A sensitive approach based on electrospray ionization tandem mass spectrometry has been employed to profile membrane lipid molecular species in Arabidopsis undergoing cold and freezing stresses. Freezing at a sublethal temperature induced a decline in many molecular species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) but induced an increase in phosphatidic acid (PA) and lysophospholipids. To probe the metabolic steps generating these changes, lipids ofArabidopsis deficient in the most abundant phospholipase D, PLDα, were analyzed. The PC content dropped only half as much, and PA levels rose only half as high in the PLDα-deficient plants as in wild-type plants. In contrast, neither PE nor PG levels decreased significantly more in wild-type plants than in PLDα-deficient plants. These data suggest that PC, rather than PE and PG, is the majorin vivo substrate of PLDα. The action of PLDα during freezing is of special interest because Arabidopsis plants that are deficient in PLDα have improved tolerance to freezing. The greater loss of PC and increase in PA in wild-type plants as compared with PLDα-deficient plants may be responsible for destabilizing membrane bilayer structure, resulting in a greater propensity toward membrane fusion and cell death in wild-type plants.

We determined the role of Phospholipase Dα1 (PLDα1) and its lipid product phosphatidic acid (PA) in abscisic acid (ABA)-induced production of reactive oxygen species (ROS) in Arabidopsis thaliana guard cells. The pldα1 mutant failed to produce ROS in guard cells in response to ABA. ABA stimulated NADPH oxidase activity in wild-type guard cells but not in pldα1 cells, whereas PA stimulated NADPH oxidase activity in both genotypes. PA bound to recombinant Arabidopsis NADPH oxidase RbohD (respiratory burst oxidase homolog D) and RbohF. The PA binding motifs were identified, and mutation of the Arg residues 149, 150, 156, and 157 in RbohD resulted in the loss of PA binding and the loss of PA activation of RbohD. The rbohD mutant expressing non-PA-binding RbohD was compromised in ABA-mediated ROS production and stomatal closure. Furthermore, ABA-induced production of nitric oxide (NO) was impaired in pldα1 guard cells. Disruption of PA binding to ABI1 protein phosphatase 2C did not affect ABA-induced production of ROS or NO, but the PA–ABI1 interaction was required for stomatal closure induced by ABA, H2O2, or NO. Thus, PA is as a central lipid signaling molecule that links different components in the ABA signaling network in guard cells.

Phospholipase D (PLD) and protein phosphatase 2C (PP2C) both play a role in mediating plant responses to abscisic acid (ABA). In this article, we show that PLDα1 and its product, phosphatidic acid (PA), regulate a PP2C, ABI1, which is a negative regulator of ABA responses in Arabidopsis . Leaves from a T-DNA insertional mutant of PLD α 1 and PLD α 1 -antisense plants lose more water than do wild-type plants. The stomatal closure of PLD α 1 -null leaves is insensitive to ABA but is promoted by PA. ABA treatment promotes an increase in PA from phosphatidylcholine in wild type but not in PLD α 1 -null cells. PLDα1-derived PA binds to ABI1; the PA–ABI1 binding is demonstrated by coprecipitating PA with ABI1 from plant cells, measuring binding of PA from vesicles to ABI1, and assaying ABI1 bound to PA immobilized on a filter. Deletion and site-specific mutational analyses show that arginine 73 in ABI1 is essential for PA–ABI1 binding. PA binding decreases the phosphatase activity of ABI1. The lack of ABA-induced production of PA in PLD α 1 -null cells results in a decrease in the association of ABI1 with the plasma membrane in response to ABA. These results indicate that PA produced by PLDα1 inhibits the function of the negative regulator ABI1, thus promoting ABA signaling. The identification of ABI1 as a direct target of the lipid messenger PA provides a functional link between the two families of important signaling enzymes, PLD and PP2C.

Terrestrial plants lose water primarily through stomata, pores on the leaves. The hormone abscisic acid (ABA) decreases water loss by regulating opening and closing of stomata. Here, we show that phospholipase Dα1 (PLDα1) mediates the ABA effects on stomata through interaction with a protein phosphatase 2C (PP2C) and a heterotrimeric GTP-binding protein (G protein) in Arabidopsis. PLDα1-produced phosphatidic acid (PA) binds to the ABI1 PP2C to signal ABA-promoted stomatal closure, whereas PLDα1 and PA interact with the Gα subunit of heterotrimeric G protein to mediate ABA inhibition of stomatal opening. The results reveal a bifurcating signaling pathway that regulates plant water loss.