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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.
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This content will become publicly available on October 15, 2026
Guard Cell-Enriched Phosphoproteome Reveals Phosphorylation of Endomembrane Proteins in Closed Stomata
ABSTRACT Control of the stomatal aperture is multifaceted, involving a complex interplay of environmental cues and intracellular signaling pathways. It is well established that changes in ion gradients drive water movement into and out of the guard cell, thereby altering cell volume and modulating the opening or closing of the stomatal pore. These rapid responses are often regulated by phosphorylation cascades to efficiently transmit environmental status and either reduce water loss or enhance carbon assimilation. The role of endomembrane trafficking networks in stomatal dynamics is not well characterized. Here, we investigated the regulation of stomatal opening and closing by generating a proteome and phosphoproteome of guard cell-enriched tissue. This deep proteome captured a protein profile that was similar to previously characterized guard cell proteomes. The guard cell-enriched tissue with closed stomata showed greater levels of phosphorylation of proteins related to endomembrane trafficking and vacuoles when compared to both whole leaf tissue with closed stomata and guard cell-enriched tissue with open stomata. These results support the hypothesis that phosphorylation of endomembrane proteins may contribute to the regulation of stomatal movements.
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- Award ID(s):
- 1918746
- PAR ID:
- 10643675
- Publisher / Repository:
- bioRxiv
- Date Published:
- Subject(s) / Keyword(s):
- Plant, vacuole, phosphoproteome, guard cell
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract BackgroundLike all plant cells, the guard cells of stomatal complexes are encased in cell walls that are composed of diverse, interacting networks of polysaccharide polymers. The properties of these cell walls underpin the dynamic deformations that occur in guard cells as they expand and contract to drive the opening and closing of the stomatal pore, the regulation of which is crucial for photosynthesis and water transport in plants. ScopeOur understanding of how cell wall mechanics are influenced by the nanoscale assembly of cell wall polymers in guard cell walls, how this architecture changes over stomatal development, maturation and ageing and how the cell walls of stomatal guard cells might be tuned to optimize stomatal responses to dynamic environmental stimuli is still in its infancy. ConclusionIn this review, we discuss advances in our ability to probe experimentally and to model the structure and dynamics of guard cell walls quantitatively across a range of plant species, highlighting new ideas and exciting opportunities for further research into these actively moving plant cells.more » « less
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SUMMARY Stomata are pores at the leaf surface that enable gas exchange and transpiration. The signaling pathways that regulate the differentiation of stomatal guard cells and the mechanisms of stomatal pore formation have been characterized inArabidopsis thaliana. However, the process by which stomatal complexes develop after pore formation into fully mature complexes is poorly understood. We tracked the morphogenesis of young stomatal complexes over time to establish characteristic geometric milestones along the path of stomatal maturation. Using 3D‐nanoindentation coupled with finite element modeling of young and mature stomata, we found that despite having thicker cell walls than young guard cells, mature guard cells are more energy efficient with respect to stomatal opening, potentially attributable to the increased mechanical anisotropy of their cell walls and smaller changes in turgor pressure between the closed and open states. Comparing geometric changes in young and mature guard cells of wild‐type and cellulose‐deficient plants revealed that although cellulose is required for normal stomatal maturation, mechanical anisotropy appears to be achieved by the collective influence of cellulose and additional wall components. Together, these data elucidate the dynamic geometric and biomechanical mechanisms underlying the development process of stomatal maturation.more » « less
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Abstract Phosphorylation of the penultimate residue, threonine (pen-Thr), of plasma membrane (PM) H+-ATPase is essential for its activation and blue light (BL)-induced stomatal opening. However, the regulatory mechanism of action of PM H+-ATPase pen-Thr phosphorylation is not completely understood. Here, we performed screening using a protein kinase inhibitor library and found that tyrphostin AG126 inhibited phosphorylation of PM H+-ATPase pen-Thr in guard cells in response to light and fungal toxin fusicoccin (FC), in addition to inhibition of light- and FC-induced stomatal opening. Analysis of the structure–activity relationship using AG126 derivatives revealed the hydroxyl group at the C-5 position of the compound to be essential for its activity. We further characterized one AG126 derivative, AGD-1, which effectively suppressed BL-induced stomatal opening with a half-inhibitory concentration of 2.0 μM. AGD-1 inhibited PM H+-ATPase pen-Thr phosphorylation in guard cells in response to BL and FC. In addition, AGD-1 suppressed FC-induced PM H+-ATPase pen-Thr phosphorylation in mesophyll cell protoplasts, implying that the effect of AGD-1 is not specific to guard cells. Furthermore, to improve the permeability of AGD-1, we synthesized acetylated AGD-1 (AcAGD-1), which was found to suppress BL- and FC-induced stomatal opening. AcAGD-1 suppressed light-induced PM H+-ATPase pen-Thr phosphorylation, but not Thr881 phosphorylation, in leaf discs, which is important for guard cell PM H+-ATPase activation in addition to pen-Thr phosphorylation. This study identified a novel stomatal opening inhibitor capable of specifically inhibiting PM H+-ATPase pen-Thr phosphorylation.more » « less
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{"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."]}more » « less
