Genome-Wide Analysis of the Type-B Authentic Response Regulator Gene Family in Brassica napus

The type-B authentic response regulators (type-B ARRs) are positive regulators of cytokinin signaling and involved in plant growth and stress responses. In this study, we used bioinformatics, RNA-seq, and qPCR to study the phylogenetic and expression pattern of 35 type-B ARRs in Brassica napus. The BnARRs experienced gene expansion and loss during genome polyploidization and were classified into seven groups. Whole-genome duplication (WGD) and segmental duplication were the main forces driving type-B ARR expansion in B. napus. Several BnARRs with specific expression patterns during rapeseed development were identified, including BnARR12/14/18/23/33. Moreover, we found the type-B BnARRs were involved in rapeseed development and stress responses, through participating in cytokinin and ABA signaling pathways. This study revealed the origin, evolutionary history, and expression pattern of type-B ARRs in B. napus and will be helpful to the functional characterization of BnARRs.

As reported in Arabidopsis and rice, the ARRs are generally divided into two subgroups, A-and B-type ARRs, which have also been reported as A-, B-, C-type, and Arabidopsis pseudoresponse regulators (APRR) in a few reports [13][14][15][16]. The type-A ARRs are known as negative regulators in cytokinin signaling, and they contain a short C-terminal extension besides the receiver domain [15]. Hitherto, type-A ARRs in Arabidopsis have been reported in controlling circadian period (ARR3/4), regulating meristem maintenance and regeneration (ARR4/7/8/15), root growth (ARR3), seed germination, and seedling growth (ARR4/5/6/7/15/16), as well as stomatal lineage ground cell division (ARR16/17) [17][18][19][20][21][22][23][24]. Unlike the A-ARRs, type-B ARRs in Arabidopsis contain a long C-terminal extension and a receiver domain in the N-terminal. They are positive regulators of phosphorelay signal transduction and transcriptional activators for A-ARRs [11,25]. The C-terminal extensions evolution, structure, and expression profile of 35 type-B ARRs in B. napus. It is valuable for functional analysis of BnARRs in regulating rapeseed development and stress responses in the future.

Gene Ontology (GO) Analysis
The GO annotation of type-B ARRs were obtained from the rapeseed genome database. The three GO categorization of BnARRs, molecular function (MF), biological process (BP), and cellular component (CC), were analyzed by Omicshare (http://www.omicshare.com/ tools/, accessed on 9 June 2022) with a corrected p (FDR) < 0.05.

Quantitative Real-time PCR (qPCR) Analysis
Total RNA from above samples were extracted with the RNAprep Pure Plant kit (TIANGEN BIOTECH, Beijing, China) and used for cDNA synthesis with HiScript ® II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, Nanjing, China). qPCR primers of BnARRs listed in Table S1 were designed by Primer Premier 5.0 and synthesized by TSINKE Biotech (Beijing, China), BnActin7 was the internal control. qPCR analysis was performed on a StepOnePlus TM Real-time PCR system (Thermo, Waltham, MA, USA) using PowerUp SYBR Green Master Mixes (Thermo, USA). The BnARR expression level was calculated using 2 −∆∆Ct method [78].

Statistical Analysis
One-way ANOVA or t-test of SPSS version 19.0 (IBM, NewYork, NY, USA) was used for significant difference analysis of multiple samples or two samples at p < 0.05, respectively.

Identification of Type-B ARRs in B. napus
In rapeseed, we identified 157 and 1046 proteins with the response regulator domain and MYB_DNA-binding domain, respectively. Only 45 proteins contained both response regulator and MYB binding domain, including ten orthologs of AtAPRRs that were excluded. Thus, a total of 35 B. napus proteins were taken as type-B ARRs for further analysis. Meanwhile, a total of 11, 15, and 18 type-B ARRs with both domains were identified in A. thaliana, B. rapa and B. oleracea. Interestingly, most type-B AtARRs were identified with orthologs in B. napus, except for AtARR13. The type-B ARRs (BnARR1~BnARR35) in rapeseed were named in serial numbers according to their chromosomal locations (Table S2). Accord-ing to the triplication and duplication history of B. napus, we found~47% B-ARRs were lost or rearranged during rapeseed evolution. Furthermore, we found BnARRs ranged from 187 (BnARR17) to 748 (BnARR18) amino acids (AA), the Mw ranged from 21.09 (BnARR17) to 83.04 (BnARR18) kDa, and the pI ranged from 4.84 (BnARR13) to 10.2 (BnARR2). Most BnARRs were acidic proteins since 80% of them were predicted with pI <7. All the BnARRs were predicted with nuclear localization (Table S2).

Sequence Alignment and Evolution Analysis of Type-B ARRs in B. napus
Multi-protein sequence alignment confirmed that B-ARRs in rapeseed contained two main conserved domains ( Figure 1). The 120 AA receiver domain (responsive regulator) in the N-terminal has a phosphorylated Asp residue in the center ( Figure 1A). The DNA binding domain (~60 AA), also named B motif, was similar to the MYB_DNA-binding motif and is distinguished from other types of ARRs ( Figure 1B). and MYB_DNA-binding domain, respectively. Only 45 proteins contained both response regulator and MYB binding domain, including ten orthologs of AtAPRRs that were excluded. Thus, a total of 35 B. napus proteins were taken as type-B ARRs for further analysis. Meanwhile, a total of 11, 15, and 18 type-B ARRs with both domains were identified in A. thaliana, B. rapa and B. oleracea. Interestingly, most type-B AtARRs were identified with orthologs in B. napus, except for AtARR13. The type-B ARRs (BnARR1~BnARR35) in rapeseed were named in serial numbers according to their chromosomal locations (Table  S2). According to the triplication and duplication history of B. napus, we found ~47% B-ARRs were lost or rearranged during rapeseed evolution. Furthermore, we found BnARRs ranged from 187 (BnARR17) to 748 (BnARR18) amino acids (AA), the Mw ranged from 21.09 (BnARR17) to 83.04 (BnARR18) kDa, and the pI ranged from 4.84 (BnARR13) to 10.2 (BnARR2). Most BnARRs were acidic proteins since 80% of them were predicted with pI <7. All the BnARRs were predicted with nuclear localization (Table S2).

Sequence Alignment and Evolution Analysis of Type-B ARRs in B. napus
Multi-protein sequence alignment confirmed that B-ARRs in rapeseed contained two main conserved domains ( Figure 1). The 120 AA receiver domain (responsive regulator) in the N-terminal has a phosphorylated Asp residue in the center ( Figure 1A). The DNA binding domain (~60 AA), also named B motif, was similar to the MYB_DNA-binding motif and is distinguished from other types of ARRs ( Figure 1B). The phylogenetic tree of type-B ARRs in B. napus, B. rapa, B. oleracea, and A. thaliana classified them into seven groups (Class I~VII), each group contained 5, 3, 4, 7, 3, 6, and 7 BnARRs, respectively ( Figure 2). In Class IV, 16 ARRs from Brassicas were orthologs of AtARR1 and AtARR2. The nine ARRs clustered in Class I were orthologs of AtARR10 and AtARR12. These BnARRs might play important roles in cytokinin response processes since AtARR1/2/10/12 have been reported with multiple functions in cytokinin signaling and plant development [3,4,32,35,37,79,80]. The phylogenetic tree of type-B ARRs in B. napus, B. rapa, B. oleracea, and A. thaliana classified them into seven groups (Class I~VII), each group contained 5, 3, 4, 7, 3, 6, and 7 BnARRs, respectively ( Figure 2). In Class IV, 16 ARRs from Brassicas were orthologs of AtARR1 and AtARR2. The nine ARRs clustered in Class I were orthologs of AtARR10 and AtARR12. These BnARRs might play important roles in cytokinin response processes since AtARR1/2/10/12 have been reported with multiple functions in cytokinin signaling and plant development [3,4,32,35,37,79,80].

Gene Structure and Conserved Motifs of Type-B ARRs in B. napus
Gene structure is correlated with expression and function divergence, the coding regions responsible for various gene functions may be due to the alterations in exon-intron structure and/or amino acid substitutions [81,82]. We found the intron number of BnARRs ranged from 2 to 11, of which 23 BnARRs contained four or five introns. The exon number ranged from 3 to 12 ( Figure 3). Furthermore, the exon number in Class IV and VII was 3~12 and 9~12, respectively. The exon length and number was more consistent in Class I, II, III, VI, and V. In general, BnARRs in the same phylogenetic branch showed similar structures, but the intron length varied in some groups, such as BnARR4/BnARR24, which may contribute to the functional differentiation of duplicated ARRs.
Nine conserved motifs of BnARRs were identified, ranging from 15 to 50 AA (Figures 3 and S1). Motifs 2, 3, and 5 were located in the receiver domain, and motifs 1 and 4 were located in the DNA-binding domain. In addition, motif 6 was presented in 32 BnARRs (80%) in seven groups, and motif 7 was existed in 26 BnARRs. Motif 9 was mainly identified in Class II and VII, and also found in two members of Class III and IV. Motif 8 was specific to Class VII. The specific motif patterns may also lead to functional divergence of BnARRs in different groups. Genes 2022, 13, x FOR PEER REVIEW 6 of 18

Gene Structure and Conserved Motifs of Type-B ARRs in B. napus
Gene structure is correlated with expression and function divergence, the coding regions responsible for various gene functions may be due to the alterations in exon-intron structure and/or amino acid substitutions [81,82]. We found the intron number of BnARRs ranged from 2 to 11, of which 23 BnARRs contained four or five introns. The exon number ranged from 3 to 12 ( Figure 3). Furthermore, the exon number in Class IV and VII was 3~12 and 9~12, respectively. The exon length and number was more consistent in Class I, II, III, VI, and V. In general, BnARRs in the same phylogenetic branch showed similar structures, but the intron length varied in some groups, such as BnARR4/BnARR24, which may contribute to the functional differentiation of duplicated ARRs.
Nine conserved motifs of BnARRs were identified, ranging from 15 to 50 AA ( Figures  3 and S1). Motifs 2, 3, and 5 were located in the receiver domain, and motifs 1 and 4 were located in the DNA-binding domain. In addition, motif 6 was presented in 32 BnARRs (80%) in seven groups, and motif 7 was existed in 26 BnARRs. Motif 9 was mainly identified in Class II and VII, and also found in two members of Class III and IV. Motif 8 was specific to Class VII. The specific motif patterns may also lead to functional divergence of BnARRs in different groups.

Chromosomal Location and Synteny of BnARRs
A total of 31 BnARRs (19 genes on A subgenome and 16 on C subgenome) were physically localized on rapeseed chromosomes, except for four members located on Ann_random and Cnn_random due to the incomplete B. napus genome (Figure 4). We found all the BnARRs with two to seven homologs on A and C subgenomes, such as BnARR2/21/22 and BnARR10/11/29/30.
Duplication is important to plant evolution and adaptation [83]. Based on the synteny analysis, we found the BnARRs have experienced different types of duplication events. Twenty-eight BnARRs (80%) were derived from WGD/segmental events, only one BnARR derived from a tandem event, two BnARRs derived from proximal events, and four genes resulted from dispersed events ( Figure 5, Table S3). In addition, 26 paralogous gene pairs were identified, indicating gene duplication, especially WGD/segmental events, was the main force driving type-B ARR expansion in B. napus.
The expansion and evolution of ARRs in Brassicaceae was revealed by synteny analysis among B. napus, A. thaliana, B. rapa, and B. oleracea ( Figure 6, Table S4). About 82.9% (29/35) of BnARRs had syntenic relationship to ARRs in other species. Specifically, 27, 27, and 22 BnARRs were predicted with synteny to B. rapa, B. oleracea, and A. thaliana, respectively. We found 29 BnARRs were inherited from B. rapa or B. oleracea, while the remaining six BnARRs were novel members after genome duplication. The detailed motif structure is showed in Figure S1. (C) Gene structure of BnARRs. UTR, untranslated region; CDS, coding sequence.

Chromosomal Location and Synteny of BnARRs
A total of 31 BnARRs (19 genes on A subgenome and 16 on C subgenome) were physically localized on rapeseed chromosomes, except for four members located on Ann_random and Cnn_random due to the incomplete B. napus genome (Figure 4). We found all the BnARRs with two to seven homologs on A and C subgenomes, such as BnARR2/21/22 and BnARR10/11/29/30.   The detailed motif structure is showed in Figure S1. (C) Gene structure of BnARRs. UTR, untranslated region; CDS, coding sequence.

Chromosomal Location and Synteny of BnARRs
A total of 31 BnARRs (19 genes on A subgenome and 16 on C subgenome) were physically localized on rapeseed chromosomes, except for four members located on Ann_random and Cnn_random due to the incomplete B. napus genome (Figure 4). We found all the BnARRs with two to seven homologs on A and C subgenomes, such as BnARR2/21/22 and BnARR10/11/29/30.  Duplication is important to plant evolution and adaptation [83]. Based on the synteny analysis, we found the BnARRs have experienced different types of duplication events. Twenty-eight BnARRs (80%) were derived from WGD/segmental events, only one BnARR derived from a tandem event, two BnARRs derived from proximal events, and four genes resulted from dispersed events ( Figure 5, Table S3). In addition, 26 paralogous gene pairs were identified, indicating gene duplication, especially WGD/segmental events, was the main force driving type-B ARR expansion in B. napus.  Table S4). About 82.9% (29/35) of BnARRs had syntenic relationship to ARRs in other species. Specifically, 27, 27, and 22 BnARRs were predicted with synteny to B. rapa, B. oleracea, and A. thaliana, respectively. We found 29 BnARRs were inherited from B. rapa or B. oleracea, while the remaining six BnARRs were novel members after genome duplication. Based on the Ka (non-synonymous substitutions per site), Ks (synonymous substitutions per site), and Ka/Ks ratio, we predicted the selective pressure of ARR gene pairs in B. napus, B. rapa, B. oleracea, and A. thaliana (Table S4, Figure S2). The Ka/Ks ratio of gene pairs in B. napus-B. napus, B. napus-B. rapa, B. napus-B. oleracea, and B. napus-A. thaliana were 0.4476, 0.4588, 0.4465, and 0.3370, respectively. As reported, Ka/Ks ratio >1, =1, and Based on the Ka (non-synonymous substitutions per site), Ks (synonymous substitutions per site), and Ka/Ks ratio, we predicted the selective pressure of ARR gene pairs in B. napus, B. rapa, B. oleracea, and A. thaliana (Table S4, Figure S2). The Ka/Ks ratio of gene pairs in B. napus-B. napus, B. napus-B. rapa, B. napus-B. oleracea, and B. napus-A. thaliana were 0.4476, 0.4588, 0.4465, and 0.3370, respectively. As reported, Ka/Ks ratio >1, =1, and <1 represented positive selection, neutral mutation, and purifying selection, respectively [84]. This indicated that most BnARR pairs experienced strong purifying selection. Furthermore, the type-B ARR gene pairs between B. napus-B. napus, B. napus-B. rapa, B. napus-B. oleracea, and B. napus-A. thaliana were diverged 0.1596, 0.0979, 0.1031, and 0.2671 million years ago (Mya), respectively. Thus, the ARRs in B. napus-A. thaliana were diverged earlier than in other comparisons.

GO Enrichment and Expression Profiles of Type-B ARRs in B. napus
To acknowledge the putative function of type-B ARRs in B. napus, we enriched these genes with the GO terms, and the top 20 enriched terms included biological process of cytokinin-activated signaling pathway (GO: 0009736), cellular response to cytokinin stimulus (GO: 0071368), and response to cytokinin (GO: 0009735). This indicated that type-B ARRs participated in the cytokinin signaling process of B. napus. Moreover,~42.22% of BnARRs were enriched in the molecular function of phosphorelay response regulator activity (GO: 0000156), agreeing with the function of the receiver domain in BnARRs ( Figure 7A, Table S5).

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BnARR12/23/33 expressed in 14 DAP silique and 21 DAP seed. The different BnARR expression pattern might be related to their functional diversification, and the ARRs with high expression level in specific tissue/organ may take part in the processes of plant development. We analyzed the BnARR expression under abiotic stresses and hormone treatments, but not all the members could respond to these treatments. Most BnARRs with response to abiotic stresses were down-regulated under cold, mannitol, salt, and PEG treatments, such as BnARR1/4/7/8/9/20/24/28/31/34. However, BnARR20 was up-regulated by salt stress, BnARR13 and 27 were up-regulated by cold stress, and BnARR23 was up-regulated by drought stress ( Figure 8A). Furthermore, the members in class III were down-regulated under IAA and SL treatments, such as  Based on the RNA-seq data of different tissues and organs representing B. napus development, we found~74.29% of the type-B ARRs were expressed in one or more tissues/organs with FPKM value > 1, while the rest BnARRs (e.g., BnARR13, BnARR16, and BnARR17) were non-expressed genes ( Figure 7B, Table S6). BnARR2, BnARR4, BnARR9, and BnARR24 were expressed in more than 12 tissues/organs. Eight BnARRs were expressed in three or fewer tissues/organs, such as BnARR14/18 expressed in 28 DAP seed, BnARR12/23/33 expressed in 14 DAP silique and 21 DAP seed. The different BnARR expression pattern might be related to their functional diversification, and the ARRs with high expression level in specific tissue/organ may take part in the processes of plant development.
We analyzed the BnARR expression under abiotic stresses and hormone treatments, but not all the members could respond to these treatments. Most BnARRs with response to abiotic stresses were down-regulated under cold, mannitol, salt, and PEG treatments, such as BnARR1/4/7/8/9/20/24/28/31/34. However, BnARR20 was up-regulated by salt stress, BnARR13 and 27 were up-regulated by cold stress, and BnARR23 was up-regulated by drought stress ( Figure 8A). Furthermore, the members in class III were down-regulated under IAA and SL treatments, such as BnARR10/11/29/30. BnARR2/21/22 in class VI were up-regulated under GA treatment. BnARR7/26 in class I, BnARR1/19/20 in class II, and BnARR4/9/24/28/31 in class IV were up-regulated under GA, IAA, and SL treatments (Figure 8B).

Cis-Acting Elements in Type-B BnARR Promoters
Gene promoters contain a large number of cis-acting elements that can specifically bind to proteins involved in the initiation and regulation of gene transcription [85]. Here, we analyzed the type-B BnARR promoters, and classified the cis-acting elements into four types, abiotic responsiveness, hormones responsiveness, plant growth and development, and other basic promoter elements like TATA-box ( Figure 9, Table S7). The circadian control (circadian motif), zein metabolism regulation (O2-site motif), meristem expression (CATbox), endosperm expression (GCN4 motif), phytochrome down-regulation expression, seed-specific regulation (RY element), endosperm-specific negative expression (AACA motif), root-specific elements (motif I), differentiation of the palisade mesophyll cells (HD-Zip 1 motif), and light-responsive elements (TCT motif, G-box, GT1 motif, and AE-box) were related to plant growth and development. Furthermore, the light-responsive elements were ubiquitous in the BnARR promoters, indicating that BnARRs may be involved in the light-regulated plant development. As to the hormone-responsive elements, we found the cytokinin and abscisic acid responsive elements were enriched in BnARR promoters. The existence of anaerobic induction (ARE motif), defense and stress responsiveness (TCrich motif), low-temperature responsiveness (LTR motif), MYB binding site involved in drought-inducibility (MBS motif), anoxic-specific inducibility (GC motif), and wound responsiveness (WUN motif) indicated that BnARRs may also regulate plant response to abiotic stresses. involved in the light-regulated plant development. As to the hormone-responsive elements, we found the cytokinin and abscisic acid responsive elements were enriched in BnARR promoters. The existence of anaerobic induction (ARE motif), defense and stress responsiveness (TC-rich motif), low-temperature responsiveness (LTR motif), MYB binding site involved in drought-inducibility (MBS motif), anoxic-specific inducibility (GC motif), and wound responsiveness (WUN motif) indicated that BnARRs may also regulate plant response to abiotic stresses.

The Expression Pattern of Type-B BnARRs under Cytokinin and ABA Treatments
As mentioned above, the cis-acting elements associated with ABA and CTK responses were enriched in BnARR promoters. Thus, we used qPCR to analyze the BnARR expression pattern under different time of ABA and CTK treatments. The BnARR4/5/30 were down-regulated under CTK treatment, while BnARR18/19/23 were strongly up-regulated. BnARR18 and BnARR23 were obviously up-regulated after 12 h and 1 h of CTK treatment, respectively. BnARR19 was consistently up-regulated during 1~6 h of CTK treatment. In addition, 12 BnARRs were slightly induced or repressed by CTK ( Figure 10). Under ABA treatment, BnARR2/9/15/28/35 were up-regulated, but with the highest expression level at different hours after ABA treatment. Moreover, ABA repressed the expression level of BnARR4 and BnARR5 with the minimum expression at 12 h of ABA treatment. We found eight BnARRs were slightly up-regulated (e.g., BnARR6 and BnARR7) or down-regulated (e.g., BnARR30) after ABA treatment ( Figure 11). These results indicated the BnARRs might be involved in CTK and ABA signaling pathways.
Under ABA treatment, BnARR2/9/15/28/35 were up-regulated, but with the highest expression level at different hours after ABA treatment. Moreover, ABA repressed the expression level of BnARR4 and BnARR5 with the minimum expression at 12 h of ABA treatment. We found eight BnARRs were slightly up-regulated (e.g., BnARR6 and BnARR7) or down-regulated (e.g., BnARR30) after ABA treatment ( Figure 11). These results indicated the BnARRs might be involved in CTK and ABA signaling pathways.

Discussion
Cytokinin is important in regulating plant development and response to biotic and abiotic stresses [1]. Among the two main types of ARRs (A-and B-ARRs) involved in cytokinin signaling [37], type-B ARRs are transcription factors that can be activated by phosphorylation of the Asp residue in the receiver domain [1]. Hitherto, the B-ARRs have been studied in Arabidopsis, rice, tomato, tobacco, and peach, but not in oil crop B. napus [11,[52][53][54][55]. In this study, 35 B-ARRs were identified in rapeseed based on the two conserved domains. The gene structure, chromosomal location, duplication event, cis-acting element, and expression patterns of these BnARRs were analyzed. The type-B BnARRs were divided into seven classes, which were consistent with that in Arabidopsis [86]. The gene structure and conserved motifs of BnARRs in the same class were similar but differed among different groups. The different intron-exon structure of BnARRs might be due to chromosome rearrangement and translocation during polyploidization. Recently, introns have been proved with important functions in regulating gene expression [87,88]. We found the intron number varied a lot among BnARRs, which might be valuable to BnARR evolution. Figure 11. Type-B BnARRs expression in response to ABA treatment. The data was represented as mean ± standard deviation (n = 3).

Discussion
Cytokinin is important in regulating plant development and response to biotic and abiotic stresses [1]. Among the two main types of ARRs (A-and B-ARRs) involved in cytokinin signaling [37], type-B ARRs are transcription factors that can be activated by phosphorylation of the Asp residue in the receiver domain [1]. Hitherto, the B-ARRs have been studied in Arabidopsis, rice, tomato, tobacco, and peach, but not in oil crop B. napus [11,[52][53][54][55]. In this study, 35 B-ARRs were identified in rapeseed based on the two conserved domains. The gene structure, chromosomal location, duplication event, cis-acting element, and expression patterns of these BnARRs were analyzed. The type-B BnARRs were divided into seven classes, which were consistent with that in Arabidopsis [86]. The gene structure and conserved motifs of BnARRs in the same class were similar but differed among different groups. The different intron-exon structure of BnARRs might be due to chromosome rearrangement and translocation during polyploidization. Recently, introns have been proved with important functions in regulating gene expression [87,88].
We found the intron number varied a lot among BnARRs, which might be valuable to BnARR evolution.
Based on the expression profile of B-ARRs during rapeseed development, we found three putative pseudogenes (BnARR13/16/17) that were not expressed in all analyzed tissues/organs. Ten BnARRs were expressed throughout rapeseed development and might be important to rapeseed growth. The different expression pattern of BnARRs might be caused by the neo-, sub-, and non-functionalization after gene duplication. Moreover, we identified a few BnARRs (e.g., BnARR4/7/8/9/24/28/31) that were down-regulated under cold, mannitol, salt, and PEG stresses, while BnARR13/20/23/27 were up-regulated by salt, cold, or drought stress. Furthermore, the BnARRs in class III were down-regulated by IAA and SL, the members of class VI were up-regulated by GA. A few members in class I (BnARR7/26), class II (BnARR1/19/20), and class IV (BnARR4/9/24/28/31) were up-regulated by GA, IAA, and SL. Cis-acting elements are important in regulating gene expression [85]. The abiotic responsive elements, hormone-responsive elements, plant growth-related elements, and development-related elements were enriched in the type-B BnARR promoters. We confirmed that 17 and 16 BnARRs were induced or repressed by CTK and ABA, respectively. These genes may participate in hormone-regulated plant development and stress responses, since ABA and CTK are important endogenous messengers in plants and play vital roles in regulating plant development and adaptation [1,97]. However, only six BnARRs were significantly up-or down-regulated by cytokinin. In Arabidopsis, A-ARR expression was more induced by cytokinin than the B-ARRs [49]. GO analysis also enriched the type-B BnARRs in CTK-related terms, such as CTK-activated signaling pathway, cellular response to CTK stimulus, and response to CTK. As reported, B-ARRs were activated through phosphorylation in the receiver domain after CTK treatment and bound to the target genes in a CTK-dependent manner [35]. AtARR1/10/12 were negative regulators in plant response to drought stress; the triple mutants were drought-tolerant compared with the wild type [49]. In plants, ABA regulates numerous biological processes, including seed dormancy and germination, lateral root formation, and stress responses. It could broadly regulate the expression of stress-responsive genes [98]. In this study, the BnARRs (e.g., BnARR2/9/15/28/35) with significant expressional changes under ABA treatment may also be involved in plant response to abiotic stresses.

Conclusions
We comprehensively analyzed the 35 type-B ARRs in B. napus. This gene family experienced expansion and loss during rapeseed polyploidization, and these BnARRs were grouped into seven classes. The GO enrichment, temporospatial expression pattern, and response to abiotic and hormone treatments suggest that type-B BnARRs played important roles in rapeseed growth, development, and stress responses, especially via ABA and cytokinin signaling pathways. In general, these findings will be helpful to the further functional investigation of type-B BnARRs.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes13081449/s1, Figure S1: Amino acid sequence logos of conserved domains in type-B ARRs of B. napus; Figure S2: Box-plot of Ka, Ks, Ka/Ks value and divergence time of type-B ARR gene pairs. Mya, million years ago; Table S1: The qPCR primers used in this study; Table S2: The information of type-B ARR family members in B. napus; Table S3: Duplication type of type-B ARRs in B. napus; Table S4 Table S5: GO enrichment analysis of type-B ARRs in B. napus; Table S6: The RNA-seq data (FPKM) of type-B BnARRs in different tissues and developmental stages; Table S7: Cis-acting element analysis of type-B BnARRs.