Journal of Crop Breeding

Extended Abstract
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 end-of-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/threonine-phosphatase, 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 stimuli and stress, response to endogenous stimuli, proteolysis, phosphorylation, RNA metabolism and biosynthesis, the protein metabolic process, and serine family amino acid metabolism and transport. At the level of molecular function, the groups of catalytic activity and connection assigned the largest number of genes to themselves for all the genes with differential expression. Other molecular functions identified for genes responsive to drought stress include protein binding, nucleotide binding, transport activity, DNA binding, transferase, kinase, hydrolase, and pyrophosphatase activity. In addition, these increased genes expressed specifically had the functions of message transmission, transcription factor, enzyme regulation, molecular transport, and receptor activities. The binding positions of transcription factors in genes with differential expression were classified into 64 families. The highest percentage of binding sites in the up-expressed genes belongs to ERF/AP2 transcription factors, followed by the most abundant binding sites belonging to the transcription factor family of bZIP, bHLH, DOF, and GATA. Furthermore, the most abundant binding sites in the down-expressed genes included AP2/ERF, BES1, EIL, TCP, Myb/SANT, GATA, and DOF.
Conclusion: By evaluating the gene expression under end-of-season drought stress, aspects of the resistance mechanism of barley to drought stress were identified that are related to the metabolic and biosynthetic activities of the plant in the reproductive stage. The results show that diverse and complex gene networks play a role in the response of the barley plant to end-of-season drought stress, which mainly decreased the biological processes related to photosynthesis and the production of precursor metabolites and increased the metabolic processes. Additionally, the response process to the stimulus was observed in both sets of increased and decreased expressed genes.