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D P Verma

Publications and source records attributed to D P Verma.

At least 19 recordsLinked to original sources
PubMed2001

Plant callose synthase complexes.

Synthesis of callose (beta-1,3-glucan) in plants has been a topic of much debate over the past several decades. Callose synthase could not be purified to homogeneity and most partially purified cellulose synthase preparations yielded beta-1,3-glucan in vitro, leading to the interpretation that cellulose synthase might be able to synthesize callose. While a rapid progress has been made on the genes involved in cellulose synthesis in the past five years, identification of genes for callose synthases has proven difficult because cognate genes had not been identified in other organisms. An Arabidopsis gene encoding a putative cell plate-specific callose synthase catalytic subunit (CalS1) was recently cloned. CalS1 shares high sequence homology with the well-characterized yeast beta-1,3-glucan synthase and transgenic plant cells over-expressing CalS1 display higher callose synthase activity and accumulate more callose. The callose synthase complex exists in at least two distinct forms in different tissues and interacts with phragmoplastin. UDP-glucose transferase, Rop1 and, possibly, annexin. There are 12 CalS isozymes in Arabidopsis, and each may be tissue-specific and/or regulated under different physiological conditions responding to biotic and abiotic stresses.

Gene Expression Regulation, Enzymologic↗
PubMed2001

A cell plate-specific callose synthase and its interaction with phragmoplastin.

Callose is synthesized on the forming cell plate and several other locations in the plant. We cloned an Arabidopsis cDNA encoding a callose synthase (CalS1) catalytic subunit. The CalS1 gene comprises 42 exons with 41 introns and is transcribed into a 6.0-kb mRNA. The deduced peptide, with an approximate molecular mass of 226 kD, showed sequence homology with the yeast 1,3-beta-glucan synthases and is distinct from plant cellulose synthases. CalS1 contains 16 predicted transmembrane helices with the N-terminal region and a large central loop facing the cytoplasm. CalS1 interacts with two cell plate--associated proteins, phragmoplastin and a novel UDP-glucose transferase that copurifies with the CalS complex. That CalS1 is a cell plate--specific enzyme is demonstrated by the observations that the green fluorescent protein--CalS1 fusion protein was localized at the growing cell plate, that expression of CalS1 in transgenic tobacco cells enhanced callose synthesis on the forming cell plate, and that these cell lines exhibited higher levels of CalS activity. These data also suggest that plant CalS may form a complex with UDP-glucose transferase to facilitate the transfer of substrate for callose synthesis.

Amino Acid Motifs↗
PubMed2001

A novel UDP-glucose transferase is part of the callose synthase complex and interacts with phragmoplastin at the forming cell plate.

Using phragmoplastin as a bait, we isolated an Arabidopsis cDNA encoding a novel UDP-glucose transferase (UGT1). This interaction was confirmed by an in vitro protein--protein interaction assay using purified UGT1 and radiolabeled phragmoplastin. Protein gel blot results revealed that UGT1 is associated with the membrane fraction and copurified with the product-entrapped callose synthase complex. These data suggest that UGT1 may act as a subunit of callose synthase that uses UDP-glucose to synthesize callose, a 1,3-beta-glucan. UGT1 also interacted with Rop1, a Rho-like protein, and this interaction occurred only in its GTP-bound configuration, suggesting that the plant callose synthase may be regulated by Rop1 through the interaction with UGT1. The green fluorescent protein--UGT1 fusion protein was located on the forming cell plate during cytokinesis. We propose that UGT1 may transfer UDP-glucose from sucrose synthase to the callose synthase and thus help form a substrate channel for the synthesis of callose at the forming cell plate.

Arabidopsis↗
PubMed2000

Phragmoplastin polymerizes into spiral coiled structures via intermolecular interaction of two self-assembly domains.

Phragmoplastin, a high molecular weight GTPase belonging to the dynamin superfamily of proteins, becomes associated with the cell plate during cytokinesis in plants. Growth of the cell plate requires continuous fusion of vesicles, and phragmoplastin appears to play a role in the formation of vesicle-tubule-vesicle structures at the cell plate. In this study, we have demonstrated that two self-assembly domains (SA1 and SA2) are involved in polymerization of phragmoplastin. SA1 is about 42 amino acids long and is located near the N terminus overlapping with the GTP-binding region. SA2, containing at least 24 amino acids, is located in the middle of the molecule outside the GTP-binding domain. Peptides containing either SA1 or SA2 interact efficiently with the full-length phragmoplastin. The SA1 domain of one phragmoplastin molecule also binds to SA2 of another as confirmed in vitro by using radiolabeled peptides. This interaction leads to the formation of polymers with a staggered contoured spiral structure. Electron microscopy studies revealed that helical arrays of phragmoplastin can be induced by reducing salt concentration. Our results suggest that phragmoplastin may assemble into helical arrays that wrap around and squeeze vesicles into vesicle-tubule-vesicle structures observed on the forming cell plate.

Amino Acid Sequence↗
PubMed2000

Removal of feedback inhibition of delta(1)-pyrroline-5-carboxylate synthetase results in increased proline accumulation and protection of plants from osmotic stress.

The Delta(1)-pyrroline-5-carboxylate synthetase (P5CS; EC not assigned) is the rate-limiting enzyme in proline (Pro) biosynthesis in plants and is subject to feedback inhibition by Pro. It has been suggested that the feedback regulation of P5CS is lost in plants under stress conditions. We compared Pro levels in transgenic tobacco (Nicotiana tabacum) plants expressing a wild-type form of Vigna aconitifolia P5CS and a mutated form of the enzyme (P5CSF129A) whose feedback inhibition by Pro was removed by site-directed mutagenesis. Transgenic plants expressing P5CSF129A accumulated about 2-fold more Pro than the plants expressing V. aconitifolia wild-type P5CS. This difference was further increased in plants treated with 200 mM NaCl. These results demonstrated that the feedback regulation of P5CS plays a role in controlling the level of Pro in plants under both normal and stress conditions. The elevated Pro also reduced free radical levels in response to osmotic stress, as measured by malondialdehyde production, and significantly improved the ability of the transgenic seedlings to grow in medium containing up to 200 mM NaCl. These findings shed new light on the regulation of Pro biosynthesis in plants and the role of Pro in reducing oxidative stress induced by osmotic stress, in addition to its accepted role as an osmolyte.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed1997

Dynamics of phragmoplastin in living cells during cell plate formation and uncoupling of cell elongation from the plane of cell division.

The cell plate is formed by the fusion of Golgi apparatus-derived vesicles in the center of the phragmoplast during cytokinesis in plant cells. A dynamin-like protein, phragmoplastin, has been isolated and shown to be associated with cell plate formation in soybean by using immunocytochemistry. In this article, we demonstrate that similar to dynamin, phragmoplastin polymerizes to form oligomers. We fused soybean phragmoplastin with the green fluorescence protein (GFP) and introduced it into tobacco BY-2 cells to monitor the dynamics of early events in cell plate formation. We demonstrate that the chimeric protein is functional and targeted to the cell plate during cytokinesis in transgenic cells. GFP-phragmoplastin was found to appear first in the center of the forming cell plate, and as the cell plate grew outward, it redistributed to the growing margins of the cell plate. The redistribution of phragmoplastin may require microtubule reorganization because the microtubule-stabilizing drug taxol inhibited phragmoplastin redistribution. Our data show that throughout the entire process of cytokinesis, phragmoplastin is concentrated in the area in which membrane fusion is active, suggesting that phragmoplastin participates in an early membrane fusion event during cell plate formation. Based on the dynamics of GFP-phragmoplastin, it appears that the process of cell plate formation is completed in two phases. The first phase is confined to the cylinder of the phragmoplast proper and is followed by a second phase that deposits phragmoplast vesicles in a concentric fashion, resulting in a ring of fluorescence, with the concentration of vesicles being higher at the periphery. In addition, overexpression of GFP-phragmoplastin appears to act as a dominant negative, slowing down the completion of cell plate formation, and often results in an oblique cell plate. The latter appears to uncouple cell elongation from the plane of cell division, forming twisted and elongated cells with longitudinal cell divisions.

Antibodies↗
PubMed1996

Reciprocal regulation of delta 1-pyrroline-5-carboxylate synthetase and proline dehydrogenase genes controls proline levels during and after osmotic stress in plants.

Plants generally accumulate free proline under osmotic stress conditions. Upon removal of the osmotic stress, the proline levels return to normal. In order to understand the mechanisms involved in regulating the levels of proline, we cloned and characterized a proline dehydrogenase (PDH) cDNA from Arabidopsis thaliana (AtPDH). The 1745 bp cDNA contains a major open reading frame encoding a peptide of 499 amino acids. The deduced amino acid sequence has high homology with both Saccharomyces cerevisiae and Drosophila melanogaster proline oxidases and contains a putative mitochondrial targeting sequence. When expressed in yeast, the AtPDH cDNA complemented a yeast put1 mutation and exhibited proline oxidase activity. We also determined the free proline contents and the delta 1-pyrroline-5-carboxylate synthetase (P5CS) and PDH mRNA levels under different osmotic stress and recovery conditions. The results demonstrated that the removal of free proline during the recovery from salinity or dehydration stress involves an induction of the PDH gene while the activity of P5CS declines. The reciprocal regulation of P5CS and PDH genes appears to be a key mechanism in the control of the levels of proline during and after osmotic stress. The PDH gene was also significantly induced by exogenously applied proline. The induction of PDH by proline, however, was inhibited by salt stress.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed1996

Phragmoplastin, a dynamin-like protein associated with cell plate formation in plants.

Cytokinesis in a plant cell is accomplished by the formation of a cell plate in the center of the phragmoplast. Little is known of the molecular events associated with this process. In this study, we report the identification of a dynamin-like protein from soybean and demonstrate that this protein is associated with the formation of the cell plate. Plant dynamin-like (PDL) protein contains 610 amino acids showing high homology with other members of the dynamin protein family. Western blot experiments demonstrated that it is associated with the non-ionic detergent-resistant fraction of membranes. Indirect immunofluorescence microscopy localized PDL to the cell plate in dividing soybean root tip cells. Double labeling experiments demonstrated that, unlike phragmoplast microtubules which are concentrated on the periphery of the forming plate, PDL is located across the whole width of the newly formed cell plate. Based on the temporal and spatial organization of PDL in the phragmoplast, we termed this protein 'phragmoplastin'. The data suggest that phragmoplastin may be associated with exocytic vesicles that are depositing cell plate material during cytokinesis in the plant cell.

Amino Acid Sequence↗
PubMed1996

Biogenesis of the peribacteroid membrane in root nodules.

An infected root nodule cell may contain several thousand rhizobial symbionts, each enclosed in a membrane envelope, the peribacteroid membrane (PBM). The PBM is derived from the host plasma membrane, but shares properties with the vacuolar membrane and contains several nodule-specific proteins (nodulins) that perform unique functions for symbiosis.

Biological Transport↗
PubMed1995

A rice HAL2-like gene encodes a Ca(2+)-sensitive 3'(2'),5'-diphosphonucleoside 3'(2')-phosphohydrolase and complements yeast met22 and Escherichia coli cysQ mutations.

A plant homolog of yeast HAL2 gene (RHL) was cloned from rice (Orizya sativa L.). The RHL cDNA complemented an Escherichia coli cysteine auxotrophic mutant, cysQ, and the yeast HAL2 mutant, met22. The latter is a methionine auxotroph and cannot use sulfate, sulfite, or sulfide as sulfur sources but exhibits wild-type activities of the enzymes necessary to assimilate sulfate and has normal sulfur uptake system. These results demonstrated that HAL2, cysQ, and RHL genes encode proteins with similar function in sulfur assimilatory pathway. The RHL cDNA expressed a 40-kDa protein that was shown to catalyze the conversion of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) to adenosine 5'-phosphosulfate (APS) and 3'(2')-phosphoadenosine 5'-phosphate (PAP) to AMP. The enzyme activity is Mg(2+)-dependent, sensitive to Ca2+, Li+, and Na+ and activated by K+. The inhibition by Ca2+ depends on the Mg2+/Ca2+ ratio and is reversible by high Mg2+ concentration. The substrate specificity and kinetics of RHL enzyme are very similar to the Chlorella 3'(2'),5'-diphosphonucleoside 3'(2')-phosphohydrolase (DPNPase). Our evidence suggests that this enzyme regulates the flux of sulfur in the sulfur-activation pathway by converting PAPS to APS. Several residues that are essential for the activity of this enzyme were identified by site-directed mutagenesis, and the possible role of DPNPase in salt tolerance is discussed.

Adenosine Phosphosulfate↗
PubMed1995

Removal of feedback inhibition of delta 1-pyrroline-5-carboxylate synthetase, a bifunctional enzyme catalyzing the first two steps of proline biosynthesis in plants.

delta 1-Pyrroline-5-carboxylate synthetase (P5CS) catalyzes the first two steps in proline biosynthesis in plants. The Vigna aconitifolia P5CS cDNA was expressed in Escherichia coli, and the enzyme was purified to homogeneity. The Vigna P5CS exhibited two activities, gamma-glutamyl kinase (gamma-GK) and glutamic acid-5-semialdehyde (GSA) dehydrogenase. The gamma-GK activity of the P5CS was detected by the hydroxamate assay and by a [14C]glutamate assay. The native molecular mass of the P5CS was 450 kDa with six identical subunits. The Vigna P5CS showed a Km of 3.6 mM for glutamate, while the Km for ATP was 2.7 mM. The gamma-GK activity of the P5CS was competitively inhibited by proline, while its GSA dehydrogenase activity was insensitive to proline. In addition, a protein inhibitor of the P5CS was observed in the plant cell. Western blot showed that the level of the P5CS was enhanced in Vigna root under salt stress. A single substitution of an alanine for a phenylalanine at amino acid residue 129 of the P5CS resulted in a significant reduction of proline feedback inhibition. The 50% inhibition values of gamma-GK activity of the wild type and the mutant P5CS were observed at 5 mM and 960 mM proline, respectively. The other properties of the mutant P5CS remained unchanged. These results may allow genetic manipulation of proline biosynthesis and overproduction of proline in plants for conferring water stress tolerance.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed1995

Control of de novo purine biosynthesis genes in ureide-producing legumes: induction of glutamine phosphoribosylpyrophosphate amidotransferase gene and characterization of its cDNA from soybean and Vigna.

Soybean (Glycine max) and mothbean (Vigna aconitifolia) cDNA clones encoding glutamine phosphoribosylpyrophosphate amidotransferase (PRAT), the first enzyme of the de novo purine biosynthesis pathway, have been isolated from nodule cDNA libraries. The amino acid sequence deduced from soybean clone showed > 85% homology to the PRAT sequence of mothbean and 33-47% homology to those of bacteria, yeast, chicken, rat and human. The soybean clone encodes a protein with an N-terminal sequence resembling a plastid-targeting peptide. Downstream from this peptide is a sequence similar to the 11 amino acid propeptide found in the Bacillus subtilis, chicken, rat and human PRAT proteins. The mothbean cDNA, although lacking most of the plastid presequence, encodes the putative propeptide and efficiently complements purine auxotrophy in an Escherichia coli purF mutant. Both the soybean and mothbean clones encode characteristic cysteine residues that are known to be involved in the assembly of a [Fe-S] cluster near the C-terminus of this protein. Levels of PRAT mRNA in mothbean nodules were found to increase steadily as the nodules matured from 13 days to 23 days. PRAT mRNA was not detectable in uninfected root tissue but a low level of transcript was detected in leaves. Treatment of uninfected root with L-glutamine induced the PRAT mRNA transcript suggesting that glutamine produced as a result of assimilation of fixed nitrogen is funnelled into the de novo purine biosynthesis and controls the expression of this pathway in root nodules.

Amidophosphoribosyltransferase↗
PubMed1994

A phosphatidylinositol 3-kinase is induced during soybean nodule organogenesis and is associated with membrane proliferation.

Phosphatidylinositol 3-kinase (PI3K) is an important component of various receptor tyrosine kinase complexes in mammalian cells and a key enzyme required for cell division and vacuolar protein sorting in yeast. To our knowledge, this enzyme has not been characterized in plants. We report the cloning and characterization of soybean PI3K cDNAs and present evidence for the induction of a distinctive form of this enzyme specific to nodule organogenesis. Expression of the root form of PI3K is repressed during nodule organogenesis and is reinduced in mature nodules. Primer-extension results showed that the gene encoding the nodule form of PI3K is highly expressed in young (12-15 day old) root nodules in parallel with membrane proliferation but is repressed in mature nodules. The root form of the PI3K cDNA (SPI3K-5) encodes a peptide of 814 amino acids and the nodule form (SPI3K-1) encodes a peptide of 812 amino acids. Both cDNAs share 98% sequence identity in the coding region but differ in the noncoding regions. The polypeptides encoded by soybean PI3K cDNAs show significant sequence homology (50-60% similarity and 20-40% identity) to both PI3Ks and phosphatidylinositol 4-kinases from mammalian and yeast cells. Escherichia coli expressed soybean PI3K phosphorylated phosphatidylinositol specifically at the D-3 position of the inositol ring to generate phosphatidylinositol 3-phosphate. The temporal increase of a specific PI3K activity during membrane proliferation in young nodules suggests that PI3K plays a pivotal role in development of the peribacteroid membrane forming a subcellular compartment.

Amino Acid Sequence↗
PubMed1994

Nodulin-24 follows a novel pathway for integration into the peribacteroid membrane in soybean root nodules.

Nodulin-24 is a nodule-specific protein of the peribacteroid membrane (PBM) in soybean. It has an apparent molecular mass of 33 kDa while its full-length cDNA encodes a polypeptide of only 24 kDa. In vitro transcription of nodulin-24 cDNA followed by translation resulted in a peptide translocated into microsomal membranes with cleavage of a signal sequence. The cleavage site of the signal sequence in nodulin-24 was determined to be between Ala (A25) and Arg (R26) by microsequencing of the [3H]leucine-labeled processed peptide. Fusion of the signal sequence of nodulin-24 with the beta-glucuronidase peptide prevented co-translational cleavage of the signal sequence although the translocation of the fused protein into microsomes occurred co-translationally. Trypsin treatment of membrane-translocated nodulin-24 did not result in any alteration in size suggesting that the newly synthesized peptide is fully protected in the membrane vesicle. Fusion of nodulin-24 with beta-glucuronidase also showed no change in size following trypsin treatment, suggesting that nodulin-24 has no membrane-spanning region. In addition, in vitro synthesized nodulin-24 was present in the supernatant fraction after sonication of microsomal membranes. Mature nodulin-24, on the other hand, is not solubilized from PBM by sodium carbonate (pH 11) or EGTA and is soluble only in detergent. These data suggest that nodulin-24 is synthesized as a lumenal protein in the endoplasmic reticulum and post-translationally attached to the membranes en route to the PBM. This processing results in a significant increase in the apparent molecular mass of nodulin-24 which may be due to the attachment of membrane lipids as this protein shares characteristics with membrane lipoproteins of many pathogenic bacteria.

Amino Acid Sequence↗
PubMed1994

Structural organization of de novo purine biosynthesis enzymes in plants: 5-aminoimidazole ribonucleotide carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide synthetase cDNAs from Vigna aconitifolia.

Nodules of tropical legumes generally export symbiotically fixed nitrogen in the form of ureides that are produced by oxidation of de novo synthesized purines. To investigate the regulation of de novo purine biosynthesis in these nodules, we have isolated cDNA clones encoding 5-aminoimidazole ribonucleotide (AIR) carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide (SAICAR) synthetase from a mothbean (Vigna aconitifolia) nodule cDNA library by complementation of Escherichia coli purE and purC mutants, respectively. Sequencing of these clones revealed that the two enzymes are distinct proteins in mothbean, unlike in animals where both activities are associated with a single bifunctional polypeptide. As is the case in yeast, the mothbean AIR carboxylase has a N-terminal domain homologous to the eubacterial purK gene product. This PurK-like domain appears to facilitate the binding of CO2 and is dispensable in the presence of high CO2 concentrations. Because the expression of the mothbean PurE cDNA clone in E. coli apparently generates a truncated polypeptide lacking at least 140 N-terminal amino acids, this N-terminal region of the enzyme may not be essential for its CO2-binding activity.

Amino Acid Sequence↗
PubMed1993

Cloning of ornithine delta-aminotransferase cDNA from Vigna aconitifolia by trans-complementation in Escherichia coli and regulation of proline biosynthesis.

Proline prototrophy was restored to an Escherichia coli proBA proline auxotroph by ornithine and a mothbean (Vigna aconitifolia) cDNA expression library. This novel strategy, "trans-complementation," allowed isolation of a cDNA encoding ornithine delta-aminotransferase (delta-OAT). This enzyme transaminates ornithine to glutamic-gamma-semialdehyde (GSA), thereby bypassing the block in GSA synthesis from glutamate in the proBA mutant. The identity of the mothbean enzyme was confirmed by its high sequence homology to mammalian and yeast delta-OATs as well as to a family of bacterial and fungal omega-aminotransferases and an absence of significant homology to various alpha-aminotransferases. The V. aconitifolia OAT cDNA encodes a polypeptide of 48.1 kDa. The native enzyme expressed in E. coli appears to be a monomer with Km of 2 mM for ornithine and 0.75 mM for alpha-ketoglutarate. Levels of mRNA in V. aconitifolia for delta 1-pyrroline-5-carboxylate synthetase (P5CS) and delta-OAT, the two key enzymes for proline synthesis, were monitored under different physiological conditions. Salt stress and nitrogen starvation induced P5CS mRNA levels and depressed OAT mRNA levels. Conversely, OAT mRNA level was elevated in plants supplied with excess nitrogen while the P5CS mRNA level was reduced. These data suggest that the glutamate pathway is the primary route for proline synthesis in plants during conditions of osmotic stress and nitrogen limitation whereas the ornithine pathway assumes prominence under high nitrogen input.

1-Pyrroline-5-Carboxylate Dehydrogenase↗