Genome-Wide Identification, Expression Profile of the TIFY Gene Family in Brassica oleracea var. capitata, and Their Divergent Response to Various Pathogen Infections and Phytohormone Treatments

TIFY, a plant-specific gene family with the conserved motif TIF[F/Y]XG, plays important roles in various plant biological processes. Here, a total of 36 TIFY genes were identified in the Brassica oleracea genome and classified into JAZ (22 genes), TIFY (7 genes), ZML (5 genes), and PPD (2 genes) subfamilies based on their conserved motifs, which were distributed unevenly across nine chromosomes with different lengths (339–1077 bp) and exon numbers (1–8). Following phylogenetic analysis with A. thaliana and B. rapa TIFY proteins, ten clades were obtained. The expression of these TIFY genes was organ-specific, with thirteen JAZ genes and two PPD genes showing the highest expression in roots and leaves, respectively. More importantly, the JAZs showed divergent responses to various pathogen infections and different phytohormone treatments. Compared with the susceptible line, most JAZs were activated after Plasmodiophora brassicae infection, while there were both induced and inhibited JAZs after Fusarium oxysporum or Xanthomonas campestris infection in the resistance line, indicating their probably distinct roles in disease resistance or susceptibility. Further, the JAZs were all upregulated after MeJA treatment, but were mostly downregulated after SA/ET treatment. In summary, these results contribute to our understanding of the TIFY gene family, revealing that JAZs may play crucial and divergent roles in phytohormone crosstalk and plant defense.


Introduction
The TIFY family is plant-specific and encodes transcription factors (TFs), which was previously annotated as zinc-finger (C-X 2 -C-X 20 -C-X 2 -C) protein expressing in the inflorescence meristem (ZIM) family [1]. AT4G24470, which encodes a putative TF that contains the CCT domain and the C2C2-GATA zinc finger domain, was the first gene to be characterized as having a ZIM domain [2]. Then, 29 different Arabidopsis loci encoding proteins containing GATA-like zinc fingers were identified by BLAST searches [3]. However, ZIM and two ZIM-like proteins (ZML proteins), ZML1 (AT3G21175) and ZML2 (AT1G51600), belonged to a different group from the other typical GATA-type proteins, as determined by phylogenetic analysis [3,4]. In addition, the ZIM family was uncharacterized in Pfam or InterPro the role of JAZ genes in phytohormone crosstalk and plant defense, and provide valuable information for future functional studies.
Based on the annotation information regarding the gene structure, the CDS sequences of all cabbage genes were extracted from the genome and translated into protein sequences by using TBtools [39]. Two methods were used for the identification of TIFY family genes in this work. First, the Hidden Markov Model (HMM) profiles of the TIFY domain (Pfam: PF06200) were downloaded from Pfam (http://pfam.xfam.org/) and used for protein screening in HMMER 3.2.1 (e-value < 0.01) [40]. The first part of the candidate TIFY family proteins was obtained. In addition, BLAST analyses were conducted using 18 Arabidopsis [7] and 36 Brassica rapa [26] TIFY protein sequences as queries on the D134 genome by using the BLASTP program, thus obtaining the second part of the candidate TIFY proteins in B. oleracea. Subsequently, the two candidate sets were merged, the redundant proteins were removed, and their conserved domains were further identified by using NCBI-CDD (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) [41]. Finally, cabbage TIFY family genes were obtained from the D134 genome and used for subsequent analysis.

Chromosomal Location and Tandem Duplication Analysis
Based on the genomic sequence annotation file provided by the D134 genome database, the chromosomal position of the cabbage TIFY family genes were obtained and drew maps using TBtools. MCscanX software (http://chibba.pgml.uga.edu/mcscan2/) was used to search tandem and duplicated genes. The Ka/Ks analysis of the TIFY family genes was also conducted using TBtools.

Gene Structure, Conserved Motif, and Phylogenetic Analyses
To investigate the intron-exon organization of the cabbage TIFY proteins, the coding sequences with corresponding genomic sequences were aligned, and the results were obtained online using the Gene Structure Display Server (GSDS, http://gsds.cbi.pku.edu.cn/index.php) [42]. The conserved motifs in full-length TIFY proteins were identified by MEME (http://meme-suite.org/) [43]. The hylogenetic analysis of B. oleracea, B. rapa, and A. thaliana TIFY proteins was generated by using the Molecular Evolutionary Genetics Analysis software package, Version 6 (MEGA 6) with the neighbour-joining (NJ) algorithm [44]. Bootstrap analysis with 1000 replications was performed to assess group support.

Expression Pattern Analysis of Cabbage TIFY Genes Using RNA-Seq Data
The RNA-seq data of various cabbage tissues (bud, callus, root, stem, leaf, flower, and silique) were downloaded from the DDBJ/EMBL/GenBank Sequence Read Archive (SRA) database (GSE42891) and used for the expression analysis of the cabbage TIFY genes. To determine the expression patterns of JAZ subfamily genes after different pathogen infections, the RNA-seq data sets of cabbage clubroot resistance (XG) and susceptible (JF) lines (28 days after P. brassicae inoculation and the control mock-inoculated with sterile water, https://www.ncbi.nlm.nih.gov/sra/SRP144315), and Fusarium wilt resistance (96-100) and susceptible (01-20) lines (0 and 3 days after F. oxysporum inoculation, https://www.ncbi.nlm.nih. gov/bioproject/PRJNA548392) were downloaded from SRA database. The data sets of cabbage black rot resistance (Fuji early) and susceptible (87-534) lines (0 and 6 days after X. campestris inoculation) were obtained from relevant research in our laboratory (unpublished). All high-quality reads of each sample that passed the quality control were mapped to the B. oleracea reference genome, and the uniquely mapped reads were used for expression analysis. The fragments per kb per million (FPKM) method was conducted to normalize and calculate the gene expression levels of TIFY genes in different tissues or JAZ subfamily genes after different pathogen inoculations [45]. The negative binomial (NB) distribution test in the DESeq software package (http://bioconductor.org/packages/release/bioc/html/DESeq.html) was used to test the significance of differences (Fold Changes ≥ 2 and p-value ≤ 0.05). The heat maps of hierarchical clustering were constructed in TBtools.

Phytohormone Treatment and Quantitative Real-Time PCR Analysis
The cabbage cultivar 'Zhonggan No. 21 provided by the Cabbage and Broccoli Research Group, the Institute of Vegetables and Flowers, the Chinese Academy of Agricultural Sciences (IVFCAAS), was used for the expression analyses after different phytohormone treatments. In a greenhouse, cabbage seedlings were cultivated at 28 • C with 14 h of light/10 h of dark under artificial light until the three-leaf stage. Salicylic acid (SA) and MeJA were dissolved in 100% ethanol, and ethephon was dissolved in sterile distilled water to suitable concentrations as stock solutions and diluted with sterile distilled water containing Tween 20 [0.1% (v/v)] for foliar spraying. Sterile distilled water containing Tween 20 [0.1% (v/v)] was used as the mock control. The cabbage seedlings were sprayed with 100 µM SA, 100 µM MeJA and 500 mg/L ethephon. There were three repetitions in every treatment, and each repetition consisted of 10 plants. After two hours, the leaf samples of every treatment were taken and frozen in liquid nitrogen and stored at −80 • C for RNA extraction.
Total RNA of the leaf samples was extracted using a FastPure Plant Total RNA Isolation Kit (Vazyme Biotech Co., Nanjing, Jiangsu Province, China) following the manufacturer's instructions. The quantity and purity of RNA were estimated using an ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA). First-stand cDNAs were synthesized by reverse transcription using HiScript ® III RT SuperMix (Vazyme Biotech) following the manufacturer's instructions and diluted to 50 ng/µL for downstream processing. The specific primers of JAZ genes were designed using Premier 6 software. The sequences, amplification length, and locations of each primer have been listed in Table S1, and the specificity of the amplification products was test by agarose gel electrophoresis ( Figure S1). Each reaction contained 1.0 µL of cDNA, 0.4 µL of forward and reverse primer (10 µM), 10.0 µL of 2× ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech), and 8.2 µL double-distilled H 2 O in a total reaction volume of 20 µL and was conducted in a Bio-Rad CFX96 Real-Time PCR System (Bio-Rad Laboratories, Hercules, CA, USA) with three technical replicates by using hard-shell PCR plates (HSP9601, Bio-Rad Laboratories). Conditions for the reaction were as follows: 95 • C for 3 min, followed by 45 cycles of 95 • C for 10 s, 60 • C for 30 s, and 72 • C for 20 s. The delta-delta Ct (2 −∆∆Ct ) algorithm was used to analyse the relative gene expression levels [46,47]. Actin (GenBank accession number XM_013731369.1, Table S1) was used as the internal control to normalize the expression of the target genes. Between phytohormone treated and control samples, statistical analysis to find significant differential expression was determined using a two-tailed Student's t-test in Microsoft Office Excel 2017 (p-values < 0.05α-level).

Subcellular Localization
The pCAMBIA1300-GFP vector was used for the subcellular localization test and digested with two restriction endonucleases (XbaI and KpnI) to insert the target genes. The CDS sequences of six JAZ genes were amplified with specific primer pairs with homologous arms (Table S2), and the amplification products were recovered using the FastPure Gel DNA Extraction Mini Kit (Vazyme Biotech). Through homologous recombination, the six JAZ genes were connected to the pCAMBIA1300-GFP vector by using the ClonExpress ® Ultra One Step Cloning Kit (Vazyme Biotech). Then, the recombinant plasmids were transferred into Agrobacterium tumefaciens strain GV3101. The transformed Agrobacterium tumefaciens was cultured for 24 h at 28 • C in L-broth supplemented with 50 µg/mL kanamycin, sedimented by centrifugation at 5000× g for 10 min at room temperature and resuspended in sterile distilled water containing 10 mM MgCl 2 and 150 µg/mL acetosyringone to an optical density (OD600) of 1.0. After standing for 2 h, cells were infiltrated into the abaxial air spaces of Nicotiana benthamiana plants [48]. Forty-eight hours after infiltration, the expression position of the JAZ proteins was observed by a Leica SP8 laser confocal microscope (Leica Microsystems, Inc., Buffalo Grove, IL, USA) using filter blocks to select for spectral emission at 488 nm (matching the GFP), and the empty vector was used as a control.

Genome-Wide Identification of the TIFY Family Genes in Cabbage
On the basis of HMMER search results, 34 TIFY proteins were identified in the D134 genome (Table S3) and were taken as the first part of the candidate TIFY proteins. Then, 79 and 89 homologous proteins were obtained according to the BLASTP search using 18 A. thaliana TIFY proteins and 36 B. rapa TIFY proteins (Table S3), respectively. Subsequently, all the candidate TIFY proteins were merged and scanned using NCBI-CDD for the identification of their conserved domains. Finally, a total of 36 non-redundant TIFY genes were identified in the D134 genome of B. oleracea, including 22 JAZ, 2 PPD, 5 ZML and 7 TIFY proteins. The gene locus IDs of the 36 TIFY genes in the D134 genome are shown in Table 1, and the homologous loci in the 02-12 genome were present simultaneously (4 TIFY genes have no homologous genes in the 02-12 genome). The nucleotide and amino acid sequences of these TIFY genes are summarized in Table S4. The length of these TIFY proteins ranged from 113 (Boc04g01322) to 359 (Boc03g00474) amino acid (aa) residues with an average length of 236 aa. The molecular weight ranged from 11939.03 Da to 38823.97 Da, and the pI values varied from 4.68 to 10.07. Subcellular location prediction showed that all TIFY proteins were predicted in the nucleus.

Chromosomal Location and Gene Duplication Analysis of the Cabbage TIFY Genes
All 36 TIFY genes were assigned to nine chromosomes of B. oleracea (Figure 1), and the distribution of the TIFY genes on each chromosome was uneven. Chromosome 3 contained the largest number of TIFY genes (6 genes), followed by chromosomes 2, 5, 6, and 8, which contained 5 genes. Only one TIFY gene was located on chromosome 7. Based on the chromosomal location and the subfamily classification, the 36 TIFY genes in B. oleracea were renamed (BoJAZ1-BoTIFY7) ( Figure 1 and Table 1).  Gene duplication is one of the most important characteristics of plant genomic structure, which usually contributes to the expansion of gene families. Due to the importance of gene duplication in the evolution of gene families in plants, the duplication patterns of 36 putative TIFY genes were analysed in the cabbage genome. A total of 26 duplicated gene pairs were identified by whole genome duplication (WGD) (Figure 2). The ratios of Ka/Ks can be used as an indicator for the selection pressure of a gene during evolution. The values of all the duplicated TIFY gene pairs were less than 1 in B. oleracea ( Table 2), indicating that the TIFY genes primarily evolved under the influence of purifying selection.

Gene Structure, Conserved Motif, and Phylogenetic Analysis of the Cabbage TIFY Genes
The divergence of the exon-intron organization played a critical role in the evolution of multiple gene families. To study the structural diversity of the cabbage TIFY genes, untranslated regions (UTRs), exons and intron organization of each TIFY gene were investigated ( Figure 3A). The majority of the TIFY genes contained more than two exons. BoPPD1 and BoPPD2 have the largest number of exons at 8, whereas two TIFY subfamily genes (BoTIFY4 and BoTIFY5) had only one exon and no intron. The length and position of exons and introns of cabbage TIFY genes were varied. In addition, the conserved motifs were examined using the MEME motif search tool, five consensus motifs (tify, Jas, PPD, CCT, ZnF_GATA) were detected in the cabbage TIFY genes, and the distribution of these conserved motifs was further analysed ( Figure 3B). All 36 cabbage TIFY genes contained the tify motif, and these genes in the same subfamily have consistent motifs. Seven TIFY subfamily genes only contained the tify motif, while five ZML subfamily genes had the extra CCT and ZnF_GATA motifs, 2 PPD genes had the PPD motif, and the largest JAZ subfamily had 22 members with the C-terminal Jas motif.  Based on the amino acid sequences of full-length TIFY proteins in A. thaliana (18) [7], B. oleracea (36) and B. rapa (36) [26], the phylogenetic tree was constructed using the neighbour-joining method in MEGA 6 software. The 91 TIFY proteins were grouped into ten clades (groups 1-10 with different background colours) (Figure 4). Among these clades, group 5 was formed with 13 ZML proteins (3 of A. thaliana, 5 of B. rapa, 5 of B. oleracea), and six PPD proteins (2 of A. thaliana, 2 of B. rapa, 2 of B. oleracea) were gathered together in group 6. Groups 1, 3, 7, and 9 were four JAZ subfamily clades, including 14 (2 of A. thaliana, 6 of B. rapa, 6 of B. oleracea), 6 (2 of A. thaliana, 2 of B. rapa, 2 of B. oleracea), 12 (3 of A. thaliana, 5 of B. rapa, 4 of B. oleracea) and 5 (1 of A. thaliana, 2 of B. rapa, 2 of B. oleracea) JAZ proteins, respectively. Groups 4 and 8 were two TIFY subfamily branches, including 2 (1 of B. rapa, 1 of B. oleracea) and 5 (1 of A. thaliana, 2 of B. rapa, 2 of B. oleracea) TIFY proteins, respectively. However, group 2 and group 10 were two mixed branches, containing both JAZ and TIFY subfamily members. In group 2, there were 11 JAZ subfamily proteins (2 of A. thaliana, 5 of B. rapa, 4 of B. oleracea) and 1 cabbage TIFY subfamily protein (BoTIFY7). In group 10, there were 8 JAZ subfamily proteins (2 of A. thaliana, 2 of B. rapa, 4 of B. oleracea) and 7 TIFY subfamily proteins (4 of B. rapa, 3 of B. oleracea). The same protein number and similar phylogenetic classification of TIFY proteins in B. oleracea and B. rapa may reveal the parallel evolutionary relationship of this family between the two species [49].

Expression Patterns of the Cabbage TIFY Family Genes in Various Tissues
To explore the expression pattern of the TIFY family genes, RNA-seq data from seven cabbage tissues (root, stem, leaf, bud, flower, callus, and silique) were used ( Figure 5 and Table S2). Because these RNA-seq data were obtained from the 02-12 transcriptome sequence data, the four TIFY genes (BoJAZ5, BoJAZ14, BoZML3, BoTIFY7) without homologous loci in the 02-12 genome were not analysed. Lower expression levels of ten JAZ genes in three clades (BoJAZ6/BoJAZ8/BoJAZ10, BoJAZ2/BoJAZ13/BoJAZ17/BoJAZ20, BoJAZ9/BoJAZ18/BoJAZ19) were observed in leaves, buds, flowers, and siliques, while these JAZ genes showed the highest expression level in roots. BoJAZ3 and BoJAZ7 were in the same clade with BoJAZ15, BoJAZ17, and BoJAZ22, while the first two had the highest expression in buds and the last three in roots. In addition, BoJAZ1, BoJAZ12, and BoJAZ16 in another clade were highly expressed in silique. For TIFY subfamily genes, the expression levels were diverse in different tissues. Three TIFY subfamily genes (BoTIFY3, BoTIFY4, BoTIFY6) in one clade showed lower expression levels in leaves, buds, flowers, and siliques, while BoTIFY1, BoTIFY2 and BoTIFY5 in another clade were different. Two PPD genes (BoPPD1 and BoPPD2) showed the highest expression in leaves, indicating that they may be involved in the coordination of leaf growth like AtPPD1 and AtPPD2 in A. thaliana [14]. In addition, the four cabbage ZML genes may perform similar biological functions with relatively consistent expression levels in all tissues.

Expression Profiles of the Cabbage JAZ Genes induced by Different Pathogen Infection
Jasmonates and related signalling compounds regulate a wide range of biological processes in plants, not only sexual reproduction and development but also host immunity [20,22]. JAZ proteins act as key repressors of JA signalling linking COI1 and downstream transcription factors, suggesting that these proteins may also play key roles in plant defense responses [21,50]. To investigate the expression patterns of cabbage JAZ genes after pathogen infection in both resistant and susceptible materials, RNA-seq data sets of cabbage clubroot, Fusarium wilt, and black rot were used to explore their expression differences. Thirteen JAZ genes were significantly upregulated in the cabbage clubroot-resistant line after P. brassicae inoculation, especially BoJAZ10 ( Figure 6A and Table S5). For the susceptible line, in addition to BoJAZ4 and BoJAZ21, other JAZ genes were all downregulated to various degrees, and twelve of them were significantly downregulated, especially BoJAZ15 ( Figure 6A and Table S5). The opposite responses of JAZ genes between cabbage clubroot-resistant line and susceptible line indicate that JAZ genes may play an important role in the resistant reaction of cabbage to P. brassicae. Similar to the reaction of the cabbage-resistant line for P. brassicae, sixteen JAZ genes in the cabbage Fusarium wilt-resistant line were upregulated after F. oxysporum inoculation, and ten of them were significantly upregulated ( Figure 6B and Table S5). However, BoJAZ12 and ten other JAZ genes were also upregulated in the susceptible line. After the inoculation of X. campestris, a bacterium causing black rot of cabbage, we also found that many JAZ genes were upregulated in both the resistant line and susceptible line ( Figure 6C and Table S5). Although many JAZ genes were induced whether inoculated with P. brassicae, F. oxysporum or X. campestris, the specific JAZ genes were different among them. For example, BoJAZ15 and BoJAZ16 were downregulated after F. oxysporum inoculation but upregulated after X. campestris inoculation. It was suggested that the mechanism of these JAZ members responding to different pathogen infections were different and highly complex, and both redundancy and antagonism were observed.

Expression Patterns of the Cabbage JAZ Genes after Exogenous Phytohormone Treatment
Recent studies have revealed that JAZ proteins may play a role in the regulation of diverse phytohormone signalling pathways involved in defence and plant growth [51,52]. In this work, qRT-PCR was conducted to evaluate the responses of cabbage JAZ genes after different phytohormone (JA, SA, ET) treatments. After treatment with MeJA, the transcription of all JAZ genes in cabbage seedlings was induced compared to the control (Figure 7), which was consistent with the results in A. thaliana and B. rapa [21,26]. Among the JAZ genes, fifteen were significantly upregulated, and six (BoJAZ6, BoJAZ8, BoJAZ15, BoJAZ16, BoJAZ18, and BoJAZ20) were upregulated more than 10-fold, while BoJAZ11 was upregulated less than twofold and not significantly, showing the different responses of these JAZ genes to MeJA. In response to SA, we found that most cabbage JAZ genes were downregulated, and five JAZ genes (BoJAZ5, BoJAZ6, BoJAZ7, BoJAZ11, and BoJAZ20) were upregulated. Among these genes, BoJAZ6 and BoJAZ10 had the highest and lowest expression levels, respectively. Similar cases also occurred after ethylene treatment. In addition, we found specific BoJAZ6, which was concurrently induced by the three phytohormones. Figure 7. The relative expression of cabbage JAZ genes in the control and exogenous MeJA, SA, and ethylene treatments. Relative expression of JAZ genes was analysed by quantitative real-time qPCR using cabbage actin as a control. Error bars indicate standard deviation, and asterisks indicate significant differences between the control and phytohormone treatment, * p < 0.05, ** p < 0.01.

Subcellular Localization of Cabbage JAZ Genes
Based on the results of the phylogenetic analyses of 91 TIFY genes (Figure 4), twenty-two cabbage JAZ subfamily genes were grouped into six clades (1,2,3,7,9,10), and all of them were predicted to be located in the nucleus by using ProtComp and Plant-mPLoc (Table 1). We recombined the pCAMBIA1300-GFP vector with the CDS sequences of six cabbage JAZ genes (BoJAZ1, BoJAZ2, BoJAZ3, BoJAZ4, BoJAZ5, and BoJAZ6), which participated in the six clades of the phylogenetic tree. The GFP signal of all six JAZ-GFP fusion proteins was observed exclusively in the nucleus (Figure 8), which was consistent with the prediction results.

Discussion
Arabidopsis thaliana, one member of the Brassicaceae family, was the first genome sequenced [53]. Subsequently, the whole-genome sequences for other Brassicaceae members were completed, such as Arabidopsis lyrate, Brassica oleracea, Brassica rapa, Brassica napus, Camelina sativa, and Raphanus sativus [54]. The availability of these genomes improved our understanding of phylogenetic relationships in the Brassicaceae family and laid a solid foundation for genome-wide gene identification and functional research [55,56]. Even in closely related species, the genome structure, size and copy number were vary considerably [57], and the variations were restricted to repetitive sequences and affected specific gene families involved in different plant physiological processes [58]. Members of the TIFY family have been demonstrated to be putative TFs with various responsiveness in plant development and defense [59,60]. Hence, we performed genome-wide identification and expression profiling analysis of the TIFY gene family in Brassica oleracea. A total of 36 TIFY family genes were identified in the D134 genome of B. oleracea and included 22 JAZ, 2 PPD, 5 ZML and 7 TIFY subfamily genes. The total number of TIFY genes and the number of the four subfamily genes in B. oleracea were the same as those in B. rapa. This result indicated that the evolution rate of the TIFY genes was similar between the two Brassica species.
Gene duplication, including tandem, segmental, and whole genome duplication, plays an important role in the evolution of various species and contributes to the expansion of gene families [61,62]. Previous whole genome identification found a total of 1825 gene clusters containing 4365 tandemly duplicated in B. oleracea, and there were similar numbers in B. rapa (5181) and A. thaliana (4170) [34]. In this study, 26 duplicated gene pairs of 36 TIFY genes in B. oleracea caused by WGD were identified. WGD is one of the important mechanisms of species evolution, producing new genomic regions and making it more complex and diverse [63]. Similar conditions have been found in other species, such as 19 of the 24 poplar TIFY genes produced by WGD [64]. The large proportion of duplicated genes in the TIFY family revealed that WGD made a large contribution to the generation of the TIFY gene family. Although the duplicated TIFY genes may have a common ancestor, their functions and expression patterns were complex, since duplicated genes can undergo substantial changes in their structures and/or regulatory mechanisms to assume novel roles [62,63]. For example, in the duplicated gene pair of BoJAZ1/BoJAZ19 identified in this work, BoJAZ1 has five exons with 241 amino acid residues, while BoJAZ19 has three exons with 292 amino acid residues, and their expression levels were different in various cabbage tissues.
JA regulates large-scale changes in gene expression to exert its many effects in such processes as plant defense response, cell division, photomorphogenesis, and sexual reproduction [65,66]. JAZ proteins repress the activity of transcription factors that execute responses to JA [20][21][22]. JAZ proteins contain two highly conserved sequence regions: the C-terminal Jas domain, which plays a key role in destabilizing the repressor for the response of JA-Ile, and the ZIM/TIFY domain, which mediates homo-and heteromeric interactions between most JAZs [7,67]. However, a non-TIFY JAZ protein (JAZ13, encoded by At3g22275) was also demonstrated as a functional repressor of JA-mediated responses in Arabidopsis [68]. In this work, 22 JAZ genes were identified in B. oleracea, with the largest number being observed among the four TIFY subfamilies, and there were higher expression levels of most JAZ genes in cabbage root compared with other tissues. In view of the important role of JA in regulating root growth [69], we deduced that JAZ proteins may play a key role in this process and other root-related traits.
Phytohormone signalling networks are extensively involved in the process of plant interactions with pests and pathogens, and numerous studies have shown that JAZ targets appear to be mainly TFs associated with hormone regulation, revealing the crosstalk between JA and other plant hormones to some degree [70]. Salicylic acid (SA) is the major signalling molecule associated with the hypersensitive response (HR) and implicated in plant resistance to (hemi)biotrophic pathogens [71], while defenses against necrotrophic pathogens have been linked to JA and ethylene (ET) signalling [72,73]. However, antagonistic relationships between SA and the JA/ET pathway were most often reported [74]. EIN3 and EIL1, two nuclear transcription factors that initiate downstream transcriptional cascades for ET responses, are capable of interacting with JAZ proteins [75]. Meanwhile, EIN3 and EIL1 repressed SID2, a gene encoding an isochorismate synthase required for SA biosynthesis [76]. RGA, a DELLA protein involved in the regulation of gibberellin (GA) signalling, interacted with JAZ proteins to compete for the JAZ-MYC2 interaction, and the 'relief of suppression' model was built [76][77][78][79]. In addition, SA-inducible genes were not constitutively expressed in the quadruple-DELLA mutant, indicating that DELLA has a negative effect on SA signalling [80]. In this study, most cabbage JAZ genes were upregulated after MeJA treatment, and different expression levels were also found after SA or ethylene treatment, particularly BoJAZ6. It was indicated that the JAZ genes can respond to JA, SA and ET signalling simultaneously. These results further proved that JAZ proteins may play a key role in the crosstalk among different phytohormones, especially JA, SA and ET.
Since phytohormones play a key role in signal transduction when plants encounter pathogens, we investigated the expression of the JAZ genes in cabbage-resistant and cabbage-susceptible lines after pathogen inoculation. We found that many cabbage JAZ genes were induced after inoculation with both fungal and bacterial diseases. The most obvious response was the cabbage response to P. brassicae; thirteen JAZ genes were upregulated in the resistant line, and eighteen JAZ genes were downregulated in the susceptible line. The role of SA and JA signalling in the resistance response to biotrophic clubroot has been investigated. In A. thaliana, through the expression analysis of SA-and JA-responsive genes, the determination of SA and JA levels and exogenous phytohormone application, both SA and JA pathways were found to contribute to the inhibition of clubroot development [81]. In addition, glucosinolates (GSLs) play roles in plant defense response against microbial pathogens [82]. In Chinese cabbage, the increased content of JA has been proven to mediate the accumulation of aliphatic GSLs and is involved in clubroot during the secondary infection stage [83]. In view of the role of JA in the development of clubroot and the higher expression levels of cabbage JAZ genes in root tissue, and the distinct responses of JAZ genes between cabbage clubroot-resistant and susceptible lines after P. brassicae inoculation, functional studies of cabbage JAZ genes for clubroot resistance are warranted. However, the activation of JAZ genes was also observed both in cabbage resistant and susceptible lines after F. oxysporum and X. campestris inoculation, suggesting that the mechanism by which JAZ genes participate in cabbage disease resistance may be complex and diverse, and there may be functional redundancy and antagonism among them.

Conclusions
In summary, 36 TIFY genes (22 JAZ, 2 PPD, 5 ZML and 7 TIFY) were identified in the B. oleracea genome through genome-wide analysis. The number and length of exons and introns of these TIFY genes were varied, and the conserved motifs of these TIFY genes were consistent in the same subfamily. The expression of these TIFY genes was organ-specific, and a larger number of JAZ genes were activated after different pathogen infections and MeJA treatment. These results presented in this report lay the foundation for further functional characterization of TIFY genes, and improve our understanding of the JAZ genes in plant development and disease resistance through the JA signalling pathway.