The specific W-boxes of GAPC5 promoter bound by TaWRKY are involved in drought stress response in wheat.

Drought is one of the most common abiotic stresses, and can limit wheat yield, crops and productivity. GAPCs play vital roles under drought stress conditions in multiple species. The aim of this experiment was to determine the regulatory mechanism of TaGAPC5 under drought stress. In this study, the genes and promoters of TaGAPC5 in diverse drought-tolerant cultivars were cloned. The amino acid sequences were conserved, while the promoter fragments were not identical. Under abiotic stress, the expression level of TaGAPC5 was substantially different among the diverse drought-tolerant cultivars and the promoter activities were significantly improved. The yeast one-hybrid system and Electrophoretic mobility shift assay (EMSA) demonstrated that TaWRKYs bound to specific W-boxes: TaWRKY28, TaWRKY33, TaWRKY40 and TaWRKY47 bind to G/ATGACG/C/A, C/G/ATGACG, C/ATGACC and C/ATGACC/G, respectively. By analyzing different 5' deletion mutants of these promoters, it was determined that these W-boxes in CW-TaGAPC5 promoter (-1262, -1202, -904, -880 and -207) and ZY-TaGAPC5 promoter (-697 and -220) bound by these four TaWRKYs and were functional under drought stress. The deletion or addition of specific W-boxes in the promoter fragments significantly restrained or advanced the promoter activity under drought stress, and these results further confirmed that these W-boxes play vital roles in improving transcription levels under drought stress. The W-boxes in CW-TaGAPC5P (-1262, -1202, -904, -880 and -207) and ZY-TaGAPC5P (-697 and -220) were identified as the key cis-elements for responding to drought stress and were bound by the transcription factor TaWRKY.

Cytosolic glyceraldehyde-3-phosphate dehydrogenase 2/5/6 increase drought tolerance via stomatal movement and reactive oxygen species scavenging in wheat.

Drought is a major threat to wheat growth and crop productivity. However, there has been only limited success in developing drought-hardy cultivars. This lack of progress is due, at least in part, to a lack of understanding of the molecular mechanisms of drought tolerance in wheat. Here, we evaluated the potential role of three cytosolic glyceraldehyde-3-phosphate dehydrogenases (TaGAPC2/5/6) under drought stress in wheat and Arabidopsis. We found that TaGAPC2/5/6 all positively responded to drought stress via reactive oxygen species (ROS) scavenging and stomatal movement. The results of yeast co-transformation and electrophoretic mobility shift assay showed that TaWRKY33 acted as a direct regulator of TaGAPC2/5/6 genes. The dual luciferase reporter assay indicated that TaWRKY33 positively activated the expression of TaGAPC2/5/6. The results of bimolecular fluorescence complementation and yeast two-hybrid system demonstrated that TaGAPC2/5/6 interacted with phospholipase Dδ (PLDδ). We then demonstrated that TaGAPC2/5/6 positively promoted the activity of TaPLDδ in vitro and in vivo. Furthermore, lower PLDδ activity in RNAi wheat could lead to less PA accumulation, causing higher stomatal aperture sizes under drought stress. In summary, our results establish a new positive regulatory mechanism of TaGAPCs which helps wheat fine-tune their drought responses.

TaWRKY40 transcription factor positively regulate the expression of TaGAPC1 to enhance drought tolerance.

BACKGROUNDS: Drought stress is one of the major factors that affects wheat yield. Glyceraldehyde-3-Phosphate dehydrogenase (GAPDH) is a multifunctional enzyme that plays the important role in abiotic stress and plant development. However, in wheat, limited information about drought-responsive GAPC genes has been reported, and the mechanism underlying the regulation of the GAPC protein is unknown. RESULTS: In this study, we evaluated the potential role of GAPC1 in drought stress in wheat and Arabidopsis. We found that the overexpression of TaGAPC1 could enhance the tolerance to drought stress in transgenic Arabidopsis. Yeast one-hybrid library screening and EMSA showed that TaWRKY40 acts as a direct regulator of the TaGAPC1 gene. A dual luciferase reporter assay indicated that TaWRKY40 improved the TaGAPC1 promoter activity. The results of qRT-PCR in wheat protoplast cells with instantaneous overexpression of TaWRKY40 indicated that the expression level of TaGAPC1 induced by abiotic stress was upregulated by TaWRKY40. Moreover, TaGAPC1 promoted H2O2 detoxification in response to drought. CONCLUSION: These results demonstrate that the inducible transcription factor TaWRKY40 could activate the transcription of the TaGAPC1 gene, thereby increasing the tolerance of plants to drought stress.

The specific MYB binding sites bound by TaMYB in the GAPCp2/3 promoters are involved in the drought stress response in wheat.

BACKGROUND: Drought stress is one of the major abiotic stresses that affects plant growth and productivity. The GAPCp genes play important roles in drought stress tolerance in multiple species. The aim of this experiment was to identify the core cis-regulatory elements that may respond to drought stress in the GAPCp2 and GAPCp3 promoter sequences. RESULTS: In this study, the promoters of GAPCp2 and GAPCp3 were cloned. The promoter activities were significantly improved under abiotic stress via regulation of Rluc reporter gene expression, while promoter sequence analysis indicated that these fragments were not almost identical. In transgenic Arabidopsis with the expression of the GUS reporter gene under the control of one of these promoters, the activities of GUS were strong in almost all tissues except the seeds, and the activities were induced after abiotic stress. The yeast one-hybrid system and EMSA demonstrated that TaMYB bound TaGAPCp2P/3P. By analyzing different 5' deletion mutants of these promoters, it was determined that TaGAPCp2P (- 1312~ - 528) and TaGAPCp3P (- 2049~ - 610), including the MYB binding site, contained enhancer elements that increased gene expression levels under drought stress. We used an effector and a reporter to co-transform tobacco and found that TaMYB interacted with the specific MYB binding sites of TaGAPCp2P (- 1197~ - 635) and TaGAPCp3P (- 1456~ - 1144 and - 718~ - 610) in plant cells. Then, the Y1H system and EMSA assay demonstrated that these MYB binding sites in TaGAPCp2P (- 1135 and - 985) and TaGAPCp3P (- 1414 and - 665) were the target cis-elements of TaMYB. The deletion of the specific MYB binding sites in the promoter fragments significantly restrained the drought response, and these results confirmed that these MYB binding sites (AACTAAA/C) play vital roles in improving the transcription levels under drought stress. The results of qRT-PCR in wheat protoplasts transiently overexpressing TaMYB indicated that the expression of TaGAPCp2/3 induced by abiotic stress was upregulated by TaMYB. CONCLUSION: The MYB binding sites (AACTAAA/C) in TaGAPCp2P/3P were identified as the key cis-elements for responding to drought stress and were bound by the transcription factor TaMYB.