Chrysanthemum indicum miRNA159a targeting MYB33 raises drought sensitivity in Arabidopsis thaliana

Chrysanthemum indicum, a wild relative of cultivated chrysanthemum, exhibits stronger environmental adaptability and thus holds great potential for breeding. As an important genetic resource, it offers a promising entry point for identifying stress-resistance genes and developing high-quality chrysanthemum cultivars. Although microRNA159 (miR159) is known to be involved in various biological processes, including flowering, leaf development, and stress responses; its role in drought adaptation in chrysanthemum remains unclear. In this study, we found that the expression level of cin-miR159a was significantly downregulated after 2 h of drought stress, indicating that cin-miR159a may play a key role in the early response to drought stress in this species. Using degradome sequencing, we identified CiMYB33 as a direct target of cin-miR159a in C. indicum. Phylogenetic and structural analyses confirmed its evolutionary conservation and functional relevance. To assess the cin-miR159a-CiMYB33 role in drought tolerance, we cloned cin-miR159a and CiMYB33 genes from C. indicum and generated transgenic Arabidopsis thaliana, then subjected them to drought stress. cin-miR159a-overexpressing ( cin-miR159a-OE) plants exhibited lower relative water content (RWC), higher malondialdehyde (MDA) accumulation, and increased relative electrolyte leakage (REL), indicating greater membrane damage. In contrast, CiMYB33-overexpressing ( CiMYB33-OE) plants displayed higher RWC, lower MDA, reduced REL, and increased proline accumulation, enhancing drought resilience. These results reveal that cin-miR159a negatively regulates drought tolerance by repressing CiMYB33, while CiMYB33 enhances drought resistance. This study investigates the function of the cin-miR159a-CiMYB33 regulatory module in drought stress adaptation, providing insights for improving stress tolerance in C. indicum.