MLKs kinases phosphorylate the ESCRT component FREE1 to suppress abscisic acid sensitivity of seedling establishment

The FYVE domain protein required for endosomal sorting 1 (FREE1), which was previously identified as a plant-specific component of the endosomal sorting complex required for transport machinery, plays an essential role in endosomal trafficking. Moreover, FREE1 also functions as an important negative regulator in abscisic acid (ABA) signalling. Multiple phosphorylations and ubiquitination sites have been identified in FREE1, hence unveiling the factors involved in posttranslational regulation of FREE1 is critical for comprehensively understanding FREE1-related regulatory networks during plant growth. Here, we demonstrate that plant-specific casein kinase I members MUT9-like kinases 1-4 (MLKs 1-4)/Arabidopsis EL1-like 1-4 interact with and phosphorylate FREE1 at serine residue S582, thereby modulating the nuclear accumulation of FREE1. Consequently, mutation of S582 to non-phosphorylable residue results in reduced nuclear localization of FREE1 and enhanced ABA response. In addition, mlk123 and mlk134 triple mutants accumulate less FREE1 in the nucleus and display hypersensitive responses to ABA treatment, whereas overexpression of the nuclear-localized FREE1 can restore the ABA sensitivity of seedling establishment in mlks triple mutants. Collectively, our study demonstrates a previously unidentified function of MLKs in attenuating ABA signalling in the nucleus by regulating the phosphorylation and nuclear accumulation of FREE1.