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Ornithine delta-aminotransferase (OAT) is an important enzyme in proline biosynthetic pathway and is implicated in salt tolerance in higher plants. OAT transaminates ornithine to pyrroline 5-carboxylate, which is further catalyzed to proline by pyrroline 5-carboxylate reductase. The Vigna aconitifolia OAT cDNA, encoding a polypeptide of 48.1 kDa, was expressed in Escherichia coli and the enzyme was partially characterized following its purification using (NH(4))(2)SO(4) precipitation and gel filtration techniques. Optimal activity of the enzyme was observed at a temperature of 25 degrees C and pH 8.0. The enzyme appeared to be a monomer and exhibited high activity at 4mM ornithine. Proline did not show any apparent effect but isoleucine, valine and serine inhibited the activity when added into the assay mixture along with ornithine. Omission of pyridoxal 5'-phosphate from the reaction mixture reduced the activity of this enzyme by 60%. To further evaluate these biochemical observations, homology modeling of the OAT was performed based on the crystal structure of the ornithine delta-aminotransferase from humans (PDB code 1OAT) by using the software MODELLER6v2. With the aid of the molecular mechanics and dynamics methods, the final model was obtained and assessed subsequently by PROCHECK and VERIFY-3D graph. With this model, a flexible docking study with the substrate and inhibitors was performed and the results indicated that Gly106 and Lys256 in OAT are the important determinant residues in binding as they have strong hydrogen bonding contacts with the substrate and inhibitors. These observations are in conformity with the results obtained from experimental investigations.

The symbiotically reduced nitrogen is assimilated in the host cytoplasm with the help of cytosolic glutamine synthetase. We have shown that this enzyme is induced directly in response to ammonia. The glutamine formed in the cytoplasm is funneled to the plastids where it is converted to purines. The latter are synthesized via de novo biosynthesis pathway. The purines are then oxidized into ureides inside the uninfected cells of root nodules. We have demonstrated that the de novo purine biosynthesis pathway is induced prior to the onset of nitrogen fixation, apparently due to high demand for purines for DNA endoreduplication in the infected cells. We have also demonstrated that glutamine enhances the expression of PRAT gene, encoding the first enzyme of the pathway. Because the level of uricase increases prior to that of PRAT, it suggests that purine catabolism may begin prior to the increase in de novo purine biosynthesis and the commencement of nitrogen fixation. The access of purines, exported to the uninfected cells, apparently induce peroxisome proliferation for the oxidation of uric acid to ureides. We have demonstrated that purines are able to induce peroxisome proliferation in yeast. Using urate as a source of nitrogen, we have been able to isolate several yeast mutants defective in peroxisome proliferation/assembly. We have complemented one of these mutants with a soybean gene encoding a putative peroxisome proliferation transcriptional factor with similarity to G-box binding proteins. In addition, we have determined two independent pathways for the conversion of uric acid to ureides in root nodules and have isolated corresponding genes involved in these pathways.

Rhizobia in the infected cells of root nodules are compartmentalized inside a membrane, peribacteroid membrane (PBM), where they are differentiated into bacteroids and begin fixing nitrogen. Since bacteria are never free inside the plant cell during symbiosis, the PBM serves as an interface between the two organisms and its integrity is vital for symbiotic nitrogen fixation. As bacteria are released from the infection thread they are endocytosed by the plasma membrane of the host and this membrane proliferates to enclose bacteria individually or in a group to eight per envelope. This necessitates a tight coordination of PBM biogenesis with the bacterial cell division and the number of bacteroids per envelope reflects when cell division ceases following endocytosis. This event is determined by the host. Although derived from the plasma membrane (PM), the PBM undergoes significant chandes and becomes a mosaic membrane having properties in common with the vacuolar and the PM (Verma, 1992). Thus, vesicular traffic in root nodules must be unique to direct appropriate PBM nodulins and the peribacteroid fluid proteins to their proper sites. The plant genetics data (Tikhonovich et al., 1995) is consistent with this interpretation. The mechanism controlling PBM biogenesis and proliferation is poorly understood. Recent studies on plant and bacterial genetics have shed some lights on biogenesis of this compartment and suggest that both bacteria and the host plant control the synthesis of PBM and its effectiveness in symbiosis.

GS (EC 6.3.1.2) occupies a key position in controlling nitrogen metabolism, a pathway directly affecting plant productivity. It catalyzes the first step in the assimilation of ammonia in higher plants, converting inorganic ammonia produced by different metabolic routes into organic nitrogen. GS is encoded by a small gene family in higher plants, the members of which are expressed in different tissues, cell types, and subcellular compartments under the control of both metabolic status and developmental programs of the plant (Hirel et al., 1993). This temporal and spatial regulation of GS genes meets the requirements of ammonia assimilation under various physiological conditions. In legume-Rhizobium symbiosis, the symbiotically reduced ammonia is assimilated by host root nodule-specific and/or root/nodule-specific GS. Both nodule-specific (alfalfa, Dunn et al., 1988; and soybean, Roche et al., 1993) and nodule-enhanced (French bean, Cullimore et al., 1984) GS isoforms have been reported. In pea no nodule-specific GS has been found (Tingey et al., 1987). In addition to the nodule-specific GS, soybean also contains a root/nodule GS, the expression of which is strongly enhanced in nodules. The latter occurs in direct response to the availability of ammonia (Hirel et al., 1987; Mia0 et al., 1991). We report here the isolation of a full-length cDNA sequence encoding mothbean (Vigna aconitifolia) GS, expression of which appears to be nodule specific. Mothbean is a diploid tropical legume that can be easily transformed by Agrobacterium-mediated transformation methods (Lee et al., 1993) and regenerated (our unpublished results). Thus, mothbean is a plant of choice toward understanding the regulation and role of specific GS isoforms in plant nitrogen metabolism. Using a soybean cytosolic GS sequence (GS20; Mia0 et al., 1991) as a probe, we isolated severa1 GS cDNA sequences from a mothbean nodule cDNA library (Table I). One of these sequence, VGSn-1, contains an open reading frame of 1068 nucleotides and encodes a GS polypeptide of 356 amino acids. Sequence homology comparison showed that VGSn-1 shares more than 85% similarity at the amino acid level

Nodulin-26 is an intrinsic membrane protein of the peribacteroid membrane in soybean root nodules. This protein contains six transmembrane domains and forms an ion channel translocating specific molecules to the bacteroids. We have determined the topology of this protein by co-translational processing and protease protection experiments. Our results suggest that the carboxy and amino termini of this protein face the cytoplasm while the glycosylation site at amino acid position 150 is located on the surface facing the bacteroids. Two transmembrane domains are sufficient for the integration of nodulin-26 into the membrane as revealed by deletion analysis. Nodulin-26 is phosphorylated by a kinase located in the peribacteroid membrane. The phosphorylation mechanism may allow nodulin-26 to be an active channel.

Leghaemoglobin genes in soybean (Glycine max) are present as a moderately reiterated family of sequences. Since there are identical restriction site patterns of these sequences in DNA isolated from leaf, root, or nodule tissue, the data suggest that no major changes in the organization or methylation of leghaemoglobin genes occur during their induction. Cloned soybean leghaemoglobin–cDNA cross hybridized with RNA from root nodules of kidney bean (Phaseolus vulgaris), and to a lesser extent, of pea (Pisum sativum) indicating sequence homology in the leghaemoglobin genes of these species. Hybridization to the genomic DNA restriction fragments of two other species, Glycine soja and Vicia faba, also indicated interspecies sequence homologies. Several restriction fragments appear to be common to all the species examined. The induction of these genes occurs following infection of the plant by Rhizobium and is independent of the appearance of nitrogenase activity in the nodules. The level of expression is, however, influenced by various mutations in Rhizobium that result in the development of ineffective (nonnitrogen fixing) nodules.

A suspension culture obtained from peanut (Arachis hypogaea L.) cotyledons was passed through three standard sieves of 0.15, 0.5, and 2.0 mm and the subsequent four fractions were studied for morphological differences and molecular heterogeneity in peroxidase isozymes. Quantitative and qualitative differences occur in this enzyme between groups of cells of different sizes. The appearance of two new peroxidase isozymes is correlated with cellular differentiation as the cell mass enlarges from 0.5 to 4.0 mm in diameter.

In a suspension culture of peanut cotyledons (Arachis hypogaea L.) the rate of protein synthesis decreased as the cell mass increased in diameter, with a decrease in free amino acids except in a cell mass of 0.15–0.5 mm. A high level of catalase and low level of peroxidase activity was related to a higher rate of protein synthesis in single cell ( 0.5 mm in diameter. Another peroxidase isozyme appeared in a cell mass of 2–4 mm in diameter. Quantitatively there was a significant increase in peroxidase with a decrease in catalase activity between cell clumps of increasing sizes.

Protein synthesis and respiration of 3 day old V-56R peanut (Arachis hypogaea, L.) cell-suspension cultures are reduced to 45-55% of controls immediately after a massive dose (500 Krad) of radiation. However, they apparently recovered during 3-4 days of postirradiation period. Recovery in protein synthesis and respiration continued and exceeded the control levels one week after radiation. Analysis of the proteins synthesized during the postirradiation period indicated that the distribution of radioactivity in labeled proteins is changed and large molecular weight proteins are directly or indirectly broken down to low molecular weight fractions. A significant increase in endogenous free amino acids is observed during the postirradiation period. Both respiration and protein synthesis started to decline 2 weeks after irradiation and levelled well below the controls during subsequent postirradiation period. Although quantitatively protein synthesis showed an apparent recovery after massive irradiation, the pr...