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

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

A representative MSAP gel using the primer combination <i>Eco</i>RI-ACG/<i>Msp</i>I-AATC.

<p>Both control and salinity stressed root and shoot of rice genotypes, IR29, Nipponbare (Nipp), Pokkali (Pokk), Geumgangbyeo (Geum) were used for MSAP analysis. EH and EM refer to digestion with <i>Eco</i>RI+<i>Hpa</i>II and <i>Eco</i>RI+<i>Msp</i>I, respectively. RC: root control; RS: root stress; SC: shoot control; SS: shoot stress.</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

BLAST results of ten randomly selected polymorphic methylated fragments and their location on rice genome.

<p>BLAST results of ten randomly selected polymorphic methylated fragments and their location on rice genome.</p

DNA methylation changes in shoot and root at seedling stage under non-stress and salinity stress conditions.

a<p>MSAP (%)  =  [(II+III+IV)/(I+II+III+IV)]×100;</p>b<p>Fully methylated bands (%) = [(III+IV)/(I+II+III+IV)]×100;</p>c<p>Hemimethylated bands (%) = [(II)/(I+II+III+IV)]×100.</p><p>Type I indicated absence of methylation due to the presence of bands in both <i>EcoR</i>I/<i>Hpa</i>II and <i>Eco</i>RI/<i>Msp</i>I digest; type II bands appeared only in <i>EcoR</i>I/<i>Hpa</i>II digestion but not in the <i>Eco</i>RI/<i>Msp</i>I digest; type III generated bands obtained in <i>Eco</i>RI/<i>Msp</i>I digest but not in the <i>EcoR</i>I/<i>Hpa</i>II digest; and type IV represents the absence of band in both enzyme combinations.</p

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Resumen basado en el de la publicaciónSe centra en conocer y analizar la autopercepción que el alumnado que empieza el Grado de Maestro de Educación Primaria tiene de la dimensión comunicativa dentro de su competencia digital. Los resultados indican que los futuros maestros se consideran muy competentes en el uso del correo electrónico, herramienta que utilizan con mucha frecuencia. En cambio, muy pocos son capaces de crear, editar o modificar wikis; en general se muestran poco competentes en el trabajo colaborativo y las herramientas relacionadas. Las redes sociales tienen un enorme impacto. Las carencias en trabajo colaborativo son importantes en futuros docentes.ES

Additional file 6: of Genome and Transcriptome sequence of Finger millet (Eleusine coracana (L.) Gaertn.) provides insights into drought tolerance and nutraceutical properties

Validation of differentially expressed drought responsive genes through qRT-PCR. (PDF 116 kb