TaWRKY24 interacts with TaERFL1a
Our previous study found that the TaERFL1a played important and positive roles in wheat responses drought stress (Gao et al., 2018). In order to elucidate the molecular regulator mechanism of TaERFL1a-mediated drought tolerance, Y2H was used to screen its interaction factors by using yeast cDNA library (Fig. S1), which derived from leaves of drought-stressed wheat. WRKY24 (LOC109755167), a WRKY family transcription factor, was screened from TaERFL1a-interaction proteins (Table S2). Then, CDS of WRKY24 was screened in wheat IWGSC database (http://www.wheatgenome.org/), and found that WRKY24 is located on the chromosome 1 and contains TraseCS1A02G121500.1 (TaWRKY24-1A) and TraesCS1D02G122400.1 (TaWRKY-1D) two homologous copies (Fig. S2). The qPCR measured their transcription levels found that TaWRKY24-1D had more highly expressed than TaWRKY24-1A under drought stress (Fig. 1), suggesting that TaWRKY24-1D could play important roles under drought stress. Thus, TaWRKY24-1D was used to explore the function of TaWRKY24 under drought stress in wheat.
TaWRKY24-1D, contains one exon and encodes a putative protein with 194-amino acids (possesses WRKYGQK sequence and a C2HC zinc finger motif) (Fig. S3A and S3B), were cloned and inserted into pGADT7 yeast vector to confirm the interaction between TaWRKY24-1D and TaERFL1a. Results showed that the combination of BD-TaERFL1a + AD-TaWRKY24-1D was grown well under SD mediums of -Trp/-Leu/-His/-Ade, while the combinations of BD-TaERFL1a + AD, BD + AD-TaWRKY24-1D, and BD + AD were not grown (Fig. 2A), suggested that TaWRKY24-1D and TaERFL1a was interacted in yeast cells. And their interaction was further validated by using the BiFC, results found that co-expression of YN-TaEFRL1a and YC-TaWRKY24-1D was co-localized in the nuclear of tobacco leaves (Fig. 2B). These results demonstrated that TaWRKY24-1D interacts with TaERFL1a.
Silencing of TaWRKY24 reduces drought tolerance in wheat seedlings
To verify the function of TaWRKY24 under drought stress, the silencing of TaWRKY24 two copies in wheat genome was performed by using BSMV-VIGS. To simultaneously silence of TaWRKY24 gene, a high similarity cDNA fragment of TaWRKY24 two copies (Fig. S2) was selected and constructed into BSMV-VIGS-TaWRKY24 vectors. And then, two-weeks-old wheat plants were inoculated with the BSMV-VIGS-TaWRKY24 or BSMV-VIGS-GFP (control) vectors, the wheat plants showed exhibited growth or photobleaching phenotype after inoculation for 8 days (Fig. S4A). Compare with the control, the expression levels of TaWRKY24 were remarkably decreased by 51.6% in BSMV-VIGS-TaWRKY24-inoculated wheat plants (Fig. S4B), suggested that TaWRKY24 was successfully silenced in wheat. Moreover, the expression levels of TaERFL1a were also significantly decreased by 67.6% in above silenced of TaWRKY24 wheat plants (Fig. S4C), suggested that TaWRKY24 regulated the expression of TaERFL1a.
Subsequently, the successfully silenced TaWRKY24 wheat plants were treated with or without 20%PEG in Hoagland solutions. After 3d drought stress, BSMV-VIGS-TaWRKY24-inoculated wheat plants exhibited more curled and wilted leaves than the control (Fig. 3A and 3B), and the relative water content was significantly decreased by 22.7% compared with control (Fig. 3C). Besides these symptoms, the biomass of fresh or wry weight was significantly reduced by 39.1% or 21.6% in BSMV-VIGS-TaWRKY24-inoculated wheat seedlings compared with the BSMV-GFP-inoculated seedlings, respectively (Fig. 3D and 3E). Moreover, the expression levels of TaWRKY24 and TaERFL1a were also significantly decreased by 89.3% and 76.9% in BSMV-VIGS-TaWRKY24-inoculated wheat seedlings (Fig. 3F and 3G), respectively.
Silencing of TaWRKY24 increases the ROS accumulation and decreases the antioxidant activity of wheat seedlings under drought stress
After 5 days drought stress, the ROS accumulation was determined by using the DAB and NBT staining, which represent the content of O2 and H2O2. Under drought stress, the leaves of BSMV-VIGS-TaWRKY24-inoculated wheat seedlings were deeper ROS staining than those in BSMV-GFP-inoculated seedlings (Fig. 4A and 4B). Moreover, contents of MDA and H2O2, which acted as the stress marker, were significantly increased by 62.65% and 24.45% in leaves of BSMV-VIGS-TaWRKY24-inoculated wheat seedlings (Fig. 4C and 4D).
Furthermore, the activities of APX, SOD, POD, and CAT were measured in these BSMV-VIGS-TaWRKY24-inoculated wheat seedlings. compared to control, the activities of APX, SOD, and CAT were decreased by 50.2%, 16.6%, and 9.9% in leaves of BSMV-VIGS-TaWRKY24-inoculated wheat seedlings after drought stress, excepted to POD (Fig. 5A ~ 5D). Similar patterns were found in the ASA and GSH content, which decreased by 36.2% and 20.2% in leaves of the above BSMV-VIGS-TaWRKY24-inoculated wheat seedlings (Fig. 5E and 5F), respectively.
Silencing of TaWRKY24 changes the expression of stress response genes in wheat seedlings under drought stress
To investigate the molecular role of TaWRKY24 gene under drought tolerance, some stress response genes, which had been studied and associated with the drought tolerance (Kang et al., 2013), were selected and measured by using qPCR. Compare with the control, the expression levels of ASA-GSH system genes were significantly decreased in leaves of BSMV-VIGS-TaWRKY74-inoculated wheat plants under drought stress, respectively. Such as, the expression levels of APX, DHAR, MDHAR, GR, GSH, GST, GPX1, and GPX2 were separately decreased by 44.7%, 99.9%, 65.9%, 36.0%, 80.2%, 44.7%, 67.6%, and 16.2% in leaves of the BSMV-VIGS-TaWRKY24-inoculated wheat plants (Fig. 6), respectively. These results suggested that TaWRKY24 regulated ASA-GSH system gene expression under wheat drought stress, especially of DHAR gene.
TaWRKY24 and TaERFL1a binds to the promoter of DHAR and actives its expression under drought stress
To confirm the regulatory relationship between TaWRKY24 and ASA-GSH system genes, the Y1H and dual-luciferase assays were separately performed in this study. Interestingly that there was three WRKY core motifs in DHAR promoter, and others ASA-GSH system genes only had one or two WRKY core motifs(Fig. S5). Thus, the binding between TaWRKY24 and DHAR promoter was used for next experiment. The promoter of DHAR was cloned into yeast pHIS2.0 vector, and the TaWRKY24 or TaERFL1a was inserted into pGADT7. And then, the DHAR promoter and TaWRKY24 or TaERFL1a were cotransformed in Y187. Results showed that the yeast cells of cotransformed with TaWRKY24-AD and pHIS2.0 vector were inhibited growth on SD/-Trp/-Leu/-His media by 50 mM 3-aminotriazole (3-AT) (Fig. 7A), which a inhibitor of His synthase, while those growth of TaWRKY24 and DHAR promoter were grown well, suggested that TaWRKY24 could be bind the promoter of DHAR. Moreover, the cotransformed of TaERFL1a-AD and pHIS2.0 vector was not completely inhibited by 50 mM 3-AT, but they were less growth than that in TaERFL1a-AD and DHAR promoter, suggested that TaERFL1a could also be bind the promoter of DHAR.
Moreover, the DHAR promoter and TaWRKY24 and TaERFL1a were co-transformed in tobacco leaves by using LCI and LUC assays. Results shown that the LUC fluorescence signals of TaWRKY24 + TaERFL1a + Pro-DHAR were higher than only TaWRKY24 + Pro-DHAR or TaERFL1a + Pro-DHAR (Fig. 7B). Moreover, the LUC fluorescence signals of TaWRKY24 + Pro-TaDHAR were also higher than TaERFL1a + Pro-DHAR (Fig. 7B). Consistently, the ratios of LUC/REN was highest in TaWRKY24 + TaERFL1a + Pro-TaDHAR, which was higher 2.42-folds and 1.84-folds than TaWRKY24 + Pro-TaDHAR and TaERFL1a + Pro-DHAR (Fig. 7C), respectively. These results suggested that TaWRKY24 was more regulated DHAR expression than TaERFL1a by binding its promoter under drought stress.