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Article

Genome-Wide Identification and Transcriptional Expression Profiles of Transcription Factor WRKY in Common Walnut (Juglans regia L.)

1
College of Forestry, Northwest A&F University, Xianyang 712100, China
2
College of Animal Science and Technology, Northwest A&F University, Xianyang 712100, China
3
Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi’an 710069, China
4
Qinling National Forest Ecosystem Research Station, Huoditang, Ankang 711600, China
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Submission received: 18 August 2021 / Revised: 7 September 2021 / Accepted: 17 September 2021 / Published: 19 September 2021
(This article belongs to the Section Plant Genetics and Genomics)

Abstract

:
The transcription factor WRKY is widely distributed in the plant kingdom, playing a significant role in plant growth, development and response to stresses. Walnut is an economically important temperate tree species valued for both its edible nuts and high-quality wood, and its response to various stresses is an important factor that determines the quality of its fruit. However, in walnut trees themselves, information about the WRKY gene family remains scarce. In this paper, we perform a comprehensive study of the WRKY gene family in walnut. In total, we identified 103 WRKY genes in the common walnut that are clustered into 4 groups and distributed on 14 chromosomes. The conserved domains all contained a WRKY domain, and motif 2 was observed in most WRKYs, suggesting a high degree of conservation and similar functions within each subfamily. However, gene structure was significantly differentiated between different subfamilies. Synteny analysis indicates that there were 56 gene pairs in J. regia and A. thaliana, 76 in J. regia and J. mandshurica, 75 in J. regia and J. microcarpa, 76 in J. regia and P. trichocarpa, and 33 in J. regia and Q. robur, indicating that the WRKY gene family may come from a common ancestor. GO and KEGG enrichment analysis showed that the WRKY gene family was involved in resistance traits and the plant-pathogen interaction pathway. In anthracnose-resistant F26 fruits (AR) and anthracnose-susceptible F423 fruits (AS), transcriptome and qPCR analysis results showed that JrWRKY83, JrWRKY73 and JrWRKY74 were expressed significantly more highly in resistant cultivars, indicating that these three genes may be important contributors to stress resistance in walnut trees. Furthermore, we investigate how these three genes potentially target miRNAs and interact with proteins. JrWRKY73 was target by the miR156 family, including 12 miRNAs; this miRNA family targets WRKY genes to enhance plant defense. JrWRKY73 also interacted with the resistance gene AtMPK6, showing that it may play a crucial role in walnut defense.

1. Introduction

WRKY transcription factors are ubiquitous among higher plants, and they harbor a highly conserved WRKYGQK amino acid sequence that is followed by a zinc-finger motif at the N-terminal domain [1,2]. In important crops, WRKY genes have been examined in their related genomes, including 75 in peanut [3], 174 in soybean [4], 148 in Brassica oleracea [5], 92 in quinoa (Chenopodium quinoa) [6], 126 in Raphanus sativus [7], 79 in potato (Solanum tuberosum) [8], 112 in Gossypium raimondii and 109 in Gossypium arboreum WRKY [9], in cotton (116 in G. raimondii and 102 in Gossypium hirsutum) [10], 89 in rice [11], 63 in Dendrobium officinale [12] and 86 in Barley [13]. In woody plants, WRKY genes have been examined in their related genomes, namely 147 genes in Musa acuminate and 132 in Musa balbisiana [14], 58 in castor bean [2], 54 in pineapple [15], 56 in tea [16], 51 in Citrus sinensis [16], 48 in Citrus clementina, 79 in Citrus unshiu [17], 69 in Dimocarpus longan [18], 58 in moso bamboo [19], 85 in Salix suchowensis [20], 59 in peach [21], 95 in Dendrobium officinale [22], 71 in Fragaria vesca [23], 53 in Elaeis guineensis [24], 71 in sesame [25] and 53 in Caragana intermedia [26]. The WRKY gene is an important factor in the regulation of plant growth and development, as well as in a plant’s response to different kinds of stress [27], including drought, dehydration and salt stress [28]; however, the most important aspect of the WRKY gene is its ability to respond to abiotic stresses. This has been seen in peanut WRKY1 and WRKY12 genes, which were upregulated with salt (SA) and jasmonate (JA) treatment [28], while two abiotic stresses (salt and cold) were observed in Raphanus sativus in relation to heat, salinity and heavy metals [7]. In rice, WRKY gene family members with roles in drought tolerance and transgenic crops [29] showed a response to cold stress and methyl jasmonate (MeJA) treatments [22]. In Brassica rapa, WRKY gene family members act against abiotic and biotic stresses [30,31].
The common walnut (Juglans regia L.), i.e., the English walnut, is one of the most important hardwood trees in the world, and it is famous for its economic value, edible nuts and nutritional value [32,33,34]. Walnut oil, a high-valued oil product, is extracted from walnut kernel and used widely in food and health care industries [32,33,34]. There is no previous study regarding the WRKY gene in common walnut (J. regia) [33,34]. In a recent publication of high-quality common walnut genome data, some gene families and transcript factors were reported in common walnut, such phenylalanine ammonialyase (PAL), F-box, fatty acid desaturase (FAD), heat stress transcription factors (HSFs), nascent polypeptide-associated complex protein (NAC) and repressor of GAI; gibberellic acid-insensitive RGA and scarecrow SCR (GRAS) were also reported [35,36,37,38,39,40]. However, comprehensive information regarding the functional characterization of the WRKY gene family in common walnut is still unclear.
In this study, based on the whole-genome sequencing of walnut, we performed a genome-wide identification of the transcription factor WRKY in J. regia. We systematically characterized WRKY transcription factors in common walnut. We revealed the phylogenetic tree, structural features, duplication and conserve motifs of JrWRKYs. To understand the expression profiles of JrWRKYs, we studied the transcriptional levels of JrWRKYs in anthracnose-resistant F26 (AR) and anthracnose-susceptible F423 (AS) fruits. Our results provide useful theoretical support for the functional characterization of these JrWRKY transcription factors that are involved in resistance in common walnut.

2. Materials and Methods

2.1. Bioinformatics Analysis of Putative WRKY from Walnut

An entire protein sequence of common walnuts was downloaded from NCBI (https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/genome/?term=Juglans+regia accessed on 20 December 2020) [41]. Arabidopsis WRKY family members were downloaded from the Arabidopsis Information Resource website (TAIR, https://www.arabidopsis.org/index.jsp accessed on 21 December 2020) using a basic local alignment search tool (BLAST) to search for prevalent walnut protein sequences, including the Arabidopsis WRKY sequence as a query sequence while considering those with an E value less than 1 × 10−10 as a typical walnut WRKY sequence. The Profile Hidden Markov Model (HMM) introduced in HMMER v3.2.1 (http://hmmer.org/download.html/ accessed on 28 December 2020) and Protein Family (Pfam) database (http://pfam.xfam.org/ accessed on 28 December 2020) with default parameters were used to search for prevalent walnut WRKY with WRKY domains. The WRKY sequence name and position information was acquired through BLAST with the parameters E-value < 10–15 and ID % > 50% [42]. The WRKY sequences were predicted on the Plant-mPLoc website to predict subcellular localization of plant proteins, including those with multiple sites [43]. The theoretical isoelectric point and molecular weight were predicted in a ProtParam tool (https://web.expasy.org/protparam/ accessed on 5 September 2021) [44].

2.2. Protein Alignment, Phylogenetic Analysis, Pfam Domain Detection and Chromosome Location Analysis of Walnut WRKY Genes

The complete WRKR sequence of walnut was aligned by MEGA7.0 (State College, PA, USA) software with default parameters [45]. Subsequently, an unrooted alignment-based phylogenetic tree was constructed with pairwise deletion of 1000 bootstrap and Poisson models with MEGA7.0 software [45,46]. The Pfam web server (http://pfam.xfam.org/ accessed on 5 January 2021) was used to identify prospective domains in each sequence. We split these sequences into 3 separate subfamilies based on specific domains discovered in these WRKY sequences and used TBtools [47].

2.3. Motif Analysis, Gene Structure and Protein Structure of Walnut WRKY Genes

Feature coordinates (exon-intron boundaries) were extracted from the GFF3 annotation files of walnut. The exon-intron structure was illustrated by TBtools [47]. Identification of patterns using various pattern alignments with the default pattern-initiated (MEME) program parameters was conducted with the maximum number of patterns set to 20, and the optimum pattern width was set to 15–20 [48]. Protein structure information was predicted by an online web server of a conserved domain (https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/Structure/bwrpsb/bwrpsb.cgi, accessed on 5 January 2021) (CDD-search) [49].

2.4. Synteny Analysis and Calculating Ka, Ks and Ka/Ks Values of Duplicated Gene Pairs

BLASTP was used to identify potential pairs of homologous genes across multiple genomes (Evalue < 1×10−5, top 5 matches) [42]. The homologous gene pairs were used to identify syntenic chains through MCScanX [50]. The detected duplicate gene pairs were detected byusing MCScanX, which included whole-genome duplication (WGD), tandem duplication, segmental and other types of gene pairs [50].

2.5. Plant Materials, Treatments and Collections

To analyze the expression levels of WRKY in common walnut, 17 transcriptome data were downloaded, including a total of 17 tissues from PJ Martínez-García et al., 2016 (PRJNA291087) [51], 10 anthracnose-resistant F26 fruits from the B26 clone (AR) and 10 anthracnose-susceptible F423 fruits from the 4–23 clone (AS) of walnut [52] (PRJNA612972) (Table S1). Cufflinks was used to [53] to quantify these gene expression levels based on fragments per million base readings per million mapped read (FPKM) values with default parameters, and expression level was calculated using Hemi 1.0 software with default parameters [54]. The DESeq R package (1.10.1) was used to identify differential gene expression (DESeq) with an adjusted p-value <0.05.

2.6. qRT-PCR Analysis of WRKY Genes

We then verified the JrWRKY transcript factors expression profiles in common walnut by quantitative real-time PCR (qRT-PCR) reactions in different tissues (immature fruit, pistillate flower, mature pistillate flower, embryo, somatic embryo, vegetative bud, callus exterior, catkins, hull cortex, immature hull, young hull, mature hull, hull peel, immature leaves, young leaf, mature leaves, root) and AR and AS fruits and leaves [51,52] (samples and primer see details in Table S2). The primary specificities and associated melting curves were verified before the experiment. In each experiment, three replicates were performed. Real-time amplification responses were performed on an Applied Biosystems (USA) 7500 quick real-time PCR system. The relative concentration of expression of each gene was calculated using the 2-Ct method [55] (Table S2).

2.7. miRNA Predicted in Walnut WRKY Family Genes and the Interaction Network of JrWRKY Proteins

All of the genome sequences of the common walnut WRKY family genes were submitted as candidate genes to predict potential miRNAs by searching against the available walnut reference of miRNA sequences using the psRNATarget Server with default parameters [56]. We visualized the interactions between the predicted miRNAs and the corresponding target walnut WRKY genes using Cytoscape software with default parameters [57]. Persian walnut WRKYmatched a homologous Arabidopsis WRKY in the BLASTP program with an E value of 1 × 10−5 [58]. Regarding the Arabidopsis WRKY proteins that represented the walnut WRKY, 102 were uploaded to the STRING website to predict protein interactions (https://string-db.org/ accessed on 6 September 2021) [59].

3. Results

3.1. Identification and Classification of WRKY Genes

In this study, we detected a total of 103 WRKY genes in common walnut (Figure 1; Table 1). Based on the similar domain in which walnut WRKY genes were identified, the WRKY genes were classified into four groups, and the fourth group had the largest number of genes, including 51 members. These walnut WRKYs ranged in length from 281 to 760 amino acids, with a molecular weight from 0.76 Da to 0.69 Da and isoelectric points ranging from 4.97 to 9.72. Subcellular localization analysis indicated that all 103 walnut WRKY genes were localized in the nucleus (Figure 1; Table 1 and Table S3).

3.2. Phylogenetic Tree, Motif Composition, Conserved Domain and Gene Structure of WRKY Genes

According to the phylogenetic tree and motif composition, these gene families were divided into seven subfamilies (Figure 1). According to the gene structure and conserved motif distribution, WRKY genes showed diverse sequence structures (Figure 2, Table S4). In the present study, 15 conserved motifs were detected in WRKY proteins, and motif 2 was observed in most proteins as a subgroup that contained the most motifs (7), while the fewest motifs were found in subgroup 6, which contained only 3 motifs (Figure 2a,b). All WRKY genes contained at least one WRKY conserved domain, but subgroup 3 WRKY genes contained 2 WRKY domains; only two genes (JrWRKY76 and JrWRKY77) contained WRKY and CCCC73 domains (Figure 2c). In addition, WRKY genes were diverse in terms of gene structure, where various intron-exon numbers were observed (Figure 2d), which proved the validity of the phylogenetic tree and motif composition. The structure of WRKY genes in common walnut has different exon-intron organizations between subfamilies (Figure 3; Table S4). Intron numbers 2 to 6 were found in all WRKY genes (Figure 2d). Subgroup 1 contained 5 to 6 exons; subgroup 2 contained 3 to 4 exons; subgroup 3 contained 5 to 6 exons; subgroup 4 contained 4 to 5 exons; subgroup 5 contained 2 exons; subgroup 6 contained 3 exons; and subgroup 7 contained 3 to 4 exons (Figure 2d).

3.3. Chromosome Distribution and Synteny Analysis of WRKY Genes

All WRKY genes could be mapped onto 12 chromosomes of walnut. Chromosome 10 contained the highest number of WRKY genes (10), whereas the fewest WRKY genes were located on chromosome 5 (1) (Figure 4). The results showed a high synteny rate within WRKY genes of walnut (Figure 4, Table S5). A total of 49 showed collinear relationships between WRKY genes of walnut, indicating that these genes were WGD events (Figure 4, Table S5). In total, 20 duplicated gene pairs were found in WRKY gene walnut genomes, including two duplicate modes: whole-genome duplication (WGD)/segmental duplication and tandem duplication (Figure 3). WGD/segmental duplications and tandem duplications were only observed in walnut WRKY genes (Figure 3 and Figure 4; Table S5). Additionally, we found 22 gene pairs were under selection—17 gene pairs were under positive selection and 5 were under negative selection, indicating that these genes were under selection in evolution (Table S6). The syntenic relationships within Juglans showed that, between J. microcarpa and J. mandshurica, we identified pairs of homologs: 71 between J. regia and J. microcarpa and 70 between J. regia and J. mandshurica, indicating that WRKY genes were highly conserved among the Juglans species (Figure 5; Table S7). Multiple colinear gene pairs were found in some selected species, namely P. trichocarpa, A. thaliana, Olea europaea and Quercus robur, which inferred that the genetic copies underwent lineage-specific expansion (Figure 6; Table S7). These findings reveal closer relationships in J. mandshurica species compared to other selected species, which is consistent with their evolutionary distance. Furthermore, our results imply that continuous colinear gene pairs were found in P. trichocarpa, A. thaliana, Olea europaea and Quercus robur; therefore, we suggest that the WRKY gene might have come from the same ancestor (Figure 3, Figure 5 and Figure 6; Tables S5–S7).

3.4. GO and KEGG Enrichment Analysis of WRKY Gene Family in Walnut

We also investigated the function annotation of the WRKY gene family in walnut. GO enrichment analysis showed that the top five GO terms were response to heat, pollen-pistil interaction, recognition of pollen, response to temperature stimulus and multicellular organism processes of the biological process (Figure 7a; Table S8); however, KEGG enrichment analysis showed that the most prevalent term was the plant-pathogen interaction pathway (Figure 7b). Combining these two analytic approaches shows that the WRKY gene family might play an important role in a plant’s response to biotic and abiotic stresses (Figure 7; Table S8).

3.5. Three Genes (JrWRKY83, JrWRKY73 and JrWRKY74) May Be Involved in Resistance Traits of Walnut, Based on Transcriptome Data and qPCR

The GO and KEGG enrichment analysis results showed that JrWRKYs were differently expressed in different tissues, indicating that these genes have a variable function (Figure 8a; Table S9). In total 5 of 102 walnut WRKY members were expressed highly in peel compared to other tissues, particularly JrWRKY93, JrWRKY94, JrWRKY83, JrWRKY73 and JrWRKY74 (Figure 8a; Table S9). A mean box plot shows that there were three genes (JrWRKY83: 546-fold, JrWRKY73: 307-fold and JrWRKY74: 1920-fold) highly expressed in anthracnose-resistant F26 fruits (including 10 replicates), while these were lowly expressed in anthracnose-susceptible F423 fruits (10 replicates) (Figure 8b; Table S10; Figures S1 and S2). The morphology of leaves and fruits can be seen in the Xiangling and Shaanhe 5 cultivars, as shown in Figure 8c,d. To verify the transcriptome data, we found that there were three genes that were highly expressed in the Xiangling cultivar. We performed a qPCR analysis to verify these results. Based on our real-time PCR results, we observed that JrWRKY83 (23-fold), JrWRKY73 (10-fold) and JrWRKY74 (11-fold) were highly expressed in resistance traits compared to non-resistance traits, including leaf and fruit (Figure 8e,f; Table S11).

3.6. MicroRNA Targeting and WRKY Interaction Network

To understand the underlying regulatory mechanism of miRNAs involved in the regulation of WRKYs, we identified 206 putative miRNAs targeting 45 common walnut WRKY genes (Figure 9a; Tables S12–S14). The most target genes were JrWRKY65 and JrWRKY67, containing 197 miRNAs, while the least targeted gene was JrWRKY55, containing 62 miRNAs (Figure 9a; Tables S12–S14). Based on transcriptome profile and qPCR results, we selected JrWRKY73 and the related 85 miRNAs to construct a relationship network using Cytoscape software (Figure 9a; Table S15). Of these 85 miRNAs, we found that the miRNA family with the closest relationship was JrWRKY73, which was targeted by the Jre-miR156 family, including 12 miRNAs (Jre-miR156a, Jre-miR156b, Jre-miR156c, Jre-miR156d, Jre-miR156e, Jre-miR156f, Jre-miR156g, Jre-miR156h, Jre-miR156i, Jre-miR156j, Jre-miR156k, Jre-miR156l) (Figure 9a; Table S15). Each JrWRKYswas in close association with at least one WRKY protein from Arabidopsis. Some JrWRKYs proteins were closely aligned with the same WRKY protein in Arabidopsis. We downloaded WRKYs from Arabidopsis to detect the predicted role of highly expressed genes in the fruits and leaves of AR of Persian walnut. A previous study claimed that these genes regulate the development of fruits and are responsible for stress. Therefore, we detected the interaction relationship between these genes, and the results indicate a strong relationship between JrWRKY73s and AtCYP78A9, AtMPK6, AtMPK10, AtARF19 and AtCYP78A9 (Figure 9b).

4. Discussions

4.1. The Gene Family Member among Plants

WRKY plays a critical role in plant growth, development and resistance. Recently, there have been reports on the functional analysis of WRKY genes in plants [3,7,8,10,22,30,31]. However, their complex polyploidy and lack of genomic information have limited further study. WRKY is a large gene family in the plant kingdom, and the number of genes in the family varies from 48 to 148. This study demonstrated that the WRKY gene family contained 103 members in walnut. Comparative analysis showed that the number of WRKY genes in each plant was not determined the genome size of each plant; for instance, the maize genome was about 2300 Mb, the Arabidopsis genome was about 125 Mb and the rice genome was about 389 Mb, while the common walnut was about 584 Mb. As such, genome size was not the main determiner of the number of gene families [60,61] (Figure 1, Figure 2 and Figure 4). The WRKY gene family in walnut may also have a basic capacity to resist stress from cold, salt and disease [31]. Recently, some studies have shown that the WRKY gene is localized to the nucleus [62,63]. Our results also show that all 103 WRKY genes were predicted to be in the nucleus of common walnut (Table 1).

4.2. The Evolution of WRKY Gene Family in Walnut

The WRKY gene family in walnut can be divided into four groups, similar to the classification of WRKY genes in Musa acuminate and Musa balbisiana, Castor bean, pineapple, soybean, C. sinensis, C. clementina and C. unshiu, Eucalyptus grandis, Quinoa, Dimocarpus longan, Raphanus sativus, potato, moso bamboo, G. raimondii and G. arboretum, Cassava, willow, Oryza officinalis, peach and Dendrobium officinale. The division of the family in such a manner suggests that the results of our classification were reasonable and reliable [25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40]. In view of the conserved motifs, motifs 5, 9, 1, 2, 8, 10 and 7 were the typical motifs of group I; motifs 1 and 2 were the typical motifs of group II; and motifs 4, 1 and 2 were the typical motifs of group III. This result is consistent with our pervious study (Figure 2) [2]. Despite strong conservation of their DNA-binding domain, the overall structures of WRKYs are highly divergent and can be categorized into distinct groups, which reflects their different functions [62]. The intron and exon structures of a gene family always provide clues that demonstrate its evolution [63]. The coding sequence position and length of a single gene are always determined by intron positions, which contribute to the protein diversity caused evolution [63,64,65,66]. Duplication modes of genes, such as WGD or segmental duplication, tandem duplication and dispersed duplication, are characteristic features of the evolution of eukaryotic genomes [67]. Tandem and segmental duplication events played a critical role in the expansion of the WRKY gene family [8,9,24,28,68]. Whole-genome duplication events are common during angiosperm evolution and usually lead to the expansion of gene families [5,60]. In walnut, we found no tandem or segmental duplication events. This strongly indicates that the WRKY gene family members of walnut are predominantly influenced by WGD events, a finding that is consistent with previous studies [8,9,24,28,60,61] (Figure 3, Figure 5 and Figure 6).

4.3. The Function of WRKY Gene Family

The WRKY gene family is involved in many important biology processes, though the most important are response to abiotic stresses, pathogen defense, senescence and trichome development [3,7,8,10,22,30,31,63]. There were 20% (4/20) GO biological process pathways and 50% (1/2) KEGG pathways that were enriched in resistance pathways, which is consistent with previous studies claiming that WRKY genes work against abiotic and biotic stresses [30,31]. We were interested in WRKY genes that regulate the development of resistance traits; therefore, we analyzed public transcriptome data for J. regia (Table S1, Figure 8) and discovered that many family members were highly expressed in the hull. The hull is always impacted by stress [52,69], indicating that WRKYs may be responsible for conferring stress resistance in walnut (Figure 8) [30,31]. With the transcriptome data of AR fruits and AS fruits, we investigated the expression profile between these two fruits; three genes, JrWRKY83, JrWRKY732 and JrWRKY74, were highly expressed in AR fruits, indicating that these three genes increased their expression level when infected by the stress of Colletotrichum gloeosporioides, which predominantly affects walnut anthracnose through C. gloeosporioides can cause leaf scorches or defoliation, as well as fruit gangrene, which is currently the most challenging disease in walnut production [52,69,70]. In line with previous studies, we collected leaves and fruits infected by C. gloeosporioides. Regarding our real-time PCR results, JrWRKY83, JrWRKY732 and JrWRKY74 were induced by C. gloeosporioides stress in the leaves and root tissues of walnut cultivars. JrWRKY83, JrWRKY732 and JrWRKY74 were more upregulated in response to C. gloeosporioides stress. In recent years, many studies have shown that miRNAs in plants respond primarily to stress by regulating the expression of genes associated with stress [71]. In terms of WRKY, some researchers have reported that Md-miR156ab and Md-miR395 resulted in a significant reduction in MdWRKYN1 and MdWRKY26 expression [72]. HaWRKY6 is a particularly divergent WRKY gene exhibiting a putative target site for the miR396; thus, the possible post-transcriptional regulation of HaWRKY6 by miR396 was investigated [73]. In our study, we found that 12 miRNAs of the miRNA156 family targeted the potentially resistant gene JrWRKY73, and we also reported that Md-miR156ab targeted WRKY transcription factors to influence apple resistance to leaf spot disease [72]. The diverse patterns of microRNA targeting WRKY genes indicate that the networks of microRNA156 and JrWRKY73 may be key regulator networks for the WRKY gene family in common walnut. The results of the interaction indicate a strong relationship between JrWRKY73 and AtCYP78A9 [74], AtMPK6 [75], AtMPK10 [76] and AtARF19 [77]. AtCYP78A9 induces large and seedless fruit in Arabidopsis, indicating that JrWRKY73 may participate in the development of walnut fruits [74]. JrWRKY73 interacts with AtMPK10 and AtMPK13, while AtMKK6 and AtMPK4 activate AtMPK13 and interact with AtMPK12 in yeast cells, indicating that they may have the same function [76]. JrWRKY73 interacts with the activator of a cholera toxin known as AtARF19, indicating that JrWRKY73 may have the same function [77]. JrWRKY73 showed a higher expression level in AS fruits when induced by C. gloeosporioides stress, which was consistent with previous studies reporting that ATMPK6 was involved in distinct signal transduction pathways responding to these environmental stresses [75]. These protein interactions showed that JrWRKY73 may play a key role in fruit development, yeast cells, activation of a cholera toxin and resistance in walnut. However, when combined with the expression profile, miRNA-targeted network and protein interacted network, the results showed that JrWRKY73 played critical role in walnut defense. Moreover, these findings could lay a theoretical foundation for the functional study of JrWRKYs and the further construction of common walnut resistance regulation networks.

5. Conclusions

In this study, we identified 103 WRKY genes in walnut. Phylogenetic analysis showed that the WRKY genes could be grouped into four groups (Figure 1). These walnut WRKY genes are distributed on 16 different chromosomes (Figure 2). A phylogenetic analysis and synteny analysis showed that this gene family was conserved in evolution (Figure 3, Figure 5 and Figure 6). Tissue expression profiles of the WRKY genes demonstrated that the WRKY gene family might play a vital role in resistance traits (Figure 8). Three genes (JrWRKY83, JrWRKY73 and JrWRKY74) were highly expressed in resistant cultivars compared to susceptible varieties (Figure 8). Furthermore, 206 putative miRNAs targeting 45 common walnut WRKY genes, especially JrWRKY73, were targeted by the Jre-miR156 family, including 12 miRNAs, and it was reported that this miRNA family could target WRKY genes to enhance disease resistance in plants. JrWRKY73 interact with four genes AtCYP78A9, AtMPK6, AtMPK10 and AtARF19, indicating that JrWRKY73 plays a crucial role in plant defense (Figure 9).

Supplementary Materials

The following are available online at https://0-www-mdpi-com.brum.beds.ac.uk/article/10.3390/genes12091444/s1, Figure S1. Expression profiles of the walnut WRKY genes among 17 tissues; Figure S2. Expression profiles of the walnut WRKY genes between anthracnose-resistant F26 and anthracnose-susceptible F423 were used in this study; Table S1. The transcriptome data were used in this study; Table S2. Primers used for quantitative real-time PCR; Table S3. The sequence of WRKY gene family. Table S4. The gene structure information of WRKY genes; Table S5. The collinearity in J.regia and their related information; Table S6. The ka/ks value of WRKY genes in walnut; Table S7. The collinearity in J. regia, A. thaliana, J. mandshurica, J. microcarpa, P. trichocarpa and Q. robur and their related information; TableS8. The KEGG enrichment analysis of WRKY genes in J. regia; Table S9. The WRKY genes expression level in different tissues of J. regia; Table S10. The WRKY genes expression level in AR and AS fruits; Table S11. The qPCR data of three genes; Table S12. The predicted miRNA sequence target WRKY genes; Table S13. The target WRKY genes sequence was targeted by miRNAs; Table S14. The predicted miRNA and target genes interaction network; Table S15. The regulatory network relationships between putative miRNAs and JrWRKY73; Table S16. One-to-one orthologous relationships between Juglans regia and Arabidopsis thaliana.

Author Contributions

S.Z. conceived and designed the experiments; F.H. and G.Y. conducted the experiments; F.H., D.L. and J.Y. collected and analyzed the data; H.Z., P.Z. and F.H. carried out the experiments and utilized the software; F.H. and G.Y. wrote the manuscript; F.H., G.Y. and P.Z. reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Operating Services of Qinling National Forest Ecosystem Research Station financed by Ministry of Science and Technology of China, National Natural Science Foundation of China “Respiration flux of downed log and its influence mechanism in the Qinling Mountains” (31800372), and Natural Science Basis Research Plan in Shaanxi Province of China “Microbial community characteristics of fallen trees during decomposition process at Huoditang forest region in the Qinling Mountains” (2019JQ-641).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data sharing not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. The phylogenetic tree of WRKY gene family in walnut based on the latest genome (NCBI version: GCA_001411555.2 Walnut 2.0). These 103 sequences were used to construct a neighbor-joining (NJ) tree. The tree was divided into four subfamilies; the names of different groups are displayed.
Figure 1. The phylogenetic tree of WRKY gene family in walnut based on the latest genome (NCBI version: GCA_001411555.2 Walnut 2.0). These 103 sequences were used to construct a neighbor-joining (NJ) tree. The tree was divided into four subfamilies; the names of different groups are displayed.
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Figure 2. Phylogenetic analyses of the motifs in proteins and gene structures of WRKY genes, (a) phylogenetic tree, (b) conserved motif distribution of WRKY genes, (c) the conserved domains and (d) intron-exon distribution of WRKY genes.
Figure 2. Phylogenetic analyses of the motifs in proteins and gene structures of WRKY genes, (a) phylogenetic tree, (b) conserved motif distribution of WRKY genes, (c) the conserved domains and (d) intron-exon distribution of WRKY genes.
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Figure 3. Chromosome location and tandem analysis of WRKY genes. The blue boxes represent chromosomes of walnut; black lines represent the tandem relationships of WRKY genes in walnut.
Figure 3. Chromosome location and tandem analysis of WRKY genes. The blue boxes represent chromosomes of walnut; black lines represent the tandem relationships of WRKY genes in walnut.
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Figure 4. Chromosome location and synteny analysis of WRKY genes within walnut genome. The blue boxes represent chromosomes of walnut; red lines represent the syntenic relationships of WRKY genes in walnut.
Figure 4. Chromosome location and synteny analysis of WRKY genes within walnut genome. The blue boxes represent chromosomes of walnut; red lines represent the syntenic relationships of WRKY genes in walnut.
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Figure 5. Syntenic analysis of WRKY genes in Juglans species, including J. microcarpa and J. mandshurica. (a) the syntenic analysis of WRKY genes between J. regia and J. microcarpa. (b) the syntenic analysis of WRKY genes between J. regia and J. mandshurica.
Figure 5. Syntenic analysis of WRKY genes in Juglans species, including J. microcarpa and J. mandshurica. (a) the syntenic analysis of WRKY genes between J. regia and J. microcarpa. (b) the syntenic analysis of WRKY genes between J. regia and J. mandshurica.
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Figure 6. Syntenic analysis of WRKY genes with other species. Pt indicates P. trichocarpa; At indicates A. thaliana; Oe indicates Olea europaea; Qr indicates Quercus robur.
Figure 6. Syntenic analysis of WRKY genes with other species. Pt indicates P. trichocarpa; At indicates A. thaliana; Oe indicates Olea europaea; Qr indicates Quercus robur.
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Figure 7. GO and KEGG enrichment analysis of WRKY gene family in walnut. (a) the GO enrichment analysis of WRKY genes in walnut. (b) the KEGG enrichment analysis of WRKY genes in walnut. The red marked indicates that the concerned terms in this study.
Figure 7. GO and KEGG enrichment analysis of WRKY gene family in walnut. (a) the GO enrichment analysis of WRKY genes in walnut. (b) the KEGG enrichment analysis of WRKY genes in walnut. The red marked indicates that the concerned terms in this study.
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Figure 8. The expression profile of WRKY gene family in walnut: (a) the highly expressed genes among different tissues of walnut; IF6: immature fruit; FL3: pistillate flower; FL6: mature pistillate flower; EM8: embryo; SE7: somatic embryo; VB5: vegetative bud; CE5: callus exterior; CK3: catkins; HC2: hull cortex; HU3: immature hull; HL1: immature hull; HL6: young hull; HP3: hull peel; LY2: immature leaf; LY7: young leaf; LE5: mature leaves; RT6: root. (b) Mean box plot of WRKY members between anthracnose-resistant F26 fruits (AR) and anthracnose-susceptible F423 fruits (AS); each black circle represents each sample. (c,d) The morphology of walnut leaf (resistance (Cultivar Xiangling) and non-resistance (Cultivar Shanhe5)) and fruit (resistance (Cultivar Xiangling) and non-resistance (Cultivar Shanhe5)). (e,f) Relative expression levels of WRKY genes in walnut leaf (resistant cultivar Xiangling and non-resistant cultivar Shanhe5, and fruit resistant cultivar Xiangling and non-resistant cultivar Shanhe5).
Figure 8. The expression profile of WRKY gene family in walnut: (a) the highly expressed genes among different tissues of walnut; IF6: immature fruit; FL3: pistillate flower; FL6: mature pistillate flower; EM8: embryo; SE7: somatic embryo; VB5: vegetative bud; CE5: callus exterior; CK3: catkins; HC2: hull cortex; HU3: immature hull; HL1: immature hull; HL6: young hull; HP3: hull peel; LY2: immature leaf; LY7: young leaf; LE5: mature leaves; RT6: root. (b) Mean box plot of WRKY members between anthracnose-resistant F26 fruits (AR) and anthracnose-susceptible F423 fruits (AS); each black circle represents each sample. (c,d) The morphology of walnut leaf (resistance (Cultivar Xiangling) and non-resistance (Cultivar Shanhe5)) and fruit (resistance (Cultivar Xiangling) and non-resistance (Cultivar Shanhe5)). (e,f) Relative expression levels of WRKY genes in walnut leaf (resistant cultivar Xiangling and non-resistant cultivar Shanhe5, and fruit resistant cultivar Xiangling and non-resistant cultivar Shanhe5).
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Figure 9. (a) A schematic representation of the regulatory network relationships between the putative miRNAs and their targeted walnut WRKY genes. (b) JrWRKYs interaction network. JrWRKYs interaction network was constructed using Arabidopsis homologous WRKYs. Proteins are represented by network nodes. The 3D protein structure is displayed inside the nodes. Edges represent associations of proteins.
Figure 9. (a) A schematic representation of the regulatory network relationships between the putative miRNAs and their targeted walnut WRKY genes. (b) JrWRKYs interaction network. JrWRKYs interaction network was constructed using Arabidopsis homologous WRKYs. Proteins are represented by network nodes. The 3D protein structure is displayed inside the nodes. Edges represent associations of proteins.
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Table 1. Information on the WRKY gene family in common walnut.
Table 1. Information on the WRKY gene family in common walnut.
Protein IDGene NameChrStartEndSubcellular LocationMolecular Weight (Da)Theoretical pI
Jr01_03140_p1JrWRKY1Chr0122577712258097Nucleus56,541.738.09
Jr01_03150_p1JrWRKY2Chr0122575002257520Nucleus49,890.376.37
Jr01_03780_p1JrWRKY3Chr0128461902846322Nucleus12,027.768.89
Jr01_06810_p1JrWRKY4Chr0146353104635698Nucleus21,804.489.57
Jr01_15550_p1JrWRKY5Chr011336154013361835Nucleus26,093.398.57
Jr01_15790_p1JrWRKY6Chr011375722213757429Nucleus21,922.286.52
Jr01_15800_p1JrWRKY7Chr011375722213757429Nucleus22,078.476.95
Jr01_18750_p1JrWRKY8Chr011735596717356096Nucleus59,268.475.68
Jr01_22930_p1JrWRKY9Chr012775967827759839Nucleus41,188.188.45
Jr02_00640_p1JrWRKY10Chr02765843766023Nucleus17,790.989.72
Jr02_04990_p1JrWRKY11Chr0249863794986534Nucleus65,659.196.77
Jr02_16420_p1JrWRKY12Chr022967209929672723Nucleus40,484.075.53
Jr02_17180_p1JrWRKY13Chr023043540730435516Nucleus35,003.515.69
Jr02_17190_p1JrWRKY14Chr023043540730435516Nucleus34,916.435.69
Jr02_25510_p1JrWRKY15Chr023692871936929192Nucleus64,334.267.65
Jr02_25520_p1JrWRKY16Chr023692821136928718Nucleus56,824.616.88
Jr02_26760_p1JrWRKY17Chr023781813437818221Nucleus80,308.265.73
Jr03_08810_p1JrWRKY18Chr0366879546688183Nucleus27,753.915.29
Jr03_11540_p1JrWRKY19Chr0389686288969068Nucleus68,711.716.71
Jr03_11550_p1JrWRKY20Chr0389686288969104Nucleus69,981.27.41
Jr03_15160_p1JrWRKY21Chr031250660112506994Nucleus36,299.276.66
Jr03_17800_p1JrWRKY22Chr031664340316644049Nucleus49,731.135.09
Jr03_22520_p1JrWRKY23Chr032882294228823420Nucleus51,969.628.97
Jr03_23890_p1JrWRKY24Chr033079233830792633Nucleus38,321.415.58
Jr03_26460_p1JrWRKY25Chr033367527733675552Nucleus63,267.428.37
Jr03_27180_p1JrWRKY26Chr033446203534462179Nucleus35,255.416.05
Jr04_00630_p1JrWRKY27Chr04535739535935Nucleus33,737.696.05
Jr04_00640_p1JrWRKY28Chr04535288535738Nucleus23,556.396.47
Jr04_01100_p1JrWRKY29Chr0411798651180318Nucleus63,547.66.45
Jr04_02750_p1JrWRKY30Chr0432276713228181Nucleus34,373.425.01
Jr04_03880_p1JrWRKY31Chr0453903705390567Nucleus51,825.99.37
Jr04_03890_p1JrWRKY32Chr0453900365390075Nucleus51,825.99.37
Jr04_10710_p1JrWRKY33Chr042346525823465703Nucleus36,869.576.5
Jr04_13770_p1JrWRKY34Chr042740497927405225Nucleus67,672.36.24
Jr04_15560_p1JrWRKY35Chr042903465929034819Nucleus28,695.915.15
Jr04_15570_p1JrWRKY36Chr042903465929034819Nucleus23,041.439.4
Jr05_06670_p1JrWRKY37Chr0560640236064340Nucleus28,782.995.15
Jr06_10190_p1JrWRKY38Chr061666019216660378Nucleus17,033.675.12
Jr06_10200_p1JrWRKY39Chr061666014116660378Nucleus18,792.405.07
Jr07_07490_p1JrWRKY40Chr0786240368624525Nucleus37,173.785.45
Jr07_07550_p1JrWRKY41Chr0787327868732963Nucleus23,041.439.4
Jr07_08410_p1JrWRKY42Chr071016909610169403Nucleus40,140.445.71
Jr07_11780_p1JrWRKY43Chr071674573816745973Nucleus37,411.325.68
Jr07_12950_p1JrWRKY44Chr071812705518127153Nucleus59,910.246.89
Jr07_32310_p1JrWRKY45Chr074714668747146990Nucleus34,545.668.75
Jr07_38970_p1JrWRKY46Chr075212478952125284Nucleus42,522.335.96
Jr07_38980_p1JrWRKY47Chr075212970752130044Nucleus35,255.135.36
Jr07_38990_p1JrWRKY48Chr075212970752130044Nucleus35,168.055.36
Jr08_00860_p1JrWRKY49Chr08742451742489Nucleus30,859.848.4
Jr08_00870_p1JrWRKY50Chr08753017753211Nucleus39,081.656.87
Jr08_00880_p1JrWRKY51Chr08753017753037Nucleus33,228.877.11
Jr08_00890_p1JrWRKY52Chr08753017753037Nucleus31,222.717.63
Jr08_11260_p1JrWRKY53Chr081000211810002312Nucleus34,664.948.7
Jr08_11410_p1JrWRKY54Chr081015126110151340Nucleus29,684.26.08
Jr08_14560_p1JrWRKY55Chr081750855717508602Nucleus48,352.444.97
Jr08_14570_p1JrWRKY56Chr081750973217509923Nucleus78,751.495.48
Jr09_02120_p1JrWRKY57Chr0987674408767490Nucleus54,475.989.02
Jr09_09980_p1JrWRKY58Chr091914872619148857Nucleus40,245.485.64
Jr09_10570_p1JrWRKY59Chr091965883319659206Nucleus20,577.389.51
Jr09_10580_p1JrWRKY60Chr091966515419665485Nucleus35,303.855.81
Jr09_16000_p1JrWRKY61Chr092369716323697517Nucleus63,785.547.13
Jr09_16010_p1JrWRKY62Chr092369716323697516Nucleus63,698.467.13
Jr10_01810_p1JrWRKY63Chr1011042911104561Nucleus56,392.698.07
Jr10_02340_p1JrWRKY64Chr1014845981484699Nucleus58,693.616.74
Jr10_04760_p1JrWRKY65Chr1030872513087411Nucleus16,281.599.68
Jr10_04770_p1JrWRKY66Chr1030868653087250Nucleus20,646.359.24
Jr10_04790_p1JrWRKY67Chr1030999663100126Nucleus16,281.599.68
Jr10_10930_p1JrWRKY68Chr1085535888553895Nucleus27,001.359.41
Jr10_11120_p1JrWRKY69Chr1087407348740998Nucleus24,938.487.2
Jr10_11130_p1JrWRKY70Chr1087407348740998Nucleus25,197.87.71
Jr10_13870_p1JrWRKY71Chr101247968212480398Nucleus67,557.196.53
Jr10_25600_p1JrWRKY72Chr103724149237241717Nucleus52,902.215.65
Jr11_16130_p1JrWRKY73Chr112574672825746820Nucleus31,410.15.4
Jr11_16150_p1JrWRKY74Chr112574616025746727Nucleus31,219.885.4
Jr11_16330_p1JrWRKY75Chr112589013725890461Nucleus34,342.426.67
Jr11_17180_p1JrWRKY76Chr112657355026573846Nucleus59,310.688.76
Jr11_17190_p1JrWRKY77Chr112657355026573807Nucleus58,058.198.62
Jr11_30170_p1JrWRKY78Chr113640116336401522Nucleus35,567.937.58
Jr11_30180_p1JrWRKY79Chr113642116936421419Nucleus29,474.948.99
Jr12_04410_p1JrWRKY80Chr1255222455522483Nucleus36,853.455.3
Jr12_04430_p1JrWRKY81Chr1255524435552620Nucleus24,150.488.82
Jr12_10150_p1JrWRKY82Chr121944921419449375Nucleus36,367.456.67
Jr12_20670_p1JrWRKY83Chr122836482728365189Nucleus35,713.88.87
Jr12_25170_p1JrWRKY84Chr123137446231374933Nucleus42,430.396.48
Jr13_05050_p1JrWRKY85Chr1337168453717165Nucleus60,666.516.13
Jr13_12290_p1JrWRKY86Chr1388571998857376Nucleus20,751.249.18
Jr13_14610_p1JrWRKY87Chr131102797311028382Nucleus33,873.065.86
Jr13_14850_p1JrWRKY88Chr131122796111228243Nucleus59,330.497.29
Jr13_16070_p1JrWRKY89Chr131273456312734607Nucleus63,019.028.13
Jr13_30130_p1JrWRKY90Chr133881256838813261Nucleus42,829.335.52
Jr13_30630_p1JrWRKY91Chr133930238239302974Nucleus47,896.25.55
Jr14_02960_p1JrWRKY92Chr1421747902174952Nucleus25,660.056.99
Jr14_08090_p1JrWRKY93Chr1461851076185468Nucleus66,068.987.37
Jr14_08100_p1JrWRKY94Chr1461851076185461Nucleus65,679.577.37
Jr14_22100_p1JrWRKY95Chr142846541828465452Nucleus17,747.016.51
Jr15_12650_p1JrWRKY96Chr151930313619303172Nucleus56,662.565.47
Jr15_12660_p1JrWRKY97Chr151930284819303172Nucleus37,1559.31
Jr15_12670_p1JrWRKY98Chr151930313619303172Nucleus56,790.695.47
Jr16_00890_p1JrWRKY99Chr1611332441133569Nucleus43,423.975.82
Jr16_12390_p1JrWRKY100Chr162041976420420258Nucleus56,611.546.41
Jr16_12590_p1JrWRKY101Chr162062277320623229Nucleus22,661.499.54
Jr16_14290_p1JrWRKY102Chr162225150422251576Nucleus20,667.069.3
Jr16_19690_p1JrWRKY103Chr162654596226546702Nucleus61,636.636.06
Note: protein ID, gene ID and CDS (coding sequence) ID indicate that the accession numbers of the WRKY gene family member sequences were downloaded from the National Center for Biotechnology (NCBI). Da indicates Daltons (unified atomic mass unit); pI indicates isoelectric point.
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MDPI and ACS Style

Hao, F.; Yang, G.; Zhou, H.; Yao, J.; Liu, D.; Zhao, P.; Zhang, S. Genome-Wide Identification and Transcriptional Expression Profiles of Transcription Factor WRKY in Common Walnut (Juglans regia L.). Genes 2021, 12, 1444. https://0-doi-org.brum.beds.ac.uk/10.3390/genes12091444

AMA Style

Hao F, Yang G, Zhou H, Yao J, Liu D, Zhao P, Zhang S. Genome-Wide Identification and Transcriptional Expression Profiles of Transcription Factor WRKY in Common Walnut (Juglans regia L.). Genes. 2021; 12(9):1444. https://0-doi-org.brum.beds.ac.uk/10.3390/genes12091444

Chicago/Turabian Style

Hao, Fan, Ge Yang, Huijuan Zhou, Jiajun Yao, Deruilin Liu, Peng Zhao, and Shuoxin Zhang. 2021. "Genome-Wide Identification and Transcriptional Expression Profiles of Transcription Factor WRKY in Common Walnut (Juglans regia L.)" Genes 12, no. 9: 1444. https://0-doi-org.brum.beds.ac.uk/10.3390/genes12091444

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