The Plastidial Glyceraldehyde-3-Phosphate Dehydrogenase Is Critical for Abiotic Stress Response in Wheat

Abstract

Plastidial glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GAPCp) are ubiquitous proteins that play pivotal roles in plant metabolism and are involved in stress response. However, the mechanism of GAPCp's function in plant stress resistance process remains unclear. Here we isolated, identified, and characterized the TaGAPCp1 gene from Chinese Spring wheat for further investigation. Subcellular localization assay indicated that the TaGAPCp1 protein was localized in the plastid of tobacco (Nicotiana tobacum) protoplast. In addition, quantitative real-time PCR (qRT-PCR) unraveled that the expression of TaGAPCp1 (GenBank: MF477938.1) was evidently induced by osmotic stress and abscisic acid (ABA). This experiment also screened its interaction protein, cytochrome b6-f complex iron sulfite subunit (Cyt b6f), from the wheat cDNA library using TaGAPCp1 protein as a bait via the yeast two-hybrid system (Y2H) and the interaction between Cyt b6f and TaGAPCp1 was verified by bimolecular fluorescence complementation assay (BiFC). Moreover, H₂O₂ could also be used as a signal molecule to participate in the process of Cyt b6f response to abiotic stress. Subsequently, we found that the chlorophyll content in OE-TaGAPCp1 plants was significantly higher than that in wild type (WT) plants. In conclusion, our data revealed that TaGAPCp1 plays an important role in abiotic stress response in wheat and this stress resistance process may be completed by H₂O₂-mediated ABA signaling pathway.

Keywords: BiFC; TaGAPCp1; abscisic acid (ABA); hydrogen peroxide (H2O2); yeast two-hybrid system (Y2H).

Figure 1

Sequence analysis. (a) Alignment of the Amino acid sequence of TaGAPCps. The identical and 75% amino acid sequence similarity are separately indicated by mazarine and green color. Red underlined, Glyceraldehyde 3-phosphate dehydrogenase, NAD(P) binding domain (IPR0208); green underlined, Glyceraldehyde 3-phosphate dehydrogenase, catalytic domain (IPR0208). (b) Schematic diagram for exons/introns and upstream/downstream structures of TaGAPCp1. Exons, introns, upstream/downstream are indicated by yellow boxes, black horizontal lines, and blue boxes, respectively.

Figure 2

Expression profiles of TaGAPCp1 in wheat. (a–e) expression patterns of TaGAPCp1 in wheat leaves in response to various abiotic stresses: PEG, ABA (abscisic acid), H₂O₂, 4 °C, NaCl. (f–j) Expression patterns of TaGAPCp1 in wheat roots in response to various abiotic stresses: PEG, ABA (abscisic acid), H₂O₂, 4 °C, NaCl. Significant differences were detected by SPSS analysis. * p < 0.05, ** p ≤ 0.01.

Figure 3

Effects of ABA and H₂O₂ inhibitors on the expression of TaGAPCp1 in wheat and the effects of ABA and H₂O₂ on the stomatal of transgenic arabidopsis leaves. (a) expression of aGAPCp1 gene in wheat seedling leaves under PEG stress after pretreatment with H₂O₂ inhibitor (dimethylthiourea (DMTU)). (b) expression of TaGAPCp1 gene in wheat seedling leaves under PEG stress after ABA inhibitor (tungstate) pretreatment. Bars are shown as the mean ± Standard Deviation (SD). Significant differences were detected by SPSS analysis. * p < 0.05, ** p ≤ 0.01. (c,d) Stomatal size of WT and OE-TaGAPCp1 plants. Stomatal closure was observed after incubation of mature leaves in ABA (0, 5 and 10 μM) and H₂O₂ (0, 10, and 100 μM) buffers for 2 h, respectively. Each experiment was performed in triplicate. Bar = 10 μm.

Figure 4

Subcellular localization of TaGAPCp1 in tobacco cells. (a) Structure of TaGAPCp1-GFP fusion expression vector. (b) The images showed the expression of the pCaMV35s: TaGAPCp1-GFP fusion protein in tobacco cells. All of the images were obtained using a confocal microscope. Bar = 20 μm.

Figure 5

Screening of yeast two-hybrid. (a) Gel electrophoresis analysis of colony PCR about the Y2HGold Yeast Strain transformed with pGBKT7-TaGAPCp1 plasmids (number 1–6) and pGBKT7 plasmids (number 7). Mark is DL 2000 molecular marker and from top to bottom are 2000, 1000, 750, 500, 200 and 100. (b) Determination of autoactivation and toxicity of the bait vector in Y2HGold Yeast Strain on different selection mediums. The first line was the result of transforming pGBKT7-TaGAPCp1 into Y2HGold Yeast Strain alone; the second line (converting pGBKT7 to Y2HGold Yeast Strain) was used as a negative control; the third line was to convert pGBKT7-53 and pGADT7-T into Y2HGold Yeast Strain as positive control. (c) Screening diagram of TaGAPCp1. Blue clones indicated positive results, whereas white or absent clones were negative; (d) PCR amplification of positive prey plasmids by T7 primers. Lane P1–P9: PCR products amplified from the positives P1–P9 (responding to prey vectors P1–P9); Lane Mark: DL 2000 marker.

Figure 6

Identification of prey proteins interacting with TaGAPCp1. (a) Confirmation of true positive clones by small-scale Y2H assay. First row and second row: pGBKT7-TaGAPCp1 plasmid and the respective prey 1 to prey 7 plasmids (P1–P7) was co-transformed into Y2HGold Yeast Strain then staked on QDO/X/A plates and DDO/X/A plates, respectively. Third row and fourth row: pGBKT7-TaGAPCp1 plasmid and pGBKT7 empty bait was co-transformed into Y2HGold Yeast Strain then staked on QDO/X/A plates and DDO/X/A plates, respectively. PC indicates a positive control, NC indicates a negative control. (b) BiFC assay of the interaction between TaGAPCp1 and Cyt b6f proteins in tobacco leaf protoplasts. The pSPYNE-TaGAPCp1 and pSPYCE-Cyt b6f constructs were co-infiltrated in tobacco by Agrobacterium. The YFP fluorescence was detected by confocal laser scanning microscopy. Co-transformants of pSPYNE-TaGAPCp1 and pSPYCE as well as pSPYNE and pSPYCE-Cyt b6f were used as negative controls. Bar = 20 μm.

Figure 6

Identification of prey proteins interacting with TaGAPCp1. (a) Confirmation of true positive clones by small-scale Y2H assay. First row and second row: pGBKT7-TaGAPCp1 plasmid and the respective prey 1 to prey 7 plasmids (P1–P7) was co-transformed into Y2HGold Yeast Strain then staked on QDO/X/A plates and DDO/X/A plates, respectively. Third row and fourth row: pGBKT7-TaGAPCp1 plasmid and pGBKT7 empty bait was co-transformed into Y2HGold Yeast Strain then staked on QDO/X/A plates and DDO/X/A plates, respectively. PC indicates a positive control, NC indicates a negative control. (b) BiFC assay of the interaction between TaGAPCp1 and Cyt b6f proteins in tobacco leaf protoplasts. The pSPYNE-TaGAPCp1 and pSPYCE-Cyt b6f constructs were co-infiltrated in tobacco by Agrobacterium. The YFP fluorescence was detected by confocal laser scanning microscopy. Co-transformants of pSPYNE-TaGAPCp1 and pSPYCE as well as pSPYNE and pSPYCE-Cyt b6f were used as negative controls. Bar = 20 μm.

Figure 7

Chlorophyll content in Arabidopsis plants and Expression patterns of Cyt b6f in wheat leave. (a) Chlorophyll content in OE-TaGAPCp1 plants and wild-type plants. OE represents OE-TaGAPCp1 plants; WT represents wild-type plants. (b) Expression patterns of Cyt b6f in wheat leaves in response to various abiotic stresses: PEG, ABA and H₂O₂. Bars are shown as the mean ± SD. Significant differences were detected by SPSS analysis. * p < 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Figure 8

Model of how TaGAPCp1 responds to drought stress. Drought stress and H₂O₂ stress significantly increased the expression levels of TaGAPCp1 and Cyt b6f, respectively. TaGAPCp1 plays an active role in response to drought stress by H₂O₂₋dependent ABA signaling pathway. The interaction between TaGAPCp1 and Cyt b6f has been confirmed. TaGAPCp1 also promotes the biosynthesis of chlorophyll, which is a natural component of Cyt b6f. Cyt b6f can reduce the accumulation of reactive oxygen species (ROS) and then respond to abiotic threats. Among them, (+) represents promotion and (−) represents inhibition. Straight lines represent direct effects and dashed lines represent indirect effects.