B-BOX genes: genome-wide identification, evolution and their contribution to pollen growth in pear (Pyrus bretschneideri Rehd.)

Background The B-BOX (BBX) proteins have important functions in regulating plant growth and development. In plants, the BBX gene family has been identified in several plants, such as rice, Arabidopsis and tomato. However, there still lack a genome-wide survey of BBX genes in pear. Results In the present study, a total of 25 BBX genes were identified in pear (Pyrus bretschneideri Rehd.). Subsequently, phylogenetic relationship, gene structure, gene duplication, transcriptome data and qRT-PCR were conducted on these BBX gene members. The transcript analysis revealed that twelve PbBBX genes (48%) were specifically expressed in pear pollen tubes. Furthermore, qRT-PCR analysis indicated that both PbBBX4 and PbBBX13 have potential role in pear fruit development, while PbBBX5 should be involved in the senescence of pear pollen tube. Conclusions This study provided a genome-wide survey of BBX gene family in pear, and highlighted its roles in both pear fruits and pollen tubes. The results will be useful in improving our understanding of the complexity of BBX gene family and functional characteristics of its members in future study. Electronic supplementary material The online version of this article (10.1186/s12870-017-1105-4) contains supplementary material, which is available to authorized users.


Background
Zinc-finger protein is one of the important transcription factors, which play important roles in plant growth, development and response to environmental changes. The zinc-finger protein whose three-dimensional is stabilized by binding zinc ions [1], could interact with DNA, RNA, and proteins involved in plant cell life activities [2]. B-BOX gene family belongs to the zinc-finger protein family. In addition to the conserved B-BOX domain, some of the B-BOX members contain other family-specific domains, such as CCT (CONSTANS, CO-like and TOC1) domain. The B-BOX gene family could be divided into five subfamilies according to the number of B-BOX domains or the CCT domain they contained [2]. After the first identification of the B-BOX member from Xenopus laevis [3], its ortholog (CONSTANS: CO) in plant was cloned from Arabidoosis thaliana, with function in the photoperiod regulation of plant flowering time [4]. Subsequent researches further revealed that the B-BOX transcription factors in plants played very pivotal roles in mediating various life activities, such as seed germination [5], flowering [6], shade avoidance response [7], biological or abiotic stress response [8] and plant hormone signal transduction [9]. Recent studies have shown that as a negative regulator in brassinosteroid signaling pathways, BBX20 (AtBZS1) from A. thaliana could attach to E2 by recognition of COP1 (Constitutively Photomorphogenic 1), a key factor in light signal transduction, then for degradation by 26S proteasomes [9].  stated that AtBBX25 is involved in the negative regulation of plant photo-morphogenesis by forming a dimer with HY5 (Protein long hypocotyl 5) and suppressing its function [10]. Studies in apples found that MdBBX22 (MdCOL11) involved in the UV-B-induced synthesis of the anthocyanin synthesis [11]. Based on these results, it was implied that B-BOX gene members might be associated with COP1-HY5-mediated optical signal transduction pathway, and further involved in plant light morphogenesis, regulating flowering, secondary metabolite synthesis and regulation of a variety of life activities.
It has been proved that some transcription factor family should play an important role in the development of pear fruits, such as MYB and heat shock factor gene family [12,13]. Two MYB family members, PbMYB25 and PbMYB52 were found to be the candidate genes involved in the regulation of lignin synthesis within pear fruits [12]. Although much knowledge on the function of B-BOX gene family, such as responses to biotic and abiotic stresses and involvement in light signal transduction pathway, has been advanced [14][15][16], there is less research on their roles in pear pollen and fruit development. The completed pear genome sequencing [17] provided useful information for comprehensive analysis of the pear BBX gene family. In this study, 25 nonredundant members were identified in the pear BBX family. Subsequently, the detailed phylogenetic and expression pattern analyses of these BBX genes were carried out. The present results will be useful for further functional characterization of BBX genes in pear.

Identification of BBX genes in pear
To obtain BBX proteins in pear genome, the published Arabidopsis BBX proteins were employed as a query to search against the local pear genome database by using DNAtools software. After removing the redundant and repeated sequences, a total of 25 putative BBX protein sequences were confirmed in pear. For the sake of consistency, these BBX genes were sequentially named after PbBBX1 to PbBBX25. The detailed information on the gene identifier, chromosome location, protein structure and the characteristics of the corresponding PbBBX proteins were listed in Table 1. The length of the amino acid sequence sequences ranged from 142 (PbBBX23) to 859 (PbBBX7). The pear BBX genes encode proteins with predicted theoretical isoelectric points of 4.48-9.02 and molecular weights from 15.63900 (PbBBX23) to 93.60447 (PbBBX7) kDa (Table 1).  Table 1). The conserved structures of PbBBX members, were found with B-Box 1 sequence (CDXCXXXXAXVYC XADEAALCXXCDXXVHXANKLAXRHXH, X represents any amino acid) and B-Box 2 (CDICXXXXAXXXCXXD XAXLCXXCDXXVHXXXXXXHXRXXL) (Fig. 1). Additionally, the CCT domain was highly conserved among the PbBBXs (Fig. 1). The logos of these domains, including B-BOX1, B-BOX2 and CCT domain, were illustrated in Fig. 1, as well as the correspondence positions shown in Fig. 2.
To gain further insights into the phylogenetic relationship and divergence of the BBX family, the phylogenic tree, including BBXs from Brachypodium distachyon, Oryza sativa, A. thaliana, Populus trichocarpa and pear, was constructed. Based on the phylogenetic analysis, this tree could be divided into five clades, and consistent with the previous studies [2,18]. As shown in Fig. 3, most BBX members from poplar, Arabidopsis and pear were more closely than pear and Oryza sativa, Brachypodium distachyon. Among them, the members from clades I, II, III, contained two B-BOX domains plus a CCT domain, implying that these genes contained CCT-domain might play a crucial role in the control of flowering [4,19]. On the contrary, the members from clade VI and clade V lacked CCT-domain and only contained one or two B-BOX domain. Previous studies have shown that the B-BOX domains (CX 2 CX 8 CX 7 CX 2 CX 4 HX 8 H) in the N-terminal region, and the conserved Cysteine (C) and Histidine (H) residues in B-BOX domain are predicted to be crucial for BBX protein-protein [2]. Interestingly, the Cysteine (C) and Histidine (H) residues was also found to be conserved at B-BOX domain in the C-terminal region of the BBX members from clades I, II and IV, respectively ( Fig. 1). In summary, the structure analyses of theses B-BOX proteins were basically consistent with the phylogenetic relationship.

Gene structure and gene duplication
The previous studies implied that gene structural diversity can lead to the evolution of multi-gene families. To better characterize and understand the structural diversity of the PbBBX genes, gene exon-intron analysis was carried out (Fig. 4). As shown in Fig. 4, the number of exons was ranged from 1 to 17, with PbBBX7 containing the highest amounts of exons (17) among the PbBBXs, 12 of PbBBXs containing two exons, and 3 only one exon, respectively. Additionally, pear BBX genes were clustered in the same clade with the highly similar exonintron structure, for example, eight PbBBXs within the clades I and III (containing two exons), and most members belonging to clade IV (having three exons). Likewise, three genes in clade V only contained one exon, except for PbBBX24 and PbBBX25. These results deduced that exon-loss or -gain had occurred during the evolution of the PbBBX gene family and resulted in the functional divergence among the closely related PbBBXs (Fig. 4).
Up to data, the information about the expansion events of the BBX gene family in pear was still unclear. To further reveal how PbBBX genes were evolved, the chromosomal location and gene duplication events of PbBBX genes were investigated. The chromosome locations and distributions of 25 PbBBX genes were found among the 12 pear chromosomes (total of 17 chromosomes) (Additional file 1: Figure S1). Among them, the chromosomes 15 and 17 both contained highest number of PbBBX genes (5); followed by chromosome 5 contained three genes; chromosomes 3 and 10 both had two genes; while the chromosomes 6, 8, 9, 11, 13, 14 and 16 only had one genes. Gene family expansion was usually achieved by tandem duplication and segmental duplication. In present study, we did not identify any of tandem duplication pairs. However, 13 segmental duplication gene pairs were found in pear genome by using MCScanX software (Fig. 5). Subsequently, the divergence time between these gene pairs was calculated with the period varied from 6.15 to 253.08 million years (Mya) (Additional file 2: Table S1).
To determine the selection pressure in duplication of PbBBX genes, the non-synonymous (Ka)/synonymous (Ks) values were calculated for the 13 gene pairs. The  Table S1), indicating that they were under strong purifying selective during their evolution and a conserved evolutionary pattern was shared among BBX genes.

Expression patterns of pear BBX genes
The pollen germination and pollen tube growth in many higher plants have been known to play a crucial role in sexual reproduction. Previous studies suggested that pollen tube via tip-growth rapidly extended and then underwent senescence within 15 h (P4: stopped-growth pollen tubes) in vitro [20]. In our study, to further understand the roles of BBX family genes in pear pollen growth, expression patterns of PbBBX genes were analyzed by transcriptome sequencing data. As shown in Figure  (Additional file 3: Figure S2), 13 of PbBBX genes (52%) were not found to be expressed during the different developmental stages of pear pollen, implying that these genes might express in root, stem, leaf, or/and under special conditions. On the contrary, 12 PbBBX genes (48%) were detected to be expressed in a development-dependent pattern in pear pollen. For example, 5 PbBBXs (PbBBX6, 7,9,11,12) were specifically expressed at P1 stage (mature pollen grains), while 2 (PbBBX8 and PbBBX10) at P2 stage (hydrated pollen grains) (Additional file 3: Figure S2). The high number of PbBBX genes differentially expressed during pollen growth suggests that they are important proteins for signaling in this process. In addition, the expression patterns of PbBBX genes were validated by using qRT-PCR during pear pollen tube growth (Additional file 4: Figure S3). We found that qRT-PCR results were almost consistent with that of transcriptome sequencing data, except PbBBX6 and PbBBX7. The reason for this divergence may be the lowest expression levels during pollen tube growth. Remarkably, compared with other periods, the expression levels of PbBBX5 gene reached its peak at P4 (stopped-growth pollen tubes), implying that this gene might play potential role in plant reproductive development, such as the senescence of pollen tubes.
Subsequently, the expression profiles of these PbBBX genes in different tissues or organs were also surveyed by using qRT-PCR (Fig. 6). Results showed that except for PbBBXs 6,8,9,11  highly expressed in roots. Furthermore, the expression levels of PbBBXs 12, 17 and 25 was higher in root, stem or leaf than that those in different developmental stages of pear fruit. Interestingly, PbBBXs 6, 8, 9, and 11 were tissue-specifically expressed during different developmental stages of pear pollen. Additionally, we found PbBBX19 was not expressed in root, stem, leaf and fruits, these results recommended that the functions need to be further studied at other tissues or special conditions (Fig. 6).

Analysis of subcellular localization of PbBBX4, PbBBX5 and PbBBX13
The nuclear localization of transcription factors is very important for its regulatory function. Previous studies reported that BBX proteins were predominantly located on the nucleus, such as SlBBX5, SlBBX7 and SlBBX15 in tomato [21]. The expression levels of PbBBX4, PbBBX5 and PbBBX13 observed that they play an important role in the development of pear pollen tubes or fruits. To further understand these three proteins characteristics, subcellular localization experiment was carried out. Subsequently, we introduced GFP control and the PbBBX4-GFP, PbBBX5-GFP and PbBBX13-GFP fusion constructs (Fig. 7a) by CaMV 35S promoter into N. tabacum epidermal cells. As indicated in Fig. 7b, green fluorescence signals from the expressed fusion PbBBX4-GFP, PbBBX5-GFP and PbBBX13-GFP were specifically distributed within the nuclei as confirmed by DAPI (DNA dye 4, 6-diamidino-2phenylindole) staining. However, the control GFP protein was observed throughout the whole cell (Fig. 7b). These results suggested that PbBBX4, PbBBX5 and PbBBX13 were nuclear proteins, and consistent with the previous results [21].

Discussion
Although the BBX gene family has been identified in several model plants, such as rice [22], and Arabidopsis [2], its function and evolution was still unclear in pear. In this study, a comprehensive analysis of pear BBX gene family was performed, including analyses of phylogeny, chromosome localization, gene duplication, sequence feature, and expression pattern.
A total of 25 BBX genes were identified from pear genome. The number of BBX genes in pear was fewer, compared to their orthologs in tomato (29) [21], Arabidopsis (32) [2], and rice (30) [22]. Noteworthy, the pear genome size (512 Mb) [17] was larger than those of rice (403 Mb) [23] or Arabidopsis (125 Mb) [24], although smaller than the tomato genome size (960 Mb) [25]. These results indicated that the BBX gene family members may not be directly related to the genome sizes in different plants. Although the difference in number was not significant, however, the type of BBX gene was  Figure. The scale represents the length of the DNA sequence different among species. In tomato, the numbers of BBX members two tandem B-BOXes plus the CCT domain, BOX1 plus CCT, two tandem B-BOXes, and B-BOX1 only were 8, 5, 10 and 6, respectively [21]. In Arabidopsis, the corresponding numbers were 13, 4, 8, and 7 [2]. And in pear were 7, 4, 9 and 6. These results indicated that BBX genes may have a common ancestor among different species, and were independently expanded after the divergence of the dicots and the monocots. In addition, to elucidate how the BBX gene family evolved, a phylogenic tree of plant BBX genes from monocots (rice and Brachypodium distachyum) and dicots (pear, poplar and Arabidopsis) was constructed. Within the phylogenic tree, BBXs were divided into five clades: I, II, III, VI, and V. We found that most of the BBX genes from the dicot were clustered together, implying that these genes might be orthologous genes as reported by previous studies [2,18].
During the course of plant evolution, gene duplication plays an important role for generating novel genes. Gene duplication in plants has two main duplication patterns, including segmental duplication and tandem duplication [26], which had been demonstrated to play a key role in the expansion of gene family members in many species, such as the families WOX, MYB, PRX and 4CL in pear [12,[27][28][29], the CHS in maize [30]. To further reveal the potential mechanism of evolution of the BBX gene family, both the segmental and tandem duplication events were analyzed in pear. In present study, none of the PbBBX genes were located in tandem. And 16 PbBBX genes were identified to be arranged in segmental duplication regions of pear chromosomes. These results indicated that segment duplications were the main driver force for the expansion of pear BBX gene family members. In addition, previous studies reported that tandem duplication often occurred in the large and rapidly evolving gene family, such as NBS-LRR gene family [31], whereas, segmental duplication usually occurred in the slowly evolving gene family, such as MYB gene family [31]. The present results indicated that pear BBX gene family should be classified as a gene family with slow evolutionary characteristics. The collected transcriptome data showed that B-BOX gene family was involved in the pollen tube growth. Twelve of the pear B-BOX gene family members were found to be involved in the development of pear pollen tubes. The previous report by Gangappa et al. [32], has shown that during the photo-morphogenesis in Arabidopsis, some of BBX family members could competitively interact with protein and further regulate HY5 activity, leading to the fine regulation of the pollen tube development. Similarly, during the growth of pear pollen tube, the BBX family members might regulate the development process. Altogether, our results suggested that this gene family was not only involved in the development of floral organs, but also in the development of pollen tubes. The latter might be fulfilled by several genes (PbBBX6, 8, 9, and 11) which were specifically expressed in pear pollen tubes. Previous studies by Gao et al. [20] suggested that as the extension of cultural duration to 15 h (P4: stopped-growth pollen tubes), pear pollen tubes growth became slow and exhibited some characteristics of senescence at P4 post-cultured in vitro, implying that the senescence of the pear pollen tubes might occur at the P4 period. In the present study, the expression level of PbBBX5 in the pear pollen tubes was significantly increased, and its expression pattern was basically consistent with the previous report [20]. The increased expression level of PbBBX5 suggested that it might play a role in the regulation of pollen tube senescence.
Additional, the expression patterns of both PbBBX4 and PbBBX13, were consistent with the content dynamics of fruit lignin: at the early to middle stages the concentration of these contents increased, while at the mature stage showed less concentration [27,33,34]. These results suggested that these two genes might regulate the lignin synthesis in pear fruits. As reported Fig. 6 Expression levels of PbBBX genes in different plant tissues. 15 DAF (days after flowering), 39DAF, 55DAF, 79 DAF and 145 DAF correspond to five different developmental stages of pear fruit. In addition, root, stem and leaf were represented by R, S and L, respectively. The value on the left Y-axis indicates the relative gene expression levels by our previous studies, the content of stone cells was thought to be an important factor affecting the quality of pear fruit [33,34]. Due to closely relationship between the development of stone cell and the biosynthesis of lignin [33,34], it is possible that PbBBX4 and PbBBX13 could be applied for the improvement of pear fruits using genetic engineering.

Conclusions
In the present study, a systematic analysis of the PbBBX gene family was carried out, including conserved domain, gene structure, phylogenetic relationship, chromosome location, gene duplication and expression pattern analysis. The PbBBX genes were divided into five clades: I (4 genes), II (4 genes), III (3 genes), IV (9 genes), V (5 genes), which were supported by gene structural and conserved domain analysis. Gene duplication analysis suggested that the segmental duplications have driven expansion of the pear BBX gene family. Transcriptome sequencing and qRT-PCR analysis revealed that the PbBBX genes play an important role in different pollen tube and fruit developmental stages. Further analysis revealed that PbBBX4 and PbBBX13 might regulate the synthesis of pear fruit lignin, and PbBBX5 might play a role in the senescence of pollen tubes.

Gene structure, chromosomal location, and duplication analysis
The exons and introns of the BBX genes were identified according to the pear genome annotation file. And exonintron map was generated by using Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/) [40]. The chromosome location image of the pear BBX genes on chromosomes or scaffolds was drawn using the MapInspect software according to the physical positions on the pear chromosomes. The MCScanX software (http://chibba.pgml.uga.edu/ mcscan2/) [41] was used to identify the duplications of PbPRXs. The Calculator 2.0 software [42] was used to estimate the nonsynonymous (Ka) and synonymous (Ks) substitution rates of the different gene duplication pairs. The Ks values were used to estimate the approximate date of every duplicated event occurred in pear, seeing the formula: T = Ks/2λ × 10 -6 Mya (λ =6.5 × 10 −9 ) [17,43].

Plant material
The samples were collected from ten of healthy, 40-yearold pear trees (Pyrus bretschneideri cv. Dangshan Su), which have been managed under the same irrigation and fertilization in the orchard at Dangshan County, Anhui province, China. These pear samples under the same developmental period were grown toward the middle southern direction and collected on early April, 2016. Roots, stems, and leaves were collected at the fifteen the day after flowering (DAF). 40 [44].

RNA-seq expression analysis
The raw RNA-seq reads from pear pollen were download from the NCBI database (PRJNA299117) [44]. The pear pollen samples were as follows: P1: mature pollen grains, P2: hydrated pollen grains, P3: growing pollen tubes, and P4: stopped-growth pollen tubes. The analysis of raw RNA-seq data was according to previous method [45], and the RPKM (Reads Per Kilobase per Million mapped reads) values were used to estimate the gene expression level. The heatmap of PbBBX genes was exhibited using R software (http://www.bioconductor.org/).

qRT-PCR analysis
The TIANGEN RNAprep pure (Tiangen, Beijing, China) was used to extract the total RNA according to the manufacturer's instructions, followed by DNaseI (Tiangen, Beijing, China) digestion to eliminate any contaminating DNA. For qRT-PCR analysis, the first-strand cDNAs was synthesized from the 1 μg RNA using the Reverse Transcriptase M-MLV System (Tiangen, Beijing, China) according to the manufacturer's instructions. The Beacon Designer 7 software was used to design and check the gene-specific primers (Additional file 5: Table S2). The pear tubulin gene (forward primer: 5′ -AGAACAAGAAC TCGTCCTAC-3′; reverse peimer: 5′-GAACTGCTCGCT CACTCTCC-3′) was used as reference gene [46]. The qRT-PCR was carried out using SYBR® Premixm Ex Taq™ (TaKaRa, Japan) with the CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, USA). For each sample, we executed three biological replicates. The 2 -ΔΔCT method was used to estimate the relative expression level [47].

Subcellular localization analysis
The expression vectors of 35S:PbBBX5-GFP, 35S: PbBBX4-GFP, and PbBBX13-GFP were constructed by insertion of cDNA PbBBX5, PbBBX4 and PbBBX13, into pCAM-BIA1304 vector, respectively. After electroporation of these construction into Agrobacterium tumefaciens EHA105, using pCAMBIA1304 vector as negative control [48], the transformed bacterial cells were infected into the leaf tissue of Nicotiana tabacum as the method described by Sparkes et al. (2006) [49]. The transient expression of PbBBX-GFP was observed using a laser confocal microscope (Zeiss LSM700, Germany), the DNA dye 4,6-diamidino-2-phenylindole (DAPI) was used to visualize the nucleus.

Additional files
Additional file 1: Figure S1. Chromosomal locations and duplication events of BBX genes in the pear genome. The chromosome number is implied at the top of each chromosome. The segmental duplicated genes are connected by color lines and marked by corresponding color boxes. The scale on the left represents the megabases (Mb). (TIFF 3910 kb)