ABSTRACT CONTROL OF NITRATE AND NITRITE REDUCTION IN BARLEY ALEURONE LAYERS BY Thomas Enrico Ferrari Nitrate induces the formation of nitrate reductase activity in barley (Hordeum vulgare L. cv. Himalaya) aleu- rone layers. Previous work has demonstrated dg.ggzg_syn- thesis of alpha-amylase by gibberellic acid in the same tissue. Aleurone layers therefore provide a convenient tissue for the study of both substrate- and hormone-induced enzyme formation. As measured in cell—free extracts, the nitrate-induced increase in nitrate reductase activity is inhibited by cycloheximide and 6-methylpurine, but not by actinomycin D. Nitrate does not induce alpha-amylase syn- thesis and it has no effect on the gibberellic acid—induced synthesis of alpha-amylase. Also, there is little or no direct effect of gibberellic acid (during the first 6 hours of induction) or of abscisic acid on the nitrate- induced formation of nitrate reductase. Gibberellic acid does interfere with nitrate reductase activity during long- term experiments (greater than 6 hours). However, the time Thomas Enrico Ferrari course of this inhibition suggests that the inhibition may be a secondary one. Nitrate reductase activity in aleurone layers has also been determined in intact tissue, using two different methods. The first method measures the rate of appearance of H2180 produced during the reduction of KN1803. The “ ’1 second assay measures released nitrite resulting from nitrate reduction under anaerobic conditions. Nitrite a i I i production in this anaerobic, intact-tissue assay was de- pendent upon the presence of phosphate (pH 7.5) and was increased by ethanol and bisulfite. After ten hours of nitrate induction, nitrate reductase activities measured by the KNlBO3 assay are one- sixth, and those measured by the anaerobic intact—tissue assay are one-third, of those observed in cell-free ex- tracts of aleurone layers. Addition of ethanol to the anaerobic intact-tissue medium increased the rate of ni- trate reduction to a level greater than that found in the cell-free assay. Oxygen inhibited nitrite release in the anaerobic intact-tissue assay. However, under aerobic conditions and in the presence of 2-heptyl-4-hydroxyquinoline N-oxide (HOQNO) or antimycin A, nitrate reduction increased to rates comparable to those observed under anaerobiosis. Neither of these electron transport inhibitors affected anaerobic nitrate reduction. Thomas Enrico Ferrari A method was devised for the detection and measure— ment of nitrite reductase in aleurone layers. Nitrite reductase activity of aleurone layers was determined by measuring nitrite disappearance with time. The method also allowed simultaneous determination of nitrite uptake by the tissue. Enzyme activity was induced by nitrate, but meas- urable activity was present in noninduced tissue. Induced activity was inhibited by cycloheximide but not by actino- mycin D. Activity in induced layers was inhibited by 2,4-dinitrophenol, antimycin A, 2-n-heptyl-4-hydroxyquino- line N-oxide, and anaerobiosis. Nitrite uptake was rela- tively insensitive to all inhibitors tested. Nitrite uptake was rapid at pH 4.5 and negligible at pH 7.5. Nitrite accumulated anaerobically at pH 4.5, was rapidly released when transferred to medium at pH 7.5 --the pH of the anaerobic intact-tissue assay for nitrate reductase. Accumulated nitrite was released by the tissue whether held under anaerobic or aerobic conditions. Nitrate-induced and noninduced aleurone layers were able to reduce low levels of nitrite anaerobically. But these rates (5 to 10 nmoles/layer/hour) were consid- erably lower than rates (25 nmoles/layer/hour) of nitrite production observed during the nitrate reductase anaerobic intact-tissue assay (with ethanol present). CONTROL OF NITRATE AND NITRITE REDUCTION IN BARLEY ALEURONE LAYERS BY Thomas Enrico Ferrari A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1970 Q, ._ 815:5 / .1; /~ 3 .,, o -. “if! ACKNOWLEDGMENTS The author is greatly indebted to Dr. J. E. Varner for his counsel and assistance throughout this study and during the development of the manuscripts contained herein. Appreciation is also extended to other members of the graduate committee including Drs. A. A. DeHertogh (chair- man), D. R. Dilley, M. J. Bukovac and N. E. Tolbert. Special gratitude is expressed to Drs. P. Filner, N. E. Good and L. L. Bieber for donating some of the inhibitors and chemicals, and to Dr. R. A. Nilan for donating the Himalaya barley seeds used in this study. The author is also grateful to Mrs. Donna Anderson for typing the second manuscript for publication. This research was supported in part by a grant. from the Atomic Energy Commission's Plant Research Labora- tory, contract no. AT(ll-l)-1338. It was conducted in the Bulb Physiology Laboratory maintained by the Department of Horticulture, Michigan State University, under the direc- tion of Dr. A. A. DeHertogh. Thank you Sheila for maintaining the garden while I was away. ii TABLE OF CONTENTS Page LIST OF TABLES O O O O O O I O O O O O O O O O O O O V LIST OF FIGURES O O O O C O O O O O O O O O I O O O O Vii Introduction . . . . . . . . . . . . . . . . . . . . 1 Literature Review . . . . . . . . . . . . . . . . . . 3 Properties of Nitrate Reductase . . . . . . . . . . 3 Effects of Metabolites on Nitrate Reduction . . . . 4 Regulation of Nitrate Reductase by Nitrate Uptake . . . . . . . . . . . . . . . . . . . . . . 6 Nitrate Reduction and Source of Reducing Equivalents . . . . . . . . . . . . . . . . . . . . 7 Nitrate Reduction and the Generation of Reductant via Photosynthesis . . . . . . . . . . 8 Nitrate Reduction and the Generation of Reductant via Respiration and Photosynthesis --Use of Inhibitors . . . . . . . . . . . . . . . . 12 Nitrate Reduction and the Generation of Reductants via Respiration . . . . . . . . . . . . l3 Nitrite Reduction . . . . . . . . . . . . . . . . . 14 Literature Cited . . . . . . . . . . . . . . . . . 17 Section I. Substrate Induction of Nitrate Reductase in Barley Aleurone Layers . . . . . . . . . 23 Introduction . . . . . . . . . . . . . . . . . . 24 Materials and Methods . . . . . . . . . . . . . . . 25 Results 0 I O O O O O O O O O O O O O O O O O O O O 28 DiSCUSSiOn o o o o ' o o o o o o o o o o o o o o c o 42 Literature Cited 0 O O O O O O O O O I O O O I O C 4 4 Section II. Control of Nitrate Reductase Activity in Barley Aleurone Layers . . . . . . . . . 45 Introduction . . . . . . . . . . . . . . . . . . 47 Materials and Methods . . . . . . . . . . . . . . . 50 Results 0 O O O O O I O O O O O O O I O O O O O O O 53 iii Discussion . . . . . . . . . Literature Cited . . . . . . Section III. Nitrite Reductase Barley Aleurone Layers . . . . Introduction . . . . . . Materials and Methods . . . . Results . . . . . . . . . . . Discussion . . . . . . . . . Literature Cited . . . . . . Dissertation Summary . . . . . APPENDIX C O O O O O O O O O 0 iv Activity in Page 69 72 74 76 79 101 104 106 109 Table LIST OF TABLES Section I 1. 2. 3. Effect of GA, nitrate, and GA + nitrate on the induction of nitrate reductase and alpha-MYlaSe o o o o o o o o o o o o o o 0 Inhibition of nitrate reductase and alpha- amylase induction by abscisic acid . . . . Effect of metabolic inhibitors on nitrate reductase synthesis . . . . . . . . . . . . Section II 1. Nitrate reductase activity as determined by the cell-free and intact-tissue assay methOdS O I O O O O O O O O O O O O O I O 0 Dependence of nitrate reductase activity upon components of the cell-free assay media 0 O O O O O O O O O O O O O I O O O O Dependency of nitrate reductase activity upon phosphate or arsenate in the anaerobic intact-tissue assay . . . . . . . . . . . . Effect of ethanol on nitrate reductase in the anaerobic intact-tissue assay . . . . . Effect of HOQNO and antimycin A on oxygen uptake 0 O O O O O I O O O C O C I C O O 0 Effect of KNlBO3 or KN16O3 on nitrate reductase . . . . . . . . . . . . . . . . . Section III 1. Temperature coefficients for uptake and disappearance of nitrite by nitrate- induced and noninduced tissue . . . . . . . V Page 34 38 39 54 54 56 56 62 7O 87 Table Page 2. Effect of protein synthesis inhibitors on the uptake and disappearance of nitrite in noninduced and nitrate- induced aleurone layers . . . . . . . . . . . 88 3. Effect of respiratory inhibitors on the disappearance of nitrite by aleurone layers I I I I I I I I I I I I I I I I I I I 89 4. Effect of respiratory inhibitors on the uptake of nitrite by aleurone layers . . . . 90 5. Nitrite accumulation by aleurone layers at pH 4.5 O I I I I I I I I I I I I I I I I I 92 6. Release of accumulated nitrite under aerobic and anaerobic conditions at pH 7.5 I I I I I I I I I I I I I I I I I I I 93 7. Nitrite production and disappearance by nitrate-induced and noninduced tissue assayed in the presence or absence of low nitrite concentration . . . . . . . . . . 96 8. Effect of pentachlorophenol and ioxynil on nitrite uptake and disappearance by nitrate-induced and noninduced aleurone layers . . . . . . . . . . . . . . . . . . . 93 9. Effect of DNP on nitrate reduction . . . . . . 100 vi LIST OF FIGURES Figure Section I 1. Kinetics of nitrate reductase induction . . 2. Effect of substrate concentration on the induction of nitrate reductase . . . . . 3. Effect of GA on nitrate reductase synthesis 4. Mid-course inhibition of nitrate reductase synthesis by 6—methylpurine . . . . . . . Section II 1. Concentration curve of ethanol-enhanced nitrite release in the anaerobic intact- tissue assay . . . . . . . . . . . . . . 2 and 3. Antimycin and HOQNO-enhanced nitrite release under aerobic conditions . . . . 4. Nitrate reductase induction kinetics as determined by four different assay methods 5. Decay kinetics of nitrate reductase .'. . . Section III 1. Effect of pH on nitrite uptake . . . . . . 2. Kinetics of nitrite uptake . . . . . . . . 3. Kinetics of nitrite disappearance . . . . . 4 and 5. Effect of 2,4-dinitrophenol on nitrite uptake and disappearance . . . . . . . . 6. Release of accumulated nitrite by aleurone layers . . . . . . . . . . . . . . . . . vii Page 29 31 35 40 57 6O 64 66 80 82 82 85 94 Guidance Committee: This thesis is divided into sections I, II and III. Each was intended for publication, and was written in for- mats suited for publication in the Journal of Plant Phys- iology or the Proceedings of the National Academy of Science. The first two sections have already appeared in print. viii INTRODUCTION "'Nitrate assimilation' represents the biological conversion of nitrate to ammonia or to the amino acid or amide level for the ultimate synthesis of nitrogen con— taining cell constituents (48)." Approximately 10 billion tons of nitroqen are incorporated into plant life on a world wide basis per year (7). Although some of this fixed nitrogen comes from nitrogen fixing bacteria living symbiotically with plants, the bulk of fixed nitrogen arises from the reduction of nitrates by plants after being taken up from the soil. The importance of nitroqen as a plant constituent is reflected by its occurrence in a wide variety of bio- logically prominent molecules 2121' proteins, nucleic acids, vitamins. Yet, unlike most other elements, nitrate nitrogen cannot be utilized directly by plant or animal cells; it must first undergo an eight electron reduction prior to its functioning in the cell's metabolism. Thus, considerable research has been devoted by investigators in an attempt to understand the reduction process and its regulation by environmental and cellular factors. Detailed information on nitrate reductase--the first enzyme of the nitrate assimilating pathway—-can be found in several excellent reviews (7, 37, 46, 47, 48, 55). The following review will concentrate on how environmental and cellular factors control the activity of the nitrate reducing pathway. -_- LITERATURE REVIEW Properties of Nitrate Reductase In 1896, a nitrate reducing factor was detected in potato extracts by Each (6). Eight years later, the enzy- matic nature of the reduction of nitrate by crude extracts of plants was provided by Kastle and Elvove (33). These workers showed that potato and certain other plants contain a reducing substance or substances capable of effecting the reduction of nitrate to nitrite. Furthermore, they showed that reduction occurred most rapidly at 40 to 45°C, was augmented by an increase in the concentration of ni- trate and reductant, was inhibited by certain metabolic poisons, was accelerated by specific substances, and that the reducing property of the extract was completely lost by boiling. Except for an occasional observation (63), nitrate reductase has been found in all green and non-green plant tissues in which it has been sought. Though its presence in crude cell—free extracts was demonstrated as early as 1904 (33), Kessler (37) has credited Evans, Nason and Nicholas with identifying and characterizing nitrate re- ductase as the enzyme catalyzing the nitrate to nitrite reduction. Nitrate reductase in both plants and microorganisms appears to be an adaptive enzyme.* Its formation can be induced by nitrate, and molybdenum appears to be necessary for activity (7). Induction is preceded by a lag period as short as 20 minutes or as long as 25 hours, depending on plant material (11, 30). The enzyme appears to have a turnover rate (half-time) of 3 to 4 hours (2, 57). Evi- dence suggests that light either increases the rate of synthesis or prevents its breakdown (13, 25, 64). Based on studies from a wide variety of tissues (7, 37), nitrate reductase has been found to be a metallo- flaVOprotein of molecular weight 500,000 to 600,000. The enzyme contains flavin adenine dinucleotide, molybdenum, iron and active sulfhydryl groups. Reduced nicotinamide adenine dinucleotide serves as hydrogen donor. The labil- ity of the enzyme during extraction has hampered detailed characterization of the protein. Effects of Metabolites on Nitrate Reduction Feedback control at the molecular level is an im- portant regulatory mechanism of enzyme activity in many organisms (5), and there is increasing evidence that ni- trate reductase in plants is under such control. For example, the induction of nitrate reductase in tobacco cells grown in suspension culture may be repressed or de- repressed by a variety of amino acids (20). Amino acids with repressor-like activity included alanine, asparagine, glycine, methionine, proline, threonine, valine, aspartate, glutamate, histidine and leucine. In the presence of any of these repressors, arginine and lysine were found to act as derepressors. Except when in the presence of methionine or alanine, cysteine and isoleucine also derepressed. In addition, casein hydrolysate inhibited enzyme formation. In corn seedlings, several secondary metabolites of inorganic nitrogen metabolism were evaluated as inhibi- tors of nitrate reductase induction (56). 0f nine differ- ent phenylpropanoid compounds tested, only coumarin, trans- cinnamic acid and trans-o-hydroxycinnamic acid inhibited nitrate induction. In this same study, carbamyl phosphate and cyanate, were found to be competitive inhibitors (with nitrate) of nitrate reductase. The effects of carbamyl phOSphate and cyanate are interesting in that they indicate regulation by directly interacting with the enzyme. Cell- free preparations of apple roots contain a potent inhibitor of nitrate reduction (24). The compound(s) was removed by dialysis against phosphate buffer or passage through Sephadex. The significance of this finding to the in zizg activity of nitrate reduction is uncertain. Nitrate reductase activity extracted from sunflower leaves is increased two and three times by treatment of the seedlings with adenine and uracil, respectively (53). In cauliflower, serine is capable of stimulating production of nitrate reductase activity (1). Regulation of Nitrate Reductase bnyItrate Uptake Nitrate is necessary for the induction of nitrate reductase. The idea that nitrate has to be present in the cell to "turn on" the machinery necessary for enzyme syn- thesis, subjects this process to an important and often overlooked cellular control mechanism--regulation of ni- trate uptake by the cell. There is considerable direct and indirect evidence in support of such an hypothesis, and this regulatory aspect may involve an active control mechanism. Evidence for such a control mechanism is not un- common in nature. For example, nitrate reductase in Escherichia coli is constitutive, but there exists an adaptive system for nitrate uptake (19). In the algae Ankistrodesmus braunii there appears to be at low nitrate concentrations a 2,4—dinitrophenol (DNP)-sensitive nitrate uptake mechanism (3). The authors propose that at low concentrations of nitrate, uptake is active. But, as con- centrations are increased, passive uptake (DNP insensitive) accounts for a greater percentage of nitrate uptake. There is evidence that nitrate uptake in higher plants also involves an active uptake mechanism. In ex- cised wheat roots, nitrate uptake is increased by glucose and inhibited by galactose and salicylic acid (58). In rye seedlings, light was shown to promote nitrate uptake and this was followed by increased enzyme activity (13). Whereas ammonium inhibited nitrate uptake by nitrogen starved wheat seedlings, it had no significant effect on the enzymatic reduction of accumulated nitrate (43). Ex- periments with tobacco cell suspensions indicate that nitrate was accumulated against a concentration gradient, and that this uptake was inhibited by cyanide, DNP and threonine (30). In addition, there appeared to be little or no passive uptake of nitrate into tobacco cells. Of particular interest was the finding that all nitrate in the cell is not available for enzyme induction. When fully induced tobacco cells were transferred to nitrateless media, nitrate reductase activity declined despite the presence of high nitrate concentrations within the cells. Nitrate Reduction and Source of Reducing Equivalents Several in_xitgg_systems, often employing elaborate experimental conditions, have been devised which attempt to correlate an enzymatic reaction with the reduction of nitrate to nitrite (17, 18, 27, 39, 41). Without excep- tion, each system studied utilizes two common components: nitrate reductase and an NADH-generating reaction(s). Nitrate reductase is an obligate component in all nitrate- reducing systems described. It appears that nitrate reduc- tion in 21252 may be coupled to any of a variety of light- or dark-mediated NADH-generating reactions e.g., ethanol + alcohol dehydrogenase (17, 27), 3-phosphoglyceraldehyde + glyceraldehyde-3-phosphate dehydrogenase (39), aldehyde + aldehyde oxidase (9), hydrogen + a bacterial hydrogenase (15), chloroplasts or grana + light (18, 41). Thus, two nitrate reducing systems might operate in intact cells: one dependent on light-mediated genera- tion of reducing equivalents and the other dependent on dark reactions. Nitrate Reduction and the Generation of Reductant Via Photosynthesis Light has long been known to stimulate nitrate assimilation in green leaves and algae (8, 12, 35, 36, 42, 59). Since this discovery by Warburg and Neglein in 1920 (64), the explanation of this effect is still not yet agreed upon. Originally, Warburg and Neglein attributed the increase of nitrate assimilation by light to a perme- ability effect; that is, substrate becomes more readily available. They believed that in both the light and dark the reduction of nitrate to ammonia was coupled to respi— ration, and that extra carbon dioxide production observed during nitrate assimilation in the dark came from in- creased carbohydrate breakdown. Also, an indirect effect of light, due simply to the ample production of carbon compounds by photosynthesis was considered. The carbohy- drate thus produced would--via respiration--serve as the electron donors required for the reduction of nitrate, just as they do in the dark. A more direct consequence of light is possible. van Niel, Allen and Wright (60) proposed that light acted by providing increased amounts of reductant from photosyn- thetic electron transport. Extra oxygen production ob- served in the light during nitrate reduction was postulated to come from the water-splitting reaction of photosynthesis. And increased rates of electron flow occur as a result of the electrons being shuttled off to reduce nitrate after saturation of the carbon dioxide-fixing system. In support of this latter hypothesis, Chlorella cells exposed to high light intensity and incubated in the presence of nitrate were found to evolve more oxygen com- pared to cells incubated in the absence of nitrate (60). This observation, coupled with the finding that the gen- eration of reductants by isolated chloroplasts could be coupled to the reduction of nitrate by nitrate reductase (18, 32, 41), provides the major impetus for support of van Niel's hypothesis. In addition, light has been found to increase the amount of reductant in spinach chloroplasts 111m (29). The required stoichiometry of the photochemical reduction of nitrate or nitrite may be expressed by the following equations: \ (a) HNo3 + H20 /,NH3 + 202 (b) 2HNo2 + 2H20—————+;>2NH3 + 302 10 The equations predict a ratio of oxygen evolved to nitrate or nitrite reduced (assimilated) of 2 and 1.5, respectively. There are several reports in the literature in which such ratios for nitrate (10, 14, 66) and nitrite reduction (10, 26, 61) were measured. The major obstacle for such an approach toward explaining the action of light on nitrate l reduction is that the same ratios can be derived by ex- ‘ panding equations (a) and (b) to the following for nitrate (c and d) and nitrite reduction (e and f): (c) 2co2 + 2H20 light > 2(CH20) + 202 (a) (d) HN03 + 2(CH20)——§E£E—;>NH3 + H20 + 2co2 light \\ (e) 3C02 + 3H20 ,/3(CH20) + 302 (b) dark :1 (f) ZHNO + 3(CH20) j/2NH3 + H20 + 3C02 2 In both cases, the ratio of nitrate or nitrite reduced to oxygen is the same as in equations (a) and (b). Since carbon dioxide production and utilization (via respiration and photosynthesis, respectively) are equal, only catalytic quantities of carbon dioxide would be required to permit reactions (0) through (f) to occur. And there is evidence that greater than 90% of carbon dioxide produced during anaerobiosis can be reutilized in photosynthesis (50, Table IV). Thus, one cannot unequivocably distinguish between the two sets of equations based on the reported ratios of oxygen evolved to nitrate or nitrite reduced. 11 Despite in_zitrg evidence in favor of light— mediated reduction of nitrate, several observations tend to detract from such an hypothesis. Perhaps the most im- portant is the carbon dioxide requirement for light-stim- ulated nitrate reduction (10, 14, 22, 23). If nitrate were directly accepting electrons from photosynthetic light reactions, one would not expect a requirement for carbon dioxide. One cannot exclude at this time that electron transport is dependent in some way upon catalytic quantities of carbon dioxide, as appears to be the case in some algae (31). The observation that oxygen evolution is unaffected by the presence of nitrate, even at photosyn- thetically saturating light intensities (10) is in dis- agreement with van Niel's hypothesis which requires that at sufficiently high light intensities the rate of oxygen production would be enhanced by the addition of extra oxidant--namely nitrate (60). Furthermore, that only a loose "coupling" exists between light-generated reductants and nitrate reduction is indicated by (a) the findings that oxygen evolution, carbon fixation, the Hill reaction, and nitrate reduction show differential sensitivity to 3-(4-chloropheny1)-l,l-dimethy1 urea (CMU) (26), (b) the failure to obtain the theoretical ratios of oxygen evolved to nitrate assimilated by some investigators (22, 26, 64) (c) the observation that oxygen evolution remains the same in the presence or absence of nitrate at all light 12 intensities (10) and (d) the finding that nitrate reduc— tion is saturated at relatively low light intensities (10, 22, 26). Most arguments concerning the involvement of light in the reduction of nitrate have overlooked a significant study reported in 1955 by Good (21). In this study, mass spectroscopy was used to independently measure oxygen and carbon dioxide production by Chlorella cells incubated in the light with a helium atmosphere. Upon addition of ferricyanide to the reaction medium carbon dioxide evolu- tion occurred; then, several minutes later, oxygen evolu- tion began to occur. Ferricyanide also stimulated carbon dioxide evolution in the dark. By analogy to ferricyan— ide, these results suggest that nitrate might act as an electron acceptor of reductants generated via glycolysis, fermentation or the tricarboxylic acid cycle. The carbon dioxide thus produced by the operation of these pathways, could in turn act as a final electron acceptor in photo- synthesis--thereby causing, indirectly, oxygen evolution to occur. Therefore it seems likely that there may be an indirect interaction between the processes of photosyn- thesis and respiration in nitrate assimilation. Nitrate Reduction and the Generation of Reductant via Respiration and Photosynthesis--Use of Inhibitors The use of metabolic inhibitors has indicated a relationship between the generation of reductants from l3 respiration or photosynthesis and nitrate reduction in both higher and lower plants. With suspensions of the marine chlorOphyte Dunaliella tertiolecta, CMU reduced the rate of nitrate reduction in the light to rates com- parable to those occurring in the dark in the absence of inhibitor (22). CMU had little or no effect on nitrate reduction in the dark. Alternatively, iodoacetate was without effect on nitrate reduction in the light, yet it abolished nitrate reduction in the dark. Similar results for iodoacetate were obtained with tomato leaf disks (42). In this species, 15N-nitrate as- similation was inhibited by malonate and iodoacetate in the dark but not in the light. Iodoacetate inhibition was overcome by succinate and citrate, indicating that nitrate reduction in the dark is dependent on some product(s) of glycolysis or the tricarboxylic acid cycle. The accumula- tion of nitrite in intact wheat root tips was also inhib- ited by iodoacetate (45). Thus, the differential effects of CMU and iodoacetate with respect to light regime indi- cates that nitrate reduction is influenced by two different electron generating systems. Nitrate Reduction and the Generation of Reductants via Respiration Nitrate assimilation requires an extra reductive step compared to nitrite assimilation. Therefore, if respiration were involved in the generation of reductant 14 and energy in these processes, one would expect a greater amount of carbon dioxide evolution in the dark for nitrate assimilation compared to that for nitrite. This has been shown for intact cells of Q. tertiolecta, where the ratio of carbon dioxide evolved to nitrogen assimilated was 2.0 for nitrate and 1.5 for nitrite (23). Similarly, respira- tory quotients of barley roots incubated in the presence of nitrate and nitrite were 1.3 and 1.1, respectively (67). Coupling reducing equivalents to nitrate reduction via respiration is also indicated by an increased carbon dioxide production in the dark when intact root tips of barley (67) or cells of Q. tertiolecta (22) are incubated in the presence of nitrate. Intermediates of the tri- carboxylic acid cycle also increased 15N-nitrate assimila- tion in tomato leaf disks (42) and in cell-free prepara- tions from a variety of plants (16). Finally, in tomato leaf disks, carbohydrate depleted tissue was found to assimilate less nitrate than non-depleted tissue (42). No difference could be detected between depleted and non- depleted tissue when placed in the light. Nitrite Reduction In addition to nitrate reduction, evidence indi- cates that nitrite reduction is also affected by light. With cells of 2, tertiolecta (22), leaves of wheat (61), tobacco and broad bean (62), nitrite assimilation in the 15 light is approximately 2 to 20 times as great as rates observed in the dark. Light-stimulated nitrite assimila- tion was found to be dependent on carbon dioxide for Chlorella (14) and Q. tertiolecta (22). In the absence of carbon dioxide, nitrite reduction in the light with Q. tertiolecta occurred at rates similar to dark rates (22). Nitrite reduction in the light by Ankistrodesmus was ac- celerated by carbon dioxide (26). With Chlorella, glucose could overcome the requirement for carbon dioxide in the light (14). Light-stimulated reduction of nitrite by Q. tertiolecta and Anabaena gylindrica was partially inhibited by CMU whereas dark reduction was unaffected (22, 26). However, nitrite assimilation by E. tertiolecta was inhib- ited by iodoacetate in both the light (35%) and dark (60%) (22). For nonphotosynthetic tissues, one would expect an increase in the respiratory quotient during nitrite reduc- tion if respiration was involved. This was found to be the case for barley roots growing in nitrite-containing solutions (67). The accumulation of nitrite by plants fed nitrate in the dark under anaerobic conditions also indi- cates a requirement for electron transport or oxidative phosphorylation in nitrite reduction (40, 44, 52). The ability to inhibit nitrite reduction by DNP in a variety of higher and lower plants suggests energy is the required factor (3, 26, 34, 54, 62). Because other uncouplers of l6 oxidative phosphorylation also inhibit nitrite reduction (4, 38), it seems unlikely that DNP is exerting its effect by alternatively accepting reducing equivalents as has been shown to be the case with isolates of Pseudomonas denitrificans (51) and reductants generated by spinach chlorOplasts (65). 10. LITERATURE CITED Afridi, M. M. R. K. and E. J. Hewitt. 1965. The in- ducible formation and stability of nitrate reduc- tase in higher plants. II. Effects of environ- mental factors, antimetabolites, and amino acids on induction. J. Exp. Bot. 16:628-645. _-_——A 3-? l" Afridi, M. M. R. K. and E. J. Hewitt. 1964. The in- ducible formation and stability of nitrate reduc- tase. J. Exp. Bot. 15:251-271. Ahmad, J. and I. Morris. 1967. Inhibition of nitrate and nitrite reduction by 2,4—dinitrophenol in - Ankistrodesmus. Archiv. Mikrobiol. 56:219-224. Ahmad, J. and I. Morris. 1968. 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Some effects of sodium on nitrate assimilation in anabaena cylindrica. Plant Physiol. 42:915-921. Burnstrom, H. 1943. Photosynthesis and assimilation of nitrate by wheat leaves. K. Lanterogsk. Annlr. II, 1. [Cited by Grant (8)]. Chen, T. M. and S. K. Ries. 1969. Effect of light and temperature on nitrate uptake and nitrate re- ductase activity in rye and oat seedlings. Can. J. Bot. 47:341—343. Davis, E. A. 1953. Nitrate reduction by Chlorella. Plant Physiol. 28:539-544. Del Campo, F. F., A. Paneque, J. M. Ramirez and M. Losada. 1965. Nitrate reduction with molecular hydrogen in a reconstituted enzymic system. Nature (Lond.) 205:387-388. Delwiche, C. C. 1952. Reduction of nitrite and nitrate ions by preparations obtained from higher plants. Fed. Proc. 11:201-202. Egami, F., K Omachi, K. Lida and S. Taniguchi. 1957. Nitrate reducing systems in cotyledons and seed- lings of bean seed embryos of Vi na ses ui edalis during the germinating stage. 310 him. 22:115- 127. Evans, H. J. and A. Nason. 1953. Pyridine nucleotide nitrate reductase from extracts of higher plants. Plant Physiol. 28:233-254. Farkos-Hinsley, H. and M. Artman. 1957. Studies on nitrate reduction by Escherichia coli. J. Bac- teriol. 74:690-692. Filner, P. 1966. Regulation of nitrate reductase in cultured tobacco cells. Biochim. Biophys. Acta 118:299-310. Good, N. 1955. Reduction of ferricyanide by algal suspensions. Plant Physiol. 30:483-484. Grant, B. R. 1967. The action of light on nitrate and nitrite assimilation by the marine chlorophyte Dunaliella tertiolecta (Butcher). J. Gen. Micro- biol. 48?379-389. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 19 Grant, B. R. 1968. The effect of carbon dioxide.con- centration and buffer system on nitrate and nitrite assimilation by Dunaliella tertiolecta. J. Gen. Microbiol. 54:327-336. Grassmanis, V. O. and D. J. D. Nicholas. 1967. A nitrate reductase from apple roots. Phytochem- istry. 6:217-218. Hageman, R. H. and D. Flesher. 1963. Nitrate reduc- tase activity in corn seedlings as affected by light and nitrate content of nutrient media. Plant Physiol. 35:700-708. Hattori, A. 1962. Light induced reduction of nitrate, nitrite and hydroxylamine in a blue green algae, Anabaena cylindrica. Plant Cell Physiol. 3:355- 369. Hattori, A. and J. Myers. 1966. Reduction of nitrate and nitrite by subcellular preparations of Anabaena cylindrica. I. Reduction of nitrite to ammonia. Plant Physiol. 41:1031-1036. Hattori, A. and J. Myers. 1967. Reduction of nitrate and nitrite by subcellular preparations of Anabaena c lindrica. II. Reduction of nitrate to nitrite. PIant CelI Physiol. 8:327-337. Heber, V. W. and K. A. Santarius. 1965. Compartment- ation and reduction of pyridine nucleotides in relation to photosynthesis. Biochim. Biophys. Acta 109:390-408. Heimer, Y. 1970. Control of nitrate assimilation in cultured tobacco cells. Ph.D. Thesis. Michigan State University, East Lansing, Michigan. Heise, J. J. and H. Gaffron. 1963. Catalytic effects of carbon dioxide in carbon dioxide assimilating cells. Plant and Cell Physiol. 4:1-11. Huzisige, H., K. Satoh, K. Tanaka, and T. Hayasida. 1963. Photosynthetic nitrite reductase II. Fur- ther purification and biochemical properties of the enzyme. Plant and Cell Physiol. 4:307-322. Kastle, J. H. and E. Elvove. 1904. On the reduction of nitrates by certain plant extracts and metals, and the accelerating effect of certain substances on the progress of the reduction. Amer. Chem. J. 31:606-641. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 20 Kessler, E. 1955a. Uber die Wirkung von 2,4-Dinitro- phenol auf Nitratereducktion und Atmung von Grfinalgen. Planta (Berl.) 45:94-105. Kessler, E. 1955b. Role of photochemical processes in the reduction of nitrate by green algae. Nature 176:1069-1070. Kessler, E. 1959. Reduction of nitrate by green algae. Soc. Exp. Biol. Symp. 13:87-105. Kessler, E. 1964. Nitrate assimilation by plants. Annu. Rev. Plant Physiol. 15:57-72. Kessler, E. and W. Bucher. 1960. Uber die Wirking von Arsenat auf Nitratreduktion, Atmung und Photo- synthese von Grfinalgen. Planta (Berl.) 55:512-524. Klepper, L. and R. H. Hageman. 1969. Dependence of nitrate reduction on generation of reduced nico- E tinamide adenine dinucleotide by glycolytic metab- olism of leaf tissue. 11th Int. Bot. Congr. Abstr. Kumada, H. 1953. The nitrate utilization in seed embryos of Vigna sesquipedalis. J. Biochem. 40: 439-450. Losoda, M., J. M. Ramirez, A. Paneque and F. F. Del Campo. 1965. Light and dark reduction of nitrate in a reconstituted chloroplast system. Biochim. Biophys. Acta 109:86-96. ' Mendel, J. L. and D. W. Visser. 1951. Studies on nitrate reduction in higher plants. I. Arch. Biochem. Biophys. 32:158-169. Minotti, P. L., D. C. Williams and W. A. Jackson. 1969. The influence of ammonium on nitrate reduc- tion in wheat seedlings. Planta 86:267-271. Nance, J. F. 1948. The role of oxygen in nitrate assimilation by wheat roots. Amer. J. Bot. 35: 602-606. Nance, J. F. 1950. Inhibition of nitrate assimilation in excised wheat roots by various respiratory poisons. Plant Physiol. 25:722-735. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 21 Nason, A. 1956. Enzymatic steps in the assimilation of nitrate and nitrite in fungi and green plants, pp. 109—136. W. D. McElroy and B. Glass. Sympos- ium Inorganic Nitrogen Metabolism. Johns Hopkins Press, Baltimore. Nason, A. 1962. Symposium on metabolism of inorganic compounds II. Enzymatic pathways of nitrate, nitrite, and hydroxylamine metabolisms. Bacteriol. Rev. 26:16-41. Nason, A. and McElroy. 1963. Modes of action of the essential mineral elements, Vol. III, p. 490. F. C. Steward. Plant Physiology, Academic Press, New York. Nightingale, G. T. 1937. The nitrogen nutrition of green plants. Bot. Rev. V(III):85-l74. _ Nishida, K. 1962. Studies on the re-assimilation of respiratory C02 in illuminated leaves. Plant Cell Physiol. 3:111-124. Radcliff, B. C. and D. J. D. Nicholas. 1968. Some properties of nitrite reductase from Pseudomonas denitrificans. Biochim. Biophys. Acta 153:545-554. Randall, P. J. 1969. Changes in nitrate and nitrate reductase levels on restoration of molybdenum to molybdenum-deficient plants. Aust. J. Agr. Res. 20:635-642. Ratner, E. I. and S. A. Samoilova. 1966. Influence of exogenous ribonucleic acid and some of its nitrogenous bases on the reduction of nitrates in plant tissue and the symbiotic fixation of atmos~ pheric nitrogen in soya. [Transl. from Russian] Sov. Plant Physiol. 13:914-924. Ritenour, G. L., K. W. Joy, J. Bunning and R. H. Hage- man. 1967. Intracellular localization of nitrate reductase, nitrite reductase, and glutamic acid dehydrogenase. Plant Physiol. 42:233-237. Robinson, M. E. 1929. The protein metabolism of the green plant. New Phytol. 28:117-149. Schrader, L. E. and R. H. Hageman. 1967. Regulation of nitrate reductase activity in corn (Zea ma 5 L.) seedlings by endogenous metabolites. PIEHt P ys- iol. 42:1750-1756. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 22 Schrader, L. E., G. L. Ritenour, G. L. Eilrich, and R. H. Hageman. 1968. Some characteristics of nitrate reductase from higher plants. Plant Physiol. 43:930-940. Stenlid, G. 1957. Some differences between the accu- mulation of chloride and nitrate ions in excised wheat roots. Physiol. Plant. 10:922-936. Syrett, P. J. 1962. Nitrogen assimilation, pp. 171- 188. Ig_R. R. Lewis, Physiology and Biochemistry of Algae. Acad. Press, New York. van Niel, C. B., M. B. Allen and B. S. Wright. 1953. On the photochemical reduction of nitrate by algae. Biochim. Biophys. Acta 12:67-74. Vanecko, S. and J. E. Varner. 1955. Studies on ni- trite metabolism in higher plants. Plant Physiol. 30:388-390. Voskresenskaya, N. P. and G. S. Grishina. 1962. The significance of light for nitrite reduction in the green leaf. [Transl. from Russian] Sov. Plant Physiol. 1962. 9:4-10. Wallace, W. and J. S. Pate. 1967. Nitrate assimila- tion in higher plants with special reference to the cockelbur (Xanthium pennsylvanicum). Ann. Bot. 31:213-228. Warburg, O. and E. Negelein. 1920. Uber die Reduktion der Saltpetersafire in grfinnen Zellen. Biochem. Zeit. 110:66-115. Wessels, J. S. C. 1965. Mechanism of the reduction of organic nitro compounds by chloroplasts. Biochim. Biophys. Acta 109:357-371. Warburg, 0., G. Krippahl and C. Jetschmann. 1965. Widerlegung der Photolyse des H20 und Beweis der von C02. Z. Naturforsch. 206:993-996. Willis, A. J. and E. W. Yemm. 1955. The respiration of barley plants 11. Nitrogen assimilation and the respiration of the root system. New Phytol. 54:163-181. SECTION I ABSTRACT SUBSTRATE INDUCTION OF NITRATE REDUCTASE IN BARLEY ALEURONE LAYERS BY Thomas Enrico Ferrari .' Nitrate induces the formation of nitrate reductase (“r activity in barley (Hordeum vulgare L. cv. Himalaya) aleu- rone layers. Previous work has demonstrated dg_ngzg_syn- thesis of alpha-amylase by gibberellic acid in the same tissue. The increase in nitrate reductase activity is inhibited by cycloheximide and 6-methylpurine, but not by actinomycin D. Nitrate does not induce alpha-amylase synthesis and it has no effect on the gibberellic acid- induced synthesis of alpha-amylase. Also, there is little or no direct effect of gibberellic acid (during the first 6 hours of induction) or of abscisic acid on the nitrate- induced formation of nitrate reductase. Gibberellic acid does interfere with nitrate reductase activity during long-term experiments (greater than 6 hours). However, the time course of this inhibition suggests that the in- hibition may be a secondary one. Barley aleurone layers therefore provide a convenient tissue for the study of both substrate- and hormone-induced enzyme formation. 23 INTRODUCTION Gibberellic acid induces increases of alpha-amylase and other hydrolases in barley half-seeds (8, 9). For amylase (5) and for protease (6) these increases have been shown to be the result of dg_ngzg_synthesis which is de- pendent upon the continued presence of GA3 (2) and con- tinued synthesis of some unidentified kind (or kinds) of RNA (3). These increases in the hydrolases, in addition to being inhibited by protein synthesis and RNA synthesis inhibitors, are inhibited by abscisic acid (1), anaero- biosis and l to 2% ethyl alcohol. How many of these con- trols for gibberellin-induced hydrolase synthesis are characteristic of the d3.ngzg synthesis of any protein? And how many are specific for gibberellin-induced enzyme synthesis? To answer these questions we looked for the formation of a substrate-induced enzyme. More specifical- ly, we looked for the nitrate-induced formation of nitrate reductase. There is no nitrate reductase in the dry seed and it does not appear during the incubation of the half- seed or isolated aleurone layer until nitrate is added. 24 MATERIALS AND METHODS Enzyme Induction and Extraction The procedure used to prepare aleurone layers was the same as described by Jones and Varner (7) and the ex- traction of nitrate reductase (NR) was similar to that of Filner (4). Briefly, the preparation of tissue and ex- traction of NR and alpha-amylase are as follows. Embryos were excised from barley (Hordeum vulgare L. cv. Himalaya) and the half-seeds sterilized in 1% sodium hypochlorite, rinsed in sterile deionized water, and imbibed on moist sand for 3 to 5 days prior to removal of the aleurone layer. Ten isolated aleurone layers were used per treat- ment. Unless stated otherwise, the basic incubation med- ium for all experiments with aleurone layers consisted of the following in a total of 2 m1: 2 X 10-3 M acetate buffer (pH 4.8), 2 x 10’2 M CaClz, chloramphenicol, and 5 X 10"2 M KNO3. In experiments with half-seeds, the 2 ml incubation medium consisted of 1 drop 1 drop of 0.5 mg/ml of 0.5 mg/ml chloramphenicol and the concentration of KNO3 as indicated in the text. Aleurone layers were homogenized with sand, by morter and pestle in a total of 2 m1 extraction medium consisting of 0.1 M tris-HCl buffer (pH 7.5) and 1 mM 25 26 cysteine. The homogenate was clarified by centrifugation at 5000g for 10 minutes, and the supernatant fraction used as the crude enzyme. All steps in the extraction procedure were carried out at 0 to 4°C. Enzyme Assays NR activity was determined in an assay medium con- sisting of the following components in a total of 0.4 ml: 0.12 ml, 0.167 M phosphate buffer (pH 7.5); 0.04 ml, 0.1 M KNO 0.02 ml, 2 mM flavin mononucleotide; 0.02 ml 3. Na28204, 8 mg/ml, in 0.095 M NaHCO3; and 0.2 m1 crude enzyme preparation. Up to this point, tubes were kept in an ice bath. Each tube was shaken gently to mix the reac- tion components and reduce the flavin mononucleotide (evi- denced by a change in color from yellow to pale yellow). Just prior to placing the tubes in a water bath at 25°C to start the reaction, 1 drop of paraffin oil was run down the side of each tube. The paraffin formed a layer over the reaction medium, thereby preventing air oxidation of the reducing agent. After one-half hour, the reaction was stopped by shaking the reaction mixture vigorously until all the reducing agent was oxidized (evidenced by a return from the pale yellow to yellow color). Nitrite produced by enzymatic reduction of nitrate was determined by first adding 0.3 m1 of 1% sulfanilamide in 3 N HCl, followed by 0.3 ml of 0.02% N-l-naphthy1ethy1enediamine 2' IV 27 dihydrochloride. Optical density at 540 mu was measured after centrifuging at 5000g for 15 minutes. alpha-amylase activity was determined from the induction medium or in the NR enzyme extract, as described by Jones and Varner (7). RESULTS Production of Nitrate Reductase by Half-seeds From experiments with both dry and 3-day imbibed half-seeds, nitrate reductase (NR) could only be detected when incubated for 40 hours with nitrate. The optimum concentration of nitrate for induction was 5 x 10-2 M. No NR activity was detected in dry half-seeds or in half- seeds imbibed for 3 days on sand moistened with sterile deionized water. Nitrate Reductase_groduction by Isolated Aleurone Layers NR synthesized by isolated aleurone layers in re- sponse to nitrate occurs at a linear rate, after a lag period of about 30 minutes (Fig. 1), for at least 8 hours after the start of enzyme synthesis. The rate of NR for- mation is limited (Fig. 2) up to a concentration of ap- 2 2 M nitrate the rate proximately 10- M nitrate. Above 10- of enzyme synthesis decreased. No NR activity could be detected in the induction medium 24 hours after the start of the experiment, and no NR activity could be detected in aleurone layers incubated up to 24 hours without nitrate. 28 29 Figure l.--Kinetics of nitrate reductase induction. En- zyme activity was measured in a 0.4 ml aliquot of the crude enzyme extract. Samples were removed at the indicated time intervals and enzyme activity determined. 30 rim” 50 40 0 0 3 2 25.: 28338.. moz 10 302:5 INCUBATION TIME (HOURS) 31 Figure 2.--Effect of substrate concentration on the induc- tion of nitrate reductase. Nitrate reductase was measured after 16 hours incubation in 0.2 ml of crude enzyme extract. 32 3:: 33:2: moz 9.02: LOG No“; coucemanlon 33 Effect of GA on Nitrate Reductase Synthesis From the standpoint of induction specificity and control of enzyme synthesis, it seemed pertinent to deter- mine if GA could substitute for nitrate in the induction of NR and conversely, whether or not nitrate has any ef- fect on alpha-amylase induction. Experiments showed that m" (l) nitrate does not induce alpha-amylase; (2) GA does not induce NR; (3) nitrate does not prevent GA induction of alpha-amylase; and (4) GA does not directly prevent nitrate induction of NR (Table 1). GA does interfere with NR syn- thesis when incubated in the presence of nitrate for periods greater than 6 hours--the amount of inhibition being dependent on the concentration of GA used. With -6 M (Fig. 3) and 10—8 10 M GA (Table l) the amount of ni- trate reductase at 24 hours are 0% and 60% of control activities, respectively. The kinetics of GA interference of NR synthesis in a long-term (24 hours) experiment is 6 M GA has shown in Figure 3.’ It can be seen that 10- little or no effect on NR activity for the first 6 hours; however, as noted above, enzyme activity is abolished at 24 hours. The time course of this interference suggests that the interference is a secondary one. For example, it might be due to the consequences of the appearance of the GA-induced protease. 34 Table 1.--Effect of GA, N03, and GA + NO} on induction of nitrate reductase and alpha-amylase. Ten aleurone layers were incubated for 24 hours in 2 ml of 2 x 10-3 M acetate buffer (pH 4.8), 2 x 10-2 M CaC12, 1 drop of 0.5 mg/ml chloramphenicol and the concen- tration of KNO3 and GA indicated. Activityl NR of Amylase Treatment Extract of Medium % of control Control (-No', -GA) 0 100 + KNO3 (5 x 10’2 M) 100 90 + GA (10"8 M) 0 500 + mo3 + GA 60 500 lEach figure represents the average of 2 determi- nations. Control value for alpha-amylase in media repre- sents activity by-approximately 4 ug of alpha-amylase. 35 Figure 3.--Effect of GA on nitrate reductase synthesis. GA (10'6 M) was added at zero time and the amount of nitrate reductase in 0.4 ml of crude enzyme extract was assayed at indicated times. Potassium nitrate at 0.05 M was used during incubation. nMOLES NOS REDUCED/ LAYER 10 36 INCUBATION TIME (HOURS) +No3 -GA . + No; . + GA I? ° 1 I a 16 24 37 Effect of Abscisic Acid on Nitrate RedECtase NR synthesis is considerably less sensitive to abscisic acid (abscisin) than is alpha-amylase. With 10-6 M abscisic acid, NR was 80% of control activity, whereas alpha-amylase activity in the incubation medium and of the tissue extract were only 7% and 26%, respectively (Table 2). Inhibition of the order of magnitude observed with alpha-amylase was never observed with NR, even at the 5 highest concentration of abscisic acid tested (10- M) (Table 2). Sensitivity of Nitrate Reductase Synthesis to Several MetaBolic Inhibitors Cycloheximide was the most effective inhibitor of the chemicals tested (Table 3); actinomycin D (l ug/ml) 4 M inhibited stimulated activity; 6-methylpurine at 5 X 10- synthesis by 50%; and 8-azaguanine had little or no effect on production of the enzyme. The kinetics of mid-course inhibition of NR synthesis by 6-methylpurine are shown in Figure 4. 38 Table 2.--Inhibition of nitrate reductase and alpha-amylase induction by abscisic acid. Ten aleurone layers were incubated in 2 m1 of medium containing 2 X 10'3 M acetate buffer (pH 4.8), 2 x 10-2 M CaC12, 1 drop of 0.5 mg/ml chloramphenicol, 5 X 10‘2 M KNO3 or 10"8 M GA, and the concentration of abscisic acid as indicated. Activityl Time of Concn. (M) of Alpha-amylase Expt. Incubation Abscisic Acid NR Medium Extract % of control hr. control 100 100 100 I 24 10"6 80 7 26 II 6 10’6 78 --— --- III 6 10'5 66 --- --- 1Each number represents the average of 2 determi- nations. Percent control values for medium and extract represents activity of approximately 85 and 15 ug of alpha-amylase, respectively. 39 Table 3.--Effect of metabolic inhibitors on nitrate reduc- tase synthesis. Ten aleurone layers were incubated in 2 ml medium containing 2 X 10"3 M acetate (pH 4.8), 2 X 10'2 M CaC12, 5 X 10"2 M KNO , 1 drop of 0.5 mg/ml chloramphenicol and the concentration of the compound tested. Enzyme activity in a 0.4 m1 aliquot of enzyme extract was determined 6 hours after the start of each experiment. NR Activity]- mumole NOE'reduced per layer Expt. Treatment per 30 min. I Control 22 I Cycloheximide (1 ug/ml) 2 I Cycloheximide (5 ug/ml) 3 I Cycloheximide (10 ug/ml) 3 I 6-methy1purine (5 x 10-4 M) 11 I 6-methylpurine (10-4 M) 15 II Control 15 II Actinomycin D (1 ug/ml) 25 II Actinomycin D (10 ug/ml) 20 II Actinomycin D (40 ug/ml) 18 II 8-azaguanine (2.5 x 10’3 M) 16 1 Each number represents the average of 4 determi- nations. Each experiment was repeated twice. 40 Figure 4.--Mid-course inhibition of nitrate reductase syn- thesis by 6-methylpurine (3 mM). Aleurone layers were incubated in a standard induction medium containing 0.05 M potassium nitrate for 4 hours. At this time, 0.2 ml of 0.02 M 6- methylpurine was added giving a final concen- tration of 3 mM. Nitrate reductase activity was measured at 1, 2, and 4 hours following addition of the chemical. A 0.4 m1 aliquot of crude enzyme was used to determine enzyme activity. 41 50f- CONTROL /' . ,/+6-METHYLPURINE /ALEURONE LAYER u 0 I | N o 220 — W I“ —l o 2 C 10 \— __// l l I I l o 4 s 6 7 8 INCUBATION TIME (HOURS) DISCUSSION In barley aleurone layers, several differences exist between the substrate induction of nitrate reductase (NR) and the hormone induction of alpha-amylase. In con- trast to what is found with alpha-amylase, there is no detectable NR in half-seeds or in isolated aleurone layers after imbibition on sand with distilled deionized water or incubation in the absence of inducer. Unlike alpha-amylase (2), NR is not secreted from the aleurone layer. In this study no NR was-detected in the incubation medium as long as 24 hours after the start of the experiment. Also, NR synthesis is considerably less sensitive to inhibition by abscisic acid than is the synthesis of alpha-amylase. In contrast to what has been observed with alpha-amylase syn- thesis (3), 8-azaguanine has no effect on NR synthesis while actinomycin D enhances rather than inhibits NR ac- tivity. But as in alpha-amylase synthesis, 6-methylpurine and cycloheximide inhibit NR synthesis. The independence of the NR and the alpha-amylase inductions within the same tissue is further evidenced by their specificity toward the inducers. That is, nitrate does not induce alpha-amylase nor does GA induce NR. 42 43 Furthermore, neither does GA prevent the induction of NR by nitrate, nor does nitrate prevent the induction of alpha-amylase by GA. (.1...— LITERATURE CITED Chrispeels, M. J. and J. E. Varner. 1966. Inhibition of gibberellic acid induced formation of alpha- amylase by abscisin II. Nature 212:1066-67. Chrispeels, M. J. and J. E. Varner. 1967a. Gibberellic acid-enhanced synthesis and release of alpha-amy- lase and ribonuclease by isolated barley aleurone layers. Plant Physiol. 42:398-406. Chrispeels, M. J. and J. E. Varner. 1967b. Hormonal control of enzyme synthesis:. on the mode of action of gibberellic acid and abscisin in aleurone layers of barley. Plant Physiol. 42:1008-16. Filner, P. 1966. Regulation of nitrate reductase in cultured tobacco cells. Biochim. Biophys. Acta 118:299-310. Filner, P. and J. E. Varner. 1967. A test for de novo synthesis of synthesis of enzymes: density—TabeI- ing with H2180 of barley alpha-amylase induced by gibberellic acid. Proc. Natl. Acad. Sci. 58: 1520-1526. Jacobsen, J. V. and J. E. Varner. 1967. Gibberellic acid synthesis of protease by isolated aleurone layers of barley. Plant Physiol. 42:1596-1600. Jones, R. L. and J. E. Varner. 1967. The bioassay of gibberellins. Planta (Berl.) 72:155-61. Paleg, L. 1960. Physiological effects of gibberellic acid. 1. On carbohydrate metabolism and amylase activity_of barley endosperm. Plant Physiol. 35: Yomo, H. 1960. Studies on the alpha-amylase activating action of gibberellin. Hakko Kyakaishi. 18:600- 02. [Cited in Chemical Abstracts, 55:26145, 1961]. 44 SECTION I I r- ABSTRACT CONTROL OF NITRATE REDUCTASE ACTIVITY IN BARLEY ALEURONE LAYERS BY Thomas E. Ferrari Nitrate reductase activity in barley (Hordeum vulgare L. cv. Himalaya) aleurone layers has been determined in the intact tissue, using two different methods. The first method measures the rate of appearance of H2180 pro- duced during the reduction of KN1803. The second assay measures excreted nitrite resulting from nitrate reduction under anaeroboc conditions. Nitrite production in this anaerobic, intact-tissue assay was dependent upon the pres- ence of phosphate (pH 7.5) and was increased by ethanol and bisulfite. After ten hours of nitrate induction, nitrate reductase activities measured by the KN1803 assay are one- sixth, and those measured by the anaerobic intact-tissue assay are one-third, of those observed in cell-free extracts of aleurone layers. Addition of ethanol to the anaerobic intact-tissue medium increased the rate of nitrate reduction to a level greater than that found in the cell-free assay. 45 46 Oxygen inhibited nitrite release in the anaerobic intact-tissue assay. However, under aerobic conditions and in the presence of 2-heptyl-4—hydroxyquinoline N—oxide (HOQNO) or antimycin A, nitrate reduction increased to rates comparable to those observed under anaerobiosis. Neither of these electron transport inhibitors affected anaerobic ni- trate reduction, though they were effective in inhibiting oxygen uptake in separate experiments. 1r INTRODUCTION It is important to know if enzyme activities ob- served in cell-free extracts are representative of those which occur in the intact cell or tissue. For nitrate reductase, we have been able to measure its intracellular activity by two different methods, and to compare them with enzyme activity determined in cell-free extracts. The first method is based on the use of KN18 03 as substrate for nitrate reductase, the rationale of which is as follows. The reduction of nitrate to ammonia is believed to occur via a pathway consisting of four reactions (1): KN1803——> N180; ——> (HNlBO) —> (NHZIBOH)-—9 NH4+ + + + 18 18 18 H2 0 H2 0 H2 0 In the first and probably the rate limiting-step in the pathway, nitrate is reduced to nitrite by nitrate reductase. This reaction involves the release of one 180 atom as H2180. The products of nitrite reduction are not known exactly, but the second and third oxygens of nitrate are released during the final two or three steps of the pathway, or perhaps by 18 - 18 nonenzymatic exchange of the N 2, (HN180) and (NH2 OH) oxygens with oxygens of water. The oxygen atoms of KNIBO3 47 48 have a very low exchange rate with water (half—life measured in years) at all pH values: however, those of nitrite ex- change rapidly at low pH values. Thus, one—third of the 180 released as water during the reduction of nitrate to ammonia is indicative of the in yizg_activity of nitrate reductase. This is true whether the oxygens are enzymatically released from the nitrogen atom, as indicated in the above m equation, or in the nonenzymatic exchange with cellular water at the nitrite, or latter, stages of reduction. Thus, the method measures the amount of enzyme involved in nitrate reduction--not necessarily total enzyme levels-~in the pres- ence of nitrate only, without addition of any other exogenous materials and under natural (aerobic) conditions. The second, more rapid intact-tissue assay, is based on the observation (2) that under anaerobic conditions ni- trite resulting from nitrate reduction is excreted from the 18 tissue. The method differs from the KN O assay in that 3 assay conditions (anaerobiosis and high phosphate concentra- tions) are not normally what one might expect in situ. Nevertheless, it appears that this second assay technique will be especially useful as a rapid method for studying total levels of nitrate reductase in different biological materials and studying the factors which control the cellu- lar activity of this enzyme. Barley aleurone layers develop nitrate reductase activity upon exposure to nitrate (3). We have used isolated 49 aleurone layers to compare nitrate reductase activity of cell-free extracts to enzyme activity observed in the two intact-tissue assays. 1r MATERIALS AND METHODS The procedure used to prepare aleurone layers was similar to that described by Chrispeels and Varner (4). Barley seeds (Hordeum vulgare L. cv. Himalaya) were cut in half, the embryoless halves were imbibed on moist sand, and after 4 to 5 days, the aleurone layers were removed. Nitrate reductase was induced at 23 C under aseptic conditions in either of two ways: (1) For the KNIBO3 assay, a 5-ul drop of KNIBO3 (0.05 M) was added to each of 20 aleurone layers. Chlor- 18 amphenicol (10 ug/ml) was added to the sterilized KN 03 stock solution to insure against microbial growth during the subsequent induction period. The layers were placed in 5-cm, sterilized, foil-covered Petri dishes. A moistened filter paper (4.25 cm diameter) placed against the lid of the Petri dish prevented dehydration of the tissue. After a lO-hr induction period, water was sublimed (at 80 to 100 u pressure) from the tissue and collected in a trap cooled with liquid nitrogen. The sublimed water was weighed and placed in 12—ml conical Pyrex tubes containing 46 mg of NaHCO3. The tubes were stoppered with serum vial caps, frozen, and evacuated to approximately 100 u. The solution was made acidic by injection of 0.2 ml lactic acid, thereby 50 51 releasing C02. After about 24 hr equilibration at 40 C, the tubes were placed in dry ice and the atom percent excess of C1802 determined with a MAT GD 150 mass spectrometer. From the weight of extracted water and the isotOpic enrichment of CO the number of moles of nitrate reduced were computed. 2. (2) For the anaerobic intact-tissue assay, nitrate reductase was induced by placing 40-50 aleurone layers and two drops of chloramphenicol (0.5 mg/ml) in a 50—ml Erlen- meyer flask containing 5 ml of sterilized 0.05 M KNOB. The flasks were stoppered with cotton plugs and placed in a meta- bolic shaker set at 200 rpm. After 2 to 3 hr of induction the tissue was rinsed with approximately 20 ml of 0.05 M KNO3, and the effect of various conditions on enzyme activity during the assay was determined. The intact-tissue assay media contained in 2 ml: 10 aleurone layers, 0.1 M phos- phate buffer (pH 7.5), 0.02 M KNO and the treatment solu- 3. tion as indicated in the text. To start the assay, the re- .action mixture, contained in a 25-ml Erlenmeyer flask, was de-aerated by bubbling nitrogen gas through the medium for one minute, and then stoppered. Nitrite in the media was determined at zero time and after 20 or 30 minutes of incuba— tion, by adding aliquots to 0.3 ml each of l per cent sulfanilamide in a 3 N HCl and 0.02 per cent N—l— naphthylethylenediamine dihydrochloride. Optical density at 540 nm was measured after centrifuging at 2000 X g for 10 minutes. 52 Cell-free activity of nitrate reductase was deter- mined as described previously (3). Oxygen uptake was measured polarographically with a Clark oxygen electrode. The 2.7-ml reaction media con— tained 0.02 M KNOB, 0.1 M phosphate buffer (pH 7.5), 10 aleurone layers, and the test substance dissolved in ethanol. Pure ethanol was added to controls. RESULTS Comparison of Nitrate Reduction Rates in the Intact Tissue and Cell-Free Assays Enzyme activities as determined by the standard cell-free assay, the anaerobic intact-tissue assay, and the KNIBO3 in_yigg assay are shown in Table l. Nitrate reductase induced under exactly the same conditions shows 6 and 2.5 times more activity in the cell-free and anaero— bic intact—tissue methods, respectively, than in the KNIBO3 ig.yigg_assay. In an attempt to determine what factor was limiting the rate of nitrate reduction in the intact-tissue, nitrate- induced aleurone layers were assayed in the same medium as used in the cell-free assay. For this, the layers were placed in 2 m1 of media containing flavin mononucleotide (FMN), Na28204, KNOB, and phosphate buffer. The reaction was run in the presence of nitrogen to prevent oxidation of the Na28204 by air. Under these conditions nitrite was released from the tissue, and the amount of nitrate reduc- tase activity estimated by removing aliquots of the reaction medium after a suitable time period and analyzing for ni— trite. Enzyme activity in the complete system, and its de- pendence on each of the reaction components is shown in Table 2. 53 54 Table 1.--Nitrate reductase activity as determined by the cell-free and intact—tissue assay methods. For each assay data were obtained from tissue in- duced 10 hours under identical conditions by method 1 as described in Materials and Methods. Numbers represent averages of three experiments. J r Nitrate Reductase Activity Assay Method (nmoles N0; reduced/layer.hr) Cell-free 8.4 Anaerobic intact-tissue 3.2 KNlBO3 intact-tissue 1.4 Table 2.--Dependence of nitrate reductase activity upon components of the cell-free assay media. Data from tissue induced 22 hr by method 1 as described in Materials and Methods. Numbers represent averages of three experiments. L.___ ._—> — I A— — Nitrate Reductase Activity (nmole N0; formed/layer.hr) Anaerobic-intact Cell-free Tissue Assay* Assay Complete 3.6 9.2 Complete-noninduced 0.0 0.0 - FMN ' 3.7 0.0 - Na28204 3.2 0.0 - Phosphate buffer 0.9 2.3 - KNO 0.3 0.0 3 *Actually represents the anaerobic, intact-tissue assay supplemented with components of the cell-free assay. 55 Nitrate reduction in the intact layers in the presence of Na28204, FMN, and inorganic phosphate was greater than in the KNIBO3 in_yiyg assay, but only approximately one-third the rates observed in the cell-free assay. Effect of Arsenate, Ethanol, Antimycin A and HOQNO on Nitrate Reduction From the results presented in Table 2 it seemed that phosphate in some way limits nitrate reduction in the tissue under anaerobic conditions. Though it is less effective, arsenate can substitute for phosphate in enhancing nitrate reduction (Table 3). In later experiments, Tris buffer was also found to be effective; therefore, it is not possible to decide whether the enzyme's response to these compounds is a non-specific ion or pH effect. Ethanol also enhanced nitrate reduction in the anaerobic intact-tissue assay (Figure l). Ethanol increased nitrite release under both anaerobic and aerobic conditions. This increased sensitivity for measuring rates of nitrate reduction allowed the detection of low levels of nitrate reductase in noninduced tissues (Table 4). The ethanol- enhanced nitrate reduction was strongly dependent on phos- phate buffer (Table 4). Because bisulfite inhibits the reduction of acetald— ehyde during glycolysis, its effect on the anaerobic reduc- tion of nitrate was determined. Results qualitatively similar to those obtained with ethanol were obtained with 56 Table 3.--Dependency of nitrate reductase activity upon phosphate or arsenate in the anaerobic intact- tissue assay. Tissue induced for 3 hours by method 1 as described in Materials and Methods. Numbers represent an average of two experiments. The arsenate containing media were adjusted to pH 7.5 with NaOH. Nitrate Reductase Activity 1 Treatment (nmoles N0; formed/layer.hr) KNO3 - HPO4 0.3 KNO3 + HPO4 2.7 L KNO3 + A504 1.9 - Table 4.—-Effect of ethanol on nitrate reductase in the anaerobic intact-tissue assay. Tissue induced 3.5 hours by method 2 as described in Materials and Methods. Numbers represent an average of two experiments. Nitrate Reductase Activity Assay Condition (nmoles NOE formed/layer.hr) N2 - EtOH 3.6 N2 + EtOH 16.6 N2 + EtOH - tissue 0.0 Air + EtOH 2.0 Air - EtOH 0.1 N2 + EtOH - phosphate 1.0 N + EtOH noninduced 2.5 N2 - EtOH noninduced 0.3 57 Figure l.--Concentration curve of ethanol-enhanced nitrite released in the anaerobic intact- tissue assay. Tissue was induced 3 hours by method 2 as described in Materials and Methods. 58 mDOI . mw><4 omm: 2:52.; 322...: 32:2 M... m n. m. 8.5 7. 5 6.5 5.5 4.5 82 Figure 2.--Kinetics of nitrite uptake. Prior to determination of nitrite uptake, tissue was incubated 12 hours in the presence or absence of 0.05 M potassium nitrate. Up- take was measured with 10 aleurone layers incubated in 2 ml of medium containing 0.25 mM sodium nitrite and 0.1 M phosphate buffer, pH 4.5. Open and closed circles represent noninduced and nitrate-induced tissues, respectively. Figure 3.--Kinetics of nitrite disappearance. Assay conditions were as described in Figure 2. Nitrite disappearance was measured as described in Materials and Methods. Isue Ice or Up- .ayers .ng .sphate ‘cles [uced Assay re 2. AVSSV E!VVI.l (SilflNIW) OZ (TV (T9 ()2 (T7 (T9 813 No; Of MEDIUM (nMOLE) o 3 '33 o o o I I '0 007 // \o axvun ‘0“ I I ‘ M 3 Do 0. x 3' I 5 ” II 1 TOTAL NO; RECOVERED (nMOLE) 8 8 S 3 8 1 fl I 1 /‘i . i // "' I E 0 °’ :2 1, 3 u )- 3' o g n. to It 2 "' z .. " a) (5 out "I °I & b. (3 a: 84 As measured by nitrite disappearance, nitrite re— ductase activity was increased by prior incubation with nitrate, but considerable activity was present in noninduced tissue (Figure 3). The temperature coefficient (010) of nitrite uptake was approximately 1.4 compared to 2.6 and 2.4 for nitrite disappearance in induced and noninduced tissue, respectively (Table l). The increase in nitrite reductase activity by incu- bation with nitrate, was completely inhibited by cycloheximide and enhanced slightly by actinomycin D (Table 2). Nitrite i disappearance by noninduced tissue was not affected by L either inhibitor (Table 2). Enzyme activity in induced layers was inhibited by 2,4—dinitrophenol (DNP) (Figure 4), antimycin A, 2-n-heptyl- 4-hydroxyquinoline-N—oxide (HOQNO) and anaerobiosis (Table 3). Activity in uninduced tissue was insensitive to antimy- cin A and HOQNO (Table 3), and sensitive to anaerobiosis. Nitrite uptake was relatively unaffected by the inhibitors (Table 4, Figure 5). Nitrite Release by Aleurone Layers The following experiments were performed to deter— mine if aleurone layers are able to retain nitrite at pH 7.5 under anaerobiosis—-conditions under Which nitrite release occurs during anaerobic nitrate reduction (4). Since no nitrite is taken up by the tissue at pH 7.5 (Figure 1)--the t -- «in: 85 Figure 4 and 5.--Effect of 2,4—dinitrophenol on nitrite uptake and disappearance. Tissue was incubated 13 hours in the presence or absence Of 0.05 M potassium nitrate and 10 ug/ml chloromycetin. Uptake and disappear- ance of nitrite was determined for 10 aleurone layers incubated 40 minutes in 2 ml of media containing 2,4—dinitrophenol at the concentra- tion indicated, 0.25 mM sodium nitrite, 0.1 M phosphate buffer (pH 4.5), and 20 ug chloromycetin. :3 ZO_.—<¢._.ZmUZOU 320 004 86 T n- o- o Y n- o- o . . 1C“ —. _ . .HA .. oou . O o l 0* I N 333320: rm. on w I Z. .3255 m o e 8 u I can N w .0. $0933; 3 m I ou— a (u) m, w $025552. 1 W m o u n I ( I oo— _ .oon 32:... 6].; 32:32.35 oou n o It . L 87 Table l.--Temperature coefficients for uptake and disappearance of nitrite by nitrate-induced and noninduced tissue. After 6 hours induction with nitrate, nitrite uptake and disappearance was measured over a 20 and 40 minute period, respectively. Temperature coefficients were determined over a 15 to 350 range. i nitrate Q10 for Q10 for induction uptake disappearance + 1.4 2.6 - 1.5 2.4 88 Table 2.--Effect of protein synthesis inhibitors on the uptake and disappearance of nitrite in noninduced and nitrate-induced aleurone layers. ' Tissue incubated 7 hours in the presence or absence of 0.05 M potassium nitrate, cycloheximide (10 ug/ml) and actinomycin D (10 ug/ml). All treatments contained 20 ug chloromycetin. Disappearance Uptake I NOB- Induction + - + - (nmoles/40 min./10 layers) Control 170 68 344 388 Cycloheximide (10 ug/ml) 66 66 322 311 Actinomycin D (10 ug/ml) 207 65 336 312 In— 89 Table 3.--Effect of respiratory inhibitors on the disappear- ance of nitrite by aleurone layers. Tissue was induced in the presence or absence of 0.05 M potassium nitrate. After 12 hours of incubation, tissue was placed in media containing phosphate buffer, 0.1 M (pH 7.5), and the inhibitor being tested. Ethanol (final con— centration = 5 per cent) was used as solvent for the inhibi- tors. After 30 minutes incubation at 23°, the media con- taining the inhibitors was discarded and the tissue rinsed 2 times with 4 ml of 0.1 M potassium phosphate (monobasic). Nitrite uptake by the tissue over a 20 minute period was then determined as described in Materials and Methods. Nitrite disappearance Induced Activity Treatment Total % of (Total Induced - % of noninduced control noninduced) control (nmoles/10 layers/40 min.) Control 56 100 80 100 HOQNO (0.25 mM) 54 97 38 48 Antimycin A (0.5 mM) 49 88 46 58 Nitrogen Atm. 6 ll 0 0 90 Table 4.--Effect of respiratory inhibitirs on the uptake of nitrite by aleurone layers. Experimental procedure as described in Table 3. Nitrite Uptake % of % of Treatment Induced control Noninduced control (nmoles/10 aleurone layers/20 min) Control 157 100 194 100 HOQNO (0.25 mM) 125 80 129 67 Antimycin A (0.5 mM) 117 81 148 76 N Atm. 147 94 145 75 2 ail! . 91 pH of the anaerobic nitrate reduction medium-—tissue was first preloaded with nitrite at pH 4.5 and under anaerobic conditions to prevent its reduction. Nitrite of the media at the start of the preloading period, after 40 minutes, and after DMSO extraction, are shown in Table 5. No nitrite was lost during the 40-minute uptake period. When tissue was subsequently washed and placed in nitrite-less media at pH 7.5, nitrite leaked from the tissue whether incubated an additional 40 minutes under aerobic or anaerobic conditions (Table 6). The ability of tissue to reduce some nitrite during the pH 7.5 incubation, is indicated by the failure to recover at 40 minutes all of the nitrite present at the start of the pH 7.5 incubation. The kinetics of nitrite release and disappearance under aerobic conditions are indicated in Figure 6. Approximately 50 per cent of the nitrite accumu- lated in the tissue during incubation with nitrite at pH 4.5 leaked into the medium during the first 5 minutes of incubation at pH 7.5. Anaerobic Nitrite Dissppearance at Low Nitrite Concentrations Nitrite was found to disappear under anaerobic con- ditions when noninduced tissue was incubated in 25 uM nitrite (Table 7)--a ten fold lower concentration than what was used in previous experiments. DNP partially inhibited this disappearance (Table 7). On the other hand, nitrate-induced tissue showed no apparent ability to reduce nitrite when 92 Table 5.--Nitrite accumulation by aleurone layers at pH 4.5. Tissue was incubated in the presence or absence of 0.05 M potassium nitrate for 12 hours. Tissue was then incubated in a nitrogen atmosphere for 40 minutes in 2 ml of medium containing 0.1 M phosphate buffer (pH 4.5), 0.25 mM sodium nitrite, and 20 ug chloromycetin. Nitrite of the medium and tissue was determined as described in Materials and Methods. Nitrite of media Nitrite after DMSO Of extraction tissue* Assay time (min.) 0 40 40 . 40 (nmole/2 m1) (nmole/ I KNO3 induction 10 layers) + 429 139 452 313 - 436 135 427 292 * Nitrite of tissue = nitrite of media after DMSO extraction minus nitrite of media prior to DMSO extraction. 93 mucflfi om wEflu pm mufluuas ”mammau Ucm (“PEA—- Hui-.3“! "r ”(I 4- . b" '._ .om OEHD um mufiuuflc Hmuou mwtmfiv Hmuou u mocmummmmmmac muwnuazt ma vb mam mom 0 mo 5H mmm poosccwcoc mm om omm gem m om gm mam omosoca cmmouuwc Ham cmmouuflc Ham cmmouuflc Ham om om oe om oe 1.cHEV osflp momma AmumhmH oa\mmHoEcv Rmucmnmmmmmmaw wunuuez AHE N\moaoficv sauce mo mufluuflz AmummmH oa\mmHOEcv mammau mo mufluuflz Hmucoaflummxm .0 munmflm ca OOQHHUMOO mm mp3 munpmooum .w canon Scum cwxmu mmB mwuscflfi 0% no mammwp mo muauuflz .m.h mm um chHquGOO canoummcm tam Uflnonmm Hopes mufluuflc UODMHOEOUOM mo mmmmammll.m magma 94 Figure 6.--Release of accumulated nitrite by aleurone layers. Tissue was induced in the presence or absence of 0.05 M potassium nitrate for 12 hours. Tissue was then preloaded with nitrite by incubation in a nitrogen atmos- phere for 40 minutes and in 2 m1 of medium containing 0.1 M potassium phosphate (mono- basic), 0.25 mM sodium nitrite, and 20 ug chloromycetin. Tissue was next rinsed 3 times with 10 ml cold phosphate buffer (pH 7.5), 0.1 M, and placed in 2 ml of nitrite- less media containing 0.1 M phosphate buffer (pH 7.5), and 20 pg chloromycetin. Nitrite of the medium and tissue was determined at the times indicated, as described in Materials and Methods. 0 = noninduced tissue. 0 = nitrate induced tissue. nMOLES No; or MEDIA AND TISSUE 320 240 I80 120 95 TOTAL _ 0 MEDIUM 8 8 _. TISSUE i T I T . ID 20 30 40 MINUTES iNCUBATION AT pH 7.5 en ma m m m o 626 + mufiouno I am m H N N o muwuuwc I NHH mm av Hm Ha aw mZQ + OHHHHHC + m6 ma «8 oH o 84 ouflnuflc + mammau OUUSUCM mammwu Occupcwcoc coauflocoo hound AHE «\DHDEDV o8 oe o o4 oe o l.cflsv mafia momma cofluomuuxm coHpUMHuxw 6 Omzd Hmumm omza “mums 9 SOHOUE mo muauuflz ESHUDE mo ODAMUHZ .28 H.o u mza .OUOOHUAC QUH3 DUDGME H How coflumuflmmo op Hoflum push mflpmfi coflumnsocfl mnu on poops mm3 05mmaa .caumUMEOHOHSU «0 moaoso om oom Aoflmmnocos .2 H.ov Daemon mumnomonm .125 mm. oueuuflo EDHOOM R mcwcflmucoo mapmfi mo HE N ca UOUMQDUOH swap mumz mumhma mcousmam COB .kumz Umaaaumwo mo HE om hamumefixoummm £uw3 mmEHU m Ummcflu mp3 manna» .mnson o How wumuuac Eswmmmuom mo mucmmwnm may cw coflumndocw Hound .COMUDHUOUUOOU Unfiuuflc 30H mo mocmmnm no mocmmwum on» OH Omhmmmm mammau Umoaucecoc 6cm OOUOOOHIUUDHUAG ma DUOMHDUQQMMHO tam scauospoum muwnquII.h manna 97 incubated with low levels of nitrite (Table 7). When DNP was included in the assay media, nearly 2.5 times more ni— trite was found at the end of the assay than was present at the start (Table 7). When tissue was assayed in the absence of nitrite, 20 nmoles of nitrite were produced by nitrate- induced tissue (Table 7). Again, DNP increased consider- ably (3.5 fold) the amount of nitrite accumulated by nitrate-induced tissue. Little or no nitrite is present in induced tissue at the start of the assay. Thus, it appeared that nitrite was being produced from the anaerobic reduction of nitrate which had accumulated during the nitrate incuba- tion phase of the experiment. Therefore, it appears that nitrite disappearance occurred in induced tissue as well as noninduced tissue, and reduction was inhibited by DNP. Effect of PCP and onynil on the Uptake and Disappearance of Nitrite Two other uncouplers of oxidative phosphorylation, pentachlorophenol (PCP) (8) and 3,5-diiodo-4— hydroxybenzonitrile (ioxynil) (3), were tested for their ability to inhibit nitrite disappearance in aleurone layers. While causing only relatively slight inhibitions of uptake, both PCP (10"3 M) and ioxynil (10_4 M) strongly inhibited nitrite disappearance in induced and noninduced tissue (Table 8) . 98 Fu‘yfie a Q .Honpcoo on cocoa Odoumod .Aucmo umm H n coflumuucmocoo Hmcflmv mcoumum OH pm>aommap HODHQHSCA OOGHDUGOU mucmaummuu Had R mo wm mm mm OOH Mb ma Ho mm OOH hm mh mm mm OOH Aaonucoo mo ucmo Mom. Om vb mm mm OOH oH Hflcmon oH Hflcmxoe IOH Hocwnmouoanomucmm IOH HOGOSQOHOngmucmm HOHUCOU pmospcflcoc OOUDOOH mucmnmmmmmmap Duanpflz Umonpcflcoc UUUOOCH mxmuos mnauuflz IOOOEV mumnmmosm Esfimmmuom z H.o «.ooummu moflmn .mumupwc SHUOM mo HE m ca UOUDHQ pcm noun? omaawumwp mo HE om m pmmcwu can» mp3 mummwe .CAUOUNEOHOHSU HE\m5 Douanwncw Opp 0cm AUHme SE mm.o mcflcflmucoo mecca hamumsaxoummm EMAB mmfiwu OH cam mumuuwc Edwmmmpom z mo.o mo mocmmnm no mocmmmum map cw muson m Omumnbocfl mm3 USMMHB .wnmhma Ucousmam pmonocwcoc pcm pmospcfllmumnuac Mn mocmummmmmmap tam mxmums muflnuwc co Hashxow 0cm HocwsmouOHsomucmm mo UUUMMMIl.m manna 99 Effect of DNP on Nitrate Reduction The effect of DNP on the anaerobic intact-tissue assay (4) for nitrate reductase is shown in Table 9. When the assay was done in the absence of ethanol, DNP increased nitrite recovery by 65 per cent; however, DNP had little or no effect on ethanol-enhanced nitrite production. In 1P7 VfiT . i ‘I 100 Table 9.-—Effect of DNP on nitrate reduction. After 4 hours induction with nitrate, tissue was placed in 2 ml of media containing 0.1 M phosphate buffer (pH 7.5), 0.1 M potassium nitrate and nitrogen atmosphere. Nitrite of media at 30 minutes(after DMSO extraction)was determined as described in Materials and Methods. DNP = 0.1 mM. Ethanol = 5 per cent. nmoles nitrite Assay cond1tion produced/layer.hour - ethanol — DNP 4.0 - ethanol + DNP 6.6 + ethanol - DNP 25.6 + ethanol + DNP 27.0 I! u' r '1'“in “Am _ flip-aim“, W) O ‘5. . 101 Discussion When nitrate-induced tissue is incubated anaero- bically in the presence of nitrate, nitrite leaks from the tissue (4, 7, 10). Nitrite production and release was en- hanced considerably by ethanol (4). The failure of 2,4- dinitrophenol (DNP)--an inhibitor of nitrite reduction in higher and lower plafis (l, 6, 12)--to further increase ni- trite production in the presence of ethanol (Table 9), indicated that the inhibition of nitrite reduction by anaerobiosis was complete. However, DNP did increase ni- trite accumulation in tissue assayed in the absence of ethanol (Table 9). This additional nitrite accumulated may represent inhibition of the tissue's ability to reduce low levels of nitrite (Table 7) under anaerobic conditions. The failure to observe nitrite disappearance at high levels of nitrite (250 uM) might be due to the accumu— lation of toxic levels of nitrite within the cell. Assuming 0.01 ml of water and 30 umoles of nitrite accumulated (Table 5) per layer, the concentration of nitrite within an aleurone layer would be approximately 3 mM. Such concen- trations are toxic to Anabaena cylindrica (2) and cultured tobacco cells (5). Although induced and noninduced aleurone layers were able to reduce low levels of nitrite (25 uM) anaerobically, these rates (5 to 10 nmoles/layer/hour) are considerably lower than rates of nitrite production from I u n f.".\' m‘:€h“' alias-53...". 102 nitrate under anaerObic conditions in the presence of ethanol (25 nmoles/layer/hour). Thus, it appears nitrite cannot be reduced fast enough, and because nitrite cannot be retained by the tissue (Table 6) it leaks into the medium. The DNP-inhibited reduction of nitrite under aerobic conditions in aleurone layers, and in intact cells of Chlorella (6) and Ankistrodesmus (1), suggests that nitrite reduction is dependent on oxidative phosphorylation. The ability to inhibit nitrite disappearance by other uncouplers of oxidative phosphorylation (Table 8) (l, 6)--with and ‘without -N02 functional groups--argues against the possi- bility that DNP is alternatively accepting reducing equiva— lents, as has been shown to be the case with isolates of Pseudomonas denitrificans (9) and reductants generated by spinach chloroplasts (13). Evidence that nitrite disappearance in both induced and noninduced tissue is enzymatically mediated, is indi- cated by their temperature coefficients, and sensitivity to anaerobiosis and uncouplers of oxidative phosphorylation. The ability to inhibit nitrate-induced nitrite reductase activity with cycloheximide argues for the requirement for protein synthesis in the induced system. The differential sensitivity of induced and non- induced nitrite disappearance to all inhibitors tested, 103 suggests that there might be two enzymes responsible for nitrite reduction, or that one is present in different cellular compartments.) 10. 104 LI TERATURE C ITED Ahmad, J. and I. Morris. 1968. The effects of 2,4- dinitrophenol and other uncoupling agents on the assimilation of nitrite by chlorella. Biochim. Biophys. Acta 162:32-38. Brownell, P. F. and D. J. D. Nicholas. 1967. Some effects of sodium on nitrate assimilation and N2 fixation in Anabaena cylindrica. Plant Physiol. 42:915-921. Ferrari, T. E. and D. E. Moreland. 1969. Effects of 3,5-dihalogenated-4—hydroxybenzonitriles on the activity of mitochondria from white potato tubers. Plant Physiol. 44:429-434. Ferrari, T. E. and J. E. Varner. 1970. Control of nitrate reductase activity in barley aleurone layers. PNAS. 65:729-736. Kelker, H. Ph.D research. MSU/AEC Plant Research Lab, Michigan State University. Personal Communication. Kessler, E. and W. Bucher. 1960. Uber die Wirking von Arsenate auf Nitratreduktion, Atmung und Photo- synthese von Grunalgen. Planta (Berl.) 55:512-524. Kumada, H. 1953. The nitrate utilization in seed embroys of Vigna sesquipedalis. J. Biochem. 40: 439-450. Moreland, D. E. 1967. Mechanisms of action of herbi— cides. Ann. Rev. Plant Phys. 18:365-386. Radcliff, B. C. and D. J. D. Nicholas. 1968. Some properties of nitrite reductase from Pseudomonas denitrificans. Biochim. Biophys. Acta 153:545-554. Randall, P. J. 1969. Changes in nitrate and nitrite reductase levels on restoration of molybdenum to molybdenum-deficient plants. Aust. J. Agr. Res. 20:635-642. 105 ll. Snell, F. D. and C. T. Snell. Colorimetric Methods of Analysis. Vol. II. Van Nostrand, Princeton, third edition. 1949. p. 804. 12. Voskresenskaya, N. P. and G. S. Grishina. 1962. The significance of light for nitrite resuction in the green leaf. Fiziol. Rast. 9:7-15. (Translated in Soviet Plant Physiol. 1962. 9:4-10.) 13. wessels, J. S. C. 1965. Mechanism of the reduction of organic nitro compounds by chloroplasts. Biochim. Biophys. Acta 109:357-371. 106 DI SSERTATI ON SUMMARY Nitrate induced the formation of nitrate reductase activity as measured in cell-free extracts of barley. (Hordeum vulgare L. cv. Himilaya) aleurone layers. No ni- trate reductase activity was detected in dry half-seeds or in aleurone layers after imbibition or incubation in the absence of inducer. Induction of nitrate reductase activity was not affected by abscisic acid or 8—azaguanine. Actinomycin D enhanced rather than inhibited enzyme activity; however, 6-methylpurine and cycloheximide were inhibitory. In addition to determining activity in cell-free extracts, nitrate reductase activity was also measured in the intact tissue using two different methods. The first method measured the rate of appearance of H2180 produced during the reduction of KN1803. The second assay measured released nitrite resulting from nitrate reduction under anaerobic conditions. After 10 hours of nitrate induction, nitrate reductase activities measured by the KNJTBO3 assay were only one-sixth, and those measured by the anaerobic intact-tissue assay were one-third, of those observed in cell-free extracts of aleurone layers. Nitrite production in the anaerobic intact-tissue assay system was inhibited by oxygen and increased by Ixé'i J". 107 ethanol and bisulfite. Oxygen-inhibited nitrite production was reversed by 2-heptyl—4—hydroxyquinoline N-oxide (HOQNO) or antimycin A—-two inhibitors of mitochondrial electron transport. Neither of these inhibitors affected the anaerObic reduction of nitrate. A method was devised for the detection and measure- ment of nitrite reductase activity in aleurone layers. The technique involved administering nitrite to aleurone layers and measuring nitrite disappearance after a given time period. The method also allowed simultaneous measurement of nitrite uptake. Recovery of nitrite from tissue was obtained by rapid heating of the medium plus tissue in the presence of dimethyl sulfoxide. Using the above procedure, nitrite reductase activi— ty was found to be increased by prior incubation of tissue with nitrate, but considerable activity was present in noninduced tissue. The temperature coefficient (Q10) of nitrite uptake was approximately 1.4, compared to 2.6 and 2.4 for nitrite disappearance in induced and noninduced layers, respectively. Nitrite-induced activity was inhibited by cyclo- heximide but not by actinomycin D. Enzyme activity in induced layers was inhibited by 2,4—dinitrophenol (DNP), antimycin A, HOQNO and anaerObiosis. Activity in non- induced tissue was insensitive to antimycin A and HOQNO, and sensitive to anaerobiosis and DNP. 108 Nitrite uptake was rapid at pH 4.5 and negligible at pH 7.5. Nitrite accumulated at pH 4.5 anaerobically, was rapidly released when transferred to media at pH 7.5-- the pH of the anaerobic intact-tissue assay for nitrate reductase. Accumulated nitrite was released by the tissue whether held under anaerobic or aerobic conditions. Nitrate-induced and noninduced aleurone layers were able to reduce low levels of nitrite (25 uM) anaerobically. But, these rates (5 to 10 nmoles/layer/hour) were consider- _ably lower than rates (25 nmoles/layer/hour) of nitrite production observed during the nitrate reductase anaerobic intact tissue assay (with ethanol present). DNP increased nitrite accumulation during the nitrate reductase anaerobic intact-tissue assay in the absence, but not in the presence of ethanol. This additional nitrite recovery in the absence of ethanol may represent an inhibition of the tissue's ability to reduce low levels of nitrite under anaerobic conditions. Thus, it appears nitrite cannot be reduced fast enough during the nitrate reductase anaerobic intact-tissue assay; and, because nitrite cannot be retained by the tissue at neutral pH, it leaks into the medium. hi 'I d -‘ APPENDIX 109 Reproducibility1 of the nitrate reductase anaerobic intact tissue assay, and the effect of different amounts of tissue2 on enzyme activity. Number of layers Experiment (nmoles N0; produced/hour) 5 10 15 1 41.3 95.6 100.0 2 53.2 64.3 113.0 3 47.5 101.3 127.0 mean 47.3f3.5 87.0113.1 ll3.3i7.8 1 Standard deviation from the mean calculated from the formula: 2 52-4917 n(n—l) Where S = the standard deviation from the mean, d==deviation from the mean, the n = number of samples. '2Barley seeds (1965 harvest) used in this and all previous studies were obtained from Dr. R. A. Nilan, Department of Agronomy, washington State university, Pullman, Washington. )IHIIILIIINUIJ