Transcriptome analysis of common bean under salt stress and functional study of salt responsive genes

Abstract

Salinity is a major abiotic stress that impairs physiology and productivity of economically important crops such as common bean. It is an important grain legume and provides 30% of the protein diet in developing countries. Several common bean genotypes have been phenotypically classified according to its diverse responses to salinity. In this study, a comprehensive high-throughput transcriptome analysis in root and leaf tissues of common bean genotypes with contrasting salt tolerance was performed under stress in hydroponic conditions. Prior to transcriptome analysis, the stress onset time was determined as the end of 3rd day of 125mM NaCl application for tolerant Ispir and susceptible TR43477 varieties following several physiological measurements. Transcriptome analysis have generated 255 million high quality reads which were assembled into 73762 all-unigenes with a mean length of 930 bp. Among the all-unigenes, 75% (55433) were assigned with Nr annotations. A total of 12001 differentially expressed genes (DEGs) were identified. Validation of the RNA-seq quantifications (RPKM values) were performed by qRT-PCR analysis. Within the selected candidate gene pool with contrasting expression levels between the two genotypes and two tissues, five genes (Unc2, Unc4, TT12, CPRD2 and β-Glu) were cloned and transformed to Arabidopsis Col-0 ecotype to generate homozygous overexpression transgenic lines. Functional studies on these lines by both physiological assays and their impact on the expression of salt responsive genes as well as the transcription factors (TFs) regulating them, suggested a highly significant regulatory role for TT12 and CPRD2 genes in improvement of salt tolerance. Major contributions of these genes were implied for better management of osmotic and ionic imbalance most probably by imposing synthesis, accumulation and sequestration of osmolytes and reinforcement of the cell wall structure while minimizing damage through ROS scavenging. To do so, regulation by interacting ABA -independent and ABA-dependent expression of DREB2A, MYB2 TFs and downstream P5CS1 and RD29B genes were also implicated.