The O-glycosyltransferase SECRET AGENT Participates in Abscisic Acid-Induced Microtubule Remodeling and Stomatal Closure in Arabidopsis thaliana

Secret Agent (SEC) encodes the major O-GlcNAc transferase in Arabidopsis thaliana and has been implicated in stress and signaling pathways, but its role in stomatal regulation remains unknown. In this study, we investigated the function of SEC in abscisic acid (ABA)-induced stomatal closure and drought responses. Loss of SEC function in the sec-5 mutant delayed stomatal closure and increased water loss under drought, whereas SEC overexpression enhanced stomatal responsiveness and drought tolerance. Pharmacological assays revealed that sec-5 was less sensitive to microtubule-stabilizing and -disrupting drugs during ABA-induced stomatal closing, consistent with a defect in cytoskeletal dynamics. Confocal imaging and quantitative analysis of microtubule rearrangements showed that SEC loss-of-function mutation retarded microtubule reorganization in response to ABA, with abnormal microtubule bundling and density. Mechanistically, SEC physically interacted with -tubulin 4 (TUA4) in both yeast two-hybrid and bimolecular fluorescence complementation assays, suggesting tubulin as a potential substrate of SEC-modified O-GlcNAcylation. Together, these findings establish SEC as a key regulator of ABA-induced microtubule remodeling in guard cells, thereby linking O-GlcNAcylation to stomatal regulation and drought tolerance.