Acylation of non‐specific phospholipase C4 determines its function in plant response to phosphate deficiency

Non-specific phospholipase C (NPC) is involved in plant growth, development and stress responses. To elucidate the mechanism by which NPCs mediate cellular functions, here we show that NPC4 is  S -acylated at the C terminus and that acylation determines its plasma membrane (PM) association and function. The acylation of NPC4 was detected using NPC4 isolated from Arabidopsis and reconstituted  in vitro . The C-terminal Cys-533 was identified as the  S -acylation residue, and the mutation of Cys-533 to Ala-533 in NPC4 (NPC4 C533A ) led to the loss of  S -acylation and membrane association of NPC4. The knockout of  NPC4  impeded the phosphate deficiency-induced decrease of the phosphosphingolipid glycosyl inositol phosphoryl ceramide (GIPC), but introducing NPC4 C533A  to  npc4-1  failed to complement this defect, thereby supporting the hypothesis that the non-acylated NPC4 C533A  fails to hydrolyze GIPC during phosphate deprivation. Moreover, NPC4 C533A  failed to complement the primary root growth in  npc4-1  under stress. In addition, NPC4 in  Brassica   napus  was  S -acylated and mutation of the  S -acylating cysteine residue of BnaC01.NPC4 led to the loss of  S -acylation and its membrane association. Together, our results reveal that  S -acylation of NPC4 in the C terminus is conserved and required for its membrane association, phosphosphingolipid hydrolysis and function in plant stress responses.