ABA signaling orchestrates SnRK1α‐dependent phosphorylation of WRKY41 to regulate SPS4 and sugar accumulation in citrus fruit under drought conditions

Drought stress and abscisic acid (ABA) have been known to play a critical role in modulating sugar accumulation in fruit, and yet, the underlying molecular mechanisms remain elusive. In this study, we have demonstrated that drought-mimicking film mulching increased sucrose levels in Satsuma mandarin (Citrus unshiu) fruit, coinciding with upregulation of CuSPS4, which encodes the sucrose phosphate synthase (SPS), in the transcriptome profiling. CuSPS4 was further shown to be drought- and ABA-inducible and functionally essential for sucrose synthesis. Mechanistically, two transcription factors, CuWRKY41 and CuWRKY23, directly bound to and activated the CuSPS4 promoter via the W-box element, with CuWRKY41 additionally regulating CuWRKY23 expression. Consistently, both CuWRKY41 and CuWRKY23 positively regulated sucrose synthesis by upregulating CuSPS4. Meanwhile, the substrate-interacting subunit (CuSnRK1β1) and catalytic subunit (CuSnRK1α) of SUCROSE NON-FERMENTING RELATED KINASE 1 (SnRK1) interacted with CuWRKY41, triggering CuSnRK1α-mediated phosphorylation and subsequent degradation of CuWRKY41, thereby suppressing its activation. However, ABA promoted cytoplasmic translocation of CuSnRK1α and CuSnRK1β1 and reduced nuclear interaction with CuWRKY41, leading to its phosphorylation alleviation and protein stabilization, concurrent with enhanced transcription activation of CuWRKY23 and CuSPS4. Taken together, these findings reveal a sophisticated regulatory mechanism whereby drought promotes sucrose accumulation by suppressing CuSnRK1α-mediated phosphorylation and degradation of CuWRKY41, enabling its transcriptional activation of CuSPS4 directly or via CuWRKY23. Our study provides significant insights into the molecular basis of drought-induced sucrose accumulation and presents valuable regulatory components that could be targeted for fruit quality improvement.