Salt stress induced variation in DNA methylation pattern and its influence on gene expression in contrasting rice genotypes

Abstract

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.

Figure 1. A representative MSAP gel using the primer combination EcoRI-ACG/MspI-AATC.

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 EcoRI+HpaII and EcoRI+MspI, respectively. RC: root control; RS: root stress; SC: shoot control; SS: shoot stress.

Figure 2. Expression profiles of MSAP loci in rice under salt stress.

Salinity stressed shoot (A) and root (B) of six genotypes of rice, Bengal (Beng), IR29, Nipponbare (Nipp), Pokkali (Pokk), Nonabokra (Nona), and Geumgangbyeo (Geum) were used for quantifying the expression of MSAP loci relative to control. Real-time PCR analysis was performed using gene-specific primers. The expression of each gene in different RNA samples was normalized with the expression of internal control gene, rice elongation factor 1 α (eEF1 α). Fold changes in mRNA expression for each candidate gene in different genotypes were calculated relative to its control using ddCt method. The values represented are the mean of two biological replicates, each with three technical replicates. Error bars indicate the standard deviation.