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
In order to characterize a specific extracellular 21-kDa calmodulin-binding protein (named: ECBP21) from Angelica dahurica L. suspension-cultured cells, the cDNA coding for the protein has been cloned. Here, Southern blot analysis shows that there are at least two copies of ECBP21 gene in Angelica genome. Using truncated versions of ECBP21 and synthetic peptide in CaM binding assays, we mapped the calmodulin-binding domain to a 16-amino acid stretch (residues 200-215) at the C-terminal region. The ECBP21 was localized in the cell wall area by the immunogold electron microscopy and by GFP labeling method. These results define ECBP21 as a kind of an extracellular calmodulin-binding protein (CaMBP). Furthermore, using Northern blot analysis, we examined the expression dynamics of ecbp21 during the incubation of Angelica suspension-cultured cells and the treatments with some growth regulators. The above studies further provide the molecular evidence for the existence of the gene coding for extracellular CaMBPs and imply a possible role for ECBP21.
Subject(s)
Angelica/genetics , Calmodulin-Binding Proteins/genetics , Abscisic Acid/pharmacology , Amino Acid Sequence , Angelica/drug effects , Angelica/metabolism , Calmodulin/metabolism , Calmodulin-Binding Proteins/chemistry , Calmodulin-Binding Proteins/metabolism , Cells, Cultured , Cyclopentanes/pharmacology , DNA, Complementary/genetics , DNA, Plant , Gene Expression Regulation, Plant/drug effects , Gibberellins/pharmacology , Molecular Sequence Data , Oxylipins , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Binding , Salicylic Acid/pharmacology , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
Confocal laser scanning microscopy (CLSM) and whole-cell patch-clamp were used to investigate the role of Ca2+ influx in maintaining the cytosolic Ca2+ concentration ([Ca2+]c) and the features of the Ca2+ influx pathway in germinating pollen grains of Lilium davidii D. [Ca2+]c decreased when Ca2+ influx was inhibited by EGTA or Ca2+ channel blockers. A hyperpolarization-activated Ca2+-permeable channel, which can be suppressed by trivalent cations, verapamil, nifedipine or diltiazem, was identified on the plasma membrane of pollen protoplasts with whole-cell patch-clamp recording. Calmodulin (CaM) antiserum and W7-agarose, both of which are cell-impermeable CaM antagonists, lead to a [Ca2+]c decrease, while exogenous purified CaM triggers a transient increase of [Ca2+]c and also remarkably activated the hyperpolarization-activated Ca2+ conductance on plasma membrane of pollen protoplasts in a dose-dependent manner. Both the increase of [Ca2+]c and the activation of Ca2+ conductance which were induced by exogenous CaM were inhibited by EGTA or Ca2+ channel blockers. This primary evidence showed the presence of a voltage-dependent Ca2+-permeable channel, whose activity may be regulated by extracellular CaM, in pollen cells.
Subject(s)
Calcium Channels/physiology , Calcium Signaling/physiology , Calmodulin/metabolism , Lilium/physiology , Pollen/physiology , Antibodies , Calcium/metabolism , Calcium Channel Blockers/pharmacology , Calmodulin/immunology , Calmodulin/pharmacology , Cell Membrane/physiology , Chelating Agents/pharmacology , Cytosol/metabolism , Egtazic Acid/pharmacology , Extracellular Space/metabolism , Lilium/growth & development , Membrane Potentials/drug effects , Membrane Potentials/physiology , Signal Transduction/physiologyABSTRACT
The crystal structure of a potato calmodulin (PCM6) was solved by molecular replacement and refined to a crystallographic R factor of 22.8% (R(free) = 25.0%) using X-ray diffraction data in the resolution range 8.0-2.0 A. This is the first report of the three-dimensional structure of a plant Ca(2+)-calmodulin. PCM6 crystallizes in a crystal form that belongs to space group P2(1)2(1)2(1), which is different to that of most other calmodulin crystals. The main structural difference between PCM6 and the other calmodulins is in the central helix region and appears to be caused by crystal packing. The surface properties of PCM6 molecules were compared with those of animal calmodulins, which provided an explanation for the unique crystal-packing state of PCM6.
Subject(s)
Calmodulin/chemistry , Solanum tuberosum/chemistry , Amino Acid Sequence , Animals , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Sequence AlignmentABSTRACT
ECBP21 is a calmodulin binding protein (CaMBP) purified from extracellular extracts of suspension-cultured cells of Angelica dahurica, and it is the first reported extracellular CaMBP in plant kingdom. We have recently cloned the full-length cDNA for ECBP21. In this work, using recombinant ECBP21 we prepared rabbit antiserum with high specificity and high titer against ECBP21, and investigated the organ-specific distribution of ECBP21 in Angelica dahurica. ECBP21 was found in all organs examined, particularly abundant in the leaves flowers, and raches, and less in the roots. It was also found in all cells examined, and particularly enriched in the cell wall. These data support the notion that ECBP21 is specifically localized extracellularly, and imply that it may be involved in plant growth and development. In addition, using immunogold transmission electron microscopy method, we studied the subcellular localization of ECBP21 in rachis cells of Angelica dahurica. The results indicated that the ECBP21 was mainly localized in cell wall; this provided a direct evidence of the extracellular existence of ECBP21.