Molecular and enzymatic characterization of three phosphoinositide-specific phospholipase C isoforms from potato.
Many cellular responses to stimulation of cell-surface receptors by extracellular signals are transmitted across the plasma membrane by hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2), which is cleaved into diacylglycerol and inositol-1,4,5-trisphosphate by phosphoinositide-specific phospholipase C (PI-PLC). We present structural, biochemical, and RNA expression data for three distinct PI-PLC isoforms, StPLC1, StPLC2, and StPLC3, which were cloned from a guard cell-enriched tissue preparation of potato (Solanum tuberosum) leaves. All three enzymes contain the catalytic X and Y domains, as well as C2-like domains also present in all PI-PLCs. Analysis of the reaction products obtained from PIP2 hydrolysis unequivocally identified these enzymes as genuine PI-PLC isoforms. Recombinant StPLCs showed an optimal PIP2-hydrolyzing activity at 10 microM Ca2+ and were inhibited by Al3+ in equimolar amounts. In contrast to PI-PLC activity in plant plasma membranes, however, recombinant enzymes could not be activated by Mg2+. All three stplc genes are expressed in various tissues of potato, including leaves, flowers, tubers, and roots, and are affected by drought stress in a gene-specific manner.
Kopka
J
J
Max-Planck-Institut für Molekulare Pflanzenphysiologie, Golm/Potsdam, Germany.
Pical
C
C
Gray
J E
JE
Müller-Röber
B
B
D000535:Aluminum / Q000494:pharmacology; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D018515:Plant Leaves; D018517:Plant Roots; D018547:Plant Stems; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000166:cytology / Q000201:enzymology*; D014158:Transcription, Genetic; D010738:Type C Phospholipases / Q000737:chemistry* / Q000378:metabolism*
D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D000535:Aluminum; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D008274:Magnesium