Genome-wide identification, expression and functional analysis of the phosphofructokinase gene family in Chinese white pear (Pyrus bretschneideri)

Gene 702 (2019) 133–142

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Gene journal homepage: www.elsevier.com/locate/gene

Research paper

Genome-wide identification, expression and functional analysis of the phosphofructokinase gene family in Chinese white pear (Pyrus bretschneideri)

T

⁎

Hongmei Lü1, Jiaming Li1, Yuhua Huang, Mingyue Zhang, Shaoling Zhang, Jun Wu

Centre of Pear Engineering Technology Research, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Pear (Pyrus bretschneideri) Phosphofructokinase Gene expression Subcellular localization Gene function Transient transformation

Phosphofructokinase plays an essential role in sugar metabolism in plants. Plants possess two types of phosphofructokinase proteins for phosphorylation of fructose-6-phosphate, the pyrophosphate-dependent fructose-6phosphate phosphotransferase (PFP), and the ATP-dependent phosphofructokinase (PFK). Until now, the gene evolution, expression patterns, and functions of phosphofructokinase proteins were unknown in pear. In this report, 14 phosphofructokinase genes were identified in pear. The phylogenetic tree indicated that the phosphofructokinase gene family could be grouped into two subfamilies, with 10 genes belonging to the PbPFK subfamily, and 4 genes belonging to the PbPFP subfamily. Conserved motifs and exon numbers of the phosphofructokinase were found in pear and other six species. The evolution analysis indicated that WGD/Segmental and dispersed duplications were the main duplication models for the phosphofructokinase genes expansion in pear and other six species. Analysis of cis-regulatory element sequences of all phosphofructokinase genes identified light regulation and the MYB binding site in the promoter of all pear phosphofructokinase genes, suggesting that phosphofructokinase might could be regulated by light and MYB transcription factors (TFs). Gene expression patterns revealed that PbPFP1 showed similar pattern with sorbitol contents, suggesting important contributions to sugar accumulation during fruit development. Further functional analysis indicated that the phosphofructokinase gene PbPFP1 was localized on plasma membrane compartment, indicating that PbPFP1 had function in plasma membrane. Transient transformation of PbPFP1 in pear fruits led to significant increases of fructose and sorbitol compared to controls. Overall, our study provides important insights into the gene expression patterns and important potential functions of phosphofructokinase for sugar accumulation in pear fruits, which will help to enrich understanding of sugar-related bio-pathways and lay the molecular basis for fruit quality improvement.

1. Introduction Sugars are the predominant energy and carbon source for prokaryotes and eukaryotes, as well as being key signaling molecules for normal growth in higher plants. Additionally, sugar content is an important indicator of fruit quality. Energy metabolism of many organisms is based on glycolysis, and in plants, there are two types of phosphofructokinase (PFK and PFP) proteins that phosphorylate fructose-6-phosphate. The classical glycolytic pathway is regulated by

phosphofructokinase degrading glucose (Plaxton, 1996). From a chemical point of view, sugars represent a large class of metabolites, present mainly in the form of the disaccharide sucrose or monosaccharide glucose and fructose in sink organs, however, Rosaceae species also have high sorbitol in fruit (Li et al., 2015). PFK is central in the classical glycolytic pathway and is present in all three domains of life (Siebers and Klenk, 1998). PFK catalyzes the interconversion of fructose-1, 6-bisphosphate and fructose-6-phosphate (Fru6P). In plants, various types of PFK exist and have a very complex

Abbreviations: PFP, pyrophosphate-dependent fructose-6-phosphate phosphotransferase; PFK, ATP-dependent phosphofructokinase; WGD, whole-genome duplication; qRT-PCR, quantitative real-time PCR; TAIR, the Arabidopsis information resource; HMM, hidden Markov model; SMART, simple modular architecture research tool; NCBI, National Center for Biotechnology Information; ORF, open reading frame; MEME, multiple EM for motif elicitation; GSDS, gene structure display server; BLASTP, basic local alignment search tool protein; CTAB, cetyltrimethyl ammonium bromide; HPLC, high pressure liquid chromatography; YFP, yellow fluorescent protein; CDS, coding sequence ⁎ Corresponding author. E-mail address: [email protected] (J. Wu). 1 Hongmei Lü and Jia-ming Li contributed equally to this work. https://doi.org/10.1016/j.gene.2019.03.005 Received 15 January 2019; Received in revised form 4 March 2019; Accepted 5 March 2019 Available online 21 March 2019 0378-1119/ © 2019 Elsevier B.V. All rights reserved.

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2. Materials and methods

evolutionary history (Bapteste et al., 2003). Additionally, PFP, a second type of phosphofructokinase, can use pyrophosphate instead of ATP as a phosphoryl donor to catalyze the reversible phosphorylation of Fru6P to fructose-1, 6-bisphosphate. PFP catalyzes the reaction and reacts near equilibrium in both directions, while PFK is virtually irreversible in vivo (Siebers and Schönheit, 2005). The first phosphofructokinase was discovered in 1980 and PFP have been characterized in detail at the molecular and biochemical level in all three domains of life (Dunaway, 1983; Siebers and Klenk, 1998; Mustroph et al., 2007; Darikova and Sherbakov, 2009; Zhu et al., 2013a). PFP always have two different subunits: alpha and beta (Cawood et al., 1988; Zhu et al., 2013b; Qin et al., 2014). While PFP has been studied widely, plant PFK has received minimal attention because of the instability of PFK in isolation during the purification process (Turner and Plaxton, 2003). Some studies have shown that plant PFKs were found in plastids and cytosol in tomato fruit (Isaac and Rhodes, 1982); Triticum aestivum L. (Mahajan and Singh, 1992), and Ricinus communis seeds (Podestá and Plaxton, 1994). Additionally, the properties of PFKs from R. communis, wheat, potato tubers, and germinating cucumber seeds have also been studied (Cawood et al., 1988; Knowles et al., 1990; Mahajan and Singh, 1992; Teramoto et al., 2000). During the past decade, the development of sequencing technology has led to sequencing of many plant genomes. The phosphofructokinase gene family has also been identified in many plant species, such as spinach (Winkler et al., 2007), Arabidopsis (Mustroph et al., 2007), Saccharum (Zhu et al., 2013a), and rice (Mustroph et al., 2013). For gene family expansion, gene duplication is a major mechanism generating new models for evolutionary innovation in eukaryotes (Ohno, 1970; Zhang, 2003). Genome analysis has provided direct evidence indicating that gene family members come from small-scale gene duplication events (dispersed duplication, tandem duplication) and large-scale duplication events (segmental duplication and wholegenome duplication). Almost all land plants have undergone at least one WGD (Jiao et al., 2011), including the five Rosaceae species (Velasco et al., 2010; Shulaev et al., 2011; Zhang et al., 2012; International Peach Genome et al., 2013; Wu et al., 2013). The pear genome was recently sequenced (Wu et al., 2013) and gene family analysis in pear provided direct evidence that the main driving force of gene family expansion was WGD (Li et al., 2015; Qiao et al., 2015; Li et al., 2017). Large-scale duplication events (such as WGD or segmental duplication) play important roles for expansion of gene families. Pear is one of the most well-known fruits in the world and it is cultivated in all temperate zone countries of both hemispheres. Sugar is a key factor affecting pear fruit quality, therefore, improving the sugar content of pear fruit is a challenge for molecular breeding. Until now, most researchers have focused on the evolution of sugar content (Hudina and Štampar, 2000; Hudina et al., 2000; Chen et al., 2007) but only a few sugar-related genes have been cloned and studied (Keta et al., 2010; Wang et al., 2016), most likely because of limited genome sequence information. Availability of the genome of pear (Wu et al., 2013) has laid a solid foundation for genome-wide analysis of phosphofructokinase genes in pear. The importance of this enzyme for regulating metabolism prompted us to identify phosphofructokinaseencoding sequences in pear because the sequences and functions of the phosphofructokinase gene family in pear is still unknown. In this study, the pear genome database was used for identifying phosphofructokinase gene family members, and the expression of phosphofructokinase genes in different tissues and during fruit development of pear were determined using qRT-PCR. The aims of this study were to: (1) identify the phosphofructokinase gene members in pear; (2) analyze the evolution of phosphofructokinase of pear; (3) characterize the expression patterns of phosphofructokinase gene members in different tissues and fruit during development of pear; (4) determine the sub-cellular localization of phosphofructokinase genes; and (5) analyze the functions of phosphofructokinase genes in pear fruits.

2.1. Identification of phosphofructokinase proteins in pear and five other species For data retrieval, the pear protein database was downloaded from the pear genome project (http://peargenome.njau.edu.cn) and the other four Rosaceae protein databases were obtained from the Genome Database for Rosaceae (GDR; http://www.rosaceae.org), including Apple, Peach, Woodland strawberry and Japanese apricot. The Arabidopsis protein database was obtained from the TAIR website (http://www.arabidopsis.org). HMMER software, which could be used for identifying the gene family from the whole genome database (Eddy, 1998) was used to search all PFP proteins in pear and the other five species. First, the PFP domain (PF00365_seed) was downloaded from the Pfam website (http://pfam.sanger.ac.uk/) and then the seed file was used to search the pear protein database using HMMER software. The e-value cut-off was 1e−5 or less and then Pfam and SMART (http:// samrt.embl-heidelberg.de) were used to confirm that the obtained sequences had the PFK domain; sequences with the PFK domain were retained. Finally, sequence lengths that were too short or too long and/ or had obvious errors were manually deleted. 2.2. Phylogenetic tree reconstruction of the phosphofructokinase gene family Multiple alignments of phosphofructokinase proteins were performed using the MUSCLE program in MEGA7 software (Kumar et al., 2016). The best substitution model for all phosphofructokinase proteins was determined using modelgenerator, version 0.85 software (Naughton et al., 2006); the best fit was the JTT method. PhyML version 3.0 (Guindon et al., 2010) was used for maximum likelihood (ML) phylogenetic analysis with 100 bootstrap replicates. Finally, the phylogenetic tree was visualized using FigTree v1.4.3 (http://tree.bio.ed. ac.uk/software/figtree/). 2.3. Identification of conserved protein motifs, gene structure analysis, and cis-elements Conserved protein motifs were analyzed using MEME (http:// meme-suite.org) with the following parameters: expect motif sites to be distributed in sequences: zero or one per sequence; maximum number of motifs: 15; minimum motif width: 6; maximum motif width: 50; and maximum number of sites: 300. The gene structure display server 2.0 (GSDS, http://gsds.cbi.pku.edu.cn) was used to illustrate intron and exon organization for individual pear phosphofructokinase genes and genes from the other five species by comparing coding sequences with their corresponding genomic sequences. Promoter sequences (2000 bp) of pear phosphofructokinase genes were obtained from the genomic sequence, if nucleotide acid numbers were < 2000 bp between two genes, we only used the nucleic acids contained between the two genes. Finally, we submitted the nucleic acids to the PLACE web Signal Scan (http://www.dna.affrc.go.jp/htdocs/PLACE/) and analyzed the cis-elements of promoters of phosphofructokinase genes. 2.4. Identification of chromosomal location and collinearity, gene duplication modes The duplication mode of each phosphofructokinase gene was identified in our study. Briefly, the total proteins were identified by all-vs-all BLASTP search within each of the Rosaceae species genomes and Arabidopsis; the top five hits with e-value cut-off of 1e−5 were retained. Then, the potential homologous gene pairs from the all-vs-all BLASTP search were used as the input information for MCScanX (Y. Wang et al., 2012) to identify collinearity chains, and the results were evaluated using a procedure in CollinearScan (Wang et al., 2006) with an e-value 134

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Fig. 1. Phylogenetic analysis of phosphofructokinase genes of pear and five other species. The evolutionary history was inferred using maximum likelihood (ML). Prior to ML analysis, Modelgenerator, version 0.85 identified JTT as the best fit substitution model for phosphofructokinase proteins. An ML tree was created using PhyML program with 100 bootstrap replicates.

cut-off of 1e−10. Chromosome location information was obtained from the six genome annotation files. The gene duplication modes of each gene in six species were obtained from the all-vs-all BLASTP step which contain dispersed, tandem, WGD/segmental, proximal, and single under a default criterion.

2.6. Quantitative real-time PCR analysis for phosphofructokinase gene expression All primers used in this study are in Supplementary Table 5. LightCycler 480 SYBR GREEN I Master (Roche) was used for qRT-PCR analysis based on the manufacturer's protocol. Pyrus tubulin (accession number AB239681) was used as the internal control. Each reaction mixture contained 10 μl of LightCycler 480 SYBR GREEN I Master, 0.4 μl of each gene-specific primer, 1 μl of diluted cDNA, and 7.4 μl of nuclease-free water. The qRT-PCR conditions were as follows: pre-incubation at 95 °C for 10 min, and then 94 °C for 15 s, 60 °C for 30 s, 72 °C for 30 s with 40 cycles, finally, extension at 72 °C for 3 min, and then the plate for fluorescence data collection was read at 60 °C. A melting curve was performed from 60 to 95 °C in order to check the specificity to the amplified product. Finally, the average threshold cycle (Ct) was calculated per sample. The relative expression levels were calculated with the 2−ΔΔCt method. In this research, each reaction was

2.5. RNA extraction and first-strand cDNA synthesis In this study, seven pear tissues (young leaf, mature leaf, anther, filament, pistil, petal, and stem) and six pear fruit developmental stages were used to detect phosphofructokinase gene expression. Total RNA was extracted from different tissues using the CTAB method, and DNase I was used to remove genomic DNA. Finally, 1 μg of total RNA of different tissues was used for first-strand cDNA by a Rever Tra Ace-alphaFirst Strand cDNA Synthesis Kit (TOYOBO Biotech Co. Ltd.) following the manufacturer's protocol. 135

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Fig. 2. Gene structure of phosphofructokinase. A: Organization of putative conserved motifs in pear phosphofructokinase genes identified by MEME; numbered color boxes represent different putative conserved motifs. B: GSDS output of the phosphofructokinase gene structure.

2.8. Transient transformation of pear fruits

repeated three times and three independent analyses showed the same trends for each gene in different pear tissues and fruit stages.

The full-length CDS of PbPFP1 were inserted into Gateway vector pCR8/GW/TOPO. Then, PbPFP1 fragments used to generate the complementation constructs were transferred by LR recombination reactions into the Plant Gateway vector pMDC32 with a cauliflower mosaic virus (CaMV) 35S promoter. The recombinant plasmid was introduced into Agrobacterium tumefaciens strain GV3101 by heat shock and then used for transient transformation in pear fruit 110 days after full blooming (DAFB, Fig. 6A) (Yao et al., 2017).

2.7. Subcellular localization analysis of PbPFP1 Full-length cDNAs of the PbPFP1 genes without the stop codon were isolated by PCR using gene-specific primers (Supplementary Table 5), and were then cloned into the Gateway vector pCR8/GW/TOPO and confirmed by sequencing using an M13-forward primer (TGTAAAACG ACGGCCAGT). The pear PbPFP1 genomic fragments containing the CDS were used to generate the complementation construct, and the CDS fragments were LR recombined into pEarleyGate 104, which carries the YFP gene at the N terminus of the fusion and is driven by the cauliflower mosaic virus (CaMV) 35S promoter. The fusion construct and the control plasmid were mobilized into Agrobacterium tumefaciens strain GV3101 by heat shock. Then, the abaxial surfaces of Nicotiana benthamiana leaves were agroinfiltrated with the bacterial suspension (OD600 = 0.8-1.0) and kept tobacco plants in an incubator for 2 d (Yoo et al., 2007), and then checked for the presence of yellow fluorescence through a laser-scanning confocal microscopy (LSM510; Carl Zeiss). The YFP emission was detected by a photomultiplier through a 524 nm to 600 nm band-pass filter using an Achroplan 40X/0.75W objective.

3. Results 3.1. Phylogenetic relationships of the phosphofructokinase gene family in pear and other Rosaceae species In our study, a total of 55 open reading frames (ORFs) encoding putative phosphofructokinase genes were identified in pear and the other four Rosaceae species using HMMER software (Fig. 1, Supplementary Table 1). We also identified 11 putative phosphofructokinase genes from Arabidopsis. Previous research in Arabidopsis identified 11 putative phosphofructokinases, so our method for identifying the phosphofructokinase gene family was reliable. Of the 55 total genes, we 136

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phosphofructokinase genes were duplicated gene pairs from different duplication modes, and WGD/Segmental and dispersed duplications were the main duplication models for the phosphofructokinase genes of peach, woodland strawberry and Japanese apricot. However, for pear and apple, we found that the main duplication model not only included WGD/segmental and dispersed duplications, but also had proximal and tandem duplications (Supplementary Fig. 1). This result explains why there are more phosphofructokinase genes in apple and pear than in peach, woodland strawberry, and Japanese apricot.

found that there were more phosphofructokinase genes in apple (17) and pear (14) than in woodland strawberry (8), peach (9), and Japanese apricot (7) (Supplementary Table 2). This result indicated a greater expansion of the phosphofructokinase gene family in pear and apple. In order to explore the evolutionary relationships of phosphofructokinase genes in pear and the other five species, the protein sequences of all identified phosphofructokinases were aligned using ClustalX, and an ML phylogenetic tree was re-constructed using PhyML software. Considering the topology of the phylogenetic tree and bootstrap values, two subfamilies with five clades were defined in six species (PFK subfamily and PFP subfamily, Fig. 1). We named the different clades PFK_A, PFK_B, PFK_C, PFP_alpha, and PFP_beta. We found that the genes in PFP_beta and PFK_B clades only had one copy in different Rosaceae species, indicating that those genes may play important roles for plant growth and development.

3.4. Predicting cis-elements involved in the transcriptional regulation of phosphofructokinase genes In order to determine whether some common cis-elements regulating phosphofructokinase gene expression were present, a 2 kb promoter region for all phosphofructokinase genes was identified, and the PLACE website was used to identify cis-elements. Ultimately, 239 cis-elements were present in phosphofructokinase genes. Based on the cis-elements identified, we found a total of 60 common cis-elements in all promoter regions of the phosphofructokinase genes (Supplementary Table 3). The common cis-elements responded to light, CO2, low temperatures, sugar contents, or binding to MYB. This result suggested that cis-elements in the promoter region might be regulated by light, sugar contents, or binding to the MYB gene. We also checked the unique cis-element sequences present in the promoter region of unique phosphofructokinase genes, which might indicate specific regulation of gene expression. In total, 22 unique ciselements were present in nine genes (Supplementary Table 4). This result indicated that a limited number of gene specific cis-elements were concentrated in the promoter regions of some phosphofructokinase genes.

3.2. Sequence feature analysis reveals highly conserved exon-intron structure and motifs in pear phosphofructokinase To better understand the sequence and structural characteristics of phosphofructokinase genes in pear, we used the MEME website to confirm and predict conserved domains for all phosphofructokinase protein sequences. A total of 15 distinct motifs were identified in the PFK and PFP gene subfamilies. Based on the MEME conserved motifs analysis, we found that the motifs in PFK were different from those in the PFP subfamily (Fig. 2A). This might be because the gene members in the PFK and PFP subfamilies have different functions, so they have different functional domains. Next, we analyzed the exon-intron organization of different PFK and PFP. We found that most of the phosphofructokinase genes in pear had more than ten exons, with one phosphofructokinase gene only containing two exons (Fig. 2B). However, 13 out of 14 phosphofructokinase genes contained more than ten exons, suggesting that phosphofructokinase proteins had conserved exons in different species. We also analyzed the conserved domains and exon-intron organization of phosphofructokinase genes in the other four Rosaceae species and Arabidopsis and found similar results to pear (data not shown).

3.5. Expression characteristics of phosphofructokinase genes in different tissues of pear Phosphofructokinase genes play diverse functional roles in different tissues and fruit development, such as sugar accumulation and responses to biotic and abiotic stress. qRT-PCR was used determine expression and thereby characterize the functions of phosphofructokinase genes in different tissues and during fruit development of pear. The expression of all 14 phosphofructokinase genes could be detected in different tissues and during different stages of pear fruit development. As shown in Fig. 4, PbPFK3 and PbPFK5 were highly expressed in pollen, indicating that they might contribute to the nutrition supply for pollen development and growth, and PbPFK1, PbPFK2, PbPFK6, PbPFK7, PbPFP1 and PbPFP2 were had a similar pattern with different sugar contents during pear fruit development, among of them, PbPFP1 had a more similar expression pattern with sorbitol and fructose contents, in particular, it is more similar with sorbitol content patterns (Yu et al., 2019). Suggesting that PbPFP1 might play more important role for the sorbitol sugar accumulation during pear fruit development.

3.3. Chromosomal localization and collinearity analyses of phosphofructokinase genes in pear and five other species The genomic distribution of phosphofructokinase genes on chromosomes of pear and the other five species were determined. Thirteen out of 14 phosphofructokinase genes in pear were distributed throughout nine of 17 chromosomes another one gene (PbPFK5) was located in the scaffold1061.0 (Fig. 3A). Chromosomes 5 and 17 contained three and four phosphofructokinase genes, respectively. For the other five species, we also found that phosphofructokinase genes were not evenly distributed on each chromosome (Fig. 3A). Collinearity relationships of phosphofructokinase genes between pear and the other five species were also analyzed in this study. Results showed high collinearity between pear and the other five species, indicating that the phosphofructokinase genes in Rosaceae species are conserved. In order to verify the reliability of the collinearity analysis of seven species, both ends of a 100 kb region of PbPFK1, FvPFK4, PmPFK1, MdPFK12, PpPFK2, and AtPFK6 were further analyzed. The genes located at the ends of PbPFK1 had good collinearity with the genes located at the ends of FvPFK4, PmPFK1, MdPFK12, PpPFK2, and AtPFK6 genes (Fig. 3B). This result suggested that our collinearity analysis between pear and the other five species was highly reliable. Additionally, as shown in Fig. 3A, we found no collinearity between PFP and PFK genes, indicating that PFP and PFK evolved from independent ancestors, even though they belong to the same family. To explain the origin of phosphofructokinase gene family expansion, different duplication modes of phosphofructokinase genes were analyzed using MCScan software. We found that all predicted

3.6. PbPFP1 localization on the plasma membrane in cell In order to elucidate where functional gene localization. We also determined subcellular localization of PbPFP1, which might play important roles for sugar accumulation in pear fruit based on the qRT-PCR analysis. To experimentally establish subcellular localization of the PbPFP1 proteins, we constructed YFP fusion constructs (35S::YFPPbPFP1,) and the construct were transiently co-expressed in N. benthamiana leaf epidermal cells. Confocal microscopy was used to determine the subcellular localization of recombinant phosphofructokinases. As shown in Fig. 5, the empty vector, which only contained YFP as a control was located throughout the cell, including in the membrane and nucleus. For the phosphofructokinases localization, we found that PbPFP1 was localized on the plasma membrane. 137

Fig. 3. Chromosomal distribution and synteny analysis of phosphofructokinase in pear and five other species. The chromosomes of six species are depicted as several boxes with different colors. Chromosome IDs of different species are shown in the different boxes and the approximate distribution of each phosphofructokinase gene is marked with a short red line on the different boxes. Different colored lines denote the details of syntenic gene pairs between pear and the other five species. B. Syntenic relationships of phosphofructokinase genes between pear and other five species, 100 kb on each side flanking the genes were shown. The WGD/ segmental duplication gene pairs are connected by lines with different colors between different species and the target duplication gene pairs of phosphofructokinases were marked with red and connected by the red line between pear and the other five species. The scale on the circle is mega bases. The numbers of each chromosome and the name of each species are shown outside the circle of each bar. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 4. qRT-PCR analysis of the expression of 14 phosphofructokinase genes in different tissues and during fruit development of pear. The x-axes indicate the different tissues and fruit development stages of pear. Units on the y-axes indicate expression fold change. Error bars indicate three technical replicates derived from one bulked biological replicate.

fruits were collected which was injected based on YFP fluorescence and used to measure sugar and extract RNA. As shown in Fig. 6B, after overexpressed the PbPFK1 genes in pear fruits, expression of PbPFP1 (Fig. 6C) led to a significant increase in fructose and sorbitol content in pear fruits. Our result indicated that PbPFP1 could improve fructose and sorbitol content during pear fruit development.

Together, our results demonstrate that phosphofructokinase proteins were functional on the plasma membrane. 3.7. The effect of manipulation of phosphofructokinase expression on the endogenous content of sugars Sugars fulfill many essential functions in all types of plant cells. To examine the precise molecular functions of the putative phosphofructokinase genes for sugar accumulation in pear fruit, we transiently expressed the PbPFP1 genes in ‘Dangshansuli’ pear fruits 110 DAFB (Fig. 6A) by injecting Agrobacterium containing the 35S::PbPFP1-YFP construct vector. The empty vector was injected as a control at the same stage of the pear fruits. Finally, 10 d after the initial injection, the pear

Bright field

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4. Discussion 4.1. Phylogenetic analysis and duplication of the phosphofructokinase gene family in pear and five other Rosaceae species The genome, transcriptome, and proteome sequence profiles of the

Merge

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Fig. 5. Subcellular localization of 35S::YFP-PbPFP1 fusion protein in N. benthamiana leaves. A: Bright-field image of N. benthamiana leaf epidermal cells. B: Localization of the 35S::YFP fusion protein in N. benthamiana leaf epidermal cells. C: Merge of A and B. D: Bright-field image of N. benthamiana leaf epidermal cells. E: Localization of the 35S::YFP-PbPFP1 fusion protein in N. benthamiana leaf epidermal cells. F: Merge of E and F.

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Fig. 6. Transient transformation assays demonstrate that the function of PbPFP1 gene could increase the sugar content in ‘Dangshansuli’ pear fruit. The different sugar contents after transient over-expression of the PbPFP1 genes in pear fruit. The sugar contents were measured by HPLC; one star above the line indicates significant differences at the level of P < 0.05. Error bars show the SEs of the means (n = 3). A: The biomass of the injection pear fruit, which used for control and transient transformation; B: the different sugar content in control and Ox-PbPFP1 pear fruit; C: gene expression after over-expressed the PbPFP1 in pear fruit.

From an applied perspective, the identification of phosphofructokinase genes with potential roles in different tissues development and fruit sugar accumulation would benefit gene functional analysis. qRT-PCR was used to analyze phosphofructokinase genes expression in different tissues and during fruit development. Previous studies have indicated that PFP plays important roles in glycolysis during pollen development (Funaguma et al., 1992; Nakamura et al., 1992; Groenewald and Botha, 2001; Funaguma et al., 2006). In this study, we also detected that two phosphofructokinase genes (PbPFK3 and PbPFK5) were highly expressed in pollen. This result might indicate that PbPFK3 and PbPFK5 play important roles during pear pollen development. Additionally, we were more focused on the functions of genes for sugar accumulation in pear fruits and we detected high expression levels of most phosphofructokinase genes during pear fruit development. Among them, PbPFP1 had similar expression pattern to fructose and sorbitol accumulation, suggesting that the phosphofructokinase genes might contribute to sugar metabolism during pear fruit development. The expression of phosphofructokinase genes in pear tissues and during fruit development provides a substantial experimental database and reliable guide for further gene functional analysis in pear.

pear provide a large amount of useful data to explore the functional diversity of the phosphofructokinase gene family from multiple perspectives. In this report, 14 phosphofructokinase genes were identified from the pear genome. Among them, ten are PbPFK genes and four are PbPFP genes (Fig. 1). Additionally, as shown in Supplementary Table 1, there were 17 phosphofructokinases in apple, seven in Japanese apricot, eight in wild strawberry, and nine in peach. All phosphofructokinase genes in pear and the other five species could be grouped into two subfamilies (PFK and PFP) with five subgroups: PFK_A, PFK_B, PFK_C, PFP_alpha, and PFP_beta (Fig. 1). Similar results have been found in Arabidopsis (Mustroph et al., 2007), Saccharum (Zhu et al., 2013b), and rice (Mustroph et al., 2013) and the phylogenetic tree showed that the phosphofructokinase genes in those species could be grouped into the same two subfamilies, PFK and PFP. Genome evolution analysis has shown that almost all land plants have undergone at least one WGD (Jiao et al., 2011), including the five Rosaceae species (Velasco et al., 2010; Shulaev et al., 2011; Zhang et al., 2012; International Peach Genome et al., 2013; Wu et al., 2013). However, identification of the duplicate genes retained after WGD may be challenging (Van de Peer, 2004). Synteny is lost after many rearrangements in different species via fractionation and WGDs. Due to the difficulty of separating potential segmental duplications from WGD gene pairs we did not distinguish between WGD and segmental duplications. In this report, there were more phosphofructokinase genes in apple and pear than in Japanese apricot, woodland strawberry, and peach. Gene duplication analysis showed that tandem and proximal duplication events occurred in apple and pear, but not in Japanese apricot, wild strawberry, or peach, leading to the higher gene numbers in the former two species. This result suggests that the main driving force of phosphofructokinase gene family expansion in Japanese apricot, wild strawberry, and peach species is WGD/segmental duplication and dispersed duplication but that tandem and proximal duplication events also contributed to gene family expansion in apple and pear. Analysis of the sugar transporter and heat shock transcription factor gene families in pear also provided direct evidence indicating that the main driving force of gene family expansion was WGD/segmental duplication (Li et al., 2015; Qiao et al., 2015; Li et al., 2017).

4.3. PbPFP1 genes regulated sugar accumulation in pear fruits Sugars fulfill many essential functions in all types of plant development and fruit quality. High quality fruit improves economic benefits for growers. Therefore, breeders typically focus research on how to improve the sugar content of fruit. However, traditional pear breeding is very time-consuming. Molecular approaches to breeding are a shortcut but also a challenge in terms of improving the sugar content of pear fruit. In this study we found that PbPFP1 had similar expression pattern with sorbitol and fructose accumulation during pear fruit development and thus conducted further functional analysis of these genes. As shown in Fig. 6B, after transient transformation of PbPFP1, the fructose and sorbitol contents of pear fruit significantly increased. Our result indicated that phosphofructokinase contributed to sugar accumulation during pear fruit development. In loquat, Qin et al. (2014) showed that EjPFPa and EjPFPb1 were located on the cell membrane and the fructose content in over-expressed EjPFPb1 transgenic tobacco lines increased compared with wildtype (WT). Similar results were also found in sugarcane: after over-expressing PFP, hexose concentrations increased significantly in young internodes (Groenewald and Botha, 2001). Additionally, in loquat, the expression of EjPFPa and EjPFPb in the leaves of loquat seedlings was all significantly increased after 3 h of treatment with 0.5, 1.0, and 1.5 M fructose or glucose (Qin et al., 2014).

4.2. Expression analysis of phosphofructokinase genes in pear suggests possible roles in pollen development and sugar accumulation of pear fruits As is evident from phosphofructokinase gene expression patterns in other plant species, phosphofructokinase genes play diverse functional roles in different tissues and can also respond to stress (Groenewald and Botha, 2001; Mustroph et al., 2013; Zhu et al., 2013b; Qin et al., 2014). 140

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