4-’- and Ca2effects, since Cl- was maintained at the same level. Thus thedepolarizations referred to in the remainder of the paper were interpretedas predominantly due to the transport of NH4.A feature of these initial studies was the apparent repolarization ofz+1 following depolarization in the chloride solutions (Fig. 12, traces e andg) at high [Cl10. Although repolarization to the resting potential was notcomplete in 10 mM NH4C1, the extent of the initial repolarization wascomparable to that in CaCl2,where repolarization was completed. A similarspontaneous repolarization of z’P was noted in Lemna and in barley rootsfollowing depolarization of A’P by N03 (Ulirich and Novacky, 1981; Glass etal., 1992).5.4.2. Depolarization of AP by HATS and LATSAddition of ammonium chloride into -N solutions induced a rapiddepolarization of membrane potential of rice epidermal and cortical cells(Figs. 12 and 13). This was evident even at very low concentration (2 jiMNH4C1) (Fig. 13). Ullrich et al. (1984) reported that addition of NH4immediately decreased the membrane potentials of Lemna gibba.Likewise, the zP of green thallus cells of Riccia fluitans were rapidlydepolarized by [NH4C1] as low as 1 jiM (Felle, 1980). As can be seen from132Fig. 13, the time to reach initial maximum depolarization increased from0.5 to 3 mm with increasing concentrations of NH4C1.The depolarization of z’P by NH4 exhibited a biphasic concentration-dependence (Figs. 14A and 14B), similar to NH4 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 [NH4]0(Fig. 14A). Both net flux and unidirectional influx of NH4 in rice roots havebeen shown to respond to [NH4]0in a similar fashion (Youngdahl et al.,1982; Wang et al., 1991; 1993b). Estimated half-saturation values forNH4-induced depolarization (analogous to a Km value) were 21.8 ± 2.7 .tMfor G2 plants and 35.0 ± 8.0 jiM for G100 plants. These values weresomewhat lower than the Km for 1NH4 influx, 32 jiM and 90 jiM,respectively (Wang et al., 1993b). Since our studies were undertaken withthe same rice variety as employed for the‘3NH4influx experiments, thesedifferences may represent differences in growth conditions for plants usedfor the two studies, or that membrane depolarization reflects the net,rather then the unidirectional, effect of ion fluxes. Another factor, alreadyaddressed above, is the possible effect of the accompanying anions. Themaximum depolarizations (analogous to a Vmax value) were 50.6 ± 2.0 mVand 34.3 ± 1.9 mV for G2 and G100 plants, respectively. The largerdepolarizing effects of [NH4-’-]0 in G2 compared to G100 plants (Figs.. 14Aand 14B) correspond to the higher values of‘3NH4influx observed in G2compared to G100 plants (Wang et al., 1993b). Clearly the depolarization ofz’ in response to [NH4-’-]0 (<1 mM) was due to the carrier-mediated NH4uptake that exhibited Michaelis-Menten kinetics (Wang et al., 1993b).Similar saturable patterns of A’P depolarization were associated with theuptake of either NH4-’- or N03 in Lemna (Ulirich and Novacky, 1981; Ulirich133et al., 1984) and the uptake of both NH4 and CH3N in cells of Ricciafluitans (Felle, 1980).Between 1 and 40 mM, the depolarization of A’{’ increased linearlywith increasing [NH4C1]0(Fig. 14B) in a manner similar to that observed for13NH4influx (Wang et al., 1993b). Both G2 and G100 rice plants exhibitedthis linear response, but the extent of depolarization was smaller in G100plants, where 13NH4+influx was also smaller. The concentration-dependentdata for depolarization of zM’ by LATS was fitted by linear regression withr2 values of 0.94 and 0.99 for G2 and G100 rice plants, respectively. Asimilar linear response to [NH4]0was reported for net NH4 uptake byLemna at [NH4]0between 0.1 to 1 mM (Ulirich et al., 1984). However, inthis concentration range, NH4 uptake by Lemna was not associated withfurther depolarization of A’P. Ulirich et al., (1984) interpreted this patternas due to a diffusive uptake of NH4 or NH3. It is clear that NH3 influxwould not depolarize z’P. However it is not clear how NH4 uptake couldoccur without further zXi’ depolarization, unless NH4 influx was associatedwith a stoichiometric anion influx or cation efflux resulting in anelectroneutral transport.To better understand the relationship between NH4 uptake andchanges in z’P, the observed values of A’P depolarization were paired withthe data for‘3NH4influx from Wang et al. (1993b) at each [NH4-’-]0 (Fig. 8).It is evident that the depolarization of A’P was strongly correlated with‘3NH4 influx, and that the relationship was biphasic. By use of acomputer-based procedure to determine the ‘break-points’ for the biphasicpattern objectively (Rygiewicz et al., 1984), the correlation coefficientestablished a break-point at 1 mM [NH4]0.The biphasic pattern (Fig. 17)134120 (a) G2 plants (b) G100 plants10080 •r”2 = 0.9360r’2 = 0.99at 1 mM NH4 Cl4020 r”2 = 0.98r”2 = 0.930 I0 10 20 30 40 50 0 10 20 30 40 501 3 NIT4 Influx (pmol/gFW/h)Figure 17. The relationship between‘3NH4influx and z’P depolarization atthe same [NH4]0.1NH4 influx is from Figs. 8 and 10 and netdepolarization of membrane potentials is from Figs. 14 A and 14B for G2and G100 plants measured at the same [NH4J0.135indicates that NH4 influx and the depolarization of A’P are due to twodistinct systems for NH4 uptake by rice roots, i.e. a high affinity transportsystem (HATS) and a low affinity transport system (LATS). The largerslope of the lines for the low concentration range for G2 and G100 plantssuggests that the HATS is more electrogenic than the LATS. This may bedue to the increasingly electroneutral NH4 transport at high {NH4C1J0.Inthe present study, the electrophysiological evidence suggested that at high[NH4C1]0ammonium is taken up by rice roots in the cation form (NH4-’-)despite the presence of a relatively high concentration of NH3 in solution.Alternatively, it might be argued that depolarization of AP may be due tothe inhibition of the H-ATPase by NH3 at high [NH4+]0.However, the lackof a pronounced increase of ‘3N uptake at pH values approaching the pKafor NH4-’- does not support this interpretation (Wang et al., 1993b). Inaddition, the rapid repolarization of A’P following removal of external NH4(in Fig. 12, traces g and h) is unexpected considering that the ti,i2 forcytoplasmic ‘3N exchange is 7 mm (Wang et al., 1993a).5.4.3. Calculation of the free energy for NH4 transportThe average A’P values were substantially more negative in G2 plantsimpaled in 2 iM NH4-’- than in G100 plants impaled in 100 iM NH4 (Table11). Furthermore, the extent of the depolarization of A’f by NH4 wasconsistently greater for G2 plants than G100 plants at a particular [NH4--]0.The average P value was -116 mV for G2 plants and -89 mV for G100plants (Table 11). For both G2 and G100 plants, the resting potentials inthe absence of NH4-’- (AW-N) were in the range of -120 to -140 mV. In lowsalt bathing medium (0.2 mM CaSO4), the transmembrane electricalpotentials (AW02 mM CaSO4) were 25 mV more negative than A’YN and 45 mV400020000136—00G100G2.0.0 0.2 0.4 0.6 0.8-2000External ammonium concentration (mM)Figure 18. Free energy requirement for NH4 uptake as a function ofexternal [NH4].Values of cytoplasmic [NH4+] were taken from our previousstudy (Wang et al., 1993a). Arrows indicate the [NH4J0below which NH4-’-uptake is against the electrochemical potential gradient for G2 and G100plants, respectively.137more negative than &‘G2 and APG100, respectively. These differencesreflect the contributions to the membrane depolarization from the variousions present in MJNS. Since the values of APN, and M’G2 and A’PG100, weremeasured in the same basal medium (MJNS), the observed differencesmust largely be due to the [NH4]0in the bathing medium.The measured A’P, together with values for cytoplasmic {NH4÷], areneeded to estimate the electrochemical potential difference for NH4 acrossthe plasma membrane, which in turn allows us to determine the energyrequirement for transport (Findlay and Hope, 1976). Taking 3.72 mM and20.55 mM as cytoplasmic {NH4+], and -116 mV and -89 mV as steady-stateA’P for G2 and G100 roots, respectively (Wang et al., 1993a), at a series ofgiven [NH4]0the Nernst potentials (EN) were estimated for G2 and G100roots, respectively. From these values, the free energy (zSt) required totransport NH4 across the plasma membrane can be computed from thedifferences between measured membrane potentials (A’PG2 or zS’PG100) andestimated Nernst potentials at specific [NH4]0(Fig. 18). The estimated freeenergy differences (Aji) for NH4 distribution were positive at or below 42jiM for G2 and 655 jiM for G100 plants (Fig. 18). This means that belowthese concentrations, NH4 uptake by G2 and G100 roots respectively, mustbe active (Fig. 18). These concentrations represent the lower limits foractive transport under steady-state conditions. However, displacing [NH4]0to values greater than 2 or 100 jiM, respectively, will elevate the limit foractive transport because of further AW depolarization and increasedcytoplasmic [NH4]. Above these minimum levels, the uptake of NH4-- mayoccur via passive transport systems, down the electrochemical potentialgradients for NH4--. As pointed out previously (Wang et al., 1993b), thesefree energy estimations only provide a prediction of the feasibility of theuptake process occurring under the prescribed conditions. For both G2 and138G100 plants, the predicted [NH4-’-]0 for the shift from active to passiveuptake was quite a bit lower than the break-point determined by thekinetics analyses (42 jiM and 655 jiM versus 1 mM). Thus, one must becautious in identifying a specific transport system based purely onthermodynamic or kinetic considerations.5.4.4. Mechanisms of NH4 uptake by HATS and LATSThe preceding section has demonstrated that at low [NH4-’-]0 (<42 jiMfor G2 plants and 655 jiM for G100 plants), NH4 influx appears to be anactive process in roots of rice plants. However, the details of thismechanism are unknown for rice and for any higher plants. Possiblemechanisms for this active uptake via HATS include: (a). a proton : NH4symport; (b). a specific NH4 ATPase. The results of the inhibitor studies,both for the electrical potentials in the present study and‘3NH4-’- influx(Wang et al., 1993b) provide evidence for a dependence (either direct orindirect) on the proton motive force. Application of CCCP caused 89% and85% inhibition, respectively, of membrane depolarization by NH4-’- and13NH4 influx in solution containing 100 jiM NH4. The strong inhibitoryeffects of CN+SHAM on depolarization of zSP (9 1%) and on‘3NH4 influx(8 1%) confirm the dependence of these processes on a source of metabolicenergy without distinguishing the nature of the mechanisms. The effects ofDES, an inhibitor of the H-ATPase, indicated the involvement of the protonpump, 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 differentmechanisms of energy coupling. Above 42 jiM for G2 and 655 jiM for G100139plants, NH4 transport was predicted to be a passive process. Thisprediction is borne out by the generally smaller effects of metabolicinhibitors at high external [NH4-’-] than at low [NH4+]0(present study and inWang et al., 1993b), although 13NH4 influx showed greater sensitivity toinhibitors than the AP depolarization. There is virtually no informationavailable regarding the energy coupling for the LATS. Passive entry ofNH4-’- might occur via an electrogenic uniport (Kleiner, 1981; Ullrich et al.,1984). This may be a specific channel for NH4 or a shared cation channel.For example, the recently described K channel in Arabidopsis has beenshown to have an NH4 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) andNH4 was closely related. Thus low affinity NH4 transport might occur viathe K channel.5.5. SUMMARYThe transmembrane electrical potential differences (&P) weremeasured 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)NH4÷ (G2 or G100 plants, respectively). In modified Johnson’s nutrientsolution (MJNS) containing no nitrogen, A’I’ was in the range of -120 to-140 millivolts (mV). Introducing NH4 to the bathing medium caused arapid depolarization. At the steady-state, average &P of G2 and G100plants were -116 mV and -89 mV, respectively. This depolarizationexhibited a biphasic response to external [NH4] similar to that reported for‘3NH4 influx isotherms (Wang et al., 1993b). Plots of membranedepolarization versus 13NH4 influx were also biphasic, indicating distinct140coupling processes for the two transport systems, with a break-pointbetween two concentration ranges around 1 mM NH4. The extent ofdepolarization was also influenced by nitrogen status, being larger for G2plants than G100 plants, corresponding to the larger NH4 influxes in G2plants than G100 plants. Depolarization of A’P due to NH4 uptake waseliminated by a protonophore (carboxylcyanide-m-chlorophenylhydrazone), inhibitors of ATP synthesis (sodium cyanide plussalicyihydroxamic acid), or an ATPase inhibitor (diethyistilbestrol).141Chapter 6. REGULATION OF AMMONIUM UPTAKE6.1. INTRODUCTIONWhen plants are deficient in nutrients, such as PO4-,S042-, Cl-, theiruptake capacity is greatly enhanced (Lee, 1982). This phenomenon hasbeen known since the works of Brezeale (1907 in Glass, 1989) thatnutritional history of a plant can profoundly affect its subsequent capacityto absorb the same ion (see also Hoagland and Broyer, 1936; Broyer andHoagland, 1943). Such relationships between the ions provided duringplant growth and their subsequent uptake by roots or tissues was welldefined 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,1973; Hodges and Vaadia, 1964), p043- (Lefebvre and Glass, 1982; Lee,1982) S042- (Lee, 1982) and N03 (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 correlationbetween the rate of N absorption and the N-status of the plant material isnot precise (Lee and Rudge, 1986).It have been demonstrated that plants are able to adapt to availablesources of N over a wide range of concentrations (Clement et al., 1978;Wang et al., 1991). The existence of distinct transporters with differentaffinities 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 N142excess leads to down regulation of uptake. However, the underlyingmechanisms responsible for these changes are largely unknown. Severalhypotheses have been advanced concerning the sources of feedbackregulation responsible for controlling N uptake. These include theimportance of products of N assimilation (Lee and Rudge, 1986; Cooper andClarkson, 1989; Jackson and Volk, 1992), as well as the effects ofaccumulated ions (NO3-and NH4)on influx or efflux (Morgan and Jackson,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 highplant N status, reduction or suppression of net ammonium uptake may bedue to (i) low energy supply to the root system, (ii) accumulation in theroot tissue of a nitrogenous compound which exerts negative feedback onthe influx system, (iii) high efflux of endogenous NH4-1-. This accumulatedregulating effector could be ammonium ions generated by degradation oforganic nitrogenous sources within roots, or rapid accumulation ofammonium in N-depleted roots upon initial exposure to ammonium, orrelative ease of outward ammonium movement (Morgan and Jackson,1988a, 1988b). The regulation of influx may therefore reflect the interplayamong suppression of influx by a product of ammonium assimilation, theaccumulation of root ammonium and associated ammonium efflux, and astimulation by ammonium of its own uptake (Morgan and Jackson, 1992).It was found that 13NH4 influxes into intact roots of rice werenegatively correlated with the level of NH4 provision during growth andthe internal [NH4] in root tissues (Wang et al., 1993 a, 1993b). It has beensuggested that the regulation of NH4 uptake could result from feedbackeffects of accumulated NH4-’- or products of NH4-’- assimilation (Ullrich et al.,1984; Lee and Rudge, 1986; Morgan and Jackson, 1988; Lee et al., 1992;143Jackson and Volk, 1992; Wang et al., 1993a). These exert effects on bothinflux 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 ofNH4÷ uptake, I investigated the effects of the following pretreatments on13NH4 influx: (1) repleting N-depleted plants in 1 mM NH4 in thepresence or absence of MSX; (2) depleting N-repleted plants in 2 iiM NH4solution in the presence or absence of MSX; (3) elevating root glutamineconcentrations by supplying this amino acid exogenously; (4) alteringinternal concentrations of NH4, glutamine and other amino acids in roottissue of the above treatments; (5) using selected inhibitors of ammoniumassimilation to study the effect of perturbing ammonium metabolism onammonium uptake. The results of these experiments are interpreted interms of a cascade model for the regulation of NH4 influx in rice roots.6.2. MATERIALS AND METHODS6.2.1. Plant growth and ‘3N productionSection 2.2. Seed germination; Section 2.3. Growth conditions; Section2.4. Provision of nutrients; Section 2.5. Production of‘3NH4.1446.2.2. Experimental design6.2.2.1. Experiment I. Depletion and repletion studyTo investigate NH4 uptake by roots in response to changing plantN status, 13NH4 influx was measured in NH4-repleted G2 plants orNH4-1-depleted G1000 plants as well as G2 and G1000 plants under theirgrowth conditions. At designated times, the assigned G2 plants weretransferred to the G1000 medium and G1000 plants were transferred tothe 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 MSXThe objective of this study was to investigate the time course ofeffects of MSX on13NH4+influx. Either G2 or G1000 plants were pretreatedin their respective growth media in the presence of 1 mM MSX (G2+MSX orG1000+MSX) for 1, 4, 12 and 24 h before the‘3NH4influx measurement.A second set of plants was used to investigate MSX effects during repletionand depletion: plants were first transferred into growth media with MSXcontaining the same [NH4]0as they had been grown in (i.e. in G2+MSX forG2 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 G1000plants were transferred from G1000+MSX to G2+MSX at times of 1, 4, 12and 24 h. For comparison, a third set of plants was transferred fromgrowth medium to pretreatment medium i.e. G2 plants to G1000 mediumor G1000 plants to G2 medium at times of 1, 4, 12 and 24 h. In anotherexperiment, the pretreatment times for both G2 plants repleted in G1000145medium and G1000 plants depleted in G2 medium were 0, 1, 4, 12, and 24h. The influxes were measured for 10 mm in 100 jiM 13NH-’--labeledsolution without MSX. Each datum point is the mean of 6 replicates and thevertical bar represents the standard error (± Se).6.2.2.3. Experiment III. Effects of exogenous amino acids(1) Effects of pretreatment with glutamine on 1NH4 influx of riceroots: G100 plants were pretreated in G100 medium with or without 10mM glutamine for 16 h before measuring 13NH4 influx. 13NH4 influxeswere then measured in 2, 10, 25 and 100 jiM 13NH4-labeled solutionwithout glutamine. (2) The effects of various exogenously supplied aminoacids on the influx of 13NH4:G2 plants were pretreated in G2 medium orG100 medium plus 10 mM glutamate, glutamine or asparagine for 16 h,respectively. 1NH4 influxes were measured in 100 mM labeled 1NH4÷solution in the presence of the same amino acids. Each experiment wasrepeated twice, with 3 replicates.6.2.2.4. Experiment IV. Effects of selected inhibitorsInhibitors of glutamine synthesis (L-methionine DL- sulfoximine,MSX), glutamate synthesis (6-diazo-5 -oxo-L-norleucine, DON) andaminotransferases (amino-oxyacetate, AOA) were used to perturb tissueconcentrations of glutamine and glutamate to investigate the effect ofchange of these compounds on 1NH4 influx. All treatments of inhibitorswere administered for 16 h at 100 mM. 1NH4 influxes were measured ineither 100 mM or 10 mM labeled1NH4solution.6.2.3. Determination of free ammonium in root tissueSee section 2.5.1466.2.4. Determination of amino acids in root tissueSee section 2.8.6.3. RESULTS6.3.1. Experiment I. Depletion and repletion studyAs shown in Fig. 19, the initial‘3NH4 influx of nitrogen-deficientrice 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 inG1000 medium, influx increased to nearly 3 times its initial value (to 31.97iimolg4FW h4) during the first 5 h. Between 6 to 12 h of loading, influxesdeclined to about 10 p.mol g’FW h’. After three days in 1 mM NH4--solution, the ‘3NH4 influx dropped below 5 jimol g’FW h-’. When G2plants were repleted in 10 or 100 jiM NH4 solution, G2 roots respondedwith a similar pattern, but showed a delay in reaching the maximum ofinflux (data not shown).Nitrogen-sufficient rice seedlings were grown in G1000 medium forat least 13 days and transferred to G2 medium for periods varying from0.3 to 192 h, respectively, before measurement of‘3NH4influx. As shownin Fig. 20A, initial‘3NH4influx of G1000 plants was quite low (1.15 jimolg1FW h-i) in agreement with previous reports (Wang et al., 1993b). Shortterm depletion in G2 medium, for periods of 0.5 to 4 h, caused 1NH4influxes to increase almost 10 fold. Between 4 to 24 hours,‘3NH4influx ofthese N-depleted plants was close to the Vm for 13NH4 influx of G21474019A 40Repletion in G1000 Media (h)Figure 19. ‘3NH4 influx of repleted G2 plants. After repletion in G1000medium for various periods, 13NH4 influx of G2 plants was measured in100 jiM13NH4-labeled solutions. Insert 19B shows, in expanded form, thefirst 24 h of repletion. Each datum point is the mean of 3 to 6 replicatesand the vertical bar represents the standard error (± Se).1481520AjlO_________0 20 40 60 80 100 120 140 160 180 200Depletion in G2 media (h)Figure 20. 1NH4 influx of G1000 plants during depletion in G2 mediumfor various periods. The influxes were measured in 100 iM13NH4-labe1edsolution. Insert 20B shows in expanded form the data for the first 24 h ofdepletion. Each datum point is the mean of 3 to 6 replicates and thevertical bar represents the standard error (± se).14921A 60 5 10 15 20 252.—0EE0• • • I • I • I • I • I • I • I • I • I0 20 40 60 80 100 120 140 160 180 200Depletion in G2 media (h)Figure 21. Internal ammonium content of depleted G1000 roots. G1000roots were depleted in G2 medium for various periods and internalammonium content were assayed. Insert 2 lB shows in expanded form thedata for the first 24 h of depletion. Each datum point is the mean of 6replicates and the vertical bar represents the standard error (± se).150plants (—41 pmol g’FW h-i) (Fig. 20B). After 24 hours depletion, the‘3NH4 influxes declined but were still higher than those of G1000 plantsat steady-state. The results indicated, that depletion in G2 medium for upto 8 days, caused no further decline of influx, which remained at about 6imol g-’FW h-i. Meanwhile, root NH4÷ concentrations dropped rapidlyduring the first 4 h depletion of N, from 5.6 to 3.6 iimol g’FW (Fig. 21A).After 24 h depletion, internal [NH4+j remained at a low level (0.6 !Imol g1FW, in Fig. 21B). Figures 20B and 21B reveal that there was a negativecorrelation (r2 = 0.74) between [NH4]1 and 13NH4 influx during 24 hdepletion of N. Beyond 24 h of N depletion, no correlation was found.Changes of the total AA content in root tissue of G1000 plants duringdepletion in G2 medium, are presented in Fig. 22A. In the first 4-5 h ofdepletion of N, total amino acid concentration ([AA]) increased (Fig. 22B).In fact the total {AA]1 remained above the original level through 200 h ofdepletion. The contents of the major amino acids and amides, [Gln]1 {Glu]1,[Asn]1, and [Asp] were also found to have increased in the same fashion(data not shown).The phenomenon of stimulated influx observed during the first hoursfollowing exposure of G2 plants to 1000 jiM NH4 was not as pronounced inthe second experiments (open circles in Fig. 23A) as in the first experiment(Fig. 19A). This may have been due to differences of experimentalconditions. In the first experiment, the depletion/repletion was carried outin a large volume of nutrient solution (in 35-liters Plexiglas tanks) inwhich the NH4 concentrations were held relatively constant. In the secondexperiment, the same treatments were performed in a volume of 20 ml ofmedium. Such a small volume may have limited the repletion process andconsequently affected the extent of the influx response. For example,typical cytoplasmic and vacuolar [NH4+] were 0.19 and 2.19 jimol g’FW for1515._______________________________22A 2.5____:::10 15 20250 25 50 75 100 125 150 175 200Depletion in G2 media (h)Figure 22. Total amino acid concentration ([AA]) of depleted G 1000 roots.After depletion in G2 medium for various periods, G1000 roots wereassayed for tissue amino acid concentration ([AA]1). Insert 22B shows inexpanded form the data for 24 h of repletion. Each datum point is themean of 6 replicates and the vertical bar represents the standard error (±se).152G2 roots and 1.94 and 4.91 jimol g’FW for G1000 roots, respectively (seeTable 4). This means that in order to convert G1000 plants to G2 plantsthere is about 4.47 jimol NH4g4FW to be depleted either by metabolismor efflux to the external media. Assuming that rates of efflux andassimilation are equivalent at about 20% of the rate of influx (Chapter 3and Wang et al., 1993a), then the release of NH4 could elevate external[NH4] to nearly 100 tiM. In the small volume employed for thisexperiment the released NH4would readily be re-absorbed, slowing downthe change from G1000 to G2 statues.As shown in Fig. 23A, when G2 plants were repleted with NH4-- inG1000 medium, the‘3NH4 influx (closed circle) increased from 8.17 to10.00 j.tmol g’FW h’ during the first hour, then dropped to 8.61 at 4 hand 1.95 iimol g-’FW h-i after 24 h repletion. Root [NH4]1(closed square)increased rapidly in the first hour from 2.21 to 6.48 iimol g’FW andincreased only slightly to 7.13 iimol g’FW during the next 23 h of NH4repletion (Fig. 6B). By contrast, depletion of G1000 plants in G2 mediumincreased 13NH4 influx only very slightly during the first hours. Theninflux increased rapidly from 0.72 to 7.29 iimol g’FW h’ (open circle inFig. 23A). During the depletion in G2 medium, the [NH4+]1of G1000 plants(open square) decreased gradually from 6.35 to a value similar to that ofG2 at steady-state, 2.36 jimol g’FW by 12 h of depletion. During the next12 h, there was only a small further decrease of [NH4+] (Fig. 23B).The changes of tissue amino acids present different patterns forplants undergoing nitrogen depletion or repletion. During the repletionprocess, G2 plants were exposed to 1000 .tM NH4 for up to 24 h. The total15312- L0,23A — G2IG1000 23B G2IG1000—0— G1000IG2 —D G1000IG2——E 8 E:.-.U—4z. II2-EE_______________________.0 0-0 5 10 15 20 25 0 5 10 15 20 25Pretreatment time (h)Figure 23. 13NH4 influx (23A) and internal ammonium content (23B) ofrepleted G2 or depleted G1000 roots. 23A:‘3NH4influxes of G2 or G1000roots, after pretreatment for 1, 4, 12 and 24 h in G1000 or G2 medium,respectively, were measured in 100 1iM 13NH-labeled solution. 23B:Internal ammonium content of the same roots. Each datum point is themean of 6 replicates and the vertical bar represents the standard error.154030000E20001000 —s—— G2/G1000M—O---- G1000/G2MU-0 5 10 15 20 25Pretreatment time (h)Figure 24. Total [AA]1 of repleted G2 or depleted G1000 roots. Total [AAJ1of G2 or G1000 roots were assayed after pretreatment for 1, 4, 12 and 24 hin G1000 or G2 medium, respectively. Each datum point is the mean of 6replicates and the vertical bar represents the standard error (± Se).1552000- 25B Glu G2/G1000M25A G1n G2/G1000M—0-— G1000IG2M —C]— G1000/G2M1501500 -1000- 100500 - 500 u-0 5 10 15 20 25 0 5 10 15 20 25600- 600-25C Asn——— G2/G1000M 25D Asp ——— G2/G1000M° —0— G1000/G2M —ETh— G1000/G2M400- 400—E200 200-.0• 00 5 10 15 20 25 0 5 10 15 20 25Pretreatment time (h)Figure 25. Tissue amide or amino acid contents of repleted G2 or depletedG1000 roots. After pretreatment for 1, 4, 12 and 24 h in G1000 or G2media, respectively, the amino acid contents of G2 and G1000 roots wereassayed. 25A for [Gin]1; 25B for [Glu]1; 25C for [Asn]1,; 25D for [Asp]1.Eachdatum point is the mean of 6 replicates and the vertical bar represents thestandard error (± Se).156[AA]1 increased during 12 h of repletion and stayed at more or less thesame 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 Glucontent (Fig. 25B, closed circle) decreased continuously during NH4--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% duringthe first hour, then it increased nearly 39% of the initial level in the next12 h (Fig. 25C, closed circles). [Asp]1was reduced (49%) in G2 roots duringthe 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 thefour major amino acids decreased rapidly for the first hour (Figs. 24,25AD, open symbols). This is interesting because despite big changes inthese [amino acid], influx changed little. After that, the [AA], [Gln]1 [Glu]1,[Asn]1, and [Asp]1 increased 65%, 353%, 61%, 40% and 31%, respectively,within 23 h of commencing the depletion process.6.3.2. Experiment II. Effects of MSXShort periods (< 12 h) of MSX treatment increased‘3NH4influx of G2roots (closed circles in Fig. 26) from 8.17 to 16.93 jimol g1FW h’, butlonger (12 - 24 h) exposures reduced influx slightly, to 12.64 jimol g’FWh’. During 24 h pretreatment of G2 plants in G1000+MSX,‘3NH4influxes(open squares) remained essentially constant at about 10 iimol g’FW h’and were lower than those of in G2+MSX (closed circles). Likewise, G1000plants, pretreated in G2+MSX or G1000+MSX media, exhibited very low‘3NH4influx values (closed and open squares) which remained essentiallyconstant for the duration of the experiment. Fluxes of G1000 plants were157significantly lower than in G2 plants in G2+MSX or G1000+MSX (compareopen to closed symbols in Fig. 26).For G2 plants pretreated in G2+MSX, root [NH4+]1 increased rapidlyfrom 2.21 to 7.19 at the first hour and remained at the same level for theremainder of the experiment (closed circles in Fig. 27A), but pretreatmentin G1000+MSX caused root [NH4]1to increase rapidly from 2.21 to 8.49imol g’FW during the first hour, reaching a value of 9.35 after 24 hrepletion (closed squares in Fig. 27B). G1000 plants possessed a higherinitial [NH4]1(6.35 jimolg1FW) (Figs. 27A and 27B), which continuouslyincreased to 8.57 imol g1FW after 24 h during treatment of G1000+MSXmedium. Root [NH4-’-]1 in G1000 plants treated in G2+MSX declinedgradually from 7.36 at 1 h to 5.77 between 4 and 24 h (open circles in Fig.27A). The increment of [NH4-’-]1 in MSX treated plants varied with priorNH4 provision during growth and additional depletion or repletiontreatments (Figs. 27A and 27B). During the first hour, the [NH4-’-]1 of G2plants increased 230% in G2+MSX medium and 320% in G1000+MSXmedium. The [NH4]1 of G1000 plants increased 35% in G1000+MSXmedium, and 16% during the same time period in G2+MSX medium, thelatter then decreased to 9% after 24 h.The total [AA]1 of G2 or G1000 plants in the four treatmentspretreated with 1 mM MSX, remained at similar levels, respectively, overthe 24 h period (Figs. 28A and 28B). G1000 plants (open symbols) had ahigher total [AA] than G2 plants (closed symbols). Both plants showed asmall increase in the G1000+MSX treatment (Fig. 28B). Pretreatment inG2+MSX, caused the [Gin]1 of G2 roots to decline at the first hour but nofurther changes were observed during the remainder of the experiment(Fig. 29A, closed circles). The opposite effect was observed in G1000+MSX15825• G2/G2M+MSX —0— G1000/G2M+MSXG2/G1000M+MSX D G1000/G1000M+MSX20010 210 25Pretreatment time (h)Figure 26. Effect of MSX pretreatment on 1NH4 influx of rice roots. G2(closed symbols) or G1000 plants (open symbols) were pretreated with 10mM MSX for a maximum duration of 24 h including 0, 1, 4, 12, and 24 h inG2+MSX medium (open or closed circles) and in G1000+MSX medium (openor closed squares), respectively. The influxes were measured in 100 jiM13NH4-labe1ed solution without MSX. Each datum point is the mean of 6replicates and the vertical bar represents the standard error (± Se).159—• 1227A in G2M+MSX 27B in G1000M+MSX10.21 G2—a— G2—0— G1000—D— G1000• I • I • I • 0 • • I • I • I0 5 10 15 20 25 0 5 10 15 20 25Depletion or repeltion time (h)Figure 27. Effect of MSX on internal ammonium content of rice roots. Thepretreatments and symbols are same as in Fig. 26. Each datum point is themean of 6 replicates and the vertical bar represents the standard error (±se).160‘—‘ 3000 300028A in G2M+MSX 28B in G1000M+MSX—— G2— G2—0— G1000—D— G10000- 00 5 10 15 20 25 0 5 10 15 20 25Depletion or repeltion time (h)Figure 28. Effect of MSX on total [AA]1 of rice roots. The pretreatments andsymbols are same as in Fig. 9. Figures hA and 11B are for the plantspretreated in G2+MSX medium and in G1000+MSX medium, respectively.Each datum point is the mean of 6 replicates and the vertical barrepresents the standard error (± Se).161; :::z100• 100•EE ooo * G2—D—- G10000• 00 5 10 15 20 25 o 5 10 15 20 25‘ 150 15029C in G2M+MSX 29D in G1000M+MSXG2 G2100 —0—— G1000 100—U— G1000I III.. .. 25Depletion or repeltion time (h)Figure 29. Effect of MSX on amide or amino acid content of rice roots. Thepretreatments and symbols are the same as in Fig. 26. Figures 29A, 29C,29E, 29G is for [Gin]1, [Glu]1, [Asn]1, and [Asp]1 of plants pretreated inG2+MSX medium, respectively. Figs. 29B, 29D, 29F, 29H is for [Gin]1, [G1u],[Asn]1, and [Asp]1 of plants pretreated in G1000+MSX medium, respectively.162300 30029E in G2M+MSX 29F in G1000M÷MSX• G2—•— G2—0— G1000f 20O 200 I IE10000 5 10 15 20 25 00 5 10 15 20 25300 30029G in G2M+MSX 29H in G1000M+MSXII—— G2—— G2—0— G1000 —D— G1000— 00 200L1—E100 1000• U-0 5 10 15 20 25 0 5 10 15 20 25Depletion or repeltion time (h)Figure 29. (Continued).163(Fig. 29B, closed squares). The [Gin]1 of G1000 roots was reduced more inG2+MSX (Fig. 29A, open circles) than in G1000+MSX (Fig. 29B, opensquares). In the latter medium, Gln recovered slightly after 24 hpretreatment (Fig. 29B). The levels of [Glu]1 in roots declined rapidly withinthe first 4 h of pretreatment in G2+MSX and in G1000+MSX (Figs. 29C and29D) except in the G2 plants treated in G2+MSX, in that it took a longertime to achieve the same reduction (Fig. 29C, closed circles). The [Asn]1 and[Asp]1 of G2 roots were also significantly reduced in all four pretreatments(Figs. 29EH). A similar extent of reduction of [Asn]1 was reached in ashorter time period when G1000 plants were pretreated with MSX ineither repletion with or depletion of NH4 (open circles in Fig. 29C andopen squares in Fig. 29D) whereas the change of [Asp]1was more graduallyin G2+MSX (Fig. 29G) than in G1000+MSX (Fig. 29H); in the latter treatmentthe reduction occurred within 4 h of pretreatment.6.3.3. Experiment III. Effects of exogenous amino acidsPretreatment of G100 roots with 10 mM glutamine significantlyreduced ‘3NH4 influx at all concentrations tested (Fig. 30). Assays of[NH4]1revealed that glutamine pretreatment was associated with higher[NH4-1-] (6.2 ± 0.5 .tmol g’ FW) than those pretreated without glutamine(2.3 ± 0.8 pmol g-’FW). The 18 h pretreatment in 10 mM Gin raised thecontents of Gin, Glu, and Asp near 4 times and Asn 7 times (Figs. 3 1A and31B).The interaction of exogenous amino acids and nitrogen status werealso investigated. When G2 plants were treated with either 10 mM [Gin]0 or[Glu]0 for 18 h, 1NH4influxes were significantly reduced (from 8.9416460 without glutamine[NH Jo (jiM)Figure 30. Effect of exogenous glutamine on‘3NH4influx of roots. G100plants were pretreated in G100 medium with or without 10 mM glutaminefor 16 h before measuring‘3NH4influx.‘3NH4influxes were measured in2, 10, 25 and 100 jiM13NH-labe1ed solution without glutamine. Eachdatum point is the mean of 6 replicates and the vertical bar represents thestandard error (± se).Figure 31. Effect of exogenous glutamine on the contents of amides andamino acids of root tissues. The pretreatments are same as in Fig. 30. Fig.31A is total [AA]1 and Fig. 31B is [Gin]1, [Glu]1, {Asnj1, and [Asp]1. Eachdatum point is the mean of 6 replicates and the vertical bar represents thestandard error (± se).31A Total1656000 -4000-20002000100000-.031BI/+GinPretreatmentF,F?F,F—F——F—F—FF—F—F’‘FF—F——F—F‘F—FFF.‘ ,FFFF..F—‘FF•AspGluAsnLJGIn0-+Gln -Gin0- —-Gin166E——Figure 32. Effect of exogenous amides and amino acid on‘3NH4influx. G2plants 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. Theinfluxes were measured in 100 jiM13NH4-labeied solution. Each datumpoint is the mean of 6 replicates and the vertical bar represents thestandard error (±se).32B G2/G100M1210864.20T32A G2/G2M• 0T)•Im•‘Il/I,12108642-0-zControl Gin Glu Asn Control GinPretreatment-I-Glu Asn167iimol g’ FW h-i of the control to 5.12 and 2.30, respectively, Fig. 32A). Nosignificant reduction of 1-NH4 influx occurred as a result of Asnpretreatment (Figs. 32A, B). Comparisons of pretreatments in G2 mediumand G 100 medium for G2 plants, revealed that the higher concentration ofNH4 in the latter medium led to a reduction of‘3NH4 influxes of thecontrol 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).The combination of 100 iM [NH4]0and 10 mM [Gln]0 or [Glu]0 (Fig. 32A)failed to reduce 13NH4 influx further than the pretreatments of 2 jiM[NH4]0 and 10 mM [Gln]0 or [Glu]0 (Fig. 32A). Pretreatments withexogenous amides or amino acids increased [NH4]1from 1.1 to 3.6 - 5.5jimol g1 FW at low external NH4 conditions (G2 medium) (Fig. 33A). InG100 treatment, internal [NH4+] was higher for the Glu pretreatment,followed by the control, and the pretreatments with Gln and Asn (Fig. 33B).Total AA concentrations were significantly higher for plantspretreated in G100 medium than in G2 medium (Fig. 34). In both cases, thetotal AA was higher in the pretreatments of Glu and Asn (Fig. 34B). Whenexogenous amides or amino acids were provided during pretreatments,[Gln]1 was highest in the Gin pretreatment (Figs. 35A and 36A), except forthe [Glu]0 pretreatment in G2 medium that had the highest [Gln] (Fig. 34A).Compared to the control, the concentrations of Gln were doubled in bothmedia. [Glu]1 was highest in GIn pretreatments, followed by the Asnpretreatment (Figs. 35B and 36B). Both [Asn]1 and [Asp] were highest inthe Asn pretreatment (Figs. 35C, 36C, 35D and 36D).168Figure 33. Effects of exogenous amides and amino acid on internalammonium content. Details as in Fig. 32.33 B G2/G100M000-.SCECE65.4.3.2-1-0433A0 G2IG2M TT_Wf WFA Ff1III0”Control Gin Glu Asn Control Gin Glu AsnPretreatment2-169-.6Control Gin Glu AsnPretreatmentFigure 34. Effects of exogenous amides and amino acid on total amino acid34A G2IG2ME4.-.C.?12 -29-18 -34B G2/G100MC6-Liii03.Control Gin Glu Asncontent. Details as in Fig. 32.1701???1?1‘‘‘I‘—‘F‘—‘FF—,———‘F‘F,—F,———F——\\‘•___\\%______F,, FFFF,,—___\\%\IF,FFI .‘..I’ ••. •‘ ‘.1 •.. •s “ ‘.IFtFPI .tFZ_____Figure 35. Effect of exogenous amides and amino acid on contents of aminoacids in G2 roots. Pretreatments are same as in Fig. 32. Figs. 35AD is for[G1n], [Glu]1, [Asn]1, and {Asp] of plants pretreated in G2 medium,35A Gin 35B GluI‘V/CC200100-C0-300-.‘CC200_100-C0Control Gin15001000500-0-300020001000Giu Asn35C AsnControl Gin35D AspGlu AsnControl Gin Glu AsnF——,F—,—F,—,0Control GinPretreatmentGiu Asnrespectively.00—0-.S0PretreatmentFigure 36. Effect of exogenous amides and amino acid on contents of aminoacids in G100-pretreated roots. Pretreatments are same as in Fig. 32. Figs.36AD is for [Gin]1, [Giu]1, [Asnj1, and [Asp] of plants pretreated in G10036A Gint7180006000 -4000 -2000 -0-3000 -2000-1000-36B GluVA/-IControl Gin Glu Asn36C Asn80006000-40002000-06000400W20000-Control Gin Giu Asnp0c-)36D Asp —c-F—,——F———F———F—————F———FF———F FFF_F__ FFF•‘.\ ‘.\\____ F__F‘\\F___ FF‘%‘\F__F FF.‘‘.%\ \‘‘•,____ F__F____ F,,,\‘\ ‘\‘\_,F_ FF?‘%.\F__F FFF•\‘ ,\•\____ F,,,,\•\____ F,,,______‘‘,,I _,__ F,,,\‘‘‘I \•\ \‘s’.\ ‘.\\‘—‘F_I FFFF FFFF ‘FF‘‘.‘.\‘I \%‘ \\\ ,‘\•FFI FF ‘F. ?FF\‘.•% \‘‘\ ‘•\•‘IControl Gin Glu Asn Control Gin Giu Asnmedium, respectively.1726.3.4. Experiment IV. Effects of selected inhibitorsG100 plants were treated with inhibitors of glutamine synthesis,MSX, glutamate synthesis, DON, and aminotransferases, AOA, for 16 h,respectively. The‘3NH4 influxes were measured in either 100 jiM or 10mM labeled 1NH4 solution without inhibitors. The largest effect of theinhibitors of NH4 assimilation was associated with AOA pretreatment (Fig.36A). The13NH4 influx due to HATS (high affinity transport system) andLATS (low affinity transport system) were reduced by 68% and 32%,respectively (Figs. 37A and 37B). MSX reduced‘3NH4÷ influx by the LATS25% and by the HATS 19%. DON treatment produced only a slight reductionof1NH4influx (16% for LATS and 4% for HATS).As can be seen in Fig. 38A, MSX significantly increased [NH4j1almost3 fold. The level of [NH4]1 was 1.9 times higher as a result of AOApretreatment, while rice roots treated with DON actually had a lower[NH4]than the control. The total [AA]1 was doubled by the AOA treatment(Fig. 38B). While slightly increased by MSX, the total [AA1 was greatlyreduced by DON treatment. Looking at the four major amides and aminoacids, (as shown in Figs. 39A, B, C, D), the pretreatment of AOA significantlyincreased 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 fourmajor amino acids were reduced to about half that of controls aftertreating plants with DON (Figs. 39A, B, C, D).17354.———+.1.zo • . 0-Control MSX DON AOA Control MSX DON AOAPretreatmentFigure 37. Effect of MSX, DON and AOA on1NH4influx. G100 plants werepretreated with MSX, DON, and AOA for 16 h, respectively. The influxeswere measured in either 100 1iM (Fig. 37A) or 10 mM (Fig. 37B) labeled13NH4 solution without inhibitors. Each datum point is the mean of 6replicates and the vertical bar is the standard error (± Se).37A Uptake in 0.1 mM1537B Uptake in 10 mM10__F:V//4’174-.S—E______:iCFigure 38. Effect of MSX, DON and AOA on internal ammonium and totalamino acid content. Pretreatments are same as in Fig. 37. Fig. 38A is forinternal ammonium and Fig. 38B is for total amino acid content.38B Total AA321284.038A AmmoniumPd/”IIControl MSX DON0AOA Control MSXPretreatmentDON AOAPretreatmentFigure 39. Effect of MSX, DON and AOA on major amino acid contents.Pretreatments are same as in Fig. 37. Figs. 39AD is for [Gin]1, [Glu]1, [Asn]1,and [Asp], respectively. Each datum point is the mean of 6 replicates and,IflhI39B Glu1751000’80060040020039A GinI II300200100’CCC,)-.SCControl MSX DON AOA“7400’20039C AsnUControl MSX DON AOA600-39D Asp‘\,‘‘I,,%‘\\... , , F‘,.‘., ,,,\\\\‘,‘f400 -‘.‘\\, ,.,,‘.‘‘..., ,,,‘‘‘\‘/_f‘‘\‘f—f,%,‘f_f‘‘f_f‘f_f%‘‘‘f_f2“'‘U) /,,, tf\\\f T7\•\,,,,\%\\f_f, ,,__ ,f,f\‘‘.\,,,, f_f,\\s\,,,, ,,,,‘.‘‘.\ \\ ,%\‘f_f, ,,__ ,f__ ,,f,\\\,,,, ,,,, f_f,\s\% %\‘\ \‘.•..\,,,, ,,,, ,,,_ ,,,,• ‘\s ‘. — \\ .‘Control MSX DON AOA0Control MSX DON AOAthe vertical bar is the standard error (± Se).1766.4 DiscussioN6.4.1. Negative feedback on NH4 uptake by NH4 assimilatesNH4 uptake is probably regulated continuously in response to the Nstatus of the plant, but it is not clear how this is achieved. Increase inammonium influx upon nitrogen limitation and decrease in influx as cellnitrogen status rises have commonly been observed (McCarthy andGoldman, 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). Feedbackinhibition of NH4 uptake by nitrogenous effectors has been implicated inorganisms like Lemna, algae, yeast and higher plants (Kleiner, 1985; Ulirichet 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 actas the negative feedback factors for the NH4 uptake process (Cook andAnthony, 1978b; Breteler and Siegerist, 1984; Wiame et al., 1985; Revillaet al., 1986; Lee and Rudge, 1986; Morgan and Jackson, 1988a). In thereview by Clarkson and Luttge (1991) a central role for glutamine inregulating the uptake of N by fungi and microalgae was presented.Glutamine or asparagine are the low molecular weight N-containingcompounds stored or translocated by plants in the family of Poaceae(Gramineae) (Marschner, 1986). Lee and Rudge (1986) showed sizableincreases in NH4 uptake by barley following N-depletion, and theincreased capacity for NH4 uptake was inversely related to the reduced-N177status of the root tissue. In tobacco cells cultured on nitrate, urea, orammonium, Gln is the first major organic product of assimilation of‘3NH4(Skokout et al., 1978). It is also true for rice, because glutamine andglutamate were the primary products of ammonium assimilation in riceroots (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 ofN uptake.In the present study, evidence supporting a central role forglutamine or other amino acids in controlling NH4 influx was equivocal.When plants were maintained at 2 tM or 1000 iM NH4 respectively,‘3NH4 influx was inversely correlated with [Glfl]i (closed symbolscompared to open symbols in Figs. 29A and 29B). Likewise, when theinternal concentrations of Gln and other amino acids were increased bypretreatment with Glu,‘3NH4influx declined (Figs. 30, 31B and 35A). Theresults indicated that Glu had an inhibitory effect on‘3NH4influx, greaterthan Gln or Asn (Figs. 32A and 36A). This point was supported by theresults of the AOA treatment. After treating plants with AOA, under theconditions 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). Thisincrement was associated with a significant reduction of1NH4influx (Fig.20A). It must be pointed out that the above mentioned reductions of13NH4 influx in rice also coincided with a significant increase of [NH4÷]1(Figs. 33A and 38A). Pretreatment with 10 mM Gln doubled the [NH4]jfrom 2.30 to 6.10 iimol g4FW (also in Fig. 33A) and decreased 1NH4influx.178In the depletion experiment shown in Fig. 23A transfer of G1000plants to G2 solution failed to increase NH4÷ influx until 4 h had elapsed.Yet, the amino acid analysis indicated strong reduction of total [AA] and[Gin], [Giu] and [Asp] (Figs. 24, 25AD). Strong reductions of amino acidswere not correlated with 13NH4 influx. Therefore, it is not entirely clearwhich N derivative is responsible for limiting influx.Although applying organic N to the growth media has been found toincrease crop yield (Mon et al., 1977; Mon and Uchino, 1977), thetreatment of organic N suppresses the uptake of inorganic N. For example,maize roots pretreated with Gin or Asn exhibited reduced net uptake ofNH4 and N03 (Lee et al., 1992). The uptake of‘5N03 by barley roots wasdepressed by pretreatment with Arg and His (Mon et al., 1979). It wassuggested that transport activity for ammonium was controlled byintracellular rather then extracellular metabolites (Jayakumar and Barner,1984).6.4.2. Effect of MSX: reduced amino acid pooiMSX inhibited the activity of glutamine synthetase in plant roots, andstopped the ‘5N labeling of free amino acids, particularly glutamine andglutamate in roots of barley or rice (Arima and Kumazawa, 1977; Lewis etal., 1983). Preventing the assimilation of newly absorbed NH4 or releasingNH4 from the catabolism of internal N-containing compounds rapidlyincreased the NH4 concentration in roots (Arima and Kumazawa, 1977;Lewis et al, 1983; Lee et al., 1992). Two major effects are expected: theamino acid pool is reduced and NH4 pool is increased. After treating withMSX, tissue [Gin]1 is typically decreased (Steward and Rhode, 1976; Fentem179et al., 1983a, 1983b) and consequently the amide donor to Asn synthesis isdecreased, since the concentrations of Gin and Asn closely correlated (Leeet al., 1992). When products of ammonium assimilation were reduced bytreatment of MSX, NH4 influx was increased (Jackson et al., 1993), thoughNO3- influx was not stimulated (Lee et al., 1992).MSX increased the cytoplasmic ammonium concentration in roottissue of rice (Arima and Kumazawa, 1977), Datura (Probyn and Lewism1979), barley (Lewis et al., 1983; Fentem et al., 1983b; Morgan andJackson, 1988a, 1988b); wheat (Morgan and Jackson, 1988a, 1988b), maize(Lee and Ratcliffe, 1991; Lee et al., 1992). A ten fold increment of thecytoplasmic pooi was reported in maize roots compared to the control (Leeand Ratcliffe, 1991; Lee and Ayling, 1993). This increase is due to twoeffects: (a) the assimilation of NH4 into amino acids is blocked, and (b) theproduction of NH4 from breakdown of amino derivatives remainsunaffected. It has been claimed that release of NH4 from this degradationpath occurs at a rate which is 50% higher than the rate of NH4 influx(Jackson et al., 1993). As a result, ammonium appeared in the xylem sap(Lee and Ratcliffe, 1991) and net NH4 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 theouter membrane of plasma membrane of root cells, through whichammonium with a high 15N abundance permeates from the externalsolution. MSX treatment might enlarge this ammonium compartment nearthe membrane.Another explanation for the enhanced NH4 influx by MSX treatmentis that MSX enlarged the cytoplasmic and vacuolar NH4 pools of root tissueseveral times (Jackson et aL, 1993; Lee and Ayling, 1993). The enlarged180NH4 pools in cell enhanced influx of1NH4in maize and barley (Lee et al.,1992; Lee and Ayling, 1993). According to Lee and Ayling (1993) thisresulted in a large value of NH4 influx because what was measured underthese circumstances was a true value of influx. By contrast, under ‘normal’circumstances (they claim) even short‘3NH4 influx measurements arecompromised by a significant efflux. The results of studies on rice andbarley (Siddiqi et al., 1991; Wang et al., 1993a) repudiate thisinterpretation because the half-life of the cytoplasmic compartment is toolong (7-8 mm) and the efflux term is too small (10%- 30%) compared toinflux (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 todifferent extents (Figs. 29A-H), accompanied by an increased [NH4-’-]1.Theincrement of [NH4]was varied with NH4 provision and additionaldepletion or repletion treatments (Figs. 27A and 27B). However thetreatment with MSX in this experiment failed to increase the 13NH4+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 repletionby the same plants in the absence of MSX (Fig. 23A). However, G2 plantstreated with G2+MSX conditions revealed a significant increase of influx(Fig. 26). When the same G2 plants were treated in G1000 medium plusMSX there was no decline of influx of the sort observed in the absence ofMSX (Fig. 23A). This is consistent with an important role of amino N indown-regulating influx in low-N plants. The lack of an increased influxwhen G1000 plants were transferred to G2 medium with MSX (Fig. 26)argues that internal [NH4-’-] is important in maintaining low NH4 fluxes inhigh-N plants. This has also been claimed by Causin and Barneix (1993) in181wheat. Thus the results of these experiments indicated that both [NH4]and [AA]1 may play important role in regulating NH4 fluxes.6.4.3. Effect of short-term N depletionIt has been recognized that the nitrogen (both ammonium andnitrate) uptake capacity of plant roots is enhanced when plants undergonitrogen 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). NH4 uptake shows aparticularly 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 andIngversenm, 1971; Lee et al., 1992), barley (Lee and Rudge, 1986), andoats (Morgan and Jackson, 1988a, 1988b).In the present study, rice also responded to nitrogen depletion withenhanced NH4 influx (Fig. 20). The short-term depletion of highNH4+-grown plants (G1000) in low N medium (G2 medium) stimulatedNH4 influx during the first 4 to 5 h of depletion. 13NH4 influx remainedhigh for the next 20 h, then declined to a relatively lower rate for the next20 h of depletion (Fig. 20B). Similar rapid initial increases of NH4 uptakewere 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).182A likely explanation for this enhancement is the removal of a factorwhich exerts negative feedback regulation on NH4 uptake. Both NH4-- andits 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 isdue to enhanced influx and reduced efflux (Morgan and Jackson, 1988a,1988b). Substantial ammonium cycling occurred during net ammoniumuptake (Jackson et al., 1993), yet plants grown in low N possess a low NH4efflux. For G2, G100 and G1000 plants at steady-state with respect to[NH4-’-]0,the effluxes of NH4were 10%, 20% and 29%, respectively, of influx(Wang et al., 1993a). However, changes of these relatively smallproportions may not account for the large increases of NH4 uptake such aswere observed in the present study.In the present study, [NH4]1was negatively correlated with influxduring 4 h of depletion (Figs. 20B and 21B). It was also observed that theVmax for NH4 influx was negatively correlated with internal NH4 (Wang etal., 1993b). When plants were subjected to N depletion, the tissue contentof NH4 (Fig. 2 1B) dropped rapidly to lower levels and possibly resulted ina relief of N-suppression of the uptake process. [NH4]c is a likely candidatefor negative feedback regulation since the free NH4 pools (cytoplasmicand vacuolar) will be drained in two opposite directions: efflux out of thetissue and metabolism into amino acids. In such a short time, [NH4]willbe the first fraction to be drained to a minimum. Therefore internal NH4 isa likely factor to exert a negative signal on NH4 transport across theplasma membrane (also in section 6.4.5.).It is generally believed that short periods of N depletion, less than 24to 48 h, would not cause a decline of growth rate (Siddiqi et al., 1989;183Jackson and Volk, 1992). Though it was reported that enhanced uptakereached a maximum after 3 days of depletion when nitrogen stress wasnot severe enough to alter the RGR significantly (Lee and Rudge, 1986),longer N depletion may not sustain the maximum enhanced uptake ratedue to possible adjustment of the RGR. For 8-d-old maize plants grown on5 mM NO3-, NH4 uptake rates increased steadily, and within 72 h of N-depletion, rates of NH4 uptake initially increased followed by a declineand a subsequent increase (Jackson and Volk, 1992). This enhanced NH4uptake or NH4 influx may be due to a relief of the uptake process from N-suppression. As suggested by Morgan and Jackson (1992), this type ofresponse reflects the interplay of suppression by a product of ammoniumassimilation, the accumulation of root ammonium and associatedammonium efflux, and a stimulation by ammonium of its own uptake.6.4.4. Stimulated NH4 influx after long-term N depletionWhen N-depleted roots are first exposed to elevated levels of NH4-l-there is an initial increase of NH4 influx for the first few hours ofexposure to NH4 (Goyal and Huffaker, 1986; Morgan and Jackson, 1988a).The above workers observed a 25-35% increase of influx in wheat duringthe period from 2-10 h after exposure to NH4; further exposure causedinflux to decline. This phenomenon was found in wheat but not oat(Morgan and Jackson, 1988a). In the present study, an even greater effectwas observed when G2 plants were repleted in G1000 medium. Within thefirst two hours repletion with NH4-’-, 13NH4 influx increased rapidly from11.10 imol g’FW h’ to 31.97 iimol g’FW h’ (Fig. 19B). Then, influxdropped to the initial rate of about 10 p.mol g’FW h1 after 8 h morerepletion. A smaller stimulation can also be seen in Fig. 6A.184There are at least two possible explanations suggested for thisphenomenon (Morgan and Jackson, 1988a). First, a second system forammonium influx may be initiated (induced?) as N-depleted plants areexposed to ammonium for a short period before negative feedback becomeactive. Another possibility is that there are two effectors (positive andnegative) to regulate a single transport system. The positive effector couldbe NH4 and the negative one may be a product of NH4 assimilation(Morgan and Jackson, 1988a). Ammonium concentrations were related tothe stimulation in influx whereas a product of ammonium assimilation wassubsequently responsible for its reduction/inhibition (Wiame et al., 1985;Cook and Anthony, 1978a, 1978b).The initial increase of NH4 influx may be resulted from provision ofN to synthesize more transporters that are sacrificed when plants areunder N stress. In this sense, NH4 would exert an effect as a source of Nfor transporters and also as a transport regulator. It was observed in aseparate study (Fig. 43 in Chapter 7) that rice plants grown in low N andlow K doubled their 86Rb influx after preloading in 1 mM NH4 for 2 h. Insuch a situation, it may be hypothesized that immediately after exposureto NH4, more transporters are synthesized. This may not necessarilyinvolve the synthesis of a different carrier for K system. Re-supplyingNH4-- provides the ‘building blocks’ to assemble more transporters topromote uptake and meet plant demand for N and K. Subsequently,negative feedback mechanisms begin to exert their regulation.1856.4.5. Negative feedback on 13NH4 influx from internal NH4As discussed above, the theory of amides or amino acids as N uptakeregulators can not explain all the observed results on the regulation of13NH4influx. The data seem to indicate that internal NH4 may able play arole in regulating NH4 influx. It has been reported that ammoniumtransport is repressed by intracellular ammonium per se but not by itsassimilates or de novo protein synthesis (Rai et al., 1986; Franco et al.,1987, 1988). The active, specific transport of1NH4 and‘4C-MA in bothwild type and mutant cells of Aspergillus nidulans is regulated by theconcentrations of internal ammonium (Pateman et al., 1973, 1974).One of the major reported reasons for excluding NH4 as a negativefeedback factor was that there was not an exact parallel between rootammonium concentrations and net NH4 influx (Lee and Rudge, 1986) oreffiux (Morgan and Jackson, 1988a). Therefore endogenous NH4-- in rootsappeared to exert no effect on uptake of either NH4 (Lee and Rudge, 1986;Morgan and Jackson, 1988) or N03 (Rufty et al., 1982a; Chaillou et al.,1991; Vessey et al., 1990a). Despite this claim by the above workers, therewere negative correlations between NH4 absorption and tissueconcentration. It was reported that when plants were depleted of nitratefor a week, net NH4 uptake was increased 5 to 10-fold (Morgan andJackson, 1988a) “because of low internal NH4 (1.—2 iimol g’)” (Morgan andJackson, 1988a, 1988b, 1989). But this appears to agree that [NH4-1-] iscorrelated (negatively) with NH4 uptake. While there is a positivecorrelation between the N provision during growth and the internalcontent of NH4 in root tissue, the Vmax of‘3NH4 influx was negativelycorrelated with these two conditions (Wang et al., 1993a). Nitrogendepletion rapidly altered the N-status of the plants, particular the tissue186concentration (Vose and Bresse, 1964; Lee and Rudge, 1986). In thepresent study, within 4 to 12 hours of depletion of G1000 roots in G2medium, ‘3NH4 influxes increased and were closely correlated withdecreases of internal NH4 content (Figs. 20B, 21B, 23A, and 23B).Lee and Ratcliffe (1991) argued that at steady-state, cytoplasmicammonium concentration would be not in the millimolar range because theactivity of GS was considerable higher than the uptake rate of NH4-’- (4jimol g’FW h’). Glutamine synthetase from higher plants has a highaffinity for ammonium (Km 20 iiM) (Steward et al., 1980; Milflin and Lea,1976). It would seem that if NH4 is not accumulated to a certain level inthe cytosol it would not be necessary to invoke a possible regulatory rolefor this N form. However, most reported estimates of cytoplasmic NH4-’-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 thepresent study, the indirect estimation of cytoplasmic NH4 concentrationswould give 0.8 mM as the lowest value (Fig. 20A). However lowconcentrations in the cytoplasm may be due to its rapid movement into thevacuole. It was calculated that half of the total free NH4 was in a ‘storagepool’ in the roots (Fentem et al., 1983a). In rice roots, it was estimated, thatabove 70% of NH4 was stored in the root vacuoles (Wang et al., 1993a).The proportional distribution of newly absorbed NH4 to N assimilation andto storage may depend on the balance between the gradient across thetonoplast and, the capacity of the GS/GOGAT system, which is probablyinfluenced by whole plant N status. Since high external NH4 repressed theactivity of GS reversibly (Rhodes et al., 1976; Arima and Kumazawa, 1977)and NR (Siddiqi et al., 1993; King et al., 1993), NH4 should have a role inregulating the NH4 transport across plasma membrane but not the overall187N assimilation, which would include transport across the plasmamembrane, metabolism, translocation and utilization (as discussed insection 6.4.6.).Second, the rapid dispersion of NH4 may be the reason it is sodifficult to reveal the contribution of NH4 to the regulation process. At lowexternal [NH4], NH4 entering across the plasma membrane is rapidlymetabolized by GS/GOGAT at a rate that is potentially faster than influx(Lee and Ratciiffe, 1991), or is transferred to the vacuole for storage. Theremay be only limited opportunity for NH4 per se to exert any directregulation on NH4 influx (transport step) under these conditions. Underconditions of elevated NH4 supply, when the GS/GOGAT system andvacuole are relatively saturated, internal NH4 may increase to a levelwhich enables it to exert a negative feedback on the transport step. Undersuch condition, there may be a good correlation between [NH4]1 andaccumulated primary products such as Gin. Ideally, the treatment withMSX blocks the assimilation of NH4 into Gin therefore leading to increased[NH4]and decreased [amino acids] in roots. As a result one might expectthe influx to be increased. This was observed in the present study.Pretreatment of G1000 plants with MSX resulted in a decrease of all majorprimary products of NH4 assimilation (open symbols in Figs. 29AD).However these changes did not result in the enhanced‘3NH4-’- influxes aswould be expected. Internal [NH4] remained at essentially the same levelthough there was a trend to reduce [NH4]1 in G2+MSX after 24 hpretreatment (open circles in Fig. 27A). This may be the reason‘3NH4influxes increased during the first hour and remained at the same levelthereafter (closed symbols in Fig. 26).188A comparison of the G2 plants treated with MSX in 2 mM or 1000mM solutions (Fig. 26) revealed a significantly higher influx in the G2 planttreated in G2+MSX than in the G2 plant treated in G1000+MSX at 4 and 12h. Yet the amino concentrations in the G2 plant treated in G1000+MSXshowed no significant change during this period (Fig. 29). However [NH4--]1appeared to be higher in the G2 plant treated in G1000+MSX (Figs. 27A and27B), consistent with an inhibitory effect of [NH4--]1 on 13NH4 influxwhenever [NH4÷] is elevated; either by growth in high N condition or as aresult of MSX treatment.6.4.6. Cascade regulation system of nitrogen uptakeThe process of NH4 uptake may be sensitive to regulation fromseveral signals, related to N status of the plant. These may include internalN pools (NH4,NO3-, AA), the GS/GOGAT system, translocation (andrecycling) and utilization. Clearly all these processes interact strongly. Toimagine that only single cytosolic substrate (e.g. glutamine) might regulatethe critical uptake step, may be naive. Therefore, there may be a cascadesystem with many levels of negative feedback regulation on NH4-’- uptake.In addition to N signals, nitrogen (NH4+) uptake may be limited by thesupply of carbohydrate from shoots (Kleiner, 1985). This could beconsidered as an important component of the regulation at the whole plantlevel. The ambient conditions such as light intensity and temperature willeffect the production of carbohydrates. It was found, for example, that netNH4 uptake rates oscillate between maximum and minimum with aperiodicity co-ordinated with intervals of leaf emergence (Tolley andRaper, 1985; Tolley-Henry et al., 1988; Henry and Raper, 1989a; Rideout etal., 1994). At the time of emergence and early expansion of a new leaves189there is a requirement for large amount of nitrogen (Radin and Boyer,1982; Steer et al., 1984), and carbohydrate (Turgeon, 1989). Therefore newleaves become the sink of photosynthate (Turgeon, 1989) and the flux ofcarbohydrate to roots is reduced. Nitrogen uptake depends on andcompetes (with other growth process) for soluble carbohydrate from theshoot (Raper et al., 1978; Lim et al., 1990; Henry and Raper, 1991), sincecarbohydrates provide metabolic energy for nitrogen uptake andtranslocation (Minotti and Jackson, 1970; Penning de Vries et al., 1974;Jackson et al., 1976). Translocation of carbohydrate from shoot to roots isresponsive to concentration of carbohydrate in the shoot pool (Wann et al.,1978; Granato and Raper, 1989; Lim et al., 1990). Since NH4 is assimilatedrapidly and almost exclusively in roots as it is absorbed (Given, 1979;Chaillou et al., 1991), this source of carbon skeletons is equally importantfor NH4 uptake and assimilation. It appears that regulation of both NH4-’-and N03 uptake at the whole-plant level is subject to commonmechanisms that influence diverse processes within the root and aredifferentially affected by nitrogen stress (Rideout et al., 1994).The next level of this cascade may be nitrogen assimilation and themajor regulators responsible for controlling NH4 uptake would be activeinside root cells. These might include amides and some major amino acids(Pelley and Bannister, 1979; MacFarlane and Smith, 1982; Wright andSyrett, 1983; Ulirich, 1984; Kleiner, 1985; Thomas and Harrison, 1985;Wiame et al., 1985; Lee and Rudge, 1986; Morgan and Jackson, 1988b). Asthe primary product of NH4-’- assimilation, glutamine is the primarycandidate for negative effector (Cook and Anthony, 1978a, 1978b; Duboisand Grenson, 1979; Wiame et al., 1985). Within the N cycling of plants, thesimultaneous movement of N-compounds from root to shoot, and fromshoot to root (Cooper and Clarkson, 1989; Larsson et al., 1991) may enable190N absorption to be regulated to match the demand imposed by plantgrowth (Drew and Saker, 1975; Edwards and Barber, 1976). Theconcentrations of amides (Gin and Asn) in the roots will be the result of thebalance between their synthesis from absorbed inorganic N (NH4+ or NO3-),their import via the phloem, and their export via the xylem (Lee et ai.,1992).Internal NH4 has not been considered as a negative feedbackeffector for NH4 uptake (Lee and Rudge, 1986; Morgan and Jackson,1988a, 1988b; Raper et al., 1992), because it is claimed that there is nocorrelation between cumulative uptake of NH4 and endogenous NH4-- inroots (Chaillou et ai., 1991; Vessey et ai., 1990a). One may consider NH4÷ tobe at the center of a vital process of uptake and metabolism. Unlike K-i-,NH4-’- will be rapidly consumed into amino acids within the root. Therefore,tissue [NH4-’-] is not an ideal indicator of N status. A second reason is that, itwas observed by Morgan and Jackson (1988b) that during the first twodays of N-deprivation, root NH4 concentration and NH4 uptake wereclosely correlated. After 5 d of N-deprivation, the root NH4 concentrationswere found increased slightly and the rate of NH4÷ uptake was continuedto increase. Based on present studies, NH4 would be expected to be thenegative effector when internal NH4 levels increase beyond a certainlevel. Below this level one may assume that any free NH4 would beimmediately drawn into the metabolic process to meet the high demandfor plant growth. There may be a critical nitrogen status below which thesystem is impaired and above which it is subject to repression and/orinhibition (Breiman and Barash, 1980).It is proposed, therefore, that internal NH4 represents a third levelof control, operating whenever internal [NH4+] is elevated. The site(s) for191its putative effects may include the transport step at the plasmamembrane, or the transcriptional level involving the genes coding for NH4-’-transport.In view of the different effects of internal NH4 on NH4 influx ofN-repleted G2 plants and on N-depleted G1000 plants, it is possible thatnegative feedback regulation of NH4 uptake may be facilitated by eitherNH4 or its assimilates. In low N-grown roots the up-regulation of influxmay be exerted through products of NH4÷ assimilation, while in highN-grown roots, internal NH4 may participate in the down-regulation ofNH4 uptake systems.In the case of the up-regulation of 13NH4 influx following transfer ofG1000 plants to G2 medium (Fig. 20A), the [NH4]1dropped during the firsttwo hours of depletion (Fig. 20B) and then decreased gradually to a valuesimilar to that of G2 plants at steady-state. I consider that cytoplasmic[NH4j may be the controlling effector here. This is based upon thefollowing additional observations: first, similar negative correlations werefound in the 24 h depletion experiment (Figs. 23A, 23B), however‘3NH4influxes were negatively correlated with the [NH4+] (Figs. 23A, 23B) butnot the content of amides or amino acids (Figs. 24, 25A-D); Second, whenthe GS-GOGAT pathway was blocked by MSX, ‘3NH4 influx remained atlow rate (Fig. 26) due to higher [NH4](Figs. 27A and 27B) despite a largedecrease of four major amides and amino acids (Figs. 29A-H). Third, 13NH4influxes were different when G2 plants were pretreated with MSX for thesame 24 h (Fig. 26), but transferred to either G2 or G1000 medium, whichresulted in higher {NH4+]1 for plants in G1000+MSX than in G2+MSXmedium (Fig. 27). Since estimated half-life for cytoplasmic NH4 exchangeis <10 mm (Wang et al., 1993a), it would be expected that this component192of internal [NH4]would respond more dynamically to change of external[NH4-’-] than the vacuolar [NH4-’-].In contrast, the observed declines of1NH4 influxes were related tohigh [NH4+] and major amino acids (Figs. 23B, 24, and 25A-D). Iinterpreted this result to indicate that the decline of ‘3NH4÷ influxnormally observed when G2 plants are loaded in G1000 medium, dependsupon products of NH4 assimilation. This conclusion was supported by theresults of glutamine pretreatment (Fig. 30), which reduced 13NH4influx atall concentrations tested. Further proof to this effect is provided by ouramino acid analyses. Figure 25A and 25C show that transfer from G2 toG1000 medium caused [Gln]1 and [Asn]1 to increase several times while inthe presence of MSX this increase was prevented (Figs. 29A and 29C). Inaddition the 1NH4 influx was strongly correlated (negatively) withincreased Gln, Glu, Asn, and Asp after treatment with AOA (Figs. 37A and39A-D).193Chapter 7. INTERACTION BETWEEN K+ AND NH4÷7.1. INTRODUCTIONPotassium uptake has been well studied in higher plants (Glass,1975; 1976, 1978; Glass et al., 1981; Kochian and Lucas, 1982, 1988; Glassand Fernando, 1992). Likewise, the kinetics of ammonium transport havealso been characterized (Becking, 1956; Fried et al., 1965; Ullrich et al.,1984; Wang et al., 1993a, 1993b). Despite the similarities between K andNH4, such as charge, hydrated ion diameter and some aspects of transportprocesses (Haynes and Goh, 1978), the interaction of these two cations ispoorly understood.The interaction between K and NH4 may be examined at differentlevels, such as the bioavailability in soils, effects on plant growth, andeffect on plant roots’ uptake/transport of these ions. Mutual beneficialeffects of K and N on plant growth have often been described. An adequateK supply has been shown to enhance NH4 uptake and assimilation (Ajayiet al., 1970; Barker and Lachman, 1986; Scherer and MacKown, 1987) andis very important for nitrogen use efficiency. On the other hand, NH4 maypromote K-I- stress in rice (Noguchi and Sugamara, 1966) or reduce the Kconcentration of plants (Claassen and Wilcox, 1974; Faizy, 1979; Lamond,1979).A number of studies have been carried out to investigate theinteractions of K and NH4 at the transport level. In short-termexperiments, the uptake of K was significantly reduced by the presence ofNH4 in the uptake solution (Deane-Drummond and Glass, 1983b; Rosen194and Carison, 1984; Morgan and Jackson, 1988). However the influence of K-’-on NH4-’- uptake has not been consistent. In most cases, the uptake of NH4-’-by plant roots has appeared to be independent of K-’- levels in the uptakesolution and the K status of the plants (Rufty et al., 1982; Rosen andCarison, 1984; Scherer and MacKown, 1987). Nevertheless, Bange et al.,(1965) reported that K is capable of inhibiting NH4 uptake in barleyplants.The objective of this study was to investigate the interactionsbetween K-’- and NH4 at the membrane transport step, and the influencesof tissue K and N status on these ion fluxes, using 86Rb and‘3NH4,respectively, as tracers.7.2. METHoDs AND MATERIALS7.2.1. Plant growth and ‘3N productionSection 2.2. Seed germination; section 2.3. Growth conditions; section2.4. Provision of nutrients; section 2.5. Production of 13NH4.7.2.2. Experimental designThree experimental variables were employed in this study involvingN and K supply. These were (i) provision during three-week-growthperiods or less as designated; (ii) pretreatment for up to three days priorto flux measurement; and (iii) presence in the uptake solutions. Testmaterials were 3-week-old rice seedlings. Each experiment was repeated195twice with three replicates. Both influxes of 13NH4 and 86Rb werecalculated based on root fresh weight and 10 mm uptake periods, except inexperiment I, where the net fluxes of NH4 and 86Rb were calculated from30 mm uptake periods. Before and after transfer into or out of theradioactive isotopic labeled uptake solution, plant roots were prewashedand postwashed in an identical unlabeled solution for 5 and 3 mm,respectively. These time periods were based on a previous study (Wang etal., 1993a, 1993b).7.2.1.1. Experiment I: Effects of K and NO3- in pretreatment, K and NH4in uptake solutions on net K and NH4 fluxes.Plants were grown in MJNS containing 200 iM K plus 1.5 mM N03for 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 1iM KH2PO4,-K 100 iiM Ca(H2P04);+N = 0.75mM Ca(N03)2;and -N = 0.75 mM CaC12.The 86Rb influxes were measuredfrom radioisotope-labeled MJNS (+K*+N, +K*N) containing 200 jiM K-’- withor without 200 jiM NH4. Net NH4 fluxes were measured from MJNScontaining 200 j.tM NH4with or without 200 jiM K (+K+N, -K+N).7.2.1.2. Experiment H: Effects of NH4 provision during growth and of Kand NH4 in pretreatment and uptake solutions on 86Rb÷ (K+) influxes.Plants were grown in MJNS containing 200 jiM K plus 10, 50 or 100j.tM NH4,hereafter referred as Gb, G50, or G100 plants, respectively. Theplants were transplanted for three days to MJNS with or without additionsof K and N, in which +K = 200 jiM KH2PO4;-K = 100 jiM Ca(H2P04);and +N =10, 50 or 100 jiM NH4C1; -N = 5, 25 or 50 jiM CaC12 for G2, Gb, or G100plants, respectively. The 86Rb-’- influxes were measured from radioactive196isotopic labeled uptake solutions (MJNS containing 200 iiM K with orwithout 100 iM NH4+).7.2.1.3. Experiment III: Effects of NH4 provision during growth andpresence in uptake solution upon influx isotherms for 86Rb÷ (K÷).Plants were grown in four different growth media containing 2 or100 pM NH4 plus either 2 or 200 .tM K, hereafter referred as G2/2,G2/200, G100/2, G100/200 plants, respectively. The 86Rb influxes weremeasured in MJNS containing 2, 10, 50, 75, 100, 250 or 500 iM K,respectively, plus 2 .tM NH4 for G2/2 and G2/200 plants, or 1.00 jiM NH4-’-for G100/2 and G100/200 plants.7.2.1.4. Experiment 1½ Effects ofNH4provision during growth and short-term pretreatment upon 86Rb÷ (K÷) influx.Plants were pretreated for 0, 2, 4, 8, 24 h in 1 mM NH4 plus 2 jiM Kfor G2/2 and G100/2 plants, or in 1 mM NH4 plus 200 jiM K-’- for G2/200and G100/200 plants. 86Rb+ influxes were measured during 10 mm inuptake solution containing 100 jiM NH4 and 200 jiM K.7.2.1.5. Experiment V: Effect of NH4 concentrations present in uptakesolution upon influx isotherms for 86Rb+ (K).The 86Rb influxes of G2/2, G2/200, G100/2, G100/200 plants weremeasured in MJNS containing 2, 25, 50, 100, or 200 jiM K, plus 2, 25, 50,or 100 jiM NH4. The translocations of 86Rb into plant shoots were alsoestimated based on the radioactivity recorded from plant shoots.1977.2.1.6. Experiment VI: Effects ofK provision during growth and presencein uptake solutions upon influx isotherms for1NH4.The 13NH4influxes of G2/2, G2/200, G100/2, G100/200 plants weremeasured in uptake solutions (a) containing 2, 10, 50, 100, or 200 jiM NH4plus either 0, or 200 jiM K; (b) containing 100 jiM or 10 mM NH4 plus 2,20, 200, or 2000 jiM K.7.3. RESuLTS7.3.1. Experiment I: Effects of K-’- and NO3- in pretreatment, K-- and NH4-’-in uptake solutions on net K and NH4 fluxes.Pretreatment with NH4 during three days prior to the uptakemeasurement generally increased 86Rb (K+) uptake. Only in -K+N, -K-Ntreatments was there no increase of 86Rb uptake; all other treatmentsincreased influx by 1.35 times (-K+N, -K-N) and 3.4 times (+K+N, +K-N)when NH4-’- was absent from the uptake solution and by 1.85 times (+K+N,+K-N) when NH4-’- was present (Table 13). Yet, the means for +K+N and -K+Nwere not significantly different at the 5% level of probability when NH4was present in the uptake solution. When NH4 was absent, the means(3.29/0.96 for +K+N/+K-N and 8.72/6.44 for -K+N/-K-N) were statisticallydifferent at the 1% level.The removal of K during pretreatment caused a much greater effecton 86Rb accumulation, increasing 86Rb÷ (K+) uptake by 2.65 and 6.7 timeswhen it was absent from uptake solution, and by 5.25 and 10.2 times198Table 13. Net 86Rb+ flux measured with or without ammonium. Riceplants were grown in MJNS containing 1.5 mM NO3- and pretreated 3 daysin 4 different solutions with or without either 200 ji,M K (+K or -K) or 1.5mM NO3- (+N or -N). The net flux of 86Rb was measured in the followinguptake solutions: +K+N or +K-N (N = 200 jiM NH4 and K 200 p.M K÷)labeled with S6RbC1. Fluxes were calculated based on 30 mm uptakeperiods.Uptake solutionPretreatment +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 RangeTest were performed, separately, on the data of net s6Rb+ flux. Means having acommon letter are not significantly different at the 5% significance level for smallletter.199Table 14. Net NH4 flux measured with or without potassium. Rice plantswere grown in MJNS containing 1.5 mM NO3- and pretreated 3 days in 4different solutions with or without either 200 jiM K (+K or -K) or 1.5 mMNO3- (+N or -N). The net NH4 flux was measured in the uptake solutions(+K+N, N = 200 jiM NH4 and K = 200 jiM K; or -K+N) for 30 mm uptake.Uptake solutionPretreatment +K+N -K+N(jimolg4FW h-i)+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 RangeTest were performed, separately, on the data of net NH4 flux. Means having acommon letter are not significantly different at the 5% significance level for smallletters.200when NH4 was present (Table 13). In these -K plants the presence orabsence of NH4 during pretreatment caused only a much smaller effect(compare fluxes for -K+N and -K-N pretreatments). Clearly, the presence ofNH4 in the uptake solution caused a large reduction of 86Rb+ (K-’-) uptake,regardless of the pretreatments.The net NH4 fluxes were reduced by 3 days of pretreatment withNH4 in all treatments (Table 14). Removing K from pretreatmentsolutions caused small increases in NH4 uptake as they had for 86Rb+ (K+)uptake, but these differences were not significant at the 5% level ofprobability. The presence of K in the uptake solutions caused statisticallynon-significant reductions in NH4 uptake in all pretreatments except -K-Nwhen NH4 uptake actually decreased. Here again, however, the differencewas not statistically significant.7.3.2. Experiment II. Effects of NH4 provision during growth, and of Kand NH4 in pretreatment and uptake solutions on 86Rb (K--) influxes.The effects of three factors (N provision during the growth period,3 d of K and NH4 pretreatment, and the presence or absence of NH4-’- inthe uptake solution) on 86Rb (K) uptake were examined in Exp II. 86Rbinflux was increased in virtually all treatments by increased levels of NH4provision during the growth period (see Figs. 40A and 40B). In thoseexperiments where NH4 was present during influx measurement thenoted positive effect of NH4 pretreatment was reduced or absent at thehighest level of NH4-’- (100 riM) but was still pronounced between 10 and50 iM NH4. As in Experiment I, provision of NH4 during the 3 dpretreatment caused the greatest increase of 86Rb (K) influx in low K20110 5.A. uptake without N] B. uptake with N1—A--— -K+N A—h-— -K-N::0 50100 0 50100Ammonium levels for plant growth (jiM)Figure 40. Effects of NH4 in the growth media, pretreatment and uptakesolutions on 86Rb influx. Gb, G50, and G100 plants were pretreated for 3days in four solutions including +K+N (closed squares), +K-N (open squares),-K+N (closed triangles), -K-N (open triangles). The 86Rb+ influxes weremeasured in MJNS containing 200 j.tM K without NH4 (Fig. 40A) or with100 iM NH4 (Fig. 40B). Data points are the average of three replicateswith ±se as vertical bars.20212 -10- G10012— 8-—E I:i‘V—’ 6-G21242’G100/200A0- G212000 100 200 300 400 500[K Jo (riM)Figure 41. Effects of NH4 and K in growth media and uptake solutions on86Rb÷ influx. The 86Rb influxes of G2/2, G2/200, G100/2, G100/200 plantswere measured for 10 minutes in 86Rb labeled MJNS, containing 2, 10, 50,75, 100, 250 or 500 iiM K--, respectively, plus 2 j.tM NH4 for G2/2 (opencircle) and G2/200 (open triangle), or plus 100 iM N}l4 for G100/2 (closedcircle) and G100/200 (closed triangle). Data points are the average of threereplicates with ±se as vertical bars.203plants when NH4 was absent from the uptake solutions (Fig. 40A) and theleast effect in high K plants in the presence of NH4-’- during uptake (Fig.40B). However, as in Experiment I, the presence of NH4 during fluxmeasurements, reduced 86Rb (K+) influx in all treatments. Again,removing K from the pretreatment solution caused increased 86Rb (K)influx, and this effect was more pronounced when adequate N wasprovided (compare squares and triangles to note the K’- effect, and closedand open triangles to note the N effect).7.3.3. Experiment III: Effects of NH4 provision during growth, andpresence in uptake solution upon influx isotherms for86Rb+ (K-’-).Figure 41 presents the 86Rb influx isotherms for plants grown underG2/2, G2/200, G100/2 and G100/200 conditions. The data were fitted toMichaelis-Menten equations. The kinetics of 86Rb uptake were influencedby the provision of both NH4 and K during three weeks growth. The 86Rbinflux curves for G2/200 and G100/200 plants (grown in higher externalK+) revealed a low Vmax, 0.34 and 0.59 j.imol g’FW h-’, respectively. Bycontrast, plants grown in low K (2 riM), exhibited much higher Vmax value(3.74 and 9.58 jimol g4FW h-i for G2/2 and G100/2 plants, respectively).As in the previous experiments the provision of NH4 during the growthprior to influx measurements caused a significant positive effect; Vmax for86Rb (K-’-) influx was increased 3 fold. The estimated values of Km werealso higher for plants grown in higher K conditions (15.02 jiM for G2/200and 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 betweenestimated kinetic parameters and measured tissue K concentrations clearlyindicated the operation of negative feedback inhibition of 86Rb influx.20412108E 64.E20• I • I • I0 20 40 60Internal [K + 1 (mM)Figure 42. Relationship between estimated kinetic parameter of 86Rbinflux (Vmax) and the assayed roots internal [K+J. The vertical bars arestandard errors for Vmax and the horizontal bars are standard errors for[K].2058’6’4,2’—E—“C— G10012—A--— G100/200G212zr G21200-0-0’ I • I • I • I • I • I0 5 10 15 20 25 30Growth condition (NH /K )Figure 43. Effect of short-term NH4 pretreatment on 86Rb influx. Plantswere pretreated in 1 mM NH4 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. 86Rb influxes were measured for 10 minutes in 86Rb labeledMJNS containing 200 jiM K and 100 jiM NH4.Data points are the averageof three replicates with ± se as vertical bars.206Figure 42 showed a strong negative correlation between Vmax values andinternal [K] values.7.3.4. Experiment IV: Effects of NH4 provision during growth, and short-term pretreatment upon 86Rb (K÷) influx.When the N status of plants was changed by short-term exposures toNH4-’-, 86Rb+ influxes were also altered, as shown previously for 3 daysexposures to NH4 (Table 13, Figs. 40 and 41). For plants grown in higher N(G100/2 or G100/200) the 86Rb influxes were affected little by loading in1 mM NH4 for various periods (Fig. 43). For plants grown in low N, theresults of pretreatment in 1 mM NH4 varied according to the differencesin the K status. The 86Rb influxes of G2/2 plants were greatly increasedduring the first 4 h pretreatment in 1 mM NH4. In contrast, 86Rb influxesof G2/200 declined slightly after the first 4 h.7.3.5. Experiment V: Effect of NH4 concentrations present in uptakesolution upon influx isotherms for 86Rb (Kj.To further understand the inhibitory effect of NH4 in uptakesolutions, 86Rb influxes were measured at five [K+]0 levels in the presenceof four levels of [NH4-’-]0.Generally, 86Rb influxes for G100/2 were higherthan for G2/2 and G100/200. G2/200 plants had the lowest rates ofpotassium uptake. Generally, S6Rb+ influx decreased with increasing[NH4-’-]0in the uptake solutions, but the effect on G100/2 is not so evident(Fig. 44). Even at 2 p.M [K-’-]0, the inhibitory effect of NH4 was evident.Table 15 presents estimated Michaelis-Menten parameters for all 86Rb207G21200 —------16.0012.00YEoo5oioo xG2/2z16.0012.008.00 Y4.00 001 000.00 50/ ;‘ 25225 250100 XG100/20016.0012.008.004.000.0025100 XG100I2z16.0012.008.00 y4.001000.00 J./50‘25225 250 XFigure 44. Effects of NH4 and K in growth media and uptake solutions on86Rb influx. The 86Rb influx of G2/2, G2/200, G100/2, or G100/200plants, respectively, were measured in MJNS containing 2, 10, 50, 100, or200 jiM K-’- plus 2, 25, 50, or 100 jiM NH4,respectively. In plots: X = [NH4](jiM), Y= [K] (jiM), Z = 86Rb influx (j.tmol K-’- g’FW root h-i), respectively.208Table 15. Michaelis-Menten kinetic parameters for 86Rb+ influx for fourgroups of plants (G2/2, G2/200, G100/2 or G100/200). Based on the dataof Fig. 44, the parameters were estimated by nonlinear procedure onreplicated influx data (n=2).[NH4i0 Vmax ± se Km ± se r2(riM) (pmol g’FW h-i) (iiM)G2/2 plants2 11.07± 1.04 11.82± 5.90 0.8225 7.83 ± 0.78 13.23 ± 7.59 0.7950 8.27 ± 0.80 23.36 ± 8.73 0.86100 5.83 ± 0.81 18.39 ± 11.34 0.86G100/2 plants2 14.97 ± 1.81 8.63 ± 9.05 0.8925 14.02 ± 1.77 10.99 ± 7..47 0.7850 15.79 ± 1.80 49.53 ± 16.08 0.93100 12.34± 0.89 11.58± 4.66 0.82G2/200 plants2 1.02 ± 0.13 3.09 ± 1.57 0.1725 0.41 ± 0.06 105.93 ± 31.03 0.9650 0.41 ± 0.04 136.18 ± 21.07 0.90100 0.55 ± 0.12 186.75 ± 70.79 0.98G100/200 plants2 7.36 ± 0.55 17.60 ± 5.61 0.8025 4.06 ± 0.54 36.25 ± 15.26 0.8150 2.18 ± 0.32 17.68 ± 10.73 0.81100 2.31± 0.58 63.88± 41.43 0.95G2/200z16.0012.0044 Y8.004.00 00“ 00500.00252 2550100 XG212Jz16.0012.008.004.00i/50250.00225 2100G1 00/20Q]16.00\Y004.00-r5500.00x100Gi00/2100209Figure 45. Effects of NH4 and K in growth media and uptake solutions on86Rb÷ translocated to shoots. (Details as in Fig. 44) In plots: X = [NH4+] (.tM),Y= [K] (jiM), Z = S6Rb+ translocated (nmol K g’FW shoot h’), respectively.210influxes isotherms. Generally, Vmax values decreased with increasing[NH4+]0in the uptake solutions. In contrast, Km values tended to increasewith increasing {NH4+]0in the uptake solutions for G2/200 and G100/200plants (Table 15). However, Km values remained relatively constant forG2/2 and G100/2 plants. Similar inhibitory effects were true for thetranslocation of K(86Rb) to shoots (Fig. 45). It was evident that higherrates 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 makecomparisons of the effects of growth conditions. There were statisticallysignificant differences among total fresh and dry weights of plants(G100/200 > G2/200 > G100/2 > G2/2) although the ratios of dry:freshweight were relatively constant (Table 16). Both fresh or dry shoot weightsof plants grown in well-supplied media (G100/200) were significantlyhigher than for other types of plant. With inadequate supply of either Kor NH4-’-, plants (G2/200 or G100/2) plants had smaller biomass but thesewere still significantly higher than that of G2/2 plants. However, thedifferences of root weight indicated that K played a more important role inroot growth than did NH4 (compare G100/200 to G2/200). When K wasadequately supplied, plant roots grew better. Under K-’- stress, NH4-’- seemedto have little effect on root biomass.7.3.6. Experiment IV: Effects of K-’- provision during growth and presencein uptake solutions upon influx isotherms for 13NH4.The effects of K in the uptake solutions on the‘3NH4 influx wereexamined using G2/2, G2/200, G100/2, and G100/200 plants (Fig. 46). The211Table 16. Effects of NH4 and K on plant growth. Rice plants were grown ineither 2 1iM or 100 jiM NH4 plus either 2 or 200 jiM K (G2/2, G2/200,G100/2 or G100/200, respectively). Each value is the average of 40 samplemeans (mg per plant) with ± Se.Plants G2/2 G100/2 G2/200 G100/200Total FW (mg) 179 ± 5 d 225 ± 6 c 293 ± 14 b 368 ± 3 aTotalDW(mg) 21± 1 d 30± 1 c 38± 2 b 49± 2 aTotal D/F 0.13 ± 0.00 b 0.13 ± 0.OOAb 0.14 ± 0.01 b 0.14 ± 0.01 aStFW(mg) 114± 4c 163± 5 b 168± 8 b 258± 10 aStDW(mg) 18± ic 26± 7 b 27± 1 b 40± 2 aSt D/F 0.16 ± 0.00 a 0.16 ± 0.00 a 0.17 ± 0.01 a 0.14 ± 0.01 aRtFW(mg) 56± 2c 62± 1 c 125± 6 a 110± 4 bRtDW(mg) 4± Oc 4± 0 c 11± 1 a 9± 1 bRt D/F 0.06 ± 0.00 b 0.06 ± 0.00 b 0.09 ± 0.01 a 0.09 ± 0.01 aFW St/Rt 2.03 ± 0.00 c 2.61 ± 0.00 a 1.36 ± 0.00 D 2.38 ± 0.02 bDW St/Rt 5.43 ± 0.27 b 7.20 ± 0.23 a 2.88 ± 0.24 C 4.92 ± 0.38 b* For comparing all possible pairs of treatment means, Duncan’s Multiple Range Testwere performed, separately, on the data of net 86Rb flux. Means having a commonletter are not significantly different at the 5% significance level for small letter.21216 8G21200T G100120012 6E• • •__o 50 100 150 200 0 50 100 150 20016 8G212 G10012 I-12 Q 6‘1EEE “iZ0 50 100 150 200 0 50 100 150 200[NH Jo (riM)Figure 46. Effects of K in uptake solution on‘3NH4influx isotherm. Root13NH4 influx of G2/2, G2/200, G100/2, or G100/200 plants, respectively,were measured in MJNS containing 100 1iM NH4 in the presence of either2 (open circle) or 200 iM K-’- (closed circle). Predicted isotherms (dashedlines for 0 jiM K-’- and solid lines for 200 jiM K) were calculated from thecomputed Vm and Km for different plants (Table 17).213Table 17. Michaelis-Menten kinetic parameters for 1NH4 influx for fourplants (G2/2, G2/200, G100/2 or G100/200) derived from influx isothermsbased on 2, 25, 50, 100, or 200 jiM NH4with or without 200 jiM K-i- (+K or-K). The parameters were estimated by nonlinear procedures on replicatedinflux data.Plants 13NH4solution Vm ± se Km ± seG2/2 +K 10.07 ± 0.94 17.40 ± 3.13G2/2 -K 10.69 ± 0.91 13.62 ± 2.80G2/200 +K 13.99± 2.77 16.36± 1.09G2/200 -K 13.95 ± 2.93 16.22 ± 0.31G100/2 +K 4.60± 1.09 359.36±121.31G100/2 -K 13.07± 2.29 209.11± 57.13G100/200 +K 3.94± 0.61 37.89± 20.17G100/200 -K 4.62 ± 0.80 30.56 ± 11.93214129.—EzL63.z0—I——Figure 47. Effects of K in uptake solution on 13NH4 influx by HATS. Root‘3NH4 influxes of G2/2, G2/200, G100/2, or G100/200 plants,respectively, were measured in MJNS containing 100 iM NH4 in thepresence of 0, 20, 200, 2000 tM K. Data points are the average of threereplicates with ±se as vertical bars.• G2/200 G2/2 G1001200 G100121o 20 200 2000[K Jo (jiM)21540—zL20+• 10z0-0 200 2000[K Jo (pM)Figure 48. Effects of K in the uptake solution on 13NH4 influx byHATS+LATS. Root‘3NH4influxes of G2/2, G2/200, G100/2, or G100/200plants, respectively, were measured in MJNS containing 1000 jiM NH4 inthe presence of 0, 200, 2000 jiM K. Data points are the average of threereplicates with ±se as vertical bars.• G2/200 G2/2 • G1001200 G10012216presence of K in the‘3NH4uptake solutions failed to significantly reduce13NH4 influx except in the case of the G100/2 plants where significantdifferences were apparent. The estimated influx kinetics also showed thesame trends (Table 17). Nevertheless, there were slight reductions of‘3NH4 influx which failed to satisfy statistical evaluation in G2/2 andG100/200 plants. It was noted that plants grown at low N levels hadhigher1NH4 influxes when the K nutrition was adequate during growth(compare G2/200 and G2/2 plants).When K in the uptake solution was increased from 0, 2, 20, 200, and2000 jiM (Fig. 47), a strong inhibitory effect of K (in the uptake solutions)on 1NH4 influxes of G100/2 and G100/200 plants was evident. Bycontrast,‘3NH4 influxes were significantly increased by growth in low N(G2/2 and G2/200) with no effects of K÷ when present in the uptakesolutions. The‘3NH4influxes measured in 10 mM NH4were not changedsignificantly although the influxes were lower in the presence of 2000 jiMK (Fig. 48).7.4. DISCuSSION7.4.1. Plant growth in response to provisions of NH4 and K-Both N and K are very important to crop growth and yield. Uptake ofK and N, plant dry weight, and paddy yields of rice increased withincreasing K and N application rate (Biswas et al., 1987; Ichii and TsumuraH,1989; Fageria et al., 1990). Deficiency of either N or K in the nutrientsolution decreased the tissue content of either N or K, influenced217photosynthetic rate and translocation of carbohydrates, caused lower grainweight and therefore reduced rice yield (Grist, 1986; Dey and Rao, 1989).Reduction in photosynthetic rate may be due to impairment of stomataldiffusive conductance and decreased N content/unit leaf area (Dey andRao, 1989). High tissue K not only promoted CO2 assimilation, starchformation and the transport of the assimilates but also improved thenitrogen metabolism of the plant and nitrogen use efficiency (Kemmler,1983; Dibb and Thompson, 1985). K enhanced NH4 assimilation andreduced the toxic effects of NH4 such as stem lesions in tomato or leaflesion in corn (Ajay et al., 1970; Dibb and Welch, 1976). In a recent paperby Yong et al., (1993) the presence of K in Arabidopsis’ growth media wasresponsible for preventing toxic effects of NH4 on root growth. Supplyinghigh levels of K÷ to NH4-N grown plants stimulated shoot growth andmore vigorous root growth (Xu et al., 1992).In the present study the total fresh and dry weights weresignificantly higher in the sequence of G100/200, G2/200, G100/2 andG2/2 (Table 16). The significant difference between G2/200 and G100/200indicates the importance of K for plant growth when the N nutrition isadequate. Comparing both fresh and dry weights of roots among fourtreatments in Table 16, higher K in the growth media producedsignificantly higher root mass (G100/200 and G2/100) than growth in lowK (G2/2 and G100/2), whereas the shoot fresh and dry weights, were notsignificantly different between G2/200 and G100/2. A greater root mass ofseedlings grown in higher K indicated that K may play an important rolein facilitating root development (Beaton and Sekhon, 1985; Xu et al., 1992).There was a significant positive correlation between total root weight andK uptake (Table 16). Total root length and dry weight increased as cropgrowth advanced and N supply increased (Chamuah and Dey, 1988).218However, the root number was negatively correlated with NH4-N uptakein lowland rice (Ichii and Tsumura,1989).As shown in Table 16, the Shoot:Root ratios for both fresh and dryweights were higher for G100/2 than G200/200 or G2/2. G2/200 plantshad the lowest Shoot:Root ratio. It has been reported that N deficiencydecreased S/R ratios of seedling plants (Zsoldos et al., 1990). N stressreduces plant growth, particularly shoot growth, through severalmechanisms operating on different time scales. The possible signals maybe related to N stress-induced changes of abscisic acid and cytokinins(Goring and Mardanov, 1976; Sattelmacher and Marschner, 1978; Chapin etal., 1988a, 1988b; Kuiper et al., 1989). This lower ratio of shoot:root mayalso due to higher root mass in higher K condition as discussed above.7.4.2. Effect of plant N status on K (86Rb+) uptakeThe nitrogen status of plants had a significant influence on K(86Rb)uptake. Typically, S6Rb+ influxes of Gb, G50 and G100 plants wereincreased with increasing [NH4je levels in growth media (Figs. 40A and40B). The presence of NH4 during the pretreatment period also causedincreased 86Rb influx (Figs. 40A and 40B). 86Rb uptake by roots exposedto +K+N and -K+N pretreatments were significantly higher than that for +KN or -K-N pretreatments (Table 13). This positive effect of N status on Kuptake may be related to protein synthesis for K transport. The long termregulation of ion uptake probably involves induction or derepression ofcarrier synthesis. It is known that plants respond to K deprivation rapidlyby synthesizing novel polypeptides in the plasma membrane (Fernando etal., 1992) which are believed to form part of the high affinity K-’- transport219system (Glass and Fernando, 1992). When plants were grown in low K (21iM), with sufficient N supply (100 1iM NH4+), K(86Rb+) influx waspromoted (Figs. 41, 44). However, when the supply of nitrogen was limitedduring plant growth, the synthesis of K transporters in the cell membranemay be limited. In the present study, when G2/2 plants were pretreatedwith 1 mM NH4 for 4 hours, more transporters could be synthesized andthe 86Rb+ influx was significantly increased and remained relative highduring the 24 h pretreatment (Fig. 43). This raises an important questionconcerning the ‘induction’ of increased NH4 uptake observed when low Nplants are first exposure to NH4 (Goyal and Huffaker, 1986; Morgan andJackson, 1989; Wang et al., 1993b; in Chapter 6). The observation thatexposure to NH4 also increased K uptake on a similar time scale indicatesthat this NH4-’- effect is not specific as for example the induction of N03uptake by exposure. Rather, it appears that the so-called ‘induction’ maybe general positive N effect associated with N-depleted plants.Another possible explanation for the positive effect of NH4 may bedue to the effect of N supply on growth rate. The influx of ions into rootsmay be negatively correlated with the internal concentration of aparticular ion, such as Cl- (Cram, 1973); K (Young et al., 1970; Pitman andCram, 1973; Glass, 1975; Glass and Dunlop, 1978); NO3- (Siddiqi et al.,1992), and S042- (Smith, 1975). Figure 3 showed that the Vm for 86Rb+influx was negatively correlated with internal K levels in agreement withprevious reports (Glass, 1975; Clarkson, 1983; Pettersson, 1986; Zsoldos etal., 1990). Vmax decreased and Km increased exponentially with increasedtissue K concentration (Dunlop et al., 1979; Glass, 1976, 1977, 1978). Inthe present study, the 86Rb influx was increased in the sequence ofG100/2, G2/2, G100/200, and G2/200 (Figs. 41 and 44) and coincides withthe sequence of [K]1 of these roots. Higher 86Rb influxes also resulted220from three days pretreatment in minus K solution (Figs. 40A and 40B).Therefore high N supply, resulting in increased plant growth, would causethe opposite effect on tissue [K] and K(86Rb) influx, i.e. a biologicaldilution effect. This may explain why NH4 supplement to rice plantspromotes K stress (Noguchi and Sugawara, 1966), or reduced Kconcentration of plants (Claassen and Wilcox, 1974; Faizy, 1979; Lamond,1979).7.4.3. Effect of NH4 in the uptake solution on K-’-(86Rb) uptakeDespite the positive effect of NH4 provided during the growth periodand the pretreatment period, NH4 has been shown to strongly inhibit theabsorption of K in short-term experiments (Bange et al., 1965; Moraghanand Porter, 1975; Breteler, 1977; Munn and Jackson, 1978; Rosen andCarison, 1984; Scherer et al., 1984). In the present study, 86Rb influxeswere inhibited by the presence of NH4-’- in the uptake solution (Figs. 40A,40B and 44, Table 13 and 15). The inhibition of 86Rb influx increased withincreasing [NH4-’-] in the uptake solutions (Table 15). The uptake of K byexcised rice roots decreased markedly with increasing concentrations ofNH4 in the uptake solution (Scherer et al., 1987). Greater inhibition of Kuptake was exerted by 1000 jiM NH4 than 100 jiM NH4 (Rosen andCarison, 1984), and the inhibition by 1000 jiM NH4 occurred after 90 mmtreatment and the inhibition by 100 jiM NH4 took about 240 mm(Jongbloed et al., 1991).Since this inhibitory effect of NH4 on K(86Rb+) influx isindependent of K provision or pretreatments, it is probably exerted on thetransport processes at the plasma membrane. It is suggested that certain221solutes are bound to, or associated with, a particular transporter. When anion of a particular species is attached to this transporter, another similarion (of the same or a different species) may compete for the same bindingsite and reducing its uptake. Mixed competitive and non-competitiveinhibition between K and NH4-’- has been reported for tobacco (Scherer etal., 1984) and barley (Dean-Drummond and Glass, 1983). Although NH4-’-may not always inhibit K uptake competitively, NH4÷ often has a loweraffinity 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 NH4supplementation in ectomycorrhizal fungi (Boxman et aL, 1986; Jongbloedet al., 1991).There was a considerable K efflux induced by NH4 influx duringNH4 uptake by roots of corn, wheat or oat (Becking, 1956; Morgan andJackson, 1989). NH4 markedly inhibits K uptake in many speciesincluding wheat (Tromp, 1962), barley (Bange et al., 1965; Meijer, 1970).maize (Rufty et al., 1982) and tobacco (Scherer et al., 1984). It was alsoreported that exposure of seedlings of Scots pine and Douglas fir to NH4induced a loss of K (Boxman and Roelofs, 1986; Bledsoe and Rygiewicz,1986). The need to maintain cation-anion balance may explain someaspects of this inhibitory effect. For example, the presence of monovalentcations (NH4-’-, K, Na) in the uptake solution depressed 45Ca2 influx dueto stimulated Ca2 extrusion (Siddiqi and Glass, 1984). Generally plantssupplied with NH4-’--N contain lower concentrations of inorganic cationssuch as Ca2,Mg2, K-’- (Kirkby and Mengel, 1967; Barker and Maynard,1972; Harada et al., 1968; Moraghan and Porter, 1975; Magalhaes andWilcox,, 1983; Scherer et al., 1984; Siddiqi and Glass, 1984). It was foundthat K content of white mustard leaves was reduced to near half that ofN03--N grown by growth on NH4-’--N (Kirkby, 1968). Similar competitive222effects were also found in maize and sugar beet when grown on eitherurea or NH4-N (Beusichem and Neeteson, 1982).Although NH4 may stimulate the leakage of K-’-, it may not be themain mechanism responsible for the inhibition of K-’- influx. It is wellknown that NH4 uptake is associated with H-’- efflux and acidification ofgrowth 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 commonpractise 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 NH4-’-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 Hefflux (Mitchell, 1970; Glass et al., 1981). The K:H exchangestoichiometries were almost consistently greater than 2:1 (Glass andSiddiqi, 1982). Last, but not least, the efflux of K was not significant inuptake regulation (Glass, 1983) compared to the importance of K influx(Johansen et al., 1970; Yong and Sims, 1972). Since the presence of NH4 insolution inhibited K-’- uptake to a greater extent in K-loaded plants than inK-starved plants (Rosen and Carlson, 1984), the efflux of K may notaffected by the addition of NH4 (Jongbloed et al., 1991).7.4.4. Effect of K-’- on NH4 uptakeThe uptake of NH4 by young rice plants, as well as tomato and plumwas not competitively affected by the K concentration of the nutrientmedium (Mengel et aL, 1976, 1978; Rosen and Carlson, 1984) or by plant K223status (Rosen and Carison, 1984; Scherer and Mackown, 1987). However itwas found that the addition of high concentrations of K caused a reductionin methylamine transport rate in Anacystis nidulans (Boussiba et al.,1984).There is a synergistic behavior between N and K in the scope of cropgrowth and production (Mengel, 1989). Plant NH4-N nutrition wasimproved by supplying K-’- (Mengel et aL, 1976; Dibb and Thompson, 1985).For example, barley response to increasing N concentrations wasdependent on levels of K in the whole plant sample (MacLeod, 1969). Themuch higher N and K uptake with the higher K supply rate suggested thatthere might be a complementary uptake effect between NH4 and K-’- (Dibband Thompson, 1985). Lee and Rudge (1986) found that both K-’- and NH4uptake were stimulated to the same extent in N-starved roots. Ingreenhouse tests, K application tended to increase grain N content and totalN uptake by rice plants (Chakravorti, 1989). Tomato plants grown in sandculture with high NH4 appeared to display symptoms of NH4 toxicityrelated to increased ethylene synthesis that declined as K supply increased(Corey and Barker, 1989).In the present study, 1NH4 influxes of G100/2, G100/200 andG2/2plants were reduced by the presence of K in the uptake solution.Clearly K was most inhibitory to NH4 influx when plants were Nsufficient (Figs. 46 and 48) and K-deficient, especially at high [K-’-]0 (Fig.47). In the former condition, the NH4 influx would be relative low andprobably mediated by the high affinity transport system (Wang et al.,1993b). Studies on rice and tomato showed that K had inhibitory effectsbut did not compete with NH4-’- for selective binding sites in the absorptionprocess (Ajay et al., 1970; Dibb and Welch, 1976; Mengel et al., 1976).2247.4.5. Shared transport and different feedback signal?It is known that at low external concentrations, both NH4-’- 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; Fischerand Luttge, 1980; Kochian and Lucas, 1982; Luttge and Higinbotham, 1982;Wang et al., 1993b). The rapidity of the inhibitory effects of NH4-’- and Kon each other observed in the present studies indicated that inhibitionprobably occurred at the level of membrane transport although thisinhibition may not be a competitive one. Similar results were reported formaize roots (Shaff et al., 1993). This suggests that NH4 and K may share acommon transport pathway, such as an ion channel (Wang et al., 1992b,1993b; Shaff et al., 1993) and this hypothesis is supported by molecularevidence. In a recently cloned K channel from Arabidopsis, the NH4conductance was determined to be 30% of the K conductance for the KAT1K channel (Schachtman et al., 1992).Uptake of both NH4 and K caused depolarization of plasmamembrane electrical potentials (Kochian and Lucas, 1989; Ulirich et al.,1984; Wang et al., 1992b). Since the influx of both cations may be drivenby the proton motive force (at high external concentration), diminishingmembrane potential may lead to reduced ion uptake by influencing theproton motive force. It has been reported that the depolarization of theplasma membrane by NH4 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 notexplain why NH4-’- inhibited K÷ uptake in all four nutrient treatments225(G2/2, G2/200, G100/2, G100/200) and K only inhibited NH4-’- influx athigh N/low K plant status.‘3NH4influx and its kinetic parameters (Vm and Km) of N-deficientplants (G2/2) were not significantly affected by the presence of K inuptake solution except as noted above for the G100/2 plants. Also theinhibition of K (86Rb+) influx by NH4 was lower when plants were Kstarved. The uptake of K by excised rice roots decreased markedly withincreasing concentrations of NH4 in the uptake solution, while the uptakeof NH4was little affected by the concentration of K-’- in the uptake solution(Scherer et al., 1987). K uptake was suppressed during rapid NH4-’- uptakeby N-starved plants (Tromp, 1962), but K-starvation did not produce thesame effect as N-starvation on the transport of NH4 (Tromp, 1962; Leeand Rudge, 1986). This biased inhibitory effect between NH4 and K maysuggest that NH4 and K share a common transport pathway, but theregulation signal for these two ions may arise from separate sources. Thesuperior competitive behavior of NH4 over K is similar to the inhibitoryeffect of NH4 on N03 uptake which has also been linked to thedepolarizing effects of NH4 on 1P (see Lee and Draw, 1989 for discussion).Yet it is clear that, although K causes a depolarization of AP similar to thatcaused by NH4, it is not inhibitory to N03 uptake, nor is it as effectiveinhibiting NH4-’- uptake. Hence it is unlikely that the inhibitory effect ofNH4-’- is due to membrane depolarization/dissociation of pmf. The basis ofNH4 inhibitory effect remains to be resolved.226Chapter 8. GENERAL CONCLUSIONSThis study has identified and characterized the ammonium uptakesystem in rice roots in terms of cellular compartmentation (Chapter 3),kinetics (Chapter 4), energetics, electrophysiology (Chapter 5) andbiochemistry (Chapter 6). The interaction between NH4 and K on theplant growth and ion uptake was also examed (Chapter 7).Ammonium is absorbed by rice roots in the cation form even atelevated [NH4+]0.Newly absorbed NH4 is either stored in the root cellvacuoles or rapidly metabolized to amino acids in roots. Amino acids, butnot NH4, are consequently translocated to the shoots. Cytoplasmic [NH4-’-]may range from 3 to 38 mM according to the N provision during growth.The concentration dependence of NH4 uptake demonstrated that, atleast, two individual systems, HATS and LATS, operate at the plasmamembrane to transport NH4 into root cells. A saturable pattern of1NH4-’-influxes is due to HATS and a linear relationship between 13NH4÷ influxand [NH4+]0is mediated by LATS. HATS and LATS are not only kineticallydifferent, but also different in energy dependence and stoichiometry ofmembrane potential depolarization.Significant efflux of NH4 was observed even when plants weregrown at lower level of [NH4-’-]0,2 1iM. Efflux increased as [NH4+]0increasedfrom 2 to 100 and 1000 1iM, correspoding to 10, 20 30% respectively ofinflux at these [NH4]0.NH4-’- uptake is subjected to negative feedback regulation by bothNH4 and its metabolites.The effects of pretreatment with exogenous Gln,227Glu and Asn were found to reduce influx to differing extents. A cascaderegulation system is proposed to explain the regulation of ammoniumuptake in response to changes of internal NH4 and its metabolites. Thisinvolves regulation at many levels, from the whole plant down to themolecular level.The results of NH4 and K interaction studies at the level of plantgrowth and uptake gave quite different results. 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Reported studies on using radioactive isotope ‘3NYear Author N Species Objective MaterialBerkeley, U.S.A.1940 Ruben et al, 13N2 N2-Fixation Non-legumeEngland1961 Nicholas et al, 13N2 N2-Fixation BacteriaManitoba, Canada1967 Campbell et al, 13N2 N2-Fixation MicroorganismMichigan, U.S.A.1974 & 76 Wolk et al, 13N2 N2-Fixation Blue-green algae1975 & 77 Thomas et al, 13N2 -Fixation Blue-green algae1977 & 78(a) Meeks et al, 13N2 N2-Fixation Blue-green algae1978 (b) Meeks et al, 13N2 -Fixation Soybean1978 Skoukit et al, 13NO,H4 N assimilation Tobacco cells1979 Hanson et al, 13NH3 Translocation Barley1979 & 81 Tiedje et al, 13N Denitrification soils1981 Schubert et al, 13NH4 N2-Fixation Non-legumeDavis, U.S.A.1976 Gersberg et al, 13N03 Denitrification Flooded rice soil1978 Gersberg et al, 13N N assimilation Phytoplankton1982 Thayer&Huffaker 13N03 N03 transport Kiebsiella1985 Meeks et al, 13N03 Translocation CynobacteriaLower Hutt, New Zealand1981 McCallum et al, Denitrification Soils1983 & 85 McNaughton et al, 13N03H4+ N uptake, Flux Maize1984 & 86 Presland et al, 13N03 N uptake, Flux MaizeQuebec, Canada1984 Caidwell et at, ‘N0,’H4t2N2-Fixation AlfalfaVancouver, Canada1985 Glass et al, 13N03 N uptake, Flux Barley1989 Siddiqi et al, 13N03 N uptake, Flux BarleyWantage, England1986 (a,b) Lee et al, 13N03 N uptake, Flux BarleyStockholm, Sweden1987 Oscarson et al., 13N03 N uptake, Flux Pisum1988 (a,b) Ingemarsson et al., 13N03 N uptake, Flux LemnaNew York, U.S.A.1989 Calderon et al, 13N-glutamine assimilation Neurospora crassaHouston, U.S.A.1990 Hole et at, 13NO3 NO3 transport MaizeJülich, Germen.1992 Wieneke 13NO3 NO3 transport Squash263Appendix B Reported values of half-life (t172) and ion content (0) ofvarious compartments of root cells.Superficial Free space Cytoplasm Vacuolet 1/2(sec) (mm) (mm) (h)K onion 15 - 43 3.3 - 7.3 82 - 103 80 - 108barley 25- 75 14- 30barley 29 - 30 11 - 23Na onion 18 - 19 2.8 - 3.7 18 - 23 326 - 362barley 20- 22 77-231barley 17- 25 35-390Ca onion 12 - 13 1.3 - 1.5 54- 56 12- 30Mg onion 18 3.2 74 49- 71Cl- onion 18-52 9.5-17.9 90-104 68-137N03 barleybarleyQ(p,mol g-’)K-’- onion 0.2-1.4 0.3-1.0 0.8-1.3 72.7-73.1barley 11.0-22.0 28.0-95.6Na onion <0.1- 0.8 <0.1 - 0.3 <0.4 - 0.1 34.3 - 34.9barley 0.6 - 3.6 37.9 - 76.0barley 0.5-2.8 25.0-46.0Ca onion 1.2 - 1.3 0.4 - 0.6 1.6 - 2.5 5.4- 5.9Mg onion 0.23 0.05 0.32 11.1 - 11.2Cl- onion <0.1 - 0.4 <0.1 - 0.1 <0.1 - 0.1 68 - 137N03 barleybarley

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