Spatiotemporal formation of the large vacuole regulated by the BIN2-VLG module is required for female gametophyte development in Arabidopsis

In Arabidopsis thaliana, female gametophyte (FG) development is accompanied by the formation and expansion of the large vacuole in the FG; this is essential for FG expansion, nuclear polar localization, and cell fate determination. Arabidopsis VACUOLELESS GAMETOPHYTES (VLG) facilitates vesicular fusion to form large vacuole in the FG, but the regulation of VLG remains largely unknown. Here, we found that gain-of-function mutation of BRASSINOSTEROID INSENSITIVE2 (BIN2) (bin2-1) increases VLG abundance to induce the vacuole formation at stage FG1, and leads to abortion of FG. Loss-of-function mutation of BIN2 and its homologs (bin2-3 bil1 bil2) reduced VLG abundance and mimicked vlg/VLG phenotypes. Knocking down VLG in bin2-1 decreased the ratio of aberrant vacuole formation at stage FG1, whereas FG1-specific overexpression of VLG mimicked the bin2-1 phenotype. VLG partially rescued the bin2-3 bil1 bil2 phenotype, demonstrating that VLG acts downstream of BIN2. Mutation of VLG residues that are phosphorylated by BIN2 altered VLG stability and a phosphorylation mimic of VLG causes similar defects as did bin2-1. Therefore, BIN2 may function by interacting with and phosphorylating VLG in the FG to enhance its stability and abundance, thus facilitating vacuole formation. Our findings provide mechanistic insight into how the BIN2-VLG module regulates the spatiotemporal formation of the large vacuole in FG development.