Proline antagonizes GABA-induced quenching of quorum-sensing in Agrobacterium tumefaciens.

Plants accumulate free L-proline (Pro) in response to abiotic stresses (drought and salinity) and presence of bacterial pathogens, including the tumor-inducing bacterium Agrobacterium tumefaciens. However, the function of Pro accumulation in host-pathogen interaction is still unclear. Here, we demonstrated that Pro antagonizes plant GABA-defense in the A. tumefaciens C58-induced tumor by interfering with the import of GABA and consequently the GABA-induced degradation of the bacterial quorum-sensing signal, 3-oxo-octanoylhomoserine lactone. We identified a bacterial receptor Atu2422, which is implicated in the uptake of GABA and Pro, suggesting that Pro acts as a natural antagonist of GABA-signaling. The Atu2422 amino acid sequence contains a Venus flytrap domain that is required for trapping GABA in human GABA(B) receptors. A constructed atu2422 mutant was more virulent than the wild type bacterium; moreover, transgenic plants with a low level of Pro exhibited less severe tumor symptoms than did their wild-type parents, revealing a crucial role for Venus flytrap GABA-receptor and relative abundance of GABA and Pro in host-pathogen interaction.

Identification and characterization of GABA, proline and quaternary ammonium compound transporters from Arabidopsis thaliana.

Arabidopsis thaliana grows efficiently on GABA as the sole nitrogen source, thereby providing evidence for the existence of GABA transporters in plants. Heterologous complementation of a GABA uptake-deficient yeast mutant identified two previously known plant amino acid transporters, AAP3 and ProT2, as GABA transporters with Michaelis constants of 12.9 +/- 1.7 and 1.7 +/- 0.3 mM at pH 4, respectively. The simultaneous transport of [1-14C]GABA and [2,3-3H]proline by ProT2 as a function of pH, provided evidence that the zwitterionic state of GABA is an important parameter in substrate recognition. ProT2-mediated [1-14C]GABA transport was inhibited by proline and quaternary ammonium compounds.

The simultaneous measurement of low rates of CO2 and O2 exchange in biological systems.

An instrument for measuring low rates of biological O2 exchange using an open-flow gas analysis system is described. A novel differential O2 sensor that is capable of measuring as little as 0.4 Pa O2 against a back-ground of ambient air (20,900 Pa O2), yet has a dynamic range of +/- 2000 Pa O2 (i.e., +/- ca. 2% O2) is described. Baseline drift was typically less than 0.025 Pa min-1. The differential O2 sensor was incorporated into a respiratory quotient/photosynthetic quotient analyzer that contained other environmental sensors for atmospheric pressure, absolute O2 and CO2 concentration, temperature of the differential O2 sensor block, and differential pressure between reference and sample streams. Protocols for how these sensors can be used to calibrate the differential O2 sensor and to improve its stability with time are described. Together, the differential O2 sensor, the environmental sensors, and the simple calibration techniques allow for simultaneous, noninvasive, and accurate measurements of O2 and CO2 exchange in tissues with metabolic rates as low as about 0.1 mumol O2 or CO2 h-1. Example data are provided in which O2 differentials of 3 to 41 Pa O2 were measured in an open-flow system.

Calcium/Calmodulin Activation of Soybean Glutamate Decarboxylase.

Recently, we provided preliminary evidence for calcium (Ca2+)/calmodulin (CaM) stimulation of plant glutamate decarboxylase (GAD; EC 4.1.1.15). In the present study, a detailed characterization of the phenomenon is described. GAD was partially purified from various soybean (Glycine max L. Merr.) tissues (developing seed coat and cotyledons, leaf, and root) in the presence of EDTA by a combination of ammonium sulfate precipitation and anion-exchange fast protein liquid chromatography. GAD activity showed a sharp optimum at pH 5.8, with about 12% of maximal activity at pH 7. It was stimulated 2- to 8-fold (depending on the tissue source) in the presence of Ca2+/CaM at pH 7 but not at pH 5.8. Furthermore, when the protease inhibitor phenylmethylsulfonyl fluoride was omitted from the purification procedure, GAD activity was insensitive to Ca2+/CaM but was similar in magnitude to CaM-stimulated activity. The stimulation by Ca2+/CaM was fully inhibited by the CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfon-amide and trifluoperazine. With saturating CaM or Ca2+, the concentrations of Ca2+ and CaM required for half-maximal stimulation were about 7 to 11 [mu]M and 25 nM, respectively. The effect of Ca2+ and CaM appeared to be through a 2.4-fold stimulation of Vmax and a 55% reduction in Km. The results suggested that GAD is activated via Ca2+ signal transduction.

Molecular and biochemical analysis of calmodulin interactions with the calmodulin-binding domain of plant glutamate decarboxylase.

We previously provided what to our knowledge is the first evidence that plant glutamate decarboxylase (GAD) is a calmodulin (CaM)-binding protein. Here, we studied the GAD CaM-binding domain in detail. A synthetic peptide of 26 amino acids corresponding to this domain forms a stable complex with Ca2+/CaM with a 1:1 stoichiometry, and amino acid substitutions suggest that tryptophan-485 has an indispensable role in CaM binding. Chemical cross-linking revealed specific CaM/GAD interactions even in the absence of Ca2+. However, increasing KCI concentrations or deletion of two carboxy-terminal lysines abolished these interactions but had a mild effect on CaM/GAD interactions in the presence of Ca2+. We conclude that in the presence of Ca(2+)-hydrophobic interactions involving tryptophan-485 and electrostatic interactions involving the carboxy-terminal lysines mediate CaM/GAD complex formation. By contrast, in the absence of Ca2+, CaM/GAD interactions are essentially electrostatic and involve the carboxy-terminal lysines. In addition, a tryptophan residue and carboxy-terminal lysines are present in the CaM-binding domain of an Arabidopsis GAD. Finally, we demonstrate that petunia GAD activity is stimulated in vitro by Ca2+/CaM. Our study provides a molecular basis for Ca(2+)-dependent CaM/GAD interactions and suggests the possible occurrence of Ca(2+)-independent CaM/GAD interactions.

Subcellular Compartmentation of the 4-Aminobutyrate Shunt in Protoplasts from Developing Soybean Cotyledons.

The subcellular localization of enzymes involved in the 4-ami-nobutyrate shunt was investigated in protoplasts prepared from developing soybean [Glycine max (L.) Merrill cv Maple Arrow] cotyledons. Protoplast lysate was fractionated by differential and continuous Percoll-gradient centrifugation to separate organelle fractions. Glutamate decarboxylase (EC 4.1.1.15) was found exclusively in the cytosol, whereas 4-aminobutyrate:pyruvate transami-nase (EC 2.6.1.19) and succinic semialdehyde dehydrogenase (EC 1.2.1.16) were associated exclusively with the mitochondrial fractions. Mitochondrial fractions also catabolized [U-14C]4-aminobu-tyrate to labeled succinate.

Analysis of a soluble calmodulin binding protein from fava bean roots: identification of glutamate decarboxylase as a calmodulin-activated enzyme.

The identity of a soluble 62-kD Ca(2+)-dependent calmodulin binding protein (CaM-BP) from fava bean seedlings was determined. Using 125I-CaM overlay assays, a class of soluble CaM-BPs was detected in extracts of tissues comprising the axis of 1.5-week-old seedlings, excluding the root tip and emergent leaves. The size of these CaM-BPs was not uniform within all parts of the plant; the apparent molecular masses were 62 kD in roots, 60 kD in stems, and 64 kD in nodules. The root 62-kD CaM-BP was purified, and internal microsequence analysis was performed on the protein. A tryptic peptide derived from the CaM-BP consisted of a 13-residue sequence corresponding to a highly conserved region of glutamate decarboxylase (GAD), an enzyme that catalyzes the alpha-decarboxylation of glutamate to form the stress-related metabolite gamma-aminobutyrate. Activity assays of partially purified, desalted, root GAD revealed a 50% stimulation by the addition of 100 microM Ca2+, a 100% stimulation by the addition of 100 microM Ca2+ plus 100 nM CaM, and no appreciable stimulation by CaM in the absence of added Ca2+. The demonstration that plant GAD is a Ca(2+)-CaM-stimulated enzyme provides a model in which stress-linked metabolism is modulated by a Ca(2+)-mediated signal transduction pathway.

The production and efflux of 4-aminobutyrate in isolated mesophyll cells.

The pathway of 4-aminobutyric acid (GABA) production and efflux was investigated in suspensions of mesophyll cells isolated from asparagus (Asparagus sprengeri Regel) cladophylls. Analysis of free amino acids demonstrated that, on a molar basis, GABA represented 11.4, 19, and 6.5% of the xylem sap, intact cladophyll tissue, and isolated mesophyll cells, respectively. l-Glu, a GABA precursor, was abundant in intact cladophylls and isolated cells but not in xylem sap. When cells were incubated with l-[U-(14)C]Glu, intracellular GABA contained less than 10% of the radioactivity found in intracellular Glu. However, GABA in the medium contained 78% of the radioactivity found in extracellular l-Glu metabolites. Incubation with l-[1-(14)C]Glu resulted in the appearance of unlabeled GABA, demonstrating its production through decarboxylation at carbon 1. GABA released to the medium from cells incubated with l-[U-(14)C]Glu had a specific activity of 18 nanocuries per nanomole, whereas GABA remaining in the cell had a specific activity of 2.25 x 10(-1) nanocuries per nanomole. In the presence of exogenous l-Glu, amino acid analysis and cell volume measurements indicated intracellular Ala and GABA concentrations of 4.2 and 1.4 millimolar, respectively. In the medium, however, the corresponding concentrations were 2 and 57 micromolar. The data indicate that l-Glu entering the cell is decarboxylated to GABA, and that specific and passive efflux is from this pool of recently synthesized GABA and not from a previously synthesized unlabeled pool of GABA.

Distribution and metabolism of xylem-borne ureido and amino compounds in developing soybean shoots.

Pulse-chase feeding (30-120 minutes) of (14)C-labeled nitrogenous compounds to cut transpiring shoots was used to investigate the early fate of the major xylem-borne solutes in N(2)-fixing soybean (Glycine max) plants at the V(4) growth stage. By comparison with the foliar distribution of [(14)C]inulin (a xylem marker), it was determined that the phloem supply of allantoin, allantoic acid, asparagine, glutamine, aspartate, and arginine, respectively, provided about 20, 10, three, two, five, and 20 times the (14)C delivered to the developing trifoliolate in the xylem stream. Recovery of unmetabolized asparagine, aspartate, and arginine in this indicator trifoliolate, and significant declines in the percentage of (14)C from allantoic acid and allantoin recovered in the first trifoliolate, provided some support for the direct xylem-to-phloem transfer of these compounds, but did not preclude the involvement of indirect transfer. Data on stem retention and foliar distribution, expressed as a function of the relative xylem sap composition, indicated that ureides provide the major sources of nitrogen to all plant parts. There was no consistent distinction in distribution patterns between pairs of similar anionic and neutral compounds. The extent of xylem-to-phloem transfer among the ureido or the amino compounds was inversely related to its prominence in xylem sap.

Xylem-to-Phloem Transfer of Organic Nitrogen in Young Soybean Plants.

Xylem-to-phloem transfer in young vegetative soybean (Glycine max [L.] Merr.) plants (V4 stage) was identified as the difference in the distribution of [(14)C]inulin, a xylem marker, and [(14)C]aminoisobutyric acid (AIB), a synthetic amino acid, fed via the transpiration stream. Since [(14)C]AIB was retained in the stem to some extent, whereas [(14)C]inulin was not, the distribution of these marker compounds in each leaf was expressed as a percentage of the total [(14)C] radioactivity recovered in the foliage. The developing third trifoliolate was a consistent and reliable indicator of xylem-to-phloem transfer. The phloem stream provided to the developing trifoliolate up to fourfold the relative proportion of solute received from the xylem stream; this was markedly reduced by increased light intensity and consequently water flow through the xylem. Evidence from heat girdling experiments is discussed with respect to the vascular anatomy of the soybean plant, and interpreted to suggest that direct xylem-to-phloem transfer in the stem, in the region of the second node, accounted for about one-half of the AIB supplied to the developing trifoliolate, with the remainder being provided from the second trifoliolate. Since AIB is not metabolized it seems likely that rapid transfer within the second trifoliolate occurred as direct veinal transfer rather than indirect cycling through the mesophyll. This study confirmed that xylem-to-phloem transfer plays a major role in the partitioning of nitrogen for early leaf development.

Arginine Metabolism in Developing Soybean Cotyledons: III. Utilization.

Tracerkinetic experiments were performed using l-[guanidino-(14)C]arginine, l-[U-(14)C]arginine, l-[ureido-(14)C]citrulline, and l-[1-(14)C]ornithine to investigate arginine utilization in developing cotyledons of Glycine max (L.) Merrill. Excised cotyledons were injected with carrier-free (14)C compounds and incubated in sealed vials containing a CO(2) trap. The free and protein amino acids were analyzed using high performance liquid chromatography and arginine-specific enzyme-linked assays. After 4 hours, 75% and 90% of the (14)C metabolized from [guanidino-(14)C]arginine and [U-(14)C]arginine, respectively, was in protein arginine. The net protein arginine accumulation rate, calculated from the depletion of nitrogenous solutes in the cotyledon during incubation, was 17 nanomoles per cotyledon per hour. The data indicated that arginine was also catabolized by the arginase-urease reactions at a rate of 5.5 nanomoles per cotyledon per hour. Between 2 and 4 hours (14)CO(2) was also evolved from carbons other than C-6 of arginine at a rate of 11.0 nanomoles per cotyledon per hour. It is suggested that this extra (14)CO(2) was evolved during the catabolism of ornithine-derived glutamate; (14)C-ornithine was a product of the arginase reaction. A model for the estimated fluxes associated with arginine utilization in developing soybean cotyledons is presented.The maximum specific radioactivity ratios between arginine in newly synthesized protein and total free arginine in the (14)C-citrulline and (14)C-ornithine experiments indicated that only 3% of the free arginine was in the protein precursor pool, and that argininosuccinate and citrulline were present in multiple pools.

Arginine metabolism in developing soybean cotyledons : I. Relationship to nitrogen nutrition.

The free and protein amino acid composition of Glycine max (L.) Merrill cotyledons was determined for the entire developmental period using high performance liquid chromatography. Arginine constituted 18% of the total protein nitrogen throughout development, and there was a linear arginine nitrogen accumulation rate of 1212 nanomoles per cotyledon per day between 16 and 58 days after anthesis. Arginine and asparagine were major constituents of the free amino acid pool, constituting 14 to 62% and 2 to 41% of the total free amino acid nitrogen, respectively. The urea cycle intermediates, citrulline, ornithine, and argininosuccinate were also detected in the free pool. A comparison of the amino acid composition of cotyledonary protein and of seedcoat exudate suggested that 72% of the cotyledon's arginine requirement is satisfied by in situ biosynthesis, and that 20% of the transformed nitrogen is incorporated into arginine. Also, [1-(14)C]glutamate and [U-(14)C]glutamine were fed to excised cotyledons. After 4 hours, (14)C was incorporated into protein and released as (14)CO(2), but none was incorporated into the C-1 and C-6 positions of free and protein arginine, determined using arginine-specific enzyme-linked assays. It is not currently known whether arginine biosynthesis in the cotyledon involves glutamate delivered from the mother plant or glutamate derived in situ.

Arginine Metabolism in Developing Soybean Cotyledons : II. Biosynthesis.

Tracer kinetic experiments were performed using [ureido-(14)C] citrulline, [1-(14)C]ornithine, and isotope trapping techniques to determine if arginine is synthesized via the urea cycle in developing cotyledons of Glycine max (L.) Merrill. Excised cotyledons were injected with the (14)C-solution and incubated in sealed vials containing a CO(2) trap. The free and protein amino acids were analyzed using high performance liquid chromatography and arginine-specific enzyme-linked assays. In the (14)C-citrulline feeding experiment argininosuccinate was the most highly labeled compound after 5 minutes and it was the first compound to lose (14)C later in the time course. Carbon-14 was also recovered in free arginine, protein arginine, and CO(2) up to 4 hours after introduction of label. All of the (14)C in free and protein arginine could be accounted for in the C-6 position. Metabolism of (14)C-ornithine resulted in (14)C-incorporation into citrulline and free and protein arginine and the evolution of (14)CO(2). Citrulline was the most highly labeled compound after 15 minutes and was the first compound to reach a steady state level of (14)C. With the addition of 800 nanomoles unlabeled citrulline to the (14)C-ornithine feeding solution citrulline was the only compound labeled after 5 minutes and the steady state level of (14)C-citrulline increased 12-fold. The appearance of (14)C in free arginine and protein arginine was also delayed. In both (14)C-ornithine feedings all of the (14)C in free and protein arginine could be accounted for in the C-1 position. Together, the data support the reaction sequence: ornithine --> citrulline --> argininosuccinate --> arginine --> protein arginine.

Ureide metabolism in leaves of nitrogen-fixing soybean plants.

In leaf pieces from nodulated soybean (Glycine max [L] Merr cv Maple Arrow) plants, [(14)C]urea-dependent NH(3) and (14)CO(2) production in the dark showed an approximately 2:1 stoichiometry and was decreased to less than 11% of the control (12-19 micromoles NH(3) per gram fresh weight per hour) in the presence of 50 millimolar acetohydroxamate, a urease inhibitor. NH(3) and CO(2) production from the utilization of [2-(14)C] allantoin also exhibited a 2:1 stoichiometry and was reduced to a similar extent by the presence of acetohydroxamate with a concomitant accumulation of urea which entirely accounted for the loss in NH(3) production. The almost complete sensitivity of NH(3) and CO(2) production from allantoin and urea metabolism to acetohydroxamate, together with the observed stoichiometry, indicated a path of ureide assimilation (2.0 micromoles per gram leaf fresh weight per hour) via allantoate, ureidoglycolate, and glyoxylate with the production of two urea molecules yielding, in turn, four molecules of NH(3) and two molecules of CO(2).

Purification and properties of inosine monophosphate oxidoreductase from nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp).

Using ammonium sulfate precipitation, gel filtration, and affinity chromatography, inosine monophosphate (IMP) oxidoreductase (EC 1.2.1.14) was isolated from the soluble proteins of the plant cell fraction of nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp). The enzyme, purified more than 140-fold with a yield of 11%, was stabilized with glycerol and required a sulfydryl-reducing agent for maximum activity. Gel filtration indicated a molecular weight of 200,000, and sodium dodecyl sulfate-gel electrophoresis a single subunit of 50,000 Da. The final specific activity ranged from 1.1 to 1.5 mumol min-1 mg protein-1. The enzyme had an alkaline pH optimum and showed a high affinity for IMP (Km = 9.1 X 10(-6) M at pH 8.8 and NAD levels above 0.25 mM) and NAD (Km = 18-35 X 10(-6) M at pH 8.8). NAD was the preferred coenzyme, with NADP reduction less than 10% of that with NAD, while molecular oxygen did not serve as an electron acceptor. Intermediates of ureide metabolism (allantoin, allantoic acid, uric acid, inosine, xanthosine, and XMP) did not affect the enzyme, while AMP, GMP, and NADH were inhibitors. GMP inhibition was competitive with a Ki = 60 X 10(-6) M. The purified enzyme was activated by K+ (Km = 1.6 X 10(-3) M) but not by NH+4. The K+ activation was competitively inhibited by Mg2+. The significance of the properties of IMP oxidoreductase for regulation of ureide biosynthesis in legume root nodules is discussed.

Effects of short-term n(2) deficiency on N metabolism in legume nodules.

The study aimed to test the hypothesis that ammonia production by Rhizobium bacteroids provides not only a source of nitrogen for growth but has a central regulatory role in maintaining the metabolic activity and functional integrity of the legume nodule. Production of ammonia in intact, attached nodules was interrupted by short-term (up to 3 days) exposure of the nodulated root systems of cowpea (Vigna unguiculata L. Walp cv Vita 3: Rhizobium CB 756) and lupin (Lupinus albus L. cv Ultra: Rhizobium WU 425) to atmospheres of argon:oxygen (80:20; v/v). Treatment did not affect nodule growth, levels of plant cell and bacteroid protein, leghaemoglobin content, or nitrogenase (EC 1.7.99.2) activity (acetylene reduction) but severely reduced (by 90%) synthesis and export of the major nitrogenous solutes produced by the two symbioses (ureides in cowpea, amides in lupin). Glutamine synthetase (EC 6.3.1.2) and NAD:glutamate oxidoreductase (EC I.4.1.2) were more or less stable to Ar:O(2) treatment, but activities of the glutamine-utilizing enzymes, glutamate synthase (EC 2.6.1.53), asparagine synthetase (EC 6.3.5.4) (lupin only), and de novo purine synthesis (cowpea only), were all markedly reduced. Production and export of nitrogenous solutes by both symbioses resumed within 2 hours after transferring Ar:O(2)-treated plants back to air. In each case the major exported product of fixation after transfer was initially glutamine, reflecting the relative stability of glutamine synthetase activity. Subsequently, glutamine declined and products of its assimilation became predominant consistent with resurgence of enzymes for the synthesis of asparagine in lupin and ureides in cowpea. Enzymes not directly involved with either ammonia or glutamine assimilation (purine synthesis, purine oxidation, and carbon metabolism of both bacteroids and plant cells) also showed transient changes in activity following interruption of N(2) supply. These data have been interpreted to indicate a far-reaching effect of the production of ammonia by bacteroids on a wide range of enzymes, possibly through control of protein turnover, rather than a highly specific effect of ammonia, or some product of its assimilation, on a few enzyme species.

Effects of n(2) deficiency on transport and partitioning of C and N in a nodulated legume.

Nodulated root systems of white lupin (Lupinus albus L. cv Ultra: Rhizobium strain WU425) were exposed to Ar:O(2) (80:20, v/v) or Ar:N(2):O(2) (70:10:20, v/v/v) and C and N partitioning were examined over a 9- or 10-day period in comparison with control plants with nodulated roots retained in air. Accumulation of N ceased in plants exposed to Ar:O(2) or was much reduced in plants exposed to Ar:N(2):O(2), but net C assimilation rates and profiles of C utilization remained similar to those of control N(2)-fixing plants. There was, however, a proportional reduction in CO(2) evolution from nodulated roots of the Ar:O(2) treatment. Xylem N levels fell rapidly after application of Ar:O(2). C:N ratios of phloem sap of petioles and of stem base rose during the first day of Ar:O(2) treatment and then fell progressively back to levels close to that of control plants as leaf reserves of N became available for loading of phloem. Stem top phloem sap increased progressively in C:N ratio throughout Ar:O(2) treatment, presumably due to increasing shortage of xylem derived N for xylem to phloem exchange. Reexposure of Ar:O(2)-treated nodulated root systems to air prompted a rapid recovery of N(2) fixation and restoration of plant N status. Rates of N(2) fixation in plants whose roots were exposed to a range of N(2) concentrations indicated an apparent K(m) of 10% N(2) for the attached intact white lupin nodule.

Nitrogen nutrition and the development and senescence of nodules on cowpea seedlings.

Cowpea (Vigna unguiculata (L.) Walp cv. Vita 3) seedlings inoculated with Rhizobium strain CB756 were cultured with their root systems maintained in air or in Ar: O2 (80:20, v/v) during early nodule development (up to 24 d after sowing). Compared with those in air, seedlings in Ar:O2 showed progressive N deficiency with inhibited shoot growth, reduced ribulose-1,5-bisphosphate carboxylase and total protein levels and loss of chlorophyll in the leaves. Nodule initiation, differentiation of infected and uninfected nodule tissues and the ultrastructure of bacteriod-containing cells were similar in the air and Ar: O2 treatments up to 16 d after sowing. Thereafter the Ar: O2 treatment caused cessation of growth and development of nodules, reduced protein levels in bacteroids and nodule plant cells, and progressive degeneration of nodule ultrastructure leading to premature senescence of these organs. Provision of NO 3 (-) (0.1-0.2 mM) to Ar: O2-grown seedlings overcame the abovementioned consequences of N2 deficiency on nodule and plant growth, but merely delayed the degenerative effects of Ar: O2 treatment on nodule structure and senescence. Treatment of Ar: O2-grown seedlings with NO 3 (-) greatly increased the protein level of nodules but the increase was largely restricted to the plant cell fraction as opposed to the bacteroids. By contrast, NO 3 (-) treatment of air-grown seedlings increased protein of bacteroid and host nodule fractions to the same relative extents when compared with air-grown plants not supplemented with NO 3 (-) . These findings, taken together with studies of the distribution of N in nodules of symbiotically effective plants grown from (15)N-labeled seed, indicate that direct incorporation of fixation products by bacteroids may be a critical feature in the establishment and continued growth of an effective symbiosis in the cowpea seedling.

Nitrogen nutrition and the development of biochemical functions associated with nitrogen fixation and ammonia assimilation of nodules on cowpea seedlings.

During early development (up to 18 d after sowing) of nodules of an "effective" cowpea symbiosis (Vigna unguiculata (L.) Walp cv. Vita 3: Rhizobium strain CB756), rapidly increasing nitrogenase (EC 1.7.99.2) activity and leghaemoglobin content were accompanied by rapid increases in activities of glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), enzymes of denovo purine synthesis (forming inosine monophosphate) xanthine oxidoreductase (EC 1.2.3.2), urate oxidase (EC 1.7.3.3), phosphoenolpyruvate carboxylase (EC 4.1.1.31) and led to increased export of ureides (allantoin and allantoic acid) to the shoot of the host plant in the xylem. Culturing plants with the nodulated root systems maintained in the absence of N2 (in 80 Ar: 20 O2, v/v) had little effect on the rates of induction and increase in nitrogenase activity and leghaemoglobin content but, in the absence of N2 fixation and consequent ammonia production by bacteroids, there was no stimulation of activity of enzymes of ammonia assimilation or of the synthesis of purines or ureides. Addition of NO 3 (-) (0.1-0.2 mM) relieved host-plant nitrogen deficiency caused by the Ar: O2 treatment but failed to increase levels of enzymes of N metabolism in either the bacteroid or the plant-cell fractions of the nodule. Premature senescence in Ar: O2-grown nodules occurred at 18-20 d after sowing, and resulted in reduced levels of nitrogenase activity and leghaemoglobin but increased the activity of hydroxybutyrate oxidoreductase (EC 1.1.1.30).