G Protein Activation Stimulates Phospholipase D Signaling in Plants.

Abstract

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.

Phosphatidic Acid Binds to Cytosolic Glyceraldehyde-3-phosphate Dehydrogenase and Promotes Its Cleavage in Arabidopsis

“…The seedlings of GAPC2-knockouts and overexpression lines, as well as EV and WT, were treated with n-butyl alcohol that diverts part of PLD-produced PA to phosphatidylbutanol (44). n-Butyl alcohol at 0.4% substantially reduced growth of all lines as reported previously (45), but there was no significant difference among WT and GAPC2-altered seedlings (supplemental Fig.…”

Enhanced induction of microspore embryogenesis after n-butanol treatment in wheat (Triticum aestivum L.) anther culture

“…PLD has the unique ability to transfer the phosphatidyl group of its substrate, instead of water, to a primary alcohol forming phosphatidyl alcohols (Dawson 1967;Yang et al 1967). Among the three alcohols used in this study, only n-butanol can be used by PLD as a transphosphatidylation substrate (Munnik et al 1995). This occurs at the expense of PA formation, which is known to act as a second messenger in several processes (Laxalt and Munnik 2002;Wang et al 2006).…”

“…Treatment of Arabidopsis seedlings with n-butanol also produced cortical MT disruption, and inhibited seed germination and seedling growth (Gardiner et al 2003). The effect of n-butanol on plant MTs, is thought to be mediated by its known ability to activate phospholipase D (PLD) (Munnik et al 1995). This enzyme is tightly associated with plasma membrane and with the MT cytoskeleton (Gardiner et al 2001), and its stimulation has been correlated with microtubular rearrangements (Dhonukshe et al 2003).…”

“…Both the primary alcohol, n-butanol, and the secondary alcohol, sec-butanol, can activate PLD via G-proteins (Munnik et al 1995), but only the former produces MT detachment from the plasma membrane. The tertiary alcohol, tert-butanol, neither activates PLD nor acts on MT cytoskeleton, and therefore can be used as a control for any non-specific effect of butyl-alcohols (Munnik et al 1995;Dhonukshe et al 2003).…”

“…The tertiary alcohol, tert-butanol, neither activates PLD nor acts on MT cytoskeleton, and therefore can be used as a control for any non-specific effect of butyl-alcohols (Munnik et al 1995;Dhonukshe et al 2003).…”