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
• Názvy látek
• aktiny MeSH
• fosfolipasa D MeSH
• izoenzymy MeSH

Phospholipase D (PLD) forms the major family of phospholipases that was first discovered and cloned in plants. In this report we have shown, for the first time, that C2 phosphatidylinositol-4,5-bisphosphate (PIP2)-dependent PLD(s) from 5 day hypocotyls of Brassica oleracea associated with plasma membrane is covalently modified-phosphorylated. Pre-incubation of the plasma membrane fraction with acid phosphatase resulted in concentration-dependent inhibition of PIP2-dependent PLD activity. Using matrix-assisted laser desorption/ionization time of flight mass spectrometry of tryptic in-gel digests, the BoPLDgamma(1,2) isoform was identified. Comparing the spectra of the proteins obtained from the plasma membrane fractions treated and non-treated with acid phosphatase, three peptides differing in the mass of the phosphate group (80 Da) were revealed: TMQMMYQTIYK, EVADGTVSVYNSPR and KASKSRGLGK which possess five potential Ser/Thr phosphorylation sites. Our findings suggest that a phosphorylation/dephosphorylation mechanism may be involved in the regulation of plant PIP2-dependent PLDgamma activity.

• MeSH
• Brassica enzymologie   MeSH
• fosfatidylinositol-4,5-difosfát metabolismus   MeSH
• fosfolipasa D metabolismus   MeSH
• fosforylace MeSH
• kyselá fosfatasa metabolismus   MeSH
• membránové proteiny metabolismus   MeSH
• peptidové fragmenty MeSH
• rostlinné proteiny metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• fosfatidylinositol-4,5-difosfát MeSH
• fosfolipasa D MeSH
• kyselá fosfatasa MeSH
• membránové proteiny MeSH
• peptidové fragmenty MeSH
• rostlinné proteiny MeSH