Abscisic acid stimulation of phospholipase D in the barley aleurone is G-protein-mediated and localized to the plasma membrane

Abstract

We have previously determined that phospholipase D (PLD) is activated by abscisic acid (ABA), and this activation is required for the ABA response of the cereal aleurone cell. In this study, ABA-stimulated PLD activity was reconstituted in vitro in microsomal membranes prepared from aleurone protoplasts. The transient nature (20 min) and degree (1.5- to 2-fold) of activation in vitro were similar to that measured in vivo. Stimulation by ABA was only apparent in the membrane fraction and was associated with a fraction enriched in plasma membrane. These results suggest that an ABA receptor system and elements linking it to PLD activation are associated with the aleurone plasma membrane. The activation of PLD in vitro by ABA was dependent on the presence of GTP. Addition of GTPgammaS transiently stimulated PLD in an ABA-independent manner, whereas treatment with GDPbetaS or pertussis toxin blocked the PLD activation by ABA. Application of pertussis toxin to intact aleurone protoplasts inhibited the ability of ABA to activate PLD as well as antagonizing the ability of ABA to down-regulate gibberellic acid-stimulated alpha-amylase production. All of these data support the hypothesis that ABA stimulation of PLD activity occurs at the plasma membrane and is mediated by G-protein activity.

Figure 1

The effect of varying SDS and protein concentration on PLD activity in microsomal membrane preparations in the presence or absence of ABA. PLD assays were carried out in the presence of various SDS (A) and protein (B) concentrations ± 10 μm ABA as described in “Materials and Methods.” In A the protein concentration used was 112.5 μg/mL per assay, in B the SDS concentration used was 55 μm. The results show the means ± se of three separate experiments for each graph. Asterisks show statistically different points (*, P < 0.05; **, P < 0.025; ***, P < 0.01, t test).

Figure 2

Time course of ABA stimulation of PLD activity in vitro. PLD assays were carried out using microsomal membrane preparations for various times ± 10 μm ABA. The stimulation by ABA was calculated by division of the PLD activity measured in assays run + ABA by that of the equivalent − ABA control. The results show the means ± se of three separate experiments.

Figure 3

The stimulation of PLD by ABA is specific for this hormone and is dependent on the ABA concentration. PLD assays using microsomal membrane preparations were carried out in the presence of a range of plant hormones (all 10 μm) (A) and a range of ABA concentrations (B). The results show the means ± se of three separate experiments. BA, Benzyl adenine.

Figure 4

The effect of non-hydrolyzable GTP analogs on ABA-stimulated PLD activity. PLD assays of microsomal membrane preparations were conducted using ±10 μm ABA in the presence of various nucleotides (10 μm). The results show the means ± se of three separate experiments. Asterisks show statistically different points (between control and + GTPγS; **, P < 0.025, t test).

Figure 5

Time course of GTPγS stimulation of PLD activity in vitro. PLD assays of microsomal membrane preparations were carried out for various times ±10 μm GTPγS and ±10 μm ABA. The stimulation by GTPγS was calculated by division of the activity in assays run + GTPγS ± ABA by those from assays −GTPγS and −ABA. The results show the means ± se of three separate experiments.

Figure 6

The effect of G-protein agonists and antagonists on ABA-stimulated PLD activity and in vivo effect of PTX and CTX on ABA-stimulated PtdOH levels. A, In vitro PLD assays of microsomal membrane preparations were conducted ±10 μm ABA in the presence of a range of G-protein-related peptides (1 or 0.5 μg/mL for protomer A or oligomer B of PTX) and P-ethanol measured. B, Aleurone protoplasts were loaded with fluorescently labeled phosphatidylcholine and, after application of ABA (10 μm) and toxins (1 or 0.5 μg/mL for protomer A or oligomer B of PTX) for 15 min, lipids were extracted and fluorescent PtOH produced in vivo was quantified. The results show the means ± se of three separate experiments. For A, basal in vitro PLD activity was 0.5 ± 0.05 nmol P-ethanol mg⁻¹ min⁻¹. For B, basal in vivo fluorescent PtdOH production was 1.3 ± 0.15 arbitrary fluorescent units. The data are expressed as the ratio of P-ethanol (A) or PtdOH (B) produced with the treatment in the presence versus absence of ABA. None of the treatments (Mas, CTX, PTX, PTX-A, or PTX-B) significantly affected either PLD activity (P-ethanol produced, A) or production of fluorescent-PtdOH (B) in non-ABA-treated samples (P < 0.05, t test). Mas, Mastoparan; control, activation in presence of ABA without additional treatment.

Figure 7

The effect of PTX and CTX on α-amylase activity. Aleurone protoplasts were treated with combinations of GA (1 μm), ABA (10 μm), PTX, and CTX (1 or 0.5 μg/mL for individual subunits of PTX). After 48 h, secreted α-amylase activity was assayed. The results show the means ± se of three separate experiments.

Figure 8

ABA stimulation of PLD activity is localized to a plasma membrane-enriched fraction and is GTP dependent. A, Microsomal membrane preparations were processed through phase partitioning and the resulting upper and lower phases, plus the initial microsomal and soluble fractions were assayed for PLD activity (in the presence of 10 μm GTP) ± 10 μm ABA. B, The upper phase was assayed for PLD activity in the absence or presence of 10 μm ATP and/or GTP. The results show the means ± se of three separate experiments.