Phospholipase D- and Phosphatidic Acid-Mediated Signaling in Plant Response to Abscisic Acid and Reactive Oxygen Species

Arabidopsis genome has 12 phospholipase D (PLD) genes that are classified into six types, PLDalpha, PLDbeta, PLDgamma, PLDdelta, PLDepsilon, and PLDzeta, based on sequence similarities, domain structures, and biochemical properties. Phosphatidic acid (PA) produced by PLDs has been identified as important lipid signaling molecule in cell growth, development, and stress responses in both plants and animals. This study was undertaken to determine the role of PLD and PA in plant response to abscisic acid (ABA) and reactive oxygen species (ROS). The lipid mediator PA was found to interact with sphingosine kinases (SPHKs) in Arabidopsis. Two unique SPHK cDNAs were cloned and expressed. Both SPHKs are catalytically active, phosphorylating various long-chain sphingoid bases (LCBs). PA binds to and stimulates both SPHKs, and the interaction promotes lipid substrate binding to the catalytic site of the enzyme. SPHK -deficient and PLDalpha1-deficient mutants were employed to determine the cellular and physiological functions of the PA-SPHK interaction in plants. Compared to wild-type (WT) plants, SPHK and PLDalpha1 mutants all displayed decreased sensitivity to ABA-promoted stomatal closure. The data indicate that SPHK and PLDalpha1 act together in ABA response and that SPHK and phytosphingosine-1-phosphate (phyto-S1P) act upstream of PLDalpha1 and PA in mediating the ABA response. On the other hand, PA is involved in the activation of SPHK, forming a positive loop in signaling plant response to ABA. In addition, another PLD, PLDdelta, was found to be involved in the ROS and ABA signaling pathways. PLDdelta functions downstream of PLDalpha1 and H2O2 to mediate the ABA-induced stomatal closure. Furthermore, the study has identified that cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC) acts as a molecular link between H2O 2 and PLDdelta activation. H2O2 inhibited GAPC activity but promoted GAPC-PLDdelta interaction and the PLDdelta activity. The loss of both GAPCs decreased plant insensitivity to ABA- and H2O 2-induced stomatal closure, like PLDdelta. In addition, GAPC -deficient plants produced less PA than wild-type in response to ABA and H2O2. These results indicate that GAPCs mediate H2O2-activation of PLDdelta in Arabidopsis response to ABA. The interaction of a cytosolic metabolic enzyme GAPC and a membrane-associated PLDdelta transduces ROS signals in plant response to ABA and oxidative stress. The physiological functions of GAPC were characterized using the GAPC T-DNA insertion lines. There are two GAPC genes in Arabidopsis, which are involved in the glycolytic pathway, are potentially important to plant growth and development. GAPC deficiency did not cause growth inhibition or development problems for the plants. Instead, the GAPC mutants displayed larger size and accumulated more biomass when grown under normal condition. However, GAPC- and PLDdelta-deficient mutants were less tolerant to salt and freezing stresses. In addition, GAPC double knockouts had a 4-5% decrease in seed oil content. These results indicate the importance of GAPC in plant stress tolerance and metabolism pathway.