Mutual regulation of plant phospholipase D and the actin cytoskeleton.
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.
Institute of Experimental Botany, Academy of Sciences of the Czech Republic, v.v.i., Rozvojová 263, 165 02 Prague 6, Czech Republic.
Roman
R
Pleskot
Martin
M
Potocký
Premysl
P
Pejchar
Jan
J
Linek
Radek
R
Bezvoda
Jan
J
Martinec
Olga
O
Valentová
Zuzana
Z
Novotná
Viktor
V
Zárský
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003599:Cytoskeleton / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D055785:Gene Knockdown Techniques; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*; D053205:Pollen Tube / Q000254:growth & development; D020539:Sequence Analysis, Protein; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics
D000199:Actins; D007527:Isoenzymes; D010739:Phospholipase D