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SnRK2 and MLKs orchestrate FREE1 nuclear retention through phosphorylation to regulate ABA signaling in Arabidopsis
Zien Li1, Fanfan Qiao1, Fei Rao1
1Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, MOE Key Laboratory & Guangdong Provincial Key Laboratory of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China.
Abstract:
Previous studies have shown that SNF1-related protein kinase 2 (SnRK2s) phosphorylate FREE1 (FYVE domain protein required for endosomal sorting 1) at serine residue S530S533 and MUT9-like kinase (MLKs) phosphorylate FREE1 at serine residue S582 in response to ABA signaling. This study aims to elucidate the synergistic regulation of FREE1 protein phosphorylation and ABA signaling by SnRK2s and MLKs kinases. Using bimolecular fluorescence complementation (BiFC) and transient expression, we found that SnRK2s associate with MLK4 and show significant co-localization in the nucleus. The enhanced ABA sensitivity observed in the snrk2.2/2.3/mlk134 quintuple mutant relative to the snrk2.2/2.3 background points to a genetic interaction where MLKs are epistatic to SnRK2s, revealing a hierarchical relationship in this regulatory pathway. Furthermore, MLKs phosphorylated FREE1 in the snrk2.2/2.3 mutant indicated that SnRK2s and MLKs regulate FREE1 phosphorylation independently. Additionally, compared with overexpressing FREE1(S530D/S533D/S582A), overexpression of the FREE1(S530A/S533A/S582D) variant resulted in enhanced nuclear localization and rescued the ABA hyposensitivity in free1-ctmut and mlks mutant backgrounds. Together, our findings unveil a novel regulatory layer of ABA signaling and establish a framework for understanding the synergistic action of SnRK2 and MLK kinases.
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