Malireddy K. Reddy

Transgenic tobacco plants overexpressing glyoxalase enzymes resist an increase in methylglyoxal and maintain higher reduced glutathione levels under salinity stress

The mechanism behind enhanced salt tolerance conferred by the overexpression of glyoxalase pathway enzymes was studied in transgenic vis‐à‐vis wild‐type (WT) plants. We have recently documented that salinity stress induces higher level accumulation of methylglyoxal (MG), a potent cytotoxin and primary substrate for glyoxalase pathway, in various plant species [Yadav, S.K., Singla‐Pareek, S.L., Ray, M., Reddy, M.K. and Sopory, S.K. (2005) MG levels in plants under salinity stress are dependent on glyoxalase I and glutathione. Biochem. Biophys. Res. Commun. 337, 61–67]. The transgenic tobacco plants overexpressing glyoxalase pathway enzymes, resist an increase in the level of MG that increased to over 70% in WT plants under salinity stress. These plants showed enhanced basal activity of various glutathione related antioxidative enzymes that increased further upon salinity stress. These plants suffered minimal salinity stress induced oxidative damage measured in terms of the lipid peroxidation. The reduced glutathione (GSH) content was high in these transgenic plants and also maintained a higher reduced to oxidized glutathione (GSH:GSSG) ratio under salinity. Manipulation of glutathione ratio by exogenous application of GSSG retarded the growth of non‐transgenic plants whereas transgenic plants sustained their growth. These results suggest that resisting an increase in MG together with maintaining higher reduced glutathione levels can be efficiently achieved by the overexpression of glyoxalase pathway enzymes towards developing salinity stress tolerant plants.

Redox homeostasis, antioxidant defense, and methylglyoxal detoxification as markers for salt tolerance in Pokkali rice

To identify biochemical markers for salt tolerance, two contrasting cultivars of rice (Oryza sativa L.) differing in salt tolerance were analyzed for various parameters. Pokkali, a salt-tolerant cultivar, showed considerably lower level of H2O2 as compared to IR64, a sensitive cultivar, and such a physiology may be ascribed to the higher activity of enzymes in Pokkali, which either directly or indirectly are involved in the detoxification of H2O2. Enzyme activities and the isoenzyme pattern of antioxidant enzymes also showed higher activity of different types and forms in Pokkali as compared to IR64, suggesting that Pokkali possesses a more efficient antioxidant defense system to cope up with salt-induced oxidative stress. Further, Pokkali exhibited a higher GSH/GSSG ratio along with a higher ratio of reduced ascorbate/oxidized ascorbate as compared to IR64 under NaCl stress. In addition, the activity of methylglyoxal detoxification system (glyoxalase I and II) was significantly higher in Pokkali as compared to IR64. As reduced glutathione is involved in the ascorbate–glutathione pathway as well as in the methylglyoxal detoxification pathway, it may be a point of interaction between these two. Our results suggest that both ascorbate and glutathione homeostasis, modulated also via glyoxalase enzymes, can be considered as biomarkers for salt tolerance in Pokkali rice. In addition, status of reactive oxygen species and oxidative DNA damage can serve as a quick and sensitive biomarker for screening against salt and other abiotic stresses in crop plants.

Genome-wide transcriptomic analysis of cotton under drought stress reveal significant down-regulation of genes and pathways involved in fibre elongation and up-regulation of defense responsive genes

Cotton is an important source of natural fibre used in the textile industry and the productivity of the crop is adversely affected by drought stress. High throughput transcriptomic analyses were used to identify genes involved in fibre development. However, not much information is available on cotton genome response in developing fibres under drought stress. In the present study a genome wide transcriptome analysis was carried out to identify differentially expressed genes at various stages of fibre growth under drought stress. Our study identified a number of genes differentially expressed during fibre elongation as compared to other stages. High level up-regulation of genes encoding for enzymes involved in pectin modification and cytoskeleton proteins was observed at fibre initiation stage. While a large number of genes encoding transcription factors (AP2-EREBP, WRKY, NAC and C2H2), osmoprotectants, ion transporters and heat shock proteins and pathways involved in hormone (ABA, ethylene and JA) biosynthesis and signal transduction were up-regulated and genes involved in phenylpropanoid and flavonoid biosynthesis, pentose and glucuronate interconversions and starch and sucrose metabolism pathways were down-regulated during fibre elongation. This study showed that drought has relatively less impact on fibre initiation but has profound effect on fibre elongation by down-regulating important genes involved in cell wall loosening and expansion process. The comprehensive transcriptome analysis under drought stress has provided valuable information on differentially expressed genes and pathways during fibre development that will be useful in developing drought tolerant cotton cultivars without compromising fibre quality.

The Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein Is Phosphorylated and Localizes in the Cytoplasm by 14-3-3-Mediated Translocation

ABSTRACT The severe acute respiratory syndrome coronavirus(SARS-CoV) nucleocapsid (N) protein is one of the four structural proteins of the virus and is predicted to be a 46-kDa phosphoprotein. Our in silico analysis predicted N to be heavily phosphorylated at multiple residues. Experimentally, we have shown in this report that the N protein of the SARS-CoV gets serine-phosphorylated by multiple kinases, in both the cytoplasm and the nucleus. The phosphoprotein is stable and localizes in the cytoplasm and coprecipitates with the membrane fraction. Also, using specific inhibitors of phosphorylation and an in vitro phosphorylation assay, we show that the nucleocapsid protein is a substrate of cyclin-dependent kinase (CDK), glycogen synthase kinase, mitogen-activated protein kinase, and casein kinase II. Further, we show that the phosphorylated protein is translocated to the cytoplasm by binding to 14-3-3 (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein). 14-3-3 proteins are a family of highly conserved, ubiquitously expressed eukaryotic proteins that function primarily as adapters that modulate interactions between components of various cellular signaling and cell cycle regulatory pathways through phosphorylation-dependent protein-protein interactions. Coincidentally, the N protein was also found to downregulate the expression of the theta isoform of 14-3-3 (14-3-3θ), leading to the accumulation of phosphorylated N protein in the nucleus, in the absence of growth factors. Using short interfering RNA specific to 14-3-3θ we have inhibited its expression to show accumulation of phosphorylated N protein in the nucleus. Thus, the data presented here provide a possible mechanism for phosphorylation-dependent nucleocytoplasmic shuttling of the N protein. This 14-3-3-mediated transport of the phosphorylated N protein and its possible implications in interfering with the cellular machinery are discussed.

Expression of a finger millet transcription factor, EcNAC1, in tobacco confers abiotic stress-tolerance.

NAC (NAM, ATAF1-2, and CUC2) proteins constitute one of the largest families of plant-specific transcription factors and have been shown to be involved in diverse plant processes including plant growth, development, and stress-tolerance. In this study, a stress-responsive NAC gene, EcNAC1, was isolated from the subtracted stress cDNA library generated from a drought adapted crop, finger millet, and characterized for its role in stress-tolerance. The expression analysis showed that EcNAC1 was highly induced during water-deficit and salt stress. EcNAC1 shares high amino acid similarity with rice genes that have been phylogenetically classified into stress-related NAC genes. Our results demonstrated that tobacco transgenic plants expressing EcNAC1 exhibit tolerance to various abiotic stresses like simulated osmotic stress, by polyethylene glycol (PEG) and mannitol, and salinity stress. The transgenic plants also showed enhanced tolerance to methyl-viologen (MV) induced oxidative stress. Reduced levels of reactive oxygen species (ROS) and ROS-induced damage were noticed in pot grown transgenic lines under water-deficit and natural high light conditions. Root growth under stress and recovery growth after stress alleviation was more in transgenic plants. Many stress-responsive genes were found to be up-regulated in transgenic lines expressing EcNAC1. Our results suggest that EcNAC1 overexpression confers tolerance against abiotic stress in susceptible species, tobacco.

Isolation and characterization of expressed sequence tags (ESTs) from subtracted cDNA libraries of Pennisetum glaucum seedlings

Pearl millet (Pennisetum glaucum), used as forage and grain crop is a stress tolerant species. Here we identify differentially regulated transcripts in response to abiotic (salinity, drought and cold) stresses from subtracted cDNA libraries by single-pass sequencing of cDNA clones. A total of 2,494 EST sequences were clustered and assembled into a collection of 1,850 unique sequences with 224 contigs and 1,626 singleton sequences. By sequence comparisons the putative functions of many ESTs could be assigned. Genes with stress related functions include those involved in cellular defense against abiotic stresses and transcripts for proteins involved in stress response signaling and transcription in addition to ESTs encoding unknown functions. These provide new candidate genes for investigation to elucidate their role in abiotic stress. The relative mRNA abundance of 38 selected genes, quantified using real time quantitative RT-PCR, demonstrated the existence of a complex gene regulatory network that differentially modulates gene expression in a kinetics-specific manner in response to different abiotic stresses. Notably, housekeeping and non-target genes were effectively reduced in these subtracted cDNA libraries constructed. These EST sequences are a rich source of stress-related genes and reveal a major part of the stress-response transcriptome that will provide the foundation for further studies into understanding Pennisetum’s adaptability to harsh environmental conditions.

Functional genomics of fuzzless-lintless mutant of Gossypium hirsutum L. cv. MCU5 reveal key genes and pathways involved in cotton fibre initiation and elongation

BackgroundFuzzless-lintless cotton mutants are considered to be the ideal material to understand the molecular mechanisms involved in fibre cell development. Although there are few reports on transcriptome and proteome analyses in cotton at fibre initiation and elongation stages, there is no comprehensive comparative transcriptome analysis of fibre-bearing and fuzzless-lintless cotton ovules covering fibre initiation to secondary cell wall (SCW) synthesis stages. In the present study, a comparative transcriptome analysis was carried out using G. hirsutum L. cv. MCU5 wild-type (WT) and it’s near isogenic fuzzless-lintless (fl) mutant at fibre initiation (0 dpa/days post anthesis), elongation (5, 10 and 15 dpa) and SCW synthesis (20 dpa) stages.ResultsScanning electron microscopy study revealed the delay in the initiation of fibre cells and lack of any further development after 2 dpa in the fl mutant. Transcriptome analysis showed major down regulation of transcripts (90%) at fibre initiation and early elongation (5 dpa) stages in the fl mutant. Majority of the down regulated transcripts at fibre initiation stage in the fl mutant represent calcium and phytohormone mediated signal transduction pathways, biosynthesis of auxin and ethylene and stress responsive transcription factors (TFs). Further, transcripts involved in carbohydrate and lipid metabolisms, mitochondrial electron transport system (mETS) and cell wall loosening and elongation were highly down-regulated at fibre elongation stage (5–15 dpa) in the fl mutant. In addition, cellulose synthases and sucrose synthase C were down-regulated at SCW biosynthesis stage (15–20 dpa). Interestingly, some of the transcripts (~50%) involved in phytohormone signalling and stress responsive transcription factors that were up-regulated at fibre initiation stage in the WT were found to be up-regulated at much later stage (15 dpa) in fl mutant.ConclusionsComparative transcriptome analysis of WT and its near isogenic fl mutant revealed key genes and pathways involved at various stages of fibre development. Our data implicated the significant role of mitochondria mediated energy metabolism during fibre elongation process. The delayed expression of genes involved in phytohormone signalling and stress responsive TFs in the fl mutant suggests the need for a coordinated expression of regulatory mechanisms in fibre cell initiation and differentiation.

Unraveling Regulation of the Small Heat Shock Proteins by the Heat Shock Factor HvHsfB2c in Barley: Its Implications in Drought Stress Response and Seed Development

The rapid increase in heat shock proteins upon exposure to damaging stresses and during plant development related to desiccation events reveal their dual importance in plant development and stress tolerance. Genome-wide sequence survey identified 20 non-redundant small heat shock proteins (sHsp) and 22 heat shock factor (Hsf) genes in barley. While all three major classes (A, B, C) of Hsfs are localized in nucleus, the 20 sHsp gene family members are localized in different cell organelles like cytoplasm, mitochondria, plastid and peroxisomes. Hsf and sHsp members are differentially regulated during drought and at different seed developmental stages suggesting the importance of chaperone role under drought as well as seed development. In silico cis-regulatory motif analysis of Hsf promoters showed an enrichment with abscisic acid responsive cis-elements (ABRE), implying regulatory role of ABA in mediating transcriptional response of HvsHsf genes. Gene regulatory network analysis identified HvHsfB2c as potential central regulator of the seed-specific expression of several HvsHsps including 17.5CI sHsp. These results indicate that HvHsfB2c is co-expressed in the central hub of small Hsps and therefore it may be regulating the expression of several HvsHsp subclasses HvHsp16.88-CI, HvHsp17.5-CI and HvHsp17.7-CI. The in vivo relevance of binding specificity of HvHsfB2C transcription factor to HSE-element present in the promoter of HvSHP17.5-CI under heat stress exposure is confirmed by gel shift and LUC-reporter assays. Further, we isolated 477 bp cDNA from barley encoding a 17.5 sHsp polypeptide, which was predominantly upregulated under drought stress treatments and also preferentially expressed in developing seeds. Recombinant HvsHsp17.5-CI protein was expressed in E. coli and purified to homogeneity, which displayed in vitro chaperone activity. The predicted structural model of HvsHsp-17.5-CI protein suggests that the α-crystallin domain is evolutionarily highly conserved.

A POLYCOMB group gene of rice (Oryza sativa L. subspecies indica), OsiEZ1, codes for a nuclear-localized protein expressed preferentially in young seedlings and during reproductive development

The SET domains are conserved amino acid sequences present in chromosomal proteins that contribute to the epigenetic control of gene expression by altering regional organization of the chromatin structure. The SET domain proteins are divided into four subgroups as categorized by their Drosophila members; enhancer of zeste (E(Z)), trithorax (TRX), absent small or homeotic 1 (ASH1) and supressor of variegation (SU(VAR)3-9). Homologs of all four classes have been characterized in yeast, mammals and plants. We report here the isolation and characterization of rice (Oryza sativa L. subspecies indica) cDNA, OsiEZ1, as a monocot member of this family. The OsiEZ1 cDNA is 3133 bp long with an ORF of 2799 bp, and the predicted amino acid sequence (895 residues) corresponds to a protein of ca. 98 kDa. All the characteristic domains known to be conserved in E(Z) homologs (subgroup I) of SET domain containing proteins are present in OsiEZ1. In the rice genome, a 7499 bp long OsiEZ1 sequence is split into 17 exons interrupted by 16 introns. Southern analysis indicates that OsiEZ1 is represented as single copy in the rice genome. Expression studies revealed that the OsiEZ1 transcript level was highest in rice flowers, almost undetectable in developing seeds of 1-2 days post-fertilization but increased significantly in young seeds of 3-5 days post-fertilization. The OsiEZ1 transcript was barely detectable in mature zygotic embryos, but its levels were significantly higher in callus derived from rice scutellum, somatic embryos and young seedlings. The OsiEZ1/GUS recombinant protein was confined to the nucleus in living cells of particle-bombarded onion peels. The expression of OsiEZ1 complemented a set1Delta Saccharomyces cerevisiae mutant that is impaired in telomeric silencing. We suggest that the nuclear-localized OsiEZ1 has a role in regulating various aspects of plant development, and this control is most likely brought about by repressing the activity of downstream regulatory genes.

Molecular cloning and characterization of gene encoding for cytoplasmic Hsc70 from Pennisetum glaucum may play a protective role against abiotic stresses

Molecular chaperones (Hsps) have been shown to facilitate protein folding or assembly under various developmental and adverse environmental conditions. The aim of this study was to unravel a possible role of heat-shock proteins in conferring abiotic stress tolerance to plants. We isolated a cDNA encoding a cytoplasmic Hsp70 (PgHsc70) from Pennisetum glaucum by screening heat-stress cDNA library. PgHsc70 cDNA encoding 649 amino acids represents all conserved signature motifs characteristic of Hsp70s. The predicted molecular model of PgHsc70 protein suggests that the N-terminus ATP-binding region is evolutionarily conserved, in comparison to C-terminus peptide-binding domains. A single intron in ATPase domain coding region of PgHsc70 exhibited a high degree of conservation with respect to its position and phasing among other plant Hsp70 genes. Recombinant PgHsc70 protein purified from E. coli possessed in vitro chaperone activity and protected PgHsc70 expressing bacteria from damage caused by heat and salinity stress. Nucleotide sequence analysis of 5′ flanking promoter region of PgHsc70 gene revealed a potential heat-shock element (HSE) and other putative stress-responsive transcription factor binding sites. Positive correlation existed between differentially up-regulated PgHsc70 transcript levels and the duration and intensity of different environmental stresses. Molecular and biochemical analyses revealed that PgHsc70 gene was a member of the Hsp70 family and suggested that its origin was from duplication of a common ancestral gene. Transcript induction data, presence of several putative stress-responsive transcription factor-binding sites in the promoter region of PgHsc70 and the presence of a protective in vitro chaperone activity of this protein against damage caused by heat and salinity, when expressed in E. coli, suggest its probable role in conferring abiotic stress tolerance to this plant.