Abstract

Phosphatidylcholine (PC) is a crucial membrane phospholipid involved in both cellular processes and stress responses. CTP: phosphocholine cytidylyltransferase 1 (CCT1) is considered to catalyze a key regulatory step in primary PC de novo biosynthesis, but its functions and regulation are yet to be well elucidated. This study explored the physiological functions of CCT1 in Arabidopsis (Arabidopsis thaliana) (AthCCT1) in PC biosynthesis under normal growth conditions and in root development under osmotic stress, as well as its regulation by phosphorylation. Arabidopsis cct1 knockdown cct2 knockout lines exhibited significantly decreased PC intensities under normal growth conditions and impaired root growth under osmotic stress, which was rescued by AthCCT1 overexpression. Moreover, based on our previous findings that AthCCT1 is phosphorylated at Serine 187 (S187), we further investigated how this phosphorylation affects its biochemical and biological functions. The S187D phosphomimetic protein mutant of AthCCT1 exhibited reduced lipid-induced conformational changes and decreased enzymatic activity compared to the native protein. Molecular dynamics simulations of the S187D protein mutant revealed that the auto-inhibitory region, a conserved regulatory domain across CCT enzymes, remained closer to the ?E helix, maintaining a constrained interaction between them. Consistent with the results of the in vitro analyses, overexpression of AthCCT1-S187D did not rescue stress-induced short-root phenotypes in cct1 knockdown cct2 knockout Arabidopsis lines. Taken together, the results revealed that AthCCT1 regulates PC biosynthesis under normal conditions and root development under osmotic stress, with its phosphorylation state at S187 playing an important role in modulating its enzymatic activity and physiological functions. © The Author(s) 2025.