More Like this

1. Model-based inference of a dual role for HOPS in regulating guard cell vacuole fusion

   https://doi.org/10.1093/insilicoplants/diae015  

   Hodgens, Charles; Flaherty, D T; Pullen, Anne-Marie; Khan, Imran; English, Nolan J; Gillan, Lydia; Rojas-Pierce, Marcela; Akpa, Belinda S (August 2024, in silico Plants)

   Zhu, Xin-Guang (Ed.)

   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. 

   more » « less
2. Data from: Regulation of vacuole fusion in stomata by dephosphorylation of the HOPS subunit VPS39

   https://doi.org/10.5061/dryad.905qftv05  

   Pullen, Anne-Marie; Billings, Grant; White, Giselle; Hodgens, Charles; Akpa, Belinda; Rojas-Pierce, Marcela (January 2025, Dryad)

   {"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
3. Guard Cell-Enriched Phosphoproteome Reveals Phosphorylation of Endomembrane Proteins in Closed Stomata

   https://doi.org/10.1101/2025.10.15.682613  

   Pullen, Anne-Marie; Lyons, Scott; Mordant, Angie; Herring, Laura; Akpa, Belinda S; Rojas-Pierce, Marcela (October 2025, bioRxiv)

   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. 

   more » « less
4. Young guard cells function dynamically despite low mechanical anisotropy but gain efficiency during stomatal maturation in Arabidopsis thaliana

   https://doi.org/10.1111/tpj.16756  

   Jaafar, Leila; Chen, Yintong; Keynia, Sedighe; Turner, Joseph_A; Anderson, Charles_T (April 2024, The Plant Journal)

   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
5. VPS45 is required for both diffuse and tip growth of Arabidopsis thaliana cells

   https://doi.org/10.3389/fpls.2023.1120307  

   Mugume, Yosia; Roy, Rahul; Agbemafle, William; Shepard, Gabriella N.; Vue, Yee; Bassham, Diane C. (February 2023, Frontiers in Plant Science)

   IntroductionVPS45 belongs to the Sec1/Munc18 family of proteins, which interact with and regulate Qa-SNARE function during membrane fusion. We have shown previously thatArabidopsis thalianaVPS45 interacts with the SYP61/SYP41/VTI12 SNARE complex, which locates on thetrans-Golgi network (TGN). It is required for SYP41 stability, and it functions in cargo trafficking to the vacuole and in cell expansion. It is also required for correct auxin distribution during gravitropism and lateral root growth. ResultsAsvps45knockout mutation is lethal in Arabidopsis, we identified a mutant,vps45-3, with a point mutation in theVPS45gene causing a serine 284-to-phenylalanine substitution. The VPS45-3 protein is stable and maintains interaction with SYP61 and SYP41. However,vps45-3plants display severe growth defects with significantly reduced organ and cell size, similar tovps45RNAi transgenic lines that have reduced VPS45 protein levels. Root hair and pollen tube elongation, both processes of tip growth, are highly compromised invps45-3. Mutant root hairs are shorter and thicker than those of wild-type plants, and are wavy. These root hairs have vacuolar defects, containing many small vacuoles, compared with WT root hairs with a single large vacuole occupying much of the cell volume. Pollen tubes were also significantly shorter invps45-3compared to WT. DiscussionWe thus show that VPS45 is essential for proper tip growth and propose that the observed vacuolar defects lead to loss of the turgor pressure needed for tip growth. 

   more » « less