Desh Pal S. Verma

Overexpression of a Δ1-pyrroline-5-carboxylate synthetase gene and analysis of tolerance to water- and salt-stress in transgenic rice

Abstract A Δ 1 -pyrroline-5-carboxylate synthetase (P5CS) cDNA from mothbean ( Vigna aconitifolia L.) was introduced into rice ( Oryza sativa L.) genome by the biolistic method. Expression of this P5CS transgene under the control of a stress-inducible promoter led to stress-induced overproduction of the P5CS enzyme and proline accumulation in transgenic rice plants. Second-generation (R 1 ) transgenic rice plants showed an increase in biomass under salt-stress and water-stress conditions as compared to the non-transformed control plants.

Arabidopsis TARGET OF RAPAMYCIN Interacts with RAPTOR, Which Regulates the Activity of S6 Kinase in Response to Osmotic Stress Signals

TARGET OF RAPAMYCIN (TOR) kinase controls many cellular functions in eukaryotic cells in response to stress and nutrient availability and was shown to be essential for embryonic development in Arabidopsis thaliana. We demonstrated that Arabidopsis RAPTOR1 (a TOR regulatory protein) interacts with the HEAT repeats of TOR and that RAPTOR1 regulates the activity of S6 kinase (S6K) in response to osmotic stress. RAPTOR1 also interacts in vivo with Arabidopsis S6K1, a putative substrate for TOR. S6K1 fused to green fluorescent protein and immunoprecipitated from tobacco (Nicotiana tabacum) leaves after transient expression was active in phosphorylating the Arabidopsis ribosomal S6 protein. The catalytic domain of S6K1 could be phosphorylated by Arabidopsis 3-phosphoinositide-dependent protein kinase-1 (PDK1), indicating the involvement of PDK1 in the regulation of S6K. The S6K1 activity was sensitive to osmotic stress, while PDK1 activity was not affected. However, S6K1 sensitivity to osmotic stress was relieved by co-overexpression of RAPTOR1. Overall, these observations demonstrated the existence of a functional TOR kinase pathway in plants. However, Arabidopsis seedlings do not respond to normal physiological levels of rapamycin, which appears to be due its inability to bind to the Arabidopsis homolog of FKBP12, a protein that is essential for the binding of rapamycin with TOR. Replacement of the Arabidopsis FKBP12 with the human FKBP12 allowed rapamycin-dependent interaction with TOR. Since homozygous mutation in TOR is lethal, it suggests that this pathway is essential for integrating the stress signals into the growth regulation.

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-β-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.

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

Abstract 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 Δ1-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.

Roles of plant homologs of Rab1p and Rab7p in the biogenesis of the peribacteroid membrane, a subcellular compartment formed de novo during root nodule symbiosis.

The peribacteroid membrane (PBM) in legume root nodules is derived from plasma membrane following endocytosis of Rhizobium by fusion of newly synthesized vesicles. We studied the roles of plant Rab1p and Rab7p homologs, the small GTP‐binding proteins involved in vesicular transport, in the biogenesis of the PBM. Three cDNAs encoding legume homologs of mammalian Rab1p and Rab7p were isolated from soybean (sRab1p, sRab7p) and Vigna aconitifolia (vRab7p). sRab1p was confirmed to be a functional counterpart of yeast Ypt1p (Rab1p) by complementation of a yeast ypt1‐1 mutant. Both srab1 and vrab7 genes are induced during nodulation with the level of vrab7 mRNA being 12 times higher than that in root meristem and leaves. This induction directly correlates with membrane proliferation in nodules. Antisense constructs of srab1 and vrab7, under a nodule‐specific promoter (leghemoglobin, Lbc3), were made in a binary vector and transgenic nodules were developed on soybean hairy roots obtained through Agrobacterium rhizogenes‐mediated transformation. Both antisense srab1 and vrab7 nodules were smaller in size and showed lower nitrogenase activity than controls. The antisense srab1 nodules showed lack of expansion of infected cells, fewer bacteroids per cell and their frequent release into vacuoles. In contrast, antisense vrab7 expressing nodules showed accumulation of late endosomal structure and multivesicular bodies in the perinuclear region. These data suggest that both Rab1p and Rab7p are essential for the development of the PBM compartment in effective symbiosis.

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-β-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.

A soybean gene encoding Δ1-pyrroline-5-carboxylate reductase was isolated by functional complementation in Escherichia coli and is found to be osmoregulated

SummaryWe have isolated several cDNA clones encoding Δ11-pyrroline-5-carboxylate reductase (P5CR, l-proline: NAD(P)+ 5-oxidoreductase, EC 1.5.1.2) which catalyzes the terminal step in proline biosynthesis, by direct complementation of a proC mutation in Escherichia coli with an expression library of soybean root nodule cDNA. The library was constructed in the λ ZapII vector, converted to a plasmid library by in vivo excision of recombinant pBluescript phagemids, and used for transformation of the E. coli mutant. Complementing plasmids contained inserts of about 1.2 kb which hybridized to a 1.3 kb RNA transcript in nodules, uninfected roots and leaves. DNA sequence analysis of one full length cDNA clone showed that it encoded a 28 586 Mr polypeptide with 39% amino acid identity to the E. coli P5CR sequence. Genomic analysis showed that there are two to three copies of the P5CR gene in the soybean genome. The steady-state level of P5CR mRNA in root nodules was twice as high as in uninfected roots and about five times higher than in leaves. Subjecting young seedlings to osmotic stress by watering with 400 mM NaCl resulted in an almost six-fold increase in the level of root P5CR mRNA, suggesting that this gene may be osmoregulated.

Signals in Root Nodule Organogenesis and Endocytosis of Rhizobium.

The rhizobia comprise a diverse group of organisms that elicit hypertrophic growth on the roots of legume plants to form a new organ, the root nodule, which they inhabit to fix nitrogen. This endosymbiotic association makes legume plants auto- trophic for external nitrogen, an essential nutrient for plant growth. Because the interaction with rhizobia is highly benefi- cial, legume plants have evolved a set of genes encoding nodulekpecific proteins (nodulins; Legocki and Verma,

A unified nomenclature for Arabidopsis dynamin-related large GTPases based on homology and possible functions

Z. Hong1, S.Y. Bednarek2, E. Blumwald3, I. Hwang4, G. Jurgens5, D. Menzel6, K.W. Osteryoung7, N.V. Raikhel8, K. Shinozaki9, N. Tsutsumi10 and D.P.S. Verma1,∗ 1Department of Molecular Genetics and Plant Biotechnology Center, Ohio State University, Columbus, OH 43210, USA (∗author for correspondence; e-mail verma.1@osu.edu; 2Department of Biochemistry, University of Wisconsin, 433 Babcock Drive, Madison, WI 53706, USA; 3Department of Pomology, University of California, One Shields Ave., Davis, CA 95616, USA; 4Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, 790-784, Korea; 5Lehrstuhl fur Entwicklungsgenetik, Universitat Tubingen, 72076 Tubingen, Germany; 6Zellbiologie der Pflanzen, Botanisches Institut, Universitat Bonn, Kirschallee 1, Bonn, 53115, Germany; 7Department of Plant Biology, 166 Plant Biology Building, Michigan State University, East Lansing, MI 48824, USA; 8Department of Botany and Plant Sciences and Center for Plant Cell Biology, University of California, Riverside, CA 92521, USA; 9Laboratory of Plant Molecular Biology, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba 305-0074, Japan; 10Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Expression of callose synthase genes and its connection with Npr1 signaling pathway during pathogen infection

Callose synthesis occurs at specific stages of plant cell wall development in all cell types, and in response to pathogen attack, wounding and physiological stresses. We determined the expression pattern of “upstream regulatory sequence” of 12 Arabidopsis callose synthase genes (CalS1–12) genes and demonstrated that different callose synthases are expressed specifically in different tissues during plant development. That multiple CalS genes are expressed in the same cell type suggests the possibility that CalS complex may be constituted by heteromeric subunits. Five CalS genes were induced by pathogen (Hyaloperonospora arabidopsis, previously known as Peronospora parasitica, the causal agent of downy mildew) or salicylic acid (SA), while the other seven CalS genes were not affected by these treatments. Among the genes that are induced, CalS1 and CalS12 showed the highest responses. In Arabidopsis npr1 mutant, impaired in response of pathogenesis related (PR) genes to SA, the induction of CalS1 and CalS12 genes by the SA or pathogen treatments was significantly reduced. The patterns of expression of the other three CalS genes were not changed significantly in the npr1 mutant. These results suggest that the high induction observed of CalS1 and CalS12 is Npr1 dependent while the weak induction of five CalS genes is Npr1 independent. In a T-DNA knockout mutant of CalS12, callose encasement around the haustoria on the infected leaves was reduced and the mutant was found to be more resistant to downy mildew as compared to the wild type plants.