Loss of ZmDMP increases phosphatidic acid production and disrupts lipid homeostasis in maize sperm cells

Abstract

Haploid embryo seed production has been applied to accelerate plant breeding efficiency. Several genes, including DUF679 domain membrane protein (DMP) and phospholipase D3 (PLD3), are involved in maternal haploid induction in maize (Zea mays L.) and other plant species. However, how these gene variants trigger haploid induction and how they functionally interact remain largely unknown. Here, we generated CRISPR-induced DMP-knockout and examined the effects of DMP loss on the lipid composition of pollen and sperm cells in maize. Disruption of DMP led to a pronounced increase in phosphatidic acid (PA) accompanied by a decrease in phosphatidylcholine in sperm cells, with similar but weaker effects in pollen. Consistently, dmp mutants exhibited elevated transcript and protein levels of ZmPLDs, enzymes that hydrolyze phospholipids to produce PA, in both pollen and sperm cells. Immunoblot and PLD activity assays using isolated sperm cell proteins demonstrated the presence of active PLD enzymes in sperm cells and that DMP suppresses PLD activity. Manipulating sperm cell lipid composition further showed that increased PA levels, as well as the addition of DMP, enhance membrane fusogenicity. Structurally, DMP contains an N-terminal intrinsically disordered region and C-terminal transmembrane domains, and the full-length protein is required to suppress PLD activity and to promote membrane fusion. Together, these results indicate that DMP and PA have additive and compensatory effects on membrane fusion, while DMP suppresses PLD expression and PA production. These findings reveal a previously unrecognized role of DMP and its regulatory interplay with PLD in maintaining lipid homeostasis and modulating membrane fusion, providing mechanistic insights into maternal haploid induction.