Positive feedback loop between RAF12 and ABI5 reinforces ABA-mediated suppression of Arabidopsis seed germination.

Abstract

ABA-INSENSITIVE 5 (ABI5) is a key transcriptional regulator mediating abscisic acid (ABA)-induced suppression of seed germination. However, the downstream regulatory network through which ABI5 exerts its function remains incompletely understood. Here, by integrating ChIP-seq and RNA-seq analyses, we identify RAF12, a member of the B2 Raf-like kinase subfamily, as a direct transcriptional target of ABI5. ABI5 binds to the RAF12 promoter and activates its expression. Loss-of-function raf12 mutants exhibit reduced sensitivity to ABA during seed germination, suggesting a negative regulatory role for RAF12 in this process. Conversely, RAF12 interacts with and phosphorylates ABI5, thereby enhancing its transcriptional activity. Further analysis showed that RAF12 regulates its own kinase activity through autophosphorylation. Mutations at its phosphorylation sites significantly weaken its ability to enhance ABI5's transcriptional activity. Together, these findings uncover a positive feedback loop wherein ABI5 transcriptionally activates RAF12, which in turn reinforces ABI5 activity through phosphorylation. This module may function in parallel with the canonical SnRK2s-ABI5-mediated ABA signaling cascade, offering new mechanistic insights into the fine-tuning of ABA responses during seed germination.