G Protein Activation Stimulates Phospholipase D Signaling in Plants

Abstract

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.

Identification and Characterization of a Novel Plant Phospholipase D That Requires Polyphosphoinositides and Submicromolar Calcium for Activity in Arabidopsis

“…In plants, most reports on PLD functional studies have dealt with its possible effects on membrane deterioration in relation to stress injuries, senescence, aging, and pathogenesis (4 -6). Recent studies have, however, indicated a role of PLD in signal transduction processes in response to environmental cues (7)(8)(9).…”

Molecular Cloning and Functional Analysis of Polyphosphoinositide-dependent Phospholipase D, PLDβ, from Arabidopsis

“…PLD in plants was originally proposed to be important in phospholipid catabolism, initiating a lipolytic cascade in membrane deterioration during senescence and stress injuries (1,2). Recent studies in plants, animals, and yeast indicate that PLD hydrolysis plays a pivotal role in transmembrane signaling and cellular regulation (3)(4)(5)(6)(7)(8)(9). Activation of PLD has been proposed to mediate many cellular processes including cell proliferation, membrane trafficking, meiosis, and responses to external and internal stimuli.…”

Arabidopsis Phospholipase Dα1 Interacts with the Heterotrimeric G-protein α-Subunit through a Motif Analogous to the DRY Motif in G-protein-coupled Receptors

“…Most of the studies have involved the use of potential G-protein activators and inhibitors, such as toxins, nonhydrolyzable guanine nucleotide analogues, and alcohols (16,17). In barley aleurone cells, a PLD␣-like activity has been suggested to be associated with a G-protein on the plasma membrane to mediate ABA signaling (17).…”