Genome Wide Identification and Characters of TCP Family Genes in Brassica juncea var. tumida

Teosinte branched1/Cycloidea/Proliferating cell factor (TCP) proteins are plant-specific transcription factors, in So far, TCP proteins function in tumorous stem mustard has not been reported. . Here we identified and characterized the entire TCP protein family members in the tumorous stem mustard. Results We identified fifty-four TCP genes in Brassica juncea var. tumida, containing thirty-three Class I subfamily members and twenty-one Class II subfamily members. Fifty-three TCP genes are distributed on 15 chromosomes. Gene structure and conserved motif analysis showed that the same clade genes have similar gene intron/exon structure and conserved motifs. Cis-acting element results showed that the same clade genes also have similar cis-element, however subtle differences also imply the different regulated pathway. More than twice paralogs genes relation to diploid species in some members imply gene duplication events in evolution. The members of BjTCP18s are low expressed in DY strains and un-swelling stage of YA strains. After treatment with GA and SA, it was detected that the expression levels of multiple TCP genes were affected by these two hormones. we perform The results


Abstract
Background Teosinte branched1/Cycloidea/Proliferating cell factor (TCP) proteins are plant-specific transcription factors, which widely involved in leaf development, flowering, shoot branching, and circadian rhythm.
So far, TCP proteins function in tumorous stem mustard has not been reported. . Here we identified and characterized the entire TCP protein family members in the tumorous stem mustard.

Results
We identified fifty-four TCP genes in Brassica juncea var. tumida, containing thirty-three Class I subfamily members and twenty-one Class II subfamily members. Fifty-three TCP genes are distributed on 15 chromosomes. Gene structure and conserved motif analysis showed that the same clade genes have similar gene intron/exon structure and conserved motifs. Cis-acting element results showed that the same clade genes also have similar cis-element, however subtle differences also imply the different regulated pathway. More than twice paralogs genes relation to diploid species in some members imply gene duplication events in evolution. The members of BjTCP18s are low expressed in DY strains and un-swelling stage of YA strains. After treatment with GA and SA, it was detected that the expression levels of multiple TCP genes were affected by these two hormones.

Conclusion
In this study, we perform the first genome-wide analysis of the tumorous stem mustard TCP gene family. The results provide valuable information for understanding the classification and functions of TCP genes in tumorous stem mustard.
Base on their conserved domain, the TCP proteins are divided into two subfamilies: Class I and Class II [7,9]. Class I TCPs bind the DNA sequence GGNCCCAC and Class II TCPs bind GTGGNCCC [12]. In Arabidopsis, TCP2-5, TCP10, TCP13, TCP17 and TCP24 were related to lateral organ organogenesis and controlled leaf development [13][14][15][16]. TCP1, TCP12 and TCP18 are similar homology genes in one subfamily, for the reason that Branched1(TCP18) and Branched2(TCP12) control branch outgrowth [17][18][19][20]. TCP18 interacts with the florigen proteins FLOWRING LOCUS T(FT) and modulates its activity in the axillary buds to repress the floral transition of axillary meristems [21]. TCP21 is involved in circadian clock through interacts with TIMING OF CAB EXPRESSION 1(TOC1) and In recently years, TCP proteins were demonstrated to be related with defence responses. TCP13, TCP14 and TCP19 were found to be directly targeted by effectors form both Pseudomonas syringae and Hyaloperonospora arabidopsidis [25]. Kim et al. found TCP8, TCP13, TCP15, TCP20, TCP22 and TCP23 can interact with the Arabidopsis immune adaptor SUPPRESSOR OF rps4-RLD1 (SRFR1) that is a negative regulator of effector-triggered immunity [26].
TCP gene family has been identified in many plant species, such as, 24  industry. The growth of Tumorous stem mustard are divided into four stages: germination, seedling, stem swelling and flowering. Stem swelling is a key character of tumourous stem formation. The balance between stem swelling and flowering were directly related to the quality and yield of tumourous mustards. Tumourous stem mustard is an annual plant, and the stem does not swell except in plants sown between mid-September and mid-October in Chongqing and the other valleys of the Yangtze River, China. Thus, the production period of edible stems is limited. As above reports, TCP proteins were extensively involved in branching, flowering and a series development processes, forming the shape of plant architecture [7,17,18,21,24,[34][35][36][37][38][39][40][41][42][43][44][45][46]. However, no report on tumorous stem mustard TCP family exists, and whether TCP family proteins control the stem swelling and flowering development of tumorous stem mustard are still unknown.
As the whole genome of tumorous stem mustard is sequenced[47], a genome wide analysis of TCP genes is performed for the first time in the current study. Fifty-four BjTCP genes were identified in the tumorous stem mustard genome, and their phylogenetic relationship, gene structure, protein motifs, chromosome location, and expression profile in different tissue were analysed. The results provide information on the classification and detail of BjTCPs and lay the foundation on the stem swelling and flowering regulation mechanism of TCP proteins in tumorous stem mustard.

Identification of members of the TCP family in Brassica juncea var. tumida
Fifty-four TCP family genes were identified in Brassica juncea var. tumida, based on the similarity with Arabidopsis thaliana homology genes, the tumorous stem mustard TCP genes were named with BjTCP1a-BjTCP27b (Table 1). The coding amino acids were 171-457, with the molecular weight (MW) of 18.6-50.08 kDa and the isoelectric point (pI) of 5.5-10.18. Except BjTCP27b anchored in contig6125, TCP genes were located on 15 of 20 chromosomes. There are one TCP gene on chromosomes A10, B01and B06, two TCP genes on A06, as well as three to seven members on the other chromosomes ( Figure 1). Except BjTCPa-c, other BjTCP proteins all are localized in nuclear (Table 1), that is a typical feature of transcription factors.

The phylogenetic tree of BjTCPs and AtTCPs
Multiple sequence alignment of TCP proteins showed the conserved region mainly focused on the TCP domain ( Figure S1).
To assess the phylogenetic relationships of the TCP family, the predicted TCP proteinsequences in Brassica juncea var. tumida and Arabidopsis thaliana were used to construct a phylogenetic tree. The results indicated that all the TCP proteins were divided into two groups, Class Ⅰ and Class Ⅱ TCPs ( Figure 2a). In the Class II TCPs cluster, these proteins were further divided into three subgroups, CYC, TB1 and CIN. The CYC subgroup was mainly cluster with AtTCP1 and AtTCP12, containing four AtTCP1 homology proteins BjTCP1a-d and two AtTCP12 homology proteins BjTCP12a-b. The TB1 subgroup was composed of AtTCP18 and four homology TCP proteins BjTCP18a-d. In CIN cluster, no proteins were found to be homology with AtTCP2-4 and AtTCP10, the other TCP proteins were found two or three homology proteins like AtTCP24 (three homology proteins BjTCP24a-c), AtTCP13 (three homology proteins BjTCP13a-c), AtTCP27 (two homology proteins BjTCP27a-b) and AtTCP5 (three homology proteins BjTCP5a-c). In the Class I TCPs cluster, except AtTCP6, AtTCP11, AtTCP16, AtTCP23 cannot found homology proteins in Brassica juncea var. tumida, the other TCP proteins all have multiple homology proteins, such as AtTCP15 and AtTCP21 even have six homology proteins, respectively.
The BjTCP proteins of mustard have a typical bHLH motif in all other identified TCP proteins, (

The gene structures and conserved motifs analysis of BjTCPs
The intron-exon gene structure results showed that most BjTCP genes only have one exon except BjTCP18s, BjTCP12s, BjTCP20b, BjTCP13b containing two and more exons. The genetic structure and evolutionary relationships of all TCP family members of stem mustard are also closely related. Genes within the same subfamily often have similar gene structures. BjTCP12a and BjTCP12b genes were composed of two exons, and BjTCP18a-d were more than three exons ( Figure 3A and B). The conserved motifs in these BjTCPs also showed the similar characters in the same subgroup, such as three similar motifs in all BjTCP15 homology proteins and five similar motifs in all BjTCP21 homology proteins ( Figure 3C).

The gene duplication of BjTCP members
The gene duplication events were further analysed in the tumorous stem mustard TCP gene family. As shown in Table 2, 67 paralogous and/or orthologous pairs were identified. The Ka/Ks ratio is widely applied to measure genetic evolution and selection pressure. The ratios of forty-eight homologous pairs were greater than 1, in contrast other nineteen homologous pairs were smaller than 1, indicating positive selection and negative selection during evolution, respectively (Table 2).
Meanwhile, the divergence times of the TCPs were also calculated. The results indicated that these BjTCPs underwent duplication events from ∼2.65 to 37.39 million years ago (MYA).

The promoter cis-acting element analysis of BjTCPs
The cis-acting element in the promoter of a gene usually regulated the expression and function.
Multiple cis-acting elements were found in these TCP gene promoters, such as plant hormone responses element, light responses element, stress responses element, meristem expression, circadian control, low-temperature and wound responses element and so on ( Figure 4).
For hormone-related cis-acting elements, the abscisic acid (ABA) responsive elements were identified at least two or more ABRE cis-acting elements in the tumorous stem mustard TCP gene promoters expect BjTCP5s, BjTCP18s, BjTCP19 and BjTCP24s. Auxin responsive elements include AuxRP and TGA elements. AuxRP has a relative small number of components, mainly in the BjTCP5, BjTCP19 and Under GA treatment, the levels of BjTCP12, BjTCP17 and BjTCP20 cannot be detected. In contrast, the expression of other genes were induced at the early stages but then decreased to a low level mainly at 8h ( Figure 6).

Discussion
As plant-specific TFs, the TCP TFs play a various function role in plant growth and development processes. In tumorous stem mustard, 54 family members were found. As other plants, the general organization of these TCP family are also conserved and significantly more members in the class I subfamily than those in the class II[28-33, 49-52]. As a tetraploid plant, the number of TCP proteins in tumorous stem mustard is significantly more than twice as abundant as Arabidopsis (24 TCP proteins), which suggest that some genes are doubled in the process of evolution.
In addition, the exon/intron structure, conserved motif distribution patterns and domain of BjTCP homology genes often showed high similarity, such as BjTCP21a-f, BjTCP12a/b and so on. These similarities cluster homology genes members might play similar function during tumorous stem mustard growth and development.
Interesting, a series of genes such as BjTCP15b, c and BjTCP1b and BjTCP22b are located on the same chromosome A07. But the homology gene of these genes can be found (BjTCP15b and BjTCP15f, BjTCP15e and BjTCP15c, BjTCP1b and BjTCP1c, BjTCP22b and BjTCP22d) in series and showed the same order on the corresponding B03 chromosome (Figure 1 Figure S2). These results also indicted that the division of BjTCP15b and BjTCP15c may be earlier than the formation of tetraploids.
There are 24 TCP genes in Arabidopsis, whereas some corresponding homologous TCP genes cannot find in tumorous stem mustard, such as TCP2-4, 6, 11 and 16, which may due to occurred losing event during the evolution process. Tumorous stem mustard is a tetraploid plant that belongs to the cruciferous near-source species of Arabidopsis. In theory, each of the Arabidopsis TCP genes has two orthologous genes in the stem mustard. However, some TCP genes can find out more than two paralogous genes, such as BjTCP1 (four homology genes), BjTCP18 (four homology genes), BjTCP21 (six homology genes), BjTCP15 (six homology genes) and so on. These genes may be caused by multiple gene doubling events, the function of these paralogous genes have similarity and gradual differentiation in the process of evolution. First of all, from the characteristics of the cis-elements of these genes, most of the paralogous genes have similar cis-acting elements, but there are also a few differences, such as the four paralogs of BjTCP18 are no ABA and auxin cis-acting elements, but BjTCP18b is the only member of the four genes with circadian regulatory elements, suggesting that the BjTCP18b gene may be involved in circadian rhythm ( figure 4). Correspondingly, the expression patterns of these paralogous genes are also different ( Figure 5).
Previous studies demonstrated that B.juncea genome underwent genome duplication events about 0.039−0.055 million years ago [47]. In this study, all the duplication pair gene was occurred before 2.65 MYA, suggesting that the gene duplication events occurred before the B.juncea forming. This results also proved the guess above that the BjTCP genes clustered together on the chromosome A07 and B03 were formed before the formation of tetraploids. But four BjTCP18s homology genes in tumorous stem mustard, which may form by gene duplication.
Functional differentiation may occur between the four TCP18 genes. From the perspective of their expression patterns, the four genes are not consistent during tissue development. In this study, the expression profiles of tumorous stem mustard seedlings and tumour stems were used, and the expression levels of these four genes were low in these tissues. The expression levels of the four genes gradually decreased with the swelling of the tumorous stems ( Figure S3). The flowering stage of the tumorous stem mustard is mainly characterized by the swelling of the tumorous stem. At this time, there will be a bolting and flowering phenomenon similar to Arabidopsis. Since BRC1 has the function of inhibiting branching and flowering, the gradual down-regulation of its mRNA levels may reflect the gradual decrease ability in inhibition of branching and flowering. The results also indicated that the tumorous mustard is about to enter the period of reproductive growth.
According to reports, there are 16 varieties of mustard species identified, which the mainly difference of these vegetables tissues are used for food and shape, including root, stem, leaf and branch[57]. BRC1 gene controls plant branching and flowering, and four identified BjBRC1 may imply the further function differentiation of branch development and flowering during the development of mustard.
Among these BjTCP genes, multiple BjTCP15s and BjTCP21s in DY and/or the early stage of seedling and tumour stem per-swelling stage were higher expression than swelling stage. DY is a no swelling mutant line tissues sample, and YA1 and YA2 are also not begin to swelling. This result may indicate that these genes are involved in the process of stem swelling in tumorous stem mustard.
Increasing evidence verified that TCP proteins are involved in responses to plant hormone[37, 58,59].
In this study, most of tumorous stem mustard TCP genes appear to be regulated by SA and GA ( Figure   6). In Arabidopsis thaliana, several TCPs interact with the SA biosynthetic enzyme ISOCHORISMATE SYNTHASE 1 gene and enhanced the gene's expression through binding to the TCP-binding motif in its promoter region [60]. In our results, there are many SA-related cis-elements in the promoter regions of BjTCPs (Figure 4), and the expression levels of several BjTCP genes significantly increased after the SA treatment (Figure 6), indicating that BjTCPs may be involved in the signal transduction of SA. In addition, most BjTCP genes have polytype gibberellin (GA) responsive elements GARE, P-box and TATC-box in their promoters (Figure 4), which may lead to more complex regulation of their expression and more diverse expression patterns ( Figure 6).
This study was the first to identify 54 BjTCP gene family members in tumorous stem mustard and to investigate their roles in stem development. Our results provide a foundation to further determine the molecular mechanisms of TCP genes in the development of tumorous stem.

Conclusions
We performed a genome-wide analysis and identify 54 TCP genes in Brassica juncea var. tumida.
These genes are divided into two subfamilies, 33 Class I and 21 Class II. Chromosomal mapping showed that 53 BjTCP genes were heterogeneously distributed on 15 chromosomes. Structural analyses of BjTCP genes showed that 44 genes had no introns, most of the BjTCP genes in the same cluster had similar patterns of exon length, intron number, and conserved motifs. We identified several genes that are highly expressed in development of tumorous stem mustard and branching relation genes were low expression in swelling stage of vegetative growth.

Sequence and phylogenetic analyses
Multiple alignments of TCP protein sequences from Brassica juncea var. tumida and Arabidopsis thaliana were performed using the ClustalW programme [66]. Phylogenetic tree were constructed with the MEGA 7.0 software using the neighbour joining method and a boostrap test replicated 1000 times [67]. The gene structure diagram was drawn using the online software of the GSDS2.0 server (http://gsds.cbi.pku.edu.cn/) [68]. The physical location data of BjTCP genes were retrieved from the Brassica juncea var. tumida genomes. The mapping of these TCP genes was subsequently performed using MapInspect soſtware. Default parameters were used for the Multiple Em for Motif Elicitation (MEME, http://meme-suite.org/) programme for the identification of conserved protein motifs and a maximum number of 12 motifs. Subcellular localizations of BjTCPs were predicted using ProComp9.0.
The 2000 bp of the 5′ sequence as the promoter domain of each TCP genes were used to analyse the cis-acting elements using the online soſtware PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [69].

Chromosomal location and gene duplication
The physical location data of BjTCP genes were retrieved from the Brassica juncea var. tumida genomes. The mapping of these TCP genes was subsequently performed using MapInspect soſtware.
Gene duplication was defined according to the criteria described in previous studies: the aligned region of two sequences covers over 80% of the longer sequence, and the similarity of the aligned region is over 80%. In addition, the KaKs_Calculator soſtware was employed to calculate Ka (nonsynony-mous substitution rate) and Ks (synonymous substitution rate), The divergence time was calculated with the formula T = Ks/2r, the r was taken to be 1.5◊10 -8 synonymous substitutions per site per year for dicotyledonous plants.

The expression profile of TCP genes
The RNA-seq data were we previously reported and can downloaded from NCBI SRA database

RNA extraction and real-time quantitative PCR analysis
The total RNA was extracted from different plant materials using RNA plant plus reagent (Tiangen Biotech Co., Ltd., Beijing, China). The total RNA samples were treated with DNase I (Takara, Qingdao, China) to remove contaminating genomic DNA. First-strand cDNA was synthesized from the total RNA using a Hiscript II 1 st strand cDNA synthesis Kit (Vazyme, Nanjing, China). Real-time qPCR (qRT-PCR) was performed using TB Green™ Premix Ex Taq™ II (Tli RNaseH Plus) (Takara, Qingdao, China).
BjActin was used as the internal reference gene for qRT-PCR, and the gene-specific primers are listed in Supplementary Table S1

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download. Figure S2.tif Table S1.xlsx Figure S3.pdf Figure S1.pdf