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The fidelity of signal transduction requires spatiotemporal control of the production of signaling agents. Phosphatidic acid (PA) is a pleiotropic lipid second messenger whose modes of action differ based on upstream stimulus, biosynthetic source, and site of production. How cells regulate the local production of PA to effect diverse signaling outcomes remains elusive. Unlike other second messengers, sites of PA biosynthesis cannot be accurately visualized with subcellular precision. Here, we describe a rapid, chemoenzymatic approach for imaging physiological PA production by phospholipase D (PLD) enzymes. Our method capitalizes on the remarkable discovery that bulky, hydrophilic trans -cyclooctene–containing primary alcohols can supplant water as the nucleophile in the PLD active site in a transphosphatidylation reaction of PLD’s lipid substrate, phosphatidylcholine. The resultant trans -cyclooctene–containing lipids are tagged with a fluorogenic tetrazine reagent via a no-rinse, inverse electron-demand Diels–Alder (IEDDA) reaction, enabling their immediate visualization by confocal microscopy in real time. Strikingly, the fluorescent reporter lipids initially produced at the plasma membrane (PM) induced by phorbol ester stimulation of PLD were rapidly internalized via apparent nonvesicular pathways rather than endocytosis, suggesting applications of this activity-based imaging toolset for probing mechanisms of intracellular phospholipid transport. By instead focusing on the initial 10 s of the IEDDA reaction, we precisely pinpointed the subcellular locations of endogenous PLD activity as elicited by physiological agonists of G protein-coupled receptor and receptor tyrosine kinase signaling. These tools hold promise to shed light on both lipid trafficking pathways and physiological and pathological effects of localized PLD signaling.
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This content will become publicly available on June 24, 2027
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.
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- PAR ID:
- 10697126
- Publisher / Repository:
- Plant Physiology
- Date Published:
- Journal Name:
- Plant Physiology
- ISSN:
- 0032-0889
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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