Next Article in Journal
Novel Splicing Mutation in MTM1 Leading to Two Abnormal Transcripts Causes Severe Myotubular Myopathy
Next Article in Special Issue
Genetic Mechanism Study of Auditory Phoenix Spheres and Transcription Factors Prediction for Direct Reprogramming by Bioinformatics
Previous Article in Journal
The Inflammasome Activity of NLRP3 Is Independent of NEK7 in HEK293 Cells Co-Expressing ASC
Previous Article in Special Issue
A Uniquely Stable Trimeric Model of SARS-CoV-2 Spike Transmembrane Domain
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 23
Issue 18
10.3390/ijms231810270
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Identification of the WRKY Gene Family and Characterization of Stress-Responsive Genes in Taraxacum kok-saghyz Rodin

by
Yifeng Cheng
†,
Jinxue Luo
†,
Hao Li
,
Feng Wei
,
Yuqi Zhang
,
Haiyang Jiang
* and
Xiaojian Peng
*
National Engineering Laboratory of Crop Stress Resistance Breeding, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2022, 23(18), 10270; https://doi.org/10.3390/ijms231810270
Submission received: 28 July 2022 / Revised: 25 August 2022 / Accepted: 29 August 2022 / Published: 7 September 2022
(This article belongs to the Collection Feature Papers in Molecular Informatics)
Browse Figures
Review Reports Versions Notes

Abstract

:
WRKY transcription factors present unusual research value because of their critical roles in plant physiological processes and stress responses. Taraxacum kok-saghyz Rodin (TKS) is a perennial herb of dandelion in the Asteraceae family. However, the research on TKS WRKY TFs is limited. In this study, 72 TKS WRKY TFs were identified and named. Further comparison of the core motifs and the structure of the WRKY motif was analyzed. These TFs were divided into three groups through phylogenetic analysis. Genes in the same group of TkWRKY usually exhibit a similar exon-intron structure and motif composition. In addition, virtually all the TKS WRKY genes contained several cis-elements related to stress response. Expression profiling of the TkWRKY genes was assessed using transcriptome data sets and Real-Time RT-PCR data in tissues during physiological development, under abiotic stress and hormonal treatments. For instance, the TkWRKY18, TkWRKY23, and TkWRKY38 genes were significantly upregulated during cold stress, whereas the TkWRKY21 gene was upregulated under heat-stress conditions. These results could provide a basis for further studies on the function of the TKS WRKY gene family and genetic amelioration of TKS germplasm.

1. Introduction

The essence of TFs is DNA-binding proteins, which can specifically interact with upstream/downstream genes to regulate gene transcription [1]. This is incredibly plant specific, sometimes even in high proportion, for WRKY TFs in higher plants [2,3]. WRKY domains consisted of approximately 60 amino acid residues, mainly comprising a conserved domain of WRKYGQK at the N-terminus, followed by a zinc finger motif C2H2 (C-X4–5-C-X22–23-H-X-H) or C2HC (C-X7-C-X23-H-X-C) at the C terminal [4,5,6]. WRKY proteins were divided into three groups determined by the WRKY domains. All proteins containing two WRKY domains were divided into Group I, whereas the remaining two groups contain only one. The slight difference between the remaining two groups was the type of their zinc finger motifs. Group II contained a C2H2 type motif whereas the C2HC type belonged to Group III [7].
Now, a growing stream of research suggests that the WRKY gene family plays important roles in plant development and substance metabolism. Many WRKY proteins have been certified in the plant kingdom, which are mainly related to seed dormancy and germination [8,9], embryogenesis [10,11], trichome development [12,13], pollen development [14,15], root development [16], modulation of flowering time [17], leaf senescence [18], plant nutrient utilization [19], and biosynthesis of secondary metabolites [20]. Although they play important roles in these situations, more important are their biological functions and molecular regulatory mechanisms in plant responses to biological and abiotic stresses [21]. The growth of the knockout lines of the ATWRKY4 gene is significantly inhibited under salinity or Me-JA stress. The ATWRKY3/WRKY4 knockout transgenic plants also exhibit an increase in relative electrolyte leakage (REL) and a significant decrease in antioxidant enzyme activity [22]. For overexpression of rice transcriptional repressor OsWRKY76, which is expressed in rice plants, the tolerance to cold stress is increased [23]. The tolerance of transgenic Arabidopsis to high temperature is increased by the overexpression of ZmWRKY106 involved the ABA signaling pathway, and it reduces the reactive oxygen species in transgenic lines by enhancing the activity of superoxide dismutase and catalase under drought stress [24]. The four wheat WRKY TFs, including TaWRKY142, TaWRKY112, TaWRKY92, and TaWRKY49, are usually upregulated in hybrid necrosis and hybrid chlorosis. The expression of either TaWRKY142, TaWRKY112, TaWRKY92, or TaWRKY49 will increase the tolerance to salinity and osmotic stress. These proteins serve as a comprehensive hub of multiple stress-signaling pathways, and they play a critical role in hybrid necrosis and hybrid chlorosis induced by autoimmune response [25]. SlWRKY8 can activate W-box-dependent transcription in tomato, which plays a positive regulatory role in immunity against pathogen infection and response to salinity stress [26].
TKS is considered as an important potential alternative source for the production of natural inulin and rubber [27,28,29]. Pictures of TKS1151 at different stages are as follows (Figure 1). TKS roots contain latex and can produce rubber, which is used to make general rubber products. TKS leaves are rich in chicory acid, which has a potential antioxidant application and can be used as a nutritional component in Chinese herbal medicine and healthy feed production [30]. Susceptibility to allergic reactions and the spread of fungal pathogens of South American leaf blight disease have both reduced the production of Hevea brasiliensis, so TKS is of strategic and economic importance as a potential alternative source of natural rubber [31]. Compared with other rubber-producing plants, TKS has the ideal agronomic characteristics and can be used as an annual crop for high-efficiency production, including rapid development and high biomass production. In addition, TKS has several advantages, such as a wide growth range, high content of natural rubber, rubber with no protein allergy, relatively simple genome, and easy genetic transformation and gene editing [32,33]. Therefore, from the perspective of genomics, it proposes suggestions for further study of rubber plants. This provides a good idea and an important research direction for the cultivation of high-quality TKS.
The WRKY gene family has been identified and studied in many plants except TKS. With the publication of the draft genome, it becomes possible to identify the WRKY TFs of TKS at the genome-wide scale. In this study, 72 TKS WRKY genes were identified and divided into three groups. Further comprehensive analysis was made from the exon-intron structure, motif composition, phylogeny, and cis-acting elements. Some genes that may play critical roles in abiotic stress and plant hormone signaling pathways through global expression analysis were identified. This study aims to provide new ideas for the application and popularization of WRKY genes in TKS, and some genes of this gene family may be used for TKS quality improvement and become resources for resistance breeding in crops.

2. Result

2.1. Identification of the WRKY Protein Family in TKS

To identify the WRKY proteins in TKS, a BLASTP search was performed using the conserved WRKY domain in Arabidopsis. The putative WRKY proteins were validated by Pfam and SMART programs. At the same time, 72 putative WRKY proteins were identified using the InterProScan Database with the hidden Markov model (PF03106). According to the genome database information, they were named TkWRKY1 to TkWRKY72, corresponding to the size of the accession number.
The physicochemical properties of the TkWRKY proteins were analyzed. Among the 72 TkWRKY proteins, TkWRKY8 (67 aa) was the smallest protein whereas the largest protein was TkWRKY23 (676 aa). The MW of the proteins ranged from 7.74 kDa to 75.09 kDa. The pI ranged from 4.88 (TkWRKY54) to 10.07 (TkWRKY8), and half of the proteins had isoelectric points greater than 7. The instability index and grand average of hydropathicity (GRAVY) of all the proteins were greater than 40 and less than 0, respectively. The Aliphatic index had a maximum of 85.2 (TkWRKY70) and a minimum of 38.09 (TkWRKY9). The subcellular location prediction results indicated that 71 TkWRKY proteins were located in the nucleus region and only 1 TkWRKY protein was located in the mitochondria (Table S1). In conclusion, TKS WRKY proteins may be instable and hydrophilic.

2.2. Multiple Sequence Alignment, Structural Analysis of the WRKY Variants, and Phylogenetic Analysis of the TkWRKY Genes

The phylogenetic relationship of the WRKY protein domain in TKS was studied by multiple sequence alignment. In total, 70 amino acid domains of the TKS WRKY protein domain were intercepted, and we then selected the WRKY domain of 7 different Arabidopsis WRKY proteins (ATWRKY58, ATWRKY18, ATWRKY61, ATWRKY13, ATWRKY17, ATWRKY65, and ATWRKY46) from each group or subgroup as representatives for further comparison. The sequence in the WRKY domain was highly conserved, whereas four TkWRKY proteins (TkWRKY6, TkWRKY24, TkWRKY58, and TkWRKY41) were variants in the WRKY domain (Figure 2). Many WRKY domain variants have been observed in plant species such as Camelina and legumes [34,35]. The core motif WRKY of the variants was replaced by other amino acids. A zinc-finger structure unlikely formed due to the lack of specific cysteine or histidine. However, the proportion of these variants was very small in the TKS WRKY protein.
Phylogenetic analysis of Taraxacum kok-saghyz Rodin and Arabidopsis was conducted based on the WRKY domain (Figure 3). TKS WRKY domains were divided into three main groups, namely, Groups I, II, and III, where the second group contained five subgroups [5,6]. Sixteen TKS WRKY genes (TkWRKY2, TkWRKY11, TkWRKY13, TkWRKY18, TkWRKY25, TkWRKY29, TkWRKY31, TkWRKY35, TkWRKY36, TkWRKY38, TkWRKY42, TkWRKY44, TkWRKY47, TkWRKY48, TkWRKY64, and TkWRKY67) with two WRKY domains belonged to Group I, which comprised a zinc-finger motif of C2H2. Based on the phylogenetic relationship between TKS and Arabidopsis expressed by the tree branch graph of the evolutionary tree, the other 43 TKS WRKY genes with the C2H2 types were assigned to Group II, which contained only one WRKY domain. The 43 Group II TKS WRKY genes were distributed into five subgroups: Group II-a (two: TkWRKY52 and TkWRKY71); Group II-b (eleven: TkWRKY5, TkWRKY12, TkWRKY14, TkWRKY17, TkWRKY19, TkWRKY21, TkWRKY26, TkWRKY34, TkWRKY40, TkWRKY53, and TkWRKY69); Group II-c (seventeen: TkWRKY3, TkWRKY4, TkWRKY16, TkWRKY20, TkWRKY23, TkWRKY24, TkWRKY28, TkWRKY30, TkWRKY41, TkWRKY46, TkWRKY49, TkWRKY55, TkWRKY57, TkWRKY58, TkWRKY61, TkWRKY65, and TkWRKY66); Group II-d (five: TkWRKY7, TkWRKY22, TkWRKY51, TkWRKY56, and TkWRKY63); and Group II-e (eight: TkWRKY1, TkWRKY6, TkWRKY8, TkWRKY9, TkWRKY37, TkWRKY50, TkWRKY62, and TkWRKY68). Unlike the other two groups, Group III genes contained a C2HC type and included twelve WRKY genes in TKS (TkWRKY10, TkWRKY15, TkWRKY27, TkWRKY32, TkWRKY33, TkWRKY39, TkWRKY43, TkWRKY45, TkWRKY59, TkWRKY60, TkWRKY70, and TkWRKY72).
The phylogenetic tree is divided into groups I, II-a, II-b, II-c, II-d, II-e, and III with green, dark yellow, dark blue, orange, red, yellow and blue marking the clusters, respectively.

2.3. Gene Structure and MOTIF Composition of TKS WRKY Gene Family

Since the diversity of the gene structure drives the evolution of multi-gene families, the exon-intron structure of the identified 72 TkWRKY genes were analyzed (Figure 4). It is obvious that Group I contained 2–5 introns, Group II-a contained 3–4 introns, Group II-b contained 2–5 introns, and Group II-c contained 0–5 introns, half of which contained three introns. Group II-d and Group II-e all contained 2 introns, excluding TkWRKY1. Group III varied from 1 to 4 introns (Figure 4b). There were significant differences in the number of introns and exons in the different groups, indicating that the TkWRKY gene structure was relatively complex and may have undergone domain shuffling after genome replication. Genes in the same group usually had similar structures. For example, all TkWRKY genes in Group II-d contained 3 exons and 2 introns [36,37].
To further study the structure and phylogenetic relationships of TKS WRKYs, the conserved motifs were obtained, and schematic diagrams of all TkWRKY proteins were constructed according to the motif analysis results. WRKY motifs in the same group contained similar structures. Motif 1 and 4 were WRKY domains; Motif 4 was only present in Group I whereas Motif 1 was distributed the whole groups. Motif 10 was unique to Groups II-a and II-b, whereas Motif 8 to Group II-b and Motif 15 to Group II-c (Table S2). Clustered TkWRKY pairs, such as TkWRKY25/29 and TkWRKY10/43, exhibited a highly similar motif distribution (Figure 4c). Although some proteins contained unknown functions, the motif structure of most TkWRKY proteins in the same subgroup was similar, indicating that the protein structure of this family was conserved in a specific subfamily. All in all, the similar gene structure, conservative motif arrangement, and phylogenetic analysis of the same group of proteins can be used as an important basis for protein classification.

2.4. Evolutionary Analysis of Group III WRKY Genes in TKS and Several Different Species

The WRKY III gene in plants originated after the differentiation of monocots and dicots, and it seemed to play a critical role in plant adaptation and evolution [38]. To study the evolution of the TKS Group III WRKY gene, MEGA7 was used to construct four monocotyledonous plants (rice, maize, Brachypodium distachyon, and pineapple) and five dicotyledonous plants (Arabidopsis, grapes, peaches, poplars, and Taraxacum kok-saghyz Rodin) of Group III WRKY protein phylogenetic trees. All the WRKY family members of Group III was divided into 8 clades (Figure 5). The species that aggregated in the same clade were closely related. Most proteins in dicotyledons were clustered on Clade 1, 4, and 7, whereas most proteins in monocotyledons were clustered on Clade 1, 2, 3, 5, 6, and 8, which strongly indicated that the evolutionary relationships among the WRKY members of the same clade were similar. Clade 1 contained proteins from all nine species, suggesting that these proteins may be orthologs from a single ancestor gene. Motif 1 and 11 were WRKY domains, and Motif 2 was a C2HC zinc-finger motif. Interestingly, Motif 1 and 2 were found in all proteins, indicating these two motifs may play different functions in different species. Motif 15 and 16 were only found in the dicotyledonous plant, Arabidopsis, where Motif 3 was only found in TKS and Motif 8 was only found in dicotyledonous plants and pineapples (Figure 5 and Table S3). In general, the conserved motif composition and similar gene structure of the WRKY members in the same group, as well as the results of the phylogenetic analysis, strongly supported the reliability of the population classification.

2.5. Stress-Related Cis-Elements in TkWRKY Promoter Regions

To further study the potential regulatory mechanism of WRKY TFs involved in stress response, 12 representative cis-elements were selected for quantitative and functional analysis (Figure 6). At the same time, according to their functions, they were divided into three categories, namely, related to environmental stress, hormone response, and development; the number of each cis-element is counted in Table S4.
In the current study, all TkWRKY TFs contained 2–9 cis-elements associated with stress response except TkWRKY43 and TkWRKY44, probably because their protein sequences were too short. Many TkWRKY promoter regions contained identified elements related to hormone regulation, such as ABRE, CGTCA−motif, GARE−motif, TCA−element, and TGA−element. In total, 63 TkWRKYs (87.5%) contained one or more ABREs, indicating that there was a potential abscisic acid reaction under pressure [39]. CGTCA motifs were involved in the MeJA response in 56 TkWRKYs (77.8%). TCA−element, TGA−element, and GARE−motif were found in 25 (34.7%), 26 (36.1%), and 9 (12.5%) TKWRKYs, which were the response elements of salicylic acid, auxin and gibberellin, respectively. Among the environmental stress-related elements in the TkWRKY gene family, about half of the genes included WUN−motif, W−box, and TC−rich repeats. They play critical roles in plant defense and stress responsiveness and response to mechanical damage, respectively. In addition, about half of the genes included LTR and MBS, which contribute significantly to plant responses to low temperature and drought induction, respectively. Significantly, the only development-related element was circadian, which was associated with responsiveness to meristem development and controls circadian rhythms [40]. These results play a decisive role in the profound cognition of stress-related cis-elements in TkWRKY.

2.6. Expression Profiling of TKS WRKY Genes with RNA-Seq

To determine the expression pattern of 72 TkWRKY genes at different developmental stages in TKS tissues, we conducted a hierarchical clustering heat map with the public transcriptome data obtained from the NGDC Genome Warehouse (Table S5). Twelve tissues were obtained from the database, including flowers (FL), latex (LA), peduncle (PE), seed (SE), mature leaf (ML), mature lateral root (MLR), mature main root (MMR), mature stem (MS), young leaf (YL), young lateral root (YLR), young main root (YMR), and young stem (YS) [29]. Among the 72 TkWRKY genes, the expression of TkWRKY61 in all detected samples was almost 0, which seemed to be a pseudogene or a special spatiotemporal expression pattern that was not detected in our library. In total, 55 TkWRKY genes were found to be expressed in all 12 tissues (FPKM > 0), and 20 of them showed constitutive expression, such as TkWRKY1, TkWRKY35, TkWRKY6, TkWRKY56, TkWRKY7, TkWRKY60, TkWRKY48, and TkWRKY67 (Figure 7a). Obviously, the expression pattern of the TkWRKY genes in TKS was tissue specific. For instance, TkWRKY34 was induced only in young lateral root, whereas TkWRKY45 in leaves. The root and stem tissues were gathered into a large clade. Leaf, seed, and pedicels were clustered into one clade whereas the pedicel is more closely related to stem. Flower and latex belonged to separate branches, but latex was more closely related to roots. In general, the expression level of reproductive tissue (flower and seed) in TKS was generally lower than that of vegetative tissue (leaf, root, peduncle, and stem). The expression levels of many TkWRKY genes in mature lateral roots, mature stems, young lateral roots, young main roots, and young stems were higher than that in other tissues (Figure 7b). For example, TkWRKY3, TkWRKY49, TkWRKY59, TkWRKY37, TkWRKY65, TkWRKY18, TkWRKY62, TkWRKY71, TkWRKY64, TkWRKY50 and TkWRKY17. TkWRKY38, TkWRKY63, TkWRKY64, and TkWRKY71 exhibited high transcription abundance in almost all tissues and clustered into a group, whereas TkWRKY14, TkWRKY20, TkWRKY32, TkWRKY45, TkWRKY53, TkWRKY54, and TkWRKY55 exhibited extremely low expression levels in all tested tissues. Interestingly, the expression level of several genes showed significant trends at different developmental stages. For instance, with the maturity of the main root, lateral roots, and stems, TkWRKY72, TkWRKY36, and TkWRKY18 were depressed during the process. The expression level of TkWRKY71 was lower in the seed sample, but higher in young and mature tissues. TkWRKY27 expression increased with stem maturation (Figure 7a).

2.7. Expression Patterns of TKS WRKY Genes in Response to Different Treatments

To further investigated the changes in the TkWRKY gene expression levels under different abiotic stress and hormone treatments, 14 genes were carefully selected with high expression levels in the expression profile or specific expression in tissues. Real-time RT-PCR was further performed to detect the expression patterns under different treatments. In general, there were multiple TkWRKY genes that can be induced simultaneously by a single treatment. Eight TkWRKY genes (TkWRKY4, TkWRKY10, TkWRKY13, TkWRKY18, TkWRKY23, TkWRKY28, TkWRKY38, and TkWRKY71) were induced by cold stress (Figure 8a). Seven genes (TkWRKY4, TkWRKY6, TkWRKY13, TkWRKY21, TkWRKY23, TkWRKY27, and TkWRKY38) were induced by heat stress (Figure 8b). Five genes (TkWRKY6, TkWRKY10, TkWRKY18, TkWRKY27, and TkWRKY39) were induced by NaCl treatment (Figure 8c). Five genes (TkWRKY6, TkWRKY10, TkWRKY18, TkWRKY27, and TkWRKY39) were induced by PEG treatment (Figure 8d), whereas three genes (TkWRKY10, TkWRKY13, and TkWRKY21) were induced by ABA treatment (Figure 9a). Three genes (TkWRKY18, TkWRKY38, and TkWRKY27) were induced by MeJA treatment (Figure 9b). Three genes (TkWRKY18, TkWRKY38, and TkWRKY71) were induced by SA treatment (Figure 9c). Conversely, a single TkWRKY can be significantly induced/suppressed under a variety of treatments. For instance, both TkWRKY18 and TkWRKY38 belonged to the same group and significantly responded to MeJA treatment, SA, and cold treatments, suggesting that genes from the same populations may have similar functions. Moreover, TkWRKY21 was induced by ABA treatment and heat stress. TkWRKY10 was induced by NaCl, PEG, and ABA treatments. TkWRKY71 was induced by cold stress and SA treatment. Interestingly, the transcript levels of several TkWRKYs, such as TkWRKY21, TkWRKY23, and TkWRKY39, were repressed intensively by the SA and MeJA treatments. This demonstrated that they may be involved in negative regulation of the SA pathway. Several genes exhibited the opposite or identical expression patterns under different treatments. For example, TkWRKY23 was significantly upregulated by heat stress, whereas it was repressed by SA treatment. The expression level of TkWRKY38 was downregulated under ABA and PEG treatments, but it was upregulated after the cold and heat stresses. Both TkWRKY21 and TkWRKY23 were repressed by the MeJA and SA treatments. Furthermore, the expression level in several genes fluctuated under a complete single-treatment process. For instance, the expression of TkWRKY13 was rapidly upregulated at 9 h after cold stress but repressed at 16 and 24 h. Similarly, TkWRKY23 was induced by heat and cold stresses, but the turning points were the 16 h and 9 h timepoints, respectively. TkWRKY18 under heat stress was repressed at 3 and 6 h, but induced at 9 and 16 h, and then repressed at 24 h. Notability, TkWRKY27 was induced by NaCl, PEG, and SA treatments at the 3 h timepoints and then repressed over the rest of the process. It was indicated that this gene can quickly respond to various abiotic stresses and hormone treatments. These results primarily revealed the diverse mechanisms controlling TKS WRKY gene responses to various intensities of abiotic stresses and hormonal treatments (Figure 8 and Figure 9).

3. Discussion

As reported, genome-wide- analysis of the WRKY gene family has been studied for multiple plants, including maize [41], Helianthus annuus L. [42], sweet Osmanthus [43], white pear [44], upland cotton [45], and switchgrass [46]. In this study, 72 WRKY genes were identified based on TKS genome sequences.
Although the WRKY motif is almost conserved in many plants with the WRKYGQK domain, there were many kinds of variants in the WRKY domain reported in previous research. Grapes have been reported to have two variants (WRKYGKK and WKKYGQK) [47] and eggplant has seven variants: WIKYGQK, WRKYGEK, WRKYGKK, WKKYEIG, WRKYGQN, WHKFGQK, and WDKFGQK [48]. Moreover, five variants have been observed in poplars (WRKYGKK, FWRKYGQK, WKKYGQK, WRKYGRK, and WRKYGEK) [36] and 1 in cucumber (WRKYGKK) [49]. In this study, domain recognition of the TKS WRKY domains revealed that three variants with WRKYGKK belonged to Group II-c (TkWRKY24, TkWRKY41, and TkWRKY58) and one (TkWRKY6) with WKKYGEK belonged to Group II-e (Figure 2). Furthermore, the uniqueness of the TkWRKY6 domain and the group to which it belonged revealed that TkWRKY6 may possess a different biological function. Interestingly, TKS contained three WRKYGKK variants in Group II-c and similar variants were also found in Group II-c of Helianthus annuus L. [42]. It seemed that this mutation was indeed common in Asteraceae. The existence of the WRKYGQK motif variants may increase the diversity of WRKY protein binding to target genes. Perhaps more binding elements will bind to proteins with more mutants, not just W-box elements [5].
Domain loss is a normal phenomenon, which is considered to be the divergent power of the gene family expansion [4,50]. In the current study, the Group I proteins in TKS all contained two WRKY domains with no domain loss event found, whereas some genes in Group II-c, II-e, and III had domain deletions, indicating that these groups performed different functions during their evolution. In total, 72 WRKY TFs were identified from 46,731 TKS annotated genes (Figure 2). Compared with the number of genes of Helianthus annuus L. (90) and rice (100), the size of the WRKY family in TKS is small. Similar to other WRKY gene families, such as Arabidopsis and Helianthus annuus L., the WRKY genes in Group II-a account for a small proportion and seemed to play various roles in dealing with abiotic stress [51,52]. Interestingly, phylogenetic and comparative genomics studies have indicated that Group II-a was the last to evolve and appears to be derived from Group II-b [6]. Although the whole-genome sequence of TKS was obtained, the information about the chromosomal location of the TkWRKY genes is still unknown; therefore, analysis of the synteny and gene-duplication events of this species could not be performed in this work.
Gene structure and motif analysis of the TKS WRKY gene family show that genes on the same branch usually have a similar structure or domain composition. These results can be used as an important basis for WRKY protein classification (Figure 4). Group III WRKY genes are considered to be the most advanced, dynamic, and adaptable gene group [38]. The diversity in the number of WRKY gene families is due to the difference in Group III in different species. For instance, TKS WRKY III contains 12 genes, fewer than rice or maize, but more than pineapple or grape, so the number of WRKY III may represent the total number of genes in the family to some extent (Figure 5). Group III WRKY genes formed monocotyledonous and dicotyledonous plant-specific subbranches, indicating that the WRKY gene evolved independently after monocotyledonous plant-dicotyledonous plant division. Therefore, Group III WRKY genes not only originated from the differentiation of monocotyledons and dicotyledons, but also evolved independently after the division of them, which proved their advanced nature and strong adaptability [38].
WRKY stress-related cis-elements play critical roles in several plant processes under ever-changing environmental conditions by regulating the plant complex defense signal transduction pathways [53]. WRKY TFs interacted with the cis-elements downstream of gene promoters to regulate the expression of target gene and caused a series of responses, consequently enhancing the resistance to stress of plants [49,54,55,56,57]. WRKY transcription factors can recognize and bind w-box elements to regulate the expression of downstream genes [4]. In this study, the 12 cis-elements were ABRE (cis-acting element involved in the abscisic acid responsiveness), CGTCA−motif (cis-acting regulatory element involved in the MeJA-responsiveness), GARE−motif (gibberellin-responsive element), LTR (cis-acting element involved in low-temperature responsiveness), MBS (MYB binding site involved in drought-inducibility), MBSI (MYB binding site involved in flavonoid biosynthetic genes regulation), TC−rich repeats (cis-acting element involved in defense and stress responsiveness), TCA−element (auxin-responsive element), TGA−element (auxin-responsive element), W−box (WRKY transcription factor binding site in defense responses), WUN−motif (mechanical damage response element), and circadian (cis-acting regulatory element involved in circadian control) (Figure 6). In total, 32 TKS WRKY genes contained W−box elements, which may interact with other WRKY transcription factors. The TKS WRKY promoter also contained several hormone-responsive elements, such as ABRE, CGTCA−motif, GARE−motif, TCA−element, and TGA−element, suggesting that they may be involved in a wide variety of hormone-mediated signaling pathways. The MYB-binding sites MBS and MBSI are also present in TKS WRKY promoter domains, suggesting being involved in drought-inducibility and flavonoid biosynthetic gene regulation (Figure 6).
WRKY genes play a critical role in plant development. As reported, AtR8 lncRNA defective mutants and ATWRKY53 and ATWRKY70 respond to the induction of exogenous SA. The experimental results revealed that the wrky53 and wrky70 mutant lines had longer roots than the wild type and mutants of SA [58]. In the current study, the orthologous genes of the two WRKY proteins were TkWRKY70 and TkWRKY27, respectively, which were highly expressed in young and mature roots of TKS (Figure 3 and Figure 7a). In particular, TkWRKY27 was expressed in all tissues, suggesting that the function of this gene was activated at any stage of TKS growth. ATWRKY13 gene have made outstanding contributions in Arabidopsis stem development. The mutants of this gene exhibited a weaker stem phenotype, with the number of thick-walled tissue cells, stem diameters, and vascular bundles being reduced [59]. Similarly, the orthologous protein of ATWRKY13, named TkWRKY57, was specifically expressed in young and mature stems (Figure 7a). This research indicated TkWRKY57 may be involved in regulating the growth and development of TKS stems. Moreover, ATWRKY75 TFs regulate the acquisition of phosphate and root development in Arabidopsis. RNAi silencing inhibited the expression of ATWRKY75, which made plants susceptible to phosphorus stress. ATWRKY75 also cause early accumulation of anthocyanins and increase the length and number of lateral roots and root hairs [60]. TkWRKY28 and TkWRKY30, which have high expression levels in roots, were orthologous genes of ATWRKY75 (Figure 3). These results revealed the significant contribution of TkWRKY28 and TkWRKY30 in the development of roots. Interestingly, TkWRKY28 and TkWRKY30 may be closely related to the amount of latex produced by TKS because of their higher expression in latex (Figure 7a).
In addition to playing a critical role in plant development, WRKY TFs also play an important role in giving plants tolerance to abiotic stresses, including salinity, osmotic, heat, cold, and injury [54,61]. As everyone knows, temperature, osmotic, and saline-alkali are important factors affecting crop growth, so it is necessary to study the tolerance of TKS to abiotic stresses. We planted some TKS1151 in a greenhouse and treated them with various abiotic stresses and hormones (Figure 1). When salinity and osmotic stress act on plants, it will cause the accumulation of ABA content, consequently activating downstream WRKY genes. According to reports by Han et al., ATWRKY18 and ATWRKY60 in Arabidopsis have a positive effect on the ABA sensitivity of plants, inhibiting seed germination and root growth and enhancing the sensitivity of plants to salinity stress; in turn, ATWRKY40 is antagonistic to ATWRKY18 and ATWRKY60. This effect suggests that ATWRKY18 is a weak transcriptional activator that can be induced to function under salt stress [62]. In the current study, the homologous protein TkWRKY71 of ATWRKY18 was induced under salinity stress and its expression level increased (Figure 3 and Figure 8c). Therefore, it can be inferred that TkWRKY71 may also participate in the salt stress response mechanism of plants by regulating the activity of some hormone-like proteins involved in the ABA signal transduction pathway, thus enhancing the salt tolerance of TKS. The Arabidopsis TFs WRKY46, WRKY54, and WRKY70 are very important in the regulation pathway of brassinosteroids (BRs). The triple mutant has obvious growth defects and is drought tolerant. More importantly, ATWRKY54 can interact with the BR master regulator BES1 to co-regulate downstream genes. Moreover, ATWRKY54 is phosphorylated by GSK3-like kinase, which destroys the protein structure and leads to its instability [63]. Similarly, after PEG treatment, the homologous protein TkWKRY27 in TKS can also be induced into expression; it likely can also interact with some unknown binding protein or regulatory factor to regulate the expression of target genes (Figure 3 and Figure 8d). Interestingly, TkWKRY27 was also activated under salinity stress, so there may be a synergistic effect between these two different stresses. As previously reported, Arabidopsis wrky25wrky26wrky33 triple mutant is extremely sensitive to heat stress. The germination rate of mutant seeds becomes very low under high-temperature treatment. It was demonstrated that these three WRKY TFs are involved in the cooperation between ethylene activating proteins and HSP-related signaling pathways and mediate the heat-stress response [58]. TkWRKY38 was close to ATWRKY26 in phylogeny, and the expression level of TkWRKY38 was increased after heat-induced and cold stress in TKS (Figure 3 and Figure 8a,b). TkWRKY38 may be involved in the regulation of similar reactions and play a certain role in improving crop heat tolerance. Notably, under the treatment of ABA, MeJA, and SA, the expression of some genes were significantly downregulated, such as TkWRKY4, TkWRKY23, and TkWRKY39 (Figure 9a–c). A similar situation was reported in pineapples, where the transcript levels of many AcWRKY genes were downregulated by heat-stress treatment [64]. These results indicate that some WRKY genes were negatively regulated.
Due to the systematicity of genomics technology, the signaling pathway of TKS in response to stress and the glue-producing pathway of glue-producing plants can be studied in a more comprehensive and in-depth way, providing a more reliable theoretical basis and data support for the biosynthesis mechanism of natural rubber. As Lin et al. assembled high-quality genome sketches of TKS using SMRT, the difficulty of cloning and obtaining key genes for natural rubber biosynthesis was greatly reduced, which was very important for further exploring the gene functions of TKS [29]. Several genes of TKS showed many positive characteristics in the process of coping with adversity. For example, TkUBC2, an E2 ubiquitin-binding enzyme gene cloned from TKS, was upregulated under salt stress and UV radiation, but downregulated under drought stress and osmotic stress. This suggested that the gene responded to salt, drought, and osmotic stress in TKS. Another study found that the expression of the TkJAZ9 gene was upregulated by 2.16 times after 6 h of salt stress in 3-month-old TKS, indicating that this gene responded to salt stress in TKS. In addition to the above cases, TkDREB2 was selected after a differential expression profile analysis was performed on the transcriptomes treated with MeJA. It was proven by an experiment that TkDREB2 can increase the drought tolerance of transgenic tobacco and actively respond to MeJA signal transduction regulation of tobacco drought resistance. However, there are very few reports on the TKS gene family, and only functional reports of a single TKS gene can be found. Therefore, this study provides a reference for further study of transcriptomics of TKS under stress.
In addition to studying the signaling pathway of TKS in response to stress, there has been a clear pathway for the rubber biosynthesis of TKS. Genes with high expression in roots and rubber are of particular concern, such as TkHMGR1, TkHMGR2, and 9 TkSRPP genes [29]. In this study, the TkWRKY38, TkWRKY63, TkWRKY64, and TkWRKY71 proteins showed high expression patterns in roots and latex of TKS, which is beneficial to further study the potential relationship between these proteins and root development, especially rubber production (Figure 7a). In conclusion, a comprehensive analysis revealed the potential functional role of TKS WRKY genes to a certain extent, and contributed to the further characterization of the functions of TKS candidate WRKY gene families, and provided new ideas for agronomic genetic improvement and cultivation of high-quality, stress-tolerant, and high-yielding TKS varieties.

4. Materials and Methods

4.1. Gene Identification

The genome of TKS was downloaded from NGDC Genome Warehouse (https://ngdc.cncb.ac.cn/gwh/ (accessed on 20 September 2021)). A Hidden Markov Model (HMM) profile of the WRKY DNA-binding domain (PF03106) was used to BLASTP. The Pfam databases (http://pfam.sanger.ac.uk/ (accessed on 27 October 2021)) were used to validate putative WRKY proteins. To verify the conservative domain, those potential sequences were further queried in the InterProScan database (http://www.ebi.ac.uk/interpro/search/sequence-search/ (accessed on 3 November 2021)) and SMART databases (http://smart.embl-heidelberg.de/ (accessed on 4 November 2021)). The physicochemical properties of the putative WRKYs, including the number of amino acids (No. of aa), molecular weight (MW), isoelectric point (pI), aliphatic index, instability index, and GRAVY, were obtained by using the ExPasy tools (https://web.expasy.org/protparam/ (accessed on 9 November 2021)). The subcellular location prediction results were obtained by using tools from Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/ (accessed on 15 November 2021)).

4.2. Sequence Analysis and Alignment

ClustalX software (version 1.81, Desmond G. Higgins and Paul Sharp, Dublin, Ireland) was used to perform multi-sequence alignment on the TKS WRKY protein sequence domain with the default settings, and the results were saved in MSF file format. The core sequences were then manually adjusted and colored. The intron-exon structures of the TKS WRKY genes were analyzed by the online tool Gene Structure Display Server (GSDS, http://gsds.gao-lab.org/index.php/ (accessed on 1 December 2021)) [65]. The MEME online program for protein sequence analysis (http://meme.nbcr.net/meme/intro.html/ (accessed on 4 December 2021)) was used to identify the conserved motifs in the identified TKS WRKY protein [66].

4.3. Phylogenetic Analysis

MAFFT version 7 (https://mafft.cbrc.jp/alignment/server/ (accessed on 8 December 2021)) was used to perform multi-sequence alignment on the TKS WRKY protein sequence domain with G-INS-1 (progressive method with an accurate guide tree) settings. The phylogenetic tree was constructed by MEGA software (version 7.0, Mega Limited, Auckland, New Zealand) with the neighbor-joining (NJ) method based on the comparison between TKS and Arabidopsis proteins; then we used Illustrator software (version 2019, Adobe, San Jose, CA, USA) to beautify and add color. The sequences of the WRKY protein from Arabidopsis, maize [50], rice [61,67], grape [47], Brachypodium distachyon [68], pineapple [64], peach [69], and poplar [36] were obtained according to the corresponding literature and instructions downloaded from the Phytozome databases (https://phytozome.jgi.doe.gov/ (accessed on 27 December 2021)).

4.4. Analysis of Cis-Acting Elements in TkWRKY Promoter Regions

The upstream sequences (2000 bp) of the TKS were obtained from NGDC Genome Warehouse. PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 5 January 2022)) was utilized to identify the twelve cis-elements of the TkWRKY promoter regions [70].

4.5. Plant Materials and Treatments

The dandelion line 1151 was planted in the greenhouse of Anhui Agricultural University, Hefei, China (31°87′ N 117°26′ E). TKS1151 was grown in 16/8 h light/dark at 22 ± 2 °C and 76% humidity. The seeds were germinated and grown in black soil and vermiculite mixture in a ratio of 1:1. TKS at the seven-leaf stage were subjected to 40 °C and 4 °C for heat and cold treatments, respectively, and the leaves were collected at 3, 6, 9, 16, and 24 h for both treatments. For salinity and osmotic treatments, seedlings were sprayed with 150 mM NaCl and 15% PEG6000 solutions for 3, 6, 9, 16, and 24 h. Finally, the leaves were sprayed with 300 μM ABA, 100 μM SA, and 50 μM MeJA for hormone treatment; all hormones were dissolved in sterile water [53], and sterile water was sprayed on leaves from the same batch as the negative control. In contrast, leaves were collected at 3, 9, 16, 24, and 48 h after treatments.

4.6. RNA Extraction and Real-Time RT-PCR Analysis

Total RNA was extracted using the Trizol method [71,72]. All samples were tested for the concentration, A260/A280 ratio, and A260/A230 ratio by a Thermo SCIENTIFIC spectrophotometer. HiScript® III RT SuperMix (Vazyme, Nanjing, China) was used to remove residual genomic DNA contamination and reverse transcription to obtain cDNA for qPCR. The Real-Time RT-PCR was carried out with a Roche Lightcyler® 480 instrument using SYBR qPCR Master Mix in 20 μL volumes. The stable and reliable housekeeping TKS β-actin gene and GAPDH gene were used as internal controls. The Real-Time RT-PCR primers were designed by Primer Premier software (version 5, Premier, Vancouver, BC, Canada) (Table S6). The standard amplification procedure of the Real-Time RT-PCR was as follows: first, the holding stage is the reaction at 95 °C for 30 s, followed by the cycle stage, which runs 40 cycles at 95 °C for 5 s and 60 °C for 34 s, and finally the melting curve stage. The expression level was expressed as a change in relative multiples of 0 h (control) set to 1. Each reaction was performed in three biological replicates and the Real-Time RT-PCR data were analyzed by 2−ΔΔCT method. The bar graph was drawn by GraphPad Prism (version 8.0.2, GraphPad Software, San Diego, CA, USA).

4.7. Expression Profiling of TkWRKYs in Different Tissues Based on the Public RNA-seq Data Sets

RNA-seq data sets of 12 different TKS tissues, including flower (FL), latex (LA), peduncle (PE), seed (SE), mature leaf (ML), mature lateral root (MLR), mature main root (MMR), mature stem (MS), young leaf (YL), young lateral root (YLR), young main root (YMR), and young stem (YS), were obtained from NGDC Genome Warehouse. The gene expression level was obtained by FPKM value [73]. A heat map was obtained from HemI1.0 and generated based on a log2 (FPKM + 1) value conversion [74]. The boxplot was drawn using Origin software (version 2018, OriginLab, Northampton, MA, USA).

5. Conclusions

In this study, 72 genes encoding WRKY TFs in Taraxacum kok-saghyz Rodin (TKS) were identified. TkWRKYs were divided into three main populations. Many identified WRKY proteins have close evolutionary relationships with Arabidopsis proteins, suggesting possible similarity of WRKY evolutionary patterns in dicotyledons. The WRKY domain is conserved in TKS, and the gene structure and motif analyses showed that the same group contained a highly similar exon-intron structure and motif. The analysis of cis-elements of the TKS promoter gene provides valuable clues for understanding the evolutionary characteristics of the TKS WRKY gene. According to the tissue-expression pattern and abiotic-stress quantitative expression results, WRKY genes play important roles in TKS development and stress resistance, such as cold-stress response (TkWRKY18, TkWRKY23, and TkWRKY38), heat-stress response (TkWRKY21 and TkWRKY38), salt-stress response (TkWRKY71), and osmotic stress (TkWRKY27). However, whether these WRKY genes can participate in the plant hormone signal transduction pathway or interact with other TFs in TKS needs further study.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms231810270/s1.

Author Contributions

Investigation, Y.C. and F.W.; data curation, Y.C., J.L., H.L. and Y.Z.; funding acquisition, H.J. and X.P.; writing—original draft, Y.C.; writing—review and editing, Y.C. and X.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Postgraduate Science Research Project of Anhui Province, grant number YJS20210228.

Institutional Review Board Statement

The study did not require ethical approval.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data are displayed in the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Rushton, P.J.; Somssich, I.E.; Ringler, P.; Shen, Q.J. WRKY transcription factors. Trends Plant Sci. 2010, 15, 247–258. [Google Scholar] [CrossRef]
  2. Bakshi, M.; Oelmüller, R. WRKY transcription factors: Jack of many trades in plants. Plant Signal. Behav. 2014, 9, e27700. [Google Scholar] [CrossRef] [PubMed]
  3. Ulker, B.; Somssich, I.E. WRKY transcription factors: From DNA binding towards biological function. Curr. Opin. Plant Biol. 2004, 7, 491–498. [Google Scholar] [CrossRef] [PubMed]
  4. Brand, L.H.; Fischer, N.M.; Klaus, H.; Oliver, K.; Dierk, W. Elucidating the evolutionary conserved DNA–binding specificities of WRKY transcription factors by molecular dynamics and in vitro binding assays. Nucleic Acids Res. 2013, 41, 9764–9778. [Google Scholar] [CrossRef] [PubMed]
  5. Eulgem, T.; Rushton, P.J.; Robatzek, S.; Somssich, I.E. The WRKY superfamily of plant transcription factors. Trends Plant Sci. 2000, 5, 199–206. [Google Scholar] [CrossRef]
  6. Rinerson, C.I.; Rabara, R.C.; Tripathi, P.; Shen, Q.J.; Rushton, P.J. The evolution of WRKY transcription factors. BMC Plant Biol. 2015, 15, 66. [Google Scholar] [CrossRef] [PubMed]
  7. Zhang, Y.; Wang, L. The WRKY transcription factor superfamily: Its origin in eukaryotes and expansion in plants. BMC Evol. Biol. 2015, 5, 1. [Google Scholar]
  8. Huang, Y.; Feng, C.; Ye, Q.; Wu, W.; Chen, Y. Arabidopsis WRKY6 Transcription Factor Acts as a Positive Regulator of Abscisic Acid Signaling during Seed Germination and Early Seedling Development. PLoS Genet. 2016, 15, e1005833. [Google Scholar] [CrossRef]
  9. Zhou, C.; Lin, Q.; Lan, J.; Zhang, T.; Liu, X.; Miao, R.; Mou, C.; Nguyen, T.; Wang, J.; Zhang, X.; et al. WRKY Transcription Factor OsWRKY29 Represses Seed Dormancy in Rice by Weakening Abscisic Acid Response. Front. Plant Sci. 2020, 11, 691. [Google Scholar] [CrossRef]
  10. Grunewald, W.; De Smet, I.; De Rybel, B.; Robert, H.S.; van de Cotte, B.; Willemsen, V.; Gheysen, G.; Weijers, D.; Friml, J.; Beeckman, T. Tightly controlled WRKY23 expression mediates Arabidopsis embryo development. EMBO Rep. 2013, 14, 1136–1142. [Google Scholar] [CrossRef]
  11. Yang, Y.; Wang, N.; Zhao, S. Functional characterization of a WRKY family gene involved in somatic embryogenesis in Panax ginseng. Protoplasma 2020, 257, 449–458. [Google Scholar] [CrossRef]
  12. Johnson, C.S.; Kolevski, B.; Smyth, D.R. TRANSPARENT TESTA GLABRA2, a trichome and seed coat development gene of Arabidopsis, encodes a WRKY transcription factor. Plant Cell. 2002, 14, 1359–1375. [Google Scholar] [CrossRef]
  13. Zhao, J.; Wang, Y.; Yao, D.; Zhu, W.; Chen, L.; He, H.; Pan, J.; Cai, R. Transcriptome profiling of trichome–less reveals genes associated with multicellular trichome development in Cucumis sativus. Mol. Genet. Genom. 2015, 290, 2007–2018. [Google Scholar] [CrossRef]
  14. Guan, Y.; Meng, X.; Khanna, R.; Lamontagne, E.; Liu, Y.; Zhang, S. Phosphorylation of a WRKY Transcription Factor by MAPKs is Required for Pollen Development and Function in Arabidopsis. PLoS Genet. 2014, 10, e1004384. [Google Scholar] [CrossRef]
  15. Zheng, Y.; Deng, X.; Qu, A.; Zhang, M.; Tao, Y.; Yang, L.; Liu, Y.; Xu, J.; Zhang, S. Regulation of pollen lipid body biogenesis by MAP kinases and downstream WRKY transcription factors in Arabidopsis. PLoS Genet. 2018, 14, e1007880. [Google Scholar] [CrossRef]
  16. Xu, Y.; Lu, J.; Zhang, J.; Liu, D.; Wang, Y.; Niu, Q.; Huang, D. Transcriptome revealed the molecular mechanism of Glycyrrhiza inflata root to maintain growth and development, absorb and distribute ions under salt stress. BMC Plant Biol. 2021, 21, 599. [Google Scholar] [CrossRef]
  17. Li, W.; Wang, H.; Yu, D. Arabidopsis WRKY Transcription Factors WRKY12 and WRKY13 Oppositely Regulate Flowering under Short–Day Conditions. Mol. Plant. 2016, 9, 1492–1503. [Google Scholar] [CrossRef]
  18. Kang, G.; Yan, D.; Chen, X.; Yang, L.; Zeng, R. HbWRKY82, a novel IIc WRKY transcription factor from Hevea brasiliensis associated with abiotic stress tolerance and leaf senescence in Arabidopsis. Physiol. Plant. 2021, 171, 151–160. [Google Scholar] [CrossRef]
  19. Hu, Z.; Wang, R.; Zheng, M.; Liu, X.; Meng, F.; Wu, H.; Yao, Y.; Xin, M.; Peng, H.; Ni, Z.; et al. TaWRKY51 promotes lateral root formation through negative regulation of ethylene biosynthesis in wheat (Triticum aestivum L.). Plant J. 2018, 96, 372–388. [Google Scholar] [CrossRef]
  20. Lloyd, A.; Brockman, A.; Aguirre, L.; Campbell, A.; Bean, A.; Cantero, A.; Gonzalez, A. Advances in the MYB–bHLH–WD Repeat (MBW) Pigment Regulatory Model: Addition of a WRKY Factor and Co–option of an Anthocyanin MYB for Betalain Regulation. Plant Cell Physiol. 2017, 58, 1431–1441. [Google Scholar] [CrossRef]
  21. Zheng, J.; Liu, F.; Zhu, C.; Li, X.; Dai, X.; Yang, B.; Zou, X.; Ma, Y. Identification, expression, alternative splicing and functional analysis of pepper WRKY gene family in response to biotic and abiotic stresses. PLoS ONE. 2019, 14, e0219775. [Google Scholar] [CrossRef] [Green Version]
  22. Li, P.; Li, X.; Jiang, M. CRISPR/Cas9–mediated mutagenesis of WRKY3 and WRKY4 function decreases salt and Me–JA stress tolerance in Arabidopsis thaliana. Mol. Biol. Rep. 2021, 48, 5821–5832. [Google Scholar] [CrossRef]
  23. Yokotani, N.; Sato, Y.; Tanabe, S.; Chujo, T.; Shimizu, T.; Okada, K.; Yamane, H.; Shimono, M.; Sugano, S.; Takatsuji, H.; et al. WRKY76 is a rice transcriptional repressor playing opposite roles in blast disease resistance and cold stress tolerance. J. Exp. Bot. 2013, 64, 5085–5097. [Google Scholar] [CrossRef]
  24. Wang, C.; Ru, J.; Liu, Y.; Li, M.; Zhao, D.; Yang, J.; Fu, J.; Xu, Z. Maize WRKY Transcription Factor ZmWRKY106 Confers Drought and Heat Tolerance in Transgenic Plants. Int. J. Mol. Sci. 2018, 19, 3046. [Google Scholar] [CrossRef]
  25. Kuki, Y.; Ohno, R.; Yoshida, K.; Takumi, S. Heterologous expression of wheat WRKY transcription factor genes transcriptionally activated in hybrid necrosis strains alters abiotic and biotic stress tolerance in transgenic Arabidopsis. Plant Physiol. Biochem. 2020, 150, 71–79. [Google Scholar] [CrossRef]
  26. Gao, Y.; Liu, J.; Yang, F.; Zhang, G.; Wang, D.; Zhang, L.; Ou, Y.; Yao, Y. The WRKY transcription factor WRKY8 promotes resistance to pathogen infection and mediates drought and salt stress tolerance in Solanum lycopersicum. Physiol. Plant. 2020, 168, 98–117. [Google Scholar] [CrossRef]
  27. Stolze, A.; Wanke, A.; van Deenen, N.; Geyer, R.; Prufer, D.; Gronover, C.S. Development of rubber–enriched dandelion varieties by metabolic engineering of the inulin pathway. Plant Biotechnol. J. 2017, 15, 740–753. [Google Scholar] [CrossRef] [PubMed]
  28. Wieghaus, A.; Prufer, D.; Gronover, C.S. Loss of function mutation of the Rapid Alkalinization Factor (RALF1)–like peptide in the dandelion Taraxacum koksaghyz entails a high–biomass taproot phenotype. PLoS ONE. 2019, 14, e0217454. [Google Scholar] [CrossRef] [PubMed]
  29. Lin, T.; Xu, X.; Ruan, J.; Liu, S.; Wu, S.; Shao, X.; Wang, X.; Gan, L.; Qin, B.; Yang, Y.; et al. Genome analysis of Taraxacum kok–saghyz Rodin provides new insights into rubber biosynthesis. Natl. Sci. Rev. 2018, 5, 78–87. [Google Scholar] [CrossRef]
  30. Molinu, M.G.; Piluzza, G.; Campesi, G.; Sulas, L.; Re, G.A. Antioxidant Sources from Leaves of Russian Dandelion. Chem. Biodivers. 2019, 16, e1900250. [Google Scholar] [CrossRef]
  31. Van Beilen, J.B.; Poirier, Y. Guayule and Russian dandelion as alternative sources of natural rubber. Crit. Rev. Biotechnol. 2007, 27, 217–231. [Google Scholar] [CrossRef]
  32. Salehi, M.; Cornish, K.; Bahmankar, M.; Naghavi, M.R. Natural rubber–producing sources, systems, and perspectives for breeding and biotechnology studies of Taraxacum kok–saghyz. Ind. Crops Prod. 2021, 170, 113667. [Google Scholar] [CrossRef]
  33. Salehi, M.; Bahmankar, M.; Naghavi, M.R.; Cornish, K. Rubber and latex extraction processes for Taraxacum kok–saghyz. Ind. Crops Prod. 2022, 178, 114562. [Google Scholar] [CrossRef]
  34. Song, Y.; Cui, H.; Shi, Y.; Xue, J.; Ji, C.; Zhang, C.; Yuan, L.; Li, R. Genome–wide identification and functional characterization of Camelina sativa WRKY gene family in response to abiotic stress. BMC Genom. 2020, 21, 786. [Google Scholar] [CrossRef]
  35. Song, H.; Sun, W.; Yang, G.; Sun, J. WRKY transcription factors in legumes. BMC Plant Biol. 2018, 18, 243. [Google Scholar] [CrossRef]
  36. He, H.; Dong, Q.; Shao, Y.; Jiang, H.; Zhu, S.; Cheng, B.; Xiang, Y. Genome–wide survey and characterization of the WRKY gene family in Populus trichocarpa. Plant Cell Rep. 2012, 31, 1199–1217. [Google Scholar] [CrossRef]
  37. Wu, K.; Guo, Z.; Wang, H.; Li, J. The WRKY family of transcription factors in rice and Arabidopsis and their origins. DNA Res. 2005, 12, 9–26. [Google Scholar] [CrossRef]
  38. Wang, Y.; Feng, L.; Zhu, Y.; Li, Y.; Yan, H.; Xiang, Y. Comparative genomic analysis of the WRKY III gene family in populus, grape, Arabidopsis and rice. Biol. Direct. 2015, 10, 48. [Google Scholar] [CrossRef]
  39. Diao, W.; Snyder, J.C.; Wang, S.; Liu, J.; Pan, B.; Guo, G.; Ge, W.; Dawood, M.H.S.A. Genome–Wide Analyses of the NAC Transcription Factor Gene Family in Pepper (Capsicum annuum L.): Chromosome Location, Phylogeny, Structure, Expression Patterns, Cis–Elements in the Promoter, and Interaction Network. Int. J. Mol. Sci. 2018, 19, 1048. [Google Scholar] [CrossRef]
  40. Li, M.; Hou, L.; Liu, S.; Zhang, C.; Yang, W.; Pang, X.; Li, Y. Genome–wide identification and expression analysis of NAC transcription factors in Ziziphus jujuba Mill. reveal their putative regulatory effects on tissue senescence and abiotic stress responses. Ind. Crops Prod. 2021, 173, 114093. [Google Scholar] [CrossRef]
  41. Hu, W.; Ren, Q.; Chen, Y.; Xu, G.; Qian, Y. Genome–wide identification and analysis of WRKY gene family in maize provide insights into regulatory network in response to abiotic stresses. BMC Plant Biol. 2021, 21, 427. [Google Scholar] [CrossRef] [PubMed]
  42. Li, J.; Islam, F.; Huang, Q.; Wang, J.; Zhou, W.; Xu, L.; Yang, C. Genome–wide characterization of WRKY gene family in Helianthus annuus L. and their expression profiles under biotic and abiotic stresses. PLoS ONE. 2020, 15, e0241965. [Google Scholar] [CrossRef] [PubMed]
  43. Ding, W.; Ouyang, Q.; Li, Y.; Shi, T.; Li, L.; Yang, X.; Ji, K.; Wang, L.; Yue, Y. Genome–wide investigation of WRKY transcription factors in sweet osmanthus and their potential regulation role in aroma synthesis. Tree Physiol. 2020, 40, 557–572. [Google Scholar] [CrossRef]
  44. Huang, X.; Li, K.; Xu, X.; Yao, Z.; Jin, C.; Zhang, S. Genome–wide analysis of WRKY transcription factors in white pear (Pyrus bretschneideri) reveals evolution and patterns under drought stress. BMC Genom. 2015, 16, 1104. [Google Scholar] [CrossRef]
  45. Gu, L.; Dou, L.; Guo, Y.; Wang, H.; Li, L.; Wang, C.; Ma, L.; Wei, H.; Yu, S. The WRKY transcription factor GhWRKY27 coordinates the senescence regulatory pathway in upland cotton (Gossypium hirsutum L.). BMC Plant Biol. 2019, 19, 116. [Google Scholar] [CrossRef]
  46. Rinerson, C.I.; Scully, E.D.; Palmer, N.A.; Donze–Reiner, T.; Rabara, R.C.; Tripathi, P.; Shen, Q.J.; Sattler, S.E.; Rohila, J.S.; Sarath, G.; et al. The WRKY transcription factor family and senescence in switchgrass. BMC Genom. 2015, 16, 912. [Google Scholar] [CrossRef]
  47. Guo, C.; Guo, R.; Xu, X.; Gao, M.; Li, X.; Song, J.; Zheng, Y.; Wang, X. Evolution and expression analysis of the grape (Vitis vinifera L.) WRKY gene family. J. Exp. Bot. 2014, 65, 1513–1528. [Google Scholar] [CrossRef]
  48. Yang, Y.; Liu, J.; Zhou, X.; Liu, S.; Zhuang, Y. Identification of WRKY gene family and characterization of cold stress–responsive WRKY genes in eggplant. PeerJ. 2020, 8, e8777. [Google Scholar] [CrossRef]
  49. Chen, C.; Chen, X.; Han, J.; Lu, W.; Ren, Z. Genome–wide analysis of the WRKY gene family in the cucumber genome and transcriptome–wide identification of WRKY transcription factors that respond to biotic and abiotic stresses. BMC Plant Biol. 2020, 20, 443. [Google Scholar] [CrossRef]
  50. Wei, K.; Chen, J.; Chen, Y.; Wu, L.; Xie, D. Molecular Phylogenetic and Expression Analysis of the Complete WRKY Transcription Factor Family in Maize. DNA Res. 2012, 19, 153–164. [Google Scholar] [CrossRef]
  51. Rushton, D.L.; Tripathi, P.; Rabara, R.C.; Lin, J.; Ringler, P.; Boken, A.K.; Langum, T.J.; Smidt, L.; Boomsma, D.D.; Emme, N.J.; et al. WRKY transcription factors: Key components in abscisic acid signalling. Plant Biotechnol. J. 2011, 10, 2–11. [Google Scholar] [CrossRef] [PubMed]
  52. Shang, Y.; Yan, L.; Liu, Z.; Cao, Z.; Mei, C.; Xin, Q.; Wu, F.; Wang, X.; Du, S.; Jiang, T.; et al. The Mg–Chelatase H Subunit of Arabidopsis Antagonizes a Group of WRKY Transcription Repressors to Relieve ABA–Responsive Genes of Inhibition. Plant Cell 2010, 22, 1909–1935. [Google Scholar] [CrossRef] [PubMed]
  53. Walley, J.W.; Dehesh, K. Molecular mechanisms regulating rapid stress signaling networks in Arabidopsis. J. Integr. Plant Biol. 2010, 52, 354–359. [Google Scholar] [CrossRef] [PubMed]
  54. Dhatterwal, P.; Basu, S.; Mehrotra, S.; Mehrotra, R. Genome wide analysis of W–box element in Arabidopsis thaliana reveals TGAC motif with genes down regulated by heat and salinity. Sci. Rep. 2019, 9, 1681. [Google Scholar] [CrossRef]
  55. Tak, H.; Negi, S.; Ganapathi, T.R. 5′–upstream region of WRKY18 transcription factor from banana is a stress inducible promoter with strong expression in guard cells. Physiol. Plant. 2021, 4, 1335–1350. [Google Scholar] [CrossRef]
  56. Chi, Y.; Yang, Y.; Zhou, Y.; Zhou, J.; Fan, B.; Yu, J.; Chen, Z. Protein–protein Interactions in the Regulation of WRKY Transcription Factors. Mol. Plant. 2013, 6, 287–300. [Google Scholar] [CrossRef]
  57. Jiang, J.; Ma, S.; Ye, N.; Jiang, M.; Cao, J.; Zhang, J. WRKY transcription factors in plant responses to stresses. J. Integr. Plant Biol. 2017, 59, 86–101. [Google Scholar] [CrossRef]
  58. Li, S.; Nayar, S.; Jia, H.; Kapoor, S.; Wu, J.; Yukawa, Y. The Arabidopsis Hypoxia Inducible AtR8 Long Non–Coding RNA also Contributes to Plant Defense and Root Elongation Coordinating with WRKY Genes under Low Levels of Salicylic Acid. Non–Coding RNA 2020, 6, 8. [Google Scholar] [CrossRef]
  59. Li, W.; Tian, Z.; Yu, D. WRKY13 acts in stem development in Arabidopsis thaliana. Plant Sci. 2015, 236, 205–213. [Google Scholar] [CrossRef]
  60. Devaiah, B.N.; Karthikeyan, A.S.; Raghothama, K.G. WRKY75 transcription factor is a modulator of phosphate acquisition and root development in Arabidopsis. Plant Physiol. 2007, 143, 1789–1801. [Google Scholar] [CrossRef]
  61. Sharma, R.; Singh, G.; Bhattacharya, S.; Singh, A. Comparative transcriptome meta–analysis of Arabidopsis thaliana under drought and cold stress. PLoS ONE 2018, 13, e0203266. [Google Scholar] [CrossRef] [PubMed]
  62. Chen, H.; Lai, Z.; Shi, J.; Xiao, Y.; Chen, Z.; Xu, X. Roles of Arabidopsis WRKY18, WRKY40 and WRKY60 transcription factors in plant responses to abscisic acid and abiotic stress. BMC Plant Biol. 2010, 10, 281. [Google Scholar] [CrossRef] [PubMed]
  63. Chen, J.; Nolan, T.M.; Ye, H.; Zhang, M.; Tong, H.; Xin, P.; Chu, J.; Chu, C.; Li, Z.; Yin, Y. Arabidopsis WRKY46, WRKY54, and WRKY70 Transcription Factors are Involved in Brassinosteroid–Regulated Plant Growth and Drought Responses. Plant Cell 2017, 29, 1425–1439. [Google Scholar] [CrossRef]
  64. Xie, T.; Chen, C.; Li, C.; Liu, J.; Liu, C.; He, Y. Genome–wide investigation of WRKY gene family in pineapple: Evolution and expression profiles during development and stress. BMC Genom. 2018, 19, 490. [Google Scholar] [CrossRef]
  65. Hu, B.; Jin, J.; Guo, A.Y.; Zhang, H.; Luo, J.; Gao, G. GSDS 2.0: An upgraded gene feature visualization server. Bioinformatics 2015, 31, 1296–1297. [Google Scholar] [CrossRef]
  66. Bailey, T.L.; Boden, M.; Buske, F.A.; Frith, M.; Grant, C.E.; Clementi, L.; Ren, J.; Li, W.W.; Noble, W.S. MEME SUITE: Tools for motif discovery and searching. Nucleic Acids Res. 2009, 37, w202–w208. [Google Scholar] [CrossRef]
  67. Shen, Q.J.; Yu, D.; Jeon, J.S.; Piffanelli, P.; Abbruscato, P.; Guo, Z.; Zhang, Y.; Itoh, T.; Lee, S.S.; Buell, C.R.; et al. Nomenclature report on rice WRKY’s—Conflict regarding gene names and its solution. Rice 2012, 5, 3. [Google Scholar]
  68. Wen, F.; Zhu, H.; Li, P.; Jiang, M.; Mao, W.; Ong, C.; Chu, Z. Genome–Wide Evolutionary Characterization and Expression Analyses of WRKY Family Genes in Brachypodium distachyon. DNA Res. 2014, 21, 327–339. [Google Scholar] [CrossRef]
  69. Gu, Y.; Ji, Z.; Chi, F.; Qiao, Z.; Xu, C.; Zhang, J.; Zhou, Z.; Dong, Q. Genome–wide identification and expression analysis of the WRKY gene family in peach. Acta Agric. Boreali–Sin. 2017, 38, 254–270. [Google Scholar]
  70. Lescot, M.; Dehais, P.; Thijs, G.; Marchal, K.; Moreau, Y.; Van de Peer, Y.; Rouze, P.; Rombauts, S. PlantCARE, a database of plant cis–acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002, 30, 325–327. [Google Scholar] [CrossRef]
  71. Salehi, M.; Karimzadeh, G.; Naghavi, M.R.; Badi, H.N.; Monfared, S.R. Expression of key genes affecting artemisinin content in five Artemisia species. Sci. Rep. 2018, 8, 12659. [Google Scholar] [CrossRef]
  72. Ma, J.; He, Y.; Wu, C.; Liu, H.; Hu, Z.; Sun, G. Cloning and Molecular Characterization of a SERK Gene Transcriptionally Induced During Somatic Embryogenesis in Ananas comosus cv. Shenwan. Plant Mol. Biol. Rep. 2012, 30, 195–203. [Google Scholar] [CrossRef]
  73. Li, B.; Dewey, C.N. RSEM: Accurate transcript quantification from RNA–Seq data with or without a reference genome. BMC Bioinform. 2011, 12, 323. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  74. Deng, W.; Wang, Y.; Liu, Z.; Cheng, H.; Xue, Y. HemI: A Toolkit for Illustrating Heatmaps. PLoS ONE. 2014, 9, e111988. [Google Scholar] [CrossRef]
Ijms 23 10270 g001 550
Figure 1. Morphologies of Taraxacum kok-saghyz at different growth stages. (a) Morphologies of Taraxacum kok-saghyz at young leaves stage. Bar = 1 cm. (b) Morphologies of Taraxacum kok-saghyz at mature leaves stage. Bar = 5 cm.
Figure 1. Morphologies of Taraxacum kok-saghyz at different growth stages. (a) Morphologies of Taraxacum kok-saghyz at young leaves stage. Bar = 1 cm. (b) Morphologies of Taraxacum kok-saghyz at mature leaves stage. Bar = 5 cm.
Ijms 23 10270 g001
Ijms 23 10270 g002 550
Figure 2. Domain recognition in the TKS WRKY protein sequences. The ATWRKY domain is color-coded.
Figure 2. Domain recognition in the TKS WRKY protein sequences. The ATWRKY domain is color-coded.
Ijms 23 10270 g002
Ijms 23 10270 g003 550
Figure 3. The phylogeny of Arabidopsis and Taraxacum kok-saghyz Rodin TFs.
Figure 3. The phylogeny of Arabidopsis and Taraxacum kok-saghyz Rodin TFs.
Ijms 23 10270 g003
Ijms 23 10270 g004 550
Figure 4. Phylogeny (a), gene structure (b), and putative motif analysis (c) of each TkWRKY gene and WRKY protein. Exons, introns, and upstream/downstream are represented by yellow, black, and blue lines, respectively. The length and proportion are measured with a ruler below. The length and position of the 20 motifs are represented by differently colored boxes.
Figure 4. Phylogeny (a), gene structure (b), and putative motif analysis (c) of each TkWRKY gene and WRKY protein. Exons, introns, and upstream/downstream are represented by yellow, black, and blue lines, respectively. The length and proportion are measured with a ruler below. The length and position of the 20 motifs are represented by differently colored boxes.
Ijms 23 10270 g004
Ijms 23 10270 g005 550
Figure 5. Phylogenetic relationships (a) and putative motifs (b) of Class III WRKY proteins in nine plants. The proteins are grouped into eight branches. The length and position of the 20 motifs are represented by differently colored boxes.
Figure 5. Phylogenetic relationships (a) and putative motifs (b) of Class III WRKY proteins in nine plants. The proteins are grouped into eight branches. The length and position of the 20 motifs are represented by differently colored boxes.
Ijms 23 10270 g005
Ijms 23 10270 g006 550
Figure 6. Location distribution of different types of cis-elements in the TkWRKY promoter region; the 12 differently colored lines correspond to 12 different cis-elements.
Figure 6. Location distribution of different types of cis-elements in the TkWRKY promoter region; the 12 differently colored lines correspond to 12 different cis-elements.
Ijms 23 10270 g006
Ijms 23 10270 g007 550
Figure 7. Expression profiling of TKS WRKY genes in 12 tissues of flower (FL), latex (LA), peduncle (PE), seed (SE), mature leaf (ML), mature lateral root (MLR), mature main root (MMR), mature stem (MS), young leaf (YL), young lateral root (YLR), young main root (YMR), and young stem (YS): (a) phylogenetic relationship clustering of the expression profiles of the WRKY genes from 12 tissues; (b) boxplots showing the dispersion of the WRKY genes expression profile in 12 tissues. Red and yellow represent the median line and average line, respectively.
Figure 7. Expression profiling of TKS WRKY genes in 12 tissues of flower (FL), latex (LA), peduncle (PE), seed (SE), mature leaf (ML), mature lateral root (MLR), mature main root (MMR), mature stem (MS), young leaf (YL), young lateral root (YLR), young main root (YMR), and young stem (YS): (a) phylogenetic relationship clustering of the expression profiles of the WRKY genes from 12 tissues; (b) boxplots showing the dispersion of the WRKY genes expression profile in 12 tissues. Red and yellow represent the median line and average line, respectively.
Ijms 23 10270 g007
Ijms 23 10270 g008a 550Ijms 23 10270 g008b 550
Figure 8. Modulation of gene expression of 14 TkWRKY genes at 0 (control), 3, 6, 9, 16, and 24 h after (a) cold, (b) heat, (c) salinity, and (d) osmotic stress. Data were normalized to β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Vertical bars indicate the standard deviation whereas an asterisk indicates the summary of the p value of the independent sample T-test of the corresponding gene compared with the control (* p < 0.05, ** p < 0.01).
Figure 8. Modulation of gene expression of 14 TkWRKY genes at 0 (control), 3, 6, 9, 16, and 24 h after (a) cold, (b) heat, (c) salinity, and (d) osmotic stress. Data were normalized to β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Vertical bars indicate the standard deviation whereas an asterisk indicates the summary of the p value of the independent sample T-test of the corresponding gene compared with the control (* p < 0.05, ** p < 0.01).
Ijms 23 10270 g008aIjms 23 10270 g008b
Ijms 23 10270 g009a 550Ijms 23 10270 g009b 550
Figure 9. Modulation of gene expression of 14 TkWRKY genes at 0 (control), 3, 9, 16, 24, and 48 h after (a) ABA, (b) MeJA, and (c) SA treatments. Data were normalized to β-actin and GAPDH. Vertical bars indicate the standard deviation whereas an asterisk indicates the summary of the p value of the independent sample T-test of the corresponding gene compared with the control (* p <0.05, ** p < 0.01).
Figure 9. Modulation of gene expression of 14 TkWRKY genes at 0 (control), 3, 9, 16, 24, and 48 h after (a) ABA, (b) MeJA, and (c) SA treatments. Data were normalized to β-actin and GAPDH. Vertical bars indicate the standard deviation whereas an asterisk indicates the summary of the p value of the independent sample T-test of the corresponding gene compared with the control (* p <0.05, ** p < 0.01).
Ijms 23 10270 g009aIjms 23 10270 g009b
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Cheng, Y.; Luo, J.; Li, H.; Wei, F.; Zhang, Y.; Jiang, H.; Peng, X. Identification of the WRKY Gene Family and Characterization of Stress-Responsive Genes in Taraxacum kok-saghyz Rodin. Int. J. Mol. Sci. 2022, 23, 10270. https://doi.org/10.3390/ijms231810270

AMA Style

Cheng Y, Luo J, Li H, Wei F, Zhang Y, Jiang H, Peng X. Identification of the WRKY Gene Family and Characterization of Stress-Responsive Genes in Taraxacum kok-saghyz Rodin. International Journal of Molecular Sciences. 2022; 23(18):10270. https://doi.org/10.3390/ijms231810270

Chicago/Turabian Style

Cheng, Yifeng, Jinxue Luo, Hao Li, Feng Wei, Yuqi Zhang, Haiyang Jiang, and Xiaojian Peng. 2022. "Identification of the WRKY Gene Family and Characterization of Stress-Responsive Genes in Taraxacum kok-saghyz Rodin" International Journal of Molecular Sciences 23, no. 18: 10270. https://doi.org/10.3390/ijms231810270

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop