Genome-Wide Analysis of Auxin Receptor Family Genes in Brassica juncea var. tumida

Transport inhibitor response 1/auxin signaling f-box proteins (TIR1/AFBs) play important roles in the process of plant growth and development as auxin receptors. To date, no information has been available about the characteristics of the TIR1/AFB gene family in Brassica juncea var. tumida. In this study, 18 TIR1/AFB genes were identified and could be clustered into six groups. The genes are located in 11 of 18 chromosomes in the genome of B. juncea var. tumida, and similar gene structures are found for each of those genes. Several cis-elements related to plant response to phytohormones, biotic stresses, and abiotic stresses are found in the promoter of BjuTIR1/AFB genes. The results of qPCR analysis show that most genes have differential patterns of expression among six tissues, with the expression levels of some of the genes repressed by salt stress treatment. Some of the genes are also responsive to pathogen Plasmodiophora brassicae treatment. This study provides valuable information for further studies as to the role of BjuTIR1/AFB genes in the regulation of plant growth, development, and response to abiotic stress.


Introduction
Auxin is one of most important phytohormones, playing a crucial role in most physiological and biochemical processes of plant growth and development, such as embryo and fruit development, tissue differentiation, root initiation, plant gravitropism, and plant response to biotic and abiotic stresses [1][2][3][4]. The transport inhibitor response 1/auxin signaling f-box protein (TIR1/AFB) family genes perceive the auxin signal as auxin receptors, which then trigger the auxin response signaling pathway [5,6]. It has been reported that TIR1 and the AFBs are located in the nucleus and bind auxin [5,6]; the nuclear localization of GmTIR1 and GmAFB3 was also reported in soybean [7]. After auxin molecules bind to TIR1/AFB proteins, the activated TIR1/AFB proteins react with auxin/indole-3-acetic acid (Aux/IAA) family proteins [8]. The Aux/IAA family genes play important roles in the regulation of auxin response factor genes (ARFs). These ARF family genes are notable for their ability to directly activate or inhibit auxin responsive genes at the transcription level [9][10][11]. This TIR1/AFB-Aux/IAA-ARF cascade comprises a typical auxin signaling pathway.
At conditions of low auxin concentration in the plant, the Aux/IAA proteins could bind to ARFs, which results in the inhibition of the transcriptional activity of ARFs. When auxin molecules accumulate patterns during plant growth and development. This study will be useful as a starting point for more functional investigations of TIR1/AFBs in tumorous stem mustard.

Plant Materials and Growth Conditions
In this study, tumorous stem mustard cultivar Yong An XiaoYe was used for gene expression pattern analysis. The seeds were sowed into 2:1 vermiculite:turfy soil, cultured under constant light at 22 • C with a 16/8 h light/dark regime in culture room. For the salt stress assay, the seeds of Yong An Xiao Ye were sowed in pots in a culture room, and two-week-old seedlings were irrigated by nutrient solution with or without 200 mM NaCl for 3, 6, 12, 24 h, respectively, and the roots were collected for real-time quantitative PCR analysis. For pathogen treatment, two-week-old seedlings were then inoculated with a 5 mL resting spore suspension of Plasmodiophora brassicae (OD 600 = 0.07) for the indicated time points.

The Identifications of Transport Inhibitor Response 1/Auxin Signaling F-Box Protein in Tumorous Stem Mustard and Phylogenetic Analysis
The TIR1, AFB1, AFB2, AFB3, AFB4, and AFB5 sequence of A. thaliana were obtained from The Arabidopsis Information Resource website (https://www.arabidopsis.org/). Using the TIR1/AFB genes as the query sequence to blast relative homologs in the B. juncea genome database (http://brassicadb. org/brad/). The gene structure diagram was drawn using the online software of the GSDS2.0 server (http://gsds.cbi.pku.edu.cn/), the phylogenetic analysis was done using MEGA5 software [26] with the neighbor-joining method. The bootstrap value was 1000 replicates.

RNA Extraction and Real-Time Quantitative PCR Analysis
The total RNA was extracted from different plant materials using Trizol reagent (Tiangen Biotech Co., Ltd., Beijing, China). The total RNA samples were treated with DNase I (Invitrogen, Carlsbad, CA, United States) to remove contaminating genomic DNA. First-strand cDNA was synthesized from the total RNA using a FastQuant RT Kit (Tiangen Biotech (Beijing) Co., Ltd.). Real-time qPCR (qRT-PCR) was performed using SuperReal PreMix Plus (SYBR Green; Tiangen Biotech (Beijing) Co., Ltd.). BjuACTIN3 was used as the internal reference gene for qRT-PCR [27], and the gene-specific primers are listed in Supplementary Table S1.

Statistical Analysis
All data were analyzed using SigmaPlot 10.0 (Systat Software, Inc., Chicago, IL, United States) and SPSS 16.0 software. The averages and standard deviations of all results were calculated, one-way ANOVA followed by the Dunnett test was used.

The Identification of TIR1/AFB Homologs in Brassica juncea var. tumida
Eighteen genes were identified as homologs of TIR1/AFB genes in B. juncea var. tumida; they were named BjuTIR1 and BjuAFBs, and the CDS sequences of those genes can be found in Supplementary File S2. The open reading frame of BjuTIR1/AFBs varied between 1566 bp to 1830 bp, coding 521 to 609 amino acids; the molecular weight was 58.58-68.13 and the isoelectric point (pI) were 5.22-8.19 (Table 1). With the exception of BjuO006283 contig, which could not be anchored in the chromosome of B. juncea var. tumida, the left 17 BjuTIR1/AFBs genes located are in 11 of 20 B. juncea chromosomes. Each of the chromosome A03, A09, and B06 contains three genes, and the other eight genes were located at different chromosomes ( Figure 1). BjuTIR1A, BjuTIR1D, and BjuTIR1F were located at the same chromosome of B06 ( Figure 1).  (Table 1). With the exception of BjuO006283 contig, which could not be anchored in the chromosome of B. juncea var. tumida, the left 17 BjuTIR1/AFBs genes located are in 11 of 20 B. juncea chromosomes. Each of the chromosome A03, A09, and B06 contains three genes, and the other eight genes were located at different chromosomes ( Figure 1). BjuTIR1A, BjuTIR1D, and BjuTIR1F were located at the same chromosome of B06 ( Figure 1).

The Phylogenetic Analysis and Gene Structures of BjuTIR1/AFBs
The TIR1/AFBs protein sequences of A. thaliana and B. juncea var. tumida were used for phylogenetic analysis, and six clades were obtained in the neighbor-joining phylogenetic tree ( Figure 2). According to the phylogenetic analysis, six TIR1 homologs were identified, and named

The Phylogenetic Analysis and Gene Structures of BjuTIR1/AFBs
The TIR1/AFBs protein sequences of A. thaliana and B. juncea var. tumida were used for phylogenetic analysis, and six clades were obtained in the neighbor-joining phylogenetic tree ( Figure 2). According to the phylogenetic analysis, six TIR1 homologs were identified, and named BjuTIR1A to BjuTIR1F; four AFB1 homologs were identified, and named BjuAFB1A to BjuAFB1D; one AFB2 homolog was identified, and named BjuAFB2; four AFB3 homologs were identified, and named BjuAFB3A to BjuAFB3D; one AFB4 homolog was identified, and named BjuAFB4; and two AFB5 homologs were identified, and named BjuAFB5A to BjAFB5B ( Figure 2, Table 1). We also analyzed the phylogenic relationships of TIR1/AFB genes between B. juncea var. tumida and Brassica rapa, and the results showed that there was high sequence similarity between those genes ( Figure S3). Interestingly, many TIR1/AFB genes are located at same chromosomes in each genome, respectively. For example, BjuTIR1C had highest sequence identity with Bra003518, and they were both located at chromosome A07 in their genome, respectively ( Figure S3). According to gene structure analysis, most of the BjuTIR1/AFBs genes had similar gene structure to Arabidopsis TIR1/AFBs, which includes three exons and two introns. The exceptions were BjuAFB1D and BjuAFB3D, both of which contain four exons and three introns, as well as BjuAFB1B with two exons and one intron ( Figure 2). The protein sequence of each gene was used for the alignment, and the phylogenic neighbor-joining tree was constructed using MEGA5 phylogenetic analysis software.

The Alignment of BjuTIR1/AFB Proteins and Secondary Domain Prediction
The BjuTIR1/AFBs protein sequences were aligned by Clustalx [28], and the results showed that the sequences of those proteins were conserved ( Figure 3). The sequence identification was more than 83.67% among the BjuTIR1 proteins. Similar results were found for BjuAFBs proteins: there was more than 90.77%, 89.21%, and 98.36% sequence identification among BjuAFB1, BjuAFB3, and BjuAFB5 family proteins, respectively. In particular, the peptide sequences were highly conserved in the F-box domain region (Figure 3).
The secondary domains of BjuTIR1 and BjuAFBs were predicted, the results showed that all the BjuTIR1, BjuAFB1, BjuAFB2, and BjuAFB3 proteins contained an F-box domain in the N-terminus, indicating that those genes might function as E3 ubiquitin ligases. However, no F-box domains were found in the BjuAFB4 and BjuAFB5 proteins, suggesting that their genes function in a different manner. There were several LRR domains in all the BujTIR1 and BjuAFB proteins; those LRR domains were related to the interaction between the BjuTIR1/AFBs with other proteins (Figure 4).

The Cis-Element Prediction of BjuTIR1/AFB Promoters
The 2 kb DNA region upstream BjuTIR1/AFB sequences were chosen as promoters of those genes, and cis-elements were predicted by the PLACE software. Some auxin-responsive elements were found in most of BjuTIR1/AFBs' promoters, such as ARFAT (TGTCTC) [29] and GMSAUR Figure 2. The phylogenetic analysis and gene structure of TIR1/AFBs in Arabidopsis thaliana and Brassica juncea. The protein sequence of each gene was used for the alignment, and the phylogenic neighbor-joining tree was constructed using MEGA5 phylogenetic analysis software.

The Alignment of BjuTIR1/AFB Proteins and Secondary Domain Prediction
The BjuTIR1/AFBs protein sequences were aligned by Clustalx [28], and the results showed that the sequences of those proteins were conserved (Figure 3). The sequence identification was more than 83.67% among the BjuTIR1 proteins. Similar results were found for BjuAFBs proteins: there was more than 90.77%, 89.21%, and 98.36% sequence identification among BjuAFB1, BjuAFB3, and BjuAFB5 family proteins, respectively. In particular, the peptide sequences were highly conserved in the F-box domain region (Figure 3). might specifically play roles in B. juncea var. tumida.  The secondary domains of BjuTIR1 and BjuAFBs were predicted, the results showed that all the BjuTIR1, BjuAFB1, BjuAFB2, and BjuAFB3 proteins contained an F-box domain in the N-terminus, indicating that those genes might function as E3 ubiquitin ligases. However, no F-box domains were found in the BjuAFB4 and BjuAFB5 proteins, suggesting that their genes function in a different manner. There were several LRR domains in all the BujTIR1 and BjuAFB proteins; those LRR domains were related to the interaction between the BjuTIR1/AFBs with other proteins (Figure 4).

The Transcriptional Expression Pattern of BjuTIR1/AFBs in Different Tissue
The seedlings of Yong An Xiao Ye were cultivated in a field for six months, and the root, stem, swollen stem, leaf, flower, and pod were harvested for RNA extraction. The real-time quantitative PCR were performed to detect the expression pattern of BjTIR1/AFB family genes; the raw data of the qPCR results can be found in the Supplementary Materials (File S4). The results showed that BjuTIR1A, BjuTIR1D, and BjuAFB1B were highly expressed in most tissues, and moderate expression levels were found for BjuTIR1C, BjuAFB1B, BjuAFB3A, and BjuAFB3C ( Figure 6). BjuAFB1D, BjuAFB2, BjuAFB3D, and BjuAFB5A demonstrated lower expression levels in all tissues, suggesting that those four genes had limited function during plant growth and development ( Figure  6). As shown in Figure 6, BjuTIR1A was expressed at much higher levels in the flower than in any other tissues, indicating this gene might be involved in flower development. Many genes, such as BjuTIR1B, BjuTIR1C, BjuTIR1D, BjuTIR1F, BjuAFB1B and AFB3C, showed their highest expression levels in pods, suggesting that those genes might be responsible for pod formation. All the BjuTIR1 genes were highly expressed in the pod except BjuTIR1A, which indicates that BjuTIR1 family genes mainly function in pod formation and development ( Figure 6). Besides that, the high expression of BjuTIR1A, BjuTIR1D, BjuAFB1B, and AFB3A in the stem as well as the observed swollen stem suggests that those genes might play important roles in the function of stem development ( Figure 6).

The Transcriptional Expression Pattern of BjuTIR1/AFBs in Different Tissue
The seedlings of Yong An Xiao Ye were cultivated in a field for six months, and the root, stem, swollen stem, leaf, flower, and pod were harvested for RNA extraction. The real-time quantitative PCR were performed to detect the expression pattern of BjTIR1/AFB family genes; the raw data of the qPCR results can be found in the Supplementary Materials (File S4). The results showed that BjuTIR1A, BjuTIR1D, and BjuAFB1B were highly expressed in most tissues, and moderate expression levels were found for BjuTIR1C, BjuAFB1B, BjuAFB3A, and BjuAFB3C ( Figure 6). BjuAFB1D, BjuAFB2, BjuAFB3D, and BjuAFB5A demonstrated lower expression levels in all tissues, suggesting that those four genes had limited function during plant growth and development ( Figure 6). As shown in Figure 6, BjuTIR1A was expressed at much higher levels in the flower than in any other tissues, indicating this gene might be involved in flower development. Many genes, such as BjuTIR1B, BjuTIR1C, BjuTIR1D, BjuTIR1F, BjuAFB1B and AFB3C, showed their highest expression levels in pods, suggesting that those genes might be responsible for pod formation. All the BjuTIR1 genes were highly expressed in the pod except BjuTIR1A, which indicates that BjuTIR1 family genes mainly function in pod formation and development ( Figure 6). Besides that, the high expression of BjuTIR1A, BjuTIR1D, BjuAFB1B, and AFB3A in the stem as well as the observed swollen stem suggests that those genes might play important roles in the function of stem development ( Figure 6).

The Transcriptional Expression Pattern of BjuTIR1/AFBs Under Salt Stress Treatment
To investigate candidate BjuTIR1/AFB genes' interaction with plant response to salt stress, the gene expression levels were tested. Two-week-old seedlings of Yong An Xiao Ye were treated by 200 mM NaCl for 3, 6, 12, and 24 h, respectively, and the roots were collected for RT-qPCR analysis. The

The Transcriptional Expression Pattern of BjuTIR1/AFBs Under Salt Stress Treatment
To investigate candidate BjuTIR1/AFB genes' interaction with plant response to salt stress, the gene expression levels were tested. Two-week-old seedlings of Yong An Xiao Ye were treated by 200 mM NaCl for 3, 6, 12, and 24 h, respectively, and the roots were collected for RT-qPCR analysis. The results showed that BjuTIR1A was significantly induced by salt treatment, especially after 3 and 6 h treatment, as the transcriptional expression levels of BjuTIR1B and BjuAFB1C increased at 3 and 6 h, respectively (Figure 7). In contrast, the expression of most BjuTIR1/AFB family genes were inhibited by salt treatment, including BjuTIR1C, BjuTIR1D, BjuTIR1E, BjuAFB1A, BjuAFB1B, BjuAFB3A, BjuAFB3B, BjuAFB3C, and BjuAFB4 (Figure 7). Those results suggest that most of the BjuTIR1/AFB genes were repressed by salt treatment, and might be involved in plant responses to salt stress. Three independent biological repeats were performed, and all data points were the means of three biological replicates ± standard error (SE). Significant differences: *, p < 0.05; **, p < 0.01.

The Transcriptional Expression Pattern of BjuTIR1/AFBs under Plasmodiophora brassicae Treatment
Plasmodiophora brassicae is a main pathogen of B. juncea var. tumida, which could lead to club root of cruciferous plants. To test which gene may be involved in the plant response to pathogen attack, the expression patterns of BjuTIR1/AFB family genes were detected under P. brassicae treatment. Two-week-old seedlings of Yong An Xiao Ye were treated by P. brassicae (OD600 = 0.07) for 6, 12, 24, Figure 7. The expression patterns of BjuTIR1/AFB genes under salt stress. The expression pattern of BjuTIR1/AFB homologous genes were analyzed by qPCR. BjuActin3 was used as the internal control. Three independent biological repeats were performed, and all data points were the means of three biological replicates ± standard error (SE). Significant differences: *, p < 0.05; **, p < 0.01.

The Transcriptional Expression Pattern of BjuTIR1/AFBs under Plasmodiophora brassicae Treatment
Plasmodiophora brassicae is a main pathogen of B. juncea var. tumida, which could lead to club root of cruciferous plants. To test which gene may be involved in the plant response to pathogen attack, the expression patterns of BjuTIR1/AFB family genes were detected under P. brassicae treatment. Two-week-old seedlings of Yong An Xiao Ye were treated by P. brassicae (OD 600 = 0.07) for 6, 12, 24, 48, and 72 h, respectively, and the roots were collected for (RT-qPCR) analysis. The results showed that BjuTIR1A and BjuAFB3B were severely induced by P. brassicae treatment; BjuTIR1D, BjuTIR1E, BjuAFB1A, BjuAFB1B, BjuAFB2, BjuAFB3D, BjuAFB4, and BjuAFB5B were induced moderately by P. brassicae treatment (Figure 8). Other genes showed similar expression levels during P. brassicae treatment, and nearly no gene was inhibited by this biotic stress treatment. Experiment results show the BjuTIR1/AFB family genes induced by P. brassicae could be involved in plant response to this pathogen-especially BjuTIR1A (Figure 8). The expression pattern of BjuTIR1/AFB homologous genes were analyzed by qPCR. BjuActin3 was used as the internal control. Three independent biological repeats were performed, and all data points were the means of three biological replicates ± SE. Significant differences: *, p < 0.05; **, p < 0.01.

Discussion
B. juncea var. tumida is an allotetraploid species formed by hybridization between the diploid ancestors of B. rapa and Brassica nigra, followed by genome duplication [41]. In A. thaliana, six auxin The expression pattern of BjuTIR1/AFB homologous genes were analyzed by qPCR. BjuActin3 was used as the internal control. Three independent biological repeats were performed, and all data points were the means of three biological replicates ± SE. Significant differences: *, p < 0.05; **, p < 0.01.

Discussion
B. juncea var. tumida is an allotetraploid species formed by hybridization between the diploid ancestors of B. rapa and Brassica nigra, followed by genome duplication [41]. In A. thaliana, six auxin receptor genes were identified [5,6,[16][17][18]. According to our results, 18 putative auxin receptors were found in the genome of B. juncea var. tumida. Except for AtAFB2 and AtAFB4, each Arabidopsis auxin receptor was homologous to 2-6 genes in the B. juncea var. tumida genome ( Table 1). The multiple homologs of auxin receptors in B. juncea var. tumida may be from the result of genome duplication. Among the 18 auxin receptors, nine genes were located in the A sub-genome and eight in the B sub-genome (BjuAFB5A could not be mapped to a genome). The comparable auxin receptor gene numbers in the A or B sub-genome indicates that the genome of B. juncea var. tumida experienced co-linearity in the gene duplication process. Most of the auxin receptor genes contained three exons and two introns, the exceptions being BjuAFB1D and BjuAFB1D, both of which contained four exons and three introns (Figure 2). The gene structure showed that more exons attributed to a partial exon coding sequence converted to a non-coding sequence. This exon-intron splicing arrangement is present in other species, such as rice and maize [42,43].
Abnormal phenotypes of enlarged flag leaf inclination, more tillers, early flowering and a lower tolerance to salt were found due to reduction of OsTIR1 and OsAFB2 in rice [44,45]. However, the expression levels of BjuAFB2 was low in all tissues of B. juncea var. tumida (Figure 8), suggesting that BjuAFB2 might not play an important role as a main auxin receptor. In contrast, the high expression levels of BjuTIR1A and BjuTIR1D indicate that these two genes might be the main auxin receptors in B. juncea var. tumida. According to the results of the gene expression pattern, tissue-specific gene expressions were identified, such as BjuTIR1A demonstrating influence on the regulation of flower development, and BjuTIR1F may specifically influence pod development regulation.
It has been reported that auxin signaling plays an important role in plant resistance to abiotic stress, especially at high salinity [3]. In this study, the expression levels of most of auxin receptor genes was inhibited under salt treatment (Figure 7). In tobacco, overexpression of AtmiR393a could repress the expression levels of auxin receptors like TIR1, leading to the inhibition of plant growth and enhanced resistance to salt stress [46]. The reduced expressions of BjuTIR1/AFB family genes under salt treatment were consistent with the previous study. The exception was BjuTIR1A-the expression of this gene was significantly induced by salt treatment, which might result from feedback of the signaling transduction.
The auxin signaling pathway mediated by TIR1/AFBs is usually involved in plant resistance or tolerance to pathogen attacks, such the anthracnose disease in cassava [47], Zea mays defense against Rhizoctonia solani [48], or Arabidopsis resistance to the bacterium Pseudomonas syringae [25]. In Arabidopsis, AtTIR1 and AtAFB1 are transcriptionally upregulated in clubroots, and the mutants of tir1, afb1-3, and afb1-3 afb2-3 all resulted in more susceptible reactions to the root pathogen P. brassicae [49]. In this study, similar results were found. The transcriptional level of BjuTIR1A was severely induced by P. brassicae treatment, especially at 6 and 12 h after inoculation. Besides that, all the BjuTIR1s were induced by this pathogen treatment, except for BjuTIR1B (Figure 8). For BjuAFB1 genes, the expression levels of AFB1A, AFB1B, and AFB1C also increased at 48 h after pathogen inoculation (Figure 8). The induction of those genes suggested that BjuTIR1s and BjuAFB1s might play similar roles to AtTIR1/AFB1s in the processes of plant defense against P. brassicae invasion.

Conclusions
In this study, a total of 18 BjuTIR1/AFB genes were identified, and they have similar gene structures and protein domains to the auxin receptors of Arabidopsis. Several cis-elements related to plant response to phytohormones, biotic stresses, and abiotic stresses were found in the promoter of BjuTIR1/AFB genes, indicating those genes may play a role in the regulation of plant development and defense against biotic and abiotic stresses, which could benefit from a more targeted follow-up study. Gene expression analysis showed that some BjuTIR1/AFB family genes exhibit a special expression pattern, and most genes are responsive to salt stress treatment. Together, our data provides a useful foundation for future research regarding the function of auxin receptors in B. juncea var. tumida.