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Genome-wide identification and analysis of WRKY gene family in jute(Corchorus capsularis)

Background WRKY transcription factor is a kind of transcription factor which plays an important role in plant response to biotic, abiotic stress, plant growth and development. However, little information was available about the WRKY genes in jute ( Corchorus capsularis ).Results In the present study, 43 jute WRKY (CcWRKY) genes were identified by using Pfam database domain search and BLAST homology alignment based on the transcriptome data of jute. And the gene structure, phylogeny, conserved domain and three-dimensional structure of protein were also analyzed by GSDS2.0, MEGA7.0, DNAMAN5.0, WebLogo 3 and SWISS-MODEL bioinformatics tools. According to the WRKY conserved domain features and the evolution analysis with Arabidopsis thaliana , 43 members were divided into three classes: I, II and III containing 9, 28, 6 members, respectively. According to the evolutionary relationship, class II further divided into five subclasses: II-a (2), II-b (7), II-c (7), II-d (6) and II-e (6). Genetic structure analysis showed that exon and intron number of CcWRKY genes had high variability (3-11 exons), even within the same subgroup. Most of the CcWRKY genes were expressed in different tissues, but they were mainly expressed in stem bark and stem stick. After GA 3 stress, the expression of most WRKY genes in GA 3 -sensitive variety "Aidianyehuangma" was significantly different from that of normal variety "Huangma 179". These results indicated that CcWRKY genes play an important role in gibberellin biosynthesis pathway and fiber development.Conclusions CcWRKY proteins are highly conserved, the length of the gene sequence and the number of introns varied widely, all WRKY genes showed a variety of expression patterns in different tissues, most of the WRKY genes responded to GA 3 stress, which play an important role in gibberellin

3 biosynthesis pathway and bast fiber development.
background The WRKY gene family is a transcription factor that exists only in plants. It is mainly involved in transcriptional regulation and signal transduction processes in plants [1].In the transcriptional regulatory network, WRKY transcription factors bind specific DNA sequences to activate or repress transcription of multiple target genes [2,3].The conserved WRKY domain contains approximately 60 amino acid residues. In the WRKY domain, a conserved WRKYGQK hexapeptide sequence is usually followed by a C2H2-or C2HC-type zinc finger structure. According to the number of WRKY domains and the type of zinc finger structure, the WRKY family can be divided into 3 groups: Group Ⅰ, Group Ⅱ and Group Ⅲ [4]. Group II could be further divided into five subgroups: II-a, II-b, II-c, II-d and II-e. Group Ⅰ contains two WRKY domains and C 2 H 2 zinc finger structure, Group Ⅱ contains a WRKY domain and a C 2 H 2 zinc finger structure, and Group Ⅲ contains a WRKY domain and a C 2 -H-C zinc finger structure [5].
Since the first WRKY gene (SPF1) was cloned from sweet potato [6], WRKY genes have been found in many plants, such as Arabidopsis [7] [14], poplar [15], cotton [16], cucumber [5] and grape [17]. At present, WRKY transcription factors have been found to play an important role in plant defense response to environmental stresses and growth development. The transcription factor WRKY70 is a common component in salicylic acid (SA)-and jasmonic acid (JA)-mediated signal pathways [18]. Expression of WRKY70 is activated by SA and repressed by JA [18]. In Arabidopsis, some WRKY transcription factors are positive regulators of 4 ABA(Abscisic acid)-mediated stomatal closure and hence drought responses [19].
AtWRKY75 in Arabidopsis thaliana and OsWRKY31 in rice are both related to the growth of lateral root [20,21].The synergistic interaction of ABA-inducible WRKY genes in regulating GAMYB (GA-inducible transcriptional activator)-mediated GA (Gibberellin) signaling in aleurone cells, thereby establishing a novel mechanism for ABA and GA signaling cross-talk [22].
Jute (Corchoruscapsularis L) is a diploid (2n=14) natural fiber plant, which belongs to the Malvaceae family [23]. It is mainly cultivated in Bangladesh, India, and China.
The bast fiber of jute is mainly used to make fabrics, ropes and threads, which is an indispensable part in human's life. At present, most of the fibrin use, are mainly coming from limited petrochemical products, which are not only expensive, but also harmful to the environment due to the emission of greenhouse gases to the atmosphere. In recent years, a number of people were interested in plant fiber textiles and production technology [24].This was due to the reason that plant fibers are renewable, eco-friendly, and 100% biodegradable. Jute bast fiber has excellent moisture absorption, air permeability and strong antibacterial property, so it has great application value and development potential. In these perspectives, the processing technology and new spinning technology of jute bast fiber were constantly improving and developing, resulting in more and more refined jute products that will hugely promote space in clothing, home textile, building materials and other fields. So far, jute has become one of the most economic fiber crops in the world, known as "gold fiber". All the industries related to jute bast fiber demand sufficient and high-quality fiber. However, both quality and yield of jute are constrained by various stresses attributed to global climate change notably abiotic and biotic stresses.

5
At present, many studies have shown that WRKY genes plays an important role in plant growth, development, stress tolerance, especially in fiber development [25].
However, information about the WRKY genes in jute is poorly understood. Therefore, this was the first time to identify and analyzed the WRKY transcription factors in jute. The aims of this study were: (1) Identification and structure analysis of WRKY gene family in jute. (2) Phylogenetic analysis and expression analysis of the WRKY gene family in jute after the stressed with exogenous GA 3 . (3) the effect of WRKY genes on bast fiber development in jute. Understanding the structure and roles of WRKY gene family can provide basic information for manipulating this gene family in order to increase jute resistance to stresses and fiber yield as well.

Plant materials
Varieties, "Huangma 179" and "Aidianyehuangma", were used as plant materials in this study. These two varieties of white jute were grown in the farm of Fujian agriculture and forestry university during the growing season in May, 1st, 2017. 10 days after sowing, hypocotyl samples were independently collected from three "Huangma 179" plants. On July 1 st , 2017 (60 days after sowing), the stem bark and stem stick were independently collected from three "Huangma 179", stem bark were independently collected from three "Aidianyehuangma", separately. On September 1 st , 2017(120 days after sowing), the stem barks were independently collected from three "Huangma 179", separately.
To identify the expression of CcWRKY genes after GA 3 stress, six jute plants were treated with GA 3 stress 60 days after sowing. The GA 3 stress treatment was carried 6 out for 4 hours and 72 hours, respectively. After the GA 3 stress treatment, the stem barks of each jute were obtained from "Huangma 179" and "Aidianyehuangma". In addition, three jute plants of the same age were sampled separately as control. All samples were immediately frozen in liquid nitrogen and stored in a refrigerator at -80℃ for follow-up analysis. Three samples come from each tissue were stored separately as three biological replicates (Additional file 1: Table S1).

Identification and Analysis of Conservative Domain of CcWRKY
The assembled sequences data have been deposited at the NCBI Sequence Read

Phylogenetic analysis of CcWRKY protein
Phylogenetic and molecular evolutionary analysis was conducted using MEGA7 (http://www.megasoftware.net) [29] with pairwise distance and the neighbor-joining algorithm. The p-distance method was used to compute the evolutionary distances, which were used to estimate the number of amino acid substitutions per site. Conducting 1,000 bootstrap sampling steps[12] established the reliability of each tree. Arabidopsis WRKY proteins (AtWRKY) were added to phylogenetic analysis to facilitate the subgroup classification of CcWRKY. WRKY family sequences from Arabidopsis were downloaded from TAIR (http://www.arabidopsis.org/).

Expression analysis of CcWRKY genes at different growth stages
The expression analysis CcWRKY genes was performed according to the FRKM value of different genes in different tissues (hypocotyls-10d, leaf-60d, root-60d, stem bark-60d, stem stick-60d, stem bark-120d), the heatmaps was drawn by R language.
The data obtained were calculated by using log function.

Effects of GA 3 on the development of bast fiber
To explore the effect of GA 3 on the development of bast fiber in jute, "Huangma 179" was used as the control variety and "Aidianyehuangma" as the test variety.
According to the method of MS medium [30], the standard medium (CK) and 0.1 mg·L -1 GA 3 (GA) exogenous hormone medium were prepared. The common variety "Huangma 179" and sensitive to gibberellin variety "Aidianyehuangma" were used as experimental materials. Each pair has 3 bottles as biological repeats, 30 seeds were planted in each bottle, and the length of hypocotyl was measured when the first true leaf was grown. Base on homologous protein sequence in Arabidopsis as query sequence, the other fiber marker genes in jute were identified by local BLASTP. According to the FRKM values of fiber marker genes and CcWRKY genes in stem bark (60-day-after-sowing) before and after GA 3 treatment, the histogram was drawn with Excel. The data obtained were calculated by using log function, formula 2 -△△CT and SPSS. The upstream promoter of CcWRKYs were predicted and analyzed by PlantCARE.

RNA extraction and quantitative real-time PCR (qRT-PCR)
The total RNA was extracted from four different tissues (leaves, roots, stem bark and stem stick) of jute plant at the vigorous growth stage i.e. 60-day-after-sowing using. EZNA Plant RNA Kit (from OMEGA) following manufacturer's instructions. The cDNA was synthesized by a reverse transcription kit (from Takara) according to the manufacture's procedures. This experiment used the Actin [31] as the reference gene by qRT-PCR. qRT-PCR reaction system: 10 μl of GoTaq® qPCR Master Mix, 0.4 μl of left primer(10μM), 0.4 μl of right primer(10μM), 2μl of cDNA, and 7.2μl of Nuclease-Free Water. Amplifications were performed with an initial 10min step of 95℃ followed by 40 denaturation cycles at 95℃ for 15s and primer annealing at 60℃ for 1min. The melting curve used the default program of the instrument (ABI7500). All experiments were used in triplicate for each sample and relative gene expression levels were calculated using the formula 2 -△△CT as earlier described [32]. All data was analyzed using Microsoft Excel. The primers were designed to avoid the WRKY conserved domain.

Identifcation of full length CcWRKY genes
In this study a local BLASTP search was performed to identify complete WRKY members in jute, using Arabidopsis WRKY protein sequences as query sequences and also Pfam was used to detect their conserved domain. As result a total of 43 candidate genes containing WRKY domain (named as CcWRKY) were identified, as shown in Table 1.

Analysis of conservative domain of CcWRKY genes
The conservative domain of CcWRKY gene sequences were identified and analyzed 9 using DNAMAN5.0 software and. conservative structure prediction was performed by Weblogo. The results showed that the conserved domains of WRKY gene family in jute could be divided into three groups: I, II and III. Group I had nine members. It could be further divided into I-C and I-N subgroups. Group I contain two WRKY domains and zinc finger structures, and the zinc finger structure are CX 4 C 22-23 HXH.Group II could be further divided into subgroups II-a, II-b, II-c, II-d and II-e, with 2, 7, 7, 6 and 6 members, respectively. In II-a, II-b, II-d and II-e, the heptapeptide domain and zinc finger structure of WRKY at C-terminal were WRKYGQK and CX 5 C 23 HXH, while in II-c, the heptapeptide domain and zinc finger structure of WRKY at C-terminal were WRKYGQK and CX 4 C 23 HXH. There were six members in group III. The heptapeptide domain and zinc finger structure of WRKY at C-terminal were WRKYGQK and CX 7 C 23 HXC (Fig1). Moreover, the present results showed that there are still mutations in its protein sequence, though WRKY transcription factor has a much conserved WRKY domain. Among the 43 members of WRKY transcription factor identified in jute, the conserved domain of one gene (WRKYGQK) and the zinc finger structure of four genes were all mutated (Additional file 2: Table S2). This variation indicated that despite the structurally high conserved WRKY gene family, some variations still occur in its WRKY domain, which also illustrated that the plant WRKY gene family had diversity in the evolutionary process.

Phylogenetic analysis of CcWRKY protein in diverse species
BY comparing the known WRKY region of Arabidopsis thaliana WRKY protein with CcWRKY, the WRKY domain sequence of CcWRKY protein was clustered and analyzed using MEGA7 (Fig2). These CcWRKY proteins can be divided into three groups: I, II and III. And Group II can be divided into II-a, II-b, II-c, II-d and II-e subgroups. The classifications of phylogenetic tree analysis were consistent with the results of Figure 1 (Table 1).

Structure analysis of intron and exon of WRKY in jute
In this study, the number of exons and introns of jute WRKY gene were analyzed and the results are shown in Figure 3.

Analysis of tertiary structure of protein
The tertiary structure of protein is further coiled and folded on basis of the secondary structure. The tertiary structure of CcWRKY protein was conducted by SWISS-MODEL. The majority of the 43 amino acid sequences have the similar threedimensional structure. One representative homology modeling from CcWRKY gene family was shown in Figure S1, and consists of several beta folding. Their tertiary structure were quite similar with that of Arabidopsis thaliana [33]. It had also proved that the CcWRKY gene family is highly conserved in structure.

Expression analysis of CcWRKY genes in different tissues
Tissue specific expression of genes is often considered as markers of specific gene functions in this tissue. Since WRKY genes are related to the bast fiber development of plants [34,35], we mainly focus on the expression of CcWRKY genes at different stages of stem growth. Based on the RNA-seq data, we used R language to draw the heatmap of the expression patterns of CcWRKY genes in different stem growth stages (Additional file 4: Fig. S2). The difference of gene expression is generally represented by colors, red represents high expression and blue represents low expression. The results showed that all the CcWRKY genes were expressed in the stem of jute, and the expression of WRKY genes differ at different stem growth stages. Meanwhile, it proved that there were no pseudogenes in 43 genes.
From Figure S2, we could see that 43 genes were divided into two categories. The expressions of 13 genes were lower in the different tissues of jute, and the others were higher. Totally, 10 WRKYs were highly expressed in leaf(60d), 3 WRKYs were highly expressed in hypocotyls (10d), 2 WRKYs were highly expressed in stem stick(60d), 2 WRKYs were highly expressed in stem bark(60d), 14 WRKYs were highly expressed in root(60d), and 12 WRKYs were highly expressed in stem bark(120d). It could be seen that the WRKY genes were mainly expressed in the stem bark of jute. With the continuous growth of jute, the bast fiber of jute will gradually accumulate in the stem bark. Therefore, it is believed reasonably that the WRKY genes are involved in bast fiber development in jute. For example, Ccv40032460 was highly expressed in hypocotyls (10d), lowly expressed in stem bark (60d), and no expression in stem bark (120d). It suggests that this gene may play a negative regulatory role in jute fiber accumulation.

GA 3 stress analysis of CcWRKY genes involved in cell wall formation
According to our previous research [36], "Aidianyehuangma" is a dwarf variety that sensitive to GA3. "Huangma 179" and "Aidianyehuangma" were planted in 0.1 mg·L -grown. It could be seen that the hypocotyl length of " Aidianyehuangma " treated by GA 3 (4.58cm) is higher than that of CK (1.77cm) (Additional file 5: Fig. S3). The average lengths of hypocotyl of "Aidianyehuangma" and "Huangma 179" treated with GA 3 was 4.58cm and 4.67cm respectively, while that of "Huangma 179" without GA 3 was 4.61cm (Additional file 5: Fig. S3). The results showed that there were no significant differences in the lengths of hypocotyl among the three groups. After stressing the plant with GA 3 , it was found that the plant height of "Aidianyehuangma" could be significantly increased, which would greatly improve the fiber yield of the variety. This dwarf variety is very suitable for studying the relationship between GA 3 and fiber development.
To further explore the relationship between fiber development, CcWRKYs and GA 3 , we selected some important fiber related genes as marker genes. These genes were include,CesA1 (CesA, Cellulose synthase), CesA4, CesA7, CesA8, CCoAOMT (Caffeioyl coenzyme A methyltransferase), 4CL (4-Coumarate: Coenzyme A Ligase), Ent-copalyl diphosphate synthase, Ent-kaurene oxidase, Ent-kaurene synthase, Ent-kaurenoic acid oxidase, GA 20-oxidase, Gibberellin 2,3-hydroxylase and Gibberellin C13 oxidase. In the vigorous growth period (60 days after sowing), the stem barks were treated with GA 3 stress for "Huangma 179" and " Aidianyehuangma ". Then, the samples were taken after 4 hours and 72 hours, respectively. The samples without From these, we also found21 CcWRKY genes and most of the fiber related marker genes of "Aidianyehuangma" were sensitive to the GA 3 stress. The variations of expression of these genes in "Aidianyehuangma" were more significant than those in "Huangma 179" (Fig 4 and Fig 5).This indicated that these CcWRKY genes, were similar to that of like other fiber related marker genes, played a certain role in the increase of fiber yield of "Aidianyehuangma" under GA 3 stress. In addition, the promoters of these CcWRKYs were analyzed by PlantCARE. The results showed that the promoters of most CcWRKY genes contained elements related to gibberellin, such as GARE-motif, P-box and TATC-box ( Table 2). The CcWRKY genes responded to the stress of GA 3 and could increase the fiber yield of the "Aidianyehuangma". This suggested that WRKY genes might also be involved in the growth and development of bast fiber like other fiber genes in jute.
To verify the accuracy of the gene expression, 9CcWRKY genes were randomly selected for qRT-PCR analysis (Additional file 8: Fig. S6). The results of qRT-PCR corresponded to the results of FPKM.  [1,[37][38][39].In this study, there were at least 43 WRKY members in jute genome, and the numbers were similar to those of sugar beet (40) [40], canola (43) [41] and castor (47) [42]. However, these numbers were different from those of other plants, such as Arabidopsis thaliana(74), cotton(116) [16], soybean(188) [43], poplar(104) [15], tomato(81) [44], cabbage(145) [45], whose members were more than those of jute. By comparing species, the numbers of WRKY genes in different species is not proportional to their genome size. Nowadays, researchers have suggested that gene duplication, segmental duplication and whole genome duplication play important roles in the mass production of gene families [43].Unfortunately, due to the lack of data on jute research, genome data also only published a draft, so many problems failed to get a satisfactory answer. We suspected that jute genome WRKY genes were less than other species, perhaps because they did not experience whole genome replication as other species do. However, with the improvement of genome sequencing and assembly, upgrade and update of the search and analysis software, discovery of variable splicing in genomes and the continuous advance of jute related research, we believed that the new WRKY members also existed in the genome of jute.
In general, the locations of introns and exons in the genome may provide important evidences for their evolutionary relationships. In this study, we systematically and comprehensively analyzed the distributions and lengths of exons and introns of the members of the WRKY gene family. By analyzing the gene structures of jute WRKY, it was found that its members consisted of 3 to 11 exons, and nearly half of them were 3 exons. These results provided valuable information for the study of jute and the evolutions of the WRKY gene family in other species. Moreover, the members of 15 the WRKY family have similar three-dimensional structures, which were formed by several beta folds. It is similar to the 3D structure of the domains of Arabidopsis WRKY protein in the database [33].

CcWRKY genes involved in cell wall formation
Both Jute and cotton are important fibre crops in the world. So, we usually would compare the results of jute with that of cotton. According the expressions analysis of CcWRKY genes in different tissues, we could find that the mostly group III CcWRKY genes (Ccv40018580, Ccv40120290, Ccv40170160 and Ccv40154680) were highly expressed during the fiber development except two genes(Ccv40018590 and Ccv40064890).Theses results in jute were consistent to that in cotton [46].In recent years, the study [47] has shown that Arabidopsis WRKY transcription factor was

Availability of data and materials
The datasets supporting the results of this publication are included within the article and its additional files.  Effects of GA3 stress dwarf variety "Aidianyehuangma" on the expression of CcWRKY genes