The RNA-seq transcriptomic analysis reveals genes mediating salt tolerance through rapid triggering of ion transporters in a mutant barley

Considering the complex nature of salinity tolerance mechanisms, the use of isogenic lines or mutants possessing the same genetic background albeit different tolerance to salinity is a suitable method for reduction of analytical complexity to study these mechanisms. In the present study, whole transcriptome analysis was evaluated using RNA-seq method between a salt-tolerant mutant line "M4-73-30" and its wild-type "Zarjou" cultivar at seedling stage after six hours of exposure to salt stress (300 mM NaCl). Transcriptome sequencing yielded 20 million reads for each genotype. A total number of 7116 transcripts with differential expression were identified, 1586 and 1479 of which were obtained with significantly increased expression in the mutant and the wild-type, respectively. In addition, the families of WRKY, ERF, AP2/EREBP, NAC, CTR/DRE, AP2/ERF, MAD, MIKC, HSF, and bZIP were identified as the important transcription factors with specific expression in the mutant genotype. The RNA-seq results were confirmed at several time points using qRT-PCR for some important salt-responsive genes. In general, the results revealed that the mutant accumulated higher levels of sodium ion in the root and decreased its transfer to the shoot. Also, the mutant increased the amount of potassium ion leading to the maintenance a high ratio [K+]/[Na+] in the shoot compared to its wild-type via fast stomata closure and consequently transpiration reduction under the salt stress. Moreover, a reduction in photosynthesis and respiration was observed in the mutant, resulting in utilization of the stored energy and the carbon for maintaining the plant tissues, which is considered as a mechanism of salt tolerance in plants. Up-regulation of catalase, peroxidase, and ascorbate peroxidase genes has resulted in higher accumulation of H2O2 in the wild-type compared to the mutant. Therefore, the wild-type initiated rapid ROS signals which led to less oxidative scavenging in comparison with the mutant. The mutant increased expression in the ion transporters and the channels related to the salinity to maintain the ion homeostasis. In overall, the results demonstrated that the mutant responded better to the salt stress under both osmotic and ionic stress phases and lower damage was observed in the mutant compared to its wildtype under the salt stress.