GarWRKY5, a Member of the WRKY Transcription Factor Gene Family from a Diploid Cotton Species (Gossypium aridum L.), Is Involved in Salt Stress Response CURRENT STATUS:

Background Cotton is one of the most economically important crops in the world, and it is exposed to various abiotic stresses during its lifecycle, especially salt stress. However, the molecular mechanisms underlying cotton tolerance to salt stress are still not fully understood due to the complex nature of salt response. Therefore, identification of salt stress-tolerance-related functional genes will help us to understand key components involved in stress response and to provide valuable genes for salt stress tolerance improvement via genetic engineering in cotton. In a previous study, expression of a Group III WRKY gene family member from the diploid cotton species Gossypium aridum, GarWRKY5, was significantly induced in response to salt stress. Results In this present study, virus-induced gene silencing of GarWRKY5 in cotton showed enhanced salt sensitivity compared to wild-type plants under salt stress. Overexpression of GarWRKY5 in Arabidopsis positively regulated salt tolerance at the stages of seed germination and vegetative growth. Additionally, GarWRKY5-overexpressing plants exhibited higher activities of superoxide dismutase (SOD) and peroxidase (POD) under salt stress. The transcriptome sequencing analysis of transgenic Arabidopsis plants and wild-type plants revealed that there was enriched co-expression of genes involved in reactive oxygen species (ROS) scavenging (including glutamine S-transferases (GSTs) and SODs) and altered response to jasmonic acid and salicylic acid in the GarWRKY5-OE lines. Conclusion GarWRKY5 is involved in salt stress response by the jasmonic acid- or salicylic acid-mediated signaling pathway based on overexpression of GarWRKY5 in Arabidopsis and virus-induced gene silencing of GarWRKY5 in cotton.


Introduction
WRKY proteins comprise one of the largest transcription factor families in plants. The conserved WRKY domain contains approximately 60 amino-acid residues. The WRKY domain is defined based on the conserved WRKYGQK hexapeptide sequence is usually followed by a C2H2-or C2HC-type zinc finger motif at N-terminal end. WRKY transcription factors are classified on the basis of both the number of WRKY domains and zinc finger motifs that they contain; WRKY proteins with two WRKY domains belong to group I, whereas Group II and Group III members have only a single WRKY domain, followed by a novel zinc-finger-like motif C2H2 (C-X4-5-C-X22-23-H-X-H) and C2HC (C-X7-CX23-H-X-C), respectively [1]. WRKY proteins play diverse roles in regulating plant defense responses, and developmental and physiological processes of plants. In addition to their role in plant development, WRKY family genes are also important in regulating plant biotic and abiotic stress. For example, pathogen-induced defense pathways, drought, salt stress and others [2][3][4]. Increasing numbers of studies are reporting that WRKY genes are involved in regulating plant responses to salt stress. Their function has been elucidated using genetic and molecular approaches in different species, such as AtWRKY25 and AtWRKY33 in Arabidopsis [5], OsWRKY11 and OsWRKY45 in rice [2], GmWRKY13 and GmWRKY54 in soybean [6], and TaWRKY10 in wheat [7].
Cotton is one of the most economically important crops in the world, which endures various abiotic stresses during its lifecycle, especially salt stress. However, the molecular mechanisms underlying cotton tolerance to salt stress are still not fully understood due to the complex nature of this response. With the release of the Gossypium genome sequence [8][9][10], genome-wide identification of WRKY family genes has been conducted in G. raimondii, G.arboreum and G. aridum [11][12][13][14]. Several studies have suggested the importance of specific WRKYs in the transcriptional regulation of saltrelated genes in cotton. For example, overexpression of GhWRKY25 from G. hirsutum in Nicotiana benthamiana enhanced tolerance to salt stress [15]; GhWRKY39-1-overexpressing plants exhibited increased tolerance to salt and oxidative stress and increased transcription of antioxidant enzyme genes [16]. Overexpression of GhWRKY34 in Arabidopsis resulted in a transgenic plant with increased tolerance to salt stress [17]. The ectopic expression of the GhWRKY6-like gene significantly increased salt tolerance in Arabidopsis thaliana while silencing the GhWRKY6-like gene increased the sensitivity of cotton to abiotic stresses [18]. Despite the abovementioned reports, the molecular mechanisms by which WRKY transcription factors (TFs) regulate salt stress still remain largely unclear in cotton.
In a previous study, 109 GarWRKYs gene were identified in a salt-tolerant wild cotton species, Gossypium aridum, based on transcriptome sequencing data; meanwhile, 28 salt-responsive GarWRKY genes were identified from transcriptome data and real-time quantitative PCR analysis [14].

Results
Characterization of GarWRKY5 based on structure, evolution and expression Based on previous studies, GarWRKY5 encodes a member of Group III of WRKY. The predicted GarWRKY5 proteins and homologous genes from G. hirsutum (Gh_A02G0029/Gh_D02G0043), G. raimondii (Gorai.005G003900) and G. arboreum (Cotton_A_04316) contain an approximately 60amino-acid WRKY domain that is composed of the conserved amino acid sequence (WRKYGQK) and a zinc-finger motif (C-X 4-5 -CX 22-23 -H-X 1 -C) ( Figure 1A).. Based on the evolutionary tree, Gorai.005G003900 from the G. ramondii genome was close to Gh_D02G0043 from the Dt-subgenome of allotetraploid cotton in evolutionary relationships. The paralogous pairs had ratios of nonsynonymous to synonymous substitutions (Ka/Ks) of more than 1.0 between GarWRKY5 and the other four genes except Gh_D02G0043, indicating that they had gone through positive selection in the evolutionary process ( Figure 1B, Supplementary Table S1).. In terms of expression in vegetative and reproductive organs of G. aridum, GarWRKY5 had a higher expression level in the root than in other organs ( Figure 1C)..

Silencing GarWRKY5 in upland cotton line compromises salt tolerance.
To elucidate the role of GarWRKY5, the virus-induced gene silencing (VIGS) method was used to knockdown the expression of GhWRKY5, a homologous gene of GarWRKY5 in upland cotton. After growing plants in illumination incubator for one week, we hand-infiltrated Agrobacterium cultures carrying the VIGS vector into cotton cotyledons. Approximately 7 d after agroinfiltration, leaves of the GhCLA1-silenced plant displayed the photobleaching phenotype as expected which was uniformly distributed on the entire true leaves (Figure 2A),, Suggesting that the VIGS system can work well based on our experimental conditions. To investigate the silencing efficiency of GhWRKY5 in the tested plants, Semi-quantitative RT-PCR was used to determine the expression levels. The results showed that the GhWRKY5 expression level in the silenced plants was much lower than in the control plants. At least five GhWRKY5-silenced plants with four true leaves were treated with 300 mM NaCl solution, the distilled deionized water was used to the control. Ten days later, the tolerance of the GO enrichment analysis was performed on the 253 upregulated DEGs and 145 downregulated genes, respectively. For the upregulated DEGs, under "biological process," oxidant detoxification, response to jasmonic acid and salicylic acid, response to salt stress and osmotic stress were significantly enriched, like glucosyltransferase activity and calcium ion binding in "molecular function," and vacuole and protein-containing complex in "cellular component." For downregulated DEGs, response but no categories were significantly enriched with respect to "molecular function" and "cellular component" (Supplementary Table S3)..
Because GarWRKY5 was homologous to AtWRKY70 [14], we performed network analysis for these 19 DEGs and AtWRKY70 using the STRING database, version 11.0 (https://string-db.org/). The result showed that AtWRKY70 could regulate AT1G02930 (glutathione S-transferase F6, GSTF6) and AT5G24770 (acid phosphatase VSP2) (Supplementary Figure S1).. In addition, we analysed the promoters (1-kb upstream of the translation start sites) of the 19 DEGs using the JASPAR database [21]. Promoter region screening showed that all 19 DEGs had three to ten W-box motifs in their promoter regions, a DNA-sequence motif (T)TGAC(C/T) which could bind to a WRKY transcription factor ( Table 1).. Based on the data presented in this study, we hypothesize that GarWRKY5 may be a positive transcription regulator involved in plant response to high salinity stress through the ROSscavenging system such as by activating expression of GST and SOD genes by jasmonic acidmediated or salicylic acid-mediated signaling pathways.

Discussion
WRKY TFs are key regulators of many plant processes, including responses to biotic and abiotic stresses, senescence, seed dormancy and seed germination [22]. Recent studies have broadened our knowledge of the WRKY TF family and its functions in salt stress responses in cultivated cotton [18,23,24]. However, wild relatives of crops represent potentially valuable gene pools and are primary source of important genes. In the previous study [14], by using a D-genome diploid species (G. aridum) from the Pacific coastal states of Mexico, which shows remarkable tolerance to salt stress, we set out to perform transcriptome analysis and identified the response of 28 WRKY TFs in G. aridum to salt stress conditions. Based on overexpression of GarWRKY17 and GarWRKY104 in Arabidopsis, functional analysis indicated that these two genes could positively regulate salt tolerance in different developmental stages of transgenic Arabidopsis [14]. In the present study, we have provided GarWRKY5 shows sequence homology with OsWRKY45 in rice and AtWRKY70 in Arabidopsis [14]. The expression of rice WRKY45 (OsWRKY45) was markedly induced in response to the stress-related hormone abscisic acid (ABA) and various stress factors, e.g. application of NaCl, polyethylene glycol (PEG), mannitol or dehydration. Constitutive over-expression of OsWRKY45 conferred a number of properties to transgenic plants, including increased resistance to the bacterial pathogen, and increased tolerance to salt and drought stresses in Arabidopsis [29]. GST and cytochrome P450 genes are regulated by WRKY45 in rice [30]. In this study, network analysis for AtWRKY70 andthe 19 DEGs enriched with respect to the salt stress and osmotic stress processes by the STRING database showed that AtWRKY70 could regulate AT1G02930 (glutathione S-transferase F6, GSTF6). All these observations showed that GarWRKY5 might have regulatory mechanisms similar to those of Previous studies have demonstrated that the Group III WRKY members may play prominent roles under biotic and abiotic stress responses. For example, overexpression of a grape Group III WRKY transcription factor gene, VlWRKY48, in A. thaliana increased disease resistance and drought stress tolerance [31]. Another Group III member, AtWRKY46, functioned in both basal resistance against pathogens and tolerance to oxidative stress and aluminium toxicity to be induced by drought, salt and oxidative stresses [32]. Overexpressed OsWRKY45 in Arabidopsis increased pathogen defense, drought and salt resistance [29]. Overexpression of AtWRKY70 led to upregulation of PR genes and downregulation of PDF1.2, leading to enhanced resistance against biotrophic pathogens and enhanced susceptibility to necrotrophic pathogens. AtWRKY70, as a repressor of JA-responsive genes and an activator of SA-induced genes, integrating signals from these mutually antagonistic pathways [33]. The function of Group III WRKY members may be a node of convergence that integrates biotic and abiotic stress signals, so they have great potential for increased stress tolerance [34]. Encoding a member of the Group III WRKY family, the potential role of GarWRKY5 in mediating response to multiple stress factors needs to be further investigated.

Conclusions
Based on the data presented in this study, we hypothesize that GarWRKY5 may be a positive transcription regulator in plant response to high salinity stress through the ROS-scavenging system, such as activating expression of GST and SOD genes by the jasmonic acid-or salicylic acid-mediated signaling pathway.

Materials And Methods Plant materials and treatment conditions
The National Wild Cotton Plantation in Hainan Island, China, kindly supplied seeds from the wild Gossypium species G. aridum. The same treatment procedure was used as described by Xu et al. (2013) [35]. The G. aridum seeds were germinated in distilled deionized water, the growth conditions were 60% humidity, the day and night temperature were 28℃ and 23℃ respectively, photoperiod of 12h light/12h dark in the growth chamber. The germinated seeds were planted into nutritional soil and cultured in the plant growth chamber with the same set conditions. The uniform cotton seedlings with about 20cm in height and four true leaves were transferred into paper cup with 1×Hoagland's nutrient solution. After three days, the uniform cotton seedlings were treated for 200 mM NaCl for 0, 1, 3, 6, 12, 24 and 72 h, and untreated seedlings were used for the control. Root and leaf tissues were collected respectively at each stage under salt stress treatment. All samples were immediately frozen in liquid nitrogen and stored at -70℃. 94°C for 3 min, followed by 40 cycles at 94°C for 15 s, 60°C for 15 s and 72°C for 30 s. The relative expression levels were calculated using the 2 -ΔΔCt method with three biological replicates and three experimental replicates [38].

Analysis of salt tolerance in transgenic Arabidopsis plants
For the salt tolerance of GarWRKY5 transgenic Arabidopsis plants during the seed germination stage, 50 seeds of T 2 generation transgenic lines (three lines for GarWRKY5) were surface sterilized and sown on Murashige & Skoog (MS) medium with and without 150 mM NaCl, respectively. The wild type (WT) was used to control. After ten days, the germination rate of seeds was calculated. The experiment was repeated at least three biological replicates. For further verification overexpression of GarWRKY5 could enhance tolerance to salt stress during vegetative growth, sterilized seeds of WT and T 2 transgenic Arabidopsis were sown in soil. After 20 days, the seedlings were grown in a pot supplemented with 150 mL NaCl solution (150 mM/L) and the distilled deionized water was used for control. The phenotype of seedlings was observed after four weeks. For the determination of antioxidant enzymes activity, three-week-old seedlings from WT and T 2 generation of three GarWRKY5-overexpressing transgenic lines (GarWRKY5-1, GarWRKY5-6 and GarWRKY5-14) were soaked in 150 mM/L NaCl solution for 24 h. Leaves of at least ten seedlings were collected from the wild type and three transgenic lines, respectively. The activity of peroxidase (POD) and superoxide dismutase (SOD) was determined based on the procedure described by Liu et al. (2008) [39]. It is one unit of SOD activity that the mount of enzyme required to cause 50% inhibition of nitro blue tetrazolium (NBT) reduction. The SOD was measured at 560 nm by the ultraviolet spectrophotometer.
The activity of Peroxidase (POD) was analyzed at 470nm using guaiacol as a substrate by the ultraviolet spectrophotometer. The experiment was performed in 50 mmol/L phosphate buffer, 50 mmol/L guaiacol and 2% H 2 O 2 and 2 μl of enzyme extract were added. The data was recorded after adding 2.0 ml 20% chloroacetic acid. All the above procedures of enzyme extraction were carried out at 0-4 ℃. The enzyme assays were performed in three biological replicates.

Virus-induced gene silencing (VIGS) assays
In order to knockdown the expression of the GhWRKY5 gene, a 389-bp fragment of the GhWRKY5 cDNA from TM-1 was amplified using the VIGS primers. The resulting PCR product with double digested (XbaI and KpnI) was recombined into XbaI-KpnI-digested pTRV2 in order to produce pTRV2::GhWRKY5. The pTRV2::GhWRKY5 vector was introduced into theAgrobacterium strain GV3101 by means of electroporation (Bio-Rad, Hercules, CA, USA). For the VIGS assay, the GV3101 containing pTRV1, pTRV2 (mock-treated controls), pTRV2::GhWRKY5 and pTRV2::GhCLA1 respectively were used for VIGS experiments. The strains were grown overnight at 28°C with shaking at 150 rpm in LB broth containing two antibiotics kanamycin and rifampicin in concentrations of 50 mg/L each. The The reads from RNA-Seq were aligned to the reference genome (TAIR10 data) using Tophat v.2.0.11, which was compatible with Bowtie2 v2.2.1 [45]. All reads were allowed only one nucleotide mismatch.
Clean reads mapping to reference sequences from multiple genes were filtered out. For differentially expressed genes (DEGs) analysis, we adopted a conservative criterion by choosing consistent results of cuffdiff (ref), with |log 2 (fold change)|≥1 and significant expression with FDR < 0.05 and genes FPKM value ≥ 1.   Table   Table 1 Nineteen DEGs involved in salt stress and osmotic stress process based on GO enrichment analysis

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download.
Fig S1 prediction of function protein association network -Copy.pdf Table S3 GO enrichment of DEGs between OE 0d and WT 0d.xlsx Table S2 The details of DEGs and their function annotation -Copy.xlsx Table S1 Ka&Ks of the homologous gene -Copy.xlsx