Auxin response factor (ARF) is a member of the plant-specific B3 DNA binding superfamily. Here, we report the results of a comprehensive analysis of ARF genes in allotetraploid Brassica napus (2n = 38, AACC). Sixty-seven ARF genes were identified in B. napus (BnARFs) and divided into four subfamilies (I–IV). Sixty-one BnARFs were distributed on all chromosomes except C02; the remaining were on Ann and Cnn. The full length of the BnARF proteins was highly conserved especially within each subfamily with all members sharing the N-terminal DNA binding domain (DBD) and the middle region (MR), and most contained the C-terminal dimerization domain (PBI). Twenty-one members had a glutamine-rich MR that may be an activator and the remaining were repressors. Accordingly, the intron patterns are highly conserved in each subfamily or clade, especially in DBD and PBI domains. Several members in subfamily III are potential targets for miR167. Many putative cis-elements involved in phytohormones, light signaling responses, and biotic and abiotic stress were identified in BnARF promoters, implying their possible roles. Most ARF proteins are likely to interact with auxin/indole-3-acetic acid (Aux/IAA) -related proteins, and members from different subfamilies generally shared many common interaction proteins. Whole genome-wide duplication (WGD) by hybridization between Brassica rapa and Brassica oleracea and segmental duplication led to gene expansion. Gene loss following WGD is biased with the An-subgenome retaining more ancestral genes than the Cn-subgenome. BnARFs have wide expression profiles across vegetative and reproductive organs during different developmental stages. No obvious expression bias was observed between An- and Cn-subgenomes. Most synteny-pair genes had similar expression patterns, indicating their functional redundancy. BnARFs were sensitive to exogenous IAA and 6-BA treatments especially subfamily III. The present study provides insights into the distribution, phylogeny, and evolution of ARF gene family.
Citation: Wen J, Guo P, Ke Y, Liu M, Li P, Wu Y, et al. (2019) The auxin response factor gene family in allopolyploid Brassica napus. PLoS ONE 14(4): e0214885. https://doi.org/10.1371/journal.pone.0214885
Editor: Maoteng Li, Huazhong University of Science and Technology, CHINA
Received: January 6, 2019; Accepted: March 21, 2019; Published: April 8, 2019
Copyright: © 2019 Wen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This work was supported by the National Natural Science Foundation of China (31671727 and 31471528), the “111” Project of China (B12006), and the National Basic Research Program of China (973 Program; 2015CB150201).
Competing interests: The authors have declared that no competing interests exist.
Auxin response factors (ARF) are a group of transcription factors generally consisting of an N-terminal DNA-binding domain (DBD), a middle region (MR), and a C-terminal dimerization domain (PBI) [1,2]. The DBD was further classified into three distinct structural domains based on crystal structure analysis: a B3 domain showing similarity to the DNA-contacting domain of bacterial endonucleases, two dimerization domains (DD1 and DD2) allowing ARF dimerization, and a Tudor-like ancillary domain (AD) of unknown function which might be involved in an interaction with the DD . The DBD of ARFs fulfills a critical role for a transcription factor: recognition of a DNA motif, called the auxin-responsive element (AuxRE). The MR enriched in glutamine (Q), serine (S), and leucine (L) residues is identified as the activation domain, while those enriched in S, proline (P), L, and glycine (G) residues are identified as repression domains . The PBI consists of domain III and domain IV. The N-terminal III region consists of an antiparallel β-sheet (β1–β2) and α1. The C-terminal IV region contains a second antiparallel β-sheet (β3–β5) and two α-helices (α2 and α3). The PBI domain is the most optional one and is similar to motifs III and IV of the Aux/IAA protein family , which is not present in certain ARF proteins such as AtARF3 and AtARF17 [5,6]. The auxin response is not fully dependent on PBI . Transient expression assays and domain swaps have demonstrated that each of the three domains can act in isolation .
After the first ARF gene (AtARF1) was cloned from Arabidopsis , a large gene family containing 23 ARFs has been isolated, also from Arabidopsis [9,10]. Many homologous genes that play important roles in diverse biological processes in plants, such as controlling plant growth, and developmental and physiological processes in the whole life cycle of plants have been functionally characterized [11–13]. For instance, AtARF17 plays an essential role in anther development and pollen formation in Arabidopsis ; AtARF2-4 and AtARF5 are essential for female and male gametophyte development in Arabidopsis ; SlARF3 plays a key role in the formation of epidermal cells and trichomes in tomato . Unlike most plant transcription factors, ARFs can act as both activators and repressors. ARF activators bind to TGTCTC auxin response elements (AuxRE) in promoters of auxin response genes to mediate auxin-dependent transcriptional regulation . ARF repressors may squelch the transcriptional activity of activator ARFs either by heterodimerizing with them or through competition for DNA binding sites . The PBI is not required for DNA-binding or activation but facilitates the formation of ARF-ARF, ARF-Aux/IAA, and Aux/IAA-Aux/IAA homo- and hetero-oligomers [18–20]. Owing to the increasing amount of available plant genome data, the ARF gene family were identified in some species at genome-wide level. For example, 23 genes were identified in Arabidopsis (AtARFs), 22 genes in tomato, and 28 genes in chickpea [10,21,22]. These studies provide the fundamental information needed for the functional analysis of ARF genes. However, only one gene, named ARF18, has been functionally analyzed in Brassica napus which affects seed weight and silique length . It is clear that the genome-wide identification and functional analysis of this gene family in B. napus will provide fundamental information for further functional assays of ARF genes in this species.
In the present study, we carried out systematic identification and phylogenetic analysis of the ARF genes in the B. napus genome. Gene structure and protein motif analysis provided an additional criterion for our classification based on the phylogenetic tree. Cis-element analysis of the promoters of candidates indicated that they were involved in light signaling response. The whole genome-wide duplication (WGD) and segmental duplication mainly contributed to the rapid expansion of this gene family in B. napus, whereas no tandem duplication was observed. Expression profile analysis showed that B. napus ARF genes have wide expression profiles across the vegetative and reproductive organs during different developmental stages, and that some members were extra-hormone mediated. This study provides valuable information regarding the evolution, radiation, and functional diversification of the ARF gene family in B. napus, which will facilitate further analysis of their functional traits.
Material and methods
Identification of ARF family genes and chromosome location in B. napus
The sequences of 23 Arabidopsis ARF genes were downloaded from the TAIR Arabidopsis genome (http://www.arabidopsis.org/) . We performed a BLASTP search of the GNOSCOPE genome database (http://www.genoscope.cns.fr/brassicanapus/) using the full protein sequences of Arabidopsis ARF genes as queries. To verify the reliability of our results, the protein functional and structural domains were predicted with PROSITE profiling (http://www.expasy.org/tools/scanprosite/)  to confirm that each protein had the B3 domain. We acquired B. oleracea and B. rapa ARF protein sequences from Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html) and BRAD (http://brassicadb.org/brad/index.php) by the same method.
The biochemical properties of the candidates were predicted by ExPASy . Subcellular location was investigated using Cell-PLoc . The gene locations of candidates were extracted from the GNOSCOPE genome database. MapChart v2.3 software was used to view the chromosome locations of candidates . The physical and chemical characteristics of the candidate BnARFs proteins were predicted by Protparam tool (http://web.expasy.org/) , received the theoretical pI/Mw for the BnARFs sequence.
Phylogenetic and synteny analysis of B. napus ARF proteins
To examine the evolutionary history of the B. napus ARF gene family, we constructed a neighbor-joining (NJ) tree based on the multiple alignment of the ARF domains using MEGA 5.1 . To determine the statistical reliability, we applied bootstrap analysis with 1000 replicates, and the main parameters were p-distance and pairwise deletion. The tree file was viewed and edited using FigTree v1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/). Using the same method, we constructed a NJ tree of B. napus, B. rapa, B. oleracea, and Arabidopsis ARF proteins. Synteny relationships of ARF genes from Arabidopsis, B. napus, B. oleracea, and B. rapa were acquired from CoGe (https://genomevolution.org/coge/).
Protein sequence analysis and construction of the protein interaction network
To reveal the sequence features of BnARF proteins, we performed a multiple alignment analysis of the full protein sequences with MAFFT version 7 using the default parameters (https://mafft.cbrc.jp/alignment/server/). To obtain optimized alignments, the deduced amino acid sequences were adjusted manually in MEGA 5.0 with default parameters. MEME version 4.11.1  was used to identify potential protein motifs using the following parameters: distribution of motifs, maximum number of motifs, 20; minimum width of motif, six; maximum width of motif, 50. Only motifs with an e-value ≤ 1e-10 were retained for further analysis. The protein–protein interactions (PPI) network of AtARFs was acquired based on publicly available data in STRING . Then we predicted the PPI network of BnARFs based on orthology analysis with AtARFs and visualized with Cytoscape version 3.4.0 .
Analyses of gene structure, miRNAs, and cis-elements
The intron insertion pattern (including the distribution, position, and phases) and DBD and PBI encoding regions in the B. napus and Arabidopsis ARF genes were analyzed and viewed by Gene Structure Display Server software  (http://gsds.cbi.pku.edu.cn/). Plant small RNA targeted gene prediction was performed by psRNATarget (http://plantgrn.noble.org/psRNATarget/analysis/) . To identify the putative cis-elements, the 1.5-kb upstream DNA sequences of all B. napus ARF genes were analyzed by the online PlantCARE web tool (http://bioinformatics.psb. ugent.be/webtools/plantcare/html/).
Expression analysis of B. napus ARF genes at different developmental stages and under hormone treatments
The temporal and spatial expression patterns of candidate BnARFs were analyzed using the RNA-seq data (BioProject ID: PRJNA358784) from 50 different roots, stems, leaves, flowers, seeds, and siliques tissues of the B. napus cultivar ‘Zhongshuang 11’ (ZS11) at germination, seedling, budding, initial flowering, and full-bloom stages. The expression information of candidates in seedling roots of ZS11 under five extra hormone inductions (e.g, auxin, gibberellin, cytokinin, abscisic acid, and ethylene) were collected for analysis. The data were analyzed by Cluster 3.0  and the heatmap was drawn using Java Treeview software .
For qRT-PCR analysis, seeds of ZS11 were obtained from the College of Agriculture and Biotechnology, Southwest University, Chongqing, China and germinated on Petri dishes. At the five-leaf stage, the seedlings were treated with Hoagland's liquid medium, which contained phytohormones (75 μM 6-benzyladenine (6-BA) and 10 μM Indole-3-acetic acid (IAA)) and grown in an artificial climate chamber at 25°C under a 14-h/10-h (day/night) photoperiod. Root tissues were harvested 0, 1, 3, 6, 12, and 24 h after treatment, and then were immediately frozen in liquid nitrogen and stored at −80°C for RNA isolation.
The extraction of total RNA from root samples and subsequent cDNA synthesis were performed as described previously . Total RNA was extracted using Eastep total RNA Extraction kit, according to the manufacturer’s instructions, and treated with DNase I (Promega, USA). First-strand cDNA synthesis was performed using an oligo (dT) primer (S1 Table) and 2 μg of total RNA in a 20-μl reaction volume with the M-MuLV RT kit (Takara Biotechnology, Japan), according to the manufacturer’s instructions. The real-time PCR thermocycling parameters were as follows: initial denaturation for 3 min at 95°C, followed by 45 cycles of denaturation at 95°C for 15 s and then annealing at 58°C for 20 s. Fluorescence was measured after the extension step using the CFX Connect Real-Time System (Bio-Rad). After the thermocycling reaction, the melting step was performed from 65°C to 95°C, with increments of 0.5°C each 0.05 s. Each PCR was replicated three times for verification.
Sequence identification and chromosomal locations of B. napus ARF genes
A total of 67 BnARFs were identified in the B. napus genome, consisting of 62 typical ARF genes with a complete open reading frame (ORF) and five putative genes with short fragment deletions in the BDs (BnaC03g57150D, BnaAnng13910D, BnaC09g52440D, BnaC09g23080D, and BnaA06g14090D). This is the largest ARF gene family reported to date (Table 1). Finally, the 67 candidate BnARFs were classified and relevant nomenclature was applied according to their positions on B. napus chromosomes.
In summary, the full-length coding sequences of BnARFs ranged from 1389 bp (BnARF64) to 7574 bp (BnARF14) with the deduced proteins varying from 182 (BnARF64) to 1058 (BnARF44) amino acids (Table 1). The predicted molecular weights of the 67 deduced BnARF proteins ranged from 20.76 kDa (BnARF64) to 124.92 kDa (BnARF37) and the isoelectric points (pIs) ranged from 5.26 (BnARF47 and BnARF49) to 9.67 (BnARF11) (Table 1). As determined previously in other plant species [39,40], the wide range of pIs suggests that B. napus ARF proteins can function in different subcellular environments. Subcellular localization results showed that 63 BnARFs were localized in the nucleus, three in the mitochondrion nucleus, and one in the chloroplast nucleus. To further explore the evolutionary relationship in the B. napus ARF gene family after the formation of allopolyploids, 29 and 33 ARF genes were also obtained from the ancestors, B. oleracea and B. rapa genomes, respectively, using the same method. Detailed information regarding the candidates is listed in S2 Table.
Chromosomal location analyses revealed that 61 of the 67 BnARFs were mapped on 18 of the 19 chromosomes, i.e. three genes on A01, all except C02 (Fig 1). The remaining genes were in Ann and Cnn. The distribution of BnARFs in An- and Cn-subgenomes were nearly even, with 32 genes from the An-subgenome and 35 from the Cn-subgenome. However, the candidates on each chromosome were uneven. For example, C09 had the largest number (eight genes), while A02 had only one.
In total, 67 BnARF genes were mapped to 18 chromosomes. The gray background represents the genes originating from B. rapa; the charcoal gray background represents the gene originating from B. oleracea; the black border represents the gene originating from segmental duplication, the black line represents the origin of the duplication; ★: homologous exchange (HE) event; ○: segmental exchange (SE) event.
Phylogenetic analysis and duplication of the B. napus ARF proteins
To explore the phylogenetic relationships among candidate ARF proteins, an unrooted NJ tree of the 152 ARF family members from Arabidopsis (23 genes), B. oleracea (29 genes), B. rapa (33 genes), and B. napus (67 genes) was constructed (Fig 2).
The NJ tree shows that the 23 Arabidopsis, 29 Brassica oleracea, 33 Brassica rapa, and 67 B. napus ARF proteins were clustered into four subfamilies (I–IV). Subfamilies I, II, III, and IV marked with red, yellow, green, and blue backgrounds, respectively. Genes in the four species labeled in different colors.
Similar to the results of previous studies, such as on Arabidopsis and rice [10,41], Salvia miltiorrhiza , and Eucalyptus grandis , all 152 ARF proteins were grouped into subfamilies I–IV containing 69, 13, 48, and 22 members, respectively. In the phylogenetic tree, members of each subfamily or clade were commonly found in the four species tested herein as sister-pairs. Except for the Ib clade in subfamily I, in which the ARF genes from Arabidopsis and the three Brassica species were clustered into two clearly separated subclades: Ib-1 and Ib-2 (Fig 2). Subclade Ib-1 was found only in Arabidopsis containing a fake gene (AtARF23) and seven tandem duplicated genes (AtARF12–AtARF15 and AtARF20–AtARF22 ); whereas, subclade Ib-2 contained B. napus, B. rapa, and B. oleracea genes. The Ib homologs were also not found in other plants, such as rice, maize, tomato, grape, or Eucalyptus [41,44–47], implying that this clade was formed of Brassicaceae-specific functions. Subfamily III had five AtARFs, 11 B. rapa ARF genes (BrARFs), 11 B. oleracea ARF genes (BoARFs), and 21 BnARFs. There were twice the number of BrARFs and BoARFs than AtARFs in this subfamily, whereas the number of BnARFs was likely to be the sum of BrARF and BoARF homologs. Similarly, subfamily IV had three AtARFs, five BrARFs, five BoARFs, and nine BnARFs, and there were around three times the number of BnARFs than AtARFs. However, this situation was different in the last two subfamilies. Subfamily I had 13 AtARFs, 14 BrARFs, 10 BoARFs, and 32 BnARFs, which indicates that the BoARFs and BrARFs in this subfamily tended to be lost compared to subfamilies III and IV. Similarly, subfamily II had two AtARFs, three BrARFs, three BoARFs, and five BnARFs, suggesting that the expansion of this subfamily in Brassicaceae was not as extensive as it was in the other subfamilies.
Based on syntenic analyses, a total of 59 BnARFs have syntenic relationships with B. oleracea or B. rapa homologs, resulting in 98 orthologous pairs among 32 BrARFs and 51 BnARFs, 63 orthologous-pairs among 22 BoARFs and 41 BnARFs, and 65 synteny-pairs within the B. napus genome (S3 Table). Among the 59 genes, 10 were derived from segmental duplication, the other 49 were derived from heterologous doubling; no tandem genes were observed (S4 Table). Moreover, 32 of the 49 heterologous doubling genes were derived from B. rapa, including 23 from the An-subgenome (13 from orthology events, two from segmental exchange (SE) events, eight from homologous exchange (HE) events), and nine genes were transferred to the Cn-subgenome by SE or HE events. Seventeen genes were derived from B. oleracea including 14 from the Cn-subgenome and three were transferred to the An-subgenome by HE events. These results indicated that gene loss following WGD is biased, with genes from B. rapa being retained and those from B. oleracea being readily lost. This confirms that the An-subgenome replaced more of the Cn-subgenome after WGD and featured more dominantly in each chromosome . Given that B. napus evolved ~7500 years ago by hybridization (allopolyploidy) between B. rapa and B. oleracea, it was evident that WGD and segmental duplication contributed to the rapid expansion of ARF genes in B. napus, which may be independent of tandem duplication.
Sequence features of B. napus ARF proteins
To explore the sequence features of candidate BnARFs, we further analyzed protein sequences of the N-terminal DBD, MR, and PBI. The DBDs of BnARFs generally consisted of three domains: DD (DD1 and DD2), B3, and AD . The sequence features were quite different across these three domains. B3 and AD had conserved amino acid content and length while the DD was relative divergent. It was reported that some amino acid residues in the B3 domain play a vital role in the binding of ARF proteins to target DNA (AtARF5: H170, P218, R215, S165, S174, T227, and S230) .
In Arabidopsis, the mutant H170 residues of AtARF5 lead to its DNA binding feature being significantly reduced. The mutants of P218 or R215 residues lead to its sequence-specific binding being lost; whereby S165, S174, T227, and S230 residues are involved in interactions with the DNA backbone, and the mutants of these four residues will reduce the binding ability of AuxRE . It was reported that three residues (G279, A282 and A287) in the DD of AtARF5 control the dimerization of the DD because substitutions at these sites resulted in functional differences . In the present study, we found that the corresponding amino acids in these two domains were absolutely conserved in subfamily III and were highly conserved in subclade Ia (except the sequence inaccuracy of BnARF11 and BnARF14), only the AtARF2 and its homologs have one site substitution (from T227 to S227) (Fig 3). However, there are some conserved substitutions at these sites in other subfamilies as well, e.g, the H170 and G279 residues in the B3 and DD of subfamily IV were conserved in terms of being substituted into glycine (G) and glutamic acid (E), respectively. In subfamily II, the G279 and A282 residues were substituted into S279/N279 and S282, respectively. Moreover, there exists some non-conserved substitutions in subfamily IV (A282 and A287) and subclade Ib (H170, S174, T227, and S230) as well. Together, these results imply a possible role for the corresponding residues in functional diversification during evolution.
Triangle indicates the positions of key residues. The residues in the box represent substitution sites. The subfamily information is marked on the left.
In the MR, the N-terminal is relatively conserved, but the C-terminal is not. According to the distribution and number of Q at its C-terminal, it could be divided into two groups. The first group which is rich in Q residues was suggested to contain activators, whereas those with fewer Q residues were suggested to be repressors. In Arabidopsis, AtARF5-8 and 19 function as transcriptional activators [1,2,5,49–51], the rest function as transcriptional repressors [2,5]. In B. napus, 21 BnARFs of subfamily III which were rich in Q residues (about twice that of others) were suggested to be activators, and the remaining 46 BnARFs in the other three subfamilies were suggested to be repressors (S1 Fig). In addition, both transcriptional activators and repressors of BnARFs have large amounts of S as well. Moreover, we found that B. napus transcriptional repressors have three- and two-times the amount of E and lysine (K) compared to transcriptional activators. This may also be important for gene functions.
The PBI domain mediates interactions with Aux/IAA proteins . We found that 10 BnARFs (8, 10, 65, 47, 49, 19, 11, 14, 64, and 50) lack the PBI domain (Fig 4). Among which, BnARF8, 10, and 65 were the homologs of AtARF3, BnARF47, 49 and 19 were the homologs of AtARF17. As both AtARF3 and AtARF17 lack the PBI domain, it is suggested that homologs of these genes have conserved sequence features during evolution. Arabidopsis homologs and their homologs in B. rapa (BrARF13) have a conserved fragment deletion in the PBI domain, implying that the deletions in BnARF11 and BnARF14 originated from the same ancestor. While the fragment deletion of the PBI domain in BnARF64 and BnARF50 may be due to inaccurate sequencing or gene mutation, as their ancestor proteins were complete.
A: Gene structures of BnARFs; exon indicated by yellow boxes, B3 domain by fuchsia boxes, PBI domain by green (domain III) and red (domain IV) boxes, and the spaces between the colored boxes correspond to introns. B: The motifs identified in BnARF proteins by MEME analysis.
The MEME online software was further applied to identify motifs in the 67 BnARFs and 23 AtARFs. Altogether, 20 motifs were isolated (Fig 4). Among them, motifs 7, 20, 4, 2, 16, 1, 9, 10, 5, 15, 6, 11, 12, and 13 consisted of the DBD; motif 7, 20, and 4 consisted of the DD1; motifs 2, 16, 1, 9, and 10 consisted of the B3 domain; motifs 5 and 15 consisted of the DD2; motifs 6, 11, 12, and 13 consisted of the AD; motifs 17 and 14 consisted of the MR; while motif 18, 8, 3, and 19 consisted of the PBI domain. No other new motifs were observed in this study. This suggested that the structures of the ARF gene family are relatively conservative, especially within the same subfamily. Taken together, the homologous genes between Arabidopsis and B. napus commonly had the same sequence features, indicating functional conservation. The sequence features of subfamilies I, II, and III were similar, but those of subfamily IV were different.
Gene structure analysis and small RNA predictions of B. napus ARFs
As the positions, lengths, and phases of introns were generally consistent with the phylogenetic relationships of a gene family , a comparison of the full-length cDNA and DNA sequences of each BnARF was performed to determine the numbers and positions of exons and introns. It was shown that there were many intron insertions in the coding regions of BnARFs, and the number of introns was different (from 1–17) across distinct subfamilies (Fig 4). For example, members in subfamily I generally had 13 intron insertion sites (except for a few genes with incomplete sequences). Members in subfamily II usually have eight or 11 introns; members in subfamily III have 10 or 12 introns, while those in subfamily IV have one–four introns (Fig 4). Interestingly, unlike other transcription factor gene families , the DNA-binding domain, intron insertion site, phase, and even the number of introns are highly conserved in BnARFs within a given subfamily or clade, which provides evidence supporting the reliability of our phylogenetic classification.
Among the three major domains (DBD, MR, and PBI) of ARF proteins, the intron patterns in the DBD and PBI domain were relatively more conserved than those in the MR (Fig 4). Moreover, the intron patterns in the DBD and PBI domain were the same across subfamilies I–III, but not in subfamily IV. For instance, nearly all the members in subfamilies I–III have three conserved intron insertion sites in B3 and two in the PBI domain. However, no introns were detected in B3 of subfamily IV, and only one conserved intron insertion site, which was the same as the second conserved intron insertion site in the PBI domain of the other subfamilies, was present in the PBI domain of some members. Therefore, although the number of intron insertions between subfamily IV and other subfamilies is different, the insertion site is conserved. However, the intron patterns in the MR were less conserved across different subfamilies, but it was relatively conserved within a given subfamily or clade.
Previous studies have revealed that several ARF genes are targets for small RNAs. For example, AtARF6 and AtARF8 in subfamily III are miR167 targets ; AtARF10, AtARF16, and AtARF17 in subfamily IV are miR160 targets . Accordingly, we found that all homologs of AtARF6, AtARF8, AtARF10, AtARF16, and AtARF17 in B. napus may be potential targets for miR160 or miR167 (S5 Table). Moreover, the complementarity of the small RNA targets in subfamily IV is from 85–90%, while in subfamily III this is from 55–65% (S5 Table). This result suggests that target sequences of small RNAs have undergone differentiation and mutation in B. napus during evolution.
Promoter region analysis of B. napus ARF genes
Transcription factors are known to bind to specific cis-elements and modulate the expression of target genes. Therefore, analysis of cis-elements in the upstream regulatory region of a gene can provides insights regarding the regulatory regime it follows. Therefore, we analyzed the 1.5 kb upstream sequence of all BnARFs (S2 Fig). In total, 106 cis-elements were identified in promoters of the 67 BnARFs, such as ARF binding sites, ethylene responsive elements, and drought responsive elements (ACCGAGA). Three putative auxin response cis-elements, TGA-box, TGA-element, and AuxRR-core were also identified, which are involved in 9, 9, and 27 BnARFs, respectively. Moreover, putative cis-elements involved in light signaling perception were observed in nearly all promoters of BnARFs, which may be the reason of the strong crosstalk between auxin and light signals [6,55]. Moreover, putative cis-elements involved in hormones, such as abscisic acid (ABA), gibberellin acid (GA), ethylene, and methyl jasmonic acid were found in a series of BnARF promoters. In addition, putative cis-elements associated with biotic and/or abiotic stress, such as dehydration, low temperature-, and heat stress-response elements were identified in most promoters of BnARFs (S6 Table). Overall, the evidence of diverse cis-elements in promoter regions of BnARFs suggested that ARF genes may be linkers between auxin response and other important processes, and thus, their regulatory networks should be focused on in future research [56,57].
Interaction network of B. napus ARF genes
According to the visualization of the interaction relationship in STRING, 53 interaction proteins of 21 of the 23 AtARFs were obtained, including 26 Aux/IAA proteins, 11 ARF proteins, and 16 other proteins (such as TPR1/2/3, SWI3A/3B/3C and BIN etc.) (S6 Table). We observed that members of subfamily IV interacted with those of subfamily I, II, and III, while subfamily II could interact with subfamily III, implying that ARF proteins from different subfamilies commonly interact with each other. Moreover, AtARF4 and AtARF9 widely interacted with members of the other four subfamilies.
In B. napus, based on orthology analysis, 14 of the 21 AtARFs (except the genes belonging to the Ib-1 subclade) were found to have syntenic relationships with 51 BnARFs including 16 BnARFs from subfamily I, five from subfamily II, 21 from subfamily III, and nine from subfamily IV (Fig 5) (S6 Table). Furthermore, 49 of the 53 Arabidopsis interaction proteins have 169 homologs in B. napus. GO analysis showed that up to 96 of the 169 B. napus putative interaction proteins were IAA related proteins, 30 were ARF proteins, and 43 belonged to other proteins. Moreover, we found that members of subfamily I interacted with 70 IAA proteins, eight ARF proteins, and 15 other proteins; subfamily II interacted with 31 IAA proteins, four ARF proteins, and 17 other proteins; subfamily IV interacted with 25 IAA proteins, nine ARF proteins, and 17 other proteins, whereas subfamily III merely interacted with 45 IAA proteins and six ARF proteins. This evidence supports the functions of subfamily III, which interacts with IAA to regulate auxin response. Furthermore, we found that many interaction proteins were shared by different subfamilies, such as subfamilies I and III both interacting with 14 IAA proteins; subfamilies I and IV both interacting with nine IAA proteins and four other proteins; subfamilies I, II, and III all interacting with 17 IAA proteins. There were 14 IAA proteins that could interact with the proteins in all subfamilies simultaneously (Fig 5). Similar to the situation in Arabidopsis, the homologs of AtARF4 (BnARF61 and BnARF28) and AtARF9 (BnARF02, BnARF05, BnARF34, and BnARF52) widely interacted with the BnARFs in the other four subfamilies.
The ARF proteins in the four subfamilies are represented by dots of different colors. Red: subfamily I; green: subfamily II; blue: subfamily III; yellow: subfamily IV. The Aux/IAA genes are represented by green triangles, and the other type of interaction proteins are represented by aquamarine blue quadrilaterals.
Overall, our results showed that ARF proteins are likely to functions in interaction relationships, and that most interaction proteins are IAA-related proteins. The ARF proteins from different subfamilies generally shared many common interaction proteins and tended to interact with each other as well.
Expression profiles of B. napus ARFs during different developmental stages
Gene expression patterns are generally related to gene functions; therefore, the expression of the 67 BnARFs in 50 tissues during different stages was investigated, based on the RNA-Seq data (Bio Project ID PRJNA358784). The genes with FPKM values of <1 were not included in the analysis. As shown in Fig 6, the candidate BnARFs have wide expression profiles across different vegetative and reproductive organs during different stages in the development of B. napus. In general, the expression patterns between different subfamilies and even different clades of a given subfamily were different but were very similar within the same clade. In subfamily I, the candidates were divided into two major clades: the genes in the Ib clade nearly had no expression value in all tissues investigated, whereas the genes in the Ia clade (16 BnRAFs) were widely expressed in various tissues. In the Ia clade, the five homologs of AtARF2 were highly expressed in various tissues, such as leaves, roots, stems, and flowers, which is similar to those of Arabidopsis . The four homologs of AtARF9 were greatly expressed in roots; the three homologs of AtARF1 had high expression levels in roots, stems, and cotyledon; however, the two homologs of AtARF11 had divergent expression patterns, where one gene was highly expressed in seeds, seed coating, and cotyledon, and the others exhibited low expression in all tissues.
Genes and their corresponding clades are presented on the right. Tissues used for expression analysis are indicated at the top of each column. GS, germinate seed; Hy, hypocotyl; Ao, anthocaulus; Ro, root; St, stem; Le, leaf; Cal, calyx; Cap, capillament; Pe, petal; Sta, stamen; Pi, pistil; SP, silique; Se, seed; SC, seed coat; Em, embryo; Co, cotyledon. s, seedling stage; b, bud stage; i, initial flowering stage; and f, full-bloom stage. The 24, 48, and 72 h indicate the time after seed germination. The 3, 19, 21, 30, 40, and 46 d indicate the days after pollination. The color bar represents log2 expression values (FPKM ≥ 1).
In subfamily II, all five BnARFs had a similar expression pattern, and most of them were highly expressed in roots and stems. A few genes were partially expressed in flowers. In subfamily III, the homologs of AtARF6/8 were mainly expressed in flowers; the homologs of AtARF19 were highly expressed in roots and stems; the homolog of AtARF7 (BnARF04) was highly expressed in roots, stems, leaves, flowers, and silique, whereas the homologs of AtARF5 were mainly expressed in roots and seed tissues in B. napus. In subfamily IV, the homologs of AtARF17 were mainly expressed in roots, stems, and seed coating, and the homologs of AtARF10/16 were mainly highly expressed in flowers and/or seed tissues. Taken together, the expression profile analysis showed that BnARFs may be involved in multiple processes throughout B. napus development, and that the functions of homologs in a given clade might be redundant as they share similar expression patterns.
Expression analysis of B. napus ARFs under hormone induction
As ARF genes are well-known for their roles in the auxin-response process, which is important for root development, we also explored the expression patterns of BnARFs in B. napus roots under five exogenous hormone treatments (IAA, ACC, ABA, GA3, and 6-BA) (Fig 7).
IAA: indoleacetic acid, ACC: 1-aminocyclopropanecarboxylic acid, ABA: abscisic acid, GA3: gibberellin acid 3, 6-BA: cytokinin. The “1”, “3”, “6”, “12”, and “24” represent hours after treatment. Genes with no or weak expression levels (FPKM <1) are not included. The color bar at the top represents log2 expression values: green represents low expression, black represents mean expression level, and red represents high expression.
For the IAA treatment, eight genes from subfamily I (BnARF42, BnARF11, and BnARF14), III (BnARF22, BnARF55, and BnARF04), and IV (BnARF53 and BnARF06) were up-regulated while one gene in subfamily I (BnARF02) was suppressed. For the GA3 treatment, five genes from subfamily I (BnARF11 and BnARF14) and III (BnARF22, BnARF55, and BnARF23) were up-regulated, and no genes were suppressed. For the ABA treatment, four genes from subfamily I (BnARF11 and BnARF14) and III (BnARF22 and BnARF13) were up-regulated, and one gene from subfamily III (BnARF12) was suppressed. For the ACC treatment, three genes from subfamily I (BnARF11 and BnARF14) and III (BnARF04) were up-regulated, and one gene from subfamily IV (BnARF41) was suppressed. For the 6-BA treatment, seven genes from subfamily I (BnARF42, BnARF11, and BnARF14), II (BnARF08), and III (BnARF27, BnARF40, and BnARF54) were up-regulated, and two genes from subfamily I (BnARF02) and IV (BnARF01) were suppressed.
Overall, two genes (BnARF11 and BnARF14) were commonly up-regulated under these five hormone treatments. Two genes (BnARF22 and BnARF55) were up-regulated by IAA and GA3 treatments (BnARF22 was also up-regulated by the ABA treatment). One gene (BnARF42) was up-regulated by IAA and 6-BA, and one gene (BnARF02) was suppressed by IAA and 6-BA. It has been previously reported that the expression of AtARF4, AtARF16, and AtARF19 genes could be up-regulated by IAA treatment in Arabidopsis [59, 60]. Accordingly, in B. napus, we found that the homologs of AtARF19 (BnARF22 and BnARF55) and AtARF16 (BnARF06 and BnARF53) were also up-regulated by the IAA treatment (Fig 7). Taken together, our results suggest that the genes in subfamily III are more susceptible to hormones than those in other subfamilies, and that these BnARFs were more sensitive to exogenous IAA and 6-BA treatments.
To further verify the results of the RNA-seq analyses, twelve BnARFs (BnARF01, BnARF02, BnARF04, BnARF05, BnARF06, BnARF08, BnARF22, BnARF27, BnARF55, BnARF40, BnARF42, and BnARF54) with complete coding sequences were selected to investigate their responses to IAA and 6-BA treatments using qRT-PCR. The results were similar to those of our RNA-seq analysis and the candidates were evidently differentially regulated by IAA and 6-BA treatments (Fig 8). Under IAA treatment, six genes (BnARF04, BnARF22, BnARF42, BnARF55, BnARF06, and BnARF53) were upregulated, among which the expressions of BnARF22 at 6 and 12 h, BnARF42 at 24 h, BnARF06 at 6 h, and BnARF53 at 1 and 24 h were more than two-fold upregulated compared to 0 h. One gene (BnARF02) was significantly downregulated under the IAA treatment. Under the 6-BA treatment, five genes (BnARF08, BnARF40, BnARF42, BnARF54, and BnARF53) were upregulated, two genes (BnARF01 and BnARF02) were downregulated, and one gene (BnARF27) was downregulated at 1 h and then upregulated at 24 h. Moreover, as shown in Fig 6, up to 10 genes of the 12 candidate BnARFs (BnARF01, BnARF02, BnARF04, BnARF08, BnARF22, BnARF27, BnARF40, BnARF42, BnARF54, and BnARF55) were highly expressed in root tissues; only two genes (BnARF06 and BnARF53) have no detectable expression levels in roots but were obviously upregulated by IAA and 6-BA inductions in roots. Together, these results identify these genes as candidates for further study on the potential roles of BnARFs in hormone response in B. napus root.
ARF gene increase, functional redundancy, and differentiation in B. napus
In the present study, we systematically predicted and identified 67 ARF members in the B. napus genome, which represents the largest ARF gene family identified in plants to date. Considering that B. napus originated through a natural doubling of chromosomes following the hybridization of B. rapa and B. oleracea, and that Brassicaceae species experienced a common whole genome triplication (WGT) event, it was expected that the 23 Arabidopsis ARF genes may be expanded to ~70 and ~140 genes in B. rapa/B. oleracea and B. napus genomes, respectively. However, only 29, 33, and 67 genes were found in these three species, respectively. In B. napus, the number of genes in the An- and Cn- subgenomes were almost the same as that in their diploid progenitors B. rapa and B. oleracea. These findings indicate that there many ARF genes were lost after the Brassicaceae WGT, with 58% of ARF genes in B. oleracea and 52% of ARF genes in B. rapa being lost. However, most of the duplicated ARF genes were retained after the WGD event. Thus, the WGD is the main source of gene expansion in B. napus. Together, the different retention ratios between these two large expansion events (WGD and WGT) may be because the WGT event occurred ~9–15 Ma [61,62] or 28 Ma [63–65], whereas the WGD event occurred only ~7500 years ago, which may be too short for the gene loss experienced by their older relatives. The WGD event duplicated many genes in B. napus. It would be interesting to further explore the fates of these duplicates, such as gene loss , neofunctionalization , and subfunctionalization . Accordingly, many BnARFs have very similar expression patterns, such as BnARF07/66, BnARF19/47, BnARF27/46, BnARF12/44, etc. (S7 Table).
Then, we calculated the sequence similarity and sequence identity of B3, full protein, DNA, and the promoter sequence, and analyzed the expression pattern of 65 synteny-pairs in B. napus. We found that all synteny-pairs had highly similar B3 domains (the sequence similarity was > 90%), while only three synteny-pairs (BnARF14/45, BnARF14/56, and BnARF14/23) had low similarity in all sequences, which may due to the large fragment deletion in BnARF14. The sequence similarity of the full length of the proteins was a little reduced, and 51 synteny-pairs (78%) shared over 80% sequence similarity. However, the DNA sequence similarity was obviously reduced, and there were 27 synteny-pairs (42%) sharing over 80% sequence similarity. The promoter sequences of the synteny-pairs had the lowest sequence similarity with only one pair (BnARF19/47) having sequence similarity >80% (91.5%). These results indicate that neofunctionalization of duplicated genes may occur in nucleic acid sequences firstly, especially in the promoter regions.
Pearson correlation coefficient analysis of the expression patterns of 65 synteny-pairs in B. napus (S7 Table) showed that 19 of the 65 (29%) pairs had almost the same expression pattern, 18 pairs (28%) had similar expression patterns, and 21 (32%) pairs had different expression patterns. Seven pairs were not expressed in B. napus. Accordingly, we can speculate that most synteny-pair genes are functionally redundant, and that some synteny-pair genes have undergone sequence differentiation leading to differentiation of expression. These results suggest that the differences in expression patterns may result from neofunctionalization of duplicates during evolution.
Activation and repression roles of ARF genes in the auxin signaling pathway
Auxin is a key regulator of virtually every aspect of plant growth and development from embryogenesis to senescence by controlling gene expression via the ARF gene family. In this process, ARF proteins represent the core of auxin signaling . The key components of this pathway are the transport inhibitor resistant 1/auxin signaling f-box (TIR1/AFB) F-box proteins, the transcriptional co-regulators auxin/indole-3-acetic acid (Aux/IAA), and ARFs. Among them, the roles of ARF genes are divided into two types: transcriptional activator and transcriptional repressor. The activity of ARF activators is enabled through the auxin-dependent degradation of the Aux/IAA repressors . In the absence of auxin, Aux/IAA proteins and ARF activators form heterodimers to prevent ARF-mediated transcription [4,17,69]. In the presence of auxin, auxin binds to the pocket in the TIR1/AFB protein, and the affinity for Aux/IAA proteins increases . Aux/IAA proteins are subsequently ubiquitinated  and degraded in the 26S proteasome [72,73]. Therefore, ARF activity is depressed and numerous auxin-mediated transcriptional changes occur [19,68, 71].
Although ARF activators conform to the established ARF signaling pathway, the role of ARF repressors remains less clear . It is still not clear how the ARF repressors regulate gene repression and how other transcription factors and signaling proteins interact with ARF proteins. Consequently, functions of ARF repressors have been proposed, such as squelching the transcriptional activity of ARF activators either by heterodimerizing with them or through competition for DNA binding sites . Alternatively, some ARF repressors might homodimerize or heterodimerize and recruit co-repressor such as TPL/TPR (TOPLESS and related) proteins . Little information is available to distinguish between these alternatives, as the full catalogue of potential in planta ARF dimers/oligomers is not available. However, the co-expression of ARF activators and repressors in the same tissue  is probably functionally relevant for auxin-dependent transcription. It was proposed that ARF repressors are involved in auxin signaling in both a competitive and cooperative fashion with ARF activators . The activator/repressor categorization correlates with the divisions in subfamilies (Fig 2).
In B. napus, those ARFs identified as activators belong to subfamily III, while ARFs associated with subfamilies I, II, and IV are repressors. According to our prediction, subfamily III only interacts with Aux/IAA and ARF proteins, while the other subfamilies can interact with Aux/IAA proteins and many other types of proteins, such as ARF, BIN2, TPR1/2/3, and SWI3A/3B/3C. These results demonstrate the different mechanisms of ARF activators and repressors where the activators regulate auxin response by interacting with Aux/IAA. Conversely, the repressors act in a competitive role in the same pathway, and the activate or suppress functions of these ARFs on target genes creates an equilibrium that is critical in during auxin reactions.
A total of 67 ARF genes were identified in the B. napus genome, which were further classified to four subfamilies (I–IV) with conserved intron patterns and protein motifs in each subfamily or clade. The full-length protein structures of BnARFs were highly conserved in each subfamily or clade. The evolutionary relationship between B. napus, B. rapa, B. oleracea, and Arabidopsis demonstrated that WGD and segmental duplication can be attributed to the rapid expansion of ARF genes in B. napus, and that this may be independent from tandem duplication. The duplicates derived from B. rapa tend to be retained in B. napus compared to those from B. oleracea. Also, most of the synteny-pairs in B. napus have similar expression patterns indicating functional redundancy. Meanwhile, there are some synteny-pairs that underwent expression divergence and are likely to occur primarily in the promoter regions. The results of the present study provide valuable information regarding the expansion and evolution of the ARF gene family in B. napus following allopolyploidy.
S1 Fig. Amino acid composition at the C-terminal region of the MR of the transcriptional repressors and activators in B. napus.
S2 Fig. Classification of the cis-elements in BnARF promoters.
S1 Table. List of the primers used for the Real-time PCR analyses.
S2 Table. ARF gene family in Arabidopsis, B. oleracea, and B. rapa genomes.
S3 Table. The orthologous-pairs between AtARF–BnARF, BrARF–BnARF and BoARF–BnARF genes.
S4 Table. Duplications of ARF genes in B. napus, B. rapa, and B. oleracea.
S5 Table. Potential miRNA targets of B. napus ARF genes.
S6 Table. List of the interaction proteins in Arabidopsis and B. napus.
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