Identification of BGLU genes in five Rosaceae species
The BGLU family conserved domain (Pfam:PF00232) was used as the target query sequence, and the corresponding hidden Markov model was obtained from the Pfam website (http://pfam.xfam.org/) were used to identify the BGLU members. SMART (http://smart.embl-heidelberg.de/) was used to check whether there are characteristic domains, finally removed those redundant and repeated sequences. As a result, in total, we identified 343 BGLU genes from 5 Rosaceae species (Pyrus bretschneideri 50, Prunus mume 52, Malus domestica 132, Prunus avium 78, Fragaria vesca 31). The detailed information on the gene ID, gene name, chromosome location, protein structure and the characteristics of the corresponding BGLU proteins were listed in Additional file 1: Table S1.
Phylogenetic conserved motif and Exon–Intron Structures analyses of BGLU genes in Chinese white pear
For all PbBGLU genes, The phylogenetic tree was constructed with MEGA 7.0 software using the neighbor-joining (NJ) (bootstrap = 1,000), and then divide them into 8 groups(I,II,III,IV-A,IV-B,IV-C,V-A,V-B)with supported bootstrap values(Fig. 2),We used the MEME online software to analyze the amino acid sequence of PbBGLU genes, and identified 20 Motifs in all sequences (Additional file 1:Table S2, Fig. 2), All the PbBGLUs contain Motif1 (CTGGBSATEPYJVAHHQLLAHAAAVKLYREKYQA), only two genes do not contain Motif9 (WFEPASESKEDKAAALRALDF), Speculate that Motif1 and Motif9 are conserved gene motifs of PbBGLUs family. Only group I and group II contain Motif19, the other groups do not, and all members of group IV-C do not contain Motif2. It is speculated that these Motifs may have special significance. Previous studies implied that gene structural diversity can lead to the evolution of multi-gene families [37]. To better characterize and understand the structural diversity of the PbBGLUs, gene exon-intron analysis was carried out༈Fig. 2༉, except that PbBGLU23 and PbBGLU24 have only one exon, the rest contain 7 or more exons, of which PbBGLU33 contains the most exons (21) and most gene exons range between 10–15. These results indicating the possible occurrence of alternative splicing during gene expression, which in turn leads to functional differences between these closely related PbBGLUs [37].
In order to more clearly understand the evolutionary relationship and related functions of PbBGLUs, the phylogenic tree, including BGLUs from Arabidopsis thaliana, Poplar [1, 19]and pear was constructed (Fig. 3). We have marked the position of the 8 groups in the pear on this phylogenic tree and found that group I contains only PbBGLUs but not AtBGLUs and PtrBGLUs, indicating that some changes occurred among BGLUs of different species during the evolutionary process [38]. PbBGLUs Group II were clustered together with Poplar PtrBGLU9 and PtrBGLU11, however the functions of these two genes are unclear. Arabidopsis AtBGLU12-17 was related to the use of Arabidopsis flavonoids, AtBGLU15 can catalyze the hydrolysis of Q3G7R, K3G7R and flavonol 3-O-glucosides [4], these genes and PbBGLU19 (group III) were clustered together, and it is speculated that PbBGLU19 may be involved in the hydrolysis of flavonoid glycosides in pear. PbBGLUs IV-A was clustered together with AtBGLU(40,41,43,44) and some poplar BGLU genes, but the functions of these genes have not yet been explored. The functions of AtBGLU45, AtBGLU46, AtBGLU47 in Arabidopsis and PtrBGLU6 in Poplar had been clarified, they can catalyze the hydrolysis of monolignol glucosides during lignification [16,17,19 ], the three genes PbBGLU1, PbBGLU15 and PbBGLU16 in the PbBGLUs group IV-B were clustered together with these functional genes, speculating that PbBGLU1, PbBGLU15 and PbBGLU16 may be able to affect the lignification by catalyze the hydrolysis of monolignol glucosides in pear fruit, which in turn affects the development of pear stone cells. PbBGLUs Group IV-C clustered together with AtBGLU42, AtBGLU42 is a myb72-dependent key regulator of rhizobacteria-induced systemic resistance and modulates iron deficiency responses in Arabidopsis roots [12]. AtBGLU1-6, which plays a role in the accumulation of flavonoids in Arabidopsis [2], was clustered together with PbBGLUs group V-A, speculating that PbBGLU28 and PbBGLU29 may have similar functions in pears. PbBGLUs group V-B clustered together with Arabidopsis AtBGLU34-39, AtBGLU34-39 function as myrosinases for chemical defense against herbivores and pathogen attacks. [10, 11]
Chromosome location and collinearity analysis of BGLUs family members of five Rosaceae species
To further reveal how the BGLU genes family evolved, we mapped the BGLU genes to their respective chromosomes (Additional file 2: Fig. S1), 40 PbBGLUs (10 genes are not located on any chromosome) were unevenly distributed on 10 chromosomes (a total of 17 chromosomes of pear),Chromosome 7,10,13 had only one chromosome distribution, chromosome 11 had the most chromosome distribution (11). 40 PmBGLUs (12 unlocated) in Prunus mume were located on 8 chromosomes, except for only one gene was located on chromosomes 4 and 6, the other PmBGLU genes were relatively evenly located on the rest chromosome. Apple 114 (9 unlocated) MDBGLUs were located on 17 chromosomes, among which chromosome 3 and chromosome 11 are more distributed. The 42 PaBGLU (36 unlocated) genes of Prunus avium were located on 8 chromosomes, chromosome 3, 7, 8 had less PaBGLUs distributed, the remaining chromosomes had more PaBGLUs distributed and relatively uniform. The 31 FvBGLUs of strawberry were located on 7 chromosomes and were unevenly distributed. Among them, FvBGLUs on chromosomes 3 and 5 were concentrated located and the others chromosomes were only located a small amount.
Species homologous genes are divided into orthologous and paralogous, orthologous genes refer to the genes that exist in the ancestral genome and are subsequently transferred to different species because of species differentiation, these genes often have a high degree of similarity in structure and function༎Paralogous genes refer to homologous genes in the same genome due to gene duplication, because homologous genes generated by replication do not have the same selection pressure effect, some mutations are prone to occur, which may lead to functional mutations [39].298 syntenic pairs were found in intra- and inter-species collinear analysis of five Rosaceae species (Fig. 4, Additional file 1:Table S 3),A total of 12 syntenic pairs were found between pears, among which PbBGLU3/PbBGLU7 gene pairs were on the same chromosome, PbBGLU7 and PbBGLU50 were collinear with PbBGLU3, PbBGLU19 and PbBGLU25 were collinear with PbBGLU33, PbBGLU44 and PbBGLU13 were collinear with PbBGLU22, these genes may be paralogous and may formed by the whole genome replication of pears a million years ago. Pear and apple have more syntenic pairs (41) than pear and plum (19), Pear and strawberry (15), Pear and cherry (11), these gene pairs between pear and other Rosaceae species may be the orthologous caused by the differentiation of species in the ancestral genome. Both pears and apples contain 17 chromosomes, and the number of chromosomes is double that of strawberries, cherries, and Mei. Previous studies have shown that the genome-wide replication (GWD) may be occurred greater than 50 million years ago, resulting in Pear and Apple chromosomes from 9 ancestral chromosomes to 17 chromosomes. Data from a three-way sequence alignment between predicted gene space in apple (~ 84 Mb) and experimentally derived EST data from pear (~ 14.9 Mb), predicted gene spaces of apple and pear were compared, a value of 96.35% nucleotide identity was calculated between these two species of the tribe Pyreae. When the frequency of transitions and transversions was considered, the ratio R (transitions/transversions) was similar for apple-specific and pear-specific mutations [40], these may be the reasons why pears and apples have more syntenic pairs.
PbBGLU genes replication event analysis
To determine the selection pressure in duplication of PbBGLUs genes, the non-synonymous (Ka)/synonymous (Ks) values were calculated for the 12 gene pairs (Additional file 1: Table S4). The pear divergence time between these gene pairs was calculated with the period varied from 0.27-154.25 million years (Mya). It is generally believed that synonymous mutations are not subject to natural selection, while non-synonymous mutations are subject to natural selection. If Ka/Ks > 1, it is considered to have a positive selection effect. If Ka /Ks = 1, then there is a neutral selection. If Ka/Ks < 1, it is considered to have purification selection effect. We calculated Ka/Ks values of 12 gene pairs, only one group of genes PbBGLU3/PbBGLU7 Ka/Ks > 1, the replication mode is tandem, and the remaining 11 pairs of genomes Ka/Ks < 1, the replication mode is segmental. The result suggested that the evolution of the BGLU gene pairs in the pear may be mainly due to fragment replication under purification selection and with limited functional divergence after the duplication events.
We performed sliding window analysis on these 12 gene pairs(Additional file 3: Fig. S2), It was found that PbBGLU3/PbBGLU7 had a large number of Ka/Ks ratios of coding sites more than 1, experience strong positive choices during the evolution, while PbBGLU12/PbBGLU42, PbBGLU25/PbBGLU33, PbBGLU33/PbBGLU19, PbBGLU44/PbBGLU22 and PbBGLU49/PbBGLU20 had a small amount of Ka/Ks ratios of coding sites more than 1, suggested that these gene pairs may also experience some positive choices During the evolution.
Promoter Regions of PbBGLU Genes
The promoter sequence of 2000 bp upstream of the members of the PbBGLUs family was obtained from pear GigaDB database PLACE database, and then the promoter region was analyzed using the online software PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) Cis-acting element [41].The results shown that (Fig. 5, Additional file 1: TableS5): A total of 9 regulatory element including MBS, MRE, AC, LTR, HSE, TC-rich repeat, ABRE, ERE, CGTCA motifs were identified. Among them, the MBS element related to drought stress is the most, only 8 PbBGLU genes do not contain this element. Minimum number of AC elements, only 4 PbBGLU genes contain AC elements. Most genes contain light response element MRE, low temperature stress response element LTR, heat shock stress response element HSE, and defense response element TC-rich repeat. In addition, cis-acting elements related to hormone response (ABA response element ABRE, Ethylen response element ERE, and MeJR response element CGTCA motif) were also found in many genes of PbBGLU. These results suggested that in addition to its normal function, PbBGLUs may also participate in light response, stress response, plant self-defense response and hormone response process.
Expression characteristics of Chinese white pear BGLU genes
We had built the phylogenic tree, including BGLUs from Arabidopsis thaliana, Poplar [1, 19]and pear, and found out that PbBGLU1, PbBGLU15 and PbBGLU16 in the PbBGLUs IV-B of pears were clustered together with these functional genes AtBGLU45, AtBGLU46, AtBGLU47 and PtrBGLU6 which can hydrolysis monolignol glucosides during Lignification [16, 17, 19]. In order to verify whether PbBGLU1, PbBGLU15 and PbBGLU16 have similar functions. We selected all PbBGLU genes in IV-B, and also selected all PbBGLU genes in IV-B, IV-C, V-A, V-B which were relatively close to IV-B on the phylogenic tree (a total of 25 PbBGLUs) to do qRT-PCR analysis. The purpose was to explore these genes in different tissue parts of pears (leaves, buds, stems, flowers) and pear fruit at different developmental stages (15,39,47,55,63,79,145 days after flowering) expression profiles (Fig. 6). It was found that PbBGLU36 and PbBGLU42 were expressed neither in different tissue parts nor in different developmental stages of fruit. The expression levels of the other 23 PbBGLUs were generally higher in leaves, buds, flowers and young fruits (15, 39, 47, 55 days after flowering), this result is consistent with previous research that the β-glucosidase can be found mainly in young vegetative parts [42, 43]. Compared with other genes, PbBGLU38 and PbBGLU43 are more uniformly expressed in all tissue parts and all stages, and may perform a certain indispensable function in pear fruit trees. The expression levels of PbBGLU28 and PbBGLU29 gene in flowers, 79 DAF (when the lignification stop in fruit) [31, 35, 36] and 145 DAF (when the fruit was fully mature) [31, 35, 36] relatively high. Moreover, these two genes were clustered together with AtBGLU1-6 which related to the accumulation of flavonoids in Arabidopsis [2], so PbBGLU28 and PbBGLU29 may participate in the accumulation of flavonoids in pear. The expression levels of the three genes PbBGLU1, PbBGLU15 and PbBGLU16 have similar characteristics, they are relatively high in leaves and flowers, and relatively low in buds and stems, all three genes were expressed at different stages of fruit development, and its expression shows a trend of rising first and then falling, with similar trend of the content of lignin and stone cells in “Dangshan Su” pear fruit[35, 36], and the three genes had similar expression patterns with the key genes involved in regulation of the lignin synthesis pathway [44]. These results suggested that the three genes PbBGLU1, PbBGLU15, and PbBGLU16 in PbBGLUs IV-B may be functional genes involved in the hydrolysis of monolignol glucosides in the pear lignin synthesis pathway.
Subcellular Localization of PbBGLU1, PbBGLU15 and PbBGLU16
As early as 1995, it was found that P. contorta β-glucosidase activity was located in the cell wall of secondary xylem tissue [14]. AtBGLU45 and AtBGLU46 of Arabidopsis thaliana were also located in the cell wall [16].The rice Os4BGlu16 tagged with a C-terminal GFP was transiently expressed in tobacco leaf epithelial cells and found that the GFP signal was exclusively localized to the plasma membrane and extracellular space in both intact and plasmolysed cells, consistent with an apoplastic or cell wall localization [18]. In this study, in order to explore the PbBGLU1, PbBGLU15, PbBGLU16 subcellular localization, PbBGLU-GFP expression vectors were constructed and transformed into N. tabacum. As shown in Fig. 7, green fluorescence signals from the expressed fusion PbBGLU1-GFP, PbBGLU15-GFP and PbBGLU16-GFP genes were specifically distributed in the cell wall.