Understanding plant breathing:

Plants constantly exchange oxygen (O₂) and carbon dioxide (CO₂) with the environment, an essential process for photosynthesis. This breathing process occurs thanks to a very specific structure: the stoma. Stomata (from Greek στόμα, "mouth") are pores found on the surface of leaves that control the gas exchange between the plant and the external atmosphere. Control of stomata aperture is essential to regulate photosynthesis and water use efficiency, and the overall plant physiology. Hence, a precise regulation of stomata dynamics is crucial to understand changes in photosynthesis and yield when plants face environmental challenges, and can have an impact in the agricultural sector.

In this work, published in Nature Communications, the group led by the CSIC researcher at the Center for Research in Agricultural Genomics (CRAG) Elena Monte, has deciphered the molecular mechanism that regulates the rhythmic movements of stomata along the day.

Stomata consist of two paired guard cells (GC) and the pore between them, the size of which depends on the swelling or shrinking of the cells, hence increasing or decreasing the stomatal aperture. Optimal stomatal apertures are determined by the integration of different environmental and internal factors. In general, closing occurs in the dark and under water stress in response to the stress hormone ABA (abscisic acid) to prevent water loss, whereas light induces stomata opening to allow CO₂ uptake and O₂ release during the day.

To fully understand the mechanisms controlling stomatal movements, CRAG researchers studied the model plant Arabidopsis thaliana in periods of controlled light and dark to monitor stomata aperture and to identify the proteins and genes involved, using molecular biology methods as well as bioinformatics analyses.

Researchers found that a family of proteins named PIFs (Phytochrome Interacting Factors) accumulate at the end of the night period, and that this is a crucial step to induce stomata opening in the morning. PIFs are transcription factors that control the expression of certain genes and, in this case, researchers determined that accumulation of PIFs triggers the expression of KAT1 gene, a guard cell-specific potassium (K⁺) channel that controls the amount of ions and, hence, the ammount of water entering these cells. In the morning, the presence of light activates KAT1 protein, triggering the intake of potassium ions and the swelling of guard cells, therefore causing stomata to open.

Nil Veciana and Arnau Rovira, co-first authors of the work, highlight the importance of the findings in an experimental set-up “with no water restrictions and, thus, endogenous ABA levels”, conditions in which there is rhythmic stomata aperture and the regulatory response to dark/light cycles can be evaluated.

Actually, this interplay between PIF function and endogenous ABA signaling in the guard cells is key for the dynamic regulation of stomata. During the night, endogenous ABA is necessary to maintain stomata closed, while in the morning, a reduction in ABA is necessary for light-induced and PIF-regulated stomata opening.

Although stomata were known to have rhythmic movements and the proteins responsible for opening-closing were already known, this is the first time that the exact transcriptional mechanism regulating stomata aperture during day/night is elucidated. Moreover, identification of PIF and KAT1 as essential players in this regulation opens up the possibility to new research avenues and possible biotechnology approaches.

Understanding how the light/dark cycle regulates stomatal aperture could be used to identify targets to optimize plant yield and plant adaptation to different stressors, for example in a water restrictive environment and drought conditions.

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Reference Article: Arnau Rovira, Nil Veciana, Aina Basté-Miquel, Martí Quevedo, Antonella Locascio, Lynne Yenush, Gabriela Toledo-Ortiz, Pablo Leivar & Elena Monte. PIF transcriptional regulators are required for rhythmic stomatal movements. Nature Communications, https://doi.org/10.1038/s41467-024-48669-4

About the authors and funding of the study: This work was supported by grants from FEDER/Ministerio de Ciencia, Innovación y Universidades – Agencia Estatal de Investigación (Project References BIO2015-68460-P, PGC2018-099987-B-I00, and PID2021-122288NB-I00 to E.M.; PID2019-10454GB-I00 to L.Y.), from the CERCA Programme/ Generalitat de Catalunya (Project Reference 2017SGR-718) to E.M, and from the Daiwa Anglo Japanese Foundation (1019/13721) and the Royal Society (IEC\R1\180047) to G.T-O. We acknowledge financial support from the Spanish Ministry of Economy and Competitiveness, through the ‘Severo Ochoa Programme for Centres of Excellence in R&D’ 2016–2019 (SEV-2015-0533) and CEX2019-000902-S funded by MCIN/AEI/ 10.13039/501100011033. A.B.-M. was supported by the predoctoral program AGAUR-FI ajuts (2023 FI-1 00910) Joan Oró of the Secretariat of Universities and Research of the Department of Research and Universities of the Generalitat of Catalonia and the European Social Plus Fund, and M.Q. received postdoctoral funding from the European Union ’s Horizon 2020 research and innovation programme under the Marie Sk łodowska-Curie grant agreement no. 945043.

About the Centre for Research in Agricultural Genomics (CRAG): CRAG is a centre that forms part of the CERCA system of research centres of the Government of Catalonia (Spain), and which was established as a partnership of four institutions: the Spanish National Research Council (CSIC), the Institute for Agri-Food Research and Technology (IRTA), the Autonomous University of Barcelona (UAB) and the University of Barcelona (UB). CRAG’s research spans from basic research in plant and farm animal molecular biology, to applications of molecular approaches for breeding of species important for agriculture and food production in close collaboration with industry. In 2020, CRAG was recognized for the second time as a "Severo Ochoa Centre of Excellence” by the Spanish Ministry of Economy and Competitiveness.

Images:

• 1_NatComms_Researchers.jpeg: Some of the authors of the study in a chamber with Arabidopsis thaliana plants. From left to right: Nil Veciana, Elena Monte, Arnau Rovira (Credit: CRAG).
• 2_NatComms_Stomata.jpg: Images of leaves of the model plant Arabidopsis thaliana with stomata mainly closed (left) and open (right) (marked with arrow heads) (Credit: CRAG).
• 3_NatComms_Infographics_ENG.jpg: Scheme summarising the main findings of the study (Credit: CRAG).

Images can be dowloaded here: https://tuit.cat/0ozfZ

For more information and interviews:

Muriel Arimon

Communication Department

Centre for Research in Agricultural Genomics (CRAG)

+34 93 563 66 00 Ext 3033

+34 600 008 159

email: muriel.arimon@cragenomica.es