The Apple microR171i-SCARECROW-LIKE PROTEINS 26.1 Module Enhances Drought Stress Tolerance by Integrating Ascorbic Acid Metabolism

Drought stress severely restricts crop yield and quality. Small non-coding RNAs (snRNAs) play critical roles in plant growth, development, and stress responses by regulating target gene expression, but their roles in drought stress tolerance in apple (Malus domestica) are poorly understood. Here, we identified various snRNAs and their targets from the wild apple species Malus sieversii via high-throughput sequencing and degradome analysis. Forty known microRNAs (miRNAs) and eight new snRNAs were differentially expressed in response to 2 or 4 h of drought stress treatment. We experimentally verified the expression patterns of five selected miRNAs and their targets. We established that one miRNA, mdm-miR171i, specifically targeted and degraded SCARECROW-LIKE PROTEINS 26.1 (MsSCL26.1) transcripts. Both knockout of mdm-miR171i and overexpression of MsSCL26.1 improved drought stress tolerance in the cultivated apple line 'GL-3' (M. domestica Borkh.) by regulating the expression of antioxidant enzyme genes, especially that of MONODEHYDROASCORBATE REDUCTASE (MsMDHAR), which functions in ascorbic acid (AsA) metabolism under drought stress. Transient expression analysis demonstrated that MsSCL26.1 activates MsMDHAR transcription by positively regulating the activity of the P1 region in its promoter. Therefore, the miR171i-SCL26.1 module enhances drought stress tolerance in apple by regulating antioxidant gene expression and AsA metabolism.