Mutual regulation of plant phospholipase D and the actin cytoskeleton.

Pleskot R., Potocký M., Pejchar P., Linek J., Bezvoda R., Martinec J., Valentová O., Novotná Z., Žárský V.
PLANT JOURNAL 62: 494-507, 2010

Keywords: phospholipase D, actin, signaling, pollen tube, tip growth, Nicotiana tabacum
Abstract: 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 NtPLDβ1 as a regulatory partner of actin, by both activity and in vitro interaction assays. Similarly to published data, the activity of tobacco PIP2-dependent PLD (PLDβ) is specifically enhanced by F-actin and inhibited by G-actin. We then identified the NtPLDβ1 domain responsible for actin interactions. Using sequence- and structure-based analysis, together with site-directed mutagenesis, we identified Asn323 and Thr382 of NtPLDβ1 as the crucial amino acids in the actin-interacting fold. The effect of antisense-mediated suppression of NtPLDβ1 or NtPLDδ on pollen tube F-actin dynamics shows that NtPLDβ1 is the active partner in PLD–actin interplay. The positive feedback loop created by activation of PLDβ 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.
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IEB authors: Jan Martinec, Přemysl Pejchar, Roman Pleskot, Martin Potocký, Viktor Žárský