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.

中文翻译：

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BIN2-VLG模块调控的大液泡的时空形成是拟南芥雌配子体发育所必需的

在拟南芥中，雌配子体（FG）的发育伴随着FG中大液泡的形成和扩张；这对于 FG 扩展、核极性定位和细胞命运决定至关重要。拟南芥无空配子体（VLG）促进囊泡融合，在 FG 中形成大液泡，但 VLG 的调控仍然很大程度上未知。在这里，我们发现油菜甾醇INSENSITIVE2 (BIN2) (bin2-1) 的功能获得性突变增加了VLG 丰度，从而诱导FG1 阶段的液泡形成，并导致FG 流产。BIN2 及其同源物 (bin2-3 bil1 bil2) 的功能丧失突变降低了 VLG 丰度并模仿了 vlg/VLG 表型。敲低 bin2-1 中的 VLG 降低了 FG1 阶段异常液泡形成的比例，而 VLG 的 FG1 特异性过表达模仿了 bin2-1 表型。VLG 部分挽救了 bin2-3 bil1 bil2 表型，表明 VLG 作用于 BIN2 下游。被 BIN2 磷酸化的 VLG 残基的突变改变了 VLG 的稳定性，而 VLG 的磷酸化模拟物会导致与 bin2-1 类似的缺陷。因此，BIN2可能通过与FG中的VLG相互作用并磷酸化VLG来发挥作用，以增强其稳定性和丰度，从而促进液泡形成。我们的研究结果为 BIN2-VLG 模块如何调节 FG 发育中大液泡的时空形成提供了机制见解。