ABSTRACT
A cDNA encoding the catalytic site of a phosphatidylinositol-specific phospholipase C (PI-PLC) was isolated from Coffea arabica suspension cells. The cDNA (designated CaPLC) encodes a polypeptide of 308 amino acids, containing the catalytic X and Y domains, and has 99% identity to the soybean gene. Recombinant CaPLC protein was expressed in Escherichia coli, purified, and used to produce a polyclonal antibody. The peptide has a molecular mass of 27 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analyses. Immunoblots revealed the presence of PLC-like proteins in the tissues of different plant species.
ABSTRACT
The cascade of phospholipid signals, is one of the main systems of cellular transduction, and is related to other signal transduction mechanisms. These include the interaction between the generation of second messengers and different proteins such as ionic channels, protein kinase proteins, signaling proteins and transcription factors, among others. The result of this interaction could alter cellular metabolism. This phospholipid signal cascade is activated by the changes on the environment such as phosphate starvation, water and saline stress, as well as plant-pathogen interactions.Because aluminum has been considered a main toxic factor for agriculture carried out in acid soils, many researches have focused on aluminum toxic mechanism in plants.1,2 We contribute by researching on the aluminum effects on phospholipids signalling. We focused on phosphatidic acid (PA), because its relevant role in signal cascades in plants. Also PA is the precursor of most of the phospholipids in their de novo biosynthesis. Our results show a dramatic inhibitory effects by aluminum on PA. The most important PA formation routes in plant signalling are: phospholipase C (PLC)/diacylglycerol kinase (DGK) and phospholipase D (PLD).3 We investigated which one of the pathways was affected by aluminum treatment and found that aluminum affects mainly the PLC/DGK route of PA formation. We conclude that Al(3+) not only could generate a signal cascade in plants, but that it can also modulate other signal cascades generated by others stress. The aim of this addendum is to discuss the possible involvements of other phospholipids in the aluminum toxicity in plant cells.
ABSTRACT
The properties of phospholipase C (PLC) partially purified from Catharanthus roseus transformed roots were analyzed using substrate lipids dispersed in phospholipid vesicles, phospholipid-detergent mixed micelles, and phospholipid monolayers spread at an air-water interface. Using [(33)P]phosphatidylinositol 4,5-bisphosphate (PIP(2)) of high specific radioactivity, PLC activity was monitored directly by measuring the loss of radioactivity from monolayers as a result of the release of inositol phosphate and its subsequent dissolution on quenching in the subphase. PLC activity was markedly affected by the surface pressure of the monolayer, with reduced activity at extremes of initial pressure. The optimum surface pressure for PIP(2) hydrolysis was 20 mN/m. Depletion of PLC from solution by incubation with sucrose-loaded PIP(2) vesicles followed by ultracentrifugation demonstrated stable attachment of PLC to the vesicles. A mixed micellar system was established to assay PLC activity using deoxycholate. Kinetic analyses were performed to determine whether PLC activity was dependent on both bulk PIP(2) and PIP(2) surface concentrations in the micelles. The interfacial Michaelis constant was calculated to be 0.0518 mol fraction, and the equilibrium dissociation constant of PLC for the lipid was 45.5 &mgr;M. These findings will add to our understanding of the mechanisms of regulation of plant PLC.
ABSTRACT
Evidence was obtained on the occurrence of protein threonine, serine, and tyrosine (Tyr) kinases in developing coconut (Cocos nucifera L. ) zygotic embryos, based on in vitro phosphorylation of proteins in the presence of [gamma-32P]ATP, alkaline treatment, and thin-layer chromatography analysis, which showed the presence of [32P]phosphoserine, [32P]phosphothreonine, and [32P]phosphotyrosine in [32P]-labeled protein hydrolyzates. Tyr kinase activity was further confirmed in extracts of embryos at different stages of development using antiphosphotyrosine monoclonal antibodies and the synthetic peptide derived from the amino acid sequence surrounding the phosphorylation site in pp60(src) (RR-SRC), which is specific for Tyr kinases. Anti-phosphotyrosine western blotting revealed a changing profile of Tyr-phosphorylated proteins during embryo development. Tyr kinase activity, as assayed using RR-SRC, also changed during embryo development, showing two peaks of activity, one during early and another during late embryo development. In addition, the use of genistein, a Tyr kinase inhibitor, diminished the ability of extracts to phosphorylate RR-SRC. Results presented here show the occurrence of threonine, serine, and Tyr kinases in developing coconut zygotic embryos, and suggest that protein phosphorylation, and the possible inference of Tyr phosphorylation in particular, may play a role in the coordination of the development of embryos in this species.
