Overexpression of a Plasma Membrane Protein Gene, SaPMP3, from Spartina alterniflora L. Enhances Salinity Tolerance in Rice (Oryza sativa L.)

Salinity continues to be a major abiotic stress limiting crop productivity. As rice is staple food for nearly half of the world population, improvement in its salt tolerance will have a major impact on global food security. Compared to rice and other field crops, halophytes have evolved special physiological mechanisms to withstand high salinity. The overall goal of this study was to characterize plasma membrane protein 3 genes, SaPMP3-2 and SaPMP3-1, from a halophyte, Spartina alterniflora L., and evaluate their potential in single gene as well as pyramided transgenic plants in combination with the vacuolar ATPase subunit c1 (SaVHAc1) gene in improving salt tolerance in cv. Cocodrie background. Both genes, SaPMP3-2 and SaPMP3-1, enhanced the ability of E. coli to survive at 600 mM NaCl. Genetic complementation of the mutant yeast strain and enhanced salt tolerance in wild type yeast strain by SaPMP3-2 indicated its conserved functional role in salt tolerance. Subsequently, enhanced salt tolerance in transgenic rice plants was demonstrated through overexpression of SaPMP3-2 and SaPMP3-1 independently as well as the combination of SaPMP3-1 and SaVHAc1. Chlorophyll retention and relative water content were higher in transgenic plants compared to Cocodrie under salt stress during the vegetative stage. The transgenic plants survived wilting and drying symptoms with enhanced growth and higher K+/Na+ ratio at 100 mM NaCl stress during early seedling stage in hydroponic conditions. Salt stress screening during reproductive stage revealed that the single gene and the pyramided transgenic plants had better grain filling whereas only the pyramided plants showed significantly higher grain yield per plant and higher test weight compared to Cocodrie. The improvement in salt tolerance in transgenic rice plants could be due to the role played by SaPMP3-2 and SaPMP3-1 through maintenance of ion homoestasis by restricting uptake of salts. The impact of SaPMP3 gene was further amplified when combined with SaVHAc1 in pyramided transgenic plants, which showed better growth, vigor, and enhanced salt tolerance at all stages of crop growth compared with Cocodrie. Our study provided evidence that S. alterniflora could be a potential source for mining genes to enhance salt tolerance in rice and other cereal crops

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. © The American Genetic Association 2013

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

Transgene Pyramiding of Salt Responsive Protein 3-1 (SaSRP3-1) and SaVHAc1 From Spartina alterniflora L. Enhances Salt Tolerance in Rice

The transgenic technology using a single gene has been widely used for crop improvement. But the transgenic pyramiding of multiple genes, a promising alternative especially for enhancing complexly inherited abiotic stress tolerance, has received little attention. Here, we developed and evaluated transgenic rice lines with a single Salt Responsive Protein 3-1 (SaSRP3-1) gene as well as pyramids with two-genes SaSRP3-1 and Vacuolar H+-ATPase subunit c1 (SaVHAc1) derived from a halophyte grass Spartina alterniflora L. for salt tolerance at seedling, vegetative, and reproductive stages. The overexpression of this novel gene SaSRP3-1 resulted in significantly better growth of E. coli with the recombinant plasmid under 600 mM NaCl stress condition compared with the control. During early seedling and vegetative stages, the single gene and pyramided transgenic rice plants showed enhanced tolerance to salt stress with minimal wilting and drying symptoms, improved shoot and root growth, and significantly higher chlorophyll content, relative water content, and K+/Na+ ratio than the control plants. The salt stress screening during reproductive stage revealed that the transgenic plants with single gene and pyramids had better grain filling, whereas the pyramided plants showed significantly higher grain yield and higher grain weight compared to control plants. Our study demonstrated transgenic pyramiding as a viable approach to achieve higher level of salt tolerance in crop plants

Data_Sheet_1_Transgene Pyramiding of Salt Responsive Protein 3-1 (SaSRP3-1) and SaVHAc1 From Spartina alterniflora L. Enhances Salt Tolerance in Rice.PDF

The transgenic technology using a single gene has been widely used for crop improvement. But the transgenic pyramiding of multiple genes, a promising alternative especially for enhancing complexly inherited abiotic stress tolerance, has received little attention. Here, we developed and evaluated transgenic rice lines with a single Salt Responsive Protein 3-1 (SaSRP3-1) gene as well as pyramids with two-genes SaSRP3-1 and Vacuolar H+-ATPase subunit c1 (SaVHAc1) derived from a halophyte grass Spartina alterniflora L. for salt tolerance at seedling, vegetative, and reproductive stages. The overexpression of this novel gene SaSRP3-1 resulted in significantly better growth of E. coli with the recombinant plasmid under 600 mM NaCl stress condition compared with the control. During early seedling and vegetative stages, the single gene and pyramided transgenic rice plants showed enhanced tolerance to salt stress with minimal wilting and drying symptoms, improved shoot and root growth, and significantly higher chlorophyll content, relative water content, and K+/Na+ ratio than the control plants. The salt stress screening during reproductive stage revealed that the transgenic plants with single gene and pyramids had better grain filling, whereas the pyramided plants showed significantly higher grain yield and higher grain weight compared to control plants. Our study demonstrated transgenic pyramiding as a viable approach to achieve higher level of salt tolerance in crop plants.</p

Analysis of DNA methylation patterns under salinity stress with respect to control condition in the shoot and root of seedlings of rice varieties, IR29, Nipponbare (Nipp), Pokkali (Pokk), and Geumgangbyeo (Geum).

<p>A score of 1 and 0 represents presence and absence of bands, respectively. Values in parentheses indicate percentage of bands in each pattern which was determined by dividing number of bands in each pattern by total number of bands in all three patterns.</p

Genetic Analysis And Traits Association Study In Marker-Assisted Multi-Drought-Traits Pyramided Genotypes Under Reproductive-Stage Moisture Stress In Rice (Oryza Sativa L.)

Abstract Reproductive-stage drought-stress is a major production constraint in rainfed rice ecosystem. Emergence of marker-assisted breeding strategies for developing drought-tolerant rice varieties are being optimized through exploiting adaptive-traits for their increased contribution towards grain-yield under recurring-drought. Grain-yield is a complex-trait; requires knowledge of genetics and association among yield contributing component-traits. Current study was undertaken using 21 marker-assisted multi-drought-traits pyramided genotypes responses for genetic variability and association of traits for grain-yield under aerobic and reproductive-stage drought conditions. Field evaluation was carried-out in two seasons and data was collected on various parameters. Path-coefficient analysis was used as a selection criterion to select yield contributing-traits and found nine phenotypic traits were having a positive direct-effect on grain-yield during both and/or at least one season under both moisture-regimes. The data from summer and Kharif seasons have been pooled within their respective moisture-regimes due to the non-significance of Levene’s test of homogeneity of variances and estimated BLUP values. ANOVA based on BLUP values revealed significant differences for moisture-conditions and also among genotypes. Phenotypic variation via. box-plots and histogram depicted mean phenotypic differences of traits under two moisture-regimes. Majority of the traits possessed high PCV and GCV with high heritability and GAM indicating higher trait expression and additive gene action lead to effectiveness of selection under drought/moisture stress. Grain-yield possessed a positive correlation with all the component-traits under consideration during both moisture-regimes. Selection of genotypes based on these component-traits were rewarding and seems to be better selection-criteria. Finally, we can end-up with superior-genotypes suitable for intermittent-drought conditions.</jats:p