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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 partialfulfilment of the requirements for an advanceddegree at the University of British Columbia,I agree that the Ubrary shall make itfreely available for reference andstudy. I further agree that permission for extensivecopying of this thesis for scholarly purposes maybe granted by the head of mydepartment or by his or her representatives, Itis understood that copying orpublication of this thesis for financial gain shallnot be allowed without my writtenpermission.Department of__________________The University of BritishColumbiaVancouver, CanadaDateDE-6 (2/88)ABSTRACT13NH4 uptake was studied using 3-week-old rice plants(Oryzasativa L. cv. M202), grown hydroponically in modified Johnson’snutrientsolution containing 2, 100 or 1000!IMNH4 (referred to hereafter as G2,G100 or G1000 plants, respectively). At steady-state, the influxand effluxof 13NH4was increased as NH4-’- provision during growthwas increased.The half-life of cytoplasmic‘3NH4exchange was calculatedto 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 NH4was located inthe vacuole. During a 30 minute period G100 plants metabolized19% of thenewly absorbed 13NH4-’- and the remainder was partitionedamong thecytoplasm (41%), vacuole (20%) and efflux (20%). Of the metabolized13N,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 G1000roots 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 NH4provisionduring 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%. EstimatedQ10values 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).‘3NH4influx 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) reducedthelevels of ammonium assimilates but did not increase 13NH4 influxprobably because internal [NH4-’-] was increased. Short-term nitrogendepletion stimulated1NH4influx, but long-termN depletion caused NH4influx to be reduced probably due to N limitation of carriersynthesis. Acascade regulation system is proposed to explain the multi-levelregulationof NH4 influx.111The interaction between ammonium and potassiumshowed thatwhen N is adequate, K promoted NH4 uptake and utilization.Likewise,proper N nutrition promoted K-’- uptake but the presence ofNH4 in uptakesolution strongly inhibited the K(86Rb+) uptake at the transportstep. Theresults indicated that NH4 andK-I-may share the same channel but areregulated by different feedback signals.ivTABLE OF CONTENTSAbstractiiTable of ContentsvList of abbreviationxiiList of TablesxivList of FiguresxvDedicationxviiiAcknowledgmentXixChapter 1. RESEARCH BACKGROUND11.1. General Introduction11.1.1. Rice11.1.2. Essentiality of nitrogen11.1.3. Necessity of N fertilization21.1.4. Bio-availability of nitrogen21.2. Ammonium Uptake31.2.1. Importance of transport research31.2.2. Transport of NH4by lower plants41.2.3. Transport of NH4 by higher plants51.2.3.1. Carrier-mediated transport51.2.3.2. Concentration-dependent kinetics61.2.3.3. Depolarization of membrane potential61.2.3.4. Energy dependence71.3. Major Factors Affecting Ammonium Uptake71.3.1. Effects of photosynthesis71.3.1.1. Dependence on soluble carbohydrates71.3.1.2. Periodic variations of light and growth81.3.1.3. Ambient environmental factors91.3.2. Effects of root temperature101.3.2.1. Short-term perturbation101.3.2.2.Qiovalue for NH4 uptake101.3.2.3. Long-term low temperature effects111.3.3. Effects of pH on NH4 uptake121.3.3.1. Acidification of rhizosphere byNH4 uptake 12V1.3.3.2. Retarded plant growth in acidic medium131.3.3.3. NH4 toxicity and acidic damage131.3.4. NH4 fluxes at the plasma membrane141.3.4.1. Net flux141.3.4.2. Influx141.3.4.3. Efflux151.3.4.4. Balance of fluxes151.3.4.5. N cycling in the whole plant161.3.5. Regulation of ammonium uptake171.3.5.1. Negative feedback regulation171.3.5.2. Enhanced NH4 uptake171.3.6. Interaction between NH4 and K181.3.6.1. Mutual beneficial effects between N and K181.3.6.2. Inhibition of K uptake by NH4181.3.6.3. Inhibition of NH4 uptake by K191.4. Research Objectives19Chapter 2. MATERIALS AND METHODS222.1. Plant Growth222.1.1. Seed germination222.1.2. Growth conditions222.1.3. Provision of nutrients232.2. N Isotopes For Studying N Uptake242.2.1. Isotopic tracer242.2.2. Nitrogen Isotopes242.2.3. Stable ‘5N techniques252.2.4. Radioactive isotope, 13N262.2.4.1. Use in biological studies262.2.4.2. Production of 1N272.2.4.3. Advantages of the use of 13N in biologicalstudies 292.2.4.4. Considerations of using ‘3N in nitrogen uptake302.2.4.5. Use of 13N in nitrogen transportstudies 312.2.4.6. Use of ‘3N in nitrogen assimilation322.2.4.7. Use of 1N in denitrification332.2.5. Protocol for‘3NH4production inpresent study 33vi2.3. Measurement Of NH4 Fluxes352.3.1. Influx of 13NH4352.3.2. Effluxof’3NH4-’-352.3.3. Net flux of NH4÷362.4. Compartmental (Efflux) Analysis362.4.1. Compartmentation of plant cells362.4.2. Development of theory372.4.3. Models for compartmental analysis382.4.4. The general procedureof compartmental analysis422.4.5. Procedures for compartmentalanalysis in the presentstudy442.5. Determination Of Ammonium462.6. Preparation Of Metabolic Inhibitors462.7. Electrophysiological Study472.7.1. Transmembrane electrical potentialmeasurement 472.7.2. Single impalement and membranepotential 532.7.3. Setup for measuring membranepotential 542.8. Determination of amino acids in root tissue55Chapter 3. FLUXES AND DISTRIBUTION OF 13NH4INCELLS 573.1. Introduction573.2. Materials And Methods593.2.1. Plant growth and ‘3N production593.2.2. Measurement of fluxes593.2.2.1. ‘3NH4influx593.2.2.2. Net NH4 flux593.2.2.3. Time course of 13NH4uptake603.2.3. Compartmental analysis603.2.4. Partition of absorbed1NH4603.2.4.1. Separation of‘3N-compounds in planttissue 603.2.4.2 Chemical assay of NH4 in roottissue 613.2.5. Calculation of flux to vacuole(Øcv)61vii3.3. Results623.3.1. Compartmental analysis623.3.2. Metabolism and translocation of‘3N 713.3.3. Time course of‘3NH4influx in rice roots713.4. Discussion753.4.1. The half-lives of 13NH4÷exchange753.4.2. Fluxes of‘3NH4into root cells783.4.3. The NH4 pools in roots823.4.4. Model of‘3NH4uptake by rice plants833.5. SUMMARY864. KINETICS OF‘3NH4INFLUX884.1. Introduction884.2. Materials And Methods904.2.1. Plant growth and 1N production904.2.2. Relative growth rate904.2.3. Influx measurement914.2.4. Kinetic study914.2.5. Metabolic inhibitor study924.2.6. Temperature study934.2.7. pH profile study934.3. Results944.3.1. Kinetics of 13NH4influx944.3.2.1. HATS944.3.1.2. LATS984.3.2. Effect of metabolic inhibitors on the influx of13NH4 984.3.3. Effect of root temperature on‘3NH4influx1014.3.4. Effect of solution pH on‘3NH4influx1044.4. Discussion1044.4.1. Kinetics of ammonium uptake1044.4.2. Energetic of ammonium uptake1074.4.3. Effect of pH profile on ammoniumuptake 1114.4.4. Regulation of ammonium uptake112viii4.5. Summary114Chapter 5. ELETROPHYSIOLOGICAL STUDY1155.1. Introduction1155.2. Materials And Methods1165.2.1. Growth of plants1165.2.2. Measurements of cell membrane potential1175.2.3. Experimental treatments1185.2.3.1. Effect of [NH4+]0on A’F1185.2.3.2. Effect of accompanying anion on A’P1185.2.3.3. Effects of metabolic inhibitors onNH4-induced APdepolarization1195.3. Results1205.3.1. Transmembrane electrical potentials of riceroots 1205.3.2. Contribution of the accompany anions to AW1205.3.3. Effect of [NH4C1]0on A’P1235.3.4. Effect of metabolic inhibitors on Z’{’1265.4. Discussion1305.4.1. Anion effect1305.4.2. Depolarization of A’P by HATS and LATS1315.4.3. Calculation of the free energy for NH4-’-transport 1355.4.4. Mechanisms of NH4 uptake by HATS andLATS 1385.5. Summary 139Chapter 6. REGULATION OF AMMONIUM UPTAKE1416.1. Introduction1416.2. Materials And methods1436.2.1. Plant growth and 13N production1436.2.2. Experimental design1446.2.2.1. Experiment I. Depletion and repletionstudy 1446.2.2.2. Experiment II. Effects of MSX1446.2.2.3. Experiment III. Effects of exogenous aminoacids 1456.2.2.4. Experiment IV. Effects of selectedinhibitors 1456.2.3. Determination of free ammonium in roottissue 1456.2.4. Determination of amino acids in root tissue1466.3. Results1466.3.1. Experiment I. Depletion and repletionstudy 1466.3.2. Experiment II. Effects of MSX156lx6.3.3. Experiment III. Effects of exogenous amino acids1636.3.4. Experiment IV. Effects of selected inhibitors1726.4 Discussion1766.4.1. Negative feedback on NH4 uptakeby NH4assimilates1766.4.2. Effect of MSX: reduced amino acid pool1786.4.3. Effect of short-term N depletion1816.4.4. Stimulated NH4 influx after long-term Ndepletion 1836.4.5. Negative feedback on 1NH4 influx frominternalNH4-I-1856.4.6. Cascade regulation system of nitrogen uptake188Chapter 7. INTERACTION BETWEEN K AND NH41937.1. Introduction1937.2. Materials And Methods1947.2.1. Plant growth and 1N production1947.2.2. Experimental design1947.2.1.1. Experiment I: Effects of K and NO3-in pretreatmentand K-- and NH4÷ in uptake solutions on net K+andNH4 fluxes1957.2.1.2. Experiment II: Effects of NH4 provision duringgrowth and ofK--and NH4 in pretreatmentanduptake solutions on 86Rb (K+) influxes1957.2.1.3. Experiment III: Effects of NH4 provisionduringgrowth and presence in uptake solutionupon influxisotherms for 86Rb (K+)1967.2.1.4. Experiment IV: Effects of NH4 provisionduringgrowth and short-term pretreatmentupon 86Rb(K) influx1967.2.1.5. Experiment V: Effect of NH4 concentrationspresentin uptake solution upon influxisotherms for 86Rb(K-I-)1967.2.1.6. Experiment VI: Effects of K provision duringgrowthand presence in uptake solutionsupon influxisotherms for1NH41977.3. Results1977.3.1. Experiment I: Effects of K andN03 in pretreatmentandK-I-andNH4-1-in uptake solutions on net K+andNH4 fluxes197x7.3.2. Experiment II. Effects of NH4 provisionduring growthand of K and NH4 in pretreatment anduptakesolutions on86Rb+(K) influxes2007.3.3. Experiment III: Effects of NH4provision duringgrowth and presence in uptake solution uponinfluxisotherms for86Rb+(K)2037.3.4. Experiment IV: Effects of NH4provision duringgrowth and short-term pretreatmentuponS6Rb+(K) influx2067.3.5. Experiment V: Effect of NH4 concentrationspresent inuptake solution upon influx isothermsfor 86Rb(K)2067.3.6. Experiment IV: Effects of K provision duringgrowthand presence in uptake solutions upon influxisotherms for1NH42107.4. Discussion2167.4.1. Plant growth in response to provisions of NH4and K 2167.4.2. Effect of plant N status on K(86Rb)uptake 2187.4.3. Effect of NH4 in the uptake solution onK(86Rb+)uptake2207.4.4. Effect of K on NH4 uptake 2227.4.5. Shared transport and different feedback signal? 224Chapter 8. GENERAL CONCLUSIONS226REFERENCES228APPENDIX A. Reported studies on using radioactiveisotope ‘3N 262APPENDIX B. Reported values of half-life (t1/2)and ion contnt(Q.)ofvarious compartments of root cells263xiAbbreviationsAA 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;massrate of assimilation of‘3NH4in roots;flux across the tonoplast into vacuole;translocation of 13N labeled metabolitesto xylem (shoots);Poc, ‘P,andnetinward, outward and net fluxes(iimolg’FWh-i)across the plasmalemma, respectively;G2, G100 and G1000 plants rice seedlings grown in MJNScontaining2, 100 or 1000 jiM NH4,respectively;G2M, GlOOM and G1000M MJNS containing 2, 100 or1000 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 NH4transport systems,respectively;Kmthe external ion concentration giving half ofthe 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 externalmedia andcytoplasmic compartments, respectively;Vmaxthe calculated maximum rate of ion influx(jimol g-’FW h-’);[NH4]cytoplasmic ammonium concentration(iiMor 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 compoundsby cation exchangecolumn.64Table 2. Estimated half-lives of 1NH4exchange for threecompartments of root cells.66Table 3. Comparison of1NH4fluxesacross the plasmalemmaofroot cells.67Table 4. Size of ammonium pools in rootcells at steady-state. 70Table 5. Calculation of the flux (P,) from cytoplasminto vacuole. 72Table 6. Distribution of newly absorbed 13Nin shoot and roottissues.73Table 7. Kinetic parameters for‘3NH4 influxof G2, G100, G1000plants.96Table 8. Reduction of‘3NH4-’- influx by metabolicinhibitors. 102Table 9.Qovalues for‘3NH4influx by the HATS orLATS. 103Table 10. Effect of uptake solution pH on 13NH4influx. 105Table 11. Membrane potentials of G2 and G100plants measured indifferent bathing solutions.121Table 12. Effect of metabolic inhibitors on thedepolarization of A’P. 129Table 13. Net 86Rb flux of rice plants grown withor without eitherpotassium and ammonium.198Table 14. Net NH4 flux of rice plants grown withor without eitherpotassium and ammonium.199Table 15. Michaelis-Menten kinetic parametersfor 86Rb influx ofplants grown in different levels ofNH4-’- and K. 208Table 16. Effects of NH4-- and K on plant growth.211Table 17. Michaelis-Menten kinetic parametersfor‘3NH4-’- influx ofplants grown in different levelsof potassium andammonium.213xivList of FiguresFigure 1. Scheme of‘3NH4convertion in laboratory.34Figure 2. Diagrame of the setup for measuring cellmembranepotential.56Figure 3. A represented pattern of 13NH4released intactroots. 65Figure 4. Fluxes of G2, G100 and G1000 plantsat steady-state. 69Figure 5. Cumulative uptake of1NH4by G2 andG100 roots. 74Figure 6. Influxes of‘3NH4 into G2 andG100 roots at steady-state.76Figure 7. Proposed model for ammonium uptakeandconpartmentation in rice roots.84Figure 8. Concentration dependence of 13NH4influx at low range(<1 mM).95Figure 9. Relationship between kinetic parametersof NH4 uptakeand root ammonium concentrations of rice seedlings.97Figure 10. Concentration dependence of 13NH4influx at lowrange(>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 cellby NH4C1. 124Figure 14. Concentration dependence of net A’Pdepolarization ofroot cells.125Figure 15. Effects of metabolic inhibitors on A’Fdepolarization 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 uptakeas a function ofexternal {NH4+J.136Figure 19. 13NH4influx of repleted G2 plants.147Figure 20. 1NH4influx of depleted G1000 plants.148Figure 21. Internal ammonium contentof repleted G2 plants. 149Figure 22. Total amino acid concentration ([AA]1)of repleted G2plants.151Figure 23.13NH4+influx (23A) and internal ammoniumcontent(23B) of repleted G2 or depleted G1000 roots.153Figure 24. Total [AA]1 of repleted G2 or depletedG1000 roots. 154Figure 25. Tissue amide or amino acid contents ofrepleted G2 ordepleted G1000 roots.155Figure 26. Effect of MSX on1NH4influx of rice roots.158Figure 27. Effect of MSX on ammonium content of riceroots. 159Figure 28. Effect of MSX on total [AA]1 of rice roots.160Figure 29. Effect of MSX on root content of amide oramino acid. 161Figure 30. Effect of exogenous glutamine on root 13NH4influx.164Figure 31. Effect of exogenous glutamineon root contents of amideand amino acid.165Figure 32. Effects of exogenous glutamine on‘3NH4influx. 166Figure 33. Effects of exogenous amides andamino acid on rootammonium content.168Figure 34. Effects of exogenous amides andamino acid on totalamino acid content.169Figure 35. Effects of exogenous amides andamino acid on aminoacid content.170Figure 36. Effects of exogenous amidesand amino acid on aminoacid content.171Figure 37. Effects of MSX, DON and AOAon 13NH4influx. 173xviFigure 38. Effects of MSX, DON andAOA on internal ammonium andtotal amino acid content.174Figure 39. Effects of MSX, DON and AOAon major amino acidscontent.175Figure 40. Effect of NH4 in the growthmedia, pretreatment anduptake solutions on 86Rb influx.201Figure 41. Effects of NH4÷ and K in growthmedia and uptakesolutions on 86Rb influx.202Figure 42. Relationship between estimatedVmof 86Rb influx androots internal {K÷].204Figure 43. Effect of short-term NH4 pretreatmenton 86Rb influx. 205Figure 44. Effects of NH4 and K in growthmedia and uptakesolutions on 86Rb uptake isotherm.207Figure 45. Effects of NH4 and K in growthmedia 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‘3NH4influxby HATS 214Figure 48. Effect of K in uptake solution on‘3NH4 influx ByHATS+LATS.215xviiTo my wife, Xiao Gefor her love, understanding andsacrificexviiiACKNOWLEDGMENTSMy 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 ofthe‘13N brigade’: Mala Fernando, BryanJ.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 alsolike to thankDrs. J.W.Huang andP. Ryan, Ms.L. ArmstrongandMr. T. Toulemondefor assistanceand discussions.I am gratefulto fellowgraduatestudentsas wellas staffand facultymembersof theBotanydepartmentfor theirsupportand friendship.Iwish toexpressmy gratitudeto formercolleaguesin Soiland FertilizerInstitute,ZheijiangAcademyof AgriculturalSciences.A veryspecialthanks isto all myfamily membersback homeinChina.I am gratefulto all myfriends, particularto familiesof Bill andKris,Jame andJill, Warrenand Liz,who treatedme likea brotherand gavemeand myfamilystrong supportin manyaspects.At last,but notleast, myspecialgratitudemust beexpressedto mywife, Xiaoge andmy son,Li ren fortheir loveand supportall throughthisprogram.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 isgrown in over 100countries on every continent except Antarctica, extendingfrom 5 3°N to35°S latitude, from sea level to 3000 m altitude (Luand Chang, 1980;Mikkelsen and De Datta, 1991). Rice grows either asan 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 riceis produced in Asia(IRRI, 1988). Rice is the staple food and the energysource for about 40% ofthe world’s population (De Datta, 1981, 1986b);it supplies the energysource for more than half of the world’s populationand 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 ofamino acids, proteins,nucleic acids and many secondary plant productssuch as alkaloids. It isinvolved in the whole life cycle of plants;in enzymes for biochemical2processes, in chlorophyll for photosynthesis, and in nucleoproteinsfor thecontrol of hereditary and developmental processes. SinceN is present in somany essential compounds, it is not surprisingthat growth withoutsufficient N is slow. Nitrogen is the single most important chemicalelementlimiting crop yield.1.1.3. Necessity of N fertilizationProper application of N increases both yield and proteincontent ofrice (Patrick et al., 1974; Gomez and De Datta, 1975; Allenand Terman,1978). The intensification of rice production has involved atremendousincrease in the use of nitrogen fertilizers and the selectionof high yieldingvarieties that are highly responsive to nitrogen. However, researchon theeffects of nitrogen fertilizers on rice production has focused mainlyon theagronomic context, in terms of grain yield, carbohydratemetabolism,growth patterns or morphological characteristics. Informationconcerningphysiological and biochemical aspects of nitrogen uptakeby rice as well asother higher plants, is limited, which is unfortunate since thesedetails mayprove to be important for the production of new varietieswith improvednitrogen utilization.L1.4. Blo-availability of nitrogenAmmonium is the predominant and most readilybio-availablenitrogen form in paddy soil; it is the preferred nitrogenspecies taken upby rice plants (Sethi, 1940; Sasakawa and Yamomoto, 1978;Goyal andHuffaker, 1984). Besides NH4,rice roots alsoabsorb 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 transportsystem(s) of root cells ofrice, and their regulations, is meagre. Moreover, therelationships amonguptake, assimilation and other metabolicprocesses are not as wellunderstood as is the case for other plant nutrients. Tounderstand theammonium transport system(s), generally, it is necessaryto characterizetheir kinetics, energetic and genetic properties.In order to achieve this,fluxes should be measured in response to variationof concentration,temperature and pH, and the effects of metabolic inhibitorsshould bedetermined. Where transport mutants are available, thegenetic basis ofthe transport system(s) can be evaluated(Kleiner, 1981, 1985; Glass,1988). This information satisfies more than theresearcher’s curiosity; itprovides a better understanding of ammoniumuptake for thedevelopment of better fertilization practice andimproved 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 electrochemicalpotentialgradients, resulting in significant ammonium concentrationwithin plantcells (Smith and Walker, 1978; Pelley and Bannister, 1979;Kleiner, 1981;Boussiba et al., 1984). NH4 uptake is concentration dependentand itsisotherm in the low range of external concentrationconformed 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 beenclaimed to occur via anelectrogenic uniporter which depolarizes membrane electricalpotentialdifferences (Barr et al., 1974; Haines and Wheeler, 1977;Slayman, 1977;Raven and Smith, 1976; Smith et al., 1978; Smithand Walker, 1978;Walker et al., 1979a, 1979b; Laane, 1980; Raven,1980; Smith, 1980;Kleiner and Fitzke,1981; Berti et al., 1984; Ulirichet al., 1984). TheQovalue for NH4+ uptake has been reported to be 2.0(Hackette et al., 1970)and ATP may be involved in the transport step, hencethe uptake systemis inhibited by anaerobiosis or several metabolicinhibitors (Stevenson andSilver, 1977; Cook and Anthony, 1978a; Felle, 1980).The responses of NH4uptake to pH changes is complex (Hackette et al., 1970; Roonet al., 1977;Kleiner, 1981). The optimum pH was 67 forbacteria and fungi. Theexistence of specific proteinaceous carriers forNH4 uptake is supportedby biochemical, kinetic and physiologicalevidences. Moreover, NH4transport mutants have been isolated andsome transport genes have beenidentified and cloned (Arst and Page, 1973;Castorph and Kleiner, 1984;Holtel and Kleiner, 1985; Franco et al., 1987; Reglinskiet al., 1989).51.2.3. Transport of NH4 by higher plantsThere is a limited literature available regardingNH4÷ transport inhigher plants (Highinbotham et al., 1964), althougha 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 plantsare very similar tothose in lower plants. Ammonium transport islocalized perhaps at theplasma membrane and possible other membranes(Kleiner, 1981; Churchilland Sze, 1983). Evidence from kinetic studies ofammonium uptake byplant roots indicates that NH4 transport isa carrier-mediated process(Nissen, 1973; Joseph et al., 1975). There areseveral lines of evidence thatsupport the existence of the proteinaceous carriersto be discussed in thefollowing sections.1.2.3.1. Carrier-mediated transportEvidence indicating that ammonium transportis a carrier-mediatedprocess (Nissen, 1973; Joseph et al., 1975) comesfrom determining kineticparameters for NH4 accumulation in cells (Kleiner,1985). The uptake ofNH4 by barley, rice, ryegrass, tomato,and wheat is concentrationdependent and follows Michaelis-Mentenkinetics, (Tromp, 1962;Lycklama, 1963; Fried et al., 1965;Cox and Reisensuer, 1973; Rao andRains, 1976; Bloom and Chapin, 1981; Youngdahlet al., 1982; McNaughtonand Presland, 1983; Bloom, 1985;Deane-Drummond and Thayer, 1986;Smart and Bloom, 1988). Preslandand 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 continuouslyflowing nutrientsolution system, NH4 uptake ratesof intact rice plants were fitted toaMichaelis-Menten model (Friedet al., 1965; Youngdahl et al., 1982).61.2.3.2. Concentration dependent kineticsA biphasic pattern of NH4 uptake, with both saturableand 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 withKm 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 0.061and 0.017 mmol kg-’ s’ were obtained for4-week-old and9-week-old rice plants, respectively, (Youngdahl et al., 1982).The secondsystem, above 1 mM [NH4], failed to correspond toMichaelis-Mentenkinetics (Ullrich et al., 1984; Presland and McNaughton,1986). Generally,uptake studies at high external concentration havebeen achieved onlywith some difficulty, because depletion of the externalsolution is so small.1.2.3.3. Depolarization ofmembrane potentialThe inward movement of ammonium occurs as thecation NH4-’-(Walker et al., 1979a, 1979b; MacFarlane and Smith, 1982;Kleiner, 1985;Deane-Drummond, 1986). Only one report measuring AW in riceroots hasappeared in the literature: Usmanov (1979) reported AP to be-160 mV. Asearly as 1964, Higinbotham et al., noted the markeddepolarizing effect of[NH4+]0on coleoptile cells AP in oats. Ullrichet al., (1984) found that, inLemna, depolarization of zSM’ by NH4 below0.2 mM [NH4]0 wasconcentration-dependent and both NH4 uptake and z’Pdepolarizationresponded in a saturable fashion with half saturationvalues of 17 pM forboth processes. From 0.2 to 1 mM, net uptakeof NH4÷ responded linearlyto [NH4+]0,with no further AP depolarization.Since NH4-’- is the main speciestaken by plant roots, it mustbe taken up via active transport and/or7facilitated diffusion. Both processes are coupled to an energysource, eitherdirectly (the former) or indirectly (the latter).1.2.3.4. Energy dependenceMetabolic energy is important to NH4uptake. Macklonet al., (1990)has shown that NH4 absorption by excised root segments ofAllium cepa L.was an active process. The uptake of ammonium at hightemperature (25-30°C) is closely associated with metabolism (Sasakawaand Yamamoto,1978), and the uptake process was also decreased whencarbohydratelevels were reduced (see Section 1.3.1.1.) orwhen temperatures werelowered (see Section 1.3.2.1.).1.3. MAJOR FACTORS AFFECTING AMMONIUM UPTAKEBesides the mechanism and kinetics of ammoniumuptake, researchon ammonium uptake has also included otherrelated issues such as theeffect of energy status, nitrogen cycling within theplant, the effects of rootpH and temperature. It must be emphasizedthat when environmentalfactors are concerned, one must be aware ofthe root’s capacity to adaption uptake in response to changedconditions, especially in long-termexperiments.1.3.1. Effects of photosynthesis1.3.1.1. Dependence on soluble carbohydratesOf major importance in the uptake ofammonium is the energy statusof the plant. The energystatus of rice plants had a substantialinfluence on8the uptake of NH4-’- and on its conversion into highmolecular weight Ncompounds (Mengel and Viro, 1978). The high demandfor carbohydrate isin order to achieve active transport of NH4 at low externalconcentration,and to supply carbon skeletons for the rapid assimilationof NH4-’- as it isabsorbed by roots (Givan, 1979; Fentem et al.,1983a, 1983b). When theavailability of carbohydrate is low, the assimilationof NH4-’- is also low(Breteler and Nissen, 1982), and consequentlya high efflux rate of NH4may result. A general relationship exists betweenthe proportion of totalnitrogen absorbed as NH4 from mixed N sourcessuch as NH4NO3and theavailability of soluble carbohydrates in roots.(Raper et al., 1992). Theconcentration of soluble carbohydrates in theleaves of NH4-fed plantswas greater than that of N03--fed plants, but waslower in roots of NH4-fed plants, regardless of pH (Chaillou et al., 1991).The study of NH4 uptake isotherms in Chiorellarevealed thatpreincubation with glucose drastically increasedVmax(5-fold), with nochange ofKm (Schlee and Komor, 1986). It was reported that glucoseinduced a glucose transport system and two specificamino acid transportsystems (Cho et al., 1981). Glucose also induced the transportsystems forammonium, nitrate and urea (Schleeet al., 1985). Removal of theendosperm of rice seedling suppressed NH4uptake markedly (Sasakawaand Yamamoto, 1978), while the additionof 30 mM sucrose restoreduptake. In higher plants, provisionof carbon skeletons in the form ofa-ketoglutarate increased uptake andassociation of NH4 in Lemna(Monselise and Kost, 1993).1.3.1.2. Periodic variations of light and growthThere is a great variation in NH4 assimilationrates between day andnight during the tillering stageof rice plants (Ito, 1987). Thisis probably9related to the diurnal changes in carbohydrate flux fromshoot to rootresulting from changes in relative source-sink activity ofshoots (Rufty etal., 1989; Lim et al., 1990). This periodic variationof carbohydrate supplyis also influenced by morphological variations ofplant growth (Henry andRaper, 1989a; Vessey et al., 1990b). The net rate ofNH4 uptake oscillatedbetween a maximum and a minimum with a periodicityco-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 observeddifferences of net NH4-’-uptake (Henry and Raper, 1989).1.3.1.3. Ambient environmental factorsThe ability of the plant root to absorb nitrogen wasaffected byprevious growth conditions of the examined plants (Monet al., 1979),since environmental factors will influence thecarbohydrate status.Susceptibility of plants to NH4 toxicity is alsorelated to plantcarbohydrate status (Nightingale, 1937; Prianishnikov,1941; Givan, 1979).The soluble carbohydrate concentration in rootsincreased with increasingroot temperature (Clarkson et al., 1975; Macduffet al., 1987a) and withnitrogen deprivation (Rufty et al., 1988; Henryand Raper, 1991), anddecreasing rhizospheric pH (Chaillouet al., 1991). High ambient C02concentration increased total plant N andtotal 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 plasmamembrane is sensitive totemperature. Although ion accumulationat 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 perturbationstudy, it was found that theuptake and assimilation of ammonium were profoundlyaffected in bothIndica and Japonica rice plants (Ta and Ohira, 1981). Thismight beexplained by the dependence of the NH4 uptakesystem on the rate ofmetabolism (Raven and Smith, 1976), or effects oflow temperature onenzymes of NH4-’- assimilation (Shen, 1972). The effectof temperature onion uptake may also be due to physical changes indifferent parts of thecell membrane (e.g. membrane fluidity) instead ofon the transport process(Clarkson and Warner, 1979).1.3.2.2.Qovalue for NH4 uptakeQiovalues can be used to indicate the temperaturedependence ofion transport. When temperature is loweredor increased by 10°C, the ratioof the two transport rates can be calculatedby equation:LnQjo= {(t2-t1)/10] Ln(V2/V1)[1]where t1 and t2 are the temperature beforeand after the change, andVi.and V2 are the transport rates at respectivetemperatures. WhenQoisclose to 1, the transport rates are the sameat the different temperatures,and ion transport is insensitive to temperature.AQovalue greater than 2is often considered as indicating themetabolic dependence of a11physiological process such as ion transport. In a seven hoursperturbationof root temperature, Sasakawa and Yamamoto(1978) found that theuptake of ammonium by 9-days old rice seedlings was closelyassociatedwith metabolism. TheQovalues between 9 24°C were> 2.5for 15NH4÷absorption by rice roots estimated from Ta and Ohira’s (1981)data. LowQiovalues (1.0 1.5) were reported for net ammoniumuptake 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 varieswith thetreatment duration. Plants may adjust rates of iontransport in the long-term so that net uptake is independent of externalvariables such astemperature (Clarkson, 1976). As a result of plantadaptation to low roottemperatures, NH4 is absorbed more readily thanN03 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 rootsoccurred even attemperatures as low as 0°C (Yoneyama et al., 1977).In both Indica and Japonica rice plants ammoniumand nitrateuptake and assimilation were strongly affectedby temperature (Ta andOhira, 1981). The uptake as well as assimilationof the two forms ofnitrogen were greatly inhibited at low temperatureand low light intensity.At low root temperature, uptake of NH4was higher than that of N03.Theproportion of NH4-’- absorbed from mixed NH4-’- and N03solution wasincreased as root temperature decreasedfrom 13 to 3°C (Macduff andWild, 1989). Likewise, transferring cornroots from 30°C to 0°C, reduced12‘5N03-uptake more drastically than 1NH4 uptake(Yoneyama et al.,1977).The lower sensitivity ofNH4+uptake to reduced temperature(compared to NO3-uptake) might be explained by a lesserdependence ofNH4+uptake on the rate of metabolism and energyproduction (Raven andSmith, 1976), or less effect of low temperatureon enzymes ofNH4+assimilation (GS-GOGAT) compared to those enzymes ofNO3-uptake andreduction (NR and NiR).1.3.3. Effects of pH on NH4 uptakeIt has frequently been reported that NH4+ uptakeis higher atelevated pH while NO3-uptake is stimulated at low pH (vanden Honertand Hooysman, 1955; Fried et al., 1965; Jungk,1970). When plants aregrown in medium containing NH4÷ as the solo source ofN, the inevitableacidification of the medium may cause damageto the roots and even deathof plants (Loo, 1931; Raven and Smith, 1976).Moreover root growth maybe restricted in NH4 medium even whenthe pH of the medium iscontrolled between 6.0 and 6.5 (Lewis et al., 1987).1.3.3.1. Acidification of rhizosphereby NH4 uptakeA major factor in N uptake is the change ofrhizosphere pH associatedwith NH4 uptake and its effect on plantgrowth, root morphology andcapacity for ion uptake. It is well known thatNH4 uptake will causeacidification of the growth medium (Ravenand Smith, 1976). At high NH4concentrations an enhanced NH4-’- uptake byectomycorrhizal fungi causedan accelerated medium acidificationthat indirectly inhibited growth13(Jongbloed and Borst-Pauwels, 1990). NH4 has greater detrimentaleffectson plant roots than on shoots (Loo, 1931; Raven andSmith, 1976). Plantssupplied with moderate concentrations of NH4 generallygrow poorlycompared with plants supplied with other sources of nitrogen(Rufty et al.,1982b) or mixed N03-/NH4-’- supplies. Increased proportionsof NH4 inmixed NH4 and NO3-nutrient solutions increasedshoot:root ratios at alllevels of root-zone pH (Vessey et al., 1990).When NH4 and NO3- weresupplied together, cumulative uptake of total nitrogenwas 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 growthand NH4÷uptake. Root growth was restricted by increased aciditybetween 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 andN03 uptake increased(Vessey et al., 1990). It was reported that the growthrate of soybeanshoots and roots was reduced by increasing pH (Ruftyet 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 growthwith 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 NH4as anitrogen source as long as root-zone pH is strictlycontrolled and a balanceis maintained between carbohydrateavailability and acquisition of NH4(Rufty et al., 1983). It was suggested thatthe inhibition of plant growth atlow pH was due to a decline in NH4-1- uptakeand a consequential limitationof growth by N stress (Vesseyet al., 1990).141.3.4. NH4 fluxes at the plasma membrane1.3.4.1. Net fluxNET FLUX(net)describes the ‘net’ rate of ion uptakeby roots. The netion uptake from the medium (outside) into thecytoplasm is determined bythe balance between influx and efflux. In practice, net fluxPnet = Poc - Øco[2]is measured by the disappearance of tested ionin the uptake solution.1.3.4.2. InfluxINFLUX()is defined as the rate of inward movementof soluteacross a particular membrane. Strictly speaking influx shouldrefer to theunidirectional movement measured during a very shortperiod, shortenough to discount the efflux. NH4÷ influx is negativelycorrelated withplant N status in lower plants (Silver and Perry,1981; Hartmann andKleiner, 1982; Wiegel and Kleiner, 1982; Boussibaet al., 1984; Mazzuccoand Benson, 1984; Rai et al., 1984; Jayakumaret al., 1985), and higherplants (McCarthy and Goldman, 1979; Pelleyand 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 seedlingsdecreased after pretreatment withglutamine and NH4 and increased afterpretreatment with asparagine(Deane-Drummond, 1986).1.3.4.3. EffluxEFFLUX(Ø)is the rate of outward solute flow fromcytoplasm acrossthe plasma membrane. Efflux of ionsfrom plant roots wasidentified 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 andSim, 1976, 1981;Behi and Jeschke, 1982; Jeschke, 1982; Lazof and Cheeseman,1986; Siddiqiet al., 1991).Continuous NH4efflux may be a common feature of net NH4uptakeby roots of higher plants (Morgan and Jackson, 1973). In astudy usingintact ryegrass,‘4N03--grown roots were equilibrated in a‘5N03-solutionenriched with ‘5N (97.5 atmo %). The results suggested thatthere was asimultaneous occurrence of the influx of‘5N03-and effluxof‘4N03(Morgan et al., 1973). Moreover, careful measurements of 14NH4effluxrevealed that there must have been generation of NH4 bybreakdown ofnitrogen compounds during the course of the experiment.There was excessquantity of4NH effluxes compared with the initial contentin the roots(Morgan and Jackson, 1988a). There is even an‘4NH efflux from14N03-grown roots (Morgan and Jackson, 1988b).1.3.4.4. Balance of fluxesThere is thought to be an ammonium cycle acrossthe root cellplasma membrane (Morgan and Jackson, 1988b).It was reported thatendogenous NO3-effluxes to the unstirred layers wererecycled throughNO3-influx (Morgan et al., 1973). The same couldbe expected for NH4efflux. Substantial ammonium cycling occurredduring net ammoniumuptake (Jackson et aL, 1993), yet plantsgrown under low N conditionspossess a low NH4-’- efflux. Morgan and Jackson(1988a) suggested that the16regulation of NH4 uptake by roots of higher plantsmay involve changes ofboth influx and efflux in response to plant nitrogenstatus. It was foundthat net 15NH4influx was increased and net‘4NH effluxwas decreasedin nitrogen depleted wheat and oat seedlings (Morganand Jackson, 1988a),and net NH4-’- uptake of barley and maize plantspreviously 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 treatmentwas claimed to bedue to the enlargement of cytoplasmic and vacuolarNH4 pools of roottissue several times (Jackson et al., 1993; Lee andAyling, 1993) whichappeared enhance the influx of‘3NH4 of (maizeand barley) plants byreducing isotopic efflux (Lee et al., 1992; Lee and Ayling,1993). However,the enlarged [NH4]1 was also advanced to explainthe enhanced effluxobserved in their system (Morgan and Jackson, 1988b).1.3.4.5. N cycling in the whole plantWithin the plant, N cycling, the simultaneousmovement of N-compounds from root to shoot, and from shootto root (Cooper andClarkson, 1989; Larsson et al., 1991)may enable N absorption to beregulated to match the demand imposedby plant growth (Drew and Saker,1975; Edwards and Barber, 1976). The concentrationsof amides (Gln andAsn) in the roots will be the result of the balancebetween their synthesisfrom absorbed inorganic N (NH4or N03), theirimport via the phloem, andtheir export via the xylem (Lee et al., 1992).171.3.5. Regulation of ammonium uptakeFeedback inhibition of NH4 uptakeby nitrogenous effectors hasbeen implicated in lower plants (Kleiner, 1985; Ullrichet 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; Morganand Jackson,1988a). There is, however, only limited informationavailable concerningthe possible mechanism(s) of regulating NH4-’- uptakeby either NH4per seor its primary assimilates.1.3.5.1. Negative feedback regulationAt high nitrogen status, plant NH4-’- uptake couldbe suppressed dueto (i) low energy supply to the root system, (ii) accumulationin the roottissue of nitrogenous compounds that exerts negativefeedback on thetransport system, or (iii) high efflux ofendogenous NH4 (Morgan andJackson, 1988b). Repression of NH4uptake may be due to continualgeneration of ammonium from degradationof organic nitrogenous sourceswithin roots and rapid accumulation of ammoniumin 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 asglutamine, are more likely negativeeffectors on NH4-’- uptake.1.3.5.2. Enhanced NH4 uptakeNegative correlation between ammoniumuptake 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; Morganand Jackson,1988a, 1988b; Clarkson and Luttge, 1991). It has been recognizedthat thecapacity for nitrogen uptake is enhanced in N-depletedplants 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 andKN and K are essential plant nutrients, required for healthyplantgrowth and high yield production (Ajayi etal., 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 andN on plant growth haveoften been described. An adequate K supplyhas 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 biologicaldilution effect of better plantgrowth (Noguchi and Sugawara, 1966;Kirkby, 1968; Claassen and Wilcox,1974; Faizy, 1979; Lamond, 1979; Beusichemand Neeteson, 1982).1.3.6.2. Inhibition of K÷ uptake by NH4However, NH4 has been shown to stronglyinhibit the absorption ofK-’- in short-term experiments in manyspecies including wheat, barley,maize and tobacco (Breteler, 1977; Munnand Jackson, 1978; Ruftyet al.,191982; Rosen and Carison, 1984; Scherer et aL, 1984).There was a negativecorrelation between the external NH4 concentrationsand K uptake (Rosenand Carlson, 1984; Scherer et al., 1987; Jongbloedet al., 1991), and netammonium uptake was correlated with potassiumefflux (Morgan andJackson, 1989).The inhibitory effect of NH4 on K uptake has beenclaimed to beindependent of K-i- provision or pretreatments; it is probablyexerted on thetransport processes at the plasma membrane.Insufficient evidence isavailable to draw a conclusion regardingthe inhibition of K uptake byNH4 in terms of competitive and non-competitiveeffects (DeaneDrummond and Glass, 1983b; Scherer et al., 1984).K uptake wassuppressed during rapid NH4-’- uptake by N-starvedplants (Tromp, 1962),but K-starvation did not produce the same effectas 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 plantswas not reduced by K inthe nutrient medium (Mengel et al., 1976; Rosen and Carison, 1984;Scherer and Mackown, 1987). However, the influenceof K÷ on NH4 uptakehas not been consistent. It was reportedthat K had inhibitory effects butdid not compete with NH4 for selective bindingsites in the absorptionprocess (Ajayi et al., 1970; Dibb and Welch, 1976;Mengel et al., 1976).1.4. REsEARCH OBJECTIVESThe objective of this studywas to investigate the mechanisms andcharacteristics of ammonium uptakeby rice plants. In particular, the20studies have emphasized short-term responsesof fluxes to changesinambient conditions. This particular goal was achievedby using the short-lived radioisotope 1N (t1/2 = 9.98 mm), addressingfive different areas:(1). By measuring NH4 influx and efflux,the exchange of N at the plasmamembrane and the relationships betweenthese fluxes werequantified.Subcellular distribution of absorbed NH4 was alsoestimated. The resultsof these studies are interpreted in termsof a root cell model in Chapter3.(2). To describe the kinetics of NH4uptake and the pattern(s) ofitsconcentration dependence, NH4 influx was measuredin perturbationexperiments in plants grown in different levelsof N. By altering ambientconditions such as medium pH, root temperature,and by treating rootswith various metabolic inhibitors, theenergetic of NH4 uptake wasinvestigated. These are described in Chapter 4.(3). By measuring electrical potential differencestogether with assayingcytoplasmic [NH4], the electrochemicalpotential gradient for NH4between external solution and cytosol were definedin order to explore themechanisms of NH4 uptake. Membrane electricalpotential differences ofrice roots were recorded as a function of externalNH4 concentration. Thisinformation is incorporated with data dealing withbiochemical, kinetic andenergetic aspects of NH4-’- uptake to formulatea model for the mechanismsof NH4-’- uptake (Chapter 5).(4). Without informationon the regulation of NH4 uptake, the uptakemodel is incomplete. NH4 influxwas measured as a function of root Nstatus. Internal[NH4-’-l was determined as well as the concentrations ofindividual amino acids. In Chapter6, the results are discussed in referenceto existing reports to develop amodel of the regulation of NH4-’-uptake.21(5). Chapter 7 deals with the interactions betweenNH4 and K-’- at theuptake level and explores the effects of priorexposure to these ions onsubsequent ion uptake.22Chapter 2. METHODS AND MATERIALSIn this chapter, the general methods usedin this study are described.Method(s) used in a particular experiment willbe addressed in thecorresponding chapter.2.1. PLANT GROWTH2.1.1. Seed germinationRice seeds (Oryza sativa L. cv. M202) were surfacesterilized in 1%NaOC1 for 30 mm and rinsed several times with de-ionizeddistilled water.Seeds were imbibed overnight in aerated dc-ionized distilledwater at3 8°C, then placed on plastic mesh mounted on Plexiglas discs.The discswere set in a Plexiglas tray filled with dc-ionized distilledwater just abovethe level of the seeds, and seeds were allowedto germinate in a growthchamber in the dark (at 38°C) for 4 d. Duringthe 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 Plexiglastanks.2.1.2. Growth conditionsPlants were grown hydroponically in 40-L Plexiglastanks located ina walk-in growth room, in which growth conditionswere 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 whenthey were usedfor most experiments unless specifically indicated.2.1.3. Provision of nutrientsThe growth medium was modified based on therecipe 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 ofnitrogen (except forsome specific experiments as specifically indicated) and siliconwas addedas Na2SiO3.5H0.This modified Johnson’s nutrient solution(hereafterreferred to as MJNS) was also the medium usedto 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 ammoniumconcentration ([NH4÷]0)was varied as indicated at the appropriate places. Generally plantsweregrown in MJNS containing 2, 100, or 1000 jiM [NH4+]0,referredto hereafteras G2, G100, G1000 plants, respectively. The concentrations ofnutrients ingrowth medium were maintained by infusion ofappropriate stocksolutions, through peristaltic pumps (Technicon ProportioningPump II,Technicon Inst. Corp.). Generally 2 liters perday of stock solution weresupplied and stock concentrations were determinedfrom daily chemicalanalyses of medium samples. Solutions weremixed continuously bycirculating pumps (Circulator Model IC-2, BrinkmannInst., Inc.), andaerated continuously. The pH of growth mediumwas maintained at 6.0 ±0.5 by adding powdered CaCO3 (13 g/tank), accordingto measured pHvalues, 12 times daily.242.2. N ISOTOPES FOR STUDYING N UPTAKE2.2.1. Isotopic tracerThere is now widespread use ofisotopic 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 employedtodetermine the kinetics of transport andtransformation of these elementsin living systems. Measurementsof radioisotopic influxand/or efflux havebeen used to obtain an estimateof the unidirectional fluxes ofthe stableisotope of the ion at the plasmalemmaand tonoplast and to estimatetheseparate amounts of the stable isotopes inthe cytoplasm and vacuole(Cooper, 1977; Thain, 1984).The utility of radiochemical techniquesis afforded by (i) their greatsensitivity compared to other analytical methods.Radioisotopic tracersmay offer 108-fold increased detection sensitivityover stable isotopemethods (Cooper, 1977; Krohn and Mathis,1981); (ii) the fact that they“label” the atoms of molecules withoutsignificantly altering their chemicalproperties (Cooper, 1977; Boyer, 1986).2.2.2. Nitrogen IsotopesThere are six isotopes ofnitrogen known, ranging in massnumberfrom 12 to 17 (Kamen, 1957).The stable isotopes of nitrogenare ‘4N and15N, the latter being presentto the extent of 0.365 atomper cent.Radioactive isotopes 12N and‘3N are positron emitters withhalf-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, 17Nalsoemits neutrons. The longest-lived radioactive isotope of nitrogenis ‘3Nwhich is the only radioactive isotope that has beenused in tracer research(Kamen, 1957; Krohn and Mathis, 1981; Bremnerand Hauck, 1982). Theuse of 1N (Burns and Miller, 1941) in biological studies startedas 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 hasbeen 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 N03and NH4-’- uptakeprocesses of plants (Fried et al., 1965; Yoneyamaand Kaneko, 1989;Yoneyama et al., 1991) and tracing themetabolism of nitrogen in plantcells (Yoneyama and Kumazawa, 1975; Arima andKumazawa, 1977). 1N isalso widely used in studyingN2-fixationin 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 denitrificationof soil N (Mosier and Schimel,1993).15N-labeled nitrogen fertilizer has alsobeen used in the study of fertilizeruse efficiency (Azam et al., 1991).26Stable N isotope techniques have severaladvantages over techniquesusing radionuclides. As a biochemical tracer, ‘5Noffers the advantages ofbeing relatively inexpensive, widely available, freeof radiation hazard andless limiting in terms of experiment duration. Theadvantages of using ‘5Nalso embodies a major disadvantage in its useas a tracer: a sizablebackground, present in all nitrogenous materials,against which addedtracer must be measured (Cooperet al., 1985). In order to measuresignificant enrichment of 5N in specific metaboliccompartments,investigators have to administer a large amountof‘5N-labelednonphysiological precursors to biological systems(Cooper et al., 1985). Inaddition it requires tedious preparation to convert samplesto 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 artificiallyby induction of radioactivity inotherwise stable elements (boron) by bombardment withparticles emittedby polonium (Joliot and Curie, 1934). Itwas first used as a biological tracerin studying theN2-fixation of non-legume barleyplants (Ruben et al.,1940), which was one year earlier than the firstreport of using 15N2 tostudy N2 fixation (Burns and Miller, 1941).Much of the early tracer work in biochemistrywas carried out withpositron-emitting radionuclides, suchas “C, and to a lesser extent13N, butwith the introduction of‘4C and 15N, their importance declined over aperiod of two decades. Onlyin the past 10 years or so, have theseshort-27lived isotopes again become important as tracersparticularly 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 acceleratedparticle (Cooper et al., 1985). The10B(o,n)13N;‘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 allbeen used to make ‘3N (Krohn andMathis, 1981; Tilbury, 1981). The methodmost widely used at present forthe production of‘3N-ammonia is the protonirradiation 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; Chaskoand Thayer, 1981;Cooper et al., 1985).An example flow scheme of ‘3N2 productionbased on nuclearreactions of‘6O(p,c’3Nis as follows: (Meekset 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 withan 10 jiAproton beam of high energy (>19 MeV) could yield36 mCi pA4 20 miw1(Vaalburg et al., 1975). The ‘3N species,‘3N0,1NO2 and1NH4,arepresent in the radioactive sample. The relative concentrationsof thesespecies is dependent upon the irradiation doseas well as on other factorssuch as the previous irradiation history of the targetfoil (Tilbury and Dahi,1979). The study of the effect of integrateddose showed that at low dose‘3NH4-’- is greater than‘3N02 and at high dose 13NH4isless 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=1.8h),150(t1/2=2.0 mm), and48V(t1/2=16.2 d). Both 18F and 48V produce noproblems with‘3NH4since these radloisotopes do not distil.Sincel8Fisfrom the reaction of‘80(p,n)’F,its contamination canbe minimized bydepleting180in water (Skokut et al., 1978). Though15Qcan be detected in‘3N-ammonia solution, it will disappear during preparationslasting morethan 20 mm (Vaalburg et al., 1975).The 13N isotope disintegrates by emission ofa positron(E3,1.2 MeVof maximum emission energy) giving riseto ‘3C (Meeks, 1993). Inannihilation reaction between a positron andan electron, two gammaphotons are formed each of 0.511 MeVenergy traveling in nearly oppositedirections (Cooper et al., 1985; Meeks,1993). Therefore the detection ofradioactive decay in the sample is accomplishedin 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 gammacounter and allcounts are decay-corrected to a common time. Theadmitted 13N in planttissues can be observed by placement of multipleGieger-Mueller tubesalong the plant axis (McNaughton andPresland, 1983; Caidwellet al.,1984), by autoradiography on X-ray film betweenblocks 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 biologicalstudiesThe use of‘3NH4-N in biological studies ofnitrogen nutrition hasseveral advantages:(1) The chief advantage is that such nuclide canbe prepared at a very highspecific activity increasing sensitivity for detectionapproximately108-fold, to trace rapid kinetics and metabolicpathways (Krohn andMathis 1985). Because of the greatsensitivity of the radioactive isotopetechnique, 13N has proved to be of value inelucidating biologicalmechanism over very short time intervals.(Hanck, 1982).(2) In order to measure the initial eventsin biological processes it may benecessary to determine eventson a time scale of seconds to minutes. Highspecific activity tracers whichare detected with high efficiency (e.g. 13N)make possible such measurements. Itis clear that time resolution of atracer-influx experiment is crucial forsubsequent interpretation of thefluxes. In short term experiment,by using‘3N0,one is able to monitornet uptake and disappearance of‘3N0 simultaneously, thus increasingthe experimental resolutioncompared with experiments where plants30have to be sampled and further preparedbefore assay (Oscarson et aL,1987).(3) The isotope decays rapidly (t1/2 =9.98 mm). After allowing sufficienttime for decay, repeat studies canbe carried out in the same systemwithout interference from previously administeredtracer (Cooper et al.,1985). In tissue dissection or in vitro studies, thetotal quantity of tracerpresent in rather large specimens canbe determined rapidly andaccurately, with little sample preparation, by gammacounting techniques.(4) 13N is inherently less hazardousto use in comparison withconventional, much longer lived tracers. Theproblem of radioactive wastedisposal is eliminated (Cooper et al., 1985).Nevertheless, the disadvantages are alsorelated to its short half-life.It is only available at relatively few research centerslocated close to thecyclotron. Its production requiresa suitable accelerator and acorrespondingly large capital investment(Cooper et al., 1985). Its shorthalf-life limits the period over which it canbe 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 appropriatelyrapid (Fuhrman et al.,1988).2.2.4.4. Considerations of using 13Nin nitrogenuptakeTo study nitrogen uptake, especially ammoniumuptake by plantroots, several facts have to be considered:(1) Membrane fluxes of nitrogen are of utmostimportance for the over-allnitrogen utilization in plant growth.31(2) Ammonium is rapidly metabolized to amino acids and amideswithinthe root before transport to the shoot (Pate, 1973). Evidenceshowed thatthe NH4 uptake rate is also regulated by theN assimilation andtranslocation rates of the plants (Wiame et al., 1985;Morgan and Jackson,1988). Therefore it is necessary to identify the nitrogencompounds in theuptake, assimilation and transport processes.(3) It is difficult to measure the subcellular, i.e. cytoplasmic andvacuolar,pools of N03 and/or NH4 directly due to their small size and rapidturnover. It was found that the half-time for exchange ofthe 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 adaptduring a long-termexperiments in response to changes of environmentalconditions, such astemperature or pH (Macduff et al., 1987). Therefore the tracertechniquecan be chosen as a proper approach to study ammoniumuptake by riceroots in consideration of high sensitivity, rapid measurementand shortduration of experiments. Another point is that uptakeby depletion is soslow from high external concentration that it can notbe measured exceptwith ‘3N.2.2.4.5. Use of1Nin nitrogen transport studiesIn short-term experiments, ‘3Nhas been used to study nitrogenuptake by plant roots (McNaughton et al., 1983; Glasset al., 1985; Lee etal., 1986; Oscarson et al., 1987). Most reportedstudies used‘3NO’inuptake experiments; few made use of 1NH4.‘3N0 has been used toidentify and characterize the transportsystems (Thayer and Huffaker,1982; McNaughton and Presland, 1983;Siddiqi et al., 1990; Glasset al.,321990); regulation of influx (Glass et al., 1985; Oscarsonet 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 ammoniumuptake by roots ofhydroponically grown maize seedlings and thetransport of 13N to theshoot. It was found that the rate of uptake ofammonium, by Zea mays,was a function of external ammonium ion concentrationat less than 1 mM.2.2.4.6. Use of13Nin nitrogen assimilation13N has also proven useful in understandingnitrogen assimilation inplant cells. Gin is the first major organic productof‘3NH4 assimilation(Skokout et al., 1978) and the GS/GOGAT pathwayis the primary route ofassimilating fixed ‘3N (Meeks et al., 1978a). Itwas found that MSXinhibited the incorporation of‘3NH4-’- into Gln morethan into Glu. Theopposite was true for‘3N0.In tobacco cells GDH onlyplays a minor role(Skokout et al., 1978) but in non-leguminous angiospermN2-fixers, GDHmay play a major role in the assimilation ofexogenously supplied NH4(Schubert et al., 1981).Since 13NH4can be produced in hundreds of millicuries,it should bepossible to synthesize a large numberof 13N-labeled amino acids,nucleotides, amino sugars, and other metabolitesvia known enzymaticroutes (Cooper et al., 1985). Organic N-containingcompounds, such as L(13N)-glutamate and L-(amide-’3N)-glutamine, are also synthesized from13NH4and used in studies of NH4 and glutamineassimilation pathways(Suzuki et al., 1983; CalderOnet al., 1989). It was found in Neurosporacrassa that(13N)-Gln is metabolizedto(13N)-Glu by GOGAT and to 13N}I4by the glutamine transaminase-o-amidasepathway. Then released1NH4is reassimilated by both GDHand GS (Calderón et al., 1989). Extracted‘3N-33labeled amino acids or amides can be separatedby HPLC andelectrophoresis (Cooper et al., 1979; Meeks, 1993). Itwas found thattranslocation of N compounds can also be traced by 1N.Barley leavesexposed to‘3NH gas for 30 mm, incorporated 1N mainly intofree Gin andGlu and 1 to 3% of these were exported to the sheaths throughthe phloem(Hanson et al., 1979).2.2.4.5. Use of 13Nin denitrificationIn addition 1N has also been used to study denitrificationin soils(Gersberg et al., 1976; Tiedje et al., 1979; Bremnerand Hauck, 1982). Useof ‘3N allows the direct quantitative measurementsof denitrification ratesover short time intervals, without changingthe concentration of N03 inthe soil system from flooded rice fields (Gersberget al., 1976).2.2.5. Protocol for 13NH4production in present studyThe short-lived radioisotope 13N (t1/2= 9.98 minutes) was producedas described by Siddiqi et al., (1989),by 20 MeV-proton irradiation of H20on an ACEL CP42 cyclotron. Contaminants inthe‘3N0 sample (mainly18F) were removed by passing thesamples twice through a SEP-PACAlumina-N cartridge (Waters Associates).Reduction of‘3N0 to 13NH3wasachieved by using DevardaTsalloyat 70°C in a water bath (Vaalburg et al.,1975; Meeks et al., 1978); 1NH3was separated from remaining chemicalspecies by distillation at alkaline pH, and trappingin acid solution as13NH4-’-. The flow scheme for thisconversion is shown in Figure1.3413N0+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 (hereafterreferred to as‘loading’ solution) for designated periods;(b) pre-wash and post-wash:prior to and after loading, roots werepre-washed and post-washed in unlabeled MJNS (hereafter referred to as ‘washing’solution) for 5 mm and 3mm, respectively. The choice of these times isrationalized in the Discussionsection (section 3.4.). Experiments wereconducted at steady-state withrespect to [NH4--]0,i.e., the [NH4]0 of ‘washing’ solutionsand ‘loading’solutions were the same as those providedduring the growth period or inexperiments to define influx isotherms; plants wereexposed to different[NH4]0for short (perturbation) experiments. Immediatelyafter the post-wash period, plants were cut into shoots and rootsand the surface liquidadhering to the roots was removed by astandard 30 sec spin in a slow-speed table centrifuge (International ChemicalEquipment, Boston). Rootsand shoots were introduced intoseparate scintillation vials andimmediately counted in a gamma counter(MINAXIy-5000,Packard). Thefresh weights of roots andshoots were recorded immediately aftercounting.2.3.2. Efflux of 13NH4Roots of rice seedlings were immersed inthe1NH4labeled ‘loading’solution for 30 mm. Atthe end of this time plants weretransferred to an36elusion vessel and tracer leaving the roots in exchangefor‘4NH in theun-labeled identical ‘washing’ solution. This solutionwas collected atprescribed interval in 20-ml scintillation vials forcounting.2.3.3. Net flux of NH4Net NH4 flux was measured in uptake solutionsby the depletionmethod. Solution samples (S1 and S2) were taken at different times(t1 andt2), and the difference of assayed [NH4]was used to calculatenet NH4flux. Net NH4 flux can also be estimated by subtractingefflux from influxof the same roots.2.4. CoMPARTMENTAL (EFFLux) ANALYsIs2.4.1. Compartmentation of plant cellsPlant cells are highly compartmentalized. Theyare surrounded bythe cell wall, and the plasma membrane encloses thecytoplasm, in whichare found the vacuole, mitochondria, nucleus, plastidsand other organelles.Up to 80% or more of cell volume is occupiedby the vacuole which isenclosed by the tonoplast (Salisbury andRoss, 1985). The cytoplasm is thevital part of cell. The major functions ofthe vacuole are to maintain turgorwhich contributes to cell shape and tostore solutes. The compartmentationof the cell has important consequencesfor nutrient uptake, unidirectionalfluxes, assimilation, distribution andtranslocation. Because higher plantcells are too small to dissect andthe size of the compartmentsis even37smaller, it seems technically impossible to obtain informationon thecomposition of each compartment. However, throughvarious methods,such as NMR, ion-specific electrodes, EDX, compartmentalanalysis, orfluorescent dyes, the ion concentration of one ormore particularcompartments, or fluxes between compartments canbe estimated.Compartmental analysis is the only systematic methodof investigatingtransport processes and estimating the size ofcompartments and toanalyze the kinetics of movement of ions to or froma tissue (Cram, 1968).Therefore it has been established as a tool for characterizingthe exchangeproperties of multicompartment systems.2.4.2. Development of theoryCompartmental analysis was first usedby Fourier in 1822 todescribe the relationships between heat flow andtemperature 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 transportprocesses in a higherplant. Compartmental analysishas mostly been used by plant physiologiststo calculate the fluxes, characterize internal ion poolsizes and membranekinetic parameters for ion exchange.The basic assumption of this methodology isthat the system is atsteady state, or at equilibrium. Additionalassumptions include that (1) thesubstance of interest flows into and fromthe separate compartments ofthe system; (2) the flux is proportionalto the quantity (or concentration) of38the substance in the compartment from which thematerial flows. It isassumed that the material under study is neitherdestroyed norsynthesized in any compartment, and thateach compartment ishomogeneous, or well stirred; (3) the concentrationof an ion species or itsflux is described by a first-order linear differentialequation with constantcoefficients which are independent of elapsed timeand of the conjugate(Zierler, 1981). For higher plant systems, the additionalassumption is thatthe relevant compartments of the experimental systemare functionally inseries with each other (Walker and Pitman, 1976; Cheeseman,1986). Theseassumptions may not always be valid (Lazof andCheeseman, 1986). It issuggested that compartmental efflux analysis should notbe used alone, butintegrated with other methods such as influx measurements(Cheeseman,1986).2.4.3. Models for compartmental analysisThe testing model or the analysis processcan be varied with theresearch subject (excised tissue or intact plant), numberof compartments(2, 3, or more), nutrition status (steady ornon-steady) (Walker andPitman, 1976). The conventional compartmentalanalysis is suited todetermine unidirectional fluxes and compartmentalcontents 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 tobe small in excised roots(Macklon, 1975), the xylem transport in intact plantswas not included inthis conventional model (Pitman, 1963;Etherton,1967; Pallaghy et al.,1970). However, the method was modifiedby Pitman (1971, 1972) tostudy Cl- uptake and transport in barleyroots. Tracer efflux from the39cortical cell surface and the transport of tracer into thexylem weremeasured and analyzed separately. A three compartmentmodel, includingxylem transport, was tested in the study of unidirectionalfluxes of Na inroots of intact sunflower seedlings (Jeschke and Jambor,1981). In twocompartment models, xylem transport was also consideredin studies of‘3N0 fluxes in roots of intact barley seedlings (Lee andClarkson, 1986;Siddiqi et al., 1986). The two compartments included thecell 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 compartmentalanalysis(Walker and Pitman, 1976) is based on the assumptionthat (1) thecytoplasm and vacuole are in series; (2) the cytoplasmiccontent is verymuch less than the vacuolar content; (3) the tissue is ina steady state(Cram, 1968). Therefore one may expect that at steady-stateconditions ofroots:S = S0 (1- ekct)[3]when roots are exposed to a radioisotope-labeled mediumwith specificactivity S0, the radioisotope content of thecytoplasm S increasesexponentially with time (t) and the rate of tracerexchange of thecytoplasm(kc)is given by the relationship(kc=O.693/tl/2).The quantity ofradioactivity inside the cellQis given byQc*AtcpocSc[4]where A is a cross sectionconstant andpis the flux from outside tocytoplasm. The fluxes in oppositedirections, between cytoplasm andvacuole are considered to be equalat steady state:40Øcv =[5]then the flux into the cytoplasmPoc=Øco + - xc;(if4<< 0 it may be neglected)[6]therefore net uptake of an ionJoc = Poc - Pco [7]and the transport of ion from root to shootthrough xylem would beJox = - Pxc [8]if roots were uniformly labeled after 16-24hours loading:S,= S= S0[9]and the specific activity in the xylem can be estimated fromthe transportrate of tracer (cI(t)) and transport rate of ion(J0(t))with the assumptionthat the symplasm behaves like a rapidly mixedphase and has a uniformspecific activitySc= I(t) /J0(t) [10]Based on these relationships, one is ableto estimate unidirectional fluxesand other parameters for each ofthe compartments.A biphasic efflux pattern suggests two phases,outside and inside theplasma membrane (Luttge andHiginbotham, 1979). Since the fastestcomponent was found in bothliving tissue and chloroform-killed tissue,Cram (1965) concluded thatthe fastest component of efflux of tracer Clfrom carrot tissue probablycorresponded to the apparent freespace(AFS). After treating barleyroots 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 (Siddiqiet al., 1991).Therefore this rapid efflux component probably correspondsto the AFS.Another approach has been to use different sizes ofmolecules to confirmthe AFS phase. It was found that [1,2-3H] polyethyleneglycol(3H-PEG) istoo large to diffuse into AFS, but D-[1-’4C] mannitol isable to diffuse freelyin the AFS without been absorbed by root cells (Shoneand Flood, 1985).After loading with a mixture of3H-PEG and D-[1-’4CJmannitol, plant rootswere washed in unlabeled solution. Since the ratio of 3Hand ‘4C should besame from the surface film of ‘loading’ solution carriedover 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 thatthere was an initialrapid release of 90% of H and ‘4C within the first 1 mmbut more ‘4C wassubsequently released (Lee and Clarkson, 1986). Thereforethe rapidlyreleased radioactivity during early efflux is probably fromthe AFS.A tricompartmental efflux pattern (including theapparent freespace) were reported for Cl- in carrot root slides or isolatedcorn rootcortex (Cram, 1968; 1973), and excised or intactbarley roots (Pitman,1963, 1971); and for Na and K÷ in intact barleyroots (Poole, 1971a,1971b; Jeschke, 1982). Based on the resultsof compartmental analysis andother studies, Cram (1965) concluded that, in additionto the fastest effluxfrom the AFS, the two slower componentswere considered to besubcellular in origin, the cytoplasm andthe vacuole. Further quantitativeconsiderations and model fitting suggestedthat the cytoplasm and thevacuole are arranged in series withdirect connection between theexternalsolution and the cytoplasm,but not between the external solutionand thevacuole (MacRobbie, 1964;Cram, 1965).42Also a third small symplastic kinetic compartmentmay exist inaddition to the bulk cytoplasm and vacuole (Luttgeand Higinbotham,1979; Lazof and Cheeseman, 1986). Ina study of sodium transport inSpergularia marina, Lazof and Cheeseman (1986) found.that the rapidfluxes involved only a very small portion of the totalNa in the roots butthe authors were unable to identify the physicalentity corresponding tothe compartment identified. There were also severalsimilar reports inother transport studies. The additional compartmentcould be the smallportion of the bulk cytoplasm connecting to the vacuole (Pitman,1963); orthe cytoplasm can exchange with both vacuoleand plastids (Walker andPitman, 1976); or the possible involvement ofvesicles 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 compartmentalanalysisThe general procedure for compartmentalanalysis has beendescribed in detail (Walker and Pitman,1976; Zierler, 1981; Rygiewicz etal., 1984). Several radioisotopes havebeen used in compartmentalanalyses,36C1,82Br, 42K or86Rb+,22Na, 45Ca, and 28Mg. One partofthis technique involves theuse of radioisotopic tracers to measure influxand efflux, the separate components ofthe net flux. The second part is amore systematic method to analyse thekinetics of movement of ionsto orfrom different compartments (Cram,1968). The basic assumptionof thisprocedure is that radioisotope loadedinto different compartmentswill bewashed out with different rate constants.43After allowing plant tissues,cells or roots to loadwith radioactivetracer for a designated duration,the efflux of this radioisotopeis measuredfor a prescribed period oftime. Dependingon the type of ion studied,thereare two ways to count theradioactivity. Fornonmetabolized ions,such asCl-, Br-, K-’-, Na, and Mg+,the radioactivityremaining in thetissue atthe end of elution canbe counted. By countingthe eluatesat differenttimes the counts remainingin the tissue at thesetimes can be estimated.For metabolized ions, however,counts remaining inthe tissues wouldbemisleading becausethey consist of the radioactiveion under examinationand the metabolic products ofits assimilation. Inthe latter case therate ofefflux, rather than countsremaining must beestimated as a functionof theduration of elusion. However,even this methodrequires thatthe identityof the effluxed ion beconfirmed.Plotted as a functionof time on a semi-logarithmicplot, the activitydata (e.g. cpm remainingin system or effluxrate) are resolvedintodifferent linear phaseswhich have been interpretedas correspondingtodifferent compartmentswithin the cells. Oneflaw in this methodhas beenthe subjective basisof line fitting (curve-peeling)of data which hasimplications for the numberof exponential terms andtheir coefficients. Toimprove the method,Rygiewicz et al. (1984)proposed a microcomputermethod in which maximizationof r2 for linear regressionserves as thecriterion to determinedata points belongingto each compartment. Thisdevelopment greatlyincreased the accuracyof parameter estimation(Rygiewicz et al.,1984) and the objectivityof the estimatedresults(Cheeseman, 1986).Selected parametersobtained fromcompartmentalanalysis fromseveral sources areshown in AppendixB. It was reportedthat the half-44lives of C1 exchange for apparent free space, cytoplasmand vacuole were1.4 mm, 10 mm and 300 h, respectively for carrot root tissue(Cram, 1968).In excised barley roots, a slow, vacuolarcompartment, was not visibleeven after 10 h of exchange (Behl and Jeschke, 1982).It must be kept inmind that compartmental analysis alone does notallow one to identifyeach compartment (Luttge and Higinbotham, 1979), one mustinterpret theresults with necessary caution and verify these correlationsindependently.For example, several techniques are availableto identify and quantify thevacuole (Clarkson and Luttge, 1984).2.4.5. Procedures for compartmental analysisin the present studyFor better time control of the separation of ‘washing’solutions fromthe 13NH4-labeled roots during the effluxprocess and to reducedisturbance of roots, I devised a simple apparatusin which to perform theefflux study. The spout of aplastic funnel (100 mm diameter)was cut tofit into the barrel of a 25cc plastic syringe, into which it was sealed.Alength of rubber tubing replacedthe needle end of the syringe and a metalspring clip on the tubing functionedas drainage control. A small holewasdrilled in the wall of the syringe barrelnear the bottom, and a needleintroduced through this hole to provide foraeration. This technique alsoresulted in good mixing of the ‘washing’ solution.Roots of rice seedlings used forcompartmental analysis wereimmersed for 30 mm in the ‘loading’ solution.These pre-labeled rootswere carefully introduced into the syringebarrel for elution. Samplesof 20ml ‘washing’ solution were poured intothe efflux-funnel and allowedtoexchange with the 13N-labeled roots.After prescribed intervals,this45solution was drained from the funnel directlyinto a 20-ml scintillationvial, by opening the drainage clip. Fresh‘washing’ solution was poured intothe efflux-funnel from the top of the funnel, immediatelyafter closing thedrainage clip. The duration of successivewashes 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 thelast wash, the plants werecut into shoots and roots and introduced into separatescintillation vials.The radioactivities of all samples werecounted immediately. In order tobeassured that the13Nspecies that had effluxed from theroots was 13MI4rather than any metabolic products, twoother sets of13NH4-labeled rootswere effluxed for 30 mm in 750 ml ‘washing’ solution.Two 20-ml samplesof the efflux solution from each beaker were takenand separated by theCEC procedure (see below) and counted forradioactivities. Theradioactivities released from intact rice roots intoefflux solutions during18 mm efflux experiments, were counted, convertedto efflux rates andplotted versus time in semi-log plots (see Fig. 2 insection 3.3.1.). Thismethod of analysis is required because NH4 is rapidlymetabolized in riceroots (Yoneyamo and Kumazawa, 1974), and convertedinto amino acidsand proteins. As a consequence, standardmethods of compartmentalanalysis (Walker and Pitman, 1976),based on semi-log plots of cpmremaining in the tissue plotted against timeare not appropriate. Hence thevalues of log of rate 13NH4released againsttime 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 extractedfrom rice roots by use ofa CationExchange Column (CEC) separation basedon the methods of Fentemet al.,(1983a) and Belton et al., (1985) and determinedby the indophenol bluecolorimetric method (Solorzano, 1969).The procedure was as describedinWang et al., (1993a): in brief, after desorbing inNH4-free MJNS for 3 mmto remove NH4 in the cell wall, the rootswere cut, weighed, and groundwith liquid nitrogen in a pre-cooled porcelainmortar and extracted with10 ml of 10 mM sodium acetate buffer(pH 6.2). The resulting slurrywaspassed through a Whatman #1 filter paperand then washed 3 timeseachwith 5 ml of the same buffer solution. The filtratewas passed through theCEC filled with 3 ml of resin (Dowex-50,200-400 mesh, Na form). TheNH4 adsorbed on the CEC column was eluted using250 mM KC1. Theconcentration of NH4 in solution was determined bythe indophenol bluecolorimetric method (Solorzano, 1969).2.6. PREPARATIoN OF METABoLIC INHIBITORSThe same metabolic inhibitors were usedin the 1NH4influx study(Chapter 5) and electrophysiological study(Chapter 6). The inhibitors usedwere as follows: (1) CCCP (10iiM):carbonylcyanide m-chlorophenyihydrazone dissolved in ethanol;(2) CN- plus SHAM (1 mM): NaCNplus salicylhydroxamic acid dissolvedin water. The resulting alkaline pHwas adjusted by titration withH2504 to pH 6; (3) DES(50 1iM):diethylstilbestrol dissolved in ethanol;(4) DNP (0.1 mM): 2,4-dinitrophenoldissolved in ethanol; (5) Mersalyl(50iiM):Mersalyl acid dissolved inwater; (6) pCMBS (1 mM):p-chloromercuribenzene-sulfonatedissolved in47water. The acidic pH was adjusted by titration with Ca(OH)2to pH 5.8.Ethanolic solutions of CCCP, DES and DNP wereadded to the nutrientsolutions to give a final ethanolic concentration of1%. Control solutionswere treated with ethanol at the same concentration.2.7. ELEcm0PHYsI0L0GIcAL STUDY2.7.1. Transmembrane electrical potentialmeasurementUsually plant cell transmembrane potential differencesare in therange of -100 to -200 mV negative inside (Higinbothan,1973; Tester,1990). In the early 1930’s, Umrath started to usemicroelectrodes tomeasure the membrane potential across the tonoplast(Findlay and Hope,1976). Since then, other electrical properties of plantcells have also beenstudied such as membrane capacitance (Curtis and Cole,1938), membraneconductances (Cole and Curtis, 1939), andmembrane resistance(Higinbotham et al., 1964; Spanswick,1970; Anderson et al., 1974). Thecontemporary climax of electrophysiologyoccurred when Neher andSakmann (1976) developed of patch-clampingtechniques. The combinationof molecular gene cloning and patch-clamp analysis(Hedrich et al., 1987)represents a particularly powerful meansof 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 workand can be expressedby theequation [11]:48iO+RTlna+z1FVP+mgh[11]where j.i,j is the chemical potential of the ion fin joulesmol-1 and is the’standard state chemical potential of 1 mole of the ionsfper liter at 0°C; Ris the gas constant (8.314Jmol” °K’); T is absolute temperaturein °K (°K= 273 + [°C]);a3 is the activity of the ion; Z3 is its valency; F isthe Faradayconstant (9.65 x 1OJmol1V);V is the electrical potential involts; 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 smalland h is generallynegligible. When the concentration(C1) of the solute is low so that theactivity and concentration are close, concentration C1 (molrn-3)can be usedin place of the activity a1 (a1= yjC3),whereyjis the activity coefficient.Simple diffusion is a non-mediated transport processwhereby thesolute moves along the free energy gradient. In additionto the lipidcomposition, the difference of ion concentrationjust inside and outside theplasma membrane determines the diffusion of soluteacross a membrane.Ion diffusion through membranes may be describedby the permeabilitycoefficient which is the flux per unit driving force(in its originalconception, the concentration gradient). Forthe diffusion of smallnoncharged molecules such as NH3 and H20, thechemical potential=+ RT ln C1[12]can be expressed as in equation [12]. Since the drivingforce is only due tothe concentration gradient from highto low (negative sign), the net fluxJ3(mol m2-1)is expressed as in equation [13]:J3= K1J(-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/dx)[15]Equation [15] is Fick’s First Law of diffusion, whereK1 is the proportionalcoefficient or the mobility of the ionj,and D1 is the diffusion coefficient ofspecies fin m2 s. If P1 (m s-i) is the permeability coefficientof themedium or the membrane for ionj,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) isa measure of theability of the species of small non-electrolyte topass through a membrane.The permeability coefficient for isopropanol orphenol is10-6m sec1across the plasma membrane (Nobel, 1983).The diffusion of most ions across the membraneis very low due totheir low permeability compared to non-electrolytes.In addition to theconcentration gradient, the electrical potentialgradient 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 electrochemicalpotential gradient(11*])determinesthe potential for passive ion flux. Atequilibrium both outside and insideelectrochemical potentials are thesame:50=-= 0[19]combining equation [18] and [19]= (RT in C1 + zF’{ - (RT in C0 + zF%)[20]where : z$C10 is the electrochemicalpotential difference across themembrane; and C1 are the electrochemicalpotential outside and insidethe cell membrane respectively, ‘P1 and ‘P0 representthe inside and outsideelectrical potentials, respectively, measuredas V; C1 and C0 are theconcentration (mM or mol m3) inside and outside thecell membrane,respectively.Because of the selective and permeable nature of membranesandthe existing concentration asymmetry, theelectrical potential difference atzero net flux, when zs.i = 0, is definedas the Nernst potential(‘PN)as inequation [21]:RT C0=------ ln( ) [21]zF C1This is the Nernst equation whichdescribes the electrochemical potentialof an ion distributed at thermodynamicequilibrium between two phasesseparated by a cell membrane. Consideringmonovalant 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 themeasured membrane electrical potentialdifferences acrossthe membrane in volts(‘-PM= ‘P-•‘fe),normally this potentialdifferenceacross the plasma membrane is large for plantcells (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 whichmay 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-) whichmay have differentmembrane permeability (PK÷, PNa andPcii. Presuming that there isinitially no electrical asymmetry across the membrane, whenions movealong their chemical potential gradient, differentmobilities of cations andanions result in charge separation which createsan electrical potentialdifference, known as a diffusion potential(‘PD).It can be assessed by theGoldman voltage equation:RTPK[K]o + PNa[Nalo + Pi[Cl]j +ln( )[24]FPK[KJj + PNa[Na]j + Pi[C1]o +The second source of membrane potentialis the Donnan potential,though the contribution is relativelysmall. Inside the plant cell, there aremany large organic molecules, suchas protein and other large polymers(RNA and DNA), with a large numberof immobile carboxyl, phosphate andamino groups from which H can dissociate.The asymmetrical distributionof diffusible cations leads toa small negative potential across the plasmamembrane (negative inside)(Nobel, 1983).52Thirdly, a major component is a metabolically-driven potentialdueto the operation of an electrogenic ion pump - the H pump.The H-’- pump(H-translocating ATPase) carries a net positive chargeacross themembrane and contributes directly to the membranepotential (Poole,1973; Sza, 1984). The activity of H pump depends on thehydrolysis ofATP catalyzed by a plasma membrane ATPase (Hodges, 1973;Poole, 1978;Spanswick, 1981). From equation [20], one can obtain anequation whichcalculates the electrochemical potential difference for protonat 25°C:AtH= A’P + 59 zpH[25]A proton concentration difference (ApH) and an electricalpotentialdifference (AP) are two related entities that make up theelectrochemicaldifference 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 necessaryto 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 ofprotons 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 electricalpotentialdifference (equation [18]), it is used to describe the freeenergy status of asolute in a particular location. It is assumed thata difference of freeenergy between two points of a systemrepresents the driving force for apassive flux of ions from one point to another.When the resultant chemicalpotential difference is just balanced bythe resultant electrical potentialdifference(AI*jo= 0), there is no net flux of solute bypassive forces.53Alternatively it can be stated that no energy isexpended in moving ionsbetween the two locations.2.7.2. Single impalement and membrane potentialMicroelectrodes are commonly preparedfrom a micropipette filledwith electrolyte solution. It is a filament-containingor single-barreledborosilicate glass capillary tube with the fine-tipwhich is pulled witheither a vertical or horizontal electrode puller (Purves,1960; Findlay andHope, 1976). The external diameter of the tip shouldbe 0.5% or less of thediameter of the plant cell which itis to impale (Purves, 1960). Forcytoplasmic insertion, a tip diameter of 1 to 2 jimis usually satisfactory(Findlay and Hope, 1976). A tip diameter of <0.5jim has often been used(Kochian et al., 1989; Ullrich and Novacky, 1990;Glass et al., 1992).However, the smaller the tip diameterthe higher the tip potential orelectrical resistance (Findlay and Hope, 1976).Membrane potential difference can be easilyexpressed in a numberof equations (refers to section 2.7.1.), such as theNernst potential (Eq.[21]), or electrochemical potential (Eq.[24]), or the Goldman diffusionpotential (Eq. [21]). When the potential difference ismeasured by insertedmicroelectrode, the value is an apparent restingpotential 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 dissimilaritybetween the indifferentelectrode and the electrode which contactsthe microelectrode’s fillingsolution. The latter can be compensatedby the offset control of theoscilloscope amplifier. The liquid junctionpotential occurs between themicroelectrode’s filling solution and the electrolyteoutside the tip. It can54be decreased by use of 3 M KC1 as filling solution sincethe diffusioncoefficients of K and of C1 are almost identical.The tip potential is due to the characteristics of glass wall, electrolyteconcentration difference between inside andoutside of the tip ofmicropipette and can be eliminated by filling themicropipette with low pHsolution or other treatments (Purves, 1960). A tip potentialof -5 to -30mV was recorded for the microelectrode filled with 0.5 M KC1plus 0.1 MMes (pH 5) (Ulirich and Novacky, 1990). The electrolyte solutioncould 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 technologycan beregarded as a succession of attempts to minimize tipdiameter andresistance simultaneously.2.7.3. Setup for measuring membrane potentialThe fundamental setup for measuring electrical potentialdifferencebetween two aqueous phases (cell ambient and cytoplasm), isan electricalcircuit which should be connected by a salt-bridge,i.e. Hg2C1 plus KC1(Willians and Wilson, 1981). The microelectrodeis 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. Theelectrical signals are amplifiedthrough a preamplifier (or electrometer),and are sent to the outputdevices such as the oscilloscope,the tape recorder, the penrecorder the55digital voltmeter or the audio monitor (Findlayand Hope, 1.976). Since theplant cells are tiny, vivid and fragile,the impalement the cell throughthecell wall and cell membraneis operated bythree-waymicromanipulators (Kochian et al.,1989; Glass et al., 1992)under themicroscope on an anti-vibrationtable (Purves, 1960). A diagramof such asetup is shown in Figure 2.2.8. Determination of amino acidsin 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 liquidN2 in a porcelain mortar and extractedwith 80% aqueous ethanol. After centrifugation(IEC Clinic Centrifuge), thesupernatant was transferred to an evaporatingflask. The extraction andcentrifugation were repeated5 times. Pooled extracts were evaporatedunder vacuum at 35°C ona flash evaporator (Buchler Evapomix).The crudeextracts were then re-suspendedinto 5 ml of distilled deionizedwater.After mixing 5 ml of chloroformwith the crude extract,the supernatant(aqueous phase) was collected intoan Eppendorf tube (1.5ml) for furthercentrifugation and lyophilization.The extracts were derivatizedwithphenylisothiocyanate (PTC) automaticallyon an Amino Acid Analyzer(ABI,Model 402A) equippedto derivatize and hydrolyze appliedsamples, andthen separated by HPLC analysis(Separation system, ABI130A). Theamino acid concentrationswere determined by the AminoAcid Analyzerand analyzed by means ofan ABI 920A data analysismodule. Thechemicals used as amino acidstandards were fromSigma.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 membraneelectrical potential.57Chapter 3. FLUXES AND DISTRIBUTIONOF 13NH4IN CELLS3.1. INTRODUCTIONThe short-lived radioisotope ‘3N (t172= 9.98 mm) has been used as atracer in studies of the fluxes of NO3-andNH4 into intact roots ofcorn andbarley plants (McNaughton and Presland, 1983;Glass et al., 1985; Lee andClarkson, 1986; Hole et al., 1990; Siddiqiet al., 1991). It provides amethodology for the measurement ofunidirectional fluxes (influx orefflux) across biological membranes over extremelyshort 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 gammacounting techniques(McNaughton and Presland, 1983; Cooper etal., 1985; Meeks, 1992).The major emphasis in studies of N uptakehas been upon N03,reflecting the widely held perceptionthat N03 is the predominant form ofN available to crop species. Relativelyless is known about the uptake andsubcellular partitioning of NH4 in higherplants. Nevertheless in ricecultivation (Sasakawa and Yamomoto, 1978), in forestecosystems (Lavoieet al., 1992), in Arctic tundra (Chapinet al., 1988) and even in wintervarieties of cereals growing in cold soils (Bloom andChapin, 1981), NH4may represent the more important formof available nitrogen.It was demonstrated that net fluxes of NH4into rice roots graduallyacclimated between 0.1 and 1 mM external[NH4+] so that net flux atsteady-state varied little betweenplants grown in these concentrations(Wang et al., 1991). Nevertheless,there is a lack of information about58fluxes between subcompartments in relation to acclimationor to themechanism(s) of NH4 uptake. For example, Preslandand McNaughton(1986) failed to observe‘3NH4 efflux from maizeroots. By contrast, asizable net efflux of endogenous 14NH4was reportedin wheat, oat, andbarley upon transfer to‘5NH4 solution, although therewas no exactcorrelation between root ammonium concentration andnet 14NH4efflux(Morgan and Jackson, 1988a, b).The internal NH4-’- concentration of plant rootscan readily beassayed, after extraction, by methods based on colorimetry orion-specificelectrodes (Fentem et al., 1983a; Morgan and Jackson, 1988a,1988b;Roberts and Pang, 1992). However, such analyses failto provideinformation on the subcellular distribution of NH4.On the basis ofbiochemical analysis, it was concluded that more thanone intracellularpool of NH4 existed in roots of rice (Yoneyama and Kumazawa,1974,1975; Arima and Kumazawa, 1977). Two othermethods have beenemployed to determine subcellular NH4 distribution,namely, effluxanalysis (Macklon et al., 1990) and the nuclearmagnetic resonancespectroscopy (Lee and Ratcliffe, 1991; Roberts andPang, 1992). Thesestudies recognized several NH4-’- fractions of roots,corresponding to thoseof the superficial, water free space, Donnan freespace, the cytoplasm andthe vacuole.In this chapter, the results of compartmentalanalyses, using13NH4+efflux, are used to estimate the half-livesof NH4 exchange and the size ofmajor compartments in root cells, as wellas NH4 fluxes between thesecompartments. Together with data obtained fromchemical fractionation, itwas possible to develop a detailed analysis of theinitial fate of absorbed‘3NH4.In addition, thet1/2 values for 13NH4exchange provide essential59parameters for the design of appropriateprotocols for influx measurement,particularly the duration of‘3NH4loadingand post-wash treatments.Toevaluate the methodology of the compartmentalanalyses, influx and netflux of NH4 were also measured by independentmethods.3.2. MATERIALS AND METHODS3.2.1. Plant growth and ‘3N productionDetails of seed germination, growth conditions,provision of nutrientsand production of1NH4are described inSections 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 effluxanalysis: After ‘loading’for10, 20, and 30 mm, respectively,at steady-state conditions, influxof13NH4 was also determinedby two independent methods:(1) theaccumulation of 13N by seedlingroots (see section 2.3.2.); (2) therate ofdepletion of 13NH4-’- from ‘loading’solution.3.2.2.2. Net NH4fluxIn addition, the net flux ofNH4was also measured basedon the rateof depletion of 14NH4(seesection 2.3.4.).603.2.2.3. Time course of13NH4uptakeIn the time-course experiments, G2 or G100plants were exposed to 2iM or 100 jiM13NH4-labeled loading solutions, respectively,for durationsranging from 10 sec to 31 mm. As described in section2.3.1., roots weresubjected to a standard pre-wash, loading andpost-wash procedure.3.2.3. Compartmental AnalysisThe procedure for compartmental analysis wasfollowed asdescribed in section 2.4.5.3.2.4. Partition of absorbed ‘NH43.2.4.1. Separation of13N-compounds inplant tissue13NH4-’- was separated from its immediatemetabolic products byCation Exchange Column (CEC) Separation describedin section 2.5. Afterplants were loaded in 100 jiM‘3NH4for30 minutes, the separated, frozen13NH4-labeled shoots and roots were first countedin the gamma counterand then ground in liquid nitrogen. Afterthe filtration, the radioactivityremaining on the filter was referred toas root debris. The filtrate waspassed through the CEC filled with 3 mlof 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, containing100 120 plants each, were used.613.2.4.2 Chemical assay ofNH4in root tissueRoot NH4 contents(Qj)of G2, G100, and G1000 seedlingswereseparated and determined as described in section2.5.3.2.5. Calculation of flux to vacuole(4,)The results of CEC separation quantifiedthe un-metabolized 1NH4fraction in roots following 30 mm 1NH4loading. This amount (Q*c+v)represented the combined values ofcytoplasmic(Q*c)and vacuolar (Q*v)radioactivities that can be converted toa chemical quantity(Q+)afterdividing by the specific activity of‘3NH4÷in theexternal solution (S0):Qc+v=Q*c+v/So[261The specific activity of‘3NH4within the cytoplasm(Sc)during loading willincrease to its steady-state value accordingto 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 andt1/2 are known,S can be determined for any particulartime (t). By 30 mm of loading (equivalentto 4 cytoplasmic half-lives, seeTable 2), the specific activity ofcytoplasmic‘3NH4(Sc) is brought toapproximately 94% ofSand 13NH4 accumulatedwithin the cytoplasmalso reaches about 94% ofQ(in Table 4). Therefore, theproportion ofQ+transferred to the vacuole is givenby:62[27]and from Equation [27], the flux to the vacuole()can be roughlyestimated (Method I). The portion of Q*c+v thatis transferred to thevacuole(Q*)is given by:*_( / \ *v— ‘ +vI c+vThe accumulation of tracer in the rootvacuole is related to the chemicalflux to vacuole(Ø)and the specific activity of the cytoplasmat eachinterval:* _fCv (t) — ‘I’cv •-‘c (t)andQ*Oi*ES(t)[30]The sum of tracer accumulation within the vacuole Q*(Q*v(t))is givenby Equation [28], and ESc (t)can be calculated for each minutefromEquation [3]. Therefore, by means of Method II, it ispossible to estimateØ,more rigorously from Equation [30].3.3. RESULTS3.3.1. Compartmental analysisAnalysis of the ‘3N released into ‘washing’solutions duringcompartmental analysis revealed that99.5% of the radioactivity wasretained on the CEC (Table 1). Since positivelycharged amino acids(arginine, histidine and lysine) representedonly 5% of total amino acidsin633-week-old rice roots (Yoneyama and Kumazawa,1974), I interpretedthisresult to indicate that‘3NH4was the predominantN species released fromroots and adsorbed on the cation exchangeresins.The influence of [NH4]0on compartmentalanalyses was investigatedby using G2, G100, or G1000 plants,to represent inadequate, adequateandexcess N supply, respectively, priorto efflux measurements. Arepresentative sample of such data (18mm efflux) for G1000plants isshown in Fig. 3. Three distinct phases, havingdifferent slopes with high r2values were found for each of the threetypes of plants tested (G2, G100and G1000). These compartmentswere tentatively definedascorresponding to: (I) the superficial solutionadhering to roots, (II)the cellwall and (III) the cytoplasm, respectively.The half-lives for exchange(t1/2)of these compartments were calculatedto be 3 sec, 0.5 to 1 mm,and 7 to 8.5 mm, respectively(Table 2). According to Duncan’smultiplerange test, there were no significantdifferences for these values amongplants grown under differentconcentrations of NH4,exceptfor the cell-wall fraction of G2 plants.One important part of the compartmentalanalysis was to calculatethe fluxes of NH4 acrossthe plasma membrane of rootcells. Thesecalculated fluxes are in goodagreement with the values obtainedby moredirect methods using thesame root material (Table3). Influx(%)variedwith the NH4 levelprovided during the growthperiod. Average NH4influx values for G2, G100and G1000 plants were estimatedto 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 subtractingthe estimated values of‘3NH4-- efflux(derived from efflux analysis)from the influx of 13NH4,or by measuring64Table 1. Separation of13N-labeled compounds by cationexchange column.The loading solution, efflux solution and shoot extractwere 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•.—E6-C.? .5.4.‘5-,3.——‘-,2-z10- • - I • I •0 5 10 15 20Efflux time (mm)Figure 3. A representative pattern of 13NH4released fromintact 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 correlationcoefficient.66Table 2. Estimated half-lives of 13NH4exchange for threecompartments 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 wereloaded in 13N-labeledMJNS for 30 mm and effluxed in un-labeled identicalMJNS for 18 mm atsteady-state conditions with regards to [NH4]0.Each mean isthe averageof 4 individual efflux tests ± Se.Compartments G2 G100G1000I. Superficial(s)a3.42 ± 1.00 a 3.83 ± 0.24 a3.38 ± 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 ± 0.12a 8.33 ± 0.60 aaDuncan’s multiple range test was used to compare the meansof each compartment.Only means followed by a different letter are significantly differentat the 5% levelof significance.67Table 3. Comparison of‘3NH4fluxes across the plasmamembrane of rootcells. Each mean13NH4+fluxes (influx, efflux, and net flux) isthe averageof 3 or 4 replicates with ± Se.Methods G2G100 G1000Net flux(Pnet):NH4efflux analysisa1.06 ±0.0714NH4-’- depletion of mediuma1.11 ± 0.04Efflux():(7) 1NH4effluxanalysisa(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 analysisa1.20 ± 0.07(2) 1NH4accumulated in rootsb1.39 ±0.02(3) NH4depletion of mediumb1.37 ±0.02(4) 1NH4depletion of mediumb1.33 ± 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 ±0.17aBased on 30-mm uptake;bBased on 10-mm uptake.68net depletion of‘4NH from the uptake solution. Bothmethods 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 4fold higher than those ofG2 plants (Table 3). Fluxes of G1000 plantswere about 1.5 times thevalues of G100 plants. Efflux values, expressed as percentagesof influx,were 11%, 20%, and 29% for G2, G100 and G1000 plants,respectively, (Fig.4).Since the volumes of subcellular compartments arevery different(Steer, 1981; Patel, 1990), it is necessary to distinguishbetween NH4-’-content(Q)expressed as moles per unit weightof roots (pmol g’), andNH4-’- concentration ([NH4]) expressed as moles per unitvolume of acompartment (jiM or mM). The results of estimatedcytoplasmic NH4concentration([NH4]), chemically assayed total root NH4 contents(Q)ofG2, G100 and G1000 plants, as well as calculatedvalues of [NH4--]1,[NH4],QandQ,are presented together in Table 4. Values of [NH4-’-]1and[NH4-’-Jwere higher with higher levels of NH4provision. The values of[NH4-’-]c,were 5 to 6 fold higher in G100, and 10 foldhigher in G1000 plants thanin G2 plants. The values for the vacuolarpool were based on thedifferences between the total NH4 content inthe roots (Qj) and thecytoplasmic pool (O). Of the total NH4 of the roots,92% was localizedwithin the vacuole in G2 plantsand about 72% to 76 % in G100 and G1000plants. Chemical and radioisotopicquantities for various compartmentsused in calculating are presented in Table5. The specific activity ofcytoplasm(S(t))was calculated for each minute fromt=1 to 30 mm. BothEQ*v(t)and E S(t)were used for estimatingcv. TheP,estimated bymethods I and II are given in Table5.69.Efflux11%:.iIIralIJIiIdlIl90 Efflux 20%EffluxNet Flux 899k Net Flux 8O9 Net Flux 719kG2 G100G1000PlantsFigure 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 Table2.70Table 4. Size of ammonium pools in root cells. Ammoniumpools in rootcells of G2, G100 and G1000 plants at steady-state. The contentsof 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 20.553.86G1000 6.85 1.94 (28%) 4.91 (72%) 14.41 38.085.78aThe values of[NH4]and [NH4]were estimated from compartment analysis withfour replicates each and [NH4 was estimated fromQ3.bThe values ofQwere obtained from chemical NH4 assay with three replicates eachand are the same as the values of [NH4+].CThe values ofQwere calculated fromENH4]based on the assumption that thecytoplasm only had 5% of total cell volume.dThe values forQ,are based on the difference betweenQandQand the assumptionthat the vacuole occupies 85% of cell volume. In parenthesis,QorQ,respectively,are presented as percentages ofQ.713.3.2. Metabolism and translocation of 13NVirtually none of the‘3NH4absorbed by rice rootswas translocatedto the shoots (Table 1). It is improper to express thetranslocation of ‘3N(to the shoot) as j.tmol NH4 per gram fresh weight ofroots because (a) ‘3Nis transported from the root in the formof amino acids and (b) the specificactivities of these amino acid poolswere unknown. Therefore thetranslocation was expressed as a percentage of thetotal radioactivity (cpmaccumulated in roots plus shoots duringthe loading period). This totalradioactivity is equivalent to net absorptionof 1NH4.Furtherfractionation of root tissues of G100 plantsby the CEC separation revealedthat about 8.6% of the radioactivity provided byinflux 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 basedon the total cpm remaining in roots(Table 6).3.3.3. Time course of‘3NH4influx in rice rootsThe results of steady-state‘3NH4-’- uptakeby G2 and G100 plants,establishing the pattern of‘3NH4accumulationin rice roots, are shown inFig. 5. The accumulation of‘3NH4appearedto be linear for the duration ofthe 30 mm uptake experiments; the coefficientof determination of theselines (0.87 and 0.99 for G2and G100 plants, respectively) were high.In allcases, the intercept on the ordinatediffered significantly from zero(at 5%significance level). G100 plants hada higher accumulation ratethan 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+v238982 cpmg’1.46 imolg40.97 jimolg’Q__v0.49iimol 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 androot tissues. After30 minutes loading in 13N-labeled MJNS containing100 jiM NH4-’-,Fractionation of radioactivity in shoots and rootsof G100 plants werecarried out according to sections 2.5. and 3.2.4.1.Radioactivities areexpressed as percentages of total cpm in plants. Eachanalysis 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) basedon 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 3035Uptake time (mm)Figure 5. Cumulative uptake of‘3NH4by G2 and G100 roots. Timecoursestudy 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 wereused 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 influxof G100 plants appearedto be about 20 to 30% higher than the steadyvalue of influx. Beyond5 to10 mm, influx in both G2 and G100 plantsremained 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) withhalf-lives for 13NfI4÷exchange of approximately 3 sec, 1 mm and8 mm, respectively, wereidentified by means of compartmental analysis (Table2). Phase I isprobably due to the surface solution on rootscarried-over from the‘loading’ solution (Fig. 3). The second phaseis attributed to the cell wallfraction, or the apparent free space (AFS) which is the sumof the WaterFree Space (WFS) and the Donnan FreeSpace (DFS) (McNaughton andPresland, 1983 and references therein). The half-lifeof this phase (0.5 to 1mm) was shorter than the equivalent phasereported 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 of13NH4into G2 and G100 roots were measured in the timecourse study.Symbols are the same as in Fig. 4. Influx is expressed as (p.molg-’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 thecytoplasm. The half-livesofcytoplasmic exchange for G2, G100 and G1000plants ranged from 6.9 to8.3 mm, but the differences were statistically insignificant,although thecytoplasmic pool sizes varied accordingto the provision of NH4-- duringgrowth (Table 2). Siddiqi et al., (1991) showed thatbarley roOts, treatedwith SDS or pretreated by immersionin water at 70°C for 30 mm,accumulated and released significantlyless‘3N0 from phase III, butphase II appeared unaffected. These results wereconsistent with phase IIIbeing the cytoplasm. In studies of 1NH4 effluxfrom spruce roots,Kronzucker, H. (personal communication)has found that elevated [Ca2]0jthe loading and washing solutions reducedthe extent of phase II for‘3NH4 exchange in spruce roots, (which had similarhalf-lives to thoseobserved in rice) as would be expected if thisphase 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 parametersby efflux analysis using‘3N. Using 15NH4,Macklon et al., (1990)estimated the half-lives forcytoplasmic and vacuolar exchangeto 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 valuesfor cytoplasmic‘3NH4exchange, ranging from 4 to 10 mm in roots ofwheat. The latter values aremuch closer to those obtained in the presentstudies, i.e. 6.9 to8 mm(Table 2). The longer tl,/2 values reportedby Macklon et al., (1990) mayhave arisen from species differences and/ordifferences of methodology.In order to select appropriatedurations for the loadingand washingperiods employed in influx studies, itis important to estimate the halflives for 13NH4-’- exchangebetween different compartments(Cram, 1968).78The choice of a 10 mm loading time, used in the presentstudy and insubsequent13NH4+influx studies, was arrived at fromconsidering 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 of13N retained bythe plant roots or transported to the stem becomes as highas ±15% afterabout 40 mm (McNaughton and Presland, 1983); (2) ifthe loading time islong, compared to thet1/2 for cytoplasmic exchange for‘3NH4,the specificactivity of the cytoplasmic pool may approach saturation andthe‘3NH4--efflux term(4c0)will be maximized. The measured 13NH4influx underthese conditions would approximate the net flux(Pnet = Øoc - ‘Pco);(3)although the over-estimation of influx (see below) wasminimized by 20minutes, 10 minutes loading reduced that over-estimationto less then 10%(Fig. 6). The duration of the loading period and thepost-wash period is acompromise (Lee and Clarkson, 1986). Since the goalwas to measure theunidirectional flux across the plasma membrane(Ø),13N present in thecell wall should be removed during the post-washperiod. Based on theestimatedt1/2 of the cell wall fraction, a short post-washperiod of 3 mm(corresponding to 3 to 6 half-lives, Table 1) was adoptedin all influxexperiments. In order to equilibrate the cell wall fractionto any changes of[NH4--]0,rice roots were, therefore, always pretreated for5 mm in identicalun-labeled MJNS before loading in 13N-labeled MJNS.3.4.2. Fluxes of 13NH4into root cellsThe results of the present study showedthat13NH4+appeared to beaccumulated at a constant rate (r2 = 0.874and 0.997, respectively) during7930 mm loading of G2 and G100 plants understeady-state conditions (Fig.5). Moreover,‘3NH4accumulation increased withincreasing [NH4+]0of theloading solution. This observation is similarto previous reports indicatingthat the accumulation of ‘3N (eitheras‘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 arealso presented as plots ofinflux versus time (Fig. 6). Influx valuesbased upon very short exposuresto‘3NH4-’- were accompanied by large errors probablyassociated with thelower counts accumulated and a large multiplicativefactor involved incalculating influx on a per hour basis. Nevertheless,the data indicated thatinitial influx values were 20 to 30% higher than thoserecorded after 2 to5mm. After loading for more than5 mm, the influxes were 1 and 7.5 jtmolg1FW h4 for G2 and G100 plants respectively,and notwithstanding somevariation, remained reasonably constant forthe next 25 mm. Presland andMcNaughton (1984) noted a higher rate of1NH4accumulationin maizeroots during the first 2 mm that they attributedto apoplasmic filling. Inthe present study, although the roots weresubjected to a 3 mm post-wash,any tracer uptake by rice roots duringthe post-wash period wouldrepresent an over-estimate. The impact ofthese additional counts wouldbe to over-estimate the calculated influx valuesat shorter loadingintervals due to the multiplicative effectin calculating fluxes on a per hourbasis. This effect, which decreases as theduration of the influx periodincreased, was minimized at about20 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 anunder-estimate of influx duetorelease of absorbed ‘3N or‘5N as cytoplasmic specific activityreachessteady-state. I question thisinterpretation because: (1)the t172 for80exchange of1NH4from the cytoplasmic phasewas 8 mm for rice rootsgrown at various nitrogen conditions (Table 2); (2) the absolutevalue 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 measuredinflux wouldresult from efflux of tracer during the short durationof these exposures.Values for influx efflux(Ø)and net flux(Ønet)of 13NH4determined by efflux analyses corresponded very well withthose obtainedby other (more direct) methods (Table 3). This closecorrespondence allowsus to accept the parameters derived from‘3NH4compartmental analysiswith some degree of confidence. Influxesof 13NH4into rice roots understeady-state conditions increased accordingto the levels of [NH4+]0in thegrowth media (Table 3). A similar trendwas shown for net fluxesdetermined either by efflux analysis or by depletionmethods. Net uptake(Pnet)tended to show only a small increase as [NH4]0increasedfrom 100to 1000 1iM (Table 3). This confirms my previous reportthat net uptake ofNH4was acclimated to [NH4+]0in growth media, althoughthe acclimationwas not achieved by G2 plants (Wanget al., 1991). These resultsdemonstrated that NH4 fluxes are closely relatedto the nitrogen status ofplants, which is determined by plant growthconditions.Estimated effluxes of NH4 from rice rootswere about 10, 20 and29% of the influx values for G2, G100 andG1000 plants, respectively(Table 3 and Fig. 4). In addition, effluxwas positively correlated with the[NH4-’-] (Table 3 and 4). This result agrees with the suggestion thatcontinuous NH4 efflux may be a commonfeature of net NH4 uptake byroots of higher plants (Morgan and Jackson,1988a). Nitrogen efflux (eitherNH4 or NO3-) has been reported tobe quite significant, particularly at81elevated concentrations of N (Morgan et al., 1973; Bretelerand Nissen,1982). Indeed, Deane-Drummond and Glass (1983a,b) suggested thatnitrate efflux might regulate net uptake by means of a typeof ‘pump andleak’ mechanism. By contrast, Lee and colleagues haveemphasized theimportance of influx in the regulation of net uptakeof nitrate, althoughnitrate efflux was equivalent to almost 40% of nitrate influx inbarley roots(Lee and Clarkson, 1986; Lee and Drew, 1986). Morganand 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 rootammoniumconcentrations and net 14NH4efflux was not observed.Although plasmamembrane influx determines the maximum rate of net uptake(Lee andClarkson, 1986), efflux certainly makes a significant contributiontodetermining net uptake.Because of its short half-life, ‘3N is unsuitable for thedeterminationof vacuolar parameters by efflux analysis. Nevertheless,the combination of‘3NH4efflux analysis and the CEC separation of‘3N products enabled us toestimateØ,using two methods. Both results give values forin therange from 1 to 1.5 jimol g-’FW h-1. Method(I) is based on the estimated13NH4 accumulation during 30 mm loading, whilemethod (II) involvedthe use of S values estimated minute by minute froma knowledge of thehalf-life of cytoplasmic exchange (see section 3.2.5.).Therefore method (II)is probably more refined than the value derived frommethod I. Thesevalues are somewhat lower than those obtained by effluxanalysis in onion(Macklon et al., 1990), however the Macklon’sstudy was undertaken at 2mM [NH4+10,compared to my analyses undertakenwith G100 plants at 10082jiM NH4.The differences may also reflect the methodologyand plantsspecies employed.3.4.3. The NH4 pools in rootsIn the present study, the values ofQwere 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 in9-d-oldbarley roots grown in 1 mM NH4.For barley, wheat and oat grown inNO3-or N-free conditions, the value of [NH4+]1was in the range of0.4 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 from6 to 35 jimol g‘FW (Lee and Ratcliffe, 1991; Morgan and Jackson, 1988a). Therelativelylow intracellular NH4 content, particularly, under steadystate conditions,may reflect the efficiency of NH4 assimilation (Goyal andHuffaker, 1984).Irrespective of the [NH4-’-]0provided during the growthperiod, 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 NH4concentrations of riceroots estimated in the presentstudy (Table 4) were in the range ofreported values for wheat, maize, barley and onion (Fentemet al., 1983b;Cooper and Clarkson, 1989; Macklon et al., 1990;Lee and RatcIiffe, 1991).On the basis of NMR studies of NH4 distribution inroot tip of maize,cytoplasmic [NH4+] ranging from 3 to 438jiM were reported (Roberts and83Pang, 1992). However, in that study, lower values mightbe expected sinceroot tips were excised from 2-day-old maize seedlingsand maintainedwithout an exogenous source of NH4 during estimationof[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 to36 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 analysisto investigatecompartmentation of non-metabolized ions,e.g. Cl- (Cram, 1968), Na(Jeschke and Jambor, 1981), and K (Memonet al., 1985), relatively fewstudies have been undertaken using metabolizableions such as PO4(Lefebvre and Clarkson, 1984), NO3-(Presland andMcNaughton, 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 (threein the roots and one in the shoot)based upon the distribution of‘3N among these tissues in maize plants.Using 1NH4efflux analysis with excisedonion 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-cellularcompartments and one extracellular compartment for 13NH4in rice roots. The biochemicalfractionation approach was alsoused to identify different compartments84PlasmakmmaRoot cellStele —‘I..CytoplasmMetabolitesI[13.OC 1 i5.78J[_[_]A551% cI1O%NH4 0cxc(?)‘S “S ‘.S5 “S ‘S ‘SFigure 7. Proposed model for ammonium uptake andcompartmentation inrice G100 roots. The bold values in parenthesesare estimated fluxes ofabsorbed 13NH4(jimol g”FW h”). Thepercentages represent the relativedistributions of ‘NH4 among the compartmentsas a proportion of theisotope entering the cell during the30 mm loading.oc,from outsideplasmalemma to cytoplasm;øco,from cytoplasm tooutside plasmalemma;4cv,from cytoplasm to vacuole;vc,from vacuole to cytoplasm;‘Icx,metabolites translocation fromroot to shoot;‘Pxc,metabolites translocationfrom shoot to root; cIASS, assimilationrate;ØDEG,degradation rate;prepresents chemical flux and 1 representsradioisotopic flux. Fluxesaccompanied by (?) indicate fluxes forwhich data are not available fromthe present study.85for NH4 assimilation. By using 15NH4,threecompartments were foundcorresponding to different cell types and a storagepool in barley roots(Fentem et al., 1983a) or different organelles (Rhodeet al., 1980). Spatialdifferences in the activities of enzymes involvedin NH4-’- assimilation arealso found along the root (Fentem et al., 1983a). In additionto this form ofheterogeneity, there are distinct isozymes of glutaminesynthetase, locatedwithin the cytosol and within plastids (Miflin and Lea,1980).Much less information is available concerningthe partitioning ofnewly absorbed ammonium between these compartments, particularlyconcerning the partitioning between metabolizedand un-metabolizedfractions in the root and translocation to the shoot.In the presentexperiments, nearly 90% of absorbed ‘3N remained inthe 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 analysisof this ‘3N by ion-exchangechromatography (Table 1) revealed a virtualabsence of 13NH4.Theremaining metabolized fractions consisted of 5.5% thatfailed to be held onthe CEC, presumed to be amino acids and/or solubleprotein, and 3.9%,which was not soluble and remained associated withthe ‘Root debris’.Calculations derived from results ofboth efflux and chemical analysesshowed that un-metabolized NH4 in the cytoplasm(Q)constituted only8% of Qj for G2 roots and 30% for G100and G1000 roots, respectively,(Table 4). Taking G100 plantsas an example, a model describing the spatialand biochemical compartmentation ofnewly absorbed NH4-’- uptakeby riceroots is given in Fig. 7. About 24%of un-metabolized NH4wasallocated tothe cytoplasm and 76% to the vacuole.Based on the influx of 13NH4÷into86roots, 21% and 40% of ‘3N remained in the cytoplasmic andvacuolarcompartments, respectively, along with 20% that was effluxed and19%that was assimilated. Of the 19% assimilated, roughly half (10% ofinflux)was translocated to shoots. This assimilation rate was based on total13Ntransported across the plasmalemma and may underestimate thetrueassimilation rate because during the loading period, the cytoplasmic‘3NH4pool would not have reached steady state.3.5. SUMMARYUptake of 13NH4by roots and distribution of 13NH4among 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 and10.11 jimolg’FW h’, respectively, for G2, G100 and G1000 plants; effluxwas 11, 20,and 29%, respectively, of influx. The NH4 flux to the vacuole wascalculated to be between 1 to 1.4 jimol g’FWh-i.By means of 1NH4÷efflux analysis, three kinetically distinct phases (superficial,cell wall, andcytoplasm) were identified, with half-lives fori3NH4+exchange of 3seconds, 1 and 8 minutes, respectively. Cytoplasmic [NH4]was estimatedto be 3.72, 20.55, and 38.08 mM for G2, G100 andG1000 plants,respectively. These concentrations were higher thanvacuolar [NH4], yet72% to 92% of total root NH4 was located in thevacuole. Distributions ofnewly absorbed 13NH4between plant parts and amongthe compartmentswere also examined. During a 30 minute periodG100 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 ‘3NH4INFLUX4.1. INTRODUCTIONDespite the potential benefits of nitratefor the growth of rice plants,especially under anaerobic conditions (Malavolta,1954; Bertani et al.,1986), ammonium is the predominantand most readily bio-availablenitrogen form in paddy soil (Yu, 1985). It is thepreferred nitrogen speciestaken up by rice (Fried et al., 1965; Sasakawaand Yamomoto, 1978), andin terms of the efficiency of fertilizer ultilization,ammonium is superior tonitrate in paddy soil (Craswell and Vlek,1979).Ammonium uptake systems havebeen well defined as concentrative,energy-dependent and carrier-mediatedin algae (Smith and Walker,1978), fungi (Kleiner, 1981), bacteria (Kleiner,1985), and cyanobacteria(Boussiba and Gibson, 1991). Howevercompared to the extensiveinvestigations of N03 uptake, the kineticsand energetics of ammoniumtransport in higher plants have receivedrelatively little attention. In bothrice plants and Lemna NH4uptake followed a bi-phasic pattern,with asaturable carrier-mediatedsystem operating at low external NH4([NH4]0)and either a second saturatingsystem (Fried et al., 1965) ora lineardiffusive component at elevated[NH4]0(Ullrich et al., 1984). InN-starvedLemna both NH4 uptakeby the saturable system anddepolarization ofplasma membrane potential werefound to exhibit the same concentrationdependence(KmT5 for both processes were 17 jiM). At higher [NH4-’-]0theuptake by the linear systemwas not accompanied by furtherdepolarization of membrane potential(Ullrich et al., 1984). The saturable89component of NH4 uptake was sensitive to some metabolicinhibitors(Sasakawa and Yamamoto, 1978) and to changes of roottemperature(Bloom and Chapin, 1981). In addition, NH4-’- uptakeis subject to negativefeedback, supposedly from N metabolites (Lee andRudge, 1986; Morganand Jackson, 1989; Clarkson and Luttge, 1991). Youngdahlet al., (1982)demonstrated that NH4 uptake in rice decreased with plantage. However,despite these studies, the mechanism(s) of NH4-’- uptake by rootsof higherplants remain unclear. In particular, the highconcentration systemrepresents virtually unexplored territory.Ammonium is unique among inorganiccations, because followingabsorption by plant roots, it is rapidly assimilated into organicpoois. Thishas made the analysis of uptake and thesubsequent 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-term13NH4influx 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 respondingto the imposed conditions.By contrast, net uptake measurement, oftenobtained by means of long-term depletion experiments, actuallymeasures the difference betweeninflux and efflux. This is especially relevantbecause nitrogen (either NH4or NO3-) efflux has been reportedto 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-livesfor exchange of the subcompartments of the root (Lee andClarkson, 1986; Presland andMcNaughton, 1986; Siddiqi et al., 1991;Wang et al., 1993), it is possible to90measure the plasma membraneinflux as opposedto other fluxes (tovacuole or to stele) which resultfrom long-term experiments(Cram, 1968).The objective of this studywas to investigate themechanisms andcharacteristics of ammoniumuptake by rice plants.I have particularlyemphasized short-term responsesof 13NH4influxes to changesin [NH4]0of uptake solutions overa wide range of externalconcentrations, in orderto define the transport mechanismsresponsible for influxacross theplasma membrane. I have examinedthe influence of prior NH4provisionupon the kinetic parametersfor influx by both componentsof the biphasic system for NH4transport. In addition thesensitivities of thesefluxes to metabolic inhibitors,short-term variations intemperature and pHwere determined with a view to clarifyingthe mechanisms ofthese fluxes.4.2. METHoDS AND MATERIALS4.2.1. Plant growthand 1N productionSee section 2.2. Seed germination;section 2.3. Growth conditions;section 2.4. Provision of nutrients;and section 2.5. Productionof13NH4+4.2.2. Relative growthrateRice seedlings were grownin 2, 100, and 1000 iMNH4 (designated,hereafter, as G2, G100 andG1000 plants, respectively)to representinadequate, adequateand excess nitrogen provision.Total fresh weights of91plants were recorded for three treatments atages 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-’-]0from 2 pMto 40 mM in perturbationexperiments. Perturbation experiments aredefined as those in whichplants are grown at one particular [NH4+]0,andinfluxes 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 methodusing 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 whileKm(.tM) represents [NH4-’-]0giving half ofthemaximum influx; b and a are constantscharacterizing the linear phase. Ateach concentration tested, influxes weredetermined in two to six separate92experiments with three or fourreplicates. Each replicateconsisted of about20 rice seedlings.Based on the results of thekinetics studies(see Results), measuredNH4influx from < 1 mM [NH4+]0appeared to result froma saturable highaffinity transport system (hereafterreferred to as HATS).Since the influxby the HATS had saturatedbetween 0.1 and 1.0 mM[NH4-’-]0,influx from0.1 mM [NH4+]0was selectedas a concentration representativeof the HATSin the following studies.Above 1 mM [NH4+]0,measured NH4influxappeared to result from theparticipation of boththe HATS anda lowaffinity transport system(hereafter referredto as LATS). Therefore,thedifference between measuredinflux at concentrations >1mM [NH4]0andthe saturated values ofthe HATS were taken torepresent fluxesdue to theLATS.4.2.5. Metabolic inhibitorstudyInfluxes were measuredin MJNS containing representativelevels ofeither 0.1 mM to estimatethe activities of the HATS,or 20 mM NH4C1forthe HATS plus LATS,in the presence orabsence of differentmetabolicinhibitors. The inhibitorsused were as follows:(1) 10iiMCCCP; (2) 1 mMCN plus SHAM; (3) 50 j.tM DES;(4) 0.1 mM DNP;(5) 50 iM Mersalyl;(6) 1mM pCMBS. Details ofpreparation refersto Section 2.9.In this study, both 3-week-oldG2 and G100 plantswere used. Beforelabeling with radioisotope,rice roots were treatedwith un-labeled MJNScontaining the same concentrationsof CN- plus SHAMfor 30 mm. Therewere no pretreatmentsfor the other inhibitors.Measurements ofinflux93were undertaken as in the kinetic study.Each inhibitor experiment wasrepeated twice with three replicatesfor each treatment. Eachreplicateconsisted of about 20 seedlings. Thereforethe means for influxes andstandard errors were calculated from sixreplicates and representedthemean for approximately 120 seedlings.4.2.6. Temperature studyRice plants were grown under the sameconditions as describedpreviously, so that they were adapted to 20± 2°C. Influxes weresubsequently measured in MJNS with either 0.1 mMor 20 mM NH4C1 atsolution temperatures of 5, 10, 20 and 30°C. During thepre-wash, uptakeand post-wash, solutions were maintainedat the designated temperatures.The measurements of influx were undertakenas in the kinetic study.4.2.7. pH profile studyRice plants were grown in MJNS containing2 jiM NH4under theconditions described in METHODS ANDMATERIALS and adaptedto growthmedium at pH 6. Uptake solutionswere adjusted to pH values of3.0, 4.5,6.0, 7.5 and 9.0 by additions of HC1 orNaOH, respectively. To examine theeffects of solution pH upon‘3NH4influx, roots were exposedto thedesignated pH levels during 5 mmpre-wash, 10 mm influxas well as 3mm post-wash. Influxes of‘3NH4÷weremeasured in either 0.1mM or 10mM NH4 solution. The choice of 10mM NH4,rather than20 mM wasdictated by the desire to minimize additionsof HC1 or NaOH inadjusting pHlevels in the uptake solutions.944.3. RESuLTS4.3.1. Kinetics of NH4influxInfluxes of‘3NH4in response to externalconcentrations in the rangefrom 0.002 to 40 mM [NH4]0were resolved into two distinctphases,presumably mediated by two separate transportsystems; at low [NH4]0 (<1 mM), a saturable high affinitytransport system (HATS);and at high[NH4]0(>1 mM), the combined activities of a saturatedHATS 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 orG1000 rice plants conformed to MichaelisMenten kinetics (Fig. 8). The kineticparameters ofVmaxandKmwereestimated using non-linearregression analysis (Table 7)to fit theMichaelis-Menten equation. Analysisby means of a more comprehensiveequation (see equation [32] insection 4.2.4.) gave similar trendsalthoughactual values ofVm and Km were slightly different (data not shown). Withincreasing provision of NH4 from2, through 100 to 1000 jiM inthe periodof two weeks prior to uptake measurements,root [NH4]increased from2.37, through 4.31 up to 6.85jimoles g’FW, respectively.As shown in Fig.9, increasing [NH4]1was associatedwith decreasingVmvalues, from 12.8through 8.2 down to 3.4 jimolg’FW h’, and increasingKmvalues, 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-NH4influxat low [NH4]0.Influxof 13NH4into rice roots was measured in perturbationexperiments. Riceseedlings were grown at 2, 100 or 1000 jiM NH4(G2 (A), G100 (0) orG1000 (x), respectively). Each datum point isthe mean of 16 replicateswith standard error as a vertical bar. Thesolid lines are estimated fromVmandKmvalues (Table 7) of G2, G100 and G1000 plants,respectively.96Table 7. Kinetic parameters for saturable and linear‘3NH4influx of G2,G100 or G1000 roots as functions of [NH4J0.Therelationships between‘3NH4 influx and [NH4+]0 of uptake solution wereestimated fromMichaelis-Menten kinetics for influx measuredbetween 2 to 1000 tM[NH4]0 and for linearity in the range of 1 to 40mM, where ‘a’ is theintercept and ‘b’ is the slope.G2 G100G1000HATSaVm12.8 ± 0.2b8.2 ± 0.7 3.4 ± 0.2Km 32.2 ± 2.1 90.2 ± 23.2 188.1 ± 34.5HATS+LATS a 13.21 10.144.59b 0.67 0.79 1.30r2 0.97 0.970.99LATS a 0.41 1.941.19b 0.67 0.79 1.30r2 0.98 0.960.98aHATS represents the high affinity transport system, measuredbelow 1 mM [NH4]0.Influx measured at concentrations above 1 mM [NH4]0is considered to be thecombined contributions of both high and low affinitytransport systems(HATS+LATS). LATS represents the low affinity transport systemand is estimated bysubtracting HATS from HATS+LATS.bVmax and Km were estimated by non-linearregression with ± se,9715 250-20010E—0— Vmax& Km-100E5-50•G2 G1OOGi000. •0.0 2.0 4.0 6.0 8.0 10.0Root ammonium concentration (mM)Figure 9. Relationship between kinetic parameters of NH4uptake androot ammonium concentrations ([NH4]1)of rice seedlings.The values ofVmax (0)andKm (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 13NH4influx was linear (Fig. bA). TheY intercepts of theselines (13.21, 10.14 and 4.59 for G2,Gb00 and G1000, respectively)decreased according to the ammonium provisionduring the growth andagreed well with the correspondingVm forthe HATS (Table 7). Thusit isconcluded that the measured fluxesat elevated [NH4+]0result from thecombined activities of the HATS and the LATS. Toevaluate the effect ofprior NH4provision on the LATS for‘3NH4influxwithout the influence ofthe HATS, theVmaxvalues for HATS were subtractedfrom the measuredinfluxes at elevated [NH4]0 values. The derivedLATS values were replotted accordingly (Fig. lOB). As shown in Fig. lOB,13NH4influx by LATSis higher for G1000 than for G100 orG2. 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 linearrelationships at high[NH4]0 were confirmed by means of F-tests forlinearity (Zar, 1974).Statistical analyses revealed that theslope of the G1000 line wassignificantly different from the slopesof the G2 and G100 lines (data notshown).4.3.2. Effect of metabolic inhibitors on theinflux of‘3NH4In most cases 13NH4 influxes ofG2 plants were reduced by thepresence of metabolic inhibitors inthe uptake solutions asshown in Fig.11. Net reductions of influxes, listed inTable 8, were calculatedby usingthe influx of the control as zero reduction(0%). The HATS for NH4 influx99701OA60lOBG1000I30•20xGl0000 G100:o 511015 20 2’5 30G2External Ammonium Concentration(mM)Figure 10. Influx of‘3NH4into riceroots 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 theVrnax of the HATS of G2, G100or G1000, respectively, fromthe corresponding measured influxes(in 9-A).These plotted lines of LATShave the same slopes as their correspondinglines in 9-A but with slightly differentvalues of the intercept,0.53, 1.96and 0.99 for G2, G100, andG1000 plants, respectively.10030HATS+LATSEJHATSE:L!1LATS-- 20:i____I1LrControl CCCP CN+SHAM DES DNP Marselyl pCMBSInhibitors in uptake solutionFigure 11. Effect of metabolic inhibitorson 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 presenceor absence of aspecific metabolic inhibitor. Each datumpoint 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): mersalylacid; 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 inhibitorsof ATP synthesis(DNP). These three treatments reduced theLATS by only 31 to 51%.ATPase inhibitor DES reduced‘3NH4influxdue to the HATS by 51% buthad negligible effects on LATS. External proteinmodifiers of themembrane surface, pCMBS and Mersalyl, reduced13NH4÷influx of HATS byabout 40% with slightly less or similar reductionsof LATS (22 to 46%).These patterns of inhibition were also observed forG100 plants (data notshown).4.3.3. Effect of root temperature on‘3NH4influxShort-term perturbations of root temperaturesignificantly affectedthe influx of13NH4+into rice roots that were adaptedto the growthtemperature of 20°C (data not shown). Table9 shows the calculatedQj.ovalues for G2 and G100 plants in the temperature range from5°C to 30°C.In this temperature range theQj.o values for HATS fell from> 2.4 between5 to 10°C to 1.25 between 20 to 30°C. The resultsof F-tests in conjunctionwith Duncan’s Multiple Range Testsdemonstrated thatQovalues for thedifferent temperature rangeswere significantly different for the HATS (P>0.05). In contrast, there were no significant differencesbetween theQiovalues for LATS in the same three temperatureranges for both G2 andG100 plants (P > 0.05). NeverthelessQjo values for the LATS weresignificantly greater than 1.102Table 8. Reduction of‘3NH4 influx into rootsof G2 plants by variousmetabolic inhibitors.Treatment Inhibitor %Reduction ofLevelHATSaLATSbControl None0 0CCCP 10 mM 84.5830.72CN+SHAM 1 mM 80.8443.20DES 50 mM 53.964.00DNP 0.1mM 86.7250.55Mersalyl 50 mM 41.9722.40pCMBS 0.5mM 41.3346.11aThe 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%.bReduction of LATS (%) was calculated by first determining the influxdue to LATS bysubtracting the influx values measured at 0.1 mM NH4from that at 20 mM NH4-- forcontrol and for each inhibitor treatment, respectively. The reductionof influx valuedue to LATS under control conditions was then set at 0%.103Table 9. CalculatedQjovalues for 13NH4influx by theHATS or LATS ofrice plants grown at 20°C with 2 or 100 iM NH4C1(G2 and G100 plants).Temperature G2 PlantsaDMRTbG100 Plants DMRTRange(a)HATS: 5-10°C 2.48±0.04a 2.59±0.21a10 - 20°C 1.79 ± 0.08 b1.68 ± 0.22 b20-30°C 1.25±0.16 c1.44±0.16 b(b) LATSC:5 - 10°C 1.41 ± 0.21 1.54± 0.2710 - 20°C 1.49 ± 0.061.90 ± 0.4620-30°C 1.56±0.061.33±0.12aEach value (± Se) is the average of three means from duplicateexperiments; eachmean is derived from three replicates.bDMRT 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% significancelevel.CBoth F-tests andDMRT indicated that means for the LATS were not significantlydifferent 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 controlwas computed on the basisofthe influx value at pH 6.0 for either HATSor LATS (Table 10). In thiscase,a Least Significant Difference test (LSD) wasused for making pairwisecomparisons between the control andother treatments. In the rangefrom4.5 - 9.0, solution pH had only a small effect ti 13NI-J4influx from 0.1 mM[NH4]0,whereas13NH4+influx by LATS decreased verysignificantly withincreasing ambient pH beyond pH 6.0. By contrast,reduction of solution pHdown to 3.0 drastically reduced 13NH4influx by HATSas 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 thecell 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 10mm exposures to13NH4+and 3 mm post-washes, therefore, estimates of plasmamembrane influxesrather than net flux or quasi-steady fluxes to vacuolewere obtained (seeCram, 1968). The results of thepresent study revealed that NH4-’- influxacross the plasma membrane into riceroots exhibits a bi-phasic pattern: inthe low range (below 1 mM [NH4÷]0),influxoccurred via a saturable highaffinity transport system (HATS); whilefrom 1 to 40 mM [NH4+]0a second,low affinity, non-saturable transport system(LATS) became apparent. This105Table 10. Effect of uptake solution pH on1NH4influx 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--]0at either 0.1 mM for the HATS or 10 mM for theHATS+LATS. Thevalue of LATS was obtained by subtracted the valuesof HATS fromHATS+LATS of each treatment.pH InfluxaLSDb(%) of ControlC(a)HATS: 3.0 6.91±1.43*534.5 12.02±0.46 ns876.0 13.22±0.27 control1007.5 14.51±0.39 ns1099.0 12.94 ± 0.30 ns95(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*51aEach value (± Se)(i.tmol g’FW h’) is the average of four means of duplicateexperiments. Each mean is derived from three replicates.bLSD stands for LeastSignificant Difference test, used for making pairwisecomparisons between thecontrol at pH 6.0 and other treatments.*= significant at 5% level andns = notsignificant.CThe percentages of control were calculatedusing the NH4 influxmeasured at pH=6.O as 100%.106bi-phasic pattern of uptake has been reportedfor NH4 uptake by Lemna(Ulirich et al., 1984), for K uptakeby corn roots (Kochian andLucas,1982), and for NO3-uptake by barley roots (Siddiqiet al., 1990).Plasma membrane ‘3NH4 influxat low [NH4+]0 conformedtoMichaelis-Menten kinetics (Table 7)in accord with earlier studiesof netNH4 uptake by rice (Youngdahl et al.,1982; Wang et al., 1991).This hasalso been found to be the case for rootsof other species, includingcorn(Becking, 1956), rye-grass (Lycklama,1963), and barley (Bloom andChapin, 1981), where net NH4 uptake rates saturatedin the range from100 to 1000 iM [NH4]0.The significance of this HATSfor NH4 in rice rootsis that it allows plants to absorbsufficient nitrogen (NH4--) from very lowlevels in the rhizosphere to meet the minimum requirementfor plantgrowth. In the present experiments, for example,by three weeks, therelative growth rates were independent of [NH4]0from100 to 1000 pMNH4.The relative growth rates calculated from total freshweight of bothG100 and G1000 plants were at —0.16 d’ for the thirdweek of growthwhile for G2 the value was 0.06 d’. By the fourth weekthe differences inRGR had diminished to 0.05, 0.06, and 0.06 d’, respectivelyfor G2, G100and Gl000 plants. The reduced growth rates ofG2 plants wereaccompanied by increased root:shoot ratios, and leaveswere slightly palerthan those of plants grown at higher [NH4]0.At the higher range of [NH4+]0 (1to 40 mM), a linear, low affinitytransport system (LATS) also participated in NH4 uptakeby rice roots, asis the case for other ions and plant species (Kochianand 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 withthe correspondingVmax107values for the HATS (Table 7), whichsuggests that the two distincttransport systems (HATS and LATS) areadditive.Despite the importance of NH4 as principalsource of N for manyplant species and the increasing availabilityof techniques forthemeasurement of short-term 13NH4 and 15NH4influxes, few detailedinflux isotherms (as distinct from netuptake isotherms) have beenreported for NH4 influx into roots of higher plants.Nevertheless, UllrichetaL, (1984) were able to demonstrate linear kineticsof NH4 uptake byLemna between 0.1 to 1.0 mM [NH4]0 usinga depletion method. Thequestion of the saturation of this apparently linearsystem at higherconcentrations remained unresolved. Clearly,it is difficult to measure netfluxes by employing concentration depletionmethods at high externalconcentrations without extending the uptakeexperiment for long periodsof time. By using short-lived radioisotopes,such as ‘3N, it has beenpossible to measure unidirectional fluxes ofN03 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 concentrationsas 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 clearlydependent on metabolicenergy. In the present study metabolicinhibitors, CCCP, DNP or CN plusSHAM, diminished‘3NH4influxes of HATSby more than 80% (Table 8).The effects of these inhibitors onthe LATS were much smaller (31to 51%inhibition). Further evidence from the0110values (Table 9) supported the108notion of energy dependence. AQovalue greater than 2 isconsidered toindicate the metabolic dependence of physiologicalprocesses such as iontransport. Short-term perturbations oftemperature between5 to 10°C,significantly increased theQovalues for HATS up to 2.5 comparedto1.5 between 20 to 30°C. Ina 7 h concluded that the uptake of ammoniumby 9-day old rice seedlings was closelyassociated with metabolism.However, such long-term studies probablymeasure theQofor NH4assimilation rather than the transportprocess. The values ofQoestimatedfrom Ta and Ohira’s data (1982) providedvalues larger than 2.5for 15NH4-’-absorption by rice roots between 9 to 24°C.LowerQiovalues (1.0 to 1.6)were reported for net ammonium uptakeof low-temperatureadaptedryegrass (Clarkson and Warner, 1979);barley (Bloom and Chapin, 1981);and oilseed rape (Macduff et al., 1987)indicating that NH4-’- transporthadacclimated to the low temperature growth conditions.Consistent with theresults of the metabolic inhibitor studies,the presentQ10study indicatedthat LATS was less sensitive to changes ofroot temperature than the HATS(Table 9).The apparent energy-dependence of the HATS maynot necessarilymean that NH4 uptake is an activetransport process, although activetransport systems for ammonium have beenproposed in bacteria, fungiand algae (Kleiner, 1981; Schlee and Komor,1986, Singh et al., 1987). Theaccumulation of NH4 against its concentrationgradient could be achievedby active or passive uptake mechanisms:the former, by direct use ofmetabolic energy to carry a soluteacross a membrane towards a region ofhigher electrochemical potential; while thelatter, by solute flux acrossamembrane along the electrochemical potentialgradient, a process that maybe only indirectly related tometabolic 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 potentialdifference for G2 plantsin ‘MJNS’ minus Nitrogen solutions (Wanget al., 1992), predictions derivedfrom the Nernst equation indicated thatnet ammonium uptake wouldbeactive only when [NH4+]0falls below 125jiM. This is rather similarto thevalue of 67 jiM calculated for Lemna (Ulirichet al., 1984). However, thiscalculation only serves to predict the feasibility ofthe process occurringunder the prescribed conditions. The preciserelationship between thecalculated electrochemical potential difference for anion and the putativetransport systems, predicted on thebasis of concentration-dependentinflux curves, are difficult to realize. In the presentcase, for example,there are no discontinuities in the uptake curvecorresponding to thepredicted concentration at which the switchbetween active and passivetransport (-425 jiM [NH4]0)occurs. This issue is raisedto warn against atoo literal interpretation of the thermodynamicpredictions. While onthermodynamic grounds influx is uphill below125 jiM and downhillbeyond this level, the kinetic data revealno apparent change of transportmechanism.The characteristics of the two transportsystems for NH4 influx havesignificant features in common withthose described for K-i- uptake in which(incidentally) there is yet no clear consensusregarding the mechanisms ofinflux into higher plant roots. Likewise,the mechanism of the apparentlyactive transport of ammonium below 125jiM is unknown. It might occurby means of a specific ATPase or a secondarytransport system such as anNH4:H symport that is drivenby the proton motive force(pmf). As110proposed for K’- uptake by Neurospora,for each K entering, one H-’-is cotransported and 2H are extruded by theproton pump (Rodriquez-Navarroet al., 1986). The net result is thereforea 1:1 K/H-’- exchange. Is itpossiblethat NH4 influx is mediated by an analogoussystem? It has long beendocumented that NH4uptake is associatedwith strong acidificationof theexternal medium (e.g. Becking, 1956).Likewise in the present study,whenpH was not adjusted daily in the initialgrowth experiments, externalpHdropped so low that plants failed to grownormally.So far as the passive uptake of ammoniumis concerned at higherconcentrations, several authors have proposed thatNH4-’- influx may occurby an electrogenic uniport in responseto the electrical gradient (Kleiner,1981; Ullrich, 1984). When ambient concentrationis beyond the predictedthreshold for active uptake, the concentrative NH4uptake may be due toa facilitated transport system driven by theelectrochemical potentialdifference for NH4.This has two components; thedifference in chemicalpotential of NH4(L$LNH4÷) between cytoplasm and outside and theelectrical potential difference (z\’-P) generatedin part by proton effluxacross the transducing membrane. The actualmechanistic link, if oneexists, between NH4 influx and the pmfacross the plasma membrane isunclear at present. Certainly the results of thetreatments with theprotonophore (CCCP) or the un-coupler of ATP formation(DNP and CN plusSHAM), which caused greater than 81% reductionof influx due to HATS,are consistent with a dependence ofNH4 influx on transmembrane pmf.Further support for this hypothesis is providedby the effect of ATPaseinhibitor, DES, which reduced‘3NH4influx due to HATS by 54%but hadnegligible effects on LATS.1114.4.3. Effect of pH profile on ammoniumuptakeIn the present study, influx by the HATSwas strongly reduced belowpH 4.5. By contrast, in the range frompH 4.5 to 9.0, 1NH4influxby theHATS appeared to be relatively insensitiveto pH. 13NH4 influx by theLATS actually decreased with increasingambient pH beyond pH6.0. It hasbeen reported for several species that thespecific uptake rate of NH4--canbe reduced by short-term decreases inpH below 6.0 (Munn andJackson,1978; Marcus-Wyner, 1983; Vessey,1990) and even terminatedall-together at pH 4.0 (Tolly-Henry and Raper,Jr., 1986). Tanaka (1959)suggested that rice is very sensitive to pHbelow 4. Most probably thisreflects a general detrimental effect ofsuch acidic conditionson thetransport systems. In addition, it has beenobserved that when plants weregrown at such low pH values over extendedperiods of time, the rootsbecame stunted and discolored. It has beensuggested that both high pHand/or high ammonium concentration of solutionmay result in high ratesof NH3 uptake due to increased NH3 concentrationand the higherpermeability of cell membranes to NH3 than NH4(see Macfarlane andSmith, 1982). However, in many studies thisexpectation has not beenobserved, and uptake failed to increase at elevatedpH (MacFarlane andSmith, 1982; Deane-Drummond, 1984; Schleeand Komor, 1986). Likewise,in the present study, influxes of1NH4due to the LATS were reduced by25 - 35% at higher pH (7.5 - 9.0), despitea predicted increase of [NH3]from less then 0.1% of total [NH4+ NH3] at pH 6.0, to 36% at pH9.0according to the pKa for NH4 (9.2 5).Furthermore, membrane electricalpotentials of rice roots have been shownto be depolarized by elevatedammonium concentrations (Wang et al.,1992). These observations indicatethe entry of cation (NH4+) ratherthan neutral ammonium (NH3).The112evidence from our electrophysiological study of rice roots indicated alinear relationship between depolarization of membrane potentialandinflux 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 thoughnet 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 increasedand NH4influx decreased (Fig. 9). It is noteworthy that in the present case, negativefeedback regulation appeared to affect bothVmandKmvalues (Table 7,Figs. 8 and 9). It has commonly been observed thatVmaxis strongly andunequivocally influenced by the level of nutrient supplied during growth.By contrast, an effect onKmhas rarely been observed (Lee, 1982). Only inthe case of K (Glass, 1976) was theKmstrongly influenced by K statusalthough other ions such as CF do show small changes (Lee, 1982). In thepresent study, the values ofKmwere strongly influenced by the prior levelof NH4-’- supply, and are positively correlated with [NH4-’-]1.113Contrary to expectation,‘3NH4influxesdue to the LATS were higherin plants previously maintained at 1000jiM NH4 than in those maintainedat 2 jiM NH4-’-. The reverse was foundto be the case for‘3N0-influxinbarley (Siddiqi et al., 1990). This positivecorrelation between provisionofNH4-’- and 13NH4influxes at high [NH4+]0mayindicate that the LATS maynot be subject to regulation by negativefeedback. Another possibleexplanation is that better nitrogen nutrition mayprovide more buildingmaterials (protein?) for constructing transporters. However,exposures tohigh [NH4-’-]0(>1 mM) were brief and in longerexposures NH4 influx maybe down-regulated in accord with expectation.The present study has demonstratedthe strong negative down-regulation of influx by the HATS in responseto elevated NH4 supplyduring growth. At present the mechanism(s) and signalsresponsible forthis down-regulation of uptake are unclear. Feedbacksignals may resultfrom un-metabolized ammonium of rootcells or reduced nitrogen (Lee,1982; Morgan and Jackson, 1989). Lee and Rudge(1986) have suggestedthat in barley the uptake of NH4 andN03 are under common negativefeedback control from a product of NH4-’- assimilation ratherthan NH4and/or N03 accumulation per Se. Reduced N pools whichcycle in xylemand phloem from root to shoot have been implicatedin the whole plantregulation of N uptake by plant roots(Cooper and Clarkson, 1989).However, Siddiqi et al. (1990)have suggested that in the case ofN03influx, vacuolar accumulation of N03 perse may also, at least indirectly,participate in flux regulation. Further supportfor this proposal has comefrom studies of nitrate reductase mutantsof barley that are capable ofnormal induction of N03 uptake andappear to show diminished‘3N0influx as N03 accumulates (Kinget al., in press). In the present study,also,114there was a close negative correlation between NH4influx and [NH4-’-j Inroot tissues (Fig. 9). However, the altered NH4statusin G2, G100, andG1000 plants was probably also associated with changes inorganic Nfractions. Since efflux was estimated to be 10 to 30% of influxfor G2, G100and G1000 plants, respectively (Wang et al., 1993), negativefeedback actsvery strongly on the influx step of the HATS,but since efflux alsoincreased with increasing [NH4]0,this flux will exertsignificant effectsupon net uptake.4.5. SUMMARYThe work described provides the first detailed characterizationofNH4influx across the plasma membrane of rice roots. Ammoniuminflux isbi-phasic, mediated by two discrete transport systems.Metabolic inhibitorstudies andQodeterminations indicated that both systems were energy-dependent, although the HATS consistently showedgreater 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 electrochemicalgradient. It is highlyunlikely that the LATS is active. The HATS was foundto be extremelysensitive to prior exposure to ammonium as indicatedby the alteredvalues ofKm and Vmax. General insensitivity of influx to pH in the rangefrom 4.5 to 9.0 argues strongly against significant entryof NH3 across theplasma membrane even at high [NH4+]0.115Chapter 5. ELECTROPHYSIOLOGICALSTUDY5.1. INTRODUCTIONAmmonium influx by rice roots (Oryza sativaL. cv. M202) has beenshown to exhibit a biphasic dependence on[NH4]0 (Wang et al., 1991,1992b; 1993b). At low [NH4+10,influx is mediatedby a saturable HATSwhich exhibits highQiovalues between 10 and 30 °C anda significantsensitivity to metabolic inhibitors (Wanget al., 1993b). At elevated [NH4]0(between 1 and 40 mM), NH4 influx increases ina linear fashion withincreasing [NH4]0,and though still exhibitingenergy-dependence, thisLATS was shown to be less responsive to metabolic inhibitors(Wang et al.,1993b). A biphasic pattern of NH4 uptakeof this sort, with both saturableand linear phases, was first reported in Lemna,by Ullrich et al., (1984).In order to make a definitive evaluationof the thermodynamics ofNH4 influx (passive versus active transport), itis essential to determinethe chemical potential difference for NH4between the cytoplasm andexternal media, and &P across the plasmamembrane. In Chapter 3,compartmental analysis was used to estimate cytoplasmic[NH4]. So far asI am aware, only one report measuring A’P in riceroots has appeared inthe literature: Usmanov (1979) reported AWto be -160 mV. As early as1964, Higinbotham et al. noted the marked depolarizingeffect of [NH4j0coleoptile cell z’P in oats. Likewise, Walker et al. (1979a, b)demonstratedthe transport of ammonium andmethylamine across the plasmamembrane of Chara, and the depolarizing effects of these cations.The most116detailed study of the concentration dependence of A’Pdepolarization byNH4-’- was undertaken by Ullrich et al. (1984), usingLemna. Below 0.2 mM[NH4]0both NH4 uptake and A’P depolarizationresponded in a saturablefashion with half-saturation values of 17 j.tM for bothprocesses. From 0.2to 1 mM, net uptake of NH4 responded linearlyto [NH4]0,with no furtherP depolarization. On the basis of this observation,Ulirich et al. (1984)concluded that the linear system might result fromdiffusion of NH4 orNH3 across the plasma membrane.The present study was initiated, therefore, toestimate M’ in intactrice roots, under conditions correspondingto those employed to estimatecytoplasmic [NH4j in our previousstudy, and to determine theconcentration dependence of the depolarizing effectof [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 surfacesterilized in 1%NaOCI for 30 mm and rinsed with deionized water. Seeds wereimbibedovernight in aerated deionized water at 38°C beforeplanting 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 polyethylenevessel withthe solution level just above the seeds. Seeds wereallowed to germinate inthe dark (at 20°C) for 4 days. At day5, rice seedlings were exposed to lightand MJNS containing the designated levels ofNH4C1. The composition of117MJNS, growth conditions, nutrient supplyand pH adjustmentwere thosedescribed in Section 2.1.2. Thegrowth medium in the 1-litrepolyethylenevessels were completely replaced on alternatedays and the nutrientlevelswere topped up with concentratedstock solutions daily. Rice plantsused inthe experiments were 3-week-oldG2 or G100 plants respectively.5.2.2. Measurements of cell membrane potentialPlasma membrane P of rice roots weremeasured as describedbyKochian et al. (1989) and Glass et al. (1992).In short, rice plants weresecured in the larger part of a flow-throughPlexiglas impalementchamber, and one intact root was carefullyplaced over the platinum pinsin a narrow section of the chamber.This root was held firmlyduring theimpalement by two short lengths of Tygontubing, from each of which asmall wedge had been cut. The tubingwas placed on either sideof theimpalement zone to clamp the root inplace. All impalements were madeina region about 1 to 3 cm behind the root tip,using a hydraulically driven,three-dimensional micromanipulator (ModelMO-20, Narashige, USA). Boththe Plexiglas impalement chamberand micromanipulator were mountedon the microscope stage. Microelectrodes(including impaling, referenceand grounding electrodes) were madefrom 1.0 mm single-barreledborosilicate glass tubing pulled to a tip diameter of0.5 iM and filled with3M KC1 (adjusted to pH 2 to reduce tip potentials).Measured membranepotentials of root cells, which are the voltagedifferences between theimpaling and reference electrode, wereamplified and recorded ona stripchart recorder. During impalement, solutionswere continuously deliveredfrom an air-pressured reservoir to thechamber through tygontubing atcontrolled flow rates (7.5 ml minl).1185.2.3. Experimental treatmentsAt the beginning of each experiment, theimpalement was made onG2 or G100 roots bathed in their growthmedia (MJNS containing 2 or100iM NH4C1, respectively) and the membranepotential was recorded(A.PG2orA’I’GlOO). MJNS without NH4 is referred to throughout as the -N solution.Before applying each treatment, the -N solutionwas introduced to obtain aresting membrane potential,MN, as the point of reference. Roots wereallowed to equilibrate for at least 3 to5 mm in this -N solution to reach theresting potential before introducing subsequenttreatment solutions.5.2.3.1. Effect of[NH4+]0Roots were exposed to {NH4J0of2, 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. Whenroots were exposedto several different [NH4]0 during a single impalement,G2 or G100medium was flushed through the chamber beforeeach change of NH4concentration. When ‘P returned to its original(A’PG2 or A’’G1OO) value, itwas satisfied that the physiological status of the roothad returned to itsoriginal condition.5.2.3.2. Effect of accompanying anion on 4’i’To evaluate the contribution of the accompanyinganion to theobserved depolarization of AP by NH4-saltsin the low concentrationrange, A’P were measured in the following solutions insequence: (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) 5mM (NH4)2S0.These119concentrations were chosen to provideequivalent anioncharge in alltreatments.5.2.3.3. Effects of metabolic inhibitors onNH4-induced Af’ depolarizationThe same metabolic inhibitorsused in the 13NH4 influxstudy(Section 2.9.), were used to investigateeffects on NH4-induceddepolarization of AP. These included 1mM NaCN plus 1 mM SHAM,10iiMCCCP, 50 jiM DES, and 1 mM pCMBS. Thisstudy involved three steps:(1) the responses of z’P to additionsof 0.1 or 10 mM NH4C1weredetermined in sequence;(2) the inhibitor to be evaluatedwas first introduced in-N solution. Whenz’P had reached a new steady-state,this solution was replacedwith theinhibitor plus 0.1 or 10 mM NH4C1 insequence;(3) the solution containing inhibitorplus NH4C1 was replaced by -Nsolution.When a new steady value ofL\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 inMJNS, were selected asrepresentativelevels for the operation of the HATS orthe 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 cellsof 3-week-oldrice roots (Table 11) were measured in 0.2 mMCaSO4alone(A’PCaSO4), or -Nsolution, or G2 and G100 media(‘‘PN, ‘PG2and‘‘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 correspondingA’PG2 or zPG100values. The depolarizingeffect of NH4C1 additions can be directlycompared in Table 11 for aparticular root type because -N and G2 or G100 media differedonly by thepresence of NH4C1 in MJNS. Therefore, bothAPG2 and z’+’oo representedthe membrane potentials of root cells adapted to theirrespective growthconditions.5.3.2. Contribution of the accompany anions to ‘i’Figure 12 reveals that there was a very small depolarizingeffect ofCa2-salts compared to NH4-salts, underconditions where theconcentration of the accompanying anion was held constant.Also there wasvirtually no difference between the depolarizing effectsof Cl- andS042.This was true also at the higher concentrations ofCa2-saltsand NH4-salts(Traces e, f, g and h in Fig. 12). In the lower concentrationrange, norepolarization of M’ was observed until the Ca2-saltsor NH4-salts werewithdrawn from the chamber. By contrast, in5 mM CaC12,completerepolarization and even hyperpolarizationwas evident within 10 mm of121Table 11. Membranepotentials ofG2 and G100plants measuredindifferent bathingsolutions. Thebathing solutionfor measurementswere0.2 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)aG2 or G100 plantswere impaled in 0.2mM CaSO4 solution;bG2 or G100 plantswereimpaled in -N solution;CG2 or G100 plants wereimpaled in MJNScontaining either2iM or 100 tM NH4C1,respectively;dAverage value ±standard error,n: number ofobservations;122Addition ofionsUa. 50 jiM CaC2V-138—b. 50 jiM CaSO4-110100 jiM NH4CId. 50 jiM(NH)2SOrNN-114e. 5 mM CaCI2f.5mMCaSO4vg.1OmMNH4CIomV{-130h. 5 mM (NH4)2S0rnnesFigure 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 fromMJNS. Eachtreatment was repeated on three separate plants.123evidence of repolarization in NH4C1 (Fig. 12, traceg) but this was onlypartial. Only after removal of the NH4-saltswas complete repolarizationobserved.5.3.3. Effect of [NH4C1]0on z\PThe addition of NH4C1 to the -N solution induceda strongdepolarization of &P (Fig. 13). This depolarizationoccurred rapidly afterthe introduction of NH4C1, even at verylow concentrations (e.g. 2 jiMNH4C1). The time required to reach the initial maximumdepolarization wasfrom 0.5 to 2 mm, increasing with increasing [NH4C1]0.The depolarization of A’P was positively correlatedwith [NH4C1]0.Asaturable pattern was evident in the range from2 to 1000 jiM NH4C1 (Fig.14A) for both G2 and G100 plants. Estimated half-saturationvalues for netdepolarization (analogous to aKm value) were 21.8 ± 2.7 jiM for G2 plantsand 35.0 ± 8.0 jiM for G100 plants, while themaximum depolarization(analogous to aVmvalue) was 50.6 ± 2.0 mV for G2 plantsand 34.3 ± 1.9mV for G100 plants. Kinetic parameters were obtainedby fitting the datato the Michaelis-Menten equation by means ofa nonlinear regressioncomputer program “Systat” (Wilkison, 1987)as used in our earlier kineticstudy of 13NH4influx (Wang et al., 1993b). Between1 to 40 mM {NH4C1]0(Fig. 14B), the magnitude of thedepolarization increased linearly withincreasing concentrations of NH4C1.This relationship was observed for bothG2 and G100 rice plants, although the extentof 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 NH4CI2MC1Figure 13. The &P depolarization of root cell by NH4C1.Representativetraces from G2 plants showing the depolarization of rootcell zVP induced byadding various concentrations of NH4C1. V NH4C1 was withdrawnfromMJNS.12560 -1 4A.—0•G210-0G100U- • I.I.I.I.I.I.I.I.I.I.I.0 100 200 300 400 500600 700 800 9001000External Ammoniumchloride (jiM)120-100- 14 B• G2 r’2=O.9420 0G100 r”2= 0.9900 5 1015 20 2530 35 4045External AmmoniumChloride (mM)Figure 14. Concentrationdependence of netA’P depolarizationof root cells.Rice seedlings were grownin either 100 jiM(G100) or 2 jiM NH4(G2). The-N media were usedas basal solutions forthe resting AP. Eachpoint is theaverage of 3 measurementsfrom each of3 individual plants. Theverticalbar is the standarderror. 14A: Low[NH4C1]0range (<1 mM);14B: Highrange (1 to 40 mM).126Figure 15. shows the effects of four metabolicinhibitors on &T’recorded in -N solutions. The largest depolarizationof z’P (95 mV), wasinduced by the protonophore, CCCP, while CN+SHAMand the ATPaseinhibitor, DES, elicited depolarizationsof 82 mV and 40 mV, respectively.The external protein modifier, pCMBS, caused onlya small depolarization(8 mV). Representative traces depicting the effectsof each of theseinhibitors on NH4-induced depolarization of A’Pare shown in Fig. 16. InTable 12, the effects of these inhibitors on theNH4-induced depolarizationof z’P are expressed as a percentage of thedepolarization under the controlconditions, in absence of the inhibitor.The data are presented as follows:(i) control: in absence of the inhibitor the reductionof NH4-induceddepolarization of A’P is zero; (ii) plus inhibitor:reduction ofNH-’--induceddepolarization of zXP varied from 0 to 91%, dependingupon the inhibitorused and [NH4+]0;and (iii) residual effect: the residualeffect after removalof the inhibitor from external solutions on NH4-induceddepolarization ofA’P. The [NH4C1J0employed were 0.1mM 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 theeffect due to IATS alone.In the presence of the various inhibitors,the depolarization of zs’1’ inducedby HATS was generally reduced by greater than50%. By contrast,depolarization of AP due to NH4 uptake throughthe LATS was onlyslightly affected by the presence of inhibitors.Table 12 also reveals thatthere was virtually no recovery from the inhibitortreatments followingremoval of the inhibitors from theexternal medium.127-3410 m V[012345/-44mmAdd/SHAMInhibitor== 80 mm)f—jFigure 15. Effects of metabolic inhibitors on AW depolarization of rootcells.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) 50iiMDES; (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 34 5mmL add 0.1 mM N1LCl (i) control(ii) inhibition (inhibitor presented)add 10 mM NEI4CI (iii)residual effect (inhibitor removed)Figure 16. Effects ofmetabolic inhibitors on NH4C1induced A’Pdepolarization. Representativetraces for the effects of NH4CIon Pdepolarization in the presenceor absence of metabolic inhibitorsin -Nmedia. Metabolic inhibitorswere those shown in Figure 15..129Table 12. Effect of metabolic inhibitorson the depolarizationof zXP due toNH4 uptake via HATS or LATS inG2 plants. The inhibitors usedwere: (A)10 jiM CCCP; (B) 1 mM CN- + 1 mMSHAM; (C) 50 jiM DES; (D) 1mM pCMBS.Inhibitor CCCPCN+SHAM DESpCMBSTreatment Reduction ofA’P depolarization (%)1. Due to NH4 uptake by HATSa(i) control0 00 0(ii) plus inhibitor89 91 7252(iii) residual effect 9168 81 902. Due to NH4uptake by LATSb(i) control0 0 00(ii) plus inhibitor9 0 14-(iii) residual effect 34- 3 -aThe values of zP were measured when rootswere 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 calculatedfrom the differences betweencontrol values for zSP induced by NH4 and depolarizationvalues in the presence ofthe inhibitor (ii) or after removal of the inhibitor(iii);bThe values of ‘P for LATSwere the differences between measured s’-P at 10mM (for HATS+LATS) and at 0.1 mMNH4C1 (for HATS). Then the percentagewere calculated as described above (a);CThecalculated values were negative due to the less zSWdepolarization of the control.1305.4. DISCuSSION5.4.1. Anion effectA perennial problem associated withattempts to evaluate theelectrical effect of a particular ion is the contributionof the accompanyingcounterion. This problem has rarely been acknowledgedin publishedstudies. However, indirect approaches, suchas comparisons of thedepolarizing effects of NO3-in N03-induced andun-induced plants havebeen employed in order to dissect outthe anion effect (Glass et al., 1992).Another approach that has proven effective is to switchfrom one anion toanother (e.g. CaC12 to Ca(N03)2without changing theaccompanying cationor its concentration. As a result, the observed changesof Af are due solelyto the anion effect (McClure et al., 1990; Glass etal., 1992). The results ofsuch studies have demonstrated thatN03 can strongly depolarize A’1’ andthese observations have formed the basis ofcurrently proposedproton/nitrate cotransport mechanisms (Ulirichand 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 smalldepolarization (Fig. 12,trace a). Replacing this solution with the sameconcentration of CaSO4 (Fig.12, trace b) confirmed that C1 was responsiblefor most of thisdepolarization. Thus when these calciumsalts were replaced by theirammonium equivalents, maintaining the sameanion concentration, thesignificant depolarization of AP could largelybe attributed to NH4.Although the depolarizing effects of thecalcium salts, presented at 5 mMwere significantly higher than at 50 jiM (Fig.12, traces e and f), the effects131of transfer to the equivalent ammonium saltscan be seen to induce a muchlarger depolarization (53 mV compareto 18 mV; Fig. 12, tracesg and e).Even though it was not possible to quantitativelyisolate the contribution ofCl- for studies of LATS, I consider that theNH4-’- effect still predominated,even at high external [NH4C1]0.In fact, the differencebetween tracesg ande (Fig. 12) can be attributed to the differencebetween NH4-’- and Ca2effects, since Cl- was maintained at the samelevel. Thus thedepolarizations referred to in the remainderof the paper were interpretedas predominantly due to the transport of NH4.A feature of these initial studies was the apparentrepolarization ofz+1 following depolarization in the chloride solutions(Fig. 12, traces e andg) at high[Cl10.Although repolarization to the restingpotential was notcomplete in 10 mM NH4C1, the extent of the initialrepolarization wascomparable to that in CaCl2,where repolarization wascompleted. A similarspontaneous repolarization of z’P was noted in Lemnaand in barley rootsfollowing depolarization of A’P by N03 (Ulirichand Novacky, 1981; Glass etal., 1992).5.4.2. Depolarization of AP by HATSand LATSAddition of ammonium chloride into -Nsolutions induced a rapiddepolarization of membrane potential ofrice epidermal and cortical cells(Figs. 12 and 13). This was evident evenat very low concentration (2 jiMNH4C1) (Fig. 13). Ullrich et al. (1984) reportedthat addition of NH4immediately decreased the membranepotentials of Lemna gibba.Likewise, the zP of green thallus cellsof Riccia fluitans were rapidlydepolarized by [NH4C1] as low as 1jiM (Felle, 1980). As can beseen from132Fig. 13, the time to reach initial maximumdepolarization increased from0.5 to 3 mm with increasing concentrationsof NH4C1.The depolarization of z’P by NH4 exhibiteda biphasic concentration-dependence (Figs. 14A and 14B), similarto NH4 influx into roots of rice(Wang et al., 1993b). In the lowconcentration range (<1 mM),depolarization of the membrane potentialsaturated in response to [NH4]0(Fig. 14A). Both net flux and unidirectionalinflux of NH4 in rice roots havebeen shown to respond to [NH4]0ina similar fashion (Youngdahl et al.,1982; Wang et al., 1991; 1993b). Estimatedhalf-saturation values forNH4-induced depolarization (analogousto a Km value) were 21.8 ± 2.7 .tMfor G2 plants and 35.0 ± 8.0 jiMfor G100 plants. These valuesweresomewhat lower than theKmfor 1NH4 influx, 32 jiMand 90 jiM,respectively (Wang et al., 1993b). Since ourstudies were undertaken withthe same rice variety as employed forthe‘3NH4influx experiments, thesedifferences may represent differences in growthconditions for plants usedfor the two studies, or that membrane depolarizationreflects the net,rather then the unidirectional, effect ofion fluxes. Another factor, alreadyaddressed above, is the possible effect of the accompanyinganions. Themaximum depolarizations (analogousto aVmaxvalue) were 50.6 ± 2.0 mVand 34.3 ± 1.9 mV for G2 and G100plants, respectively. The largerdepolarizing effects of [NH4-’-]0inG2 compared to G100 plants (Figs.. 14Aand 14B) correspond tothe higher values of‘3NH4influx observedin 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-Mentenkinetics (Wang et al., 1993b).Similar saturable patterns of A’P depolarizationwere associated with theuptake of either NH4-’- or N03 in Lemna(Ulirich and Novacky, 1981;Ulirich133et al., 1984) and the uptake of both NH4 and CH3NHin cells of Ricciafluitans (Felle, 1980).Between 1 and 40 mM, the depolarization ofA’{’ increased linearlywith increasing [NH4C1]0(Fig. 14B) in a manner similarto that observed for13NH4influx (Wang et al., 1993b). Both G2 andG100 rice plants exhibitedthis linear response, but the extent of depolarizationwas smaller in G100plants, where13NH4+influx was also smaller. The concentration-dependentdata for depolarization of zM’ by LATS was fittedby linear regression withr2 values of 0.94 and 0.99 for G2 andG100 rice plants, respectively.Asimilar linear response to [NH4]0wasreported for net NH4 uptakebyLemna at [NH4]0between 0.1 to 1 mM (Ulirichet al., 1984). However, inthis concentration range, NH4 uptake byLemna was not associated withfurther depolarization of A’P. Ulirich et al.,(1984) interpreted this patternas due to a diffusive uptake of NH4 or NH3.Itis clear that NH3 influxwould not depolarize z’P. However itis not clear how NH4 uptake couldoccur without further zXi’ depolarization, unlessNH4 influx was associatedwith a stoichiometric anion influx orcation efflux resulting in anelectroneutral transport.To better understand the relationshipbetween NH4 uptake andchanges in z’P, the observed values of A’Pdepolarization 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’Pwas strongly correlated with‘3NH4 influx, and that the relationshipwas biphasic. By use of acomputer-based procedure to determinethe ‘break-points’ for thebiphasicpattern objectively (Rygiewiczet al., 1984), the correlationcoefficientestablished 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 Cl4020r”2 = 0.98r”2 = 0.930 I0 10 20 30 40 50 0 10 20 30 40 501 3NIT4 Influx (pmol/gFW/h)Figure 17. The relationship between‘3NH4influxand z’P depolarization atthe same [NH4]0.1NH4 influx is fromFigs. 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 depolarizationof 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 G2and G100 plantssuggests that the HATS is more electrogenic thanthe LATS. This may bedue to the increasingly electroneutral NH4 transport at high{NH4C1J0.Inthe present study, the electrophysiological evidencesuggested that at high[NH4C1]0ammonium is taken up by rice roots inthe cation form (NH4-’-)despite the presence of a relatively high concentration ofNH3 in solution.Alternatively, it might be argued that depolarization of APmay be due tothe inhibition of the H-ATPase by NH3 at high [NH4+]0.However, the lackof a pronounced increase of ‘3N uptake at pHvalues approaching the pKafor NH4-’- does not support this interpretation(Wang et al., 1993b). Inaddition, the rapid repolarization of A’P following removal ofexternal NH4(in Fig. 12, traces g and h) is unexpected considering thatthe 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 inG2 plantsimpaled in 2 iM NH4-’- than in G100 plants impaledin 100 iM NH4 (Table11). Furthermore, the extent of the depolarization of A’fby 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), thetransmembrane electricalpotentials (AW02mM CaSO4)were 25 mV more negative thanA’YN and 45 mV400020000136—00G100G2.0.0 0.2 0.4 0.60.8-2000External ammonium concentration(mM)Figure 18. Free energy requirement for NH4 uptake asa function ofexternal [NH4].Values of cytoplasmic [NH4+] were taken fromour previousstudy (Wang et al., 1993a). Arrows indicate the [NH4J0below whichNH4-’-uptake is against the electrochemical potential gradient forG2 and G100plants, respectively.137more negative than&‘G2 and APG100, respectively. These differencesreflect the contributions to the membrane depolarization fromthe variousions present in MJNS. Since the values ofAPN, and M’G2 and A’PG100, weremeasured in the same basal medium (MJNS), theobserved 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 NH4acrossthe plasma membrane, which in turn allows us to determinethe energyrequirement for transport (Findlay and Hope, 1976). Taking 3.72mM 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) wereestimated for G2 and G100roots, respectively. From these values, the free energy (zSt) requiredtotransport NH4 across the plasma membrane can be computedfrom thedifferences between measured membrane potentials(A’PG2 or zS’PG100) andestimated Nernst potentials at specific [NH4]0(Fig. 18). Theestimated 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 ofNH4-- mayoccur via passive transport systems, down the electrochemical potentialgradients for NH4--. As pointed out previously (Wanget al., 1993b), thesefree energy estimations only provide a prediction of the feasibilityof theuptake process occurring under the prescribed conditions. For bothG2 and138G100 plants, the predicted [NH4-’-]0for the shiftfrom active to passiveuptake was quite a bit lower than thebreak-point determinedby thekinetics analyses (42 jiM and 655 jiM versus 1 mM).Thus, one must becautious in identifying a specific transportsystem based purely onthermodynamic or kinetic considerations.5.4.4. Mechanisms of NH4uptake by HATS andLATSThe preceding section has demonstrated thatat low [NH4-’-]0(<42 jiMfor G2 plants and 655 jiM for G100 plants), NH4influx appears to be anactive process in roots of rice plants. However,the details of thismechanism are unknown for rice and for anyhigher plants. Possiblemechanisms for this active uptake via HATS include:(a). a proton : NH4symport; (b). a specific NH4 ATPase. The resultsof the inhibitor studies,both for the electrical potentials in the present studyand‘3NH4-’- influx(Wang et al., 1993b) provide evidence for a dependence(either direct orindirect) on the proton motive force. Application ofCCCP caused 89% and85% inhibition, respectively, of membrane depolarizationby NH4-’- and13NH4influx in solution containing 100 jiM NH4.The strong inhibitoryeffects of CN+SHAM on depolarizationof zSP (9 1%) and on‘3NH4influx(8 1%) confirm the dependence of these processeson a source of metabolicenergy without distinguishing the natureof the mechanisms. The effects ofDES, an inhibitor of the H-ATPase, indicated theinvolvement of the protonpump, suggesting speculatively that H+-transportmight be involved.The results of the present and earlierstudies (Wang et al., 1993b),strongly suggest that the two systems,HATS and LATS, have differentmechanisms of energy coupling. Above 42jiM for G2 and 655 jiM forG100139plants, NH4 transport was predicted to be a passiveprocess. Thisprediction is borne out by the generally smallereffects of metabolicinhibitors at high external [NH4-’-] than at low [NH4+]0(present study and inWang et al., 1993b), although 13NH4influx showedgreater sensitivity toinhibitors than the AP depolarization. There is virtuallyno informationavailable regarding the energy coupling for theLATS. 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 sharedcation channel.For example, the recently described K channel inArabidopsis has beenshown to have an NH4 conductance that is 30%of the K conductance(Schachtman et al., 1992). Also, in the cyanobacterium Anabaenavariabiis(Avery et al., 1992), the uptake of Cs (a K analogat the uptake step) andNH4 was closely related. Thus low affinity NH4 transport mightoccur viathe K channel.5.5. SUMMARYThe transmembrane electrical potentialdifferences (&P) weremeasured in epidermal and cortical cells of intact rootsof 3-week-old rice(Oryza sativa L. cv. M202) seedlings grown in 2 or 100micromolar (jiM)NH4÷ (G2 or G100 plants, respectively). In modifiedJohnson’s nutrientsolution (MJNS) containing no nitrogen, A’I’ was inthe range of -120 to-140 millivolts (mV). Introducing NH4 to the bathingmedium 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 13NH4influx werealso biphasic, indicating distinct140coupling processes for the two transport systems,with a break-pointbetween two concentration ranges around 1 mMNH4.The extent ofdepolarization was also influenced by nitrogen status,being larger for G2plants than G100 plants, corresponding to thelarger NH4 influxes in G2plants than G100 plants. Depolarization of A’Pdue to NH4 uptake waseliminated by a protonophore (carboxylcyanide-m-chlorophenylhydrazone), inhibitors of ATP synthesis (sodiumcyanide plussalicyihydroxamic acid), or an ATPase inhibitor (diethyistilbestrol).141Chapter 6. REGULATION OF AMMONIUMUPTAKE6.1. INTRODUCTIONWhen plants are deficient in nutrients, suchas PO4-,S042-,Cl-, theiruptake capacity is greatly enhanced (Lee, 1982).This phenomenon hasbeen known since the works of Brezeale (1907 inGlass, 1989) thatnutritional history of a plant can profoundly affect itssubsequent capacityto absorb the same ion (see also Hoagland and Broyer,1936; Broyer andHoagland, 1943). Such relationships between theions provided duringplant growth and their subsequent uptake by rootsor tissues was welldefined in several species for the uptake of K (Leigh andWyn 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; MacKownet al.,1982; Glass et al., 1985; Siddiqi et al.,1989, 1990; Jackson and Volk, 1992;King et al., 1993). However, the quantitativebasis of the correlationbetween the rate of N absorption and the N-status ofthe plant material isnot precise (Lee and Rudge, 1986).It have been demonstrated that plants areable to adapt to availablesources of N over a wide range ofconcentrations (Clement et al., 1978;Wang et al., 1991). The existence of distincttransporters with differentaffinities for either nitrate or ammonium(Siddiqi et al., 1989; Wanget al.,1993b) represents an importantpart of this capacity foradaptation.Typically, nitrogen starvation leadsto elevated fluxes of nitrogen, while N142excess leads to down regulation of uptake. However, theunderlyingmechanisms responsible for these changes are largelyunknown. Severalhypotheses have been advanced concerning the sourcesof feedbackregulation responsible for controlling N uptake. Theseinclude theimportance of products of N assimilation (Lee and Rudge, 1986;Cooper andClarkson, 1989; Jackson and Volk, 1992), as well as theeffects ofaccumulated ions (NO3-and NH4)on influx or efflux (Morganand Jackson,1988a, 1988b; Siddiqi et al., 1989; King et al., 1993; Wang etal., 1993a).It has been suggested by Morgan and Jackson (1988b),that at highplant N status, reduction or suppression of net ammonium uptakemay bedue to (i) low energy supply to the root system, (ii) accumulationin theroot tissue of a nitrogenous compound which exerts negativefeedback onthe influx system, (iii) high efflux of endogenous NH4-1-. Thisaccumulatedregulating effector could be ammonium ions generatedby degradation oforganic nitrogenous sources within roots, or rapidaccumulation ofammonium in N-depleted roots upon initial exposureto ammonium, orrelative ease of outward ammonium movement (Morganand Jackson,1988a, 1988b). The regulation of influx may therefore reflectthe interplayamong suppression of influx by a product of ammoniumassimilation, theaccumulation of root ammonium and associatedammonium efflux, and astimulation by ammonium of its own uptake (Morganand Jackson, 1992).It was found that 13NH4 influxes into intact rootsof rice werenegatively correlated with the level of NH4 provisionduring growth andthe internal [NH4]in root tissues (Wanget al., 1993 a, 1993b). It has beensuggested that the regulation of NH4 uptake could resultfrom feedbackeffects of accumulated NH4-’- or products ofNH4-’- assimilation (Ullrich et al.,1984; Lee and Rudge, 1986; Morgan andJackson, 1988; Lee et al., 1992;143Jackson and Volk, 1992; Wang et al., 1993a). Theseexert effects on bothinflux and efflux although the principle effect isupon influx (Wang et al.,1993a). However, the mechanism(s) of regulation arestill unclear.In order to explore the basis of the negative feedbackregulation ofNH4÷ uptake, I investigated the effects of the followingpretreatments on13NH4 influx: (1) repleting N-depleted plants in1 mM NH4 in thepresence or absence of MSX; (2) depleting N-repletedplants in 2iiMNH4solution in the presence or absence of MSX; (3) elevatingroot glutamineconcentrations by supplying this amino acid exogenously;(4) alteringinternal concentrations of NH4,glutamine and otheramino acids in roottissue of the above treatments; (5) using selected inhibitorsof ammoniumassimilation to study the effect of perturbing ammoniummetabolism onammonium uptake. The results of these experiments areinterpreted interms of a cascade model for the regulation of NH4 influx in riceroots.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. Productionof‘3NH4.1446.2.2. Experimental design6.2.2.1. Experiment I. Depletion andrepletion studyTo investigate NH4 uptake by roots inresponse to changing plantN status, 13NH4 influx was measured inNH4-repleted G2 plants orNH4-1-depletedG1000 plants as well as G2 andG1000 plants under theirgrowth conditions. At designated times,the assigned G2 plants weretransferred to the G1000 medium andG1000 plants were transferredtothe G2 medium. The time periods of repletionwere 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 periodsof 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 investigatethe time course ofeffects of MSX on13NH4+influx. Either G2 or G1000 plants were pretreatedin their respective growth media in the presence of 1mM 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 MSXeffects during repletionand depletion: plants were first transferred into growthmedia with MSXcontaining the same [NH4]0as they hadbeen grown in (i.e. in G2+MSX forG2 plants or G1000+MSX for G1000 plants) at 24 h beforemeasurement,and then G2 plants were transferred from G2+MSX to G1000+MSXor G1000plants were transferred from G1000+MSXto G2+MSX at times of 1, 4, 12and 24 h. For comparison, a third set of plantswas transferred fromgrowth medium to pretreatment mediumi.e. G2 plants to G1000 mediumor G1000 plants to G2 medium at timesof 1, 4, 12 and 24 h. In anotherexperiment, the pretreatment times forboth G2 plants repleted in G1000145medium and G1000 plants depleted in G2 mediumwere 0, 1, 4, 12, and 24h. The influxes were measured for 10 mm in100 jiM 13NH4-’--labeledsolution without MSX. Each datum point is themean 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 1NH4influx of riceroots: G100 plants were pretreated in G100 medium withor without 10mM glutamine for 16 h before measuring 13NH4influx.13NH4 influxeswere then measured in 2, 10, 25 and 100 jiM 13NH4-labeledsolutionwithout glutamine. (2) The effects of various exogenously suppliedaminoacids on the influx of 13NH4:G2 plants were pretreated inG2 medium orG100 medium plus 10 mM glutamate, glutamine or asparaginefor 16 h,respectively. 1NH4 influxes were measured in 100 mMlabeled 1NH4÷solution in the presence of the same amino acids. Eachexperiment 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 toperturb tissueconcentrations of glutamine and glutamate to investigatethe effect ofchange of these compounds on 1NH4influx. All treatmentsof inhibitorswere administered for 16 h at 100 mM. 1NH4influxes weremeasured ineither 100 mM or 10 mM labeled1NH4solution.6.2.3. Determination of free ammonium in roottissueSee 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 influxof nitrogen-deficientrice plants (G2 plants) was 11.10 jimol g’FW h’, whichis close to theVm(12.8 jimol g’FW h-i) of G2 plants (Wang et al., 1993b). Afterrepletion inG1000 medium, influx increased to nearly3 times its initial value (to 31.97iimolg4FW h4) during the first 5 h. Between 6 to 12h of loading, influxesdeclined to about 10 p.mol g’FW h’. After threedays in 1 mM NH4--solution, the‘3NH4 influx dropped below 5 jimol g’FWh-’. When G2plants were repleted in 10 or 100 jiM NH4 solution,G2 roots respondedwith a similar pattern, but showed a delay in reachingthe maximum ofinflux (data not shown).Nitrogen-sufficient rice seedlings were grownin G1000 medium forat least 13 days and transferred to G2 mediumfor periods varying from0.3 to 192 h, respectively, before measurement of‘3NH4influx. As shownin Fig. 20A, initial‘3NH4influx of G1000 plantswas quite low (1.15 jimolg1FW h-i) in agreement with previous reports(Wang et al., 1993b). Shortterm depletion in G2 medium, for periods of0.5 to 4 h, caused 1NH4influxes to increase almost 10 fold. Between 4to 24 hours,‘3NH4influx ofthese N-depleted plants was close to theVm for 13NH4influx of G21474019A40Repletion in G1000 Media (h)Figure 19.‘3NH4 influx of repleted G2 plants. After repletionin G1000medium for various periods, 13NH4influx of G2 plantswas measured in100 jiM13NH4-labeled solutions. Insert 19B shows, inexpanded form, thefirst 24 h of repletion. Each datum point is the meanof 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. 1NH4influx of G1000 plants during depletionin G2 mediumfor various periods. The influxes were measuredin 100 iM13NH4-labe1edsolution. Insert 20B shows in expanded form the datafor the first 24 h ofdepletion. Each datum point is the mean of 3 to6 replicates and thevertical bar represents the standard error (± se).14921A60 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 depletedG1000 roots. G1000roots were depleted in G2 medium for variousperiods and internalammonium content were assayed. Insert 2 lBshows in expanded form thedata for the first 24 h of depletion. Eachdatum point is the mean of6replicates and the vertical bar represents the standard error(± se).150plants (—41 pmol g’FW h-i) (Fig.20B). After 24 hours depletion,the‘3NH4influxes declined but were stillhigher than those of G1000 plantsat steady-state. The results indicated, that depletionin G2 medium forupto 8 days, caused no further decline ofinflux, which remainedat about 6imol g-’FW h-i. Meanwhile, root NH4÷concentrations dropped rapidlyduring the first 4 h depletion of N, from5.6 to 3.6iimol g’FW (Fig. 21A).After 24 h depletion, internal [NH4+j remainedat a low level (0.6!Imol g1FW, in Fig. 21B). Figures 20B and 21B reveal thatthere was a negativecorrelation (r2 = 0.74) between [NH4]1and 13NH4 influx during 24hdepletion of N. Beyond 24 h of N depletion,no correlation was found.Changes of the total AA content in root tissueof 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 originallevel through 200 h ofdepletion. The contents of the major amino acidsand amides, [Gln]1,{Glu]1,[Asn]1,and [Asp] were also foundto have increased in the same fashion(data not shown).The phenomenon of stimulated influx observed duringthe first hoursfollowing exposure of G2 plants to 1000 jiM NH4 wasnot as pronounced inthe second experiments (open circlesin Fig. 23A) as in the first experiment(Fig. 19A). This may have beendue 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 heldrelatively constant. In the secondexperiment, the same treatments wereperformed in a volume of 20 ml ofmedium. Such a small volume mayhave limited the repletion process andconsequently affected the extent of theinflux response. For example,typical cytoplasmic and vacuolar [NH4+]were 0.19 and 2.19 jimolg’FW for1515._______________________________22A2.5____:::10 15 20250 25 50 75 100125 150 175200Depletion in G2 media (h)Figure 22. Total amino acid concentration([AA])of depleted G 1000 roots.After depletion in G2 mediumfor various periods,G1000 roots wereassayed for tissue amino acidconcentration ([AA]1).Insert 22B shows inexpanded form the datafor 24 h of repletion. Eachdatum point is themean of 6 replicates andthe vertical bar represents thestandard error (±se).152G2 roots and 1.94 and 4.91 jimol g’FW for G1000roots, respectively (seeTable 4). This means that in order to convert G1000plants to G2 plantsthere is about 4.47 jimol NH4g4FW to be depletedeither by metabolismor efflux to the external media. Assuming thatrates of efflux andassimilation are equivalent at about 20% of the rateof influx (Chapter 3and Wang et al., 1993a), then the release of NH4could elevate external[NH4]to nearly 100 tiM. In the small volumeemployed for thisexperiment the released NH4would readily bere-absorbed, slowing downthe change from G1000 to G2 statues.As shown in Fig. 23A, when G2 plants wererepleted with NH4-- inG1000 medium, the‘3NH4 influx (closed circle) increasedfrom 8.17 to10.00 j.tmol g’FW h’ during the first hour, thendropped to 8.61 at 4 hand 1.95iimolg-’FW h-i after 24 h repletion. Root [NH4]1(closedsquare)increased rapidly in the first hour from 2.21 to6.48iimol g’FWandincreased only slightly to 7.13iimolg’FW during the next 23 h of NH4repletion (Fig. 6B). By contrast, depletion of G1000plants in G2 mediumincreased 13NH4 influx only very slightly duringthe first hours. Theninflux increased rapidly from 0.72 to 7.29iimolg’FW h’ (open circle inFig. 23A). During the depletion in G2 medium, the [NH4+]1of G1000 plants(open square) decreased gradually from 6.35to 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 presentdifferent patterns forplants undergoing nitrogen depletion or repletion.During the repletionprocess, G2 plants were exposed to 1000.tM NH4for up to 24 h. The total15312- L0,23A— G2IG1000 23B G2IG1000—0— G1000IG2 —DG1000IG2— —E8E:.-.U—4z. II2-EE_______________________.00-0 5 10 15 20 250 5 10 15 20 25Pretreatment time (h)Figure 23. 13NH4influx (23A) and internal ammoniumcontent (23B) ofrepleted G2 or depleted G1000 roots. 23A:‘3NH4influxesof G2 or G1000roots, after pretreatment for 1, 4, 12 and 24 h inG1000 or G2 medium,respectively, were measured in 100 1iM 13NH4-labeledsolution. 23B:Internal ammonium content of thesame roots. Each datum point is themean of 6 replicates and the vertical bar representsthe 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 afterpretreatment for 1, 4, 12 and 24 hin G1000 or G2 medium, respectively. Eachdatum point is the mean of 6replicates and the vertical bar represents thestandard error (± Se).1552000- 25B Glu G2/G1000M25A G1nG2/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/G1000M25D Asp ——— G2/G1000M° —0— G1000/G2M—ETh— G1000/G2M400-400—E200200-.0• 00 5 10 15 20 25 0 5 10 15 20 25Pretreatment time (h)Figure 25. Tissue amide or amino acid contents ofrepleted G2 or depletedG1000 roots. After pretreatment for 1, 4, 12 and 24 h inG1000 or G2media, respectively, the aminoacid 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 verticalbar represents thestandard error (± Se).156[AA]1 increased during 12 h of repletionand 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 patternas the total [AA]1 but the Glucontent (Fig. 25B, closed circle) decreasedcontinuously during NH4--repletion. Although the reduction of [Glu] was 37%,[Gln]1increased 372%during 24 h of repletion. In contrast, [Asn]1decreased 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 whichit increased slightly.When G1000 plants were depleted in G2 medium,total [AA] as well as thefour major amino acids decreased rapidly for the firsthour (Figs. 24,25AD, open symbols). This is interesting becausedespite big changes inthese [amino acid], influx changedlittle. 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 depletionprocess.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 16.93jimol 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 atabout 10iimolg’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) whichremained essentiallyconstant for the duration of the experiment. Fluxesof G1000 plants were157significantly lower than in G2 plants in G2+MSX orG1000+MSX (compareopen to closed symbols in Fig. 26).For G2 plants pretreated in G2+MSX, root[NH4+]1increased rapidlyfrom 2.21 to 7.19 at the first hour and remainedat the same level for theremainder of the experiment (closed circles in Fig. 27A),but pretreatmentin G1000+MSX caused root [NH4]1to increase rapidlyfrom 2.21 to 8.49imol g’FW during the first hour, reaching a valueof 9.35 after 24 hrepletion (closed squares in Fig. 27B). G1000 plantspossessed a higherinitial [NH4]1(6.35 jimolg1FW) (Figs. 27A and 27B),which continuouslyincreased to 8.57 imol g1FW after 24 h during treatmentof G1000+MSXmedium. Root [NH4-’-]1 in G1000 plants treated inG2+MSX declinedgradually from 7.36 at 1 h to 5.77 between 4 and24 h (open circles in Fig.27A). The increment of [NH4-’-]1in MSX treated plants variedwith priorNH4 provision during growth and additional depletionor repletiontreatments (Figs. 27A and 27B). During the first hour,the [NH4-’-]1of G2plants increased 230% in G2+MSX medium and 320% inG1000+MSXmedium. The [NH4]1 of G1000 plants increased 35% inG1000+MSXmedium, and 16% during the same time period in G2+MSXmedium, thelatter then decreased to 9% after 24 h.The total [AA]1 of G2 or G1000 plants in the fourtreatmentspretreated 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 showedasmall increase in the G1000+MSX treatment (Fig.28B). Pretreatment inG2+MSX, caused the [Gin]1 of G2 roots to decline at the firsthour but nofurther changes were observed during the remainderof the experiment(Fig. 29A, closed circles). The opposite effect was observedin G1000+MSX15825•G2/G2M+MSX—0—G1000/G2M+MSXG2/G1000M+MSXDG1000/G1000M+MSX2001021025Pretreatment time (h)Figure 26. Effect of MSX pretreatment on 1NH4influx 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+MSX10.21G2—a— G2—0— G1000—D— G1000• I • I • I • 0• • I • I • I0 5 10 15 20 250 5 10 15 20 25Depletion or repeltion time (h)Figure 27. Effect of MSX on internal ammonium content of riceroots. Thepretreatments and symbols are same as in Fig. 26. Each datumpoint is themean of 6 replicates and the vertical bar represents the standard error (±se).160‘—‘ 3000300028A in G2M+MSX28B 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 riceroots. The pretreatments andsymbols are same as in Fig. 9. Figures hAand 11B are for the plantspretreated in G2+MSX medium and in G1000+MSXmedium, respectively.Each datum point is the mean of6 replicates and the vertical barrepresents the standard error (± Se).161;:::z100•100•EEooo* G2—D—- G10000•00 5 10 15 20 25o 5 10 15 20 25‘ 150 15029C in G2M+MSX 29D in G1000M+MSXG2G2100—0—— G1000100—U— G1000IIII.. ..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]1of plants pretreated in G1000+MSX medium,respectively.16230030029E in G2M+MSX 29F in G1000M÷MSX• G2—•— G2—0— G1000f20O200I IE10000 5 10 15 20 2500 5 10 15 20 2530030029G in G2M+MSX29H in G1000M+MSXII—— G2 —— G2—0— G1000 —D— G1000— 00200L1—E100 1000•U-0 5 10 15 20 250 5 10 15 20 25Depletion or repeltion time (h)Figure 29. (Continued).163(Fig. 29B, closed squares). The [Gin]1of G1000 rootswas reduced more inG2+MSX (Fig. 29A, open circles) than inG1000+MSX (Fig. 29B, opensquares). In the latter medium, Gln recovered slightlyafter 24 hpretreatment (Fig. 29B). The levels of [Glu]1in rootsdeclined 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 thatit took a longertime to achieve the same reduction (Fig. 29C, closed circles).The [Asn]1and[Asp]1of G2 roots were also significantly reduced in all fourpretreatments(Figs. 29EH). A similar extent of reduction of [Asn]1was reached in ashorter time period when G1000 plants were pretreatedwith MSX ineither repletion with or depletion of NH4 (open circlesin 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 thelatter treatmentthe reduction occurred within 4 h of pretreatment.6.3.3. Experiment III. Effects of exogenous amino acidsPretreatment of G100 roots with 10 mM glutaminesignificantlyreduced‘3NH4 influx at all concentrations tested(Fig. 30). Assays of[NH4]1revealed that glutamine pretreatmentwas associated with higher[NH4-1-]1(6.2 ± 0.5 .tmol g’ FW) than those pretreated withoutglutamine(2.3 ± 0.8 pmol g-’FW). The 18 h pretreatmentin 10 mM Gin raised thecontents of Gin, Glu, and Asp near 4 times and Asn 7times (Figs. 3 1A and31B).The interaction of exogenous amino acidsand nitrogen status werealso investigated. When G2 plants weretreated with either 10 mM [Gin]0or[Glu]0for 18 h, 1NH4influxes were significantlyreduced (from 8.9416460 without glutamine[NH Jo (jiM)Figure 30. Effect of exogenous glutamine on‘3NH4influxof roots. G100plants were pretreated in G100 medium with or without10 mM glutaminefor 16 h before measuring‘3NH4influx.‘3NH4influxeswere measured in2, 10, 25 and 100 jiM13NH-labe1ed solutionwithout glutamine. Eachdatum point is the mean of 6 replicates and the vertical barrepresents 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 acidon‘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, orAsn for 6 h. Theinfluxes were measured in 100 jiM13NH4-labeiedsolution. Each datumpoint is the mean of 6 replicates and the verticalbar represents thestandard error (±se).32B G2/G100M1210864.20T32A G2/G2M• 0T)•Im•‘Il/I,12108642-0-zControl Gin Glu AsnControl 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]0or [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]1was highest in the Gin pretreatment (Figs. 35A and 36A), except forthe [Glu]0pretreatment 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 amidesand amino acid on internalammonium content. Details as in Fig.32.33 B G2/G100M000-.SCECE65.4.3.2-1-0433A0 G2IG2MTT_Wf WFA Ff1III0”Control Gin Glu Asn ControlGin Glu AsnPretreatment2-169-.6Control Gin Glu AsnPretreatmentFigure 34. Effects of exogenous amides and amino acid ontotal amino acid34AG2IG2ME4.-.C.?12 -29-18 -34BG2/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 acidon contents of aminoacids in G2 roots. Pretreatments are sameas in Fig. 32. Figs. 35AD is for[G1n], [Glu]1,[Asn]1,and {Asp] of plantspretreated in G2 medium,35A Gin35B 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 andamino acid on contents of aminoacids in G100-pretreated roots. Pretreatmentsare same as in Fig. 32.Figs.36AD is for [Gin]1,[Giu]1,[Asnj1,and [Asp] of plants pretreatedin G10036A Gint7180006000 -4000 -2000 -0-3000 -2000-1000-36B GluVA/-IControl Gin Glu Asn36C Asn80006000-40002000-06000400W20000-Control Gin GiuAsnp0c-)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 ControlGin Giu Asnmedium, respectively.1726.3.4. Experiment IV. Effects of selected inhibitorsG100 plants were treated with inhibitors ofglutamine synthesis,MSX, glutamate synthesis, DON, and aminotransferases,AOA, for 16 h,respectively. The‘3NH4influxes were measured in either100 jiM or 10mM labeled 1NH4 solution without inhibitors. The largesteffect of theinhibitors of NH4 assimilation was associated with AOA pretreatment(Fig.36A). The13NH4influx due to HATS (high affinity transportsystem) andLATS (low affinity transport system) were reduced by68% and 32%,respectively (Figs. 37A and 37B). MSX reduced‘3NH4÷ influxby the LATS25% and by the HATS 19%. DON treatment producedonly 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 actuallyhad a lower[NH4]than the control. The total [AA]1was doubled by the AOA treatment(Fig. 38B). While slightly increased by MSX, the total[AA1 wasgreatlyreduced by DON treatment. Looking at the four major amidesand aminoacids, (as shown in Figs. 39A, B, C, D), the pretreatment of AOAsignificantlyincreased 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 AOAControl MSX DON AOAPretreatmentFigure 37. Effect of MSX, DON and AOA on1NH4influx. G100plants werepretreated with MSX, DON, and AOA for 16 h, respectively. Theinfluxeswere measured in either 100 1iM (Fig. 37A) or 10 mM (Fig.37B) labeled13NH4 solution without inhibitors. Each datum point is themean 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 ammoniumand 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 DON0AOAControl MSXPretreatmentDON AOAPretreatmentFigure 39. Effect of MSX, DON and AOA on major amino acidcontents.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 of6 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 inresponse to the Nstatus of the plant, but it is not clear how this is achieved.Increase inammonium influx upon nitrogen limitation anddecrease in influx as cellnitrogen status rises have commonly beenobserved (McCarthy andGoldman, 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 andLuttge, 1991). Feedbackinhibition of NH4 uptake by nitrogenous effectors has beenimplicated inorganisms like Lemna, algae, yeast and higher plants (Kleiner,1985; Ulirichet al., 1984; Pelley and Bannister, 1979; MacFarlane andSmith, 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 proposedto 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). Inthereview by Clarkson and Luttge (1991) a central role for glutamineinregulating the uptake of N by fungi and microalgaewas presented.Glutamine or asparagine are the low molecular weightN-containingcompounds stored or translocated by plants in the family ofPoaceae(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 relatedto the reduced-N177status of the root tissue. In tobacco cells culturedon nitrate, urea, orammonium, Gln is the first major organicproduct of assimilation of‘3NH4(Skokout et al., 1978). It is also true for rice,because glutamine andglutamate were the primary products ofammonium assimilation in riceroots (Arima and Kumazawa, 1977). However thestudies by Lee et al.,(1992) and by several other workers (summarized inClarkson and Luttge,1991) showed that other amino acids may participatein the regulation ofN uptake.In the present study, evidence supportinga central role forglutamine or other amino acids in controlling NH4influx was equivocal.When plants were maintained at 2 tM or 1000iM NH4 respectively,‘3NH4 influx was inversely correlated with [Glfl]i (closedsymbolscompared to open symbols in Figs. 29A and29B). Likewise, when theinternal concentrations of Gln and other aminoacids 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 supportedby theresults of the AOA treatment. After treating plants with AOA,under theconditions of the present study therewas 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 mentionedreductions of13NH4influx in rice also coincided with a significant increaseof [NH4÷]1(Figs. 33A and 38A). Pretreatment with 10 mM Glndoubled the [NH4]jfrom 2.30 to 6.10iimolg4FW (also in Fig. 33A) and decreased1NH4influx.178In the depletion experimentshown in Fig. 23A transferof G1000plants to G2 solution failed to increaseNH4÷ influx until 4 h hadelapsed.Yet, the amino acid analysis indicated strongreduction of total [AA] and[Gin], [Giu] and [Asp] (Figs. 24, 25AD).Strong reductions of aminoacidswere not correlated with 13NH4influx. Therefore,it is not entirely clearwhich N derivative is responsible for limitinginflux.Although applying organic N to the growthmedia has been found toincrease crop yield (Mon et al., 1977; Monand Uchino, 1977), thetreatment of organic N suppresses the uptake ofinorganic N. For example,maize roots pretreated with Gin or Asnexhibited reduced net uptake ofNH4 and N03 (Lee et al., 1992). The uptake of‘5N03by barley roots wasdepressed by pretreatment with Arg and His (Monet al., 1979). It wassuggested that transport activity for ammoniumwas controlled byintracellular rather then extracellular metabolites (Jayakumarand Barner,1984).6.4.2. Effect of MSX: reduced amino acid pooiMSX inhibited the activity of glutamine synthetase inplant roots, andstopped the ‘5N labeling of free amino acids, particularlyglutamine andglutamate in roots of barley or rice (Arima andKumazawa, 1977; Lewis etal., 1983). Preventing the assimilation of newlyabsorbed NH4 or releasingNH4 from the catabolism of internal N-containingcompounds rapidlyincreased the NH4 concentration in roots (Arimaand Kumazawa, 1977;Lewis et al, 1983; Lee et al., 1992). Two majoreffects are expected: theamino acid pool is reduced and NH4 pool is increased.After treating withMSX, tissue [Gin]1is typically decreased (Stewardand Rhode, 1976; Fentem179et al., 1983a, 1983b) and consequently the amide donorto Asn synthesis isdecreased, since the concentrations of Gin and Asnclosely correlated (Leeet al., 1992). When products of ammonium assimilationwere 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 ammoniumconcentration in roottissue of rice (Arima and Kumazawa, 1977), Datura(Probyn and Lewism1979), barley (Lewis et al., 1983; Fentemet al., 1983b; Morgan andJackson, 1988a, 1988b); wheat (Morgan and Jackson,1988a, 1988b), maize(Lee and Ratcliffe, 1991; Lee et al., 1992). A ten foldincrement of thecytoplasmic pooi was reported in maize rootscompared to the control (Leeand Ratcliffe, 1991; Lee and Ayling, 1993). This increaseis due to twoeffects: (a) the assimilation of NH4 into amino acids isblocked, and (b) theproduction of NH4 from breakdown of amino derivativesremainsunaffected. It has been claimed that release of NH4 fromthis degradationpath occurs at a rate which is 50% higher than therate of NH4 influx(Jackson et al., 1993). As a result, ammoniumappeared in the xylem sap(Lee and Ratcliffe, 1991) and net NH4 efflux was increasedsubstantially(Morgan and Jackson, 1988a). Arima and Kumazawa(1974, 1975, 1976,1977) proposed that most of the glutamineis synthesized adjacent to theouter membrane of plasma membraneof root cells, through whichammonium with a high 15N abundance permeatesfrom the externalsolution. MSX treatment might enlarge this ammoniumcompartment nearthe membrane.Another explanation for the enhancedNH4 influx by MSX treatmentis that MSX enlarged the cytoplasmic andvacuolar NH4 pools of roottissueseveral times (Jackson et aL, 1993; Leeand 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 measuredunderthese circumstances was a true value of influx. Bycontrast, under ‘normal’circumstances (they claim) even short‘3NH4 influxmeasurements arecompromised by a significant efflux. The results ofstudies on rice andbarley (Siddiqi et al., 1991; Wang et al., 1993a) repudiatethisinterpretation because the half-life of the cytoplasmiccompartment 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 treatingplants with MSX,the concentrations of major amides and amino acidswere all reduced todifferent extents (Figs. 29A-H), accompanied by an increased[NH4-’-]1.Theincrement of [NH4]was varied with NH4 provision andadditionaldepletion or repletion treatments (Figs. 27A and27B). However thetreatment with MSX in this experiment failed to increasethe13NH4+influxes of G1000 plants treated in either G2+MSX or G1000+MSXmedium(open symbols in Fig. 26) compared to the effects of depletion orrepletionby the same plants in the absence of MSX (Fig. 23A). However, G2plantstreated with G2+MSX conditions revealed a significant increaseof influx(Fig. 26). When the same G2 plants were treatedin G1000 medium plusMSX there was no decline of influx of the sortobserved in the absence ofMSX (Fig. 23A). This is consistent with an importantrole of amino N indown-regulating influx in low-N plants. The lackof an increased influxwhen G1000 plants were transferred to G2medium with MSX (Fig. 26)argues that internal [NH4-’-] is important in maintaininglow NH4 fluxes inhigh-N plants. This has also been claimedby Causin and Barneix (1993) in181wheat. Thus the results of these experimentsindicated that both [NH4]and [AA]1 may play important role in regulatingNH4 fluxes.6.4.3. Effect of short-term N depletionIt has been recognized that the nitrogen (bothammonium andnitrate) uptake capacity of plant roots is enhanced whenplants 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). NH4uptake shows aparticularly strong response in several species, such as wheat(Tromp,1962; Minotti et al., 1969; Jackson et al., 1976b; Morganand 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 nitrogendepletion withenhanced NH4 influx (Fig. 20). The short-term depletionof highNH4+-grown plants (G1000) in low N medium (G2 medium)stimulatedNH4 influx during the first 4 to 5 h of depletion. 13NH4influx remainedhigh for the next 20 h, then declined to a relatively lowerrate for the next20 h of depletion (Fig. 20B). Similar rapid initial increases of NH4 uptakewere observed when plants were depleted of N for the first0.25 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 theremoval of a factorwhich exerts negative feedback regulation on NH4uptake. Both NH4-- andits primary assimilate were suggested assuch factors in uptake regulation(Breteler and Siegerist, 1984; Revilla et al., 1986;Lee and Rudge, 1986;Morgan and Jackson, 1988a). Another explanationfor this enhancement isdue to enhanced influx and reduced efflux (Morganand Jackson, 1988a,1988b). Substantial ammonium cycling occurredduring net ammoniumuptake (Jackson et al., 1993), yet plants grown in lowN possess a low NH4efflux. For G2, G100 and G1000 plantsat 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 relativelysmallproportions may not account for the large increases ofNH4 uptake such aswere observed in the present study.In the present study, [NH4]1was negatively correlatedwith influxduring 4 h of depletion (Figs. 20B and 21B). It was also observedthat theVmaxfor NH4 influx was negatively correlated with internalNH4 (Wang etal., 1993b). When plants were subjectedto N depletion, the tissue contentof NH4 (Fig. 2 1B) dropped rapidly to lower levelsand possibly resulted ina relief of N-suppression of the uptake process.[NH4]c is a likely candidatefor negative feedback regulation since the freeNH4 pools (cytoplasmicand vacuolar) will be drained in two opposite directions: effluxout of thetissue and metabolism into amino acids. In sucha short time,[NH4]willbe the first fraction to be drained to a minimum.Therefore internal NH4 isa likely factor to exert a negative signal onNH4 transport across theplasma membrane (also in section 6.4.5.).It is generally believed that short periods ofN depletion, less than 24to 48 h, would not cause a decline of growthrate (Siddiqi et al., 1989;183Jackson and Volk, 1992). Though it was reported thatenhanced uptakereached a maximum after 3 days of depletion whennitrogen stress wasnot severe enough to alter the RGR significantly(Lee and Rudge, 1986),longer N depletion may not sustain the maximum enhanceduptake ratedue to possible adjustment of the RGR. For 8-d-old maize plantsgrown on5 mM NO3-, NH4 uptake rates increased steadily, and within 72h of N-depletion, rates of NH4 uptake initially increased followedby a declineand a subsequent increase (Jackson and Volk, 1992). Thisenhanced NH4uptake or NH4 influx may be due to a relief of the uptake processfrom N-suppression. As suggested by Morgan and Jackson (1992),this type ofresponse reflects the interplay of suppression by a productof ammoniumassimilation, the accumulation of root ammoniumand associatedammonium efflux, and a stimulation by ammonium of its ownuptake.6.4.4. Stimulated NH4 influx after long-term N depletionWhen N-depleted roots are first exposed to elevated levelsofNH4-l-there is an initial increase of NH4 influx for the first fewhours ofexposure to NH4 (Goyal and Huffaker, 1986; Morgan andJackson, 1988a).The above workers observed a 25-35% increase of influxin wheat duringthe period from 2-10 h after exposure to NH4;furtherexposure causedinflux to decline. This phenomenon was foundin 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-’-, 13NH4influxincreased rapidly from11.10 imol g’FW h’ to 31.97iimolg’FW h’ (Fig. 19B). Then, influxdropped to the initial rate of about 10 p.molg’FW h1 after 8 h morerepletion. A smaller stimulation can also be seenin Fig. 6A.184There are at least two possible explanationssuggested for thisphenomenon (Morgan and Jackson, 1988a). First, a secondsystem forammonium influx may be initiated (induced?) as N-depletedplants areexposed to ammonium for a short period beforenegative feedback becomeactive. Another possibility is that there are two effectors(positive andnegative) to regulate a single transport system. The positiveeffector couldbe NH4 and the negative one may be a product of NH4assimilation(Morgan and Jackson, 1988a). Ammonium concentrationswere related tothe stimulation in influx whereas a product of ammoniumassimilation wassubsequently responsible for its reduction/inhibition (Wiameet al., 1985;Cook and Anthony, 1978a, 1978b).The initial increase of NH4 influx may be resulted fromprovision ofN to synthesize more transporters that are sacrificed when plants areunder N stress. In this sense, NH4 would exert an effect as asource of Nfor transporters and also as a transport regulator. It was observedin aseparate study (Fig. 43 in Chapter 7) that rice plants grown inlow 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 notnecessarilyinvolve the synthesis of a different carrier for K system.Re-supplyingNH4-- provides the ‘building blocks’ to assemble moretransporters 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 13NH4influx from internalNH4As discussed above, the theory of amides oramino acids as N uptakeregulators can not explain all the observed resultson the regulation of13NH4influx. The data seem to indicate that internalNH4 may able play arole in regulating NH4 influx. It has been reportedthat ammoniumtransport is repressed by intracellular ammoniumper se but not by itsassimilates or de novo protein synthesis (Rai et al.,1986; Franco et al.,1987, 1988). The active, specific transport of1NH4and‘4C-MA in bothwild type and mutant cells of Aspergillus nidulans is regulatedby theconcentrations of internal ammonium (Pateman et al., 1973, 1974).One of the major reported reasons for excluding NH4as a negativefeedback factor was that there was not an exact parallel betweenrootammonium 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 aboveworkers, therewere negative correlations between NH4 absorptionand 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.—2iimolg’)” (Morgan andJackson, 1988a, 1988b, 1989). But this appearsto agree that[NH4-1-]iscorrelated (negatively) with NH4 uptake. While there isa positivecorrelation between the N provision during growth andthe internalcontent of NH4 in root tissue, theVmax of‘3NH4 influx was negativelycorrelated with these two conditions (Wanget al., 1993a). Nitrogendepletion rapidly altered the N-statusof the plants, particular the tissue186concentration (Vose and Bresse, 1964; Lee and Rudge,1986). In thepresent study, within 4 to 12 hours of depletion ofG1000 roots in G2medium, ‘3NH4 influxes increased and were closelycorrelated 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 rangebecause theactivity of GS was considerable higher than the uptake rateof NH4-’- (4jimol g’FW h’). Glutamine synthetase from higher plants hasa highaffinity for ammonium(Km20iiM)(Steward et al., 1980; Milflin and Lea,1976). It would seem that if NH4 is not accumulated to a certainlevel inthe cytosol it would not be necessary to invoke a possible regulatoryrolefor this N form. However, most reported estimates ofcytoplasmic 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 ina ‘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 NH4to 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 NH4repressed theactivity of GS reversibly (Rhodes et al., 1976; Arima and Kumazawa, 1977)and NR (Siddiqi et al., 1993; King et al., 1993), NH4 should havea role inregulating the NH4 transport across plasma membrane but notthe 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 decreaseof 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 remainedat the same levelthereafter (closed symbols in Fig. 26).188A comparison of the G2 plantstreated with MSX in 2 mMor 1000mM solutions (Fig. 26) revealed asignificantly higher influx in theG2 planttreated in G2+MSX than in the G2 plant treatedin G1000+MSX at 4 and 12h. Yet the amino concentrations in theG2 plant treated in G1000+MSXshowed no significant change during thisperiod (Fig. 29). However[NH4--]1appeared to be higher in the G2 plant treatedin G1000+MSX (Figs. 27A and27B), consistent with an inhibitory effectof [NH4--]1on 13NH4 influxwhenever [NH4÷] is elevated; either by growth inhigh N condition or as aresult of MSX treatment.6.4.6. Cascade regulation system of nitrogen uptakeThe process of NH4 uptake may be sensitiveto regulation fromseveral signals, related to N status ofthe plant. These may include internalN pools (NH4,NO3-, AA), the GS/GOGAT system,translocation (andrecycling) and utilization. Clearly allthese 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 feedbackregulation on NH4-’- uptake.In addition to N signals, nitrogen (NH4+) uptakemay be limited by thesupply of carbohydrate from shoots (Kleiner,1985). This could beconsidered as an important component ofthe regulation at the whole plantlevel. The ambient conditions such as lightintensity and temperature willeffect the production of carbohydrates.It was found, for example, that netNH4 uptake rates oscillate betweenmaximum and minimum with aperiodicity co-ordinatedwith intervals of leaf emergence(Tolley andRaper, 1985; Tolley-Henry et al., 1988; Henryand Raper, 1989a; Rideout etal., 1994). At the time of emergenceand early expansion of anew 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 imposedby plantgrowth (Drew and Saker, 1975; Edwards and Barber, 1976).Theconcentrations of amides (Gin and Asn) in the roots will be theresult 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; Morganand Jackson,1988a, 1988b; Raper et al., 1992), because it is claimed thatthere is nocorrelation between cumulative uptake of NH4 and endogenousNH4-- 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 firsttwodays of N-deprivation, root NH4 concentration and NH4 uptake wereclosely correlated. After 5 d of N-deprivation, the root NH4concentrationswere found increased slightly and the rate of NH4÷ uptakewas continuedto increase. Based on present studies, NH4 would be expectedto be thenegative effector when internal NH4 levels increase beyonda certainlevel. Below this level one may assume that any freeNH4 would beimmediately drawn into the metabolic process to meetthe high demandfor plant growth. There may be a critical nitrogen statusbelow which thesystem is impaired and above which it is subject to repressionand/orinhibition (Breiman and Barash, 1980).It is proposed, therefore, that internalNH4 represents a third levelof control, operating whenever internal[NH4+] is elevated. The site(s) for191its putative effects may include the transport step atthe plasmamembrane, or the transcriptional level involving the genescoding for NH4-’-transport.In view of the different effects of internal NH4 onNH4 influx ofN-repleted G2 plants and on N-depleted G1000 plants, it is possiblethatnegative feedback regulation of NH4 uptake may be facilitatedby eitherNH4 or its assimilates. In low N-grown roots the up-regulation of influxmay be exerted through products of NH4÷ assimilation, while inhighN-grown roots, internal NH4 may participate in the down-regulationofNH4 uptake systems.In the case of the up-regulation of 13NH4influx following transferofG1000 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[NH4jmay 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 dynamicallyto change of external[NH4-’-] than the vacuolar [NH4-’-].In contrast, the observed declines of1NH4influxeswere related tohigh [NH4+] and major amino acids (Figs. 23B, 24, and25A-D). Iinterpreted this result to indicate that the declineof‘3NH4÷ influxnormally observed when G2 plants are loaded in G1000medium, dependsupon products of NH4 assimilation. This conclusion wassupported by theresults of glutamine pretreatment (Fig. 30), which reduced13NH4influx atall concentrations tested. Further proof to this effect isprovided by ouramino acid analyses. Figure 25A and 25C show that transfer fromG2 toG1000 medium caused [Gln]1 and [Asn]1to increase severaltimes while inthe presence of MSX this increase was prevented (Figs. 29A and29C). 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 higherplants (Glass,1975; 1976, 1978; Glass et al., 1981; Kochian and Lucas, 1982, 1988; Glassand Fernando, 1992). Likewise, the kinetics of ammonium transporthavealso been characterized (Becking, 1956; Fried et al., 1965; Ullrichet al.,1984; Wang et al., 1993a, 1993b). Despite the similarities between KandNH4,such as charge, hydrated ion diameter and some aspects of transportprocesses (Haynes and Goh, 1978), the interaction of these two cationsispoorly understood.The interaction between K and NH4 may be examinedat 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 maypromoteK-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 outto investigate theinteractions of K and NH4 at the transport level.In short-termexperiments, the uptake of K was significantly reduced bythe presence ofNH4 in the uptake solution (Deane-Drummond and Glass,1983b; Rosen194and Carison, 1984; Morgan and Jackson, 1988). However theinfluence of K-’-on NH4-’- uptake has not been consistent. In most cases, theuptake of NH4-’-by plant roots has appeared to be independent of K-’- levelsin the uptakesolution and the K status of the plants (Ruftyet al., 1982; Rosen andCarison, 1984; Scherer and MacKown, 1987). Nevertheless,Bange et al.,(1965) reported that K is capable of inhibiting NH4uptake in barleyplants.The objective of this study was to investigate theinteractionsbetween K-’- and NH4 at the membrane transport step, andthe influencesof tissue K and N status on these ion fluxes, using86Rb 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 forup to three days priorto flux measurement; and (iii) presence in the uptakesolutions. Testmaterials were 3-week-old rice seedlings. Each experimentwas repeated195twice with three replicates. Both influxes of 13NH4 and86Rb werecalculated based on root fresh weight and 10 mm uptake periods,except inexperiment I, where the net fluxes of NH4 and 86Rb were calculatedfrom30 mm uptake periods. Before and after transfer into or out of theradioactive isotopic labeled uptake solution, plant rootswere prewashedand postwashed in an identical unlabeled solution for5 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, Kand NH4in uptake solutions on net K and NH4fluxes.Plants were grown in MJNS containing 200 iM K plus 1.5 mMN03for 18 days, and were transferred to pretreatment solutions for threedays.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 100iiMCa(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 measuredfrom MJNScontaining 200 j.tM NH4with or without 200 jiM K (+K+N, -K+N).7.2.1.2. Experiment H: Effects of NH4provision during growthand of Kand NH4in 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 orwithout 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 or50 jiM CaC12 for G2, Gb, or G100plants, respectively. The 86Rb-’- influxes were measured fromradioactive196isotopic labeled uptake solutions (MJNS containing 200iiMK with orwithout 100 iM NH4+).7.2.1.3. Experiment III: Effects of NH4 provision duringgrowth andpresence in uptake solution upon influx isotherms for 86Rb÷(K÷).Plants were grown in four different growth media containing2 or100 pM NH4 plus either 2 or 200 .tM K, hereafter referredas G2/2,G2/200, G100/2, G100/200 plants, respectively. The86Rb influxes weremeasured in MJNS containing 2, 10, 50, 75, 100, 250 or500 iM K,respectively, plus 2 .tM NH4 for G2/2 and G2/200 plants, or1.00 jiM NH4-’-for G100/2 and G100/200 plants.7.2.1.4. Experiment 1½ Effects ofNH4provision during growthand short-term pretreatment upon 86Rb÷ (K÷) influx.Plants were pretreated for 0, 2, 4, 8, 24 h in 1 mM NH4 plus 2jiM Kfor G2/2 and G100/2 plants, or in 1 mM NH4 plus 200jiM K-’- for G2/200and G100/200 plants. 86Rb+ influxes were measuredduring 10 mm inuptake solution containing 100 jiM NH4 and 200 jiM K.7.2.1.5. Experiment V: Effect of NH4 concentrations presentin uptakesolution upon influx isotherms for86Rb+(K).The 86Rb influxes of G2/2, G2/200, G100/2, G100/200 plants weremeasured in MJNS containing 2, 25, 50, 100, or 200 jiMK, plus 2, 25, 50,or 100 jiM NH4.The translocations of 86Rb into plantshoots 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 plantsweremeasured in uptake solutions (a) containing 2, 10, 50, 100, or200 jiM NH4plus either 0, or 200 jiM K; (b) containing 100 jiM or 10 mMNH4 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 andby 1.85 times (+K+N,+K-N) when NH4-’- was present (Table 13). Yet, the means for +K+Nand -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 muchgreater effecton 86Rb accumulation, increasing 86Rb÷ (K+) uptakeby 2.65 and 6.7 timeswhen it was absent from uptake solution, andby 5.25 and 10.2 times198Table 13. Net86Rb+flux measured with or without ammonium. Riceplants were grown in MJNS containing 1.5 mM NO3-andpretreated 3 daysin 4 different solutions with or without either 200 ji,MK (+K or -K) or 1.5mM NO3-(+N or -N). The net flux of 86Rb was measuredin the followinguptake solutions: +K+N or +K-N (N = 200 jiM NH4 and K200 p.M K÷)labeled withS6RbC1.Fluxes were calculated based on 30 mmuptakeperiods.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 ± 0.28b*For comparing all possible pairs of treatment means (±se), Duncan’sMultiple RangeTest were performed, separately, on the data of net s6Rb+ flux.Means having acommon letter are not significantly different at the 5% significancelevel for smallletter.199Table 14. Net NH4 flux measured with or without potassium.Rice plantswere grown in MJNS containing 1.5 mM NO3-and pretreated3 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 uptakesolutions(+K+N, N = 200 jiM NH4 and K = 200 jiM K; or -K+N) for30 mm uptake.Uptake solutionPretreatment +K+N -K+N(jimolg4FW h-i)+K+N 4.97 ± 0.45d*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 ± 1.40a-K-N 8.65 ±0.91 ab 7.00 ±0.85bcd*For comparing all possible pairs of treatment means (±se), Duncan’sMultiple RangeTest were performed, separately, on the data of net NH4 flux.Means having acommon letter are not significantly different at the 5% significancelevel 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 of86Rb+(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 for86Rb+(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 K201105.A. uptake without N]B. uptake with N1—A--— -K+N A—h-— -K-N::0 501000 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+ influxesweremeasured 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-—EI:i‘V—’ 6-G21242’G100/200A0-G212000 100 200 300 400 500[KJo(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 500iiMK--,respectively, plus 2 j.tM NH4 for G2/2 (opencircle) and G2/200 (open triangle), or plus 100 iM N}l4for 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-’- duringuptake (Fig.40B). However, as in Experiment I, the presence of NH4during fluxmeasurements, reduced 86Rb (K+) influx in alltreatments. Again,removing K from the pretreatment solutioncaused increased 86Rb (K)influx, and this effect was more pronounced whenadequate N wasprovided (compare squares and triangles to note theK’- effect, and closedand open triangles to note the N effect).7.3.3. Experiment III: Effects of NH4 provision duringgrowth, andpresence in uptake solution upon influx isotherms for86Rb+ (K-’-).Figure 41 presents the 86Rb influx isotherms for plantsgrown underG2/2, G2/200, G100/2 and G100/200 conditions. The data werefitted toMichaelis-Menten equations. The kinetics of 86Rb uptake wereinfluencedby the provision of both NH4 and K during three weeks growth. The86Rbinflux curves for G2/200 and G100/200 plants (grown inhigher externalK+) revealed a low Vmax, 0.34 and0.59 j.imol g’FW h-’, respectively. Bycontrast, plants grown in low K (2 riM), exhibited much higherVmax 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 duringthe growthprior to influx measurements caused a significant positiveeffect; Vmax for86Rb(K-’-) influx was increased 3 fold. The estimated valuesofKmwerealso higher for plants grown in higher K conditions(15.02 jiM for G2/200and 38.59 jiM for G100/200 plants) than for thosegrown in low K supply(18.00 jiM for G100/2 and 3.47 jiM for G2/2).The relationship betweenestimated kinetic parameters and measuredtissue K concentrations clearlyindicated the operation of negative feedbackinhibition of 86Rb influx.20412108E64.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 forVmax 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, for0, 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 correlationbetweenVmax values andinternal [K] values.7.3.4. Experiment IV: Effects of NH4 provisionduring growth, and short-term pretreatment upon 86Rb (K÷) influx.When the N status of plants was changed by short-termexposures toNH4-’-,86Rb+influxes were also altered, as shown previouslyfor 3 daysexposures to NH4 (Table 13, Figs. 40 and 41). For plantsgrown in higher N(G100/2 or G100/200) the 86Rb influxes were affected littleby loading in1 mM NH4 for various periods (Fig. 43). For plants grownin low N, theresults of pretreatment in 1 mM NH4 variedaccording to the differencesin the K status. The 86Rb influxes of G2/2 plantswere 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 NH4concentrations present in uptakesolution upon influx isotherms for 86Rb (Kj.To further understand the inhibitory effect ofNH4 in uptakesolutions, 86Rb influxes were measured at five [K+]0 levels inthe presenceof four levels of [NH4-’-]0.Generally, 86Rb influxesfor G100/2 were higherthan for G2/2 and G100/200. G2/200 plantshad the lowest rates ofpotassium uptake. Generally, S6Rb+ influxdecreased with increasing[NH4-’-]0in the uptake solutions, but the effecton G100/2 is not so evident(Fig. 44). Even at 2 p.M [K-’-]0,the inhibitoryeffect of NH4 was evident.Table 15 presents estimatedMichaelis-Menten parameters for all 86Rb207G21200 —------16.0012.00YEoo5oiooxG2/2z16.0012.008.00Y4.00001 000.0050/ ;‘ 25225250100XG100/20016.0012.008.004.000.0025100XG100I2z16.0012.008.00y4.001000.00J./50‘25225250 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 estimatedby 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 ± 7.590.7950 8.27 ± 0.80 23.36 ± 8.73 0.86100 5.83 ± 0.81 18.39 ± 11.340.86G100/2 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 ± 1.570.1725 0.41 ± 0.06 105.93 ± 31.030.9650 0.41 ± 0.04 136.18 ± 21.07 0.90100 0.55 ± 0.12 186.75 ± 70.790.98G100/200 plants2 7.36 ± 0.55 17.60 ± 5.610.8025 4.06 ± 0.54 36.25 ± 15.260.8150 2.18 ± 0.32 17.68 ± 10.730.81100 2.31± 0.58 63.88±41.43 0.95G2/200z16.0012.0044 Y8.004.0000“ 00500.002522550100XG212Jz16.0012.008.004.00i/50250.002252100G1 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 withincreasing[NH4+]0in the uptake solutions. In contrast, Km values tendedto increasewith increasing {NH4+]0in the uptake solutions for G2/200and G100/200plants (Table 15). However, Km values remained relativelyconstant forG2/2 and G100/2 plants. Similar inhibitory effects weretrue for thetranslocation of K(86Rb)to shoots (Fig. 45). It was evident thathigherrates of86Rbtranslocation were associated with growth on sufficientN(G100/200) or insufficient K (G2/2 and Gt00/2).In this experiment, plant biomass was recorded in order tomakecomparisons of the effects of growth conditions. There werestatisticallysignificant differences among total fresh and dry weightsof plants(G100/200 > G2/200 > G100/2 > G2/2) although the ratiosof dry:freshweight were relatively constant (Table 16). Both fresh or dry shootweightsof plants grown in well-supplied media (G100/200) weresignificantlyhigher than for other types of plant. With inadequate supplyof either Kor NH4-’-, plants (G2/200 or G100/2) plants had smallerbiomass but thesewere still significantly higher than that of G2/2 plants. However, thedifferences of root weight indicated that K played a more importantrole 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 growthand presencein uptake solutions upon influx isotherms for 13NH4.The effects of K in the uptake solutions on the‘3NH4influxwereexamined 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 grownineither 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 8G21200TG100120012 6E• • •__o 50 100 150 2000 50 100 150 20016 8G212G10012I-12Q6‘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. Root13NH4influx of G2/2, G2/200, G100/2, or G100/200 plants, respectively,were measured in MJNS containing 100 1iM NH4 in the presenceof either2 (open circle) or 200 iM K-’- (closed circle). Predicted isotherms(dashedlines for 0 jiM K-’- and solid lines for 200 jiM K) werecalculated from thecomputed Vm andKm for different plants (Table 17).213Table 17. Michaelis-Menten kinetic parameters for 1NH4influx 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 13NH4solutionVm ± 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 13NH4influx by HATS. Root‘3NH4 influxes of G2/2, G2/200, G100/2, orG100/200 plants,respectively, were measured in MJNS containing100 iM NH4 in thepresence of 0, 20, 200, 2000 tM K. Data points arethe average of threereplicates with ±se as vertical bars.•G2/200 G2/2 G1001200G100121o 20 200 2000[KJo(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 • G1001200G10012216presence of K in the‘3NH4uptake solutionsfailed to significantly reduce13NH4 influx except in the case of theG100/2 plants where significantdifferences were apparent. The estimated influxkinetics also showed thesame trends (Table 17). Nevertheless,there were slight reductionsof‘3NH4 influx which failed to satisfy statistical evaluationin G2/2 andG100/200 plants. It was noted that plants grownat low N levels hadhigher1NH4influxes when the K nutritionwas adequate during growth(compare G2/200 and G2/2 plants).When K in the uptake solutionwas 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 andG100/200 plants was evident. Bycontrast,‘3NH4influxes were significantly increasedby growth in low N(G2/2 and G2/200) with no effects of K÷ whenpresent in the uptakesolutions. The‘3NH4influxes measured in 10 mM NH4were not changedsignificantly although the influxes werelower in the presence of 2000 jiMK (Fig. 48).7.4. DISCuSSION7.4.1. Plant growth in response to provisionsof NH4 andK-Both N and K are very important to crop growthand yield. Uptake ofK and N, plant dry weight, andpaddy yields of rice increased withincreasing K and N application rate(Biswas et al., 1987; Ichii and TsumuraH,1989; Fageria et al., 1990). Deficiencyof either N or K in the nutrientsolution decreased the tissuecontent 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 maybe due to impairment of stomataldiffusive conductance and decreasedN content/unit leaf area (Dey andRao, 1989). High tissue K not only promotedCO2 assimilation, starchformation and the transport of the assimilatesbut also improved thenitrogen metabolism of the plant and nitrogenuse efficiency (Kemmler,1983; Dibb and Thompson, 1985). K enhancedNH4 assimilation andreduced the toxic effects of NH4 suchas 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 rootgrowth. Supplyinghigh levels of K÷ to NH4-N grown plants stimulatedshoot growth andmore vigorous root growth (Xu et al., 1992).In the present study the total freshand dry weights weresignificantly higher in the sequence ofG100/200, G2/200, G100/2 andG2/2 (Table 16). The significant difference betweenG2/200 and G100/200indicates the importance of K for plant growth whenthe N nutrition isadequate. Comparing both fresh and dry weightsof roots among fourtreatments in Table 16, higher K in the growthmedia producedsignificantly higher root mass (G100/200and G2/100) than growth in lowK (G2/2 and G100/2), whereas the shoot freshand dry weights, were notsignificantly different between G2/200and G100/2. A greater root mass ofseedlings grown in higher K indicated that K mayplay an important rolein facilitating root development (Beatonand Sekhon, 1985; Xu et al., 1992).There was a significant positive correlationbetween total root weight andK uptake (Table 16). Total root lengthand dry weight increased as cropgrowth advanced and N supply increased(Chamuah and Dey, 1988).218However, the root number was negatively correlatedwith NH4-N uptakein lowland rice (Ichii and Tsumura,1989).As shown in Table 16, the Shoot:Root ratios forboth fresh and dryweights were higher for G100/2 than G200/200or G2/2. G2/200 plantshad the lowest Shoot:Root ratio. It has been reportedthat N deficiencydecreased S/R ratios of seedling plants (Zsoldoset al., 1990). N stressreduces plant growth, particularly shoot growth,through severalmechanisms operating on different time scales. Thepossible signals maybe related to N stress-induced changesof abscisic acid and cytokinins(Goring and Mardanov, 1976; Sattelmacherand Marschner, 1978; Chapinetal., 1988a, 1988b; Kuiper et al., 1989).This lower ratio of shoot:root mayalso due to higher root mass in higherK condition as discussed above.7.4.2. Effect of plant N status on K (86Rb+) uptakeThe nitrogen status of plants had a significantinfluence 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 pretreatmentperiod also causedincreased 86Rb influx (Figs. 40A and 40B). 86Rbuptake by roots exposedto +K+N and -K+N pretreatments were significantlyhigher than that for +KN or -K-N pretreatments (Table 13). This positiveeffect of N status on Kuptake may be related to protein synthesisfor K transport. The long termregulation of ion uptake probably involves inductionor derepression ofcarrier synthesis. It is known that plantsrespond to K deprivation rapidlyby synthesizing novel polypeptides in theplasma membrane (Fernandoetal., 1992) which are believedto form part of the high affinityK-’- transport219system (Glass and Fernando, 1992). When plantswere grown in low K (21iM), with sufficient N supply (1001iM NH4+), K(86Rb+) influxwaspromoted (Figs. 41, 44). However, whenthe supply of nitrogen waslimitedduring plant growth, the synthesis of Ktransporters in the cell membranemay be limited. In the present study,when G2/2 plants were pretreatedwith 1 mM NH4 for 4 hours, more transporterscould be synthesized andthe86Rb+influx was significantly increased andremained relative highduring the 24 h pretreatment (Fig. 43).This raises an importantquestionconcerning the ‘induction’ of increasedNH4 uptake observed when lowNplants are first exposure to NH4 (Goyal and Huffaker,1986; Morgan andJackson, 1989; Wang et al., 1993b; in Chapter6). The observation thatexposure to NH4 also increased K uptake on a similartime scale indicatesthat this NH4-’- effect is not specific as for examplethe 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 positiveeffect of NH4 may bedue to the effect of N supply on growth rate. The influxof ions into rootsmay be negatively correlated with the internalconcentration of aparticular ion, such as Cl- (Cram, 1973); K (Young etal., 1970; Pitman andCram, 1973; Glass, 1975; Glass and Dunlop, 1978);NO3- (Siddiqi et al.,1992), andS042-(Smith, 1975). Figure 3 showed thattheVm for86Rb+influx was negatively correlated with internal K levelsin 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 influxwas increased in the sequence ofG100/2, G2/2, G100/200, and G2/200 (Figs. 41 and44) and coincides withthe sequence of [K]1 of these roots.Higher 86Rb influxes also resulted220from three days pretreatment inminus K solution (Figs. 40Aand 40B).Therefore high N supply, resulting inincreased plant growth,would causethe opposite effect on tissue[K] and K(86Rb) influx,i.e. a biologicaldilution effect. This may explainwhy NH4 supplement to riceplantspromotes K stress (Noguchi andSugawara, 1966), or reducedKconcentration of plants (Claassen andWilcox, 1974; Faizy, 1979;Lamond,1979).7.4.3. Effect of NH4 in the uptake solutionon K-’-(86Rb) uptakeDespite the positive effect of NH4 providedduring the growth periodand the pretreatment period, NH4has been shown to strongly inhibittheabsorption 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-’- inthe uptake solution (Figs. 40A,40B and 44, Table 13 and 15). The inhibitionof 86Rb influx increased withincreasing [NH4-’-] in the uptake solutions(Table 15). The uptake of K byexcised rice roots decreased markedlywith increasing concentrationsofNH4 in the uptake solution (Schereret al., 1987). Greater inhibition of Kuptake was exerted by 1000 jiMNH4 than 100 jiM NH4 (Rosen andCarison, 1984), and the inhibitionby 1000 jiM NH4 occurred after90 mmtreatment and the inhibition by100 jiM NH4 took about 240 mm(Jongbloed et al., 1991).Since this inhibitory effect of NH4on K(86Rb+) influx isindependent of K provision orpretreatments, it is probably exertedon thetransport processes at the plasmamembrane. It is suggestedthat certain221solutes are bound to, or associated with, a particular transporter.When anion of a particular species is attached to this transporter, anothersimilarion (of the same or a different species) may compete for thesame bindingsite and reducing its uptake. Mixed competitive andnon-competitiveinhibition between K and NH4-’- has been reported for tobacco(Scherer etal., 1984) and barley (Dean-Drummond and Glass, 1983). AlthoughNH4-’-may not always inhibit K uptake competitively, NH4÷ oftenhas a loweraffinity for the carrier than K (Conway and Duggan, 1958; Jongbloedet aL,1991). Likewise, it was found that theKmfor K was increased by NH4supplementation in ectomycorrhizal fungi (Boxmanet aL, 1986; Jongbloedet al., 1991).There was a considerable K efflux induced by NH4 influxduringNH4 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 Douglasfir 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 presenceof monovalentcations (NH4-’-, K, Na) in the uptake solution depressed45Ca2influx 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 wasreduced to near half that ofN03--N grown by growth on NH4-’--N (Kirkby,1968). Similar competitive222effects were also found in maize and sugarbeet when grown on eitherurea or NH4-N (Beusichem and Neeteson,1982).Although NH4 may stimulate theleakage of K-’-, it may not bethemain mechanism responsible for theinhibition of K-’- influx. Itis wellknown that NH4 uptake is associated withH-’- efflux and acidificationofgrowth media (Pitman, 1970; Rileyand Barber, 1971; Pitmanet al., 1975;Revan and Smith, 1976; Haynesand Goh, 1978; Bagshaw etal., 1982;Marschner and ROmheld, 1983;Nye, 1986; Youssef and Chino,1989;Jongbloed and Borst-Pauwels, 1990; Chaillouet al., 1991). It is a commonpractise to add base to neutralize the Hgenerated 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 theuptake of 1 mol NH4-’-required the excretion of 1.33 mol H-’- and0.33 mol K entered root cells(Raven, 1985). Further, K uptake is intimatelyassociated with active Hefflux (Mitchell, 1970; Glasset al., 1981). The K:H exchangestoichiometries were almost consistentlygreater than 2:1(Glass andSiddiqi, 1982). Last, but not least, the effluxof K was not significant inuptake regulation (Glass, 1983) comparedto 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 extentin K-loaded plants than inK-starved plants (Rosen and Carlson, 1984),the efflux of K may notaffected by the addition of NH4 (Jongbloedet al., 1991).7.4.4. Effect of K-’- on NH4 uptakeThe uptake of NH4 by youngrice plants, as well as tomatoand plumwas not competitively affected by theK concentration of the nutrientmedium (Mengel et aL, 1976, 1978; Rosenand Carlson, 1984) or by plant K223status (Rosen and Carison, 1984; Schererand Mackown, 1987). However itwas found that the addition of high concentrationsof K caused a reductionin methylamine transport rate in Anacystisnidulans (Boussiba et al.,1984).There is a synergistic behavior between N andK in the scope of cropgrowth and production (Mengel, 1989). PlantNH4-N nutrition wasimproved by supplying K-’- (Mengel et aL, 1976; Dibband Thompson, 1985).For example, barley response toincreasing N concentrations wasdependent on levels of K in the whole plant sample(MacLeod, 1969). Themuch higher N and K uptake with the higher K supplyrate suggested thatthere might be a complementary uptake effect betweenNH4 and K-’- (Dibband Thompson, 1985). Lee and Rudge (1986) foundthat both K-’- and NH4uptake were stimulated to the same extent inN-starved roots. Ingreenhouse tests, K application tended to increasegrain N content and totalN uptake by rice plants (Chakravorti, 1989). Tomatoplants grown in sandculture with high NH4 appeared to display symptoms ofNH4 toxicityrelated to increased ethylene synthesis that declinedas K supply increased(Corey and Barker, 1989).In the present study, 1NH4 influxesof G100/2, G100/200 andG2/2plants were reduced by the presenceof K in the uptake solution.Clearly K was most inhibitory to NH4 influxwhen plants were Nsufficient (Figs. 46 and 48) and K-deficient, especiallyat high [K-’-]0 (Fig.47). In the former condition, the NH4 influxwould be relative low andprobably mediated by the high affinitytransport system (Wanget al.,1993b). Studies on rice and tomato showed thatK had inhibitory effectsbut did not compete with NH4-’- for selective bindingsites 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 toMichaelis-Menten kinetics(Epstein, 1972; Debnam and Levin, 1975; Polley andHopkins, 1979; Fischerand Luttge, 1980; Kochian and Lucas, 1982; Luttge andHiginbotham, 1982;Wang et al., 1993b). The rapidity of the inhibitoryeffects of NH4-’- and Kon each other observed in the presentstudies indicated that inhibitionprobably occurred at the level of membranetransport although thisinhibition may not be a competitive one.Similar results were reported formaize roots (Shaff et al., 1993). This suggests thatNH4 and K may share acommon transport pathway, such as an ionchannel (Wang et al., 1992b,1993b; Shaff et al., 1993) and this hypothesis issupported by molecularevidence. In a recently cloned K channel from Arabidopsis,the NH4conductance was determined to be 30% of the K conductancefor the KAT1K channel (Schachtman et al., 1992).Uptake of both NH4 and K caused depolarizationof plasmamembrane electrical potentials (Kochian andLucas, 1989; Ulirich et al.,1984; Wang et al., 1992b). Since theinflux of both cations maybe drivenby the proton motive force (at high externalconcentration), diminishingmembrane potential may lead to reduced ionuptake by influencing theproton motive force. It has been reported that thedepolarization of theplasma membrane by NH4 may increasethe Km for K (Kleiner, 1981;Borst-Pauwels et al., 1971; Roomansand Borst-Pauwels et al.,1977;Jongbloed et a!., 1991). However the effecton membrane potential can notexplain why NH4-’- inhibited K÷ uptakein all four nutrient treatments225(G2/2, G2/200, G100/2, G100/200) and K only inhibitedNH4-’- influx athigh N/low K plant status.‘3NH4influx and its kinetic parameters(Vm and Km) of N-deficientplants (G2/2) were not significantly affectedby the presence of K inuptake solution except as noted above for the G100/2plants. Also theinhibition of K(86Rb+)influx by NH4 was lower when plants wereKstarved. The uptake of K by excised rice roots decreased markedlywithincreasing concentrations of NH4 in the uptake solution,while the uptakeof NH4was little affected by the concentration ofK-’- in the uptake solution(Scherer et al., 1987). K uptake was suppressed during rapidNH4-’- uptakeby N-starved plants (Tromp, 1962), but K-starvationdid not produce thesame effect as N-starvation on the transport of NH4(Tromp, 1962; Leeand Rudge, 1986). This biased inhibitory effect between NH4and K maysuggest that NH4 and K share a common transport pathway,but theregulation signal for these two ions may arise from separatesources. Thesuperior competitive behavior of NH4 over K is similar tothe inhibitoryeffect of NH4 on N03 uptake which has alsobeen linked to thedepolarizing effects of NH4 on 1P (see Lee and Draw, 1989for discussion).Yet it is clear that, although K causes a depolarizationof AP similar to thatcaused by NH4,it is not inhibitory to N03 uptake,nor is it as effectiveinhibiting NH4-’- uptake. Hence it is unlikely thatthe inhibitory effect ofNH4-’- is due to membrane depolarization/dissociationof pmf. The basis ofNH4 inhibitory effect remains to beresolved.226Chapter 8. GENERAL CONCLUSIONSThis study has identified and characterized theammonium uptakesystem in rice roots in terms of cellular compartmentation(Chapter 3),kinetics (Chapter 4), energetics, electrophysiology(Chapter 5) andbiochemistry (Chapter 6). The interactionbetween NH4 and K on theplant growth and ion uptake was alsoexamed (Chapter 7).Ammonium is absorbed by rice rootsin the cation form even atelevated [NH4+]0.Newly absorbed NH4 is eitherstored in the root cellvacuoles or rapidly metabolized to amino acidsin roots. Amino acids, butnot NH4,are consequently translocated to theshoots. Cytoplasmic [NH4-’-]may range from 3 to 38 mM according to theN provision during growth.The concentration dependence of NH4 uptakedemonstrated 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 linearrelationship between 13NH4÷ influxand [NH4+]0is mediated by LATS.HATS and LATS are not only kineticallydifferent, but also different in energy dependenceand stoichiometry ofmembrane potential depolarization.Significant efflux of NH4 was observedeven when plants weregrown at lower level of [NH4-’-]0,21iM. Efflux increased as [NH4+]0increasedfrom 2 to 100 and 1000 1iM, correspodingto 10, 20 30% respectively ofinflux at these [NH4]0.NH4-’- uptake is subjected to negativefeedback regulationby bothNH4 and its metabolites.The effectsof pretreatment with exogenousGln,227Glu and Asn were found to reduce influx to differing extents. A cascaderegulation system is proposed to explain the regulationof ammoniumuptake in response to changes of internal NH4 and its metabolites. Thisinvolves regulation at many levels, from the whole plantdown to themolecular level.The results of NH4 and K interaction studies at the levelof plantgrowth and uptake gave quite different results. Both cations areessentialfor plant growth, and utilization of each nutrient is optimizedwhen each isin adequate supply. At the uptake level, pretreatment with NH4-’-caused astrong stimulation of K uptake, but was inhibitory to K uptakewhen itwas present in the uptake solution. 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Reported studies on using radioactiveisotope ‘3NYear Author N Species Objective MaterialBerkeley, U.S.A.1940 Ruben et al, 13N2 N2-FixationNon-legumeEngland1961 Nicholas et al, 13N2 N2-FixationBacteriaManitoba, Canada1967 Campbell et al, 13N2N2-Fixation MicroorganismMichigan, U.S.A.1974 & 76 Wolk et al, 13N2 N2-FixationBlue-green algae1975 & 77 Thomas et al, 13N2 -FixationBlue-green algae1977 & 78(a) Meeks et al, 13N2 N2-FixationBlue-green algae1978 (b) Meeks et al, 13N2 -FixationSoybean1978 Skoukit et al, 13NO3,NH4 N assimilationTobacco cells1979 Hanson et al, 13NH3 Translocation Barley1979 & 81 Tiedje et al, 13N Denitrificationsoils1981 Schubert et al, 13NH4 N2-FixationNon-legumeDavis, U.S.A.1976 Gersberg et al, 13N03 Denitrification Floodedrice 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, 13N03,NH4+ N uptake, Flux Maize1984 & 86 Presland et al, 13N03 N uptake,Flux MaizeQuebec, Canada1984 Caidwell et at,‘N0,’NH4t’N2N2-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, FluxLemnaNew York, U.S.A.1989 Calderon et al, 13N-glutamine assimilationNeurospora crassaHouston, U.S.A.1990 Hole et at,13NO3 NO3 transport MaizeJülich, Germen.1992 Wieneke13NO3 NO3 transport Squash263Appendix B Reported values of half-life (t172) and ioncontent(0)ofvarious compartments of root cells.Superficial Free space CytoplasmVacuolet1/2(sec) (mm) (mm) (h)K onion 15 - 43 3.3 - 7.3 82 - 10380 - 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- 2535-390Ca onion 12 - 13 1.3 - 1.5 54 - 56 12- 30Mg onion 18 3.2 7449- 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 - 3.637.9 - 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 - 0.168 - 137N03 barleybarley

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