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 (PLDδ) to transduce the ROS hydrogen peroxide (H(2)O(2)) signal in Arabidopsis thaliana. Genetic ablation of PLDδ impeded stomatal response to abscisic acid (ABA) and H(2)O(2), placing PLDδ downstream of H(2)O(2) in mediating ABA-induced stomatal closure. To determine the molecular link between H(2)O(2) and PLDδ, GAPC1 and GAPC2 were identified to bind to PLDδ, 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-PLDδ interaction and PLDδ 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 PLDδ 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 PLDδ may provide a direct connection between membrane lipid-based signaling, energy metabolism and growth control in the plant response to ROS and water stress.

Figure 1.

Decreased Response of pldδ Plants to H₂O₂ and ABA. (A) ABA-induced PA production in leaf protoplasts of pldα1, pldδ, pldα1 pldδ, PLDδ-complementation (COM), and the wild type (WT). Values are means ± se (n = 3). (B) Stomatal closure induced by 25 µM ABA or 100 µM H₂O₂. Values are means ± se (n = 50). (C) Representative image of ROS production in guard cells, visualized by fluorescent dye. +ABA, epidermal peels were loaded with H₂DCF-DA for 10 min followed by addition of 25 µM ABA for 5 min; –ABA, no ABA added. Bars = 50 µm. (D) Quantification of ROS production based on fluorescence intensity (mean pixel intensity). Values are means ± se (n = 50). Columns with different letters are significantly different from each other (ANOVA, P < 0.05). [See online article for color version of this figure.]

Figure 2.

Interaction of GAPC with PLDδ. (A) Immunoblotting of proteins after coprecipitation using E. coli–expressed GST-PLDδ and His-GAPC1/2, as affected by H₂O₂ (100 μM) and DTT (100 μM). i, Coprecipitation of His-GAPC1 with GST-PLDδ. GAPC1, immunoblotting of GAPC1 using anti-His antibody for the precipitates; PLDδ, the starting GST-PLDδ used for precipitation. ii, Coprecipitation of GST-PLDδ with His-GAPC2. PLDδ, immunoblotting of PLDδ using anti-GST antibody for the precipitates. GAPC2, the starting His-GAPC2 used for precipitation. DTT was added before the addition of H₂O₂ when both were applied. (B) Immunoblotting of coprecipitated GAPC and PLDδ that were coexpressed in yeast grown in the presence or absence of added H₂O₂ (20 μM). i and ii, Reciprocal pulldown of PLDδ and GAPC1 and GAPC2, respectively. PLDδ was fused with a FLAG tag and GAPC1or GAPC2 with a cMyc tag. GAPC1 or GAPC2 band indicates immunoblotting with cMyc antibody against the sample precipitated with FLAG antibody–conjugated agarose beads. PLDδ band indicates immunoblotting with FLAG antibody against the sample precipitated with cMyc antibody for GAPC1 or GAPC2. (C) Quantitative SPR analysis of PLDδ binding to GAPC1. GAPC1 (no H₂O₂ treatment or pretreated with 100 µM H₂O₂) was first immobilized on the NTA chip followed by injection of GST or GST-PLDδ. (D) Representative confocal images of BiFC. Green color represents YFP fluorescence, indicating interaction of GAPC with PLDδ. PLDδ-YFP^C was cotransformed with GAPC1-YFP^N or GAPC2-YFP^N into tobacco leaves by infiltration. Bars = 50 µm.

Figure 3.

Oxidized GAPC Promotes PLDδ Activity. (A) H₂O₂ inhibition of GAPC1 and GAPC2 activities. (B) GAPC promotion of PLDδ activity under oxidative conditions. Equal molar ratios of PLDδ and GAPC proteins were used. PLDδ activity was assayed in the presence of GAPC1 (i) or GAPC2 (ii) under different conditions as indicated; 100 μM DTT or 100 μM H₂O₂ was used as indicated. Values are means ± se (n = 3). Different letters indicate significant differences (ANOVA, P < 0.05).

Figure 4.

H₂O₂ Effects on GAPC and PLDδ Activities. (A) RT-PCR detection of GAPC1 and GAPC2 expression in the leaves of wild-type (WT) and mutant plants. 18S rRNA was a control confirming the synthesis of cDNA. (B) GAPDH activity in the total protein extracted from the leaves of wild-type and mutant plants. (C) GAPDH activity using protein extracted from protoplasts after 1 mM H₂O₂ treatment. (D) H₂O₂-promoted PA production in protoplasts. Values are means ± se (n = 3). Different letters mark significant differences from each other (ANOVA, P < 0.05).

Figure 5.

PA Content of GAPC and PLDδ Mutant Leaves in Response to ABA. (A) Total PA content of leaves harvested at different times after spraying with ABA (100 µM). WT, the wild type. (B) PA molecular species in leaves of the wild type and mutants treated with ABA for 10 min. Values are means ± se (n = 5). Asterisks indicate significant difference from the wild type at the same time point of ABA treatment (P < 0.05, t test).

Figure 6.

Response of GAPC and PLDδ Mutants to ABA and Water Deficits. (A) Changes in stomatal aperture after ABA (25 μM) or H₂O₂ (100 μM) treatment. Values are means ± se (n = 50). Different letters mark significant differences from each other (ANOVA, P < 0.05). WT, the wild type. (B) Stomatal conductance, cumulative water transpiration, photosynthesis, and dry weight. Asterisks mark significant difference from the wild type under the same growth condition. Values are means ± se (n = 16).

Figure 7.

Increased Water Loss in GAPC-KO and PLDδ-KO Arabidopsis Plants. (A) Instant WUE of wild-type (WT) and mutant plants under 100 and 60% FC. Arabidopsis seedlings were transplanted to pots and maintained at 100% FC and 60% FC. Instant WUE was calculated as the ratio of the photosynthetic rate to stomatal conductance; measurements were taken after the first 4 d after the onset of required stress. Asterisks indicate significant difference from the wild type. Values are means ± se (n = 16; *P < 0.05, t test). (B) Increased dehydration of GAPC-KO and PLDδ-KO plants when FC was not maintained. Plants (25 d old) were fully watered and then left unwatered for 16 d when the photograph was taken. D1 and D2 are GAPC1 and 2 double KOs gapc1-1 gapc2-1 and gapc1-1 gapc2-2, respectively. T1 and T2 are GAPC1, GAPC2, and PLDδ triple KOs gapc1-1 gapc2-1 pldδ and gapc1-1 gapc2-2 pldδ, respectively. [See online article for color version of this figure.]

Figure 8.

A Proposed Model for the Role of PLD/PA in Regulating ROS Production and Response under Water Deficits. This model depicts only the known targets of PLD/PA in ABA-mediated stomatal closure; other ABA regulators are not included in this model. GAPCox refers to oxidized, catalytically inactive GAPC that interacts with PLDδ and promotes PLDδ activity. GAPCred refers to reduced, active GAPC that converts glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate (1,3-bisPG) with NADH production. PLDα1 uses preferably phosphatidylcholine (PC), whereas PLDδ prefers phosphatidylethanolamine (PE) as substrate. Solid arrows indicate established links, and dashed arrows denote putative links. PM, plasma membrane.