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Stomatal opening under high temperatures is controlled by the OST1-regulated TOT3–AHA1 module

Nature Plants volume 11, pages 105–117 (2025)Cite this article

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Abstract

Plants continuously respond to changing environmental conditions to prevent damage and maintain optimal performance. To regulate gas exchange with the environment and to control abiotic stress relief, plants have pores in their leaf epidermis, called stomata. Multiple environmental signals affect the opening and closing of these stomata. High temperatures promote stomatal opening (to cool down), and drought induces stomatal closing (to prevent water loss). Coinciding stress conditions may evoke conflicting stomatal responses, but the cellular mechanisms to resolve these conflicts are unknown. Here we demonstrate that the high-temperature-associated kinase TARGET OF TEMPERATURE 3 directly controls the activity of plasma membrane H+-ATPases to induce stomatal opening. OPEN STOMATA 1, which regulates stomatal closure to prevent water loss during drought stress, directly inactivates TARGET OF TEMPERATURE 3 through phosphorylation. Taken together, this signalling axis harmonizes stomatal opening and closing under high temperatures and/or drought. In the context of global climate change, understanding how different stress signals converge on stomatal regulation allows the development of climate-change-ready crops.

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Fig. 1: TOT3 regulates stomatal aperture and conductance under high temperatures in dicot and monocot plants.
Fig. 2: TOT3 phosphorylates AHA1 to regulate stomatal aperture under high temperatures.
Fig. 3: OST1-mediated TOT3 phosphorylation regulates stomatal aperture under high temperatures.
Fig. 4: OST1-mediated phosphorylation of TOT3 affects drought tolerance and leaf temperature under drought.

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Data availability

The data are available in the supplementary materials. The mass spectrometry proteomics data are available via ProteomeXchange with the identifier PXD044300. Source data are provided with this paper.

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Acknowledgements

We thank D. Van Damme and B. De Rybel for critical comments on the manuscript. We thank E. Farmer for providing the ost2-2D seeds, M. B. Palmgren for the aha1-6 seeds and the Eurasian Arabidopsis Stock Centre for providing various seeds. This work was supported by MCIN/AEI/10.13039/501100011033 grant no. PID2020-113100RB (to P.L.R.), Graduate School Green Top Sectors grant no. GSGT.2018.007 of the Netherlands Organization for Scientific Research (to M.P. and M.v.Z.), UGent BOF postdoctoral mandate no. 01P12219 (to L.D.V.), UGent BOF doctoral mandate no. 01CD7122 (to X.X.), China Scholarship Council grant no. 201708340063 (to R.W.), China Scholarship Council grant no. 201706350153 (to X.X.), China Scholarship Council grant no. 201806170025 (to Z.J.) and China Scholarship Council grant no. 202204910025 (to H.L.).

Author information

Author notes

  1. Xiangyu Xu

    Present address: Department of Plant and Microbial Biology, University of California at Berkeley, Berkeley, CA, USA

  2. Myrthe Praat

    Present address: Green Biotechnology, Inholland University of Applied Sciences, Amsterdam, the Netherlands

  3. Lam Dai Vu

    Present address: Cryptobiotix SA, Ghent, Belgium

  4. These authors contributed equally: Hongyan Liu, Myrthe Praat.

  5. These authors jointly supervised this work: Lam Dai Vu, Ive De Smet.

Authors and Affiliations

  1. Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium

    Xiangyu Xu, Hongyan Liu, Ren Wang, Brigitte Van De Cotte, Selwyn L. Y. Villers, Hilde Nelissen, Steffen Vanneste, Lam Dai Vu & Ive De Smet

  2. VIB Center for Plant Systems Biology, Ghent, Belgium

    Xiangyu Xu, Hongyan Liu, Ren Wang, Brigitte Van De Cotte, Selwyn L. Y. Villers, Hilde Nelissen, Steffen Vanneste, Lam Dai Vu & Ive De Smet

  3. Plant Stress Resilience, Institute of Environmental Biology, Utrecht University, Utrecht, the Netherlands

    Myrthe Praat, Zhang Jiang & Martijn van Zanten

  4. Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas–Universidad Politécnica de Valencia, Valencia, Spain

    Gaston A. Pizzio & Pedro L. Rodriguez

  5. Centre for Crop Systems Analysis, Wageningen University and Research, Wageningen, the Netherlands

    Steven Michiel Driever

  6. Department of Biomolecular Medicine, Ghent University, Ghent, Belgium

    Kris Gevaert & Lam Dai Vu

  7. VIB Center for Medical Biotechnology, Ghent, Belgium

    Kris Gevaert & Lam Dai Vu

  8. Aix Marseille University, CEA, CNRS UMR7265, Bioscience and Biotechnology Institute of Aix Marseille, Saint-Paul-lez-Durance, France

    Nathalie Leonhardt

  9. Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Chikusa Nagoya, Japan

    Toshinori Kinoshita

  10. Department of Plants and Crops, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium

    Steffen Vanneste

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  1. Xiangyu Xu
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Contributions

I.D.S. and L.D.V. conceptualized the project. P.L.R., M.P., M.v.Z., L.D.V., Z.J. and X.X. acquired the funding. X.X., H.L., L.D.V., M.P., N.L., B.V.D.C., G.A.P., Z.J., R.W., S.M.D. and S.L.Y.V. conducted the investigation. I.D.S., L.D.V., K.G., M.v.Z., P.L.R., H.N. and S.V. supervised the project. T.K. provided tools for the research. X.X., L.D.V. and I.D.S. visualized the data. X.X., I.D.S. and L.D.V. wrote the original draft of the paper. X.X., M.P., Z.J., K.G., N.L., P.L.R., M.v.Z., S.M.D., S.V., H.N., L.D.V. and I.D.S. reviewed and edited the paper.

Corresponding author

Correspondence to Ive De Smet.

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Nature Plants thanks Maija Sierla and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–29, Methods, uncropped images and references.

Reporting Summary

Supplementary Data

Statistical source data for the supplementary figures.

Supplementary Table 1

List of identified phosphosites and protein of AHA1 from tobacco infiltration IP–mass spectrometry.

Supplementary Table 2

List of identified phosphosites and protein of TOT3 from tobacco infiltration IP–mass spectrometry.

Supplementary Table 3

List of identified phosphosites and proteins from GFP–TOT3 IP–mass spectrometry data in Arabidopsis exposed to high temperatures or ABA.

Supplementary Table 4

List of identified phosphosites of TOT3 from phosphoprofiling of Arabidopsis seedlings treated with progressing drought stress (our data) or mannitol.

Supplementary Table 5

Primers used in this study.

Supplementary Table 6

Plasmids generated and used in this study.

Source data

Source Data Figs. 1–4

Statistical source data.

Source Data Figs. 2 and 3

Unprocessed western blots and/or gels.

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Xu, X., Liu, H., Praat, M. et al. Stomatal opening under high temperatures is controlled by the OST1-regulated TOT3–AHA1 module. Nat. Plants 11, 105–117 (2025). https://doi.org/10.1038/s41477-024-01859-w

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