Ratna Karan

Salt Stress Induced Variation in DNA Methylation Pattern and Its Influence on Gene Expression in Contrasting Rice Genotypes

Background Salinity is a major environmental factor limiting productivity of crop plants including rice in which wide range of natural variability exists. Although recent evidences implicate epigenetic mechanisms for modulating the gene expression in plants under environmental stresses, epigenetic changes and their functional consequences under salinity stress in rice are underexplored. DNA methylation is one of the epigenetic mechanisms regulating gene expression in plant’s responses to environmental stresses. Better understanding of epigenetic regulation of plant growth and response to environmental stresses may create novel heritable variation for crop improvement. Methodology/Principal Findings Methylation sensitive amplification polymorphism (MSAP) technique was used to assess the effect of salt stress on extent and patterns of DNA methylation in four genotypes of rice differing in the degree of salinity tolerance. Overall, the amount of DNA methylation was more in shoot compared to root and the contribution of fully methylated loci was always more than hemi-methylated loci. Sequencing of ten randomly selected MSAP fragments indicated gene-body specific DNA methylation of retrotransposons, stress responsive genes, and chromatin modification genes, distributed on different rice chromosomes. Bisulphite sequencing and quantitative RT-PCR analysis of selected MSAP loci showed that cytosine methylation changes under salinity as well as gene expression varied with genotypes and tissue types irrespective of the level of salinity tolerance of rice genotypes. Conclusions/Significance The gene body methylation may have an important role in regulating gene expression in organ and genotype specific manner under salinity stress. Association between salt tolerance and methylation changes observed in some cases suggested that many methylation changes are not “directed”. The natural genetic variation for salt tolerance observed in rice germplasm may be independent of the extent and pattern of DNA methylation which may have been induced by abiotic stress followed by accumulation through the natural selection process.

Clustered metallothionein genes are co-regulated in rice and ectopic expression of OsMT1e-P confers multiple abiotic stress tolerance in tobacco via ROS scavenging

BackgroundMetallothioneins (MT) are low molecular weight, cysteine rich metal binding proteins, found across genera and species, but their function(s) in abiotic stress tolerance are not well documented.ResultsWe have characterized a rice MT gene, OsMT1e-P, isolated from a subtractive library generated from a stressed salinity tolerant rice genotype, Pokkali. Bioinformatics analysis of the rice genome sequence revealed that this gene belongs to a multigenic family, which consists of 13 genes with 15 protein products. OsMT1e-P is located on chromosome XI, away from the majority of other type I genes that are clustered on chromosome XII. Various members of this MT gene cluster showed a tight co-regulation pattern under several abiotic stresses. Sequence analysis revealed the presence of conserved cysteine residues in OsMT1e-P protein. Salinity stress was found to regulate the transcript abundance of OsMT1e-P in a developmental and organ specific manner. Using transgenic approach, we found a positive correlation between ectopic expression of OsMT1e-P and stress tolerance. Our experiments further suggest ROS scavenging to be the possible mechanism for multiple stress tolerance conferred by OsMT1e-P.ConclusionWe present an overview of MTs, describing their gene structure, genome localization and expression patterns under salinity and development in rice. We have found that ectopic expression of OsMT1e-P enhances tolerance towards multiple abiotic stresses in transgenic tobacco and the resultant plants could survive and set viable seeds under saline conditions. Taken together, the experiments presented here have indicated that ectopic expression of OsMT1e-P protects against oxidative stress primarily through efficient scavenging of reactive oxygen species.

Metabolic engineering of sugarcane to accumulate energy‐dense triacylglycerols in vegetative biomass

Elevating the lipid content in vegetative tissues has emerged as a new strategy for increasing energy density and biofuel yield of crops. Storage lipids in contrast to structural and signaling lipids are mainly composed of glycerol esters of fatty acids, also known as triacylglycerol (TAG). TAGs are one of the most energy-rich and abundant forms of reduced carbon available in nature. Therefore, altering the carbon-partitioning balance in favour of TAG in vegetative tissues of sugarcane, one of the highest yielding biomass crops, is expected to drastically increase energy yields. Here we report metabolic engineering to elevate TAG accumulation in vegetative tissues of sugarcane. Constitutive co-expression of WRINKLED1 (WRI1), diacylglycerol acyltransferase1-2 (DGAT1-2) and oleosin1 (OLE1) and simultaneous cosuppression of ADP-glucose pyrophosphorylase (AGPase) and a subunit of the peroxisomal ABC transporter1 (PXA1) in transgenic sugarcane elevated TAG accumulation in leaves or stems by 95- or 43-fold to 1.9% or 0.9% of dry weight (DW), respectively, while expression or suppression of one to three of the target genes increased TAG levels by 1.5- to 9.5-fold. Accumulation of TAG in vegetative progeny plants was consistent with the results from primary transgenics and contributed to a total fatty acid content of up to 4.7% or 1.7% of DW in mature leaves or stems, respectively. Lipid droplets were visible within mesophyll cells of transgenic leaves by confocal fluorescence microscopy. These results provide the basis for optimizations of TAG accumulation in sugarcane and other high yielding biomass grasses and will open new prospects for biofuel applications.

Histidine kinase and response regulator genes as they relate to salinity tolerance in rice

We have previously shown that Oryza sativa L. Pokkali maintains higher levels of transcripts under non-saline conditions, which are otherwise induced under salinity in the sensitive genotype—IR64. We wanted to test this hypothesis of differential gene regulation further, within the members of a given stress responsive gene family, which share significant structural and functional similarities. For this purpose, we chose to work on the two-component system (TCS family) which plays an important role in stress perception and signal transduction under hormonal, abiotic stress, light and developmental regulation. We present data to show that all members of TCS family, including sensory histidine kinases, phosphotransfer proteins and response regulators, are having differential transcript abundance (under both non-stress and salinity stress conditions) in contrasting rice genotypes. Further, under non-stress conditions, transcript abundance for all TCS members (except RR21) was found to be higher in the salt-tolerant genotype—Pokkali. TCS transcripts are otherwise induced by salinity stress to a relatively higher level in the sensitive cultivar IR64. A few of these members were also found to be localised within important salinity-related quantitative trait loci identified earlier. Based on the above findings, we propose that the TCS members may have a significant role in salinity tolerance in rice and can serve as useful ‘candidate genes’ for raising salinity-tolerant crop plants.

Histidine kinases in plants: cross talk between hormone and stress responses.

Two-component signaling pathways involve sensory histidine kinases (HK), histidine phosphotransfer proteins (HpT) and response regulators (RR). Recent advancements in genome sequencing projects for a number of plant species have established the TCS family to be multigenic one. In plants, HKs operate through the His–Asp phosphorelay and control many physiological and developmental processes throughout the lifecycle of plants. Despite the huge diversity reported for the structural features of the HKs, their functional redundancy has also been reported via mutant approach. Several sensory HKs having a CHASE domain, transmembrane domain(s), transmitter domain and receiver domain have been reported to be involved in cytokinin and ethylene signaling. On the other hand, there are also increasing evidences for some of the sensory HKs to be performing their role as osmosensor, clearly indicating toward a possible cross-talk between hormone and stress responsive cascades. In this review, we bring out the latest knowledge about the structure and functions of histidine kinases in cytokinin and ethylene signaling and their role(s) in development and the regulation of environmental stress responses.

A stress inducible SUMO conjugating enzyme gene (SaSce9) from a grass halophyte Spartina alterniflora enhances salinity and drought stress tolerance in Arabidopsis

BackgroundSUMO (Small Ubiquitin related Modifier) conjugation is a post translational regulatory process found in all eukaryotes, mediated by SUMO activating enzyme, SUMO conjugating enzyme, and SUMO ligase for the attachment of SUMO to its target protein. Although the mechanism for regulation of SUMO conjugation pathway genes under abiotic stress has been studied to certain extent, the role of SUMO conjugating enzyme in improving abiotic stress tolerance to plant is largely unexplored. Here, we have characterized a SUMO conjugating enzyme gene ‘SaSce9’ from a halophytic grass Spartina alterniflora and investigated its role in imparting abiotic stress tolerance.ResultsSaSce9 gene encodes for a polypeptide of 162 amino acids with a molecular weight of ~18 kD and isoelectric point 8.43. Amino acid sequence comparisons of SaSce9 with its orthologs from other plant species showed high degree (~85-93%) of structural conservation among each other. Complementation analysis using yeast SCE mutant, Ubc9, revealed functional conservation of SaSce9 between yeast and S. alterniflora. SaSce9 transcript was inducible by salinity, drought, cold, and exogenously supplied ABA both in leaves and roots of S. alterniflora. Constitutive overexpression of SaSce9 in Arabidopsis through Agrobacterium mediated transformation improved salinity and drought tolerance of Arabidopsis. SaSce9 overexpressing Arabidopsis plants retained more chlorophyll and proline both under salinity and drought stress. SaSce9 transgenic plants accumulated lower levels of reactive oxygen under salinity stress. Expression analysis of stress responsive genes in SaSce9 Arabidopsis plants revealed the increased expression of antioxidant genes, AtSOD and AtCAT, ion antiporter genes, AtNHX1 and AtSOS1, a gene involved in proline biosynthesis, AtP5CS, and a gene involved in ABA dependent signaling pathway, AtRD22.ConclusionsThese results highlight the prospect of improving abiotic stress tolerance in plants through genetic engineering of the sumoylation pathway. The study provides evidence that the overexpression of SaSce9 in plant can improve salinity and drought stress tolerance by protecting the plant through scavenging of ROS, accumulation of an osmolyte, proline, and expression of stress responsive genes. In addition, this study demonstrates the potential of the halophyte grass S. alterniflora as a reservoir of abiotic stress related genes for crop improvement.

Ectopic expression of Pokkali phosphoglycerate kinase-2 (OsPGK2-P) improves yield in tobacco plants under salinity stress

Key messageOur results indicate that OsPGK2a-P gene is differentially regulated in contrasting rice cultivars under stress and its overexpression confers salt stress tolerance in transgenic tobacco.AbstractPhosphoglycerate kinase (PGK; ECxa0=xa02.7.2.3) plays a major role for ATP production during glycolysis and 1, 3-bisphosphoglycerate production to participate in the Calvin cycle for carbon fixation in plants. Whole genome analysis of rice reveals the presence of four PGK genes (OsPgks) on different chromosomes. Comparative expression analysis of OsPgks in rice revealed highest level of transcripts for OsPgk2 at most of its developmental stages. Detailed characterization of OsPgk2 transcript and protein showed that it is strongly induced by salinity stress in two contrasting genotypes of rice, i.e., cv IR64 (salt sensitive) and landrace Pokkali (salt tolerant). Ectopic expression of OsPgk2a-P (isolated from Pokkali) in transgenic tobacco improved its salinity stress tolerance by higher chlorophyll retention and enhanced proline accumulation, besides maintaining better ion homeostasis. Ectopically expressing OsPgk2a-P transgenic tobacco plants showed tall phenotype with more number of pods than wild-type plants. Therefore, OsPgk2a-P appears to be a potential candidate for increasing salinity stress tolerance and enhanced yield in crop plants.

Overexpression of a nascent polypeptide associated complex gene (SaβNAC) of Spartina alterniflora improves tolerance to salinity and drought in transgenic Arabidopsis

Salinity and drought are the most important environmental constraints limiting crop growth and productivity. Here, we have characterized a gene SaβNAC encoding the β subunit of nascent polypeptide associated complex from a halophyte Spartina alterniflora and investigated its role toward abiotic stress regulation. Expression of SaβNAC was differentially regulated by abiotic stresses, including salinity, drought, cold, and ABA in leaves and roots of S. alterniflora. Constitutive over-expression of SaβNAC in Arabidopsis exhibited normal growth under non-stress conditions but enhanced tolerance to salt and drought stresses. Transgenic SaβNAC Arabidopsis retained more chlorophyll, proline, and showed improved ionic homeostasis with less damage under stress conditions compared to WT plants. This is a first report to demonstrate the involvement of βNAC in imparting abiotic stress tolerance which might be due to protection of the newly synthesized polypeptides involved in various stress tolerance mechanisms from abiotic stress induced damage and inhibition of cell death in plant.

Mapping of Seed Shattering Loci Provides Insights into Origin of Weedy Rice and Rice Domestication

Seed shattering is an important trait that distinguishes crop cultivars from the wild and weedy species. The genetics of seed shattering was investigated in this study to provide insights into rice domestication and the evolution of weedy rice. Quantitative trait locus (QTL) analysis, conducted in 2 recombinant inbred populations involving 2 rice cultivars and a weedy rice accession of the southern United States, revealed 3-5 QTLs that controlled seed shattering with 38-45% of the total phenotypic variation. Two QTLs on chromosomes 4 and 10 were consistent in both populations. Both cultivar and weedy rice contributed alleles for increased seed shattering. Genetic backgrounds affected both QTL number and the magnitude of QTL effects. The major QTL qSH4 and a minor QTL qSH3 were validated in near-isogenic lines, with the former conferring a significantly higher degree of seed shattering than the latter. Although the major QTL qSH4 overlapped with the sh4, the presence of the nonshattering single nucleotide polymorphism allele in the weedy rice accession suggested involvement of a linked locus or an alternative molecular genetic mechanism. Overlapping of several QTLs with those from earlier studies indicated that weedy rice may have been derived from the wild species Oryza rufipogon. Natural hybridization of rice cultivars with the highly variable O. rufipogon present in different geographic regions might be responsible for the evolution of a wide range of phenotypic and genotypic variabilities seen in weedy rice populations worldwide.

Approaches to Increasing Salt Tolerance in Crop Plants

Soil salinity is widely recognized as a major threat to global food security. Salinity and other abiotic stresses, which are expected to be more frequent in future due to disturbances in global climate, pose a serious challenge for plant scientists to ensure food supply for the growing world population. Several approaches have been advocated to address the salinity problem, but the most logical solution to maintain crop productivity in salt-affected areas is to enhance salt tolerance of crop plants. Due to the genetic and physiological complexity associated with salt tolerance, efforts to breed salt-tolerant plants have met with limited success. Although progress has been made in deciphering the genetic basis of salt tolerance, sustained efforts are needed to systematically dissect and utilize the natural variability in the available germplasm for improving crop adaptation in saline environments using modern genomics tools. Wide range of variability for salt tolerance in wild relatives, cultivars of major field crops, and halophytes offers bright prospect for discovery of superior salt-tolerant alleles for crop improvement. With an enhanced understanding of molecular mechanisms and the associated genes for component traits of salt tolerance, it would be possible to breed salt-tolerant plants using an integrated approach involving conventional breeding, physiological analysis, marker-assisted selection, and transgenic technology.