Expression of TaGST1 in wheat plants suffering from Cu stress
Transcripts of the TaGST1 gene have been measured in wheat roots and leaves, and it was observed at higher expression levels in the leaves than in roots (Fig. 1A). Thus, TaGST1 expression levels were further measured in wheat leaves. Under different Cu concentrations, the transcripts of the TaGST1 gene were found to be significantly induced at Cu concentrations of 50 or 100 µM (Fig. 1B). It has been reported that induced genes or proteins could play important roles in plant responses to abiotic stresses [17], demonstrating that TaGST1 could function in the Cu stress response, especially at higher Cu concentrations.
Identification of the regulators binding to the TaGST1 promoter
To explore the regulatory mechanisms of TaGST1 under Cu stress, its promoter was cloned (Fig. S1), and then was used for Y1H screening (Fig. S2). Upon screening, positive clones were obtained and subsequently sequenced, and 28 putative proteins were identified and functionally annotated (Table S2). Among these regulators, two WRKY transcription factors have been identified. TaWRKY74 (GenBank accession number: EF368359.1), a WRKY transcription factor, exhibited the higher homology relative to AetWRKY6 in Aegilops (Table S3), and its sequence had a 98.9% similarity to Chinese Spring (CS) (Fig. S3). Thus, the TaWRKY74 transcription factor was used in following analyses.
To confirm the regulatory relationship between TaWRKY74 and TaGST1, the Y1H assay was performed using the pGADT7-TaWRKY74 and pAbAi-TaGST1 promoters. There were eight W-box core elements in the TaGST1 promoter (Fig. S1). Full-length or partially-deleted TaGST1 promoter fragments containing different W-boxes, i.e., P1 (eight W-boxes), P2 (five W-boxes), P3 (three W-boxes), P4 (three W-boxes), and P5 (one W-box) (Fig. 2A), were separately transformed into the Y1HGold yeast strain. Then, four AbA concentrations (0, 100, 150, and 200 ng/mL) were added to test the self-activation of these promoter fragments, thereby identifying positive strains (Fig. 2B). Under different AbA concentrations, the yeast strains harboring the P1, P2, and P3 fragments exhibited similar growth to those harboring the P4 and P5 fragments, which grew worse under 100~200 ng/mL AbA addition (Fig. 2B). This suggested that the former fragments had a stronger self-activation capacity than P4 and P5; therefore, P4 and P5 were better suited for further Y1H assays. Thus, the prey vector pGADT7-TaWRKY74 was transformed into Y1HGold strains containing the P4 and P5 fragments, respectively. In the presence of 100 ng/mL AbA, the P4 and P5 fragment yeast stains were activated by the TaWRKY74 prey vector (Fig. 2C and 2D), suggesting that the TaWRKY74 transcription factor could bind to the TaGST1 promoter.
Binding between TaWRKY74 and TaGST1 promoter via W-box
LUC assay was used to explore whether TaWRKY74 affected the transcriptional activity of the TaGST1 promoter. LUC reporter vectors were constructed using two different TaGST1 promoters, which contained normal or mutant W-box cis-element (Fig. 3A and B). In addition, effector vectors of 35S-TaWRKY74-GFP or 35S-GFP were also constructed (Fig. 3B). Compared to the 35S-GFP vector (control), the ratio of LUC/REN in the P5 fragment of the TaGST1 promoter was upregulated by 2.04-fold, while this ratio was insignificantly changed in the mutated P5 fragment (Fig. 3B and 3C). These suggested that TaWRKY74 interacted with TaGST1 promoter via the W-box.
Characterization and phylogenetic tree of TaWRKY74
To characterize TaWRKY74 in wheat, a blast search was performed in the IWGSC database (http://www.wheatgenome.org/) and TaWRKY74 was found to possess the highest similarity with TraesCS5D02G190800 (Fig. S4), suggesting that it localizated on 5D chromosome. Sequence analysis showed that TaWRKY74 contained two exons, one intron, and encoded a putative protein with 351-amino acids. It possessed a highly conserved WRKYGQK sequence and a C2HC zinc finger motif (Fig. S5), suggesting that it belonged to group III of the WRKY family. Phylogenetic analyses indicated that TaWRKY74 had high similarities (≥ 85%) to AetWRKY6, TaWRKY, TaWRKY5, and OsWRKY30 or OsWRKY74 (Fig. S6). However, their functions in responses to metal stresses have not been identified.
Subcellular localization and transcriptional activity of TaWRKY74
In order to measure the TaWRKY74 localization in cells, its CDS was fused with a vector, which driven by a 35S promoter and contained a GFP-encoding gene, to produce TaWRKY74-GFP protein (Fig. 4A). TaWRKY74-GFP was then transiently introduced into leaf cells of tobacco plants. The fluorescence signal of TaWRKY74-GFP was specifically observed in the nucleus compared with the control (GFP protein), which was localized in both the nucleus and cytoplasm (Fig. 4B), suggesting that TaWRKY74 was a nucleus-localized protein.
To detect the transactivation activity of TaWRKY74, its CDS was inserted into the pGBKT7 vector to construct a pGBKT7-TaWRKY74 fusion protein (Fig. 4C). Afterward, the construct was transformed into the Y2HGold yeast strain in SD medium without tryptophan (SD/-Trp). All yeast cells grew well in the SD/-Trp medium at 30 °C for 2~3 days without X-α-gal. However, when X-α-gal was added in this medium, TaWRKY74 and the positive control (pGBKT7-P53) turned blue, whereas the negative control (pGBKT7-Lam) remained unchanged (Fig. 4D). This indicated that TaWRKY74 possessed the potential of transcriptional activation.
Expression of TaWRKY74 under metal stresses
To determine whether the TaWRKY74 gene responded to different metal stresses, transcripts of TaWRKY74 were determined in leaves of wheat plants suffering from 50 µM Cu, 50 µM Cd, and 50 µM Al. qPCR results indicated that transcripts of TaWRKY74 were significantly induced by 27.64-, 12.54-, and 11.26-fold under Cu, Cd, and Al stresses, respectively (Fig. 5). Based on these results, it was speculated that the TaWRKY74 gene could function in metal-induced stresses, especially upon Cu exposure.
Function of TaWRKY74 in response to Cu stress
BSMV-VIGS experiment was used to demonstrate the role of TaWRKY74 for Cu tolerance. A 207 bp cDNA fragment, which shared high similarity (99.7%) among three copies (Fig. S7), was selected and constructed into BSMV-derived vectors to simultaneously silence three copies of the TaWRKY74 gene in wheat genome. Wheat plants inoculated with the BSMV-TaWRKY74 or BSMV-GFP (control) vectors exhibited visible photobleaching phenotype at 8 days after inoculation (Fig. S8A). Moreover, the TaWRKY74 transcription level remarkably decreased (by 43%) in leaves of BSMVS-TaWRKY74-inoculated wheat plants compared to the controls (Fig. S8B), suggesting that the TaWRKY74 gene was successfully silenced in wheat plants. The TaGST1 transcript levels also were decreased by 61% in TaWRKY74-silenced wheat plants (Fig. S8C). These results suggested that the expression of TaGST1 could be controlled by the TaWRKY74.
Subsequently, BSMV-TaWRKY74- and BSMV-GFP-inoculated wheat plants were separately transferred to Hoagland solutions supplemented with or without 100 μM Cu. After 10-d stress, the BSMV-VIGS-TaWRKY74-inoculated wheat plants exhibited more deleterious phenotypes (e.g., curled and wilted leaves, and stunted growth), compared to the BSMV-GFP-inoculated wheat plants (Fig. 6A). The transcripts of TaWRKY74 and TaGST1 were also significantly decreased in BSMV-TaWRKY74-inoculated wheat plants (Fig. 6B and C). Similar to TaGST1 expression, the GST activity and GSH content were also significantly decreased in BSMV-TaWRKY74- inoculated wheat plants (Fig. 6D and E). The biomass of the BSMV-TaWRKY74-inoculated wheat plants were also decreased, especially at Cu stress conditions (Fig. 7A), whereas both H2O2 and MDA contents were significantly increased in these wheat plants (Fig. 7B and C). Additionally, Cu content dramatically increased in root and shoot tissues of the BSMV-VIGS-TaWRKY74-inoculated wheat plants (Fig. 7D). These results showed that TaWRKY74 could be involved in Cu tolerance of wheat plants, by regulating expression of TaGST1, which could catalyze the interaction of GSH with H2O2.
To further explore the Cu tolerance of BSMV-VIGS-TaWRKY74-inoculated wheat plants, contents of other nutrient elements were also measured in above plants. Similar with Cu content, the translocation factor of Cu, which was calculated using Cu content and plant biomass, was significantly also increased to 1.76-fold in these plants (Fig. S9), suggesting that more Cu accumulation in BSMV-VIGS-TaWRKY74-inoculated plants. And the translocation factors of other nutrients, K (1.59-fold), Mn (1.48-fold), Ca (1.25-fold), and Fe (1.17-fold) were similar with Cu between these two inoculated wheat plants (Fig. S9), whilst B (0.47-fold), Mo (0.89-fold) , and Al (0.71-fold) were opposed with Cu (Fig. S9), speculating that Cu accumulation would lead to the imbalance of other nutrient elements in wheat plants.
Transiently ectopic expression assay was performed to obtain TaWRKY74 over-expressed tobacco plants, in which this wheat gene was expressed at high level in tobacco plants. Under 50 μM Cu stress, the expression of TaWRKY74 was rapidly induced and peaked at 6 h, and then declined (Fig. 8A); GSH content increased by 22.5% , while MDA content decreased by 23.2 % at 6 h in TaWRKY74 transient over-expressed plants (Fig. 8B and C). These data further suggested the role of TaWRKY74 in Cu stress response via GSH metabolism.
Effect of exogenous GSH application on Cu tolerance of wheat seedling
To determine the effect of exogenous GSH supplement on Cu toxicity. Both BSMV-inoculated wheat plants were separately transferred to Control, Cu stress, Cu + GSH, and Cu+GSH+BSO mediums. Under Cu stress, exogenous GSH application significantly alleviated Cu-stress-injured morphology in both BSMV-TaWRKY74- and BSMV-GFP-inoculated wheat plants, whereas the above phenotype was broken by exogenous application with BSO (an inhibitor of GSH biosynthesis) under Cu + GSH treatment (Figs. 9A and S10). These phenotypic results were further confirmed by quantitative analysis with the remarkably changed of plants biomass, MDA and H2O2 contents (Fig. 9B-D). In addition, the application of exogenous GSH markedly decreased the GST activity, GSH contents, and Cu contents in both the BSMV-TaWRKY74- and BSMV-GFP-inoculated wheat plants after Cu stress for 10 days (Fig.9E-G).