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PM mum; M'WTE Aflih/MTIM IN rum: presented by H“: DE MACJA 214de has been accepted towards fulfillment of the requirements for 8.9th 'V 7 _1£[LD_._degree in P/A’Nf P/WIIOIOGY W129: Major professor its .‘W’ 1’ MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 )V153I_} RETURNING MATERIALS: Place in book drop to “BRAKES... .. remove thiflgcheckout from ‘ ‘ ‘ ‘ ibur'record. “F?NES“will ‘13‘v‘vbe changed.if book is returned after the date stamped below. fix?" “a. l H '5" i. .2! IF .. F”: W .- 1 I ~ ‘ . _ JV?“ 3' j _ i -‘. a ‘ . p 2:" I! n‘\ fl is) ’.‘I "v . my. I ... 4, _ .., IS “I In. . to "'s‘":‘1l~'&3§g pd /e’/J-‘//o?a EVALUATION OF CHLORATE AS A TOOL FOR STUDYING NITRATE ASSIMILATION IN PLANTS By MARIA de GRACIA ZABALA A DISSERTATION Submitted to Michi an State University in partial ful lllment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1983 ABSTRACT EVALUATION OF CHLORATE AS A TOOL FOR STUDYING NITRATE ASSIMILATION IN PLANTS By MARIA de GRACIA ZABALA The absence of a long-lived radioisotope of nitrogen is the major obstacle to advances in understanding nitrogen transport in plants. Chlorate behaves as an analogue of nitrate in the nitrate reductase (NR) reaction, but its role as a competitor with nitrate at the level of absorption remains obscure. The objective of this study was to evaluate 3GCl-chlorate as a nitrate analogue in transport studies with the XD strain of cultured tobacco cells (Nicotiana tabacum L. cv. Xanthi) grown on nitrate, or an efficient urea-assimilating variant (14U cells) grown on urea. Chlorate uptake by 14U cells was pH dependent with a maximum at pH 5.5. 14U cells concentrated chlorate against a gradient at pH 5.5 but not at pH 6.5. Chlorate did not induce a chlorate uptake system. The rate of chlorate uptake increased with chlorate concentration. Kinetic analysis revealed both a saturable uptake system, functioning below 1 mM chorate and a linear system, responsible for rate increases at chlorate concentrations above 1 mM. The former system is shared with nitrate, while the latter is shared with chloride but not with nitrate. The nitrate-inducible nitrate transport system appears to be distinct from the high affinity chlorate transport system, in that nitrate transport is insensitive to chlorate, and chlorate transport is constitutive. It was concluded that chlorate is not an ideal analogue of nitrate for transport studies in tobacco cells. Tobacco cells’ NR could not discriminate between chlorate and nitrate. Their mutual inhibition at the level of reduction was competitive. The affinity of the enzyme for nitrate was an order of magnitude greater than that for chlorate. Chlorite was undetectable as a product of chlorate reduction. Its apparently rapid conversion to chloride may explain the intrinsic chlorate tolerance of XD cells. Chlorate, unlike nitrate, could not induce NR activity. Nitrate was antidotal to chlorate toxicity in tomato seedlings. Unexpectedly, these plants were more chlorate tolerant when grown on nitrate than on any source of reduced nitrogen. Alternative nitrogen sources had dramatic effects on tomato seedling root development. Y-Amino butyric acid in particular elicited a light dependent periodicity of root hair position which was not abolished by concomitant feeding of additional nitrogen compounds. ACKNOWLEDGMENTS I am grateful to Dr. Philip Filner, who allowed me complete freedom to pursue my own interests but gave generously of his time when I sought his advice. I would also like to thank Dr. Thomas Jacobs, Dr. David Rhodes and Dr. Michael Guy for their help and enjoyable and productive collaborations. The suggestions, concern and assistance offered by Dr. C. Peter Wolk, Dr. Norman Good, Dr. Peter S. Carlson, Dr. Hans Kende and Dr. Robert Sheffer are also gratefully acknowledged. For excellent technical assistence, I am indebted to Lloyd LeC ureux, Michael Shimei and Janet Nelsen. Finally, to many graduate students and postdocs, too numerous to list, in the Plant Research Laboratory and at the ARCO-Plant Cell, Research Institute, I extend an abrazo muy fuerte for their help and companionship. A DOE/PRL assistantship, a Noyes Foundation fellowship and an ARCO-PCRI fellowship are gratefully acknowledged. TABLE OF CONTENTS LIST OF FIGURES v LIST OF TABLES ix LIST OF ABBREVIATIONS AND TERMINOLOGY xi INTRODUCTION ‘ 1 PART I: NITRATE AND CHLORATE UPTAKE SYSTEMS IN CULTURED TOBACCO CELLS 18 MATERIALS AND METHODS 18 Plant Material 18 Growth Media 19 Growth Conditions 20 Measurement of Nitrate and Chlorate Uptake 20 Synthesis and Purification of 36Cl-Chlorate 22 Kjeldahl Procedure 24 Determination of 15N Atom Percent 26 Determination of Nitrate 29 RESULTS 3 1 Chan es in the Internal and External Nitrate Pools of XD Cel sin Culture 3 1 Changes in Km and Vmax of Chlorate Uptake with Changes in IC/IelDAge and with Time in Inducing Medium (Nitrate-less 3 1 ) 5 Effect of Nitrate on Chlorate U take 40 Concurrent Determination of 'netic Characteristics of Nitrate Inhibition of Chlorate Uptake and of Chlorate Inhibition of Nitrate U take 44 Inhibition o Nitrate Uptake by Chlorate in Urea-Grown cells 49 Effects of Ammonium and Cyc oheximide on Chlorate Uptake 54 Chlorate Uptake in 14U Tobacco Cells 56 Inhibition of Chlorate Uptake by Nitrate in 14U Tobacco Cells 61 Effect of Chloride Ion on Chlorate U take 61 Characteristics of Chloride Uptake ystem 73 ‘ iii PART II: REDUCTION OF CHLORATE BY NITRATE REDUCTASE 77 MATERIALS AND METHODS 77 Preparation of Enzyme Extracts 77 Determination of Nitrate and Chlorate Reductase Activities 78 Determination of 36Cl-Chlorite and 3GCl-Chloride 78 Determination of Total Protein 79 RESULTS 79 Inhibition of Cell Growth by Chlorate ‘ 79 Chlorate Reduction by Nitrate Reductase of Tobacco Cells 82 Kinetic Characteristics of Chlorate and Nitrate Reduction by Nitrate Reductase Pre arations 90 Inhibition by Chlorate o Nitrate Reduction 95 Inhibition by Nitrate of Chlorate Reduction 100 Induction of Nitrate Reductase by Nitrate and Chlorate 100 Effects of Urea, Chloride and Ammonium ions on Tobacco Cells’ Sensitivity to Chlorate 113 PART III: NITROGEN NUTRITION AND CHLORATE UPTAKE IN TOMATO SEEDLINGS 120 MATERIALS AND METHODS ° 120 Plant Material 120 Initiation and Maintenance of Cultures 121 Culture of Tomato Seedlings 122 Compartmental Analysis 124 Measurements of Chlorate Uptake by Tomato Seedlings 124 RESULTS 125 Nitrogen Nutrition of Tomato Tissue Cultures 125 Chlorate Toxicity in Tomato Cells 127 Chlorate Toxicity in Tomato Seedlings ‘ 13 1 Antidote Effect of Nitrate on Chlorate Toxicity 144 Effect of Nitrogen Compounds on Tomato Seedling Development 148 Y-Amino Butyric Acid Effect on Root Development 158 Compartmentalization of Labelled Chlorate in Tomato Root Cells 166 Chlorate Uptake by Tomato Roots: Effect of Nitrate 168 DISCUSSION 17 6 BIBLIOGRAPHY 192 1v 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. LIST OF FIGURES . Time course of product formation during H36C1 electrolysis under alkaline conditions. . Diagram of apparatus devised for introduction of samples into the MAT, GD150, Mass Spectrometer. . Intra- and extracellular nitrate levels in XD cells over the culture cycle. Leakage of NO3' fromXD cells cultured in the presence of 36C103“. . Culture-cycle dependence of chlorate uptake by XD cells. . Effect of nitrogen starvation on kinetic characteristics of chlorate uptake by XD cells. . Effect of nitrogen starvation on kinetic characteristics of nitrate uptake in XD cells. Effect of nitrate on the kinetics of chlorate uptake in XD cells. . Lineweaver-Burk plot of nitrate inhibition of chlorate uptake in XD cells. Dixon plot of nitrate inhibition of chlorate uptake in XD cells. Lineweaver-Burk plot of chlorate inhibition of nitrate uptake in XD cells. Dixon plot of chlorate inhibition of nitrate uptake in XD cells grown in nitrate. Dixon plot of chlorate inhibition of nitrate uptake in XD cells grown in urea. . Dixon plot of chlorate inhibition of nitrate uptake by 14U cells pretreated in nitrate induction medium. * Effect of ammonium and cycloheximide on chlorate uptake in XD cells. pH dependence of chlorate accumulation in 14U cells. pH dependence of chlorate uptake in 14U cells. pH dependence of intracellularzextracellular chlorate concen- tration ratio in 14U cells. Nitrate inhibition of chlorate uptake at pH 5.5 in 14U cells. Nitrate inhibition of chlorate uptake at pH 6.5 in 14U cells. 25 28 33 34 36 38 41 43 46 47 48 50 52 53 55 58 59 60 62 63 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. Effect of Cl" on ClOg‘ uptake by 14U cells. Combined effects of pH and 01' on ClO3' uptake in 14U cells. Combined effects of Cl' and pH on 0103‘ uptake in 14U cells. Eflfiect of counterion on C1‘ inhibition of ClO3' uptake in 14U ce 3. Effect of 0104' on C103" uptake in 14U cells. Effect of Cl' on ClO3' uptake by 14U cells. Effect ofNO3' on ClOg‘ uptake in 14U cells in the absence of Cl". Effect of chlorate and pH on C1' uptake in 14U cells. Lineweaver-Burk analysis of effect of pH and C103‘ on C1' uptake in 14U cells. Effect of chlorate on growth of (A) 14U cells in urea-MlD, pH 6.2; (B) XD cells in MID, pH 6.2; (C) XD cells in MID, pH 5.5. Dose-response curves of chlorate effect on 14U and XD cell culture growth Time course of chlorate reduction by extracts of XD cells. (A) Time dependence of nitrate reduction by extracts of XD cells. (B) Concentration dependence of NR activity from XD cells. Lineweaver-Burk plot of chlorate reduction by XD cell extracts. Lineweaver-Burk plot of inhibition by chlorate of nitrate reduction in XD cells. Dixon plot of chlorate inhibition of nitrate reduction in XD cells. Dixon plot of nitrate inhibition of chlorate reduction in XD cells. Effect of chlorate on growth of 14U cells in urea-MlD. Effest of chlorate on growth of 14U cells in urea-MlD + 2.5mM KN 3. Dose-response curve of chlorate effect on initial growth rates of 14U cells. Dose-response curve of chlorate effect on exponential growth rates of 14U cells. Dose- -response curve of chlorate effect on fresh weight accumula- tion in 14U cell cultures. Dose- -response curves of chlorate effect on growth rates of 14U and XD cells. Dose-response curves of effect of chlorate on fresh weight accumu- lation.in XD cell cultures. Dose-response curves of effect of chlorate on fresh weight accumu- lation in 14U cell cultures. Dose- -response curves of effect of chlorate on fresh weight accumu- lation by XD cells grown in urea-supplemented MlD. v1 64 66 67 68 7O 71 72 74 75 81 84 86 93 96 98 99 101 105 106 108 109 110 112 114 115 117 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. Dose-response curves of effect of chlorate on fresh weight accumu- lation in XD cell cultures supplemented with ammonium succinate. Dose-response curves of effect of chlorate on fresh weight accumu- lation in XD cell cultures in which ammonium succinate replaced nitrate. Growth of batch suspension cultures of L. esculentum cv. VF-36. Growth of L.esculentum cv. VF-36 batch suspension cultured cells in urea-MlD medium. Chlorate toxicity in batch suspension cultures of L.esculentum cv. VF-36. Growth response of tomato cell cultures to chlorate. Effect of chlorate on tomato seedling growth. Effect of tungstate on chlorate toxicity in tomato seedlings. Nitrate protection of tomato seedlings from chlorate toxicity. Effect of nitrogen source on chlorate toxicity in tomato seedlings. Effect of ureide nitrogen on tomato seedling root morphology. Effect of amide nitrogen and GABA on tomato root morphology. Effect of nitrate and GABA on tomato root morphology. Effect of GABA and nitrate on tomato seedling root hair growth. Effect of GABA analogues on tomato seedling root morphology. Comparison of GABA and 3-APSA effects on tomato seedling root morphology. Compartmentalization analysis of chlorate in tomato seedling roots: vacuole compartment. Compartmentalization analysis of chlorate in tomato seedling roots: cytoplasmic compartment. Compartmentalization analysis of chlorate in tomato seedling roots: apoplast compartment. Chlorate uptake by tomato seedlings in the presence of GABA or glutamine. Effect of nitrate on chlorate uptake in tomato seedlings. Model for interactions between ClO3', N 03' and Cl' transport systems. v11 118 119 126 130 132 133 134 145 147 . 150 154 156 160 161 164 165 167 169 170 173 175 184 9° S955"? 9° 10. 11. 12. 13 14 15 16. 17. 18. 19. 20. LIST OF TABLES . Method of determination of 15N atom % abundance from mass spectrometric data. Effect of nitrogen starvation on kinetic characteristics of chlorate uptake in XD cells. Effect of nitrogen starvation on kinetic characteristics of nitrate uptake in XD cells. Properties of chlorate and chloride transport systems. Effect of chlorate on pH of culture medium. Substrate dependence of chlorate reduction by XD cell extracts. Effect of nitrite on recovery of chlorite from chlorate reduction products of XD cell extracts. Chlorate reduction by bacteroid nitrate reductase. Chlorate reduction by NR from urea and nitrate grown XD cells. Stability of NR extracted from 14U cells. Kinetic parameters of nitrate and chlorate reduction by XD cells. Michaelis constants for nitrate and chlorate reductases of various spec1es. Induction of NR activity in 14U cells by nitrate, chlorate and urea. Effect of medium nitrogen source, chlorate and stage of culture cycle on nitrate reductase activity extractable from 14U cells. Callus growth response of tomato species to different medium nitrogen sources. (A) Nitrogen source dependence of chlorate sensitivity in tomato seedlings own in unbuffered solutions. (B) Effect of nitrogen source an chlorate on pH of tomato seedling nutrient solution. Effect of MES, ammonium and chlorate on growth and nutrient solution pH of tomato seedlings. Effect of MES, ammonium and succinic acid on growth and nutrient solution pH of tomato seedlings. Effect of MES, ammonium and succinic acid on growth and nutrient solution pH of tomato seedlings. Effect of MES, ammonium and succinic acid on growth and nutrient solution pH of tomato seedlings. .viii 30 39 42 76 83 87 88 89 90 92 94 97 102 104 129 135 138 139 140 141 21. 22. 23. 24. 25. Nitrogen source degendence of chlorate sensitivity in tomato seedlings grown in uffered solutions. Effect of nitrogen source on chlorate toxicity in tomato seedlings. Effect of ureide and amide nitrogen sources on tomato seedling growth. Effect of ureide and amide nitrogen on the pH of the nutrient solution and on growth of roots of tomato plants. Effect of nitrogen source on tomato seedling root growth. 143 149 152 157 159 LIST OF ABREVIATIONS AND TERMINOLOGY 3-APSA AS BA CCCP Cl-less N-less M1D 2,4-D EGTA GABA HEPES IAA 150 K1 Km MID MES NADH N-less MlD N-less T12 medium NR S [S] 3-amino propane sulfonic acid ammonium succinate 6-Bencylaminopurine Carbonyl cyanide, m-chlorophenyl- hydrazone N -less M1D in which the chlorides of calcium and potassium had been substituted by equimolar amounts of calciumsulfate and potassium phosphate respectively 2,4-dichlorophenoxyacetic acid Ethyleneglycol-bis-( p-amino-ethyl ether) N ,N’-tetra- acetic aci y-amino butyric acid N -2-hydroxyethylpi perazine N ’-2-ethanesulfonic acid Indole-3-acetic acid The concentration resulting in a 50% reduction in growth rate with respect to the control Dissociation constant Michaelis constant, substrate concentration at which velocity 1s half-maximal A modification of Wh1te 3 medium in which nitrate 15 the sole nitrogen source 2(N-morpholino) ethanesulfonic acid N icotinamide adenine dinucleotide,reduced form MlD medium in which the nitrates of calcium and potassium have been replaced by equimolar amounts of their respective chlorides T12 medium 1n which the nitrates of ammonium and gtassium have been replaced by equimolar amounts of Nitrate reductase Substrate Substrate concentration T12 medium TLC 14U Urea-M1D Vmax XD Culture medium consisting of the major salts of Murashige and Skoog supplemented with Nitsch’s vitamins, hormones (IAA, 2, 4-D and BA), inositol and sucrose Thin layer chromatography Urea-assimilating variant strain of XD cells N-less M1D medium to which a filter-sterilized urea solution was added to give a final urea concentration of 3 mM velocity maximum velocity Strain of cultured tobacco cells (N icotiana tabacum L. cv. Xanthi) x1 INTRODUCTION Soil nitrogen is comprised of two distinct pools with regard to its availability to higher plants: a large inaccessible pool of organic compounds and a small readily accessible pool of inorganic forms (Scarsbrook, 1965). The latter is in continuous flux as soil microorganisms generate accessible nitrogen from atmospheric nitrogen by nitrogen fixation, or from the relatively inaccessible pool by mineralization (ammonification and nitrification). Simultaneously, nitrogen is removed from the relatively accessible pool by leaching and by assimilation and denitrification by plants, animals and bacteria (Harmsen et al., 1965). The steady-state concentration of accessible nitrogen in the soil is determined by its exchange between these sources and sinks. The steady-state concentration of accessible nitrogen of any given field decreases with cultivation since the nitrogen fixation rate in the soil is insufficient to replenish the nitrogen which is removed annually by harvesting. After several years of cultivation, nitrogen fertilizer must be applied to maintain soil fertility at a level allowing adequate crop productivity. It can thus be inferred that,in general, current crop cultivars do not take up nitrogen as rapidly from low steady-state concentrations of accessible nitrogen in unfertilized, intensively cultivated fields as they can from the high concentrations in fertilized fields. One approach to overcoming this problem, besides adding increasingly costly nitrogen fertilizer to cultivated soils each year, is to increase the efficiency with which crop plants take up nitrogen at low concentrations of accessible nitrogen. Because nitrogen fixation is inhibited by high concentrations of accessible nitrogen, the lower steady-state concentration would have the important benefit of increasing the rate of natural replenishment of soil nitrogen. Inorganic nitrogen in most arable soils is mostly found in the nitrate form, regardless of past fertilization practices. Soil ammonium from whatever source is rapidly oxidized to nitrate, in most soils, by nitrifying bacteria. Accordingly, nitrate is the most important source of available nitrogen for cultivated plants. Since nitrate is anionic, it is not bound by the anionic soil clays. Thus, the total supply of nitrate in the root zone is available (Scarsbrook,1965), though subject to leaching losses. Nitrate nitrogen is incorporated into plant cell constituents by the following pathway: Nitrate Nitrate Nitrite U ptalce Reductase _Reductase NO§(outside)—>NO3(inside) )N02—) N H4 +-—»AMINO ACIDS Nitrate reductase is thought to be the rate-limiting enzyme in the pathway (Schrader, 1978). This assumption is based on the observation that plants and cultured plant cells can accumulate nitrate considerably in excess (50-100x) of the ambient concentration (Heimer and Filner, 1971; Jackson et al.,197 6) but do not accumulate nitrite. If nitrate reductase were not highly regulated, i.e., if the amount of enzyme were independent of the environment, the accumulation of nitrate, and lack of accumulation of nitrite would be strong evidence that the enzyme is rate-limiting in the pathway. Alternatively, the enzyme might be actively modulated by regulatory 3 mechanisms, so as to adjust nitrate reductase amounts or activities to achieve reaction rates compatible with the plants’ overall ability to use the nitrogen in nitrate. That is, the rate at which nitrogen is assimilated may be the cause rather than the effect of a given level of nitrate reductase activity. This model is supported by the observation that added glucose enhances the level of nitrate reductase without affecting the total nitrate in corn root tissues (Aslam and Oaks, 1975). These results were attributed to a redistribution of root nitrate, resulting in a greater portion being present in the metabolic pool, thus enhancing nitrate reductase activity (Aslam and Oaks, 197 5). Therefore the concentration of nitrate in the metabolic pool, which is primarily determined by the rate of nitrate uptake, could be the limiting factor in nitrogen assimilation. Chantarotwong and coworkers (1976) and Eisele and Ullrich (1977) have also concluded that, under normal conditions, the rate of in vivo nitrate reduction is more a function of the rate of nitrate uptake than of the amount of nitrate reductase present in the cell or tissue. Consequently, it is not unreasonable to propose that plants capable of taking up nitrate more efficiently, from soils containing low steady state concentrations of accessible nitrogen, may be able to grow more rapidly than less efficient absorbers of nitrate under such suboptimal conditions. Therefore, it is important to examine the characteristics and regulation of nitrate uptake in order to evaluate the feasibility of selecting these more efficient nitrate absorbers. Experiments on the mechanism of absorption of nitrate and ammonium ions have been less extensive and less conclusive than experiments on assimilation of most other macronutrient ions (Carter and Lathwell, 1967; Furner and Sung, 1982; Kochian and Lucas, 1982; Nissen, Schiff and Hodson, 1973; 1973; Siegel, 1977; Smith,~ 1975, 1980). At first glance, this seems 4 surprising in view of the great importance of nitrogen in soil fertility and the plant nutrition. For the investigation of ion absorption by plant tissues, radioisotopes have been indispensible because of the ease and extreme sensitivity of radioassay methods and because they provide a means of distinguishing among different pools and fluxes of the same element. However, there simply isn’t a generally useful radioisotope of nitrogen. Because 13N has a half-life of only 10.05 minutes, one must have access to a nuclear reactor or particle accelerator in which 13N can be produced, and one is limited to rather short-term experiments. As a result, experimenters have more often used the stable isotope 15N, the assay of which, by mass spectrometry, is more cumbersome, expensive and difficult to adapt to high throughputs of samples, and less sensitive than most radioassay procedures. Still more often, researchers have used chemical methods of analysis which limit them to following depletion or accumulation of chemical amounts. These methods also do not approach the radioassay in ease and sensitivity, especially for short—term experiments. Another special aspect of absorption of nitrate ions is inherent in the biology of the process and its metabolic consequences.“ Other mineral nutrients such as phosphorus and sulfur, are incorporated into organic metabolites, but none equal nitrogen in the extent, variety and complex consequences of its transformations. For this reason, absorption of the nitrate ion does not necessarily lead to accumulation of the ion within the cells. Experimentally, reduction of nitrate can be inhibited by means of tungstate. Incorporation of tungstate, rather than molybdate, into the nitrate reductase molecule renders it non-functional for reduction, and tungstate treatments often result in an increase in nitrate accumulation by the tissue(Chantarotwong et al., 1976; Eisele and Ulrich, 1977; Flores and 5 Guerrero, 1980a; Goldsmith et al., 1973;Heimer et al., 1969; Heimer and Filner, 1971; Jackson et al., 1973; Minotti and Jackson, 1970; Radin, 1975; Rao and Rains, 1976a; Schloemer and garret, 1974; Yoder and Scheffer, 1973). There are instances, however, where tungstate treatment decreased the accumulation of nitrate (Heimer et al., 1969;. Rao and Rains, 1976b). Moreover, even when increased accumulation does occur, the uptake process may still be restricted (Jackson, 1978; Rao and Rains, 1976b); it can only be concluded that uptake is not as restricted as is reduction. Use of tungstate to separate nitrate uptake from nitrate reduction may also be complicated by indirect effects. Decapitated corn seedlings grown in the absence of nitrate, upon exposure to nitrate, showed the typical pattern of apparent induction (Ezeta and Jackson, 1975; Jackson et al., 1973). The .presence of tungstate in the uptake solution resulted in the initial slow phase of uptake being extended. It also slightly lowered the rate after the accelerated phase had been initiated. It clearly cannot be assumed that tungstate affects only one component (reduction) of the nitrate assimilation pathway in root tissue (Jackson, 1978). Tungstate may at times restrict nitrate uptake (perhaps indirectly), but it seldom seems to abolish it completely. In spite of these limitations, valuable information about the nitrate uptake systems of higher plants has still been obtained. Roots of whole plants and plant cells previously grown in the absence of nitrate exhibit negligible or low uptake rates upon first exposure to nitrate; the rate increases steadily, reaching a constant rate about 5 times greater than the initial rate. This suggests that the nitrate transport across the plasmalemma of the root cells is induced by nitrate, the substrate for the transport process. This pattern is in contrast to that commonly observed for uptake of other essential elements by 6 root tissue. Such systems are ordinarily characterized by relatively linear rates. The increase in activity of the nitrate transport system in response to the substrate, nitrate, was discovered by Heimer and Filner (1971) in a study done on the XD strain of cultured tobacco cells. Since then, induction of the nitrate uptake system by nitrate has been demonstrated in corn (Ezeta and Jackson, 1975; Jackson et al., 1973; N eyra and Hageman, 1975) barley (Chantarotwong et al., 1976; Rao and Rains, 1976a), cotton and tobacco (Jackson et al., 1972), Wolffii arrhiza (Swader et al., 1975) dwarf bean (Breteler, 1979), cell suspension cultures of tobacco (Behrend and Mateles,1975), Penicillium chrysogenum (Goldsmith et al., 197 3), Neurospora crassa (Schloemer and Garret, 1974), and Lemna gibba (Ullrich et al., 1981). The nitrate transport system of cultured tobacco cells was totally absent from cells grown on urea (Heimer and Filner, 1971). Breteler et al. (1979) found that, in dwarf bean plants, started nitrate uptake at about 30 to 40% of the fully induced rate immediately after addition of nitrate to the medium. The preexisting nitrate uptake was attributed to either constitutive uptake or to the physico-chemical processes of diffusion and adsorption into the apparent free space. . The nitrate uptake system is highly specific for nitrate and is not induced by other anions (e. g. sulfate, chloride, phosphate, borate or molybdate) at concentrations commonly employed in stande nutrient solutions. Chlorate does not induce the nitrate transport system either (Jackson,1978). However, it is not at all certain that nitrate is the only inducer; nitrite will induce nitrate transport in tobacco cell suspension cultures (Heimer, 1975). Inhibitors of RNA and protein synthesis restrict development of the accelerated uptake phase in higher plants (Ezeta and Jackson, 197 5; Jackson, 7 1975; Jackson et al., 1973; Neyra and Hageman, 1975; Rao and Rains, 1976a) the fungi Neurospora crassa (Schloemer and Garret, 1974) and Penicillium chrysogenum (Goldsmith et al., 1973). This is suggestive of an inducible transport system which turns over and which requires continual protein synthesis to sustain maximal transport rates. However, these metabolic inhibitors have multiple effects on the components of the nitrate metabolic pathway and detrimental effects on the transport of other ions which do not show the apparent induction uptake pattern (Jackson, 197 8). Therefore, the use of these compounds does not give unequivocal evidence for synthesis of a specific proteinaceous component of the nitrate transport system during the apparent induction period. Consequently it is not possible at this time to conclude with certainty that the increasing nitrate uptake rate observed in root tissues or cultured cells upon first exposure to nitrate represents a true induction phenomenon, rather than the enhanced activity of a preexisting system. Plant cells maintain a negative electrical potential between their cytoplasm and the ambient medium (Higinbotham, 1973) but they can accumulate nitrate considerably in excess (50-100x) ,of the ambient concentration (Heimer and Filner, 1971; Jackson et al., 1976b). Transport of nitrate against a strong electrochemical potential gradient, as well as detrimental effects on nitrate accumulation, of low temperature, anaerobiosis, and inhibitors of oxidative phosphorylation clearly indicate a dependence on metabolic energy for sustaining nitrate uptake (Huffaker and Rains, 1978). However, movement of nitrate is not restricted to the inward direction accross the plasmalemma, even when the system is operating effectively. l5N-nitrate- fed ryegrass and wheat released previously absorbed l4N-nitrate to the ambient solution, concurrently with an inward flux of the 15N-nitrate 8 (Jackson et al., 1976b; Morgan et al., 197 3). These data have been interpreted in terms of a "pump and leak” model (Morgan et al., 1973). Its essential features include a passive bidirectional pathway, an active uptake mechanism, and a pathway for recycling of endogenous nitrate within unstirred layers from the passive pathway to the active uptake site. Low ambient hydrogen ion concentrations decrease nitrate transport and the process is restricted above pH 6 (Lycklama,1963; Rao and Rains, 1976a). However, the response to a decrease in pH below pH 6 is not uniformly observed. In some cases, the decline in nitrate uptake is not evident until pH values of 4.5 or below are attained (Minotti et al., 1969b). In the work of others, the rate of nitrate uptake started to decrease below pH 6 (Lycklama,1963). Once the nitrate uptake system is fully developed, the rate of nitrate uptake depends upon the concentration of nitrate in the medium. The uptake mechanism follows Michaelis-Menten saturation kinetics in a range of concentrations up to 1 mM or higher. The reported apparent Km values for nitrate uptake represent a wide range: A value of 0.3 mM was reported for N. crassa (Schloemer and Garret, 1974) while, it was less than 0.01 mM in P. chrisogenum (Goldsmith et al., 1973), 0.1-0.2 mM in corn (Honert and Hooymans, 1955; N eyra and Hageman, 1975), 0.033 mM in perennial ryegrass (Lycklama, 1963), 0.6 mM in rice (Fried et al., 1965), 011-02 mM in barley (Chantarotwong et al., 1976Rao and Rains, 1976a), 0.4 mM in Wolffii arrhiza (Swader et al., 1975) and 0.4 mM in the XD strain of cultured tobacco cells (Heimer and Filner, 1971). Behrend and Mateles (1975) found a Km of 0.7 mM for the same tobacco cell line cultured on a different medium. Lancaster, 1976 (cited by Huffaker and Rains, 1978) reported the Km values for a number of annual range grass species: Avena fatua L. (0.015 mM), Bromus mollis L. 9 (0.015 mM) and Lolium multiflorum Lam. (0.03 mM). He suggested that the Km value reflects the distribution of the plants in the field since L. multiflorum (Km=0.03 mM) requires relatively high soil fertility to compete with the other grasses. B. mollis (Km = 0.015 mM) on the other hand, has been found to occupy less fertile soils. However, Van de Dijk (1981) and Van de Dijk et al., (1982), indicated that the Km value is not important for the distribution of different species in the field: U rtica dioica L. (Km: 11 1.1M) is a species from nutrient-rich sites; Plantago lanceolata L. (Km=20 pM) occurs at nutrient- poor sites together with H ypochaeris radicata ssp. radicata (Km: 13 pM); Plantago major ssp major L. (Km: 15 pM) is intermediate between U. dioica and P. lanceolata; H ypochaeris radicate L. ssp ericetorum (Km: 17 pM ) occurs at extremely poor sites. In the case of Wolffia arrhiza (Swader et al., 197 5), Oryza sativa (Fried et al., 1965) and Arabidopsis thaliana (Doddema and Telkamp, 1979), when the rate of nitrate uptake was plotted against nitrate concentration in the medium, biphasic absorption curves were obtained. Such absorption kinetics have been interpreted as reflecting two mechanisms, I and II, of ion absorption (Epstein, 1972). Mechanism I acts at low ion concentrations with a high affinity for the ion. Mechanism II has a low affinity for the ion and becomes operative at higher ionic concentrations (1-5011M). This biphasic mechanism of ion absorption has been criticized extensively (Borstlap, 1981). To get such a pattern the second uptake system must be sigmoidal. This is not generally realized; If both show Michaelis-Menten kinetics there would be no inflection. More precise measurements are required to demonstrate unequivocally the existence of more than one nitrate uptake system whether they are in series (two mechanisms: one operating at the plasmalemma and the other at the tonoplast) or in parallel (two independent mechanisms operating at the 10 plasmalemma). In the case of Arabidopsis thaliana the chlorate resistant mutant BI (Oddema and Telkamp, 1979) also showed the dual-phase relationship. However, phase II did not follow Michaelis-Menten kinetics and the uptake rate of nitrate in the phase 11 concentration range was considerably lower in the BI mutant than in the wild type. It was concluded that the mutation in BI disturbed phase II of the uptake system, without affecting phase I. Thayer and Huffaker (1982), using 13N have measured initial rates of nitrate transport in Klebsiella pneumoniae at concentrations between lpM and 1 mM and their results indicated the presence of two transport processes; 4.911M and 4.2 mM were the Km values for the high- and low-affinity systems respectively. A number of genetic studies on Aerobacter (Klebsiella) sp. (Stouthamer, 1967a, b), Pseudomonas sp. (Hartinsgsveldt and Stouthamer, 1973; Sias and Ingraham, 1979; Sias et al., 1980) and Escherichia coli (Venables, 1972) have not revealed a class of mutants that would be considered permease negative. A restriction in net nitrate uptake occurs when intact plants have been exposed to prolonged high nitrate concentrations, and net leakage from the roots can occur even to nitrate-containing solutions (Jackson et al., 1976b; Minotti and Jackson, 1970; Minotti et al.,1969a). The ineffective plasmalemma transport may be caused directly by a limitation in the energy supply or indirectly by an increase in the root cytoplasmic ammonium pool as the carbohydrate supply to the root system is curtailed (Jackson et al., 1976a; Minotti and Jackson, 1970). Also, it has been shown that high levels of endogenous nitrate restrict net nitrate uptake in excised barley roots and disks of storage tissue (Cram, 1973; Smith, 1973). Glass (1976) has proposed a relevant model for allosteric regulation. of potassium transport in excised barley roots, the effect being exerted by cytoplasmic , not ambient, potassium. l 1 Ambient ammonium has been shown to restrict nitrate uptake by roots of wheat (Jackson et al., 1976a; Lycklama, 1963; Minotti et al., 1969b), rice (Fried et al., 1965; Sasakawa and Yamamoto, 1978; Shen, 1969), barley (Rao and Rains, 1976a) and corn (Butz et al., 1975; Ivanko and Ingrersen, 1971), and cell cultures of rose (Mohanty and Fletcher, 197 6) and tobacco (Heimer and Filner, 1971). Similar restricting effects have been observed with Spirodella (Ferguson and Bollard, 1969), fungi (Goldsmith et al., 1973; Schloemer and Garret, 1974), various green algae (Pistorius et al., 1978; Syrett and Leftley, 197 6; Thacker and Syrett, 1972; Tischner and Lorenzen, 197 9), blue-green algae (Flores et al., 1980b; Ohmori et al., 1977) and bacteria (Thayer and Huffaker, 1982). None of the studies with root systems, however, has revealed the mechanism of control exerted by ammonium. The effect appears to be an indirect one, rather than direct competition with nitrate for uptake. Recent experiments carried out with the blue-green algae (Flores et al., in preparation, cited by Losada et al., 1981) have shown that in the presence of specific inhibitors of ammonium assimilation (methionine sulfoximide to block glutamine synthetase, and azaserine to block glutamate synthase) ammonium does not inhibit nitrate uptake. These results suggest feedback regulation by the organic products of ammonium assimilation. Inhibitory effects of various amino acids indicate end product regulation of the nitrate transport system of tobacco cell cultures (Heimer and Filner, 1971, 1970). Inhibition, inactivation, or repression may all be involved. Relatively little nitrate needs to be absorbed for the apparent induction of nitrate uptake (Heimer and Filner, 1971) or root nitrate reductase (Oaks et al., 197 2). Transfer of fully induced tissue to solutions lacking nitrate results in a rapid decline of in vitro nitrate reductase activity of tobacco cells (Heimer and Filner, 1971) and root tissues (Jackson et al., 1973; Oaks et al., 1972). 12 These effects occur even when the tissue contains large quantities of nitrate at the time of transfer. It is clear that compartmentalization of nitrate exists in most plant tissues and that the storage nitrate pool (presumably in vacuoles (Martinoia et al., 1981)), cannot readily supply the metabolic nitrate pool (presumably in the cytoplasm) for sustained nitrate reductase induction (Aslam et al., 1976; Ferrari et al., 1973). Information on the distribution of nitrate between storage and metabolic pool is limited. Aslam et al. (197 6) reported data suggesting the existence of two mechanisms for regulating the metabolic nitrate pool: a) a transfer from the storage pool which requires light; and b) a transfer from the external medium which requires either glucose or light. In order to facilitate further studies on the characteristics and regulation of nitrate uptake, it would be desireable to make use of a non-metabolizable labelled analogue of nitrate as in Kt uptake studies using rubidium (86Rb +) (Nissen, 1973). Chlorate is a chemical analogue of nitrate and was used extensively as a non-selective herbicide during the 1930’s to 1950’s (Crafts, 1962). Its toxicity decreases with increasing soil fertility. In water culture, nitrate is the most effective nutrient for. protecting plants from chlorate absorption. In general, the more successfully a plant competes with weeds, the more sensitive it is to chlorate (Crafts, 1962). These observations suggest that nitrate and chlorate may be taken up by the same transport system, and that varietal differences in nitrate uptake should be paralleled by differences in chlorate uptake. Aberg (1947) proposed that chlorate was not itself toxic to the plants, but rather as an analogue of nitrate, it was rendered toxic by conversion to chlorite via the catalytic action of nitrate reductase. Several independent lines of evidence support this hypothesis of chlorate toxicity: 1) Resting 13 anaerobic suspensions of Escherichia coli catalyze the reduction of chlorate to chlorite in the presence of lactate and methylene blue; reduction of chlorate is competitive with nitrate and vice versa (Goksoyr, 1952). 2) Nitrate reductase isolated from A. aerogenes and M. denitriflcans (Pichinoty, 1969), Chlorella vulgaris (Solomonson and Vennesland, 1972), Chlamydomonas reinhardtii and Chlorella pyrenoidosa (Rhodes and Filner, 1979) and Lycopersicum esculentum (Hofstra, 1977) catalyze the chlorate dependent oxidation of NADH. 3) Genetic selection for chlorate resistance concomitantly selects for nitrate reductase deficiency in A. aerogenes (Stouthamer,. 1967), Escherichia coli (Piechaud et al., 1967), Chlamydomonas reinhardtii (Nichols and Syrett, 1978), Aspergillus nidulans (Core, 1976), Arabidopsis thaliana (Braaksma and Feenstra, 1975; Oostindiér-Braaksma and Feenstra, 1973). Hordeum vulgare (Kleinhofs et al., 1980), Pisum sativum (Feenstra and J acobsen, 1980), allodihaploid Nicotiana tabacum cultured cells (Muller and. Grafe, 1978), diploid Rosa damascena cultured cells (Murphy and Imbrie, 1981), haploid Datura innoxia cultured cells (King and Khanna, 1980), haploid H yoscyamus muticus mesophyll protoplasts (Strauss et al., 1981) and haploid Nicotiana plumbaginifolia protOplasts (Marton et al., 1982). Cove (1976) however, has suggested an alternative hypothesis for the mechanism of chlorate toxicity in Aspergillus nidulans. Chlorate, as an analogue of nitrate, interacts with the product of a positive regulator gene (nir A) for nitrate assimilation. This leads to reduced catabolism of nitrogen- containing compounds, producing symptoms of nitrogen starvation. Cove’s proposal is based on the following findings: a) chlorate is much more toxic to wild-type strains with some nitrogen sources than with others, b) chlorate is not toxic with certain nitrogen sources which can also act as carbon sources, when 1% D-glucose is replaced by alternative carbon sources and c) not all 14 mutations in the cm: or nia D genes which abolish nitrate reductase lead to chlorate resistance. All mutants isolated at the plant level were found to have some nitrate reducing capacity (Feenstra and Jacobsen, 1980; Kleinhofs et al., 1980; Oostindier-Braaksma and Feenstra, 1973). Fully deficient lines, however were obtained in cultured allodiploid Nicotiana tabacum cells by chlorate selection (Muller and Grafe, 1978). In this species only two types of deficient mutants were recovered, nia (structural gene for the nitrate reductase apoprotein) and cnx (molybdenum-containing cofactor). However, in Aspergillus and Neurospora the utilization of nitrate is controlled by at least 10 genes (Marzluf, 1981). In the case of Nicotiana plumbaginifolia, Marton and coworkers (1982) selected 7 clones defective in the nitrate reductase apoenzyme and 4 clones defective in the molybdenum-containing cofactor. The cofactor defective mutants could be divided into two groups. In 2 of the clones (NX1 and NX9) nitrate reductase activity was partially restored by the addition of high (0.2-1mM) molybdate. to the culture medium. The other 2 clones (NX21 and NX24) did not restore nitrate reductase activity upon addition of molybdate. They are not allelic but complemented with the N X1 and N X9 clones in somatic hybrids. Therefore, N X21 and NX24 are not allelic with the other cofactor defectives either, and are types not yet described in flowering plants. Three soybean mutants (LNR-2) have been isolated by selecting for chlorate resistance which lacked "constitutive”, or non-nitrate induced nitrate reductase activity usually evident in urea-grown soybeans. However, all had inducible activity when grown on nitrate. In the LNR-2 mutant, the lesion was a single recessive mutation (Ryan et al., 1982). Murphy and Imbrie (1981) reported that when strains of Rosa 15 damascena cultured cells are selected for chlorate resistance, only a minor fraction (15%) lacks the ability to grow on medium containing nitrate as the sole nitrogen source and probably lacks the ability to reduce chlorate to chlorite. The major fraction (85%) retains the ability to grow on nitrate medium and possesses the ability to transform chlorate to toxic products. These strains lost catalase activity following chlorate treatment, indicating that they took up and reduced chlorate. These observations suggest the operation of a mechanism for tolerating chlorate and its reduction products, rather than avoiding them. A common theme of both Aberg’s and Cove’s hypotheses of chlorate toxicity is the nature of chlorate as an analogue of nitrate. This together with Craft’s observations of the antidote effect of nitrate in relieving chlorate toxicity symptoms in chlorate-treated plants suggests that plants are in general unable to discriminate between these two molecules in certain key metabolic and regulatory processes involving nitrate. If plants are indeed unable to discriminate between chlorate and nitrate at the level of uptake, then 36Cl-chlorate could become a powerful tool in studies of nitrate transport in plant cells, thus overcoming the lack of a readily available long-lived radioisotope of nitrate. Furthermore, if nitrate transport into roots is rate-limiting for growth, then it might be possible to make use of chlorate as a screening tool to select for genetic material with superior nitrate transport properties. Screening of large populations and manipulation of nutrient availability is easier with cell cultures than with whole plants. I therefore decided to investigate in cultured cells the following questions: 1) How good an analogue of nitrate is chlorate in the process of nitrate transport? A vital question that arises when transport studies are carried out with cultured cells is: 2) Does 16 the nitrate transport system of cultured cells from a given cultivar reflect the relative transport capability of roots of that cultivar? The XD cell line of tobacco cells was the system of choice in which to seek the answer to the first question. This cell line has been maintained for about 20 years in the laboratory of P. Filner and laboratories scattered around the world (e.g., Japan, Germany , Israel). In this time about 100 papers with data on the XD cells have appeared. Among them, considerable data have been collected regarding XD cell biochemistry and physiology relevant to this study. The regulation of nitrate uptake (Heimer and Filner, 1971), nitrate compartmentalization within the cell (Ferrari et al., 197 3) and nitrate reductase (Behrend and Nateles, 197 5; Filner, 1966; Zielke and Filner, 1971) have been characterized. Regenerative activity of this cell line was demonstrated after 14 years (T. Rice, personal communication), 17 years (Traynor and Flashman, 1981), and 19 years (Jacobs and Zabala, in progress). A particularly useful feature of this cell line was its ability to grow with urea as a sole nitrogen source. This allows measurements of chlorate uptake without the interference of nitrate accumulated during the growth period previous to the uptake assays. The genus Lycopersicon, which includes the cultured tomato and its wild relatives, was chosen as the system in which to investigate the second question for several reasons: 1) rapid and uniform seed germination, 2) rapid growth and convenient seedling size, 3) well-developed genetics and physiology, 4) amenability to tissue culture and 5) availability of a large store of variable geMplasm. On the other hand, tobacco is severely deficient in 1), 2) and 3). It should be noted that plants have been observed to activate or dere press nitrate uptake as nitrate concentrations become suboptimal. Jackson et al. 17 (1976b) reported that the rate at which wheat seedlings take up nitrate from a 1mM solution increased about 5-fold as the concentration of nitrate on which the plants were precultured was decreased from 15mM to 0.25mM. In another study, activity of the sulfate uptake system of cultured tobacco cells also increased as sulfate became less available (Hart and Filner, 1969; Smith, 197 5). There is also the possibility that plants might increase their root surface area (size) under suboptimal nitrate concentration conditions (Jackson et al., 1976b). This would bring about an increase in the number of nitrate "carriers” and consequently an increase in Vmax (Frith and Nichols, 1975). These responses in either root surface area or nitrate uptake affinity may operate down to nitrate concentrations at which diffusion becomes rate-limiting. If this were indeed the case, then devising in vitro selections for cells displaying enhanced nitrate scavenging ability would be unwarranted. PART I: NITRATE AND CHLORATE UPTAKE SYSTEMS IN CULTURED TOBACCO CELLS MATERIALS AND METHODS Plant Material Two tobacco cell lines were used in this study. the XD cell line was initiated from pith explants of N icotiana tabacum L. cv. Xanthi-nc by P. Filner in 1961. Since its isolation this cell line has been maintained on M1D (a minimal medium in which nitrate is the sole nitrogen source). under these conditions, XD cultures grow with a exponential doubling time of 2 days and reach stationary phase after 10 days (Filner, 1965). The second cell line, 14U, arose spontaneously from the XD cell line after two years of serial subculturing of the cells in a medium with urea as the sole source of nitrogen (Yamaya and Filner, in preparation). The cells were cloned when urease was 4 times the level in XD cells, and they were subcloned from a variant of the clone in which urease had increased further. The urease activity of 14U cells is 14 times that of the wild type XD and they retain the high urease level in the absence of urea (Yamaya and Filner, in preparation). The mechanism giving rise. to this Permanent change is still under investigation. The growth characteristics of 14U cells grown in urea-M1D are very similar to those of XD cells grown on nitrate, having an exponential doubling time of 2.2 days and an exponential phase lasting about 10 days (Yamaya and Filner, in preparation). 18 19 Growth Media The XD cells were maintained in M1D, a modification of White’s medium described by P. Filner (1965). It contains 2.5 mM nitrate as the sole nitrogen source and limiting nutrient. The cells cease growing on this medium when they exhaust the nitrogen supply. Nitrate-less M1D (N-less M1D) medium was made by replacing the nitrates of calcium and potassium with equimolar amounts of their respective chlorides. As a result, the final chloride concentration of this N-less M1D medium becomes 2.55 mM. A variation of the N-less M1D medium called chloride-less nitrate-less M1D (Cl-less N-less M1D) medium was developed for experiments where the effects of chloride were investigated. Cl-less N -less M1D medium was prepared by substituting the chlorides of calcium and potassium with equimolar amounts of calcium sulfate and potassium phosphate respectively. These changes increased the sulfate concentration from 312 mM and phosphate from 12 mM in M1D to 398 and 178 mM, respectively in the Cl-less N -less M1D. All media were adjusted to pH 6.2 with N aOH before autoclaving at 20 psi for 20 minutes. Urea-M1D medium was prepared by adding to sterile N-less M1D an aliquot of a filter-sterilized urea solution (ultra pure grade, Schwarz /Mann). The final urea concentration in the urea-M1D medium was 3 mM. As in the case of urea, when supplemented media were required, sterile solutions of the supplements were diluted into sterile media. The supplement solutions were adjusted to the desired pH prior to filter sterilization. 20 Growth Conditions Stock cultures were grown in 1 litre erlenmeyer flasks containing 500 ml of liquid medium on a horizontal platform reciprocal shaker with a displacement of 4 cm at 80 cycles per minute. The shaker was kept in a controlled room at 28 ° C with a light regime of 12 hour day/ 12 hour night (the cells were not green, and growth was unaffected by light). Cell suspensions were subcultured by inoculating 500 ml portions of fresh media with 25-ml aliquots of lO-day-old stationary phase cultures using wide bore pipettes. The fresh weight of the initial inoculum was 0.5 to 1 g/l. Experiments were always conducted with exponentially growing cultures, 4 to 6 days following subculture. Measurement of Nitrate and Chlorate Uptake When cells growing in M1D were used to study the mechanism of nitrate and chlorate uptake, exponential phase cultures were combined and harvested by vacuum filtration onto Whatman N o 1 filter paper. Cells were then rinsed twice with N-less M1D, being kept moist at all times. The cells were then resuspended in fresh N -less M1D. Accumulated nitrate was allowed to diffuse out of the cells into the N -less medium for 30 minutes. After this period of time, the cells were again harvested and rinsed as described above and resuspended in fresh N -less M1D at a density of approximately 8 grams of cells per litre. 25-ml aliquots were then transferred to 125-ml erlenmeyer flasks by means of a wide bore pipet. Solutions of nitrogen sources and other compounds were diluted into the 125-ml flasks to give the appropriate final concentration of the respective ions. Two replicate flasks were prepared per data point. I Experimental cultures were grown on the same shaker as described for 21 maintenance of the stock cultures. The uptake reaction was stopped by harvesting the cells by filtration onto a glass microfibre filter of 2.1 cm diameter. The cell pellet was rinsed twice with 5 ml of fresh N-less M1D, weighed and transferred to scintillation vials when determinations of uptake of 36Cl-chlorate were to be made. Each vial also contained 5 ml of the scintillation cocktail described by Trombolla (1970), consisting of 30% toluene, 70% triton X-100, 8 g/l PPO and 0.35 g/l dimethyl POPOP. When measurements of nitrate uptake were to be made, the cell pellets were weighed and transferred to 30-ml Kjeldahl flasks for digestions as described below. When 14U cells growing in Urea-M1D were used, the harvesting procedure was changed to filtration by gravity. This was due to the observation made by Thom et al. (1981) that vacuum filtration temporarily damaged active transport in plant cell systems in general and in the KB cells in particular. This observation of Thom et al., (1981) was readily verified, whereupon the harvesting procedure was changed to avoid the temporary injury. Thus, 5 day old 14U cell cultures were harvestedby gravity filtration onto a 202 pm nylon mesh cloth and rinsed twice with N-less M1D. The harvested cells were carefully transferred to a graduated cylinder and the cell suspension brought to the desired volume by the addition of N-less M1D medium. The cell suspension was then transferred to a 3 or 6 litre fernbach flask to which aliquots of urea and a hydrogen ion buffer had previously been added. The buffer consisted of 20 mM 2[N-morpholino] ethanesulfonic acid (MES) and 20 mM N-2-hydroxyethylpiperazine N’-2-ethanesulfonic acid (HEPES) (final concentrations). The pH of the buffer solution was adjusted to 5.5 or 6.5 by the addition of crystalline Tris HCl. 25 ml of the supplemented cell suspensions were transferred to 125 ml erlenmeyer flasks to which 22 solutions containing the anion of interest had been added. All the manipulations described above for the 14U cells were carried out under aseptic conditions in a laminar flow hood. The remaining steps in the processing of the 14U samples were carried out as described for the XD cells. Synthesis and Purification of 36Cl-Chlorate 36Cl--chlorate can be synthesized by electrolysis of commercially available H36Cl. Under alkaline conditions possible partial reactions during the electrolytic process include the following: 01' + 20H‘ ——> ClO‘ + H20 + 2e’ 0.90 v ClO' + 20H' , 0102' + H20 + 2e' 0.59 v 0102' + ZOH' > C103“ + H20 + 2e' 0.35 v ClO3' + 2OH' ——> ClO4' + H20 + 2e" 0.17 v The lifetime of the radioisotOpe 36Cl is 3.1x105 years and the observed mode of decay is (3", (3+, EC. The decay energies are : 0.712 Mev ([3" ) and 1.14 Mev (13+, EC),i.e., higher than 14C (0.156 Mev) but lower than 32P (1.710 ’ Mev). The low specific activity of the radioisotope, 500,000 cpm/pmole represents the only disadvantage for short term uptake studies since the measured rate of chlorate uptake by tobacco cells (see Part II) is 4 nmoles/ h / g fr. wt. under unrestricted conditions. 36Cl—chlorate was synthesized and purified as described by Trombolla (1970) with some modifications. H36C1 (New England Nuclear; specific activity 0.23 pCi/pmole) was diluted to 0.02 M in a total volume of 7 ml of distilled water. The solution was made alkaline by addition of 2 M N aOH to a 23 final concentration of 0.023 M. The solution was electrolyzed at room temperature (20° C) for 3.5 hours at 50 mA constant current and approximately 12 volts. The apparatus used for electrolysis consisted of a 10 ml glass beaker covered with a plastic cap through which two platinum wire electrodes were inserted. The spacing between the electrodes was 1.5 cm. After electrolysis, the electrolysate was rotary evaporated at 30° C to a final volume of 0.2 ml and applied to the base line of a plastic thin layer chromatography plate coated with MN 300 cellulose. The products of electrolysis were chromatographed in a solvent composed of 80 ml 1-butanol, 40 ml pyridine, 80 ml H20 and 5 ml ammonia. The bands corresponding to the three products of the electrolysis (perchlorate, chlorate and chloride) were localized by autoradiography of the plate. Chlorite was undetectable. The bands for 36Cl-perchlorate, 36Cl-chlorate and 36Cl-chloride have Rf’s of 0.73, 0.54 and 0.34 respectively. The identity of those compounds with their respective standards was demonstrated by developing the spots on a TLC plate with a solution of aniline in HCl (50 g of pure aniline hydrochloride in l l of 1:3 hydrochloric acid) (Welcher, 1947). Chlorate develops a bluish green colour and chlorite a spot which is violet for about half a minute before turning bluish green. The reaction with chlorite develops soon after the spraying, whereas the chlorate spot is not visible until the paper is practically dry. Chloride and perchlorate give no colour with this reagent (Goksdyr, 1952). Perchlorate was detected by spraying first with saturated aqueous sodium acetate and, once dried, overspraying with 0.2% aqueous methylene blue. Perchlorate becomes violet against a light blue background (Harrison and Rosenblatt, 1964) 35Cl-chlorate was extracted from the chromatogram by incubating the TLC plate fragments overnight in a minimum volume of water. The recovery 24 of label as chlorate after 3.5 hours was approximately 30% and the remainder was 36Cl-perchlorate (68%) and 36Cl-chloride (2%). Purity of chlorate was better than 99%. The 36Cl-chlorate solution was stored at 4° C. Rechromatography of the preparations, and analysis of chloride and chlorite content over a period of 6 months indicated that the 36Cl—chlorate preparations were stable to storage. In most experiments 36Cl-chlorate was supplied to cells at a maximum specific activity of 500,000 cpm / pmole at a level of 7,000 cpm/ml medium or 14 pM. In addition, unlabelled chlorate was added to achieve higher chemical concentrations, with proportionally lower specific radioactivities. The time course of the electrolysis of H36Cl was determined by removing 20-pl samples from the electrolysis every 30 minutes for 4 hours. Three microliters from each one of those samples were chromatographed as described above. Strips corresponding to each time point were cut into 1 cm fragments corresponding to the Rfs of the electrolysis products. Each fragment was then counted by scintillation spectrometry. Optimum recovery of 36Cl-chlorate was achieved after 1.5 hours of electrolysis (Figure 1). However, for routine synthesis of C103" electrolysis was allowed to proceed for 3.5 hours, as suggested by Trombolla (1970). Kjeldahl Procedure Nitrate incorporated into XD cells was determined by measuring 15N abundance, using mass spectrometry after conversion of nitrogen to N2. An estimation of the total nitrogen in the plant cell was achieved by a two-step Kjeldahl procedure (Nelson and Sommers, 1973). 0.2 to 0.6 g fresh weight of tobacco cells were placed in a 30 ml Kjeldahl flask to which 2 ml of a salicylic acid-H2804 mixture (1 g salicylic acid per 40 ml of cone. H2804) were added. 25 v.08 n 3. C558 M iv "-553 n AS 3:63:58 «£13.? .895 mmmbosoflo 58m maize aoflaata 3:33 .3 $38 25,—. 3 9:65 .55. “2.... Owu 00—. Our ‘ lfi - d l./ ELDNIH Had SanOO 26 After allowing the mixture to stand at 25° C for 2 hours, 0.25 g of sodium thiosulphate was added followed by cautious heating over a burner until frothing ceased. The tube was then removed from the heating block and cooled to 25° C. 4 ml of a 5 M H2804 solution containing 20 g per litre of sodium selenate and 20 g per litre of CuSO4-5H20 were then added. The tube was swirled to mix the sample and digestion reagents and then returned to the burner. The samples were digested at the boiling point of the mixture 60 minutes past the time of clearing, removed. from the heating block, and allowed to cool to room temperature. At this point, all forms of nitrogen are converted to ammonium. The digest was them diluted with distilled water to a final volume of 15 ml. Ammonium in the digest was then released in a steam distillation apparatus by addition of 15 ml of a 5 N NaOH solution. The ammonia and water vapors were condensed and collected in an erlenmeyer flask containing 2 .ml of 0.1 M H2 $04. The excess water was evaporated to dryness on a hot plate. The concentrated (N H4)2SO4 solution was rediluted in 2 ml of water and the amount of ammonium determined by a direct colorimetric method described by McCulfough (1967). One ml each of the diluted (NH4)2SO4 solution, reagent A (10 g/l phenol and 50 mg/l of sodium. nitroprusside), and reagent B (5 g/l NaOH, 53.7 g/l N a2HPO4-12 H20 and 10 ml of 10-14% sodium hypochlorite) were mixed and incubated at 70° C for 5 minutes. The absorbance was measured at 625 nm in a Gilford spectrophotometer. Determination of 15N Atom Percent The usual way of introducing a sample into an isotope ratio mass spectrometer is as follows: 1) A vacuum-proof manifold vessel containing the ammonium sample is attached to both the mass spectrometer inlet system and 27 to a preevacuated reservoir of lithium hypobromite. 2) The sample is then preevacuated by Opening it to the vacuum system intercalated between the mass-spectrometer reservoir and the sample’s vessel. 3) The vacuum system is closed and the ammonium sample is allowed to react with an aliquot of lithium hypobromite drawn in from its reservoir. 4) The reaction mixture is frozen by surronding the sample vessel with liquid nitrogen. 5) The N2 gas formed in the reaction between ammonium sulfate and lithium hypobromite is then drawn through the mass spectrometer reservoir to the analyzer. 6) 15N percent abundance is then measured and corrected with that of the residuals previously measured. This procedure has two major disadvantages. Large amounts of sample are required and the glassware of such an inlet system is expensive, fragile and cumbersome. A procedure was devised for handling large numbers of samples quickly with inexpensive disposable glassware. One-half ml of the (NH4)2SO4 solution produced in the total nitrogen determination, was injected into a preevacuated glass vial (10x36 mm; 1 ml) covered with a rubber septum. The injection of the sample was done by allowing the vacuum in the vial to draw in the sample, minimizing contamination by air. The ammonium was then; oxidized to nitrogen gas by injection into the vial (as described for the sample) of 0.5 ml lithium hypobromite solution (60 ml cold 10% w/v lithium hydroxide (analytical grade) to which 2 ml of analytical grade bromine was added). The vial was then immersed in liquid nitrogen and as soon as the solution froze, another rubber septum was placed over the first one (Figure 2). The air space between the two septa permitted the removal of the air located between the needle used to inject the sample into the mass spectrometer (MAT GD 150) and the valve which connects to the reservoir. Once that air was removed by 28 GLASS VIAL (101130111111, 1m!) STAINLESS STEEL IIILET TUBE QOLDEIIED j 1.101111 4 :L >— 911 1 assenvom T rwo RUBBER seen SECOND SEPTIII FIRST SEPTUI a, an FROZEN SAIPLE [ I Figure 2: Diagram of apparatus devised for introduction of samples into the MAT GD 150 Mass Spectrometer. 29 the instrument’s vacuum system, the needle was forced through the first septum into the head space containing 15N2. The valve to the reservoir was opened for 6 seconds and the isotope ratio of 15N and 14N of the nitrogen gas measured. The spectra of both the residuals and a 92-93% 15N atom percent of a (15NH4)2SO4 standard sample were used to determine a factor to correct the error due to air contamination of each individual sample (see example in Table 1). 15N abundance was then calculated in one of two ways: 1) For samples with low abundance, and therefore with low probability of containing 15N-15N (mass 30).molecules, the following expression was used (Hauck and Bremner, 1976): 15N atom % = 100/2R+ 1 where R represents the quotient of the residual-oxygen corrected peak areas of l4N-14N (mass 28) divided by that of 14rN-15N (mass 29). The 15N atom%excess is then determined by subtracting the 15N atom%atmospheric(0.37) from the 15N atom%observed. 2) For samples with high 15N abundance and therefore with high probability of containing 15N-15N (mass 30) molecules, the expression 15N atom % = 100/0.5R’+ 1 was used (Hauck and Bremner, 1976). R’ corresponds to the quotient of the residual-oxygen corrected peak areas of 14N-15N (mass 29) over that of 15N- 15N (mass 30). The 15N atom%excess is then determined as described above for low 15N abundance samples. 15N enriched potassium nitrate ( 15N atom% = 99 ) was purchased from BIO—RAD laboratories. Determination of Nitrate Nitrate in the cell’s soluble compartment and in the external medium was measured by the method described by Lowe and Hamilton (1967), based on the ability of soybean nodule bacteroids to reduce nitrate to nitrite. Bacteroid suspensions prepared by Y. Heimer in 1969 and kept frozen at 30 Table 1: Method of , determination of 15N atom % abundance from mass spectrometric data. 1) Determination of correction factor (F) PEAK AREAS AT: Mass 28 Mass 29 Mass 30 Mass 32 Residuals a b c d Standard e f g h (92.5% 15N) A28=e~a ° 2 A28: e-a A32 = h - (1 Correction Factor, F 332 m 2) Correction for residuals and oxygen PEAK AREAS OF: Correction for Correction for Residuals Samples Residuals Oxygen Mass28 A G i=G-A K=i-(Fxl) MassZQ B H j=H-B L=j-(Fx l ) 1'33 Mass30 C I k=I-C M=k-(Fx l) 266 Mass 32 D J = I - D 3) Determination of 15N abundance For samples low in 15_N_ For samples high in 15N 15N abundance (28/29) 15N abundance (29/30) R = K/L R, = UM Q = 100/(2R + 1) Q = 100/(0.5R' + 1) 31 -20° C were used for this analysis. The assay mixture was composed of the following: 0.5 ml of 0.2 M potassium succinate buffer pH 6.8, 0.3 ml of the nitrate containing solution and 0.2 m1 of undiluted bacteroid suspension. The assay tubes were incubated for 30 minutes at 40° C. The reaction was stOpped ' by the addition of 1. ml of 1% w/v sulfanilamide in 3N HCl. The diazonium salt formed with the nitrous acid is reacted with 1 ml of 0.02% N-(l-naphthyl)- ethylenediamine dihydrochloride to give a colored diazoamino compound (Azo, Benzene l-Naphthalene, 4-Sulphamyl, 4’- N- Ethylene-1,2-Diamine Hydro- chloride) that absorbs at 540 nm. Before reading the absorbancy the assay mixtures were cleared by centrifugation at 15,000 x g for 10 minutes (Snell and Snell, 1949). For the assay described above, nitrate was extracted from the cells’ soluble compartment with ethanol. Cells were separated from the ethanol by filtration and the nitrate recovered by evaporating the alcohol in a rotoevaporator at 38 ° C. RESULTS Chagfies in the Internal and External Nitrate Pools of XD Cells in Culture The XD cell line was initially used to characterize the cellular nitrate and chlorate uptake systems for two reasons. First, some features of its nitrate metabolism had already been established (Heimer and Filner, 1971; Ferrari et al., 1973) and second, as mentioned in Materials and Methods, this cell line has proven to be a stable experimental system in the past. 32 Previously reported indications of the analogy between the nitrate and chlorate ions in physiological systems prompted us to examine the nitrate status of the XD cells during their growth cycle as well as the effect it could have on the analysis of chlorate uptake. Stationary-phase XD cells were subcultured into M1D and the nitrate concentration of both the cells’ nitrate pool and medium was determined over the cells’ growth cycle. The results are summarized in Figure 3. The initial medium nitrate concentration of 2.5 mM decreased to a negligible level by the time the culture entered the stationary phase. Nitrate accumulated in the cellular pool as the cells divided exponentially, reaching a maximum concentration of 24 moles nitrate per g fr. wt. at the mid-exponential phase of the culture cycle. As the cells entered stationary phase under these conditions of nitrogen limitation, the level of cellular nitrate was reduced, depleting the intracellular pool, since no further accumulation was taking place. This indicated that exponential cultures, ideally suited for physiological studies, contained high levels of nitrate in a free pool. In order to determine the effect that this nitrate pool had on chlorate uptake, the following experiment was performed. Late exponential phase cells were transferred to N-less M1D and the uptake of chlorate was measured with time. In addition, the amount of nitrate in the medium and in the cells’ nitrate pool was concurrently determined. The results, shown in Figure 4 reveal a rapid release of the cells’ nitrate into the medium. This is in agrement with the results reported by Ferrari et al. (1973). Since my working hypothesis presumed that chlorate and nitrate shared a common transport system, preincubation and washing of the cells in N-less medium was clearly indicated. A preincubation period of 30 minutes was chosen followed by 33 I I 0 v3” . ‘24- O S - 3 ‘g 3' ' I . 5 '2'2- .. 3 .\. .\. O O 2 Z O O 2 I .2 o 1 o E E I =- ) 0.. 1 o 7 14 TIMEMays) Figure 3: Intra- and extracellular nitrate levels in XD cells over the culture cycle. (0) = N03' content of cells (umoles/ g fr. wt.); (I) = medium N 03' (moles / ml). 34 10 r r 1 1 4o 1S pmoloe nitrate/g tnwt. medium“) cells“) Rate of chlorate uptakeInmolee/h/o tr.wt.)(ll O 10 TIME( h) In nitrate-loss MID Figure 4: Leakage of NO3" from XD cells cultured in the presence of 350103'. (I) = rate of 33C103' uptake, nmoles/ h / g fr. wt.; (0) = medium N03', pmoles/g fr. wt.; (A) = cell N 03‘, moles/ g fr. wt.. .35 rinsing in N -less medium and transfer of the cells to experimental solutions. The onset of chlorate accumulation followed a lag of approximately 2 hours. The rate of accumulation then increased to a constant value after 4 hours. The lag phase of chlorate uptake could be explained by the observation made by Thom et al. (1981) that vaccum filtration temporarely damaged active transport in plant cell systems. They studied arginine uptake in N icotiana tabacum XD cell line following both gentle (gravity) and vigorous (vacuum) filtration. Arginine uptake after vigorous filtration showed a lag of approximately two hours, whereas gentle filtration resulted in no appreciable - lag in transport. As this report appeared well into the progress of our studies, gravity filtration was not adopted until the beginning of the experiments with 14U cells (see below). Figure 5 shows the accumulation of chlorate in exponential (2-day-old) and early stationary phase (10-day-old) XD cells with time. These chlorate uptake measurements were carried out after a preincubation period of 30 minutes in N-less medium to correct for contamination by nitrate leaking out of the cells. This correction reduced but did not eliminate. the initial lag in chlorate uptake (Figures 7 and 14). Chapges in Km and Vmax of Chlorate Uptake with Changes in Cell Age and with Time in Inducing Medium (Nitrate-less MID). Nitrate uptake rates have been reported to be higher in nitrogen-starved than in nitrogen-fed plants (Jackson et al.,1976a and 1976b). Thus, the sensitivity of the uptake system to nitrate depends upon the system’s preexisting nitrogen nutritional status. It was therefore pertinent to know whether chlorate uptake was affected by prior nitrogen status. The kinetics of 36 l ./ / ,. nmoloe of chlorate/g Inwt. .l o I 4 1.51? o 4 1 1 O 1 2 3 4 TIME (II) Figure 5: Culture-cycle dependence of chlorate uptake by XD cells. Accumulation of 36C1' in exponential (I) and stationary (I) phase XD cells was determined after incubation with K36C103 for the times indicated. 37 chlorate uptake of cells preincubated in N -less MID for 0, 2, and 4 hours were measured. This preincubation period was in addition to the standard 30 minute rinse in N-less medium. XD cells growing in M1D were harvested by vacuum filtration, rinsed twice with N-less M1D and resuspended in fresh N- less M1D. Accumulated nitrate was allowed to diffuse out of the cells into the N-less medium for 30 minutes. The cells were then harvested, rinsed and resuspended in fresh N -less M1D at a density of approximately 8 g of cells per litre. 0.2 g of cells were then transferred to 125-ml Erlenmeyer flasks. The cells were incubated with 36Cl-chlorate (7,000 cpm/ml of medium) and unlabelled potassium chlorate (0.1 to 3.2 mM) to give a range of specific activities (60,800-2,165 cpm/pmole). 36Cl-chlorate taken up into the cells was measured after 2 hours of incubation. This analysis was repeated with cells of different ages and the results are summarized in Table 2. Figure 6 shows an example of a Lineweaver-Burk plot from which some of the Km and Vmax values reported in Table 2 were generated. This particular set of plots corresponds to the 4-day-old cells after 0, 2 and 4°hours of nitrogen starvation. The apparent affinity of the uptake system for chlorate increased with time in N-less medium, regardless of culture age. Three reasons can be proposed for this effect. First, nitrogen starvation could indeed derepress or activate a nitrate, and therefore a chlorate, transport system. Second, injury of the cells by vigorous (vacuum) harvesting could have a negative effect on the uptake system during the first hours following transfer to N -less medium. The greater affinity demonstrated by the transport system after 2 or 4 hours may simply reflect recovery from such injury (Thom et al., 1981). Finally, nitrate continues to leak out of the cells following the second transfer to N -less medium (see Figure 4; also Ferrari et al., 1973). This nitrate may compete with chlorate for a shared uptake system, resulting in the initially higher Km 38 .393 335538 303 8.333 app-nonv—Ovfitp Or I 0 ¢ N O O O V N O O 0 V N O . . d . d . . d d u . . OIIOuOfl 10S— .\_ \C x I\ I 1 r L- 1 l I 1r. 1 O L p p b . . . . . p b . 0.0.12 c. p; 0.0.12 :- ad 000.12 c. :0 O u— 09 cu =< 3:8 ON .3 £33. 3825 .«o Sawing—~35 33:3 :6 consign comet: mo Spam 6 0.33% ”mum; O/Ii/«IomflM/ 1 39 m3. ma. v mam. S a . N cam. 93. 0 ads 3 mam. 3v. v can. . mmm. a m3. m2. c ads 5 amm. mmu. v cmv. mam. a Sb. 2:. a 9mm 3 «on. own.“ v m3. v84 a and mg. o ad p 3w. mafia ¢ 5m. .23. fl a «mad man. a mfi v 33.: m2 3. A25 >88? 83:3 5332 3.2-2 3 3 EM 233:; 5 comaaaaofimum Emma? 43.—rm 0.5250 .3 mm< .38 Gun 5 83E: 33030.3 moflmfimaoauaao 0333 :o 5:35.? 59:3: .3 gamma .N 035,—. 40 values. The effect of nitrogen starvation on nitrate uptake was also examined. Six-day—old cells were preincubated in N -less medium for 0, 2, and 4 hours before the addition of 15N-KN03 at concentrations ranging from 0.1 to 1.6 mM. Cells were harvested after 2 hours of incubation, and the 15N abundance of the total cellular nitrogen was determined by mass spectrometry. The double reciprocal plots of the data obtained from those measurements are shown in Figure 7. The Km and Vmax values obtained from these measurements are presented in Table 3. Whereas the chlorate uptake system reflected changes in the state of nitrogen nutrition of the cells, the nitrate uptake system displayed no systematic effect. The Km values in all instances are close to 0.1 mM. Effect of Nitrate on Chlorate Uptake. In our first attempt to evaluate directly the interaction between chlorate and nitrate uptake, we analyzed the changes in the kinetic parameters of chlorate uptake caused by the addition of nitrate to the incubation medium. XD cells growing in M1D at a density of 2 g fr. thl were harvested and transferred to N-less M1D where they were incubated for a period of 30 minutes at a cell density of 26 g fr. wt./l. Cells were then harvested and resuspended in 600 ml of fresh N-less medium at a cell density of approximately 9 g fr. MA. This culture was divided into three portions of 100, 100 and 400 ml. A tracer amount of 36Cl-chlorate (14.5 1.1M) was added to each of the lOO-ml portions. To one of these, nitrate was also added to give a 1 mM final concentration. Ten-ml aliquots were removed from each culture every 30 minutes during a 4.5-hour incubation period. Determination of radioactive incorporation into the cells at each time point gave a time course of chlorate 1/v(umolu of "N/h/g mm.) Figure 7: Effect of nitrogen starvation on kinetic characteristics of nitrate 0.2 0.1 .41 1 IEKNOJ um /I ,o/: o 1 1b uptake in XD cells. (I) = 0 h, (0) = 2 h, and (A) = 4h. 42 Table 3: Effect of nitrogen starvation on kinetic characteristics of nitrate uptake in XD cells. Time in N-less M1D Km Vmax (h) (mM) ([1 moles l5N/h/g fr. wt.) 0 0.08 ' 7.9 2 0.10 15.9 4 0.06 15.4 4.3 .AOV 33:: mo 3:83 05 E can A0 V «02% SE u .«o madame...“ 23 5 333: 3983“. he 83 gunmégaosoafi "O .A IV 32:: «o 258% 05 5 .33 A 0v mOZM 28 fl .3 8:803 23 5 8—39. 332:“. we mocovcaov 55.55050 ”m .A 0v 38:: mo 853a on: E 38 ADV mOva 25 H go 8:3an 23 5 5:23:38“ .3325 .«o 3.58 2:; ”4 .230 ON E 33.3: 332:“. .«o 852:: 05 no 38:: mo 80me "m 959% 551203: 5810.3: Ema: o . w . o a u p o \I\.\ II. II! I k m we" N. m on W m \ E W n .36» .3 u m . .m. 8. :1. 0 index. n m w m u. u. . \o -19 . J on . w .. a. ... e. 44 uptake in the presence or absence of nitrate (Figure 8A). The third portion (400 ml) of the initial culture was set aside on inducing medium (N-less M1D) for 4 hours. From this culture, two series of 25-ml batches were made. Each series consisted of a range of non-radioactive chlorate concentrations (0.1 to 3.2 mM) and a constant initial input of labelled chlorate (7000 cpm/ml). In addition, one series was brought to 1 mM KNO3, while the other remained N -free. The amount of chlorate accumulated by each culture was measured after 2 hours of incubation (Figure 8B). Nitrate appears to have a strong inhibitory effect on chlorate uptake. 1 mM nitrate decreased 10-fold the amount of chlorate incorporated into the cells after 4.5 hours (Figure 8A). The effects of nitrate on the apparent Km (20 mM with 1 mM KN 03 versus 1 mM without KN 03) and Vmax (0.6 moles of KClO3 / h / g fr. wt.) for chlorate uptake, deduced from the Lineweaver-Burk double reciprocal plot (Figure 8C) suggest that nitrate may act as a competetive inhibitor of chlorate uptake in this system. However, this conclusion must remain tentative, since the scatter of points on the +KNO3 line does not permit a precise determination of whether the + KN03 and - KN03 lines intersect on the ordinate. Concurrent Determination of Kinetic Characteristics of Nitrate Inhibition of Chlorate Uptake and of Chlorate Inhibition of Nitrate Uptake. To investigate further the relationship between chlorate and nitrate uptake systems, an experiment was designed to determine the Ki of inhibition of chlorate uptake by nitrate, as well as the Ki of nitrate uptake by chlorate. These measurements were made in the same experiment by using simultaneously 36Cl-chlorate and 15N-nitrate in the following way. A 7-day- old XD culture, treated as previously described for uptake studies, was divided 45 into an experimental block of 25—ml batches and incubated for 3 hours in N - less medium. To each 25-ml batch 36Cl-chlorate was added to give a final concentration of 7000 cpm/ml to different subsets of the experimental block, non-radioactive chlorate was added to final concentrations of 0 to 3.2 mM. Likewise, final 15N-KNO3 concentrations of 0.02 to 2 mM were generated in the orthogonal subsets of the block. Each member of this grid of chlorate and nitrate concentrations was incubated for 2 hours and then divided into 2 equal portions. One portion was used to measure 3SCI-chlorate accumulation and the other to determine 15N atom % by mass spectrometry. Figure 9 shows the results obtained from the 36Cl-chlorate determinations. The increase in the slope of the double reciprocal plot (and therefore the increase in the Km) resulting from an increase in nitrate concentration and the fact that the ordinate intercept remains constant (Vmax = 0.6 mole of chlorate / h / g fr. wt.) once again indicates that KN O3 behaves as a competitive inhibitor of chlorate uptake. A Dixon plot (Figure 10) of UV versus [inhibitor, KN 03] at different substrate concentrations gives a series of straight lines with slopes inversely proportional to the substrate (KC103) concentration. The intersection of these lines on the abscissa at [KN 03] = -0.1 mM gives a Ki value of 0.1 mM. The Ki value for nitrate inhibition of chlorate uptake is identical to the Km obtained for nitrate uptake after 2 hours of nitrogen starvation (see Table 3). On the other hand, when the UV values obtained from measurements of 15N abundance in the other portion of each sample were plotted against US (S = [KN03] ) for each KC103 concentration, no pattern characteristic of inhibition was apparent (Figure 11). The effect of chlorate on nitrate transport was reexamined to confirm the results described above, using a. slightly different experimental approach. 46 O b 48 0) N 1/v(pmom ot mag/lug tum)" d O 0 1 2 3 4 5 1/[KCl031mM Figure 9: Lineweaver-Burk plot of nitrate inhibition of chlorate uptake in XD cells. [N03‘] = 0.75 (O), 0.25 (a), 0.05 (e), 0 (A) mM. 47 104 78 1/v(umolu of CIO;/II/g tum)" ?. . 13V} -o.2 o 0.4 0.3 1 Figure 10: Dixon plot of nitrate inhibition of chlorate uptake in XD cells. [0103'] = 0.21 (0), 0.61 (e), 1.61 (A), and 3.21 (I) mM. K, = 0.1 mM. 48 2.4r- - 1.8 1.2 1 /v(umolu ‘ “NO;/hr./gr.fr.wt.) " 0.8 o s 10 15 20 25 Figure 11: Lineweaver—Burk plot of chlorate inhibition of nitrate uptake in XD cells. (0) = 1.5 mM, (I) = 3.2 mM, (A) = 0.6 mM, (1.) = 0.02 mM, and . , (.) = OmMKClO3. 49 Five-day-old cells preincubated in N -less medium were transferred to N-less medium containing 0.5 mM l5N-KNO3. The culture was divided into 25-ml portions to which KC103 was then added to give final concentrations ranging from 0 to 20 mM. After 2 hours, the cells were harvested and prepared for 15N atom% measurements. In this instance, the 15N abundance of the remaining medium after harvesting the cells was also measured. This determination was used to correct for the change in 15N abundance caused by leakage of 14N from the cells’ nitrate, pool. The percent decrease in medium 15N abundance ranged from 17 to 28%. The results obtained after correcting for the decrease in the medium 15N abundance are shown in Figure 12. UV values plotted against inhibitor concentration gave an almost horizontal line indicating that chlorate only slightly inhibits the uptake of nitrate, thus supporting our previous finding. Inhibition of Nitrate Uptake by Chlorate in Urea-Grown Cells. The rate of chlorate and nitrate incorporation into XD cells previously maintained in urea-M1D medium, upon transfer to N-less M1D containing either chlorate or nitrate, is the same for each of the two anions during the intial 2.5 hours. After this time, the rate of nitrate uptake increases rapidly _ while the chlorate accumulation maintains the intial rate for a longer period of time (David Rhodes, personal communication). From these results, it was hypothesized that XD cells have a constitutive uptake system that does not discriminate between chlorate and nitrate while there is an inducible system with a higher affinity for nitrate. If this were true, then nitrate uptake should be inhibited by chlorate only during the first 2.5 hours upon transfer of nitrate-free cells to nitrate supplemented medium. This hypothesis was tested by transferring the urea-grown cells to M1D 50 '1/v(umolu ot Mag/lilo tr.wt.)"x103 g 0 4 8 12 16 2O 24 [KCIOalmMfl D Figure 12: Dixon plot of chlorate inhibition of nitrate uptake in XD cells grown in nitrate. [KN03] = 0.5 mM. 51 containing 0.5 mM 15N-KN03. This suspension was- then divided into 25-ml batches to which chlorate was added to final concentrations of O to 10 mM. 15N abundance in cells harvested at 2.5 hours following inoculation was determined by mass spectrometry. A plot of the reciprocal of the intial velocity values versus the inhibitor concentration (KC103) gave a line characteristic of inhibition in support of the proposed hypothesis (Figure 13). Chlorate inhibited nitrate uptake when the measurements were done during the time period when chlorate and nitrate accumulation were equivalent. Nitrate transport is inducible by nitrate in XD cells (Heimer and Filner, 1971; Behrend and Matales, 1975). Grown in urea, XD cells are therefore uninduced for nitrate transport (Heimer and Filner, 1971). When XD cells are transferred from urea to nitrate medium, the induction of their nitrate transport systems should cause them to behave in this situation exactly like nitrate-maintained XD cells. Such a result would strengthen our interpretation of the findings reported above. This experiment was performed by preincubating XD cells, which had been previously grown on urea, for 6 hours in nitrate medium (0.5 mM KNO3). Cells were then harvested and incubated in N-less M1D for 30 minutes, harvested again and split into 25-ml portions. 15N-nitrate (0.5 mM) and chlorate (0 to 10 mM) were added as previously described. After two hours of incubation, cells were harvested and 15N atom % determined. The values obtained were corrected for abundance changes of medium 15N as before. The results shown in Figure 14 are equivalent to those shown in Figure 12 for cells maintained on nitrate. This agreement reinforces the hypothesis of a dual system for nitrate uptake, of which one carrier is constitutive and shared with chlorate, while the other is inducible and essentially exclusive for nitrate. 52 l l I 5- 4 q r ‘7 ‘3' 4*- 4 .- b O \ 3 >0 3. .. o 2 fi 0 5 2+ - O E I' 3 0 t 1.] .. F 0 4 8 12 16 Figure 13: Dixon plot of chlorate inhibition of nitrate uptake in XD cells grown in urea. [KN03] = 0.5 mM. 53 I r l B F I in 2 " A g C O O’./ \ z 8 O .\.. O z 3 1 .. .. o .2 o O E 3 > \ P 0 4 8 1 2 [KCIOal mflzl Figure 14: Dixon plot of chlorate inhibition of nitrate uptake by 14U cells pretreated in nitrate induction medium. [KN03] = 0.5 mM. 54 Effects of Ammonium and Cycloheximide on Chlorate Uptake. The effect of ammonium on chlorate accumulation was examined since it is widely recognized that ammonium prevents nitrate utilization (Buth et al., 1975; Heimer and Filner, 1971; Minotti et al., 1969a; and, Rao and Rains, 1976a). If nitrate and chlorate uptake share common uptake systems, then ammonium should affect both systems similarly. Cycloheximide was used to investigate a protein synthesis requirement or a high turnover rate of the carrier involved in chlorate uptake. Exponential-phase cells were washed and resuspended in 200 ml of N- less M1D to which 3fiCl-chlorate had been added. Five- m1 aliquots were harvested every 30 minutes, and the number of counts incorporated was measured. After 3 hours, two 50-ml batches of the cell suspension were removed. Ammonium succinate was added to one to a final concentration of 1 mM. Succinic acid was added together with ammonium because it has been shown previously that plant cells cannot grow on ammonia in the absence of an organic acid (Heimer and Filner, 1971; Ohta et al., 1981). A solution of cycloheximide to a final concentration of 20 pg/ml [sufficient to completely inhibit protein synthesis in XD cells (Heimer and Filner, 1971)] was added to the second batch. The remaining cells were used to continue measuring chlorate incorporation as a control. 5-ml aliquots were harvested from each one of the three cultures every 30 minutes, and chlorate accumulation was measured by liquid scintillation counting. Ammonium succinate does not inhibit chlorate accumulation in the cells but rather seems to have a stimulatory effect (Figure 15). It is well documented that ammonium uptake by either cultured plant cells or roots brings about a release of protons, increasing the acidity of the medium (Ohta 55 4o . ao- . ‘52 E : / "' zo- : - 3 v :/ 1'. ' °\.\ ° 0 ° 0 g 10- / .. ./O /o/ 04‘ 1 L o a 0 TIME (1:) Figure 15: Effect of ammonium and cycloheximide on chlorate uptake in XD cells. Arrow indicates time of addition of ammonium succinate (to 1 mMJID or cycloheximide (to 20 pg/ ml [0]). (O) = untreated control. 56 et al., 1981; Raven and Smith, 197 6). As discussed below, the optimum pH for chlorate uptake is 5.5. The medium used for this experiment had a pH of 6.2. Therefore, a decrease of the medium pH by ammonium incorporation into the cells could explain an increase in the rate of chlorate uptake. Cycloheximide inhibited chlorate uptake very rapidly, and no appreciable incorporation of label took place after its addition. It would appear that not only did cycloheximide inhibit 36C103' uptake but also allowed leakage of 36Cl-chlorate back into the medium. In a period of 3 hours, 35Cl-chlorate content decreased from 16 nmoles / g fr. wt. to 12.4 nmoles / g fr. wt. The high sensitivity of the system to cycloheximide is an indication that the carrier for chlorate may have a rapid rate of turnover. This is surprising since the chlorate uptake system is constitutive. However, there are reports in the literature (Jackson, 1978) of other effects of cycloheximide besides inhibiting protein synthesis. The drug may have inhibited the uptake system directly. It should be pointed out that manipulation of the cultures at 3 hours resulted in a chlorate uptake lag in all treatments, including the chlorate-only control. Chlorate Uptake in 14U Tobacco Cells. At this stage of the study, the 14U cell system became available, with growth rate characteristics on urea identical to the wild type XD cells on nitrate. The XD cell cultures grow 20% less rapidly on urea than they do on nitrate. The 14U cell line simplified procedures by obviating the need to eliminate nitrate contamination from the internal cell pool. The method of harvesting the cells was also changed from vacuum filtration to a more gentle one (gravity filtration) for reasons already described. The discovery that chlorate uptake has a sharp pH optimum at 5.5 and 57 the transport rate at pH 6.5 is one-fifth that at pH 5.5 (M. Guy, personal communication) made it desirable to repeat the chlorate uptake measurements using buffered incubation media at two different pHs: 5.5 and 6.5 (Figure 16). After 20 hours of uptake, cells incubated at pH 5.5 accumulated five times more chlorate (48 nmoles / g fr. wt.) than those incubated at pH 6.5 (9.4 nmoles/ g fr. wt.). Comparing those two values with the amount of chlorate per ml in the external medium (15 nmoles / ml), tobacco cells concentrated chlorate against a gradient at pH 5.5 but not at pH 6.5. It should also be noted that the plots of chlorate accumulation by 14U cells grown on urea do not show the lag phase seen with the XD cells grown on M1D. The possibility remains that intrinsic properties of the cell line, rather than the harvesting procedure, are responsible for the observed lag in chlorate uptake in the XD cells versus the 14U cells, where no lag was observed. The rate of chlorate uptake increases with increasing chlorate concentration, giving complex uptake kinetics (Figure 17). This indicates the existence of two systems of chlorate uptake: A saturable component functioning at concentrations below 1 mM and a linear one which accounts for net increases at concentrations above 1 mM. This linear portion of the curve could be the result of diffusion of chlorate into the cells such that the rate would be proportional to the external chlorate concentration. Another poissibility is the existence of a second potentially saturable transport system with low affinity for chlorate. Figure 18 shows the ratio of chlorate accumulated (values obtained from Figure 17) over the amount of chlorate in the external medium at different chlorate concentrations. At pH 5.5, those ratios remain above 1, supporting the previous observation that chlorate at pH 5.5 is taken up against a concentration gradient. On the other hand, at pH 6.5, this ratio is close to or 58 dd mm H AIV and In H AOV 50383850 39830 853:8 can 33:03.55 52.53 3:353:00 .3 85oz mSaumcfi on: 3:30 .mzmo D: 5 55558388 39820 .3 85.33% mm ”3 23mg .5 ma: 3 2 up a v e A . . . q q q . 0 ll.\. ll.‘ 0 #11 m 0 u. lIllllllllllllllllllllllllllllllo llllll C - -3 m. 9 u. m 1.9 O l a - -3 m. 0* é . . . . p . b p . . 59 dd mm u :3 and mm H ACV i=3 D: 5 .9339: Bane—no.3 convenes“... in "S 95mg 3.5.0.08“— 1m 1"” 3/ "78/013311” 3° 3.10"“ 60 .3 mm .19 as an n A! .38 D: E 383 5595538 .32ch 33:03.5“..."33:03.55 no 856533 in ”mg 026?; 25.0.05 o e u p «.o . a d d I + - J lllllllllllllllllllllllllllll V 101.. 11.1.4? m ll ill /0 a V / .I- I 3 .L / m 1 1a m... a 3 / .J 3 H 1 r 1 D O U V I. 3 r. m 1 1 N l- m P P b n n “ 61 below 1. This suggests that active uptake or cotransport of chlorate occurs at pH 5.5, but at pH 6.5, chlorate enters passively, perhaps by facilitated diffusion. Protons could conceivably play a role in the active process at pH 5.5. Inhibition of Chlorate Uptake by Nitrate in 14U Tobacco Cells. The pH dependence of inhibition of chlorate uptake by nitrate was examined in light of the above results. Nitrate concentrations ranged from 0 to 30 mM. Nitrate inhibited chlorate uptake at pH 5.5 (Figure 19) and 6.5 (Figure 20). The curved portion of each plot of v versus [S] becomes a straight line when the rate of chlorate uptake is measured in the presence of 15 mM nitrate. Apparently, 15 mM nitrate completely inhibits the saturable component of chlorate transport at chlorate concentrations below 1 mM (the high affinity system) but does not .inhibit transport at high chlorate concentrations (low affinity system or diffusion). The latter is evident from the parallel lines above 1 mM chlorate. Subtraction of the low affinity or the diffusion component from the v versus [5] curves in Figures 19 and 20 reveals the saturation kinetics of the high affinity system (see insets). From these plots, the Km and Vmax for chlorate uptake by the two systems at the two pHs were determined (see Table 4). Effect of Chloride Ion on Chlorate Uptake. To further analyze the discriminatory power of the chlorate uptake system, the effect of chloride on chlorate accumulation was examined. KCl at 15 mM was added to the incubation medium (urea-M1D) which already contained 2.5 mM KCl. The results at pH 5.5 are shown in Figure 21. Surprisingly, chloride dramatically inhibited chlorate incorporation into the cells. However, of greater interest is the observation that chloride inhibits the 62 .motflo :a SP... 83:23.8 33833.50: .3 553.595 88.. mas—5mm.— mozg 216:» €02: .28 ADV 3 N was .A‘V m .AIV 9m .AOV o H C57: .23“. D: 5 m6 an an 3.39. 39830.3 55335 fiahmz ”mg 0.233 Spa—”.908“— 0 Q n o a . m d I 1 1 ° M v \ F ‘ 0 «lllhlllq m m. v-IIIII-IIIII.\ C . .ll.I-III_II..\. a“ w I T a O . . - . - m .. m 19 I n. O .7 u. / a "4 - m w 63 .m0>.~=o :a 89c unecoafioo 03333-55 mo :omuoahazm 89a mama—5mm: moi—8 mkosm .2va .28 AOV 3 ES .3 m A! 3 .5 o n 20E .2? :3 5 3w ma 3 33% 382.3 a. 83325 3.32 am 2&5 3.5.905 0 V N P «.6 u u u u a \u . .H m m. \ O O . C q o m. 9 N r L p O u a u m . .4. fall...- u A m 1 .l fiDflO‘I-‘W‘b 8. m I N 64 .6: SE a: + .82 o u 3: 5x 28 3 + .82 SE B m 3 5x 25 3 + .52 a u E £8 a: 3 8.3% -85 2 -83 3am ”a 2%; 3.5.28: ....... :W\ \\ d - “3"” 'I‘I'Z/CI'JOIUO 1° .OIOW" 65 linear component of chlorate uptake, but not the high-affinity system. This is precisely opposite the effect that nitrate has on the two components of chlorate uptake. Chloride inhibition at high chlorate concentrations cannot be explained by a diffusion process. Therefore, the linear component of chlorate uptake appears to be an uptake system with low affinity for chlorate. Since chloride inhibits chlorate uptake and N -less M1D medium contains 2.5 mM chloride, the previous values obtained for chlorate uptake in this N-less M1D medium may represent an inhibited transport system. In order to correct for this possible error, chlorate uptake was measured in cells incubated in a chloride-free nitrogen-free medium (Cl-less N-less M1D) to which urea, chlorate and chloride were added. Indeed, chlorate transport by the low affinity system was enhanced at both pHs 5.5 and 6.5 (Figures 22 and 23). In the experiment plotted in Figure 22, all CaCl2 and ROI were simply omitted from the medium. However, in the following experiment (Figure 23), the missing cations were returned to the medium in the form of CaSO4 and K2HPO4. This nutritional alteration was probably responsible for the differences in uptake rates between similar treatments in the two experiments. In the absence of chloride, there was a four-fold reduction in the pH sensitivity of chlorate uptake (Figures 22 and 23). To determine whether the K01 effect (Figures 21, 22, and 23) was due to chloride or to the counterion K+, the inhibitions of chlorate transport by the K+ and N a+ salts of chloride were compared. No differences were observed in the degree of inhibition caused by the two salts at either pH 5.5 or 6.5 (Figure 24). Therefore, chloride, not the counterion K + is responsible for the inhibition. The sensitivity of the chlorate uptake system was examined further by 66 .3 as now 28 3 n :3 "3 ma .SM 28 c n A! as E .SM 28 3 u 8. as an .Bx c u A e V 2.8 a: 5 2.3% -86 8 -8 23 mg as 38% 3:358 ”mm 2:5 a... 128: mm; Emu/cumin: 1° mound 67 as E .BM 28 o u 2: as E. .SM 25 m u 2: as an 40M c u E as as .63 c u A 3 £8 a: 5 83% -86 8 ma 23 -8 a 58....» 3:388 an 2&3 258.3: a v - . «6 u I u 1 ¢ a 4\Mi\ T\\\-\\\\\-\\ m .- o\\ n m o m. \ .\ I”. ~\ m o m. .. no. n O u w w .w _ m.- H*\ u 1 has 68 .62 25 a u a: gum :5 m m A! sum o u 5 aa an 5. £8 a: 5 2.3% -86 a 8:325 -6 8 8:358... $5 an 2:3 a... 129: o v u p «.6 u q d d u a a... n u.\ \h\\ .u ‘I‘... m ‘0‘“‘I d. o |‘.I"‘||.| “ . m ‘m“““ w 9 N . i. m I o u / z .7 u. / a . w. 1 .. 0— \~ . b L p - 69 evaluating the effect of perchlorate on chlorate transport. Guy (personal communication) had shown that the kinetic characteristics of perchlorate uptake wereidentical to those of chlorate uptake in 14U cells. The results shown in Figure 25 indicated that the high-affinity component of chlorate uptake was inhibited by perchlorate at both pHs 5.5 and 6.5, suggesting an interaction between the two ions at the uptake level. The results presented thus far suggest that chloride inhibits chlorate uptake at the chlorate low-affinity transport system. An experiment was designed to determine the nature of the inhibition. Chlorate uptake was measured as a function of increasing chloride concentrations (3 to 9 mM KCl) (Figure 26). Three millimolar KCl, the lowest concentration tested, completely inhibited the chlorate low affinity transport system, suggesting either that the affinity of the chlorate low affinity transport system for chloride is very high or that chloride inhibits chlorate uptake by a mechanism other than competitive inhibition. In order to determine whether the previously observed inhibition of chlorate transport had been influenced by the presence of chloride in the medium, the effect of two nitrate concentrations (2.5 and 15 mM) on chlorate uptake was measured in the new Cl-less N-less M1D medium. As in previous ' experiments, nitrate inhibited chlorate uptake (Figure 27). With nitrate at 15 mM, the high affinity system for C103' transport was completely inhibited, and a linear rate of chlorate uptake was observed throughout the range of chlorate concentrations, with the slope being 30% lower at 15 mM NO3‘ than at O or 2.5 mM. This means that N03- can inhibit the low affinity system too, but rather weakly. 70 aa mg .06 28 a n :3 aa as £06 2.: a u 3V aa ma n56 c u 3 aa ma $.08 c n A! .2258: had Samoa ma? LO oz 38 a: 5 3.3% -86 8 -66 a swam 3.5.0.8: 0 ' N p «.0 q u u u q O a\ a \\\\I\ o m ‘9‘ m. \\\\ C ” \\\ .\ ” \\\\ C m F \%\\ 1 GP I0- \ u \ C \\ / \\\ a \\ a ' \\.—. a m... u * . ON - p n n n - 6a 932% 71 SM 28 m N 3 8a a u 5 .88 a: .3 33% -85 8 no.8 swam am 2:3,.“ V 3.38.3: b «N “'11 5/“78/ '1'30N9 5° OOIOtIllI 72 .mOZM SE 3 H A5 m”02% 28 Wm H AOV 82x c u A a V .-6 .3 833a 2:. 5 £8 a: 5 33% -86 8 -82 a spam am 2&6 -O V 1.58.03 .1 \‘ .P \ \ fi I \ 1"” 5/‘1'8/ 0‘31“!” 3° would P I I 73 Characteristics of Chloride Uptake System. . The interaction of chloride and chlorate described above raised the question of whether they share a common (low affinity) transport system. In contrast to chlorate uptake, chloride transport at pHs 5.5 and 6.5 displays saturation kinetics.(Figure 28). The double reciprocal plot (Figure 29) for chloride transport suggests the existence of two uptake systems. Chlorate at 6 mM lowered the Vmax of chloride uptake at both pHs, though proportionally more at pH 5.5 (Figure 28). Since chloride transport is relatively insensitive to pH, the greater inhibition of chloride uptake by chlorate at pH 5.5 than at 6.5 can be accounted for by the pH dependence of chlorate transport. Chlorate uptake at pH 5.5 was 4 times that at pH 6.5 in the presence of 2.5 mM chloride (Figure 22) and chlorate was 3.2 times more effective as an inhibitor of chloride uptake (Figure 28) under conditions in which chlorate uptake would have been proceeding 4 times more rapidly (Figure 22). In addition, a comparison between the double reciprocal plots (Figure 29) of chloride transport in the presence and absence of chlorate, suggests that chlorate preferentially inhibits a low-affinity chloride uptake system. That is, at low 1/[S], the + and - chlorate plots converge, but are parallel at high l/[S]. ‘ Therefore, it appears that chloride is taken up via separate high and low- affinity systems. Only the low affinity transport system is inhibited by chlorate. The type of inhibition is unclear, but seems not to be competitive (Figure 29). Table 4 summarizes the properties of chlorate and chloride transport systems. 74 aa man86 25m u :3 aa 6. .-86 c u A! aa ma . 8628a u A9 aa mg .-86 A. u A o C .88 D: 5 2.3% -6 8 ma 3:. 382% a team an 2&6 3.323: a v u p «.o - cl 1 u d 1 g u m n o a. u - m u I !|\\W\s 4;: m II"||I°r'IInI-Ilu|u-Inlnln \ " glau||||llu N n n. W I\\ W ‘ IIIIIII “"'|‘-"|--|-' ‘ “ III-lil- .— lll \ 111 H .GN - p . p n . ”r I r r 130 g“ 1/v(umolu 0‘! Cl /24|I/a I'r.wt.)"‘x10a u l L L L 0.17 1 2 5 1/[KCIJMM Figure 29: Lineweaver-Burk analysis of effect of pH and C103‘ on C1’ uptake in 14U cells. (D) = 6 mM 0103', pH 5.5; (I ) = 0 ClOa', pH 5.5; (0)6 mM 0103’. pH 6.5; (O) = 0 C103‘, pH 6.5. 76 on. on no» no» - - tog—mag... LU mo -moa 3 53335 - - mo.» 3% o: o: .. 98.3995 -noa no LO .6 5:323 .- - 336 33?. mo» 8.» 983:9: -mOAU .6 3953 3 55325 38 3.: 38 5.: - - . 9158M -5 3A: «.2 3.6 3: - - . AAA $335 3-6 22: 1:88 > -6 - - m A A mA A and and 32:: EM -mOAO - - mad :3 8A. awn A: $33.... 3.86 28. 3 23> -86 3 mg aa ma 3 ma aa EA 3 ma ma mg a 832nm fl 539$ HEBmhm $539? 983:9: 63.836 v.3 39836 .6 83.835 .v 63am. PART-II: REDUCTION OF CHLORATE BY NITRATE REDUCTASE MATERIALS AND METHODS Preparation of Enzyme Extracts Cells were harvested by vacuum filtration onto Whatman No. 1 filter paper, rinsed with deionized distilled water and weighed. The cells were resuspended in a phosphate buffer consisting of 0.1 M K2HPO4 pH 7.5, 1 mM EGTA, 1 pM L-cysteine free base (Trinity, 1981). Five milliliters of the buffer were used per gram of cells. In experiments employing urea, the concentration of L-cysteine was increased to 5 mM. The cells were homogenized at 4° C with 30 strokes of a motor driven teflon-glass homogenizer. The soluble enzymes were separated from cell debris by centrifugation at 12,000 x g in a Sorvall SS-34 rotor for 20 minutes at 4°C. Nitrate reductase was precipitated from the supernatant solution by 1 hour incubation in 50% saturated ammonium sulfate pH‘ 7.5, followed by centrifugation at 12,000 x g for 20 minutes at 4°C. The pellet was resuspended in a volume of phosphate buffer (described above) equal to that to which ammonium sulfate had been added in the earlier step. Low molecular weight substances were removed from the extract by centrifugation of 2 ml aliquots through 4 ml columns of Sephadex G-25-80 resin (particle size: 20-80p) in a 5 ml disposable plastic syringe. The centrifugation was performed at 3400 rpm for 15 seconds in a clinical centrifuge at 4°C, with each syringe hanging in a 15ml conical centrifuge tube, in a swinging bucket rotor. 77 78 Determination of Nitrate and Chlorate Reductase Activities Nitrate reductase activity was measured as the rate of nitrite formation at 25°C in the presence of 0.1 ml 0.1 M KNO3, 0.1 ml 1 mM NADH and 0.5 ml 0.1 M Kzl-IPO4 buffer pH 7.5. Final volume of 1 ml was reached by addition of 0.2 ml of enzyme extract and 0.1 ml of water (Wray and Filner, 1970). The latter was substituted by 0.1 ml of different chlorate solutions when the effect of chlorate on nitrate reduction was examined. The amount of nitrite formed after 30 minutes of incubation was determined colorimetrically as described above (pages 25 - 26). A reaction mixture stopped at zero time was used as the blank. Chlorate reductase was followed as the rate of 36Cl-chlorite and 36Cl- chloride formation at 25°C in the presence of 50 pl of approximately 2.6 mM 3GCl-chlorate and 50 pl of 1 mM N ADI-I. A final volume of 0.5 ml was attained by the addition of 0.4 ml of enzyme extract. When the effect of nitrate on chlorate reduction was examined, the volume of each of the above components was tripled and 50 pl of different nitrate solutions added. The reaction was stopped after 30 minutes of incubation as described below. Determination of 36Cl-Chlorite and 36Cl-Chloride 36C1-chlorite produced by chlorate reduction was chemically reduced to 36Cl-chloride by the addition of 0.5 ml 100 mM KI and 0.5 ml 4 M acetic acid to 0.5 ml assay mixtures (Goks¢yr, 1952). The chloride produced was precipitated with unlabeled carrier chloride by the addition of 0.5 ml of 20 mM NaCl (as carrier) and 3 ml of 100 mM Ag(NO3)2, thereby forming insoluble AgCl. In parallel tubes, enzymatically reaped 36Cl-chloride was precipitated by the addition of 0.5 ml 20 mM NaCl, 1 ml of H20 and 3 ml of 100 mM Ag(N03)2. The silver chloride precipitates were collected by centrifugation at 79 12,000 x g for 10 minutes, washed with 5 ml of water, and recentrifuged. The 36C1' in the precipitate was solubilized by addition of 1 m1 of 1 M NaCN, thereby forming soluble NaCl and AgCN. Scintillations were counted in 5 ml of the scintillation cocktail described by Trombolla (1970). 36Cl-chlorite was determined by the difference in counts between the chloride specific precipitate and the chlorite and chloride precipitate. Determination of Total Protein Total protein was determined by the methOd of Lowry et al (1951). A 1- ml aliquot of the enzyme extract was precipitated with an equal volume of 20% (w/v) trichloroacetic acid. After 2 hours at room temperature, the precipitate was collected by centrifugation and rinsed twice with 95% ethanol. After drying under nitrogen gas, the precipitate was dissolved in 1 N NaOH and assayed by the Lowry procedure. Bovine serum albumin was used as a standard. RESULTS Inhibition of Cell Growth by Chlorate According to Aberg’s hypothesis, chlorate is rendered toxic upon its reduction to chlorite by the enzyme nitrate reductase. While it is true that this hypothesis has been very useful in providing a rationale for the successful selection of nitrate-reductase-less mutants of bacteria, fungi, algae and higher plants based on chlorate resistance, the actual mechanism of chlorate action 80 has not been completely established. Prior to this work, it has not been proven that nitrate reductase of higher plants catalyzes the reduction of ClO3'. The XD and 14U cell lines, together with 36Cl-chlorate, provided a suitable system in which to examine details of the mechanism of chlorate toxicity proposed by Aberg (1947). According to the hypothesis, XD cells growing on a medium with nitrate as the sole nitrogen source and containing high levels of nitrate reductase should be more sensitive to chlorate than the 14U cells growing on a medium with urea as the sole nitrogen source and containing much lower levels of nitrate reductase. To our additional advantage, these two cell lives have equal growth rates in their respective media, greatly simplifying comparisons of their growth rates under different conditions. The toxic effect of chlorate on the XD and 14U cells was determined by measuring the inhibition of cell growth in the following way. Growth tests were begun by diluting 25 ml of stationary-phase cultures into 225 ml of M1D or urea-M1D medium containing KC103 at concentrations ranging from 0 to 100 mM. The pH of both media, M1D and urea-M1D,had been adjusted to 6.2. Since the results of our uptake experiments established a pH optimum for chlorate uptake of 5.5, an additional series of XD cultures was prepared in a medium of pH 5.5. No buffers were added to maintain pH during the period of the growth test. The cultures were kept on a reciprocal shaker in the dark at 28°C. Periodically the cells from 2 flasks from each treatment were harvested by vacuum filtration and their fresh weights determined. The pH of the medium remaining after harvesting the cells was measured in some instances. The XD cells growing on a medium containing nitrate at pH 5.5 and 6.2 were more sensitive to chlorate than were the 14U cells grown on urea at pH 6.2 (Figures 30 A, B, C and 31). The I50 values deduced from the dose response curves of Figure 31 were 54 mM for the 14U cells and 16 and 17.5 mM for the 81 40A v r GROWTH (a InwL/Il GROWTH (a flaw! ll) d o \ _ GROWTH (a “mt/ll a n O O 0 \\ 0 3 O 1 0 1 4 TIME “I!“ Figure 30: Effect of chlorate on growth of (A) 14U cells in urea-M1D, pH 6.2; (B) XD cells in M1D, pH 6.2; (C) XD cells in M1D, pH 5.5. [KClOa] = 0(0), 1 (O), 3 (A). 10 (A). 30 (D), 100 (I) mM. 82 XD cells at pH 5.5 and 6.2, respectively. Chlorate seems to be slightly more toxic to the XD cells at pH 5.5 than at pH 6.2. This pH effect might have been more prominent had the cultures been grown in buffered media. The pH of the M1D medium increased with the age cf the culture (Table 5) shifting chlorate uptake to a rate characteristic of cells grown under less acidic conditions. The difference in sensitivity to chlorate between the XD and 14U cells lines may have been overestimated. The fresh weight of the initial inoculum was 2 g/l for the 14U cells and 1.2 g/l in the case of the XD cells. As will be seen below chlorate toxicity seems to depend upon the size of the initial inoculum. Another characteristic of the response of a tobacco cell culture to chlorate is the culture’s eventual recovery from growth inhibition. Cultures in the 100 mM and 30 mM treatments in figures 30A and 30B, respectively, attained growth rates similar to that of the control after a 6-day lag. This recovery phenomenon becomes more prominent in experiments described below. Chlorate Reduction by Nitrate Reductase of Tobacco Cells The reduction of chlorate by NR was indicated by chlorate-dependent N ADH oxidation catalyzed by extracts of the alga Chlorella‘I (Solomonson and Vennesland, 1972) or tomato plants which contain NR activity (Hofstra, ‘ 1977). In this laboratory, enzyme-catalyzed reduction of 360103' has been measured directly by following 360102' + 3601' formation after reduction of 3(SCI-chlorate by partially purified NR from the algae Chlamydomonas and Chlorella (Rhodes and Filner, 197 9). In the present study, the latter approach was used to demonstrate chlorate reduction by NR of tobacco cells. Previous attempts to apply a colorimetric assay for chloriteformation to the tobacco cell system had been unsuccessful (P. Filner, personal communication). A desalted enzyme extract from exponential-phase XD cells had a NR 83 ad ad ad ad ad ad dd dd ad «ad dd vd vd wd pd ad ad dd 3 ad ad od dd dd ad ed dd ad ad dd ad ad ad ad ad ad pd 3 ad ad ad ad ad ad ed ad ad ad «d ad ad ad ad vd vd dd c 333$ :3 332% a: aaaxaeox :3 333$ a: 3323 a: eaaxaaax a: 2: on S m z c 3.33 SE 323: pea. as me .32 E 5:5 £8 ax n 33 ”we we .52 5 :38» 2.8 ON H «dQN ”ad in £32.35 E 559% m=mo D: H D: .5338 95:3 .3 mm :o 398?? do .5on "m wish. 84 .3 ma .9: 5 ax u 3. as E. dds 5 ax u 8V as we .9253 5 DE H AIV .532m 231.8 :8 GM and D: .8 east» 398—go do 323 3:38..-»ch 3m 95mg 351205 co. on o. a p - 1 d :a --—---- --’ 'IOHJJIOO =IO % ELVU HLMOUS 85 activity of 31 nmoles / min / g fr. wt. and catalyzed chlorate reduction with an initial rate of 0.75 nmoles / min / g fr. wt. This rate decreased rapidly during the assay (Figure 32). This could have been due 1) to progressive depletion of substrate [Km (C103‘) = 3.5 :t 1.7mM; Table 12] or 2) inhibition of NR by chlorite. Indeed, NR inhibition by chlorite has been previously demonstrated in Chlorella (Solomonson and Vennesland, 1972). However, upon analysis of radioactive label in silver chloride precipitates before and after conversion of chlorite to chloride by chemical reduction with KI in acetic acid, no evidence for the formation of 35Cl-chlorite was found. All the radioactivity was found to be 3GCl-chloride (Table 6). On the assumption that chlorite could be converted to chloride via an enzymatic reduction and that, as an analog of chlorite, nitrite could compete in this reaction, we determined whether nitrite could inhibit chlorite reduction, therefore allowing the detection of chlorite. From previous experiments it had been determined that the maximum amount of chlorite which could be formed by chlorate reduction under our assay conditions was 2.86 pM. Nitrite was added to the assay mixture at concentrations ranging from 0 to 50 pM. After 30 minutes of incubation, the radioactivity in the silver chloride precipitates before and after conversion of chlorite to chloride showed that nitrite did not have any effect On the recovery of counts as chlorite (Table 7). Alternatively, it seemed possible that the origin of chloride in these assays is via the non- enzymatic reduction of the powerful oxidant chlorite (Rhodes and Filner, 1979). To evaluate the generality of this phenomenon, chlorite formation upon reduction of chlorate by crude extracts from soybean nodule bacteroids was measured. Soybean nodule bacteroids are a source of abundant and easily accessible nitrate reductase (Lowe and Hamilton, 1967). 21% of the initial 86 ] I l l f 2-4- . _...- J 3 o 5 , g 18- - '5 o o a a 2 .3 12- ’ . h 2 .c o '5 o s .- - o E c 0| 1 l l l 1 O 20 4O 60 80 100 TlMEhnln) Figure 32: Time course of chlorate reduction by extracts of XD cells. 87 Table 6: Substrate dependence of chlorate reduction by XD cell extracts. Assay mixture consisted of 292 M 360103' (73,088 cpm), 0.1 mM NADH and 0.4 ml-enzyme extract. Assay Mixture Incubation C1' + C102' Cl‘ Time (min) (cpm) (cpm) Complete 0 141 ND. Complete 90 1054 1 1 14 -NADH 90 118 i N .D. -Enzyme 90 ND. 89 88 Table 7: Effect of nitrite on recovery of chlorite from chlorate reduction products of XD cell extracts. [N aN 02] C1' + ClOz' , Cl' pM (cpm) (cpm) 0 726 739 1.5 666 - 730 3.0 670 695 10.0 716 667 25.0 - 635 604 50.0 590 544 Assay mixture: 283 1.1M 360103' M (77957 cpm) 0.1 mM NADH 0.4 ml enzyme extract 50 pl NaNOZ (0, 16.5, 33, 110, 275, 550 pM) Incubation time: 30 minutes. 89 3GCl-chlorate was reduced after a period‘of 30 minutes. 19% of that 21% was recovered as 36Cl-chlorite (Table 8). Table 8: Chlorate Reduction by Bacteroid Nitrate Reductase Assay Mixture Incubation ClOg‘ + C1‘ C1' 0102' Time (min) (cpm) (cpm) (cpm) Complete 30 16,424 13,314 3110 Assay mixture: 305 pM 360103' (76,347 cpm) 0 .05 M potassium succinate 0.1 ml crude soybean bacteroid extract ~ In the results obtained with the tobacco cell extracts, only 1.4% (1054 cpm / 73,088 cpm x 100%) of the initial chlorate present was reduced. Using the bacteroid data as a guide, 19% of this reduction product would mean a recovery of only 200 cpm, too small an amount to be detected accurately in the assay used. No chlorate reduction activity was detectable in a desalted enzyme extract prepared from exponential-phase XD cells growing in urea M1D with a NR activity of only 46 pmoles / min / g fr. wt. (Table 9). These results support the view that NR is directly involved in chlorate reduction. 90 Table 9: Chlorate Reduction by NR from Urea and Nitrate Grown XD Cells N -Source 0103' Reduction (cme h / g fr. wt.) Urea 0 KN 03 ‘ 795 Assay Mixture: 300 pM 36C103' (75,000 cpm) 0.01 mM NADH 0.4 ml enzyme extract Kinetic Characteristics of Chlorate and Nitrate Reduction By N itrate- Reductase Preparations To measure the kinetic parameters of chlorate and nitrate reduction as well as their mutual inhibitory effects, it was desirable for comparative purposes to utilize for each measurement samples taken from the same ' enzyme preparation. Crude extracts containing NR can be stored at -20°C in the modified buffer (see MATERIALS AND METHODS) for up to two weeks without loss of NR activity. In Table 10, the changes with time in storage of the enzyme activity of a crude extract preparation are shown. These data indicate not only the maintenance of activity but indeed a slight increase (P. Trinity, 1981). Crude extracts still retained trace amounts of nitrate after the 50% saturated ammonium sulfate precipitation step. Desalting of the enzyme 91 preparation was required to avoid such contamination. As indicated in Table 10, the enzyme ativity was enhanced by passage through a G-25 Sephadex column. In general, the total protein recovery from a 4-ml G-25 Sephadex column is 60% (Yamaya and Filner, 1981). It has been shown that NR in the KB cells is present in an active and inactive form and that the ratio between the two forms changes during the cell’s growth cycle (Trinity, 1981). The data in Table 10 suggest that a low molecular-weight inhibitor of NR, present in crude extracts, is removed by Sephadex G-25 chromatography. The possibility of co—extraction of an inhibitor is supported by. a further observation. In one experiment, the enzyme extract was concentrated two-fold to increase its activity. When nitrate reduction by the 2X extract was measured under saturating substrate conditions, a plateau was reached after 15 minutes (Figure 33A). The enzyme activity in unconcentrated extracts is linear up to 40 minutes. In a similar experiment, the specific activity of NR was shown to decrease with increasing concentrations, of enzyme in the assay mixture (Figure 33B). The desalted extract did not show such a trend (Figure 33A and 333). The Km’s and Vmax’s of chlorate and nitrate reduction were calculated several times using fresh and stored desalted extracts from exponential-phase XD cells. To measure the kinetics of nitrate reduction, KN03 was used as the substrate at concentrations ranging from 0.02 to 0.2 mM. To determine the Km and Vmax of chlorate reduction, 36Cl-chlorate was added to the assay mixture to a final concentration of 0.3 mM. Unlabelled K0103 was added to the reaction mixture to final concentrations ranging from 0.3 to 2.0 mM. Corresponding 36Cl-chlorate specific activities ranged from 500,000 to 67,525 cpm/pmole. Tobacco nitrate reductase has a higher affinity for nitrate (Km values 92 Table 10: Stability of NR extracted from 14U cells. Assay mixture consisted of 0.1 ml 0.1 M KNO3, 0.1 m1 1 mM NADH, 0.5 ml 0.1 M K2HPO4 pH 7.5, 0.2 m1 enzyme extract, 0.1 ml H20. Crude extracts were stored at -20°C. Time in storage Enzyme act. crude ext. Enzyme act. desalted (days) (nmole/min/g fr.wt.) ext. (nmol/min/g fr.wt.) 0 66 72 1 72 76 2 72 82 3 71 79 4 69 78 5 7o 78 .33»: 3:33 um H A3 was .3955 $5.8 xw u A! .8955 $5.8 5 u ACV 6:8 QN 89c 3353 33053.. 3953 .3 851536 55655650 av 6:8 ON mo 33:38 .3 .8326?— 3653 .«o 8:353“. 25B 2. "mm 0.33% 93 3:5 30sz 0.5.5 .55 mi..— oo av on o «.o .6 no... 3... _ . . 4 o u - u u \ m y .o« w u o u m m r m. u . m V . 10' . o o 4 / .. m u 8 u u. . .3 m w. m \. o O O m x m. . .8 m 9 u .. o. .. dlld\¢ m . < Loo— . b p b p - 94 - - - - mm . mud 63 :6 a.“ as mm as and 3 66 m6 S 2... 86 Q: .3 3 «.6 mad - - - - mm . was - - - 66 3. ad - 2: c6 - - - 3.5 33.... 63585 A25 35 33.6 «Basses 9:5 20236 use> am @229 58> :2 22.5368 5.838 zoabanmm 69$; 6:25.89"... 3:38:63: :6 $8838 38 86W 6:8 Gun .3 833:8 38°36 .23 38:: mo 836883 afloag a: 056:. 95 ranged from 0.18 to 0.28 mM) than for chlorate (Km values ranged from 2.3 to 6 mM) (Table 11). Determination of Km’s for chlorate reduction was difficult because the chlorate concentrations used to determine the Km values were below the Km. An example of the double recriprocal plot from which the Km and Vmax values of chlorate reduction were calculated is shown in Figure 34. The Km values for nitrate and chlorate fall in the same range as data found for tomato plants, green algae and several bacteria (Table 12). Inhibition by Chlorate of Nitrate Reduction We have shown so far that tobacco NR preparations reduce nitrate and chlorate anions. To determine whether or not these two substrates compete for the same enzyme, the effects of chlorate on nitrate reduction and nitrate on chlorate reduction were examined. The inhibition of nitrate reduction was first analyzed by incorporating KN 03 at 0.08 to 0.4 mM and K0103 from 0 to 16 mM in the assay mixture. The reciprocal of the reaction velocity was then plotted against the reciprocal of nitrate concentration for each KC103 concentration. As the concentration of chlorate was increased in the assay, there was an increase in the Km for nitrate reduction (Figure 35). These results indicated that chlorate competitively inhibited nitrate reduction. The ' Ki value of this inhibition was determined in several experiments and ranged from 5 to 8.9 mM. These values are in the same range as the higher Km values obtained for chlorate reduction as expected for a competitive inhibitor. On this basis, the low Km 0103‘ values of 2.3 (Table 10) might be considered erroneous. An example of a Dixon plot from which these Ki values were generated is shown in Figure 36. _ 96 l l I l I C 20 d '0 o F “I L C an: 3. = 3 s 10" " E /- '0 9 o 0 C 2 Q E 3 , \ P 1 L n I -o.1e7 1 2 3 4 Figure 34: Lineweaver-Burk plot of chlorate reduction 'by XD. cell extracts. 82 58205 3 m3, :8 2%....»st :2: 339.285 $4 3.: «stowage: Loflooeogoax $3 .bochE m9: 3.: «233:3»: ”3889.32 :23 $3555 . $4. 3.: 398355: muzofiovzomm 7 «SH insight, vac :omaoESom «A M5:: atom}: 329830 9 2.3 con—E can 3:23 :.m. - oaofimozunaq 33830 2.3 .353 cam 8:95 «a: - 3:98:52 358823.830 2.3 .ahmuom :.v 3.: mag”: 8:33:93 coommcomooad Q0639 202:5: 8325 $320qu 838% £523, :o 33:33.. 398.5 EB 33:: .8: 353239 mzoasoflz "Na «Bah. 98 1/v(nmoles of "Ga/mulls fr.wt.)-‘x‘lm 27 ' 0‘? 01 15 P d o 0;. o "w 0 5 1O 1/[KN033mM Figure 35: Lineweaver-Burk plot of inhibition by chlorate of nitrate reduction in XD cells. Intersection of lines on ordinate is indicative of competitive inhibition. [K0103] = 0 (A), 2 (D), 4 (I), 8 (O) and 16 (0) mM. 99 a O I L 15- / - 1/v(nmom at Magnum/g tr.wt.)-'ix1o= p r b b Figure 36: Dixon plot of chlorate inhibition of nitrate reduction in XD cells. Ki = 5 mM KC103. [KN03] = 0.08 (D), 0.2 (I), and 0.4 (0) mM. 100 Inhibition by Nitrate of Chlorate Reduction The effect of nitrate on chlorate reduction was analyzed by supplementing the assay mixture with 500 pM 36C103', 4, 8 and 16 mM unlabelled chlorate and O to 0.4 mM KN03 (final concentrations). As in the case of chlorate inhibition of nitrate reduction, nitrate competitively inhibited chlorate reduction. The Ki values obtained for inhibition by nitrate were 0.13 and 0.2 mM, very close to the Km values for reduction of nitrate (see Table 11). An example of a Dixon plot of these data is shown in Figure 37. Induction of Nitrate Reductase by Nitrate And Chlorate Hofstra (1977 ) attempted, without success, to study the induction of nitrate reductase by chlorate using Lycopersicon esculentum leaf discs. Hackenthal et. al (1965) examined the induction of NR by a series of organic anions structurally related to nitrate in Bacillus cereus. Among the anions tested, perchlorate but not chlorate induced the enzyme. In our attempt to evaluate the use of chlorate as an analog of nitrate for metabolic studies, we made use once again of the 14U tobacco cells growing in" urea M1D to re- examine the induction of NR by chlorate. The cells from 3 one-liter flasks containing 500 mls of a 5 day old culture in urea-M1D were harvested and transferred to 3 other flasks containing 500 ml of fresh urea M1D medium. KNO3 and K0103 were added to flasks number 1 and 2 respectively to give final concentrations of 6 mM. The cells from the third flask were untreated controls. Starting at time 0, 100 ml of the cell suspension was removed from each culture every 2 hours. The cells were harvested and crude enzyme extracts were assayed for nitrate reductase activity. The results summarized in Table 13 show an induction of NR activity 101 - l I I r I. J 3. a 96° '- cl 0 \ '5 I E D D. Q 0 640 - - '5 . 3 o E u’ 3 l- .J/ 4 z I“ P ' .__ 4...... J..— : [ , 1' g n 4 1 n O 0.2 0.4 Figure 37: Dixon plot of nitrate inhibition of chlorate reduction in XD cells. Ki = 0.2 mMKNO3. [K0103] = 4(0 ), 8(I) and 16(0) mM. 102 Table 13: Induction of NR activity in 14U cells by nitrate, chlorate and urea. 6 mM KN03 and 6 mM K0103 were added in their respective treatments to basal urea-M1D medium employed throughout. NITRATE REDUCTASE ACTIVITY Time KN 03 K0103 UREA (h) nmoles/h/g fr wt. nmoles/h/g fr.wt. nmoles/h/gfw O 10 0 22 2 179 48 ,. a 132 . 4 287 O 8 6 410 1 1 0 8 315 27 54 103 by nitrate but not by chlorate or urea. The NR activity values obtained after an induction period of 6 hours in the case of nitrate are low (410 nmoles / h / g fr. wt.) compared to those reported by Heimer and Filner (1971) (1,000 nmoles / h / g fr. wt.). This difference and the irregular values of enzyme activity obtained from control cells and chlorate treatment could be explained by the presence of phenolics in the crude extracts. In this experiment, the concentration of cysteine free base used in the extraction buffer was the same as the one used to prepare crude extracts from XD cells growing in M1D medium. The amount of phenolics seems to be higher in the tobacco cells grown in urea judging from the color of the extracts. The discrepancy between our results and those of Heimer and Filner could also be due to a difference in cell type. For their induction studies, XD cells were grown in urea-M1D for a short period of time (5 days). However, in our experiments we used the 14U cells, that had been maintained on urea for over one hundred generations. To reconcile the old and current data, we measured the growth and NR induction response of 14U cells growing in urea-M1D and urea-M1D + 2.5 mM nitrate, at a range of chlorate concentrations. Such an experiment should provide an estimate of the degree of chlorate sensitivity“ as a function of induced NR enzyme activity. Ten milliliters of stationary phase 14U cells were subscultured into two series of 500 ml flasks containing 200 ml urea-M1D medium and K0103 at concentrations ranging from 0 to 100 mM. To one of the series, KN03 was added to a final concentration of 2.5 mM. Two flasks per treatment were harvested 4, 6, 8 and 12 days after the start of the experiment and the fresh weights determined. The levels of NR were measured at days 0, 4, 6, 8 and 12 for the'O and 3 mM chlorate treatments, as indicated in Table 14. The results of the growth tests are shown in Figures 38 and 39. Chlorate 104 Table 14: Effect of medium nitrogen source, chlorate and stage of culture cycle on nitrate reductase activity extractable from 14U cells. NR specific activities are expresed in terms of nmoles N02' g fr. wt. of tissue, above, and in terms of nmoles N 027mg protein, below. NIRATE REDUCTASE ACTIVITY [K0103] TIME (days) Medium (mM) 0 4 A 6 12 U-MlD 0 15 i 3 39 .t 16 30 i 13 27 i 11 3 t 2 U-MlD 3 15 i 3 45 i- 17 45 : 10 36 i: 12 1 t 1 U—M1D+N03‘ 0 15 i 3 1,105 i 501 279 i- 113 91 j: 35 49 t 6 U-M1D+N03‘ 3 15 t 3 771 .t 111 218 i 78 65 t 8 22 4.- 7 nmoles/ g fr.wt./h NIRATE REDUCTASE ACTIVITY [K0103] TIME (days) Medium (mM) 0 4 6 8 12 U-MlD 0 3:1 4.522 4:2 6:3 0.9 :06 U-MlD 3 3 x 1 4.5 i 2 4.5 i 2 6 i 2.5 0.3 t 0.4 U-M1D+N03‘ 0 3 a: 1 119 j: 24 40 2t 13 24 1: 9 17.5 t 2.3 U-M1D+N03' 3 3 i 1 71 i 17 25 i 7 12 1: 1 7 i 3 nmoles/mg prot/h 105 100 f . 10+- - GROWTH (g fr.thl) pon\ \ 0.1 fl 0 4 8 1 2 TIME (days) Figure 38: Effect of chlorate on growth of 14U cells in urea-M1D. [K0103] = 0 (O), 1 (A). 3 (I), 10 (O). 30 (A). and 100 (0) mM. ‘106 1oo ' - T / 4 ,Z/ . °\._ 0.1 1 . an , o 4 a 12 TIME (days) onowm (g tr.wi./I) Figure 39: Effect of chlorate on growth of 14U cells in urea-M1D + 2.5mMKN03. [K0103] = 0 (O). 1(A). 3 (I) . 10(0), 30 (A) and 100 (D)mM. 107 imposed a lag, after which the cells grew at rates similar to those of the controls. When the initial growth rate, expressed as a percent of the control rate, is plotted against K0103 concentrations (Figure 40), a slight increase in sensitivity to chlorate is observed when nitrate is present in the medium. This increase corresponds to increased NR activity in these cells (Table 14). The relationship is reversed however, when the log phase growth rates, rather than initial rates, are used (Figure 41). In addition, if growth is measured as total fresh weight accumulation after 8 days, the urea and nitrate-grown cells once again appear more tolerant of chlorate (Figure 42). The control cells in this experiment (14U cells growing in urea-M1D) appeared to be more sensitive to chlorate than were similar controls in the experiment shown in Figures 30 and 31. Differences in inoculum size could explain this discrepancy. In the earlier experiment, the average fresh weight of the initial inoculum was 2.0 g/l, while in the current experiment it was 0.86 g/l. The enzyme analysis summarized in Table 12 revealed the previously established induction of nitrate reductase activity by nitrate. Three millimolar chlorate reduced the level of NR induction by nitrate, perhaps by inactivation of the enzyme by the products of chlorate reduction (Solomonson and Vennesland, 1972). 3 mM chlorate in the nitrate-less medium did not induce NR activity. The fully induced NR levels seen in Table 15 are now comparable to those reported for XD cells by Heimer and Filner (1971). Our inability to achieve such levels in previous experiments (Table 13) could have been due to 1) the protection afforded by higher cysteine levels in Heimer and Filner’s conditions, or 2) a metabolic state in their cultures more ripe for nitrate induction, engendered by previous maintenance on nitrate as a sole nitrogen source. The sensitivity to chlorate of the 14U cells in urea-M1D + 2.5 mM KN 03 108 .2" 23 mm 835 see :83 35 .85. 28 3 + 82-8.3 n 3. Q3432: H AOV 3:8 33 mo 83.. £26..» 335 no acute 39830 go 023 uncommouémon "3. 953% 251205 0 p o u . 1‘ a O p .5 ‘d 9 a O M l. H w 00 l. a w» 0 J— O O N l- a r 00—. O .l 109 “mm 23 mm 835 88.. 5:3 33 .mozm 28 3 + 52-83 n 3 “ES -88: u AOV 3:8 DE mo m3“.— fisonm .asaocoqxo :o gumbo 39828 .3 2:3 omooammzémon ”3.. 933% 2510.05 0 p a p o q q n J! O 9 a O M I. H 1 cm H V I. 3 an \\Pl” 0 \\\\ IIIIC’ I: /o\\ .111... 1.. co 9 m / \ N I. o H O 1 110 GROWTH (9 tr. wt./|) o 7". 1 ' 1 L 1 0 1 3 1O 30 100 [KCIOalmM Figure 42: Dose-response curve of chlorate effect on fresh weight accumulation in 14U cell cultures. Fresh weights were measured after 8 days of growth. (0) = urea-M1D; (o) = urea-M1D + 2.5 mM KNO3, Data taken from Figures 38 and 39. 111 although greater than that of the 14U cells in the absence of KNO3, was not as great as that of XD cells in M1D medium. Either some clonal characteristic of the 14U cells or the medium (urea-M1D) itself may have been protecting the cells from more acute chlorate toxicity. To test those hypotheses, an experiment was designed in which sensitivity to chlorate was measured in the 14U cells in several media: 1) M1D, 2) urea-M1D and 3) M1D with a preincubation period in M1D. The latter treatment was added to test for the elimination of any enhancement of protection against chlorate provided by an excess of urea (or its reduction products). The sensitivity to chlorate of the XD cells in M1D was also measured for comparative purposes. Figure 43 shows the dose-response curve obtained by plotting the logarithmic growth rate (as percent of control) for each treatment against K0103 concentration. 14U cells showed higher sensitivity to chlorate in M1D than in urea-M1D and the degree of sensitivity was similar to that of the XD cells in M1D. Therefore, these results exclude the possibility of the 14U cells become intrinsically more tolerant-to chlorate than the XD cells. When 1-3 mM K0103 was present in MIDI-grown 14U cultures, the growth rate was higher than that of the control (0 mM K0103). This could be an artifact of experimental. manipulation. It may mean that chlorate stimulates growth at low concentrations. This growth ' stimulation by chlorate has been reported for cultures of Datura innoxia (King and Khanna, 1980). Also, Doddema and Telkamp (1979) reported enhanced uptake of nitrate by Arabidopsis plants in the presence of very low concentrations of chlorate. 112 .52 E ox n § ”92 5 8338595 .92 5 D: n 3 “92-83 5 a: u A! 522 E D: H ADV .27.“. Gun can :3 «o 33.. 538m .3 acute 39839 go 823 omcamouomon "av 03mg 3.5.905 8.. 0» Op 9 p 6 q q 1 at G 'IOIIJMOG SO % 31."! MORE 113 Effects of Urea, Chloride and Ammonium Ions on Tobacco Cells’ Sensitivity to Chlorate. The medium constituents that may confer some type of protection from 0103‘ toxicity upon the 14U cells are chloride, urea or the products of urea metabolism. As mentioned above, the chloride content of the urea-M1D medium is higher than that of the M1D medium. Since the results of the uptake experiments (Part I) showed that chloride inhibits chlorate uptake at high chlorate concentrations, it might be expected that this inhibition results in the protection of the 14U cells from chlorate toxicity. Urea (and the products of its reduction) was the only other addition to the standard M1D recipe in urea-M1D. The growth of XD and 14U cells in their respective maintenance media, modified as indicated, was measured to determine the effect of urea and chloride ions on the cell’s sensitivity to chlorate. Stationary phase XD cells were subcultured into M1D medium supplemented with either 2.5 mM K01 or 3‘ mM urea at chlorate concentrations ranging from 0 to 100 mM. Stationary phase 14U cells were subcultured into urea-M1D, Cl-less N -1ess M1D + 3 mM urea or N -less M1D + 2.5 mM KN03 each at chlorate concentrations ranging from 0 to 100 mM. The fresh weight of the cells was measured after 8 days. The results obtained are plotted as a percent of control’s fresh weight versus chlorate concentration (Figure 44 and 45). Chloride ions conferred a slight protection against chlorate to the KB cells when added to the M1D medium. However, removal of chloride ions from urea-M1D did not increase the sensitivity of 14U cells to chlorate. 0n the other hand, when the M1D medium was supplemented wih urea, the XD cells were less sensitive to chlorate. Also, when urea was replaced by 2.5 mM 114 do; 25m + n22 H AIV 30% 28 ad + 932 H AOV 532 .l.. 2! .3333 :8 fix E dose—38:88 Emma? 53¢ no 3983“. Ho gumbo mo mots“. annex—motomon 3% 23mg 251205 O09 'IOHLNOO :IO 95 (Imam; 6) Humous 115 .823 SE 33 + 52 32.2 n :3 32: 28 3 + as 32.2 33.6 u 3 . “was: 28 3 + 92 32.2 u 9 32333 :8 D: 5 53358333 3333 smmé :0 33.838 .3 tuba «o 3253 mmcoqfiuémon ”we Snug 5510.03 GOP 1oumoo :IO % (V'IM'JJ 5) HLMOEIS 116 nitrate in the urea M1D medium, the 14U cells became more sensitive to the toxic effect of chlorate. These results suggest that urea by itself, or the products of its reduction, rather than chloride ions, may be responsible for the observed protection. To further investigate the urea effect, an experiment was designed in which the growth response of the XD cells to chlorate was measured in M1D medium supplemented with increasing concentrations of either urea (0, 3, 6 and 9 mM) or ammonium succinate (0, 3, 6 and 9 mM NH4Cl + 0, 1.5, 3 and 4.5 mM succinic acid, respectively). As an additional treatment, the response of the KB cells to chlorate in N-less M1D supplemented with 3 mM NH401 plus 1.5 mM succinic acid was also measured. The results of this experiment did not agree with our previous observation. Urea did not protect the XD cells against chlorate at any of the concentrations tested (Figure 46). Ammonium succinate on the other hand protected the cells against chlorate toxicity (Figure 47) in a curious fashion. At 3 mM ammonium succinate XD cells were less sensitive (than controls) to chlorate at any of the tested concentrations. However, ammonium succinate at 6 and 9 mM concentrations protected the cells differently depending upon the chlorate concentration. Protection was greatest with 6 and 9 mM ' ammonium succinate at 10 mM K0103. This same effect was observed when the XD cells were grown on N-less M1D plus 3 mM ammonium succinate (Figure 48). The pH of the medium measured in several instances after harvesting the cells (values recorded on figures next to corresponding data points) follow the same pattern as the one observed for the protection response against chlorate toxicity. The basis for this apparently synergistic interaction of chlorate and ammonium succinate has yet to be resolved. 117 39:. $35 + Q32 H AOV 33.3 28 m + Q22 H AIV 33.5 28 m. + Q22 H 23 #22 H ADV .QHE 6358335339.: 5 558m 328 ON 3 con—£58383 Emma? :39... .8 33.839 .3 Soto mo 33.53 omnonmonémom new 953,.” 58190:“. 2: an GP 0 p o, (l/‘lM'H 6) HLMOHD 118 I I r t \ 5.5 ‘5. /1 c: 3 ‘\ .. 5.4 2 3,“.c‘k‘fi‘ ; I \1' \ O Y“ \ a: \ 0 o 14 1 J k 7, [KCIOalmM Figure 47: Dose-response curves of effect of chlorate on fresh weight accumulation in XD cell cultures supplemented with ammonium succinate. (O) = M1D + 3 mM ammonium succinate (AS); (A) + M1D + 6mM AS; (0) = M1D + 9 mM AS; (I) = M1D control. 119 .Safiuoam 535538 SE m. + GSA 82.2 H A. V 3:2 H A. V .3953 coon—non 3.3625 8:23:88 303.3 E mouse—so :8 Gun 5 5335833 Emma? 48¢ no 3335 .«o comma go 823 omnonmgémon— "mv 95mg 3520.05 3 a . \i 0.. Ga (l/‘IM'JI 5) HLMOUS PART III: NITROGEN NUTRITION AND CHLORATE UPTAKE IN TOMATO SEEDLINGS MATERIALS AND METHODS Plant Material. An objective of the present work was to evaluate the correlation between nitrate uptake affinity and adaptation to nitrogen poor soils, in a number of species or cultivars. To judge the feasibility of cell culture selections for altered nitrate uptake, the developmental consistency of this character must be compared in whole plants and cultured cells of an individual cultivar. Tomato plants were chosen for this part of our study for several reasons: 1) rapid and uniform seed germination, 2) rapid growth and convenient seedling size, 3) well-developed genetics and physiology, 4) amenability to tissue culture and 5) availability of a large store of variable germplasm. On the other hand, tobacco is severely deficient on 1), 2) and 3). ._ J. Fobes (ARCO-PCRI, Dublin, California) kindly supplied seeds of the ‘ following genotypes: Lycopersicon chilense, drought resistant plants with an extensive root system native to very sandy soils poor in nitrogen. L. pimpinellifolium, which competes well in corn fields. L. pennellii, drought resistant plants with small root systems surviving in very sandy soils. L. esculentum cv VF-36, a cultivated tomato. S.E. Barsel (Department of Botany and Plant Pathology, Michigan State University) generously supplied seeds of L. esculentum cv. VFN-Bush, also a cultivated tomato. 120 121 Initiation and Maintenance of Cultures. Callus cultures were induced from radicle, hypocotyl and cotyledonary leaf explants of each one of the tomato species and cultivars described above. Seeds were surface sterilized by soaking them for 30 minute in 10% commercial bleach plus 1% sodium lauryl sulfate (with periodic agitation) followed by 6 five minute rinses in sterile distilled water. The seeds were then germinated over sterile Whatman number 1 filter paper discs wetted with 2 ml of sterile water. Seeds were germinated at room temperature (22° C to 25° C) on the laboratory bench top. Portions of the cotyledons, hypocotyls and radicle were dissected from 7 day old seedlings and plated on T12, M1D and M1D supplemented with the hormones of T12 medium (see media formulae below). Callus developed from these explants was subcultured onto fresh medium every 2 weeks thereafter. Cultures of the genotypes mentioned above were initiated in January, 1981. From these callus cultures, suspension cultures were made by introducing 3 to 6 grams of friable callus of each cell line into 50 ml of liquid medium in a 125 ml erlenmeyer flask. Cultures were maintained in a regime of 12 h light and 12 h dark at 28°C on a reciprocal shaker. Subculture was achieved by diluting 25 ml of the cell suspension into 200 ml of fresh medium. The subculture schedule varied, depending on medium and genotype. T12 medium (developed by SE. Barsel) consisted of the major and minor salts of Murashige and Skoog (1962) supplemented with N itsch’s vitamins (0.5 mg/l thiamine, 2 mg/l glycine, 5.0 mg/l nicotinic acid, 0.5 mg/l pyridoxine, 0.5 mg/l folic acid and 0.05 mg/l biotin), hormones (3 mg/l IAA, 2 mg/l 2,4-D and 0.1 mg/l BA), 100 mg/l inositol and 30 g/l sucrose. SOIid medium contained 0.9% agar. The pH of the medium was adjusted to 5.8 with KOH and/or HCl 122 prior to autoclaving for 15 minutes at 20 pounds pressure. Nitrate-less T12 medium was made by replacing NH4NO3 and KN 03 with 9 mM KCl. For the experiments, this medium was supplemented with one or more of the following nitrogen sources: 1) NH4C1 (20 mM) + Na succinate (20 mM), 2) Urea (3mM) and 3) Arginine (3mM). M1D and urea-M1D medium were prepared as described in MATERIALS AND METHODS of PART I. Arginine-M1D medium was prepared by dilution of an arginine solution into nitrogen-less M1D medium to give a final arginine concentration of 3 mM. Solid media contained 0.9% agar and the pH was adjusted to 6.2 with N aOH and/or HCl prior to autoclaving for 15 minutes at 20 psi. Tomato explants were also plated on a modified M1D medium in which the hormones had been substituted by those of the T12 medium. Supplemented media were always made by addition of filter—sterilized supplements to autoclaved basal media. Culture of Tomato Seedlings. Tomato seeds were surface disinfested and germinated as described above. The petri dishes containing the germinating seeds were sealed with . parafilm and stored in the dark at room temperature. When the radicles had reached 1 to 2 cm in length, the seedlings were transfered in a laminar flow hood, to test tubes where they were grown hydroponically, as described below. Half strength modified Hoagland’s solution (Wilson et al,.1978) was supplied for the growth of seedlings. N—free Hoagland’s solution was made by replacing the nitrates of calcium and potassium with equimolar amounts of their respective chlorides. The pH of the solution was adjusted to 5.4, 5.5 or . 6.5 with N aOH and/or HCl. In several instances, MES (20 mM) was added to 123 stabilize pH. Twenty-ml aliquots of medium were dispensed into 25mm x 200 mm test tubes. A filter paper support platform was then inserted into the test tubes prior to sealing with foam plugs. Sterilization was carried out by autoclaving for 15 minutes at 20 psi. The support platform was constructed by folding a Whatman No. 4 filter paper disc (7 cm in diameter) over the mouth of a 23 mm diameter, test tube. A small hole was punctured in the center of the paper platform for insertion of the seeling radicle and an additional hole was punctured half way to the edge of the platform to be used for the addition of media supplements. When supplemented media were required, sterile solutions of the supplements were diluted into sterile media. The supplemental solutions were adjusted to the desired pH prior to filter sterilization. The seedlings were transfered to the test tubes by inserting the seedling radicle through the center hole of the paper platform. This assembly was then pushed down with sterile forceps until the skirt of the platform was in contact with the liquid medium. The bottom part of the test tube was covered with aluminum foil to darken the root environment. The seedlings were grown under gro-lights (126 microeinsteins M‘2 sec") with a photoperiod of 14 hours followed by 10 hours of darkness. The temperature inside the test tube, recorded with a thermister, was 23° C during the dark period and 27° C during the light. Three replicates were prepared per data point. The seedlings were harvested at one week intervals and their growth was determined by fresh and dry weight measurements. Fresh tissues were dried in a 60° C oven for 24 hours. After removal of the seedlings, the pH of the liquid media was also measured. Root size and morphology were recorded by spreading the roots over cellophane sheets and photocopying them on a Xerox 8200. The length of main and lateral roots was measured with a planimeter 124 (Arin, W. Germany). Compartmental Analysis. L. esculentum cv VF-36 seedlings were exposed to N-free Hoagland’s solution supplemented with 3 mM glutamine, '20 mM MES and 36Cl-chlorate for a "loading” period of 10 hours. They were then transfered to a solution containing an identical nutrient solution lacking 36Cl-chlorate for a subsequent "unloading” period of 2 hours. The nutrient solution was replaced every 4 minutes at the beginning of the "unloading” period. This interval was gradually increased to 32 minutes by the end of the "unloading” period. Such a scheme minimized reabsorption of the radioisotope The radioactivity in 3 ml aliquots from each of the rinses was counted in 12 ml of the scintillation fluid described by Trombolla (1970). Following the final rinse, the seedlings were blotted with paper towelling, the roots and shoots separated and the fresh weight of each portion measured. The amount of radioactivity remaining in these tissues was determined by counting in 15 ml of the scintillation cocktail. Measurements of Chlorate Uptake by Tomato Seedlings. Chlorate uptake was measured in three week old L. esculentum cv. VF- ' 36 seedlings grown on N -free Hoagland’s solution supplemented with either 3 mM glutamine or 3 mM Y-amino butyric acid (GABA). 36Cl-chlorate, KC103 and KNO3 solutions were delivered to the nutrient medium with a sterile syringe through the lateral hole in the paper platform. After 24 hours, the roots were blotted with a Kimwipe and rinsed for 10 minutes in fresh medium (minus chlorate and nitrate). The seedlings were then blotted dry in paper toweling and their fresh weight determined. The roots and shoots were 125 counted separately in the scintillation cocktail described by Trombolla (1970). RESULTS Nitrogen Nutrition of Tomato Tissue Cultures. All explants from all genotypes developed friable calluses when grown on T12 and ammonium succinate-T12. These calluses gave rise to finely divided cell suspensions when transfered to liquid T12 or liquid ammonium succinate-T12. L. esculentum cv. VF-36 suspensions in liquid ammonium succinate-T12 medium grew with a doubling time of about 2 days (Figure 49). This particular cell line had been subcultured 13 times at 10 day intervals prior to these growth measurements. Tomato cells grown in M1D would provide a direct comparison with results obtained with tobacco cells. However, tomato seedling explants on M1D and M1D supplemented with T12 hormones became necrotic and showed no signs of growth. To avoid nitrate carryover from the tomato cells’ maintenance medium, we sought a combined nitrogen source Other than nitrate on which to maintain tomato cells stock cultures. Ammonia was unacceptable because of its interference with nitrate transport. Tomato calluses of all cultivars developed from root explants and maintained for one year on T12, were transferred to arginine-T12 and urea- T12. At this time callus tissue from the same source was evaluated on M1D, urea-M1D and arginine-M1D. Although most of the callus tissue became 126 1 COO ' T l I I I = o o E ./ :5 ./ 3 100 .. a .5 ./ 3 O C G J l l J l l 0 2 4 6 8 1O 12 14 TIME (days) Figure 49: Growth of batch suspension cultures of L. esculentum cv. VF-36. 127 necrotic on all media tested, upon repeated transfer to the same media, rare colonies arose. Those colonies were then picked and serially transferred, selecting for cells able either to metabolize the supplied nitrogenous compounds or grow on a minimal medium (M1D). The current status of these cultures is shown in Table 15. L. esculentum cv. VF-36 calluses grown on urea-M1D medium were transfered to liquid urea-M1D medium and a cell suspension was obtained. After 8 subcultures at 13 day intervals growth kinetics of the cell suspension were determined. A doubling time of 3.8 days was computed from the growth curve shown in Figure 50. Chlorate Toxicity in Tomato Cells. Hofstra (1977) reported that tomato plants were insensitive to chlorate when grown on ammonia as the sole nitrogen source. This finding agrees with the current belief that chlorate becomes toxic only after its reduction to chlorite by the enzyme nitrate reductase (Aberg, 1947). L. esculentum cv. VF- 36 seedlings should therefore display a reduced sensitivity to chlorate when grown on media containing nitrogen sources other than nitrate. However I found that ammonia-grown seedlings were more sensitive to chlorate than ‘ those grown on nitrate (see below). In addition, lower concentrations of chlorate were required to cause growth inhibition in tomato seedlings comparable to that in tobacco cells (see below). The latter could be accounted for by chlorate being concentrated by the seedlings via transpiration. Nevertheless, an evaluation of the cell-level chlorate response of L. esculentum cv. VF-36 in ammonium succinate-T12 was needed to establish the relative sensitivity of cultured tomato cells to chlorate toxicity. Ten ml aliquots of L. esculentum cv. VF-36 tomato cells growing in 128 Table 15: Callus growth response of tomato species to different medium nitrogen sources. Root, shoot or leaf tissues from tomato germplasm sources indicated were qualitatively evaluated for callus response on indicated nitrogen sources. Overall evaluation of performance was made after 6 to 8 passages in culture: (+ ++) = prolific growth; (+ +) = fair growth; (+) = poor growth; (-) = no growth; (NT) = no tested. __,___—/ W A_,._.‘ _. __. -A M__.. A. .‘—_~ __ ,_. ‘ ___ _ _ _.. _ 129 Table 15: Callus growth response of tomato species to different medium nitrogen sources. MEDIA Source of Urea- Arg- Urea- Am.Suc. Genotype Explant M1D M1D T12 T12 T12 T12 L.esculentum root + + + + + + + + + + + + + cv VF36 shoot NT NT' NT NT + + + + + + leaf NT NT NT NT + + + + + + L.esculentum root + + NT + + + + + + + + + cv VFN-Bush . shoot NT - NT NT NT + + + + + + leaf NT NT NT NT + + + + + + L. Pennellii root + + + + (tiny + + + + + + + + colonies) shoot NT NT NT NT + + + + + + leaf NT NT NT NT + + + + + + L. Pimpinelli- root (tiny NT - (tiny + + + + + + folium colonies) colonies) shoot NT NT NT NT + + + + + + leaf NT NT NT NT + + + + + + L. chilense root NT NT NT NT + + + + + + shoot NT NT NT NT + + + + + + leaf NT NT NT NT + + + + + + 130 10° ' r ' T It I /./O s / s / g o 3 1o. . I 1. 3 O c Q G 1 . J O 4 8 12 16 20 24 28 TIME (days) Figure 50: Growth of L. esculentum cv. VF-36 batch suspension cultured cells in urea-M1D medium. 131 ammonium succinate-T12 medium from a 2 1 suspension culture were transfered to 500 ml flasks containing 200 ml of ammonium succinate-T12 medium and KC103 at concentrations ranging from 0 to 100 mM. Every three days 2 flasks per treatment were harvested and fresh weight determined. Under these conditions, the chlorate I50 (= the concentration resulting in a 50% reduction in growth rate with respect to the control) was 10 mM (Figures 51 and 52). This value is lower than that obtained for 14U tobacco cells grown in urea-M1D (150 = 40 mM, See Figure 43), indicating that, at the cellular level, tomato and tobacco do not differ significantly in their sensitivity to chlorate. Chlorate Toxicity in Tomato Seedlings. Chlorate toxicity in L. esculentum cv VF36 plants was measured by transfering 2 day old seedlings into test tubes containing 20 ml of Hoagland’s solution (7.5 mM KN03) and KC103 concentrations ranging from 0 to 100 mM. Seedlings were harvested and their fresh and dry weight determined 1, 2 and 3 weeks after transfer to the growth tubes. The I50 for the effect of chlorate on seedling growth was 620 pM (Figure 53). This value was very low compared to the concentration required to elicit the same response in tomato cells (150 = 10.5 mM, Figure 52) and tobacco cells (I50 == 13 mM, Figure 43). In a preliminary experiment, the response of tomato seedlings to chlorate was also measured in Hoagland’s solution in which KN03 had been substituted with 7.5 mM NH4C1. K0103 was supplied at 1 mM. Seedlings growing in chlorate-free Hoagland’s solution (7.5 mM nitrate) were used as controls. The data in Table 16-A indicate that: 1)ammonia-grown plants developed poorly compared to those grown on nitrate and 2) chlorate toxicity was greater, not less, in ammonia grown plants than in those grown on 132 1000 ’ r ' ’\ 100. \\ \ \\ \\ 10t 1 m 0 5 9 14 TIME (days) Figure 51: Chlorate toxicity in batch suspension cultures of L. esculentum cv. VF-36. Cultures were grown in ammonium succinate T12 medium supplemented with 0 (O), 1 (A), 3 (I), 10 (O), 30 (A), or 100 ([3) mM KC103, and harvested by vacuum filtration for fresh weight determinations on indicated days. 133 495:8 Sane—sod 2: do 38.89 a mm @3333 623.5538 3983". «o 552:... a as 63.83 mha 3 9:33 68.. 3% 526..» hot m>E .3825 3 33:3 :8 8.3.3 mo uncommon 5380 ”an 95»:— 5810.3: 2: on 2 a .p o /. a”. . . «a o u 0 . . m u \I B . ruin..-inninuulmuiuuulu.1100 u... 0 an / m. . o 000.. O 0 m m P . b . . 134 493:8? 38.8: a mm 3389:. 63.8: :33 m a :26 mmfizuomm 332%: m .3 3a.. 4.39% Sufi—$3 23 3:39am: ”Ema: scam .5398 m::cmmm 3.2.:3 :o 35.830 mo acubm ”an «Sufi 3510.05 2: on 3 a . ad to 36 I * q o . . a r u .3 a . O . m u .. u .3 a ............... w o J.— o. . .3 m o 1 o .2: 1 n b u n P b b 135 Table 16A: Nitrogen source dependence of chlorate sensitivity in tomato seedlings grown in unbuffered solutions. Seedlings were harvested and weighed after 3 weeks’ growth on the N-source/KClOa combination indicated. Data are means of three replicate measurements. Fresh Weights (% Control) KN O3 NH4Cl (7.5 mM) (7.5 mM) -KClO3 100 21 + KC103(1mM) 22 2 Table 16B: Effect of nitrogen source and chlorate on pH of tomato seedling nutrient solution. The pH of seedling nutrient solution was determined after 7 days growth in the N-Source/KC103 combination indicated. Data are means of 3 replicate samples. pH KN 03 N H401 (7.5 mM) (7 .5 mM) -KC103 6.29 3.79 + KClO3(1mM) 6.07 5.29 136 nitrate. Since the growth of the tomato seedlings was reduced dramatically when grown in ammonia, it was possible that their high sensitivity to chlorate was a consequence of their weak physiological state. Poor growth on ammonia may result from the media’s lowered pH which developed as a consequence of ammonium metabolism in growing seedlings. Table 16-B summarizes the final pH values of the media in which the seedlings were grown for the above experiment. The necessity of a buffered medium was obvious. The buffering capacity of MES and its effect on seedlings was examined by its addition at 5 and 20 mM to Hoagland and ammonim—Hoagland nutrient solutions. Fresh and dry weights of the seedlings as well as the pH changes of the media were recorded at weekly intervals. 20 mM MES maintained the pH of the Hoagland’s solution; however, its buffering Capacity is insufficient for the ammonium-Hoagland solution. These results are summarized in Table 17 and correspond to the seedlings harvested after three weeks. As in previous experiments, the ammonium grown plants showed greater sensitivity to chlorate than those grown on nitrate. Even though MES (20 mM) maintained the pH for the first 2 weeks (Table 17) we cannot rule out the possibility that the difference in response was due to a difference in pH created by nitrate and ammonia assimilation. Supplementation of ammonium-Hoagland’s with . succinic acid also achieved a measure of pH stabilization (Table 18). Still, a pH drop from 5.4 to 5.0 was observed with 15 mM succinic acid. Furthermore, plants grown in 15 mM succinic acid buffered ammonium-Hoagland’s did not attain fresh or dry weight comparable to growth on nitrate. One possibility for the poor performance of seedlings under these conditions was that 7.5 mM ammonium was toxic. Therefore combinations of MES and succinic acid and decreasing amounts of ammonium were tested. The results of these experiments (Tables 19 and 20) suggested that 4 mM 137 $232: 3 :23 «an m: 8 33:2: 225 323.8 32:3 Z 32:38 £3293“: 32:38:: N m $2 .2 32:38 £32925 H m. .m:232:::22. 2:232 m .«o :o::o 323:? “3: :23: 2:333: 3:3: 23m .3232322: 3: 32:3: :2 >33? :23st 225 mwfizomom 303232 322.38 32:3: 2.3 :23: £38m .«o 3325 m :33 3:23:22: 825 3:22:32:an .«o 3:22: >3 :3: 2mm; .mwfizuomm 0333 .«o m: 5233.: 32:3: «3: 532m :o 23:22: v:m._::2:o:::m .mmnz mo 32mm :3 2:3. 138 2.. . «a: 2.. . :n: 2.. . 5:. . 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H 3.3 m H HHH 3H H an H an 3.5 REV 322389 2303 in 2953 €on .33 3583 H GABA > Urea > KN 03 > NH4C1 Since glutamine was the overall most efficient in sustaining growth and stabilizing pH, it was chosen as the nitrogen source for the uptake studies. In a subsequent experiment, the growth of L. esculentum cv. VF-36 seedlings in nitrate-free Hoagland’s solution supplemented with asparagine and several degradation products of purines (hypoxanthine, xanthine,uric acid and allantion) was evaluated. The pH of the stock solutions of these compounds was adjusted to 5.5 before they were diluted into nitrate-free Hoagland’s solution to a final concentration of 3 mM . MES (20 mM) was used to buffer the nutrient solutions. Some difficulty was enountered in adjusting the pHs of the uric acid, xanthine and hypoxanthine solutions. Consequently, the pH of the final solutions was higher than planned (see Table 23). GABA and glutamine treatments were incorporated into this experiment to confirm previous observations. Plants grown on standard Hoagland’s solution (KNO3) . were used as controls. The effect that these nitrogen compounds had on the growth of L. esculentum cv. VF-36 seedlings is summarized in Table 23, where the fresh and dry weights of 12 day old seedlings are tabulated. As in the previous experiment, seedling growth and development were extremely sensitive to the source of nitrogen . Even though the size (Table 23) and timing of primary leaf expansion was affected by these compounds, more dramatic changes were observed in root morphology and development (Figures 57 and 58, Table 24). 152 m5. m3 - H S - H 8m 89283 3. $6 H H m S H m2 mfifiéx as was - H H .. - H 2: 823333 .3 was a H w a H mm 33 25 3 an.» o H m H H SH 53:22 3 8d H H S a H SN "0sz A88 ammzmm 3.5 8:: 33:3 83w In 3935 5.5 Emma? :35 “5.53.5. 89a 8:3va 3:08.52 sumac. 83m .mcsmoia: as 5:3 .983 moons—om 33.55: mo and docs“: .3va ”7.2: no a: 3 £3: mug—€2.38 .mwczuoom 3o .36 a :0 33355.33. 3823.. m go .820 castaway. can .808 383%.. Bav :< .538» mczvmom 32:3 no 3938 comet: 2.83 can ~39:— mo 80th "mm ~33. 153 .838 coach? 3% on... mm 350qu“. «.3835 on... :33 55on soon was 533 38.. mnzvmom 33:3 3o £33 a .«o 338 who? mgnmumouox .Qofioneofi 38 mangom 33:3 :0 some“? @293 .«o aoobm Hum 933% 154 $335308 38.. mam—comm 33:3 co comet: 3:95 we uuotm u o 62: . to 1.025.200 £Ez2 155 $338383. 83 302 mammzmtm .8 833.8 cm 3 8233.3 238333 :o 539% .88 .833 me $380va .35 .5338 we «8:8 38 a ms 88.888 @3863 33 :o 3.39% .383 25 83.3 3:830th 983 38.. 985.com $335988 .83 3m83 8o < $2822 “com 332 am in aamgaofi. .mmz 28 ca 853:8 3353.: :< .532» .mmau 3 6:“ :33 8mg 33 538m 88 mezumom Bess no 83% comes? do ”scam "mm 395. 160 59:91:08 no.8 89:8 so 3ng Ammonium Succinate) Urea > Arginine I proceeded to evaluate the growth response of tomato seedlings to several other nitrogen sources including glutamine, y-amino butyric acid (GABA), asparagine, hypoxanthine, xanthine, uric acid and allantoin. Seedling growth and development was extremely sensitive to the source of nitrogen. The size (Table 23) and timing of primary leaf expansion was somewhat affected by these nitrogen sources, but more dramatic changes were observed in root morphology and development (Figures 57, 58 'and Table 24). Treatments which resulted in larger root systems (e. g., allantoin and hypoxanthine (Table 24)) had the opposite effect on stem and leaf size and development (Table 23). It was also observed that tomato seedlings developed extensive root systems in the absence of a nitrogen source, while growth of the aerial plant parts was retarded by the lack of nitrogen (Table 25). This finding concurs with the observation by Jackson et al. (1976b) that root surface area increases with suboptimal nitrate nutritional conditions. Such a response would enable a plant to scavenge nitrogen more efficiently from a nitrogen- poor soil. GABA had a striking effect on root morphology and root hair development (Figures 59 and 60). The growth of the main and lateral roots was inhibited when tomato seedlings were grown on GABA. Both main and lateral roots of GABA-treated plants developed root hairs in a periodic pattern in which portions of the epidermis had no root hairs whereas the intervening regions developed long and numerous root hairs. The regions of the GABA- treated roots where root hairs appeared had a greater diameter than the adjacent hairless portion. In contrast, the distribution of root hairs of seedlings grown on nitrate was uniform, with a progressively smaller root diameter toward the apices of the main and lateral roots. 189 This effect was specific for GABA, since none of the other nitrogen sources tested elicited such a response. The GABA effect was "dominant”, in that seedlings grown on solutions containing mixtures of GABA and other nitrogen compounds showed root hair growth periodicity. No GABA analogue tested elicited the GABA effect (Figure 61). All inhibited growth to differing degrees, with the exception of 3—amino propane sulfonic acid, which stimulated root growth when compared with the nitrate control (Figures 61 and 62). The GABA effect was light dependent. Roots of etiolated GABA- treated seedlings did not develop lateral roots and no periodicity was evident in the main root. Finally, the GABA effect was not species or cultivar specific. The root system of L. esculentum cv. Floridale, L. esculentum cv. VF-36 and L. pennellii seedlings all responded to GABA with the characteristic root hair periodicity. The response of the tomato seedlings to GABA is of interest because little is known about the role or function of this compound in higher plants. It has been reported that GABA accumulates in plant roots under anaerobiosis (Wickremasinghe and Swain, 1965). Selman and Cooper (1978) found GABA to be distributed throughout tomato plant parts and postulated that its role was that of a nitrogen storage compound. The results obtained with tobacco cells in the first part of this study indicated that chlorate is not an ideal analogue of nitrate for transport studies. In contrast, Deane-Drummond and Glass (1982) have reported that chlorate is an excellent analogue of nitrate for studying nitrate uptake by intact barley roots. I used tomato plants to determine whether whole plants show nitrate- chlorate responses similar to those of cultured cells. The 150 for chlorate inhibition of tomato seedling growth was 0.62 mM. Seedlings treated with 3 mM 0103' showed a antidotal response to N 03' in the 7 .5 to 37.5 mM NO3' 190 range. At the highest N 03‘ concentration tested, 3 mM 0103‘ still inhibited growth by 17%. The uptake of chlorate by roots of L. esculentum cv. VF-36 seedlings growing on glutamine-Hoagland’s solution was measured in the presence and absence of nitrate. The kinetics of chlorate uptake (Figures 66 and 67) resembled those obtained with tobacco cells (Figure 15). Nitrate inhibited chlorate uptake into roots in a fashion similar to that observed with tobacco cells in vitro, in that 15 mM KN O3 reduced chlorate uptake to a linear, non- saturable response (Figure 67). However, further experiments would be required to characterize this interaction to the extent described above in the in vitro tobacco cell system. Chlorate was much more toxic to seedlings of L. esculentum cv. VF-36 grown in Hoagland’s solution (150 = 620 pM, Figure 53) than to cells of the same cultivar, grown in vitro on ammonium succinate T12 (I50 = 10.5 mM, Figure 51) or to tobacco cells cultured on M1D (150 = 13 mM, Figure 41) or the latter grown on urea-M1D. (I50 = 40 mM, Figure 43). Seedlings may concentrate chlorate in their. aerial portions via tranSpiration, thus accounting for their enhanced sensitivity. Hofstra (1977) reported that tomato plants were insensitive to chlorate when grown on ammonium as the sole source of nitrogen. This finding agrees with the predictions of Aberg’s hypothesis of chlorate becoming toxic only after its reduction to chlorite by the enzyme nitrate reductase. However, we found that alternative nitrogen sources such as ammonium succinate, urea, GABA, glutamine, and arginine increased the toxicity of chlorate over that observed when seedlings were grown on nitrate (Tables 20 and 22, Figure 56). Doddema and Telkamp (1979) also reported this unexpected phenomenon in studies using Arabidopsis plants. 191 In addition to Cove’s (1976) alternative mechanism of chlorate toxicity (see INTRODUCTION), yet another explanation for these results can be proposed, based on Aberg’s hypothesis: If these young seedlings had a constitutive nitrate reductase, this activity could reduce .chlorate to toxic chlorite without competition from medium nitrate. Tomato seedlings at this stage had a level of nitrate reductase activity (0.39 nmoles / min / g fr. wt.) two orders of magnitude below the full induced activity of cultured XD tobacco cells (see PART II). It is unlikely that such a low activity could generate cytotoxic levels of chlorite, considering the low affinity of NR for chlorate (Table 12). Furthermore, ammonium inhibits both nitrate reductase and nitrate transport in plants (see INTRODUCTION). Seedlings treated with tungstate, in order to block insertion of molybdenum into NR, developed toxicity symptoms in the presence of chlorate to the same extent as untreated ones. This, and the results discussed immediately above, suggest that chlorate toxicity results from either (i) reduction of chlorate by (not readily detectable) preexisting constitutive NR, or (ii) a mechanism not involving nitrate reductase. Any model based on this second alternative would have to account for the apparent enhancement of chlorate toxicity by ammonium, GABA, urea and glutamine in tomato ' seedlings. BIBLIOGRAPHY BIBLIOGRAPHY Aberg, B. 1947. On the mechanism of the toxic action of chlorates and some related compounds upon young wheat plants. Kingl. Lantbrukshogskol- ans. Ann. 15237-107. Ashley, D.A., W.A. Jackson, R.J. Volk. 1975. Nitrate uptake and assimila- tion by wheat seedlings during initial exposure to nitrate. Plant Physiol. 55:1102-1106. Aslam, M. and A. Oaks. 197 5. Effect of glucose on the induction of nitrate reductase in corn roots. Plant Physiol. 56:634-639. Aslam, M. A. Oaks and RC. Huffaker. 1976. Effect of light and glucose on the induction of nitrate reductase and on the distribution of nitrate in etiolated barley leaves. Plant Physiol. 58:588-591. Behrend, J .. and RI. Mateles. 1975. Nitrogen metabolism in plant cell sus- pension cultures. I. Effect of amino acids on growth. Plant Physiol. 56:584-589. -- Borstlap, A.C. 1981. Invalidity of the multiphasic concept of ion absorption in plants. Plant, Cell and Environment 4:189-195. Braaksma, F.J. and W.J. Feenstra. 197 5. Nitrate reduction in Arabidopsis thaliana. Arabidopsis Inf. Serv. 12:16-17. Breteler, H., Ch.H. Hanischtencate and P. Nissen. 1979. Time-course of nitrate uptake and nitrate reductase activity in nitrogen-depleted dwarf bean. Physiol. Plant. 47:49-55. Butz, R.G., W.A. Jackson and R.J. Volk. 197 5. Effects of ammonium on nitrate uptake and reduction by corn roots. Agron. Abstracts p.69. 192 193 Cove, DJ. 1976. Chlorate toxicity in Aspergillus nidulans. Studies of mugtants altered in nitrate assimi ation. Molec. Gen. Genet. 146:147- 15 . Crafts, AS. 1939. The relation of nutrients to toxicity of arsenic, borax, and chlorate in soils. J our. Agr. Res. 58:637-671. Crafts, AS. and W.W. Robbins. 1962. Weed Control.McGraw-Hill,N.Y. p.69. Cram, W.J. 1973. Internal factors regulating nitrate and chloride influx in plant cells. J our. Exp. Bot. 24:328-341. I Chantarotwong, W.R., R.C. Huffaker, B.L. Miller and RC. Graustedt. 1976. In vivo nitrate reduction in relation to nitrate uptake, nitrate content and in vitro nitrate reductase activity in intact barley seedlings. Plant Physiol. 57:519-522. Deane-Drummond, CE. and A.D.M. Glass. 1982. Nitrate uptake into barley (Hordeum vulgare) plants. A new approach using 36ClO3' as an analog for N03‘. Plant Physiol. 70:50-54. Dijk, Van de.S.J. 1981. Differences in nitrate uptake of species from habitats rich or poor in nitrogen when grown at low nitrate concentrations,using a new growth technique. Plant and Soil 62:265-278. Dijk, Van de.S.J.,L. Lauting, H. Lambers, F. Posthumus, I. Stulen and R. Hofstra. 1982. Kinetics of nitrate uptake by different species from nutrient-rich and nutrient-poor habitats as affected by the nutrient supply. Physiol. Plant. 55:103-110. Doddema, H. and GP. Telkamp. 197 9. Uptake of nitrate by mutants of Arabidopsis thaliana, disturbed in uptake or reduction of nitrate. 11. Kinetics. Physiol. Plant. 45 :332-338. Eisele, R., and W.R. Ullrich. 1977. Effect of glucose and C02 on nitrate uptake and coupled OH- flux in Aukistrodesmus braunii. Plant Physiol. 59:18-21. Epstein, E. (ed.) 1972. Mineral Nutrition of PlantszPrinciples and Perspectives. J. Wiley and SonsInc.,I‘T.Y. 194 Ezeta, RN. and W.A. Jackson. 197 5. Nitrate translocation by detopped corn seedlings. PlantPhysiol.56:148-l56. Feenstra, W.J. and E. J acobsen. 1980. Isolation of a nitrate reductase deficient mutant of Pisum sativum by means of selection for chlorate resistance. Theor. Appl. Genet. 58:39-42. Ferrari, T.E., O.C. Yoder, and P. Filner. 197 3. Anaerobic nitrite production by plant cells and tissues: Evidence for two nitrate pools. Plant Physiol. 51:423-431. Filner, P., 1965. Ph.D. Thesis, California Institute of Technology, Pasadena. Filner, P. 1966. Regulation of nitrate reductase in cultured tobacco cells. Biochim. Biophys. Acta 1 18:299-310. Flores, E. and M. G. Gerrero. 1980a. Properties of nitrate utilization in Anacystis nidulans attributable to a nitrate transport system. Proc. of the 2nd FESPP Congress. Santiago de Compostela. Flores, E., M.G. Guerrero and M. Losada. 1980b. Short-term ammonium inhibition of nitrate utilization by Anacystis nidulans and other cyanobacteria. Arch. MicrObiol. 128:137-144. Fried, M. F. Zsoldos,P. B. Vose andI..L Shatokhin.1965. 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