Characterization of B-BOX gene family and their expression profiles under hormonal, abiotic and metal stresses in Poaceae plants

B-box (BBX) proteins play important roles in plant growth regulation and development including photomorphogenesis, photoperiodic regulation of flowering, and responses to biotic and abiotic stresses. In the present study we retrieved total 131 BBX members from five Poaceae species including 36 from maize, 30 from rice, 24 from sorghum, 22 from stiff brome, and 19 from Millet. All the BBX genes were grouped into five subfamilies on the basis of their phylogenetic relationships and structural features. The expression profiles of 12 OsBBX genes in different tissues were evaluated through qRT-PCR, and we found that most rice BBX members showed high expression level in the heading stage compared to seedling and booting stages. The expression of OsBBX1, OsBBX2, OsBBX8, OsBBX19, and OsBBX24 was strongly induced by abiotic stresses such as drought, cold and salt stresses. Furthermore, the expression of OsBBX2, OsBBX7, OsBBX17, OsBBX19, and OsBBX24 genes was up-regulated under GA, SA and MeJA hormones at different time points. Similarly, the transcripts level of OsBBX1, OsBBX7, OsBBX8, OsBBX17, and OsBBX19 genes were significantly affected by heavy metals such as Fe, Ni, Cr and Cd. Change in the expression pattern of BBX members in response to abiotic, hormone and heavy metal stresses signifies their potential roles in plant growth and development and in response to multivariate stresses. The findings suggest that BBX genes could be used as potential genetic markers for the plants, particularly in functional analysis and determining their roles under multivariate stresses.


Background
Zinc finger transcription factors (TFs) are one of the most important families in plants. They regulate different plant growth and development processes. Zinc finger TFs are classified into several subfamilies based on the structural and functional features of their individual members. Among them, B-box proteins (BBXs) drew more attention in recent years due to their multiple functions. The BBXs contain one or two conserved B-box domains near to N-terminus and some have an additional CCT (CONSTANS, CO-like, and TIMING Of CAB1) conserved domain near to C-terminal. The B-box domains are divided into two classes, known as B-box1 (B1) and B-box2 (B2). Two B-box conserved domains are recognized on their consensus sequence and the distance between the zinc-binding residues [1]. The segmental duplication and deletion events during evolution result in the differences of the consensus sequences in the two B-box domains [2,3]. The highly conserved CCT domain is comprised of 42-43 amino acids and is important for the regulation of functional transcription and nuclear protein transport [4].
Recent genome-wide expression studies suggested that the BBX proteins are involved in plant hormone signaling responses. Abscisic acid (ABA) is a phytohormone which is activated when the plants are exposed to different stresses [5]. Microarray analysis detected that the expression pattern of BBX genes is different in response to ABA [6,7]. The microarray study also found that the expression of BBX32 was up-regulated by the cyclopentenone precursor of JA, 12-oxo-pentadienoic acid (OPDA), but not by JA or MeJA in Arabidopsis plants [8]. In addition, it was found that BZS1/BBX20 integrates signals from brassinosteroids (BR) and light pathways [9]. BRASSINAZOLE RESISTANT 1 (BZR1) is a transcription factor that triggered hypocotyl growth by directly binding to BBX20 [10]. Interestingly, GATA2, a GATA-binding zinc-finger protein stopped hypocotyl growth by reducing BR signaling action [11]. So, it can be postulated that BBX20 works together with GATA2 in facilitating light and BR crosstalk. Recently, it was reported that BBX18 play a potential role in the gibberellin (GA) signaling pathway [12]. Molecular and phenotypic studies proved that BBX18 enhances the hypocotyl growth by up-regulation of bioactive GA levels. Certainly, BBX18 promotes the activities of GA3ox1 and GA20ox1 metabolic genes but decreased the activities of GA2ox1 and GA2ox8 catabolic genes under light [12]. The involvement of BBX genes in the COP/HY5 signaling pathway indicates that BBX18 may work as an integrator of both GA and COP1/HY5 pathways [13]. In addition, the microarray database showed that the transcript level of 11 BBX genes was distinct in rice when the plants were exposed to auxin, GA, and cytokinin treatments, and the studied rice BBXs have hormone-responsive cis-acting elements in their promoters [14]. These results indicate the probable involvement of BBX proteins in hormones signaling in plants. However, the functional mechanisms of BBXs in hormonal signaling pathways are still little known.
BBXs might also play vital roles in abiotic stress tolerance of plants. The salt tolerance protein (STO, AtBBX24) enhances the growth of root under a high salinity condition in Arabidopsis [15], and was also triggered by the salt tolerance activities in yeast cells [16]. STO inoculates with CLONE EIGHTY-ONE/RADICA-L-INDUCED CELL DEATH1 (CEO/RCD1) [17], which negatively regulates a wide range of stress-related genes [18]. Another BBX gene, AtBBX18, acts as a negative regulator both in photomorphogenesis and thermotolerance in Arabidopsis [12]. Furthermore, AtBBX18 negatively regulates the expression of heat-responsive genes such as DGD1, Hsp70, Hsp101, and APX2, thereby reducing germination and seedling survival after the heat treatment [12]. In Chrysanthemum, CmBBX24 performed a dual function, delaying flowering and also increase cold or drought tolerance [19]. Moreover, the overexpression of AtBBX24 enhances salt tolerance compared to wild-type plants, and a significant increase in root length in Arabidopsis [15]. Twenty-nine out of 30 rice BBX genes possess at least one stress-responsive cis-elements such as ARE, Wbox, GC-motif, Box-W1, HSE, and MBS, signifying that these genes may express during biotic and abiotic stresses [14].
The studies on B-box proteins have emerging role in the plant development and of great interest for various researchers nowadays. Although, the BBX gene family and their expression patterns under a few hormones were previously reported in rice [14], the evolutionary relationships of BBXs especially in Poaceae not yet been clearly understood. Additionally, the roles of BBX genes in evolutionary origin and structural changes, the plant stress response and functional diversity of these proteins are still little understood in land plants. Therefore, in the present study, the BBX gene family members in five Poaceae species and their expression profiles under various hormones, abiotic and heavy metal stresses in rice were systematically investigated. The obtained results will enlighten the novel insights into their action and the evolutionary significance of their functional divergence. Furthermore, the gene expression pattern will assist to improve the potential BBX candidate genes involved in plant development regulation and multivariate stress responses.

Chromosomal localization, Exon and Intron Distribution and Conserved Motif Analysis
The corresponding genome database was used to obtain the candidate BBX gene annotations and their chromosomal locations. The exact locations of genes on chromosomes were identified by using MapDraw. The conserved and shared domains for all BBX protein sequences were created by online version 4.9.1 of the Multiple Expectation for Motif Elicitation (MEME) tool (http://meme-suite.org/) [21,22]. Gene Structure Display Server (http://gsds.cbi.pku.edu.cn) was used to construct the exon-intron structure consisting exon positions and gene length of BBX genes.

Sequence alignment and Phylogenetic analysis
Multiple alignments of BBX protein sequences were performed with DNAMAN software (Version 5.2.2, LynnonBiosoft, Canada), and the sequence logos were constructed through online Weblogo platform (http:// weblogo.berkeley.edu/logo.cgi). The candidate BBX proteins were initially multiply aligned by using the ClustalW v2.0 online tool (http://www.ebi.ac.uk/Tools/webservices/ services/msa/clustalw2_soap) to further search the evolutionary relationships of the BBX gene family and then the maximum likelihood phylogenetic tree was constructed by using the MEGA 6.06 software package with default parameters and the reliability of interior branches was assessed with 1000 bootstrap repetitions.

Tandem Duplication and Synteny Analysis
The Plant Genome Duplication Database (http://chibba.agtec.uga.edu/duplication/) was used to obtain syntenic blocks. Then circos version software (http://circos.ca/) was used to draw the diagrams. The physical location of a gene on the chromosome was used to find out the Tandem duplication of BBX gene. Genes having an adjacent homologous BBX gene on the same chromosome with no more than one intervening gene were considered to be tandemly duplicated.

Plant Material and Growth Conditions
The experimental work was performed in the field of State Key Laboratory of Crops Stress Biology for Arid Areas (Northwest A&F University, Yangling, China). First of all, the seeds were sterilized with 0.5% (w/v) sodium hypochlorite (NaClO) for 4 h, then washed thrice with distilled water. Seeds were then soaked in water for 48 h in darkness. Subsequently, the seeds were propagated on humid cheesecloth at 28°C for 72 h and wetted with deionized water each day. Healthy and uniform seedlings were selected and grown in hydroponic solution prepared in Milli-Q water [23] , 800 μmol m -2 s -1 light intensity and 60-65% relative humidity level). The solution was changed after 24 h duration and the pH was adjusted to 5.8 by using NaOH or HCl.

Stress Treatments and Sample Collection
To identify the transcript profiles of BBX genes in rice, the young seedling (two-week-old) were exposed to various abiotic stresses, phytohormones and heavy metals. For heat stress treatment, the seedlings at four-leaf stage were subjected at 40°C temperature with 60% humidity, 16 h photoperiod in the growth chamber under fluorescent light for 24 h. For cold stress, at the same stage seedlings were transferred into the cold cabinet (SANYO) under a 14-h light: 10-h dark, with light conditions of 300 μmol photons m −2 s −1 . For dehydration 20% polyethylene glycol (PEG-6000), the solution was purified by passing it through an ion exchange column to remove any impurities and was filtered using Miracloth (22-25 μm, Thomas Scientific, Swedesboro, NJ, USA). Salt (200 mM NaCl) was prepared from stock solution by dissolving in water. Then seedlings were submerged in 20% PEG6000 or 200 mM NaCl solutions for drought and salt treatments respectively. The final hormonal concentration of gibberellic acid (GA) (100 μM), abscisic acid (ABA) (100 μM), methyl jasmonate (MeJA) (100 μM) and salicylic acid (SA) (500 μM) were prepared from stock solutions, after addition of wetting agent Tween-20 at 0.05% (v/v) the individual hormone were sprayed on two weeks old rice leaves. For metals treatments, FeSO 4 (7 mM), CdCl 2 (0.5 mM), K 2 Cr 2 O 7 (1 mM), and NiCl 2 (1 mM) were prepared from stock solutions and applied into fresh nutrient solution and as [24] with exception of phosphorus (P) that prevents precipitation of lead (Pb) [25]. The whole leaf blades from the treated two-week-old rice plants were harvested at 0h, 3h, 6h, 12h and 24h time intervals after treatments. Rice plants were allowed to grow in normal condition (day/ night temperature cycle of 32°C /26°C, 16 h/8 h photo-period with 800 μmol m -2 s -1 light intensity and 60% humidity), and the different plant organs at various developmental stages (namely seedling, tillering, booting and heading stages) were collected for the analysis of tissue-specific expression. The samples were immediately frozen in liquid nitrogen and stored at -80°C until for further analysis.

Quantitative PCR analysis
The total RNA was extracted from all the samples by using the cetyltrimethylammonium bromide (CTAB) method [26]. The samples were run on 2% agarose gels to examine the intensity of ribosomal RNA (rRNA) bands, degraded products, and gDNA contamination. The residual genomic DNA was removed by treating the RNA samples with RNase-free DNase. The cDNA was synthesized through the PrimeScript RT Reagent Kit with gDNA Eraser (Takara Bio, Shiga, Japan) following the manufacturer's instructions. All the primers were designed from rice BBX sequences for real-time PCR using primer 6.0 (Additional file 1: Table S3). Each primer pair was examined through standard RT-PCR to confirm the size of the amplified product through 1% agarose gel electrophoresis. Real-time PCR was carried out in an iCycler iQ Real-Time PCR Detection System (Bio-Rad). Each reaction consisted of 5 μl SYBR Premix ExTaq (Takara, Kyoto, Japan), 2 μl cDNA samples, and 0.5 μl of each primer (10 μM) and 2 μl ddH 2 O in a reaction system of 10 μl. The thermal cycle was as follows: 95°C for 3 min, followed by 40 cycles at 94°C for 15 s, 62°C for 20 s, and 72°C for 20 s. Melting-curve analysis was performed directly after real-time PCR to verify the presence of gene-specific PCR products. This analysis was done by 94°C for 15 s, followed by a constant increase from 60 to 95°C at a 2% ramp rate. The rice actin gene (OsActin1, Gene ID: KC140126) was used as an internal control and served as a standard gene for normalizing all mRNA expression levels. The relative amount of template present in each PCR amplification mixture was evaluated by using the 2−ΔΔCt method.

Statistical analysis
The data underwent an analysis of variance. The means and standard deviation of three replications for all the treatments were compared by the least significant difference (LSD) test at the 5% level using the SPSS 11.5 software package (SPSS, Chicago, IL, USA). Graphs were designed using Origin 7.5 (Microcal Software Inc., Northampton, MA, USA).

Identification, Classification, and Annotation of BBX Family Members
The Arabidopsis BBX genes were used as quarries sequences against the Hidden Markov Model (HMM) algorithm [27] to retrieve and characterize the BBX gene family members in five Poaceae species. A total of 131 BBX genes were identified in the studied five Poaceae species. The number of BBX genes members were diverse among these plants such as 36, 30, 24, 22 and 19 BBX genes from maize (Zea mays), rice (Oryza sativa), Sorghum (Sorghum bicolor), stiff brome (Brachypodium distachyon) and Millet (Setaria italica), respectively ( Table 1). The potential domains of BBX gene family were confirmed through the conserved domain database, Pfam and SMART databases and structural integrity of these domains were drawn by Web Logo and EXPASY-PROSITE. All the putative BBX members lack transmembrane segment except ZmBBX30 (Additional file 1: Figure S1). Moreover, the physiochemical characteristics and amino acid sequence of BBX members were studied through EXPASY PROTOPARAM (http://www.expasy.org/ tools/protparam.html) online tool (Additional file 1: Table  S1). The assumed length of the BBX proteins and molecular weights vary widely, ranging from 9.51 (OsBBX20) to 52.89 kD (SbBBX10). The maximum number of Poaceae BBX proteins was acidic in nature according to their isoelectric point, which was lower than seven. However, the isoelectric point of some BBX members (OsBBX15, OsBBX20, OsBBX21, ZmBBX17, ZmBBX19, ZmBBX24, BdBBX16, SbBBX6, and SbBBX11) was greater than seven, indicating that they are alkaline proteins in nature. The present study divided the majority of Poaceae BBX genes into unstable proteins because the instability index of most of the genes of this family was greater than 40. However, the instability index of BdBBX20, OsBBX12, and OsBBX20 were less than 40, and they corresponded to stable proteins. All the BBX proteins were found to be hydrophilic except OsBBX25 based on their GRAV value. ZmBBX9 showed high negative and positive charge residues. Based on a total number of atoms, SbBBX10 contained the highest number of atoms (7281), followed by ZmBBX3 (7200). OsBBX20 was the smallest protein (1299) on the basis of atom compositions. This investigation found that 68 BBX proteins were located on the sense strand, and the remaining 63 BBX proteins were found on the antisense strand. The GC content of the majority studied BBX was above 60%. Furthermore, the aliphatic index values ranged from lowest (39.91) (BdBBX11) to 78.93 (SbBBX11). The major amino acid of the BBX proteins is Ala, followed by Ser, while other most abundant amino acids are Pro, Asp, Asn, or Thr, varied depending on the particular BBX protein (Additional file 1: Table S1).

Systematic Evolutionary Relationship, Gene Structural Diversity, and Motif Analysis
We found four different classes of BBX proteins on the basis of domain organization; BBXs containing one B-box domain, BBXs having two B-boxes domains, BBXs possessing one B-box and additional CCT domain, and BBXs with two B-boxes and additional CCT domain ( Table 2). The homologs of BBX genes from six different species were selected for the multiple sequence alignments and phylogenetic relationships analysis to study the evolutionary phylogenetic relationships and functional divergence among BBX genes (Fig. 1). We constructed an unrooted maximum-likelihood phylogenetic tree using MEGA 6.06 Software to investigate the evolutionary relationship. The present study clustered the BBX genes into five well-conserved subfamilies based on the difference of protein topological structure with high bootstrap support (Fig. 1). The phylogenetic tree divided the BBX from five Poaceae plants (maize, rice, sorghum,  (Fig. 1). Arabidopsis BBX possessing only one B-box domain was detected in subfamily II and IV. Two B-boxes domains containing AtBBXs were grouped into IV and V. AtBBXs with one B-box and additional CCT domain containing genes were detected in subfamily II, whereas two B-boxes and additional CCT possessing AtBBXs genes were noted in subfamily I and III. The conservation of gene structure in a paralogous gene is sufficient to determine the evolutionary connection between introns in various circumstances; therefore, an exon-intron diagram of the BBX genes members was constructed according to their genomic and coding sequences (Additional file 1: Figure S2). The exon-intron distribution of all the studied BBX family members was investigated through GSDS online software. The range of a number of introns was from one to seventeen (ZmBBX28) in this study. However, we also identified some BBX members without of intron, they comprised only of the exon. For instance, ZmBBX9, ZmBBX17, ZmBBX24, and ZmBBX33 genes have the only exon in maize. In rice, OsBBX2 and OsBBX25 were found without of intron. However, without of intron genes were not found in sorghum, stiff brome, and millet.
Furthermore, all the BBX proteins were run on MEME tool to investigate the motifs (Additional file 1: Figure S2). MEME analysis found a total of 10 motifs and was named 1-10. Based on width, Motif-7 was the largest motif, whereas next spots were held by motif-8 and motif-2 (Additional file 1: Table S2). We observed that motif-2 was present in 126 out of 131 BBX members, followed by the shortest motif, named motif-6 (115 BBX members). The longest motif (motif-7) was only found in 10 BBX members. Similarly, each motif-8 and 9 were found in 11 BBX members.

Chromosomal Location, Multiple Alignments and Gene Duplication of BBX Genes
The chromosome location and annotation information of the BBX genes showed that BBX genes are unevenly distributed on the chromosomes in the genome of the studied species (Additional file 1: Figure S3). In maize, all the 36 ZmBBX genes were found to be distributed on the 10 chromosomes, except for chromosomes 8 (Additional file 1: Figure S3A). However, the number of ZmBBX genes varied widely on each chromosome. A high number of ZmBBX genes (7) was localized on chromosome no. 5, whereas 6, 5, 5, 4, 4, 3, 1 and 1 ZmBBX members were identified on chromosomes 4, 6, 9, 2, 10, 1, 3 and 7 in the maize genome, respectively. In rice, all 30 OsBBX genes are distributed on chromosomes 1-9: 8 OsBBXs are located on chromosome 6, 7 rice BBX members were detected on chromosomes 2, 3 OsBBX genes on each chromosome 4, 8 and 9, 2 OsBBX genes were found on chromosome 3, while 1 each on chromosomes 1, 5 and 7 (Additional file 1: Figure S3B). In sorghum, SbBBX genes were found to be distributed on all the chromosomes except chromosome 5: 8 SbBBXs were found on chromosome 4, 6 SbBBXs on chromosome 10, 3 SbBBXs were detected on chromosome 6, 2 SbBBX members are present on each 1, 2 and 7, while one each on chromosomes 3, 8 and 9 (Additional file 1: Figure S3C). All the BdBBX genes member are distributed on all chromosomes in stiff brome genome. A maximum number of BdBBX genes are localized on chromosome 1 (8 BdBBXs) and 3 (7 BdBBXs). Remaining BdBBX members are distributed as: 3 BdBBXs on chromosome 5, while 2 BdBBX genes are located on each chromosome 2 and 4. SiBBX genes were detected on all chromosome expect on chromosome 8 (Additional file 1: Figure S3D). The number of BBX genes  on the chromosome is varied in millet genome. However, a high number of SiBBX (6) genes were observed on chromosome 1, whereas the lowest number of SiBBX genes (1) was found on chromosome 1. 4 and 3 SiBBX members are located on chromosome 4 and 7, respectively. 2 SiBBX genes were investigated on each chromosome 2 and 3 (Additional file 1: Figure S3E). Multiple online databases including Pfam, SMART, Inter Pro Scan, Conserved Domain Database (CDD), NCBI, and Scan Prosite were used to identify the conserved domains of the Poaceae BBX proteins. The family-specific domains of BBX proteins including B-box1, B-box2, and CCT conserved domains, were aligned by DNAMAN software, and their logos were constructed via Web Logo online tool (Additional file 1: Figure S4). Previous studies investigated that the CCT domains comprised are the most conserved family specific domain among B-box1, B-box2 and CCT domains (Additional file 1: Figure S5a,b,c) [4,28], and similar results were obtained for Poaceae BBX proteins. Previously, it was also postulated that B-box1 domain is the highly conserved domain than B-box2 domain and deletion event occur in the B-box2 domain. We also found that B-box1 was more conserved compared with B-box2 domain signifying that the deletion process could happen in B-box2 domains during evolution (Additional file 1: Figure S5a, b).
The duplication of individual genes, chromosomal segment, or of the entire genome itself are the major forces during the course of genome evolution in plants [29]. We identified the possibility of gene duplication in the BBX gene family in maize, rice, sorghum, stiff brome and millet (Fig. 2). A diagram constructed with the Circos program was used to draw the duplicated blocks in these plants genome. Both the segmental and tandem duplications were studied in this investigation. 25 ZmBBX pairs were located in the segmentally duplicated regions on different chromosomes in the maize genome. OsBBX pairs of the duplicated region were found in the rice genome. Only one pair of the segmentally duplicated region was identified in each sorghum and stiff brome genome, whereas two pairs of the duplicated region of BBX genes were located on the chromosome in millet genome. However, no tandem duplication was observed among the BBX family members in the studied plants. The results indicated that only segmental duplication may take part in the evolution of BBX genes in maize, rice, sorghum, stiff brome, and millet.

Developmental and Tissue-Specific Expression Profiles of Rice BBX Genes
We examined the different developmental stages/tissues to study the biological roles of BBX genes in the plant growth and development, based on a set of microarray data obtained from Genevestigator v3 and quantitative real-time polymerase chain reaction (qRT-PCR). The expression data from the microarray analysis of rice BBXs are presented in the form of a heat map, from blue to pink reflecting the percentage expression (Fig. 3). Nine tissues including seedling, shoot, leaves, seed, endosperm, embryo, anther, pistil, pre and post-emergence inflorescences, were analyzed. The 30 candidates of rice BBX genes displayed quite a similar expression profile among the tested tissues (Fig. 3). Eight members of rice BBX (OsBBX4, OsBBX5, OsBBX9, OsBBX10, OsBBX11, OsBBX12, OsBBX20, and OsBBX29) were highly expressed in seedling, shoot, leaves, seed-5 DAP, pistil, anther, pre and post-emergence inflorescences. No expression was detected for all the members of BBX genes in endosperm and seed-10 DAP except for OsBBX7, OsBBX16 and OsBBX29; however, we found 17 BBX genes members (OsBBX1, OsBBX2, OsBBX3, OsBBX4, OsBBX5, OsBBX7, OsBBX9, OsBBX10, OsBBX11, OsBBX12, OsBBX14, OsBBX16, OsBBX19, OsBBX20, OsBBX22, OsBBX24 and OsBBX29) with high transcripts in seed-5 DAP. No or extremely low transcript level was detected for OsBBX6, OsBBX18, OsBBX28, and OsBBX30 among all the studied tissues. Moreover, we observed the expression profile of two BBX genes, namely OsBBX16 and OsBBX29, among all the tissues apart from endosperm-25 DAP, seed-10 DAP and endosperm-25 DAP (replicate). This investigation found that all the BBX genes were expressed in the shoot except OsBBX15, OsBBX18, OsBBX21, OsBBX23, and OsBBX28.
Furthermore, we performed qRT-PCR of the 12 rice BBX members (OsBBX1, OsBBX2, OsBBX7, OsBBX8, OsBBX9, OsBBX12, OsBBX14, OsBBX16, OsBBX17, OsBBX19, OsBBX21 and OsBBX24) to find out the expression profiles among 14 different tissues (Fig. 4). The tissues were collected at three different stages: 1) seedling stage including leaf, stem and root; 2) booting stage consisted node-1, node-2, internode-1, internode-2, leaf sheath-1, and leaf-sheath-2; 3) heading stage including flag leaf, leaf blade, flower stage-1, flower stage-2 and flower stage-3. The transcript levels of all the studied BBX genes were high in the stem, internode-1, and flower stage-3 tissues. All the 12 BBX members showed low transcription in the root, flag leaf, and internode-2 tissues. No high expression was detected for the all the BBX genes in node-2 except for OsBBX14, OsBBX16, OsBBX21, and OsBBX24. Low transcript level was observed for OsBBX17 gene in leaf, whereas high transcript level was detected for the remaining BBX members. The expression profile of all the BBX genes was almost similar in node-2 and internode-2. High expression profile was found for OsBBX1, OsBBX2, OsBBX7, OsBBX8, OsBBX12 and OsBBX17 in leaf sheath-1 and leaf sheath-2, while the rest of BBX members showed low expression profile in these two tissues. In leaf blade and flowering stage-1, the expression profile of all OsBBX genes was maximum except OsBBX8, OsBBX12, and OsBBX17. The transcription rate of all BBX members was high in flowering stage-2 excluding OsBBX14, OsBBX17, and OsBBX19. Overall, we noted that the transcript level of most rice BBX genes was high in the heading stage, followed by booting and seedling stage based on the three stages. The present study found the expression profile (low or high) of OsBBXs in almost all the tested tissues. These findings indicated the multiple roles of BBX gene family in the development and growth of rice.

Inducible Expression Analysis of Rice BBX Genes under Abiotic stresses and hormonal applications
Gene expression analysis can provide essential clues for gene function; therefore, we carried out qRT-PCR to investigate the transcript levels of the rice BBX genes under different abiotic stresses, including drought, cold and salt. Describing the expression profiles of all rice BBX genes was exhaustively difficult; therefore, twelve BBX members (OsBBX1, OsBBX2, OsBBX7, OsBBX8, OsBBX9, OsBBX12, OsBBX14, OsBBX16, OsBBX17, OsBBX19, OsBBX21, and OsBBX24) of rice BBX gene family were assessed (Fig. 5). More than two-fold difference in transcript levels was considered to be the true difference for the genes under treatments. We found that the transcript levels of OsBBX7, OsBBX8, OsBBX9, OsBBX12, OsBBX16, and OsBBX21 were down-regulated, whereas the remaining six BBX members were up-regulated at least at one (OsBBX14, OsBBX17, and OsBBX19) or two-time points (OsBBX1, OsBBX2, and OsBBX24) under drought stress. Under cold stress, the expression profile of only one BBX gene (OsBBX12) was high at all the tested time points compared to 0 hr sample (control), whereas the expression profile of OsBBX14 and OsBBX21 was down-regulated. The expression of OsBBX1 and OsBBX2 and OsBBX19 was high at 3 hr and 6 hr time points, respectively, while the other six BBX members were up-regulated at two or three time points under cold stress. Similarly, the transcript profile of OsBBX1, OsBBX7, OsBBX8, and OsBBX16 was high at all the time points under salt stress. Moreover, some BBX members (OsBBX12, OsBBX14, OsBBX17, and OsBBX24) were down-regulated, while the rest of the four BBX genes up and down-regulated at different time points under salt stress. Altogether, we observed that transcript of most rice BBX members was significantly affected under salt and cold stresses; in addition, we also noticed that the BBX members were also up and down-regulated at some time points under drought conditions. All these results indicate the involvement of BBX gene family in plant growth and development and their response against multivariate stresses.
qRT-PCR was also used to analyze the transcript patterns of all BBX genes under GA, ABA, SA, and MeJA hormones applications, to reveal the effects of various hormones on the expression of BBX gene family members in rice (Fig. 6). We noticed that the expression levels of OsBBX1, OsBBX17, OsBBX19, and OsBBX24 were promoted in response to exogenous GA treatment at all the time points, whereas the transcripts of OsBBX9 and OsBBX21 were down-regulated. Furthermore, OsBBX2, OsBBX7, and OsBBX8 were up-regulated at 3, 6 and 24 hr. We also found low transcripts for some BBX members including OsBBX12, OsBBX14 and OsBBX16 genes under GA treatment. In contrast, the expression levels of all rice BBX gene members were very low excluding OsBBX14 under ABA treatment. Moreover, the transcript levels of OsBBX12, OsBBX17, and OsBBX19 were up-regulated at all the time points under SA hormone, whereas OsBBX21 Fig. 3 The expression profiles obtained from the ArrayExpress data, dsiplaying diverse expression levels of apple BBX genes in different tissues and organs. Relative transcript level of BBX genes members based on ArrayExpress data were presented as heat maps from green to red reflecting relative signal values; where dark green boxes represent stronger down-regulated expression and dark red boxes represents stronger up-regulation Fig. 4 Expression profile of the OsBBX genes in tested tissues. The graphs indicate tissue specific expression level in rice plant. The samples were collected in different developmental stages, and were analyzed through qRT-PCR. The x-axis indicates the tissues. The y-axis shows the relative expression level of each tissue. The error bars indicate the standard deviations of the three independent qRT-PCR biological replicates was down-regulated. We found some genes members, namely OsBBX2 and OsBBX9, with high expression profiles till 12 hr post-treatment and their expression was suddenly declined at the 24 hr time point. The expression of OsBBX1 was increased at only one time point (12 hr). We also observed a maximum number of BBX members shown up-regulation in expression at 3, 6 and 12 hr time points under SA treatment. Under MeJA hormones, most rice BBX was up-regulated at least one or two time points, however, OsBBX2 and OsBBX12 were up-regulated at all the time points. Low transcript level was detected for OsBBX1 and OsBBX8 at all the time points in response to exogenous MeJA treatment. Overall, the expressions of rice BBX genes members were highly affected by exogenous GA, SA and MeJA hormones. Additionally, the transcripts of rice BBX members were also changed by exogenous ABA treatment at a few time points. Thus, the results reveal that in response to signaling molecules the BBX genes members underwent clear variations in transcript level suggesting their hormone-induced differential responses in rice.

Expression Profiles of rice BBX genes under metals treatments
Two-week-old rice plants were exposed to four different metals stressors such as Cr, Cd, Ni, and Fe, to insight the transcriptional regulation and expression profiles of rice BBX genes, and the possible involvement of heavy metal stresses (Fig. 7). The temporal induction of rice BBX genes members at the transcriptional level at a various time point were evaluated through qRT-PCR. We found that the transcript profiles of OsBBX1, OsBBX7, OsBBX8, OsBBX17, and OsBBX19 were affected by all the four metals including Cr, Cd, Ni and Fe metals at some time points. The expression profiles of OsBBX2 and OsBBX14 genes were up-regulated under all four metal stresses apart from Cr and Cd, respectively. The transcription patterns of OsBBX9 had shown obvious changes in the expression level under Ni stress; likewise, OsBBX16 and OsBBX21 were up-regulated by Fe stress while the response of these genes to other metals such as Ni, Cr, and Cd was very low. Similarly, the expression level of OsBBX24 gene was high at 3 and 6 hr under Ni metal, while low transcript was noticed under other three metal treatments. For OsBBX12, low transcript level was observed under Ni and Cr metal, however, the expression was up-regulated under Fe and Cd metal stresses. Based on time points, we noticed that most rice BBX members were up-regulated at 12 hr time point followed by 6, 3 and 24 hr, respectively. Furthermore, based on metals, this study observed the expression of almost all the BBX members shown up-regulation at least at one time point under Fe and Ni metals excluding OsBBX9 and OsBBX21 genes, respectively. In response to Cr and Cd, rice BBX genes showed a low level of expression apart from OsBBX8, OsBBX12, and OsBBX19 and OsBBX7 and OsBBX14, respectively. Overall, the studied BBX members showed high expression profiles in Fe and Ni compared with Cr and Cd metals. The unique inducible expression patterns of the BBX gene family members under metal stresses may indicate the role of BBX genes family in response to heavy metals. However, further studies are required to investigate deeply the particular behavior role of BBX gene family in plant multivariate stresses.

Discussion
The gene clustering and evolutionary relationship mostly change due to domain shuffling and low sequence identity among the homologs proteins. Therefore, the rearrangement of domain composition, exon shuffling and gene duplication may lead to the expansion of gene families in plants during evolutionary processes [29,30]. Subsequently, the duplicated gene may promote functional variations, and possibly expand the functional characteristics of genes [31,32]. Furthermore, single gene duplication might be the main cause leading to the expansion of gene families in plants.

Identification and Evolution history of BBX family members in various plants
BBX gene family has been got more attention from the scientific community in the past couple of years. The genome-wide identification analysis of BBX genes has been already investigated in rice [14] and other important plants such as Arabidopsis, tomato, potato, pear and apple [1,14,[33][34][35][36]. In this study, we also reported the genome-wide identification of BBX genes in five Poaceae species (maize, rice, sorghum, millet, and stiff brome), and their expression analysis under abiotic (cold, drought and salt), hormones (GA, ABA, SA and MeJA) and metal stresses (Cr, Cd, Ni and Fe) in rice. Based on our results, we found a total of 131 BBX genes in the five Poaceae species genomes including 36 from maize, 30 from rice, 24 from sorghum, 22 from stiff brome and 19 from millet ( Table 1). The previous study also found a similar number of BBX genes in the rice genome [14]. The number of BBX gene family members is pretty consistent among different crop plants, such as 30, 32, 29 and 30 BBX genes members were already identified in rice, Arabidopsis, tomato, and potato, respectively [1,14,33,36]. The difference in the number of BBX genes among the crops plants is very less. However, a total of 67 BBX genes in apple [35]. The difference in the number of BBX genes between tree and crop plants may due to the large and heterozygous genome of apple. Furthermore, we also found less number of BBX members in two species of Poaceae family, 22 from stiff brome and 19 from millet. The difference may due to the genome of these two species are not fully sequenced or may small and simple genome.
Previous studies identified 4 different types of BBX proteins based on domain organization in tomato and Arabidopsis [1,36]. We also found 4 different types of BBXs (Table 2) The Arabidopsis BBX was clearly divided into five clusters on the basis of different conserved domains combinations [1]. Two B-boxes plus additional CCT domains containing BBX (AtBBX1-AtBBX13) were found in group-1 and 2; one B-box plus CCT domain containing genes (AtBBX14-17) were clustered into group-3, BBX genes containing two B-boxes (AtBBX18-25) and one B-box domains (AtBBX26-32) were observed in clade-4 and 5 in Arabidopsis, respectively [1]. Whereas, in five Poaceae species, maximum number of one and two B-boxes and additional CCT conserved domains containing BBX genes members were cluster together into subfamily I, II and III (Fig. 1), BBX genes possessing one B-box domain were detected in subfamily II, IV and V, whereas two B-boxes containing BBX genes were observed in subfamily IV and V in this study. The classification of Poaceae BBX members based on conserved domain was relatively difficult. The reason behind uneven distribution may due to a large number of genes or the small difference in the domain organization in the plant species. For instance, we noticed that 7 BBX genes possessed only one B-box domain, 8 BBX members had two B-boxes domain, 4 BBX members contained one B-box and additional CCT domain and 13 BBX genes were found with two B-boxes and additional CCT domains in Arabidopsis [1]. In contrary, 3 BBX possessed only one B-box domain, 10 BBX found having two B-boxes domains, one B-box and additional CCT domain were observed in 10 BBX members and 7 BBX genes comprised of two B-boxes and additional CCT domains in rice ( Table 2). Similar differences were also observed for B-box genes in other four studied Poaceae species. However, we also noted that the gene structure and functional characteristic of BBX genes within the subfamily was quite similar. Thus, it is assumed that BBX members share a similar gene structure and functional characteristic within the same subfamily during the evolutionary relationship. Previously, it also has been reported that FRO gene family members in rice shared similar gene structure and functional characteristic during evolution in rice [37].
Moreover, It has been already reported that CCT is the highly conserved domain [29,38]. The alignment of B-box1, B-box2 and CCT domain also indicated that the CCT domain was highly conserved compared with B-box1 and B-box2 domain (Additional file 1: Figure S5a, b, c). However, a theory has been proposed that a deletion process occurs during the evaluation that leads to making another class of BBX genes, containing only one B-box domain [3]. After detail sequence alignment of two B-box domains (B-box1 and B-box2) revealed that B-box1 domain was highly conserved compared with B-box2 in rice BBX (Additional file 1: Figure S5a, b), thus, it's postulated that deletion process could occur in the B-box2 domain and give birth to the B-box1 domain.
Large-scale duplication and tandem duplication processes are vital for the amplification of gene family members in the genome during the evolution [39]. In this study, both the tandem and segmental duplication events were analyzed to study the evaluation of the BBX genes in Poaceae. We found only segmental duplication in the BBX genes (Fig. 2) indicating that segmental duplication events took part in the expansion of the BBX gene family in Poaceae.

Tissue-Specific gene expression profiles reveal the diverse roles of BBX gene family in plant growth and development
The specific gene family members have common genes expression profile features in plants. This may coordinate and/or differ in the functional interaction of the family members. It was previously reported that BBX proteins control the diverse functions of the plant, such as photomorphogenesis, flowering and shade avoidance [40,41]. In Arabidopsis, the overexpression of a BBX gene (BBX6, COL5) promotes early flowering [42], and the overexpression COL9 (BBX7) delay the flowering under SD (short day) condition [43]. BBX homologous genes which contribute to different biological processes with obvious tissue specificity in gene expression have been functionally characterized in maize [44]. The members of BBX gene family also showed diverse expression in all the tested tissues in tomato [36]. Similarly, in potato maximum number of BBX family members was detected with distinct expression pattern among the tested organs [33]. Likewise, we investigated the expression of BBX family in 14 different tissues and the samples were collected at three different stages, seedling stage root, booting stage and heading stage (Fig. 4). We found that the expression of almost all the BBX members was high in all the tested samples apart from roots. Furthermore, we also noticed that the transcript levels of the studied BBX members were high in the heading stage. Moreover, the database searching found that BBX gene more expressed seedling, leaf, shoot and flowering-related tissues (Fig. 3). Thus, the database searching and functional prediction of BBX gene family members in various tissues and different developmental stages demonstrate that BBX gene family might play vital roles in plant growth, and some BBX genes members might have a unique function in specific developmental stages.
Pronounced but differentiated inducible expression patterns under a number of environmental, hormonal and metal stresses imply the vital contributions of BBX gene members to multivariate stress tolerance Various adverse environmental aspects such as ion toxicity, salinity, drought, extreme temperatures negatively disturb plant growth and development [45][46][47]. Among them, several abiotic stresses cause general or specific effects on growth and development and changes at the transcriptional level in plants [48][49][50]. Here, we detected that rice BBX genes are sensitive to a set of abiotic stresses, and their transcriptional expressions were greatly altered by salt, cold, drought, GA, SA, MeJA, ABA and metals stress treatments, displaying their contribution in responses to multiple stresses in rice. Several investigations have proposed that BBX genes are important for the photoperiodic regulation of flowering, seedling photomorphogenesis, shade avoidance, and responses to biotic and abiotic stresses. It has been also stated that the salt tolerance protein STO (AtBBX24) enhances the growth of root under a high salinity condition in Arabidopsis [15] and the salt tolerant activities was also triggered in yeast cells [16]. AtBBX18 acts as negative regulator both in photomorphogenesis and thermotolerance in Arabidopsis [12]. Furthermore, BBX18 negatively regulates the expression of heat-responsive genes such as DGD1, Hsp70, Hsp101, and APX2, thereby reducing germination and seedling survival after a heat treatment [12]. In Chrysanthemum, CmBBX24 performs a dual function, delaying flowering and also increasing cold or drought tolerance in the plant [19]. Moreover, some studied found that BBX proteins also involve in hormones signaling. A recent investigation found that the expression pattern of BBX genes was altered in response to ABA and cyclic ADP-ribose (cADPR) temperatures [6,7]. The involvement of BBX genes in the COP/HY5 signaling pathway indicates that BBX18 may work as an integrator of both GA and COP1/HY5 pathways [13]. Based on the previous studies, we evaluated the expression of OsBBX genes in response to numerous abiotic and hormonal stresses and found that the most rice BBX members show high expression levels under abiotic stresses (Fig. 5). The expression patterns of OsBBX1, OsBBX2, and OsBBX19 genes were affected by all the three used abiotic stresses including drought, salt and cold stresses. OsBBX7, OsBBX8, and OsBBX16 genes showed high expression under salt and cold conditions, whereas OsBBX17 and OsBBX24 genes were up-regulated in response to drought and cold. In addition, we found that most rice BBX genes were up-regulated under the cold and salt condition, while, less transcript level was observed for most rice BBX genes in response to drought. The members of rice BBX gene family also showed maximum expression levels in response to different hormones (Fig. 6). The expression of OsBBX2, OsBBX7, OsBBX17, OsBBX19, and OsBBX24 genes were strongly triggered in response to GA, SA and MeJa hormones. Similarly, OsBBX1 and OsBBX16 genes displayed high expression under GA and MeJa hormones, respectively. Moreover, the transcript levels of OsBBX8 and OsBBX14 were promoted under GA, ABA, SA and MeJa hormones. Although most rice BBX genes were up-regulated at different points under GA, SA and MeJA hormones, the transcripts of the BBX gene family were less effected by ABA. Furthermore, the transcript levels of most BBX members were significantly stimulated by heavy metal stresses even though somewhat unique responses occurred for some members under certain metals (Fig. 7). For example, the transcript profiles of OsBBX1, OsBBX7, OsBBX8, OsBBX17, and OsBBX19 members were greatly affected by Fe, Ni, Cr, and Cd metals, however, the transcription activity of OsBBX24 was significantly changed in response to all the applied metals apart from Cr metal. Similarly, the transcript profile of OsBBX14 was enhanced in response to all used metals except Cd metal. Furthermore, we also found some BBX genes which showed high expression profile in response to only one metal, for instance, OsBBX9 was highly expressed under Ni metal. Overall, the results obtained here suggest that BBX gene family may perform several functions in plant growth and development and in response to abiotic, metal stresses and hormonal applications although their exact role remains unclear. Further experiments need to be done to investigate the exact role of BBX gene family in plant growth and development.

Conclusions
Over a long evolutionary relationship of plants, BBX genes had shown consistency in their common characteristics and functional behavior. In this context, the differential expression patterns of BBX genes in Poaceae plants play a vital role in the plant growth regulation. The regulatory mechanism and transcriptional variation of BBX genes are highly responsive to external factors, thus, the multivariate stresses and hormonal application substantially triggered the up-regulation of the differentially expressed genes, thereby participating the beneficial allocation and potential role of these genes in plants. We suggest that the specific role of particular BBX gene should be a target for defining the stress response, functional divergence and possible crosstalk in plants such as rice.