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ABSTRACT Understanding how plants regulate water loss is important for improving crop productivity. Tight control of stomatal opening and closing is essential for the uptake of CO₂while mitigating water vapor loss. The opening of stomata is regulated in part by homotypic vacuole fusion, which is mediated by conservedhomotypic vacuoleproteinsorting (HOPS) and vacuolar SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) complexes. HOPS tethers apposing vacuole membranes and promotes the formation oftrans-SNARE complexes to mediate fusion. In yeast, HOPS dissociates from the assembled SNARE complex to complete vacuole fusion, but little is known about this process in plants. HOPS-specific subunits VACUOLE PROTEIN SORTING39 (VPS39) and VPS41 are required for homotypic plant vacuole fusion, and a computational model predicted that post-translational modifications of HOPS may be needed for plant stomatal vacuole fusion. Here, we characterized a viable T-DNA insertion allele ofVPS39which demonstrated a critical role of VPS39 in stomatal vacuole fusion. We found that VPS39 has increased levels of phosphorylation when stomata are closed versus open, and that VPS39 function in stomata and embryonic development requires dynamic changes in phosphorylation. Our data are consistent with VPS39 phosphorylation altering vacuole dynamics in response to environmental cues, similar to well-established phosphorylation cascades that regulate ion transport during stomatal opening. SIGNIFICANCE STATEMENTVacuole fusion is important for stomata opening but how it is regulated in response of stomata opening signals is not characterized. This research demonstrated the role of the HOPS complex in vacuole fusion in stomata, and it identified phosphorylation sites in the HOPS subunit VPS39 that are critical for vacuole fusion. One Ser residue was enriched in closed stomata and represents a putative site for control of vacuole fusion downstream of stomata opening signals. 

{"Abstract":["Understanding how plants regulate water loss is important for improving\n crop productivity. Tight control of stomatal opening and closing is\n essential for the uptake of CO2 while mitigating water vapor loss. The\n opening of stomata is regulated in part by homotypic vacuole fusion, which\n is mediated by conserved homotypic vacuole protein sorting (HOPS) and\n vacuolar SNARE (soluble N-ethylmaleimide sensitive factor attachment\n protein receptors) complexes. HOPS tethers apposing vacuole membranes and\n promotes the formation of trans-SNARE complexes to mediate fusion. In\n yeast, HOPS dissociates from the assembled SNARE complex to complete\n vacuole fusion, but little is known about this process in plants.\n HOPS-specific subunits VACUOLE PROTEIN SORTING39 (VPS39) and VPS41 are\n required for homotypic plant vacuole fusion, and a computational model\n predicted that post-translational modifications of HOPS may be needed for\n plant stomatal vacuole fusion. Here, we characterized a viable T-DNA\n insertion allele of VPS39 which demonstrated a critical role of VPS39 in\n stomatal vacuole fusion. We found that VPS39 has increased levels of\n phosphorylation at S413 when stomata are closed versus open, and that\n VPS39 function in stomata and embryonic development requires dynamic\n changes in phosphorylation. Among all HOPS and vacuolar SNARE subunits,\n only VPS39 showed differential levels of phosphorylation between open and\n closed stomata. Moreover, regions containing S413 are not conserved\n between plants and other organisms, suggesting plant-specific mechanisms.\n  Our data are consistent with VPS39 phosphorylation altering\n vacuole dynamics in response to environmental cues, similar to\n well-established phosphorylation cascades that regulate ion transport\n during stomatal opening."],"TechnicalInfo":["# Data from: Regulation of vacuole fusion in stomata by dephosphorylation\n of the HOPS subunit VPS39 --- The methods for this dataset are described\n in detail in our manuscript. These compressed files contain: Raw images\n (.czi) for vacuoles from roots (Root_vacuole_data.zip) used for Figure 1C.\n Raw images (.czi) for stomata vacuoles (Stomata_Vacuole_Data.zip) used for\n Figure 1D-E and Figure 3D-E. Images (.jpg) of siliques used for\n quantification of Figure 3A-C (Siliques_Data.tar). Genotypes associated\n with each plant number on each slide are listed in an Excel file. qRT-PCR\n data (.xlsx) from seedlings corresponding to Figure 1B\n (Seedling_qRT_PCR_vps39-2.xlsx). qRT-PCR data (.xlsx) from guard\n cell-enriched tissue corresponding to Figure 1F\n (Guard_Cell_enriched_RT_qPCR.xlsx). ## Description of the data and file\n structure ### **Root Vacuole image data files** This includes confocal raw\n image files captured with a Zeiss LSM980 with Airy scan microscope. Images\n are organized in folders by date of image acquisition (set 1 to set 6).\n Within each set, images are organized by genotype (WT,\n *vps39-2* or *vps39-2* VPS39-RFP/+). Each image includes green channel for\n BCECF fluorescence detection and red channel for VPS39-RFP detection. ###\n Stomata vacuole image data files This includes confocal raw image files\n captured with a Zeiss LSM980 with an Airy scan microscope. Data is\n organized in folders based on data of image acquisition. Each folder is\n subdivided by genotype: wild type (WT), *vps39-2*\n mutant, or *vps39-2* mutant complemented with VPS39-S-A-GFP (v*ps39-2*\n VPS39-S-A-GFP) or VPS39-S-D-GFP (v*ps39-2* VPS39-S-A-GFP). Within each\n genotype, images are sorted by box numbers, where each box corresponds to\n a leaf fragment from a different plant. ### **Silique image data** This\n contains all the images from siliques as captured with a Leica Thunder for\n Model Organisms dissecting scope. Images are organized in folders by date\n of data acquisition. Within each date, data is sorted by genotype. Within\n each genotype, each image includes multiple siliques from 1 or more\n plants. Each silique is marked with a genotype number as part of the\n image. An Excel sheet is included to match a plant number to a specific\n plant genotype for each image. ### **qRT-PCR files** These files contain\n raw data from gene expression studies. **Date (when included):** Date when\n qRT-PCR run was performed. **Well:** The plate position of the reaction on\n the qRT-PCR plate. **Fluor:** The fluorescence channel used for detection\n (SYBR GREEN was always used). **Target:** Specific gene transcript\n amplified for that reaction. **Content:** The reaction type as designated\n in the run file (e.g., Unknown sample, Standard, NTC, etc.). This labels\n the functional role of the well in the experiment, including NTC (no DNA\n Template Control) or NRT (no RT reaction) controls. This column was not\n specified for wells containing samples, and therefore, these were marked\n as "Unkn" by the qPCR machine. All other empty wells (not used)\n are marked as "Unkn". **Sample:** The sample ID corresponding to\n the biological sample loaded in the well. Genotypes used were either wild\n type (WT) or vps39-2 mutant (39). For each of these, biological replicates\n are indicated as A, B, C, and D, and technical replicates with numbers\n (1-4).  **Cq:** The quantification cycle (Ct) value is automatically\n calculated by the instrument. Empty cells indicate that no Ct value was\n generated due to an unused well. "N/A" indicates that no\n fluorescence was detected, and these cells correspond to non-template\n controls. Other cells left blank correspond to wells intentionally not\n used in the plate layout (no-template, no-primer, or unassigned wells).\n These were left blank because the instrument does not output data for\n unused wells."]} 

Abstract Guard cell movements depend, in part, on the remodelling of vacuoles from a highly fragmented state to a fused morphology during stomata opening. Indeed, full opening of plant stomata requires vacuole fusion to occur. Fusion of vacuole membranes is a highly conserved process in eukaryotes, with key roles played by two multi-subunit complexes: HOPS (homotypic fusion and vacuolar protein sorting) and SNARE (soluble NSF attachment protein receptor). HOPS is a vacuole tethering factor that is thought to chaperone SNAREs from apposing vacuole membranes into a fusion-competent complex capable of rearranging membranes. In plants, recruitment of HOPS subunits to the tonoplast has been shown to require the presence of the phosphoinositide phosphatidylinositol 3-phosphate. However, chemically depleting this lipid induces vacuole fusion. To resolve this counter-intuitive observation regarding the role of HOPS in regulating plant vacuole morphology, we defined a quantitative model of vacuole fusion dynamics and used it to generate testable predictions about HOPS-SNARE interactions. We derived our model by using simulation-based inference to integrate prior knowledge about molecular interactions with limited, qualitative observations of emergent vacuole phenotypes. By constraining the model parameters to yield the emergent outcomes observed for stoma opening—as induced by two distinct chemical treatments—we predicted a dual role for HOPS and identified a stalled form of the SNARE complex that differs from phenomena reported in yeast. We predict that HOPS has contradictory actions at different points in the fusion signalling pathway, promoting the formation of SNARE complexes, but limiting their activity.