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An Arabidopsis mutant displaying impaired stomatal responses to CO 2 , cdi4, was isolated by a leaf thermal imaging screening. The mutated gene PECT1 encodes CTP:phosphorylethanolamine cytidylyltransferase. The cdi4 exhibited a decrease in phosphatidylethanolamine levels and a defect in light-induced stomatal opening as well as low-CO 2 -induced stomatal opening. We created RNAi lines in which PECT1 was specifically repressed in guard cells. These lines are impaired in their stomatal responses to low-CO 2 concentrations or light. Fungal toxin fusicoccin (FC) promotes stomatal opening by activating plasma membrane H + -ATPases in guard cells via phosphorylation. Arabidopsis H + -ATPase1 (AHA1) has been reported to be highly expressed in guard cells, and its activation by FC induces stomatal opening. The cdi4 and PECT1 RNAi lines displayed a reduced stomatal opening response to FC. However, similar to in the wild-type, cdi4 maintained normal levels of phosphorylation and activation of the stomatal H + -ATPases after FC treatment. Furthermore, the cdi4 displayed normal localization of GFP-AHA1 fusion protein and normal levels of AHA1 transcripts. Based on these results, we discuss how PECT1 could regulate CO 2 - and light-induced stomatal movements in guard cells in a manner that is independent and downstream of the activation of H + -ATPases. [Correction added on 15 May 2023, after first online publication: The third sentence is revised in this version.]., (© 2023 Society for Experimental Biology and John Wiley & Sons Ltd.)