Genome-wide identification of the LBD transcription factor genes in common bean (Phaseolus vulgaris L.) and expression analysis under different abiotic stresses

ABSTRACT Lateral organ boundary Domain (LBD) proteins are plant-specific transcription factors that play a key role in plant lateral organ development and stress tolerance. However, LBD gene has not been identified in the common bean (Phaseolus vulgaris L.). Here, a total of 47 common bean LBD genes (PvLBDs) were identified. Members of the same subfamily had similar genetic structures. Synteny analysis indicated that LBDs in the common bean genome have greater collinearity with soybean (Glycine max L.) than with Arabidopsis and rice (Oryza sativa L.). Additionally, 9 pair of segmental duplication genes were identified by collinearity analysis. Phytozome data analysis showed significant differences in PvLBD gene expression abundance between different developmental stages of the same tissue. The qRT-PCR results showed that NaCl, CdCl2, and HgCl2 stresses up-regulated 19% and down-regulated 81% of the PvLBD genes. This study provides a basis for further analysis of the function of the PvLBD gene family.


Introduction
Lateral organ boundary Domains (LBD), also known as AS2/ LOB gene family, are plant-specific transcription factors that regulate lateral organ development, morphogenesis, and metabolism (Iwakawa et al. 2002;Majer and Hochholdinger 2011). The LBD gene was first identified at the base of the lateral organs of Arabidopsis through the insertion of enhancer traps (Iwakawa et al. 2002;Shuai et al. 2002). LBD transcription factors have three specific conserved structural domains arranged from the N to the C terminus, including the zinc finger domain (C-block, CX2CX6CX3C), the Gly-Ala-Serblock (GAS-block), and the leucine zipper-like module (LX6LX3LX6L). The C-block is essential for binding to DNA, the GAS-block affects DNA binding activity, and the leucine zipper-like module is associated with protein dimerization (Lee et al. 2009;Matsumura et al. 2009;Majer and Hochholdinger 2011). The LBD gene family can be classified into two subfamilies based on the characteristics of its structural domain: class I and class II. Class I members contain a conserved C-block, a GAS-block, and relatively complete leucine zipper-like motifs, while Class II members contain an incomplete leucine zipper-like domain. Most LBD family members belong to class I and play a key role in plant organ development and signal transduction (Majer and Hochholdinger 2011;Yu et al. 2020a). Class II LBD proteins are mainly involved in synthesizing secondary plant metabolites, such as anthocyanins (Zhang et al. 2020).
The LBD gene family members have been identified in many plants, including 43 in Arabidopsis, 44 in maize, 35 in rice, 57 in poplar, 46 in tomato, and 90 in soybean (Yordanov et al. 2010;Wang et al. 2013;Zhang et al. 2014;Guo et al. 2016;Yang et al. 2017Yang et al. , 2006. Previous studies have shown that the LBD gene family plays a crucial role in plant organ boundaries, especially in early morphogenesis of plant embryos, roots, leaves and flowers, tissue regeneration, and stress responses (Semiarti et al. 2001;Iwakawa et al. 2007;Goh et al. 2019). In Arabidopsis, AtLOB/AtASL4 is specifically expressed in the proximal base of lateral tissues and participates in early leaf development (Shuai et al. 2002). AtLBD3/AtASL9 is regulated by cytokinin and is implicated in the regulation of plant development in Arabidopsis (Naito et al. 2007). AtLBD6/AtAS2 inhibits cell proliferation in the axial region by regulating KNOX gene expression. It is also involved in the formation of the symmetric leaf lamina (Semiarti et al. 2001). AtLBD15 participates in apical meristem cell differentiation by regulating Wuschel (WUS) gene . AtLBD16 and AtLBD18 participate in the initiation and emergence of Arabidopsis lateral root formation and interact with downstream TFs ARF7 and ARF19 (Lee et al. 2009). AtLBD20 plays a key role in response to plant pathogen invasion by regulating the jasmonic acid (JA) signaling pathway during Fusarium oxysporum infection (Thatcher et al. 2012). In rice, OsARL1 encodes a protein with an LBD domain and is an auxin response factor in adventitious root formation (Liu et al. 2005). OsIG1, an LBD-like gene in rice, regulates the occurrence of empty-glume, abnormalities of various floral organs, and female gametophyte development (Zhang et al. 2015;Li et al. 2016a). Moreover, the overexpression of OsLBD3-7 induces adaxially-rolled leaves in rice. In addition, LBD gene family members have been implicated in stress responses in many plant species. GmLBD12 is involved in the response of soybean to abiotic stresses (drought, salt, cold) and hormones treatment indole acetic acid (IAA), abscisic acid (ABA), and salicylic acid (SA) treatment (Yang et al. 2017a). MaLBD5 mediates transcriptional activation of jasmonate biosynthesis gene MaAOC2 in regulating cold tolerance of banana fruit (Ba et al. 2016). All 30 LBD gene family members in grape have been implicated in stress response (He et al. 2018).
Common bean (Phaseolus vulgaris L.) is an important leguminous plant that is rich in nutrients, such as protein, fat, carbohydrate, and dietary fiber. The planting area of common bean has been expanding worldwide in recent years. However, abiotic stresses, such as saline-alkali stress, significantly affect the growth and quality of common bean (Talaat 2014). Therefore, research into the molecular mechanisms of stress response in common bean is an important topic in agricultural science. The structure and function of LBD family genes have been explored and analyzed in some plant species, and their roles in regulating plant growth and development and stress response have been verified. However, the molecular characteristics and functions of LBD family members in common bean have not been fully elucidated. In this study, we first identified the LBD gene family in the common bean using an in silico genomewide screening analysis. Then, the phylogenetic relationship, gene structure, chromosome distribution, evolutionary relationship, cis-regulatory elements, gene replication, collinearity, and spatio-temporal expression patterns of the putative common LBD genes (PvLBDs) were systematically analyzed. This study lays a foundation for further analysis of the function of PvLBD gene family in common bean and other crops and provides insights into the breeding of common bean varieties with high-stress resistance.
Phylogenetic, gene structure, and conserved motifs, and promoter predictions of PvLBD genes The LBD proteins of Arabidopsis, soybean, and maize were obtained from Ensembl plants using the LOB domain. All the LBD proteins, including PvLBDs, were used for multiple sequence alignment via ClustalX (Larkin et al. 2007) with default parameters. A phylogenetic tree with 1,000 bootstrap replications was modified and constructed using the Maximum Likelihood method of MEGA X based on the JTT + G model. The structure of PvLBD genes was obtained from the GSDS platform (http://gsds.cbi.pku.edu.cn/) (Guo et al. 2007). The correspondence between DNA and protein sequences was determined using GeneWise (Birney et al. 2004). An in-house Perl script was used to convert the coordinates in the LOB domain of the protein sequence to those in the nucleotide sequence. The MEME tool (http://meme. nbcr.net/meme/) (Bailey et al. 2009) was used to predict the conserved motifs with the following parameters: the width of characteristic motifs was between 10 and 50 residues, and the E value was less than 1e -20 . The upstream 1.5 kilobases (kb) genomic DNA sequences of the PvLBDs were retrieved from the common bean genome. The sequences were then submitted to the PlantCare database (http://bioinformatics.psb.ugent.be/webtools/plantcare/) to identify the putative cis-regulatory elements in the promoter regions.

Collinearity analysis
Multiple Collinearity Scan toolkit (MCScanX) was used to examine the synteny and collinearity of LBD genes (Wang et al. 2012). A visualization tool Circos (Version 0.69) (Krzywinski et al. 2009), was then used to express the collinearity of the duplicated genes.

Growth conditions and treatments
Common bean seeds (Longjiang Ziyun) were obtained from the National Coarse Cereals Engineering Research Center (Daqing, Heilongjiang, China). The seeds were placed on Petri dishes, then 11 mL of distilled water was added to each petri dish and incubated at 28°C in darkness to germinate. After germination, the seedlings were separately exposed to different stress treatments on the sixth day. Seedlings cultured with distilled water were used as controls (CK). Salt stress was induced by treating the seedlings with 70 mmol/L (NaCl) , while heavy metal stress was simulated by exposing the seedlings to 0.5 mg/L (CdCl 2 ) (Zhao et al. 2020) and 60 mg/L (HgCl 2 ) (Mohammadi et al. 2021) for 24 h.
RNA extraction, complementary DNA synthesis, and gene expression analysis RNA isolater Total RNA Extraction Reagent (Vazyme Biotech, China) was used to isolate total RNA from the samples. RNA quantity and integrity were determined by measuring the optical density of the extracted RNA samples at 260 nm and via 1.0% agar gel electrophoresis, respectively. Single-stranded cDNA was synthesized from the RNA samples using Evo M-MLV RT Premix for qPCR (AG11706, Accurate Biology, Hunan, China). The Light Cycler system (Roche 480II, Roche, Switzerland) and Trans-Start® Top Green qPCR SuperMix (AQ131-04, TransGen Biotech, Beijing) were used for qRT-PCR reactions. The related expressions were determined using the 2 -△△t method. The reference gene ACTIN-11 was used as the control . The primers used in this study are listed in Table S1. Each treatment had three biological replicates. Each sample also had three technical replicates.

Statistical analysis
Office Excel 2013 and SPSS version 17.0 (SPSS Inc., Chicago, IL, USA) were used for all data analyses (Li et al. 2018).
The 47 PvLBD genes are distributed on 11 chromosomes ( Figure 1). There are 9 PvLBDs (PvLBD28-36) on Chromosome 8, while chromosomes 2, 6, and 10 have two PvLBDs genes each. The chromosomal localization showed that the distribution and density of the PvLBDs are uneven and clustered. Most PvLBD genes are distributed at both ends of the chromosome, with a few in the middle.

Phylogenetic analysis and conserved sequence alignment
To clarify the evolutionary relationships between common bean PvLBD proteins and LBD proteins of other plant species, we constructed a phylogenetic tree based on multiple protein sequence alignment of 47 PvLBDs proteins, 90 LBDs proteins of leguminous crops (soybean, GmLBDs), 47 LBDs proteins of the dicotyledonous model plant (Arabidopsis, AtLBDs), and 44 LBDs proteins of monocotyledon plants (maize, ZmLBDs) ( Figure 2).
Phylogenetic analysis suggested that the LBDs could be classified into two major clades (class I and II). Class I had 216 members: 43, 85, 46 and 42 in common bean (91.5%), soybean (94.5%), Arabidopsis (97.9%), and maize (95.5)respectively. Class II had 12 members: 4, 5, 1, and 2 in common bean (8.5%), soybean (5.5%), Arabidopsis (2.1%), and maize (4.5)respectively. Moreover, class I could be subdivided into eleven sub-classes (Ia-Il), and sub-classes (II), respectively (Figure 2 and Figure S1). Phylogenetic relationships indicated that the LBD proteins of common bean are more homologous to soybean LBD proteins than those of Arabidopsis or maize. Multiple sequence alignments were created for the 47 PvLBD proteins to investigate the presence of conserved protein domains (Figure 3). The results indicated that all the PvLBDs have a conserved cysteine-rich C-motif (CX2CX6CX3C). 43 PvLBDs had complete cysteine-rich C-Motif, GAS-block, and leucine zipper-like structure and were classified as class I, while the remaining 4 PvLBDs had incomplete leucine-zipper structures and belonged to Class II.

Gene structure and conserved motif analysis
To further investigate the evolutionary relationships among the 47 PvLBDs, we utilized full-length PeLBD protein sequences to construct a second phylogenetic tree. The results showed that the 47 PvLBDs were classified into two major groups, class I and class II. Furthermore, class I was divided into 11 subclasses, including Ia, Ib, Ic, Id, Ie, If, Ig, Ih, Ii, Ij, Ik, and Il with 3, 4, 2, 5, 1, 2, 9, 2, 4, 5, 2, and 4 members, respectively. While Class II had 4 members ( Figure 4A). The MEME program identified 10 motifs according to PvLBDs sequence characteristics ( Figure 4B). All LBDS contained motifs 1 and 2, constituting a highly conserved part of the LOB structural domain. The relative positions of motifs in most sequences were similar. However, each subgroup had specific motifs, indicating that the PvLBD gene family members have different evolutionary processes.

Collinearity analysis and the tandem replication of PvLBDs
Gene duplication events are common in all species and can generate new functional genes and drive evolution. Herein, nine gene pairs were identified as duplicated gene pairs in common bean PvLBD gene family, including PvLBD36/ PvLBD27, PvLBD37/PvLBD19, PvLBD30/PvLBD20, Figure 2. Phylogenetic analysis of PvLBD proteins in common bean (Phaseolus vulgaris L.s), soybean (Glycine max), maize (Zea mays), and Arabidopsis thaliana. MEGA X was used to construct a Maximum-likelihood phylogenetic tree with the JTT + G model. The yellow, blue, green, and red circles indicate soybean, maize, Arabidopsis thaliana, and common bean, respectively.
PvLBD30/PvLBD5, PvLBD31/PvLBD20, PvLBD25/PvLBD2, PvLBD20/PvLBD5, PvLBD18/PvLBD33, PvLBD7/ PvLBD11. Notably, the nine gene pairs belong to fragment replication ( Figure 5A). The orthologous relationship between PvLBD genes in common bean and Arabidopsis, soybean, and maize genomes was assessed to clarify the evolutionary differences between LBD genes. The LBD family genes in common bean were collinear with 24 genes in Arabidopsis, 51 in soybean, and four in maize ( Figure 5B). Therefore, there was higher collinearity between the common bean and soybean genomes than between the common bean and monocotyledon genomes. Furthermore, the most soybean LBD genes had corresponding orthologs in moso bamboocommon bean, and most of them had more than two orthologs, suggesting that moso bamboo has undergone additional WGD event(s) during its evolution.
Selection pressure analysis of coding sequences was expressed using the non-synonymous mutation rate (Ka) to synonymous mutation rate (Ks) ratio. Ka/Ks < 1 represents purification selection or negative selection, while Ka/Ks > 1 represents Darwinian selection or positive selection. PvLBD30/PvLBD5 and PvLBD30/PvLBD20 underwent purification selection with the Ka/Ks < 1, while PvLBD20/ PvLBD5 and PvLBD18/PvLBD33 underwent positive selection with the Ka/Ks > 1 (Table S3).

Analysis of Cis-regulatory element in PvLBD promoters
Cis-acting elements are non-coding DNA sequences in gene promoter sequences that regulate gene transcription. Genomic sequences (1.5-kb) genomic sequences (1.5-kb) upstream from 5'-UTR of 47 PvLBDs were extracted from common bean genome sequences to investigate the cis-acting elements of PvLBDs. Fifteen cis-regulatory elements belonging to hormone-, resistance-, and development-related elements, were identified using Plant CARE software ( Figure 5 and Table S2). Several plant hormone response elements were found in the promoter of PvLBDs. Particularly, multiple copies of abscisic acid elements(ABRE) were found in most PvLBD promoter regions. Additionally, three resistance-related elements (MBS: drought stress inducibility element, LTR: low-temperature responsiveness, and ARE: anaerobic responsiveness) were identified. MBS was identified in 13 PvLBD genes, LTR in seven PvLBD genes, and ARE in 36 PvLBD genes. Development-related elements were also identified, including CAT-box (meristem expression element) and RY-element (seed-specific regulation element). The CATbox was found in 14 PvLBD genes and RY-element in three PvLBD genes. These results suggest that PvLBD family may be involved in hormone response, defense response, and growth regulation.

Tissue-specific expression of PvLBD genes
The transcriptional profiles from the Phytozome database were used to explore the expression patterns of PvLBD genes in nine different tissues, including flower buds, flowers, mature green pods, leaves, nodules, root, stem, young pods, and young trifoliate. The 47 PvLBD genes had different expression patterns among the tissues (Figure 7). Seventeen PvLBD genes were detected in all tissues, suggesting their involvement in the development or physiology of multiple tissues. However, four PvLBD genes (PvLBD1/9/31/36) were undetectable in the tissues. Three genes (PvLBD6/11/28) were highly expressed in flower buds, flowers, mature green pods, and pods, suggesting that they potentially participate in the development and function of common bean reproductive organs. Additionally, two genes (PvLBD37/47) were highly abundant in young trifoliate, one gene (PvLBD13) was highly abundant in leaves, five genes (PvLBD3/21/23/26/44) were highly abundant in roots, five genes (PvLBD16/20/22/31/32) were highly abundant in root nodules, and two genes (PvLBD10/45) were highly expressed in stems.
There was a significant difference in PvLBD gene expression abundance between different developmental stages of the same tissue. The expression abundances of  PvLBD1/17/41 in flower buds were significantly higher than in flowers, while the expression abundances of PvLBD40/15/ 35 were significantly lower than in flowers. The expression abundances of PVLBD6/11/18/28 in mature green pods were significantly higher than in young pods, while the expression abundances of PVLBD 8/13/14/17/46 were significantly lower than in young pods. The expression abundances of PVLBD 37/47 in young trifoliate were higher than in leaves, while the expression abundances of PVLBD 13/20 were lower than in leaves.

Stress-induced expression patterns of PvLBD genes
The expression abundances of the 47 PvLBDs in cotyledon, hypocotyl, and radical of common bean under NaCl, CdCl 2, and HgCl 2 stress were detected using qRT-PCR to evaluate the roles of PvLBDs in response to abiotic stress (Figure 8). The PvLBD1 /6 /11 /20 /31 /34 /38 /40 /41 genes in common bean cotyledon, hypocotyl, and radical were significantly up-regulated under NaCl, CdCl 2, and HgCl 2 treatments. In contrast, the other 36 PvLBDs were down-regulated under the abiotic stress. Notably, the PvLBD6/11 gene expression in common bean cotyledon, hypocotyl, and radical was higher under NaCl stress than CdCl 2 and HgCl 2 stresses. Moreover, the expression abundances of PvLBD1/40 genes in common bean cotyledon, hypocotyl, and radical were higher under CdCl 2 stress than under NaCl and HgCl 2 treatments. The expression abundances of PvLBD20/31/34/38/41 genes in common bean cotyledon, hypocotyl, and radical were higher under HgCl 2 stress than under other stress treatments. It's worth noting that the expression levels of PvLBD1/6/11/ 20/31/34/38/40/41genes were up-regulated by all the three stress treatments.

Discussion
Herein, 47 PvLBD genes were identified in the common bean genome and classified into two classes, class I (40, 85%) and class II (7, 15%). In addition, class I genes were further divided into 13 subclasses (Figure 2). Previous studies have shown 86% of AtLBD genes in Arabidopsis, 82% of GmLBD genes in soybeans, and 84% of ZmLBD genes in maize belong to class I (Shuai et al. 2002;Zhang et al. 2014;Yang et al. 2017). Similarly, in this study, the number of LBD members was significantly higher in class I than in class II. A total of 223 LBD genes from the 4 species were further divided into 13 subclasses (Ia -Il and II), and their phylogenetic relationships were consistent with those reported in previous studies (Shuai et al. 2002;Zhang et al. 2014;Yang et al. 2017). Furthermore, the expression abundance of homologous PvLBD gene pairs in the same tissue or under the same stress was similar (Figures 7 and 8), suggesting that the PvLBD gene pairs in common bean may have mainly led to functional redundancy.
Gene structure analysis showed that the conserved domains differed between class I and II (Figures 3 and 4). Moreover, some members of the same subclass had structural differences. This difference may be because members of different subclasses underwent splicing or insertion of gene fragments during evolution (Staiger and Brown 2013;Li et al. 2016b). Similar conserved sequences and gene structures in the LBD subclass often imply that these genes have similar biological functions, an indication that the motifs of the LBD family have been widely conserved throughout the evolutionary process.
The LBD gene family in common bean is larger than in Arabidopsis (Figure 2), possibly due to the expansion of PvLBD genes, like in soybean (Yang et al. 2017) and maize (Zhang et al. 2014). Gene replication produces differentiation in gene function, a critical factor in environmental adaptation and speciation (Conant and Wolfe 2008). Functional differentiation of duplicated gene pairs may occur during evolution, resulting in neo-functionalization, subfunctionalization, or non-functionalization (Prince and Pickett 2002). Gene duplication is the most common evaluation mechanism for gene family expansion. Gene family expansion in plants mainly occurs through tandem and fragment duplications (Cannon et al. 2004). Tandem replication mainly occurs in the recombination region of chromosomes. The gene family members formed by tandem replication are usually closely arranged on the same chromosome, forming a gene cluster with similar sequence and similar function. In contrast, genes formed via fragment duplication are usually far apart or even located on different chromosomes (Holub 2001). The duplicated genes remain in the plant genome and play an essential role in adaptation to the environment (Hanada et al. 2008;Jiang et al. 2010). In this study, 9 pairs of PvLBD genes were formed through fragment duplication and thus located on different chromosomes ( Figure 5A). Furthermore, 24, 51, and 4 common bean PvLBD genes homologous to Arabidopsis, soybean, and Arabidopsis LBD genes, respectively, were detected using a phylogenetic tree ( Figure  5B). Gene duplication analysis did not detect any tandem duplication in PvLBD gene family, indicating that tandem duplication does not significantly affect the evolution of common bean LBD gene family, similar to other plant species Chen et al. 2020). Therefore, fragment repetition may play a dominant role in the expansion of the PvLBD gene family. Gene replication and collinear analysis confirmed that although PvLBD genes did not undergo positive selection, they underwent strong purification selection, which was consistent with LBD genes in soybean (Yang et al. 2017) and maize (Zhang et al. 2014).
Cis-acting elements located in gene promoters play a crucial role in signal transduction and gene expression regulation. In this study, we found that the PvLBD promoters contain numerous motifs related to hormone response pathways, including those of gibberellin, abscisic acid, salicylic acid, and auxin ( Figure 6). Thus, we concluded that PvLBD genes might participate in the plant growth regulation and stress response. In particular, the response elements of abiotic stress such as drought stress, low-temperature, anaerobic were found on PvLBD genes. Nonetheless, further molecular analysis should be performed to verify the specific expression patterns of different LBD genes.
LBD proteins regulate the development of plant branches and the formation of flowers, stems, leaves, and roots (Semiarti et al. 2001;Iwakawa et al. 2002;Majer et al. 2012;Cabrera et al. 2014;Yu et al. 2020aYu et al. , 2020b. Tissue-specific expression of genes can provide insights into their functions in growth and development (Xiao et al. 2019). In this study, PvLBD6/ 11/28 genes were highly expressed in the flowers and pods of common bean, implying involvement in the development of reproductive organs (Figure 7). Furthermore, PvLBD10 and PvLBD45 were specifically expressed in the stem of common bean, suggesting that they may regulate the growth and development of common bean. PvLBD3/21/23/26/44 genes were specifically expressed in roots, suggesting that these genes participate in root development. Further analysis showed that PvLBD genes in class I were highly expressed in specific tissues. This is similar to the findings of previous that showed that different LBD genes regulate plant growth in different periods and different tissues. For example, PeLBD20/38 genes were upregulated in bamboo shoots during rapid growth and development period, indicating that they are involved in the rapid growth and development of bamboo shoots (Huang et al. 2021). AtLBD13/16/18/19 are highly expressed in A. thaliana roots and regulate lateral root development (Cannon et al. 2004;Goh et al. 2019;Lee et al. 2019). RA2, an ortholog of LOB, can regulate reproductive growth and participate in maize inflorescence morphogenesis (Vollbrecht et al. 2005;Bortiri et al. 2006).
The accumulation of Cd, Ni, Hg, and other heavy metal ions due to climate change and modern industrial development has led to soil heavy metal pollution and significantly affected plant growth and production. In this study, Na + , Cd 2+, and Hg 2+ stresses regulated all PvLBD genes, of which 17% of the genes were up-regulated while 83% were down-regulated (Figure 8). These results indicate that PvLBD genes are extensively involved in various abiotic stress responses in common bean, similar to previous studies that showed that LBD genes regulate resistance to stress (Kong et al. 2017;Zhang et al. 2019;Wang et al. 2021). Notably, the expression levels of PvLBD1/6/11/20/31/34/38/40/ 41genes were up-regulated by all the three stress treatments, indicating that these genes were participate in response to multiple heavy metal stresses. Previous studies have also shown that LBD genes were differentially expressed under different abiotic stresses. For instance, StLBd2-6 and StLBD3-5 are up-regulated in potato under drought stress . In wheat, Ta-6B-LBD81, Ta-4B-LBD51, and Ta-U-LBD90 are up-regulated under salt stress, Ta-4A-LBD40 and Ta-4D-LBD62 are up-regulated under cold stress, while Ta-2A-LBD13, Ta-2B-LBD15, and Ta-2D-LBD18 are up-regulated under drought stress (Wang et al. 2021). In this study, PvLBD6 and PvLBD11 were highly up-regulated under NaCl stress; PvLBD1 and PvLBD40 were highly upregulated under CdCl 2 stress; PVLBD20/31/34/38/41 were highly up-regulated under HgCl 2 stress (Figure 8). These results indicate that PvLBD genes play different roles in regulating different metal stress responses in the common bean.

Conclusions
In this study, 47 putative PvLBDs were identified in common bean and classified into two classes and thirteen subclasses. Gene structure and phylogenetic analysis showed that each subclass has a similar gene structure and motif composition, suggesting that PvLBDs have remained conserved throughout evolution. Furthermore, the PvLBD gene family probably completed the genome expansion through the fragment repetition. The promoter of PvLBD contains a large number of cis-acting elements related to plant development, hormone and abiotic stress response. Tissue-specific expression pattern revealed that LBD genes have diverse functions and are involved in multiple tissue and developmental processes of common bean. Most LBD gene family members had significant responses to NaCl, CdCl 2, and HgCl 2 stresses. This study provides candidate genes for further assessing the function of PvLBD gene family in common bean, and insights for exploring the regulatory role of LBD genes on growth and stress resistance in common bean.

Author contributions
QZ, YZ and JD conceived and designed the experiments. YD, QZ, YG and WL performed the experiments; WL, JG, JZ and SL validated the data; QZ, XY, and SL performed the formal analysis; YD and QZ participated in original draft preparation; YD, QZ, YZ and JD revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Disclosure statement
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.