Functional Prediction of AT5G35460 Reveals Its Regulatory Role in Reproductive Development and Lipid Remodeling in Arabidopsis thaliana

Membrane lipid remodeling plays a pivotal role in regulating plant growth, reproductive development, and adaptive responses to environmental stress. However, several lipid-modifying enzymes remain uncharacterized in Arabidopsis thaliana. Here, we provide the first comprehensive in silico functional characterization of the unannotated gene AT5G35460, integrating domain architecture, AlphaFold-supported structural validation, and phylogenetic, expression, and regulatory analyses. Domain architecture and conserved DUF2838 signatures, together with transmembrane topology and validation using AlphaFold-predicted structural data, support its identity as a glycerophosphocholine acyltransferase (GPCAT1). Phylogenetic reconstruction showed that GPCAT1 clustered closely with its orthologs of major angiosperms, suggesting deep evolutionary preservation. Expression profiling revealed over a tenfold higher transcript abundance in mature pollen, detected 6–8 times more than during leaf senescence, indicating strong developmental control. Co-expression network analysis revealed links to the lipid metabolism genes (CDS2, LACS8, and SBH1) as well as factors involved in response to stress, indicating that AT5G35460 may act at the level of phosphatidylcholine remodeling, membrane resistance and stress response. Analysis of the promoter sequences showed AACTAAA, ABRE and G-box elements (pollen-specific, ABA-responsive and stress-inducible motif respectively), suggesting appropriate transcriptional regulation consistent with its expression profile. As a whole, the findings revealed that AT5G35460 is an unexplored membrane-localized acyltransferase involved in lipid maintenance during reproductive development and environmental responses. This study serves as a basis for subsequent functional characterization and identifies AT5G35460 as a potential target for modifying pollen viability, senescence kinetics and stress tolerance in plants.