Sudhir K. Sopory

Role of DREB transcription factors in abiotic and biotic stress tolerance in plants

Abiotic and biotic stresses negatively influence survival, biomass production and crop yield. Being multigenic as well as a quantitative trait, it is a challenge to understand the molecular basis of abiotic stress tolerance and to manipulate it as compared to biotic stresses. Lately, some transcription factor(s) that regulate the expression of several genes related to stress have been discovered. One such class of the transcription factors is DREB/CBF that binds to drought responsive cis-acting elements. DREBs belong to ERF family of transcription factors consisting of two subclasses, i.e. DREB1/CBF and DREB2 that are induced by cold and dehydration, respectively. The DREBs are apparently involved in biotic stress signaling pathway. It has been possible to engineer stress tolerance in transgenic plants by manipulating the expression of DREBs. This opens an excellent opportunity to develop stress tolerant crops in future. This review intends to focus on the structure, role of DREBs in plant stress signaling and the present status of their deployment in developing stress tolerant transgenic plants.

Transgenic Tobacco Overexpressing Glyoxalase Pathway Enzymes Grow and Set Viable Seeds in Zinc-Spiked Soils

We reported earlier that engineering of the glyoxalase pathway (a two-step reaction mediated through glyoxalase I and II enzymes) enhances salinity tolerance. Here we report the extended suitability of this engineering strategy for improved heavy-metal tolerance in transgenic tobacco (Nicotiana tabacum). The glyoxalase transgenics were able to grow, flower, and set normal viable seeds in the presence of 5 mm ZnCl2 without any yield penalty. The endogenous ion content measurements revealed roots to be the major sink for excess zinc accumulation, with negligible amounts in seeds in transgenic plants. Preliminary observations suggest that glyoxalase overexpression could confer tolerance to other heavy metals, such as cadmium or lead. Comparison of relative tolerance capacities of transgenic plants, overexpressing either glyoxalase I or II individually or together in double transgenics, evaluated in terms of various critical parameters such as survival, growth, and yield, reflected double transgenics to perform better than either of the single-gene transformants. Biochemical investigations indicated restricted methylglyoxal accumulation and less lipid peroxidation under high zinc conditions in transgenic plants. Studies employing the glutathione biosynthetic inhibitor, buthionine sulfoximine, suggested an increase in the level of phytochelatins and maintenance of glutathione homeostasis in transgenic plants during exposure to excess zinc as the possible mechanism behind this tolerance. Together, these findings presents a novel strategy to develop multiple stress tolerance via glyoxalase pathway engineering, thus implicating its potential use in engineering agriculturally important crop plants to grow on rapidly deteriorating lands with multiple unfavorable edaphic factors.

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.

Chemical signaling under abiotic stress environment in plants

Many chemicals are critical for plant growth and development and play an important role in integrating various stress signals and controlling downstream stress responses by modulating gene expression machinery and regulating various transporters/pumps and biochemical reactions. These chemicals include calcium (Ca2+), cyclic nucleotides, polyphosphoinositides, nitric oxide (NO), sugars, abscisic acid (ABA), jasmonates (JA), salicylic acid (SA) and polyamines. Ca2+ is one of the very important ubiquitous second messengers in signal transduction pathways and usually its concentration increases in response to the stimuli including stress signals. Many Ca2+ sensors detect the Ca2+ signals and direct them to downstream signaling pathways by binding and activating diverse targets. cAMP or cGMP protects the cell with ion toxicity. Phosphoinositides are known to be involved both in transmission of signal across the plasma membrane and in intracellular signaling. NO activates various defense genes and acts as a developmental regulator in plants. Sugars affect the expression of many genes involved in photosynthesis, glycolysis, nitrogen metabolism, sucrose and starch metabolism, defense mechanisms and cell cycle regulation. ABA, JA, SA and polyamines are also involved in many stress responses. Cross-talk between these chemical signaling pathways is very common in plant responses to abiotic and bitotic factors. In this article we have described the role of these chemicals in initiating signaling under stress conditions mainly the abiotic stress.

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.

Enhancing salt tolerance in a crop plant by overexpression of glyoxalase II

Earlier we have shown the role of glyoxalase overexpression in conferring salinity tolerance in transgenic tobacco. We now demonstrate the feasibility of same in a crop like rice through overproduction of glyoxalase II. The rice glyoxalase II was cloned in pCAMBIA1304 and transformed into rice (Oryza sativa cv PB1) via Agrobacterium. The transgenic plants showed higher constitutive activity of glyoxalase II that increased further upon salt stress, reflecting the upregulation of endogenous glyoxalase II. The transgenic rice showed higher tolerance to toxic concentrations of methylglyoxal (MG) and NaCl. Compared with non-transgenics, transgenic plants at the T1 generation exhibited sustained growth and more favorable ion balance under salt stress conditions.

Induction of haploidy from pollen grains in angiosperms - the current status.

SummarySince the successful induction of haploids from anthers cultured in vitro in 1964, a great deal of attention has been given to this problem by those interested in obtaining pure lines and mutants for crop improvement and biochemical genetics. In the last 16 years the anther culture technique has been refined and extended to over one hundred and fifty different species. More recently, isolated pollen culture — which is a refinement of the original anther culture technique — has also been developed. In this review we have made an effort to critically examine existing reports with the objective of analysing the effects of various factors — e.g. culture medium, the cultural conditions, and the effect of genotype and physiological state of the parent plant on pollen induction — and to speculate on the mechanism of action of different factors in order to throw some light on the process of haploid induction.

Transcriptome map for seedling stage specific salinity stress response indicates a specific set of genes as candidate for saline tolerance in Oryza sativa L.

Oryza sativa L. cv IR64 is a widely cultivated, salt-sensitive indica rice, while Pokkali is a well-known, naturally salt-tolerant relative. To understand the molecular basis of differences in their salinity tolerance, three subtractive cDNA libraries were constructed. A total of 1,194 salinity-regulated cDNAs are reported here that may serve as repositories for future individual gene-based functional genomics studies. Gene expression data using macroarrays and Northern blots gives support to our hypothesis that salinity tolerance of Pokkali may be due to constitutive overexpression of many genes that function in salinity tolerance and are stress inducible in IR64. Analysis of genome architecture revealed the presence of these genes on all the chromosomes with several distinct clusters. Notably, a few mapped on one of the major quantitative trait loci – Saltol – on chromosome 1 and were found to be differentially regulated in the two contrasting genotypes. The present study also defines a set of known abiotic stress inducible genes, including CaMBP, GST, LEA, V-ATPase, OSAP1 zinc finger protein, and transcription factor HBP1B, that were expressed at high levels in Pokkali even in the absence of stress. These proposed genes may prove useful as “candidates” in improving salinity tolerance in crop plants using transgenic approach.

Cloning and characterization of CBL-CIPK signalling components from a legume (Pisum sativum)

The studies on calcium sensor calcineurin B‐like protein (CBL) and CBL interacting protein kinases (CIPK) are limited to Arabidopsis and rice and their functional role is only beginning to emerge. Here, we present cloning and characterization of a protein kinase (PsCIPK) from a legume, pea, with novel properties. The PsCIPK gene is intronless and encodes a protein that showed partial homology to the members of CIPK family. The recombinant PsCIPK protein was autophosphorylated at Thr residue(s). Immunoprecipitation and yeast two‐hybrid analysis showed direct interaction of PsCIPK with PsCBL, whose cDNA and genomic DNA were also cloned in this study. PsCBL showed homology to AtCBL3 and contained calcium‐binding activity. We demonstrate for the first time that PsCBL is phosphorylated at its Thr residue(s) by PsCIPK. Immunofluorescence/confocal microscopy showed that PsCBL is exclusively localized in the cytosol, whereas PsCIPK is localized in the cytosol and the outer membrane. The exposure of plants to NaCl, cold and wounding co‐ordinately upregulated the expression of PsCBL and PsCIPK genes. The transcript levels of both genes were also coordinately stimulated in response to calcium and salicylic acid. However, drought and abscisic acid had no effect on the expression of these genes. These studies show the ubiquitous presence of CBL/CIPK in higher plants and enhance our understanding of their role in abiotic and biotic stress signalling.

Genome-wide analysis of rice and Arabidopsis identifies two glyoxalase genes that are highly expressed in abiotic stresses

Glyoxalase pathway, ubiquitously found in all organisms from prokaryotes to eukaryotes, consists of glyoxalase I (GLY I) and glyoxalase II (GLY II) enzymes, which detoxify a cytotoxic molecule, methylglyoxal (MG). Increase in MG has been correlated with various diseases in humans and different abiotic stresses in plants. We have previously shown that overproduction of GLY I and/or GLY II enzymes in transgenic plants provide tolerance towards salinity and heavy metal stresses. We have identified nineteen potential GLY I and four GLY II proteins in rice and twenty two GLY I and nine GLY II proteins in Arabidopsis. An analysis of complete set of genes coding for the glyoxalase proteins in these two genomes is presented, including classification and chromosomal distribution. Expression profiling of these genes has been performed in response to multiple abiotic stresses, in different tissues and during various stages of vegetative and reproductive development using publicly available databases (massively parallel signature sequencing and microarray). AtGLYI8, OsGLYI3, and OsGLYI10 expresses constitutively high in seeds while AtGLYI4, AtGLYI7, OsGLYI6, and OsGLYI11 are highly stress inducible. To complement this analyses, qRT-PCR is performed in two contrasting rice genotypes, i.e., IR64 and Pokkali where OsGLYI6 and OsGLYI11 are found to be highly stress inducible.