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Regulation of vacuole fusion in stomata by dephosphorylation of the HOPS subunit VPS39

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Pullen, Anne‐Marie, Billings, Grant, Hodgens, Charles, White, Gisele, Akpa, Belinda S., Rojas‐Pierce, Marcela·2025·The Plant Journal
DOI 10.1111/tpj.70640PMC PMC12716901

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Abstract

SUMMARY: 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 CO 2 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.

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Figure 1 The HOPS subunit VPS39 regulates vacuole fusion in Arabidopsis. (A) Map of VPS39 with the location of T‐DNA insertions in vps39 alleles. Exons are represented in blocks, and green represents CDS. T‐DNA is located 2 bp upstream of the translation start site in vps39‐2. (B) Relative gene expression levels of VPS39 in 14‐day‐old seedlings by RT‐qPCR. Data are presented as relative normalized expression ±95% confidence interval from n = 30 seedlings per genotype. Gene expression was normalized to PEX4 (UBC21) and expressed relative to WT. Significant differences by one‐way ANOVA are indicated; P = 6.73 × 10−7 (***). (C) Mild vacuole fragmentation phenotype in vps39‐2 roots. Asterisk represents the location of root hair primordia, and a cell with fragmented vacuoles is highlighted in red. The number of seedlings with at least one cell with fragmented vacuoles is shown. Roots were stained with BCECF. Scale bar: 50 μm. (D) Vacuole phenotype of vps39‐2 stomata under opening conditions. Leaf epidermal peels of WT and vps39‐2 were exposed to stomatal opening conditions and stained with BCECF. Images of WT and vps39‐2 stomata are shown, with vps39‐2 exhibiting variable vacuole numbers. (E) Vacuole quantification from stomatal opening assays as in (D). A violin plot shows the distribution of the data along with a box and whisker plot with the median noted as a white dot and outliers as black diamonds. Statistical analysis was performed using a generalized linear mixed model comparing vacuoles per guard cell. The model containing genotype was favored (P = 0.01). n = 97–139 guard cells per genotype from 5 to 8 plants per genotype. (F) RT‐qPCR relative normalized expression of VPS39 in guard cell‐enriched tissue from 5‐week‐old WT or vps39‐2 plants. n = 12 plants per genotype. Data are represented as relative normalized expression ±95% confidence interval. Gene expression was normalized to PEX4. Statistical analysis was performed using the Mann–Whitney U test; P = 4.66 × 10−5 (***).
Figure 1 The HOPS subunit VPS39 regulates vacuole fusion in Arabidopsis. (A) Map of VPS39 with the location of T‐DNA insertions in vps39 alleles. Exons are represented in blocks, and green represents CDS. T‐DNA is located 2 bp upstream of the translation start site in vps39‐2. (B) Relative gene expression levels of VPS39 in 14‐day‐old seedlings by RT‐qPCR. Data are presented as relative normalized expression ±95% confidence interval from n = 30 seedlings per genotype. Gene expression was normalized to PEX4 (UBC21) and expressed relative to WT. Significant differences by one‐way ANOVA are indicated; P = 6.73 × 10−7 (***). (C) Mild vacuole fragmentation phenotype in vps39‐2 roots. Asterisk represents the location of root hair primordia, and a cell with fragmented vacuoles is highlighted in red. The number of seedlings with at least one cell with fragmented vacuoles is shown. Roots were stained with BCECF. Scale bar: 50 μm. (D) Vacuole phenotype of vps39‐2 stomata under opening conditions. Leaf epidermal peels of WT and vps39‐2 were exposed to stomatal opening conditions and stained with BCECF. Images of WT and vps39‐2 stomata are shown, with vps39‐2 exhibiting variable vacuole numbers. (E) Vacuole quantification from stomatal opening assays as in (D). A violin plot shows the distribution of the data along with a box and whisker plot with the median noted as a white dot and outliers as black diamonds. Statistical analysis was performed using a generalized linear mixed model comparing vacuoles per guard cell. The model containing genotype was favored (P = 0.01). n = 97–139 guard cells per genotype from 5 to 8 plants per genotype. (F) RT‐qPCR relative normalized expression of VPS39 in guard cell‐enriched tissue from 5‐week‐old WT or vps39‐2 plants. n = 12 plants per genotype. Data are represented as relative normalized expression ±95% confidence interval. Gene expression was normalized to PEX4. Statistical analysis was performed using the Mann–Whitney U test; P = 4.66 × 10−5 (***).CC BY · PubMed Central
Figure 2 VPS39‐GFP phosphorylation mutants do not display altered protein localization. (A) Predicted Arabidopsis VPS39 structure from Alphafold 2.0. (B) Domain structure of VPS39. A Conserved Citron Homology (CNH, blue), a VPS39/TGFβR association domain 1 (orange) and VPS39‐associated zinc finger (red) are predicted by Uniprot. Two predicted intrinsically disordered regions (green) are indicated as predicted by MobiDB. The positions of S406, S413, S806 (pink) used for phosphomutant analysis are indicated with arrowheads. (C) Localization of intact or phosphomutant versions of VPS39‐GFP in the roots of complemented vps39‐3, WT, or vps39‐2. Images are representative from 16 to 23 seedlings per genotype. Scale bar corresponds to 5 μm.
Figure 2 VPS39‐GFP phosphorylation mutants do not display altered protein localization. (A) Predicted Arabidopsis VPS39 structure from Alphafold 2.0. (B) Domain structure of VPS39. A Conserved Citron Homology (CNH, blue), a VPS39/TGFβR association domain 1 (orange) and VPS39‐associated zinc finger (red) are predicted by Uniprot. Two predicted intrinsically disordered regions (green) are indicated as predicted by MobiDB. The positions of S406, S413, S806 (pink) used for phosphomutant analysis are indicated with arrowheads. (C) Localization of intact or phosphomutant versions of VPS39‐GFP in the roots of complemented vps39‐3, WT, or vps39‐2. Images are representative from 16 to 23 seedlings per genotype. Scale bar corresponds to 5 μm.CC BY · PubMed Central
Figure 3 Phosphomutant versions of VPS39‐GFP at S406, 413, and 806 are not functional. (A) Embryo abortion phenotype in siliques from WT, complemented vps39‐3 VPS39‐GFP, vps39‐1/+ heterozygotes, and vps39‐1/+ carrying the VPS39SD‐GFP or VPS39SA‐GFP constructs. Arrows denote aborted embryos. Arrowheads denote unfertilized ovules or very early aborted embryos. Scale bar: 1 mm. (B) Quantification of aborted embryos from (A). Statistical analysis was performed with a Kruskal–Wallis test followed by post‐hoc Dunn's test with Šidák correction. Significant differences are shown using compact letter display (P < 0.0051). n = 30–42 siliques/genotype from 6 to 11 plants/genotype. (C) Quantification of the apparent unfertilized ovules of phosphomutant lines from (A). Kruskal–Wallis followed by post‐hoc Dunn's test with Šidák correction for multiple comparisons P < 0.0051. n = 30–42 siliques per genotype from 6 to 11 plants per genotype. (D) Vacuole fusion phenotypes of vps39‐2 mutants expressing phosphomutant VPS39‐GFP in stomata. Epidermal peels from WT, vps39‐2, and vps39‐2 expressing VPS39SD‐GFP or VPS39SA‐GFP were exposed to opening conditions for 2 h, stained with BCECF, and imaged by confocal microscopy. Examples of highly fragmented vacuoles of phosphomutant lines are shown. Scale bar: 5 μm. (E) Quantification of the number of vacuoles per guard cell as seen in (D). Statistical analysis was performed using a generalized linear mixed model comparing vacuoles per guard cell followed by bootstrap analysis. Significant difference found in the bootstrap analysis is denoted by compact letter display. The model containing genotype was favored (P = 1.30 × 10−7). n = 97–436 guard cells from 9 to 26 plants per genotype. This model includes stomata data from Figure 1E. Data distributions in (B), (C), and (E) are plotted as violin plots along with box and whisker plots with the median noted as a white dot and outliers as black dots.
Figure 3 Phosphomutant versions of VPS39‐GFP at S406, 413, and 806 are not functional. (A) Embryo abortion phenotype in siliques from WT, complemented vps39‐3 VPS39‐GFP, vps39‐1/+ heterozygotes, and vps39‐1/+ carrying the VPS39SD‐GFP or VPS39SA‐GFP constructs. Arrows denote aborted embryos. Arrowheads denote unfertilized ovules or very early aborted embryos. Scale bar: 1 mm. (B) Quantification of aborted embryos from (A). Statistical analysis was performed with a Kruskal–Wallis test followed by post‐hoc Dunn's test with Šidák correction. Significant differences are shown using compact letter display (P < 0.0051). n = 30–42 siliques/genotype from 6 to 11 plants/genotype. (C) Quantification of the apparent unfertilized ovules of phosphomutant lines from (A). Kruskal–Wallis followed by post‐hoc Dunn's test with Šidák correction for multiple comparisons P < 0.0051. n = 30–42 siliques per genotype from 6 to 11 plants per genotype. (D) Vacuole fusion phenotypes of vps39‐2 mutants expressing phosphomutant VPS39‐GFP in stomata. Epidermal peels from WT, vps39‐2, and vps39‐2 expressing VPS39SD‐GFP or VPS39SA‐GFP were exposed to opening conditions for 2 h, stained with BCECF, and imaged by confocal microscopy. Examples of highly fragmented vacuoles of phosphomutant lines are shown. Scale bar: 5 μm. (E) Quantification of the number of vacuoles per guard cell as seen in (D). Statistical analysis was performed using a generalized linear mixed model comparing vacuoles per guard cell followed by bootstrap analysis. Significant difference found in the bootstrap analysis is denoted by compact letter display. The model containing genotype was favored (P = 1.30 × 10−7). n = 97–436 guard cells from 9 to 26 plants per genotype. This model includes stomata data from Figure 1E. Data distributions in (B), (C), and (E) are plotted as violin plots along with box and whisker plots with the median noted as a white dot and outliers as black dots.CC BY · PubMed Central
Figure 4 Phosphoproteomics analysis of guard cell‐enriched tissue reveals VPS39 phosphorylation changes between open and closed stomata. (A) Depiction of HOPS‐specific (green), CORVET‐specific (purple), and shared core (gray) subunits detected in the proteome (▲) or the phosphoproteome (♦) from guard cell‐enriched tissue. (B) Lollipop representation of phosphorylation sites mapped onto the VPS39 and VPS41 primary sequences. Each lollipop denotes a modified residue. The X‐axis represents the residue position along each protein sequence, and the Y‐axis represents Log2FC of phosphorylated residues between closed and open samples. Increased phosphorylation was detected in the closed condition for both proteins (Log2FC >0.5), but only S413 on VPS39 was significant (*, Absolute Log2FC >0.5 and P < 0.05).
Figure 4 Phosphoproteomics analysis of guard cell‐enriched tissue reveals VPS39 phosphorylation changes between open and closed stomata. (A) Depiction of HOPS‐specific (green), CORVET‐specific (purple), and shared core (gray) subunits detected in the proteome (▲) or the phosphoproteome (♦) from guard cell‐enriched tissue. (B) Lollipop representation of phosphorylation sites mapped onto the VPS39 and VPS41 primary sequences. Each lollipop denotes a modified residue. The X‐axis represents the residue position along each protein sequence, and the Y‐axis represents Log2FC of phosphorylated residues between closed and open samples. Increased phosphorylation was detected in the closed condition for both proteins (Log2FC >0.5), but only S413 on VPS39 was significant (*, Absolute Log2FC >0.5 and P < 0.05).CC BY · PubMed Central
Figure 5 Proposed model for regulation of stomatal vacuole fusion by phosphorylation of HOPS. We predict that the HOPS subunit VPS39 accumulates in its phosphorylated form in closed stomata, which may promote HOPS association with the vacuolar SNAREs at the tonoplast. Dissociation of HOPS is necessary to complete fusion, and this could be triggered by VPS39 dephosphorylation in response to stomatal opening signals such as blue light. Kinases and phosphatases involved in this process are unknown. Image generated in BioRender (Biorender.com).
Figure 5 Proposed model for regulation of stomatal vacuole fusion by phosphorylation of HOPS. We predict that the HOPS subunit VPS39 accumulates in its phosphorylated form in closed stomata, which may promote HOPS association with the vacuolar SNAREs at the tonoplast. Dissociation of HOPS is necessary to complete fusion, and this could be triggered by VPS39 dephosphorylation in response to stomatal opening signals such as blue light. Kinases and phosphatases involved in this process are unknown. Image generated in BioRender (Biorender.com).CC BY · PubMed Central

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Metadata

Authors
Pullen, A., Billings, G., Hodgens, C., White, G., Akpa, B. S., & Rojas‐Pierce, M.
Affiliations
  • North Carolina State University (US)
  • University of Tennessee at Knoxville (US)
  • University of Tennessee System (US)
Corresponding author
Rojas‐Pierce, Marcela
ORCID
Venue
The Plant Journal
Publisher
Wiley
Published
2025-12
Volume
124
Issue
6
Pages
e70640-e70640
ISSN
  • 0960-7412 (ISSN-L)
  • 1365-313X
DOI
10.1111/tpj.70640
PMC
PMC12716901
OpenAlex
W4417514813
Cited by
1
References
4
Citations by year
2026: 1
FWCI
0.50
Type
journal article
Language
en
Open access
Licence
Locations
  • The Plant Journal (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
Funders
  • U.S. Department of Agriculture (Non‐Assistance Cooperative Agreement 58‐6066‐0‐058)
  • National Institute of Food and Agriculture (HATCH 7005574)
  • National Science Foundation (MCB‐1918746)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Molecular Biology Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Photosynthetic Processes and Mechanisms
Keywords
HOPS complex, VPS39, VPS41, phosphorylation, trans-SNARE complex, stomatal opening, water vapor loss, post-translational modification
MeSH terms
Membrane Fusion, Phosphorylation, Vacuoles, Arabidopsis, Arabidopsis Proteins, Vesicular Transport Proteins, SNARE Proteins, Plant Stomata

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