Abstract
We determined the role of Phospholipase Dalpha1 (PLDalpha1) and its lipid product phosphatidic acid (PA) in abscisic acid (ABA)-induced production of reactive oxygen species (ROS) in Arabidopsis thaliana guard cells. The pldalpha1 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 pldalpha1 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 pldalpha1 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, H(2)O(2), or NO. Thus, PA is as a central lipid signaling molecule that links different components in the ABA signaling network in guard cells.
Highlights
The plant hormone abscisic acid (ABA) participates in diverse physiological processes, such as stomatal movement, seed dormancy and germination, vegetative growth, and response to abiotic and biotic stresses (Schroeder et al, 2001; Finkelstein et al, 2002; Assmann, 2003; Xiong and Zhu, 2003; Hirayama and Shinozaki, 2007)
H2DCFDA is an effective dye for measuring reactive oxygen species (ROS) in our system because ABA-induced DCF fluorescence was shown to be inhibited by a suicide substrate inhibitor of NADPH oxidases, diphenylene iodonium (DPI)
Plant phospholipase D (PLD) and derived phosphatidic acid (PA) are involved in ABA-mediated stomatal movement by interaction with the a-subunit of G protein, through PA regulation of the inwardly rectifying K+ channel, and/ or ABI1 PP2C localization and activity (Jacob et al, 1999; Zhang et al, 2004; Mishra et al, 2006)
Summary
The plant hormone abscisic acid (ABA) participates in diverse physiological processes, such as stomatal movement, seed dormancy and germination, vegetative growth, and response to abiotic and biotic stresses (Schroeder et al, 2001; Finkelstein et al, 2002; Assmann, 2003; Xiong and Zhu, 2003; Hirayama and Shinozaki, 2007). Lipid mediators generated by phospholipase D (PLD), phospholipase C (PLC), and sphingosine kinase have been identified as integral parts of the complex signaling cascades in the ABA response (Fan et al, 2004; Zhang et al, 2005; Wang et al, 2006). PLDa1-produced PA binds to the ABI1 protein phosphatase 2C, a negative regulator of ABA action. This interaction inhibits the function of ABI1 by inhibiting its phosphatase activity and tethers it to the plasma membrane. On ABA inhibition of stomatal opening, PLDa1 binds to GPA1 (a-subunit of heterotrimeric G protein) and regulates its function by promoting the conversion of GTP-bound Ga to a GDP-bound Ga, producing PA that acts upstream of Ga to inhibit stomatal opening (Mishra et al, 2006)
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