Chrysanthemum indicum miRNA159a targeting MYB33 raises drought sensitivity in Arabidopsis thaliana

Chrysanthemum indicum (C. 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 hours 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.