Function Analysis of Drought Resistance Related Gene TaGAPCs and TaWRKYs in Wheat

Backgrounds: Wheat (Triticum aestivum L.) is one of the most important food crops in the world. It faces various abiotic stresses during its growth. Drought is one of the main factors limiting the growth and development of wheat. Severe drought stress will Lead to a decline in wheat production. Cytoplasmic glyceraldehyde-3-phosphate dehydrogenase (GAPC) is an important member of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) family, which is widely present in plant cytoplasm. Plants play an important role in the process of primary metabolism and stress resistance. Result: In this study, a comparative transcriptomic analysis of the TaGAPCs-RNAi strain of Changwu 134 and the wild-type wheat seedlings of Changwu 134 under natural drought conditions was carried out. A total of 30067 differentially expressed genes were screened in RNAi strains and wild-type strains, of which 19,959 genes were up-regulated in RNAi strains and 10,108 genes were down-regulated in transcription. GO analysis shows that differential genes are mainly enriched in biological regulation, cellular processes, metabolic processes, and responses to stimuli. KEGG analysis showed that the differential genes were mainly concentrated in the biosynthesis of phenylpropane, plant hormone signal transduction and avonoid biosynthesis pathways. By analyzing the expression levels of differential transcription factors, the signicantly down-regulated transcription factor WRKY family member TaWRKY2 / 22/28/29/33/40/47/52 in wheat was screened out. The TaWRKY28/33/40/47 gene silencing line was successfully obtained using the barley stripe mosaic virus (BSMV-VIGS) technology. The plants with TaWRKY28/33/40/47 gene silenced were subjected to natural drought treatment, and physiological and biochemical index tests were carried out. The results showed that the growth status of gene-silenced plants was worse than that of wild-type plants, and the relative water content and chlorophyll content decreased. The content of MDA, H 2 O 2 and superoxide anion increases, the activity of antioxidant enzymes (SOD, POD, CAT) decreases, and the main osmotic adjustment substances in plants. When plants are under drought stress, it can remove excess active oxygen accumulated in plants due to drought, and has a protective effect on the integrity of cell membranes This experimental study shows that when subjected to drought stress, the antioxidant enzyme activity in plants will have an upward process. When subjected to drought stress, the TaWRKY28/33/40/47 gene-silencing line showed lower CAT SOD, POD activity than MOCK plants. The antioxidant enzyme activity of TaWRKY28/33/40/47 gene-silencing lines is lower than MOCK, which may reect the stronger oxidative damage of gene-silencing lines under drought stress, but the weaker ability of scavenging reactive oxygen species. These results indicate that silencing TaWRKY28/33/40/47 affects the antioxidant system, and TaWRKY28/33/40/47 may be involved in the ROS-mediated wheat response to drought signaling pathway. as fold compared with BSMV-γ using the 2 −ΔΔCT three biological and three technical replicates were conducted in the transcript proles of genes. Three independent experiments were conducted.

WRKY transcription factors (WRKY TFs) play an important role in the response of plants to abiotic stress.
WRKY can participate in the stress resistance of plants by binding to the speci c W-box on the promoter of the target gene to regulate the expression of the target gene. For example, Arabidopsis WRKY6 speci cally binds to the W-box on the RAV1 promoter, RAV1 decreased expression level promotes seed germination and early seedling development [13]. Arabidopsis TTG2 (a WRKY transcription factor) is involved in the regulation of GL2 transcription during epidermal cell differentiation [14]. After drought and salt stress, IbWRKY2 in sweet potatoes can up-regulate the expression of IbVQ4 and improve the abiotic stress tolerance [15]. The interaction between AtWRKY8 and AtVQ10 increases the resistance of Arabidopsis plants to gray mold. The interaction between AtWRKY8 and AtVQ9 plays an important role in the drought tolerance of Arabidopsis. The interaction between AtVQ16 and AtVQ23 and AtWRKY33 positively regulates the defense of plants against adversity [16][17][18].
Studies have shown that some members of the wheat transcription factor TaWRKY1/2/19/33/46/93 are overexpressed in Arabidopsis, signi cantly increasing the resistance of transgenic lines to abiotic stresses (drought, osmosis, salt and heat) [19][20][21][22]. The detached leaves of TaWRKY40-D VIGS (virusinduced gene silencing) wheat plants showed a green phenotype, while the Arabidopsis plants overexpressing TaWRKY40-D showed premature leaf senescence after JA and ABA treatment. Therefore, TaWRKY40-D may positively regulate leaf senescence by changing the biosynthesis and signal transduction of JA and ABA pathway genes [23]. The Arabidopsis strains overexpressing the TaWRKY142 gene are signi cantly more resistant to the fungal pathogen Colletotrichum, and this increased resistance is due to the increased expression of the JA signal marker gene AtPDF1.2 [24]. TaWRKY51 controls the formation of lateral roots by regulating ethylene biosynthesis [25]. In summary, the WRKY transcription factor in wheat is involved in a variety of biological processes in plants.
The gene silencing induced by barley stripe mosaic virus (BSMV) was determined to be the rst system to induce VIGS in monocots [26]. BSMV is a positive RNA virus. its genome consists of three RNA strands: α, β, and γ. The RNA α chain encodes the virus's replicase large subunit αa, and the RNA β chain encodes the coat protein and the tripartite movement protein TGB1, TGB2 and TGB3, RNA γ chain encodes replicase γa and multifunctional protein γb [26,27]. By constructing multiple cloning sites downstream of the γb gene of the γ chain, the foreign gene fragments can be inserted successfully. A BSMV viral vector for silencing the target gene was constructed [28]. BSMV-VIGS technology can be used to study the function of genes in plant growth, development and biotic and abiotic stresses. Zhang and colleague showed that wheat plants infected with BSMV-VIGS/TaNAC35 have an effect on wheat rust (Puccinia Triticina Pt) increased resistance to the pathogen THTT, indicating that TaNAC35 can act as a transcriptional activator and negatively regulate wheat Pt resistance [29]. The R2R3 MYB transcription factor TaMpc1-D4 located on the D chromosome of wheat was used to silence TaMpc1-D4 in wheat using VIGS technology, the results showed that the relative water content (RWC), proline content, and proline content of the TaMpc1-D4 gene silenced wheat were compared with the wild type the activity of antioxidant enzymes is signi cantly increased, which veri es that TaMpc1-D4 plays a negative regulatory role in wheat drought stress [30]. Li and colleague used the barley stripe mosaic virus-induced silencing (BSMV-VIGS) method to silence the calcium-dependent protein kinase 34 (TaCPK34) gene in the wheat genome, after 14 days of drought stress, compared with the wild type, the TaCPK34 gene was silenced seedlings have signi cantly reduced biomass and relative water content, and increased soluble sugar and MDA content [31]. In addition, BSMV-VIGS was used to verify the barley S-adenosylmethionine synthase 3 gene (HvSAMS3) actively regulates the tolerance of wild barley (Tibetan wild barley) to drought and salt stress [32]. Using BSMV-VIGS technology to silence TabZIP74, TabZIP74 silenced wheat plants have fewer lateral roots than control wheat seedlings, indicating that TabZIP74 is involved in the development of wheat roots [33].
The previous research of our research group found that TaGAPCs-RNAi strains have reduced drought tolerance, while the transgenic Arabidopsis overexpressed TaGAPC5 has signi cantly enhanced drought resistance. In order to further study the biological functions of wheat GAPC, the TaGAPCs-RNAi mutant strain of Chang Wu 134 and the wild-type strain of Chang Wu 134 were sequenced by transcriptome. The WRKY family member TaWRKY28/33/40/47, a member of the WRKY family that signi cantly downregulates transcription factors, was screened out in wheat. By studying the function of the droughtresistant transcription factor WRKY, further reveal the function of TaGAPC, and provide abundant genetic resources for subsequent transgenic breeding.

Results
GO and KEGG enrichment analysis of DEG in TaGAPCs-RNAi silencing strains and wild-type strains after drought stress Through the analysis of transcriptome data, a total of 30067 gene expression differences were obtained. Compared with the wild type, RNAi strains signi cantly up-regulated 19,959 genes and down-regulated 10,108 genes (Fig. 1a).
The GO enrichment analysis analysis of the differentially expressed genes between the gene-silencing line and the wild-type after drought treatment showed that the differentially expressed genes are involved in molecular function, cellular component and biological process. There are 49 sub-class molecular functional classi cations are involved under the three major categories. In molecular function, there are 9558 differential genes that are mainly enriched in binding function, and there are 9053 differential genes in catalytic activity function. In terms of cell components, there are 6601 differential genes mainly enriched in the cell composition, and 7133 differential genes in the cell membrane. In the biological process, there are 2210 differential genes in the biological regulation process, 5155 differential genes in the cellular process, and 4529 differential genes in the metabolic process. There are 1973 different genes in response to stimulus (Fig. 1b).

Analysis of differentially expressed transcription factors
After comparing the effective read sequence obtained by sequencing with the reference genome, the gene families encoding transcription factors were classi ed and counted. A total of 5,631 transcription factors were obtained, which were divided into 34 categories. Among them, the transcription factor families with a large number of genes are MYB, ABI3VP1, AP2 -EREBP, WRKY, etc. Through the analysis of differential transcription factor families, 8 transcription factor families related to drought stress were screened, and the number of differential genes, differential gene expression and the classi cation of the main KEGG pathways were analyzed. As the research team selected some wheat WRKY transcription factors through yeast one-hybridization in the early stage, this experiment analyzed the TaWRKY family of differential transcription factors of TaGAPC-RNAi strains, and the results showed that the number of down-regulated genes in the TaWRKY transcription factor family is the number of up-regulated genes. After comparing and analyzing the genes with signi cant differences in down-regulation, the wheat TaWRKY2/22/28/29/33/40/47/52 genes were screened for signi cant down-regulation (Table 1). These genes will be important candidates for subsequent experimental veri cation. Quantitative real-time PCR con rms gene expression pro les In order to verify the accuracy of the RNA-Seq results, 15 potentially important functions were randomly selected for qPCR analysis. The fold change data of RNA-Seq and qRT-PCR after 15 days of drought stress showed that among the 15 tested genes, the trend of drought-induced transcript accumulation changes was same (Fig. 2).
BSMV-mediated TaWRKY28/33/40/47 gene silencing decreases resistance to drought in wheat VIGS vector BSMV:PDSandBSMV: TaWRKYs were constructed as Fig. 3a ,linearize the recombinant vector. In vitro synthesis of RNA in linear plasmids (Fig. 3b). The bleaching and virus symptoms were observed after the wheat seedlings were inoculated for 10 days. The wheat leaves inoculated with Fes buffer (MOCK) remained green, and the leaves inoculated with BSMV:PDS virus appeared bleached symptom, while the leaves inoculated with BSMV:γ, BSMV: TaWRKY28/33/40/47 virus showed slight stripe chlorotic virus symptoms. It indicated that the VIGS system was successful (Fig. 3c). Through qPCR experiment, the gene expression of TaWRKY28 .4e-f). The content of proline and the antioxidant enzyme system (POD, SOD, and CAT enzymes) were signi cantly decreased in three transgenic lines after drought treatment as compared to the Mock and BSMV:γ ( Supplementary Fig. S2).

Discussion
Through the enrichment analysis of the differential gene GO, the differentially expressed genes of wheat after drought stress mainly perform molecular functions of binding and catalytic activity. Some differentially expressed genes are involved in the composition of cell membranes, organelles, and cells, and are involved in the process of cell metabolism and response to stimuli. Through the analysis of the enriched GO entries, the results show that drought stress induces the combination and catalysis of certain substances in the plant body, and responds to drought stress by protecting the organelles and cell membranes to maintain the integrity of the cell structure, maintain the normal function of the cell, and reduce The damage to the plant body maintains the normal physiological activity of the plant body by regulating the cell process and metabolic process to improve the adaptability of wheat to drought stress [34,35].  [37]. Dendrobium o cinale avonol synthase is overexpressed in Arabidopsis. DoFLS1 enhanced avonol accumulation and abiotic stress tolerance [38]. Transcriptome sequencing results in this experiment showed that 4CL and avonol synthase were signi cantly down-regulated in TaGAPCs silenced lines. TaGAPCs silencing in wheat may reduce phenylpropane biosynthesis and avonoid biosynthesis by regulating 4CL and avonol synthase, Which reduces the drought resistance of silent strains.
Transcription factor is the main regulator of transcription reprogramming. The expression of many TF genes is affected by drought, including auxin-responsive transcription factor (ARF), zinc nger protein transcription factor (C 2 H 2 ), GRAS, LOB, MYB Transcription factors such as, NAC, TCP, WRKY, and bZIP, which participate in plant response to drought stress by regulating downstream genes. In this experiment, transcription factors that respond to drought stress have up-regulated expression in TaGAPCs-RNAi strains. There are also down-regulated transcription factors. Through the analysis of transcriptome data, the number of down-regulated WRKY transcription factor family is 2.67 times the up-regulated number. Some signi cantly different WRKY transcription factors have been screened out. Studies have shown that the overexpression of SlWRKY8 in tomato is enhanced. In view of its tolerance to drought stress [39], WRKY46, WRKY54 and WRKY70 in Arabidopsis play a negative regulatory role in drought response [40]. The WRKY transcription factor was studied to further reveal the molecular mechanism of TaGAPCs in response to drought.
In this experiment, we successfully constructed the BSMV:TaWRKY28/33/40/47 silencing vector, and inoculated the recombinant vector in vitro transcription product into the two-leaf and one-heart stage Chinese spring wheat seedlings. The phenotype of the plants after the inoculation was analyzed and veri ed by real-time uorescent quantitative PCR. Indicating that the TaWRKY28/33/40/47 gene silencing line was successfully obtained.
After drought stress, it will affect the growth and photosynthesis of plants. The water status of plant tissues has a certain relationship with their physiological functions. The relative water content (RWC) index can re ect the characteristics of changes in plant water status. The reduction of chlorophyll content under drought stress is also considered a typical symptom of oxidative stress, which may be the result of pigment photooxidation and chlorophyll degradation. The loss of chlorophyll seriously affects the photosynthetic rate of plants [41]. In this experiment, the relative water content and chlorophyll content of the TaWRKY28/33/40/47 gene-silencing line were lower than the control MOCK after drought stress. The results showed that the water retention capacity of the wheat leaves of the TaWRKY28/33/40/47 genesilencing line was weak and Photosynthesis is also affected. MDA is the nal breakdown product of lipid peroxidation, which can re ect damage to plant membrane system and plant resistance [42]. The results of this experiment showed that the VIGS gene silencing line accumulated more MDA than the control MOCK line under drought treatment, indicating that TaWRKY28/33/40/47 gene silencing promoted plant lipid oxidation. Drought may lead to the accumulation of reactive oxygen species (ROS) and the damage related to ROS. This experiment detects that the TaWRKY28/33/40/47 gene-silencing line after drought stress accumulates more superoxide anions and hydrogen peroxide than MOCK Excessive superoxide anion will affect DNA replication and protein synthesis, and also damage the membrane system that maintains cell morphology. Excessive production of H 2 O 2 can cause leaf cell death. In order to combat reactive oxygen damage, antioxidant enzymes are widely present in plants Systems, such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) are ROS scavenging enzymes, which can further convert H 2 O 2 into H 2 O, etc., and play an important role in regulating the content of H 2 O 2 in cells Role [43,44]. In addition, proline is one of the main osmotic adjustment substances in plants. When plants are under drought stress, it can remove excess active oxygen accumulated in plants due to drought, and has a protective effect on the integrity of cell membranes [45]. This experimental study shows that when subjected to drought stress, the antioxidant enzyme activity in plants will have an upward process. When subjected to drought stress, the TaWRKY28/33/40/47 gene-silencing line showed lower CAT SOD, POD activity than MOCK plants. The antioxidant enzyme activity of TaWRKY28/33/40/47 gene-silencing lines is lower than MOCK, which may re ect the stronger oxidative damage of gene-silencing lines under drought stress, but the weaker ability of scavenging reactive oxygen species. These results indicate that silencing TaWRKY28/33/40/47 affects the antioxidant system, and TaWRKY28/33/40/47 may be involved in the ROS-mediated wheat response to drought signaling pathway.

Methods plant materials
The wild-type strain of Chang Wu 134 wheat is a resistant to drought and Chang Wu 134 TaGAPCs-RNAi mutant strain is a susceptible variety to drought, which is a 333-bp conserved TaGAPC fragment was ampli ed with speci c primers to construct the wheat GAPC RNA interference (RNAi) vector. The ampli ed PCR product was puri ed and recombined in antisense and sense orientations to ank the 500bp rice Adh gene intron of PTCK303, forming the TaGAPCs-RNAi vector. Using particle bombardment technology to genetically transform the TaGAPCs-RNAi vector into Changwu 134 wheat and obtain a stable genetic transgenic line. The wheat Changwu 134 TaGAPCs-RNAi strain and Changwu 134 wildtype seeds were planted, and the wheat seedlings were grown to the two-leaf and one-heart stage and subjected to natural drought stress. On the 15th day, wheat leaf tissues were taken as experimental samples. The Changwu 134 TaGAPCs-RNAi strain and the Changwu 134 wheat RNAi group and WT group were each set up with 3 biological replicates, and the collected fresh samples were quick-frozen with liquid nitrogen and placed in a refrigerator at -80°C for later use.
RNA extraction and preparation of the cDNA library Total RNA was extracted using TRIzol from wheat seedlings and converted to cDNA using the PrimeScript™ RT reagent kit (TaKaRa, Japan). The preparation of the cDNA library was completed by Huada Biological Company using the BGISEQ-500 platform and machine sequencing with the HiSeq 4000 sequencing system.

RNA-seq analysis of wheat leaves after drought
The raw reads obtained from Trimmomatic were ltered after obtaining clean reads. Clean data (clean reads) were obtained by removing reads containing adapters, reads containing poly-N and low-quality reads from the raw data. Thereafter, clean sequence reads were mapped to the available wheat genome.
We used HISAT (Hierarchical Indexing for Spliced Alignment of Transcripts) to align the clean reads to the reference genome sequence.
Gene ontology (GO) and KEGG analysis of DEGs As described by Anders and Huber, the DESeq R (1.18.0) package was used to analyse the differential expression between the treated and control transcripts. DEGs were de ned as genes that had absolute values of log 2 -fold change (Log 2 FC) ≥ 2 or Log 2 FC ≤ − 2 and a false discovery rate (FDR) ≤ 0.05.
According to the GO and KEGG annotation results and the o cial classi cation, we functionally classi ed the DEGs and used the phyper function in R software for enrichment analysis. FDR correction was then performed on the p values. The functions with Q value ≤ 0.05 were considered signi cantly enriched.

Validation of RNA-Seq results by qRT-PCR
To verify the accuracy of the RNA-Seq results, 15 DEGs with potentially important functions were randomly selected from drought for 15d for qPCR and RNA-SEq. cDNA was synthesized by reverse transcription. QPCR primers were designed using Primer5 software, and primer speci city was evaluated by blasting primer sequences against the NCBI database (Supplementary Table S1). Three technical replicates for each of three biological replicates were performed. BSMV-mediated TaWRKY28/33/40/47 gene silencing in wheat Plasmids (α, β, γ, and γ-PDS) used for VIGS system were constructed according to [46]. Four segments (196 bp,187bp,187 bp and 186 bp) of TaWRKY28/33/40/47 were cloned using the primers with the restriction enzymes PacI and NotI (Supplementary Table S2). The wheat phytoene desaturase (PDS) gene was replaced with four speci c TaWRKY28/33/40/47 sequences. The RNA was synthesized in vitro from linearized plasmids following the instructions of RiboMAX™ Large Scale RNA Production System and Ribo m7G Cap Analog Kits (Promega, Madison, Wisconsin, USA).
The BSMV inoculum was combined 10 µL of α, β and four modi ed γ transcripts (BSMV:γ, BSMV:PDS, BSMV: TaWRKY28, BSMV: TaWRKY33, BSMV: TaWRKY40 and BSMV: TaWRKY47, respectively) with 70 µL of Fes buffer (viral inoculation buffer). The viral inoculation solution was inoculated on the second leaves of 10-day-old wheat seedlings by the sliding friction with gloved ngers according to the method of [47]. Control inoculations (MOCK) were performed using Fes buffer. BSMV:γ and BSMV:PDS were used as negative and positive controls for BSMV infection, respectively. After inoculation, the wheat seedlings were cultured in a growth chamber set at 25℃ for 24 h in the dark, and then shifted to 16 h/8 h light/dark cycle at 25℃. About 10 days after virus inoculation, the symptoms were examined.
QRT-PCR analysis to assay silencing e ciency For assays of silencing e ciency, total RNA was extracted from the leaves of silenced plants on the 14th day using Trizol reagent (TIANGEN), and cDNA synthesis was conducted using an M-MLV reverse transcriptase kit (Takara) according to the manufacturer's instructions. Real-time PCR reactions were performed in a total volume of 20 µL containing cDNA (100 ng µL − 1 ) 1.0 µL, 0.5 µL of each primer (10 mM), and 10 µL of BeyoFast™ SYBR Green qPCR Mix (2X). At the end of the reaction, the melting curve was observed to ensure that the product was speci cally ampli ed. The relative expression level of the target gene was presented as fold change compared with the BSMV-γ using the 2 −ΔΔCT method, three biological and three technical replicates were conducted in the transcript pro les of genes. Three independent experiments were conducted.

Physiological and biochemical tests of gene silencing plants under drought stress
The TaWRKY28/33/40/47 gene silencing wheat plants were withheld water for 15 days. Drought symptoms of wheat were photographed after drought treatment. The fourth leaves of seedlings were sampled to do further experiments after drought treatment. The leaves were harvested to determine the contents of chlorophyll (Chl), MDA, H 2 O 2 , proline, and relative water content (RCW),. The activities of POD (EC 1.11.1.7), SOD (EC 1.15.1.1), CAT (EC 1.11.1.6) enzymes were also measured. The Chl content of the leaves was extracted in 96 % ethanol for 24 h until the green of the wheat leaves fades to colorless as described by Jia [48]. The content of MDA was assessed using the thiobarbituric acid method [49]. The H 2 O 2 content was measured as described by Negi [50].The proline content was measured by the ninhydrin reaction method [51].To determine the O 2 − content according to the method of wang [52].The RCW detection method as described by Nauš [53].The activity of POD was estimated following the method of Huyskens-Keil [54]. The activity of SOD was examined by monitoring the inhibition of photochemical reduction of NBT [55]. The activity of CAT was determined according to Sousa [56]. Three biological replicates were analyzed and three independent experiments were conducted.

Statistical analysis
The data were rst to analyze using the Microsoft O ce Excel 2019. The error bars represented standard error (SE). The analysis of the signi cance level was performed according to Duncan's method at *P < 0.05, and **P < 0.01, through the SPSS Statistics 20.0 software. The gures were generated using the Origin 9.0 and Adobe Photoshop software.

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Open   RT-qPCR veri cation of differential gene expression results of transcriptome sequencing. Supplementary Files