Characterization and Expression Analysis of Common Bean Histone Deacetylase 6 during Development and Cold Stress Response

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 that HDA6 is involved in plant development and stress response. Differential expression of HDA6 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 of HDA6 gene during cold stress implies its prominent role in abiotic stress. Furthermore, the HDA6 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 an HDA6 specific antibody and recombinant protein overexpressed in Escherichia coli showed HDAC activity in vitro. To our knowledge, this is the first report in the agriculturally important crop common bean describing the functional characterization and biological role of HDA6.


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 2 International Journal of Genomics 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 Nterminal tails of histones [6]. Recent studies have revealed that some HDACs deacetylate nonhistone proteins besides histones [7]. HATs and HDACs are among the most wellcharacterized 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). The HDA6 homolog 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. In Arabidopsis, the RPD3 family of HDACs apart from HDA6 includes HDA7, HDA9, and HDA19.
A number of HATs and HDACs have been identified in the genome of various plant species including Arabidopsis 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 in Arabidopsis [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 of HDA6 in gene silencing have been enumerated [13].
HDA6 is involved in flower, seed, and leaf development in Arabidopsis. Mutants of HDA6 in Arabidopsis showed late flowering time [14,15]. In addition, reports show that HDA6 is implicated in silencing transgenes and controls Arabidopsis ribosomal 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 that HDA6 associates with small interfering RNAs from the RNA-directed DNA methylation pathway in gene silencing in Arabidopsis [19]. Additionally, HDA6 together with methyltransferase 1 (MET1) are needed to silence centromeric regions in Arabidopsis [20].
HDACs are also known to respond to several biotic and abiotic stresses. For example, the Arabidopsis RPD3 group in the HDAC family has been implicated in stress signal response [21], while HDA6 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 bean HDA6 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 and HDA6 in regulating light-controlled chromatin of Nuclear Organization Regions (NORs) [23]. HDA6 is also known to be involved in salt and cold tolerance in Arabidopsis [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 to Arabidopsis (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 of HDA6. Despite the involvement of HDACs in a myriad of biological processes, little is known about common bean HDACs. Therefore, we isolated HDA6 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 the HDA6 gene and demonstrating differences in HDA6 expression during development and abiotic stress (cold stress) in common bean.

Plants and Growth
Conditions. Phaseolus vulgaris cultivar "Sierra" (Mesoamerican and resistant to Uromyces 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 to Uromyces 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.

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 of HDA6. Stems, roots, flowers, and pods were also collected for spatial expression analysis. Samples from three independent biological replicates were International Journal of Genomics 3 collected for each study. All tissues collected were flash frozen in liquid nitrogen and stored in −80 ∘ C for processing.

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.

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. Firststrand cDNA synthesis was performed using 10 g of DNasetreated 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.

Gene
Cloning. The amino acid sequence of the Arabidopsis 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 of HDA6 was subsequently cloned from common bean and named PvHDA6. The coding region of HDA6 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 bean HDA6, 50 L PCR reaction contained 1 g 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 Blunt5 TOPO vector (Invitrogen Life Technologies, Grand Island, NY, USA) for subsequent sequencing.

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 bean HDA6 with homologs in other plant species was analyzed using online software CLUSTALW (European Molecular Biology Laboratory EMBL-EBI, Hinxton, Cambridge shire, 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 plant HDA6 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.

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 1 g/ L. Quantitative PCR was performed to study the temporal and spatial expression of HDA6 using an ABI 7500 real-time PCR system and SYBR Green Kit (Applied Biosystems, Grand Island, USA). Twenty-five ls of the PCR reactions contained 1 g of 1st-strand cDNA, 2x Power SYBR Green Master Mix, and 0.15 L of HDA6-specific primers (Supplementary Table 2, in Supplementary Material available online at https://doi.org/10.1155/2017/2502691), and the constitutive cons7 gene was used as an internal control. Relative expression was determined from three biological and three technical replicates using ΔΔC T method (Applied Biosystems, Foster City, CA).

Recombinant HDA6 Production in E. coli.
For recombinant protein expression in E. coli the HDA6 gene, which was previously subcloned into the Zero Blunt TOPO vector (above), was digested with BamH1 and Not1 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 control pET24) 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) harboring pET24-HDA6 or pET24a and 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 (OD 600 ) reached 0.4-0.6. Protein expression was induced by adding 1 mM of isopropyl -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.   [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 the HDA6-specific antibody developed by GeneScript (Piscataway, NJ, USA) from the cloned HDA6 (Hayford, Osena, and Kalavacharla 2014, unpublished). The antibody was diluted 1 : 1000 in TBST + 5% milk (http://www.bio-rad.com/webro -ot/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.

Histone Deacetylase Enzyme Assays.
To understand the real physiological role of common bean HDA6, the recombinant protein extracted from E. coli was purified using HIS-Select Affinity Gel (SIGMA-ALDRICH, St Louis, MO, USA) following the manufacturer's protocol. In vitro activity of the HDA6 was determined using colorimetric assay following manufacturer's protocol (BioVision Inc., Milpitas, CA, USA) with slight modification and the assay was repeated three times.

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 significant -tests, the Tukey multiple comparison procedure for all treatment pairs was used to separate the means [SAS] [36].

Gene Cloning and Sequence
Analysis. The Arabidopsis genome contains 12 HATs and 18 HDACs [9]. To find HDAC homologs in common bean, the amino acid sequence of the Arabidopsis 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 the Arabidopsis HDA6 protein (value = 0.0). The nucleotide sequence of PvHDA6 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.
The HDA6 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 putative HDA6 from both Sierra and Olathe are identical with the reference sequence from the cultivar G19833. A BLAST search of the cloned common bean HDA6 sequences at NCBI returned the highest matches (99% identity with -value = 0.0) with known HDA6 genes, confirming that the gene cloned from common bean is indeed a homolog of the plant HDAC superfamily and was hence named PvHDA6. The amino acid sequences of PvHDA6 showed very high similarity with homologs in other plants such as Glyma.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 dicotyledonous HDA6 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).

Expression Analysis of HDA6 Gene at Different Developmental Stages.
To understand whether PvHDA6 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) the PvHDA6 is constitutively expressed at all developmental stages. However, transcript accumulation increased significantly with age ( value < 0.0007). PvHDA6 is expressed higher at pod stage followed by flowering stage while expression at the seedling stage was lower compared to both pod and flower stages.
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.

Tissue-Specific Expression of HDA6.
To study the spatial expression of PvHDA6, 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 ( < 0.05) only for the pod.

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 on PvHDA6 expression, two-weekold plants were subjected to cold stress treatment at 4 ∘ C, and gene expression was studied for 72 h. Interestingly, PvHDA6 expression was significantly induced ( < 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 elementbinding 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 ( < 0.001) induced by cold stress (Figure 6). Hence, increases in expression of PvHDA6 may suggest that HDA6 is a cold responsive gene.

Recombinant Protein Production and In Vitro Deacetylase Activity.
To deduce the physiological function of the common bean HDA6, the coding sequence was cloned into pET24a (+) vector for subsequent expression in E. coli (Supplementary Figure 3). The recombinant protein was extracted and purified using HIS-Select Ni-affinity gel and used for assaying deacetylase activity. The purified HDA6 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 the HDA6 proteins in the presence of TSA. This decrease in International Journal of Genomics deacetylase activity in the presence of TSA is consistent with increased deacetylation, suggesting that the common bean HDA6 is likely a functional member of the HDAC protein superfamily.

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 of PvHDA6 and temporal and spatial expression of the gene as well as determination of its possible physiological function using recombinant protein produced in E. 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.

Members of the HDAC Superfamily in Common Bean.
In this study several members of the HDAC superfamily were identified in the common bean genome based on homology search using the Arabidopsis 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 plantspecific 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.

Temporal and Spatial Expression of HDA6.
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 the HDA6 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 the HDA6 is constitutively expressed in all of the developmental stages. However, its expression was significantly induced ( < 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 that HDA6 may play a specific role at later developmental stages of the plants.
Previous reports have shown that expression of HDA6 in other plant species such as Arabidopsis is higher in reproductive tissues [3,40]. Consistent with these reports, differential expression of HDA6 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 the Arabidopsis 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 bean HDA6 during the reproductive phase as well as in reproductive tissues may suggest a possible role of HDA6 in regulating flower initiation and development. Our suggestion is consistent with previous reports showing that downregulation of the Arabidopsis AtRPD3B (HDA6) delays flowering [42] while mutation of HDA9 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 with flowering 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 including Constans (CO), Early flowering 3 (ELF3), flowering locus D, Gigantia (GI), LEAFY (LFY), and Terminal Flower (TFL) [46]. Therefore, it remains to be understood whether PvHDA6 modulates flowering time by activating or repressing expression of flowering locus D or other genes regulating the flowering traits. Furthermore, this study also revealed higher level of HDA6 expression in the pods suggesting the possible role of HDA6 in pod or seed development [47].

Possible Involvement of HDA6 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 of PvHDA6 with prolonged exposure to cold temperature, suggesting that PvHDA6 may be involved in cold responsive pathways as previously reported for Arabidopsis 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 of HDA6 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 in Arabidopsis [51].

Recombinant HDA6 Possesses HDA6
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 including Arabidopsis HDA1 and HDA5 displayed varying degrees of histone deacetylase activity in vitro [52,53]. Other members of the family including HDA6, 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 host E. coli or in the assay reaction [54].
In this study the recombinant PvHDA6 expressed in E. 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 bean HDA6 is a functional homolog of the HDAC superfamily with domains essential for deacetylase activity [9].

Conclusions
We isolated PvHDA6 from common bean which showed high similarity with HDACs in other plant species. Recombinant HDA6 produced in E. 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 the PvHDA6 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 by PvHDA6 in planta. Furthermore, studies involving CHIP-seq, yeast twohybrid 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.

Disclosure
The funding agencies Delaware EPSCoR or NSF were not involved in designing and implementing the experiments.
helped in design of the study, and writing and drafting the manuscript. Kalpalatha Melmaiee helped to design the study, and in primer design, analyses of results, and editing of the manuscript. Venu (Kal) Kalavacharla assisted in conceiving the study, supervised the whole project, and contributed to the drafting and editing of the manuscript.