# Characterization and Expression Analysis of Common BeanHistone Deacetylase 6 during Development and Cold Stress Response

Authors: Rita Kusi-Appiah Hayford; Ayalew Ligaba-Osena; Mayavan Subramani; Adrianne Brown; Kalpalatha Melmaiee; Khwaja Hossain; Venu (Kal) Kalavacharla
Journal: International Journal of Genomics
 (2017)

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## Abstract

Histone deacetylases (HDACs) are important regulators of gene transcription thus controlling multiple cellular processes. Despite its essential role in plants,HDA6 is yet to be validated in common bean. In this study, we show thatHDA6 is involved in plant development and stress response. Differential expression ofHDA6 was determined in various tissues and the expression was seen to be upregulated with plant age (seedling < flowering < maturity). Higher expression was observed in flowers and pods than in stem, leaf, and root. Upregulation ofHDA6 gene during cold stress implies its prominent role in abiotic stress. Furthermore, theHDA6 gene was isolated from three common bean genotypes and sequence analyses revealed homology with functionally characterized homologs in model species. The 53 kDa translated product was detected using anHDA6 specific antibody and recombinant protein overexpressed inEscherichia coli showed HDAC activityin vitro. To our knowledge, this is the first report in the agriculturally important crop common bean describing the functional characterization and biological role ofHDA6.


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## Body

## 1. Introduction

In eukaryotes, epigenetic mechanisms regulate gene expression and play a significant role in many aspects of development [1]. The expression and activity of genes in eukaryotes are not only dependent on the genome but also dependent on the regulation conferred due to epigenetic marks such as DNA methylation and modification of histones. Histones are modified by the process of acetylation, phosphorylation, methylation, ubiquitination, ADP-ribosylation, glycosylation, or sumoylation [2] which subsequently affects interactions with DNA and thus gene expression. DNA methylation, histone modification, and chromatin remodeling regulate the accessibility of DNA to transcription factors and other regulatory proteins which affect transcriptional activities and gene expression. Chromatin is made up of DNA and histones as well as nonhistone proteins, which condenses DNA into the nucleus of a cell [3]. Approximately 147 base pairs of DNA wrap around an octamer of core histone proteins made up of two molecules each of H2A, H2B, H3, and H4. The N-terminal tails of histones provide the sites for histone modifications [4].Chromatin in a closed state becomes inaccessible to transcription factors and other regulatory proteins. During acetylation, the positive charge of the lysine residues at the amino terminal tail is removed by addition of acetyl groups which reduces the electrostatic charge between the positively charged histone and negatively charged DNA molecule. In an open state, chromatin allows access of transcription factors to the promoter regions causing gene expression to occur, while the reverse occurs during deacetylation when transcription is inhibited resulting in a closed chromatin state [5]. Histone acetyltransferases (HATs) and deacetylases (HDACs) are, respectively, the enzymes that catalyze the addition and removal of acetyl groups from lysine residues on the histone N-terminal tails of histones [6]. Recent studies have revealed that some HDACs deacetylate nonhistone proteins besides histones [7]. HATs and HDACs are among the most well-characterized histone modification enzymes as compared to other histone modifiers and are grouped into distinct families. HDACs have been classified into three major distinct classes based on their homology to yeast HDACs, including Reduced Potassium Dependency-3/Histone Deacetylases 1 (RPD3/HD1), Silent Information Regulator 2 (SIR2), and the plant-specific Histone Deacetylase 2 (HD2). TheHDA6homolog in common bean which is the subject of this study belongs to the RPD3 superfamily and this family of HDACs has been widely studied among other families of HDACs. InArabidopsis, the RPD3 family of HDACs apart fromHDA6 includes HDA7, HDA9, and HDA19.A number of HATs and HDACs have been identified in the genome of various plant species includingArabidopsis thaliana, maize (Zea mays), rice (Oryza sativa), soybean (Glycine max), and barley (Hordeum vulgare) [8] and have been characterized. A total of 12 HATs and 18 HDACs have been identified inArabidopsis [9]. In the rice genome seven HATs and 19 HDACs have been identified [10]. Likewise, 13 genes that encode HDACs have been identified in grapes (Vitis vinifera) and their expression analysis revealed specific functions during plant development [11]. Besides regulating chromatin structure, HDACs are also involved in plant growth and development [12]. Among the HDACs,HDA6 is well studied and has also been shown to be involved in the control and maintenance of the genome, and the mechanisms and some targets ofHDA6 in gene silencing have been enumerated [13].HDA6 is involved in flower, seed, and leaf development inArabidopsis. Mutants ofHDA6 inArabidopsis showed late flowering time [14, 15]. In addition, reports show thatHDA6 is implicated in silencing transgenes and controlsArabidopsisribosomal RNA [16]. There are reports on the involvement of the maize ZmRpd31 (RPD3-like enzyme, a member of the HDAC family) and ZmRBR1 (maize related protein) in regulating cell cycle progression, senescence, flowering, and suppression of embryonic features [17, 18]. Current reports also suggest thatHDA6 associates with small interfering RNAs from the RNA-directed DNA methylation pathway in gene silencing inArabidopsis [19]. Additionally,HDA6 together with methyltransferase 1 (MET1) are needed to silence centromeric regions inArabidopsis [20].HDACs are also known to respond to several biotic and abiotic stresses. For example, theArabidopsis RPD3 group in the HDAC family has been implicated in stress signal response [21], whileHDA6 and HDA19 are involved in plant-pathogen interactions and associated with the jasmonic acid pathway [16]. A recent study from our group showed upregulation of common beanHDA6 during inoculation with the bean rust pathogen,Uromyces appendiculatus race 53 [22].HDA6 regulates chromatin in response to a number of environmental stresses. There are reports indicating the role of light detector Phytochrome B andHDA6 in regulating light-controlled chromatin of Nuclear Organization Regions (NORs) [23].HDA6 is also known to be involved in salt and cold tolerance inArabidopsis [2, 24]. In relation to this work, cold temperatures cause considerable damage to plants and are one of the major abiotic stresses that affect the production of many crop plants including common bean [25].Common bean is the most important food legume consumed worldwide; however biotic (62%) and abiotic (37–67%) factors affect the production of this crop causing significant yield losses [26]. The common bean genome has been sequenced and 473 Mb out of the ~587 Mb genomes have been assembled. It is a diploid with relatively large genome size compared toArabidopsis (of ~135 Mbp) and serves as a model crop for other legumes. There are two major gene pools of common bean; Mesoamerican and Andean regions have distinct genetic and morphological characteristics [27, 28]. Given the availability of the genome sequence, it is important to understand the temporal and spatial expression as well as the physiological role ofHDA6. Despite the involvement of HDACs in a myriad of biological processes, little is known about common bean HDACs. Therefore, we isolatedHDA6 from common bean and studied its temporal and spatial expression as well as its expression under cold stress. To our knowledge this is the first study characterizing theHDA6gene and demonstrating differences inHDA6 expression during development and abiotic stress (cold stress) in common bean.


## 2. Materials and Methods

### 2.1. Plants and Growth Conditions

Phaseolus vulgaris cultivar “Sierra” (Mesoamerican and resistant toUromyces appendiculatus race 53), which was previously used for studying transcriptome and genome-wide histone binding analysis and global leaf HDAC activity [22, 29], was also used throughout the study.Phaseolus vulgaris genotypes “Olathe” (Mesoamerican and susceptible toUromyces appendiculatus race 53) and “G19833” (Andean origin and reference genotype in Phytozome, http://www.phytozome.org) were used for cloning and sequence analysis. Seeds were germinated for five days on Petri dishes and irrigated with distilled water for another three days at room temperature. The seedlings were then transferred to poly pots filled with Promix soil. The plants were allowed to grow in the greenhouse under natural light until pod formation according to Ayyappan et al. 2015 [29], with samples collected as per the experimental protocol below.


### 2.2. Sample Collection for Spatial and Temporal Expression Analysis

Leaves were collected from the plants at seedling, flowering, and pod developmental stages when common bean plants were 2, 6, and 8 weeks old, respectively. Leaf tissues collected at the three different stages were used to study temporal expression ofHDA6. Stems, roots, flowers, and pods were also collected for spatial expression analysis. Samples from three independent biological replicates were collected for each study. All tissues collected were flash frozen in liquid nitrogen and stored in −80°C for processing.


### 2.3. Cold Stress Treatment

Cold stress was imposed on two-week-old seedlings. One-half of the plants were treated with cold (4°C) while the other half was allowed to grow at an ambient growth condition (28/20°C) for 0, 24, and 72 h after which leaf tissues were collected. Samples from three independent biological replicates were collected for each treatment. All samples collected were immediately frozen in liquid nitrogen and subsequently stored in −80°C.


### 2.4. RNA Preparation and 1st-cDNA Synthesis

Total RNA was purified from various plant tissues and at different developmental stages using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. The concentration and purity of the RNA samples were determined using a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The integrity of all RNA samples was evaluated by gel electrophoresis. To eliminate contaminating genomic DNA, the RNA samples were treated with Deoxyribonuclease I (Invitrogen) following the manufacturer’s instructions. The RNA samples were stored at −80°C until being used for downstream experiments. First-strand cDNA synthesis was performed using 10μg of DNase-treated total RNA using SuperScript III First-Strand Synthesis System (Invitrogen Life Technologies, Grand Island, NY, USA) following manufacturer instructions. The concentration of the cDNA samples was also checked spectrophotometrically.


### 2.5. Gene Cloning

The amino acid sequence of theArabidopsis HDA6 (AtRPD3B, Acc.# BAB10553) (Pandey et al. 2002) obtained from the National Center for Biotechnology Information (NCBI) was used as a query sequence to search homologs in the common bean (Andean genotype G19833) genome (http://www.phytozome.org) [30]. Fourteen hits were found in the genome of G19833 of which Acc. # BAB10553, which matched Phvul.003G203800.1, was the best hit. The sequence ofHDA6 was subsequently cloned from common bean and namedPvHDA6. The coding region ofHDA6 was amplified from the cDNA using high fidelity Phusion Hot Start II DNA polymerase (Thermo Fisher Scientific, Inc., Grand Island, NY, USA) and gene-specific sense (Supplementary Table 1) primers designed by using DNASTAR software (DNASTAR, Madison, WI, USA). For amplifying common beanHDA6, 50 μL PCR reaction contained 1 μg of the cDNA, 400 nM of each forward and reverse primers, and one unit of the DNA polymerase. After initial denaturation at 98°C for 30 s, amplification was performed in 35 cycles of temperature gradient of 98°C for 30 s, 63°C for 30 s, and 72°C for 45 s and final extension for 10 minutes at 72°C and an infinite hold at 4°C. The PCR product was separated on agarose and purified using QIAquick gel extraction kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. Since the polymerase produces blunt-end products, gel purified cDNA was cloned into the Zero Blunt® TOPO vector (Invitrogen Life Technologies, Grand Island, NY, USA) for subsequent sequencing.


### 2.6. Sequence Analysis

The nucleotide sequence of the cDNA was determined using Sanger sequencing method [31]. The sequences were compared with the reference sequence in Phytozome database (Phvul.003G203800.1.) using the SeqMan Pro module in DNASTAR Lasergene 10 Core Suite (DNASTAR, Madison, WI). The amino acid sequence was compared with the NCBI database using the Basic Local Alignment Search Tool (BLAST). Similarity of the common beanHDA6 with homologs in other plant species was analyzed using online software CLUSTALW (European Molecular Biology Laboratory EMBL-EBI, Hinxton, Cambridgeshire, UK). Identity and similarity of amino acid residues were determined by BOXSHADE software (http://ch.embnet.org/software/BOX_form.html) with black shading representing identical amino acids while gray shading showing similar amino acids. The amino acid substitutions are found in the gray region as shown in Figure 1. The coding sequences of plantHDA6 sequences in FASTA format were retrieved from the NCBI database. Prior to the phylogenetic analysis, the Molecular Evolutionary Genetic Analysis version 6.0 (MEGA6) was used to align the sequences based on conserved domains using CLUSTALW. The evolutionary history was inferred using the neighbor-joining method with 1000 bootstrap replicates by MEGA6 as shown in Figure 2 [32]. The evolutionary distance was computed using the Poisson correction method which is estimated by the number of amino acid substitutions per site. The coding data was translated assuming a standard genetic code table. All positions containing alignment gaps and missing data were eliminated only in pairwise sequence comparisons.Figure 1
Multiple sequence alignment of putativeHDA6 from common bean cultivar Sierra with homologs in model plants. The alignment was generated using CLUSTALW and BOXSHADE and the amino acid sequences ofHDA6inPhaseolus used for the alignment are amino acids from Sierra. Highly conserved regions or identical amino acid regions from the BOXSHADE results are highlighted in black with white letters; gray regions are conserved amino acid substitutions. Four motifs were identified within the protein sequences (Pandey et al. 2002) and the three amino acids with star are conserved in common beanHDA6 as inArabidopsis HDA5 and they could be responsible for HDAC activity (Luo et al. 2015).Figure 2
Phylogenetic analysis ofHDA6 in monocots and dicots. The neighbor-joining tree was constructed with mRNA sequences of plantHDA6 using MEGA6. The tree indicates the evolutionary relationship between common beanHDA6 and other dicot and monocotHDA6.The bootstrap analysis was deduced from 1000 replicates and Poisson correction method from MEGA version 6 software. The scientific names for the monocots and dicots used to construct the tree are as follows:Glycine max (Gm), Arachis hypogaea (Ah), Cicer arietinum (Ca), Elaeis guineensis (Eg), Solanum lycopersicum (Sl), Sorghum bicolor (Sb), Hordeum vulgare (Hv), Sesamum indicum (Si), Oryza sativa (Os), Solanum tuberosum (St), Zea mays (Zm), Cucumis sativus (Cs), Morus notabilis (Mn), Tarenaya hassleriana (Th), Nicotiana sylvestris (Ns), Cucumis melo (Cm), Brassica rapa (Br), Citrus sinensis (Cs), Arabidopsis thaliana (At),andPhaseolus vulgaris (Sierra). The monocots are considered as the outgroups for the phylogenetic analysis.


### 2.7. Quantitative PCR (qPCR)

First-strand cDNA was synthesized from DNase I-treated total RNA extracted from leaf tissues collected at seedling, flowering, and pod developmental stages as well as from plants subjected to cold stress. The concentration of the cDNA samples used for qPCR was normalized to 1μg/μL. Quantitative PCR was performed to study the temporal and spatial expression ofHDA6using an ABI 7500 real-time PCR system and SYBR Green Kit (Applied Biosystems, Grand Island, USA). Twenty-five μls of the PCR reactions contained 1 μg of 1st-strand cDNA, 2x Power SYBR Green Master Mix, and 0.15 μL ofHDA6-specific primers (Supplementary Table 2, in Supplementary Material available online at https://doi.org/10.1155/2017/2502691), and the constitutivecons7 gene was used as an internal control. Relative expression was determined from three biological and three technical replicates using ΔΔCT method (Applied Biosystems, Foster City, CA).


### 2.8. RecombinantHDA6 Production inE. coli

For recombinant protein expression inE. coli theHDA6 gene, which was previously subcloned into the Zero Blunt TOPO vector (above), was digested withBamH1 andNot1 restriction enzyme and ligated to a similarly digested pET24a plasmid (Novagen, Madison, USA), in frame with the C-terminus His-Tag coding sequence under the control of a strong bacteriophage T7 transcription and translation signals. The construct (pET24a-HDA6)and the control (empty vector controlpET24) were then introduced into BL21 (DE3)E. coli competent cells (EMD-Millipore) suitable for high-level production of proteins from target genes cloned into pET vectors. Five milliliters of LB media containing 30 mg/L kanamycin was inoculated with a single colony of the BL21 (DE3) harboringpET24-HDA6 orpET24aand allowed to grow overnight at 37°C. One milliliter of the overnight culture was used to inoculate 180 mL of LB containing 30 mg/L kanamycin. The cultures were allowed to grow until the optical density (OD600) reached 0.4–0.6. Protein expression was induced by adding 1 mM of isopropyl β-D-1-thiogalactopyranoside(IPTG) (Thermo Fisher Scientific, Grand Island, NY). Extraction of recombinant proteins was performed using the BugBuster protein extraction kit (Novagen, EMD-Millipore, Darmstadt, Germany) following recommended procedures. The concentration of the crude recombinant protein was determined by the Lowry method using Spectra Max® M5 reader (Molecular Devices LLC, VWR Corporate Headquarters, USA) [33].


### 2.9. Antibody Development and Western Blot

Total protein was isolated from leaves of plants at the developmental stages using trichloroacetic acid (TCA) acetone precipitation followed by SDS and phenol extraction as outlined by Wu et al. (2014) [34]. The protein concentration was measured using the Bradford method [35]. Equal amount of the proteins was loaded onto a SDS PAGE gel and transferred onto nitrocellulose membrane for western blot with theHDA6-specific antibody developed by GeneScript (Piscataway, NJ, USA) from the clonedHDA6 (Hayford, Osena, and Kalavacharla 2014, unpublished). The antibody was diluted 1 : 1000 in TBST + 5% milk (http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6376.pdf). meanwhile the secondary antibody-goat-anti-IgG (H+L) AP conjugate (Bio-Rad, USA) was diluted 1 : 5000 in TBST + 5% milk. The blot was developed using 25x AP Color developer (Bio-Rad Laboratories Inc., Hercules, CA, USA). The molecular weight of the protein was determined based on protein precision standard of known molecular weight.


### 2.10. Histone Deacetylase Enzyme Assays

To understand the real physiological role of common beanHDA6, the recombinant protein extracted fromE. coli was purified using HIS-Select Affinity Gel (SIGMA-ALDRICH, St Louis, MO, USA) following the manufacturer’s protocol.In vitro activity of theHDA6 was determined using colorimetric assay following manufacturer’s protocol (BioVision Inc., Milpitas, CA, USA) with slight modification and the assay was repeated three times.


### 2.11. Statistical Analyses

All experiments were repeated at least twice with three biological replicates. Statistical tests of significance of gene expression and deacetylase activity was performed using the PROC GLM procedure in SAS (Cary, NC, USA). Following significantF-tests, the Tukey multiple comparison procedure for all treatment pairs was used to separate the means [SAS] [36].


## 2.1. Plants and Growth Conditions

Phaseolus vulgaris cultivar “Sierra” (Mesoamerican and resistant toUromyces appendiculatus race 53), which was previously used for studying transcriptome and genome-wide histone binding analysis and global leaf HDAC activity [22, 29], was also used throughout the study.Phaseolus vulgaris genotypes “Olathe” (Mesoamerican and susceptible toUromyces appendiculatus race 53) and “G19833” (Andean origin and reference genotype in Phytozome, http://www.phytozome.org) were used for cloning and sequence analysis. Seeds were germinated for five days on Petri dishes and irrigated with distilled water for another three days at room temperature. The seedlings were then transferred to poly pots filled with Promix soil. The plants were allowed to grow in the greenhouse under natural light until pod formation according to Ayyappan et al. 2015 [29], with samples collected as per the experimental protocol below.


## 2.2. Sample Collection for Spatial and Temporal Expression Analysis

Leaves were collected from the plants at seedling, flowering, and pod developmental stages when common bean plants were 2, 6, and 8 weeks old, respectively. Leaf tissues collected at the three different stages were used to study temporal expression ofHDA6. Stems, roots, flowers, and pods were also collected for spatial expression analysis. Samples from three independent biological replicates were collected for each study. All tissues collected were flash frozen in liquid nitrogen and stored in −80°C for processing.


## 2.3. Cold Stress Treatment

Cold stress was imposed on two-week-old seedlings. One-half of the plants were treated with cold (4°C) while the other half was allowed to grow at an ambient growth condition (28/20°C) for 0, 24, and 72 h after which leaf tissues were collected. Samples from three independent biological replicates were collected for each treatment. All samples collected were immediately frozen in liquid nitrogen and subsequently stored in −80°C.


## 2.4. RNA Preparation and 1st-cDNA Synthesis

Total RNA was purified from various plant tissues and at different developmental stages using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. The concentration and purity of the RNA samples were determined using a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The integrity of all RNA samples was evaluated by gel electrophoresis. To eliminate contaminating genomic DNA, the RNA samples were treated with Deoxyribonuclease I (Invitrogen) following the manufacturer’s instructions. The RNA samples were stored at −80°C until being used for downstream experiments. First-strand cDNA synthesis was performed using 10μg of DNase-treated total RNA using SuperScript III First-Strand Synthesis System (Invitrogen Life Technologies, Grand Island, NY, USA) following manufacturer instructions. The concentration of the cDNA samples was also checked spectrophotometrically.


## 2.5. Gene Cloning

The amino acid sequence of theArabidopsis HDA6 (AtRPD3B, Acc.# BAB10553) (Pandey et al. 2002) obtained from the National Center for Biotechnology Information (NCBI) was used as a query sequence to search homologs in the common bean (Andean genotype G19833) genome (http://www.phytozome.org) [30]. Fourteen hits were found in the genome of G19833 of which Acc. # BAB10553, which matched Phvul.003G203800.1, was the best hit. The sequence ofHDA6 was subsequently cloned from common bean and namedPvHDA6. The coding region ofHDA6 was amplified from the cDNA using high fidelity Phusion Hot Start II DNA polymerase (Thermo Fisher Scientific, Inc., Grand Island, NY, USA) and gene-specific sense (Supplementary Table 1) primers designed by using DNASTAR software (DNASTAR, Madison, WI, USA). For amplifying common beanHDA6, 50 μL PCR reaction contained 1 μg of the cDNA, 400 nM of each forward and reverse primers, and one unit of the DNA polymerase. After initial denaturation at 98°C for 30 s, amplification was performed in 35 cycles of temperature gradient of 98°C for 30 s, 63°C for 30 s, and 72°C for 45 s and final extension for 10 minutes at 72°C and an infinite hold at 4°C. The PCR product was separated on agarose and purified using QIAquick gel extraction kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. Since the polymerase produces blunt-end products, gel purified cDNA was cloned into the Zero Blunt® TOPO vector (Invitrogen Life Technologies, Grand Island, NY, USA) for subsequent sequencing.


## 2.6. Sequence Analysis

The nucleotide sequence of the cDNA was determined using Sanger sequencing method [31]. The sequences were compared with the reference sequence in Phytozome database (Phvul.003G203800.1.) using the SeqMan Pro module in DNASTAR Lasergene 10 Core Suite (DNASTAR, Madison, WI). The amino acid sequence was compared with the NCBI database using the Basic Local Alignment Search Tool (BLAST). Similarity of the common beanHDA6 with homologs in other plant species was analyzed using online software CLUSTALW (European Molecular Biology Laboratory EMBL-EBI, Hinxton, Cambridgeshire, UK). Identity and similarity of amino acid residues were determined by BOXSHADE software (http://ch.embnet.org/software/BOX_form.html) with black shading representing identical amino acids while gray shading showing similar amino acids. The amino acid substitutions are found in the gray region as shown in Figure 1. The coding sequences of plantHDA6 sequences in FASTA format were retrieved from the NCBI database. Prior to the phylogenetic analysis, the Molecular Evolutionary Genetic Analysis version 6.0 (MEGA6) was used to align the sequences based on conserved domains using CLUSTALW. The evolutionary history was inferred using the neighbor-joining method with 1000 bootstrap replicates by MEGA6 as shown in Figure 2 [32]. The evolutionary distance was computed using the Poisson correction method which is estimated by the number of amino acid substitutions per site. The coding data was translated assuming a standard genetic code table. All positions containing alignment gaps and missing data were eliminated only in pairwise sequence comparisons.Figure 1
Multiple sequence alignment of putativeHDA6 from common bean cultivar Sierra with homologs in model plants. The alignment was generated using CLUSTALW and BOXSHADE and the amino acid sequences ofHDA6inPhaseolus used for the alignment are amino acids from Sierra. Highly conserved regions or identical amino acid regions from the BOXSHADE results are highlighted in black with white letters; gray regions are conserved amino acid substitutions. Four motifs were identified within the protein sequences (Pandey et al. 2002) and the three amino acids with star are conserved in common beanHDA6 as inArabidopsis HDA5 and they could be responsible for HDAC activity (Luo et al. 2015).Figure 2
Phylogenetic analysis ofHDA6 in monocots and dicots. The neighbor-joining tree was constructed with mRNA sequences of plantHDA6 using MEGA6. The tree indicates the evolutionary relationship between common beanHDA6 and other dicot and monocotHDA6.The bootstrap analysis was deduced from 1000 replicates and Poisson correction method from MEGA version 6 software. The scientific names for the monocots and dicots used to construct the tree are as follows:Glycine max (Gm), Arachis hypogaea (Ah), Cicer arietinum (Ca), Elaeis guineensis (Eg), Solanum lycopersicum (Sl), Sorghum bicolor (Sb), Hordeum vulgare (Hv), Sesamum indicum (Si), Oryza sativa (Os), Solanum tuberosum (St), Zea mays (Zm), Cucumis sativus (Cs), Morus notabilis (Mn), Tarenaya hassleriana (Th), Nicotiana sylvestris (Ns), Cucumis melo (Cm), Brassica rapa (Br), Citrus sinensis (Cs), Arabidopsis thaliana (At),andPhaseolus vulgaris (Sierra). The monocots are considered as the outgroups for the phylogenetic analysis.


## 2.7. Quantitative PCR (qPCR)

First-strand cDNA was synthesized from DNase I-treated total RNA extracted from leaf tissues collected at seedling, flowering, and pod developmental stages as well as from plants subjected to cold stress. The concentration of the cDNA samples used for qPCR was normalized to 1μg/μL. Quantitative PCR was performed to study the temporal and spatial expression ofHDA6using an ABI 7500 real-time PCR system and SYBR Green Kit (Applied Biosystems, Grand Island, USA). Twenty-five μls of the PCR reactions contained 1 μg of 1st-strand cDNA, 2x Power SYBR Green Master Mix, and 0.15 μL ofHDA6-specific primers (Supplementary Table 2, in Supplementary Material available online at https://doi.org/10.1155/2017/2502691), and the constitutivecons7 gene was used as an internal control. Relative expression was determined from three biological and three technical replicates using ΔΔCT method (Applied Biosystems, Foster City, CA).


## 2.8. RecombinantHDA6 Production inE. coli

For recombinant protein expression inE. coli theHDA6 gene, which was previously subcloned into the Zero Blunt TOPO vector (above), was digested withBamH1 andNot1 restriction enzyme and ligated to a similarly digested pET24a plasmid (Novagen, Madison, USA), in frame with the C-terminus His-Tag coding sequence under the control of a strong bacteriophage T7 transcription and translation signals. The construct (pET24a-HDA6)and the control (empty vector controlpET24) were then introduced into BL21 (DE3)E. coli competent cells (EMD-Millipore) suitable for high-level production of proteins from target genes cloned into pET vectors. Five milliliters of LB media containing 30 mg/L kanamycin was inoculated with a single colony of the BL21 (DE3) harboringpET24-HDA6 orpET24aand allowed to grow overnight at 37°C. One milliliter of the overnight culture was used to inoculate 180 mL of LB containing 30 mg/L kanamycin. The cultures were allowed to grow until the optical density (OD600) reached 0.4–0.6. Protein expression was induced by adding 1 mM of isopropyl β-D-1-thiogalactopyranoside(IPTG) (Thermo Fisher Scientific, Grand Island, NY). Extraction of recombinant proteins was performed using the BugBuster protein extraction kit (Novagen, EMD-Millipore, Darmstadt, Germany) following recommended procedures. The concentration of the crude recombinant protein was determined by the Lowry method using Spectra Max® M5 reader (Molecular Devices LLC, VWR Corporate Headquarters, USA) [33].


## 2.9. Antibody Development and Western Blot

Total protein was isolated from leaves of plants at the developmental stages using trichloroacetic acid (TCA) acetone precipitation followed by SDS and phenol extraction as outlined by Wu et al. (2014) [34]. The protein concentration was measured using the Bradford method [35]. Equal amount of the proteins was loaded onto a SDS PAGE gel and transferred onto nitrocellulose membrane for western blot with theHDA6-specific antibody developed by GeneScript (Piscataway, NJ, USA) from the clonedHDA6 (Hayford, Osena, and Kalavacharla 2014, unpublished). The antibody was diluted 1 : 1000 in TBST + 5% milk (http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6376.pdf). meanwhile the secondary antibody-goat-anti-IgG (H+L) AP conjugate (Bio-Rad, USA) was diluted 1 : 5000 in TBST + 5% milk. The blot was developed using 25x AP Color developer (Bio-Rad Laboratories Inc., Hercules, CA, USA). The molecular weight of the protein was determined based on protein precision standard of known molecular weight.


## 2.10. Histone Deacetylase Enzyme Assays

To understand the real physiological role of common beanHDA6, the recombinant protein extracted fromE. coli was purified using HIS-Select Affinity Gel (SIGMA-ALDRICH, St Louis, MO, USA) following the manufacturer’s protocol.In vitro activity of theHDA6 was determined using colorimetric assay following manufacturer’s protocol (BioVision Inc., Milpitas, CA, USA) with slight modification and the assay was repeated three times.


## 2.11. Statistical Analyses

All experiments were repeated at least twice with three biological replicates. Statistical tests of significance of gene expression and deacetylase activity was performed using the PROC GLM procedure in SAS (Cary, NC, USA). Following significantF-tests, the Tukey multiple comparison procedure for all treatment pairs was used to separate the means [SAS] [36].


## 3. Results

### 3.1. Gene Cloning and Sequence Analysis

TheArabidopsis genome contains 12 HATs and 18 HDACs [9]. To find HDAC homologs in common bean, the amino acid sequence of theArabidopsis HDA6 (AtPRD3B, Acc. # BAB10553), obtained from the NCBI was used as a query sequence to search for homologs in the common bean genome at the Phytozome database (http://www.phytozome.org) using the BLAST tool. A total of 14 proteins belonging to the HDAC superfamily were found including Phvul.003G203800.1, which showed very high similarity to theArabidopsis HDA6 protein (E-value = 0.0). The nucleotide sequence ofPvHDA6 obtained from Phytozome was 1428 bp encoding a 475 amino acids’ protein (Supplementary Figure 1). The gene was amplified from common bean cDNA synthesized from leaves of cultivar Sierra, Olathe, and G19833 followed by gel purification of the PCR product (Supplementary Figure 2) and cloning into Zero Blunt TOPO cloning vector for subsequent sequencing.TheHDA6 homolog cloned from the cultivar Sierra and Olathe (both of Mesoamerican origin) contained six and three SNPs, respectively, as compared to the reference genome from cultivar G19833 of Andean origin (sequences are found in Supplementary Figure 4). However, these SNPs did not result in a change to the amino acid sequence. The amino acid sequence of the putativeHDA6 from both Sierra and Olathe are identical with the reference sequence from the cultivar G19833. A BLAST search of the cloned common beanHDA6 sequences at NCBI returned the highest matches (99% identity with e-value = 0.0) with knownHDA6 genes, confirming that the gene cloned from common bean is indeed a homolog of the plant HDAC superfamily and was hence namedPvHDA6. The amino acid sequences of PvHDA6 showed very high similarity with homologs in other plants such asGlyma.05G040600.1(90.9%),AtRPD3B or AtHDA6(77%), maize HD2 (74%), and rice HD2 (72%) (Figure 1). Furthermore, phylogenetic analysis established an evolutionary relationship between common bean and selected monocotyledonous and dicotyledonousHDA6 genes.PvHDA6 is very closely related to homologs in other legumes such as soybean, chickpea (Cicer arietinum), and peanut (Arachis hypogea) while it distantly related to that of cereals including rice, barley, sorghum (Sorghum bicolor), and maize (Supplementary Figure 4).


### 3.2. Expression Analysis ofHDA6 Gene at Different Developmental Stages

To understand whetherPvHDA6 expression is affected by plant age, we studied the gene expression profile of this gene at different developmental stages (seedling, flowering, and pod) in the cultivar Sierra using qPCR. As shown in Figure 3(a) thePvHDA6is constitutively expressed at all developmental stages. However, transcript accumulation increased significantly with age (p value < 0.0007).PvHDA6is expressed higher at pod stage followed by flowering stage while expression at the seedling stage was lower compared to both pod and flower stages.Figure 3
Validation of relative expression level ofHDA6 by qPCR using leaf tissues at different developmental stages of common bean. (a) Relative expression ofHDA6 gene in leaves at the seedling, flowering, and pod developmental stages of common bean. Three biological and three technical replicates were used for the analysis. The alphabets in the figure show that the expressions ofHDA6 in the leaf tissue at the different developmental stages are statistically significant at p<0.05. (b) Diagram illustrating the common bean specificHDA6 polyclonal antibody raised for protein validation. The 15 amino acids’ peptide indicated in faded lines with red arrow is one of the three peptides selected for the epitope. (c) Western blot analysis using common bean specificHDA6 antibody. The figure further validates the role ofHDA6 in common bean development.
(a) (b) (c)To confirm the expression at the protein level, we performed western blotting using anti-HDA6 antibody on crude protein samples extracted from the leaf at seedling, flowering, and pod developmental stages. Expression at the protein level is consistent with that observed at the transcript level (Figure 3(b)). A translation product of 53 kDa was detected at all developmental stages with an increase in intensity in the order of seedling < flowering < pod stages.


### 3.3. Tissue-Specific Expression ofHDA6

To study the spatial expression ofPvHDA6, RNA samples were isolated from various plant tissues (leaves, stems, roots, flowers, and pods) and used for synthesizing cDNAs, which were used for both traditional (Figure 4(a)) and quantitative PCR (Figure 4(b)).PvHDA6 is constitutively expressed in all plant tissues. However, expression in the reproductive tissues (flower and pods) is higher than expression in other tissues. Expression in stem and the leaf is lower than the other tissues although the difference is statistically significant (p<0.05) only for the pod.Figure 4
Spatial expression profile ofHDA6 in five tissues of common bean. (a) Traditional PCR was conducted usingHDA6 primers designed for qPCR. Each of the cDNA samples were normalized to 200 ng/μL. Regular PCR was conducted usingHDA6 real-time primers designed for qPCR. The cons7 primer which is a constitutive gene was used as a control. (b) Determination of the relative expression ofHDA6 transcript in different tissues of common bean by qPCR. The alphabets indicate significant difference between common bean tissues at p<0.05. (c) Western blot analysis usingHDA6 specific antibody with proteins isolated from empty pET vector (1) as a negative control, (2) leaf, (3) flower, and (4) pod.
(a) (b) (c)


### 3.4.HDA6 Expression in Response to Cold

Previous reports have shown that abiotic stresses such as drought, cold, and salt stress as well as phytohormones modulate expression of HDAC genes in various plant species [37, 38]. To understand the effect of cold stress onPvHDA6 expression, two-week-old plants were subjected to cold stress treatment at 4°C, and gene expression was studied for 72 h. Interestingly,PvHDA6 expression was significantly induced (p<0.001) after 24 h of cold stress, while 72 h of cold stress further increased gene expression (Figure 5). To determine whether the plants are really experiencing cold stress condition, we also studied the expression of a DREB (dehydration-responsive element-binding proteins), which is a well-characterized transcription factor responsive to drought, salt, and cold stresses [35, 39]. Interestingly, expression of the DREB was also significantly (p<0.001) induced by cold stress (Figure 6). Hence, increases in expression ofPvHDA6 may suggest thatHDA6 is a cold responsive gene.Figure 5
Expression analysis ofHDA6 after subjecting common bean plants to cold treatment at 4°C at different time points. Leaf tissues were collected from control and cold treated plants 24 and 72 hours after treatment. (a) PCR performed after RT-PCR of samples usingHDA6andActin primers. (b) qPCR to determine relative expression ofHDA6 during cold stress. Three biological and three technical replicates were used for the analysis. Data were analyzed by analysis of variance (ANOVA) using [SAS] statistical software. Differences between treatments were assessed by Tukey’s Studentized Range (HSD) Test for experiment. There was significant difference between control and cold treated samples at p<0.0001. (c) Coomassie stained gel of proteins extracted from control and cold treated samples. (d) Western blot analysis usingHDA6 specific antibody with proteins isolated from control and cold treated samples.
(a) (b) (c) (d)Figure 6
Expression analysis of DREB1A, a cold responsive gene after subjecting common bean plants to cold treatment at 4°C at different time points. Leaf tissues were collected from control and cold treated plants 24 and 72 hours after treatment. (a) cDNA PCR performed after RT-PCR of samples using DREB 1A andActin primers. (b) qPCR to determine relative expression of DREB 1A during cold stress. Three biological and three technical replicates were used for the analysis. Data were analyzed by analysis of variance (ANOVA) using [SAS] statistical software. Differences between treatments were assessed by Tukey’s Studentized Range (HSD) Test for experiment. The different letters A, B, and C indicate significant difference in gene expression between times of cold treatment at p<0.0001. Values with the same letters are not statistically different.
(a) (b)


### 3.5. Recombinant Protein Production andIn Vitro Deacetylase Activity

To deduce the physiological function of the common beanHDA6, the coding sequence was cloned into pET24a (+) vector for subsequent expression inE. coli(Supplementary Figure 3). The recombinant protein was extracted and purified using HIS-Select Ni-affinity gel and used for assaying deacetylase activity. The purifiedHDA6 protein and a positive control HeLa nuclear extract, containing components of HDACs, were incubated with HDAC colorimetric substrate which contains an acetylated lysine side chain. The assay was carried out with or without trichostatin A (TSA), which is an organic compound that serves as an antifungal antibiotic and selectively inhibits class I and II mammalian histone deacetylase (HDAC) families of enzymes. As shown in Figure 7, the deacetylase activity of the recombinant PvHDA6 is lower than the positive control with or without TSA. Interestingly, deacetylase activity was proportionally inhibited in both the HeLa control and theHDA6 proteins in the presence of TSA. This decrease in deacetylase activity in the presence of TSA is consistent with increased deacetylation, suggesting that the common beanHDA6 is likely a functional member of the HDAC protein superfamily.Figure 7
Histone deacetylase activity of recombinantHDA6 protein from common bean. Thein vitro activity was conducted with or without the addition of TSA (T) to purified recombinantHDA6 protein (P) and positive control; HeLa nuclear extract (H). The alphabets indicate significant difference between protein and HeLa nuclear extract with TSA and without TSA (p<0.05).


## 3.1. Gene Cloning and Sequence Analysis

TheArabidopsis genome contains 12 HATs and 18 HDACs [9]. To find HDAC homologs in common bean, the amino acid sequence of theArabidopsis HDA6 (AtPRD3B, Acc. # BAB10553), obtained from the NCBI was used as a query sequence to search for homologs in the common bean genome at the Phytozome database (http://www.phytozome.org) using the BLAST tool. A total of 14 proteins belonging to the HDAC superfamily were found including Phvul.003G203800.1, which showed very high similarity to theArabidopsis HDA6 protein (E-value = 0.0). The nucleotide sequence ofPvHDA6 obtained from Phytozome was 1428 bp encoding a 475 amino acids’ protein (Supplementary Figure 1). The gene was amplified from common bean cDNA synthesized from leaves of cultivar Sierra, Olathe, and G19833 followed by gel purification of the PCR product (Supplementary Figure 2) and cloning into Zero Blunt TOPO cloning vector for subsequent sequencing.TheHDA6 homolog cloned from the cultivar Sierra and Olathe (both of Mesoamerican origin) contained six and three SNPs, respectively, as compared to the reference genome from cultivar G19833 of Andean origin (sequences are found in Supplementary Figure 4). However, these SNPs did not result in a change to the amino acid sequence. The amino acid sequence of the putativeHDA6 from both Sierra and Olathe are identical with the reference sequence from the cultivar G19833. A BLAST search of the cloned common beanHDA6 sequences at NCBI returned the highest matches (99% identity with e-value = 0.0) with knownHDA6 genes, confirming that the gene cloned from common bean is indeed a homolog of the plant HDAC superfamily and was hence namedPvHDA6. The amino acid sequences of PvHDA6 showed very high similarity with homologs in other plants such asGlyma.05G040600.1(90.9%),AtRPD3B or AtHDA6(77%), maize HD2 (74%), and rice HD2 (72%) (Figure 1). Furthermore, phylogenetic analysis established an evolutionary relationship between common bean and selected monocotyledonous and dicotyledonousHDA6 genes.PvHDA6 is very closely related to homologs in other legumes such as soybean, chickpea (Cicer arietinum), and peanut (Arachis hypogea) while it distantly related to that of cereals including rice, barley, sorghum (Sorghum bicolor), and maize (Supplementary Figure 4).


## 3.2. Expression Analysis ofHDA6 Gene at Different Developmental Stages

To understand whetherPvHDA6 expression is affected by plant age, we studied the gene expression profile of this gene at different developmental stages (seedling, flowering, and pod) in the cultivar Sierra using qPCR. As shown in Figure 3(a) thePvHDA6is constitutively expressed at all developmental stages. However, transcript accumulation increased significantly with age (p value < 0.0007).PvHDA6is expressed higher at pod stage followed by flowering stage while expression at the seedling stage was lower compared to both pod and flower stages.Figure 3
Validation of relative expression level ofHDA6 by qPCR using leaf tissues at different developmental stages of common bean. (a) Relative expression ofHDA6 gene in leaves at the seedling, flowering, and pod developmental stages of common bean. Three biological and three technical replicates were used for the analysis. The alphabets in the figure show that the expressions ofHDA6 in the leaf tissue at the different developmental stages are statistically significant at p<0.05. (b) Diagram illustrating the common bean specificHDA6 polyclonal antibody raised for protein validation. The 15 amino acids’ peptide indicated in faded lines with red arrow is one of the three peptides selected for the epitope. (c) Western blot analysis using common bean specificHDA6 antibody. The figure further validates the role ofHDA6 in common bean development.
(a) (b) (c)To confirm the expression at the protein level, we performed western blotting using anti-HDA6 antibody on crude protein samples extracted from the leaf at seedling, flowering, and pod developmental stages. Expression at the protein level is consistent with that observed at the transcript level (Figure 3(b)). A translation product of 53 kDa was detected at all developmental stages with an increase in intensity in the order of seedling < flowering < pod stages.


## 3.3. Tissue-Specific Expression ofHDA6

To study the spatial expression ofPvHDA6, RNA samples were isolated from various plant tissues (leaves, stems, roots, flowers, and pods) and used for synthesizing cDNAs, which were used for both traditional (Figure 4(a)) and quantitative PCR (Figure 4(b)).PvHDA6 is constitutively expressed in all plant tissues. However, expression in the reproductive tissues (flower and pods) is higher than expression in other tissues. Expression in stem and the leaf is lower than the other tissues although the difference is statistically significant (p<0.05) only for the pod.Figure 4
Spatial expression profile ofHDA6 in five tissues of common bean. (a) Traditional PCR was conducted usingHDA6 primers designed for qPCR. Each of the cDNA samples were normalized to 200 ng/μL. Regular PCR was conducted usingHDA6 real-time primers designed for qPCR. The cons7 primer which is a constitutive gene was used as a control. (b) Determination of the relative expression ofHDA6 transcript in different tissues of common bean by qPCR. The alphabets indicate significant difference between common bean tissues at p<0.05. (c) Western blot analysis usingHDA6 specific antibody with proteins isolated from empty pET vector (1) as a negative control, (2) leaf, (3) flower, and (4) pod.
(a) (b) (c)


## 3.4.HDA6 Expression in Response to Cold

Previous reports have shown that abiotic stresses such as drought, cold, and salt stress as well as phytohormones modulate expression of HDAC genes in various plant species [37, 38]. To understand the effect of cold stress onPvHDA6 expression, two-week-old plants were subjected to cold stress treatment at 4°C, and gene expression was studied for 72 h. Interestingly,PvHDA6 expression was significantly induced (p<0.001) after 24 h of cold stress, while 72 h of cold stress further increased gene expression (Figure 5). To determine whether the plants are really experiencing cold stress condition, we also studied the expression of a DREB (dehydration-responsive element-binding proteins), which is a well-characterized transcription factor responsive to drought, salt, and cold stresses [35, 39]. Interestingly, expression of the DREB was also significantly (p<0.001) induced by cold stress (Figure 6). Hence, increases in expression ofPvHDA6 may suggest thatHDA6 is a cold responsive gene.Figure 5
Expression analysis ofHDA6 after subjecting common bean plants to cold treatment at 4°C at different time points. Leaf tissues were collected from control and cold treated plants 24 and 72 hours after treatment. (a) PCR performed after RT-PCR of samples usingHDA6andActin primers. (b) qPCR to determine relative expression ofHDA6 during cold stress. Three biological and three technical replicates were used for the analysis. Data were analyzed by analysis of variance (ANOVA) using [SAS] statistical software. Differences between treatments were assessed by Tukey’s Studentized Range (HSD) Test for experiment. There was significant difference between control and cold treated samples at p<0.0001. (c) Coomassie stained gel of proteins extracted from control and cold treated samples. (d) Western blot analysis usingHDA6 specific antibody with proteins isolated from control and cold treated samples.
(a) (b) (c) (d)Figure 6
Expression analysis of DREB1A, a cold responsive gene after subjecting common bean plants to cold treatment at 4°C at different time points. Leaf tissues were collected from control and cold treated plants 24 and 72 hours after treatment. (a) cDNA PCR performed after RT-PCR of samples using DREB 1A andActin primers. (b) qPCR to determine relative expression of DREB 1A during cold stress. Three biological and three technical replicates were used for the analysis. Data were analyzed by analysis of variance (ANOVA) using [SAS] statistical software. Differences between treatments were assessed by Tukey’s Studentized Range (HSD) Test for experiment. The different letters A, B, and C indicate significant difference in gene expression between times of cold treatment at p<0.0001. Values with the same letters are not statistically different.
(a) (b)


## 3.5. Recombinant Protein Production andIn Vitro Deacetylase Activity

To deduce the physiological function of the common beanHDA6, the coding sequence was cloned into pET24a (+) vector for subsequent expression inE. coli(Supplementary Figure 3). The recombinant protein was extracted and purified using HIS-Select Ni-affinity gel and used for assaying deacetylase activity. The purifiedHDA6 protein and a positive control HeLa nuclear extract, containing components of HDACs, were incubated with HDAC colorimetric substrate which contains an acetylated lysine side chain. The assay was carried out with or without trichostatin A (TSA), which is an organic compound that serves as an antifungal antibiotic and selectively inhibits class I and II mammalian histone deacetylase (HDAC) families of enzymes. As shown in Figure 7, the deacetylase activity of the recombinant PvHDA6 is lower than the positive control with or without TSA. Interestingly, deacetylase activity was proportionally inhibited in both the HeLa control and theHDA6 proteins in the presence of TSA. This decrease in deacetylase activity in the presence of TSA is consistent with increased deacetylation, suggesting that the common beanHDA6 is likely a functional member of the HDAC protein superfamily.Figure 7
Histone deacetylase activity of recombinantHDA6 protein from common bean. Thein vitro activity was conducted with or without the addition of TSA (T) to purified recombinantHDA6 protein (P) and positive control; HeLa nuclear extract (H). The alphabets indicate significant difference between protein and HeLa nuclear extract with TSA and without TSA (p<0.05).


## 4. Discussion

Given that the HDACs are known to play a crucial role in plant growth and development as well as plant responses to environmental stimuli, we were interested in isolating and characterizing HDACs from common bean. In this study we report the isolation ofPvHDA6 and temporal and spatial expression of the gene as well as determination of its possible physiological function using recombinant protein produced inE. coli. This will widen our understanding of the regulatory mechanisms involved in growth and development of plants including responses to environmental stresses such as biotic and abiotic stresses.


### 4.1. Members of theHDAC Superfamily in Common Bean

In this study several members of theHDAC superfamily were identified in the common bean genome based on homology search using theArabidopsis HDA6. HDAC proteins possess conserved histone deacetylase domains [9], suggesting that members of this family may be involved in histone deacetylation in plants. In common bean, nine distinct members of the HDAC superfamily have been identified (Supplementary Figure 5). Two of the sequences (PvSIR1 and PvSIR2) belong to the SIR2 family of NAD-dependent HDAC proteins, while one of the sequences (PvHD1) belongs to the plant-specific HD2 family. The remaining six proteins belong to the RPD3/HDA1 superfamily of HDACs which are further grouped into several classes (for review see Pandey et al. 2002 [9]). The presence of multiple proteins with HDAC conserved domains may suggest functional redundancy; alternatively, these proteins may have specific roles in various physiological processes or responses to environmental stimuli. However, the physiological function of each protein remains to be understood.


### 4.2. Temporal and Spatial Expression ofHDA6

There is considerable evidence showing that HDACs regulate transcription and play significant roles in various plant developmental processes and response to environmental factors [40, 41]. To identify if theHDA6 expression varies with plant age, we determined its expression profile at three developmental stages (seedling, flowering, and pod) using the quantitative PCR. Our findings revealed that theHDA6 is constitutively expressed in all of the developmental stages. However, its expression was significantly induced (p<0.001) with age (Figure 3). Gene expression was higher at the flowering stage as compared to the seedling stages. This level of expression was further increased as the plants reached maturity (pod stage). In line with the transcript data, the abundance of a ~53 kDa translation product increased with age. This suggests thatHDA6 may play a specific role at later developmental stages of the plants.Previous reports have shown that expression ofHDA6in other plant species such asArabidopsis is higher in reproductive tissues [3, 40]. Consistent with these reports, differential expression ofHDA6 in five tissues of common bean showed higher expression in the flowers and pod as shown in Figure 4. A tomato (Solanum lycopersicum) homolog of theArabidopsis HDA6 gene,SlHDA3, was more highly expressed in flowers though it was constitutively expressed in all stages of tomato development [4]. Enhanced expression of the common beanHDA6during the reproductive phase as well as in reproductive tissues may suggest a possible role ofHDA6 in regulating flower initiation and development. Our suggestion is consistent with previous reports showing that downregulation of theArabidopsis AtRPD3B (HDA6) delays flowering [42] while mutation ofHDA9 lead to early flowering [43], suggesting that the HDAC genes may function independently to regulate the flowering phenotype. The mechanism by which HDACs regulate flowering is not well understood. However,Arabidopsis HDACs have been shown to interact with various transcription factors to repress the expression of genes regulating developmental processes (for review see Zhao et al. 2016 [44]).Arabidopsis HDA6 has also been shown to interact withflowering locus D to regulate flowering time [45]. Furthermore, a number of genes and transcription factors implicated in flowering time and reproductive transition have been identified in common bean includingConstans (CO),Early flowering 3 (ELF3),flowering locus D,Gigantia (GI), LEAFY (LFY), andTerminal Flower (TFL) [46]. Therefore, it remains to be understood whetherPvHDA6 modulates flowering time by activating or repressing expression offlowering locus D or other genes regulating the flowering traits. Furthermore, this study also revealed higher level ofHDA6 expression in the pods suggesting the possible role ofHDA6in pod or seed development [47].


### 4.3. Possible Involvement ofHDA6 in Cold Stress Response

Exposure of some plants to cold or low temperatures has been reported to induce epigenetic changes which may lead to changes in flowering time through vernalization [48]. The control of gene expression by HOS15 (WD4-repeat protein) through deacetylation associated with cold tolerance has been reported [49].Arabidopsis HDA6 has been implicated in cold acclimation and freezing tolerance [24]. Likewise, our findings revealed upregulation ofPvHDA6 with prolonged exposure to cold temperature, suggesting thatPvHDA6 may be involved in cold responsive pathways as previously reported forArabidopsis HDA6 [24]. To substantiate this finding we studied the expression of DREB, which is a hallmark of stress-responsive gene expression [50]. We found that expression of DREB was also induced with cold stress (as shown in Figure 6). Upregulation ofHDA6 under cold stress may suggest its role in cold tolerance via direct regulation of tolerance genes or through interacting partners. For example, previous studies using the yeast two-hybrid system identified a number of interacting partners for HDA705 including RSS3, RHSF10, and GAMYB-binding protein, which are implicated in stress responses and hormone signaling inArabidopsis[51].


### 4.4. RecombinantHDA6 PossessesHDA6 Activity

It has been reported that members of the RPD3/HDA1 family possess the deacetylase catalytic domain [4]. However, to date recombinant proteins of only a few members of RPD3/HDA1 includingArabidopsis HDA1 and HDA5 displayed varying degrees of histone deacetylase activityin vitro [52, 53]. Other members of the family includingHDA6, HDA9, HDA15, and HDA17 (for review see [53]) did not show any HDAC activity which is suggested to be due to the absence of posttranslational protein modification machineries in the hostE. coli or in the assay reaction [54].In this study the recombinant PvHDA6 expressed inE. coli displayed HDAC enzymatic activity (Figure 7). Similar to HDA5 [53], enzymatic activity of the PvHDA6 was significantly inhibited by TSA. Luo et al. (2015) reported that site-directed mutagenesis revealed three critical amino acid residues (D198, D291, and H200) whose mutation abolished deacetylase activity of the recombinant HDA5. These three residues appear to be conserved among the HDAC proteins including PvHDA6. The residues fall within motif 2 (D198 and H200) and motif 3 (D291) as reported in Pandey et al. (2002) [9]. It remains to be determined whether these residues are also critical for PvHDA6. Taken together, our finding reveals that the common beanHDA6 is a functional homolog of the HDAC superfamily with domains essential for deacetylase activity [9].


## 4.1. Members of theHDAC Superfamily in Common Bean

In this study several members of theHDAC superfamily were identified in the common bean genome based on homology search using theArabidopsis HDA6. HDAC proteins possess conserved histone deacetylase domains [9], suggesting that members of this family may be involved in histone deacetylation in plants. In common bean, nine distinct members of the HDAC superfamily have been identified (Supplementary Figure 5). Two of the sequences (PvSIR1 and PvSIR2) belong to the SIR2 family of NAD-dependent HDAC proteins, while one of the sequences (PvHD1) belongs to the plant-specific HD2 family. The remaining six proteins belong to the RPD3/HDA1 superfamily of HDACs which are further grouped into several classes (for review see Pandey et al. 2002 [9]). The presence of multiple proteins with HDAC conserved domains may suggest functional redundancy; alternatively, these proteins may have specific roles in various physiological processes or responses to environmental stimuli. However, the physiological function of each protein remains to be understood.


## 4.2. Temporal and Spatial Expression ofHDA6

There is considerable evidence showing that HDACs regulate transcription and play significant roles in various plant developmental processes and response to environmental factors [40, 41]. To identify if theHDA6 expression varies with plant age, we determined its expression profile at three developmental stages (seedling, flowering, and pod) using the quantitative PCR. Our findings revealed that theHDA6 is constitutively expressed in all of the developmental stages. However, its expression was significantly induced (p<0.001) with age (Figure 3). Gene expression was higher at the flowering stage as compared to the seedling stages. This level of expression was further increased as the plants reached maturity (pod stage). In line with the transcript data, the abundance of a ~53 kDa translation product increased with age. This suggests thatHDA6 may play a specific role at later developmental stages of the plants.Previous reports have shown that expression ofHDA6in other plant species such asArabidopsis is higher in reproductive tissues [3, 40]. Consistent with these reports, differential expression ofHDA6 in five tissues of common bean showed higher expression in the flowers and pod as shown in Figure 4. A tomato (Solanum lycopersicum) homolog of theArabidopsis HDA6 gene,SlHDA3, was more highly expressed in flowers though it was constitutively expressed in all stages of tomato development [4]. Enhanced expression of the common beanHDA6during the reproductive phase as well as in reproductive tissues may suggest a possible role ofHDA6 in regulating flower initiation and development. Our suggestion is consistent with previous reports showing that downregulation of theArabidopsis AtRPD3B (HDA6) delays flowering [42] while mutation ofHDA9 lead to early flowering [43], suggesting that the HDAC genes may function independently to regulate the flowering phenotype. The mechanism by which HDACs regulate flowering is not well understood. However,Arabidopsis HDACs have been shown to interact with various transcription factors to repress the expression of genes regulating developmental processes (for review see Zhao et al. 2016 [44]).Arabidopsis HDA6 has also been shown to interact withflowering locus D to regulate flowering time [45]. Furthermore, a number of genes and transcription factors implicated in flowering time and reproductive transition have been identified in common bean includingConstans (CO),Early flowering 3 (ELF3),flowering locus D,Gigantia (GI), LEAFY (LFY), andTerminal Flower (TFL) [46]. Therefore, it remains to be understood whetherPvHDA6 modulates flowering time by activating or repressing expression offlowering locus D or other genes regulating the flowering traits. Furthermore, this study also revealed higher level ofHDA6 expression in the pods suggesting the possible role ofHDA6in pod or seed development [47].


## 4.3. Possible Involvement ofHDA6 in Cold Stress Response

Exposure of some plants to cold or low temperatures has been reported to induce epigenetic changes which may lead to changes in flowering time through vernalization [48]. The control of gene expression by HOS15 (WD4-repeat protein) through deacetylation associated with cold tolerance has been reported [49].Arabidopsis HDA6 has been implicated in cold acclimation and freezing tolerance [24]. Likewise, our findings revealed upregulation ofPvHDA6 with prolonged exposure to cold temperature, suggesting thatPvHDA6 may be involved in cold responsive pathways as previously reported forArabidopsis HDA6 [24]. To substantiate this finding we studied the expression of DREB, which is a hallmark of stress-responsive gene expression [50]. We found that expression of DREB was also induced with cold stress (as shown in Figure 6). Upregulation ofHDA6 under cold stress may suggest its role in cold tolerance via direct regulation of tolerance genes or through interacting partners. For example, previous studies using the yeast two-hybrid system identified a number of interacting partners for HDA705 including RSS3, RHSF10, and GAMYB-binding protein, which are implicated in stress responses and hormone signaling inArabidopsis[51].


## 4.4. RecombinantHDA6 PossessesHDA6 Activity

It has been reported that members of the RPD3/HDA1 family possess the deacetylase catalytic domain [4]. However, to date recombinant proteins of only a few members of RPD3/HDA1 includingArabidopsis HDA1 and HDA5 displayed varying degrees of histone deacetylase activityin vitro [52, 53]. Other members of the family includingHDA6, HDA9, HDA15, and HDA17 (for review see [53]) did not show any HDAC activity which is suggested to be due to the absence of posttranslational protein modification machineries in the hostE. coli or in the assay reaction [54].In this study the recombinant PvHDA6 expressed inE. coli displayed HDAC enzymatic activity (Figure 7). Similar to HDA5 [53], enzymatic activity of the PvHDA6 was significantly inhibited by TSA. Luo et al. (2015) reported that site-directed mutagenesis revealed three critical amino acid residues (D198, D291, and H200) whose mutation abolished deacetylase activity of the recombinant HDA5. These three residues appear to be conserved among the HDAC proteins including PvHDA6. The residues fall within motif 2 (D198 and H200) and motif 3 (D291) as reported in Pandey et al. (2002) [9]. It remains to be determined whether these residues are also critical for PvHDA6. Taken together, our finding reveals that the common beanHDA6 is a functional homolog of the HDAC superfamily with domains essential for deacetylase activity [9].


## 5. Conclusions

We isolatedPvHDA6 from common bean which showed high similarity with HDACs in other plant species. RecombinantHDA6 produced inE. coli displayed histone deacetylase activity. Future study will determine which of the conserved amino acid motifs are critical for its function using site-directed mutagenesis. Additionally, expression of thePvHDA6 in common bean increases with age, predominately in reproductive tissues, and the expression is also induced by cold stress. Taken together,PvHDA6 may be involved in regulating developmental processes and stress tolerance in common bean. However, further studies involving up- or downregulation of this gene in common bean may lead to deciphering the precise function and extent of regulation byPvHDA6 in planta. Furthermore, studies involving CHIP-seq, yeast two-hybrid or bimolecular fluorescent complementation would lead to identification of novel PvHDA6 interacting partners involved in key pathways in the regulation of common bean growth and development as well as biotic and abiotic stress tolerance.



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*Source: 2502691-2017-01-03.xml*