Abstract: Abscisic acid (ABA) promotes sugar accumulation during the ripening and quality formation of non‐climacteric fruits; however, the underlying molecular mechanisms remain incompletely understood. Here, we identified that CsSWEET16, a plasma membrane‐localized transporter, was upregulated during the late developmental stages of citrus fruit and contributes to enhanced sugar accumulation in juice sacs under ABA regulation. The regulatory mechanism revealed that two transcription factors in the ABA signaling pathway, CsABF3 and CsABI5, synergistically activated CsSWEET16 expression. Conversely, during the early stage of fruit development, the transcription of CsSWEET16 was repressed by the transcription factor CsMYB35, which was dephosphorylated and stabilized by phosphatase CsPP2C58. However, during fruit ripening and with the increase in endogenous ABA levels, the expression of CsPP2C58 was downregulated, whereas CsSnRK2.6 phosphorylated CsMYB35, resulting in its degradation and thereby alleviating the suppression on CsSWEET16 transcription. Two cooperation pathways, phosphorylation dynamics for CsMYB35 stability and CsABF3/CsABI5 transcriptional activity, were elucidated through which ABA signaling enhances CsSWEET16 expression and promotes sugar accumulation in citrus. These findings not only expand our understanding of novel cooperative regulatory mechanisms within the ABA signaling network but also offer valuable insights for optimizing the sweetness and taste quality of citrus and other non‐climacteric fruits.

Key words: abscisic acid, Citrus sinensis, phosphorylation, sugar transporter, transcriptional regulation