University of Guilan Cereal Research 2252-0163 14 4 2025 01 20 Identification of key genes and miRNAs involved in drought tolerance in rice (Oryza sativa L.) Identification of key genes and miRNAs involved in drought tolerance in rice (Oryza sativa L.) 347 362 8377 10.22124/cr.2025.29246.1844 FA Sahar Shojaee Ph. D. Candidate, Department of Agricultural Biotechnology, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran Mohammad Mohsenzadeh Golfazani Associate Professor, Department of Agricultural Biotechnology, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran Habibollah Samiezadeh Lahiji Professor, Department of Agricultural Biotechnology, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran Maryam Pasandideh Arjmand Ph. D. Graduate, Department of Agricultural Biotechnology, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran (Researcher, BioGenTAC Inc, Technology Incubator of Agricultural Biotechnology Research Institute of Iran-North Branch (ABRII) Journal Article 2024 12 14 Introduction Plants are often exposed to a various biotic and abiotic stresses. Salinity, drought, and temperature stresses are abiotic factors that negatively affect on physiological and biochemical characteristics of plants and reduce crop growth and productivity. Identifying key characteristics related to biotic and abiotic stresses is important in understanding plant responses at the molecular and cellular levels. Studying miRNA components in crop plants is an effective tool for early detection of stresses, physiological changes, and regulation of gene expression. The objective of this study was to identify key genes and miRNAs involved in drought stress tolerance and introduce them to improve rice plants for drought stress tolerance. Materials and methods In this study, microarray data of two drought-tolerant (Dagad Deshi) and drought-sensitive (IR20) rice varieties under drought stress were collected in three replications from the NCBI database and differentially expressed genes in these genotypes were identified using the GEO2R tool. Genes with LogFC ≥ 3 and LogFC ≤ -3 were considered as genes with increased and decreased expression, respectively. To determine the common genes between the sensitive (IR20) and tolerant (Dagad Deshi) genotypes, the VENNY tool was used, and to select key genes responsive to drought stress, genes that were expressed only in the drought-tolerant genotype were identified using the Cytoscape software, with the CytoHubba plugin and the MCC (Maximal Clique Centrality) method. The biological processes of key genes and differentially expressed gene pathways were assessed by the DAVID tool and the most important pathways were identified using the path analysis by the KEGG database. The psRNATarget tool version 2017 was also used to identify miRNAs regulating key genes. Research findings Microarray data analysis revealed that under drought stress conditions, significant up-expressed and down-expressed genes were observed in 33% and 26% of genes in the tolerant cultivar (Dagad Deshi) and 7% and 6% of genes in the sensitive cultivar (IR20), respectively. Also, in both tolerant and sensitive cultivars, 766 and 340 genes exhibited significant increase and decrease in gene expression, respectively. The most significant biological processes, cellular components and molecular functions among the up-expressed genes in the tolerant cultivar (Dagad Deshi), included response to salicylic acid, cytoplasm and carboxylase activity, respectively, and among the down-expressed genes included monomethylation of peptidyl lysine, large mitochondrial ribosomal subunit and long-chain acyl-CoA reductase activity forming alcohol, respectively. KEGG pathway enrichment analysis revealed that the up-expressed and down-expressed genes in the tolerant cultivar were enriched in 18 and 10 pathways, respectively. Among the altered expression genes, 15 genes including several genes from the MCM (Minichromosome Maintenance Complex) gene family, were selected as key genes involved in the drought stress tolerance pathway in rice. These genes included MCM5, Os05g0358200, MCM4, MCM7, NAC037, CDC6, OS11G0128400, RPA2A, OS12G0124700, MCM3, POLA, Os07g0406800, MCM6, Os05g0160800 and PCNA genes. Finally, 247 miRNAs including osa-miR172c, osa-miR1849, osa-miR2925, osa-miR397a and osa-miR414 were identified and introduced for genes involved in the drought stress tolerance pathway in rice. Conclusion Based on the results of this study, 15 genes including several genes from the MCMs gene family as well as 247 miRNAs were identified as key genes and miRNAs involved in the drought stress tolerance pathway in rice. The genes and miRNAs identified in this study can be used to improve and produce drought-tolerant plants in rice. Introduction Plants are often exposed to a various biotic and abiotic stresses. Salinity, drought, and temperature stresses are abiotic factors that negatively affect on physiological and biochemical characteristics of plants and reduce crop growth and productivity. Identifying key characteristics related to biotic and abiotic stresses is important in understanding plant responses at the molecular and cellular levels. Studying miRNA components in crop plants is an effective tool for early detection of stresses, physiological changes, and regulation of gene expression. The objective of this study was to identify key genes and miRNAs involved in drought stress tolerance and introduce them to improve rice plants for drought stress tolerance. Materials and methods In this study, microarray data of two drought-tolerant (Dagad Deshi) and drought-sensitive (IR20) rice varieties under drought stress were collected in three replications from the NCBI database and differentially expressed genes in these genotypes were identified using the GEO2R tool. Genes with LogFC ≥ 3 and LogFC ≤ -3 were considered as genes with increased and decreased expression, respectively. To determine the common genes between the sensitive (IR20) and tolerant (Dagad Deshi) genotypes, the VENNY tool was used, and to select key genes responsive to drought stress, genes that were expressed only in the drought-tolerant genotype were identified using the Cytoscape software, with the CytoHubba plugin and the MCC (Maximal Clique Centrality) method. The biological processes of key genes and differentially expressed gene pathways were assessed by the DAVID tool and the most important pathways were identified using the path analysis by the KEGG database. The psRNATarget tool version 2017 was also used to identify miRNAs regulating key genes. Research findings Microarray data analysis revealed that under drought stress conditions, significant up-expressed and down-expressed genes were observed in 33% and 26% of genes in the tolerant cultivar (Dagad Deshi) and 7% and 6% of genes in the sensitive cultivar (IR20), respectively. Also, in both tolerant and sensitive cultivars, 766 and 340 genes exhibited significant increase and decrease in gene expression, respectively. The most significant biological processes, cellular components and molecular functions among the up-expressed genes in the tolerant cultivar (Dagad Deshi), included response to salicylic acid, cytoplasm and carboxylase activity, respectively, and among the down-expressed genes included monomethylation of peptidyl lysine, large mitochondrial ribosomal subunit and long-chain acyl-CoA reductase activity forming alcohol, respectively. KEGG pathway enrichment analysis revealed that the up-expressed and down-expressed genes in the tolerant cultivar were enriched in 18 and 10 pathways, respectively. Among the altered expression genes, 15 genes including several genes from the MCM (Minichromosome Maintenance Complex) gene family, were selected as key genes involved in the drought stress tolerance pathway in rice. These genes included MCM5, Os05g0358200, MCM4, MCM7, NAC037, CDC6, OS11G0128400, RPA2A, OS12G0124700, MCM3, POLA, Os07g0406800, MCM6, Os05g0160800 and PCNA genes. Finally, 247 miRNAs including osa-miR172c, osa-miR1849, osa-miR2925, osa-miR397a and osa-miR414 were identified and introduced for genes involved in the drought stress tolerance pathway in rice. Conclusion Based on the results of this study, 15 genes including several genes from the MCMs gene family as well as 247 miRNAs were identified as key genes and miRNAs involved in the drought stress tolerance pathway in rice. The genes and miRNAs identified in this study can be used to improve and produce drought-tolerant plants in rice.

https://cr.guilan.ac.ir/article_8377_1508a7fbbc81928b5ebc22f96db0d6fa.pdf