Analysis of DNA methylation patterns associated with drought stress response in faba bean (Vicia faba L.) using methylation-sensitive amplification polymorphism (MSAP)

The effects of drought on water relations, gas exchanges and epigenetic alterations were studied in two faba bean (Vicia faba L.) genotypes with contrasting levels of drought tolerance. The drought-tolerant Bachar genotype was less affected by water deficit in comparison with the drought-sensitive F177 in terms of photosynthetic function and water status in plants as indicated by less reduction in net photosynthesis (A), transpiration rate (E), stomatal conductance (gs) and relative water content (RWC). In this study, the methylation-sensitive amplification polymorphism (MSAP) technique was used to profile the DNA methylation patterns of Bachar and F177 under drought and control conditions. Overall, the amount of methylation was higher in leaves than in roots and the contribution of fully methylated loci was always higher than that of hemimethylated loci. Under control and drought treatment, the total methylation level in leaf tissues was 37.43% and 30.62% in Bachar, 41.23% and 38.16% in F177, indicating a decrease of 6.81% and 3.07% in Bachar and F177 respectively, due to drought exposure. The results revealed that DNA methylation in root tissues was decreased by 3.63% (from 23.43% to 19.80%) in Bachar and increased by 0.66% (from 16.53% to 17.19%) in F177 under drought stress. Differentially displayed DNA fragments in MSAP profiles were cloned and sequenced. A sequence analysis identified six potentially drought stress-related differentially methylated regions (DMRs). Five of these have high homology to previously identified or putative proteins found in other plants, including lipoxygenase (LOX), calcium-dependent protein kinase (CDPK), ABC transporter family (ABC), glycosyl hydrolase (GH) and phosphoenolpyruvate carboxylase (PEPC) and were chosen for further characterization. Quantitative reverse transcription PCR analysis revealed that VfLOX, VfCDPK, VfABC and VfGH varied under drought stress; the expression level of these studied genes was higher in Bachar than in F177. This could suggest their possible role in faba bean drought stress tolerance. Overall, the genome-wide epigenetic changes are probably an important regulatory mechanism for faba bean response to drought and as well to other environmental stresses.