In addition to regulating growth and development, the most important function of microRNAs (miRNAs) in plants is the regulation of a variety of cellular processes underlying plant adaptation to environmental stresses. To gain a deep understanding of the mechanism of drought tolerance in rice, genome-wide profiling and analysis of miRNAs was carried out in drought-challenged rice across a wide range of developmental stages, from tillering to inflorescence formation, using a microarray platform. Among the 30 miRNAs identified as significantly down- or up-regulated under the drought stress, 11 down-regulated miRNAs (miR170, miR172, miR397, miR408, miR529, miR896, miR1030, miR1035, miR1050, miR1088, and miR1126) and eight up-regulated miRNAs (miR395, miR474, miR845, miR851, miR854, miR901, miR903, and miR1125) were revealed for the first time to be induced by drought stress in plants, and nine (miR156, miR168, miR170, miR171, miR172, miR319, miR396, miR397, and miR408) showed opposite expression to that observed in drought-stressed Arabidopsis. The most conserved down-regulated miRNAs were ath-miR170, the miR171 family, and ath-miR396, and the most conserved up-regulated miRNAs were ptc-miR474 and ath-miR854a. The identification of differentially expressed novel plant miRNAs and their target genes, and the analysis of cis-elements provides molecular evidence for the possible involvement of miRNAs in the process of drought response and/or tolerance in rice.

Salinity is one of the most important abiotic stress affecting the world rice production. The cultivation of salinity-tolerant cultivars is the most cost-effective and environmentally friendly approach for salinity control. In recent years, CRISPR/Cas9 systems have been widely used for target-site genome editing; however, their application for the improvement of elite rice cultivars has rarely been reported. Here, we report the improvement of the rice salinity tolerance by engineering a Cas9-OsRR22-gRNA expressing vector, targeting the OsRR22 gene in rice. Nine mutant plants were identified from 14 T₀ transgenic plants. Sequencing showed that these plants had six mutation types at the target site, all of which were successfully transmitted to the next generations. Mutant plants without transferred DNA (T-DNA) were obtained via segregation in the T1 generations. Two T2 homozygous mutant lines were further examined for their salinity tolerance and agronomic traits. The results showed that, at the seedling stage, the salinity tolerance of T2 homozygous mutant lines was significantly enhanced compared to wild-type plants. Furthermore, no significantly different agronomic traits were found between T2 homozygous mutant lines and wild-type plants. Our results indicate CRISPR/Cas9 as a useful approach to enhance the salinity tolerance of rice.

Rice is the staple food and rice production consumes about 50% of the fresh water resources in China. In addition, drought is one of the most important constraints in rice resulting in large yield losses and limiting the average yield increase of the country. There is an urgent need to enhance water-saving (W) capacity or drought resistance (DR) of rice. WDR varieties can be developed through introgressing the water-saving and drought resistance capacity mainly from the traditional upland to the commercialized paddy rice cultivars. The breeding target is a high yield potential under irrigation, an acceptable grain quality, and water consumption reduced by about 50% compared with paddy rice. In a water-limited environment, a higher level of drought resistance and reduced yield loss by drought stress are required. In recent years, the field drought-resistance screening facility was established and the evaluation standard was developed. Some DR rice varieties were identified and used in both molecular mapping and breeding programmes. Several WDR varieties were developed and released to farmers. This article describes our initial achievement towards this goal and provides some details on the rationale and the specific steps and methods used.

Drought is a major abiotic stress factors that reduces agricultural productivity. GRAS transcription factors are plant-specific proteins that play diverse roles in plant development. However, the functions of a number of GRAS genes identified in rice are unknown, especially the GRAS genes related to rice drought resistance have not been characterized. In this study, a novel GRAS transcription factor gene named OsGRAS23, which is located in a drought-resistant QTL interval on chromosome 4 of rice, was isolated. The expression of OsGRAS23 was induced by drought, NaCl, and jasmonic acid treatments. The OsGRAS23-GFP fused protein was localized in the nucleus of tobacco epidermal cells. A trans-activation assay in yeast cells demonstrated that the OsGRAS23 protein possessed a strong transcriptional activation activity. OsGRAS23-overexpressing rice plants showed improved drought resistance and oxidative stress tolerance as well as less H2O2 accumulation compared with the wild-type plants. Furthermore, microarray analysis showed that several anti-oxidation related genes were up-regulated in the OsGRAS23-overexpressing rice plants. The yeast one hybrid test indicated that OsGRAS23 could bind to the promoters of its potential target genes. Our results demonstrate that OsGRAS23 encodes a stress-responsive GRAS transcription factor and positively modulates rice drought tolerance via the induction of a number of stress-responsive genes.

Epigenetic mechanisms are crucial mediators of appropriate plant reactions to adverse environments, but their involvement in long-term adaptation is less clear. Here, we established two rice epimutation accumulation lines by applying drought conditions to 11 successive generations of two rice varieties. We took advantage of recent technical advances to examine the role of DNA methylation variations on rice adaptation to drought stress. We found that multi-generational drought improved the drought adaptability of offspring in upland fields. At single-base resolution, we discovered non-random appearance of drought-induced epimutations. Moreover, we found that a high proportion of drought-induced epimutations maintained their altered DNA methylation status in advanced generations. In addition, genes related to transgenerational epimutations directly participated in stress-responsive pathways. Analysis based on a cluster of drought-responsive genes revealed that their DNA methylation patterns were affected by multi-generational drought. These results suggested that epigenetic mechanisms play important roles in rice adaptations to upland growth conditions. Epigenetic variations have morphological, physiological and ecological consequences and are heritable across generations, suggesting that epigenetics can be considered an important regulatory mechanism in plant long-term adaptation and evolution under adverse environments.

Drought is a major abiotic stress factor that influences the yield of crops. Basic leucine zipper motif (bZIP) transcription factors play an important regulatory role in plant drought stress responses. However, the functions of a number of bZIP transcription factors in rice are still unknown. In this study, a novel drought stress-related bZIP transcription factor, OsbZIP62, was identified in rice. This gene was selected from a transcriptome analysis of several typical rice varieties with different drought tolerances. OsbZIP62 expression was induced by drought, hydrogen peroxide, and abscisic acid (ABA) treatment. Overexpression of OsbZIP62-VP64 (OsbZIP62V) enhanced the drought tolerance and oxidative stress tolerance of transgenic rice, while osbzip62 mutants exhibited the opposite phenotype. OsbZIP62-GFP was localized to the nucleus, and the N-terminal sequence (amino acids 1–68) was necessary for the transcriptional activation activity of OsbZIP62. RNA-seq analysis showed that the expression of many stress-related genes (e.g., OsGL1, OsNAC10, and DSM2) was upregulated in OsbZIP62V plants. Moreover, OsbZIP62 could bind to the promoters of several putative target genes and could interact with stress/ABA-activated protein kinases (SAPKs). OsbZIP62 is involved in ABA signalling pathways and positively regulates rice drought tolerance by regulating the expression of genes associated with stress, and this gene could be used for the genetic modification of crops with improved drought tolerance.