Membrane lipids and cytoskeleton dynamics are intimately inter-connected in the eukaryotic cell; however, only recently have the molecular mechanisms operating at this interface in plant cells been addressed experimentally. Phospholipase D (PLD) and its product phosphatidic acid (PA) were discovered to be important regulators in the membrane-cytoskeleton interface in eukaryotes. Here we report the mechanistic details of plant PLD-actin interactions. Inhibition of PLD by n-butanol compromises pollen tube actin, and PA rescues the detrimental effect of n-butanol on F-actin, showing clearly the importance of the PLD-PA interaction for pollen tube F-actin dynamics. From various candidate tobacco PLDs isoforms, we identified NtPLDbeta1 as a regulatory partner of actin, by both activity and in vitro interaction assays. Similarly to published data, the activity of tobacco PIP(2)-dependent PLD (PLDbeta) is specifically enhanced by F-actin and inhibited by G-actin. We then identified the NtPLDbeta1 domain responsible for actin interactions. Using sequence- and structure-based analysis, together with site-directed mutagenesis, we identified Asn323 and Thr382 of NtPLDbeta1 as the crucial amino acids in the actin-interacting fold. The effect of antisense-mediated suppression of NtPLDbeta1 or NtPLDdelta on pollen tube F-actin dynamics shows that NtPLDbeta1 is the active partner in PLD-actin interplay. The positive feedback loop created by activation of PLDbeta by F-actin and of F-actin by PA provides an important mechanism to locally increase membrane-F-actin dynamics in the cortex of plant cells.
- MeSH
- aktiny metabolismus MeSH
- cytoskelet metabolismus MeSH
- fosfolipasa D genetika metabolismus MeSH
- genový knockdown MeSH
- izoenzymy metabolismus MeSH
- klonování DNA MeSH
- molekulární sekvence - údaje MeSH
- mutageneze cílená MeSH
- pylová láčka růst a vývoj MeSH
- regulace genové exprese u rostlin MeSH
- sekvence aminokyselin MeSH
- sekvenční analýza proteinů MeSH
- tabák enzymologie genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
