Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenases Interact with Phospholipase Dδ to Transduce Hydrogen Peroxide Signals in the Arabidopsis Response to Stress

Abstract

Reactive oxygen species (ROS) are produced in plants under various stress conditions and serve as important mediators in plant responses to stresses. Here, we show that the cytosolic glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenases (GAPCs) interact with the plasma membrane-associated phospholipase D (PLDd) to transduce the ROS hydrogen peroxide (H 2 O 2 ) signal in Arabidopsis thaliana. Genetic ablation of PLDd impeded stomatal response to abscisic acid (ABA) and H 2 O 2 , placing PLDd downstream of H 2 O 2 in mediating ABA-induced stomatal closure. To determine the molecular link between H 2 O 2 and PLDd, GAPC1 and GAPC2 were identified to bind to PLDd, and the interaction was demonstrated by coprecipitation using proteins expressed in Escherichia coli and yeast, surface plasmon resonance, and bimolecular fluorescence complementation. H 2 O 2 promoted the GAPC-PLDd interaction and PLDd activity. Knockout of GAPCs decreased ABA-and H 2 O 2 -induced activation of PLD and stomatal sensitivity to ABA. The loss of GAPCs or PLDd rendered plants less responsive to water deficits than the wild type. The results indicate that the H 2 O 2 -promoted interaction of GAPC and PLDd may provide a direct connection between membrane lipid-based signaling, energy metabolism and growth control in the plant response to ROS and water stress.

Phosphatidic Acid Binds to Cytosolic Glyceraldehyde-3-phosphate Dehydrogenase and Promotes Its Cleavage in Arabidopsis

“…We also identified known PA-binding proteins, such as annexin, a PA-binding protein found in animals (35), and some isoforms of heat shock protein (Hsp81-3), and tubulins (␣2/4) previously identified (36). Among the protein candidates identified, GAPDH was particularly of interest because it was recently found to interact directly with PLD␦ (29), an enzyme responsible for PA production. In addition, GAPDH is one of the key enzymes involved in the glycolytic pathway that is critical for energy production and biosynthesis for various compounds, including acetyl-CoA and lipids (Fig.…”

“…In addition, our recent result indicates that oxidized GAPCs bind stronger to PLD␦ (29). To test in what state GAPC binds to PA, we performed a PA-GAPC binding assay in the presence of hydrogen peroxide (H 2 O 2 ) and DTT as an oxidant and reductant, respectively.…”

“…Arabidopsis has two cytosolic GAPDHs, GAPC1 and GAPC2, and a recent study showed that both GAPCs interact with phospholipase D␦ (PLD␦), which directly produces signaling PA by hydrolyzing common membrane phospholipids (29). These observations raise intriguing questions about the function and significance of PA-GAPC interactions.…”

“…The knock-out mutants were screened for homozygotes as described previously (29). Transgenic lines overexpressing GAPC2 were generated by cloning GAPC2 (At1g13440) cDNA into p35S-FAST/eYFP vector and transforming the DNA constructs into Arabidopsis by floral dipping.…”

“…Gene Cloning, Protein Purification, and Activity Assay-GAPC was cloned into pET-28a-c(ϩ) vector, expressed in BL21(DE3)pLysS, and purified using nickel-nitrilotriacetic acid-agarose (Qiagen) as described previously (29). An NADdependent GAPDH activity assay was performed by spectrophotometric quantification (340 nm) of NADH produced in the reaction according to the method described previously (32).…”