RrSPL3 confers drought tolerance in Rosa roxburghii by activating flavonol biosynthesis

Abstract

Rosa roxburghii is a perennial flowering shrub producing edible fruits with exceptional nutritional and antioxidant properties, yet its cultivation and productivity are severely constrained by drought stress. Squamosa promoter-binding protein-like (SPL) transcription factors are key regulators of plant development and stress responses. However, their roles in coordinating early drought perception with downstream metabolic adaptation in R. roxburghii remain largely unexplored. Here, we systematically identified 16 SPL genes in the R. roxburghii genome and characterized their evolutionary relationships, structural features, and expression patterns under drought stress. Among them, RrSPL3 was rapidly induced during the early phase of drought stress and localized to the nucleus. Functional analyses demonstrated that overexpression of RrSPL3 enhanced drought tolerance in both Arabidopsis thaliana and R. roxburghii, as evidenced by maintenance of chlorophyll content, enhanced antioxidant enzyme activity, and reduced membrane damage. Combined DAP-seq, transcriptomic, EMSA, and dual-luciferase analyses further demonstrated that RrSPL3 directly activates RrFLS1 under drought stress by binding to the GTAC-containing motif in its promoter, leading to increased flavonol accumulation and enhanced reactive oxygen species scavenging. Together, our findings uncover a previously unrecognized SPL-FLS-flavonol regulatory module that links early drought signaling to metabolic antioxidant defense, providing mechanistic insight and potential molecular targets for improving drought resilience in R. roxburghii and horticultural species.