Paul J. Storer

The extrafloral nectaries of cowpea (Vigna unguiculata (L.) Walp.) II. Nectar composition, origin of nectar solutes, and nectary functioning

Nectar was collected from the extrafloral nectaries of leaf stipels and inflorescence stalks, and phloem sap from cryopunctured fruits of cowpea plants. Daily sugar losses as nectar were equivalent to only 0.1–2% of the plants current net photosynthate, and were maximal in the fourth week after anthesis. Sucrose:glucose:fructose weight ratios of nectar varied from 1.5:1:1 to 0.5:1:1, whereas over 95% of phloem-sap sugar was sucrose. [14C]Sucrose fed to leaves was translocated as such to nectaries, where it was partly inverted to [14C]glucose and [14C]fructose prior to or during nectar secretion. Invertase (EC 3.2.1.26) activity was demonstrated for inflorescence-stalk nectar but not stipel nectar. The nectar invertase was largely associated with secretory cells that are extruded into the nectar during nectary functioning, and was active only after osmotic disruption of these cells upon dilution of the nectar. The nectar invertase functioned optimally (phloem-sap sucrose as substrate) at pH 5.5, with a starting sucrose concentration of 15% (w/v). Stipel nectar was much lower in amino compounds relative to sugars (0.08–0.17 mg g-1 total sugar) than inflorescence nectar (22–30 mg g-1) or phloem sap (81–162 mg g-1). The two classes of nectar and phloem sap also differed noticeably in their complements of organic acids. Xylem feeding to leaves of a range of 14C-labelled nitrogenous solutes resulted in these substrates and their metabolic products appearing in fruit-phloem sap and adjacent inflorescence-stalk nectar. 14C-labelled asparagine, valine and histidine transferred freely into phloem and appeared still largely as such in nectar. 14C-labelled glycine, serine, arginine and aspartic acid showed limited direct access to phloem and nectar, although labelled metabolic products were transferred and secreted. The ureide allantoin was present in phloem, but absent from both types of nectar. Models of nectary functioning are proposed.

Reexamination of the Intracellular Localization of de Novo Purine Synthesis in Cowpea Nodules.

Sucrose and Percoll density gradient centrifugation were used to separate organelles from the central zone tissue of cowpea (Vigna unguiculata L. Walp. cv Vita 3: Bradyrhizobium strain CB 756) nodules. Enzyme activity analysis has shown that both plastids and mitochondria have a full complement of enzymes for de novo purine synthesis. In vitro activities of individual component enzymes (glycinamide ribonucleotide synthetase, EC 6.3.4.13; glycinamide ribonucleotide transformylase, EC 2.1.2.2; aminoimidazole ribonucleotide synthetase, EC 6.3.3.1; aminoimidazole carboxamide ribonucleotide transformylase, EC 6.3.2.6; and adenylosuccinate-AMP lyase, EC 4.3.2.2) as well as of the whole purine pathway (from ribose-5-phosphate to inosine monophosphate) were similar in the two organelles. No significant cytosolic or bacteroidal activity of any of the purine pathway enzymes was detected on assay. These findings are contrary to earlier studies (M.J. Boland, K.R. Schubert [1983] Arch Biochem Biophys 220: 179–187; B.J. Shelp C.A. Atkins, P.J. Storer, D.T. Canvin [1983] Arch Biochem Biophys 224: 429–441) that concluded that enhanced expression of purine synthesis in nodules of ureide-forming species is localized to plastids. Significantly increased recovery of activity of key pathway enzymes (particularly of labile aminoimidazole ribonucleotide synthetase) coupled with improved assay methods and the use of Percoll in addition to sucrose for gradient centrifugation have together contributed to much higher reaction rates and more definitive analyses of particulate fractions.

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 NO3-(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).

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.

Purification and Properties of Purine Nucleosidase from N2-Fixing Nodules of Cowpea (Vigna unguiculata)

Summary Purine nucleosidase (nucleoside hydrolase; EC 3.2.2.1) has been purified to homogeneity from the soluble plant cell fraction of N 2 -fixing nodules of the tropical legume cowpea ( Vigna unquiculata L. Walp.). The purification procedure involved ammonium sulphate precipitation, chromatography on DEAE-Sephacel, Guanosine-Affinity Sepharose and AH-Sepharose-4B followed by preparative polyacrylamide gel electrophoresis. The enzyme was purified more than 400 fold with a yield of 21%, showed a broad pH optimum around pH 8.0, and a maximum specific activity of 432nmol xanthosine hydrolysed·min -1 ·mg -1 protein. Gel filtration indicated a native molecular weight of 160,000 daltons with a single polypeptide subunit of 30,600 daltons determined by SDS-PAGE. The purified enzyme hydrolysed both purine (xanthosine, inosine, guanosine and adenosine) as well as pyrimidine (uridine, thymidine and cytidine) nucleosides to their bases. Among the purine nucleosides the V max values were in the ratio 28:7:1:0.4 for xanthosine, inosine, adenosine and guanosine respectively. K m values were similar for xanthosine and inosine (0.80 and 0.83 mM respectively). Inosine, adenosine, guanosine and uridine were competitive inhibitors of xanthosine hydrolysis (Ki = 0.78, 1.10, 0.36 and 1.20 mM respectively). Xanthosine and adenosine were also competitive inhibitors of inosine hydrolysis (Ki = 0.80 and 1.05 mM), but guanosine inhibition showed non-competitive kinetics (Ki = 0.98 mM).

Effect of oxygen pressure on synthesis and export of nitrogenous solutes by nodules of cowpea

Nodules of cowpea plants (Vigna unguiculata (L.) Walp. cv. Vita 3 :Bradyrhizobium CB756) cultured for periods of 23 d with their root systems maintained in atmospheres containing a range of partial pressures of O2 (pO2; 1–80%, v/v, in N2) formed and exported ureides (allantoin and allantoic acid) as the major products of fixation at all pO2 tested. In sub-ambient pO2 (1 and 2.5%) nodules contained specific activities of uricase (urate: O2 oxidoreductase; EC 1.7.3.3) and allantoinase (allantoin hydrolyase; EC 3.5.2.5) as much as sevenfold higher than in those from air. On a cell basis, uninfected cells in nodules from 1% O2 contained around five times the level of uricase. Except for NAD: glutamate synthase (EC 1.4.1.14), which was reduced in sub-ambient O2, the activities of other enzymes of ureide synthesis were relatively unaffected by pO2. Short-term effects of pO2 on assimilation of fixed nitrogen were measured in nodules of air-grown plants exposed to subambient pO2 (1, 2.5 or 5%, v/v in N2) and15N2. Despite a fall in total15N2 fixation, ureide synthesis and export was maintained at a high level except in 1% O2 where formation was halved. The data indicate that in addition to the structural and diffusional adaptations of cowpea nodules which allow the balance between O2 supply and demand to be maintained over a wide range of pO2, nodules also show evidence of biochemical adaptations which maintain and enhance normal pathways for the assimilation of fixed nitrogen.

Effect of Short-Term N2 Deficiency on Expression of the Ureide Pathway in Cowpea Root Nodules

Root systems of 28-d-old cowpea (Vigna unguiculata L. Walp cv Vita 3: Bradyrhizobiumsp. strain CB756) plants bearing nitrogen-fixing nodules in sand culture were exposed to an atmosphere of Ar:O2 (80:20, v/v) for 48 h and then returned to air. Root systems of control plants were maintained in air throughout. Nodules were harvested at the same times in control and Ar:O2-treated root systems. Activities of two enzymes of de novo purine synthesis, glycinamide ribonucleotide transformylase (GART; EC 2.1.2.2), aminoimidazole ribonucleotide synthetase (AIRS; EC 6.3.3.1), uricase (EC 1.7.3.3), and phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) were measured together with the protein level of each using immune-specific polyclonal antibodies. AIRS activity and protein both declined to very low levels within 6 h in Ar:O2 together with a decline in transcript level of pur5, the encoding gene. GART activity, protein, and transcript (pur3) levels were relatively stable. Uricase activity declined in Ar:O2 as rapidly as AIRS activity but the protein was stable. PEPC activity showed evidence of increased sensitivity to inhibition by malate but the protein level was stable. The data indicate that the flux of fixed N from bacteroids (N2-fixing nodule bacteria) is in some way associated with transcriptional control over pur5and possibly also catabolism of AIRS protein. In contrast, there is limited posttranslational control over GART and PEPC and close posttranslational control over uricase activity. The significance of these different levels of regulation is discussed in relation to the overall control of enhanced expression of plant enzymes in the cowpea symbiosis.

Effect of Short-Term N2 Deficiency on Nitrogenase Activity and Ureide Biosynthesis in Cowpea (Vigna unguiculata L. Walp) Nodules

Aminoimidazole ribonucleotide synthetase (AIRS), glycinamide ribonucleotide transformylase (GART) and uricase are important enzymes in the de novo synthesis of purines and the oxidation of purines to ureides in cowpea (Vigna unguiculata L. Walp.) nodules (Atkins et al., 1997). Ureides are the main products of N2 fixation exported from the nodules of cowpea. When the roots of well nodulated cowpea plants were exposed to an atmosphere of 80%Ar:20%O2(v/v), de novo purine synthesis and ureide export declined (Atkins et al., 1984). Here we show that the decline in purine synthesis is associated with a rapid and dramatic loss of AIRS activity and protein from the nodules and a less rapid, although equally dramatic, loss of uricase activity but no loss of uricase protein.