PvB3s: Genome-wide and Transcriptome-wide Identification and Analysis Reveals the B3 Family Regulate Auxin to Resist Salt Stress at the Sprout Stage in Common Bean(P. Vulgaris L. ) .


 Background: B3 gene family is a transcription factor family unique to plants, which play an important role in plant growth and development by binding specific DNA sequences. However, data on the B3 genes in the common bean and participate in many abiotic stresses especially salt stress are limited. Result: A total of encoding 100 proteins were identified in common bean. Phylogenetic analysis showed that PvB3s were classified into 4 subgroups, and these clusters were supported by several group-specific features, including exon/intron structure, MEME motifs, and predicted binding site structure. Collinearity analysis showed the connection of PvB3s in the same species and different species. The genes expression pattern showed that PvB3s expressed with a tissue-specific manner during sprout stage. Through RNA-seq and qRT-PCR analysis, it was found that there were differences in expression in extreme materials under salt stress. The determination of auxin content and the analysis of PvB3s expression in the enriched pathway showed that PvB3s would respond to auxin to enhance salt tolerance in common bean sprouting stage. Conclusion: The results provided useful and rich resources of PvB3s for the functional characterization and understanding of B3 transcription factors (TFs) in common bean, which further provides insights that PvB3s may respond to auxin to enhance salt tolerance of common bean.

P. vulgaris L. is a valuable legume crop and a source of livelihood for many people [21,22]. Besides, the crop is rich in proteins and micro-nutrients, which are essential for human health. There has been a slow improvement in the yield of the crop, particularly in tropical regions. However, favorable yields have been realized in temperate countries as a result of the development of new cultivars [23]. Studies have shown that PvB3s participate in auxin response, which may have in uence related to various abiotic stresses in several plant species. However, the B3 family in P. vulgaris has not been characterized. To screen the potential salt tolerance genes in common bean sprouting stage, we identi ed the members of B3 gene family at the genome level. Here in, we identi ed 110 PvB3 members according to P. vulgaris L. genome sequence. Furthermore, we determined their gene distribution on chromosome and studied their conserved domain and collinearity. We evaluated PvB3s expression in multiple varieties of the plant. Also, we analyzed the expression pattern of PvB3s in various tissues at sprout stage and explored the possible regulation of speci cally expressed PvB3s under salt stress and other stress conditions. These ndings offer crucial information for studying the evolutionary relationship and functional differentiation of B3 gene family in common bean.

Identi cation of B3 Family in Common bean
In total, 118 protein sequences with B3 domains were identi ed by HMM pro le analysis from the common bean genome. Furthermore, InterPro and SMART analyses indicated that 110 members could be assigned as common bean B3 family, which were named PvB3-001 ~ PvB3-110 accordingly (Table S1). Among them, 110 PvB3 members were located on the 11 chromosomes (Chrs) of common bean (Fig. 1).
Speci cally, most of the PvB3s were located in Chr 3,which 15 members on, followed by Chr 8 with 14 members on, while minimum genes were distributed on Chr 4, Chr 5, which only have 3 and 6 members.
The position of PvB3 members can be mapped to each chromosome can be seen in Fig. 1. The denser the blue, the higher the gene density in the middle of that area.

Phylogenetic Analysis of PvB3s
A comprehensive phylogenetic tree comprising 110 PvB3 protein sequences of common bean (Fig. 2).The phylogenetic tree was generated using the Maximun Likelihoodphy using WAG + G model in MEGA software (MEGAX) with the B3 sequences. Based on the phylogenetic tree, the predicted PvB3 family clustered into 4 subgroups, which is -. Subgroups and have the most PvB3 members, compared to subgroups and .
The motif and gene structure conserved PvB3 family members We employed the MEME software to analyze the conservative motifs of 110 PvB3s in 4 subgroups in celery. Figure 3b shows the symbols of the motifs and the composition of every B3 family members in P.vulgaris.L. Each sub-group contains some differences in the motif, but the members within each subfamily have similar motif structures. For example, in the , , and subgroups all have motif 4, only members of subgroup 4 and a few members of subgroup 1 own motif 8, and the subgroup contains many different motifs, but every member of the subgroup owns these motifs.
The gene structure of exon and intron of the PvB3s were examined to reveal a certain difference in the gene structure among the 4 subfamilies, regardless of the length of their genetic structure, the number of introns and exons, including the number of CDS, but the structure of PvB3 in each subfamily is relatively consistent No matter the number of their CDS and introns, they are even similar to the position of B3 domain in their gene structure.

Cis-regulatory elements analysis of PvB3s
Cis-regulatory elements was used to be analyzed by PlantCARE software, according to the promoters of PvB3s according to the celery genome database, and the related hormone formation seed germination related elements are represented in Fig. 4b. Red boxes are hormone-related elements. These elements revealed that the PvB3s may respond to hormones in response to stress; The blue elements represent elements that respectively encounter low-temperature and drought, indicating that the PvB3s have a very important role in abiotic stress; yellow represents elements that are related to plant germination or sprout stage.

Collinearity analysis and tandem replication of PvB3s
The results of collinear analysis within common bean B3 family gene species and with legume crops and Arabidopsis thaliana are shown in Fig. 5. Only 14 pairs of bean B3 family genes were collinear and distributed in 11 LGs, of which 9 had the largest number of PvB3 with 4 members, indicating that PvB3s were not randomly and uniformly distributed on the linkage group (Fig. 5a). In legume species, there was collinearity between PvB3s and soybean 88 genes (G. max.L), adzuki bean 35 genes (V. angularis) and mung bean 50 genes (V. radiata.L) (Fig. 5b). However, only 23 genes in the B3 family of common beans are collinear with Arabidopsis thaliana,and only 5 genes in rice (O. sativa L.) (Fig. 5c). In the selection pressure analysis of PvB3s (Table S3), only 3 pairs of tandem replication genes in PvB3 had Ka/Ks < 1, while the Ka/Ks > 1 of 9 pairs of PvB3s.
Analysis of PvB3 members and the genes containing B3 domain in Arabidopsis The same Pfam identi ed proteins and genes containing B3 conserved domains in Arabidopsis, and conducted evolutionary analysis of mixture species with PvB3 members, as well as motif and gene structure analysis (Fig. 6). After analysis, it was found that the B3 family genes of the two species are also divided into 4 subgroups, as PvB3s in. The genetic structure and motif of each subfamily are relatively similar, indicating that each subfamily is traveling The functions are relatively consistent.

Expression analysis of PvB3s in Different Tissues of Common bean in sprout stage
Successively, we examined the transcriptional patterns of PvB3s in many tissues using high-throughput sequencing information from Phytozome database, that included ower buds, owers, leaves, root, trifoliates, nodules, stem, pod, seed, and other tissues (Fig. 7a), PvB3s are expressed in various tissues, and the expression differences are relatively large. To elucidate the functions of the PvB3 genes in P. vulgaris at sprout stage, we choose 6 PvB3 genes randomly, used qRT-PCR for three tissues (cotyledon, hypocotyl, radicle) in common bean R sprout stage to assess the expression of the PvB3 genes in sprout stage diverse tissues. As shown (Fig. 7b), among the 6 PvB3 genes randomly selected, The relative expression of some B3 family genes in the radicle is relatively high, and some PvB3s have relatively high expression in the hypocotyl.

Expression analysis of PvB3s in extreme materials under salt stress
From a laboratory basis, we tested two kinds of extreme common bean materials under water treatment and salt stress treatment (R is salt-tolerant variety, and N is salt-sensitive variety). The assembled unigene dataset was used as a reference for further analyses, and it can be found at the National Centre for Biotechnology Information under the accession number PRJNA558376 (http://www.ncbi.nlm.nih.gov/bioproject/). Through the determination of transcriptome, we found that there was a signi cant difference in PvB3s' expression, but many PvB3s of the fourth subfamily are not expressed (Fig. 8a). 9 PvB3 genes were randomly selected for qRT-PCR analysis, and it was found that under W (water treated) and S (salt stress treated), the transcriptome results of PvB3 genes expression in extreme materials R and N were consistent (Fig. 8b). It is suggested that under salt stress, the differential expression of PvB3s in extreme materials may affect the salt tolerance of plants.

PvB3s expression analysis of IAA enrichment pathway in extreme materials under salt stress
The KEGG analysis of PvB3s showed that PvB3s were enriched into the pathway "Plant hormone signal transduction" (pvu04075) in common bean, and its Corrected P-Value was 1.81e -14 (Fig. 9a). The result showed that PvB3s could adjust plant stomata in response to IAA. The IAA content of extreme materials was determined, and it was found that the IAA content of salt-resistant variety R itself was higher and increased sharply under salt stress, while the change of IAA content of salt-sensitive variety N was relatively small (Fig. 9b). The results of qRT-PCR analysis of radicles of two varieties showed that, compared with S treatment, some PvB3s the change of gene expression of R variety was larger than that of N variety, which indicated that the change of IAA content did affect the change of PvB3s under salt stress, which may further affect the salt tolerance of common bean at sprout stage (Fig. 9c).
The Relationship between IAA, salt stress and PvB3s After IAA and salt treatment, the sensitive salt variety N has been alleviated a lot compared with S treatment, such as the number of lateral roots of bean sprouts increased signi cantly (Fig. 10a). Exogenous IAA could signi cantly increase the content of endogenous IAA in N varieties (Fig. 10b), and 8 PvB3s, were randomly selected and veri ed by qRT-PCR analysis (Fig. 10c). It was found that the variable of these genes increased or decreased signi cantly in N, indicating that PvB3s will respond to the changes of IAA to resist salt stress in plants.

Characterization of B3 Family Members in Common bean
In previous studies, the genes of the B3 family have been reported in citrus (C. sinensis and C. grandis) [36], Savoy cabbage (B. oleracea) [37], and grapes (V. vinifera L.) [38], but no related articles have been reported in common beans. Through evolutionary tree analysis, the B3 transcription factor family has been divided into 4 Subgroups, and the motifs, cis elements, and gene structures within each sub-group are very similar, indicating that each sub-group has PvB3s with similar functions; but the PvB3s among the four subgroups are very large In the analysis of the B3 transcription factors of Arabidopsis and the common bean (P. vulgaris L.) two species, they are divided into 4 subgroups according to the genetic relationship, and the members in each subgroup are similar in terms of motif or gene structure. This conclusion is consistent among citrus (C. sinensis and C. grandis) [36] and grapes (V. vinifera L.) [6].In tissue-speci c analysis, more PvB3s are expressed in the bud stage (whether ower buds, plant buds, or young pods) compared to other tissues. The same conclusion is found in grapes (V. vinifera L.) [37], citrus (C. sinensis and C. grandis) [36] and tobacco [38] (Nicotiana tabacum L.).

Analysis collinear genes of PvB3s
In order to determine the relationship between PvB3s and potential repetitive events in evolution, the genomic information of less-studied biological genome taxonomic units was transferred to the biological taxonomic units that have been deeply studied [39]. Another use of collinear analysis provides theoretical and practical value for studying loci that control common traits among species. In 2013, Chen [40] identi ed the Rice (O. brachyantha.L) local duplications, insertions, and inversions by constructing a collinearity map of the whole genome comparison of rice and rice. The analysis further revealed the mechanism of rice genome changes, gene family expansion, gene migration and transformation. For common bean PvB3s perform collinear analysis between PvB3s and multiple species.By analyzing the collinear genes of family genes within species, LG 9 contains the most collinear genes, which can be inferred that in the process of evolution, gene replication events are most likely to occur in LG9; In the collinear analysis with three legume crops, the collinear genes in soybeans are signi cantly higher than that in adzuki bean (V. angularis) and mung bean (V. radiata.L), indicating that there may be more collinear genes due to soybean containing a larger genome; In the analysis of collinearity with Arabidopsis and Rice (O. sativa L.), the limit of collinear genes is reduced. Monocotyledonous Rice even has fewer collinear genes than dicotyledonous Arabidopsis, with only 5 paired genes, which may cause few collinear genes due to differences between species. Among 23 Arabidopsis genes which are collinear with PvB3s, as shown in Table 1. 14 of these genes have clearly stated that they are related to auxin or respond to IAA, 8 genes have been reported to be related to sprout stage or seedlings; the other 4 genes have been described to be related to stress resistance.These results predict that PvB3s is likely to be closely related to auxin response, plant seedling stage and stress resistance. In selective pressure analysis, this study found that most Ka/Ks are greater than 1, and only 3 pairs are less than 1, which indicated that 3 groups of genes chose pure selection in the face of evolution, and they maintained the changes of their ancestors under the requirements of evolution, but most PvB3s in common bean chose adaptive forward evolution in the process of evolution, thus deriving new functions. The relationship between PvB3s, IAA and salt stress in common bean sprouts The previous paragraph has shown that the B3 family gene and auxin response is very large, whether in the bud stage or other growth periods. Both in grapes and citrus have been reported to identify the relationship with auxin. But it also appears to be related to plant stress resistance in collinear genes. There are also many auxin-responsive genes, especially the B3 family genes of certain species, and their relationship with plant stress, especially salt stress; Rhizobacteria Pseudomonas Putida and Novosphingobium Sp can enhance the secretion of IAA to enhance the salt tolerance of citrus [59] ;MicroRNA390/ TRANS-ACTING SHORT INTERFERING RNA3 in poplar (Populus spp.), which can regulate the auxin pathway can be used to enhance the stress resistance of plants, especially the salt tolerance [60];The auxin pathway can play a role in maintaining the morphology and physiology of the Brassica napus seedlings in response to stress [61];IAA-Asp, a combination of IAA and amino acids, can directly and speci cally affect pea (P. Sativum L) response to abiotic stress [62].These all indicate that IAA has a very close relationship with salt stress, and auxin responsive gene IbARF5 has also been reported to enhance the salt tolerance of sweetpotato [63]. In this experiment, through identi cation and analysis of the common bean B3 gene family, PvB3s and auxin regulation pathways were found to be closely related by KEGG enrichment. Auxin was applied externally to observe the growth of common bean sprouts and the response of PvB3s, and further reveal PvB3s with the relationship between auxin and coercion (Fig. 11).

Conclusion
Despite the importance of B3 transcription factors for plant growth, development, and abiotic stress responses, little is known about the PvB3s in common bean. We used the previously published common bean reference genome to perform a comprehensive analysis of common bean B3s. The PvB3s and genes which have B3 domain in Arabidopsis were clearly divided into phylogenetic clades. These clades were supported by various group-speci c sequence characteristics, including exon/intron structure, MEME motif composition, Intra-species collinearity and inter-species collinearity. By analyzing expression of different tissues in PvB3s expression during sprout stage and extremely varieties in response to salt stress, we characterized the overall expression of PvB3s in sprouts.Transcriptomic, qRT-PCR analysis and KEGG enrichment revealed some PvB3s that may be important for the salt stress response by responding to IAA, and the application of IAA exogenously determined that the growth of common beans under salt stress could be changed, and the expression level of PvB3s could also be changed.. These informations generated in this study could facilitate further research on B3 family and other gene families in common bean.

Identi cation and Phylogenetic Analysis of the B3 Gene Family in common bean
The HMMER software (http://hmmer.janelia.org/) and the Pfam protein family database (http://pfam.sanger.ac.uk/) [24] were employed to identify the candidate B3 proteins at the B3 domain (PF02362). The protein annotation le was retrieved from the website of Esembl plants (http://plants.ensembl.org/index). Subsequently, InterPro (http://www.ebi.ac.uk/interpro/) [25] and SMART (http://smart.embl-heidelberg.de/) [26,27] programs were applied to verify the integrity of the B3 domain. Lastly, Interpro (http://prosite.expasy.org/),WoLF PSORT (https://wolfpsort.hgc.jp/), P3DB (http://www.p3db.org/) and ExPASy Proteomics Server (http://prosite.expasy.org/) were employed to verify the integrity of B3 domain in candidate genes. Each PvB3 was named according to their precise position on the chromosomes.All of the other species like Arabidopsis B3 protein sequences were obtained from Esembl plants, also used B3 domain. All these protein sequences of common bean and other species. PvB3s were imported into MEGA X [28], and multiple sequence alignments were performed Maximun Likelihoodphy using WAG + G model, which MEGA predicted, 1000 replicates were used to produce bootstrap values. MEGA X was utilized to edit and construct the phylogenetic tree.

Analyses of Gene Structure, Conserved Motif, Promoter
The exon/intron structure of PvB3s was analyzed and displayed using the GSDS platform (http://gsds.cbi.pku.edu.cn/) [29]. Gene wise [30] was used to determine the correspondence on coordinates between DNA (containing exon and intron together) and protein sequences. Then, the coordinates of B3 domain in protein sequence were transformed to that in gene sequence using in-house perl scripts. The intron splicing phase within the basic and hinge regions of B3 domains from all PvB3s were characterized and divided into different types.The MEME tool (http://meme.nbcr.net/meme/) [31] was employed to detect the additional motifs outside the B3 domain of protein sequences. The motifs with 10-50 amino acids in length and E-value less than 1e − 20 were characterized. All the motifs were compared among PvB3s to identify the group-conserved or group speci c signatures. These motifs were numbered according to their order in the protein sequences.

Availability of data and material
The raw RNA-seq data are available at the National Centre for Biotechnology Information under the accession number PRJNA558376 (http://www.ncbi.nlm.nih.gov/bioproject/) . The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Competing interests
The authors declare that they have no competing interests.      The Arabidopsis members and the common bean members containing the B3 domain, 4 different colors represent 4 subfamilies, the outer column represents the genetic structure of the B3 gene family of Arabidopsis and the common bean, the middle is the B3 protein phylogenetic tree of two species according to ML with WAG+G model, the inner The layer is the motif structure of genes of two species.