| The ERECTA family of receptor-like kinases (RLKs) plays a crucial regulatory role in plant stomatal development and responses to abiotic stresses. However, the functional mechanism of ERECTA in barley (Hordeum vulgare),a typical monocot cereal crop, remains unclear. This study therefore focused on investigating the barley HvERECTA gene, aiming to clarify its regulatory roles in these biological processes and provide potential targets for barley drought resistance breeding. To achieve the above objective,we first cloned the HvERECTA gene from barley and conducted comprehensive bioinformatics analyses to characterize its sequence and protein properties. Subsequently, we constructed a heterologous overexpression vector carrying the HvERECTA gene and transformed it into Arabidopsis thaliana. For the transgenic Arabidopsis lines, we systematically determined stomatal phenotypic traits, expression levels of key related genes, and physiological indicators under drought stress conditions to evaluate the gene function. The bioinformatics analysis results showed that the full-length coding sequence (CDS) of HvERECTA is 2934 bp, encoding a protein of 977 amino acids. The HvERECTA protein is localized to the plasma membrane and contains a typical serine/threonine protein kinase domain, which is a hallmark of the receptor-like kinase family. Sequence alignment and phylogenetic analysis revealed that HvERECTA is highly conserved among gramineous plants, with the highest homology to those from wheat (Triticum aestivum) and ryegrass (Lolium perenne). Additionally, analysis of cis-acting elements in the HvERECTA promoter region indicated that it is rich in regulatory elements associated with abiotic stress responses and plant hormone signaling, such as abscisic acid (ABA)-responsive elements (ABRE) and drought-responsive elements (MBS). These elements suggest that HvERECTA may participate in integrating multiple signaling pathways to mediate plant responses to environmental stresses. Phenotypic observations on the transgenic Arabidopsis lines showed that overexpression of HvERECTA significantly reduced stomatal density compared with the wild-type control. Molecular analysis demonstrated that the expression levels of genes encoding key stomatal development transcription factors, namely SPCH (SPEECHLESS), MUTE, and FAMA, were all downregulated by more than 50% in the transgenic lines. In contrast, the expression level of YODA, a core gene in the mitogen-activated protein kinase (MAPK) cascade pathway that is involved in stomatal development regulation, was upregulated by 50%-80% in the transgenic plants. This gene expression pattern is consistent with the known regulatory model where ERECTA mediates the MAPK signaling pathway to inhibit stomatal formation. Under drought stress conditions, the transgenic Arabidopsis lines showed enhanced drought resistance-related physiological performance. Specifically, the activities of three key antioxidant enzymes—superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in the transgenic lines were increased by 25%-40% compared with the control. Meanwhile, the content of malondialdehyde (MDA), which is an indicator of lipid peroxidation and cellular damage under stress, was decreased by approximately 30% in the transgenic lines. Additionally, the water loss rate of the transgenic lines was reduced by 20%, and their relative water content was higher than that of the control plants. Furthermore, the expression levels of stress-responsive genes, including AtKIN2, AtABF3, and AtRD22, showed no abnormal changes under normal growth conditions but were significantly upregulated under drought stress in the transgenic lines. In conclusion, the barley HvERECTA gene enhances plant drought resistance by regulating the MAPK signaling pathway to reduce stomatal density and by improving the activity of the plant antioxidant defense system. These two regulatory pathways collectively enhance the water retention capacity of plants under drought stress, thereby improving their stress tolerance. This study clarifies the important regulatory role of HvERECTA in stomatal development and abiotic stress response, and provides an important functional gene resource for drought-resistant molecular breeding of barley. |
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