A new insight into the evolution and functional divergence of FRK genes in Pyrus bretschneideri

In plants, plant fructokinases (FRKs) are considered to be the main gateway of fructose metabolism as they can phosphorylate fructose to fructose-6-phosphate. Chinese white pears (Pyrus bretschneideri) are one of the popular fruits in the world market; sugar content is an important factor affecting the quality of the fruit. We identified 49 FRKs from four Rosaceae species; 20 of these sequences were from Chinese white pear. Subsequently, phylogenic relationship, gene structure and micro-collinearity were analysed. Phylogenetic and exon–intron analysis classified these FRKs into 10 subfamilies, and it was aimed to further reveal the variation of the gene structure and the evolutionary relationship of this gene family. Remarkably, gene expression patterns in different tissues or different development stages of the pear fruit suggested functional redundancy for PbFRKs derived from segmental duplication or genome-wide duplication and sub-functionalization for some of them. Additionally, PbFRK11, PbFRK13 and PbFRK16 were found to play important roles in regulating the sugar content in the fruit. Overall, this study provided important insights into the evolution of the FRK gene family in four Rosaceae species, and highlighted its roles in both pear tissue and fruits. Results presented here provide the appropriate candidate of PbFRKs that might contribute to fructose efflux in the pear fruit.

YC, 0000-0001-5976-2382 In plants, plant fructokinases (FRKs) are considered to be the main gateway of fructose metabolism as they can phosphorylate fructose to fructose-6-phosphate. Chinese white pears (Pyrus bretschneideri) are one of the popular fruits in the world market; sugar content is an important factor affecting the quality of the fruit. We identified 49 FRKs from four Rosaceae species; 20 of these sequences were from Chinese white pear. Subsequently, phylogenic relationship, gene structure and micro-collinearity were analysed. Phylogenetic and exonintron analysis classified these FRKs into 10 subfamilies, and it was aimed to further reveal the variation of the gene structure and the evolutionary relationship of this gene family. Remarkably, gene expression patterns in different tissues or different development stages of the pear fruit suggested functional redundancy for PbFRKs derived from segmental duplication or genome-wide duplication and subfunctionalization for some of them. Additionally, PbFRK11, PbFRK13 and PbFRK16 were found to play important roles in regulating the sugar content in the fruit. Overall, this study provided important insights into the evolution of the FRK gene family in four Rosaceae species, and highlighted its roles in both pear tissue and fruits. Results presented here provide the appropriate candidate of PbFRKs that might contribute to fructose efflux in the pear fruit.
2018 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. 1

. Introduction
Sucrose, fructose and glucose are the major soluble sugars in fruits [1]; however, sucrose is unstable and easily hydrolysed into glucose and fructose under the action of invertase. Hexose metabolism is a basis for photosynthetic products to enter another metabolism during fruit development stages. Though hexokinase (HXK) and fructokinase (FRK) could catalyse phosphorylation of fructose, the affinity of FRK to fructose is much higher than that of HXK [2], signifying that FRK plays an important role in fructose metabolism. Fructose could be subjected to glycolysis and oxidized pentose pathways under the catalysation of FRK via phosphorylation, which could also be used for starch synthesis. In addition, FRK can be used as a plant hexose receptor to regulate plant growth sugar signalling. Some previous studies reported that phosphorylated fructose can help to establish sink tissue [3,4], therefore, the study of fructose metabolism and FRK gene expression pattern in the fruit can help to expose the mechanism of sugar metabolism in the pear, and provides a scientific basis for improving fruit quality.
The FRK gene belongs to the phosphofructokinase B (pfkB) gene family, and the region where ATP binds to sugar is highly conserved [5]. In the higher plant tissues, the protein encoded by the FRK gene is present as a dimer. In the plant kingdom, the first time the FRK gene was isolated and identified was in tomatoes, and four family members were named LeFRK1-4 [6][7][8][9]. LeFRK2 and LeFRK3 were expressed in most tissues, LeFRK3 showed the highest expression in leaves and apical tissues, and LeFRK4 was only expressed in stamens. Subsequently, more and more FRK was studied and reported in other plants, such as Arabidopsis thaliana [10][11][12], rice [13], poplar [14] and loquat [15] etc. In recent years, the research progress on the plant FRK gene has been accelerated. More and more plant genome sequencing has been completed, which provides more data support for the research of the FRK gene family. For example, the FRK gene family has been systematically and comprehensively studied in sugarcane and Arabidopsis genomes [16,17]. Subsequently, the functionally divergent FRK gene family members were identified in sugarcane, such as SsFRK2 and SsFRK7 presenting lower expression levels in response to drought stress than the other SsFRK genes; SsFRK7 was under neutral selection, whereas SsFRK2 was under strong positive selection [16].
Pear, peach, mei and strawberry, belonging to the Rosaceae family, have important economic value [18][19][20]. Owing to its economic prominence, the peach [21], mei [22], strawberry [23] and pear genomes [24], as well as the proteome and transcriptome of pear fruit development were sequenced. Among the main fruit varieties of China's foreign exports, the Chinese pear (Pyrus bretschneideri) accounts for more than 60% of the world pear production. Sugar content is an important factor affecting the taste and quality of the fruit [25]. To further expand the knowledge of FRK-catalysed phosphorylation, and understand the structure and evolutionary history of the FRK gene family, we investigated members of the FRK gene family in pear, peach, mei and strawberry. Gene structure, phylogenetic, microcollinearity and positive selection analyses were conducted on the FRK gene family, and their effects on function are further discussed. Finally, the pear genome information was used and the results were verified by quantitative real-time polymerase chain reaction (qRT-PCR) experiments. The current study provides valuable information about FRK members, and highlights the potential candidates for further functional analysis of this important gene family, which may regulate sugar content in fruits.

Identification of fructokinase genes
In the present study, the FRK domain (PF00294) and A. thaliana FRK proteins were used as queries to search against the genomes, and then 56 FRK genes were identified in four Rosaceae species (i.e. pear, peach, mei and strawberry). After filtering redundant genes and scanning the complete FRK domain as tested by the Pfam database, SMART database and HMMER website, 49 genes were identified as FRK genes for further analysis (table 1). The FRK genes from these four species were renamed as PbFRK01-PbFRK20, PmFRK01-PbFRK15, PpFRK01-PbFRK08 and FvFRK01-FvFRK06, respectively. Remarkably, we found more FRK genes in pear than in mei, peach and strawberry, suggesting a greater expansion in the pear FRK genes. This phenomenon could be owing to the recent genome-wide duplication that occurred in the pear, but not in mei, peach and strawberry. However, we also noticed more FRK genes in mei than in peach and strawberry, despite the lack of the recent genome-wide duplication, implying that other duplication modes might play a role in the expansion of PmFRK genes.

PmFRK12
Pm021646 chr6 419 6.4          Based on the above findings, the chromosome location of 49 FRK genes were randomly placed on chromosomes of the four Rosaceae species (figure 1). The PpFRK genes were dispersed on five chromosomes (chr 1, 2, 3, 6 and 8), the FvFRK genes were distributed on four chromosomes (chr 1, 2, 4 and 6) and the PmFRK genes mainly found on six chromosomes (chr 1, 2, 3, 4, 5 and 6), while PbFRK genes were distributed on twelve out of 17 chromosomes (chr 1, 2, 3, 5, 7, 8, 9, 10, 11, 13, 15 and 17). Among these genes, the longest protein was encoded by PbFRK03, which contains 667 amino acids (aa). However, PmFRK06 encodes the shortest protein (130 aa). The significant difference was observed in the aa sequence length between the members. These results might be owing to the different length of the N-terminal sequence (with unknown function) or different numbers of additional insertions. As previously reported, FRKs active in plants are between 319 aa (Solanum tuberosum FRK, whose GenBank accession number is CAA78283) and 389 aa long (Solanum lycopersicum FRK3, whose GenBank accession number is NP_001234396 and includes a chloroplast transit peptide), whereas SlFRK2 (GenBank accession number: XP_004239035) is greater than 600 aa long [16,26]. Common characteristics might be shared by these proteins and active FRKs, while the additional N-terminal sequences and insertions may generate loops that might interfere with fructose binding, which has been reported by Riggs and Callis [27]. We also found that the theoretical pI values of most FRK proteins were less than 7, indicating that they were acidic, whereas four proteins by the other FRK genes (PbFRK03, PbFRK20, PmFRK04 and PmFRK06) were alkaline (greater than 7). Additionally, the average molecular weight of these proteins was 42.0 kDa, with a range from 14.4 to 73.1 kDa (table 1)

Phylogenetic analyses
To evaluate phylogenetic relationships of FRK genes in these four Rosaceae species (peach, mei, strawberry and pear) and A. thaliana, a phylogenetic analysis was performed using the MEGA 5.0 software with the neighbour-joining (NJ) method. In A. thaliana, AtFRK genes have been described as the FRKs, which are essential for plant growth and development [28]. In our study, the NJ tree can be divided into two main classes (classes I-II) and 10 subfamilies (A-J), based on the topology and bootstrap values of the phylogenetic tree ( figure 2). Remarkably, we found that class II only contained pear and mei FRK genes, except for PpFRK08 ( figure 2). This might be the reason why there are more pear and mei FRK genes than peach, strawberry and Arabidopsis FRK genes. At the same time, FRK genes from pear and mei are more closely related according to the evolutionary relationship, which is supported by a previous report showing the closer relationship between pear and mei versus pear and peach/strawberry [29].

Sequence feature analyses
In the present study, the exon-intron organization of FRK genes was variable in both exon size and number among the examined plant species. The exon numbers range from 1 (PbFRK05) to 17 (PbFRK03) as shown in figure 3. Furthermore, we found that the genes of each subfamily contained similar gene structures. For example, the subfamily B contained seven exons except for FvFRK05, which had six exons; the subfamily C contained five exons except for PmFRK06, which had three exons ( figure 3). These results demonstrate that both exon gain and loss has occurred after the split of monocots and dicots during the evolution of the FRK genes, which might clarify the functional diversity of  closely related genes from the FRK gene family (figure 3). The FRK paralogous or/and orthologous gene pairs' exon-intron structure was further investigated. The number of exons in eight gene pairs has changed, including PbFRK08/PbFEK02, PbFRK03/PmFRK07, PmFEK06/PpFRK07, FvFRK06/FvFRK01, FvFRK04/PpFRK06, PmFRK15/PpFRK02, PbFRK14/PbFRK05 and PbFRK09/PmFRK10. Among these gene pairs, we found that FvFRK06, PbFRK14 and PbFRK09 gained one exon during the long evolutionary period, while FvFRK01, PbFEK05 and PmFRK10 lost one exon. The cause of these events may be a single intron loss or gain during the evolutionary process.
To further understand the diversity and structure characteristics of FRK genes, the MEME web server was used to predict the conserved domains which were shared with the 56 FRK proteins of five species (figure 3 and the electronic supplementary material, table S1). Twenty distinct motifs were found, whose distribution matched the FRK clustering pattern in the phylogenetic tree. The Pfam and SMART database were used to annotate each of the putative motifs. Motif 1, motif 2, motif 3, motif 5, motif 8, motif 9, motif 10, motif 16, motif 18 and motif 19 were authenticated to encode the PfkB carbohydrate kinase domain, while the other motifs' function requires further investigation (electronic supplementary material, table S1). In addition, we found motif 1 and motif 3 mainly distributed in class I, and motif 8 distributed in class II, indicating they are necessary for the FRK proteins in these classes. To further understand the function of the different FRK proteins, we searched the gene ontology (GO) database, which provided clues on the function of 49 FRK proteins. Subsequently, we found that most FRK genes have some common functions,  such as FRK activity, cellular metabolic process, transferase activity, biological process and molecular function (electronic supplementary material, table S2).

Micro-collinearity analyses
In our study, 29 paired micro-collinearity relationships were identified among four Rosaceae species. Among them, in pear and other species were found 20 micro-collinearity gene pairs (electronic supplementary material, table S3). Based on the result of MCSCANX, these micro-collinearity relationships are formed by genome-wide duplication or segmental duplication. Remarkably, pear and mei micro-collinear gene pairs are much larger than those of pear and peach/strawberry; this conclusion is supported by previous reports that the relationship between pear and mei is much closer than that between pear and peach/strawberry [24,29]. At the same time, we also investigated micro-collinearity relationships among the same species. We identified six micro-collinearity gene pairs in pear, two microcollinearity gene pairs in mei and one micro-collinearity gene pair in peach. To further understand the micro-collinearity relationships of pear FRK genes, the micro-collinearity diagram was generated using the MCSCANX software. As shown in figure 4, all of the FRK gene micro-collinearity occurs in the sense direction among the pear genome.

Strong purifying selection for fructokinase genes in pear
The results of the previous section indicated that the expansion of the pear FRK gene family occurred by gene duplication. To better understand the evolutionary constraints acting on the FRK gene family, Ka/Ks values of six duplicated gene pairs were calculated among the pear FRK gene family. In general, Ka/Ks less than 1, equal to 1, and greater than 1 indicates purifying selection, neutral selection and positive selection, respectively. In the present study, we discovered that all the Ka/Ks values of six pear FRK duplicated gene pairs were less than 0.3 (electronic supplementary material, table S4). These results indicated that the FRK gene family had undergone a strong purifying selection, resulting in a slow evolution of the pear FRK gene family at the protein level. Remarkably, all the Ka/Ks ratios of 29 FRK duplicated gene pairs were less than 1, further indicating that this FRK gene family has undergone a purifying selection during evolution (electronic supplementary material,  FRK duplicated gene pair. As presented in the electronic supplementary material, figure S1, many sites or/and regions experience neutral selection, or strong negative/purifying selection. Consequently, we found that most Ka/Ks values across the coding sites/regions were much lower than 1; however, one or several distinct peaks (Ka/Ks > 1) were observed in the electronic supplementary material, figure S1. Surprisingly, the domains of FRK genes generally had higher Ka/Ks ratios (peaks) than the regions outside of them (valleys). A sliding-window analysis of PbFRK13/PbFRK04 duplicated gene pairs revealed sites with higher Ka/Ks ratios (peaks: Ka/Ks values greater than 1) in FRK domains, implying positive selection in this site/region. Additionally, although positive selection contributes to higher values of Ka/Ks, it does not mean or ensure that the gene-average Ka/Ks ratio exceeds 1. To sum up, the above Ka/Ks values and sliding-window analyses strongly suggests that purifying selection might play a key role in the pear FRK gene family during evolution.

Cis-acting element analysis
Cis-acting elements play key roles in plant growth and development, such as determining the tissuespecific or stress-responsive expression patterns of genes. In our study, we identified the three categories of cis-acting elements, including plant growth and development, phytohormone responses, and biotic and abiotic stress responses (electronic supplementary material, table S5). In the promoter regions, numerous cis-acting elements were widespread, such as Skn-1-motif and GCN4_motif for endosperm expression, Box 4 and MRE for light responsiveness, and CAT-box and CCGTCC-box for meristem expression. Remarkably, there was a higher amount of cis-acting elements for plant growth and development, contrasting with the cis-acting elements of phytohormone responses and biotic and abiotic stress responses. These findings suggest that a number of the FRK gene family may have a special role in plant growth and development.

Expression profiles of pear fructokinase genes in different tissues
It is well known that gene identification makes it possible to explore the potential function of gene families through gene expression analyses. To further characterize the pear FRK gene functions, we performed comparative gene expression analyses during different developmental stages of fruits and three different tissues (root, stem and leaf) by qRT-PCR experiments. In the present study, we found that some PbFRK genes were highly expressed in specific tissues or fruit development stages. For example, the expressions of nine genes (PbFRK01, PbFRK02, PbFRK03, PbFRK04, PbFRK06, PbFRK08, PbFRK09, PbFRK10 and PbFRK19) were highly expressed in roots (PbFRK01, PbFRK03, PbFRK04, PbFRK06 and PbFRK08 were upregulated by more than 2.5-fold and PbFRK01 showed the greatest upregulation (by more than five-fold)); PbFRK07, PbFRK12, PbFRK13, PbFRK15, PbFRK17 and PbFRK20 had high expression in leaves (PbFRK12, PbFRK15 and PbFRK17 were strongly upregulated (by more than 2.8-fold, 2.9-fold and 2.0-fold)); while PbFRK05, PbFRK14 and PbFRK18 peaked in stems (PbFRK18 were highly expressed by more than 9.8-fold). These results indicated that different PbFRK genes may play key roles in the development of specific tissues ( figure 5). Interestingly, PbFRK11, PbFRK13 and PbFRK16 were detected as downregulated during pear fruit development (electronic supplementary material, figure  S2). These results showed a negative correlation with fructose levels, implying that these genes may play important roles for fructose efflux and regulate the sugar content in fruit. Moreover, some PbFRK genes were highly expressed at 133 days after flowering than other genes, such as PbFRK03 and PbFRK04, which were upregulated more than 14-fold, and PbFRK20, which presented strong upregulation by more than 2000-fold, signifying that these genes may contribute to maturation in fruit development (electronic supplementary material, figure S2).

Identification and phylogenetic analysis in the fructokinase gene family
It is well known that the FRK genes play an important role in the development of various plant organs, in fructose metabolism and in response to abiotic stresses [16,17,31,32]. Previously, the FRK gene family has been reported in Arabidopsis and Saccharum [16,17]. However, FRK genes were exclusive owing to the complex Rosaceae genome, especially in pear. In this report, we identified 49 FRK genes from four Rosaceae species; 20 of these sequences were from Chinese white pear, and six, eight and 15 FRK genes from strawberry, peach and mei, respectively. In pear, we identified a comparatively large gene family, which contained 20 FRK gene family members. Compared with mei, peach and strawberry, the pear genome has undergone two genome-wide duplication events [24,33]. It is believed that the expansion of the FRK gene family in pear might have occurred in a recent genome-wide duplication event.
Based on the topology and bootstrap values of the phylogenetic tree, all FRK genes were divided into two classes with 10 subfamilies (A-J). As shown in the phylogenetic tree, we observed differences between different subfamilies, suggesting that they have different biological functions. Among them, class I clustered together with the known function of the Arabidopsis FRK genes, whereas class II only contained the FRK genes from four Rosaceae species. These results indicated that we still need to explore in the future the function of these genes for different subfamilies. In addition, the pear FRK genes (PbFRK16-PbFRK17) from the same chromosome (no. 15) cluster together, implying the similarity of these genes. Remarkably, this duplication gene pair was not formed by tandem duplication events, because the distance between the two genes on the chromosome was greater than 200 kb [34].

Micro-collinearity analyses in the fructokinase gene family
Based on the micro-collinearity analyses, we identified 15761 micro-collinearity gene pairs in the pear genome to each other, 21119 gene pairs between the pear and mei genome, and 21773 gene pairs between the pear and peach genome (data not shown). These results have also been confirmed by previous reports [35], indicating strong micro-collinearity between segments of some chromosomes. Additionally, we found that a segment of chr 15 in the pear genome was thought to be micro-collinear with chr 8, and micro-collinear with chr 2 in the mei genome. The FRK genes PbFRK17 and PbFRK16 on chr 15 were micro-collinear with PbFRK10 on chr 8; and PbFRK17 and PbFRK16 on chr 15 were micro-collinear with PmFRK02 on chr 2 of mei. Interestingly, 14 micro-collinearity gene pairs were discovered between pear and mei, which were more than that between pear and peach (five), and pear and strawberry (zero), findings consistent with those of previously published manuscripts, in which the divergence of pear, mei and peach occurred after the divergence of strawberry from the common ancestor of pear, mei and peach [24,29]. At the same time, the current manuscript may provide novel resources for the study of the evolution of the FRK genes among different species.

Expression analyses of fructokinase genes in pear
Promoter elements are closely associated with potential gene functions and regulatory mechanisms, such as tissue-specific expression patterns. Previous studies have shown that FRK genes play an important role in fruit and seed development and play diverse functional roles in different tissues [16,28,[36][37][38]. Chen et al. [16] found that most Saccharum FRK genes were expressed in different tissues and different developmental stages of seedlings; however, expression of several FRK genes was low in specific tissues. They also found that low-expressed genes in the tested tissues (such as leaf and stem) may not play the main role in fructose metabolism, such as SsFRK2 and SsFRK7 [16]. In the current study, we also found that some PbFRK genes showed low expression levels in leaves and stems, such as PbFRK01, which was consistent with it having fewer cis-acting elements for plant growth and development. Additionally, PbFRK18, which had three Skn-1 motifs and one GCN4_motif, was highly expressed in fruits and stems, suggesting that this gene might primarily regulate the development of these tissues, which is consistent with tomato SlFRK3, which can reduce the stem xylem area and contribute to stem development [39].
Remarkably, the current study shows that almost all PbFRKs have low expression in roots, compared to other tissues (i.e. stem, leaf and flower). Similar results have been verified for FRKs from other plants, such as Solanum lycopersicum and Cassava, which participate in the development of roots [16,40]. The expression of PbFRK01 and PbFRK10 was detected by qRT-PCR, to be mainly in stems and roots, with lower expression in leaves. This result was similar to that of the FRK1 and FRK2 genes in maize [41], indicating that these genes may play an important role in the metabolism of sink tissues. In general, the expression pattern of duplicated gene pairs should be similar [42]. In our study, we found that some of the paralogous gene pairs (PbFRK02/-08, PbFRK11/-19 and PbFRK10/-16) have similar expression patterns, indicating that these genes were found to be non-divergent after duplication and might retain redundant functions in regulating tissue development (figure 5). However, three duplicate gene pairs (PbFRK04/-13, PbFRK10/-16 and PbFRK16/-17) were divergent, implying that these genes displayed sub-functionalization for some of them (figure 5).
Pear is one of the most important commercial fruit crops in the world. Sugar content is an important factor measuring fruit quality, which directly affects the flavour of the fruit. FRK genes have been reported to affect fructose accumulation in fruits [43,44]. Sugar transport is essential for plant development, and organisms can control sugar influx and efflux depending on supply and demand [45]. The activity and expression of FRK gradually decreased, while the sugar content gradually increased with the development of fruit [46]. As previously reported, sugar efflux and influx are regulated by related genes [47]. Here, PbFRK11, PbFRK13 and PbFRK16 were found to have a negative correlation with fructose levels during pear fruit development. These results indicated that these genes may play a key role in the balance of fructose levels during fruit development. To sum up, our study provides new ideas and directions for FRK gene function in the pear.
In the present study, 49 FRK genes from four Rosaceae species were identified; a systematic analysis including their physical locations, phylogenetic relationships, gene structure, micro-synteny and the expression patterns of pear FRK genes was carried out. Based on the phylogenetic analysis, all FRK genes were divided into two classes with 10 subfamilies. Additionally, the characteristics and composition of motif and exon/intron structure were relatively conserved in each subfamily. Our study suggested that the vast majority of PbFRK genes were expanded by segmental duplication or genome-wide duplication. Subsequently, these genes were found to undergo strong purifying selection, indicating their evolution at the protein level was slow. Furthermore, the expression patterns of PbFRK genes identified that these genes play key roles during pear fruit development. 4. Material and methods 4. 1