Introduction

Among various transcription factor families, dehydration responsive element (DRE) binding factor, ERF (ethylene responsive factors), WRKY, MYB, bZIP and basic helix-loop-helix (bHLH) families play crucial roles in coordination of regulatory networks related with stress responses (Zhou et al. 2009; Cui et al. 2013; Lata et al. 2014; Yan et al. 2014). The latter is a universal transcription factor found in all eukaryotes. Although, most eukaryotes carry the proteins of this family, the biological functions of the most bHLH family members are not deeply evaluated in plants (Liu et al. 2014). Up to now, studies related with function of plant bHLH proteins were relatively limited when compared with their animal orthologs (Song et al. 2014). There are a few reports showing the role of bHLH genes under salt stress in plants such as Arabidopsis, rice, and tomato (Zhou et al. 2009; Feng et al. 2013; Liu et al. 2014). The bHLH proteins are characterized with specific signature domain consisting of about 60 amino acids. Each domain has two distinct regions having different function. The first segment, which consist of about 15 amino acids with typical six basic residues that they have role in binding to DNA, is called as basic region, which settled in the N-terminus (Atchley and Fitch 1997). With the increasing number of completed and drafted genomes, a numbers of identified bHLH gene families have been also increased. For instance, a total of 225, 480 and 206 bHLH-encoding genes from Arabidopsis, soybean, and potato have been reported in plant transcription factor databases, respectively (Jin et al. 2014). It has been also reported that plants includes 26 subfamilies of bHLH proteins according to the phylogenetic analyses (Pires and Dolan 2010).

Soil salinity is one of the most important environmental stress factors affecting crop yield worldwide (Munns and Tester 2008). High salinity effects wide range of developmental process and cause to retardation of growth, decrease in biomass, and leaf senescence (Munne-Bosch and Alegre 2004). Due to their sessile nature, plants are exposed to any of abiotic stresses use different physiological, biochemical and molecular mechanisms including reprogrammed transcriptome pattern to cope with the detrimental effects of those conditions (Yamaguchi-Shinozaki and Shinozaki 2006). Molecular and physiological responses against diverse stress factors are controlled by the genetic regulation at transcriptional level, which are mainly regulated by transcription factors (TF) (Płażek et al. 2013; Dou et al. 2014; Thamilarasan et al. 2014). TF is defined as a group of proteins involved in regulation of specific gene expression either positively or negatively by binding to promoter or enhancer region of DNA (Latchman 1997).

Pulse crops have a crucial role in sustainable agriculture because of theirs nitrogen fixing ability with rhizobia. Common bean or Phaseolus vulgaris L., one of the most consumed pulse crops, is an important source for human diet by providing an important part of proteins, micronutrients and calories of daily needs (Petry et al. 2015). Common bean is available as dry, canned, and frozen forms in market. The dried seeds have more economical value than others. The annual global bean production is about 23 million metric tons. With an annual production of 3.3 million tons, India is in the first place (Food and Agriculture Organization of the United 2014). The complete genome sequence of common bean, a true diploid with genome size estimated to be 587 Mbp, was recently published (Schmutz et al. 2014) and publicly accessible (http://www.phytozome.net/commonbean.php). It will be easier to do genomic and comparative analyses, such as genome-wide identification of transcription factors, by using this genome data (Schmutz et al. 2014). However, analysis of bHLH in common bean has not been carried out at genome-wide level against different stress conditions.

In this study, we conducted a comprehensive genome-wide analysis of bHLH TFs in common bean. We also characterized genomic structures, chromosomal locations and sequence homologies of all common bean bHLH TF genes. The expression analysis of selected bHLH genes was conducted using qRT-PCR and previously uploaded RNAseq data in root and leaf tissues under salt stress conditions.

Materials and methods

Sequence retrieval, analysis and phylogenetic tree construction

The bHLH amino acid sequences belong to P. vulgaris were identified by the combination of three approaches. Firstly, bHLH encoding amino acid sequences pertaining to 13 different plants (Arabidopsis thaliana, Carica papaya, Brachypodium distachyon, Cucumis sativus, Physcomitrella patens, Oryza sativa subsp. japonica, Populus trichocarpa, Sorghum bicolor, Glycine max, Medicago truncatula, Vitis vinifera, Lotus japonicus and Zea mays) were downloaded from plant transcription factor database 3.0 (http://planttfdb.cbi.pku.edu.cn) (Jin et al. 2014). Totally, 2736 amino acid sequences encoding bHLH transcription factors were retrieved. In order to determine homologous peptides from common bean, a BLASTP search at PHYTOZOME v10.1 database (http://www.phytozome.net) were carried out using default parameters (Goodstein et al. 2012). Additionally, the keyword search was performed with bHLH in PHYTOZOME v10.1 database (http://www.phytozome.net). Moreover, The Hidden Markov Model (HMM) profiles of the bHLH domains in the Pfam database (http://pfam.sanger.ac.uk) were searched against the PHYTOZOME v10.1 database (http://www.phytozome.net) of P. vulgaris. All hits with expected values less than 1.0 were downloaded as fastq files and redundant sequences were removed using the decrease redundancy tool (http://web.expasy.org/decrease_redundancy) and elimdupes (http://hcv.lanl.gov/content/sequence/elimdupes/elimdupes.html). Every non-redundant sequence identified was subsequently checked for the presence of the conserved bHLH domain by SMART (Letunic et al. 2012) and Pfam (http://pfam.sanger.ac.uk) searches to confirm that it belong to bHLH transcription factors family.

The final amino acid sequences of bHLH proteins were aligned in ClustalW by using CLC Genomics Workbench 7.0 software (CLC bio, Aarhus, Denmark) with default parameters. Then, a phylogenetic trees of aligned common bean bHLH proteins was constructed using MEGA 6.0.1 and CLC Genomics Workbench via the Neighbor-Joining (NJ) method with the following parameters: WAG protein substitution model, gamma distribution, and bootstrap (1000). The phylogenetic tree was visualized with ITOL (Letunic and Bork 2007). The density of bHLH was determined as the ratio of number of bHLH to genome size in Mb (Song et al. 2014). The conserved motifs in full-length bHLH proteins were identified CLC Genomics Workbench 7.0.

Gene ontology (GO) and functional annotation

The functional characterization and annotation of bHLH sequences were performed using CLC Genomics Workbench Blast2GO plugin with default parameters. First, the common bean amino acids sequences were used as queries in a BLASTP search launched from CLC Genomics Workbench. The BLAST search is run against the non-redundant (NR) database at NCBI with an e-value of 1xE-10 and we used the top 50 alignments for each sequence. The resulting Multi Blast data collection is then converted into a Blast2GO Project. GO annotation is carried out by applying the Blast2GO annotation rule, which computes an annotation score for each candidate GO term.

Chromosomal location, gene-structure prediction and genomic distribution of bHLH genes

The exact positions of bHLH genes on common bean chromosomes were determined by BLASTN search of the P. vulgaris sequences against the PHYTOZOME v10.1 database using default settings (http://www.phytozome.net). The genes were aligned separately onto eleven bean chromosomes based on their ascending order of physical position (bp), from the short-arm telomere to the long-arm telomere and finally displayed using MapChart v2.2 (http://www.wageningenur.nl/en/show/Mapchart.htm) (Voorrips 2002). The exon–intron structure of each bHLH gene was determined by aligning the full-length cDNA sequence or predicted coding sequence (CDS) with the genomic sequence in the bean genome database (http://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Pvulgaris). The gene schematic structure was drawn by the Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/index.php) (Hu et al. 2014). Tandem and segmental duplications were also identified according to the method of Plant Genome Duplication Database (Tang et al. 2008). In detail, adjoining genes of same sub-family located within 30 kbp of each other were characterized as tandem duplication (Du et al. 2013; Shiu and Bleecker 2003). Firstly, BLASTP search was performed against all predicted peptide sequences of common bean for segmental duplications and top five matches with ≤1e−05 was identified as potential anchors. Then, MCScan was used for evaluation of collinear blocks and finally alignments with ≤1e−10 were considered as significant matches (Tang et al. 2008; Du et al. 2013).

Estimation of synonymous and non-synonymous substitution rates

To understand the expansion of this important gene family, the ratios of non-synonymous (Ka) versus synonymous (Ks) substitution rates (Ka/Ks) were estimated. The amino-acid sequences tandem and segmental duplicated protein-encoding bHLH genes were aligned using CLUSTALW according to multiple sequence alignment tool. For estimation of the synonymous (Ks) and non-synonymous (Ka) substitution rates, alignments of the amino-acid sequences and their respective original cDNA sequences of bHLH genes were performed in the CODEML program of PAML interface tool of PAL2NAL (http://www.bork.embl.de/pal2nal) (Suyama et al. 2006). Time of duplication (million years ago, Mya) and divergence of each bHLH genes were calculated using a synonymous mutation rate of λ substitutions per synonymous site per year, as T = Ks/2λ (λ = 6.5 × 10e−9) (Lynch and Conery 2000; Yang et al. 2008).

3D protein homology modeling and protein properties

First of all, BLASTP search with the default parameters was performed in the Protein Data Bank (PDB) (Berman et al. 2000) with all bHLH proteins for identification of the best template having similar sequence and known three-dimensional structure. Using ‘intensive’ mode in Protein Homology/Analogy Recognition Engine (Phyre2) (Kelley and Sternberg 2009), the data was analyzed for prediction of protein structure of bean bHLHs. The theoretical isoelectric point (pI) and protein statistics were analyzed using ExPASy (Gasteiger et al. 2003) and Sequence Manipulation Suite (http://www.bio-soft.net/sms/), respectively. The PROSOII program (http://mips.helmholtz-muenchen.de/prosoII/prosoII.seam) was used to predict the sequence based solubility of proteins (Smialowski et al. 2012).

In silico identification of miRNAs targeting the bHLH genes

The perfect or closely perfect complementarity between miRNAs and their target genes can be used for identification of miRNAs specifically targeted a gene (Rhoades et al. 2002). To predict potential miRNAs targeting the bHLH genes, the web-based psRNA Target Server (http://plantgrn.noble.org/psRNATarget) was used with default parameters. Additionally, identified miRNA targets were further analyzed using BLASTX searches with ≤1e−10 against P. vulgaris EST sequences at NCBI database. The interaction between identified miRNAs and their bHLH target transcripts were visualized by using CYTOSCAPE v3.2.1 (http://www.cytoscape.org/).

Expression analysis of PvbHLH genes using transcriptome data

To evaluate the common bean bHLH gene expression patterns, we used the Illumina RNA-seq data downloaded from Sequence Read Archive (SRA), following accession number SRR957668 (salt-treated leaf), SRR957667 (control leaf), SRR958472 (salt-treated root and SRR958469 (control root) that were reported previously (Hiz et al. 2014). Original RNA-seq data includes 52,858, 60,590, 51,564 and 59,510 unigenes in control leaf, control root, salt-treated leaf and salt-treated root, respectively. All raw reads, downloaded as single SRA files, was spitted into two paired-end files and converted to “fastq” format by the NCBI SRA Toolkit’s fastq-dump command. The quality of fastq files were evaluated with FASTQC and low-quality reads [Phred quality (Q) score <20] were trimmed with CLC Genomics Workbench 7.0. After final quality check, the reads were aligned to Phaseolus vulgaris genome (v1.0), downloaded from PHYTOZOME V10.1 database (http://www.phytozome.net), by using CLC Genomics Workbench with default parameters.

Uniquely mapped reads were used in the expression level determination. Normalization of the gene expression values were carried out by the reads per kilobase of exon model per million mapped reads (RPKM) algorithm (Mortazavi et al. 2008). To identify differentially expressed bHLH genes, a FDR-value ≤0.001, fold change (RPKM-tr/RPKM-cont) ≥2 and the absolute ratio of log2 (RPKM-tr/RPKM-cont) ≥1 were used as threshold values. Finally, the heat maps of hierarchical clustering were visualized with PermutMatrix (Caraux and Pinloche 2005).

Plant materials, growth conditions, and salt stress applications

The salt tolerant “Ispir” variety was used for gene expression analysis (Dasgan and Koc 2009). Surface sterilization of seeds was carried out by applying 10 % (v/v) hypochlorite solution for 15 min and rinsing three times with distilled water. The seeds were germinated in vermiculite containing plug trays at 24/20 °C cycle under a 16 h/8 h photoperiod with 350 μmol m−2 s−1 light intensity, and 50–60 % relative humidity. They watered daily with Hoagland’s solution (Hoagland and Arnon 1950) in a growth chamber up to trifoliate leaf stage. During salt treatment, seedlings were kept at same conditions and watered with same solution including 150 mM NaCl. After 9 days, root and leaf tissues were collected from salt treated and control plants in order to conduct physiological and gene expression analyzes.

RNA extraction and quantitative real-time PCR analysis

Total RNAs of leaf and root tissues were isolated by RNeasy Plant Mini Kit (Qiagen, Valencia, California, USA) according to the manufacturer’s protocol and quantified using NanoDrop 2000D UV–VIS spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and quality-checked using Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The first strand of cDNA was synthesized by using 1 µg of DNAse I treated total RNA in a 20 µl reaction volume with RevertAid™ First Strand cDNA Synthesis Kit (Thermo Scientific, USA).

qRT-PCR experiments were conducted in order to measure tissue-specific expression levels of the salt responsive PvbHLH TF family genes that showed differentially upregulated expression pattern in RNA-seq analysis upon salt stress. qRT PCR analysis was performed by following the previously reported method (Turktas et al. 2013) using SYBR Green I Master Kit (Roche Germany) on LightCycler 480 Instrument II (Roche, Germany). Primers were designed individually for root and leaf tissues in P. vulgaris cv. İspir. Leaf specific primers were PvbHLH-54, PvbHLH-148, PvbHLH-69, PvbHLH-53, PvbHLH-98, PvbHLH-28, PvbHLH-124, PvbHLH-82, PvbHLH-128 and root specific primers were PvbHLH-54, PvbHLH-148, PvbHLH-69, PvbHLH-81, PvbHLH-104, PvbHLH-04, PvbHLH-133, PvbHLH-116, PvbHLH-119 and PvbHLH-97. Besides PvbHLH-54, PvbHLH-148 and PvbHLH-69 were designed for both leaf and root tissues since they showed higher expression level in RNA-seq analysis. The common bean 18S rRNA gene was used as an internal control (Budak et al. 2013; Wang et al. 2013). A list of the primers used in qRT-PCR is given in Table S1. The qRT-PCR was carried out in 96-well optical plates. PCR reactions were performed in a total volume of 20 μl containing 0.1 μl reverse and forward primers (100 pmol), 2 μl of cDNA, 10 μl FastStart SYBR Green I Master Mix and nuclease-free water was added up to 20 μl. The qRT-PCR conditions were set up as follows: preheating at 95 °C for 5 min; followed by 55 cycles of 95 °C for 10 s; 53 °C or 55 °C for 20 s; and 72 °C for 10 s. The melting curves were adjusted to 95 °C for 5 s and 55 °C for 1 min and then cooled to 40 °C for 30 s. All reactions were repeated three times with triple biological replicates. The expression levels were calculated as the mean-signal intensity across the three replicates.

MDA and proline analysis

To assess membrane damage because of salinity stress treatments, lipid peroxidation in terms of malondialdehyde (MDA) content was measured with spectrophotometer by using the method of Ohkawa et al. (1979). For measurement of membrane damage, about 0.2 g fresh shoot tissues were homogenized with liquid nitrogen by the addition of 1 ml of 5 % trichloroacetic acid (TCA). The homogenates were transferred to fresh tubes and centrifuged at 11,200 g for 15 min at room temperature. Equal volumes of supernatant and 0.5 % thiobarbituric acid (TBA) in 20 % TCA (freshly prepared) were put into eppendorf tubes and incubated for 25 min at 96 °C. The tubes were placed in ice bath after incubation period and then centrifuged at 7800g for 5 min. Absorbance of the supernatant was determined at 532 nm and the correction for non-specific turbidity was performed by subtracting the absorbance at 600 nm. MDA contents were calculated using an extinction coefficient of 155 mM−1 cm−1.

Proline contents of salt treated and control samples were estimated according to the method of Bates et al. (1973). 0.3 g of each collected sample was grinded with liquid nitrogen and the homogenized extracts were suspended with 1 ml of 3 % sulphosalicilic acid. Supernatants were obtained with centrifugation at 15,000g for 5 min at 4 °C and 0.1 ml of supernatants were mixed with 0.2 ml acid ninhydrin, 0.2 ml 96 % acetic acid and 0.1 ml 3 % sulphosalicilic acid. Toluene was added to this mixture after the incubation at 96 °C for 1 h. In order to achieve phase separation, another centrifugation was performed at 15,000g for 5 min at 4 °C. Absorbance measurements were carried out with upper phases at 520 nm. In order to determine the proline amounts in samples, proline standard curve was used. The curve was formed from the measurement of absorbance of proline standard (1, 10, 50, 100, 150, 200 and 300 µM) suspended in 3 % sulphosalicilic acid.

Results

Identification of PvbHLH genes and their phylogeny

A total of 155 bHLH genes encoding bHLH domain was identified in common bean genome (Table S2). The protein coding sequences in the representative genomes of 13 plant species was searched for comparative genomic analyses (Table S3). Finally, 2060 genes encoding bHLH domain were identified in these selected plant species. The density of PvbHLH is about 0.2640 which is lower than most of the analyzed plants. Zea mays (0.098), Physcomitrella patens (0.2304) and Lotus japonicus (0.2038) has lower density when compared to ratio of the number of PvbHLH to genome size (Mb). Unrooted phylogenetic trees, constructed by CLC Genomics Workbench by ClustalW (Thompson et al. 1997) alignment by the Neighbor-Joining method (Saitou and Nei 1987) and bootstrap analysis (1000 replicates), was used in order to elucidate the phylogenetic relationships between PvbHLH domain sequences. The conserved motifs were predicted by the CLC Genomics Workbench 7.0 (Sup. Figure 1). The alignment indicated that the residues Arg-20, Arg-21, Leu-31 and Pro-37 were completely conserved in bHLH domains of all identified PvbHLH proteins. In general, bHLH proteins clustered in together shared similar motif composition. Based on the conserved motifs and phylogenetic tree, we identified 8 major groups of PvbHLH consisting of 21 subfamily (Fig. 1).

Fig. 1
figure 1

Relationship of neighbor-joining phylogenetic tree of the PvbHLH domains

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The identified members of PvbHLH genes have significantly different size, fundamental chemical and physical characters. Although, the bHLH domains generally located close to N-terminal, location of the domains within the protein also differs. Protein length of PvbHLHs is changed from 90 to 725 amino acids. According to an instability index (II), most of the PvbHLH proteins were estimated as unstable in a test tube. EXPASY analysis revealed that the PvbHLH protein sequences had large variations in isoelectric point (pI) values (ranging from 4.67 to 10.18) and molecular weight (ranging from 10.365 to 79.679 kDa). Sequence based solubility prediction was performed and almost half of the PvbHLH proteins were found as soluble in Escherichia coli. The details of PvbHLH protein sequences were summarized in Table S2.

Chromosomal distribution of bHLH genes

PvbHLH genes were placed on 11 chromosomes of common bean based on BLASTN analysis. The exact position (in kp) of each bHLHs on bean chromosome is shown in Fig. 2. Among all, chromosome 3 contains the highest number of bHLH genes, 24/155 (15.5 %) and chromosome 4 contains the least, 5/155 (3.2 %) (Sup. Figure 2). Distribution arrangement of bHLH genes on individual chromosomes also indicated certain physical regions with a relatively higher accumulation of gene clusters. Common bean bHLH genes are mainly located on both ends of the chromosomes. For example, bHLH genes found on chromosomes 1, 2, 3, 7 and chromosomes 10 appear to be congregate at the lower end and upper ends of the arms. In addition, some bHLH genes are located near the telomere regions of the chromosomes, especially on chromosome 5, 6, 8 and 9.

Fig. 2
figure 2

Distribution of 155 PvbHLH genes onto eleven bean chromosomes. The chromosomes are numbered between 1 and 11 and shown at the top of each chromosome (Chr represented as bars). Chromosomal distances are given in Mbp. Black regions showed telomere regions of the chromosomes

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Duplication and divergence rate of the bHLH genes

Tandem and segmental duplication of PvbHLH gene members in common bean were determined. A total of 11 tandem duplicate PvbHLH genes were detected (Table S4), which means that a small portion of tandem duplication events have caused gene expansion of bHLH family members in bean. PvbHLH genes located on chromosome 10 showed the highest numbers of tandem duplications. Several direct tandem repeats were also found on chromosome 5 (PvbHLH-65, PvbHLH-66, PvbHLH-67 and PvbHLH-68). Totally, 61 segmental duplicated bean bHLH genes have been detected, accounting for around 39 % (61/155) of total PvbHLH genes (Table S5). The most surprising aspect of the data is high segmental duplicated ratio which is firstly shown for PvbHLH genes in common bean.

The relationships between Darwin’s positive selection in divergence and duplication of PvbHLH genes were also examined to better understand family expansion of these important family members. For this, the ratios of non-synonymous (Ka) versus synonymous (Ks) substitution rates (Ka/Ks) were estimated for 11 tandem (Table S4) and 61 segmentally (Table S5) duplicated gene-pairs. The ratios of Ka/Ks for tandem duplication ranged from 0.02 to 1.11 with an average of 0.38, whereas Ka/Ks for segmentally duplicated gene-pairs varied from 0.05 to 0.54 with an average of 0.22. If Ka/Ks ratio is below 1, it is generally suggested that genes are found intense selection pressure. Therefore, average Ka/Ks ratios indicated that tandem and segmental duplicated genes of bean bHLHs are under strong purifying selection pressure. In addition, tandem and segmental duplication events of these genes may be estimated to have occurred around 4–14 and 6–20 Mya, respectively (Sup. Figure 3).

Exon–intron organization of PvbHLH genes in bean

Gene structure analysis of the 155 PvbHLHs was performed to gain some information about exon–intron organization (Sup. Figure 4). A total of 11 bHLH genes without intron were found, which accounts for 7 % of total PvbHLH genes. Most of these intronless genes were clustered into the Cluster 6 and Cluster 7c. Intron organization and numbers of bHLH genes in common bean showed different variation and distribution into different clusters. According to exon–intron organization of bHLH genes in bean, phylogenetically related proteins exhibited a closely related gene structure, in terms of intron number or exon length.

3D homology modeling of PvbHLH proteins

To construct 3D homology model for bHLH proteins, BLASTP search was performed against the PDB. Twelve PvbHLH proteins (PvbHLH-23-50-59-62-74-78-79-80-86-103-105-123) with high homology rate were selected. To predict homology modeling in Phyre2, which utilizes the alignment of hidden Markov models via HMM–HMM search (Soding 2005), the detection rate method was used. The intensive mode was selected in Phyre 2 to increase accuracy of alignment. In addition, it integrates a new ab initio folding simulation termed as Poing (Jefferys et al. 2010) to model areas of proteins without any significant homology for known structures. The 3D protein modelling of selected 12 bHLH proteins are predicted at >90 % confidence and the percentage residue varied from 80 to 100 (Fig. 3). The secondary structures were predominantly constituted of α helices and having rare incurrence of β sheets. Thus, all suggested protein structures are assessed to be highly reliable which offers a preliminary basis for understanding the molecular function of PvbHLH proteins.

Fig. 3
figure 3

Predicted 3-D structures of bHLH proteins in bean. The structure of 12 bean bHLH proteins with >90 % confidence level are shown

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GO annotation terms

The identified common bean bHLH genes were classified into the three Gene ontology (GO) categories, which were Cellular Component (5 %), Biological Process (28 %), and Molecular Function (67 %) (Fig. 4, Table S6). Within each three main categories, genes that annotated for “binding” (155), “developmental process” (20), “single-organism process” (21), “response to stimulus” (17) and “multicellular organismal process” were the most abundant (Fig. 4, Table S6).

Fig. 4
figure 4

Gene ontology (GO) patterns for the bHLH proteins. The CLC Genomics Workbench-BLAST2GO-ANNEX categorized the gene ontology as cellular component, biological process, and molecular function

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In silico identification of bHLH gene targeting miRNAs

The miRNAs targeting the PvbHLH genes were predicted. Totally, 100 PvbHLH genes targeted by 86 plant miRNAs were identified in this study. Most of the PvbHLH genes were targeted by 3 or more plant miRNAs. The most abundant transcripts, which were targeted by all 18 plant miRNAs, were belonging to PvbHLH-22 and PvbHLH-44 genes (Sup. Figure 5). miR396, miR530 and miR165 are one of the most important miRNAs found by targeting and BLASTn analysis.

bHLH gene expression profiling

We analyzed expression levels of bHLH genes in two different tissue samples of common bean. According to normalized RPKM values, expression of 20 bHLH genes (PvbHLH-01, PvbHLH-02, PvbHLH-04, PvbHLH-150, PvbHLH-143, PvbHLH-120, PvbHLH-105, PvbHLH-108, PvbHLH-90, PvbHLH-66, PvbHLH-67, PvbHLH-68, PvbHLH-39, PvbHLH-40, PvbHLH-46, PvbHLH-17, PvbHLH-80, PvbHLH-27, PvbHLH-30 and PvbHLH-141) were not detected in reads belong to either root and leaf tissues. A total of 63 PvbHLH genes were differentially expressed in at least one tissue. Among the 155 PvbHLHs, 16 PvbHLHs were differentially upregulated in leaf tissue, including PvbHLH-28, PvbHLH-53, 82 and PvbHLH-69. In root tissue, a total of 11 PvbHLHs including PvbHLH-69, PvbHLH-104, PvbHLH-119 and PvbHLH-116 showed higher expression pattern. Interestingly, three of total PvbHLHs (54, 148 and 69) were highly expressed both in leaf and root tissues. Genome-wide expression analysis also showed that there were a total of 55 up-regulated PvbHLHs and 63 down-regulated PvbHLHs in leaf tissues after salt stress (Table S7). Likewise, there were 69 up-regulated PvbHLH genes and 54 down-regulated PvbHLH genes in root tissues. An expression profile of all identified PvbHLH genes was shown as heat map in Fig. 5.

Fig. 5
figure 5

Heat map of bHLH genes expressed in both tissues

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Gene expression levels of salt responsive bHLH transcription factor family genes

Expression levels of 9 bHLH family members (PvbHLH-54, PvbHLH-148, PvbHLH-69, PvbHLH-53, PvbHLH-98, PvbHLH-28, PvbHLH-124, PvbHLH-82, PvbHLH-128) and 10 bHLH family members (PvbHLH-54, PvbHLH-148, PvbHLH-69, PvbHLH-81, PvbHLH-104, PvbHLH-04, PvbHLH-133, PvbHLH-116, PvbHLH-119 and PvbHLH-97) in salt-stressed leaf and root tissues were measured, respectively. qRT-PCR results revealed that all nine selected genes were up-regulated in common bean leaf tissues after salt treatment. Among them, PvbHLH-53 and PvbHLH-148 showed relatively higher expression level when compared to control sample. Therefore, we validated all the 9 PvbHLHs in leaf tissue by using qRT-PCR. But we found that the genes PvbHLH-69, PvbHLH-81, PvbHLH-104, PvbHLH-133, and PvbHLH-116, were down-regulated and PvbHLH-54, PvbHLH-148, PvbHLH-04, PvbHLH-119 and PvbHLH-97 were up-regulated after salt treatment in root tissue. Among the 3 PvbHLHs (PvbHLH-54, PvbHLH-148, PvbHLH-69), which was differentially up-regulated in transcriptome data of both root and leaf tissues, PvbHLH-54 and PvbHLH-148 were up-regulated in both tissues according to the qRT-PCR results (Fig. 6).

Fig. 6
figure 6

Relative expression levels of bHLH TF family genes for root and leaf tissues under salt stress conditions (salt stress treatment) and untreated (control) in Phaseolus vulgaris (P. vulgaris cv. İspir)

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MDA and proline analysis

In this study, we measured MDA content of both root and leaf tissues in control and salt-treated plants. We observed that there was no significantly difference between control root and salt-treated root tissues. Likewise, there was no significant change in spite of an increase in MDA content in control and salt-treated leaf tissues (Table S8). Additionally, we measured proline, an osmoprotectant, in order to compare control and salt-treated common bean plants. Both root and leaf tissues showed significantly increased proline content when compared to control groups under salinity stress (Table S8). Therefore, we confirmed that accumulation of proline in stress tolerant plant species under salt stress has been correlated with stress tolerance.

Discussion

Common bean is an important crop for human diet being a rich source of proteins, micronutrients and calories of daily needs. In spite of its importance, there is scarce information on genome-wide characterization of transcription factors. Likewise, there is a few reports related with genome-wide identification of bHLH transcription factors in plants compared to metazoans. Up to date a total of 150 and 45 bHLH gene family members were identified in Arabidopsis and soybean by Toledo-Ortiz et al. (2003), Pires and Dolan (2010) and Osorio et al. (2012), respectively. Among the plant bHLH transcription factors, the highest number of bHLH transcription factor genes was discovered in Chinese cabbage which contains 230 BrabHLH genes in Chinese cabbage genome (Song et al. 2014). In a recent study, Sun et al. (2015) detected 159 bHLH protein-encoding genes (SlbHLH) in tomato. We have also identified 155 bHLH genes located on 11 chromosomes of common bean. The phylogenetic analysis showed that PvbHLHs were separated into the eight groups consist of 21 subfamily consistent with previous finding (Sun et al. 2015). Although, number of common bean PvbHLH genes is similar to Arabidopsis and tomato, they are divergent in different plant species. So, it can be concluded that a similar gene evolution of bHLH transcription factor genes might occur in bean, Arabidopsis and tomato.

While there is no report related with genome-wide identification of bHLH under salt stress conditions, OrbHLH2 and AtbHLH122 genes were identified and functionally characterized in terms of resistance to high concentration of salt (Zhou et al. 2009; Liu et al. 2014). Here, we identified salt responsive bHLH genes at transcriptome level in common bean. Gene expression patterns can provide important clues for gene function. Therefore, a total of 19 genes (9 for leaf and 10 for root tissues) were selected for further confirmation via qRT PCR. PvHLH-148, an orthologs of AtbHLH122, showed higher expression in both tissues after salinity stress in consistent with previous report (Liu et al. 2014). Likewise, we observed higher expression of PvHLH-154, another bHLH122 like gene, only in salt stressed leaf tissues As a result, expression of all selected PvbHLH genes were validated in leaf tissues and a strong consistency with RNA-seq data was obtained. On the other hand, half of the measured PvbHLH genes were consistent with transcriptome analysis of root tissues. The inconsistency might be resulted due to the quality of RNA-seq data or growing conditions of plants (Fang and Cui 2011).

Although expression of selected PvbHLH genes was mostly validated in leaf and root tissues and they showed strong consistency with RNA-seq data, we also evaluated the physiological responses of common bean by measuring MDA and Proline content under salt stress. Increased proline concentration was generally evaluated as a good indicator of stress tolerance in many plants. Stress-tolerant plants accumulate more proline compared to sensitive species upon stress conditions (Ashraf and Foolad 2007; Kavas et al. 2013). As a results of MDA and Proline assays, we observed a meaningful relationship between these two substances in salt-stress-tolerant common bean cultivar. Finally, we concluded that stress tolerant common bean cultivar (Ispir) might have well established physiological and molecular stress tolerance mechanism including higher proline accumulation and regulated expression pattern of PvbHLHs, respectively.

Segmental and tandem duplications play a crucial roles in the evolution and expansion of gene families in plants (Cannon et al. 2004). These results match those observed in earlier studies which indicate that segmental duplication has a major impact for the expansion of gene families (Baloglu 2014). Gene duplication occur frequently and leads to evolution of related genes in organisms (Mehan et al. 2004). Studies in rice and Arabidopsis show that tandem and segmental duplication events of bHLH genes were observed. So, this supported a prominent role for expansion of this important gene family (Heim et al. 2003; Toledo-Ortiz et al. 2003; Li et al. 2006). A total of 14 tandem duplication were determined in tomato bHLH genes (Sun et al. 2015). The findings observed in this study are consistent with these studies that have examined prominent role for genome segments and tandem duplication in the expansion of bHLH gene family (Heim et al. 2003; Toledo-Ortiz et al. 2003; Li et al. 2006). This observation may support the idea that evolution of those genes might arise from two ways including specific gene duplications or integration into genomic region following a reverse transcription (Lecharny et al. 2003). Our findings provide further support for another idea that duplicated genes have different functions, which provides evolutionary novelty for organisms (Nam et al. 2004; Nei and Rooney 2005).

Whole-genome tandem and segmental duplications cause formation of multiple copies of genes in many plant TF gene-families such as bZIP, NAC, MBF as well as in HSPs (Cannon et al. 2004; Jain et al. 2007; Nijhawan et al. 2008; Wang et al. 2011; Puranik et al. 2012; Baloglu et al. 2014). The ratios of Ka/Ks were below 1 for tandem and segmentally duplicated gene-pairs for bHLH transcription factor members in bean. All duplicated bean PvbHLHs genes were found under strong purifying selection pressure. These results are consistent with those of other studies and suggest that the segmental and tandem duplication events have an important role in evolution, for shaping and expansion of gene families in different organisms (Nijhawan et al. 2008; Wang et al. 2011; Baloglu et al. 2014).

In order to obtain information about gene structures, detection of exon–intron organization have been widely used method (Baloglu 2014). We have found that phylogenetically related proteins have an identical gene structure. Similar cases have also been observed in Arabidopsis, poplar, rice, moss and algae (Li et al. 2006; Carretero-Paulet et al. 2010). Intron distribution pattern and its correlation with phylogenetic classification were also detected in Chinese cabbage BrabHLH (Song et al. 2014) and tomato SlbHLH genes (Sun et al. 2015). These findings may help us to understand evolutionary conservation of bHLH genes in different organisms. Amino acid motifs of PvbHLHs also show strong correlation with other plant`s bHLHs such as tomato and chinese cabbage.

MicroRNAs (miRNAs) are short (aprox. 21 nucleotides), single-stranded, noncoding RNAs characterized in different organisms including plants and animals and have functions in regulation of expression of their target (Bartel 2004). They play critical roles in plant development as well as response to environmental stress. Up to now, several salt responsive miRNAs such as miR159, miR394, miR396 and miR1866 have been identified (Liu et al. 2008; Ding et al. 2009). In the context of in silico identification of miRNAs target genes, we found that 100 PvbHLH genes targeted by 86 plant miRNAs. One of the identified miRNA was miR396, which was previously reported for salt stress response (Liu et al. 2008; Eren et al. 2014). In addition to the targeting of salt responsive genes, this family of miRNAs also target growth regulating factor (GRF) transcription factors, rhodenase-like proteins, and kinesin-like protein B.

In conclusion, a comprehensive genome-wide characterization of the common bean bHLH family genes was performed and their expression levels were analyzed in response to excess salt concentration for the first time. A total of 155 PvbHLH genes were identified by using in silico analysis. This analysis of the bHLH family in common bean with regard to other plant species reveals some common features, such as family size and global structure. Expression patterns of 19 PvbHLH genes have been analyzed under salt stress with qRT-PCR. Analysis of expression patterns shows that bHLH subfamily genes were expressed in both root and leaf tissues. Additionally, genomic structures, chromosomal locations, gene duplication and sequence similarity analyses of bHLH genes were reported. Our results suggest that chromosomal segment duplications may be the main factors for the expansion of the bHLH gene family in common bean. The results of this study revealed the importance of bHLH genes during salt stress response in roots and leaves. It may serve in a key role in elucidating bHLH family gene function in protein interactions, signaling pathway regulations and defense responses under salt stress conditions. Additionally, this results may become useful as tools to engineer stress-tolerant plants.