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 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 monocotsLucknow Investment. 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]Surat Investment. 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 tandemBangalore Investment. 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 tubesHyderabad Investment. 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.