Identification of Gene Pathways Involved in Tolerance to End-of-Season Drought Stress in the Dayton/Ranney Barley Genotype using Transcriptomics

Background: The expression of genes changes under the influence of different developmental stages and various environmental factors.Drought stress at the flowering and seed-filling stage, which is known as end-of-season drought stress, can lead to a sharp decrease in yield or complete failure of crop production.Genetic analysis of drought resistance in the reproductive stage is necessary to understand the mechanism of plant response to drought conditions in the face of the challenges of maintaining food security.Assessment of the transcript profile of genes in different tissues and developmental stages under different conditions of environmental stress can provide insight into the molecular mechanisms and plants' reactions to stress.Barley is known as a model plant for deciphering the mechanisms of drought tolerance, and the study of molecular mechanisms of barley is important for breeding crops because it can tolerate water limitations at the flowering and grain-filling stages.This research aimed to identify differentially expressed genes in barley under end-of-season drought stress using the RNA-Seq technique.Based on the study of Amini et al. on 13 genotypes of spring two-row barley under drought stress, the Dayton/Ranney genotype (modified by ICARDA) was identified as a drought-tolerant genotype.Thus, they were used in this study to investigate the gene expression profile of barley under endof-season drought stress.Methods: The Dayton/Ranney spring barley genotype was subjected to drought stress treatment (70% available water depletion) at the stage of flag leaf emergence.Total RNA was extracted from the leaves of the control and drought-treated plants, followed by qualifying the extracted RNA.After sequencing and analyzing, the expression profiles of differentially expressed genes were obtained under end-of-season drought stress.Moreover, the differentially expressed genes were functionally investigated using gene ontology enrichment analysis.The binding site of transcription factors in the promoter sequence of differentially expressed genes was identified using PlantPAN 3.0 online software, and the frequency of binding sites was reported as a percentage of all identified sites.Results: Under end-of-season drought stress, 2920 and 2290 genes showed significant increases and decreases in expression, respectively, in barley plants.The identified genes were involved in the processes of photosynthesis, carbohydrate and lipid metabolism, regulatory processes, response to abiotic stimuli and stress, seed development, and maturation.Based on gene ontology analysis, these genes were involved in the metabolic and biosynthetic processes of carboxylic acid, sucrose, and glucan cellular metabolism, proteolysis, phosphorylation, RNA metabolism and biosynthesis, and serine family amino acid metabolism.Among the genes with the highest increase in expression under drought stress are the family of abundant proteins in late embryogenesis, a phenylpropanoid pathway gene called anthranilate N-benzoyltransferase protein 1, the xyloglucan-endotrans-glucosylate/hydrolase gene, protein serine/threoninephosphatase, a mitochondrial arginine transporter, an endonuclease gene, laccase enzyme, and several transcription factors.Besides, the genes that showed the most significant decrease in expression under drought stress include an L-type lectin-containing receptor kinase (Hv-LecRK), a ribonuclease III-like gene, the HEC1-like transcription factor, methyljasmonate II -inducible lipoxygenase, glucan endo-1,3-beta-glucosidase GIII, PIP2;5 aquaporin, 70-kDa heat shock protein (HSP70), and an aspartic proteinase nepenthesin-1 gene.Moreover, two unknown genes 2HG0195510 and 4HG0389440 showed significant increases in expression.These genes are involved in the metabolic and biosynthetic processes of carboxylic acid, response to abiotic