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N T HES‘S This is to certify that the dissertation entitled NITROGEN METABOLISM IN SOYBEANS: THE BIOSYNTHESIS OF UREIDES IN SEEDLINGS AND THE PARTITIONING OF N INTO VEGETATIVE AND REPRODUCTIVE TISSUE presented by Deborah Ann PoTayes has been accepted towards fulfillment of the requirements for Ph.D. degfieh, Biochemistry professor Date ézfl /‘/, [2&3 MSUis an Affirmative Action/Equal Opportunity Inxlilun'an 0-12771 PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE MSU Is An Affirmative Action/Equal Opportunity Institution chin: pins-9.1 NITROGEN METABOLISM IN SOYBEANS: THE BIOSYNTHESIS 0F UREIDES IN SEEDLINGS AND THE PARTITIONING OF N INTO VEGETATIVE AND REPRODUCTIVE TISSUE By Deborah Ann PoIayes A DISSERTATION Submitted to Michigan State University in partial fquiIIment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1983 It,“ "' Jl‘lk ABSTRACT NITROGEN METABOLISM IN SOYBEANS: THE BIOSYNTHESIS 0F UREIDES IN SEEDLINGS AND THE PARTITIONING OF N INTO VEGETATIVE AND REPRODUCTIVE TISSUES By Deborah Ann Polayes Two aspects of nitrogen metabolism in soybeans have been examined. The first has dealt with the partitioning of nitrogen derived from N2 fixation and N-lS-depleted nitrate or ammonium utilization into vegeta- tive and reproductive tissues. The rates of N2 fixation and the relative contribution of each nitrogen source to total nitrogen were determined by mass spectral analysis of the atom %15N of samples. The nodulated roots of soybeans grown on nitrate or ammonium were enriched in [14N] when compared to shoots, suggesting that recently-fixed nitrogen is not used directly for root and nodule development. The percentage of nitrogen derived from N2 fixation in the seeds paralleled the contribution of N2 fixation to total nitro- gen in the vegetative tissue. This similarity in the contribution of N2 fixation to total nitrogen of vegetative and reproductive tissues suggests that nitrogen loading into the pods is indiscriminate with respect to the original nitrogen source. Deborah Ann Polayes The production of ureides in the developing soybean seedling was also examined. Ureides are the major nitrogenous substances trans- ported in NZ-fixing soybeans. However, ureides were found to accumu- late in the cotyledons, roots and shoots of soybean seedlings independ- ent of both the symbiosis and supplemental nitrogen. The patterns of activity for uricase and allantoinase, enzymes involved in ureide syn- thesis, were positively correlated with the accumulation of ureides in roots and cotyledons. Allopurinol and azaserine inhibited ureide pro- duction in 3-day-old cotyledons while no inhibition was observed in the roots. Incubation of 4-day-old seedlings with [14C]serine showed that in the cotyledons ureides arose via g3 ggyg_synthesis of purines. The source of ureides in both 3 and 4-day-old roots was probably the cotyledons. The inhibition by allopurinol but not azaserine in 8-day-old cotyledons suggested that ureides in these older cotyledons arose via nucleotide breakdown. Incubation of 8-day-old plants with [14C]serine showed that the roots had acquired the capability to synthesize ureides via gg,ngyg_synthesis of purines. These data indi- cate that g3 DEER purine synthesis is involved in the production of ureides in young soybean seedlings. To Mom and Dad ii ACKNOWLEDGMENTS I would like to acknowledge Dr. Karel R. Schubert for his support throughout my graduate career. I would like to thank my committee members, old and new, for their help along the way: Drs. P. Filner, S. Ferguson-Miller, N.E. Tolbert, J. Tiedje, P. Fraker and J. Speck. Special thanks to Dr. Jim Tiedje for the use of his ratio-isotope mass spectrometer without which the first project would never have been completed. My friends within Biochemistry have made the stay at Michigan State enjoyable. Special thanks to Ellen Keitelman and Anita Klein for their friendship and long talks. To Carol Fenn no words could express my appreciation for her excellent typing ability; may she never have to type another superscript and subscript again. I would like to acknowledge the financial support from the Jesse Noyes Foundation, NSF and the Biochemistry Department, MSU. Finally, I would like to extend all my love and warmest thanks to Alan Christensen for his love, understanding and encouragement over the last four years. TABLE OF CONTENTS Page List of Tables . ....... . . . . . . . . . . . . . . . . . . vii List of Figures. . . . . . . . . . . . . . . . . . . . . . . . . . ix List Of Abbrev'iat‘ionSo . O O O O O O O O O O O O O O O O O O O O 0 Xi Introduction . . . . . . . ...... . ............ . l l_iterature Review. . . . ..................... 4 Biological N2 Fixation. . . . . . . . . . . . . . . ..... 4 Reduction of Nitrate and Dinitrogen: Enzymes and Energy Costs. . . . . . . . . . . . . . . . . . . . . . . . . . 5 The Effect of Nitrate and Ammonium on N2 Fixation . . . . . . 9 Nitrogen Metabolism . . . . . . . . . . . . . . . . . . . . . l3 Ammonium Assimilation. . . . . . . . . . . . ...... 13 Ureide Synthesis . . . . . . . . . . . . . ....... l5 Translocation and Utilization of Ureides . . . . . . . . 22 (2r1apter I. The Effects of Nitrate or Ammonium on N2 Fixation and Partitioning of N into Vegetative and Reproductive Tissues Introduction. . . . . . . . . ...... . . . ....... 25 Materials and Methods . . . . . . . . . . . . Materials. . . . . . . . . . . . . . . . Preparation of K14N03. . . . . . . . . . Growth of Rhizobium japonicum. . . . . . Growth of Plants for Mass Spectral Analys O O O O Nitrogenase Assay. . . . . . . . . Root Respiration . . . . . . . . . . Nitrate Determination. . ....... Total N Determination. . . . . . . . . . Ammonium Determination . . . . . . . . . Mass Spectral Analysis of Total N Samples. 0 O O O O 0 do. 0 O O M O O O O O O 0 O O O O 0 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O (A) 0 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Methodology for Measurements of N2 Fixation and Accumulation of Combined N by Mass Spectral Analysis 34 Effect of the Application of Ammonium on Early Seedling Development. . . . . . . . . . . . . . . . . . . . . 40 iv The Pattern of Dry Matter Accumulation in Vegetative Plant Parts. 0 O O O O O O O O O O O O O O O O O 0 Effect of Combined N on Nitrogenase Activity and Root Respiration. . . . ..... . . . . . . . . . . . The Contribution of Dinitrogen and Combined N to Total N Accumulation . . . . . . . . . . . . . . . Partitioning of N from N2 Fixation, Utilization of Combined N and NO§ Within Vegetative Tissue. . . . Partitioning of Nitrogen During Reproductive Phase of Growth . . . . . . . . . . . . . . . . . . . . . . DiSCUSSion. O O O 0 O O O O O O O O ......... O O 0 Chapter II. Studies on the Biosynthesis of Ureides in Developing Soybean Seedlings IntrOdUCtiono C O O O O O O 0 O O O O O O O O 0‘ O O O 0 O 0 Materials and Methods . . . . . . . . ...... . . . . . Materials. ...... . . . . . ........... Growth of Seedlings for Ureide Studies ........ Extraction of Plant Tissue for Ureide Determination. . Ureide Determination . . . . . . . . . . . . . . . . . Determination of Total N . . . . . . . . . . . . . . . Preparation of Extracts for Uricase and Allantoinase Assa says . . ..... . . . . ...... Handling of [2-14C]Uric Acid ........... . . Uricase Assay. . . . . . . . . . . . Allantoinase Assay . . . . . . . . . . . . . ..... Protein Determination. . . . . . . . . . Inhibition Studies . . . ............... Azaserine Treatment. . . . . . .......... Allopurinol Treatment. . . . . . . . . . . . . . . Determination of Xanthine Concentration ...... Labeling Studies . . . . . . . . . . . . . . . . . . . Incubation Conditions. . . . . . . ........ Extraction of Tissue . . . . . .......... Analysis of 14C-labeled Products ......... Results . . . . . . . . . . . . . . . . . . . . . . . . . . Total N and Dry Weight Levels. . . . . . . . . . . . . Ureide Determination on Control Plants and Plants Grown on l0 mM Nitrate . . . . . . . . . . . . . . . . . Patterns of Uricase and Allantoinase Activities During Page 42 46 56 59 63 7o 96 Seedling Development . . . . . . . . . . . . . . . . 101 Inhibition Studies . . . . . . . . . . . . . . . . . . . llZ Labeling Studies . . . . . . . . . . . . . . . . . . . . l16 Discussion. . .......... . ............. l30 Page Appendices Appendix I. Derivation of Equation Used for Mass Spectral Ana-'ySiS O O O O O O O O O O O O O O O O O O O O O O O O 138 Appendix II. Total N of Roots and Shoots as a Function of Nitrogen Source. . . . . . . . . . . . . . . . . . . . . I40 Appendix III. Amount of Stored N03 in the Roots and Shoots as a Function of Nitrogen Source. . . . . . . . . 141 References ........ . ...... . ........ . . . . I42 vi 2. 9. I0. 11. I2. 13. 14. 15. LIST OF TABLES Composition of Nitrogen-Free nutrient solution. . . . . . . Digestion of Ammonia Standards by a Semi-micro Kjeldahl Procedure 0 O O O O O O O O O O O O O O O O O O O O O O O 0 Conversion of Nitrate to Ammonium During Kjeldahl Digestion in the Presence of Plant Material . . . . . . . . . . . . . Comparison of Atom %15Nobs and Corrected Atom %15N. . . . . Ammonium Concentration in the Roots of 25-day-old Soybeans. Percentage of Total Dry Weight of Plant in Root Tissue. . . Dry Weight of Roots and Shoots as a Function of Nitrogen source. 0 O O O O O O O O O O O O O O O O 0 O O O O O O 0 0 Rates of C02 Evolution and CZHZ Reduction of Intact and Excised Nodulated Roots of Soybeans . . . . . ..... Estimates of Nitrogen Derived from N2 Fixation Calculated from CZHZ Reduction Data, Mass Spectral Analysis and Ureide Content of Xylem Exudate . . . . . . . . . . . . . . Percentage of Total N Derived from N2 Fixation in Roots and Shoots. . . . . . . . . . . . . . . . ......... Total N Contents for Vegetative and Reproductive Plant Parts at 69, 90 and 127 Days. . . . . . . . . . . . . . . . Percentage of Total Plant N Derived from N2 Fixation for Vegetative and Reproductive Tissue. . . . . . . . . . . . . Relationships Between Source of Nitrogen and Accumulation of Dry Matter and Nitrogen in Seeds . . . . . . . . . . . . Chromatography of Uric Acid, Allantoic Acid and Allantoin on PEI-Cellulose TLC Plates . . . . . . . . . . . . . . . . Chromatography of Purines, Ureides, and Serine on PEI-Cellulose F TLC Plates. . . . . . . . . . . . . . . . . vii Page 29 35 36 39 43 47 48 49 55 62 65 67 69 86 95 16. 17. I8. I9. 20. 21. 22. 23. A Comparison of Two Methods for Determining Uricase ACtiVityo O O O O O O O 0 O O O O O O O O O O O O O O O O 0 Inhibition of Total Ureide Accumulation by Allopurinol and Azaseri ne 0 O O O O O O O O O O O O O O O O O O O O O O O O Accumulation of Xanthine in Plants Treated with Allopurinol Decrease in Total Ureides for Plants Treated with Allopurinol . . . . . . . . . . . . . . . . . . .». . . . . Time Course of Labeling Cotyledons and Roots of 5- day- -old Soybeans with [14CJGlycine. . . . . . . . ..... . . . . Total Radioactivity in Roots, Shoots and 4Cotyledons of 4 and 8- day-old Soybeans Incubated with [14C]Serine . . . . . Distribution of Total Radioactivity in 4the Soluble Fraction of 4- -day- -old Seedlings Treated with [14CJSerine . . . . . . Distribution of Total Radioactivity in the Soluble Fraction of 8- -day- -old Seedlings Treated with [14CJSerine . . . . . . viii Page 104 II3 115 117 118 120 124 I28 LIST OF FIGURES Page I. Reaction sequence for the reduction of N2 by the nitrogenase complex . . . . . . . . . . . . . . ...... 7 2. Purine biosynthetic pathway ....... . . . ...... l6 3. The origin of the purine ring of inosine monOphosphate (IMP) O O O O O O O O O O O O O O O 0 O O O O O O O O O O O 17 4. The pathway of purine oxidation . . . . . . . . . . . . . . l8 5. Theoretical and experimental values for atom %15N . . . . . 38 6. Ammonium concentration in the roots of l4-day-old soybeans grown on nitrate and/or ammonium. . . . . . . . . . . . . . 41 7. Dry weights of nodules as a function of nitrogen source . . 45 8. Effect of combined N on nitrogenase actvity and root respiration O O O O O O O O O O O O O O O O O O O O O O O O 52 9. Estimates of nitrogen accumulated from N2 fixation calculated from CZHZ reduction data and total N ana1ys‘i50 O O O O O O O O O O 0 O O O O O O O O O O O O O O 54 ID. The contribution of N2 fixation to total N in nodulated soybean grown on combined N . . . . . . . . . . . . . . . . 58 11. The contribution of combined N to total N in nodulated soybeans grown on combined N. . . . . . . . . . . . . . . . 61 12. The percentage of total plant N present as N03 in roots and Shoots. O O O O O O O O O O O O O O O O O 0 O O O 64 13. A model for the transport of N within the soybean . . . . . 78 I4. Measurement of time delay between UV and radioactivity detectors 0 O O O O O O O O O O O O O O O O O 0 O O O O O 0 92 IS. Separation of purine standards by ion-paired, reverse-phase HPLCO O O O O O O 0 O O O O O O O O 0 O 0 O O O O O O O O O 94 I6. Values for dry weight of developing soybean seedlings . . . 98 ix I7. I8. 19. 20. 21. 22. 23. 24. 25. Total N levels in developing soybean seedlings. . . . . Total ureide concentrations in developing soybean seedlings ..... . .......... . ........ Specific activity of uricase in the cotyledons and roots of developing soybean seedlings. . . . . . . . . . . . . . . . Total uricase activity in the cotyledons and roots of developing soybean seedlings. . . . . . . . . . . . . . . . Total allantoinase activity in cotyledons and roots of developing soybean seedlings. . . . . . . . . . . . . . . . Standard curve for xanthine determination ......... The effect of allopurinol treatment on the distribution of label from [14C]serine within 4-day-old cotyledons ..... The effect of allopurinol treatment on the distribution of label from [14C]serine within 8-day-old-cotyledons. . . . . A model for synthesis and transport of ureides in 4 and 8-day-01d soybeans. O 0 O O O O O O O O O O O O O O O 0 O O Page ICC 103 107 I09 111 114 I22 I27 I37 AICAR asp atom %15N obs combined N FeMo-co Fe protein FGAR FH4 fr. wt. gln glu HPLC . mCi MoFe protein NR PEI-cellulose POPOP PPO PRPP PRPP synthetase RT TLC uE LIST OF ABBREVIATIONS 5-aminoimidazole-4-carboxamide ribotide aspartate atom percent 15N in sample nitrate and/or ammonium iron-molybdenum cofactor dinitrogenase reductase formylglycinamide ribotide tetrahydrofolic acid fresh weight glutamine glutamate high performance liquid chromatography millicurie dinitrogenase nitrate reductase polyethyleneimine-cellulose l,4-bisEZ-(4-methyl-5-phenyloxazolyl)J-benzene 2,5-diphenyloxazole phosphoribosylpyrophosphate ribose phosphate pyrophosphokinase retention time thin layer chromatography microEinstein xi INTRODUCTION A major limitation to plant growth is the availability of nitrogen (1,2). Normally, plants absorb nitrogen in the form of nitrate which is reduced and assimilated in the roots and/or leaves (3). Plants are also capable of utilizing ammonium; however, the levels of free ammoni— um in the soil are usually low due to nitrification and the binding of ammonium to soil particles (1). The search for ways to increase food production has led to the large scale application of nitrogen fertili- zer to agricultural fields. However, in recent years, the realization that the production of nitrogen fertilizers relies on the availability of an exhaustible energy supply has led to renewed interest in those plants (legumes) which are capable of utilizing N2 through the sym- biotic association with the bacterium, Rhizobium (4). Recent studies have indicated that a major limitation of N2 fix- ation is the availability of photosynthate (5-10). Photosynthesis sup- plies the energy (ATP and low potential electrons) necessary for nitro- genase activity and the carbon skeletons needed for ammonia assimila- tion (5). Estimates of the amount of photosynthate consumed by the nodule indicate that from l5 to 35% of the net photosynthate of the host may be consumed to support nodule activities (11,12). Due to the energy intensive nature of N2 fixation, a number of investigators have suggested that the yields of legumes grown in symbiotic associa- tion with Rhizobium may be reduced compared with legumes grown on abundant nitrate (6,8,13). The yields of legumes grown symbiotically have been compared with those of legumes grown on applied nitrate. In contrast to corn (14), the application of high levels of nitrate to nodulated legumes does not result in positive yield increases (13,15- 20). The ability of soybeans to utilize dinitrogen, nitrate and/or ammonium offers a unique opportunity to examine and compare N metabo- lism within a plant. A better understanding of N assimilation, trans- port, partitioning and remobilization within a soybean may aid in the determination of the factors which govern seed yield. The assimilation of dinitrogen, nitrate and/or ammonium leads to different organic nitrogenous compounds transported within soybeans (21). Soybeans grown in symbiotic association with Rhizobium transport recently fixed N in the form of the ureides, allantoin and allantoic acid (see the following reviews: 22,23). The growth of soybeans in the presence of nitrate or ammonium results in the transport of the amides, asparagine and glutamine (21-23). McNeil gt a1. (24) have suggested that the form of organic nitrogen transported within plants may serve as the basis for the selective partitioning and utilization of N. This hypothesis is addressed in Chapter 1. The relative contribu- tion of N derived from dinitrogen, nitrate and ammonium to total plant N and seed N was examined. The partitioning of N into vegetative and reproductive tissues was also investigated. We were particularly interested in whether the seeds of nodulated soybeans supplied with ni- trate or ammonium would selectively acquire N from one of the sources. The second aspect of my research has dealt with N metabolism in the developing soybean seedling. Prior to the onset of N2 fixation, legume seedlings appear to go through a period of N stress as a result of the depletion of seed N reserves (25,26). During this period, soy- bean seedling cotyledons have been reported to accumulate ureides (27). Although ureide production is primarily associated with symbiotic N2 fixation (see 28 for a review of this area), the soybean seedling appears to be capable of synthesizing ureides without the aid or pres- ence of the microsymbiont. Ureide synthesis within the nodule appears to arise via the biosynthesis and subsequent degradation of purines (29-33). The role of ureides in the vegetative and reproductive stages of growth of symbiotically-grown soybeans is well documented (21,35). Much less is known about the role of ureides in the nitrogen economy of developing soybean seedlings and in the pathways and sites of ureide biosynthesis. We were interested in determining whether the synthesis of ureides in the seedling was via a similar pathway as observed in the nodule. In the course of this research, the ability of developing soy- bean seedlings to utilize nitrate and the subsequent effect of nitrate application on ureide synthesis was examined. A better understanding of the metabolism of N in the soybean seedling is important in the determination of factors that affect the overall growth and development of a viable and healthy plant. LITERATURE REVIEW Biological N2 Fixation. . Dinitrogen is the most abundant compound in the atmosphere, however, few organisms are capable of utilizing this form of nitrogen for growth. Some prokaryotic organisms are capable of biological N2 fixation, the enzymatic process by which N2 is reduced to NHZ (1,35). These microorganisms can be divided into two groups, the free—living and the symbiotic Nz-fixing bacteria. One class of symbiotic Nz-fixing bacteria, Rhizobium, exist in nodules on the roots of legumes. This species-specific symbiotic association between rhizobia and legumes allows the plant to utilize N2 as the sole source of nitrogen for growth (4). The establishment of the legume/Rhizobium symbiosis involves a complex series of events. One of the first steps is the binding of bacteria to the root hairs (see 36 and 37 for a review). The bacteria induce curling of the root hairs and the formation of an infection thread. The rhizobia are carried within the infection thread as the thread branches and penetrates root cortical cells. Rhizobia are released from the tip of the infection thread into the cytoplasm of the plant cell and are surrounded by envelopes of host plasma membrane (peribacteroid membrane) (37). After being released from the infection thread, the bacteria differentiate into bacteroids, the symbiotic form of the bacterium. The nitrogenase complex, the enzyme that reduces N2 to NHX, is synthesized within the bacteroid. This enzyme is readily inacti- vated by 02 (38), however, the requirement of 02 for ATP generation in the bacteroids is quite high (39). The requirement for a low par- tial pressure of free dissolved 02 in the nodule as well as a high flux of 02 for bacteroid respiration has been accomplished via a mechanism involving leghemoglobin (40,41). Addition of purified leg- hemoglobin to dense bacteroid suspensions (l2% 02) greatly enhanced nitrogenase activity (42). A large flux of leghemoglobin-bound 02 through the cytosol supports the efficient formation of ATP in the bac- teroids (43). Leghemoglobin has a high affinity for 02 resulting in little free-dissolved 02 being present in the nodule (44). Leghemo- globin is produced through the combined efforts of both the bacteria and the host plant. The bacteria produce the heme moiety (45) while the plant synthesizes the globin apoprotein (46). Reduction of Nitrate and Dinitrogen: Enzymes and Energy Costs. The nitrogenase complex consists of two disassociating protein components (see 36,47 and 48 for a review). Both protein components are essential for activity. Dinitrogenase reductase (Fe protein) is a dimer of identical subunits and transfers electrons to dinitrogenase. Dinitrogenase (MoFe protein) which reduces substrate (N2 and H+) is a tetramer of two dissimilar subunits (a282) complexed with an FeMo cofactor (49). The FeMo cofactor (FeMo-co) consists of 8 non-heme irons and 6 acid labile sulfurs per molybdenum (49). Addition of FeMo-co to an inactive, molybdenum-free MoFe protein resulted in the production of an active MoFe protein, suggesting that the FeMo-co is essential for substrate reduction (50). A reaction sequence for the association of the two protein compon- ents and subsequent reduction of substrate presented in Figure 1 is modeled after Mortenson and Thorneley (38). The Fe protein is reduced by a ferredoxin or flavodoxin. The reduced Fe protein binds 2 MgATP which results in a conformational change necessary for the binding of the MoFe protein to occur. The hydrolysis of ATP results in the transfer of one low potential electron from the Fe protein to the MoFe protein (51). The reduced MoFe protein transfers an electron to the substrate. N2 appears to be reduced in steps that include enzyme- bound dinitrogen hydride intermediates (38). The energy requirements for N2 reduction by the nitrogenase com- plex are intensive (12,52). Under optimal conditions four molecules of ATP are hydrolyzed per electron pair transferred (47). The transfer of 6 electrons from the low potential reductant is also required (38). Thus, the overall reaction for N2 reduction can be written as: M2+ N2+12ATP+6e-+8H+—-g-—» 2NHZI+I2ADP+12P1~ The nitrogenase complex reduces a number of other substrates besides N2, i.e., N20 to N2 + H20, C2H2 to C2H4, N§ to NH3 + N2 and H+ to H2 (38). All these reductions require both protein compo- nents and the hydrolysis of ATP. The reduction of H+ to H2 occurs concomitantly with the reduction of N2 (12). The evolution of H2 by nitrogenase has been found in all Nz-fixing microorganisms examined (53)- The FEdUCtION 0f H+ to H2 appears to require the hydrolysis of 4 molecules of ATP: 2H++28'+4ATP—-—9H2+4ADP+4P1 .umccmemcmcp c_ma cocuum_m cog uo~xpocvxg ¢H< yo mwpzum_oe a men memgp .:_mpoca one: .mmmcmmocuwcwu ”cwmpocg we .ommuozumc mmmcmmoca_:_u mud .:_xouo>m_e Lo :wxoumecmu mxo .umeu_xo mum; .umozwmm .Ammv ampmccogh ecu :omcmpcoz soc» um_e_coe m_ Emcmmwo .xmpano wmmcmmocpw: mg» mg «2 4o =o_uo:uwc as» Low mocmacmm =o_uuomm "H «gnaw; :3: a a: a: 9.. a .... ........ 5223...: . NZ 4 4 Acute cwaooea mac: x0mao< mzN.=LmSOLa mag N Aumtv uaawwv Axov :wmpoca mmoz . umgmmh< mzN.:_wpoca emu N //1AHN- Axov um . mzzm #1 mgmcucamowoza Amxv aa< a: a For every N2 reduced a minimum of l H2 is evolved (54). Therefore, the overall reaction for the reduction of N2 by the nitrogenase com- plex can be rewritten: 142+ N2+I6ATP+8e‘+10H+——g—-> 2NH1+ H2+I6ADP+I6P1 Some Nz-fixing microorganisms have evolved an uptake hydrogenase which is capable of reoxidizing H2 to H+ (55-58). The presence of an uptake hydrogenase may enable the organism to recover reducing power which can be used to drive oxidative phosphorylation. This would result in increasing the efficiency of N2 fixation in both free- living and symbiotic Nz-fixing organisms (38,55,57,58). Comparison of the energy requirements for N2 fixation and nitrate reduction are difficult because N03 can be reduced in the roots and shoots (12,59). The reduction of N03 to NHK requires the action of two enzymes. Nitrate reductase (NR) reduces N03 to N02, while nitrite reductase (NiR) reduces N02 to NHI (see 60 for a review). The overall reaction for the reduction of N03 to NHI can be summarized as follows: N0§+l0 H++8e'—-)NHfi+3H20 The theoretical cost for N03 reduction is l2 ATP equivalents per NHK produced (60). This is based on the assumptions that l molecule of NAD is reduced per electron pair transferred and is equiva- lent to 3 molecules of ATP (52). Based on the similar theoretical assumptions, l4 ATP equivalents are required per NHX produced for N2 fixation (61). From these types of calculations, N2 fixation and nitrate reduction have similar theoretical energy requirements (61). However, nitrate reduction is viewed as a relatively energy free process if reduction occurs in the leaves, due to the direct utiliza- tion of photosynthetically produced reductant and ATP (5,8). Estimates of the energy costs for N0§ and N2 reduction have been made based on the assumption that the total C02 respiratory efflux from the roots and nodules reflects the overall energy costs (8,9). These types of respiratory studies have yielded conflicting results. Depend- ing on the legume system, N2 fixation either has similar (10,61) or greater energy requirements than N03 reduction (7-9,63). The Effect of Nitrate and Ammonium on N9 Fixation. The effect of combined N (ammonium and/or nitrate) on N2 fixa- tion is dependent on the time of application and the concentration applied (64). Low levels of combined N have been shown to stimulate N2 fixation (64,65). The addition of low levels of combined N to seedlings is believed to alleviate nitrogen stress, thus, leading to increased photosynthesis and levels of soluble sugars necessary for nodule development (64). Pea plants (26 day old) grown in the presence of 2 mM NHZ were found to have higher rates of N2 fixation and photosynthesis than plants solely dependent on N2 fixation for growth (65). Application of l mM N0§ to clover roots led to an increase in the relative levels of both trifoliin, a clover carbohydrate-binding protein, and rhizobial adsorption to clover root hairs (66). The stim- ulatory effect of low levels of N03 on bacterial attachment is not well understood. 10 High levels of combined N have been found to inhibit N2 fixation (18,21,65,67,68). If combined N is applied while the symbiosis is still developing, further development is altered (see Reviews, 36,37). The levels of trifoliin in developing clover roots appear to be regu- lated by combined N, however, the mechanism of this regulation is not known (66). If combined N is added after the establishment of N2 fixation, nitrogenase activity is inhibited and the senescence of nodules is induced (21,68). A 40% decrease in nitrogenase activity, as measured by CZHZ reduction, was found in peas 24 h after treatment with KNO3 (68). Similar results have been observed in soybeans treated with either N0§ or NH1 (64). Evidence suggests that the combined N source may compete with N2 fixation for photosynthate (64,69,70). Changes in the availability of photosynthate has been shown to alter the rates of N2 fixation (5,6, 71-73). Increasing the light intensity (6,71) or the C02 environment around legumes (5) results in higher rates of photosynthesis and N2 fixation. Upon removal of competing sinks (i.e., pods) a greater per- centage of the carbon transported was found in the roots and nodules, thus allowing N2 fixation to continue longer (72). Shading, defolia- tion and pod filling all result in a reduced level of photosynthate available for the nodule, therefore rates of N2 fixation are lower (5,73). Similar decreases in the rates of N2 fixation have been reported for legumes grown in the presence of combined N (69,70). Small and Leonard (69) have observed a decrease in the amount of photosynthate transported to the nodules of peas upon application of N03. A decrease in N2 fixation and eventual nodule senescence were also 11 reported (69). Latimore gt 31. (70) performed similar experiments with soybeans grown on N03 and NHX. Nodulated soybeans were incubated with 14C02 for IS min and harvested l8 h after expo- sure. Application of both N03 and NH1 significantly decreased the 14C accumulated in the nodules (70). A direct rela- tionship between photosynthate flow to the nodules and N2 fixation by soybeans was reported. However, there are data which would tend to disprove the hypothesis that competition for photosynthate is the mech- anism by which combined N inhibits N2 fixation (68). Chen and Philips (68) found that when N03 reduction occurs in the leaves, sufficient photosynthate reaches the nodules. Also, increasing the C02 environment around the plant did not delay nodule senescence in the presence of N03. Therefore, Chen and Philips concluded that either N03 or a product of N03 reduction was responsible for the inhibition of N2 fixation in the presence of N03. The metabolism of N03 appears to be necessary for the inhi- bition of N2 fixation to occur (74). Nitrate did not inhibit N2 fixation in free-living, Nz-fixing cultures of Rhizobium "cowpea" 32HI (75). Nitrate also had no effect on N2 fixation when soybeans were grown in the presence of tungstate, an inhibitor of nitrate reduc- tase (NR) (74). Harper and Nicholas (74) concluded that a product of N0§ reduction was responsible for the inhibitory effect of N0§. Recently, Streeter (76) has suggested that N02 is responsible for the inhibition of N2 fixation. Partially purified nitrogenases from A; vinelandii and_§; pastuerium were inhibited by nitrite but not nitrate (77). In free-living, Nz-fixing bacteria the synthesis of nitrogenase was inhibitedoin the presence of NH1 and 12 amino acids (78-81). However, these compounds do not appear to inhibit _in‘vitrg nitrogenase activity (79,82). In the symbiotic association, NHI was found to inhibit nitrogenase activity whereas the synthe- sis of the protein was unaffected (82,83). Houwaard (84) reported a 40% decrease in C2H2 reduction activity in the presence of NH1 while the amount of nitrogenase protein remained constant. The amount of leghemoglobin in nodules of plants treated with combined N was reduced (68,81). This suggested that combined N was affecting leghemoglobin synthesis which would result in the inactivation of nitrogenase. Nitrogenase activity in pea nodules was not changed by the addition of NHZ in the presence of methionine sulfoximine, an inhibitor of glutamine synthetase (83). Houwaard (83) concluded that the assimilation of NH1 was necessary for the inhibition of N2 fixation. Others have suggested that the inhibition of N2 fixation in the presence of N03 is due to the production of nitrite (84). Nitrite is a potent inhibitor of nitrogenase activity (85). Origi- nally, the NR within the bacteroid was believed to be responsible for the reduction of N03 to N02. However, Gibson and Pagan (86) and Manhart and Wong (87) demonstrated that nodules containing NR-deficient mutants of Rhizobium were still inhibited by NO§. Recently, Streeter (84) proposed that a plant cytosol NR converts N03 to NO§ and the N02 produced by this enzyme is responsible for the inhibition of N2 fixation. Direct evidence for the inhibition of N2 fixation by N02 derived from the reduction of N03 in the nodule has not been shown. 13 Nitrogen Metabolism. Ammonium Assimilation. Ammonia, the first stable product of N2 fixation (88,89), diffuses out of the bacteroids into the plant cytoplasm where it is assimilated (90). Results from early studies using 15N2 showed that glutamate was the first amino acid formed (90,91). The formation of glutamate was concluded (92) to result from the assimilation of ammonia by glutamate dehydrogenase (GDH): GDH NH3 + 2-oxoglutarate + NAD(P)H---+’glutamate + NAD(P)+ Because of the low affinity for NHfi associated with glutamate de- hydrogenase, Miflin and Lea (98) concluded that this is not the primary pathway for NHX assimilation. The nodule also appears to contain two other enzymes involved in ammonia assimilation (93-95). Glutamine synthetase (GS) catalyzes the addition of NH1 to glutamate to form glutamine: GS Glutamate + NHX + ATP —————e~glutamine + ADP + Pi The second enzyme, glutamate synthase (GOGAT), catalyzes the transfer of the amido-N of glutamine to 2-oxoglutarate with the formation of 2 glutamates: , GOGAT + Glutam1ne + NADH + 2-oxoglutarate---4 2 glutamate + NAD Recently, short-term labeling studies with 13N2 (96) and 15N2 (97) have shown that glutamine is the first organic product of N2 fixation. Label was also recovered in glutamate (96). The appearance of 13N in glutamate was directly correlated with the decline in the 14 proportion of 13N in glutamine (96). These data support the con- clusion of Miflin and Lea (98) that the coupled activity of glutamine synthetase and glutamate synthase constitutes the major pathway for ammonia assimilation. Although glutamine, the initial organic product of N2 fixation, may be exported from the nodule, it is rarely the major nitrogenous constituent transported to the shoot via the xylem (22). Analysis of the xylem exudate from nodulated roots of legumes has led to the iden- tification of the forms of N transported from the nodule (22,99). Legumes which are fixing N2 can be divided into two classes: those that transport amides and those that transport ureides. In legumes which transport amides (i.e., lupins, peas and broad beans), the major- ity of the N is transported in the form of asparagine (100,101). Some glutamine or substituted amides such as X-methylene glutamine are also transported (22). In Lupinus albus asparagine accounts for 60-80% of the xylem nitrogen throughout plant development (101). The ureides, allantoin and allantoic acid, represent 60-99% of the N transported in the xylem exudate of nodulated COWpeas (94,124) and soybeans (21). Studies in which 15N2 was supplied to the root system of nodulated soybeans (102-104) or cowpeas (94) have shown rapid labeling of ureides in the nodules and xylem exudate. In detached nodules, allantoin and allantoic acid are readily labeled from 15N2 (97). High levels of ureides in the xylem exudate are posi- tively correlated with effective nodulation and high rates of N2 fix- ation (21,105). Application of nitrate or ammonium to nodulated soy- bean roots resulted in a decrease in the level of ureides transported (21,34,106). Soybeans grown on nitrate transport N predominantly in 15 the form of amides and free nitrate (21,34). The inhibitory effect of nitrate on N2 fixation (18,21,65,67,68) and the concomitant decrease in ureide production further supports the conclusion that the nodules are the site of ureide production (102-105). Ureide Synthesis. The pathway for ureide synthesis in the nodule is only now being elucidated (29-33,l07-l09). An early hypothesis (110) that allantoic acid arose via the condensation of urea and glyox- ylate has not been substantiated experimentally (111,112). There is mounting evidence that ureides arise from the biosynthesis and subse- quent degradation of purines (29-33,107-109). Enzymological and [14CJ-labeling studies with embryonic axes of soybeans (ll3-ll5), wheat seeds (112) and tobacco protoplasts (116) have shown that the pathway for g; EQXQ purine synthesis in plants is similar to that dis- covered in other organisms (117,118). I The formation of the purine ring requires glycine, two amide groups from glutamine, the amino group from aspartate and two activated C1 groups (Figure 2,3). The first committed step of purine synthesis is the transfer of the amido-N of glutamine to phosphoribosylperphos- phate (PRPP). This reaction is catalyzed by the enzyme, phosphoribosyl amidotransferase (118). In the next step glycine reacts with 5-P-ribo- sylamine in the presence of ATP to form glycine amide-S-ribotide (118). This compound is further metabolized to IMP, the branch point for adenine and guanine nucleotide synthesis (ll8). The origin of the purine ring of IMP is shown in Figure 3. The conversion of IMP to ureides may occur by one of 2 pathways (Figure 4, see 22,ll9 for reviews). The conventional pathway results in the formation of inosine, hypoxanthine, xanthine, uric acid and ureides. Alternatively, ribose-5- 13>? AMP swea- (Q —‘ : TP RPP synthetase gln phosphoribosyl amidotransferase 16 5-P-ribosylamine ADP< To (:> gln glu + ADP < ATP ADP Jrr"C02 ii fumarate + ADP ‘T—“T IMP Figure 2. Purine biosynthetic pathway. eg + ATP N5N10-methenyl FH4 + ATP sp + ATP Nlo-formyl FH4 Ribosephosphate pyrophosphokinase (PRPP synthetase), the first enzyme of purine synthesis, catalyzes the formation of phosphoribosyl pyrophosphate (PRPP). The abbreviations used: gln, glutamine; glu, glutamate; FH4, tetrahydrofolic acid; gly, glycine; asp, aspartate; IMP, inosine monophosphate. glutamine, (X). l. Azaserine inhibits the transfer of the amido-N from 17 HN I O 9\7/\ a/\§/ ribose P ~ HC\ 2 \O Fi ure 3. The origin of the purine ring of inosine monophOSphate (IMP) Numbers shown give origin of N and C based on a numbered ring. Th8 origin of the C and N of IMP are as follows: N-l, aspartate; C-2, -formyl FH4; N-3 and N-9, glutamine; C-4, C-5 and N-7, glycine; C-6, C02; and C-8, N ,NlO-methenyl FH4. 18 IMP a XMP inosine l 1 xanthosine hypoxanthine b NV xanthine b .L uric acid c allantoin d allantoic acid Figure 4. The pathway of purine oxidation. IMP dehydrogenase (a) catalyzes the oxidation of IMP to form XMP. Xanthine dehydrogenase (b) is an NAD requiring enzyme. Uricase (c) catalyzes the formation of allantoin. Allantoinase (d) catalyzes the formation of allantoic acid. 19 IMP may be oxidized to XMP with subsequent hydrolysis to xanthine. Xanthine and hypoxanthine also serve as intermediates in the synthesis of secondary purine derivatives in plants (120,122). The addition of [14C]xanthine or hypoxanthine to tea leaves results in the forma- tion of caffeine as well as the formation of allantoin and allantoic acid (122). The pathway for ureide synthesis in nodules has been elucidated by the identification and localization of enzymes involved in purine metabolism (94,123-125) and bY.lfl vivo and in vitro labeling studies (29-33). The enzymes for purine oxidation have been localized in the plant cytoplasm (125). The nodules of ureide-producing legumes contain significant levels of the enzymes of purine oxidation: xanthine dehydrogenase (125), uricase (123) and allantoinase (123). The nodules of legumes which transport amides were found to contain negligible enzymatic activity for purine oxidation (101). The subcellular locali- zation of the enzymes of purine oxidation has also been reported (123). Xanthine dehydrogenase was found to be a soluble enzyme while uricase was localized in the peroxisomes (123). The compartmentation of allan- toinase was found to be different between plants and animals (123,126, 127). Allantoinase is associated with the endoplasmic reticulum in soybean nodules (123) while in fish liver the enzyme was found in the soluble fraction (127). The association of plant allantoinase with the endoplasmic reticulum has been suggested to play a role in the trans- port of allantoic acid from the nodule (123,127). Allopurinol, an inhibitor of xanthine dehydrogenase, inhibited ureide formation in the nodules of cowpeas and soybeans (29,128). Addition of allopurinol to the nodulated roots of intact cowpeas 20 resulted in a decrease in the level of ureides in the xylem exudate and an accumulation of xanthine in the nodule (29). Both 14C-labeled and unlabeled XMP and IMP were readily metabolized to ureides by cell- free extracts of ureide-producing nodules (30,125). The rate of ureide synthesis via purine oxidation in these studies (30,125) is comparable to those observed for N2 fixation in vivg (107). Synthesis of ureides from [14C]hypoxanthine or [14C]guanine has also been demonstrated using cell-free extracts of cowpea nodules (31). The presence of purine oxidation enzymes in the nodule, the inhibitory effect of allopurinol on ureide production and the metabolism of XMP and IMP to ureides are all indicative of purine oxidation being directly linked to ureide synthesis. The presence of the first two enzymes involved in gg.ggvg purine synthesis has been demonstrated in soybean nodules (124,129). The activities of PRPP synthetase and phosphoribosyl amidotransferase have been positively correlated with N2 fixation (124,129). The levels of activity of these enzymes are greater in soybean nodules than in nod- ules of legumes which transport amides suggesting these enzymes are involved in ureide synthesis (22). Incubation of nodule slices from cowpea with [14Cngycine resulted in the incorporation of label into allantoin and allantoic acid (29). This incorporation suggested the involvement of phosphoribosyl glycinamide synthetase, the third enzyme of purine synthesis (29). The presence of phosphoribosylamino- imidazole carboxylase, the enzyme that catalyzes the addition of 002 to form C5 of the purine ring, has been demonstrated in soybean nod- ules (33). The preferential incorporation and accumulation of 14C from 14C02 into C5 of xanthine has been reported (33). Further 21 studies with cell-free extracts from cowpea nodules have shown the incorporation of label from [14Cngycine into FGAR, AICAR and IMP (32). These data are all indicative of a gg_ngvg purine synthetic pathway in ureide-producing nodules. The subcellular localization of some enzymes involved with gg.ggvg purine synthesis has been reported (109). PRPP synthetase was found in the soluble fraction of the plant cytosol while phosphoribosyl amido- transferase was localized in the proplastid fraction. The enzymes phosphoglycerate dehydrogenase, serine hydroxymethylase, and methylene FH4 (tetrahydrofolic acid) dehydrogenase, involved in the production of glycine and N5,N10-methenyl FH4 have also been found in the proplastid fraction of soybean nodules (109). Incubation of a proplas— tid-containing fraction with [14C]glycine resulted in the incorpor- ation of label into IMP (108). Upon incubation of proplastids with [14C]serine and NADP+, label was recovered in XMP (108). These data are consistent with the suggestion that purine synthesis in the nodule occurs in the proplastid (108). The compartmentation of ureide synthesis in the nodule is further complicated by the fact that soybean nodules contain two different cell types: the larger, bacteroid-containing cells (infected cells) and the smaller uninfected cells. Newcomb and Tandon (130) observed an enlargement of peroxisomes and an increase in the amount of smooth endoplasmic reticulum in the uninfected cells of soybean nodules. They suggested (130) that the uninfected cells may play a role in ureide synthesis. Hanks gt El. (131) have reported the localization of uri- case and allantoinase in the uninfected cells of soybean nodules. They concluded that purine oxidation was the exclusive function of the 22 uninfected cells (131). The localization of purine synthesis in the various cell types has not been accomplished. Further work is neces- sary to identify the cellular site of purine synthesis (either infected or uninfected cells) as well as the metabolite(s) transported between the two types of cells in nodules. Translocation and Utilization of Ureides. The fully expanded leaves are the main sink for ureides transported in the xylem stream of soybeans and cowpeas (22). A significant amount of ureides has also been found in the stems of these plants (22,27,94). A detailed analy- sis of the transport of ureides into fruits, roots and developing leaves has not been possible. TranSport of ureides into roots and fruits is probably via the phloem and not the xylem (94). The amount of a particular substrate which is directly transferred to the phloem for redistribution within the plant can only be obtained by collection of the phloem sap (22). The inability to recover phloem sap from cow- peas and soybeans has hindered these types of analyses. The transfer of [14C] or [15N] substrates to the phloem of lupins, an amide-transporting legume which bleeds from the phloem, has been examined (24,98,132). The partitioning and metabolism of differ- ent products of N2 fixation by the shoots of lupins has also been analyzed by these types of labeling studies (22). Asparagine is rapid- ly transferred in an unmetabolized form to the fruit of lupins (132). The labeled aspargine which remains in the leaf is also unchanged (24). Glutamine, glutamate and aspartate, however, are readily metabo- lized in the leaves (24). Aspartate and glutamate appear to be the nitrogen source for the mesophyll cells of leaves (24). Asparagine and 23 glutamine are important in the general transport of N and C to meri- stems and fruits in lupins (24). The enzymatic pathway for the metabolism of allantoic acid to a N form readily assimilated into amino acids and protein has not been elu- cidated. Allantoicase activity, the enzyme which catalyzes the hydrol- ysis of allantoic acid to glyoxylate and two ureas, has been observed in the cotyledons of germinating peanuts (133). The presence of in v_i_t_rc_) allantoicase activity in other legumes has not been reported. Incubation of soybeans seedlings with [14CJallantoin resulted in the release of 14002 (127). Although this suggested allantoin was readily metabolized in the seedling, no in vitro enzymatic activity could be detected (127). Recently, the metabolism of ureides in cowpeas has been examined using allantoin labeled with 14C and 15N (134). Extensive metabolism of allantoin by vegetative and reproductive tissues of nodu- lated cowpeas and soybeans was observed (24). Amino acids were found to be differentially labeled with 15N as opposed to 14C sug- gesting that ureide-N was important in the pathway of N assimilation in shoots (24). The translocation of [14C]allantoin within the phloem was examined in cowpeas and soybeans using aphids (134). The aphid stylet was found to be located in the phloem cells, therefore the labeled compounds recovered in aphid extracts were assumed to arise from phloem (134). Following application of [14C]allantoin to leaflets, [14C] ureides were found to represent 27% of the total radioactivity in extracts from aphids feeding on fruits (134). A large proportion of the label recovered from aphids was in compounds other than allantoin suggesting extensive metabolism of ureides in the leaves 24 prior to phloem transport (134). Ureolytic activity in the extracts of cowpea and soybean leaves was demonstrated (134). However, the enzymes and the intermediates involved in this metabolism have not been elucidated. CHAPTER I The Effects of Nitrate or Ammonium on N2 Fixation and Partitioning of N into Vegetative and Reproductive Tissues INTRODUCTION The effects of the application of combined N (nitrate and/or ammonium) on the rates of N2 fixation, the pattern of dry matter and total N accumulation, and seed yields of Nz-fixing plants have been examined (3-8). Unfortunately, in many of these studies the rates of N2 fixation were estimated indirectly using the rates of acetylene reduction which is quantitatively inadequate (8,15-18,l35). In addi- tion, the concentration of combined N was continuously declining or only provided at specific and limited periods during growth. These experimental protocols do not allow for the examination of the contri- butions of dinitrogen and combined N to total N or the partitioning of N from each source between the various plant parts. The assimilation of N from combined N or dinitrogen leads to different forms of N trans- ported within the plant (2l). McNeil gt 31. have suggested that the form of organic N transported within plants may serve as the basis for the selective partitioning and utilization of N in the shoots (24). In order to study the partitioning of N, natural abundance N2 and N-l5-depleted potassium nitrate and ammonium sulfate were used. The use of N-lS-depleted sources of combined N has been shown to offer an accurate and inexpensive means of analyzing the relative contribu- tions of multiple N sources to total plant N (136). The development of the methodology used in the analysis of N-lS-depleted nitrogen samples, the contribution of each N source to total plant N and seed N, and the 25 26 partitioning of N into vegetative and reproductive tissue of soybeans are discussed in this Chapter. MATERIALS AND METHODS Materials 14NH414N03 and (14NH4) (0.01 atom %15N) were acquired through a grant from the Stable Isotope Committee, Los Alamos Scientific Laboratory, NM. Rhizobium japonicum strain 311b ll0 was obtained as a gift from D. Weber, USDA, Beltsville, MD. Soybean seeds (Glycine max [L] Merr. cv. Amsoy 7l) were purchased from the Michigan Seed Foundation. Bromine was purchased from Fisher Scientific, Pittsburgh, PA. CaCz (technical grade) was from Sargent Nelch, Skokie, Ill. Porapak N was obtained from Waters Associates, Milford, MA. Yeast extract was purchased from Difco Laboratory, Detroit, MI. All other chemicals were reagent grade. Methods Preparation of K14NQ3. KN03 depleted of 15N was prepared from 14NH414N03 by evaporating to dryness a l M solution of 14NH414N03 in a rotary evaporator after addition of 0.l volume of l0 N KOH. The dried K14N03 was redissolved in distilled water and redried until the levels of NH4+ remaining were not detectable (<20 nmoles/ml) (l37). The NH4+ distilling over was trapped in lN H2804. The K14N03 solution 27 28 was dried, resuspended in cold (4°C) 95% ethanole20 (v/v), and allowed to recrystallize at 4°C for 10 h. The crystals were filtered on Whatman no. 1 filter paper and dried at 60°C for 48 h. Growth of Rhizobium japonicum. A liquid culture of yeast mannitol broth was inoculated with Rhizobium japonicum strain 3Ilb ll0. Yeast mannitol broth consists of 0.02 g KH2P04, 0.03 g KzHP04, 0.02 g MgSO4'7H20, 0.0l 9 NaCl, 10 g mannitol and 29 Difco yeast extract per liter of redistilled water. Cultures were placed on a Labline rotary shaker at 28°C. Growth of Plants for Mass Spectral Analysis: Experiment l. Soybean seeds were inoculated with a liquid culture of Rhizobium iaponicum strain 3Ilb ll0 or soaked in redistilled water (uninoculated) and planted in 20-cm plastic pots (l2 seeds per pot) containing Perlite. Plants were thinned to 8 plants after 2 weeks. The inoculated plants were maintained on N-free nutrient solution (Ref. No. 138, Table l). Uninoculated plants were maintained on N-free nutrient solution supplemented with l0 mM K14N03. The nutrient solution was prepared with redistilled water which contained less than 0.4 ppm nitrate. The greenhouse was maintained on a photoperiod of l6 h light at 26°C and 8 h dark at 20°C. Illumination at pot level was 200 uE'm‘z's‘l. Plants were harvested at 7 day intervals beginning at day l3 after planting and continuing until the onset of flowering (day 41). Three pots from each treatment were removed at every harvest. The remaining pots were randomized within the block. Harvested plants were separated 29 Table l: Composition of Nitrogen-Free Nutrient Solution Compound Amount per 50 litera I. Macronutrients K2504 I4 9 MgSO '7H20 24 g KHZP 4 7 9 CaSO4°2H20 50 g 1.25M CaClz 20 ml II. Micronutrients H3303 750 mg/I MnSO4°4H20 750 mg/l ZnSO4°7H20 I50 mg/I 25 ml CuSO4°5H20 40 mg/l Na2M004-2H20 20 mg/l CoClz 300 mg/l 8 ml Ferric'EDDHAb 4 g/l so ml pH = 605 - 607 (a) Nutrient solution was made up with redistilled water. (b) Fe EDDHA is technical grade sodium ferric ethylene diamine di(o-hydroxyphenyl acetate) which was purchased from AH Hummert, St. Louis, MO. 30 into nodulated roots, and stems and leaves. Plant material was dried at 60°C for 72 h and then ground using a portable Wiley Mill (20-mesh screen). Experiment 2. Soybean seeds were inoculated with a liquid culture of Rhizobium japonicum strain 3Ilb ll0 and planted in 20-cm plastic pots (8 plants per pot) containing Perlite. Plants were thinned to 5 plants per pot after 4 weeks. The plants were grown in a greenhouse with supplemental fluorescent lighting (200 uE m'zs‘1 at pot level). A photoperiod of l6 h light was maintained until day 50 at which time the photoperiod was decreased to l4 h to stimUlate flowering. Plants in all treatments were flowering by day 55 and had pods by day 69. Nitrogen-free nutrient solution (Ref. No. 138, Table l) was prepared with redistilled water and applied to plants for the first 5 days after planting. After day 5, approximately l liter of N-free nutrient solution or N-free nutrient solution supplemented with 2,4 or l0 mM K14N03 or 2 or 4 mM (14NH4)2804 was applied daily to plants. Pots were flushed with distilled water at 9:00 p.m. the night before each harvest and on weekends. The pH of the nutrient solution flushed from pots was between 6 and 6.5. Three pots per treatment were harvested at l3,20,27,34,4l,55,69,90 and l27 days after planting. Plant tissue from each replicate was divided into stems and leaves, nodulated roots, and pods. Samples were dried at 60°C for 72 h, weighed, and ground using a portable Wiley Mill (20-mesh screen). Nitrogenase Assay. Nitrogenase activity was estimated by measur- ing the reduction of acetylene to ethylene (73). The roots (3-4) were excised from plants and placed in 250-ml Erlenmeyer flasks. The flasks 31 were sealed with a rubber stopper. Air (0.l vol) was removed and re- placed with acetylene (generated from CaCz and water). Samples (0.5 ml) were removed at 5, l5 and 30 min after addition of acetylene. Gas samples were analyzed on a gas chromatograph (Varian-3700) equipped with a flame ionization detector and a 2 m column containing Porapak N. Helium was used as the carrier gas at a flow rate of 30 ml/min. The column temperature was maintained at 60°C, the injector port at l00°C and the detector at l20°C. Upon completion of the assay, nodules were removed from roots and both roots and nodules were dried at 60°C for 72 h. Root Respiration. Root respiration was determined by measuring the amount of C02 produced over 30 min (7). Plants were prepared as described for nitrogenase activity. Gas samples (0.5 ml) were removed at 5, 15 and 30 min and analyzed for C02 by gas chromatography using a thermal conductivity detector. The detector temperature was l00°C. All other conditions were as described for nitrogenase activity. Nitrate Determination. Dried plant tissue (l00 mg) was resus- pended in l0 ml of glass distilled water and incubated for l h at 45°C. Samples were centrifuged at l2,000 x g for l0 min. The procedure for the determination of nitrate from dried plant material was that de- scribed by Cataldo §t_gl. (139) with the following modification: The supernatant fluid (0.l ml) was analyzed for nitrate by the addition of 0.4 ml of 0.5% salicylic acid:36N H2304. After 20 min at 20°C, 4.5 ml of 5 N NaOH was added. Samples were cooled to room temperature and the absorbance was measured at 4l0 nm. Duplicate determinations were made for each replicate. The assay was linear between 5 to 50 ug of ' nitrate. 32 Total N Determination. Total N was determined by a semi-micro Kjeldahl procedure (140). Two samples of dried plant material (0.2-0.3 g) from each replicate were digested in 4 ml of digestion so- lution (0.7 M Na2304 and l2.7 mM selenium in 36 N H2304) for 6 h at 300°C. After cooling samples were diluted to l0 ml with glass distilled water and an aliquot was removed for ammonium determination (l37). Ammonium Determination. The amount of ammonium in the diluted di- gestion fluids was determined by the method of McCullough (l37). An aliquot (l ml) of the digested samples was mixed vigorously with l ml of reagent A (l0 9 phenol and 50 mg sodium nitroprusside per l liter of H20) and l ml of reagent 8 (5 g NaOH, 53.7 g NaHP04°l2H20 and 20 ml of commercial bleach in l liter of H20). Samples were incu- bated at 70°C for 5 min. The incubation mixture was cooled to room temperature and the absorbance was measured at 625 nm. The assay was linear between 0-200 nmoles NH4Cl with l00 nmoles NH4Cl giving an absorbance of 0.680 : 0.0l0. Mass Spectral Analysis of Total N Samples. The ammonia in the samples digested by the semi-micro Kjeldahl procedure was distilled into Erlenmeyer flasks containing 4 ml of 0.l N HCl (140). Samples were evaporated to near dryness, transferred to vials (l5 x 45 mm) and evaporated until dry. The vial was attached to a VG Micromass MM 622 Isotope Ratio Mass Spectrometer and evacuated by the use of the auxil- iary pumps attached to the mass spectrometer. The NH4Cl was con- verted to N2 with the dr0pwise addition of a solution of lithium hypobromite (6 g LiOH in 60 ml H20 (0°-5°C), and 2 ml Brz) (140). The sample was frozen with liquid N2 and allowed to diffuse into the 33 mass spectrometer. The atom percent 15W in the sample (% 15Nobs) was determined from the ratio of peak mass 28 to peak mass 29. The equation used to calculate the amount of N derived from N2 fixation is given in the Appendix I. RESULTS Methodology for Measurements of N9 Fixation and Accumulation of Combined N by Mass Spectral Analysis. A semi-micro Kjeldahl procedure was used for the preparation of samples for mass spectral analysis. In the absence of plant material, this procedure resulted in no conver- sion of nitrate to ammonium during digestion (Table 2). From 93 to 98% recovery of the added NH4Cl was observed after digestion for 6 h (Table 2). Up to 25 mg of nitrate was completely converted in the presence of 300 mg plant material (Table 3). With concentrations of nitrate greater than 25 mg per 300 mg of plant material only a fraction of the nitrate was converted. The maximum amount of nitrate in the plants sampled was 15 mg nitrate-(300 mg roots)"1 and 7 mg nitrate- (300 mg shoots)‘1. Thus, it was assumed that all the nitrate in the samples analyzed was converted to ammonium. The information gathered from mass spectral analysis of nitrogen samples was in the form of atom % 15N (%15Nobs). The %15Nobs reflected the relative contribution of seed N, N2 fixation, and utilization of combined N to total N. The relative abundance of 15N for N2 and seed N was taken to be equal to natural abundance (0°366 atom %15N). The atom %15N for KN03 and (NH4)ZSO4 was 0.0l0 atom %15N. This is the stated value given by Los Alamos Scientific Laboratory and was ver- ified experimentally. Therefore, the assimilation of N from the com- bined N source led to'a decrease in the %15Nobs. The theoretical 34 35 Table 2: Digestion of Ammonium Standards by a Semi-micro Kjeldahl Procedure. NH Cl added KNO added % recovery of NH1 Inmol) (m9) loo 0 93 x 4 I00 60 93 1'4 200 O 98 t 2 200 60 98 i 2 Standards were digested in 4 ml of Kjeldahl digestion solution for 6 h (see Materials and Methods). Amount of ammonium remaining after digestion was determined by the method of McCullough (137). Values presented are the mean of 3 replicates 1 standard error. 36 Table 3: Conversion of Nitrate to Ammonium during Kjeldahl Digestion in the Presence of Plant Material. KNO added % KNO3 converted (mg) 5 l00 l0 l00 l5 95 25 96 50 74 l00 43 250 l6 Nitrate was added to 300 mg plant material. Digestion solution (4 ml) was added and samples were digested for 6 h. Each sample contained l02 nmoles MHz-300 mg plant material‘1 (l7.8 ug NHI’ g piant-l). The % nitrate converted to ammonium was determined by measuring the amount of NH1 in each sample after digestion (137). Each value is the mean of 2 replicates. The standard error was 2 2%. 37 curve of atom %15N versus total N was calculated assuming all the N accumulated was derived from the combined N source (Figure 5). There- fore, for the theoretical line generated the %15Nobs only reflected the relative contribution of seed N and combined N to total N. The equation used to calculate the theoretical lines was as follows: %15Nobs = Seed N (0.366 atom %15N) + combined N(0.0l0 atom %15N) Total N The experimental values of atom %15N were obtained from uninocu- lated soybeans grown in the presence of l0 mM KN03 (0.010 atom %15N). Examination of the 2 curves showed there was agreement between the theoretical and experimental values of %15Nobs (Figure 5). In order to obtain an accurate estimate of the N derived from N2 fixation and utilization of combined N, the %15Nobs was corrected for the relative contribution of seed N according to the following equation: corrected atom %15N = (total N)(atom %15Nobs) - (seed N)(0.366 atom %15N) Total N - Seed N The correction of atom %15Nobs did not alter the value observed for nodulated plants grown on N-free nutrient solution, control (Table 4). At earlier harvests, a substantial correction in the atom %15Nobs due to the presence of seed N was observed for plants grown on combined N. However, at later dates, the correction was less pronounced since by then seed N accounted for less than l% of the total N. The value obtained for corrected atom %15N reflected the amount of N derived from N2 fixation and the utilization of combined N. The amount of N derived from dinitrogen (N fixed) was the product 38 0.3- . <3 2 \ 9. o\° 02- O . e \ .9. °\ 0 0.l - o'L - \a\ ‘TTJST‘TTTTTTT“‘-.§T \ O\O 50 [60 160 Total Nitrogen (mg). Figure 5: Theoretical and experimental values for atom %15N. The atom % N reflected the contribution of seed N, N2 fixation and the utilization of combined N to total N. The theoretical values (0) were obtained by assuming 1) all N accumulated WIS from combined N source and 2) combined N source was 0.0l0 atom % . The equation used to calculate the theoretical line is presented in the text of the Results section. Seed N was found to be l0 mg N/seed. The experimen- tal values( WWerf obtained from uninoculated soybeans grown on l0 mM KN03 (0. OlO (atom %5 ); see Methods, Experiment l. Each experi- mental value is the mean of 3 replicates : standard error. Table 4: Comparison of Atom %15Nobs and Corrected Atom %15N. Atom %15N Day l3 Day 34 Day 69 Treatment obs cor obs cor obs cor Control 0.367. 0.367 0.365 0.365 0.366 0.366 2 mM N03 0.343 0.244 0.293 0.268 0.3l0 0.307 2 mM NHX 0.346 0.237 0.250 0.214 0.275 0.270 l0 mM N03 0.293 0.l05 0.097 0.040 0.l02 0.088 Soybeans were inoculated with R. japonicum and maintained on N-free ngzrient solution or nutrient solution containing 2 and l0 mM K N03 or 2 mM (14NH4)ZSO4. The atom %15Nobs, (obs) was corrected for the relative contribution of seed N. (cor) was calculated as described in Results. The corrected atom %15N 40 of the ratio of corrected atom %15N to the atom %15N at natural abundance and the accumulated N (total N - seed N): N fixed (mg) = corrected atom %15N - 0.0l0 atom %15N 0.366 atom %15N - 0.010 atom %15N (total N - seed N) The mathematical derivation of this equation is presented in Appendix I. The amount of N derived from the combined N source (N comb) was calculated by difference: N comb = Accumulated N - N fixed. In plants grown on nitrate, the N comb value may include N in the form of N03 (Figure l2). Therefore, the amount of N0§-N present in the tissue was subtracted from the N comb value in order to obtain the amount of assimilated N (N assm): N assm = N comb - (N03-N). Effect of the Application of Ammonium on Early Seedling DevelOp- .EEEE° In the first attempts to grow plants on N-lS depleted KN03 and (NH4)ZSO4, addition of combined N was added on day l after plant- ing. By day l4, the plants grown on 4 and l0 mM NHfi-N were stunted and appeared to be suffering from severe ammonia toxicity. The leaf margins were necrotic and roots were stunted and thickened. The levels of ammonium accumulated in these plants was measured and com- pared to the levels for the roots of plants grown on nitrate. The ammonium concentration in the roots of plants grown on 4 mM NHX (4.5 umOIES‘g roots'l) was 3.8-fold higher than that observed for plants grown on 4 mM N03 (l.2 umoleS'g roots‘l) (Figure 6). A 5.7-fold increase in ammonium concentration was seen in the roots of plants grown on l0 mM NHfi compared with the levels in the roots of plants grown on l0 mM N03. The amount of ammonium in the leaves could not be determined due to the interference of chlorophyll with the color development of the ammonium assay. 41 o 32 84 - o o L 06' ‘ O 1’ ° 24‘ ' E 3 2- //.- .___________._.—-o o 2' 4 6 é (o Nitrogen applied (mM) Figure 6: Ammonium concentration in the roots of l4-day-old soybeans grown on nitrate and/or ammonium. Plants were maintained on N-free nutrient solution (control) or nutrient solution supplemented with 4 or l0 mM KN03 (0-0) or 2 or 5 mM (NH4)2304 (0-0) from day l after planting. Fresh tissue was harvested on day l4 after planting and homogenized in boiling distilled water (1 g tissue/6 ml) with a mortar and pestle. Extracts were filtered through 4 layers of cheesecloth and centrifuged at l7,000 x g for l5 min. The concentration of ammonium in the extracts was determined as described in Methods. 42 The levels of ammonium in the roots of plants supplied with 4 or l0 mM NHI in the nutrient solution starting on day l or day 5 after planting were compared (Table 5). Plants from both treatments were harvested 25 days after planting. The delay in application of 4 mM NHI resulted in a 4-fold decrease in the level of ammonium in these roots compared to roots of plants grown on 4 mM NHI for the whole 25 day period. In the case of l0 mM NHI application, a delay of 5 days resulted in only a l.6 fold decrease in the level of ammonium in the roots. Plants grown on l0 mM NHfi-N suffered severe ammonium toxicity even with the 5 day delay in application. The previous studies have demonstrated the effectiveness of iso- tope ratio mass spectrometry as a method for the quantitation of the 'relative contributions of nitrate and N2 reduction to total N. Fur- ther analyses using this technique were expanded to include the effect of ammonium on the overall contribution of N2 fixation to total N. The effect of ammonium on the distribution of N from each source within the various plant parts was also examined. The Pattern of Dry Matter Accumulation in Vegetative Plant Parts. The growth of soybeans in the presence of l0 mM N03 resulted in a decrease in the number and mass of nodules formed (Figure 7). The con- trol plants (nodulated soybeans grown in the absence of combined N) and plants grown on 2 mM N03 or 2 mM NHZ were effectively nodu- lated by day l3. The increases in number and mass of nodules were sim- ilar for these treatments (Figure 7). During the first phase of growth and nodule development (days l3 to 27), control plants and plants treated with 2 mM N03 or 2 mM NHZ committed more of the total biomass to growth of nodulated 43 Table 5: Ammonium Concentration in the Roots of 25-day—old Soybeans. Concentration NHI in ro ts Concentration (nmoles NHZ°g roots' ) NHfi supplied (mM) Treatment A Treatment 8 0 350 z 3 372 2 12 4 3414 i 24 863 i 57 10 4494 i 55 2722 i 29 Extracts of tissues were prepared as described in the legend of Figure 2. In treatment A, plants were maintained on the various concentra- tions of ammonium from day 1 after planting. For treatment 8, plants were maintained on N-free nutrient solution until day 5 after planting; after which time plants were maintained on 0, 4 or 10 mM NHfi-N. Values presented are the mean of duplicate samples of the same tissue 1 standard error. 44 F gure 7. Dry weights of nodules as a function of nitrogen source. In Panel A the amount of nodules from plants grown on N-free nutrient solution (controls, 0) or N-free nutrient solution supplemented with 2 mM (0), 4 mM (A) or 10 mM ([3) N03 is presented. In panel B the amount of nodules from control plants (0) or from lants maintained on N-free nutrient solution supplemented with 2 mM (0), or 4 mM (A) NHI is presented. Each value is the mean of 3 replicates 1 standard error. 45 .0 “R .0 g dry wt. nodules oplant'J 20 56 do 20 65 50 Days after Planting 46 roots than did plants given 4 or 10 mM N03 (Table 6). In general the proportion of total biomass in the roots decreased with increasing concentrations of N03 through day 55. Plants treated with N03 exhibited the greatest accumulation of dry matter in both tissue types (Table 7). Growth of plants on NHZ was reduced in relation to all other treatments until day 34, at which time root and shoot dry weights of plants treated with NHI were equivalent to or greater than those of control plants. As plants entered the stages of rapid vegetative and mid reproductive growth (days 34 to 90), there was a marked increase in both shoot and root weights over those of control plants with increasing levels of N0§. Because of the possible inhibitory effects of higher con- centrations of NH3, this pattern was less pronounced in plants treated with NHX. Effect of Combined N on Nitrogenase Activity and Root Respiration. Nitrogenase activity was estimated using the acetylene reduction tech- nique (73). The amount of energy expended by the nodules and roots can be estimated by measuring the rate of C02 evolution in nodulated roots (7). Acetylene reduction and root respiration were measured simultaneously on excised nodulated roots. In these experiments the removal of shoots prior to the onset of the assay apparently did not affect the rate of C02 evolution or acetylene reduction (Table 8). A ratio of 2 nmoles C02 evolved per 1 umole C2H2 reduced was observed for both intact and excised root systems. The rates of Csz reduction and respiration are presented in Figure 8. Similar rates of CZHZ reduction were observed for control plants and those grown on 2 mM NHI or 2 mM N0§ Table 6. Percentage of Total Dry Weight of Plant in Root Tissuel. 47 Plant Age (days) Treatment 20 27 34 41 55 Control 34 27 25 20 20 2 mM N0§ 32 26 25 19 16 4 mM N03 31 23 22 l7 17 10 mM N03 22 22 18 l7 l6 2 mM NH1 30 26 22 20 22 4 mM NH3 27 14 21 18 20 1Percentage = Eg dry wt. rootS°plant'1]°[g dry wt. (roots + shoots)'plant‘ ]‘1. The dry wt. of roots includes the g dry wt. nodules'plant‘l. Each value is the mean of 3 replicates i standard error = 1%. 48 .Loeem uemucmum w mmpmo__ame m we come esp we m:_m> zoom Amv muoozm we p;m_mz xec use .mm>mm_ use mm_o_pma .mEmNm on“ mwuzpo:_ .Hupcopa.mmpzco: p3 hen a any woozpocw Amv muooe yo u;m_mz mac ash m_.oaam._ o_.oaeN.o _o. chem. o mo.oaem.o eo.oaaN.o _o.ohmo.o _o.oaeo.o N _N.oheN.N Hm.oaNm.a mo. came. N No.OHNe._ o_.ommm.o No.oaNe.o .o.oaNN.o m #12 2e 4 m..omep._ eo.oaNN.o o_.0heo._ mo.oaNe.o eo.ompm.o No.oaN_.o _o.omN_.o N mN.oam_.N e_.oaeN.e o_.oa_e.m eo.oa0N._ oo.oao.._ No.ohom.o No.oamN.o m N12 ze N o_.oaae._ __. aN_._ mo.oaNN.o eo.oawa.o No.oaNN o eo.oamN.o No.oa__.o N N_.oaNe.m No.oaeo.e oo.oaNN.a o_.oamm.N mo.oa_m _ ...oamw.o NO.Ohmm.o m moz as o_ NO.Ohom._ No.oaom.o eo.oa.a.o mo.oaee.o No.oaNm.o «c.0hNN.o No.oaNp.o N ON.ohaN.N N_.oaeN.m _o.oamm.a o_.oam_.N No.oamN._ mo.oaNN.o No.04Nm.o m moz ze a e_.oam_._ mo.oaNN.o No.04mN.o mo.oama. o mo.oamm.o mo.oamN.o _o.oae_.o N NN.ohN_.N N_.oame.m No.OHNm.m o_.oa.o. N NO.Ohe_.P _o.oaae.o _o.oaem.o m mo: 25 N _N.oam_._ No.oamw.o ao.oame.o eo.oaNe. o _o.oaeN.o No.oa_N.o .o.oaNH.o N NN.oama.e N_.oamm.m NN.oaNN.N m_.oaNe. _ m0.Oh_N.o _o.ommm.o _o.oamm.o m _aeoeoo om me mm He em NN ON Heme “caspaaee Amxaevam< oea_e .muesom :mmocuwz mo :o_uo:=a m we mpoocm can muoom mo pgmwmz Ago .5 m_nmp 49 Table 8: Rates of 002 Evolution and C2H2 Reduction of Intact and Excised Nodulated Roots of Soybeans. pmol C02 evolved gmole 2 reduced C09 evolved Treatment g dry wt'h g dry wt°h C282 reduced Intact Plants1 156 l 76 H- m 1+ 2.05 Excised Nod. Roots2 146 H- 1+ J:- 15 72 2.03 Nodulated soybeans (27-day-old) were removed from pots and rates of C02 evolution (meI C02'g dry wt nodulated roots'1°h‘1) and acetylene reduction (umol C2H2°g dry wt nodulated roots‘l'h‘ ) were measured. In treatment 1, nodulated root system of intact soybeans were placed in 250-ml Erlenmeyer flasks (1 plant/flask). The shoot of plant was fitted through a hole in rubber stopper and any air leaks were sealed with clay. In treatment 2, shoots were removed and nodulated roots (nod roots) were placed in 250-ml Erlenmeyer flasks (4 roots/flask). Flasks were stoppered with a serum stopper. Assay was performed as described In Methods. Gas samples (0.5 ml) were removed at 0,15, 30 and 45 min after addition of acetylene. Each value is the mean of 3 replicates 2 standard error. 50 (Figure 8; a,b,d). Application of higher concentrations of N0§ led to a 40 - 50% decrease in the rate of CZHZ reduction compared to the control treatment (Figure 8; e,f). For all treatments maximum rates of C2H2 reduction were positively correlated with maximum rates of respiration. Rates of root respiration observed for control plants and those grown on 2 mM N0§ were similar. In general, the lowest rates of respiration were found in the roots of plants grown on 4 and 10 mM N03. Rates of root respiration were elevated in plants treated with ammonium when compared with other treatments, particularly during the period of maximum C2H2 reduction. Rates of CZHZ reduction can be used to estimate the amount of N derived from N2 fixation. This estimation is based on the follow- ing assumptions: 1) The measured rates of CZHZ reduction activity were constant for a 24 h period and 2) every 4 moles of CZHZ re- duced are equivalent to the reduction of 1 mole of N2 (55). The amount of N derived from N2 calculated by this method was compared with similar data determined by total N analysis (Figure 9). For the early harvests, 20, 27 or 34 days after planting, C2H2 reduction data could be used to accurately estimate the amount of plant N derived from N2 fixation. At later harvests, the values calculated from C2H2 reduction data were lower than those from total N analysis. Similar discrepancies were observed for the other treatments (Table 9). The determination of the amount of ureides in the xylem exudate is another means of estimating N2 fixation in soybeans (105). The rela- tive ureide content in the xylem exudate has been suggested to be an indicator of N2 fixation (102). The estimation is based on the fol- lowing assumptions: 1) The rate'of ureides transported in the xylem 51 Figure 8: Effect of combined N on nitrogenase activity and root respiration. Nitrogenase activity was determined by measuring the rate of acetylene reduction (O-O). Root respiratign 11 expressed as the nmoles C02 produced°g dry wt nodulated roots’ .h' (0—0). Panel A, control plants; Panel 8, plants treated with 2 mM NHI; Panel C, plants treated with 4 mM NHfi; Panel 0, plants treated with 2 mM N03; Panel E, plants treated with 4 mM N03 and Panel F, plants treated with 10 mM N03. Each value is the mean of three replicates 2 standard error. 52 A0113 .5 ‘200 . 7302 8.2.60: .3 to a. 30.605 «00 8.9:: O O O m 2 d 100 rC. db -I)- co— «1- :8. TA. ‘- d- .i. .- - 55 90 20 55 9020 55 ZOO“ ' A9): m .-c . 73:60: .3 be a . 38:35 .....No 8.9:: m 200 - 20 Days after Planting 53 Figure 9: Estimates of nitrogen accumulated from N2 fixation calculated from C2H2 reduction data and total N analysis. Plants were inoculated with B; japonicum 311b 110 and maintained on N-free nutrient solution. Measured rates of C2H2 reduction were assumed to be constant for 24 h and 4 moles of C2H2 = 1 mole of N2 (0-0). Mass spectral data was calculated as described in Results (0-0). Each value is the mean of 3 replicates i standard error. - plant‘I g . 2 mg N derived from N 8 0'1 0 1 1 54 8&0 7 .4 I I I I I I |--.—( 27 41 55 69 Days after planting I 90 55 Table 9. Estimates of Nitrogen Derived from N2 Fixation Calculated from C2H2 Reduction Data, Mass Spectral Analysis and Ureide Content of Xylem Exudate. mg N derived from N2°plant"1 Day 27 Day 55 Day 90 Treatment N C U N C U N C U Control 8 7 7 67 62 49 200 124 139 2 mM N03 3 6 6 73 57 54 196 114 186 4 mM N03 2 4 7 60 26 46 99 56 127 10 mM N0§ 0 0 2 7 6 13 43 15 35 2 mM NH1 3 5 5 69 62 41 178 121 132 4 mM NH1 0 l 5 29 27 26 141 71 133 Mass spectral data (N) was calculated as described in results. The measured rates of C2H2 reduction (C) were assumed to be constant for 24 h and 4 moles of C2H2 = 1 mole of N2. The shoots of plants were removed and xylem exudate was collected for l h. The ureide content in xylem exudate (U) was measured as described in Chapter 2 (Materials and Methods). The measured rates of ureide transported were assumed to be constant for 24 h. There are 4 moles of N per 1 mole of ureides. 56 exudate was constant for 24 h and 2) every male of ureides has 4 moles of N. The amount of N derived from N2 calculated by this method was compared with similar data determined by C2H2 reduction and mass spectral analysis (Table 9). Like the Csz reduction data, ureide determinations accurately estimated the amount of plant N derived from N2 fixation at early harvests. At later harvests this form of mea— surement led to both overestimations and underestimations of N2 fixation. The Contribution of Dinitrogen and Combined N to Total N Accumu- 123193. The amount of plant N derived from utilization of combined N and N2 fixation (Figure 10 and 11) was determined by mass spectral analysis. Values presented for days 20 through 69 were obtained from vegetative tissue only (shoots and roots). The data for days 90 and 127 included N from reproductive tissue (seeds and pads). Application of low levels of combined N (2 mM N0§ or NHfi) to nodulated soybeans did not delay the onset of N2 fixation (Figure 10). During the period 34 to 69 days after planting, the rates of N2 fixation for these plants (2.7 mg N per day) were similar to those observed for con- trol plants. However, on days 90 and 127 the amount of N derived from N2 was less in plants grown on 2 mM N03 or NHX than in control plants. Growth of plants on 4 mM N03 or 4 mM NHI resulted in a decrease in the amount of total plant N derived from N2 fixation. During 34 to 69 days after planting, treatment with 4 mM NHI appeared to reduce the amount of N derived from N2 more than treatment with 4 mM N0§. However, on days 90 and 127 the levels of N derived from N2 were similar for these two treatments. No accumulation of dinitrogen was observed for plants grown on 10 mM 57 Figure 10: The contribution of N2 fixation to total N in nodulated soybeans grown on combined N. Data presented for days 20 - 69 were for vegetative tissue only (shoots and roots). Values for days 90 and 127 included N from reproductive tissue (pods and seeds). Values obtained for plants grown on nitrate are presented in panel A; control, 0; 2 mM N03, 0; 4 mM N03, A and 10 mM N03, E]. Values presented in panel B are for lants grown on NHZ; control, I; 2 mM NH1, 0; and 4 mM NH , A. The values have been corrected for the presence of seed N. Each value is the mean of 3 replicates i standard error. Unless noted standard error values are less than or equal to the size of the symbols used. 58 A. N03 8. NH; .001 ._ - _S_ 300 -- - Q. ~ f E 200} 3: £5 8 I, d) ..>. as ‘U 2 5;” 1001— -- - l II l 1 I 20 55 90 127 20 55 90 127 Days after Planting 59 N03 until after day 4l (Figure 10a). For all the treatments, the highest rates of N2 fixation occurred during the period of pod fill (days 69 - l27). As expected an inverse relationship between N2 fixation and uti- lization of combined N was observed. Plants grown on 2 mM N03 or NHK were able to assimilate low levels of N from the combined N source (Figure 11). At all harvests, the amount of N assimilated from the combined N source was greater for plants grown on 2 mM NHX than for plants grown on 2 mM NO§. An increased utilization of N from the combined N source was observed for plants grown on 4 or 10 mM N03 and 4 mM NHI. The amounts of N assimilated from com- bined N for plants grown on 4 mM N0§ and 4 mM NH: were sim- ilar (Figure 11a,b). Plants grown on 10 mM NO§ assimilated the majority (75%) of the total N from nitrate. Partitioning of N from N7 Fixation, Utilization of Combined N and N03 within Vegetative Tissue. Differences in the partition- ing of N within the shoots and roots of plants grown on combined N were observed (Table 10, see Appendix 11). Prior to anthesis (day 55) the percentage of N derived from N2 fixation in the roots of plants treated with combined N was consistently less than that of the shoots. This difference was more pronounced in plants grown on ammonium than plants grown on comparable concentrations of nitrate. After anthesis (harvests 69 and 90), the difference in the percentage of N derived from N2 fixation in roots and shoots was reduced. On these dates approximately 80% of both root and shoot N was derived from N2 fixation in plants grown on 2 mM N03. However, for the same period a similar partitioning was not observed in the roots and shoots 60 Figure 11: The contribution of combined N to total N in nodulated soybeans grown on combined N. Data presented for days 20 - 69 were for vegetative tissue (shoots and roots). Values for days 90 and 127 included N from reproductive tissue (pods and seeds). Values obtained for plants grown on nitrate are presented in panel A; 2 mM N03, 0; 4 mM N03, A; l0 mM N03, E]. The values have been corrected for the presence of N in the form of stored N03 and N derived from the seed. Values presented in panel B are for plants grown on NH5 2 mM NH1, 0; 4 mM NHI, A. Each value is the mean of 3 replicates i standard error. Unless noted standard error values are less than or equal to the size of the symbols used. 61 \\ 8. NH; l 7A 90 l27 20 55 9O l27 Days after Planting 55 a a s. m w m. 7.53 . z 3:368 69: cos-€58 z 9: IOOF- 20 .Axmu_v Loggm ugmucmum u mmumo__amg m No came asp m_ m=FN> comm .AHH xwucmaa< :_ umucmmwga my muoogm new maoog cw z quoa asp Lo» mm:_m> szpom asp .z _Npou any EON» umuumgumnsm mm: mamm_p mzu cw moz nmgoum we pesosm asp .moz fix co czogm mucmpa yo mmmo mg» :H .mwwaENm No mwm»_m:m Pagaomam mmws he um:_eLmamu mm; mammnxom No ANW mpoog use Amv mpoocm asp cw :o_pNxN$ z Eogy um>wgmu 2 we pesosm ash 62 NN om om mm mm N4 N ON C o N22 :5 N Nm NN Nm NN mm NN om Nm N N_ N12 as N N_ o_ _ o_ o N o N o o moz :5 o_ NN QN ON om NN Nm NN 04 P N moz :5 N NN NN NN mm mm NN _© mN ON ©_ mo: :5 N - oo_ oo_ oo_ oo. oo_ oo_ oo_ oo_ co, co. .oepcou N m N m N m N m N m NNNENNNNN om Nan me has mm Nag P4 awe . NN Nag .muoosm use mpoom cw :owumxwm Nz EON; um>_gmo z _mpoh No mmmucmogma “OH mFQNp 63 of plants grown on 2 mM NHI. Although the percentage of N derived from N2 in the shoots was similar to that found in plants grown on 2 mM N03, the percentage found in the roots was reduced (57%). The data presented thus far have dealt with the partitioning and relative contribution of N2 fixation and the utilization of combined N to total N. However, for plants grown on nitrate a portion of the nitrate taken up by the plant was not metabolized but rather was stored in the roots and shoots as free N03 (Figure 12). The actual N03 levels in each tissue (mg N-N03°plant'1) are presented in Appendix III). The percentage of the total N of a tissue present as N0§ was higher in the roots than shoots of plants grown on all concentrations of nitrate. Only in the case of plants grown on 10 mM N0§ was stored leaf N0§ significant (Figure 12a). On day l3 which was prior to the onset of N2 fixation, stored NO§ accounted for approximately 12% of the total N in the roots of plants grown on 2 mM N03; thereafter, only 3-4% of the total root N was present as N03. Growth of plants on higher concentrations of N0§ resulted in greater amounts of N03 being stored. The roots of plants grown on 4 mM N03 contained 15-20% of the total N in the form of N03. In the case of plants grown on l0 mM N03, stored N03 accounted for up to 50% of the total root N after day 34 (Figure 12b). Partitioningiof Nitrogen During Reproductive Phase of Growth. During the period of pod fill (days 69 to l27), the N content of all plants was increasing (Table 11). However, the distribution of N between the vegetative and reproductive tissues was changing. On day 64 \ CI / A A .- O .. A \ °~o—o O’O‘o’°\O—"O——-o \o ...—0 1'3 3'4 55 9'013 3r4 5V5 9’0 Days after planting Figure 12: The percentage of total plant N present as N03 in roots and shoots. The amount of nitrate in each tissue was determined as described in Methods. Nodulated soybeans were grown on 2 mM (0), 4 mM (A) and l0 mM ([]) nitrate. In panel A data for shoot tissue are presented. In panel B data for roots are shown. Each value is the mean of 3 replicates 1 standard error. Unless noted standard error values are less than or equal to the size of the symbols used. 65 u ANIV xeecH .Leege egeeceam H meHeeNHeeL m we sees ecu wee neucemege meaHe> eegeum Ne eecemege ecu New eepeeegee wee mezHe> .z eemm use moz .ANNHV Nae eme>tme _N=_N Lee 2 HNHHN HeeeN\z N>Nee=e0NNeN Hme>gez ezN .meee ece meeem eeeHeeN meemmHH e>NHe=eeLeeN .meemmNH NNV e>HHeaeeLeeg meHe e>HHeHeme> eecHesee use NNV e>HHezeeeeeN .A>V e>HueHeme> New =e>Nm wee mueeHe :N eeHeH353eee z HeHeH egN NN.o ON H New NH H men m H HoH m H HHN H H me NH mmH oH H NHH wzz :5 H mN.o mH H NNm m H me m H moH HN H QMN e H NN NH H NmH m H NeH wzz :5 N mN.o NN H mom oH H mNe NH H NNH NH H mHN H H No NH H emH o H oHH moz :5 oH Nw.o HH H Nom oH H mme H H No N H NwH N H mm m H mNH o H oHH moz :5 e Nw.o e H mHH e H omm H H mm mN H me e H mm mH H mmH m H HmH moz :5 N mw.o HH H Hme N H MNm e H wN w H ooN N H me o H mmH m H mNN Hegueeo .H: N N . > N N > > NN_ Nae em Hue mmnxmm NHuuceHn.z waH z HeHeN HeeEHeegN .mxeo NNH ece om .mm He mugeN HeeHN e>HHe=eeNeeN use e>NHeueme> New museueeo z HeHeN "HH eHeeN 66 69, the amount of N in reproductive tissues accounted for only 2% of the total plant N. By day 90, approximately 20 to 30% of the plant N was now in the reproductive tissue. Increases in the amount of total N found in vegetative tissue continued between days 69 and 90. In gen- eral, though, the rate of N accumulation into vegetative tissue had declined over this period with an increasing amount of N being diverted to reproductive tissue. Plants were completely senescent and pad fill was completed by day 127. During the period 90 to l27 days, a 48% increase in total plant N was observed for control plants grown in the absence of combined N and plants grown on 2 mM N0§ (Table 11). The largest increase (64%) in total N was found in plants grown on 10 mM NO§. The majority of the total N was found in the reproductive tissue. Differences in the amount of total N remaining in the vegetative tissue were observed for the various treatments. The harvest index reflected this differ- ence and indicated the relative efficiency of mobilization of N from vegetative to reproductive tissue. The values for the harvest index for plants grown on l0 mM N03 and 2 or 4 mM NHK was similar. The harvest indices were higher for control plants and plants grown on 2 and 4 mM N03 than the other treatments. The percentage of total plant N derived from N2 fixation in the reproductive and vegetative tissue was compared (Table 12). For day 90 reproductive tissue included both pods and seeds. On this date, plants grown on 4 mM and 10 mM N03 appeared to partition a greater per- centage of N derived from N2 fixation into the reproductive struc- tures than into the vegetative tissue. However, in all other treat- ments the percentages of total plant N derived from N2 fixation in 67 Table 12. Percentage of Total Plant N Derived from N2 Fixation for Vegetative and Reproductive Tissue. Day 90 Day l27 Treatment V R V R Control 100 100 100 100 2 mM N0§ 82:10 82:4 81:1 79:1 4 mM N03 46:2 67:2 55:1 54:1 10 mM N03 17:2 24:1 19:3 24:1 2 mM NHI 74:5 74:4 67:3 72:1 4 mM NHX 58:2 58:1 50:2 51:2 The percentage N derived from N2 fixation for vegetative (V) and reproductive (R) tissue is presented for days 90 and 127. The percentage of total plant N from N2 = mg N from N2/mg accumulated N as calculated from mass spectral analysis. Total N was corrected for the presence of stored N03. Each value is the mean of 3 replicates 1 standard error. 68 the vegetative and reproductive tissue was similar. By day l27, the percentages of total plant N derived from N2 fixation in the vegeta- tive and reproductive tissues were the same for all treatments. The data for reproductive tissue on day l27 were obtained from the seeds only. The total N remaining in the pods was approximately 1 mg N per pad for all treatments. These samples were subjected to mass spectral analysis and the atom %15N was measured. The values obtained were similar to those found in the seed and would therefore not affect the percentages presented in Table 9. The seeds of control plants accumulated more N from N2 than seeds of any of the other treatment (Table 13). The amounts of N derived from N2 fixation for the seeds of plants grown on comparable concentrations of either N0§ or NHZ were similar. The total N of seeds from plants grown on NHfi was consistently greater than that of seeds from plants grown on comparable concentra- tions of N03. This increase was due solely to the greater amount of N assimilated from combined N for these plants than for plants grown on nitrate. The amount of stored nitrate in the seeds of plants grown on N03 was negligible (< 50 ug°seed’1). Application of combined N appeared to have no affect on the dry weight of seeds. A slight increase in pod number per plant was observed for plants given 4 mM and 10 mM N03, however the number of seeds per pod was the same for all treatments (approximately 2). 69 Table 13. Relationships Between Source of Nitrogen and Accumulation of Dry Matter and Nitrogen in Seeds. Dry Weight Total N Pods Treatment (mg/seed) (mg/seed) mg N (N2) Plant Control 0.17 9.4 : 0.2 9.4 21 : 1 2 mM N03 0.17 8.5 : 0.1 6.7 20 : 2 4 mM N05 0.16 7.7 : 0.2 4.2 23 : 3 10 mM N03 0.17 9.0 : 0.2 2.2 26 : 2 2 mM NHZ 0.18 9.5 : 0.2 6.8 21 : 2 4 mM NHZ 0.17 8.3 : 0.3 4.2 19 : 2 Values presented are the mean of 3 replicates : standard error. For dry weight data, the standard error was <0.004 for all treatments. The contribution of N2 fixation to seed N, mg N (N2), was calculated from data obtained by mass Spectral analysis. DISCUSSION A comparison of the growth and patterns of assimilation and parti- tioning of N derived from N2 fixation and the utilization of combined N into vegetative and reproductive tissues of soybeans was presented. The total biomass of plants grown on N03 was greater than that observed for control plants grown in the absence of combined N (Table 7). This increase was most pronounced in the aerial portion of the plants. During early nodule development, control plants and plants grown on 2 mM N0§ or 2 mM NHX committed a greater propor- tion of the total biomass to the roots than plants grown on higher con- centrations of combined N. This distribution of biomass reflected the added burden of nodule establishment for these plants. Measurements of C02 evolution from roots should also reflect the energy requirement for nodule establishment and maintenance (62). The energy consumed (ATP and electron donors) to support the reduction of N2 and N03 or the assimilation of NH1 was assumed to be derived from the complete oxidation of sugars for C02 production (10,63). At the onset of N2 fixation there was an increase in C02 evolution for all treatments, except l0 mM N03, as the energy requirement for N2 fixation was high (Figure 8). For plants grown on high levels of N03, respiration was less than observed for N2 fixing plants. This corre- lated with the lower levels of N2 fixation in these plants and the fact that N0§ was reduced in the leaves (139). If N03 was reduced in 70 71 the roots the rate of 002 evolution should equal that found in plants fixing comparable amounts of N2 since the theoretical calculations suggest the energy costs of N0§ reduction and N2 fixation are equal (61). The assimilation of NH1 occurs in the roots and necessitates the availability of large amounts of photosynthate (for energy and C-skeletons) (38). Rabie gt_al. (143) have shown that the roots of soybeans grown on NH: receive a greater percentage of the total photosynthate than roots of plants grown on N03. The amount of energy necessary to assimilate NHI is much less than that for N2 fixation (144), however the rate of 002 evolution in the roots of plants grown on NH: was greater than that observed for the other treatments (Figure 8). On day 4l, for control plants, the amount of N derived from N2 fixation was 0.74 mg N°umol 002 respired-1. For plants grown on 2 mM NHfi this value was 0.45 mg N°umal €02 respired'l. However, if the amount of N derived from NHfi was included, a value of 0.83 mg N°umole 002 respired"1 was calculated for plants grown on 2 mM NHfi. Therefore, the high rates of respiration observed in the roots of plants grown on NHI was probably due to the increase rate of NH1 assimilation, even though the energy costs are low. Prior to day 41, the high rates of respiration observed in the roots of plants grown on NHZ could not be explained by the above conclusion. The total N assimilated per 002 respired was consis- tently less for plants grown on NHZ than control plants. A more likely explanation is that NHI may be acting as an uncoupler of mitochondrial phosphorylation. Ammonia is known to uncouple 72 photophosphorylation in the chloroplast (145) resulting in increased respiration. A similar effect on root mitochondrial phasphorylation may be occurring. Differences in the partitioning of N derived from N2 fixation and the utilization of combined N source were observed for roots and shoots (Table l0). The percentage of total N derived from N2 fixa- tion in the roots of soybeans grown in the presence of combined N was less than the leaves. 0hyama and Kumazawa (l04) found that the N fixed in the nodule was distributed to the stems more than the roots. Pate _t.al, (146) have reported that the roots of Nz-fixing lupins receive a large percentage of the N for growth from the shoots via the phloem. These data suggest that the nodule does not directly supply the root with nitrogenous organic compounds. This apparent reduction in the incorporation of N derived from N2 fixation into the growing root may result from the inability of the root to directly metabolize ureides, the major form of recently- fixed N transported from the nodules of soybeans (21). Therefore, the root would be dependent upon the low levels of amino acids synthesized in the roots or amino acids synthesized from ureides in the shoots with subsequent transport to the roots via the phloem. Although the inabil- ity of the soybean roots to utilize ureide-N would explain the lack of direct incorporation of N derived from N2 fixation into this tissue, it does not explain the data obtained from lupins (146). The major form of N transported from the nodules of lupins is the amide, aSpara- gine. Since this is the same form of N derived from the assimilation of N03 or NHZ, the lupin root should be capable of directly utilizing the asparagine without transport to the shoot first. “n“— 73 Another possibility for the lack of direct incorporation of fixed N into the roots is that the root may never be exposed to the nitro- genous solutes produced within the nodule. During early N2 fixation the nodules are located around the crown of the root with most of the growing root being below the nodule region. Therefore, the organic N produced in the nodule would have to be transported down via the phloem to the growing root. However, the N assimilated within the nodule enters the xylem stream and is transported to the shoot. If there is little xylem to phloem transfer the growing root will not be exposed to the nitrogenous compounds produced within the nodule. As the plants age, the percentage of N derived from N2 fixation in the roots approached that found in the shoots (Table 10). A greater dependence an amino acids synthesized directly from ureides or indi- rectly from protein turnover within the shoot with subsequent transport to the roots of older plants could account for the changes in percent- ages. Alternatively, a decreased utilization of N03 or NHfi would result in the equilibration of N from the different sources in the roots and shoots. As the plants age, nodules can be found on the secondary roots. Thus, the roots above this region would be able to extract the nitrogenous compounds in the xylem stream as these solutes are transported to the shoot. The rate of accumulation of N derived from N2 fixation, as determined by the change in atom %15N, appeared to be biphasic (Figure 10). At early harvests, a slow rate of accumulation was noted for all treatments except plants grown on l0 mM N03. At later harvests (days 34 to l27), the rate of accumulation of N from N2 fix- ation tripled and remained constant throughout this period. This 74 pattern of N accumulation from N2 fixation observed by mass Spectral analysis was qualitatively similar to that measured by CZHZ reduc- tion activity (Figure 9). However, quantitatively the amount of N derived from N2 fixation, as calculated from C2H2 reduction data, was less than that determined by mass spectral analysis. Thus, the estimation of the rate of N2 fixation from the rates of CZHZ reduction which involves a number of assumptions and a theoretical con- version factor leads to an underestimation of N2 fixation. As the plant matured, the magnitude of this discrepancy increased, which sug- gests that even an experimentally determined conversion factor is not constant. This may have resulted from the removal of the shoots prior to the initiation of the CZHZ reduction assay. Changes in the availability of photosynthate have been shown to affect the rates of C2H2 reduction (N2 fixation) (72). The need for photosynthate by older nodules may be greater than that of young tissue. Thus, the removal of shoots will deprive the nodule of the photosynthate neces- sary to drive N2 fixation. The data presented support the idea that the use of CZHZ reduction activity in the determination of quanti- tative rates of N accumulation and total N-input from N2 is inadequate (52). The rate of N accumulation in vegetative tissue of soybeans appeared to decline after day 69 (Table 11). During the same period a rapid accumulation of N in the pods was observed. Pods have been shown to be effective sinks for transported N (94). Atkins 2E.§l- (132) have demonstrated that lupins fed [14C]asparagine rapidly translocate the asparagine to the fruit from the leaf with little metabolism occur- ring within the leaf. In the present study, the accumulation of N from 75 N2 fixation and utilization of combined N continued into the pod filling stage of growth. During this period the decreased accumulation of N into the vegetative tissue and the substantial increase into the pod suggested that the pad is a strong alternate sink for the N trans- ported from the roots and nodules. The %N of the harvested seeds ranged from 5 to 5.5%N. While this value was lower than the 6 to 7%N observed for field grown soybeans (l49) low values for %N have been reported for other greenhouse grown legumes (15,16). Under high 002 enrichment conditions, field grown soybeans were also observed to have reduced %N values for the seeds (5). Results of analysis on the seeds planted at the onset of this experiment indicated that they contain 6.4%N, consistent with values reported in the literature (l49). Therefore, results based on the semi-micro Kjeldahl procedure employed in this study for the determina- tion of total N were valid. No apparent loss of N during digestion (Table 2) or subsequent analysis was observed. Lower values of seed N have been reported for plants grown under conditions of N-limitation resulting from inoculation with an inefficient strain of Rhizobium (147). The Nz-fixing capability of a strain appears to regulate the amount of N within the plant which is stored and remabilized for repro- ductive growth. Since R;_japonicum 3Ilb ll0 is an efficient strain of Rhizobium, this does not explain the aparently low values obtained for seed N. Alternatively, the lower seed N values may have resulted from a reduced photosynthetic capacity of plants grown under less than optimal light conditions. The lower photosynthetic capacity may have regulated the amount of N fixed and assimilated which in turn con- trolled remobilization. 76 The continuous application of combined N to the soybeans in this experiment insured that changes in the relative contribution of N from N2 fixation or utilization of combined N were not due to changes in the availability of either source. The percent N derived from N2 fixation for seeds of field grown soybeans fertilized with nitrate at planting is greater than for vegetative tissue (17). The soil N was reportedly depleted by flower initiation (17) suggesting that the available sources of N had changed. Therefore, differences in the % N derived from N2 fixation between vegetative and reproductive tissue were due to the reliance on N2 fixation for the remaining N for growth. In general, the percentage of N accumulated from N2 fixation in the seeds paralleled the contribution of N2 fixation to total N in the vegetative tissue. This similarity in the contribution of N2 fixation to total N of vegetative and reproductive tissues suggests that N loading into the pods and seeds was indiscriminate with respect to the original source of N. Pods can assimilate N from newly fixed N2, newly synthesized amino acids from the utilization of combined N and the remoblized N from proteins in the leaves (Figure l3). The mobilization of N into the reproductive tissue was extensive. Less than 20% of the total N assimilated during the growing season remains in the vegetative tissue (Table ll). The fact that pods may be effec- tively assimilating newly fixed N2 does not disagree with the hypoth- esis that N loading is indiscriminate with respect to the original N source. Rather, this may reflect a reduction in ureide metabolism in the leaves allowing this pool of N to be readily available for pod- fill. The data presented suggests that high N requirement of pod-fill 77 necessitates the mobilization and transport of all available N to the seed. 78 N2 Pods protein< aaé———————-ur%ides Shoots 1V'J mobilized protein-————9residual N03 )NHI )aa( ureides a~ Ab 1N Nodule Roots _;aa<;——) p;r‘ote1n ’aa’ )ureides .L N03 - - -> NHZ —-—->aa ——-) protein 1 l 11:.— 1, ...—1. I I NO§ NHI SEZLL Fi ure 13. A model for the transport of N within the soybean. Amino acids (aa) can be produced from N03 reduction, N2 fixation or NHI assimilation. Proteins are mobilized during pod fill. The proteins that remain in the shoot after pod fill are termed residual. Dashed lines represent events that are unlikely to occur. CHAPTER II Studies on the Biosynthesis of Ureides in Developing Soybean Seedlings INTRODUCTION The ureides, alantoin and allantoic acid, are the major nitro- genous substances transported in the xylem of soybeans grown in associ- ation with the bacterium, Rhizobium japonicum (21,94). The presence of ureides in the xylem stream has been linked to the process of symbiotic Nz-fixation and is postulated to be mediated by the nodule (21,105). The production of ureides appears to arise from the biosynthesis and subsequent degradation of purines (29-33,107-109). Recently, ureide production has been noted in soybean seedlings prior to the onset of N2 fixation (27). This increase in ureide con- centration in young seedlings (2 to 5 days after planting) suggests that soybeans are capable of producing ureides without the aid or pres- ence of the microsymbiont. If purines play a major role in ureide pro- duction within young seedlings, then there are two possible explana- tions for the increased production of ureides observed. The increase in ureide concentration may result from l) ggiggvg synthesis of purines with subsequent metabolism to ureides or 2) degradation of existing nucleotides to form ureides. Fujihara and Yamaguchi (125) have found no difference in the amount of ureides in the roots of 3-day-old soy- beans after treatment with azaserine, an inhibitor of g; 3919 purine synthesis. They concluded that g§_ggvg synthesis of purines is not involved in ureide production in young seedlings. However, no data 79 80 were presented for the effects of azaserine on the levels of ureides in the cotyledons. In this chapter the following are discussed: l) the effect of applied N on ureide production, 2) the examination of enzymes involved in purine degradation, 3) the effect of inhibitors of purine metabolism on ureide production, and 4) the examination of g3 ggvg synthesis of purines and its relationship to ureide production in soybean seedlings. MATERIALS AND METHODS Materials Soybean seeds (Glycine max [L] Merr. cv. Amsoy 7l) were purchased from the Ohio Seed Foundation, Columbus, OH. [2-14C]Uric acid (57-60 mCi/mmol) was purchased from Amersham, Arlington Hts, ILL. [0-14013er1ne (140 mCi/mmol) was purchased from ICN Radiochemicals, Irvine, CA. PEI-cellulose plates (MN - polygram cel 300 PEI) used for uricase assay were obtained from Brinkman, Nestbury, NY. PEI-cellulose F plates and glass distilled methanol were purchased from E. Merck Reagents, Darmstadt, Germany. p-Dimethylaminobenzaldehyde was obtained from Aldrich Chemical Co., Milwaukee, WI. PPO and POPOP were purchased from RPI, Elk Grove Village, ILL. FLO-Scint II was obtained from Radiomatic Inst. and Chem. Co., Tampa, FL. Whatman Partisil PXS lO/25 ODS-2 column and 0.2 um nitrocellulose filters (8 mm) were purchased from Anspec, Ann Arbor, MI. Dupont Zorbax-OOS (7 um) column was pur- chased from Fisher Scientific, Pittsburgh, PA. Nitrocellulose filters (0.45 um, 25 mm, HA) were obtained from Millipore Corp., Bedford, MA. Tetrabutyl ammonium hydroxide, azaserine, allopurinol, all purine bases, nucleosides and nucleotides were purchased from Sigma, St. Louis, MO. Growth of Seedlings for Ureide Studies. Soybean seeds were washed with l% (v/v) bleach, rinsed thoroughly and soaked for l h in distilled 81 82 water. After imbibition seeds were placed on moist paper towels on trays and germinated in the dark overnight. Seeds were then planted in 20-cm plastic pots (l0 seeds per pot) containing Perlite. On day 2 after imbibition, half the plants were maintained on N-free nutrient solution (2, Table l) while the rest received N-free nutrient solution supplemented with l0 mM KNO3. Plants were harvested daily during the first 2 to l0 days after imbibition, and every other day thereafter. Data for day 0 represent seeds which had been imbibed for l h. Extraction of Plant Tissue for Ureide Determination. Fresh plant tissue was divided into cotyledons and axes (roots, shoots and leaves), weighed and frozen in liquid N2. The tissue (l g) was homogenized in 4 ml of 0.25 N HCl04 with a mortar and pestle (113). The homogenate was centrifuged at l0,000 x g for l5 min. The supernatant fluid was adjusted to pH 6.5 by the addition of 2 N KOH and centrifuged to remove the potassium perchlorate precipitate. The supernatant fluid was ana- lyzed for ureides using the differential analysis of Vogels and Van der Drift (150). Ureide Determination. The procedure for total ureide determina- tion entailed the conversion of both allantoin and allantoic acid to glyoxylate (150). Plant extracts (0.1-0.2 ml) were adjusted to 0.6 ml with distilled water. After each addition, samples were mixed vigor- ously. After each incubation, the samples were placed on ice until the next addition was made. The assay was started by adding 0.2 ml of 0.5 N NaOH and incubating the solution at l00°C for 8 min. The sample was acidified by the addition of 0.2 ml of 0.65 N HCl to convert allantoic acid to glyoxylate and incubated at l00°C for 4 min. Phenylhydrazine (0.2 ml; l00 mg/30 ml) and 0.2 ml of 0.4 M sodium phosphate (adjusted 83 to pH 7.0 with 2 N KOH) were added and the solution was incubated at room temperature for 5 min. Cold (4°C) l2 N HCl (l ml) and 0.2 ml of potassium ferricyanide (500 mg/30 ml) were added for color development. Samples were incubated at room temperature for 15 min and the absor- bance was measured at 525 nm. The assay was linear between O-lOO nmol allantoin. To determine the amount of allantoic acid in the plant extracts, the following modifications were made to the above procedure: Samples (0.l-0.2 ml) were adjusted to 0.8 ml with distilled water. The assay was started by the addition of 0.2 ml of 0.l5 N HCl and incubation of samples at l00°C for 4 min. The addition of phenylhydrazine, sodium phosphate, potassium ferricyanide and HCl were as described above. The assay was linear between O-lOO nmoles allantoic acid. Determination of Total N. Total N was determined using a micro - Kjeldahl procedure (151,152). Dried plant material (25 mg) was placed in a test tube (l0 x 75 mm) containing l.0 ml of 36 N H2S04, 0.02 ml of 34% (w/v) CuSO4°5H20, 0.02 ml of selenium (50 mg selenium in 5 ml 36 N H2504) and 0.l8 g K2804. Test tubes were placed in a sand bath at 240°C for l h. The temperature was then increased to 380°C and samples were digested for l h after solutions cleared. After cooling, samples were diluted to l0 ml with glass distilled water. An aliquot (0.5 ml) of diluted sample was placed in a test tube containing 0.1 ml of neutralization indicator (12.5 9 EDTA and l0 ml 25% (w/v) methyl red (Na-salt) per 500 ml H20, adjusted to pH l0 with 2 N NaOH). Samples were adjusted to pH 6.0 with 0.5 N NaOH (indicator turns yellow) and diluted to 5 ml with distilled water. An aliquot (l 84 ml) of the neutralized sample was removed for ammonium determination (see Chapter 1). Preparation of Extracts for Uricase and Allantoinase Assays. Fresh plant material was separated into cotyledons, roots and leaves. Tissue (l g) was homogenized in 4 ml of lO mM potassium phosphate (pH 7.6), l0% (w/w) sorbitol and l0% (w/w) PVP with a mortar and pestle. The extract was filtered through 4 layers of cheesecloth and centri- fuged at 500 g for l0 min. The supernatant fluid was saved and placed on ice (4°C) until assays were performed. Handling of [2-14CJUric Acid. Uric acid was resuspended in distilled water at a final concentration of 0.59 mM (153). The resus- pended uric acid was divided into lOO pl fractions and placed in 0.5 ml Eppendorf tubes. The fractions were frozen in dry ice and acetone, lyophilized overnight and stored at -20°C until needed. Each tube of [2-14CJuric acid was resuspended in lOO pl l50 mM CHES (pH 9.5) as needed for uricase assays. Uricase Assay. The uricase assay entailed the use of [2-14C]uric acid and the separation of products by thin layer chro- matography (150,l5l). The plates used for the separation were PEI-cel- lulose sheets (20 cm x 20 cm). Plates were predeveloped in distilled water overnight. Samples were applied 2.5 cm from the bottom and l.5 cm apart. Plates were prespotted with 5 ul each of nonradioactive l2 mM uric acid, l6 mM allantoin and 16 mM allantoic acid. The reaction mixture consisted of 20 ul of l50 mM CHES (pH 9.5), 10 61 0.59 mM [2-1401uric acid in 150 mM CHES (pH 9.5) and 10 n1 enzyme extract. The components of the assay were added to a 0.5 ml Eppendorf tube in an ice bath and the reaction was started by the 85 addition of l0 pl of enzyme. Immediately after addition of enzyme ex- tract, 5 pl of reaction mixture was removed and spotted on PEI-cellu- lose plates. The reaction mixture was incubated at 30°C and 5 ul ali- quots were removed at 5, l0 and l5 min after addition of enzyme extract and spotted on the plates. One-dimensional ascending chromatography was performed using a solvent system consisting of 0.5 M NaCl:95% ethanol (4zl). Chromato- grams were developed to a distance of l5 cm. The plates were air dried and uric acid was visualized under short wavelength UV light. Allanto- in and allantoic acid were identified by spraying with Erlich's reagent (l g p-dimethylaminobenzaldehyde, 95.4 ml 95% ethanol and 4.6 ml l2 N HCl). The relative mobilities for uric acid, allantoin and allantoic acid are presented in Table 14. The appropriate areas were circled and spots were cut out of chromatograms. Spots were placed in scintilla- tion vials containing 9 ml of cocktail (toluenezTriton X lOO: POPOP: PPO, 66:33:0.0l:05,v/v/w/w) and radioactivity was determined by liquid scintillation spectrometry. Allantoinase Assay. The allantoinase assay was as described by Hanks §t_al. (123). Enzyme extract (5-20 ul) was added to a tube con- taining l ml of 20 mM allantoin in 20 mM Tricine (pH 7.8). The reac- tion mixture was incubated at 25°C for 20 min. The reaction was stopped by the addition of l ml of 0.l5 M HCl. Samples were mixed and incubated at l00°C for 4 min. After tubes had cooled on ice, 0.8 ml of phenylhydrazine (lOO mg/30 ml) was added. Samples were mixed and incu- bated at 25°C for l5 min. The tubes were placed on ice and l.2 ml of cold (4°C) l2 N HCl and 0.8 ml potassium ferricyanide (500 mg/30 ml) was added. Samples were mixed and incubated at 25°C for 20 min. The 86 Table 14: Chromatography of Uric Acid, Allantoic Acid and Allantoin on PEI-cellulose TLC Plates. Compound Rf Uric acid 0.34 Allantoic acid 0.43 Allantoin 0.68 PEI-cellulose TLC plates were developed in 0.5 M NaCl:95% Ethanol (4:1). Uric acid was visualized under short wavelength UV Allantoin and allantoic acid were visualized by spraying with Erlich's reagent (154). The relative mobility (Rf) was determined from the middle of a spot. 87 absorbance was measured at 520 nm. The assay was linear between 0-100 nmoles allantoic acid. Protein Determination. Protein concentration was determined by a modified Lowry procedure (155). Enzyme extracts (10 - 100 pl) were added to test tubes containing 1.5 ml of water and 25 ul of 1% (w/v) sodium deoxycholate. Samples were mixed and incubated at room tempera- ture for 15 min. Trichloroacetic acid (24%; 0.5 ml) was added and sam- ples were centrifuged at 1000 x g for l h at 15°C. The supernatant fluid was removed by aspiration. The pelleted protein was solubilized by the addition of 1.5 m1 of reagent C [100 m1 of 20 g anhydrous Na2C03, 4 g NaOH and 0.2 g sodium tartrate per 1 liter H20 and 1 m1 of 5 g CuSO4°H20 per 1 liter H20]. Samples were incubated at room temperature for 10 min then 0.3 ml reagent D (0.5 N Folin-Ciocalteu reagent) was added. Absorbance was measured exactly 30 min after the addition of reagent 0. The assay was linear between 0-60 pg BSA. Inhibition Studies Azaserine Treatment. Plants grown on N-free nutrient solution were harvested at 3, 8 and 12 days after imbibition. Seedlings were washed with distilled water and placed in 125 ml Erlenmeyer flasks (4 seedlings per flask) containing 0.01 M potassium phOSphate (pH 7.0) and 0.5 mM azaserine (128). Seedlings used for controls were incubated in 0.01 M potassium phosphate (pH 7.0). The flasks were entirely covered with aluminium foil and placed on a rotary shaker (28°C) in the dark for 24 h. Incubations with azaserine were performed in the dark since azaserine was found to be light sensitive. After incubation, seedlings were washed with distilled water and separated into shoots and leaves, 88 roots, and cotyledons. Extraction of tissue and determination of ureide content within the tissue were as described previously. Allopurinol Treatment. Plants grown on N-free nutrient solution were harvested as described for azaserine treatment. Seedlings (4 seedlings per flask) were placed in flasks containing 0.01 M potassium phosphate (pH 7.0) with and without 0.5 mM allopurinol (128). The base of the flasks was wrapped in aluminium foil. Seedlings were incubated in the flasks under fluorescent lights (12 h photoperiod) for 24 h with continuous aeration. After incubation, seedlings were separated into parts as described above. Tissue extracts were prepared (as described previously) and total ureide, xanthine and allopurinol concentrations were determined. Determination of Xanthine Concentration. The amount of xanthine in the neutralized extracts was determined by ion-suppression, reverse- phase HPLC with a Nhatman Partisil ODS-2 column. All solvents were prepared with deionized water and were filtered daily through 0.45 um nitrocellulose filters (25 mm diameter). Samples were filtered through 0.2 um nitrocellulose filters (8 mm) by centrifugation at 5,000 x g for 5 min. Filtered extract (20 ul) was injected onto the column which was equilibrated with solvent A (20 mM ammonium phosphate, pH 7.5). Sam- ples were eluted from column essentially as described by Atkins gt_al. (32). The flow rate throughout the program was 1 ml/min. The column was washed for 2 min after injection with solvent A. The sample was eluted with a linear gradient from 0 to 58% solvent B (60% (v/v) metha- tnol in water) over 23 min, followed by isocratic elution at 58% solvent B for 2 min. Percent solvent B was increased to 100% in l min, fol- lowed by isocratic elution at 100% solvent B for’5 min. The column was 89 reequilibrated with solvent A for 30 min between each injection. Purine bases and nucleosides were detected at 260 nm. The following retention times (min) were observed: xanthosine (5.9), xanthine (10.8), hypoxanthine (13.2), guanine (13.6), allopurinol (15.8), guano- sine (16.3), adenosine (22.8) and adenine (24.3). Labeling Studies Incubation Conditions. Soybeans grown on N-free nutrient solution were harvested 3 and 8 days after imbibition. Four-day—old seedlings were treated with either 0.5 mM allopurinol or 0.5 mM azaserine. Seed- lings were washed with distilled water and placed in 10 m1 beakers (l seedling per beaker) containing l.ml of 0.01 M potassium phosphate (pH 7.0), 0.5 mM allopurinol or azaserine, and 20 pl [U-14C]serine (140 mCi/mmol). The controls were incubated in buffer and labeled without inhibitor. Seedlings treated with azaserine were covered with alumi- num foil and placed on a rotary shaker (28°C) for 24 h in the dark. Four-day-old seedlings treated with allopurinol were placed under fluorescent lamps (12 h photoperiod) with continuous aeration. After 2 h, 2 ml of buffer containing 0.5 mM allopurinol was added to beakers. In the case of controls, 2 ml of buffer alone was added. After 8 h, 1.5 ml of buffer with or without allopurinol and 10 pl of [U-14C]serine were added to the solution. Eight-day-old seedlings were placed in 10-ml test tubes containing 7 ml of 0.01 M potassium phosphate (pH 7.0), 0.5 mM allopurinol and 50 pl [U-14C]serine. The base of the test tube was covered with 90 aluminum foil and plants were placed under fluorescent lamps for 24 h with continuous aeration. Extraction of Tissue. Seedlings were harvested after incubation for 24 h and separated into cotyledons and radicals for 4-day-old plants and cotyledons, roots, and shoots and leaves for 8-day-old plants. Tissue was weighed, washed twice with distilled water, and frozen in liquid N2. Samples were homogenized in 0.25 N HC104 (l g/4 ml) in a mortar and pestle and centrifuged for 5 min in an Eppen- dorf microfuge (113). Extracts were neutralized with 2 N KOH and cen- trifuged for 4 min in an Eppendorf microfuge. The acid-insoluble pre- cipitate was resuspended in l N NaOH and incubated at 37°C for 16 h (115). Aliquots (10 p1) of neutralized and NaOH-solubilized samples were placed in vials containing 9 ml scintillation cocktail (see uri- case assay) and radioactivity was determined by liquid scintillation spectrometry. Neutralized extracts were stored at -20°C until analysis by HPLC and TLC. Analysis of 14C-Labeled Products. Products of labeling studies were analyzed by HPLC and TLC. Bases, nucleosides, and nucleo- tides were separated using ion-paired, reverse-phase HPLC with a Zorbax-ODS (7 pm) column. The column was equilibrated with solvent A (5 mM tetrabutyl ammonium hydroxide, adjusted to pH 6.5 with 0.85 M H3P04) and samples (20 pl) were eluted from the column as described by Atkins gt a1. (32). The sample was eluted from the column with a linear increase in solvent B (60% (v/v) methanol in water) to 92% over 45 min at 1.0 ml/min, followed by isocratic elution at 100% solvent B for 5 min. The column was reequilibrated with solvent A for 20 min at a flow rate of 1.5 ml/min between each injection. Purines were 91 detected at 260 nm. Radioactivity was determined with a radioactive flow detector (Romac, FLO—ONE Model HS). The efficiency of the flow cell was determined using 14C-labeled standards of serine and uric acid. The delay time between the UV detector and the flow counter was determined using [2-14CJuric acid (Figure 14). The retention times for standards are presented in Figure 15. Serine, allantoin, and allantoic acid were not resolved by this program. Serine, allantoin and allantoic acid were separated by TLC (156). Neutralized extracts (25 pl) were spotted on PEI-cellulose F plates (20 x 20 cm) which had been predeveloped in distilled H20. Samples were spotted 2.5 cm from the bottom and 1.5 cm apart. One-dimensional ascending chromatography was performed using a solvent system consist- ing of 0.75 M KH2P04 (adjusted to pH 3.4 with 0.75 M H3P04). Chromatograms were developed to the tap. Sample lanes were scanned for radioactivity with a gas-flow scanner (Berthold) and the appropriate areas were cut out of the plate and placed in vials containing 9 m1 scintillation cocktail (see uricase assay). Radioactivity was deter- mined by liquid scintillation spectrometry. The relative mobility of standards is presented in Table 15. 92 Absorbance 1+”) '5 10 Retention time (min) Figure 14: Measurement of time delay between UV and radioactivity detectors. Nonradioactive 1 mM uric ag1d( (20 p1) was added to 40 pl HPLC-grade H 0 and 1 pl of O. 59 mM [2 C]uric acid (57 mCi/mmol). A 20 pl sampIe was injected onto the column and eluted as described in Materials and Methods. Absorbance was measured at 280 nm. 93 .HHm>wHeeemeN .m.NN ece N.NN wee; meEHH :ewpceueg esw .ee>HemeN HHueeHewwwem pee wee; Nwueeeweee e saw; Ngemv ezweem ece E: omN He Neee eeeweeieew He meeeeeeum ecweee we :ewHeLeeem ”mH egemwm 94 leV -—== dWl :: HVOIV % VOIV t av ‘uv ‘Jes__, l 8 a (UJU 092) eouquosqv 4O 35 20 25 Retention time (min) |5 -|0 95 Table 15: Chromatography of Purines, Ureides, and Serine on PEI-Cellulose F TLC Plates. Compound .Bi Xanthinea 0.32 Uric acida 0.36 Hypoxanthinea 0.46 Xanthosinea 0.52 XMPa 0.62 Allantoin 0.68 Allantoic acid 0.78 Serine 0.94 [U-14C]Serine (0.07 nmoles; 140 mCi/mol) was spotted on PEI- cellulose F plates. For all other standards, 20 pl of a 5 mg/ml standard solution was spotted. Plates were developed in 0.75 M KH2P04 (pH 3.4). a) Compounds were visualized under short wavelength UV. Allantoin and allantoic acid were visualized by spraying plates with 1% Erlich's reagent (151). Serine was detected by scanning for radioactivity with a gas—flow scanner. The relative migration (Rf) was determined from the middle of a spot. RESULTS Total N and Dry Weight Levels. The cotyledon of a young soybean seedling is the source of carbon and nitrogen for plant development. -In these studies, the cotyledons emerged from the Perlite by day 3 after imbibition. The first true leaf emerged on day 6 and expanded by day 8 after imbibition. The changes in the patterns of dry weight (Figure 16) and total N levels (Figure 17) within the cotyledons were the same for plants grown in the presence or absence of N03. Within 2 days after imbibition a decrease in total N was observed for the cotyledons. The %N of the cotyledons, however, remained at 6 - 7% N throughout the period analyzed. Thus, equal amounts of dry weight (carbon) and nitrogen were being lost (transported) from the cotyle- dons. The roots and shoots of control plants grown on N-free nutrient solution and those grown on 10 mM N0§ had similar total of N and dry weight patterns until day 10 (Figure 16,17). After which time, the total N in plants grown on N03 appeared to increase with respect to control plants. The differences in dry weight after day 10 was not as significant (Figure 16). Ureide Determinations on Control Plants and Plants Grown on 10 mM Nitrate. The level of total ureides present in the cotyledons and in the embryonic axes, that tissue which develops into the roots and shoots of the soybean, was determined during the period 0 to 18 days after imbibition (Figure 18). Allantoic acid accounted for 96 97 Fi ure 16: Values for dry weight of developing soybean seedlings. Plants were grown on either N-free nutrient solution (open symbols) or nutrient solution supplemented with 10 mM KN03 (closed symbols). Plants were harvested and separated into cotyledons (0,0) and axes (A,A). Plants were dried for 48 h at 60°C and the dry weight was determined. Each value is the mean of 2 experiments i standard error. Each experiment was performed in triplicate. 98 o N-free, cotyledons O Nitrate, cotyledons 200' A N-free, rooialeoves A Nitrate, root 81leaves l / i 150* ~55: \ae / -— o I >“3100- / 5.5 QOINN’QAN /i 50 6% (3:13 --0 q q ‘ Days6 after lmbi2bition 99 Figure 17: Total N levels in developing soybean seedlings. Plants were grown on either N-free nutrient solution (open symbols) or nutrient solution containing 10 mM KN03 (closed symbols). Plants were harvested, separated into cotyledons (0,0) and axes (A,A), and dried at 60°C for 48 h. Values for total N were determined as described in Methods of this Chapter. The amount of stored N03 was determined as described in Chapter 1. This amount was subtracted from the total N. Each value is the mean of 2 experiments i standard error. Each experiment consisted of 3 replicates. Total N (mg N o partE') 100 5 01 0 N- tree, cotyledons O Nitrate, cotyledons A N- free, rootaleaves A Nitrate, root 81|eaves Days after lmbibition 1;\ l TEQIBR I/l/ IYN _ (VINY/ f/f\/ /4. X531) k;§ , f . ? 101 approximately 80% of the total ureides present in all tissues. The amount of total ureides in the cotyledons increased until day 6, after which time a gradual decrease in the level of ureides was observed (Figure 18). Nitrate appeared to have no effect on the pattern or level of ureides present in the cotyledons. The level of ureides in the cotyledons was greater than in the axes until day 5. At this time the level of ureides in the axes was substantially greater than that observed for the cotyledons. At the peak of ureide accumulation for both tissues, ureide-N represented 0.7% and 6% of the total N of cotyledons and axes, respectively. Nitrate appeared to have no effect on the level of ureides present in the axes prior to day 10. After day 10 the amount of ureides in the axes of plants grown on nitrate was greater than that of control plants. Patterns of Uricase and Allantoinase Activities During Seedlingfi Development. Uricase activity was not detected in seedlings using the standard assay in which a decrease in the concentration of uric acid is monitored at 293 nm (Table 16). However, uricase activity of the soy- bean nodule was easily detected with this procedure. The assay finally employed entailed the incubation of enzyme extract with [14C]uric acid and the separation of products by thin layer chromatography (153, 154). The use of [14-C3uric acid increased the sensitivity 100- fold, thus enabling the detection of uricase activity in the roots and cotyledons of the seedling (Table 16). Even with this increased sensi- tivity, no uricase activity was detectable in the developing first leaves of the seedling. Uricase activity was measured during the period 0 to 18 days after imbibition (Figure 19 and 20). The amount of uricase activity in the 102 Figure 18: Total ureide concentrations in developing soybean seedlings. Plants were grown on N-free nutrient solution (open symbols) or nutrient solution containing 10 mM KN03 (closed symbols). Plants were harvested, separated into cotyledons (0,0) and axes (A,A). Ureide determinations were made as described in Methods. The values presented for cotyledons are for a pair of cotyledons (part). Each value is the mean of 2 experiments i standard error. Each experiment was performed in triplicate. Total Ureides (umoles . part") N 'O .01 O 9' o '0 0.5 103 e N-free, cotyledons o Nitrate, cotyledons A N- free, rootaleaves A Nitrate, root 8 leaves Days after Imbi bition /4/ T C/QKSI k 1‘ ° \1\ / \9 ?\:\9 o/gf I o 6 12 18 104 Table 16. A Comparison of Two Methods for Determining Uricase Activity. nmoleS°min'1°mg protein"1 Tissue Assay 1 Assay 2 Nodulesl 155- 172 Cotyledons2 ND3 1-2 Roots2 N03 16-16.5 Extracts of tissue were prepared as described in Methods. Assay 1: Uricase activity was measured by monitoring the change in absorbance at 293 nm as described by Hanks et al. (123 AssayZ: Uricase activity was determined by measuring the conversion of [1 CJ- labeled uric acid into allantoin as described in Methods. 1. Nodules were removed from 48-day-old soybeans. For Assay 1, 20 to 50 pl aliquots of nodule extract were assayed. For Assay 2, nodule extract was diluted lOO-fold. An aliquot (10 pl) of diluted extract was assayed. 2. Cotyledons and roots were from 7-day-old plants. 3. N0 = not detectable. 105 dry seed and a seed after 1 h imbibition was determined. A value of 2.5 t 0.1 nmoleS°part"]-'min‘1 was observed in both samples (data not shown). Uricase activity was expressed as specific activity (Figure 19) or total activity (Figure 20). The specific activity of root uricase increased until day 7 after which time a steady decrease in activity was observed. Low levels of activity were found after day 10. The specific activity of uricase in the cotyledon was negligible (1-2 nmole5°mg protein'1°min'1). There was little change in activity during the period examined. Nitrate appeared to have no effect on either the pattern or level of uricase activity in the coty- ledons and roots. Expressing uricase activity as nmoleS°part"1°min‘1 (total activity) did not change the pattern observed within the roots (Figure 20b). The peak of uricase activity in the roots occurred on day 7. However, a definite difference in the pattern of uricase activ- ity in the cotyledon was observed between specific activity and total activity data (Figure 19 and 20a). Total uricase activity in the coty- ledons peaked 2 to 4 days after imbibition. The total of activity in the cotyledons during this early period was greater than that observed for the roots during the same period. Nitrate had no effect on allantoinase activity in either the roots or cotyledons of developing soybeans (Figure 21). Allantoinase activ- ity was expressed as pmoleS°part‘1°min'1. The pattern of allantoinase activity in the cotyledon was similar to that observed for uricase activity. Allantoinase activity in the cotyledon increased un- til day 4. The level of allantoinase activity in the cotyledon on day 4 was lO-fold greater than that measured in the roots. The level of 106 Figure 19: Specific activity of uricase in the cotyledons and roots of developing soybean seedlings. Plants were grown on N-free nutrient solution (open symbols) or nutrient solution containing 10 mM KN03 (closed symbols). Plants were harvested and separated into cotyledons (0,0) and axes (A,A). Each value is the mean of 2 experiments 1 standard error. Each experiment was performed in triplicate. Unless noted standard error values are less than or equal to the size of the symbol used. 107 G Uricase Activity 6 nmoles . min“l - mg protein" 01 5 3N0 \3/i I ' 6 1'2 . '18 Days after Imbibition 108 Figure 20: Total uricase activity in the cotyledons and roots of developing soybean seedlings. Plants were grown on N-free nutrient solution (apen symbols) or nutrient solution containing 10 mM KN03 (closed symbols). Plants were harvested and separated into cotyledons, panel A (0,.) and roots, panel B (A,A). Total uricase activity is expressed as nmoles ureide produced‘min‘ °part‘1. For coty- ledons a part is a pair of cotyledons. Each value is the mean of 2 experiments i standard error. Each experiment was performed in triplicate. The means of the two experiments were averaged. uricase Activity nmoles . min’l . part"l 109 A. Cotyledons 8. Roots l T l T T I o 6 12 18 o 6. .. 12 Days after Imbibition 110 Figure 21: Total allantoinase activity in cotyledons and roots of developing soybean seedlings. Plants were grown on N-free nutrient solution (open symbols) or nutrient solution containing 10 mM KN03 (closed symbols). Plants were harvested and separated into cotyledons (0,0) and roots (A,A). Total allantoinase activity was expressed as pmoles allantoic acid produced'part'1°min‘1. For cotyledons a part is a pair of cotyledons. Each value is the mean of two experiments i standard error. Each experiment was performed in triplicate. The means of the two experiments were averaged. 1.0 Allantoinase Activity umoles a part". m1n'l O .0 (D (I) .0 A 111 T § Days after lmbibition 112 allantoinase activity in the roots did not vary, in contrast to the developmental pattern observed for uricase activity in the root (Figure 19 and 20). The amount of allantoinase activity in either tissue was substantially greater than that determined for uricase activity. Leaf allantoinase activity was assayed from days 8 to 18. The level of allantoinase activity was constant (70 pmoleS°part‘1-min'1). Inhibition Studies. The amount of ureides in various tissues of plants treated with the inhibitors, allopurinol and azaserine, was examined (Table 17). Azaserine and allopurinol were found to inhibit ureide production in the cotyledons of 3-day-old soybeans, while no inhibition was observed in the roots. Azaserine had no effect on ureide accumulation in the cotyledons of 8 and lZ-day-old seedlings, however, a substantial inhibition was observed with allopurinol. A comparable decrease in ureide content in the roots was found in the presence of both inhibitors during this period. The inhibition of ureide accumulation in the shoots was similar to that observed in roots. The levels of xanthine in extracts of tissues treated with allo- purinol was examined by HPLC. A standard curve of area vs. nmoles was constructed by injecting known concentrations of xanthine onto the column and by monitoring the absorbance at 260 nm. The response was linear between 2-7 nmoles of xanthine (Figure 22). The extracts of tissues treated with 0.5 mM allopurinol were found to contain substan- tial quantities of xanthine (Table 18). The cotyledons were the only tissue that contained measurable amounts of xanthine in the absence of treatment with allopurinol. The cotyledons accumulated approximately 160 nmoles xanthine-g fr. wt.‘1 more than the amount found in 113 Table 17. Inhibition of Total Ureide Accumulation by Allopurinol and Azaserine. % of Control Plant Age Cotyledons Roots Shoots Days Az Al A2 A1 A2 A1 3 59 t 6 72 i 2 94 i 4 93 z 5 O O 8 92 i 8 80 : l 80 1 1 78 i 6 79 i l 79 i 5 12 92 z 2 64 i 1 64 i 2 68 i 5 72 i 3 68 i 4 Plants were treated for 24 h with either 0.5 mM azaserine (A2) or 0.5 mM allopurinol (Al). Controls are plants treated for 24 h with only buffer (0.01 M potassium phosphate, pH 7.0). The levels of ureides (nmole5°g tissue' ) in the control plants were as follows: For cotyledons, 600 i 50, day 3; 857 i 50, day 8; 535 i 30, day 12; For roots, 1904 i 100, day 3; 1799 t 120, day 8, 1189 i 70, day 12, and for shoots, 4734 i 80, day 8; 3628 i 100, day 12. Each value is the mean of 3 experiments 2 standard error. Each experiment was performed in triplicate. '114 l l l 1 2 3 4 5 6 7 nmol xanthine Figure 22. Standard curve for xanthine determination. Standard concentrations of xanthine were injected onto a Whatman Partisil ODS-2 column and eluted as described in Methods. Absorbance was monitored at 260 nm and the peak area was integrated by a Hewlett-Packard 3390A integrator. 115 Table 18. Accumulation of Xanthine in Plants Treated with Allopurinol. nmoles Xanthine°g fr. wt"1 Plant Age Days Cotyledons1 Roots Shoots 3 162 i 22 268 i 4 - 8 157 i 9 165 i 9 266 z 18 12 129 i 25 70 i 4 205 i 21 1The cotyledons contained measureable amounts of xanthine in the absence of treatment with allopurinol. The following amounts of xanthine were found in untreated cotyledons: 158 i 39, day 3; 80 i 16, day 8; 144 i 46, day 12. This amount was subtracted from the amount of xanthine in the cotyledons of plants treated with allopurinol. Each value is the mean of 2 experiments 3 standard error. 116 cotyledons of plants incubated for 24 h in the absence of allopurinol. The levels of xanthine in the roots of seedlings treated with allopuri- nol decreased as the plants aged. The greatest concentration of xan- thine was found in the shoots. The amount of allopurinol in the vari- ous plant parts was also measured by HPLC. The roots contained about 600 nmoles allopurinol°g fr. wt.'1 at all harvests. The cotyledons and shoots accumulated approximately 300 nmoles allopurinol°g fr. wt'l. The decrease in ureides for the cotyledons (Table 19) was compara- ble to the increase in xanthine observed. On day 3, the decrease in the amount of ureides within the roots equaled the amount of xanthine accumulated. As the plants aged, the decrease in the level of ureides was greater than the amount of xanthine accumulated within the roots. The greatest decrease in the amount of ureides accumulated was found in the shoots (Table 19). The decrease was substantially greater than the level of xanthine accumulated. Labeling Studies. The first labeling studies were performed in a detached system with [U-14Cngycine. Plants were harvested and separated into cotyledons and roots prior to incubation with the label. A time course of uptake and incorporation of [14C]glycine was determined for 5-day-old soybeans (Table 20). An increase in the total label incorporated into the cotyledons was observed with respect to time. No change in the amount of label incorporated within roots was observed with increasing time of incubation. The soluble fraction was subjected to TLC analysis to determine what compounds were labeled. The only radioactivity recovered was in the area corresponding to gly- cine. Plants of various other ages were harvested and incubated with [14C]glycine for 12 h or 24 h. In all cases, the label was 117 Table 19. Decrease in Total Ureides for Plants Treated with Allopurinol. nmoles Ureide5°g fr. wt."1 Plant Age Days Cotyledons Roots Shoots 3 171 1 20 260 1 40 - 8 177 1 4 325 1 56 920 1 15 12 187 1 26 407 1 60 1222 1 100 The decrease in total ureides due to treatment with allopurinol was calculated as follows: Decrease (nmoles ureide5°g fr. wt.‘1) = (Total ureides control) - (Total ureides allopurinol treatment). Each value is the mean of 3 experiments 1 standard error. Each experiment was performed in triplicate. 118 Table 20. Time1 Course of Labeling Cotyledons and Roots of 5- -day- -old Soybeans with [1 4C]G1ycine. Radioactivity dpm'(g fr. wt.) 1 (x10 5) Length of Soluble Insoluble Incubation (h) C R C R 6 37 61 16 29 12 61 65 24 36 24 81 56 32 30 Soybeans (5- day-old) were separated into cotyledons (C) and roots R). Tissues were incubated with O. 45 ml H20 and 25 pl [U- Cngycine (100 mCi/mmole; 5.5 pCi/ml) for the indicated time. Tissue was acid-extracted and radioactivity in the acid-soluble fraction (soluble) and acid-insoluble fraction (insoluble) was determined (see Methods). Each value is the mean of two replicates. 119 recovered in glycine. Approximately 40% of the total radioactivity was not recovered in the tissue extract, acid-insoluble fraction or the re- action mixture after incubation. Presumably some of the radioactivity may have been lost through metabolism of [14C]glycine to 14002. Labeling studies using [U-14C]serine were performed with in- tact seedlings. whole seedlings (4 and 8-day-old) were incubated with [14C]serine for 24 h. The total radioactivity incorporated into the acid-soluble and insoluble fractions of cotyledons and roots was examined (Table 21). For 4-day-old plants 95 1 3% recovery of the label added was observed while for 8-day-old plants only 60 1 5% of the label was recovered. In 4-day-old plants the cotyledons contained more label in the acid-soluble fraction than the roots. Both the cotyledons and roots incorporated the same amount of label in the acid-insoluble fraction (proteins, nucleic acids, etc.) at this time. The distribu- tion of label within detached 4-day-old cotyledons was also examined. The amount of label in the acid-soluble fraction was 24 1 3 x105 dpn'(g fr. wt)‘1 while incorporation of label in the insoluble fraction was 16 1 1 x105 dpm°(g fr. wt)'1. In 8-day-old plants, the majority of the label was present in the roots. Little incorporation of label in the cotyledons was observed. An equal dis- tribution of label between soluble and insoluble fractions was observed for the shoots. The distribution of label within the acid-soluble fraction of 4-day-old cotyledons was examined by HPLC (Figure 23). The cotyledons from soybeans were treated with allopurinol (Figure 23a) and the dis- tribution of label was compared to that for cotyledons incubated in the absence of inhibitor (controls, Figure 23b). Most of the radioactivity 120 Table 21. Total Radioactivity in RootsI Shoots, and Cotyledons of 4 and 8-Day-Old Soybeans Incubated with [ 4C]Serine. dpm°(g fr. wt.)'1 (x10‘5) Soluble Fraction Insoluble Fraction Plant Age C R S C R S 4 20 1 2 7 1 1 - l6 1 4 17 1 4 - 8 l 1 0.2 42 1 3 5.6 1 0.3 l 1 0.1 64 1 3 6.7 1 l Intact plants were incubated for 24 h with [0-1401serine (140 mCi/ mmole; 0.5 pCi/ml) as described in Methods. After incubation plants were separated into cotyledons (C), roots (R) and shoots (S). Tissues were acid-extracted and radioactivity in the acid-soluble (soluble) and acid-insoluble fraction (insoluble) was determined (see Methods). The plant age refers to the number of days after imbibition. For 4-day-old plants, 95 1 3% of the label was incorporated. For 8-day-old plants 60 1 5% of the total label was incorporated. Each value is the mean of 3 experiments 1 standard error. 121 .ceNHeeHee HHN>wHeeewee1 eH eeueeeeee :eee e>ez eye 5: omN He eeeepweee we; we AH: ONV NONNNHe e< .Ameeeedzv eea_: e NN_>_eoeeNeec eeceeeeme< .ee eee :ewueepee eeceegemee :eezuee any New meswu :ewHeeHeL mew 1e_e _._V 81_e NeHNH 611 Nod—we: .NeHeeeeizeHw e an Huw>wueeewee1 ece eweemee me empeHe ece :EeHee mooixeegeN e :e eeueewcw we: Heeguxe Umwb mm mm: 03mm?“ we cowHUMwam . NoeHv ecweemmuwflizu mcweweucee No.N :ev Newwee euegemece Ee_mmepee .< Hecee .Heeweeee e 25 m.o ece eeweem mo Hg .Lewwze :sz eeHeezecw e1 ewguwz e=HLemmu¢HH Eeew HeeeH we :ewuee.gpmwe en» ce HeeEHeeLH Heewg N Heaea .A_e\_e\: m.e .e_ass\_ee Ho.o cw eeueeze=N wee: mpceHe 1e e3 muceHN .mceeeHHHee eHenzeeie see—He we Heewwe esw .mN egzmwm EEV eEc. cozceEm Om ON 0_ O' 122 q = - = _ = — 1: IL 1 3 H1 14.. Z _ : ... . nol? 10110 T41 ...-I8?- “—_ l (o--o--o) mu 092 V O N114 116.06.4afi91111101a «I» ...: 1 N 10 {—1 ( 9-01 x) “193 123 for the controls and cotyledons treated with allopurinol was recovered in the first peak. This peak corresponded to allantoin, allantoic acid and serine which were not resolved on this column. The amount of label recovered in the peak at 13.6 min was different between plants treated with allopurinol and those incubated for 24 h in the absence of inhibi- tor. The retention time of this peak corresponded to that of uric acid. The identity of this peak was confirmed by separation on TLC plates. Label was incorporated into uric acid in the cotyledons of the plants grown in the absence of inhibitor (Figure 23b). Treatment with allopurinol led to the disappearance of the uric acid peak while the amount of label in xanthine (RT = 8.9 min) was found to increase (Figure 23a). Some other small peaks were observed in the allopurinol- treated plants that were not found in the controls. The identity of these peaks is unknown. The peak at approximately 39 min may corre- spond to XMP, however, no verification by TLC was possible. The separation of allantoin, allantoic acid and serine was achieved by TLC. The percent of total radioactivity in these compounds for 4-day-old cotyledons was determined (Table 22). In plants incu- bated in the absence of allopurinol, the ureides represented 42% of the total radioactivity in the cotyledons. Some radioactivity was found in uric acid and xanthine, with the remainder being in the serine frac- tion. Treatment with allopurinol led to the disappearance of label in uric acid and a decrease in the percentage of label now in the ureides. The majority of the label was present in serine. Treatment with azaserine resulted in the label remaining in serine. Cotyledons were detached from the plants prior to labeling and incubated as described. The distribution of label within detached 124 Table 22. Distribution of Total Radioactivity in the Soluble Fraction of 4-day-old Seedlings Treated with [14CJSerine. % Total Radioactivity Intact Seedling Detached Tissues Compounds C A1 A2 C Al A2 Xanthine 4 15 0 O 6 0 Uric acid 6 0 0 8 0 0 Allantoin 2 1 O 7 1 0 Allantoic acid 40 13 O 50 18 0 Serine 42 58 100 28 61 100 Soybeans (4-day-old) were either incubated intact (whole plant) or the cotyledons were excised (detached) and then incubated with [U- 4C]serine (140 mCi/mmol, 0.5 pCi/ml). The incubation mixture contained 0.01 M potassium phosphate buffer (pH 7.0) alone (C), or buffer plus 0.5 mM allopurinol (A1) or 0.5 mM azaserine (A2). Each value is the mean of 3 experiments. The standard error was less than 5%. Allantoin, allantoic acid and serine were separated by TLC (see Methods). Xanthine and uric acid were separated by HPLC as well as TLC. 125 cotyledons was similar to that observed for cotyledons in the intact labeling system for both allopurinol-treated and non-allopurinol- treated plants (Table 22). Greater incorporation of label into the ureides was observed for control plants of the detached labeling system compared with the intact labeling system. Again treatment with allo- purinol led to a decrease in the amount of radioactivity recovered in the ureides. Examination of the labeling pattern in 4-day-old roots was difficult since the amount of radioactivity in the extract was very low (approximately 1/3 of the radioactivity found in the cotyledons). The only measurable peak of radioactivity corresponded to the serine peak. The distribution of label within the roots of 8-day-old soybeans was also examined (Figure 24). The roots of plants incubated in the absence of inhibitor were found to have two peaks of radioactivity (Figure 24b). One corresponded to the allantoin, allantoic acid and serine peak. The other was uric acid. Treatment with allopurinol led to the complete disappearance of radioactivity in uric acid. Instead, a large amount of radioactivity was recovered in xanthine (Figure 24a). The distribution of label in the shoots and cotyledons was similar to that found in the roots (Table 23). The percent of label in uric acid for 8 day old cotyledons was substantially greater than that found for 4-day-old cotyledons. Separation of allantoin, allantoic acid and serine was performed on roots. Analysis of shoot and cotyledon extracts by TLC was not pos- sible. These samples contained very low amounts of radioactivity (20 icpn/pl, cotyledons; 50 cpm/pl, shoots). The loading of large volumes (>50 pl) onto the TLC plates generally resulted in‘a distortion of 126 .cewueepee pr>wpeeewee1 ece cewHeeHee eeeeeeemee ceezpee Ncwe H.HV NEHH HeHee egu HeeHweN op eeueeeeee :eee e>e; euee NHw>wHeeewee1 map New meswp cowpcepee ecN .NeucseeizeHw e He pr>wueeewee1 ece E: omN He eeeepwees we; eeceeeeme< .eeeweemee me eeusHe ece caeHee mooixeeeeN e :e eepuewcw we; Heeepxe we :1 eNV eeeeze eN .Ameefiezv eeezemee we we: 8:3: we 8.5623 .N 8:8 .255: me .3858 quv ecweemmu H12; mcwcweucee No.N :av Newwse muezemege Ezwmmeuee z Ho.o cw eeueezecw mew: mpceHe .ee .< Hecee .HecmeeeeHHe ze m.o eee ecweemmueHg .eewwee spwz empeeeecw wee; cheHN .mueee eHeizeeiw cwcpwz ecweemmong Eeew HeeeH we cewueewepmwe esp :e uceEueeNH HecweseeHHe we Heewwe we» .HN eeemwu 127 2:5 05:. cezcewem OV Om ON 0_ - I a,W-IT'JIKNOIQJT‘NI9101IO(3)NI10.111910. TIIOIOIOIOIOO<0 to, .91 7. I Z _ . _ . . . u. .m 1 a. La . 1 16191610101914 0101019.... 0.0.6.. TI'QIA 1.1.0.5 .4 V 1.1.0.4..) 1 .1 ..1. ...eN N < N ... N <4, .H a.— w a n 2 — 1 (—1 (2-015) Wd° 128 Table 23. Distribution of Total Radioactivity in the Soluble Fraction of 8-day-old Seedlings Treated with [14CJSerine. % Total Radioactivity Roots Cotyledons Shoots Compound C AL C AL C AL Xanthine O 37 0 53 3 50 Uric acid 39 O 44 0 33 3 Allantoin 7 l Allantoic acid 22 7 4O 4O 40 40 Serine 26 28 Soybeans (8-day-old) were incubated with [U-14C]serine (140 mCi/mmole; 0.5 pCi/ml) as described in Methods. Controls = C and allopurinol treatment = AL. Each value is the mean of 3 experiments. Each experiment was performed in triplicate. Standard error less than 5%. Xanthine and uric acid were separated by TLC and HPLC. Allantoin, allantoic acid and serine were separated by TLC. For cotyledons and shoots the level of radioactivity was not sufficient to detect allantoin, allantoic acid and serine separated by TLC. 129 relative moblities. Attempts to concentrate the samples led to variable losses in radioactivity. In the roots ureides were found to represent 29% of the total radioactivity in control plants (Table 23). Treatment with allopurinol resulted in a decrease in the percentage of label in the ureides. The rate of incorporation of serine into the ureides was similar for 4-day-old cotyledons and 8-day-old roots ( 1.0 x 106 dpm’g fr. wt.‘1°24 h'1). A lO-fold difference in the amount of label from serine incorporated into uric acid during the 24 h incubation was observed between the two tissues. Cotyledons from 4-day-old plants incorporated 0.1 x 106 dpm°g fr. wt.‘1°24 h"1 into uric acid while for 8-day-old roots 1.6 x 106 dpm'g fr. wt.'1°24 h‘1 was observed. DISCUSSION The pattern of ureide accumulation within the cotyledons (Figure 18) was similar to that reported by Matsumoto 2£.Ql° (27). Examination of ureide levels in the roots and shoots, however, revealed that these tissues also accumulated ureides. The cotyledons appeared to accumu- late ureides prior to the appearance of measureable levels of ureides in the developing axes, suggesting that early synthesis of ureides was carried out by the cotyledons. Nitrate had no effect on ureide accumu- lation in the cotyledons and only altered ureide levels in the roots and shoots after day 10. The fact that this difference was not evident until after day 10 suggested that nitrate was not being metabolized prior to this date. The total N data supported this conclusion (Figure 17). Until day 10 the roots and shoots of plants grown on nitrate had total N values comparable to those of roots and shoots of control plants. After day 10, total N increased in roots and shoots of plants grown on nitrate while the total N in controls had leveled off. The inability of soybean seedlings to utilize nitrate before day 10 was probably related to the low activity of nitrate reductase (NR). NR is primarily a leaf enzyme in soybeans (142). Since the first leaf did not emerge until day 6 and was not expanded until day 8, the reduc- tion of nitrate prior to leaf emergence was unlikely. Measureable NR activity in young soybean seedlings prior to leaf emergence (day 6) has not been observed (Wilbur Campbell, personal communication). Thus, the 130 131 effect of N03 on ureide accumulation in roots and shoots after day 10 may reflect either an increase in ureide synthesis due to higher N levels or a decrease in the utilization of ureides due to the avail- ability of another N source. The patterns of activity for 2 enzymes involved in ureide synthe- sis, uricase and allantoinase, were examined in order to determine the site of ureide synthesis and to investigate the effect of nitrate on ureide synthesis. Comparison between the enzyme activities of plants grown on nitrate or N-free nutrient solution suggested that ureide syn- thesis was not altered by nitrate (Figure 19-21). Treatment with nitrate did not change the level or developmental pattern of uricase or allantoinase activities. Differences in the patterns of uricase activity for roots and cotyledons, however, were observed. Since the cotyledons contained high levels of storage protein, total uricase activity was determined on a pair of cotyledons basis. Total uricase activity in the cotyle- dons was higher at early harvests than the activity of the root enzyme. The pattern of activities positively correlated with the accumulation of ureides observed for both tissues. A high level of uricase activity was found in the roots at a time when cotyledon activity was declining, suggesting that roots were capable of synthesizing ureides, at least in part. Uricase activity was not detected in the leaves, therefore, the ureides in this tissue presumably arose via transport from other plant tissues. The pattern of allantoinase activity in the cotyledons was similar to the pattern of uricase activity observed. However, the level of allantoinase was lOO-fold greater than the uricase activity. 'A similar 132 difference in the level of activity of these two enzymes has been reported (157). The patterns of uricase and allantoinase activity in the cotyledons of soybeans were different than the patterns reported for mustard cotyledons (157). Allantoinase activity in mustard rose rapidly beginning on day l and 2 and reached a maximum value 8 days after sowing. However, uricase activity did not reach a maximum until 12 days after sowing which corresponds to the beginning of chlorophyll breakdown (an indication of senescence). The breakdown of DNA has been associated with the latter stage of senescence within the cotyledons (158). Therefore, in mustard, uricase activity may be linked to senes- cence and thus to ureide production via nucleotide breakdown. Ureide production in wheat seedlings has also been linked to the senescence of the endosperm, the seed storage organ (112). The soybean system was unique in that the production of ureides and the patterns of allantoi- nase and uricase activity were not linked to the senescence of the cotyledons. Ureide production in the cotyledons reached a maximum on day 6 while the beginning of chlor0phyll breakdown, senescence, was not until day 13. While cotyledons and roots appear to have the capacity for purine degradation, there was no direct evidence for ureide production via gg ggxg purine synthesis previously. The ureides may arise via nucleotide breakdown. Legume cotyledons have been reported to undergo endoredup- lication during embryogenesis (158,159). Endoreduplication is the process in which the copy number of DNA increases within a cell (158). Therefore,soybean seedlings contain substantial quantities of DNA which represent a readily available source of purines for ureide synthesis. However, Dhillon and Miksche (158) reported that the DNA concentration 133 in cotyledons of soybeans did not decrease until ten days after germi- nation. Since ureide accumulation was found earlier than this period (Figure 18), it was unlikely that the ureides were arising via the breakdown of DNA. The pathway of ureide synthesis was examined by treatment with azaserine, which prevents the transamidation of the amide-N of gluta- mine to FGAR (160), and allopurinol which blocks the enzyme xanthine dehydrogenase (161). Allpurinol and azaserine inhibited ureide produc- tion in 3-day-old cotyledons (Table 17). No inhibition was observed in the roots of 3-day-old plants, again suggesting that early synthesis of ureides was carried out by the cotyledons. The inhibition by azaserine indicated that the production of ureides was not via the breakdown of preexisting purines. Fujihara and Yamaguchi (128) performed similar experiments and also observed no difference in ureide levels of 3 day old roots after treatment with the azaserine. They concluded that g; ‘ggxg purine synthesis was not involved in ureide production. However, no examination of the effect of azaserine on the level of ureides with- in the cotyledons was presented. The inhibition by allopurinol and the lack of inhibition by aza- serine in 8 and 12-day-old cotyledons suggested that the ureides in these older cotyledons may be arising via nucleotide breakdown. The inhibition of ureide production by both allopurinol and azaserine in the roots of 8 and 12-day-old plants correlated with the pattern of uricase activity observed in the roots. Roots appeared to be capable of producing ureides via de novo synthesis of purines. Azaserine and allopurinol were found to inhibit ureide accumulation in the leaves. This inhibition would suggest that the leaves were capable of ureide 134 synthesis. However, the lack of uricase activity in this tissue would indicate that the leaves do not have the capacity for purine oxidation. Since the inhibition studies were performed on an intact system, the decrease in ureide levels in the leaves may reflect the inhibition of ureide synthesis in the roots resulting in a decrease in the transport of ureides to the leaves. The accumulation of xanthine in tissues treated with allopurinol (Table 15) instead of hypoxanthine has been reported for other plant systems (117-119). In the cotyledons the accumulation of xanthine corresponded to the decrease in the level of ureides present, suggest- ing that no utilization of ureides had occurred. A similar result was observed in 3 day old roots. However, on days 8 and 12, the decrease in ureide accumulation in the roots was greater than the amount of xan- thine accumulated in this tissue. This corresponded to the period of leaf emergence. Therefore the difference between the decrease in ureides and the accumulation of xanthine probably reflected the increased transport of ureides from the roots to the shoots. The data obtained from the azaserine inhibition studies suggested that ureide production via gg ggyg synthesis of purines may occur within the cotyledons or roots at various developmental stages. To confirm the role of g; gggg synthesis of purines in the production of ureides, plants were incubated with [14Cngycine or [14C]serine. Preliminary labeling studies with [14C]glycine were unsuccessful. No incorporation of label into purine intermediates was observed. Incubation of 4-day-old seedlings with [14C]serine resulted in 42% of the total radioactivity in the acid-soluble fraction from cotyledons being in ureides. Some radioactivity was recovered in uric acid and 135 xanthine, while the remaining 42% Was unincorporated serine. Treatment with allopurinol resulted in the expected decrease in the incorporation of label into ureides and uric acid. The data for 4 day old roots sug- gested no ureide production via gg_ggyg synthesis of purines was occurring. For 8-day-old plants the majority of the label was found in the roots. Little radioactivity was recovered in the cotyledons. However, analysis of the acid-soluble fraction from cotyledons revealed that 40% of the label was uric acid. This accumulation of label in uric acid positively correlated with the decreased level of uricase activity in 8-day-old cotyledons. From the azaserine inhibition studies no gg.gggg synthesis of purine was expected in 8 day old cotyledons. The sensi- tivity of the labeling studies would enable one to monitor low levels of gg.ggyg synthesis of purines while changes in ureide concentrations due to treatment with azaserine would not be detected. _In the roots of 8-day-old plants, measurable amounts (10 nmole5°fr. wt.‘1°24 h'l) of .gg.ggyg purine synthesis were observed and recovery of label in the ureides was possible. In contrast to reports from other investigators (22,128), these data indicate that g; ggyg purine synthesis is involved in the produc- tion of ureides in young soybean seedlings (Figure 25). In 4-day-old cotyledons ureides arise via g3 ggxg synthesis of purines, as shown by the inhibition of ureide synthesis by azaserine and the incorporation of label from [14C]serine into allantoin and allantoic acid. At later harvests, the ureides may result from the breakdown of pre- existing nucleotides within the cotyledons. Low levels of uricase activity were observed in 8—day-old cotyledons. Also, azaserine no 136 longer inhibited ureide accumulation in these tissues while incubation with allopurinol resulted in a decrease in ureide levels. The source of ureides in 4-day-old soybean roots was probably the cotyledons, since the roots were found to have low levels of uricase activity, aza- serine and allopurinol did not alter the levels of ureides in this tis- sue, and label from [14C]serine was not incorporated into ureides. As the plant matured the roots were found to have high levels of uri- case activity and the accumulation of ureides in this tissue was inhib- ited by both azaserine and allopurinol. Also, label from [14C]serine was incorporated into allantoin and allantoic acid. Thus, 8-day-old roots appeared to acquire the capacity to synthesize ureides via gg novo synthesis of purines. 137 .mp:e>e HHerch Heemeeeee me:_H eezmea .mceeexem eHeiaeeiw ece e c_ meeweez we Heeemeeep eee mwmesuczm Lew Heeee < ”mN egzmwm EmeeeepeE 41111111 mmonN: 4H me>ee1 EmHHeeeHeEH/ I, - mwmezpexm .1, - mwmecucxm Emfieeeeee illl_ mafia: ..1 .ecweee TNQHNNE Al .I 1.12.23 e>e= mm A, - e>ee me a mueeN mucou— Emw Heeepea «1 EmeeeeHeE «I 11 1.11- - mwmeNHch 1.12 - mwmegueam mNoHNN: 41 1....1 11 ecwemm. _mNonN=_« ecwemm e>e= me 111! - e>e= we :zeexeege :2eexeeee..1. eewaeeHeee eewpeeHeee mceeeHapeu mceemHHHeu- mceeexem eHoiaeoim mceeeaem eHoixeoie APPENDICES w—fT' ' 1 1| ""7"” " APPENDIX I. Derivation of Equation used for Mass Spectral Analysis ° = 15 _ 15 N f1xed (mg) corrected atom % N 0.010 atom % N(Total N - Seed N) 0.366 atom %15N - 0.010 atom %15N l. The corrected atom %15N reflects the relative contribution of N2 fixation and the utilization of combined N to plant N. This value is derived from the atom %15Nobs which has been corrected for the relative contribution of seed N to plant N. The equation for this correction of %15Nobs is given in the Results section of Chapter 1. Thus, corrected atom %15N = 1N2 (0.366%) +41comb.N (0.010%) where: ipNZ = The fraction of N derived from N2 fixation 0.366 % The atom %15N at natural abundance; atomspheric dinitrogen was assumed to be at natural abundance. 11combN = The fraction of N derived from the combined N source, either NH4+ or N03 depending on treatment. 0.010% = The atom %15N of the 15N-depleted combined N source. 138 I "7.". .. 3. 139 1comb. N = 1- 1N2 Therefore: corrected atom %15N 1N2 (0.366%) + (1 - 0N2)(O.010%) 4N2(0.366 % - 0.010%) + 0.010% 4N2 (0.366% - 0.010%) = corrected atom %15N - 0.010% multiply by Ach where: Ach = Total N - Seed N (Accn)(¢N2)(0.366% - 0.010%)=[Corrected atom %15N-0.010%](Ach) (Ach)(¢N2) = N fixed = The amount of N derived from N2 fixation Therefore: N fixed (0.366% - 0.010%)=(corrected atom %15N-0.010%)(Ach) divide by (0.366% - 0.010%) N fixed = corrected atom %15N ' 0‘010%’A N) m- 0.010%“ ‘ CC substitute (Total N - seed N for Ach) N fixed = corrected atom %15N - 0.010 atom %15N, 0.366 atom %15N - 0.010 atom %15N ‘T°t°‘ N’seed “1 ...... 140 Appendix II: Total N of Roots and Shoots as a Function of Nitrogen Source. Plant Age (days) Treatment Tissue 27 41 55 69 90 mg N‘plant"1 Control Shoots 9.010.3 47.912.0 5910.7 11813 13715 Roots 4.010.3 9.310.4 1110.1 1410 2511 2 mM N03 Shoots 9.610.8 51.411.0 8717.0 12614 142111 Roots 4.41O.l 9.510.7 1310.4 1311 1910 4 mM N03 Shoots l4.212.0 60.112.0 10213.0 11316 11715 Roots 5.210.2 6.510.8 1310.3 1111 2111 10 mM N03 Shoots 18.410.6 5412.0 8013.0 11316 146111 Rots 3.210.2 4.110.l 810.6 811 1511 2 mM N01 Shoots 12.610.4 48.712.0 8714.0 14217 157111 Roots 3.010.2 - 6.710.2 1610.6 1410 1810 4 mM NHE Shoots 12.410.4 52.712.0 6312.0 10917 18215 Roots 2.510.l 6.010.3 1110.5 1711 2011 The total N in shoots and roots was determined by a semi-micro Kjeldahl procedure (see Methods). In the case of plants grown on KN03, the amount of stored N03 in the tissue was subtracted from the total N. Each value is the mean of 3 replicate 1 standard error. 141 Appendix III: Amount of Stored N0§ in the Roots and Shoots as a Function afTNitrogen Source. Plant Age (days) Treatment Tissue 20 27 34 41 55 69 90 mg N ° plant"1 Control Shoots 0.04 0.08 0 0 0 0 0 Roots 0.02 0.08 0 0 0 0 0 2 mM N03 Shoots 0.18 0.52 0.59 1.73 1.41 2.34 1.51 Roots 0.09 0.39 0.29 0.42 0.56 0.29 0.63 4 mM N03 Shoots 0.35 0.83 1.88 2.49 2.00 7.48 6.91 Roots 0.41 0.51 0.98 1.16 1.74 2.72 4.07 10 mM N0§ Shoots 2.61 4.43 7.81 8.07 11.86 11.57 16.71 Roots 1.36 1.86 3.92 3.86 8.12 8.88 5.58 The amount of N03 stored (mg N-plant‘l) was determined by the method of Cataldo gt al. (136). Plants grown on 2 and 4 mM NHI were found to have stored N03 levels comparable to the controls. Each value is the mean of 3 replicates 1 a 5% standard error. REFERENCES 10. 11. REFERENCES Date RA (1973) Nitrogen, a major limitation in the productivity of natural communities, crops and pastures in the Pacific area. Soil Biol. Biochem. 5:5-18 Hardy RWF, UD Havelka (1975) Nitrogen fixation research: A key to world food? Science 188:633-643 Pate JS (1973) Uptake, assimilation and transport of nitrogen compounds by plants. Soil Biol. Biochem. 5:109~ll9 Gibson AH, WR Scowcroft, JD Pagan (1977) Nitrogen fixation in plants: An expanding horizon. Ig_W Newton, JR Postgate, C Rodriguez-Barrueco, eds. Recent Developments in Nitrogen Fixation. Academic Press, NY, pp. 387-417 Hardy RWF, UD Havelka (1976) Photosynthate as a major factor limiting nitrogen fixation by field-grown legumes with emphasis on soybeans. 1fl_PS Nutman, ed. Symbiotic Nitrogen Fixation in Plants. Cambridge University Press, Cambridge, pp. 421-439 Mahon JD (1977) Respiration and the early requirement for nitrogen fixation in nodulated pea roots. Plant Physiol. 60:817-821 Ryle GJA, CE Powell, AJ Gordon (1978) Effect of source of nitrogen on the growth of Fiskeby soyabean: The carbon economy of whole plants. Ann. Bot. (NS) 42:637-648 Ryle GJA, CE Powell, AJ Gordon (1979) The respiratory costs of nitrogen fixation in soyabean, cowpea and white clover. I. Nitrogen fixation and respiration of the nodulated root. J. Exp. Bot. 30:135-144 Ryle, GJA, CE Powell, AJ Gordon (1979) The respiratory costs of nitrogen fixation in soyabean, cowpea and white clover. II. Comparisons of the cost of nitrogen fixation and the utilization of combined nitrogen. J. Exp. Bot. 30:145-153 Minchin FR, JS Pate (1973) The carbon balance of a legume and the functional economy of its root nodules. J. Exp. Bot. 24:259-271 Schubert KR (1982) The energetics of biological nitrogen fixation. Trends in Plant Physiology 142 12. 13. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 143 Schubert KR, GJA Ryle (1980) The energy requirements for N2 fixation in nodulated legumes. Ig_RJ Summerfield, AH Bunting, eds.) Advances in Legume Science. Royal Botanical Gardens, Kew, England, pp. 85-96 Rabie RK, Y Arima, K Kumazawa (1979) Growth, nodule activity and yield of soybeans as affected by the form and application method of combined nitrogen. Soil Sci. Plant Nutr. 25:417-424 Barker AV (1980) Efficient use of nitrogen on crop land in the northwest. Conn. Agric. Exp. Sta., Bull. 792. Summerfield RJ, PJ Dart, PA Huxley, ARJ Eaglesham, FR Minchin, JM Day (1977) Nitrogen nutrition of cowpea (Vigna unqgiculata). I. Effects of applied nitrogen and symbiotic nitrogen fixation on growth and seed yield. Expl. Agric. 13:129-142 Dart PJ, PA Huxley, ARJ Eaglesham, FR Minchin, RJ Summerfield, JM Day (1977) Nitrogen nutrition of cowpea (Vigna unquiculata). II. Effects of short-term applications of inorganic nitrogen on growth and yield of nodulated and non-nodulated plants. Expl. Agric. 13:241-252 Deibert EJ, M Bijeriego, RA Olson (1979) Utilization of 15N fertilizer by nodulating and non-nodulating soybean isolines. Agron J. 71:717-723 Richards JE, RJ Soper (1979) Effect of N fertilizer on yield, protein content, and symbiotic N fixation in fababeans. Agron J. 71:807-811 Franco AA, JC Pereira, CA Neyra (1979) Seasonal patterns of nitrate reductase and nitrogenase activities in Phaseolus vulgaris L. Plant Physiol. 63:421-424 Harper JE (1974) Soil and symbiotic nitrogen requirement for optimum soybean production. Crop Sci. 15:255-260 McClure PR, DW Israel (1979) Transport of nitrogen in the xylem of soybean plants. Plant Physiol. 64:411-416 Pate JS, CA Atkins (1981) Nitrogen uptake, transport and utilization. Ig_WJ Broughton, ed. Ecology of Nitrogen Fixation. Vol. 3, Legumes, Oxford University PreSs, UK (in press) Lea PJ, BJ Miflin (1980) Transport and metabolism of asparagine and other nitrogen compounds within the plant. 1g BJ Miflin, ed. The Biochemistry of Plants, Vol. 5, Academic Press, NY, pp. 569- 607 McNeil DL, CA Atkins, JS Pate (1979) Uptake and utilization of xylem-borne amino compounds by shoot organs of a legume. Plant Physiol. 63:1076-1081 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 144 Mahon JD, JJ Child (1979) Growth response of inoculated peas (Pisum satiuum) to combined nitrogen. Can. J. Bot. 57:1687-1693 DeJong TM, DA Philips (1981) Nitrogen stress and apparent photosynthesis in symbiotically grown Pisum satiuum L. Plant Physiol. 69:309-313 Matsumoto T, M Yatazawa, Y Yamamoto (1977) Distribution and change in the contents of allantoin and allantoic acid in developing nodulating and non-nodulating soybean plants. Plant Cell Physiol. 18:353-359 Thomas RJ, LE Schroder (1981) Ureide metabolism in higher plants. Phytochem. 20:361-371 Atkins CA, RM Rainbird, JS Pate (1980) Evident for a purine pathway of ureide synthesis in Nz-fixing nodules of cowpea (Vigna unguiculata (L) Walp). Z Pflanzenphysiol 97:249-260 Atkins CA (1981) Metabolism of purine nucleotides to form ureides in nitrogen-fixing nodules of cowpea (Vigna unquiculata L. Walp). FEBS Lett. 125:83-89 Woo KC, CA Atkins, JS Pate (1980) Biosynthesis of ureides from purines in a cell-free system from nodule extracts of cowpea (Vigna unguiculata L. Walp). Plant Physiol. 66:735-739 Atkins, CA, A Ritche, PB Rowe, E McCairns, D Sauer (1982) 03 novo purine synthesis in N-fixing nodules of cowpea (Vi na unguiculata L Walp) and Soybean (Glycine max L. merr.) Plant P ysiol. 70:55-60 Boland MJ, KR Schubert (1982) Purine biosynthesis and catabolism in soybean root nodules. Incorporation of 14C from 14002 into xanthine. Arch. Biochem. Biophys. 213:486-491 Streeter JC (1979) Allantoin and allantoic acid in tissues and stem exudate from field-grown soybean plants. Plant Physiol. 63:478-480 Brill WJ (1977) Biological nitrogen fixation. Scient Amer. 236:68-81 Bauer WD (1981) Infection of legumes by Rhizobia. Ann. Rev. Plant Physiol. 32:407-449 Dart PJ (1974) The infection process. Ig_A Quispel, ed, The Biology of Nitrogen Fixation. North-Holland Publ. Co., Amsterdam, pp. 382-428 Mortenson LE, RNF Thorneley (1979) Structure and function of nitrogenase. Ann. Rev. Biochem. 48:387-418 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 145 Tjepkema JD, CS Yocum (1973) Respiration and oxygen transport in soybean nodules. Planta 115:59-72 Appleby CA (1969) Properties of leghemoglobin lg vivo, and its isolation as ferrous oxyleghemoglobin. Biochim. Biophys. Acta 118:222-229 Appleby CA (1974) Leghemoglobin Ig.A Quispel, ed, The Biology of Nitrogen Fixation, North-Holland Publ. Co., Amsterdam, pp. 522- 554 Bergersen FJ, GL Turner, CA Appleby (1973) Studies of the physiological role of leghaemoglobin in soybean root nodules. Biochim. Biophys. Acta 292:271-282 Wittenberg JB (1980) Utilization of leghemoglobin-bound oxygen by Rhizobium bacteroids. In WE Newton, WH Orme-Johnson, eds. Nitrogen Fixation, Vol._11, University Park Press, Baltimore, pp. 53-67 Bergersen FJ, DJ Goodchild (1973) Cellular location and concentration of leghaemglobin in soybean root nodules. Aust J. Biol. Sci. 26:741-756 Nadler K0, YJ Avissar (1977) Heme synthesis in soybean root nodules. I. On the role of bacteroid 6-levulinic acid synthetase and 8-1evulinic acid dehydrase in the synthesis of the heme of leghemoglobin. Plant Physiol. 60:433-436 Dilworth MJ (1969) The plant as the genetic determinant of leghaemoglobin production in the legune root nodule. Biochim. Biophys. Acta 184:432-441 Orme-Johnson WH, LC Davis (1977) Current topics and problems in the enzymology of nitrogenase. Ig_W Lovenberg, ed., Iron-Sulfur Proteins, Vol III. Academic Press, N.Y., pp. 15-60 Eady RR, JR Postgate (1974) Nitrogenase. Nature 249:805-810 Rawlings J, VK Shah, JR Chisnell, WJ Brill, R Zimmermann, E Muncis, WH Orme-Johnson (1978) Novel metal cluster in an iron-molybdenum cofactor of nitrogenase. J. Biol. Chem. 253:1001-1004 Shah VK, WJ Brill (1977) Isolation of an iron-molybdenum cofactor from nitrogenase. Proc. Natl. Acad. Sci. USA 74:3249-3253 Ljones T, RH Burris (1978) Evidence for one-electron transfer by the Fe protein of nitrogenase. Biochem. Biophys. Res. Comm. 80:20-25 Bergersen FJ (1971) The central reactions of nitrogen fixation. Plant Soil Special Vol:511-524 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 146 Dixon R00 (1976) Hydrogenases and efficiency of nitrogen fixation in aerobes. Nature 262:173 Rivera-Ortiz JM, RH Burris (1975) Interactions among substrates and inhibitors of nitrogenase. J. Bacteriol. 123:537-545 Schubert KR, HJ Evans (1976) Hydrogen evolution: A major factor affecting the efficiency of nitrogen fixation in nodulated symbionts. Proc. Natl. Acad. Sci. USA 73:1207-1211 Schubert KR, JA Engelke, SA Russell, HJ Evans (1977) Hydrogen reactions of nodulated leguminous plants: I. Effect of rhizobial strain and plant age. Plant Physiol. 60:651-654 Schubert KR, NT Jennings, HJ Evans (1978) Hydrogen reactions of nodulated leguminous plants. II Effects on dry matter accumulation and nitrogen fixation. Plant Physiol. 61:398-401 Smith LA, S Hill, MG Yates (1976) Inhibition by acetylene of conventional hydrogenase in nitrogen fixing bacteria. Nature 262:209—210 Minchin FR, RJ Summerfield, P Hadley, EH Roberts, S Rawsthorne (1981) Carbon and nitrogen nutrition of nodulated roots of grain legumes. Plant Cell Environ. 4:5-26 Hewitt EJ, DP Hucklesby, BA Notton (1977) Nitrate Metabolism. IQ J. Banner and JE Vauner, eds, Plant Biochemistry 3rd Edition Academic Press, NY, pp. 633-681 Gibson AH (1966) The carbohydrate requirements for symbiotic nitrogen fixation: A "whole plant" growth analysis approach. Aust J. Biol. Sci. 19:499-515 Mahon JD (1977) Root and nodule respiration in relation to acetylene in intact nodulated peas. Plant Physiol. 60:812-816 Bond G (1941) Symbiosis of leguminous plants and nodule bacteria. 1. Observations on respiration and on the extent of utilization of host carbohydrates by the nodule bacteria. Ann. Bot. (NS) 5:313-337 Gibson AH (1976) Recovery and compensation by nodulated legumes to environmental stress. ‘Ig PS Nutman, ed, Symbiotic Nitrogen Fixation in Plants. Cambridge Univ. Press, Cambridge, pp. 385- 403 Bethlenfalvay GJ, SS Abu-Shakra, DA Philips (1978) Interdependence of nitrogen nutrition and photosynthesis in Pisum satiuum L. I. Effects of combined nitrogen on symbiotic fixation and photosynthesis. Plant Physiol. 62:127-130 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 147 Dazzo FB, WJ Brill (1978) Regulation by fixed nitrogen of host-symbiont recognition in the Rhizobium clover symbiosis. Plant Physiol. 62:18-21 Wang PP (1980) Nitrate and carbohydrate effects on nodulation and nitrogen fixation (acetylene reduction) activity of Lentil (Lens esculenta Moench) Plant Physiol. 66:78-81 Chen P, DA Philips (1977) Induction of root nodule senescence by combined nitrogen in Pisum sativum L. Plant Physiol. 59:440-442 Small JGC, 0A Leonard (1969) Translocation of C14-1abeled photosynthate in nodulated legumes as influenced by nitrate nitrogen. Am. J. Bot 56:187-194 Latimore M, J Gidens, DA Ashley (1977) Effect of ammonium and nitrate nitrogen upon photosynthate supply and nitrogen fixation by soybeans. Crap. Sci. 17:399-404 Bethienfalvay GJ, DA Philips (1977) Effect of light intensity on efficiency of carbon dioxide and nitrogen reduction in Pisum sativum L. Plant Physiol. 601:868-871 Lawn RJ, WA Brun (1974) Symbiotic nitrogen fixation in soybeans. I. Effect of photosynthetic source-sink manipulations. Crap. Sci. 14:11-16 Hardy RWF, RD Holsten, EK Jackson, RC Burns (1968) The acetylene- ethylene assay for N fixation: Laboratory and field evaluation. Plant Physiol. 43:11 5-1205 Harper JE, JC Nicholas (1978) Nitrogen metabolism in soybeans I. Effect of tungstate on nitrate utilization, nodulation, and growth. Plant Physiol. 62:662-664 Pagan JD, WR Scowcroft, WF Dudman, AH Gibson (1977) Nitrogen fixation in nitrate reductase-deficient mutants of cultured rhizobia. J. Bacteriol. 129:718-723 Streeter JC (1982) Synthesis and accumulation of nitrite in soybean nodules supplied with nitrate. Plant Physiol. 69:1429-1434 Castillo F, J Cardenas (1982) Nitrite inhibition of bacterial dinitrogen fixation. Z. Naturforsch. 37c:784-786 Drozd JW, RS Tubb, JR Postgate (1972) A chemostat study of the effect of fixed nitrogen sources on nitrogen fixation, membranes and free amino acids in Azotobacter chroococcum. J. Gen. Microbiol. 73:221-232 Tubb RS, JR Postgate (1973) Control of nitrogenase synthesis in Klebsiella pneumoniae. J. Gen. Microbiol. 79:103-117 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 148 Daesch G, LE Mortenson (1972) Effect of ammonia on the synthesis and function of the Nz-fixing enzyme system in Clostridium pasteurianum. J. Bacteriol. 110:103-109 Roberts GP, WJ Brill (1981) Genetics and regulation of nitrogen fixation. Ann Rev. Microbiol. 35:207-235 Houwaard F (1978) Influence of ammonium chloride on the nitrogenase activity of nodulated pea plants (Pisum sativum). Appl. Environ. Microbiol. 35:1061-1065 Bisseling T, RC van den Bos, A van Kammen (1978) The effect of ammonium nitrate on the synthesis of nitrogenase and the concentration of leghemoglobin in pea root nodules induced by Rhizobium leguminosarum. Biochim. Biophys. Acta 539:1-11 Houwaard F (1979) Effect of ammonium chloride and methionine sulfoximine on the acetylene reduction of detached root nodules of peas (Pisum sativum). Appl. Environ. Microbiol. 37:73-79 Kennedy IR, J Rigaud, JC Trinchant (1975) Nitrate reductase from bacteroids of Rhizobium japonicum: enzyme characteristics and possible interaction with nitrogen fixation. Biochim. Biophys. Acta 397:24-35 Gibson AH, JD Pagan (1977) Nitrate effects on the nodulation of legumes inoculated with nitrate-reductase-deficient mutants of Rhizobium. Planta 134:17-22 Manhart JR, PP Wong (1980) Nitrate effect on nitrogen fixation (acetylene reduction). Activities of legume root nodules induced by rhizobia with varied nitrate reductase activities. Plant Physiol. 65:502-505 Bergensen FJ, GL Turner (1967) Nitrogen fixation by the bacteroid fraction of breis of soybean root nodules. Biochem. Biophys. Acta 141:507-515 Kennedy IR (1966) Primary products of symbiotic nitrogen fixation. I. Short-term exposures of serradella nodules to 15N2. Biochem. Biophys. Acta 130:285-294 Bergensen FJ (1971) Biochemistry of symtiotic nitrogen fixation in legumes. Ann. Rev. Plant Physiol. 22:121-140 Aprison MH, WE Magee, RH Burris (1954) Nitrogen fixation by excised soybean root nodueles. J. Biol. Chem. 208:29-39 Grimes H. PF Fottrell (1966) Enzymes involved in glutamate metabolism in legume root nodules. Nature 212:295-296 Boland MJ, AM Fordyce, RM Greenwood (1978) Enzymes of nitrogen metablism in legume nodules. A comparative study. Aust J. Plant Physiol. 5:553-559 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 149 Herridge DF, CA Atkins, JS Pate, RM Rainbird (1978) Allantoin and allantoic acid in the nitrogen economy of the cowpea (Vigna ungiculata (L.) Walp). Plant Physiol. 62:495-498 McPharland RH, JG Gruevara, RR Becker, HJ Evans (1976) The Purification and properties of the glutamine synthetase from the cytosol of soybean root nodules. Biochem. J. 153:597-606 Meeks JC, CP Walk, N Schilling, PW Shaffer, Y Avigsar, WS Chien (1978) Initial organic products of fixation of (1 N) dinitrogen by root nodules of soybean (Glycine mgx). Plant Physiol. 61:980-983 0hyama T, K Kumazawa (1980) Nitrogen assimilation in soybean nodules. II. N2 assimilation in bacteroid and cytosol fractions of soybean nodules. Soil Sci. Plant Nutr. 26:205-213 Miflin BJ, PJ Lea (1977) Amino acid metabolism. Ann. Rev. Plant Physiol. 28:299-329 Pate JS (1980) Transport and partitioning of nitrogenous solutes. Ann. Rev. Plant Physiol. 31:313-340 Pate JS, BES Gunning, LG Briarty (1969) Ultrastructure and functioning of the transport system of the leguminous root nodule. Planta 85:11-34 Pate JS, CA Atkins, K Hamel, DL McNeil, DB Layzell (1979) Transport of organic solutes in phloem and xylem of nodulated legume. Plant Physiol. 63:1082-1088 Matsumoto T, M Yatazawa, Y Yamamoto (1977) Incorporation of 1 N into allantoin in nodulated soybean plants supplied with N2. Plant Cell Physiol. 18:459-462 0hyama T, K Kumazawa (1978) Incorporation of 15N into various nitrogenous compounds in intact soybean nodules after exposure to N2 gas. Soil. Sci. Plant Nutr. 242525-533 0hyama T, K Kumazawa (1979) Assimilation and transport of nitrogenous compounds originated from Nz-fixation and N03-absorption. Soil Sci. Plant Nutr. 25:9-19 McClure PR, DW Israel, RJ Volk (1980) Evaluation of the relative ureide content of xylem sap as an indicator of N2 fixation in soybeans. Greenhouse studies. Plant Physiol. 66:720-725 Pate JS, CA Atkins, ST White, RM Rainbird, KC Woo (1980) Nitrogen nutrition and xylem transport of nitrogen in ureide-producing grain legumes. Plant Physiol. 65:961—965 Atkins CA (1981) The legune - Rhizobium symbiosis: Ureide biosynthesis. '13 AH Gibson, WE Newton, eds. Current 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 150 perspectives in nitrogen fixation, Griffins Press LTD, Netley, S. Australia, pp. 271-272 Boland MJ, KR Schubert (1983) Biosynthesis of purines by a proplastid fraction from soybean nodules. Arch. Biochem. Biophys. 220:179-187 Boland MJ, JF Hanks, PHS Reynolds, DG Blevins, NE Talbert, KR Schubert (1982) Subcellular organization of ureide biogenesis from glycolytic intermediates and ammonium in nitrogen-fixing soybean nodules. Planta 155:45-51 Bollard EG (1959) Urease, urea, and ureides in plants. lg Utilization of Nitrogen and its Compounds by plants. Symp. Soc. Exp. Biol. 13:304-328 Reinbothe H, K Mothes (1962) Urea, ureides and guanidines in plants. Ann. Rev. Plant Physiol. 132129-150 Krupka RM, GHN Towers (1959) Studies of the metabolic relations of allantoin in wheat. Can. J. Bot. 37:539-545 Anderson JD (1977) Adenylate metabolism of embryonic axes from deteriorated soybean seeds. Plant Physiol. 59:610-614 Anderson JD (1977) Responses of adenine nucleotides in germinating soybean embryonic axes to exogenously applied adenine and adenosine. Plant Physiol. 60:689-692 Anderson JD (1979) Purine nucleotide metabolism of germinating soybean embryonic axes. Plant Physiol. 63:100-104 Barankieiocz J, J Paszkowski (1980) Purine metabolism in mesophyll protoplasts of tobacco (Nicotiana tabacum) leaves. Biochem. J. 186:343-350 Rowe PB, E McGairns, G Madsen, D Sauer, H Elliott (1978) 03 novo purine synthesis in avian liver. Co-purification of the enzymes and properties of the pathway. J. Biol. Chem. 253:7711-7721 Hartman SC (1970) Purine and pyrimidines. In. DM Greenberg, eds. Metabolic Pathways, Academic Press, NY 4:1-6 Vogels GD, C Van der Drift (1976) Degradation of purines and pyrimidnes by microorganisms. Bacterial. Rev. 40:403-468 Ogutuga DBA, DH Northcote (1970) Biosynthesis of caffeine in tea callus tissue. Biochem. J. 117:715-720 Suzuki T (1973) Metabolism of methylamine in the tea plant (Thea sinensis L.). Biochem. J. 132:753-763 122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133. 134. 151 Suzuki, T, E. Takahashi (1975) Metabolism of xanthine and hypoxanthine in the tea plant (Thea sinensis L.) Biochem. J. 146:79-85 Hanks JF, NE Talbert, KR Schubert (1981) Localization of enzymes of ureide biosynthesis in peroxisomes and microsomes of nodules. Plant Physiol. 68:65-69 Schubert KR 91981) Enzymes of purine biosynthesis and catabolism in Glycine mgx I. Comparison of activities with Nz-fixation and composition of xylem exudate during nodule development. Plant Physiol. 68:1115-1122 Triplett EW, DG Blevins, DD Randall (1980) Allantoic acid synthesis in soybean root nodule cytosol via xanthine dehydrogenase. Plant Physiol. 65:1203-1206 Noguchi T, Y Takada, S. Fujiwars (1979) Degradation of uric acid to urea and glyoxylate in peroxisomes. J. Biol. Chem. 254:5272-5275 Hanks JF (1982) Localization of enzymes of purine degradation in plants and animals. Ph.D. Thesis Fujihara S, M Yamaguchi (1978) Effects of allopurinol [4-hydroxypyrazolo (3,4-d) pyrimidine] on the metabolism of allantoin in soybean plants. Plant Physiol. 62:134-138 Reynolds PHS, MJ Boland, DG Blevins, KR Schubert, DD Randall (1982) Enzymes of amide and ureide biogenesis in developing soybean nodules. Plant Physiol. 69:1334-1338 Newcomb EH, SK Tandon (1981) Uninfected cells of soybean root nodules: Ultrastructure suggests key role in ureide production. Science 22:1394-1396 Hanks JF, K Schubert, NE Talbert (1983) Isolation and characterization of infected and uninfected cells from soybean nodules. Role of uninfected cells in ureide synthesis. Plant Physiol. 71:869-873 Atkins CA, JS Pate, DL McNeil (1980) Phloem loading and metabolism of xylem-borne amino compounds in fruiting shoots of a legume. J. Exp. Bot. 31:1509-1520 Singh R (1968) Evidence for the presence of allantoicase in germinating peanuts. Phytochem. 7:1503-1508 Atkins CA, JS Pate, A Ritchie, MB Peoples (1982) Metabolism and translocation of allantoin in ureide-producing grain legumes. Plant Physiol. 70:476-482 135. 136. 137. 138. 139. 140. 141. 142. 143. 144. 145. 146. 147. 148. 152 Ishuska J (1977) Function of symbiotically fixed nitrogen for grain production in soybean. Proc. Int. Sem. Soil Environ. Fert. Management in Intense Agric. pp. 618-624 Broadbent FE, AB Carlton (1980) Methodology for field trials with nitrogen-lS-depleted nitrogen. J. Environ. Qual. 9:236-242 McCullough H (1967) The determination of ammonia in whole blood by a direct colorimetric method. Clin. Chim. Acta 17:297-304 Fishbeck K, HJ Evans, LL Boersma (1973) Measurements of nitrogenase activity in intact legume symbionts jg situ using the acetylene reduction assay. Agron J. 65:429-433 Cataldo DA, M Haroon, LE Schrader, VL Youngs (1975) Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid. Comm. Soil Sci. Anal. 6:71-80 Bremmer JM (1965) Isotope-ratio analysis of nitrogen in nitrogen-15 tracer investigations. lfl CA Black, ed., Methods of Soil Analysis, Part 2, Agron 9, Am. Soc. Agron, Madison, WI pp. 1256-1286 Givan CV (1979) Metabolic detoxification of ammonia in tissues of higher plants. Phytochem. 18:375-382 Rufty TW, RJ Volk, PM McClure, DW Israel, CD Raper (1982) The relative content of N03 and reduced-N in xylem exudate as pp indicator of root reduction of concurrent absorbed N03. Plant Physiol. 692166-170 Rabie RK, Y Arima, K Kumazawa (1980) Effect of combined N on the distribution pattern of photosynthetic assimilates in nodulated soybean plant as revealed by 1 . Soil Sci. Plant Nutr. 26:79-86 Miflin BJH, PJ Lea (1980) Ammonia assimilation. 13 BJ Miflin, ed. The Biochemistry of Plants. Vol. 5, Academic Press, NY, pp. 169-202 Izawa S, NE Good (1972) Inhibition of photosynthetic electron transport and photophosphorylation. Methods Enzymol. 24:355-372 Pate JS, DB Layzell, DL McNeil (1979) Modeling the transport and utilization of carbon and nitrogen in a nodulated legume. Plant Physiol. 63:730-737 Israel DW (1981) Cultivar and Rhizobium strain effect on nitrogen fixation and remobilization. Agron J. 73:509-516 Rabie RK, Y Arima, K Kumazawa (1980) Uptake and distribution of combined nitrogen and its incorporation into seeds of nodulated soybean plants as revealed by 1 N studies. Soil Sci. Plant Nutr. 262427-436 149. 150. 151. 152. 153. 154. 155. 156. 157. 158. 159. 160. 161. 153 Pate JS, FR Minchin (1980) Comparative Studies of Carbon and Nitrogen Nutrition of Selected Grain Legumes. In_RJ Summerfield, AH Bunting, eds. Advances in Legumes Science. Royal Botanical Gardens, KEW, England, pp. 105-114 Vogels GD, C Van der Drift (1970) Differential analysis of glyoxylate derivatives. Anal. Biochem. 33:143-l57 McKenzie HA, HS Wallace (1954) Aust J. Chem. 7:55-70 Glower W (1974) J. Assoc. Offic. Anal. Chemists 57:1228-1230 Friedman TB, DH Johnson (1977) Temperature control of urate oxidase activity in Drosophila: Evidence of an autonomous timer in malpighian tubules. Science 192:477-479. Friedman TB, CR Merrill (1973) A microradiochemical assay for urate oxidase. Anal. Biochem. 55:202-296 Bensadown A, D Weinotein (1976) Assay of proteins in the presence of interfering materials. Anal. Biochem. 70:241-250 Obendorf RL, A Marcus (l974) Rapid increase in adenosine 5'—triphosphate during early wheat embryo germination. Plant Physiol. 53:779-781 Hong Y-N, P Schopfer (1981) Control by phytochrome of urate oxidase and allantoinase activities during peroxisome development in the cotyledons of mustard (Sinapis alba L.) seedlings. Planta 152:325—335 Dhillon SS, JP Miksche DNA, RNA, protein, and heterochromatin changes during embryo development and germination of soybean (Glycine max L.) submitted to J. Histological Chemistry Millerd A, PR Whitfeld (l973) Deoxyribonucleic acid and ribonucleic acid synthesis during the cell expansion phase of cotyledon development in vicia faba L. Plant Phisiol. 5l:1005—1010 Webb JL (1966) Enzyme and metabolic inhibitor. Vol. II, Academic Press, NY, p. 933 Boland MJ (1981) NAD+: xanthine dehydrogenase from nodules of navy beans: partial purification and properties. Biochem. Int. 2:567-574 . . x . y .. o AlixQ .3 m . . . . . s . ... x .1 1.113“ up." A... x », ... . . aw , o o 1 2.. w . . .. I 111?,1111}11P.P)l1r!11.3|1! . Lab 3 I? "111111111111111111111115