Genome-wide identification and functional characterization of Camelina sativa WRKY gene family in response to abiotic stress

doi:10.21203/rs.3.rs-36113/v3

Download PDF

Research article

Genome-wide identification and functional characterization of Camelina sativa WRKY gene family in response to abiotic stress

https://doi.org/10.21203/rs.3.rs-36113/v3

This work is licensed under a CC BY 4.0 License

Journal Publication

published 11 Nov, 2020

Read the published version in BMC Genomics →

You are reading this older preprint version

Read the latest preprint version →

Background: WRKY transcription factors are a superfamily of regulators involved in diverse biological processes and stress responses in plants. However, knowledge is limited for WRKY family in camelina (Camelina sativa), an important Brassicaceae oil crop with strong tolerance against various stresses. Here, genome-wide characterization of WRKY proteins is performed to examine their gene-structures, phylogenetics, expressions, conserved motif organizations, and functional annotation to identify candidate WRKYs mediating regulation of stress resistance in camelina.

Results: Total of 242 CsWRKY proteins encoded by 224 gene loci distributed uneven on chromosomes were identified, and classified into three groups via phylogenetic analysis according to their WRKY domains and zinc finger motifs. 15 CsWRKY gene loci generated 33 spliced variants. Orthologous WRKY gene pairs were identified, with 173 pairs in C. sativa and Arabidopsis genomes as well as 282 pairs for C. sativa and B. napus, respectively. 137 segmental duplication events were observed but no tandem duplication in camelina genome. Ten major conserved motifs were examined, with WRKYGQK as the most conserved and several variants existed in many CsWRKYs. Expression analysis revealed that half more CsWRKY genes were expressed constitutively, and a set of them had a tissue-specific expression. Notably, 11 CsWRKY genes exhibited significantly expression changes in plant seedlings under cold, salt, and drought stress, respectively, having preferentially inducible expression pattern in response to the stress.

Conclusions: The present described a detail analysis of CsWRKY gen family and their expression profiled in twelve tissues and under several stress conditions. Segmental duplication is the major force for large expansion of this gene family, and a strong purifying pressure happened for CsWRKY proteins evolutionally. CsWRKY proteins play important roles for plant development, with differential functions in different tissues. Exceptionally, eleven CsWRKYs, particularly five alternative spliced isoforms were found to be the key players possibly in mediating plant response to various stresses. Overall, our results provide a foundation for understanding roles of CsWRKYs and the precise mechanism through which CsWRKYs regulate high stress resistance to stress as well as development of stress tolerance cultivars for Cruciferae crops.

Epigenetics & Genomics

Camelina (Camelina sativa (L.) Crantz)

WRKY transcriptional factors

Genome-wide characterization

Expression profiles

Function analysis

Abiotic stress

Camelina (Camelina sativa (L.) Crantz), a dicotyledonous plant, belongs to the Cruciferae family. As a "low-input and environment-friendly" oil crop worldwide, camelina has been planting in many countries including Europe, Middle East and China [1]. Compared with other commercialized oil crops, C. sativa has several prominent agronomic traits, such as short life cycle (80-100 days), strong tolerance to abiotic stress (salt, drought, cold, etc.), and highly resistance to common pests and diseases infected in many cruciferous crops [2-5]. Camelina seeds accumulate high levels of oil (36%～47%) and protein (30%) as well as a variety of natural active ingredients. The seed oil contains 90% of unsaturated fatty acids of which omega-3 fatty acids are 40% or more. Enriched with such high nutrition and function in improving human immunity and protection against various diseases [5, 6], C. sativa provides the sustainable feedstock for commercial productions of food, feed, biofuel (e.g. aviation fuel and biodiesel), and other high-value industrial products [7]. Numerous studies have focused on camelina seed yield and oil quality [8, 9]. For example, over-expression of microRNA167A in C. sativa seeds reduced α-linolenic acid content and increased the seed size [10]. The expression of diacylglycerol acyltransferase 1 and glycerol-3-phosphate dehydrogenase increased seed oil yields in C. sativa [11]. Seed-specific suppression of ADP-glucose pyrophosphorylase enhanced seed size and weight in C. sativa [12]. However, there are few reports on the molecular mechanism underlying the high tolerance to various stresses in C. sativa.

Abiotic and biotic stresses seriously affect agricultural production, leading to reduction of crop yield and quality [4]. To adapt to diverse stresses, plants have evolved favorable strategies such as metabolic reconstruction, cell-tissue remodeling, and gene expression reprogramming. Transcriptional factors (TFs) can bind on cis-elements or interact with other regulatory factors to regulate expressions of the downstream defense-related genes [13]. Increasing reports show that a number of different TFs play a significant regulatory role in plant stress responses, including bHLH [14], MYB[15], bZIP [16], NAC [17], WRKY [18], and so on. Among them, WRKY proteins (WRKYs) are one of the largest TF families, with complex biological functions, and specificity to many plant species from single-celled algae to higher plants. For instance, in Arabidopsis thaliana, overexpression of AtWRKY50 promoted the production of sinapic derivatives [19]. AtWRKY46, 54, and 70 have important effect on activating the expression of brassinosteroid-mediated genes and restraining the drought gene response [20]. OsWRKY47 positively regulate both the yield and drought tolerance of rice [21]. TaWRKY33 significantly increased the wheat drought tolerance [22]. PtrWRKY18 and PtrWRKY35 enhanced the resistance to Melampsora in Populus [23].

All known WRKYs contain one or two unique DNA binding domains consisting of approximately 60 amino acids (aa) characterized by a highly conserved WRKYGQK sequence (designated as WRKY domain) at the N-terminus, followed by a C2H2 zinc-finger-like motif (C-X4-5C-X22-23-H-X1-H or C-X7C-X23-H-X1-C) at the C-terminus [13]. The WRKY domain specifically binds to the consensus W-box (a cis-acting element with the core sequence TGAC) in promoters of the target genes [24]. According to the number of WRKY domain and the type of zinc-finger-like structure, WRKY proteins are generally classified into three main groups (I-III). Group I WRKY proteins contain two WRKY domains and the zinc finger motif of C-X4-C-X22-23-H-X1-H, whereas Group II and III have only one WRKY domain, with group II proteins sharing the same zinc finger motif as group I and group III proteins bearing the unique zinc finger motif of C-X4-5-C-X23-24-H-X1-H, respectively [25]. Group II WRKYs can be further divided into several distinct subgroups (IIa-e) based on their phylogenetic relationship. In addition, some special resistance protein (R-protein) WRKYs were found in several plant species, with three R-protein WRKYs in Arabidopsis (AtWRKY16, AtWRKY19 and AtWRKY52), and one R-protein WRKY in soybean (GmWRKY1) [26] and pineapple (AcWRKY23) [27] respectively. Such R-protein WRKY may further enhance signal diversity and even shorten the speed of signal transmission with other components of signaling pathways.

Since the first WRKY gene (SPF1) was examined in sweetpotato (Ipomoea batatas) [28], numerous members of WRKY gene family have been identified in a variety of plant species, including 75 from Arabidopsis, 97 from wild rice, 83 from tomato, and 119 from corn [29, 30, 24] (for detail to see Additional file 1, Table S1). Increasing reports demonstrate that WRKY proteins function importantly in plant defense against various biotic and abiotic stresses. For example, overexpression of GmWRKY45 had a positive effect on Arabidopsis response to phosphorus and salinity stress, and also resulted in changes of fertility [31]. The expressions of GmWRKY92, 144 and 165 were greatly upregulated in soybean response to salinity stress [32]. VvWRKY30 from grape (Vitis vinifera L.) was confirmed to confer tolerance to salt stress [33]. Based on these previous findings, we hypothesize that WRKY TFs may mediate regulation of stress resistance in C. sativa despite no genome-wide detection of this gene family has been conducted in this oil crop. Thus, studying WRKY gene family would shed light on the molecular mechanism underlying strong stress tolerance in camelina, providing valuable information for genetic improvement of this oilseed and other crops.

Therefore, the current study was conducted for genome-wide characterization of WRKY family members in C. sativa. A total of 242 CsWRKY proteins were identified from camelina genome. Subsequently, a comprehensive analysis was employed to examine their physicochemical properties and conserved motifs, their genes’ chromosomal locations and duplications as well as intron-exon structures. In order to reveal their evolutionary relationship, a phylogenetic tree was constructed by combined use of WRKY proteins identified in Arabidopsis thaliana from the Cruciferae family. Moreover, the expression profiles of CsWRKY genes were extensively detected using publically available RNA-seq data, and a set of selected CsWRKY gene transcripts were also verified by quantitative Real-time RT-PCR (qRT-PCR) for camelina seedlings under stress conditions. Notably, 33 alternative splicing events were identified among CsWRKY genes, showing that different spliced variants derived from the same CsWRKY gene expressed at varied ratios in response to different stresses. Our work offers scientific resources for understanding the biological roles of CsWRKYs, particularly in plant resistance to various stresses.

Identification of 242 WRKY family members in Camelina sativa

To identify WRKY proteins encoded in camelina genome, all 75 Arabidopsis AtWRKY protein sequences were used as queries to search the publicly available genome sequences of C. sativa by BLAST (Basic Local Alignment Search Tool), and then examined using HMM (Hidden Markov Model) with the WRKY-domain (PF03106). A total of 243 putative WRKYs were initially obtained in Camelina sativa. One predicted WRKY protein (XP_019082498.1) was removed due to incomplete WRKY domain. The remaining 242 WRKYs (see Additional file 2: Table S2) were renamed from CsWRKY1 to CsWRKY242, respectively, and their characteristics were further detected, including gene locus ID, gene start and end position in the chromosomes, member classification, the protein sequence length (SL), molecular weight (MW) and isoelectric point (pI). The length of CsWRKY proteins ranged from 136 (CsWRKY136) to 1699 amino acids (CsWRKY47), with an average of 371 amino acids. Their MWs ranged from 15.98 kDa (CsWRKY136) to 190.70 kDa (PvWRKY34), with an average of 41.33 kDa. The pIs of CsWRKYs ranged from 4.13 (CsWRKY204) to 10.47 (CsWRKY107), with an average of 7.38 (Additional file 2: Table S2).

Compared with the numbers of WRKY family members reported in other plant species, CsWRKYs family with 224 gene loci coded 242 proteins is one of the largest WRKY families, just less than that in Brassica napus (287 WRKY genes) (Additional file 1: Table S1), indicating that this TF family was extensively expanded in C. sativa during evolution process.

Alternative splicing (AS) occurs among 15 CsWRKY genes, producing 33 spliced ORFs

Alternative splicing (AS) is an important post-transcriptional regulatory mechanism, which causes one gene locus to generate two or more ORFs (open reading frame) different in initiation and termination sites, as well as splice donor and acceptor sites. These alternative ORFs encode different protein variants from the same gene locus. In this study, we identified that 15 CsWRKY genes have different types of AS events such as exon skipping and intron retention, producing 33 spliced variants of CsWRKY (see Additional file 3: Table S3, Additional file 8: figure S1). All the splice variants were completely matched by their corresponding mRNAs identified in camelina. For example, Csa06g039950 had two spliced variants (CsWRKY21 and 22) while Csa10g016180 generated three spliced variants (CsWRKY41, 35, and 38). These results indicate that alternative splicing might be worked in regulating the function of WRKY family members.

Multiple sequence alignment and phylogenetic analysis of CsWRKY proteins

To investigate the evolutionary relationship among CsWRKY proteins, a multiple protein sequence alignment of all 242 CsWRKYs was conducted using ClustalW with Bioedit software. Alignment result of the randomly-selected CsWRKYs was showed in Figure 1(For details to see Additional file 9: Figure S2). Subsequently, based on the highly conserved WRKY domains of 242 CsWRKYs and 75 AtWRKYs, a phylogenic tree was built by MEGA7.0 software using the neighbor-joining method (Figure 2).

WRKYGQK heptapeptide is considered as the signature for WRKY proteins. As shown in Figure 1, however, WRKY domain amino acid sequence variants were detected among 11 CsWRKYs (all in group IIc) although WRKYGQK was the most common variant present in 231 CsWRKYs. For WRKYGQK sequence, most variations involved Q to K substitutions, with a few cases from Q to other amino acids. For example, the variant WRKYGXK sequence (X is any amino acid) was detected in CsWRKY202, but the variant WRKYGKK sequence were present in 10 CsWRKYs including CsWRKY127, 128, 135, 223, 228, 229, 230, 232, 233 and 235. All these variants were occurred in group IIc, showing that WRKYGQK sequence in subgroup IIc WRKY proteins prone to mutate, like in WRKY families from peanut [34] and soybean [32].

According to the constructed phylogenetic tree containing CsWRKYs and AtWRKYs (Figure 2), the CsWRKYs were classified into three main groups (groups I, II and III), with 56 CsWRKYs in group I, 149 CsWRKYs in group II, and 37 CsWRKYs in group III, respectively. Moreover, CsWRKYs in group II were further classified into five subgroups (groups IIa, IIb, IIc, IId and IIe), containing 12, 26, 63, 24, and 24 members of CsWRKYs, respectively. Notably, among all these groups or subgroups most members of the CsWRKYs were present in subgroup IIc, which is similar to AtWRKY proteins. Combined description above that WRKYGQK sequence variants were only present in subgroup IIc, this suggests that subgroup IIc WRKY proteins might possibly involve in a variety of biological functions.

Of the 242 CsWRKYs found, 56 CsWRKYs in group I had two conserved WRKY domains located in the N- and C-terminus of the protein, respectively, as well as the zinc-finger domains of C-X4-C-X22-23-H-X1-H. Total of 186 CsWRKYs in group II and III contained one WRKY domain. 149 CsWRKYs in group II had the zinc-finger domain of C-X4-5-C-X23-24-H-X1-H while 37 CsWRKYs in group III had the zinc-finger domain of C-X7-C-X23-H-X1-C. It was worth noting that based multiple sequence alignment, either one or two more mismatched amino acids were detected within the conserved regions of 144 CsWRKY proteins including CsWRKY100, 101, 111, 186, 201, 208, 226, 240, 241, and 242. Such annotation errors might be caused by genomic sequencing or gene prediction software.

As shown in Figure 1, each WRKY domain had at least one conserved intron structure except the N-terminal WRKY domain of group I WRKYs. Intron insertion position was also rather conserved. Two main kinds of intron structures were present in the conserved regions of CsWRKY genes, much likely to those conserved in AtWRKYs [35]. One of the introns is PR intron which was spliced at the codon of R amino acid. The other is the VQR intron. The PR intron is located in the WRKY domains of CsWRKY genes in group III, and subgroups Ic and IIc–e. However, the VQR intron exists within the zinc-finger structure (C-X4-5-C-X5-VQR-X18-19-H-X1-H) in group IIa and IIb.

A number of WRKY proteins contain both R-protein conserved domains and the typical WRKY domain, and thus named R-protein WRKYs, which are one of the most significant features of the WRKY proteins in flowering plants [25]. For example, Arobidopsis had three R protein-WRKYs (AtWRKY16, AtWRKY19 and AtWRKY52) [26]. Here, the phylogenetic analysis showed that AtWRKY19 and CsWRKY47 were clustered closely in group I while AtWRKY16, AtWRKY52, CsWRKY186 and CsWRKY220 were highly classified together into subgroup IId. This indicated that CsWRKY47, CsWRKY186 and CsWRKY220 may belong to R-protein WRKYs. Further protein architecture analysis identified three CsWRKY proteins as the R-protein WRKYs. CsWRKY47 contained the typical domain of PAH-WRKY (1-N terminus)-WRKY (1-C terminus)-NB-ARC and the protein kinase domain in the C-terminal end of the protein. CsWRKY186 had the typical domain of NB-ARC-LRR-WRKY. CsWRKY220 had the TIR-NB-ARC-LRR-WRKY domain. The three R-protein CsWRKYs might be involved in camelina responses to biotic and abiotic stress.

Ten conserved motifs were detected in CsWRKY proteins

In order to gain insight into the functional regions of CsWRKY proteins, the MEME (Multiple Em for Motif Elicitation) program was employed to reveal the conserved motifs among 242 CsWRKY proteins. A total of 10 conserved motifs were identified, namely Motif 1-10 (Figure 3). These 10 conserved motifs were shown with colored boxes according to the scale (Additional file 10: Figure S3), with sizes ranged from 15 to 50 aa residues in width (Figure 3). Among them, motif 1was examined to encode the conserved WRKY domain, motif 2 and 3 were examined to encode the conserved zinc finger structure. One or two WRKY motifs were detected in all CsWRKYs as described in the sequence alignment (Figure 1). In addition to the conserved WRKY motif, other conserved motifs (motifs 4-10) were also predicted to exist among CsWRKY proteins. Each CsWRKY protein had at least three conserved motifs, with the maximum number of seven conserved motifs for several CsWRKYs. The distributions of conserved motifs were diverse among different CsWRKY groups. For example, group I CsWRKYs had nine motifs (motif 1, 2, 3, 4, 5, 6, 8, 9 and 10), with each having at least two of motif 1, one zinc finger (motif 2 and 3), as well as motif 6 and 9 which were unique to the CsWRKY members in group I. Motif 10 only appeared in both group I and subgroup IIc. Motif 4 and 8 were present in group I, and subgroup IIb and IIc. Motif 7 existed in subgroup IId, IIe and group III. Motif 5 was only absent in subgroup IIc and IIe. However, all CsWRKY members of group IIa contained two conserved Motif 5. As a whole, the motif analysis of CsWRKYs showed that every group or subgroup of CsWRKYs had similar motif compositions, corresponding to the grouping by the phylogenetic tree analysis.

CsWRKY genes were uneven distributed on chromosomes

To determine the genomic distribution of CsWRKY genes, all 242 CsWRKY mRNAs/ORFs identified were mapped on their corresponding chromosome by BLAST against the released genome of C.sativa. A total of 224 CsWRKY gene loci were distributed across all C. sativa chromosomes visualized by MapChart (Chr1-Chr20, Figure 4), with 15 CsWRKY gene loci generating 33 alternatively spliced variants of CsWRKY ORFs (Additional file 3: Table S3) (For details to see Additional file 8: figure S1). However, the distribution and density of the WRKY genes on each chromosome were uneven. For example, the minimal number of CsWRKY genes (three loci) were located chromosome 1, 15 and 19 whereas the largest number of CsWRKY genes (20 loci) were detected on chromosome 11, which accounted for 8.3% of all CsWRKY genes. Moreover, the same number of CsWRKY genes (11 loci) existed on chromosome 2, 8, 9 and 13). One exception is that CsWRKY179 was not mapped to any chromosome since it’s present on an scaffold region. Interestingly, a few regions with a higher density of CsWRKY genes were observed on some chromosomes such as chromosome 10, 11, and 16, suggesting that there might be WRKY gene hot spots in the camelina genome.

CsWRKY gene family experienced 137 segmental duplication events, having a high synteny with WRKYs from Arabidopsis and Brassica rapa

As described above, CsWRKY gene family was expanded much greatly compared to other plant WRKY families, ranking as the second in the quantity of WRKY member tested so far. To elucidate the mechanism underlying WRKY gene family expansion in C. sativa, Blastp and MCScanX (Multiple Collinearity Scan toolkit) were employed to identify gene duplication patterns (tandem and segmental duplication), which were considered to provide genetic materials for the generation of new genes and the evolutionary formation of gene new biological functions [32, 36-39]. Results showed that 137 segmental duplication events were detected for 146 CsWRKY genes (Figure 5, Additional file 4: Table S4). However, no tandem duplication was observed for any CsWRKY gene. No tandem duplication event was further confirmed on the basis of the method by Guo et al [40]. Compared to other plant WRKY genes families tested (see Additional file 5: Table S5), CsWRKY genes were experienced the largest number of segmental duplication events. These revealed that segmental duplication was a major driving force for CsWRKY gene evolution.

The comparative synteny maps of two related genomes (C. sativa VS A. thaliana, and C. sativa VS Brassica rapa (B. rapa)) were performed in order to further explore the origin and evolution of CsWRKY genes (Figure 6, Additional file 6: Table S6). The orthologous relationships were detected between 173 CsWRKY genes and 65 AtWRKY genes, and then 173 orthologous WRKY gene pairs were identified accordingly, with most of them located on the syntenic locus in Arabidopsis and C. sativa chromosomes. Similarly, the orthologous relationships were also present between 166 CsWRKY genes and 111 BrWRKY genes, and the corresponding 282 orthologous WRKY gene pairs were built, with lots of them found on the syntenic locus in chromosomes of C. sativa and B. rapa. Remarkably, multiple CsWRKY genes were identified to be putative orthologs of a single AtWRKY gene. For instance, CsWRKY123, CsWRKY124 CsWRKY125 and CsWRKY126 were the orthologs of AtWRKY39. Such syntenic relationship detected form these WRKY genes indicates the expansion of CsWRKY genes possibly occurred after A. thaliana in evolution.

To investigate whether these orthologous WRKY genes underwent selection pressure (purifying and positive selection), the synonymous substitution rates (Ks) and non-synonymous substitution rates (Ka) of the identified orthologous CsWRKY gene pair were calculated using KaKs Calculator 2.0, followed by calculating the Ka/Ks to determine if the selective pressure acts on protein-coding CsWRKY genes or not [41]. Interestingly, Ks and Ka values for all orthologous CsWRKY gene pairs had a ω value of <1, indicating that purifying selection happen in these gene pairs, which is consistent with the reports on B. rapa by Tang et al [18].

Expression patterns of CsWRKY genes in twelve camelina tissues

Gene expression pattern can provide essential information to determine the biological function of a gene. To explore the possible functions of CsWRKY genes in C sativa growth and development, transcriptome data of C. sativa under normal growth conditions were downloaded from the publically available database [6] and used to examine the expression patterns of 202 CsWRKY genes in twelve tissues/organs, including root (R), stem (S), young leaf (YL), mature leaf (OL), flower (F), inflorescence (IF), early seed development (ESD), early-mid seed development (EMSD), late-mid seed development (LMSD), late seed development (LSD), germinating seed (GS) and cotyledon (C). The heat map illustration of expression profiles of CsWRKY genes is shown in Figure 7.

Notably, half more CsWRKY genes tested were found to express in at least one of those tissues although CsWRKY36 and 227 did not show any detectable expression. For example, 180 CsWRKY genes (89.11%) were expressed in R while 106 CsWRKY genes (52.48%) accumulated in LSD. The percent of expressed CsWRKY gene number were 88.61% in F, 81.68% in IF, 80.69% in S, 78.71% in OL, 77.23% in ESD, 76.73% in EMSD, 75.25% in GS, 68.32% in C, 67.82% in LMSD, and 64.85% in YL, respectively. 70 CsWRKY genes (34.65%) were expressed in all twelve tissues, including 28 CsWRKYs in group I, 41 CsWRKYs in group II, and one CsWRKY gene in group III. Particularly, 24 CsWRKY genes displayed high expression levels in all tissues. Furthermore, a number of CsWRKY genes exhibited a tissue-specific expression pattern. For example, genes expressed preferentially in two tissues included CsWRKY25 and 34 from group I in IF and LSD, CsWRKY174 of group IIb in GS and R, CsWRKY73 and 203 from group IIc in F and OL or GS, CsWRKY118 from group IIe in GS and S, and CsWRKY100 from group III in ESD and R. The genes expressed specifically in one tissue were CsWRKY182, 202 as well as 204 from group IIc in F, and ESD, respectively, and CsWRKY111, 208 and 226 from group III in R. Such expression analysis indicates that most of CsWRKY genes may function constitutively for plant organ development, with a number of them working differentially in different tissues.

A number of CsWRKY genes expressed in responses to various abiotic stresses

The functions of most AtWRKY genes had been verified, which can be used to infer potential roles of CsWRKY genes clustered together with the AtWRKYs in phylogenetic tree. For instance, AtWRKY25 and 33 were sensitive to NaCl stress, and overexpression of either of them enhanced tolerance to NaCl in Arabidopsis [42]. According to structural features and phylogenetic analysis of CsWRKYs and AtWRKYs, AtWRKY33 was identified to be the ortholog of CsWRKY8, 9 and 10, while AtWRKY25 was the ortholog of CsWRKY48, 49, and 50, indicating that these six CsWRKY genes may participate in plant responses to NaCl stress. Subsequently, to verify this prediction, expression profiles of the six CsWRKY genes were examined in roots and shoots under salt stress (SS) and normal conditions (NC) by quantitative RT-PCR (qRT-PCR) (Figure 8). These six genes (CsWRKY8, 9, 10, 48, 49, and 50) showed higher levels of expressions in shoots than that in roots under SS. Compared with gene expression levels in NC, expressions of three CsWRKY genes (CsWRKY8, 9, and 10) under SS were significantly down-regulated in roots, whereas the other three CsWRKY genes (CsWRKY48, 49, and 50) had no obvious change in roots. In particular, CsWRKY50 transcript was significantly up-regulated in shoots under SS. These results implied that the six CsWRKY genes may positively regulate SS response in shoot, whereas three of them (CsWRKY8, 9, and 10) may act negatively in roots.

As described above, 15 CsWRKY gene loci contain alternative structures. To investigate whether all alternative splice variants from a CsWRKY gene locus involve in plant responses to various stress, total of 5 splice transcripts derived from two CsWRKY gene loci were selected to examine their expression levels in camelina seedlings treated by cold stress (CS), drought stress (DS) and, and SS, respectively. As shown in Figure 9, these five splice isoforms had different expressions under the three stress conditions, with three of them expressing 10-fold higher than the other two. CsWRKY21 and 22, the two splice variants from gene locus Csa06g039950, both showed the highest expression at almost the same level under SS among the three treatments. However, CsWRKY21 exhibited significantly larger expression under CS than under DS. On the contrary, CsWRKY22 expression was more under DS than under CS. Similarly, CsWRKY35, 38, and 41, the three spliced variants from gene locus Csa10g016180, expressed at different levels, with CsWRKY35, 38, and 41 as the predominant transcript in response to DS, CS, and SS, respectively. More strikingly, these data led to unexpected findings that alternative slice variants from the same gene locus may function differentially in plant responses to different stresses.

Conserved and divergent features among CsWRKY family members

As one of the largest TF families in higher plants, the WRKY family members participate in plant development and in response to various stresses. In this study, we used genome and RNA-seq data to identify 224 WRKY gene loci encoding 242 CsWRKY proteins in C. sativa. Compared to quantity of WRKY family members detected in other higher plants like rice [43], soybean [32], peper [44], peanut [34], sesame [45] (See Additional file1: Table S1 for details), CsWRKY family is the second large family after Brasica napus [46] with 287 WRKY members. This indicates that a large-scale expansion of this family happened in C. sativa genome, which might be results of gene duplication (see discussion below). Subsequently, these CsWRKY proteins were characterized according to the specific conserved sequences of WRKYs and number of their WRKY zinc-finger motifs [35], following the principle used for Arabidopsis [35] and Common bean [47]. Based on phylogenetic analysis of CsWRKYs and AtWRKYs, 242 CsWRKYs were divided into three main groups: Group I, Group II (subgroup IIa, IIb, IIc, IId, and IIe) and Group III, with group II containing 149 members which accounts for the largest proportion (62%) among all the groups. The higher proportion of group II members was also detected in other plant WRKY families such as 58% in Arabidopsis, 64% in Caragana intermedia [48] and 66% in Manihot esculenta [49]. For each group, CsWRKY members exhibited the similar structure, but members in different group showed specific feature, which is in agreement with reports in other plants such as chickpea [50] and common bean [47].

Previous reports indicated that group I WRKY containing two WRKY domains was considered to be the ancient members during the WRKY evolution. The WRKYs in group IIa and IIb came from an algal single WRKY domain or the other Group 1-derived lineage [26, 50]. The members of group IIc had been evolved from the lack of N-terminal domain of WRKYs in group II. Despite of these hypotheses, it is unknown what is the origination of each type of WRKY proteins in C. sativa.

In spite of the strong conservation of WRKY domain for WRKY proteins, CsWRKY proteins showed certain degree of divergence in structure. The heptapeptide WRKYGQK is the typical domain for WRKY family. However, eleven variants including WRKYGXK, WRKYGKK, and WRKYGEK for this domain were detected in a number of CsWRKY proteins in group IIc. In supporting of this, several variants of WRKY domain were also found in in other plant species [13, 40, 51, 52], suggesting that these variants might give WRKYs multiple biological functions [47] although it is worthy of future research. In addition, we also identified several CsWRKY proteins as the chimeric proteins containing both R-protein conserved domain and WRKY domain. For example, CsWRKY47, CsWRKY186 and CsWRKY220 were newly examined as R protein-WRKYs. As reported in Arabidopsis, AtWRKY coded a NBS-LRR-WRKY protein. This protein worked as chimeric protein and the WRKY domain had the DNA-binding activity [53]. The interaction of AtWRKY52/RRS1 with R protein RPS4 protected plant from the infection of fungal and bacterial pathogens [39]. Possibly, R protein-WRKYs detected in C. sativa can work in plant resistance to disease or other stresses, like in case of Arabidopsis, but the putative function needs to be further investigated.

Segmental duplication contributed critically to expansion of CsWRKY gene family in evolution

Increasing reports indicated that gene duplication (e.g. tandem duplication, segmental duplication, and genome duplication) was the key force for gene family expansion in plant genomes [50]. Here, genome-wide identification and analysis of the WRKY gene family in C. sativa identified 137 segmental duplication events but no tandem duplication event. Furthermore, the comparative synteny map between C. sativa and A. thaliana provided more evidence for gene duplication generated during the evolution of C. sativa. Total of 173 CsWRKY genes were identified to have 65 corresponding Arabidopsis orthologs, and even multiple CsWRKY genes were found to be corresponded to a single AtWRKY ortholog. All these data demonstrate that segmental duplication is the major force for expansion of CsWRKY gene family. As the key factor, segmental duplication was detected to be related with expansion of WRKY gene family in Chickpea, Pineapple and Kiwifruit [24, 27, 50]. Therefore, we can speculate that segmental duplication events are the primary cause to drive CsWRKY gene family to expand so large members. It is worth to note that genome duplication, tandem duplication, and segmental duplication were combined together to form the evolution force for gene duplication in other plants, particularly diploidized polyploids. It was also reported that tandem duplication was the main contributor to the enlargement of BnaWRKY genes in group III [46], and the duplication events did not happen in the pepper WRKY gene family [44].

The genetic evolutionary process was reestablished probably by comparing all gene sequences within the same genome or among the different genomes [54]. Notably, according to the syntenic map of C. sativa and B. rapa, 166 CsWRKY genes were detected to have 111 corresponding B. rapa orthologs. Thus, the expansion of CsWRKY gene family probably occurred after separation of A. thaliana and B. rapa.

These duplicated CsWRKY genes probably formed new gene functions to adapt to various growth conditions. The majority Ka/Ks ratios of the above syntenic WRKY gene pairs were less than 1, which demonstrated that CsWRKY genes had undergone strongly the evolution of purify selection. Purify selection (Ka/Ks ratio < 1) usually caused the elimination of deleterious genes in evolution and favored to generate genes with conserved functions, which indicated the key conserved sequences of WRKY genes was beneficial to the plant survival and growth eventually [7, 50].

CsWRKY proteins may be major players in response to various stresses

In plants, the WRKY proteins work as one of the most important TF families and appear to regulates plant responses to biotic and abiotic stresses by acting as positive or negative regulators. Many WRKY genes were identified to respond to various stress in Arabidopsis, rice and soybean, respectively. For example, the high expression of WRKYs had the vital influence on transcription activation of downstream target genes in the plant growth and development [29]. The tissue-specific expression of WRKY genes might have great effect during the plant growth and development by regulating the transcriptional process [18]. In the present study, lots of CsWRKY genes were detected to be constitutively expressed in various tissues tested, with many of them showing a tissue-specific expression pattern. More importantly, a number of CsWRKY genes exhibited to dramatically expression change under various stress conditions. Six of such CsWRKY genes (CsWRKY8, 9, 10, 48, 49, and 50) were further selected for investigation, indicating that they positively regulate the stress response in shoot, but CsWRKY8, 9, and 10 play negatively in roots. The transgenic experiments in C. sativa are currently under way to identify their precise biological functions and to evaluate their potentials in genetic engineering for improving crop stress resistance and other agronomic traits.

One of novel findings in this study is that five splice isoforms (CsWRKY 21, 22, 35, 38 and 41) derived from two CsWRKY gene loci were differently expressed in plant seedlings under cold, salt, and drought stresses. In view of this, we can speculate that different alternative variants from same gen locus function differentially in different stress responses. However, the accurate regulatory mechanism requires to be elucidated in further detail.

In this study, a total of 242 CsWRKY proteins were firstly identified in Camelina Sativa, and classified into three main groups (I, II, III). 33 alternative splicing events were detected for 15 CsWRKY gene loci. Many orthologous WRKY gene pairs were identified between every two genomes of C. sativa, A. thaniana, and B. napus, showing a high synteny among the three genomes. Segmental duplication events were found to be the major force for great expansion of CsWRKY gene family. Moreover, camelina WRKY proteins experienced a strong purifying pressure in evolution. Half more CsWRKY genes were expressed in various organs, with many CsWRKYs having a tissue-specific expression pattern, demonstrating that CsWRKYs function critically in plant development and also act differentially in different tissues. Remarkably, eleven CsWRKYs including five alternative spliced isoforms may play crucial roles in regulating plant response to various stresses although this will require extensive additional analyses. Overall, our study establishes a functional framework to investigate CsWRKY proteins and mechanism responsible for high resistance to various stresses, facilitating the development of molecular breeding programs to enhance abiotic stress tolerance in camelina and other crops.

Sequence identification

The complete genome, proteome and CDS sequence files of C. sativa were downloaded from webpage of NCBI (ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/633/955/GCF_000633955.1_Cs/) and Genome Prairie - Prairie Gold (http://www.camelinadb.ca). The WRKY domain HMM (Hidden Markov Model) profile numbered PF03106 was extracted from the Pfam protein family database (http://pfam.xfam.org/) [47]. The candidate WRKY protein sequences were discovered by the comprehensive research of utilization of HMMER (E-value cut-off < 1E-5) and BLAST analyses (75 AtWRKYs as queries) in C. sativa whole genome protein database [49]. The AtWRKYs sequences were obtained from NCBI (https://www.ncbi.nlm.nih.gov/). These CsWRKYs sequences were identified by checking the complete WRKY conserved domain with SMART (http://smart.embl-heidelberg.de/) and InterPro (http://www.ebi.ac.uk/interpro/), the redundant sequences were manually removed. The confirmed CsWRKYs used the ExPasy online tool website (http://web.expasy.org/protparam/) to calculate physicochemical properties including SL, MW and PI.

Multiple Sequence Alignment and the Construction of Comparative Phylogenetic Tree

Multiple sequence alignment of the CsWRKY domain was performed using ClustalW with Bioedit software. Based on the alignment WRKY domains of CsWRKYs and AtWRKYs, the phylogenetic tree was constructed with MEGA 7.0 using Neighbor-Joining (NJ) method and the parameters (Poisson model, pairwise deletion, and 1000 bootstrap replications). All identified CsWRKYs were divided into different groups according to classification of AtWRKYs sequences.

Motifs Analysis

The MEME (http://meme.nbcr.net/meme/intro. html) was used to analyze CsWRKYs and searched for 10 conserved motifs. The interrelated parameters as follow: the repetitive time was any, the maximum motif number was 20 and the motif width was between 5 and 50 residues. The MEME results were displayed with the TBtool software [55].

Chromosomal Distribution

The chromosomal position of all CsWRKY genes was determined from the genome annotation file. The physical position of CsWRKY genes from short-arm to long-arm telomeres on the chromosome was mapped using MapChart [56].

Gene Duplication and Selection Pressure Analyses

Segmental duplication events within C. sativa genome were studied by using MCScanX (Multiple Collinearity Scan toolkit), the whole analytic process was utilized with default setting. Tandem duplication event (the distance of two or more genes within 200kb in equal chromosome) was checked on the basis of the method by Guo et al [40]. The synteny relation between two different genomes (C. sativa and A. thaliana, C. sativa and B. rapa) were identified and analyzed. The B. rapa data was downloaded from the Phytozome database (https://phytozome.jgi.doe.gov/pz/portal.html). The Ks and Ka of the identified CsWRKY gene pair were calculated using KaKs Calculator 2.0 [41].

Expression Profiling of CsWRKY Genes in Different Tissues

The transcriptional data of C. sativa was obtained by publically available database [6]. During twelve tissues of various developmental stages, the expression levels of CsWRKY genes were utilized to analysis, including C, EMSD, ESD, F, GS, IF, LSD, LMSD, OL, R, S and YL. The hierarchical clustering and the heatmap-based expression profiles of CsWRKY genes were performed by HemI1.0. In order to further explore the function of CsWRKY genes, we researched whether some CsWRKY genes responds to SS. the RNA-Seq data of C. sativa we used in response to SS came from publically available database [4]. C. sativa cultivar ''SC-N1'', commercially planted for 5 years in Taigu County, Shanxi province, China (E112.32°, N37.26°) was selected to our further experiments [5]. Plants were grown in a greenhouse (16-h light/8-h dark, 23°C). The 3-week-old soil-grown seedlings were treated under cold stress(CS)、drought stress(DS) and SS respectively. The seedlings were treated at 4 °C for CS. Soil moisture content is 35% ± 5 of NC for DS. The seedlings were irrigated by NaCl solutions at 150 mM in climatic chamber. The seedlings were treated for up to 3 days. Camelina seedlings were collected and frozen immediately in liquid N₂ for RNA extraction at 3 days after different treatments. Use TaKaRa RNAiso Plus kit to extract total RNA from all samples. 1 μg RNA was reverse-transcribed into cDNA with TaKaRa reverse transcription kit, and the same amount of cDNA was used as a template for qRT-PCR. The β-actin gene of C. sativa was selected as the internal reference gene [5]. All primer pairs used for qRT-PCR analysis were shown in Additional file 7: Table S7. These experiments were repeated three times.

WRKYs, WRKY proteins; TF, Transcriptional factor; qRT-PCR, quantitative Real-time RT-PCR; AS, Alternative splicing; SL, protein sequence length; MW, molecular weight; PI, isoelectric point; Ks, synonymous substitution rates; Ka, non-synonymous substitution rates; Camelina sativa, C. sativa; Arabidopsis thaliana, A. thaliana; Brassica rapa, B. rapa; C, cotyledon; EMSD, early-mid seed development; ESD, early seed development; F, flower; GS, germinating seed; IF, inflorescence; LSD, late seed development; LMSD, late-mid seed development; OL, Mature leaf; R, Root; S, stem; YL, young leaf; SS, salt stress; NC, normal conditions; CS, cold stress; DS, drought stress

Acknowledgements

Thanks to all co-authors for their dedication to this article.

Authors’ contributions

YNS and HLC conceived of the study, performed all the experiments, including the sequence alignment and phylogeny analysis and drafted the manuscript; YS, JAX, and CLJ conducted bioinformatics analysis, data mining and sample collection. CHZ, and LXY participated in research work and partially designed the study. RZL guided the project and revised the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by grants from National Natural Science Foundation of China (#31801400, 31902394), Specialized Scientific Project of Shanxi Agricultural Valley Construction (SXNGJSKYZX201906), Applying Basic Research Planning Project of Shanxi Province (201801D221250), Key Research and Development (R&D) Planning Project of Shanxi Province (201803D31063), Key Research and Development (R&D) Planning Project of Jinzhong City (201903D01100013 and Y192012), Science and Technology Innovation Planning Project of Shanxi Agricultural University (2018YJ16), the Shanxi Province Key Projects of Coal-based Science and Technology (FT-2014-01), the Project of the Key Research and Development Program of Shanxi Province, China (Grant No. 201603D312005), Graduate Education Innovation Project of Shanxi Province (2020BY056), and "1331 project" for key innovation team of Jinzhong University (Grant No. jzxycxtd2019009).

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files (from Additional file 1 to Additional file 10). The complete genome, proteome and CDS sequence files of C. sativa were downloaded from webpage of NCBI (ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/ 000/633/955/GCF_000633955.1_Cs/) and Genome Prairie - Prairie Gold

(http://www.camelinadb.ca).

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

¹Institute of Molecular Agriculture and Bioenergy, Shanxi Agricultural University, Jinzhong, Shanxi, China. ²College of Biological Science and Technology, Jinzhong University, Jinzhong, Shanxi, China

Liang, C., et al., De novo assembly and characterization of Camelina sativa transcriptome by paired-end sequencing. BMC Genomics, 2013. 14: p. 146.
Brock, J.R., et al., Phylogenetics of Camelina Crantz. (Brassicaceae) and insights on the origin of gold-of-pleasure (Camelina sativa). Mol Phylogenet Evol, 2018. 127: p. 834-842.
Abdullah, H.M., et al., Transcriptome profiling of Camelina sativa to identify genes involved in triacylglycerol biosynthesis and accumulation in the developing seeds. Biotechnol Biofuels, 2016. 9: p. 136.
Heydarian, Z., et al., Changes in gene expression in Camelina sativa roots and vegetative tissues in response to salinity stress. Scientific Reports, 2018. 8(1).
Yuan, L., et al., Characterisation of phospholipid: diacylglycerol acyltransferases (PDATs) from Camelina sativa and their roles in stress responses. Biol Open, 2017. 6(7): p. 1024-1034.
Kagale, S., et al., The developmental transcriptome atlas of the biofuel crop Camelina sativa. Plant J, 2016. 88(5): p. 879-894.
Biswas, S. and J.M. Akey, Genomic insights into positive selection. Trends Genet, 2006. 22(8): p. 437-46.
Yuan, L. and R. Li, Metabolic engineering a model oilseed Camelina sativa for the sustainable production of high-value designed oils. Frontiers in Plant Science, 2020. 11.
Malik, M.R., et al., Camelina sativa, an oilseed at the nexus between model system and commercial crop. Plant Cell Rep, 2018. 37(10): p. 1367-1381.
Na, G., et al., Enhancing microRNA167A expression in seed decreases the alpha-linolenic acid content and increases seed size in Camelina sativa. Plant J, 2019. 98(2): p. 346-358.
Abdullah, H.M., et al., Comparative transcriptome and metabolome analysis suggests bottlenecks that limit seed and oil yields in transgenic Camelina sativa expressing diacylglycerol acyltransferase 1 and glycerol-3-phosphate dehydrogenase. Biotechnol Biofuels, 2018. 11: p. 335.
Na, G., et al., Seed-specific suppression of ADP-glucose pyrophosphorylase in Camelina sativa increases seed size and weight. Biotechnol Biofuels, 2018. 11: p. 330.
Li, C., et al., Molecular cloning and expression analysis of WRKY transcription factor genes in Salvia miltiorrhiza. BMC Genomics, 2015. 16: p. 200.
Jin, C., et al., Overexpression of a bHLH1 transcription factor of Pyrus ussuriensis confers enhanced cold tolerance and increases expression of stress-responsive genes. Front Plant Sci, 2016. 7: p. 441.
Guo, H., et al., Expression of the MYB transcription factor gene BplMYB46 affects abiotic stress tolerance and secondary cell wall deposition in Betula platyphylla. Plant Biotechnol J, 2017. 15(1): p. 107-121.
Ji, C., et al., Analysis of bZIP Transcription Factor Family and Their Expressions under Salt Stress in Chlamydomonas reinhardtii. Int J Mol Sci, 2018. 19(9).
Yang, X., et al., Miscanthus NAC transcription factor MlNAC12 positively mediates abiotic stress tolerance in transgenic Arabidopsis. Plant Sci, 2018. 277: p. 229-241.
Tang, J., et al., Genome-wide fractionation and identification of WRKY transcription factors in Chinese Cabbage (Brassica rapa ssp. pekinensis) reveals collinearity and their expression patterns under abiotic and biotic stresses. Plant Molecular Biology Reporter, 2013. 32(4): p. 781-795.
Hussain, R.M.F., et al., Overexpression of AtWRKY50 is correlated with enhanced production of sinapic derivatives in Arabidopsis. Metabolomics, 2018. 14(3): p. 25.
Chen, J., et al., Arabidopsis WRKY46, WRKY54, and WRKY70 transcription factors are involved in brassinosteroid-regulated plant growth and drought responses. Plant Cell, 2017. 29(6): p. 1425-1439.
Raineri, J., et al., The rice transcription factor OsWRKY47 is a positive regulator of the response to water deficit stress. Plant Mol Biol, 2015. 88(4-5): p. 401-13.
He, G.H., et al., Drought-responsive WRKY transcription factor genes TaWRKY1 and TaWRKY33 from wheat confer drought and/or heat resistance in Arabidopsis. BMC Plant Biol, 2016. 16(1): p. 116.
Jiang, Y., et al., The WRKY transcription factors PtrWRKY18 and PtrWRKY35 promote Melampsora resistance in Populus. Tree Physiol, 2017. 37(5): p. 665-675.
Jing, Z. and Z. Liu, Genome-wide identification of WRKY transcription factors in kiwifruit (Actinidia spp.) and analysis of WRKY expression in responses to biotic and abiotic stresses. Genes Genomics, 2018. 40(4): p. 429-446.
Rushton, P.J., et al., WRKY transcription factors. Trends Plant Sci, 2010. 15(5): p. 247-58.
Rinerson, C.I., et al., The evolution of WRKY transcription factors. BMC Plant Biol, 2015. 15: p. 66.
Xie, T., et al., Genome-wide investigation of WRKY gene family in pineapple: evolution and expression profiles during development and stress. BMC Genomics, 2018. 19(1): p. 490.
Sumie Ishiguro., K.N., Characterization of a cDNA encoding a novel DNA.binding protein, SPF1, that recognizes SP8 sequences in the 5' upstream regions of genes coding for sporamin and p.amylase from sweet potato. 244(6).
Xu, H., et al., WRKY transcription factor genes in wild rice Oryza nivara. DNA Res, 2016. 23(4): p. 311-23.
Karkute, S.G., et al., Genome wide expression analysis of WRKY genes in tomato (Solanum lycopersicum) under drought stress. Plant Gene, 2018. 13: p. 8-17.
Li, C., et al., GmWRKY45 Enhances Tolerance to Phosphate Starvation and Salt Stress, and Changes Fertility in Transgenic Arabidopsis. Frontiers in Plant Science, 2020. 10.
Song, H., et al., Global analysis of WRKY genes and their response to dehydration and salt stress in soybean. Front Plant Sci, 2016. 7: p. 9.
Zhu, D., et al., VvWRKY30, a grape WRKY transcription factor, plays a positive regulatory role under salinity stress. Plant Science, 2019. 280: p. 132-142.
Song, H., et al., Genome-Wide Identification and Characterization of WRKY Gene Family in Peanut. Front Plant Sci, 2016. 7: p. 534.
Eulgem, T., et al., The WRKY superfamily of plant transcription factors. Trends Plant Sci, 2000. 5(5): p. 199-206.
Zhu, Y., et al., Soybean (Glycine max) expansin gene superfamily origins: segmental and tandem duplication events followed by divergent selection among subfamilies. Bmc Plant Biology, 2014. 14.
Vatansever, R., et al., Genome-wide identification and expression analysis of sulfate transporter (SULTR) genes in potato (Solanum tuberosum L.). Planta, 2016. 244(6): p. 1167-1183.
Zhao, J., et al., Evolutionary and expression analyses of the apple basic leucine zipper transcription factor family. Front Plant Sci, 2016. 7: p. 376.
Narusaka, M., et al., RRS1 and RPS4 provide a dual Resistance-gene system against fungal and bacterial pathogens. Plant J, 2009. 60(2): p. 218-26.
Guo, C., et al., Evolution and expression analysis of the grape (Vitis vinifera L.) WRKY gene family. J Exp Bot, 2014. 65(6): p. 1513-28.
Wang, Y., et al., MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res, 2012. 40(7): p. e49.
Li, S., et al., Arabidopsis thaliana WRKY25, WRKY26, and WRKY33 coordinate induction of plant thermotolerance. Planta, 2011. 233(6): p. 1237-1252.
Xie, Z., et al., Annotations and functional analyses of the rice WRKY gene superfamily reveal positive and negative regulators of abscisic acid signaling in aleurone cells. Plant Physiology, 2005. 137(1): p. 176-189.
Zheng, J., et al., Identification, expression, alternative splicing and functional analysis of pepper WRKY gene family in response to biotic and abiotic stresses. PLoS One, 2019. 14(7): p. e0219775.
Li, D., et al., Genome-wide analysis of WRKY gene family in the sesame genome and identification of the WRKY genes involved in responses to abiotic stresses. BMC Plant Biology, 2017. 17(1).
He, Y., et al., Genome-wide identification and expression analysis of WRKY transcription factors under multiple stresses in Brassica napus. PLoS One, 2016. 11(6): p. e0157558.
Wu, J., et al., Genome-wide investigation of WRKY transcription factors involved in terminal drought stress response in Common Bean. Front Plant Sci, 2017. 8: p. 380.
Wan, Y., et al., Identification of the WRKY gene family and functional analysis of two genes in Caragana intermedia. BMC Plant Biol, 2018. 18(1): p. 31.
Wei, Y., et al., Genome-wide identification and expression analysis of the WRKY gene family in Cassava. Front Plant Sci, 2016. 7: p. 25.
Waqas, M., et al., Genome-wide identification and expression analyses of WRKY transcription factor family members from chickpea (Cicer arietinum L.) reveal their role in abiotic stress-responses. Genes Genomics, 2019. 41(4): p. 467-481.
Yue, H., et al., Transcriptome-wide identification and expression profiles of the WRKY transcription factor family in Broomcorn millet (Panicum miliaceum L.). BMC Genomics, 2016. 17: p. 343.
Zou, Z., et al., Gene structures, evolution and transcriptional profiling of the WRKY gene family in Castor Bean (Ricinus communis L.). PLoS One, 2016. 11(2): p. e0148243.
Noutoshi, Y., et al., A single amino acid insertion in the WRKY domain of the Arabidopsis TIR-NBS-LRR-WRKY-type disease resistance protein SLH1 (sensitive to low humidity 1) causes activation of defense responses and hypersensitive cell death. Plant J, 2005. 43(6): p. 873-88.
Koonin, E.V., Orthologs, paralogs, and evolutionary genomics. Annu Rev Genet, 2005. 39: p. 309-38.
Song, M. and X. Peng, Genome-wide identification and characterization of DIR Genes in Medicago truncatula. Biochem Genet, 2019.
Voorrips, R.E., MapChart: software for the graphical presentation of linkage maps and QTLs. J Hered, 2002. 93(1): p. 77-8.
Ulker, B. and I.E. Somssich, WRKY transcription factors: from DNA binding towards biological function. Curr Opin Plant Biol, 2004. 7(5): p. 491-8.
Ding, M., et al., Genome-wide investigation and transcriptome analysis of the WRKY gene family in Gossypium. Molecular Genetics and Genomics, 2014. 290(1): p. 151-171.
Zhao, N., et al., Identification and expression analysis of WRKY gene family under drought stress in peanut (Arachis hypogaea L.). PLoS One, 2020. 15(4): p. e0231396.
Ning, P., et al., Genome-wide analysis of WRKY transcription factors in wheat (Triticum aestivum L.) and differential expression under water deficit condition. PeerJ, 2017. 5.
Schmutz, J., et al., Genome sequence of the palaeopolyploid soybean. Nature, 2010. 463(7278): p. 178-83.

Additionalfile1TableS1.xlsx
Additional file 1: Table S1. The number of WRKY genes in the reported species.
Additionalfile2TableS2.xlsx
Additional file 2: Table S2. Detailed information of all identified Camelina sativa WRKY proteins.
Additionalfile3TableS3.xlsx
Additional file 3: Table S3. Alternative splicing of WRKY genes.
Additionalfile4TableS4.xlsx
Additional file 4: Table S4. Segmental duplication of CsWRKY among C. sativa chromosomes.
Additionalfile5TableS5.xlsx
Additional file 5: Table S5. Tandem duplication and segmental duplication of some species.
Additionalfile6TableS6.xlsx
Additional file 6: Table S6. All gene pairs of two different genomes (C. sativa and A. thaliana, C. sativa and B. rapa).
Additionalfile7TableS7.xlsx
Additional file 7: Table S7. Primer pairs used for qRT-PCR analysis on the target sequences in C. sativa
Additionalfile8FigureS1.jpg
Additional file 8: Figure S1. Schematic representation of splice pattern of some CsWRKY genes.
Additionalfile9FigureS2.jpg
Additional file 9: Figure S2. Multiple sequence alignment of all CsWRKYs.
Additionalfile10FigureS3.jpg
Additional file 10: Figure S3. Phylogenetic tree (left) and motif distributions (right) of the CsWRKY proteins.

Download PDF

Journal Publication

published 11 Nov, 2020

Read the published version in BMC Genomics →

Submission checks completed at journal
12 Oct, 2020
Editorial decision: Minor revision
12 Oct, 2020

You are reading this older preprint version

Read the latest preprint version →

Genome-wide identification and functional characterization of Camelina sativa WRKY gene family in response to abiotic stress

Status:

Journal Publication

Version 3

Abstract

Figures

Background

Results

Discussion

Conclusion

Methods

Abbreviations

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 3