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I 293 10411 2556 ,. lllllllllllN!llllHIllllllllflllllllllllllHlllUllflHHIHJI This is to certify that the thesis entitled DARK CO2 FIXATION AND AMINO ACID METABOLISM IN SYMBIOTIC NZ-FIXING SYSTEMS presented by George T. Coker III has been accepted towards fulfillment of the requirements for Ph. IL degreein Biochemistry w QM Date ngl J) U (7' [52, Major professor 0-7639 MSU LIBRARIES m. RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. “£21....“ 25% l DARK CO2 FIXATION AND AMINO ACID METABOLISM IN SYMBIOTIC N -FIXING SYSTEMS. 14C and 13N-Labeled Tracers 2 Labeling Studies with By George T. Coker III A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1982 6 [WHY/0 ABSTRACT DARK C02 FIXATION AND AMINO ACID METABOLISM IN SYMBIOTIC N -FIXING SYSTEMS. 2 Labeling Studies with 14C and 13N-Labeled Tracers By George T. Coker III Amino acids constitute a greater proportion of the organic nitrogen transported from the roots of soybean (Glycine max) plants treated with N03 or NH: and from the nodulated roots of alder (Algg§_ glutinosa) than they do from the nodulated roots of soybean plants dependent solely on N2. This suggests that the metabolism of amino acids is different in these tissues. Amino acid metabolism was examined by monitoring the amino acids labeled with [14C] incorporated during dark C02 fixation and with [13N] incorporated from 13NHZ, 13No; or [13N]N2. Label from 14CO2 was directly incorporated in soybean roots and the NZ-fixing root nodules of soybeans and alders. The rate of incor- poration depended on the age and the nitrogen source for the plants. The products of dark C02 fixation were primarily amino and organic acids. The distribution of label incorporated from 14C02 into amino acids depended on the plant species and the nitrogen source. The major labeled amino acids in roots and nodules of soybean plants dependent on N2 were aspartate and glutamate; in alder nodules, citrulline; in roots of soybean plants treated with N03, asparagine; and in roots of soybean plants treated with NHZ, asparagine and glutamine. Asparagine was the major amino acid transported out of the soybean George T. Coker III root system. The pathway of asparagine synthesis was examined by T4 exposing roots either to C0 followed by a chase with 12CO2 or to 2 [2,3-3H]aspartate and [4-]4C]aspartate. The results from these two exp- eriments indicated that asparagine was synthesized directly from aspartate. 14 After exposure to C02, the specific activity of glutamine was consistently higher than that of glutamate in soybean nodules and roots of plants treated with N03. This was taken as evidence that there were two pools of glutamate, only one of which was associated with glutamine synthesis. Alder and soybean nodules and roots were incubated with 13 I3 N—labeled tracers. Those tissues incubated with NHZ had a higher ratio of [13N]glutamine to [13N]glutamate than similar tissues exposed to 13N03 or [13NjN2. An explanation for these results based on the relative rates of glutamine and glutamate synthesis is discussed. To My Family To my brother and mother who tan read this and to my father who cannot "See dad, it only took 2 more years." ACKNOWLEDGEMENTS I wish to thank my major adviser, Dr. K. R; Schubert, and the members of my guidance committee, Drs. Tolbert, Nadler, Averill and Ferguson-Miller. I also wish to thank the people that I entered graduate school with for their willingness to help me over the rough _ spots. I would also like to thank the members of the laboratory - Alan Christensen, Peter McClure and Debra Polayes, My special thanks goes to Mary Tierney who stood by me. I acknowledge the financial support of the American Cancer Society, the National Institute of Health (GM lO9l), the National Science Foundation (PCM 77-24683, PCM 80-ll736 and the National Needs Training Grant), the Michigan Agricultural Experiment Station, and the Department of Biochemistry at Michigan State University. iii TABLE OF CONTENTS Pagi LIST OF FIGURES ........................ vii' LIST OF TABLES ......................... xi LIST OF ABBREVIATIONS ..................... xi' INTRODUCTION ........................... 1 LITERATURE REVIEW ....................... 4 A. Dark CO2 Fixation .................... 4 B. Role of Dark CO2 Fixation ................ 7 C. Amino Acid Metabolism ............. _ ..... 9 1. Enzymes of NH+ Assimilation .............. 9 2. Pathway of NH4 Assimilation .............. ll 3. Asparagine Synthesis ................. l4 4. Citrulline Synthesis ................. l7 5. Allantoin and Allantoate Synthesis .......... 17 D. Effect of N03 and NHZ+on N2 Fixation ........... 18 E. Effects of NO' and NH4 on Nitrogen Transport in Soybeans . 23 F. Effects of NH4 on Amino Acid Synthesis .......... 24 MATERIALS AND METHODS ..................... 26 A. Growth of Plants ..................... 26 B. Measurement of the Rate of CZHZ Reduction ........ 27 C. Measurement of Allantoate, Allantoin, N03 and Total Amino Acids ....................... 28 D. Measurement of the Rate of O2 Consumption ........ 29 E. Production of 14CO2 ................... 29 F. Exposure of Plant Tissue to 14CO2 ............ 30 6. Measurement Of the Specific Activity of 14co2 .. ..... 30 H. Extraction of the Products of 14C02 Fixation ....... 3l I. Separation of the Products of 14CO2 Fixation ....... 32 1. High Performance Liquid Chromatography (HPLC) ..... 32 2. Ion-Exchange Chromatography .............. 33 iv . Isolation and Chromatography of 3. Thin-Layer Chromatography ............... 4. High-Voltage Electrophoresis . . . . . . ....... 5. Gas-Liquid Chromatography ............... 14c-Labe1ed 2-Oxoacids from Soybean Nodules ................... Incorporation of [2,3-3H]Aspartate and [4-14C]Aspartate into Asparagine ..................... . Distribution of Label Within Aspartate and Glutamate in 14 Soybean Nodules Exposed to CO2 ............. . Production of [IBM] ................... . Exposure of Plant Tissue to [13N] ............ . Extraction of the Labeled Products after the Assimilation of [13N] . . . ...................... . Distribution of Label in Glutamine after the Assimilation l3 + of NH4 ......................... CHAPTER l The Rate of Dark CO2 Fixation during the Development of the Soybean Nz-Fixing System ........... RESULTS ........................... A. B. CO2 Fixation Assay .................... Effect of Detachment of Nodules and Concentration of CO2 on the Rate of Dark C02 Fixation . - ........... . Effect of CO2 Concentration on 02 Consumption and CZHZ Reduction ...................... . Dark CO2 Fixation, CZHZ Reduction and Concentration of Organic Nitrogen in the Xylem Exudate during the Development of Soybean Plants .............. DISCUSSION .......................... Page 35 36 36 37 38 4O 42 44 44 45 46 47 47 48 49 52 59 L_______—---IIIlllIlIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII Page CHAPTER 2 The Products of Dark 602 Fixation in Soybean Nodules 64 RESULTS ........................... 64 A. Labeling of Organic Acids ................ 64 B. Labeling of Amino Acids ................. 66 C 4C02—Pulse 12COZ-Chase Experiment ............ 66 D. Position of [14C] within Aspartate and Glutamate ..... 68_ E. Labeling of Asparagine and Glutamine ........... 7l DISCUSSION .......................... 74 A. Initial Step of Dark CO2 Fixation in Nodules ....... 74 B. Loss of Recently-Fixed C02 from Nodules ......... 77 C. Amino Acid Metabolism .................. 80 + CHAPTER 3 The Effects of N05 and NH4 on Dark C02 Fixation and Amino Acid Metabolism in Soybean Nodules and Roots . 88 RESULTS ........................... 88 A. Effect of Inorganic Nitrogen on C2H2 Reduction and Distribution of Nitrogen in the Xylem Exudate ...... 88 B. Effects of N05 and NH: on Amino Acid Metabolism in Roots of l3-day—old Plants ................... 95 l. Concentrations of Amino Acids ............. 95 2. Products of Dark CO2 Fixation ............. 97 C. Effects of N0; and NH: on Amino Acid Metabolism in Roots of 29-day-old Plants ................... 99 l. Concentrations of Amino Acids and Organic Acids . . . . 99 2. Rates of Dark CO2 Fixation .............. 99 3. Products of Dark C02 Fixation ............. l02 4. Specific Activities of Amino Acids in Roots Exposed to 4co2 . . ' 102 0. Effects of N03 and NH: on Amino Acid Metabolism in Nodules 109 E. Rates of Incorporation of Carbon Derived from Dark CO2 Fixation into Amino Acids in Roots and Nodules ...... lll vi ¢ F. Pathway of Asparagine Synthesis in Roots ......... l. Labeling of Asparagine from 14CO2 Incorporated in Roots 2. Synthesis of Asparagine from [2, 3- 3H]Aspartate and [4 -] 4C]Aspartate ................... DISCUSSION .......................... A. Time Course of the Effect of NO3 and NH4 ......... B. Effects of NO3 and NH+ on Nodule Metabolism ....... C. Effects of NO3 and NH4 on Root Metabolism ........ 1. Dark CO2 Fixation ................... 2. Synthesis of Amino Acids ............... 3. Specific Activities of Glutamate and Glutamine D. Asparagine Synthesis ................... CHAPTER 4 Dark CO2 Fixation in Alder Nodules ......... RESULTS ........................... DISCUSSION .......................... CHAPTER 5 The Incorporation of 13N-Labeled Tracers in Soybean and Alder Nodules and Soybean Roots ......... RESULTS ........................... DISCUSSION .......................... SUMMARY ............................ REFERENCES ........................... vii Page 113 113 114 123 123 124 126 126 126 128 130 132 132 139 142 142 152 157 161 10. 11. 12. 13. 14. LIST OF FIGURES Metabolism of N2, NO3 and NH: in Nodulated Soybean Roots . . Proposed Sources of the Atoms in Allantoin and Synthesis of Tetrahydrofolate Derivatives .............. Effect of the Concentration of CO2 on the Rates of Dark CO2 Fixation in Roots and Nodules of Intact Soybean Plants . Rates of Dark CO2 Fixation and C2H2 Reduction and the Composition of the Xylem Exudate during the Development of Soybean Plants ..................... Correlation of the Concentration of Amino Acids in Xylem Exudate with the Rate of Dark CO2 Fixation in Nodules Incorporation of Label into Organic Acids and Neutral Compounds 14 in Nodules Exposed to CO 2 ................ Time Course of the Distribution of Label in Organic Products in Nodules Exposed to 14CO2 ................ Distribution of Label in Organic Compounds in Nodules during a 14coz-Pu1se 12coz-cnase .- ................ Time Course of the Specific Activities of the Amino Acids Labeled in Nodules Exposed to 14CO2 ............ Proposed Pathways for the Exchange of Label into Organic and Amino Acids of Nodules Exposed to 14co2 .......... Proposed Pathway for the Exchange of Label from 14CO2 Incorporated in Nodules into C-1 and C-4 of Oxalacetate Proposed Pathway for the Exchange of Label into Serine and Glycine in Nodules Exposed to 14CO2 ............ Proposed Pathway for the Exchange of Label into Alanine in Nodules Exposed to 14CO2 ................ Time Course of the Effect of NO- on the Distribution of 3 Nitrogen in the Xylem Exudate and the Rate of C2H2 Reduction viii Page 20 51 57 58 65 67 7O 72 76 78 82 86 9O 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. _ Page Time Course of the Effects of N03 and NH: on the Distribution of Nitrogen in the Xylem Exudate ....... 94 Effect of N03 and NH: on the Distribution of Label in Amino Acids in Roots Exposed to 14CO2 . . . . . ...... l05 Effect of N03 and NH: on the Specific Activities of Several Amino Acids in Roots Exposed to 14CO2 . .‘ ......... 108 Effects of N05 and NH: on the Amounts of Several Amino Acids in Nodules ......................... 110 Time Course of the Distribution of Label in Organic Compounds in NOS-Treated Roots Exposed to a 14COZ-Pulse ~12C02-Chase ..................... V . . . 115 Proposed Pathways for the Incorporation of Label from [2.3-3H]Aspartate and [4-]4C1A5partate into Asparagine . . . 118 Incorporation of Label and the 3WHO Ratio in Organic Products in Roots Incubated with [2,3-3H]Aspartate and [4-]4CJAspartate 122 Distribution of Amino Acids in Xylem Exudate and Extracts of Nodules of Alders ..................... 133 Time Course of the Distribution of Label in Organic Compounds in Alder Nodules Exposed to 14C02 ............. 135 Incorporation of Label into Organic Acids and Neutral Compounds in Alder Nodules Exposed to 14C02. ............. l36 Time Course of the Distribution of Label in Organic Acids in Alder Nodules Exposed to 14co2 ............... 138 Proposed Pathways for the Incorporation of Label into Citrulline in Alder Nodules Exposed to 14CO2 ............. 140 Incorporation of Label into Organic Products in Alder Nodules Incubated with 13NH; .................... 144 Time Course of Incorporation of Label into Glutamine and Glutamate in Soybean Roots and Alder Nodules Incubated with 13NH+ 146 4 OOOOOOOOOOOOOOOOOOOOOOOOOOO ix 29. 30. Page Incorporation of Label into Organic Products in Soybean Roots Incubated with 13N03 or 13NH: ............ l48 Incorporation of Label into Organic Products in Soybean 13 + 150 Nodules Exposed to [BNJN2 or NH4 ............ 10. 11. 12. 13. 14. LIST OF TABLES HPLC Elution Gradient for Amino Acid Analysis ....... Effect of the Concentration of CO2 on the Rate of Dark CO Fixation in Attached and Detached Nodules ......... Effect of the Concentration of CO2 on Nodule Respiration . . Effect of the Concentration of CO2 on the Rate of C2H2 Reduction .......................... Distribution of Label and Estimated Rates of Incorporation of Carbon Derived from Dark CO2 Fixation into Amino Acids in Nodules Exposed to 14CO2 ................ Effects of N03 and NH: on the Concentration of Amino Acids in Roots and Nodules of Young Plants ............ Effects of NO3 and NH4 on the Distribution of Label Incorporated from 14CO2 in Roots and Nodules of Young Plants Effects of N05 and NH: on the Concentration of Amino Acids in Roots of 29-day-old Plants ............... Effect of KNO3 on Concentration of Several Organic Acids . . Effects of N03 and NH: on the Rate of Dark CO2 Fixation in Roots and Nodules ..................... Estimated Rates of Incorporation of Carbon Derived from Dark CO2 Fixation into Several Amino Acids in Roots and Nodules - .......................... 3H/MC Ratio in Aspartate and Asparagine after Incubation of Roots with [2. 3-3H]Aspartate and [4-]4C]Aspartate . Effect of NH4 and Aminooxyacetate on Production of [ 3H]H2 0 from [2, 3- 3H]Aspartate and 14CO from [4- 4C]Aspartate . . . 2 Incorporation of Label into Organic Products in Soybean Roots Incubated with 13NHZor 13N03 ............ xi Page 34 50 53 54 83 96 98 100 101 103 112 119 121 149 EZ‘I aln asn asp cit citl FH fum GDH gln glu 91y GOGAT GS HPLC mal MSX N-free o.a. '1' neu. OPA PEPC POPOP PPO LIST OF ABBREVIATIONS allantoate asparagine aspartate citrate citrulline tetrahydrofolate fumarate glutamate dehydrogenase (L-glutamate:NAD(P) oxidoreductase [deaminating] EC l.4.l.3) Iglutamine -glutamate glycine glutamate synthase (L-glutamatezoxidoreductase [transaminating] cofactor NAD+ EC 1.4.1.14; NADP+ EC 1.4.1.13; ferredoxin EC 1.4.7.1) glutamine synthetase (L-glutamatezammonia ligase [ADP] EC l.4.l.3) high performance liquid chromatography malate methionine sulfoximine free of nitrogenexcept for N2 organic acids plus neutral compounds orphthalaldehyde phosphoenolpyruvate carboxylase (orthophosphate:oxalacetate carboxyl-lyase [phosphorylating] EC 4.l.l.3l) l,4-bis|2-(4-methyl-5-phenyloxazolyl)]-benzene 2,5-diphenyloxazole xii SEY‘ SUC UV serine succinate ultraviolet INTRODUCTION One of the problems the governments of the world are facing and will continue to face is how can the people of the world be fed. Up until recently, the efforts in the area of agricultural have concentrated on developing new varieties of plants that respond to increased amounts of fertilizer nitrogen (1). However, because of the increasing costs of fertilizer nitrogen that is produced in chemical factories (2), the developing countries can no longer afford to purchase the nitrogen that makes these fertilizer-intensive plants more productive than other varieties. In the future, biological N2 fixation is expected to partially replace chemical factories as the source of nitrogen for agricultural purposes. The amount of nitrogen that biological N2 fixation , contributes may be dramatically increased by understanding the processes by which N2 is reduced and is converted into usable forms of organic nitrogen. The synthesis of amino acids, in particular those most closely associated with the tricarboxylic acid cycle, is especially important in biological N2 fixation because of its role in the initial steps of converting inorganic nitrogen to organic nitrogen. The enzymes that catalyze the synthesis of these amino acids have, for some systems, been isolated and partially characterized (3,4). Recent studies using labeled tracers have partially identified the pathways by which nitrogen is incorporated into organic compounds (5,6). Further work is needed to confirm the pathways of amino acid synthesis and to determine what factors regulate amino acid metabolism in biological systems that fix N2. My research was directed towards the further understanding of the metabolism of amino acids in the soybean -Rhizobium japonicum and alder-Frankia sp. 1 systems. Previous investigators have proposed that dark CO2 fixation in lupin (Lupinus angustifolius) (7) and broad bean (Vicia faba) nodules (8) provides carbon skeletons for the synthesis of amino acids. The synthesis of amino acids, and in particular asparagine, is especially important in the nodules of these plants because amino acids may represent up to 100% of the organic nitrogen that is transported from the root to the shoot (9,10). Organic nitrogen is transported from the root system of alders primarily as the amino acid citrulline (ll) and from soybeans that are dependent on N03 primarily as asparagine (12). Therefore, carbon that is incorporated during dark CO2 fixation in these systems might be expected to be incorporated into amino acids. In contrast, soybeans that are dependent on N2 that is fixed in the nodule synthesize and transport the ureides, allantoin and allantoate, instead of amino acids (12). The dependence of soybean nodules on dark CO2 fixation might be expected to be minimal when compared to alder nodules or the root systems of soybeans that are dependent on N03. In this thesis I will address the following questions: l) Does dark CO2 fixation occur in soybean and alder nodules and soybean roots? 2) Is label incorporated into amino acids in soybean and alder nodules and soybean roots exposed to 14C02? 3) What are the effects of N03 or NH: on amino acid metabolism in alder and soybean nodules and soybean roots? I initially characterized dark CO2 fixation in soybean nodules (question l) and then determined the fate of the label that was incorporated after exposure of soybean and alder nodules to 14CO2 (question 2). The effect of N03 or NH: on amino acid metabolism in soybean roots and nodules was examined by monitoring the labeling of amino acids after exposure of tissues to 14CO2 and by determining the concentrations of a number of amino acids (question 3). In other experiments, [13 examine the metabolism of N03, NHZ, and N2 in nodulated soybean N] was used as a probe to roots and NH: in alder nodules (question 3). I have arranged the results and discussion part of this thesis into the following chapters: Chapter 1 - The Rate of Dark CO2 Fixation during the Development of the Soybean NZ-Fixing System Chapter 2 - The Products of Dark CO2 Fixation in Soybean Nodules Chapter 3 - The Effects of N0; and NH: on Dark CO2 Fixation and Amino Acid Metabolism in Soybean Nodules and Roots Chapter 4 - Dark CO2 Fixation in Alder Nodules Chapter 5 - The Incorporation of 13N-Labeled Tracers in Soybean and Alder Nodules and Soybean Roots The work that is presented in Chapter 4 was done in collaboration with Dr. P. McClure and that in Chapter 5 with Dr. K.R. Schubert. LITERATURE REVIEW Soybean plants with the bacteria Rhizobium japonicum (13) and alder plants with the bacteria Frankia sp. (14) are able to form symbioses that can use N2 as their sole source of nitrogen (15,16). During the development of these symbioses, the growth and metabolism of both the plant and the bacteria are altered. Upon infection by the bacteria, the root tissue develops into root nodules, the organ of N2 fixation. The bacteria differentiate into a form called endophytes (17,18) which are enclosed in vesicles contained within the plant cell (19). These endophytes in the R. japonicum - soybean symbiosis are called bacteroids (18,20). Both plant and bacteria are necessary for the efficient fixation of N2 in these symbioses. The plant provides the environment and the source of energy necessary for N2 fixation. The endophytes synthesize nitrogenase and nitrogenase reductase, the enzyme complex which reduces N2 to NH: (21,22). Ammonium is released from the bacteroids (23,24) and enters the host cell where it is assimilated (25). Based on both the spatial and metabolic organization of the nodule, the endophyte may be thought of as a plant cell organelle that reduces NZ to NHZ. A skematic of some of the metabolic processes occuring in soybean nodules is shown in Figure 1. A. DARK COO FIXATION Various tissues (26—30) including legume root nodules (7,8,31,32) directly incorporate CO2 in a process referred to as dark CO2 fixation. Phosphoenolpyruvate carboxylase (PEPC), which catalyzes the synthesis of oxalacetate, was proposed to be the enzyme that is primarily 4 .ucmpa esp co «005m as» ou newcoamcesp mew mcczoasoo omen» page mpeuwecw mama we new me“ cause“ moz ecu maven ocean .mmuwmc: ecu Eocm camcu mzoscm one .emucmmwsg we muoos cmmnxom umpm_=eo: :_ empmpvewmmm use sz use moz .Nz sows; An muses on“ so uwpmsmxm umwmwFQEPm < mecca eeeezom empe_=eez e2 ”:2 new moz .Nz co Emepeeeeez P deemed ‘_ boo: _ L “2241?: udzooz A . mozATmoz 5 ...—mo ._.z<._m A ...... oc_:=. .vu9. .vw¥.z ouocoooczz c.0mm...o- L-glutamine + MgADP + HPO4 The enzyme isolated from pea leaves has Km values for glutamate, + 4 Glutamine synthetase in prokaryotes is regulated by an elaborate NH and MgATP of 8 mM, 0.019 mM and 0.5 mM, respectively (65). mechanism involving the adenylylation and deadenylylation of the enzyme (66). This mechanism, however is apparently not involved in the regulation of GS in higher plants (67,68). Glutamine synthetase in plants does appear to be regulated by energy charge (3,69) and light (70). The effect of light on the activity of GS in Chlorella sorokiniana was examined by growing cells on a 7 h light: 5 h dark photoperiod and monitoring GS activity (70). The specific activity of GS from cells grown in the dark for 5 h followed by l h of light was 7 times the activity from cells examined immediately after th 5 h dark period. This increase in specific activity of GS in the light was not dependent on ge_ngyg_synthesis of GS. Rhodes et a1. (71) showed that the activity of GS purified from Lemna minor is reversibly altered by the titration of four thiol groups of the 11 enzyme with thiol reagents. The regulation of GS in_yjyg_by altering thiol groups suggests that GS activity might be modulated by the LEM (light effect modulators) system as proposed by Anderson and Avron (72) or a similar system proposed by Buchanon (73). Glutamine synthetase has recently been found to exist as several different isozymes (74-77). One of the isozymes in rice plants is localized in the chloroplasts, a second in the cytosol of leaves and a third in the roots (74). Although the kinetic analysis is preliminary, several investigators (75,76) have proposed that the isozymes have different roles in nitrogen assimilation. Glutamate synthase catalyzes the following reaction: 2-oxoglutarate + L-glutamine + NAD(P)H + H+-—-—«-2 L-glutamate + NAD(P)+ Several tissues including pea leaves (60) contain a GOGAT enzyme that uses reduced ferredoxin instead of reduced pyridine nucleotides (58). The enzyme isolated from lupin nodules has Km values for 2-oxoglutarate, glutamine, and NADH of 39 uM, 400 uM and 1.3 uM, respectively (4). The regulation of GOGAT jn_yjyg_is not completely understood; however, product inhibition may be involved. For the enzyme from lupin nodules, NAD+ is a competitive inhibitor (Ki=0.l mM) with respect to NADH and glutamate is a competitive inhibitor (Ki: 0.7 mM) with respect to 2-oxoglutarate (4). 2. Pathway of NH: Assimilation The proposal that GS/GOGAT is the primary pathway for NH: assimilation in plants is supported by two lines of evidence: 1) tracer studies with labeled nitrogen, either [13N] or [ISN] and 2) measurements of enzyme levels. If GS and GOGAT are the enzymes ‘- I 12 responsible for the assimilation of NH: then glutamine would be expected to be the initial organic compound formed from NH: and glutamate the second. Although Kennedy (6,52) indicated that glutamate and glutamine were the primary amino compounds synthesized in Nz-fixing nodules of serradella plants (Ornithopus sativus) exposed to [15N1N2, he concluded that glutamate is the primary amino acid synthesized from NH: produced during N2 fixation. The use of the radioactive isotope of nitrogen,[13N] (half-life = 10 min),has allowed other investigators to determine the products of NH: assimilation after shorter labeling periods. Glutamine accounted for 82% of the total label in the organic compounds in soybean nodules that were exposed to [13N]N2 for 20 sec, while glutamate accounted for the remaining 18% (5). The percentage of label in glutamine declined and the percentage in glutamate increased after longer periods of exposure. Similar results were obtained using tobacco cells that were incubated with 13NH: or 13N03 (78) and rice roots incubated with 15NH: (79) or 15N03 (80). The results of these studies are consistent with NH: being assimilated into glutamine and glutamate by GS and GOGAT. The pathway of NH: assimilation has been further studied using inhibitors of GS and GOGAT. Methionine sulfoximine (MSX), an inhibitor of GS, when added to a number of tissues has been shown to inhibit the incorporation of labeled-NH4+ into glutamine and glutamate (78,81,82), suggesting that GS is the enzyme primarily responsible for the initial assimilation of NH2. In several of the experiments (78,81), MSX inhibited the synthesis of glutamine more than glutamate. However,if GS is the sole enzyme that is responsible 13 for NH: assimilation, then inhibiting GS should inhibit the incorporation of labeled-NH: into glutamine and glutamate to the same extent. To explain the paradox between this hypothesis and the observed results, Skokut gt al.(78) proposed that one of two events is occurring. One, either a small portion of the 13NH: is assimilated by GDH or two, the GS activity that is not eliminated by MSX synthesizes glutamine which is preferentially used for the synthesis of glutamate. Azaserine, an inhibitor of GOGAT, inhibited the incor- poration of 13 NH: into glutamine and glutamate by 56 and 99%, respectively (78), which is consistent with GOGAT being the enzyme primarily responsible for glutamate synthesis. The second line of evidence for GS/GOGAT being the primary route for NH: assimilation comes from the measurement of enzyme levels ‘jgnyitrg. The levels of GS in nodules of 11 different species of legumes were an average of l6-times higher than the levels of GDH when both enzymes were measured with saturating amounts of substrates (83). When the relative Km values for each of the enzymes and the estimated concentration of NH: in the nodules are considered, GS would be more than 90% saturated with NH; whereas GDH would operate at about 10% of its maximum velocity (83). On this basis, Boland gt 31. (83) concluded that GS and GOGAT are responsible for the primary assimilation of newly fixed NH: produced by the bacteroid. Based on similar considerations as those above, the activity of GDH in Lemna minor can be estimated to be between 0.4 and 4.0% the activity of GS (71,84) suggesting that GS is also responsible for the assimilation of NH: in this organism as well. In summary, it is generally concluded that GS/GOGAT is the primary pathway or NH: 14 assimilation (85,86). However, the possibility that GDH or some other enzyme assimilates a portion of the NH: has not been excluded. Once NH: is assimilated into glutamine and glutamate, these amino acids are used in the synthesis of other nitrogenous compounds such as aspartate, asparagine, allantoate and citrulline. Aspartate is synthesized by aspartate aminotransferase which catalyzes the following reaction: oxalacetate + L-glutamate 4—————_'> L-aspartate + 2-oxoglutarate Several different isozymes of aspartate aminotransferases are found in soybean (87) and lupin nodules (88). The activity of a specific isozyme of aspartate aminotransferase increased during the development of lupin nodules (88). Reynolds and Farnden (88) proposed that this isozyme is specifically associated with N2 fixation. 3. Asparagine Synthesis Asparagine is one of the most prominent amino acids in many plants. It is used for the transport of nitrogen within plants (for a recent review see ref. 89) and for the storage of nitrogen in a form which is less toxic than free NH: (90). Asparagine in soybeans grown on N05 (12) and in lupins (9) accounts for up to 70% of the organic nitrogen translocated from the roots to the shoot. In the shoot the asparagine is degraded and the nitrogen and carbon are used to synthesize other compounds (91,92). Asparagine accumulates during seed germination (93-96). The carbon skeletons of the storage protein are oxidized to provide energy for the developing plant. The NH: that remains after the protein is degraded is used to synthesize asparagine. With the 15 development of the photosynthetic apparatus of the developing plant and the ability to fix carbon, the accumulated asparagine is degraded and the nitrogen is used for protein synthesis (for a review see ref. 96). Three different pathways have been proposed for asparagine synthesis: 1) direct amidation of aspartate (97-100), 2) an indirect pathway via succinate and/or fumarate (101) and 3) hydrolysis of cyanoalanine (102-104). The evidence for the first pathway includes enzymological data and labeling studies. An asparagine synthetase catalyzing the following reaction has been detected in a number of tissues (98,100,103): L-aspartate + MgATP + L-glutamine ——- L-asparagine + L-glutamate + MgAMP + PPi The asparagine synthetase in lupin seedlings has Km values for aspartate, MgATP and glutamine of 1.3 mM, 0.14 mM and 0.16 mM, respectively (100). Label from [14C]aspartate vacuum infiltrated into soybean leaves was incorporated into asparagine. This labeling of asparagine was inhibited by MSX and stimulated by glutamine (99). Stewart (99) concluded that asparagine is synthesized jn_yjyg_by a glutamine- dependent asparagine synthetase. Because uniformly labeled aspartate was used, the results do not exclude the possibility that succinate and/or fumarate are intermediates. Exogenous aspartate does not appear to be the direct precursor of asparagine in pea roots (101). On the basis of isotope-competition experiments, Mitchell and Bidwell (101) concluded that succinate and/ or fumarate are more direct precursors for asparagine synthesis than 16 exogenous aspartate. They further proposed that asparagine synthesis is compartmentalized. Lever and Butler (105) reached similar conclusions and further suggested that aspartate was an intermediate in the synthesis of asparagine from fumarate. If this proposal is true, then the first two pathways listed above are actually the result of the relative distribution of aspartate between two pools: one that is used for asparagine synthesis, and a second that is in equilibrium with succinate and/or fumarate. Streeter (106) using soybean cotyledons concluded that a fraction of the asparagine is synthesized via succinate but that another fraction is synthesized directly from aspartate. Cyanoalanine, formed by the condensation of HCN and cysteine, can be hydrolyzed to form asparagine (102-104). While the enzymes catalyzing this reaction have been isolated, the extent to which this pathway contributes to the synthesis of asparagine appears to be limited jn_ijg_because the amount of CN' that is available to the plant appears to be minimal (89). The status of asparagine synthesis in soybean nodules is not clear. Although Streeter (106) reported that he was unable to detect a glutamine-dependent asparagine synthetase in soybean nodules, two preliminary communications (107,108) have reported measuring asparagine synthetase activity in cell-free extracts of soybean nodules. The results of experiments attempting to incorporate label into asparagine in intact nodules are mixed. Asparagine accounted for 34 and 86% of the total label incorporated in soybean nodules 15 + that were vacuum infiltrated with NH4 and [amide-15N]glutamine, respectively (109). In contrast, asparagine accounted for less than 17 1% of the label incorporated in soybean nodules that were exposed to [13N]N2. Based on the incorporation of label from 15NH: and [amide-lsNJQlutamine into asparagine and the recent reports of the presence of an asparagine synthetase in soybean nodules, asparagine can be synthesized in these nodules. Whether asparagine is synthesized in the nodule from nitrogen derived from N2 is not clear. 4. Citrulline Synthesis Citrulline is the primary product of N2 fixation in alder root nodules (110). It also accounts for a large percentage of the nitrogen transported from the root to the shoot in several species of plants including alders (A130; sp.) and persimmons (Diospyros kaki) (11). The proposed pathway for citrulline synthesis in higher plants is : l) N-acylation of glutamate, 2) conversion of N-acylglutamate to N-acyl ornithine, 3) removal of the acyl group to form ornithine, 4) synthesis of carbamyl phosphate and 5) synthesis of citrulline from ornithine and carbamyl phosphate (for a review see ref. 111). 5. Allantoin and Allantoate Synthesis The ureides, allantoin and allantoate, account for up to 98% of the nitrogen that is transported to the shoot from the root nodules of several species of grain legumes, including soybeans (12), cowpeas (Vigna unguiculata; 112) and dwarf french beans (Phaseolus vulgaris; 31). Allantoate is proposed to be synthesized from purines which are synthesized gg_novo in the nodule (113,114; for a review see ref. 115,116). According to this proposal, the , W'fi I I 18 initial step is the synthesis of phosphoribosylpyrophosphate. Then in a series of reactions, the amide nitrogens of two glutamines, the amino group of aspartate, glycine, carbon from bicarbonate and two carbons from derivatives of tetrahydrofolate are added resulting in the synthesis of inosine monophosphate. This compound in another series of reactions is metabolized to form allantoin and allantoate. Figure 2 illustrates the origins of the atoms of allantoate. + 4 D. EFFECT OF NO; AND NH ON NO FIXATION The fixation of N is both inhibited (12,117-121; for 2 reviews see ref. 122,123) and stimulated (121,124) by N05 and NH1. Whether N03 or NH: inhibits or stimulates depends on the timing and the amount of nitrogen that is applied. As stimulants, N03 and NH: are suggested to alleviate nitrogen stress, especially when they are added during the early development of the plant (124). The rate of N2 fixation of 21 to 28-day-old soybean plants that had been treated with 7 mM KNO for the first 14 days after 3 they had been planted was three times that of plants that had 3 3 act to inhibit initiation and subsequent nodule development not been treated with KNO (124). As inhibitors N0 and NH; (for a review see ref. 125). The inhibitory effects of N0; and NH: on N2 fixation are also observed when the nitrogen is added to the plants after N2 fixation is established. The rate of C2H2 reduction ( an assay for nitrogenase activity) in 78-day-old soybean plants that had been treated with 7 mM KNO3 for the prior 7 days was 30% the rate of plants that had not been treated 19 mmmposcxzoFozo opepoeocexcesamp _>=m>;aoe -op .mz Amy mmmcmmocuzcmu muapoeosuxgmcumu mcmpxcums top .mz ANV mmmcmmmcmLHF>zumexxosuxc mcwsmm APV "cowuomms cmumowocw mzp mwapmpmo pas» mechm on» on some; mmmmsucmcmg cw mcmasac one .m c_ concommca mew umNmegpcxm mew Aezdv manpoeosuxcmcpmu mo mm>wum>Psmu esp mo meow sows: An mcovuommc esp .< cw umpcmmmca mew ANNF._NPV :Popcmppm cw macaw ms» co mouczom cwmoaocq one mm>_pm>wsmo mpepoeouzgespmh co mwmmgpcxm can crou:m__< cw msou< one co moossom ammoaoca N acumen 20 N mszmwd ca: 1 . VI'C . i 50 I I: 2.352: 22 oz 1... . E .22 53 inn—<2 » ao52: 0.2 oz A: z“. + .. TEE—:33 “a 03:3 :3: I I P \\\a h- - , 1 . - o/ x x o v ”Eco 1 ES 1:“. 3.352: 92 n2 :3: . In...“ a/w\,. /n_.wl\_lzn_ _ .22 .— - 1 \ . . s .1111 u xxx 2 . 000m 121/1:23am: Ea:— 21 with KNO3 (12). The time necessary for the first effects of N03 or NH: on N2 fixation to be noticeable is of the order of l to 2 days after the initial application. Two days after the addition of 7 mM KNO3 to 24-day-old soybeans, the rate of C2H2 reduction of the KNO -treated plants was 80% of that of the control plants (124). 3 Similar observations have been made using peas (120) and soybeans treated with NH4C1 (124). Two mechansims have been proposed to explain the inhibitory effects of N03 on N2 fixation. The first is that the uptake and utilization of N03 influences the distribution of photosynthate to the various organs of the plant (124,126,127). The second mechanism is that N03 or one of the products of its reduction has a direct effect on N2 fixation (124,128,129). The fixation of N2 is closely linked to the availability of photosynthate. Manipulations that result in increased rates of N2 fixation such as removing other sinks for photosynthate (130) or increasing the rate of photosynthesis (131,132) are said to have acted by increasing the supply of photosynthate to the nodules, thereby increasing the energy available for N2 fixation. Manipulations such as removing the shoot of a plant (133) or decreasing the light intensity (134) result in a reduced rate of N2 fixation presumably by reducing the flow of photosynthate to the nodules. The proponents of this mechanism argue that upon the application of N03 the sites of N03 reduction, the leaves and roots, retain the photosynthate that was previously destined to be used by the nodule for N2 fixation. The nodule, therefore, 22 has a reduced rate of N2 fixation because of the reduced supply of energy that is available. This proposed mechanism is based, in part, on the effect of N05 on the distribution of photosynthate. Small and Leonard (126) treated nodulated pea and clover plants with 14 mM KNO3 for 5 days, at which time the plants were exposed to 14 CO2 for 15 min during the photosynthetic period. Nodules from plants that were treated with KNO3 contained less label than nodules from plants that had not been treated with KN03. In contrast, the roots of plants that were treated with KN03 had higher levels of label than roots from control plants. Similar experiments (127,134) with soybeans have yielded similar results. The second proposed mechaism to explain the effect of N03 on N2 fixation is that N03 or one of the products of its reduction directly affects nodule metabolism (124,128,129). Based on the effects of tungsten on the inhibition of N2 fixation by hog, Harper and Nicholas (135) concluded that N03 must be metabolized to affect N2 fixation. This suggests that N03 does not directly affect N2 fixation, but does not eliminate the possibility that one of the metabolites of N03 reduction such as NOE, NHZ, glutamine or glutamate does so. Houwaard (l36) concluded that NH: must be assimilated to inhibit nitrogenase activity. The rate of C2H2 reduction in nodules that were treated with NH401 for 4 h was 40% the value for control nodules. If MSX was .4. 4 acids and NH: can inhibit N2 fixation by affecting the synthesis of included, NH had no effect on the rate of 02H2 reduction (136). Amino nitrogenase (137) but they do not appear to inhibit purified 23 nitrogenase (138). Nitrite, the initial product of N03 reduction, was suggested to mediate the inhibition of N2 fixation by N03 because of the low concentrations of N05 (0.1 to 0.8 mM) needed to inhibit C2H2 reduction in isolated bacteroids (139) and by purified nitrogenase (140). The N03 reductase in bacteroids was thought to be responsible for the synthesis of N05 that inhibits N2 fixation; however, Gibson and Pagan (141) and Manhart and Wong (142) concluded on the basis of their work with N03 that the bacteroid N03 reductase is not involved in the inhibition reductase mutants of rhizobia, of N2 fixation by N03. Streeter (129) in a recent preliminary report suggested that the N03 reductase in the plant portion of the nodule produces NOE which inhibits N2 fixation. Direct evidence linking N03 reduction in the nodule to the inhibition of N2 fixation is not yet available. E. EFFECTS OF NO3 AND NH: ON NITROGEN TRANSPORT IN SOYBEANS Nitrate and ammonium affect the amount of amino acids that are transported from the soybean root to the shoot, indicating that N03 and NH: affect amino acid metabolism in the root system (12,143). Amino acids account for 40% of the total nitrogen in the 3 as compared to 20% or less in the xylem exudate of plants dependent xylem exudate of plants that are dependent on NO for nitrogen on N2 fixation (12). The synthesis of asparagine, in particular, appears to be stimulated in the roots of plants treated with N03 (12,144). The effect of NH: on the transport of nitrogen in soybeans has not been well documented. In one study (143), 5-day-old 24 soybeans were treated with 10 mM NH4C1 or 10 mM KNO3 for 8 days, at which time the xylem exudate was collected and analyzed. The +. 4 contained 6-times the amount of glutamine and 2-times the amount xylem exudate of the soybeans that had been treated with NH of asparagine as found in the xylem exudate of the NOé-treated plants. F. EFFECTS OF NH} ON AMINO ACID SYNTHESIS Ammonium may affect the synthesis of amino acids either as a substrate or as a modulator of enzyme activity. In response to the addition of high levels of NHZ, plants will synthesize' large amounts of the amides, asparagine and glutamine (145). This increased synthesis is apparently because of the increased concentration of substrates, NH; for GS and glutamine for asparagine synthetase. Ammonium has been suggested to affect amino acid synthesis by directly altering the activities of certain enzymes. Several investigators (146,147), based on the levels of metabolites in Chlorella pyrenoidosa or alfalfa (Medicago sativa) leaves after the addition of NH2, proposed that NH: stimulates pyruvate kinase. This stimulation presumably results in an increase in the amount of ketoacids for amino acid synthesis. The jn_vitro activity of pyruvate kinase, which requires a monovalent cation for activity + 4 not sufficient to saturate the enzyme. Although similar results (148), can be increased by NH but only if levels of K+ are were observed in Anabaena cylindrica (149) as in C, pyrenoidosa after NH: was added (146), the in vitro activity of pyruvate kinase 25 from A, cylindrica is not stimulated by NHZ. These results suggest that NH: increases the flow of carbon through pyruvate kinase by some mechanism other than direct stimulation of the enzyme. Ammonium may also affect amino acid metabolism by regulating the synthesis or degradation of enzymes that assimilate nitrogen. The levels of GDH increase in several plants when high levels of NH: are present (62-64, 150,151). In Chlorella sorokiniana, NH: appears to regulate the activity of the NADP+-dependent isozyme of GDH by reducing the rate at which this isozyme is inactiviated (152). In contrast, the increase in GDH activity in Lemna minor (64) and oat (Avena sativa) leaves (62) after the addition of NH: appears to be the result of gg_ngy9_synthesis. Rhodes et_al,(64) suggested that glutamine, a product of NH: assimilation, rather than NH: regulates the synthesis of GDH. Although it is apparent that NH: increases the rate of synthesis of amino acids, the mechanisms by which it does so are not completely understood. MATERIALS AND METHODS A. GROWTH OF PLANTS Soybean seeds (Glycine max L. Merr. cv. Amsoy 71) were inoculated with broth cultures of Rhizobium japonicum USDA strain 3Ilb 110 (obtained as a gift from D. Weber, USDA, Beltsville, MD) and planted in 20-cm plastic pots filled with Perlite. All of the plants were irrigated with N-free nutrient solution (153) until 15 days after they were planted unless otherwise noted. The plants were then divided into four groups. One group continued to receive N-free nutrient solution while the other three groups of plants were irrigated daily with 1 liter of N-free nutrient solution that was supplemented with KNO3 to a final concentration of 3 or 10 mM or (NH4)ZSO4 to a final concentration of 1.5 mM. Plants, nodules and roots of plants that were treated with (NH4)ZSO4 will be referred to as NHZ-treated plants, MHZ-treated nodules and NHZ-treated roots. A similar format will be used for plants treated with KNO3. Unless otherwise specified NOé-treated will refer only to those plants treated with 10 mM KN03. Plants, nodules and roots of plants that were maintained solely on N-free nutrient solution will be referred to as control plants, control nodules and control roots. Young alder seedlings (Alnus glutinosa) either collected from the Kellogg Forest, Kalamazoo M1, or grown from seed (obtained as a gift from 0. Hanover, MSU, E. Lansing, MI) were planted in plastic pots filled with a commercial peat-vermiculite mixture. Stationary phase broth cultures of Frankia Avc 11 or Cp 1 (obtained 26 27 as gifts from D. Baker, Middlebury College, Middlebury, VT) or suspensions of nodules (10 g in 50 ml of H20) obtained from field-grown plants were used to inoculate the roots of 21 to 28-day-old alder plants grown from seed. Both soybean and alder plants were grown in the greenhouse under daylight extended to a photoperiod of -1 16 h with fluorescent lighting (55 pEom'gsec at pot height). 8. MEASUREMENT OF THE RATE OF C H2 REDUCTION 2 Besides reducing N2, nitrogenase is capable of reducing C2H2 to C2H4,thereby forming the basis for a convenient and sensitive assay for nitrogenase activity (154). The rate of C2H2 reduction was measured on nodules that were still attached to the roots of either intact plants or roots from which the shoots were excised or on detached alder and soybean nodules. Intact soybean plants were removed from the Perlite and each root system was placed into either a 20-ml single arm Warburg flask or a 50-ml round bottom flask. A split rubber stopper was then placed around the stem of the plant and the stopper was inserted into the top of the flask. Plasticine was used to seal around the plant, stopper and flask. Alternatively, intact plants were removed from the Perlite, the shoots were excised and each root system was placed into a 250-ml Erlenmeyer flask. The flask was sealed with a serum stopper. For other experiments, soybean or alder nodules were removed from the plant and were placed in a 10 or 50-m1 Erlenmeyer flask and the flask was sealed with a serum stopper. The assay was started by removing 0.1 volume of air and replacing it with the same volume of C2H2 which was produced from CaC (Sargent Welch) and water. Gas samples were removed 28 from each flask at various times up to l h and were injected into a gas chromatograph (Varian 3700). Ethylene was separated from C2H2 on a Porapak N column (2 m x 2 mm) with either He or N2 as a carrier gas and was detected with a flame ionization detector. The assay was ended by removing the plant tissue and placing it in a 70 C oven for 24 h. C. MEASUREMENT OF ALLANTOATE, ALLANTOIN, NO; AND TOTAL AMINO ACIDS Allantoin and allantoate were degraded to glyoxylate which was measured colorimetrically after its reaction with phenylhydra- zine and K4Fe(CN)6 (155). The assay was linear in the range that was used, 2 to 100 nmol. Nitrate was assayed either colorimetrically after its reaction with salicylic acid (156) or by its absorption of UV light after analysis by high performance liquid chromatography (HPLC) (157). The colorimetric assay was linear over the range that was used, 2 to 150 nmol, as was the HPLC method from 0.1 to 20 nmol. Total amino acids were assayed colorimetrically after their reaction with ninhydrin (158) or fluorometrically after their reaction with g¢phthalaldehyde (OPA) in a procedure adapted from Hill gt_al, (159). The colorimetric assay was linear over the range that was used, 10 to 200 nmol. In the adapted procedure, the GPA solution consisted of methanol (590 pl), saturated sodium borate (210 pl), ethanethiol (Aldrich) (50 p1), and GPA (Sigma) (30 mg). One microliter of sample, which contained 1 to 20 nmol of amino acids was added to 8 pl of OPA solution. One minute later, 2 ml of methanol were added and the solution was vortexed.’ The 29 fluorescence of the derivatized amino acids was measured with fluorometer fitted with a Corning 7-54 primary filter and Wratten 2A secondary filter. The fluorescent OPA-amino acid adduct has two excitation maxima, one at 230 nm and one at 330 nm, and one emission maximum at 460 nm. D. MEASUREMENT OF THE RATE OF 0 CONSUMPTION 2 The rate of 02 consumption of detached nodules and nodules of intact plants was measured amperometrically with a Clark oxygen electrode (YSI 4004, Yellow Springs, OH) (160). An intact plant was removed from the Perlite and the roots were trimmed so that they would fit into a 7-ml Lucite chamber in which the electrode was fitted. A split rubber stopper was placed around the stem of the plant and the stopper was inserted into the top of the chamber. Plasticine was used to seal around the stopper and the plant. Alternatively, nodules were removed from the plant and placed in the chamber which was then sealed with a rubber stopper. The system was calibrated by injecting into the chamber known volumes of O2 (Matheson Gas Products). The assay was ended by removing the plant tissue and drying it in a 70 C oven for 24 h. E. PRODUCTION OF 14CO2 Labeled CO2 was generated by adding [14C]NaHCO3 (59 mCi mmo1“; Amersham) and 1 ml of 36 N H2504 to a 10-ml flask. The flask was sealed with a serum stopper and was gently stirred and heated until the solution was boiling. The 14CO2 that was produced was removed from the flask with a l or 3-ml syringe. 30 F. EXPOSURE OF PLANT TISSUE T0 14CO 2 Roots and nodules were sealed into flask in a fashion analogous to that presented in section B. The assay for the rate of dark CO2 fixation was started by injecting 5 to 20 OCT 14 of C02 into the sealed vessel containing the plant material. For 12 some experiments, CO2 (Matheson Gas Products) was added just 14 prior to the addition of CO2 to increase the CO2 concentration in the flask. In those experiments in which the products of 14 dark C02 fixation were to be examined 10 to 100 pCi of CO 2 were added. One minute before an assay was ended, two 0.5 or l-ml gas samples were removed from the flask so that the specific activity of the 14 C02 could be determined (section G). The assay for the rate of dark C02 fixation was ended for soybean nodules and roots after a 10 min exposure and for alder nodules after 20 min. All 14CO2 fixation assays were ended by removing the plant tissue and freezing it in liquid nitrogen. 14 G. MEASUREMENT OF THE SPECIFIC ACTIVITY OF CO2 One of the two gas samples that had been removed from each incubation flask was injected into a glass scintillation vial that was sealed with a serum stopper. The vial contained 1 ml of methylbenzethonium (Sigma). One hour after the sample was injected, 10 ml of scintillation fluid (6 g PPO in 1 liter of toluene) was added and the radioactivity from the 14C02 which was absorbed by the methylbenzethonium (161) was determined by scintillation spectrometry. The CD2 of the second sample was 31 separated on a Porapak N column (2 mex 2 mm) using He as a carrier gas and was measured with a thermal conductivity detector. An analyzed gas mixture (COzzozzNz; 0.04: 1.0: 98.96, v/v/v) that was purchased from Matheson Gas Products was used as a standard. H. EXTRACTION OF THE PRODUCTS OF 14CO FIXATION 2 The plant material that had been frozen after exposure to 14CO2 was lyophilized. Nodules and roots were separated from each other, weighed and 10 to 50 mg added to a 15-ml polypropylene centrifuge tube. The dried tissue was ground with a glass stirring rod and was extracted three times with 3 ml of ethanoleZO (80:20,v/v). The slurry was vortexed and then clarified either by centrifugation at 10,000 g for 10 min or filtration through 0.45 pm Millipore filters. The clarified solutions from each extraction were combined and dried jn_yagug at 50 C either to complete dryness or to the point where the odor of ethanol was not detectable. The remaining H20 was removed by lyophilization to prevent labile compounds such as glutamine and allantoate from being destroyed by exposure to 50 C for longer than necessary. The dried material was redissolved in 0.05 to 1.0 ml of H20 or ethanoleZO (80:20,v/v). The radioactivity of an aliquot of the redissolved material was measured by scintillation spectrometry after Triton X-100 scintillation fluid (666 m1 toluene, 333 m1 Triton X-100 (Research Products Int. Corp., Mount Prospect, IL), 5 g PPO, 0.1 g POPOP) was added. 32 I. SEPARATION OF THE PRODUCTS OF 14CD FIXATION 2 1. High Performance Liquid Chromatography (HPLC) Amino acids were separated and analyzed by a procedure adapted from Hill gt al, (159). The amino acids were derivatized with OPA and ethanethiol to form a fluorescent product. The amino acid adducts were then separated by reverse-phase liquid chromatography. The amino acids in plant extracts were derivatized by adding 10 pl of a nodule or root extract (representing about 3 mg dry weight) to 25 pl methanol, 10 pl of saturated sodium borate and 5 pl of OPA solution. The OPA solution contained 0.9 m1 methanol, 0.1 m1 saturated sodium borate, 50 pl of ethanethiol and 60 mg of OPA. Approximately 1 min after the OPA solution was added to the sample, the solution containing the aminoeacids was added to a microfilter system (BioAnalytical Systems, W. Lafayette, IN) which was centrifuged at 8000 g for 2 min to remove particulate material. Twenty microliters of the resulting filtrate was injected onto the HPLC column The HPLC system consisted of two pumps, a microprocessor, a stirring chamber, an injection valve fitted with a 20 p1 sample loop, either a 5 pm Ultrasphere or a 10 pm Ultrasil C18 reverse- phase column (Beckman), a fluorometer and a fraction collector. The fluorometer was fitted with a 70 pl flow cell, a Corning 7-54 primary filter and a Wratten 2A secondary filter. The column eluate passed through the fluorometer to a fraction collector where 0.3 to 1.0 ml fractions were collected in scintillation vials. The radioactivity collected in the scintillation vials was measured by scintillation spectrometry after the addtion of Triton X-100 33 scintillation fluid. The amino acids in the plant extract were identified on the basis of the retention times of their OPA derivatives and those of standard amino acids. The standard amino acids that were used routinely were aspartate, glutamate, asparagine, glutamine, serine, glycine and alanine. Occasionally arginine, leucine, isoleucine, valine, phenylalanine, ornithine, lysine, NH: and citrulline were used as standards. The concentration of the standard amino acids in the solution that was injected onto the HPLC column varied from 0.025 to 0.50 mM per amino acid. The OPA derivatives of the amino acids were detected with a fluorometer and were quantified by an electronic integrator. To minimize the time necessary to analyze each sample, the elution gradient was modified from that described by Hill 25.91. (159) to that listed in Table l. 2. Ion-Exchange Chromatography The organic acids were separated from amino acids by ion-exchange chromatography using a procedure adapted from Stumpf and Burris (162). Dowex resins were prepared according to Atkins and Canvin (163). A plant extract was applied to a disposable pipet that contained 1 ml of Dowex 50-X8 (hydrogen, 200-400 mesh). The organic acids and neutral compounds were eluted with 2 to 3 m1 of H20. The amino acids were eluted from the Dowex 50 column with 3 to 6 m1 of 14.5 N NH4OH. The organic acid and neutral compound fraction obtained from the Dowex 50 column was _either lyophilized or applied to a 1 ml column of Dowex l-X8 (formate, 200-400 mesh). The neutral compounds were eluted from the Dowex 1 column with 2 to 3 m1 of H20 and the organic acids were eluted with 2 to 3 m1 of 16 N formic acid. All fractions were lyophilized 34 Table 1 HPLC Elution Gradient for Amino Acid Analysis Time (min) CH3CN concentration = 12% Flow rate = 1.5 ml-min'1 0 Sample injection 0 CH3CN concentration increased to 26% at 9 Flow rate increased to 2.0.ml-min'1 at 15 CH3CN concentration increased to 100% at 25 CH3CN concentration decreased to 12% at 28 Flow rate decreased to 1.0 ml-min"1 at 1 at 37 Flow rate decreased to 0 ml-min' Plant extracts, xylem exudate and standard amino acids were Comments Initial conditions Initial conditions Rate Rate Rate Rate Rate Rate l%-min'1 0.1 ml-min' 1 1 10.6%fmin' l4.7%-min- 1.0 m1~min' 1.0 ml-min' derivatized with o-phthalaldehyde and ethanethiol and injected onto a C18 reverse-phase column. The amino acid adducts were eluted with a CH3CNzNaH2PO4 (12.5 mM; pH=7.2) gradient formed by two microprocessor controlled pumps. 1 1 1 35 before they were analyzed further. The organic acids from alder nodules were separated from one another by ion-exchange chromatography. A mixed bed of Dowex l (chloride, acetate) resin was prepared according to Berl §t_al,(164). The advantage to not converting the resin completely to the acetate or chloride form is that the organic acids, which would not be retained if the column was in the chloride form, were retained by the mixed-bed column and were eluted from this column with more dilute solutions of eluant than that required if the column was completely in the acetate form. A 170-ml gradient of 0 to 1.2 N formic acid was used to elute the organic acids. The 1-ml fractions collected from the column were dried in a 100 C sand bath to remove the formic acid. Water (0.2 ml) and Trition X-lOO scintillation fluid (2 ml) were added to the dried vials and the radioactivity was measured by scintillation spectrometry. 3. Thin-Layer Chromatography Plant extracts were spotted on either a 5 x 20 cm, 0.1 mm thick or 20 x 20 cm, 0.25 mm thick, thin-layer cellulose plates (Brinkmann). The extracts were Chromatographed using a solvent system of either phenoleZO (80:20,v/v) or n-butanolzacetic acidzHZO (120:30:50, v/v/v). The phenol solvent system was used to separate amino acids from one another, and the n-butanol system was used to separate organic acids from one another. About 1 pg of allantoin and allantoate and 0.05 pCi of [14C]malate (15 mCiommol'I; Cal Atomic, Los Angeles, CA), [14C]glutamate (180 mCi-mmol-1; Cal Atomic, Los Angeles, CA) and [14C]aspartate (180 mCi-mmol'1; ICN) were applied to each plate and used as standards. The ureides, allantoate and allantoin, were detected 36 by spraying the plates with a solution containing 1 g diamino- benzaldehyde in 3 m1 of 12 N HCl and 12 ml of n-butanol (165). Amino acids were made visible either with UV light after spraying the plates with OPA (166) or by heating the plates after spraying them with 0.25% ninhydrin in n-butanol. 4. High-Voltage Electrophoresis The high-voltage electrophoresis procedure developed by Wolk gt 31, (167) was used primarily for the analysis of the products of [13N] assimilation. Samples were spotted on a 5 x 20 cm, 0.1 mm thick, thin-layer cellulose plate (Brinkmann). The plate was sprayed with 70 mM sodium borate (pH=9.2) and was subjected to electrophoresis for 6 to 12 min at 1.5 to 3 kV. Amino acids were localized by spraying the plate with ninhydrin (167) or with OPA (166). The radioactivity on the plates was detected with a gas-flow scanner (Berthold). About 5 pg of aspartate, glutamate, asparagine, glutamine, alanine and allantoate were spotted on the plates and used as standards. Allantoate was localized by spraying the plate with diaminobenzaldehyde (165). 5. Gas-Liquid Chromatography of Organic Acids The organic acids from nodule and root extracts were purified on Dowex l (formate) resins as described in section 1.2 (paragraph 1). The organic acids were converted to their trimethylsilyl derivatives using a procedure adapted from Stumpf and Burris (162). Thirty microliters of redistilled, dry pyridine and 15 pl of N,O-bis-(trimethylsi1y1)- trifluroacetamide (BSTFA; Pierce Chemical Co.) were added to the dried organic acids. The reaction was carried out at room temperature for 37 10 min in 2-ml capped vials. The derivatized organic acids (0.3 to 40 nmol per organic acid) were separated by gas-liquid chromatography on a 2 m x 2 mm, 3% SE-30 column (Supelco, Inc., Bellefonte, PA) using N2 as a carrier gas and were measured with a flame ionization detector. The temperature program was 80 to 185 C at 5 C'min']. The organic acids were identified on the basis of their retention times and quantified on the basis of the detector response as compared to the standards malonate, succinate, fumarate and malate. 14 J. ISOLATION AND CHROMATOGRAPHY OF C-LABELED 2-OXOACIDS FROM SOYBEAN NODULES The oxoacids in nodules were isolated as the 2,4-dinitrophenyl- hydrazone derivatives, prepared using a procedure adapted from Isherwood (168). About 1 g (fresh weight) of soybean nodules were 14 exposed to CO2 for 5 or 60 min, at which time the nodules were removed from the vessel containing the 14 C02 and frozen in liquid nitrogen. The frozen nodules were ground, and 5 ml of 0.6 M (HPO3)n (meta-phosphoric acid) and 0.5 ml of 1% (w/v) 2,4-dinitrophenylhydrazine in 5 N H2S04 were added. The mixture was vortexed and then incubated at 37 C. After incubation for l h, the mixture was extracted four times with 2 m1 of diethyl ether to removed the 2,4-dinitropheny1hydrzone derivatives of the oxoacids. The ether extracts were combined and extracted twice with 5 ml of saturated NaHCO3 to separate the derivatized oxoacids from the other ether soluble compounds. The NaHCO3 extracts were combined and the pH adjusted to 2 with 3 N H2S04. The acidified solutions were extracted three times with 0.5 volumes of chloroformzether (85:15,v/v). The extracts were combined and dried with a stream of air 38 to a final volume of 100 pl. One to five microliters of the chloroformzether extract was spotted on a thin-layer cellulose plate. The derivatives were separated by chromatography using n-butanolzethanolezo (70:10:20, v/v/v) as the solvent system (169). The plates were scanned for radioactivity with a gas-flow scanner (Berthold). The standards glyoxylate, pyruvate, 2-oxog1utarate, and acetone were detected by their yellow color. K. INCORPORATION OF [2,3-3HJASPARTATE AND [4-‘4CJASPARTATE INTO ASPARAGINE The pathway of asparagine synthesis in soybean roots was examined by measuring the incorporation of label from [2,3-3H]aspartate and [4-14C]aspartate into asparagine. Thirty-nine-day-Old plants that had received 10 mM KNO3 for 24 days prior to the experiment were removed from the pots in which they had been planted. About 4 g of secondary roots were harvested and cut into pieces approximately 1 cm long. About 1 g of the roots was placed into each of four 20-m1 glass scintillation vials and one of four different solutions was added to each of the vials: 0.5 mM L-aspartate 0.5 mM KCl 2.0 mM aminooxyacetate pH=7.0 1+ or 0.5 mM L-aspartate 0.5 mM NH4C1 2.0 mM aminooxyacetate pH=7.0 l+ In addition to one of the above solutions, each vial contained 26 pCi of L-[2,3-3H]aspartate (20 Ci mmoi“; ICN) and 1.7 pCi of 39 L-[4-14CJaspartate (50 mCiommol'1 ; Amersham). The vials were sealed with a serum cap and incubated at 25 C. After 1 h, the roots were removed, rinsed well with H20, patted dry and frozen. The roots were lyophilized and prepared for analysis according to the procedures discussed in section H. Just prior to Opening the scintillation vial to remove the roots, 14CO2 formed from [4-14C]aspartate, was assayed by removing 3 m1 of the gas from each vial and injecting this sample through a serum cap into a separate scintillation vial containing 1 m1 of methylbenzethonium hydroxide 90 min after the gas sample was added to the vial. The vials were then stored at -20 C for 2 h to allow the fluorescence, which is sometimes observed in this system, to decay. The radioactivity due to the 14 CO2 which was absorbed by the methylbenzethonium hydroxide was measured by scintillation spectrometry. The solution in which the roots had been incubated was assayed for 3 H20, produced from [2,3—3H]aspartate, by the microdistillation technique of Varner and Burton (170). The labeled amino acids contained in this solution were derivatized and separated by HPLC and analyzed by flurometry and scintillation spectrometry as discussed in section 1.1 and below. The amino acids extracted from the plant roots were derivatized as described in section 1.1 except that 0.2 ml of H20, 0.2 ml of saturated sodium borate, 0.5 ml of methanol and 0.1 ml of OPA solution were added to the dried extract of each root. Because of the low amount of label that was recovered from the roots in asparagine (about 3700 cpm of [14C]og fresh weight-10f roots), the entire solution containing the derivatized amino acids was injected onto the HPLCcolumn in multiple 40 injections. The CH3CN concentration in the mobile phase was zero (i.e. the mobile phase was 12.5 mM NaH2P04, pH=7.2) during the injection procedure so that the amino acids would not be prematurely eluted from the column. Once the entire solution had been injected, the CH3CN concentration was increased to 12% and the elution gradient started (Table l). The disadvantage of injecting large amounts of material onto the column is that the separation between amino acids is not as good as when smaller amounts of material are injected. The eluate from the HPLC column was collected in fractions and the radioactivity was determined by scintillation spectrometry after the addition Of Triton X-100 scintillation fluid. The radioactivity due to [3H] and to [140] was determined using two different channels of a liquid scintillation counter (Program number 4 of a Beckman LS 7000). To ensure that the CH3CNzNaH2PO4 buffer did not affect the relative counting efficiences of either isotope, several HPLC runs using unlabeled aspartate and asparagine were made. Standard [3H]aspartate and [14CJaspartate were added to the vials containing the unlabeled aspartate and asparagine eluted from the column. The counting efficiencies obtained for these standards were used to calculate the 3H/MC ratio in aspartate and asparagine that was present in the root extracts and the solution in which the roots had been incubated. L. DISTRIBUTION OF LABEL WITHIN ASPARTATE AND GLUTAMATE IN SOYBEAN 14 NODULES EXPOSED TO CO, L Root systems of soybean plants were exposed to 14 CO2 for 15 and 30 min. The amino acids were extracted and prepared as discussed in section H. Aspartate and glutamate were purified from one another 41 and the other amino acids by ion-exchange chromatography on Dowex 1 (chloride, acetate) according to the procedure of Berl et_al, (164). The fractions containing aspartate were combined and lyophilized as were the fractions that contained glutamate. Aspartate and glutamate accounted for 98% of the total amino acids in the purified aspartate and glutamate fractions, respectively, as determined by HPLC (section 1.1). The position of [14C] within aspartate and glutamate was examined by selectively removing one or more of the carbons. The carboxyl groups of aspartate and C-1 of glutamate were removed with ninhydrin by a procedure adapted from Sobel gt al. (171). Sixty microliters of ninhydrin (0.3 g.m1"1 of 95% ethanol) and 0.12 ml of l M citrate (pH=2.2) were added to 0.3 m1 of sample (about 5 nmoloml'1 of the appropriate amino acid) in a glass scintillation vial. A glass marble was placed on the top of the vial, and the vial was placed into a boiling H20 bath. After 10 min in the H20 bath, the vials were removed and 30 pl of 5 N HCl were added. The vials were then placed in a heated (70 C) vacuum desiccator and dried under reduced pressure to remove traces of [14C]HC03 produced by the decarboxylation of aspartate and glutamate. The vials were removed from the desiccator and 0.7 m1 of H20 and 7 ml of Triton X-100 scintillation fluid was added after the vials had cooled. The radioactivity which had not been lost during the treatment with ninhydrin represented the label in C-2,3 of aspartate and C-2 to 5 of glutamate. The standards were L-[4-14C]aspartate, L-[u-14C]aspartate, L-[U—14C]glutamate and L-[l-14C]ornithine. Ornithine labeled in C-l was used to determine if ninhydrin completely removed C-1 of an amino acid. 42 The amount Of label in the C-4 position of aspartate was determined by a procedure adapted from Luehr and Schuster (172). Seventy microliters of purified aspartate (5 nmol'ml'1) were added to 30 p1 of 3.8 M sodium acetate (pH=5.4) and 100 p1 of pyridoxal-H01 (Sigma) (120 mg-ml'1) and A13(SO4)2 (5 mg-ml") in a glass scintillation vial. The vials were placed in a 70 C H20 bath. After 30 min, the vials were removed and 100 p1 of 5 N HCl were added. The vials were placed in a heated (70 C) desiccator and dried under reduced pressure to remove traces of [14C]HCO§ produced by the decarboxylation of aspartate. Water (0.7 m1) and Triton X-100 scintillation fluid (7.0 ml) were added to each vial, and the radioactivity determined by scintillation spectrometry. The radioactivity remaining after the treatment with pyridoxal represented the radioactivity in C-1,2,2 of aspartate. The standards used were [U-14C]aspartate and [4-14C]aspartate. M. PRODUCTION OF [13N] Nitrogen-13 was generated by irradiation of a target for 10 min with a 12 MeV proton beam produced at the Michigan State University Heavy Ion Laboratory. The target consisted of either 18.6 mg of amorphous carbon, 97 atom % of [13C] (173) (Monsanto Research Corp., Mound Laboratory, Miamisburg, OH) or 1 ml of H20 (174). Bombardment of the [130] with protons resulted in the production of a neutron and a loss of a proton to form [13N] according to the nuclear reaction 13C(p,n)13N. Bombardment of [16O]H20 with protons resulted in the production of [13N] according to the reaction 16O(p,ot)13N. After bombardment of the target, the particular chemical form of [13N] that was desired was purified according to the schemes 43 developed in the laboratories of Tiedje (174) and Wolk (167,173,175). The [13C] target was converted to [BNJN2 by the Dumas combustion procedure of Austin gt al. (173). After bombardment, the [13C] target was mixed with 0.6 mg of fine CuO powder and 0.18 mg of KNO3 in a quartz tube. The sample was then heated to 800 C. A stream of CO2 was used to sweep the combustion products through post heater tubes filled with CuO and metallic Cu, where the nitrous oxides produced during combustion were reduced to N2. The products were then passed through a liquid nitrogen trap, to remove C02 and unreacted oxides of nitrogen , to a previously evacuated Toeppler pump. After bombardment, the [160]H20 target contained varying 13 13NH+ 13 13 amounts of N03, NH4 and N05. The N03 was purified by adding 2 to 3 drops of 10 N NH4OH to the water target which was then flash 13N H+ evaporated to remove the 4(174). One ml of H20 was added to the dried material and the solution was neutralized with 2 or 3 drops of 10 N HC1. The purity of the ‘3 N03 was determined by separating the radioactive constituents of a 10 p1 sample by HPLC using a Partisil- 10 SAX anion-exchange column (Whatman) (174). The elution buffer was 50 mM KH2P04 (pH=3.0). The 13NHZ, 13N03 13N05 eluted separately and their radioactivity was measured with a sodium and iodide coincidence detector. 13 The NH4 was purified from the H20 target as follows. After bombardment, the H20 target was placed in a flask and about 100 mg of Devardas alloy and 10 m1 of 10 N NaOH were added to reduce the 13 13N H+ (176). The flask was fitted to a steam distillation 13 N03 to apparatus (167,177) and the NH4 was transferred from the flask by 44 steam distillation under vacuum into a side arm test tube immersed in liquid nitrogen. Up to 10 mCi of 13NH: was recovered. N. EXPOSURE OF PLANT TISSUE T0 [‘3N1 Detached nodules or segments of nodulated roots were exposed to [BNJN2 in l or 3-m1 vials. The vials were briefly evacuated before the [BNJN2 (about 0.2 ml) was transferred to the sealed vial using a Toeppler pump. The pressure inside the vessel was adjusted to atmospheric pressure with a mixture of Ar:C02:O2 (80:1:10, v/v/v). Root systems of intact plants were trimmed of excess roots and placed in a test tube containing 2 to 10 mCi of 13NH4 or 13 of the labeled compounds. N0; in H20. Detached nodules were dipped in a solution 0. EXTRACTION OF THE LABELED PRODUCTS AFTER THE ASSIMILATION OF [13N] After the tissues had been exposed to [13N], they were ground and extracted twice with 1 m1 of methanoleZO (80:20,v/v). The resulting slurry was clarified by centrifugation at 10,000 g for 3 to 5 min. The supernatant fluids were combined and dried jn_vacuo at 50 C. MethanoleZO (20 to 100 p1) and amino acid standards 1 per amino acid) were (5 to 20 p1 of solution containing 1 mg-ml' added to the dried extract. The standard amino acids included aspartate, glutamate, asparagine, glutamine, alanine and arginine. For certain experiments, allantoate was also added as a standard. The labeled compounds were separated by high-voltage electrophoresis as described in section 1.3. 45 P. DISTRIBUTION OF LABEL IN GLUTAMINE AFTER THE ASSIMILATION OF 13NH: Alder nodules were incubated in a solution containing 13NH: for 2,5 and 10 min. The nodules were rinsed with H20 and labeled compounds were extracted with methanoleZO as described above. Two to three drops Of 14 N NH4OH were added to ensure that any 13NH: remaining from the incubation solution was removed during the drying of the extract. One ml of 10 mM sodium acetate (pH=4.9) containing 25 units of glutaminase (Sigma, Grade V) was added to the dried extract. After a 10 or 20 min incubation, the solution was added to a 2-ml Dowex 1 (acetate) column. The 13NH: released from the amide position of glutamine was eluted by washing the column with 3 ml of H20. The[]3N]glutamate from the [13N]glutamate that was originally present and hydrolyzed [13nglutamate was eluted from the column with 3 m1 of 2 N acetic acid (178). The radioactivity of each fraction was measured by scintillation spectrometry after Triton X-100 scintillation fluid was added. The procedure was 1 standardized with [14C1g1utamine (50 mCi-mmol' ; ICN). CHAPTER 1 THE RATE OF DARK CO FIXATION DURING THE DEVELOPMENT OF THE SOYBEAN 2 N -FIXING SYSTEM 2 Christeller 93.21-(7) proposed that dark C02 fixation was involved in the synthesis of asparagine in lupin nodules. In support of this proposal, these investigators reported that the rate of dark CO2 fixation was correlated with the rate of N2 reduction during the development of the NZ-fixing system of lupin (Lupinus angustifolius) and Rhizobium lupinus. The major form of organic nitrogen transported from nodulated lupin plants is asparagine (9), whereas allantoate is the major form transported from soybean nodules (12). Allantoate, based on the pathways proposed for its synthesis (115,116), is not expected to contain carbon that enters the metabolism of the nodule via dark CO2 fixation. Therefore, the relationship between the rate of dark CO2 fixation and N2 fixation is expected to be different in soybeans from what it is in lupins. _ In this chapter I report and discuss the following: 1) the mechanics of the assay for dark CO2 fixation, 2) the effect of several parameters on the rate of dark CO2 fixation in soybean nodules and roots and 3) changes in the rate of dark CO2 fixation and N2 reduction during the development of soybean nodules and roots. 46 RESULTS A. CO2 FIXATION ASSAY Previous workers have used [14C] in the form of 14002 and [14CJHC05 to measure the rate and/or products of CO2 fixation in 14 plants. After exposure of plant tissue to [14C]HC03 or CO 23 the labeled products were usually extracted with either an acidic solution (8,179,180) or an organic solvent such as methanol or ethanol (28,31,181). The extracts were dried, thereby removing any 14 residual CD2 or [14C]HCOS. In this study, the nodules and roots were extracted with ethanol rather than acidified solutions because some of the products of 14 C02 fixation, most notably asparagine and glutamine, are labile in acidic solutions. The efficiency of extracting labeled compounds from nodule and root tissue with ethanol was examined by extracting several tissue sample (40 mg dry weight per sample) that had been allowed to incorporate label from 14 C02, six times with 3 m1 of ethanoleZO (80:20,v/v). The first three extractions, combined, accounted for 95 to 99% Of the total radioactivity that was extracted; therefore, three extractions were used routinely. If [14C] that had not been fixed, i.e. [14C]Hco§, was present in the dried extracts, then the calculated rates of CO2 fixation would be artificially high. An extract was prepared from nodules that had been allowed to incorporate label from 14 C02. Fifty microliters of 6 N HCl or glacial acetic acid were added to a 200 pl aliquot of this extract. The acidified extract and an aliquot of the same extract without added acid were dried jn_yagug_at 55 C. The amounts of the radioactivity in the extracts with and without acid were the same, 47 48 14 demonstrating that CD2 or [14C]HCO§ was not present in the dried extracts. Prior to ending a C02 fixation assay, two gas samples were routinely removed from the container in which the plant tissue 14 was exposed to C02. Based on the amount of radioactivity measured in one sample and the total concentration of CO2 measured 1 in the other sample, the specific activity of the 4CO2 was calculated. This value together with the amount of [14C] that was recovered from the tissue was used to calculate the rate of dark C02 fixation. For different experiments the specific activity 14 of the CO2 varied from 0.5 to 25 x dpm-pmol'l. 14 The incorporation of label from CO in attached and detached 2 nodules was linear during the time period that was examined, 2 to 12 min. The external concentration of CO2 did not affect the linearity of the assay. 8. EFFECT OF DETACHMENT OF NODULES AND CONCENTRATION OF CO2 ON THE RATE OF DARK CO FIXATION 2 Based on other investigators' work (7,182,183) at least two factors needed to be considered in measuring the rate of dark CO2 fixation in soybean nodules. First, since the rate of C2H2 reduction in detached nodules is less than in intact systems (182,183), detachment of nodules might have a similar effect on the rate of dark C02 fixation. The second fact is that the rate of dark CO2 fixation in lupin nodules was affected by the concentration of CO that surrounded the nodules with high levels of CO2 resulting 2 in high rates of dark CO2 fixation (7) 49 The rates of dark C02 fixation in attached and detached soybean nodules was measured when both high and low concentrations of C02 were present. The rates of dark CO2 fixation in detached nodules appeared to be less than the rates measured in attached nodules regardless of the concentration of CO2 that was present (Table 2). Higher rates of dark CO2 fixation in nodules were measured when higher concentrations of C02 were present (Table 2). The maximal rate of dark CO2 fixation in attached nodules was attained when the external CO2 concentration was 1.5% (v/v) or greater (Figure 3). A similar saturation of the rate of dark CO2 fixation with high concentrations of CO2 was observed in detached nodules. The rate of dark CO2 fixation in roots was not affected by increasing the concentrations of CO2 around the roots from 1.0 to 3% (v/v) (Figure 3). C. EFFECT OF CO2 CONCENTRATION ON 09 CONSUMPTION AND C2H2 REDUCTION Mahon (184) reported that concentrations of CO2 between 0.1 and 3% (v/v) inhibited the respiration of nodulated pea roots. If a similar event occurs in soybean nodules, then the rate of dark CO2 fixation observed at high concentrations of 002 may not be reflective of the rate of dark CO2 fixation in nodules. Rather, the rates measured under high concentrations of CO2 may be the result of an increase in Specific activity of 14 14 CO2 within the nodule because of the reduced dilution of the ‘2 CO2 by C02 produced during respiration. The effect of high concentrations of C02 on the respiration of nodulated soybeans was examined. The rate of oxygen consumption of attached and detached nodules was measured amperometrically in the presence of high or low concentrations of 002. The initial addition of both high and low concentrations of CO2 to separate samples was 50 Table 2 Effect of the Concentration of C02 on the Rate of Dark CO2 Fixation in Attached and Detached Nodules Treatment CO2 Concentration Rate of Dark CO2 Fixation % (v/v) nmol mg dry wt of nodules'Jh.1 Attached 0.2 to 0.6 10 t 2 (9) 1.5 to 3.5 36 i 5 (9) Detached 0.2 to 0.6 4 i l (6) 1.5 to 3.5 19 i 3 (6) Nodules that were removed from the root (detached) or the entire nodulated root system of 26-day-old plants were placed into a flask. The tissue was exposed to 5 to 20 pCi of 14CO2 and either low (0.2 to 0.6%, v/v) or high (1.5 to 3.5%) concentrations of CO2 for 10 min, at which time the tissue was removed and frozen in liquid nitrogen. The values reported are t SE and the number of replicates for each determination are in parentheses. The rates of dark CO2 fixation in attached nodules in low and high concentrations of CO2 were significantly different (P5_0.10) from one another as determined by the Student's t-test. The rates for detached nodules in high and low concentrations of CO2 were also significantly different (P§O.10) from one another. 51 60- °- nodule 0 o e 1 ,2 .f 1 . . S ,_ 4O .5 3 0°C 0 u. «3‘ N ‘0 00 (3 GD 0 E o 1. 1: 2K3 ’ ' a E A A root A a G A AA . ‘ A l/z””"45"—'——u—f A A A ' A 0 L I L l L 0.5 1.0 20 3.0 CO: concentration (7.1 Figure 3 Effect of the Concentration of CO2 on the Rates of Dark C0 Fixation in Roots and Nodules of Intact Soybean Plants 2 Nodulated roots of intact 19-day-old soybean plants were incubated with 5 to 10 pCi of 14CO2 and various concentrations of 12CO2 for 10 min, at which time the tissue was removed and frozen in liquid nitrogen. The rate of'dark CO2 fixation in nodules (O) and in roots (A) was calculated by dividing the amount of label incorporated during the 10 min exposure by the specific activity of the 14CO2 that the samples were exposed to. The lines that are presented were fitted by eye. 52 to ensure that the order Of addition did not affect the results. There appeared to be no significant effect of the concentrations of CO2 on the oxygen consumption of attached nodules (Table 3). If detached nodules were initially exposed to high concentrations of C02, then the rate of oxygen consumption was greater under high concentrations of CO2 than under low concentrations. The effect of the concentration of C02 on metabolic processes in nodules was examined by measuring the rates of C2H2 reduction under high and low concentrations of C02. The rates of C2H2 reduction in attached nodules of intact plants or of plants that had had the shoot excised were not affected by the presence of high concentrations of C02. In contrast, the rate of C2H2 reduction in detached nodules was higher in the presence of high concentrations of CO2 than the rate measured in low concentrations (Table 4). D. DARK CO2 FIXATION, C.,H2 REDUCTION AND CONCENTRATION OF ORGANIC NITROGEN IN THE XYLEM EXUDATE DURING THE DEVELOPMENT OF SOYBEAN PLANTS Previously, Streeter (185) noted a decline in the amount of amino acids and an increase in ureides in the xylem exudate of developing soybeans. If dark CO2 fixation in soybean nodules provides carbon for the synthesis of amino acids translocated to the shoot as suggested for lupin nodules (7), then the rate of dark C02 fixation may be related to the transport of amino acids in soybeans. The concentration of amino acids and the ureides,allantoin and allantoate, in the xylem exudate and the rates of C02 fixation and 02H2 reduction were measured in developing soybeans. The concentrations of amino acids was greater than that of ureides in the xylem exudate of plants younger than 18 days (Figure 4, Panel B). 53 Table 3 Effect of the Concentration of CO2 on Nodule Respiration Rate of O2 Consumption in High COZ/Rate of O2 Consumption in Low CO2 Treatment Attached Nodules Detached Nodules ratio Ambient 002: 31.5% 0.92 i 0.04 (7) 0.91 i 0.03 (7) 31.5%: Ambient CO 1.11 i 0.06 (7) 1.25 i 0.03 (11) 2 The oxygen consumption of tissues incubated either in ambient or 1.5% (v/v) or greater concentrations of CO2 were measured for 10 to 15 min. To the sample originally incubated in ambient levels of C02, CO2 was added to increase the concentrations of CO2 (Ambient C02: 31.5%); the chamber of those sample that were initially incubated in high concentrations of CO2 was flushed to lower the concentration of CO2 (31.5%:Ambient 002). The oxygen consumption for each sample was measured again. The measured rates of O2 consumption werelbetween and 48 to 60 for attached and detached nodules, 126 and 180 nmol O2 consumedrmg dry weight of nodules-1 h nmolomg dry weight of nodules'L h"1 respectively. The values reported are t SE and the number of replicates for each determination are in parentheses. aThese values are significantly different (P§_0.05) from one another as determined by the Student's t-test. 54 Table 4 Effect of the Concentration of C02 on the Rate of C H 2 2 Reduction Treatment CO2 Concentration Rate of CZH2 Reduction % (v/v) nmol-mg dry wt of nodules-Jh'] Attached Nodules 0.2 to 0.6 37 s 6 (7) on intact plants 1.5 to 3.5 37 i 6 (7) Attached Nodules 0.2 to 0.6 48 i 4 (8) w1th shoots excised 1.5 to 3.5 53 i 6 (7) Detached Nodules 0.2 to 0.6 18 i 1 (7)a 1.5 to 3.5 24 s 1 (7)a Nodules, either on intact plants (attached) or removed from the roots of 26-day-old plants (detached), or attached nodules from 36-day-Old plants that had had the shoot excised just prior to the assay were placed in separate flasks and exposed to low (0.2 to 0.6%, v/v) or high (1.5 to 3.5%) concentrations of C02 and to C2H2 (10%, v/v). Periodically, gas samples from the flasks were removed and assayed for C H 2 4' for each determination are in parentheses. The values reported are t SE and the number of replicates aThese values were significantly different from one another (P50.10) as determined by the Student's t-test. 55 After the onset of N2 fixation, the ureides became the major form nitrogen in the xylem exudate while the concentration of amino acids slowly declined. The maximal observed rate of dark CO2 fixation in secondary roots was measured on day 8 (Figure 4, Panel A). Seven days later 1-h"1. the rate was 10 nmol~mg dry weight of roots' Nodules ’were first observed on day 12 and noticeable rates of dark CO2 fixation and 02H2 reduction in nodules were first measured on day 15. The maximal observed values were 117 nmol CO2 fixedo mg dry weight of nodules-1 h'1 and 138 nmol 02H2 reducedomg dry weight of nodules-1 h'1. The specific activity of C2H2 reduction remained relatively constant until day 29. In contrast, the specific activity of nodule C02 fixation declined after day 18 reaching a value of 20 nmol-mg dry weight-1 h'1 on day 35. The decline in the rate of dark CO2 fixation observed for nodules corresponded to a decrease of the concentration of amino acids in the xylem exudate. The values for the concentrations of amino acids in the xylem exudate and the rates of dark CO2 fixation for the plants from the last four sampling dates were correlated with a coefficient of 0.96 (Figure 5). 56 Figure 4 Rates of Dark CO2 Fixation, C2H2 Reduction and the Composition of the Xylem Exudate during the Development of Soybean Plants After intact nodulated roots of soybean plants that had been maintained on N-free nutrient solution were sealed in to a flask, they were exposed to either C2H2 (10%, v/v) or 5 to 10 pCi of 14C02 and saturating levels of 12CO2 (3f1.5%, v/v). The root systems that were exposed to 14CO2 were removed from the flask after 10 min and frozen in liquid nitrogen. The reported values of the rates of dark CO2 fixation in roots ([3 , Panel A) are averages of 15 to 20 replicates and in nodules (C) , Panel A) 3 to 10 replicates. The reported values Of the rates of C2H2 reduction ([3 , Panel A) are averages of 5 replicates. Bars are presented about the rate of C2H2 reduction and the rate of dark C02 fixation in nodules and roots where the SE is greater than the size of the symbOl. The xylem exudate of plants that were left in their pots was collected for 30 to 45 min and was assayed for amino acids (0, Panel B) and the ureides, allantoin and allantoate (A , Panel B). The ratio of nmoles Of amino acids to nmoles of ureides (. ------ , Panel A) in the xylem exudate is also presented. Activity (nmohmg'dny wt"oh"1 Composition of Exudate" '1 (nmol-ml 160 120 m o . A O 57 anthosis .. 4 (:sz reduction “3 i-z \ cozfixation “I é\¢ (nodule) \ ~‘fi-..---.T\2 .:.a—g- 0402 fixation (root). 0' ureides \- A \ x‘ / O\ A _ \ \ b- - - --o l 1 g L L 1 16 20 24 28 32 36 Plant Age (days) Figure 4 J lnxylom exudate [amino .a'ciOJIEiroido 58 O I: EI" ‘ o g . .. b E u _ 5. . s E 0 _:. I- 0 q 1, 6 “5,5 4 ~ 4! so - I o .5 - . 3 0 2.9L '4 p . 15 30 45 60 (nmol-mg dry wi'lh") 002 Fixation Rate Figure 5 Correlation of the Concentration of Amino Acids in Xylem Exudate with the Rate of Dark CO Fixation in Nodules The values reported in Figure 4 for the concentration of amino acids in the xylem exudate and the rate of dark CO2 fixation in nodules of plants 21-days or older are presented. The line was fitted by least squares analysis and had a correlation coefficient of 0.96. DISCUSSION The rates of dark CO2 fixation in this chapter were calculated based on the label recovered from the nodules and roots exposed to 14CO2 for 10 min. These rates, however, may not represent the rate at which net fixation of carbon occurs in the nodule, but may instead primarily reflect the rate at which carbon is exchanged through the system. Net fixation results in an increase in the amount of carbon in the system, whereas exchange results in carbon incorporated during dark C02 fixation being lost as CO2 via the respiratory activities of the nodule. The extent to which dark CO2 fixation results in net fixation or exchange cannot be determined from the data presented in this chapter. 14 The specific activity of the C02 in the gas phase surrounding the tissue was used in calculating the rate of dark CO2 fixation. The use of this specific activity may result in a calculated rate of dark CO2 fixation that does not accurately reflect the actual rate of dark C02 fixation. The specific activity that should be used in the calculations is the specific activity at the site of dark CO2 fixation within the nodule. The two specific activities are probably not the same because the 12 respiratory activities of the nodule releases CO diluting the 14 2 CO2 within the nodule. I was unable to experimentally determine the 14CO2 specific activity at the site of dark CO2 fixation; therefore, I used the value measured for the gas phase just prior to ending the incubation. During a 10 min incubation, the concentration of CO2 within a flask increased because of the respiratory activities of the plant tissue. This increase had 59 60 a minor effect (less than 10%) on the specific activity of 002, and hence the calculated rate of dark CO2 fixation, when saturating concentrations of CO2 were present. Christeller gt 31. (7) noted that for lupin nodules, high concentrations of C02 were necessary to Obtain maximal rates of dark CO2 fixation. The internal specific activity of the ‘14C02 was assumed to approach that outside the nodule only at very high external concentrations of 002. Christeller gt a1.(7) concluded that the rate of dark CO2 fixation measured at saturating concentrations of CO2 is the best estimate of the "true“ rate of dark CO2 fixation jn_yjyg, The response of the rate of dark CO2 fixation to high concentrations of CO2 was similar in soybean and lupin nodules; the rate of dark CO2 fixation in soybean roots was not affected by the concentration of C02. Unless otherwise indicated the rate of dark CO2 fixation presented represents the rate Observed at saturating concentrations of C02. High concentrations of CO2 did not inhibit respiration of nodulated soybean roots, in contrast to the inhibition of respiration noted in pea roots (184). The reason for the difference of the pea and soybean root system is not clear. Other than possible inherent differences between peas and soybeans, the method by which respiration was measured may have affected the results. I measured respiration as oxygen consumption, whereas Mahon (184) measured respiration as the efflux of 002. The addition of high concentrations of CO2 may have affected the meaurement of CO2 efflux but not oxygen consumption. Mahon (184) also performed his measurements jn_situ, whereas I removed plants from their pots and placed them 61 into a small vessel prior to the assay. The presence of high concentrations of CO2 did appear to affect the metabolism of detached nodules. The rate of C2H2 reduction in the presence of high concentrations of CO2 was significantly higher (Table 4) than the rate in the presence of low concentrations of 002. The respiration of detached nodules was altered by exposure to high concentrations of CO2 prior to exposure to ambient levels of CO2 versus the reverse treatment (Table 3). The results of this experiment do not indicate whether oxygen consumption was inhibited or stimulated by exposure to high concentrations of C02. Oxygen consumption may have been stimulated when high concentrations of CO2 were initially present or inhibited in low concentrations of CO2 after a exposure to high concentrations of C02. The differences in the rates of dark CO2 fixation, C2H2 reduction and oxygen consumption between attached and detached nodules demonstrates that detachment of the nodules even for short periods of time affects the metabolism of the nodule. Similar declines in the rates of CZH2 reduction (182,183) have been observed in nodules and nodulated root systems removed from the rest of the plants. For this reason, conclusions based on the results with detached systems should be applied with caution to the intact plant. The rate of dark CO2 fixation in attached soybean nodules 1 varied between 4 and 24 pmol-g fresh weight' oh"1 (Figure 2; assumes 5 g dry weight = 1 g fresh weight) during the development of the soybean nodule. Layzell et_al,(181) reported that the rate of dark CO2 fixation in detached cowpea and lupin nodules was 18 and 1 0.8 pmo CO2 fixed-g fresh weight of nodules-Lih' , respectively. 62 The values reported for cowpea and lupin nodules probably represent low estimates of the activity of attached nodules. The concentrations Of CO2 used in the assay for dark CO2 fixation (181) were not reported and may have been below the concentrations necessary to obtain maximal rates. Based on the rates of dark CO2 fixation (Figure 4) and respiration (Table 3) an estimated 10 to 13% of the carbon respired by the nodule could be refixed by in 1119 dark CO2 fixation in both lupin (181) and soybean nodules . Several investigators have proposed that dark CO2 fixation catalyzed by PEPC in lupin (7) and broad bean nodules (8) provides carbon for the synthesis of asparagine, the major product synthesized from N2 reduced in the nodules from these plants. Christeller et al. (7) noted that the rates of CZHZ reduction and dark 002 fixation were correlated during the development Of the lupin nodule. The major product of N2 fixation in soybeans, allantoate, does not appear to require carbon incorporated by dark CO2 fixation catalyzed by PEPC for its synthesis (115,116); therefore, a correlation between the rates of N2 fixation and C02 fixation might not be expected and was not observed in soybeans (Figure 4). On the other hand, the parallel decline of the rate of dark CO2 fixation in the nodule and the concentration of amino acids in the xylem exudate (Figure 5) suggests that there may be a relationship between dark CO2 fixation and amino acid synthesis in soybean nodules. The results, however, do not indicate whether the parallel decline was because the reduced rates of dark CO2 fixation were responsible for the reduced Concentrations of amino acids, or vice versa, or whether the decline in both measurements is caused by a third factor 63 such as the development of the nodulated root. The next chapter is concerned with the pathway and products of dark CO2 fixation in soybean nodules. CHAPTER 2 THE PRODUCTS OF DARK CO2 FIXATION IN SOYBEAN NODULES A portion of the label incorporated in broad bean (8) and dwarf french bean (31) nodules was recovered in amino acids. The amino acids accounting for the highest percentages of label in roots and nodules exposed to 14 CO2 are generally those most closely associated with the tricarboxylic acid cycle; aspartate, glutamate, asparagine and glutamine (8,31,186). However,the relative incorporation of label from 14 CO2 into these amino acids as well as other products depends on the species of plant that is examined. For example, asparagine is the major labeled amino acid in broad bean nodules (8), whereas citrulline is in alder nodules (186). In chapter 1, I demonstrated that CO2 was taken up by soybean nodules. In this Chapter, I examine the products of dark C02 fixation in soybean nodules. RESULTS A. LABELING OF ORGANIC ACIDS The incorporation of label into organic acids in nodules that were exposed to 14CO2 was examined. The fraction containing the organic acids and neutral compounds accounted for 43 to 72% of 14 the total radioactivity extracted from nodules exposed to CO 2 for 2 to 60 min. The label in this fraction was distributed among malate, which accounted for 29% of the total radioactivity extracted from nodules that had been exposed to 14 CO2 for 60 min, succinate (5%), fumarate (4%), citrate (7%), allantoate (5%) and several unidentified components (Figure 6). 64 65 ICDCJ t “- I sue tum Radioactivity (apm) (J 1!) 23¢) Distance (cm) Figure 6 Incorporation of Label into organic Acids and Neutral Compounds in Nodules Exposed to 14CO 2 Soybean plants (29-days old) were uprooted and the shoots were excised. The root systems were placed into a flask where they were exposed to 14CO2 for 60 min. The nodulated roots were removed from the flask and frozen in liquid nitrogen. The labeled products were extracted from the nodules with ethanoleZO (80:20, v/v). The extract was separated into two fractions, one containing the organic acids and neutral compounds, and a second containing amino acids. An aliquot of the former fraction was spotted on a thin-layer cellulose plate at the origin and Chromatographed with butanol:glacial acetic acidzHZO (120:30z50, v/v/v) as a solvent system. Malate (mal), citrate (cit) and allantoate (aln) were identified on the basis of co-migration with standards. Succinate (sac) and fumarate (fum) were identified on the basis of published Rf values (206). Radioactivity was detected with a gas-flow scanner. 66 B. LABELING OF AMINO ACIDS The amino acids accounted for 28 to 57% of the total 14 radioactivity extracted from nodules exposed to CO2 (Figure 7). Aspartate and glutamate accounted for the highest percentage of the label in the amino acid fraction. After a 2 min exposure 14 to C02, the percentage of the total label in aspartate was 7%, increased to 16% after a 5 min exposure, and remained at about that value for the remainder Of the experiment (Figure 7, Panel A). The percentage of label in glutamate increased until it represented 22% of the total radioactivity after a 60 min exposure of the nodules to 14 002. Lesser amounts of label were recovered in several other amino acids (Figure 7; Panels A and B). Asparagine, glutamine, alanine, serine and glycine accounted for approximately 7,1,3,3 and 2% of the total radioactivity recovered from nodules. A small 'percentages (§_5%) did not elute with the amino acids presented in Figure 7 but occasionally eluted with several unidentified amino acids. 14 12 CO -CHASE EXPERIMENT C. 2 COz-PULSE Lawrie and Wheeler (8) noted that a large proportion of 14 C0 in broad bean nodules was lost rapidly 2 from the nodules. The retention of recently-fixed 14CO2 in the recently-fixed soybean nodules was examined. The Shoots of nodulated soybeans were excised, the nodulated root systems were placed in a 250-ml flask, the flask was sealed and 14CO2 was added to the flask. 67 .mccaoaeou _mcuzmc can mewum uwcmmco ecu mcmcmmucoo cowpeas» as» cm emcm>oomc we; cmpcmmmcq mam «ecu mewom ocwsm as“ cw PmDeF to mummpcmucma may to saw on» use Noop cmmzpmn wucmcmeewu use covumppwpcmom an emszmmms mm: xum>wpumomumm vm>osmc mam; mpooc umum_:uoc ms» .mspp umpmumccw asp p< .Nou uwomn_msmz mempmzm poo; ms» N ep 2:5. :5... .uaa: a: umszecm mam: muoacoca cmpmnop esp .xgpmeocuowam .:mNocw use ow ummoaxm mew; avg» mews: xmmpw a cue? .uwmwuxm mew: mucosa we» ecu umuoocaz mam: Aepo mamuiva macmpq :meAom cue—cu ummoaxm mw_:eoz cw muozeoca uwcmmco cm Page; to compznmcumwo ms» to mmcaoo mswp N weaned 00 a? On 0. O Om nv .00 n. O o fie d a a net fiq a do a 0 11111111111 / . m 0 So /WV\ o/o - . nu . t _ ..x to D can a a... \ a \ I “a N 1 .. 1 ‘ a o_ W. - x III/III mu _ no: u/ . n. w m ..x hmm I .. T‘ ‘ . m I u... x .u .A . . ON ¢ 1 So x\x :3 I .m . .4 68 After a 3 min exposure to 14C02, the root systems were removed from the flask, flushed with a stream of air for 30 sec, and placed into another 250-m1 flask that was left open. The root systems were removed from the open flask at intervals and were frozen in liquid nitrogen. The major portion of the recently-fixed 14 12 CO2 was lost from the nodule during the chase with C02. After a 60 min chase, only 28% of the label incorporated during the 3 min exposure to 14CO2 was still present (Figure 8, Panel A). The percentage of label in aspartate remained relatively constant throughout the chase; the percentage in the organic acid plus neutral fraction declined slightly; the percentage in glutamate increased during the chase (Figure 8, Panel B). Labeled asparagine and glutamine were not detected in the extracts of the nodule because of the low amounts of radioactivity incorporated during Short exposure to 14CO2 and the low percentage of the total label incorporated into these amino acids. D. POSITION OF [14C] WITHIN ASPARTATE AND GLUTAMATE Previous investigators have proposed that dark CO2 fixation in nodules was primarily catalyzed by PEPC (7,8). The position of [14C] within the molecules of aspartate and glutamate that were isolated from nodules that had been exposed to 14 CO2 was determined to provide further evidence for this proposal. Oxalacetate that is produced from phosphoenolpyruvate and [14CJHC03 in the reaction catalyzed by PEPC should be labeled solely in C-4. Aspartate that is derived from this oxalacetate should, therefore also be labeled solely in C-4. .Labeled glutamate synthesized from labeled 69 Figure 8 Distribution of Label in Organic Compounds in Nodules 14coz-Puise 12COZ-Chase Soybean plants (29-days old) were uprooted and the shoots were excised. The root systems were placed into a flask where they were exposed to 14CO2 for 3 min at which time the nodulated roots were 14C02 and placed into a separate flask. At various times, the root systems were removed from the second flask and frozen in liquid nitrogen. The labeled products were analyzed by HPLC. Radioactivity was measured by scintillation spectrometry. Panel A, total radioactivity remaining after pulse as per cent of that present immediately after the pulse ended. Panel B, the distribution of radioactivity in organic acids and neutral compounds (D), aspartate ( A) and glutamate (C). during a removed from the flask containing the 7O 3.5qu: 33:23:51 o\.. I 1 n. 0‘ .. .. X. L L . - \- Va 8 \. - A V,» - I n. _ e s an \\ an _dr I a P n P ..J a .1. .p .Pl m n .o. a o n w ...... o £33333. .28. g. 60 30 Time (min) Figure 8 71 oxalacetate via the tricarboxylic acid cycle should be labeled solely in C-l (Figure 10 and 11). Aspartate and glutamate were isolated by ion-exchange chromatography from nodules that had been exposed to 14CO2 for 15 and 30 min. Ninety-five percent of the label in glutamate was in C-l. The C-4 and C-1 of aspartate contained 75 and 25% of the label in aspartate, respectively. E. LABELING OF ASPARAGINE AND GLUTAMINE Asparagine and glutamine are two important amino acid amides in soybeans (5,12) yet these amides account for relatively low percentages of the total radioactivity incorporated in nodules exposed to 14 CO2 when compared to their respective acids, aspartate and glutamate (Figure 7). This differenCe in the labeling of these acids and amides may exist for two reasons. Either the amides were labeled at a slower rate than the acids or that the rates of labeling were similar but there was a large difference in the amounts of the acids and amides. For example, glutamate and glutamine may be labeled to the same extent, i.e. both have the same specific activity, yet if the amount of glutamate was larger than glutamine, glutamate would account for a larger percentage of the label than glutamine. The specific activities and amounts of the amino acids in nodules exposed to 14 CO2 were measured to determine if the rate of labeling or the amounts of amino acids could account for the low percentage of label in asparagine and glutamine. The amount of glutamine in nodules was 1 nmol-mg dry weight-1 versus 22 nmol-mg dry weight.1 for glutamate. The amounts of 72 .xgumeocuumam cowpmpppucwom an umcammms we; zum>_uueo_umm .oga: An cmepmcm mew: muwum ocean umpmnmp mg» .:mmocuw: umzcv__cp :mNoLm use xmmpe mgu Ease um>osms mew; muooc umumpaeoc we» .mswp umumopccw mg» u< .NooeF ou.ummoaxm wee: mos» mews: xmm_e m opcw umuan mam: msoumam poo; esp .ummwuxm mew: muoocm as“ new umuooca: mam: Acpo mxmeuva mucoFQ :mmaxom Nou¢_ ow ummoqxu mmpaeoz cw empmnme me_o< ocws< men to mmpgw>wuo< Ovepomam any to mmczoo week a mc:m_d E5 2...: cm 0? on n. o 00 a? on m. o q 1 d d u q d d 1 q I a: m \ . a/\m pfimo x cm E/x CD C) no o I) I x 2 O J l o a: 50m . ‘ ...-O / . O . Av _ oer .. T 4 /20~_ O I 3‘ C” U "EJ- O ' <1 2 at O.\ o (D (I-'°“’“‘“’d°) MMHOV unloads am: 73 aspartate and asparagine were about the same, 11 and 8 nmol-mg dry weight-1, respectively. The concentration of these amino acids within the nodule can be estimated by making several assumptions: 1) the amino acids are evenly spread throughout the nodule, 2) 1 mg dry weight of nodule is equal to 5 mg fresh weight and 3) l g of tissue is equal to 1 ml of volume. The concentration of glutamine, glutamate, aspartate and asparagine are estimated to be 0.2 mM, 4.4 mM, 2.2 mM and 1.6 mM. The specific activity of glutamine was consistently higher than that of glutamate (Figure 9). After a 2 min exposure of 14 nodules to C02, the specific activities of glutamine and glutamate were 136 and 6 cpm-nmol'], respectively. The specific activity of glutamate increased with time reaching 80 cpm-nmol'1 after a 60 min exposure, whereas, the specific activity of glutamine slowly declined during the period from 2 to 28 min at which time it increased. The reasons for this decline and rise in the Specific activity of glutamine is not known. The specific activity of aspartate was consistently higher than that of asparagine. The specific activity of aspartate increased with increasing exposure times up to 28 min when a maximum of 123 cpmonmol'1 was reached. The maximum observed specific activity of asparagine, 51 cpm-nmol'], was attained 14 after a 60 min expoure to CO 2. DISCUSSION A. INITIAL STEP OF DARK C09 FIXATION IN NODULES I presented evidence in chapter 1 which demonstrated that 1 vivo dark CO2 fixation occurred in soybean nodules and roots and which suggested that dark CO2 fixation may be related to amino acid metabolism. In this chapter, I demonstrated that organic acids and amino acids were labeled in nodules exposed to 14 C02. The initial product(s) or step(s) of dark CO2 fixation in nodules is not known; the results from the labeling studies presented in this chapter are generally consistent with PEPC being the enzyme primarily responsible for dark CO2 fixation in soybean nodules (Figure 10). The product of the reaction catalyzed by PEPC is oxalacetate. Oxalacetate is difficult to identify and isolate because it is susceptible to spontaneous decarboxylation. I attempted to isolate oxalacetate as its 2,4-dinitropheny1- 14 hydrazone derivative from nodules exposed to 002. Less than 1000 cpmig fresh weight'1 were recovered from nodules that were exposed to 14 CO2 for l h as derivatives of 2,4-dinitrophenyl- hydrazine. Because of the low levels of radioactivity that were recovered as these derivatives,the identity of the labeled derivative(s) was not established. Oxalacetate, in most tissues, is rapidlyconverted to one of three compounds, malate, aspartate or citrate. In soybean 14 nodules exposed to C02, these three compounds accounted for 42% of the total radioactivity incorporated, which is consistent 14 with CO2 being fixed primarily by PEPC. 74 75 mmmcmmosczgmu mumscmzpm mmmsmsmcmspocwEm mumsmuzpm Aepv mmmsmszw Amv mmwpmgpcxm mcwamazpm Ampv mmmcmmosxgmo mumcwmmsm ARV mmmsmsmcmsuoc_5m mumsmuzpm mmm:_xo_;p mpmcwouzm on mmmguczm mumEmuapm mmmcmmosczzmu mumsmuapmoxouN Amv mmmcmmosexgmu mumsopapm ANFV mmmcmmosvxcmu mpmsuwmomw Aev mmmumsucxm mcwmmsmamm APPV mmmuwcoum Amv mmmsmsmcmspocwsm mumpsmamm Aopv mmmgpcam mumspwu ANV mmmcmmosuxgme mumpme Amv mmmpxxoasmm mmm>=sxapocmoggmosa AFV "cowuumms umummwezw mg» mepmumm page AmvmmEANcm mg» o“ msmsms :o_uuems :mmm smm: mmmmsucmsmq cw msmnszc mgp .APF mszmvsv mumpmumpmxo om xmmn mueseszs use mpmsmszs om mmmummmpmxo so commsm>com msa so mmzmmma umpmamp msm mumummmpmxo so mazosm szonsmm spam .cmumsoasomcw we Nouep sass HU¢FH cmsz empmnmp msm page mcoaswm mg» om smsms mxmwsmpmm ms» N8S op vmmoaxm mmpzuoz so muwm< ocws< ccm mwcmmso can? 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N N : 812w 1.12mi: 81 1o1 1o: =ng>xocoxs Amy omocmmogoxcoo opogoozpmosamogo1m Amv omopssopmoxpaocomoso on omopoco Amv omocpxxxongoo opo>=p>opocoogomoco ANV omopxxoogoo opo>=g>opocoogomoso Apv "coppoomp owpoopocp ozp mmszopoo pogp oez~=o osp op gopoc coppooop comm Loo: momocpcogoo :p Longs: och .App mpzmpmv opopooopoxo op xooo oposoEzp oco opogo23$ op opopooopoxo wo copmco>coo asp mo mmsoooo oopooop ogo opopmoopoxo yo masocm pxxoocoo spom .oopogoopoocp mp Nou N «F 83 opo>ocao 1axoceazo=nnoca 82 «znoaowzw o .11.... :«oo .8. .0. Nz.mo.".o1~.:.o :01zw :uoo: opocoo».a -.....ga-» em. aw. sop; muopu can; ompooop on op oopomoxo oco pocp mcoogoo ogp op gopoc mxmpgopmo ms» op oomooxm mopsooz cp ocpoapw oco ocpgom opcp pogo; po mucosoxm osp Lop mochom oomooopm Np opsmpm 2:33:39... 053.» 05029 «znoaokzw :o a}. . A 3812": 512.“: Nzzazo a E a . 8. .N . .z oo: 1 co: 2 co: moo: . .:. :uow. + .338 $8 «.8 «zo “w n. . .III111 N .n .. 1011111 1. N n = : nun—011w An. ... 9.ko ..N. 01w .3 I one 1w :«oo. zaoo. :«oo. . :«oo 333:9 225:3 225:3 1ogaaoga1N 1.33.3.2... 233238 ...ocoogauogo 83 Table 5 Distribution of Label and Estimated Rates of Incorporation of Carbon Derived from Dark C02 Fixation into Amino Acids l4 in Nodules Exposed to 002 Amino Acid Radioactivity Rate of Incorporation % of Total nmol of carbon-mg dry wt 1 of nodules'. h-1 Aspartate l5.8 4.8 Glutamate 22.0 6.6 Asparagine 7.0 2.2 Glutamine 1.3 0.4 Serine 3.3 0.9 Glycine 0.9 0.3 Alanine 3.0 0.9 Soybean plants (29—days old) were uprooted and the shoots were excised. The root systems were placed into a flask where they were exposed to 14002. After 60 min, the nodulated roots were removed from the flask and frozen. The amino acids were analyzed by HPLC. Radioactivity was measured by scintillation spectrometry. The rates of incorporation were estimated by multiplying the percentage of the total radioactivity that each amino acid represented (column 1) times the rate of dark C02 fixation (30 nmol CO2 incorporatedomg dry wt of nodules-1 h'I). 84 probably better estimates for those amino acids that are metabolic end products such as asparagine than they are for those amino acids that are used as substrates in other reactions, such as aspartate and glycine, because the percentage of radioactivity in each amino acid only reflects the carbon that has accumulated in that particular amino acid. It does not account for the total amount of carbon that has passed through each amino acid. An estimate of the carbon incorporated during dark C02 fixation that passed through glycine and serine can be made if it is assumed that the majority of glycine and serine synthesized in the nodules is used in the synthesis of allantoate. The percentage of label incorporated from 14 CO2 in allantoate , 5%, added to the percentages of label in glycine and serine, 0.9 and 3.3%, respectively (Table 5) represents the major portion of the label that could have passed through glycine and serine. If these values are used, the estimated rates at which carbon derived from dark C02 fixation are incorporated into glycine and serine are 1.8 and 2.4 nmol of carbon-mg dry weight of nodules-1 '1 h , respectively. The total rate of incorporation of carbon into glycine and serine can be made based on an estimated rate of allantoate synthesis. For this calculation several assumptions have to be made: l) The rate of N2 fixation equals the rate of CZHZ reduction divided by four (l90). 2) Eighty per cent of the N2 that is reduced in soybean nodules is used in the synthesis of allantoate (l2). 3) Two moles of glycine and serine are synthesized for each mole of allantoate synthesized (llS, ll6; Figure 2). Based on these assumptions and the rate of 02H2 reduction for nodules from 29—day-old soybean plants (44 nmol1mg dry weight"1 h71), the 85 estimated rate of synthesis for both glycine and serine is 8.8 nmol-mg dry weighth-h'1. Expressed on the amount of carbon that is required, the synthesis of glycine requires 17.6 nmol of carbon mg-dry weight'l h'1 and the serine requires 25.4 nmol of carbon-mg dry weight-1 h']. These values are about l0-times higher than the rates estimated above for the rates of incorporation of carbon incorporated via dark C02 fixation. If the rates that are presented are accurate, then the contribution of dark C02 fixation to the synthesis of glycine and serine, and hence allantoate is minimal. The labeling of alanine during dark €02 fixation can occur by at least two different pathways (Figure l3). Aspartate that is labeled in C-l can be decarboxylated forming alanine directly (lQl). Alternatively, pyruvate formed by the decarboxylation of labeled malate can be transaminated to form labeled alanine. There has been no published report of an enzyme catalyzing the B-decarboxylation of aspartate in nodules. Malic enzyme, which catalyzes the decarboxylation of malate, is known to occur in nodules (8); therefore, the latter pathway is the more likely route for the synthesis of labeled alanine. Glutamine is synthesized from glutamate and NH: in a reaction catalyzed by GS. The label from 14 C02 is proposed to enter into glutamine as follows: 14002 --> oxalacetate -- 2-oxoglutarate -I- glutamate ---glutamine Based on this series of reactions, the specific activity of glutamate, the direct precursor of glutamine, is expected to be greater than or equal to the specific activity of glutamine. Contrary to the expected results, the specific activity of glutamine was higher than that of 86 * - “CO: 4. CO2” phosphoenolpyruvate I fi'OPO3H2 CH2" (I) aspartate,, * * malate $02H-1 $02H _ $02H Haw-cw _‘5’ c=o ___.‘2’ HO-CH l CH2 CH2 CH2 ul id: «1| COzH 0211 COzH oxalacetate (6) (3) *cozw (4) *oozw H2N-?H ‘- $= . CH3 CH3 OIC'HHO pyruvaf. Figure l3 Proposed Pathways for the Exchange of Label into Alanine in Nodules Exposed to 14(:02 The asterisks refer to the carbons that are expected to be labeled when 14002 is incorporated. Both carboxyl groups of oxalacetate are labeled because of the conversion of oxalacetate to fumarate and fumarate back to oxalacetate (Figure ll). The number in parentheses near each reaction refer to the enzyme that catalyzes the indicated reaction: (1) phospoenolpyruvate carboxylase (2) malate dehydrogenase (3) malic enzyme (4) pyruvate aminotransferase (5) aspartate aminotransferase' . (6) aspartate beta-decarboxylase 87 glutamate in nodules exposed to 14 CO2 (Figure 9). To explain this discrepancy, I propose that there are at least two different pools of glutamate. One of these proposed pools of glutamate is assumed to be rapidly labeled with [140] from 14 002 attaining a high specific activity, while a second pool is at best only slowly labeled. It is the former pool of glutamate which is presumed to be used for the synthesis of glutamine. The differential labeling of glutamate and glutamine will be more fully discussed in chapter 3. 88 CHAPTER 3 3 AND NH: ON DARK coz FIXATION AND AMINO ACID METABOLISM IN SOYBEAN NODULES AND ROOTS THE EFFECTS OF NO For a number of years N03 and NH: have been known to affect the process of N2 fixation in nodulated legumes (l22,123). The addition of N03 also appears to increase asparagine synthesis in the root system of soybeans (12). The work presented in this chapter is concerned with two areas: the effect of N07 and NH+ 3 4 on amino acid metabolism in soybean nodules and roots, and the pathway of asparagine synthesis in the soybean roots. RESULTS £h_EFFECTS OF INORGANIC NITROGEN ON__C2H9 REDUCTION AND DISTRIBUTION OF NITROGEN IN THE XYLEM EXUDATE I initially examined the effect of inorganic nitrogen on amino acid metabolism by determining the time necessary for N03 to have an observable effect on N2 fixation and the distribution of nitrogen in the xylem exudate. One group of nodulated plants was treated daily with N-free nutrient solution while another group \was treated with nutrient solution containing 10 mM KNO3 for 8 days prior to the experiment. When the plants were 2l-days old, some of the plants that had been grown on N-free nutrient solution were now provided daily with nutrient solution containing l0 mM KNO3 until the end of the experiment. These plants are referred to as "shifted" .P7a nts. The control plants continued to receive N-free nutrient soltxtion throughtout the experiment. After 2 days of treatment with NO', the"shifted" plants had 89 Figure 14 Time Course of the Effect of N03 on the Distribution of Nitrogen in the Xylem Exudate and the Rate of CZHZ Reduction Soybean plants were irrigated with N-free nutrient solution (C)) or with nutrient solution containing 10 mM KNO3 starting when the plants were l3-days old (A) or 2l-days. old ([3 ). On the indicated days, the shoots were excised and the xylem exudate was collected for 45 min. Amino acids and N03 were analyzed by HPLC. Organic N is the sum of the nitrogen in ureides, allantoin and allantoate, and in the amino acids (12). The values reported for the concentration of N03, the ureide content and the rate of CZHZ reduction are averages for 3 plants. The average asparagine content was determined by chromatographing one sample that consisted of equal volumes of xylem exudate collected from 3 plants. Bars are presented in Panels A, B and C when the SD is greater than the size of the symbol. 9O 33 7 1N. C \. a/ . .. B /A 01—. D J 191. w. - pm - “DU h o w W M" «no 0 .. .... 5.2.3. estate-230 moz oc.oe.uno< no 2 3.395 8 1.1911. . . 1 .10... AW -Aal . an 7 1 .. 1 2 _ 70.. ‘ W A p p p b r n c b n n .b. b .Al-b l m . 9 6 3 0 O O O 0 9 6 3 .1332 .353 326;. «zuo :33: 32 25.95:. Plont'Ago (days) Figure 14 91 a higher rate of CZHZ reduction than control plants, 3.2 versus 1 h-l 1.2 nmol plant- ; after 6 days, the rate of C2H2 reduction of the “shifted" plants was the same as that measured for Nog-treated plants, both of which were considerably lower than that of control plants (Figure 14, Panel A). The concentration of N05 in the xylem exudate of the "shifted" plants, within 2 days, increased to 10 11mol~m1"1 (Figure 14, Panel B). The comparable values for Nog-treated and control plants were 14 and 0.2 umoloml'1,respectively. The ureide content (sum of allantoate and allantoin) of the xylem exudate expressed as the percentage of the organic nitrogen rose in control plants from 50 to 80%, the value that it remained at for the next 12 days (Figure 14, Panel C). In contrast, the ureide content of the "shifted" plants initially increased and then decline. The relative ureide contents of the xylem exudate of "shifted" plants after a 12-day treatment and the NOS-treated plants were similar. While asparagine is the major amino acid in the xylem exudate of plants that are dependent on N03 and N2, it makes up a larger percentage of the organic nitrogen in the xylem exudate of plants that are dependent on N03 than those dependent solely on N2 (l2). The major products of N2 fixation in soybean are the ureides, allantoin and allantoate. A decline in the ureide content and an increase in the asparagine content (the percentage of organic nitrogen in the xylem exudate as asparagine), therefore, indicates a shift of metabolism away from N2 fixation to N0; utilization. After 6 days of treatment, asparagine accounted for 92 13% of the organic nitrogen. This value was 5-times more than the levels in control plants but only 60% the levels observed in NOS-treated plants (Figure 14, Panel 0). After 12 days, the relative asparagine contents of the "shifted" and NOS-treated plants were similar. The drop in the relative asparagine content that was observed in the xylem exudate of control plants has been observed previously (114) and appears to be related to developmental process. The effect of NH: and N03 on the distribution of nitrogen in the xylem exudate was next examined. Plants (ZS-days old) that had been grown on N-free nutrient solution were irrigated with 1 liter of 1.5 mM (NH SO4 or 10 mM KNO3 at 10:00 a.m. and 12, 24 - + 3 or NH4 the plants there was a small diference in the relative ureide 4)2 and 48 h later. Twelve hours after NO was first added to contents in the xylem exudate of both the treated plants and the control plants (Figure 15, Panel A). Fourty-eight hours after the original treatment, the ureides in NH: and Nog-treate plants accounted for 80 and 68% of the organic nitrogen in the xylem exudate, respectively versus 90% in control plants. The percentage of organic nitrogen as asparagine in Nog-treated plants and asparagine and glutamine in NHZ-treated plants increased at the same time the ureide content decreased (Figure 15, Panels C and D). The concentration of N03 the first 12 h until it was l4-times higher than that of the control and NHZ-treated plants (Figure 15, Panel B). These results in the xylem exudate of NOE-treated plants increased within suggest that within 12 h and certainly within 24, N03 and NHZ affected plant metabolism. The following experiments were concerned with amino acid metabolism and dark €02 fixation in plants that 93 Figure 15 Time Course of the Effects of N03 and NH: on the Distribution of Nitrogen in the Xylem Exudate Twenty-five-day-old soybean plants that had been previously irrigated with N-free nutrient solution were irrigated either with N-free nutrient solution (0) or nutrient solution containing 10 mM KNO3 ( [5) or 1.5 mM (NH4)ZSO4 ([3 ) at 10:00 a.m. and 12, 24 and 48 h later. At each time, the shoots of 3 plants from each group were excised and the xylem exudate was collected for 45 to 60 min. Amino acids and N05 were analyzed by HPLC. Organic N is the sum of nitrogen in the ureides, allantoin and allantoate, and in the amino acids (12). The values reported for the ureide content and concentrations of N03 are averages of values for 3 plants. The asparagine and glutamine content was determined by chromatographing one sample that consisted of equal volumes of xylem exudate collected from 3 plants per treatment. Bars are presented in Panel A when the SD is greater than the size of the symbol. 94 . a J q . u . . m d J 4 AGL H 48 24 ,1 H H .4 /H /H. Ho <:_____ H B L - n p - n n \l «I. m 9 6 3 O M H :1.6~.o.=3 5:32.230 mo: 056339 no 2 2.330 8 m m .. . . u (d d d J 4 1 a d J a d 8 o.” g .. 10114.. 1.01.. - n. A Mo .4 H H 0/ AH: u m\&11 SUA/ . r AM, . b p 1 c n /-O J o O O O 9 6 O 9 7 5 .0295 no 2 2.3302. 2.30.2.3. no 2 2.3958. 95 - + were treated with N03 or NH4 for 5 to 14 days. B. EFFECTS OF NOEAND NH: ON AMINO ACID METABOLISM IN ROOTS OF 13-DAY-OLD PLANTS 1. Concentrations of Amino Acids The effect of treatments with N05 or NH: for 5 days or longer on the concentrations of amino acids and the incorporation of label from 14CO2 into amino acids was first examined in roots of l3-day-old plants that had been treated with 10 mM KNO or 1.5 mM (NH4)ZSO4 for 5 days. 3 The N03 and MHZ-treated roots had, respectively, 2.2 and 2.7-times the concentration of amino acids as the 13-day-old control roots did (Table 6). Most of this difference was accounted for by differences in the amount of asparagine. Roots of NOS-treated plants had 2.4-times the level of asparagine as l3-day-old control roots. The NHZ-treated roots had higher levels of asparagine (3.2-times) and glutamine (4.3-times) than the l3-day-old control roots. For comparision, roots of 6-day-old plants were also analyzed. They contained 7.5-times as much glutamine and 5.3- times the total amount of amino acids as the 13-day-old control roots when the values are expressed on a dry weight basis. Nodules were present on the roots of 13-day-old control plants, but were not observed on the plants that had been treated with either NO' or NHZ. When split open, the nodules were white 3 indicating that leghemoglobin was not present and that the nodules were not fixing N2 (25). The distribution of amino acids in nodules and control roots was similar except for the amount of asparagine and glutamate that was present (Table 6). In nodules the concentration of glutamate was substantially higher than that 96 ocp How mospo> och poppcoo How oopcomopo mozpo> as» ocpso popop .mpoEom Loo mpcopo N cppz mopoaom N Lop momoco>o opo mozmmpp Hospo .oposom Loo mpcopo N sppz mmposom o Ho Amm Av momopo>o ogo mpooc oco mopzoo: .oopoopoo mopoo ocpso ogp ppm Ho coppoeszm osp op mcopog mopoo .ompoogpxo mopoo oopso ogp oco opo mxoo1mp co m opoz mogp cos: oopmo>pog ogoz mpcopo asp .oomNflozzv 25 m.p co mozx zs op mcpcpopcoo coppzpom pcopgpzc .w hon co mopppopm Ho Apocpcoov pcospcoaxm ogp pzogmsoggp coppopom pcopgpzc mogp1z gmcppo sppz zppoo oopompppp ogmz mpcopo coonaom. m A No m.o A m.p o A mp m._ A a.mp m.m mp pocpcoo mopzooz HHH H.m «NH m.N N.N mp omNquzH opp o.N No o.m m.m mp mso. N A mm m.o A m._ p A mm m.o A o.N N.o A m.N mp poppcoo mom w.¢p NAN 0.3 o.o o pocpcoo mpoom 1p: xco as.posc mopo< ocps< om< popoh ocpEopzpw ocpmogoom< oposopzpm opoppoom< pcopa pcospooph mpcopa mcso> Ho mo—zooz oco mpoom op mopo< oops< mo coppocpcoocoo ocp co wzz oco moz Ho mpoowwm m opoop 97 measured in roots , 15 versus 2.4 nmolsmg dry weight-1. The reverse was true for the concentration of asparagine. Roots had a higher concentration of asparagine than nodules, 39 versus 13 nmol-mg dry weight']. 2. Products of Dark CO2 Fixation 14 The roots of 6 and 13-day-old plants were exposed to CO 2 for 5 to 28 min. The labeled compounds in these roots were then analyzed. Only the values that were obtained after a 28 min exposure to 14 CO2 are presented (Table 7), because the distribution of label did not appear. to be affected by the length of exposure. Between 70 and 86% of the total label at all times and for all tissues was recovered in the fraction containing the organic acids and neutral compounds. A larger percentage of the total label was in the amino acids of NOS-treated (30%) or MHZ-treated (23%) roots than was in the amino acids of the l3-day-old control roots (14%). In N03 and in NHZ-treated roots, asparagine accounted for 5 and 6% of the total label, whereas in control roots only 1% of the 14 total label was in asparagine. The percentage of label from C0 2 in glutamine in MHZ-treated roots was the same as in the other roots even though the concentration of glutamine in NHZ-treated roots was higher than in the other roots of the same age. Glutamate accounted for a higher percentage of the total label recovered from nodules (10%) than it did in roots (3 to 5%). 98 .csspoo cpm: ocp :o oocpopoc po: ocoz posp moczoosoo omogp op Howoc monsooeoo Poppaoc + mopo< upcomcoo .oposom Hog mpcopo N sppz moposom N so» momoHo>o pzomopoog oopcomogo mm=Po> osh .zgpoeocpooom coppoppppcpom >3 oocpEHopoo mo; app>ppooopoom .upo: >o oo~>Poco ogoz mposoopo oopooop och .:omocpp: opzopp :p coNocp oco xmopp ogp sogw oo>oeop ago; msmpmam poo; ogp .:ps mN copp< .Nou op oomooxo opmz amcp ogogz xmopw o opcp ep oooo—a osoz msmpmzm poo; onp oco .oompoxm msoz mpoogm ocp .ompoogo: ogoz xmgp .opo mxoo1mp go 0 ago; mpcopo ocp cos: .eomNAozzv :5 m.~ Lo mozx ze op mcpcpopcoo coppzpom pcopgpzc .w zoo co mopppopm Ho apogpcoov pcmsppooxm ocp paogmzossp coppopom pcopgpzc mogw1z Hosppo gppz oopompggp ago; mpcopo :oooxom mm o o op N mp poppcoo mopzooz AH m m m 8 mp vomNHezzv mm m p m m mp mozx ow o o A pp 0 _ogpcoo mpoom app>ppooopooa Popop A maoo m caooso o; so on o+ mopow wpcwmcu oopsopzpw ocpmogoom< oposopzpo opopcoom< pond; pcmspooph m mpcopa mczo> wo mopzooz oco mpoom soap oopmtoatoocp Foams Ho coppsnptpmpo asp go «I: use moz Ho mpompcu H opnap N :p coop + 1 99 - + 3 AND NH4 29-DAY-OLD PLANTS C. EFFECTS OF NO ON AMINO ACID METABOLISM IN ROOTS OF 1. Concentrations of Amino Acids and Organic Acids Starting when they were 15-days old some plants began receiving nutrient solution containing 10 mM KNO3 or 1.5 mM (NH4)ZSO4. When the plants were 29-days old, the roots were harvested and their amino acid content was determined. Nitrate and ammonium had a similar effect on the distribution of amino acids in roots of 29-day-old plants (Table 8) as they did on the distribution in the roots of l3-day-old plants. The concentrations of amino acids in Nog-treated and in NHg-treated roots were 2.4 and 3.2-times the levels in control roots. Most of the difference in the concentration of total amino acids was accounted for by larger concentrations of asparagine in N03 and NH:- treated roots and by a larger concentration of glutamine in NH:- treated roots than in control roots. The effect of N03 on the concentration of several organic acids in roots was also determined. The presence of high levels of malonate and malate in nodules and in control roots previously reported (162) was confirmed (Table 9). Control roots, however, had higher concentra- tions of malonate than either NOé-treated roots or control nodules. Nitrate-treated roots had lower amounts of all the organic acids than were measured in either nodules or control roots. 2. Rates of Dark CO2 Fixation The rates of dark CO2 fixation in roots appeared to be affected by the presence of NH2. The rate of dark CO2 fixation + in NH4-treated roots from 29-day-old plants was significantly 1OO .mpcopo a so» Amm Av mamasa>a ago aapsamaso ago pasp mazpa> asp .oapoapao mopoo ospEa asp Fpa Ho sopposszm asp op msaAas mopoa ospsa Papop .uss: xs oaprosa asaz mopoa ospsa ash .oapoaspxa mopoa ospEa asp osa opo mxao1mN asaz zasp sasz oapma>sos agaz mpsapo asp .oomNsvzzv zs m.~ so mozx :5 o_ newspapsoo coppspom psapspsc .m_ aoo so msppsapm so apospsoov psaspsaoxa asp pzosmzossp soppzpom psapspas aasA1z sppz oapampssp asaz mpsapa saasAom A A m__ a.p A o.HN N A so m.o A m.A N.o A m.m AomNHAIZH e A NN m.o A p.N m A mm .m.o A N.m ¢.o A m.m mozx m A om p.o A o.p N A pp —.o A m._ N.o A m.N pospsou P1p: ago mE.PoEs mopo< ospe< aspsapzpo aspmasaom< apasapzpw apapcaam< pcaspaasp papop mpcapa Apo-sao-am so mpoom =H mapo< ocps< Ho coppaspcaocoo asp so N12 oca moz so mpoaaaa a apnap 101 Table 9 Effect of KNO on the Concentration of Several Organic 3 Acids Treatment Malonate Malate Succinate Fumarate nmol~mg dry wt"1 Roots control 123 i 22 45 i 8 11 i 3 14 i 5 KNO3 16 i 3 23 i 2 2 i 1 1 i 1 Nodules control 33 i 5 30 i 3 12 i 2 16 i 3 Soybean plants were irrigated with N-free nutrient solution until they were lS-days old, after which time a number of the plants continued to receive N-free nutrient solution (control) while the other plants were irrigated with nutrient solution containing 10 mM KNO3. When the plants were 29-days old, the indicated tissue was extracted with ethanoleZO (80:20, v/v). The organic acids were purified by ion-exchange chromatography. The organic acids were dissolved in pyridine and derivatized with BSTFA. The trimethylsilyl derivatives of the organic acids were separated by gas-liquid chromatography on a 3%-SE 30 column with a temperature program of 85 to 185 C at 5 C-min']. Malonate, malate, succinate and fumarate were used as standards. The values reported are averages ( i SE) fbr 4 plants. 102 higher than the rates measured for the other roots of comparable age (Table 10). The rate of dark CO2 fixation in roots of 21-day-old plants for each of the treatments, however, were not significantly different from one another. 3. Products of Dark CO2 Fixation The presence of N03 or NH: had a dramatic effect on the distribution of label in 29-day-old roots exposed to 14 C02. Amino acids in control roots accounted for at most 22% of the total recovered radioactivity, with aspartate and glutamate, the two major labeled amino acids, accounting for 6 and 8% of the total label, respectively (Figure 16, Panel A). In contrast, after only a 3 min exposure, 33% of the total label recovered from MHZ-treated roots was present in the form of amino acids (Figure 16, Panel B). The percentage of label in amino acids in NHZ-treated roots continued to increase until after a 28 min exposure, asparagine and glutamine, the two major labeled amino acids accounted for 14 and 24% of the total label, respectively. In NOé-treated roots, the percentage of 14 label in the amino acids after a short exposure to CO2 was lower than that observed in MHZ-treated roots (Figure 16, Panels B and C). With longer exposures, the percentage of label in asparagine increased, representing 25% of the total label after a 60 min exposure. 4. Specific Activities of Amino Acids in Roots Exposed to 14co 2 The specific activities of glutamate and glutamine in 14 nodules after exposure to CO2 could not be explained by the simple conversion of labeled glutamate to glutamine (discussion in 103 Table 10 Effects of N03 and NH: on the Rate of Dark co2 Fixation in Roots and Nodules Roots Nodules Treatment 21-days 29-days 21-days 29-days nmolsmg dry wt']~h'1 Control 16 1 2a 16 i 3a 51 i 6a 30 i 2a KNO3 (10 mM) 12 i la 14 i la 20 i 3c 10 i lb KNO3 (3 mM) 15 i la 17 i 2a 41 : 3ab 12 i 1b (NH4)ZSO4 17 i 2a 24 i lb 34 i 3bc 14 i 2b (1.5 mM) Soybean plants were irrigated with N-free nutrient solution until they were lS-days old, after which time a number of the plants continued to receive N-free nutrient solution (control) while the other plants were irrigated with nutrient solution containing 3 or 10 mM KNO3 or 1.5 mM (NH4)ZSO4. When the plants were 21 or 29—days old, the shoots were excised and the nodulated root systems were placed into a flask where they were exposed to 14 C02. After a 10 min exposure, the nodulated roots were removed and frozen in liquid nitrogen. The amount of radioactivity that was incorporated was determined by scintillation spectrometry. The values are averages for 6 replicates (t SE). In each column, the values that are followed by the same letter are not significantly (P§_0.05) from one another according to Duncan's multiple-range test. 104 .pasaA _opop asp wo AA sasp mmap Low oapszoooo aspsaAa osa aspoApm .aspsam .moszoseoo Paspzas ssa aspoa opsamso mspspapsoo soppoasa asp sp Fasop As sow Appsaepss capszoooa was aspsap=Am osa aspmasasma .apasapzpm .apapsasma Low mazpa> asp Ao Ezm asp osa Noe» saazpas aasasaAwpo asp .Aspasospoasm soppappppspom As oaszmaas mo: App>ppoaopoam .uss: As oaNAAosa asaz mpasooss oapasop asp .apssam gas mpsaps N sppz apsEam aso so mpmAAasa psamassas sapsamaso maon> asp .Nouvp op oamosxa asaz Aasp asasz smapm a opsp oaoapa asaz mEapmAm poo; asp osa .oampoxa asaz mpoosm asp .oapoosss asaz Aasp .spo mAao1mN asaz mpso—o asp sasz .Au pasas.mozv mozx :5 oA so As pasas .wzzv AomNAsAzzv :5 m._ mspspopsoo sopp:_om psapspzs so A< _asas .pospsoov soppzpom psapspzs aasw1z sppz oapampssp asaz mpsaAs asp aEpp sopsz LapAo vo wAao1m_ asaz Aasp Apps: soppzpom psapspzs aasw1z sppz oapompssp asaz mpsaps saasAom N ouv— 0p uwmoqu mpoom sp mvpo< ospe< sp Pasas we cowpzspspmpo asp so N12 vsc moz we poamwm o_ aszmwm 105 op assmHH “..:.—.5 05:1 oa on o oa on . o oa on o - . q . q _ A A p q . p _ . o. 9 i nimj<“0\m1 0 3% Km, 114/414/ .. o \ 1 M . \« H \o 1‘ /m 1w . 1 10 ‘ ‘ a. ..r1... .- ,//, 1 \ .:.. . q 11 ‘1. f ‘ F1 . \ 11 1 N- q , 1 .:.. <\ o 11 1. W 1rd 1... J 0- 4 £00 coed a; . . aua‘ 1d moz .01. O «:2 .m 11 .8230 .< 1 vN 16111110001908 1010.1 °/. 106 chapter 2). The relationship between the specific activities of 14 glutamate and glutamine in roots exposed to CO2 was different depended on what the source of nitrogen for the roots was. The specific activities of glutamate and glutamine in control roots were about equal, as they rapidly increased reaching maximum values of 302 and 288 cpmmmoi'1 , respectively (Figure 17, Panel A). 'In +- 4 were also about equal, but they increased at a slower rate than they NH treated roots the specific activities of glutamate and glutamine did in control roots (Figure 17, Panel B). In NOé-treated roots the specific activity of glutamate (21 cpm'nmol'I) was initially higher than that of glutamine (O cpm-nmol") (Figure 17, Panel C). However, after a 5 min exposure, the specific activity of glutamine was 105 cpmonmol'1 , ZO-times that of glutamate. The maximum specific activities of glutamine and glutamate observed in NOE-treated roots were 314 and 195 cpmonmol'1, respectively. 14 After a 3 min exposure to C02, the specific activity of aspartate in control, N03 and NHZ-treated roots was 98, 36 and 1, respectively (Figure 17). The specific activity of 88 cpm «nmol- aspartate increased in control and NOE—treated roots reaching the maximum observed values after a 28 min exposure. In contrast, the specific activity of aspartate in NHZ-treated roots remained relatively constant. The specific activity of asparagine increased in all tissues although at a slower rate in control and NHZ-treated roots than it did in Nog-treated roots. 107 .mopoa ospsa scoosapm Ho amosp osa ma>ppa>psao ppapas asp Ao mpmas asp so sapmppsazo asaz mopoa ospEa ash .Aspasospoasm soppop—ppspom As oassmaas ma; App>ppoaopoom .oss: As oaNAPasa asaz mpozooss oapasap asp .apssom gas mpsaps N sppz aAsEam aso 4o mpmApasa psamassas oapsamass maapa> asp .Nou op oamosxa asaz Aasp asasz swap» a opsp oaoapo asaz msapmAm AH poo; asp use .oampoxa asaz mpoosm asp .oapooss: asaz Aasp .opo mAao1mN asaz mpsaps asp sasz .Ho paces .mozv mozx :2 o_ co Hm paces .szs Aommsezzv :5 m.p mepepepeoo coppspoA pechpse co a< chmm «POLHCOUV :OwquPOm Hcmeflzc QQLWIZ 5&5) UmHMOwLLw wLm3 main—h 0;“ meH £U_.£3 sapwa opo ono1mp asaz Aasp Apps: soppapom psapgpss aasm1z sppz oapampssp asaz mpsops saasAom Nou A op camooxu Asses ep AoHc< oeps< Haca>am to AaHAH>Hpo< oHaHoaam asp so sz sea moz Ho poaapa Hp acsmps 108 NH acsmpa ..:.... 2:; cm on o 00 on o Om on o 1 13. . o\w1 0 § 0K0 r. 1.1 1. s 4 :(.1 /‘ . 1 At 0.1 l l J .l .0 . 20 4.0 Time (min) Figure 23 Time Course of the Distribution of Labe1 in Organic Compounds in Aider Nodu1es Exposed to 14C02 Aider nodu1es were removed from the roots and were p1aced into a f1ask where they were exposed to 14C02. At the indicated time, the nodu1es were removed from the f1ask and frozen in 1iquid nitrogen. The 1abe1ed products were extracted with ethan01:H20 and were ana1yzed by HPLC. Radioactivity was measured by scintiTIation spectrometry. The o.a. + neu. refers to the fraction containing the organic acids and neutra1 compounds. Cit1. is the abbreviation for citru11ine. 136 Radlaactlvity (cpm) fltdl 755 - ' t - ' cit . 5.0- .. origin euc tum 1 1 1 - 0 .L p I (D ‘ ' l() 21) Distance (cm) Figure 24 IncorporatiOn of Labe1 into Organic Acids and Neutra1 Compounds in A1der Nodu1es Exposed to 14CO2 A1der nodu1es were removed from the roots and p1aced in a f1ask where they were exposed to 14C02. After 20 min, the nodu1es were removed and the 1abe1ed products were extracted with ethano1zH20 (80:20, v/v). The extract was separated into two fractions, one containing the organic acids and neutra1 compounds, and a second containing amino acids. An a1iquot of the former fraction was spotted on a thin-1ayer ce11u1ose p1ate at the origin and Chromatographed with butano1zg1acia1 acetic acidzHZO (120:30:50, v/v/v) as a so1vent system. Ma1ate (ma1) and citrate ( cit ) were identified on the basis of co-migration with standards. Succinate (suc) and fumarate (fum) were identified on the basis of pub1ished Rf va1ues (206). Radioactivity was detected with a gas- f1ow scanner. 137 Figure 25 Time Course of the Distribution of Label in Organic Acids in Alder Nodu1es Exposed to 14CO2 A1der nodules were removed from the roots and placed in a flask where theywere exposed to 14C02. At the indicated time, the nodules were removed and frozen. The labeled products were separated by ion-exchange chromatography into two fractions, one containing the organic acids and neutral compounds and a second that contained the amino acids. The former fraction was Chromatographed on Dowex 1 (chloride, acetate) using gradient elution with water and 1.2 N formic acid. Radioactivity was measured by scintillation spectrometry. The columns were standardized with [14C]ma1ate and [14C1citrate. Succinate was identified on the basis of its relative retention to malate and previously published separations (204,205). Based on reports in the literature (204) a portion of the label that eluted with citrate may be radioactive fumarate. Less than 2% of the radioactivity eluted in the void volume of the column. 36' Radioactivity 80 0t 0 A O 20 138 O O citrate . O . succinate 0 e’.\‘ .* ~e O 20 ' 40 Time (min) Figure 25 DISCUSSION Carbon that was incorporated during dark CO2 fixation in alder nodules was incorporated into citru11ine. There are at least two enzymes, PEPC and carbamylphosphate synthetase, that fix CO2 into products that are used as intermediates in the synthesis of citrulline in other organism (111) (Figure 26). The presence of label in glUtamate and aspartate is consistent with a part of the 14CO2 being incorporated via PEPC in alder nodules. The relationship between the percentage of label in glutamate and citru11ine (Figure 23) suggests that the [14C] was entering citrulline via glutamate. The pathway from glutamate to citrulline in other systems has ornithine as an intermediate. Labeled ornithine may not have been detected either because the amount of label fell below the detection limits of the system or because ornithine and thus citrulline may not be synthesized from labeled glutamate. Carbamylphosphate synthetase, which catalyzes the synthesis of carbamylphosphate from HCOS and [14C] incorporated during dark ATP, may be the enzyme by which the CO2 fixation is incorporated into citrulline. The change in the distribution of label in the fraction containing the organic acids and neutral compounds (Figure 27) may be explained if two pools of malate are present. MacLennan 33 31. (192) proposed that different pools of several organic acids exist in plant cells. One of the pools for each of the organic acids appears to be closely associated with the respiratory centers, whereas the other(s) is physically remote from these centers and is metabolically stable. I propose that a similar separation of pools exist in the alder nodule. The major portion of the citrate but only a minor portion 139 140 phoephoenol- glutamate ornithine citrulline pyruvate cozw . 'cozw ”00214 *00211 fi-oeoa Hz .L'lswzw-cw _‘Q... HzN-CH HzN-C'll-i CH2 “f"z’z ' (9"212 (3) ‘fH2’3 + COzH ‘ ‘ (.:Hz. NH 10 _ , l . 11cc3 Nl-iz 'c=o l 4‘ hfliz .f a ‘_ (4) qktb 2 ATP~ + NH4 + H003 ’C-OPO3H2 0' Hz" glutamine carbamylphosphate Figure 26 Proposed Pathways for the Incorporation of Label into Citrulline in A1der Nodules Exposed to 14C02 The asterisks refer to the carbons that are expected to be labeled when 14CO2 is incorporated. The numbers in parentheses refer to an enzyme or series of enzymes that catalyze the indicated reactions: (1) phosphoenolpyruvate carboxylase citrate synthase aconitase isocitrate dehydrogenase glutamate synthase (2) carbamylphosphate synthetase (3) glutamate acetyltransferase acetylglutamate kinase N-acetyl-y-glutamylphosphate reductase acetylornithine deacetylase (4) ornithine carbamyltransferase 141 of the malate is in a pool that is actively turning over; the .major portion of the malate is sequestered in a metabolically inactive pool. As label accumulates in the nodule, the percentage of label in malate would increase because the metabolically inactive pool of malate would account for a larger portion of the label. CHAPTER 5 13 THE INCORPORATION OF N-LABELED TRACERS IN SOYBEAN AND ALDER NODULES AND SOYBEAN ROOTS The experiments that were described in the previous chapters [14C] to explore amino acid metabolism. This used the tracer approach is limited, however, in that only the metabolism of the carbon skeletons can be monitored. The use of the radioactive istope of nitrogen, [13M], provides a way of tracing the path of the nitrogen of amino.acids. The following experiments were designed to follow the pathway of nitrogen assimilation in nodulated roots of soybeans using radioactive N2, N03 and NH: and to explore the assimilation of NH: in alder nodules using labeled NHZ. RESULTS The pathway of NH: assimilation in alder nodules was examined initially by incubating detached nodules with ‘3 NHZ. At intervals nodules were removed and were extracted with methanol. Labeled products were separated by high-voltage electrophoresis. Glutamine accounted for the highest percentage of label with lesser amounts in glutamate, alanine, aspartate and asparagine (Figures 27 and 28). The amide-group of glutamine accounted for 90% of the label in glutamine. Alder nodules that were still attached to the roots incorporated label from 13 NH: in a fashion similar to that of detached alder nodules. The relationship between the incorporation of [13N] into glutamine and glutamate was examined. Alder nodules were incubated 142 143 with NH: for various lengths of time, at which time they were ‘either immediately extracted or incubated with 13NH: for 5 min, washed with H20 for 3 min and then incubated for a further 10 min 14 in water containing 10 mM NH: before they were extracted. Glutamine accounted for 90 to 80% of the total radioactivity 13 + NH4 min (Figure 28). Similar results were obtained with the incorporated after exposure of the nodules to for l to 15 pulse-labeling experiment, in which glutamine accounted for 70% 14NH+. of the incorporated label even after the 10 min chase with 4 The role of GS and GOGAT in the assimilation of NH: in alder nodules was examined using an inhibitor of GOGAT, azaserine, and an inhibitor of GS, methionine sulfoximine (MSX). Nodules were incubated in water containing 1 mM azaserine for 10 min, at which time 13 NH: was added. Ten minutes later the nodules were extracted with methanol. The presence of azaserine did not have any effect on the distribution of label in glutamine and glutamate. In contrast, a pretreatment of alder nodules with l or 10 mM MSX for 10 or 20 min before the nodules were exposed to 13NH: increased the distribution of label into glutamate, aspartate and alanine in relation to the amount of label in glutamate when only 13NH: was added. Labeled glutamate and aspartate accounted for only 52% of the total radioactivity incorporated in MSX-treated nodules versus less than 15% in nodules that were not treated with MSX. The incorporation of labeled nitrogen insoybean was first examined by determining the incorporation of 13NH: and 13N03 into soybean roots. The roots of soybean plants that had been grown 144 I hD C) 1 (D In. (cpm x164.5 )(..... 9 an ° ll) ' 155 12¢) Distance (cm) Radioactivity (cpm x '0-3 H 1 ' (i in (3 «A; Figure 27 Incorporation of Label into Organic Products in Alder Nodules Incubated with 13NH: Alder nodules were removed from the plant roots and placed into H20 containing 13NHZ. Eleven minutes later, the nodules were removed and the labeled products were extracted with methanoleZO (80:20, v/v). After standard amino acids were added to the extract, an aliquot was spotted on a thin-layer cellulose plate. The plate was sprayed with 70 mM sodium borate (pH=9.2) and subjected to electrophoresis (3 kV). The radioactivity on the plate was detected with a gas-flow scanner and the standard amino acids were visualized by spraying the plate with ninhydrin. The arrows indicate the location of the standard amino acids after electrophoresis. 145 Figure 28 Time Course of Incorporation of Label into Glutamine and G1utamate in Soybean Roots and Alder Nodules Incubated with 13M”: Soybean roots (. ,I) or alder nodu1es (O ,D) were incubated in H20 containing 13NHZ‘for the indicated time. The organic products were extracted with methanoleZO (80:20, v/v). After standard amino acids were added to the extract, an aliquot was spotted on a thin-layer cellulose p1ate.‘ The plate was subjected to electrophoresis (3 kVO. The radioactivity on the plate was detected with a gas-flow scanner and the standard amino acids were visualized by spraying the plate with ninhydrin. The amount of label that co-migrated with glutamine (., O) and glutamate (I ,D) was quantified by integration of the peaks after scanning. Areas were corrected for decay. The difference between 100% and the sum of the label in glutamine and glutamate was accounted for by label in aspartate and alanine. 146 100 80 ' 6 . .4 3383.3: .28.: 15 Time (min) Figure 28 147 on N-free nutrient solution actively incorporated label from both 13NH: and 13N0; into organic products (Figure 29). The distribution of label from 13 NH: after exposure of soybean roots was similar to that observed in alder nodules (Figure 28) with glutamine accounting for about 80% of the total label incorporated. 13NHZ, the percentage of label In soybean roots incubated with in glutamine versus glutamate was higher than that in roots incubated with 13110; (Table 14). ' I Meeks gt al.(5) examined the time course of incorporation of label from [BNJN2 into the organic products of soybean nodules. One product, which migrated between glutamate and asparagine, was not identified. In an attempt to identify this compounds, we exposed soybean nodules to 13NH: for 10 min, at which time the labeled prOducts were extracted and subjected to electrophoresis. Labeled NH: was used rather than [BNJN2 because larger amounts 13NH: versus [BNJN2 (preliminary of label can be incorporated using observations ). The previously unidentified compound was identified as allantoate on the basis of its migration with standard allantoate during electrophoresis. Allantoate accounted for only a minor portion of the label (8%) in nodules incubated with 13NH: (Figure 30, Panel B) versus the 30% reported by Meeks EEJEL- (5) for the unidentified compound in nodules exposed to [‘3NJN2. The experiment performed by Meeks et_al. (5) was repeated to determine if the distribution of label in nodules exposed to 13NH: or [13NJN2 was different. Our results with detached nodules exposed to [13NJN2 (Figure 30, Panel A) were similar to those previously reported (5). Allantoate accounted for 48% of the incorporated label C) I )> I! C) 03 1 9 (I I (31:. ‘—‘* __2: 15- B '3NH1' lC)- . 91’“ ale 5 -' asp can I - Radioactivity (cpm 910's ) o ‘1 1’ 11 l o e ' . lO 15 20 Distance (cm) Figure 29 Incorporation of Label into Organic Products in Soybean Roots Incubated with 13NO; or 13NH: Soybean roots were incubated in H20 containing N03 for 12 min (Panel A) r 13NH: for 15 min (Panel B). At the indicated time, the roots were removed from the solution and the labeled products were extracted with methanoleZO (80:20, v/v). After standard amino acids were added to the extract, an aliquot was spotted on a thin-layer cellulose plate. The plate was sprayed with 70 mM sodium borate (pH=9.2) and subjected to electrophoresis (3 kV). The radioactivity on the plate was detected with a gas-flow scanner and the standard amino acids were visualized by spraying the plate with ninhydrin. The arrows indicate the location of the standard amino acids after electrophoresis. 13 149 Table 14 Incorporation of Label into Organic Compounds in Soybean Roots Incubated with 13NH: or 13N03 Treatment Glutamine Glutamate Othera % of Total Radioactivity 131m: (5) . 83 e 2 17 e 1 0 13N03 (3) 47 e 6 33 i 6 26 i 2 Soybean roots were incubated in water containing either 13NH: or 13N03 for 7 to 12 min, at which time the roots were removed and the labeled products were extracted with methanoleZO (80:20, v/v). After standard amino acids were added to the extract, an aliquot was spotted on a thin-layer cellulose plate. The plate was sprayed with 70 mM sodium borate (pH=9.2) and subjected to electrophoresis (3 kV). Radioactivity was detected with a gas-flow scanner and the standard amino acids were visualized by spraying the plate with ninhydrin. The amount of label that co-migrated with each peak was determined by integration of the peaks after scanning. The values that are reported are t SE. The number in parentheses indicates the number of experiments. aOther refers to those compounds that did not co-migrate with glutamine or glutamate. Because of the number of experiments and the poor separation of the labeled compounds,the identity of these compounds was not established. F3 I I ' A A N2 010 ’2‘ glu l g 1.5- as 1 gln -l.5 : C“ 1.0 '- _e: 5. (DJS ‘3 C) Radioactivity lcpm 1: IO.3 N 1 .ppf 1.... l (3 £5 ll) 125 1213 Dietanceicm) Figure 30 Incorporation of Label into Organic Products in Soybean Nodules Exposed to [13NJN2 or 13NH: Soybean nodules were exposed to [13N]N2 (Panel A) or were incubated in H20 containing 13NH: (Panel B). After a 10 min exposure, the nodules were removed from the vessel containing the label and the labeled products were extracted with methanoleZO (80:20, v/v). After standard amino acids and allantoate were added to the extract, an aliquot was spotted on a thin-layer cellulose plate. The plate was sprayed with 70 mM sodium borate (pH=9.2) and subjected to electrophoresis (3 kV). The radioactivity on the plate was detected with a gas-flow scanner. The standard amino acids were visualized by spraying the plate with ninhydrin and allantoate by spraying with diaminobenzaldehyde. The arrows indicate the location of the standard amino acids and allantoate (aln) after electrophoresis. 151 with lesser amounts of label in aspartate, glutamate, glutamine and alanine (Figure 30, Panel A). DISCUSSION Glutamine was the principal product of NH: assimilation in alder nodules. This result plus the observation that the amide position accounted for 90% of the label in glutamine indicates that GS was the enzyme primarily responsible for the assimilation of NH: in alder nodules. A number of investigators (78,83,85) concluded that GS plays a similar role in a number of other systems including soybean nodules. The question of whether other enzymes, such as GDH, also assimilates NH: in nodules or in other tissues has not been answered. Methionine sulfoximine, a specific inhibitor of GS, inhibited 13 the incorporation of label from NH: into glutamine more than it did into glutamate and the other amino acids labeled in alder nodules. Similar results have been observed in tobacco cells (78) and rice roots (81) that were incubated with labeled NHZ. It has been assumed + 49 then MSX should inhibit the synthesis of glutamine and glutamate to the that if GS/GOGAT is the only pathway for the assimilation of NH same extent. To explain the discrepancy between this assumption and the results obtained with tobacco cells, Skokut 33 31. (78) suggested that either a small amount of the NH: was assimilated by GDH or that the activity of GS that remained in the presence of MSX permitted the synthesis of glutamine which could be preferentially used in the synthesis of glutamate. Although similar explanations may be put forth to explain the effect of MSX on the assimilation of labeled NH: in alder nodules, it is not necessary to invoke the assimilation of NH: by GDH. Instead there may be enough residual activity of GS in both alder nodules 152 153 and tobacco cells in the presence of MSX to provide glutamine for the activity of GOGAT to remain at a substantial percentage of its activity in the absence of MSX. This assumes that the activity of GS is greater than that of GOGAT in tobacco cells and alder nodules, as is the case in a number of other systems that have been examined (83,207,208). To illustrate, the relative enzyme rates may be assumed to be similar to those in soybean nodules where the in vitro 1 activities of GS and GOGAT are 89 and 10 nmolomg protein'o min"1 (83). If the activity of GS is inhibited 95% by MSX, then the residual activity could provide 4.5 nmol of glutamineomg protein-'Jmin"1 or enough glutamine for GOGAT to have 45% of the activity it had in the absence of MSX. Methionine sulfoximine, therefore, could end up inhibiting GS activity by 95%, but inhibit GOGAT activity by only 55%. The labeled glutamine would not accumulate in the presence of MSX, but instead be used in the synthesis of glutamate. While the differential inhibition of the incorporation of labeled NH: into glutamine and glutamate by MSX can be explained in terms of differences of jn_vitro enzyme activities, further experimentation is necessary to determine if differences in enzyme activities occur jg_vivo and if these differences are indeed responsible for the differences in the labeling patterns. Further, based on the results that are now available, the possibility that this differential labeling of glutamine and glutamate is the result of GDH assimilating NH: cannot be excluded. Differences in the distribution of label in glutamine and glutamate are observed in tissues that incorporate NH: (tobacco cells, (78); rice roots, (81) and soybean nodules and roots (Figures 29 and 30)) and the same tissues that incorporated [13N]N2 (soybean nodules, 154 Figure 30 and ref. 5) or labeled N05 (tobacco cells (78); rice roots (81); and soybean roots (Figure 29)). More labeled glutamate than glutamine was formed in tissues that were exposed to [13NJN2 or labeled N03, 13 + NH4. I propose that these differences may be the result of differences in whereas the reverse was true in tissues that were exposed to the relative rates of synthesis of labeled glutamine and glutamate from labeled NO and NH+. I assume that the rate of labeled NH: 3’ "2 4 production in the tissues that were exposed to [BNJN2 and labeled N03, and the rate of glutamate synthesis in those tissues exposed to exogenous labeled NH: is lower than the rate at which exogenous labeled NH: is incorporated. Although the data are not available for all the enzymes in all the tissues, these assumptions hold for soybean nodules, if the _i_n_ v_i_t;r_g activities of GS and GOGAT are reflective of their relative activities in 1119. The estimated rate of N2 reduction is 12 nmol NH: produced-g fresh weight of nodules"1-h"1 in soybeans. This value is based on the maximum rate of CZHZ reduction that was presented in this thesis (Figure 4), 124 nmol CZHZ reduced g dry weight"1 h", and assumes that the rate of N2 reduction equals the rate of CZHZ reduction divided by 4 and that l g dry weight is equal to 5 g fresh weight. The estimated potential rate for glutamine and glutamate synthesis is 108 and 12 umolog fresh weight of nodules-1 h']. These values are based on EM activities of GS and GOGAT (83). . According to this proposal, exogenous labeled NH: is rapidly incorporated in the nodules into glutamine by GS. This 1abe1ed glutamine may then be used in the synthesis of labeled glutamate. However, the synthesis of labeled glutamate is assumed to be much slower than that of labeled glutamine because of the relative '155 activities of GS and GOGAT. The result would be a high ratio of labeled glutamine to 1abe1ed glutamate. In experiments in which labeled NH: is provided exogenously, the synthesis of glutamine is proposed to be limited by the activity of GS. In contrast, the rate at which labeled glutamine can be synthesized from [IBNJN2 is limited by the rate of N2 fixation. If the rates of N2 fixation and glutamate synthesis are assumed to be 12 and 10 nmolog fresh weight of nodules’kvh’I, respectively, as discussed above, then the rates at which labeled glutamine and glutamate would be synthesized from [BNJN2 are the approximately the same. The amount of labeled glutamine would be about the same as that of labeled glutamate. The rate of glutamine synthesis in roots treated with labeled N03 may be limited by the rate of N0; reduction in a manner similar to that discussed above. The differences in the labeling patterns when different sources of radioactive nitrogen were used was not confined to differences in the labeling of glutamine and glutamate, Allantoate accounted for 48% of the radioactivity incorporated in soybean nodu1es that had been exposed to [13N]Nz, but appeared to account for only 8% 13 of the label incorporated in nodules incubated with NH: (Figure 30). These observations confirm the work of Ohayama and Kumazawa (144), who demonstrated the preferential incorporation of label from [15N1N2 into allantoate in soybean nodules, and Fujihara and Yamaguchi 15 (109), who demonstrated that NH: was primarily incorporated into amino acids rather than allantoate. The difference in the incorporation of label from 13NH: and [13N]N2 into allantoate may be explained by differences in the 156 distribution of enzymes within the nodule. The distribution of enzymes involved in the allantoate synthesis was examined in the nodules of cowpeas (Vigna unguiculata) (209), another plant that transports nitrogen from the nodules as allantoate. Those cells that contain bacteroids and are in the center of the nodule have higher levels of these enzymes than the cells that are on the outside of the nodule (209). Thus, when nodules are incubated with 13NHZ, e 13 th NH: may be assimilated in the cells on the outside of the nodule and never reach the cells that contain the enzymes necessary 13 for allantoate synthesis. In contrast, NH: produced during the reduction of [UNJN2 would be in close proximity to the site of allantoate synthesis and therefore, would be incorporated into allantoate. In contrast to nodules, labeled allantoate was not detected in roots incubated with 13NH: or 13 N03 indicating that the synthesis of allantoate occurs primarily in the nodule. This statement is consistent with previous results (12,144,193) demonstrating that allantoate is the major form of nitrogen transported when plants are dependent on the N2 that is reduced in the nodule. SUMMARY The major results presented in this thesis are summarized - below: 1) Carbon-l4 was incorporated in soybean nodules and roots and alder nodules exposed to 14C02. 2) Most of the label incorporated in soybean nodules during exposure to 14CO2 was rapidly lost from the nodules. 3) The major labeled compounds in nodules and roots exposed to 14CO2 were closely associated with the tricarboxylic acid cycle. 4) The source of nitrogen of the plants affected the distribution of the label incorporated in nodules and roots exposed to 14C02. 13 13 5) Plant tissue exposed to NOS, NH: or [BNJN2 had different distributions of labeled products. 6) After exposure to 14C02. the specific activities of glutamine was hiaher than that of glutamate in soybean nodules and in roots of sovbean plants treated with N03. 7) After incubation of soybean roots with 3H- and 14c-iabeied aspartate, most of the labeled asparagine was recovered from the medium surrounding the roots; the remainder was recovered from the roots. The 3WHO ratio of the asparagine recovered from the medium was the same as that of the added aspartate3, the asparagine recovered from the roots had a lower 3H/MC ratio than the added aspartate. Nodules and roots of soybean plants and nodules of alder plants have a system by which CO2 can be directly taken up and enter the 157 158 metabolism of the nodules and roots. This system is referred to as dark CO fixation. The rate of dark CO2 fixation in soybean 2 nodules varied from 30 to 120 nmol-mg dry weight of nodules-L h"1 and in roots from 10 to 120 nmol-mg dry weight of roots'1 h“. The rate of dark CO2 fixation in alder nodules was similar to O that in soybean nodules. Dark CO2 fixation in soybean nodules appeared to be involved primarily in the exchange of carbon rather than the net fixation of carbon. About 70% of the recently-fixed [14C] from 14CO2 was 12 lost from the nodules after a l h chase with C02. If this label was lost via the respiratory activities of the nodules, then net fixation of carbon via dark CO2 fixation is minimal. The products of dark CO2 fixation were primarily the organic acids and amino acids clbsely associated with the tricarboxylic acid . cycle: malate, citrate, aspartate, glutamate, asparagine and glutamine and in alder nodules, citrulline. This data together with other evidence suggests that one of the major enzymes catalyzing dark CO2 fixation is phosphoenolpyruvate carboxylase. Other enzymes such as malic enzyme or carbamylphosphate synthetase may also be involved. The relative distribution of label from 14 CO2 into the above products depended in part on the source of nitrogen of the plant. The major labeled amino acids in soybean nodules and roots dependent on N2 were aspartate and glutamate. The relatively low percentage of label in asparagine in these nodules and roots is consistent with asparagine adcounting for only a small proportion of the organic nitrogen transported from the root systems of plants dependent solely on N2. In contrast, asparagine was the major labeled amino acid in 159 roots of plants treated with No; as were asparagine and glutamine in roots of plants treated with NHZ. The increased synthesis of these amino acids, as indicated by the increased incorporation of 14C02, in roots of plants treated with N03 or with NH: label from was also reflected by asparagine and glutamine accounting for an increased percentage of the organic nitrogen in the xylem exudate of these plants. While the addition of N03 or NH: appeared to increase the synthesis of amino acids in roots, it reduced the rates of dark CO2 fixation and CZH2 redUction andithe concentration of amino acids in nodules. These results are consistent with the nodules of plants treated with N03 or NH: having been deprived of photosynthate and thus energy to maintain high rates of metabolic activities. The effect of N03 and NH: on amino.acid metabolism was also 13 explored using N-labeled tracers. Differences were observed in the labeling of glutamine and glutamate inseveral different tissues exposed to 13N-tracers. In those tissues that were exposed to 13NH: - alder nodules, soybean nodules and soybean roots- the ratio of [13N]glutamine to [13N]glutamate was higher than in the tissue exposed to 13NOS, soybean roots, and [13N]N2, spybean nodules. These results were explained by assuming that the rates of glutamine and glutamate synthesis depended on the source of nitrogen. The metabolism of glutamine and glutamate was also explored by . monitoring the incorporation of label from 14 14 CO2 into these amino acids. 'After exposure to C02, the specific activity of glutamine was higher than that of glutamate in roots of soybean plants treated with NO} and in soybean nodules. These results suggest that there were 160 two pools of glutamate, only one of which was used in the synthesis of glutamine. In another experiment, the pathway of asparagine synthesis was explored by incubating soybean roots with [2.3-3H]aspartate and [4-14CJaspartate. Over 90% of the labeled asparagine was recovered from.the medium surrounding the roots, the remainder was in the roots.‘ The asparagine in the medium had the same 3H/MC ratio as that of the added aspartate whereas the asparagine in the roots had a lower ratio. These results suggest that there may be two sites of asparagine synthesis in roots, one where aspartate is directly converted to asparagine that is transported out of the roots and a second where aspartate is converted to asparagine that inretained within the roots. 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