ABSTRACT
The phosphatidylinositol-specific phospholipase C (PI-PLC) activity is detected in purified Lilium pollen protoplasts. Two PI-PLC full length cDNAs, LdPLC1 and LdPLC2, were isolated from pollen of Lilium daviddi. The amino acid sequences for the two PI-PLCs deduced from the two cDNA sequences contain X, Y catalytic motifs and C2 domains. Blast analysis shows that LdPLCs have 60-65% identities to the PI-PLCs from other plant species. Both recombinant PI-PLCs proteins expressed in E. coli cells show the PIP(2)-hydrolyzing activity. The RT-PCR analysis shows that both of them are expressed in pollen grains, whereas expression level of LdPLC2 is induced in germinating pollen. The exogenous purified calmodulin (CaM) is able to stimulate the activity of the PI-PLC when it is added into the pollen protoplast medium, while anti-CaM antibody suppresses the stimulation effect caused by exogenous CaM. PI-PLC activity is enhanced by G protein agonist cholera toxin and decreased by G protein antagonist pertussis toxin. Increasing in PI-PLC activity caused by exogenous purified CaM is also inhibited by pertussis toxin. A PI-PLC inhibitor, U-73122, inhibited the stimulation of PI-PLC activity caused by cholera toxin and it also leads to the decrease of [Ca(2+)](cyt) in pollen grains. Those results suggest that the PPI-PLC signaling pathway is present in Lilium daviddi pollen, and PI-PLC activity might be regulated by a heterotrimeric G protein and extracellular CaM.
Subject(s)
Lilium/enzymology , Phosphatidylinositol Diacylglycerol-Lyase/metabolism , Pollen/enzymology , Cloning, Molecular , DNA, Complementary , Escherichia coli/genetics , Fluorescent Dyes , Hydrolysis , Molecular Sequence Data , Phosphatidylinositol Diacylglycerol-Lyase/genetics , Phosphoinositide Phospholipase C , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
A cDNA encoding a phosphoinositide-specific phospholipase C (PI-PLC) has been isolated from Zea mays by screening a cDNA library. The cDNA, designated ZmPLC, encodes a polypeptide of 586 amino acids, containing the catalytic X, Y and C2 domains found in all PI-PLCs from plants. Northern blot analysis showed that the expression of the ZmPLC gene in roots is up-regulated under conditions of high salt, dehydration, cold or low osmotic stress conditions. Recombinant ZmPLC protein was expressed in Esch- erichia coli, purified and used to produce polyclonal antibody, this polyclonal antibody is important for further studies to assess the ultimate function of the ZmPLC gene in plants.
Subject(s)
Escherichia coli/genetics , Phosphatidylinositol Diacylglycerol-Lyase/genetics , Phosphatidylinositols/metabolism , Type C Phospholipases/genetics , Zea mays/genetics , Amino Acid Sequence , Blotting, Northern , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/genetics , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Immunoblotting , Molecular Sequence Data , Phosphatidylinositol Diacylglycerol-Lyase/metabolism , Phosphoinositide Phospholipase C , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Type C Phospholipases/metabolism , Zea mays/enzymologyABSTRACT
Two cDNAs encoding proteins, PpPLC1 and PpPLC2, with catalytic and C2 domains conserved in plant phosphoinositide-specific phospholipase C (PI-PLC) were isolated from Physcomitrella patens. The N domain, which has been identified in Arabidopsis PI-PLCs as an EF hand-like domain, was found in both isoforms, although that in PpPLC2 was a split type. At micromolar Ca2+ concentrations, PpPLC1 preferentially hydrolysed phosphatidylinositol-4,5-bisphosphate, while PpPLC2 showed no specificity. Furthermore, at millimolar Ca2+, phosphatidylinositol was hydrolysed by PpPLC2, but not by PpPLC1. Thus, PpPLC1 and PpPLC2 are typical and novel types of plant PI-PLC, respectively.
Subject(s)
Bryopsida/genetics , Phosphatidylinositol Diacylglycerol-Lyase/genetics , Base Sequence , Bryopsida/enzymology , DNA, Complementary/chemistry , DNA, Complementary/genetics , Molecular Sequence Data , Phosphatidylinositol Diacylglycerol-Lyase/metabolism , Phosphoinositide Phospholipase C , Plant Proteins/genetics , Plant Proteins/metabolism , Sequence Analysis, DNA , Sequence Homology, Nucleic AcidABSTRACT
The PIGA gene from Toxoplasma gondii has been cloned and characterized. Like mammalian PIGA, the transmembrane and C-terminal domains are sufficient to direct localization to the parasite endoplasmic reticulum. A functional copy of PIGA is required for tachyzoite viability, demonstrating that glycosylphosphatidylinositol biosynthesis is an essential process in T. gondii.