We provide evidence that phosphatidic acid (PtdOH) formed during signaling in plants is metabolized by a novel pathway. In much of this study, 32Pi-labeled Chlamydomonas cells were used, and signaling was activated by adding the G-protein activator mastoparan. Within seconds of activation, large amounts of [32P]PtdOH were formed, with peak production at about 4 min, when the level was 5-25-fold higher than the control. As the level of [32P]PtdOH subsequently decreased, an unknown phospholipid (PLX) increased in radiolabeling; before activation it was barely detectable. The chromatographic properties of PLX resembled those of lyso-PtdOH and CMP.PtdOH but on close inspection were found to be different. PLX was shown to be diacylglycerol pyrophosphate (DGPP), the product of a newly discovered enzyme, phosphatidate kinase, whose in vitro activity was described recently (Wissing, J. B., and Behrbohm, H. (1993) Plant Physiol. 102, 1243-1249). The identity of DGPP was established by co-chromatrography with a standard and by degradation analysis as follows: [32P]DGPP was deacylated, and the product (glycerolpyrophosphate, GroPP) was hydrolyzed by mild acid treatment or pyrophosphatase to produce GroP and Pi as the only radioactive products. Since DGPP is the pyrophosphate derivative of PtdOH and is formed as the concentration of PtdOH decreases, we assumed that PtdOH was converted in vivo to DGPP. This was confirmed by showing that during a short labeling protocol while the specific radioactivity of DGPP was increasing, the specific radioactivity of the 32Pi derived from DGPP as above was higher than that of [32P]GroP. DGPP was also formed in suspension cultures of tomato and potato cells, and its synthesis was activated by mastoparan. Moreover, it was also found in intact tissues of a number of higher plants, for example, carnation flower petals, vetch roots, leaves of fig-leaved goosefoot, and common persicaria and microspores of rape seed. Our results suggest that DGPP is a common but minor plant lipid that increases in concentration when signaling is activated. Possible functions of DGPP in phospholpase C and D signaling cascades are discussed.
We provide direct evidence for phospholipase D (PLD) signaling i n plants by showing that this enzyme is stimulated by the G protein activators mastoparan, ethanol, and cholera toxin. An i n vivo assay for PLD activity i n plant cells was developed based on the use of a "reporter alcohol" rather than water as a transphosphatidylation substrate. The product was a phosphatidyl alcohol, which, in contrast to the normal product phosphatidic acid, i s a specific measure of PLD activity.When 32P-labeled cells were treated with 0.1% n-butanol, 3*P-phosphatidyl butanol (32P-PtdBut) was formed in a timedependent manner. In cells treated with any of the three G protein activators, the production of 32P-PtdBut was increased i n a dose-dependent manner. The G protein involved was pertussis toxin insensitive. Ethanol could activate PLD but was itself consumed by PLD as transphosphatidylation substrate. In contrast, secondary alcohols (e.g., sec-butyl alcohol) activated PLD but did not function as substrate, whereas tertiary alcohols did neither. Although most of the experiments were performed with the green alga Chlamydomonas eugametos, the relevance for higher plants was demonstrated by showing that PLD in carnation petals could also be activated by mastoparan. The results indicate that PLD activation must be considered as a potential signal transduction mechanism in plants, just as in animals.
We provide direct evidence for phospholipase D (PLD) signaling in plants by showing that this enzyme is stimulated by the G protein activators mastoparan, ethanol, and cholera toxin. An in vivo assay for PLD activity in plant cells was developed based on the use of a "reporter alcohol" rather than water as a transphosphatidylation substrate. The product was a phosphatidyl alcohol, which, in contrast to the normal product phosphatidic acid, is a specific measure of PLD activity. When 32P-labeled cells were treated with 0.1% n-butanol, 32P-phosphatidyl butanol (32P-PtdBut) was formed in a time-dependent manner. In cells treated with any of the three G protein activators, the production of 32P-PtdBut was increased in a dose-dependent manner. The G protein involved was pertussis toxin insensitive. Ethanol could activate PLD but was itself consumed by PLD as transphosphatidylation substrate. In contrast, secondary alcohols (e.g., sec-butyl alcohol) activated PLD but did not function as substrate, whereas tertiary alcohols did neither. Although most of the experiments were performed with the green alga Chlamydomonas eugametos, the relevance for higher plants was demonstrated by showing that PLD in carnation petals could also be activated by mastoparan. The results indicate that PLD activation must be considered as a potential signal transduction mechanism in plants, just as in animals.
SecB is a cytosolic chaperone involved in protein translocation across cytoplasmic membranes in Escherichia coli. It has been shown to be required for efficient translocation of a subset of precursor proteins but is not essential for cell viability. This study investigated whether synthesis of SecB is growth rate dependent. Interestingly, the total amount of SecB synthesized in the cells was relatively small. Moreover, the levels of SecB were found to be carbon source dependent since more SecB was produced in cells grown in glycerol media than in cells grown in glucose media, regardless of the growth rate. This is in contrast to the other Sec proteins, whose synthesis is growth rate dependent and not related to glucose as a carbon source. In addition, cyclic AMP (cAMP) partially relieves the lower levels of SecB observed in glucose medium, a compensatory effect that depends on the presence of both cya and crp gene products. Thus, the glucose-dependent synthesis of SecB may be related to the cAMP-cAMP receptor protein complex-mediated activation.
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