MicroRNAs in Plant Genetic Regulation of Drought Tolerance and Their Function in Enhancing Stress Adaptation

Abstract

Adverse environmental conditions, including drought stress, pose a significant threat to plant survival and agricultural productivity, necessitating innovative and efficient approaches to enhance their resilience. MicroRNAs (miRNAs) are recognized as key elements in regulating plant adaptation to drought stress, with a notable ability to modulate various physiological and molecular mechanisms. This review provides an in-depth analysis of the role of miRNAs in drought response mechanisms, including abscisic acid (ABA) signaling, reactive oxygen species (ROS) detoxification, and the optimization of root system architecture. Additionally, it examines the effectiveness of bioinformatics tools, such as those employed in in silico analyses, for studying miRNA-mRNA interactions, as well as the potential for their integration with experimental methods. Advanced methods such as microarray analysis, high-throughput sequencing (HTS), and RACE-PCR are discussed for their contributions to miRNA target identification and validation. Moreover, new data and perspectives are presented on the role of miRNAs in plant responses to abiotic stresses, particularly drought adaptation. This review aims to deepen the understanding of genetic regulatory mechanisms in plants and to establish a robust scientific foundation for the development of drought-tolerant crop varieties.

Keywords: abiotic stress; drought stress; gene expression; mRNA; microRNA.

Figure 1

Biogenesis and processing pathway of miRNAs in plants. The miRNA gene is transcribed by RNA polymerase II into a pri-miRNA with a stem-loop structure. The pri-miRNA is processed by the DCL1 enzyme, along with auxiliary proteins such as SE and HYL1, resulting in the formation of a pre-miRNA molecule, which subsequently develops into a miRNA duplex. Then, this duplex is stabilized by methylation from the HEN1 enzyme, after which it is exported from the nucleus to the cytoplasm via the HASTY (HST1) protein for further integration into the RISC. In the cytoplasm, the mature miRNA strand is loaded into the RISC, where it guides the AGO1 protein to bind complementary target mRNA, leading to mRNA cleavage or translational repression.

Figure 2

Role of miRNAs in drought stress adaptation and tolerance. Drought stress in plants leads to stomatal closure, reducing water loss, while the accumulation of ROS at the cellular level induces oxidative damage, causing membrane disruption, hormonal imbalance, growth reduction, premature senescence, and reproductive failure. MicroRNAs regulate the interplay of phytohormones such as ABA, auxin, and cytokinin, optimizing plant adaptation to drought conditions and enhancing drought stress adaptation and tolerance.

Figure 3

Integrated workflow for studying miRNA-mediated drought tolerance in plants. (1) Drought stress significantly impacts plants, causing physiological changes such as leaf yellowing, reduced photosynthesis, and excessive accumulation of ROS due to water deficiency. During this stress, drought-responsive miRNAs are activated. (2) Bioinformatics tools such as psRNATarget, TargetFinder, miRTarBase, RNAhybrid, and MirTarget are used to predict miRNA-mRNA interactions. These tools analyze miRNA binding sites on the mRNA sequence, providing insights into their regulatory mechanisms. (3) Total RNA is extracted from plant tissues (e.g., leaves and roots), serving as the foundation for studying miRNAs and their target mRNAs. (4) Methods like qPCR and Northern blot are employed to validate the in silico predictions of miRNAs and their target mRNAs. These techniques are used to determine inverse correlations in expression levels between miRNAs and their targets. (5) Functional analysis is performed to investigate whether miRNAs repress or activate mRNA expression. These studies reveal the specific genetic pathways regulated by miRNAs. (6) Transgenic plants are developed to overexpress or suppress specific miRNAs or their target genes. These models are then studied to evaluate their ability to adapt to drought stress. (7) Drought adaptation traits in transgenic plants, such as water retention capacity, root system architecture, and ROS levels, are assessed comprehensively. These analyses help determine the plants’ drought tolerance levels. (8) As a result of these steps, strategies to improve plant drought tolerance are identified. These strategies pave the way for developing drought-resistant crop varieties.

Figure 4

RNA extraction process for miRNA profiling and analysis. (1) Collection of plant samples subjected to drought stress (leaf, root, etc.). (2) RNA extraction using guanidine thiocyanate/phenol/chloroform extraction or commercial kits. (3) Ensuring efficient extraction while preserving small RNA fractions, including miRNA. (4) RNA/miRNA quality and integrity check using spectrophotometry, gel electrophoresis, or Bioanalyzer. (5) RNA/miRNA ready for analysis (miRNA profiling, HTS, or RACE-PCR).