Identification of CsamiR2603 in cucumber and its regulatory role in drought stress response

Cucumber ( Cucumis sativus L.), a globally important vegetable crop, faces increasing threats to sustainable production due to intensifying drought stress in the face of climate change. While microRNAs (miRNAs) are known to regulate plant stress responses, their specific roles and molecular networks in cucumber drought tolerance remain largely uncharted, limiting targeted genetic improvement. Through integrated analysis of transcriptome, miRNA-sequencing, and degradome data, we identified CsamiR2603 as a novel drought-responsive miRNA in cucumber. Functional characterization, using transgenic Arabidopsis thaliana and cucumber lines, revealed that CsamiR2603 acts as a negative regulator of drought tolerance. Overexpression (OE) lines exhibited significantly lower stress tolerance: under 300 mM mannitol stress, seed germination decreased by 24.5% (OE1) and 16.3% (OE3) while cotyledon-greening rates declined by 25.5% (OE1) and 45.1% (OE3). Under natural drought, overexpression lines showed severe wilting accompanied by 16.8–32.2% reduction in superoxide dismutase (SOD) activity, 11.4–14.7% lower peroxidase (POD) activity, 38.4–50.0% lower proline accumulation, and 30.7–33.7% higher malondialdehyde (MDA) concentration. In cucumber, CsamiR2603-overexpression lines displayed greater drought sensitivity with 27.7%, 10.5%, and 13.9% reductions in SOD, POD, and catalase (CAT) activities, respectively, along with 45.0% higher MDA concentration under artificial (PEG-induced) drought stress. Conversely, short tandem target mimic (STTM)-mediated CsamiR2603-knockdown lines demonstrated significantly improved drought tolerance with favorable physiological performance. The current study reveals for the first time the functional characterization of CsamiR2603 as a negative regulator of drought tolerance in cucumber, indicating that the CsamiR2603-DAHPS2 pathway may constitute a key mechanism in the cucumber drought stress response. These findings provide crucial theoretical insights and practical breeding strategies for developing drought-tolerant cucumber varieties to address climate-induced agricultural challenges. • First identification of CsamiR2603 as a drought-responsive miRNA in cucumber • CsamiR2603 negatively regulates drought tolerance in both Arabidopsis and cucumber • Overexpression lines show reduced antioxidant activity and increased oxidative damage • STTM-mediated knockdown enhances drought tolerance with improved physiological profiles • Identifies a molecular target and strategy for breeding drought-resilient cucumber