A plant-unique protein BLISTER coordinates with core retromer to modulate endosomal sorting of plasma membrane and vacuolar proteins.

The retromer is a heteromeric protein complex that localizes to endosomal membranes and drives the formation of endosomal tubules that recycle membrane protein cargoes. In plants, the retromer plays essential and canonical functions in regulating the transport of vacuolar storage proteins and the recycle of endocytosed plasma membrane proteins (PM); however, the mechanisms underlying the regulation of assembly, protein stability, and membrane recruitment of the plant retromer complex remain to be elucidated. In this study, we identify a plant-unique endosomal regulator termed BLISTER (BLI), which colocalizes and associates with the retromer complex by interacting with the retromer core subunits VPS35 and VPS29. Depletion of BLI perturbs the assembly and membrane recruitment of the retromer core VPS26-VPS35-VPS29 trimer. Consequently, depletion of BLI disrupts retromer-regulated endosomal trafficking function, including transport of soluble vacuolar proteins and recycling of endocytosed PIN-FORMED (PIN) proteins from the endosomes back to the PM. Moreover, genetic analysis in Arabidopsis thaliana mutants reveals BLI and core retromer interact genetically in the regulation of endosomal trafficking. Taken together, we identified BLI as a plant-specific endosomal regulator, which functions in retromer pathway to modulate the recycling of endocytosed PM proteins and the trafficking of soluble vacuolar cargoes.

The plant FYVE domain-containing protein FREE1 associates with microprocessor components to repress miRNA biogenesis.

FYVE domain protein required for endosomal sorting 1 (FREE1), originally identified as a plant-specific component of the endosomal sorting complex required for transport (ESCRT) machinery, plays diverse roles either in endosomal sorting in the cytoplasm or in transcriptional regulation of abscisic acid signaling in the nucleus. However, to date, a role for FREE1 or other ESCRT components in the regulation of plant miRNA biology has not been discovered. Here, we demonstrate a nuclear function of FREE1 as a cofactor in miRNA biogenesis in plants. FREE1 directly interacts with the plant core microprocessor component CPL1 in nuclear bodies and disturbs the association between HYL1, SE and CPL1. Inactivation of FREE1 in the nucleus increases the binding affinity between HYL1, SE, and CPL1 and causes a transition of HYL1 from the inactive hyperphosphorylated version to the active hypophosphorylated form, thereby promoting miRNA biogenesis. Our results suggest that FREE1 has evolved as a negative regulator of miRNA biogenesis and provides evidence for a link between FYVE domain-containing proteins and miRNA biogenesis in plants.

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.

Shedding Light on the Role of Phosphorylation in Plant Autophagy.

Autophagy is a highly conserved quality control process that maintains cellular health by eliminating deleterious cargoes. Compared with the extensive studies in yeast and mammalian models, the molecular details and significance of post-translational modifications (PTMs) in the autophagy process in plants remain less well defined. In this review, we discuss recent progress in our understanding of phosphorylation, one of the most extensively studied PTMs, in the regulation of autophagosome biogenesis and autophagic degradation in plants. Based on the plant mass spectrometric database, we summarize the experimentally verified phosphorylation sites of the core autophagy machinery in plants. Furthermore, we put forward several approaches to test the roles of phosphorylation in the regulation of plant autophagy.