DOI: 10.5061/dryad.905qftv05
title: Data from: Regulation of vacuole fusion in stomata by dephosphorylation of the HOPS subunit VPS39

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