Identification of hexokinase family members in pear (Pyrus × bretschneideri) and functional exploration of PbHXK1 in modulating sugar content and plant growth

Gene 711 (2019) 143932

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Research paper

Identification of hexokinase family members in pear (Pyrus × bretschneideri) and functional exploration of PbHXK1 in modulating sugar content and plant growth

T

Biying Zhaoa,b, Kaijie Qia, Xianrong Yib, Guodong Chena, Xing Liua, Xiaoxiao Qia, ⁎ Shaoling Zhanga, a b

College of Horticulture, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China Guangxi Key Laboratory of Citrus Biology, Guangxi Academy of Specialty Crops, Guilin, China

ARTICLE INFO

ABSTRACT

Keywords: PbHXK1 Sugar content Pear Glucose sensor Transgenic technology

Hexokinase (HXK) is a multifunctional protein that serves as a sugar sensor for glucose signaling and a catalyst for glycolysis. It has been well studied in many species, however, there is far less information about this family in pear. To investigate the roles of HXK in the growth and development of pear fruit, we performed a genome-wide analysis and identified the HXK gene family members in pear. In addition, we functionally characterized a glucose sensor gene, PbHXK1, in P. bretschneideri. In total, 10 HXK genes were identified in pear, and a multiple sequence alignment and phylogenetic analysis showed that PbHXK1 is a Type B HXK that contains four conserved domains, phosphate 1 and 2, sugar binding and adenosine, which are specific to plant HXKs and essential for enzymatic functions. A qRT-PCR analysis revealed that the relative expression levels of PbHXK1 were negatively correlated with sugar content but significantly positively correlated with HXK activity during pear fruit development. Furthermore, the overexpression of PbHXK1 in tomatoes significantly enhanced the HXK activity and decreased the sugar content. In addition, the growth of transgenic tomato plants overexpressing PbHXK1 was inhibited, leading to shortened internodes and smaller leaves. Thus, in pear, PbHXK1 encodes HXK, which regulated the sugar content in fruit and affected the growth and development of plants.

1. Introduction Sugars are the main energy sources of life activities for organisms and a main component of cell structures. They not only play important roles in the growth and development of living organisms and affect the flavor of the fruit, but they also function as signaling molecules that regulate various signal transduction pathways and modulate gene expression (Ohto et al., 2001; Sarowar et al., 2008; Bolouri Moghaddam and Van den Ende, 2013; Cao et al., 2013). In higher plants, the carbohydrate produced by photosynthesis is mainly sucrose, which is distributed and transported through the phloem to different tissues. Then, sucrose can be directly stored and converted to hexose under the action of sucrose synthetase or invertase (Li et al., 2012). Afterwards, the hexose begins to undergo glycolysis owing to phosphorylation by hexokinase (HXK) during the respiratory metabolism of plants, thereby providing energy and metabolites for plant activities (Claeyssen and

Rivoal, 2007; Yim et al., 2012; Granot et al., 2013). Therefore, starch synthesis and the carbon cycle are inseparable from the HXK-driven phosphorylation of hexose in plants (Alonso et al., 2005; Zhao et al., 2016). During the plant growth process, HXK is not only involved in sugar metabolism but also acts as a hexose sensor (Kim et al., 2013) and signal in signaling networks to sense external nutrients, light and hormones, and regulate plant growth (Feng et al., 2015). The HXK family are structurally classified as Type A and Type B, and exists in almost all living organisms, based on their N-terminal amino acid sequences (Olsson et al., 2003). One group contains a chloroplast transit peptide of ~30 aa (Type A), while the other contains a common N-terminal hydrophobic membrane anchor domain of ~24 aa (Type B) (Olsson et al., 2003). The first higher plants HXK gene was isolated and identified using a functional complementation expression library from Arabidopsis (Minet et al., 1992) and it has now been well studied. It contains six genes, AtHXK1-3 and AtHKL1-3, which have been divided

Abbreviations: HXK, hexokinase; DAFB, day after full bloom; HMM, Hidden Markov Model; GSDS, Gene Structure Display Server; qRT-PCR, quantitative real-time reverse transcriptase PCR ⁎ Corresponding author. E-mail address: [email protected] (S. Zhang). https://doi.org/10.1016/j.gene.2019.06.022 Received 19 January 2019; Received in revised form 19 May 2019; Accepted 11 June 2019 Available online 13 June 2019 0378-1119/ © 2019 Elsevier B.V. All rights reserved.

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into four subgroups based on the evolutionary relationships of their encoded HXK protein sequences in Arabidopsis (Karve et al., 2008). Furthermore, the HXK gene family has been identified and cloned from Oryza sativa (OsHXK1-10); (Jung-Il et al., 2006), Lycopersicon esculentum (Kandel-Kfir et al., 2006), Nicotiana tabacum (Giese et al., 2005), Zea mays (Zhang et al., 2014), Physcomitrella patens (Olsson et al., 2003) and other plants (Zhao et al., 2016). HXK proteins are present in the cellular cytosol, mitochondria, plastids, nuclei and Golgi (Da-Silva et al., 2001; Olsson et al., 2003; Kandel-Kfir et al., 2006). The diversity of subcellular localizations of HXKs may reflect their roles in a variety of metabolic pathways. In addition, HXK expression patterns were analyzed and found not only in the roots but also in the leaves, flowers, kernels and stems. This further illustrates the diversity of HXK functions, which involve not only acting as a processor of plant energy metabolism, but also in regulating plant growth and development, and signal transduction. For example, three members of the HXK family phosphorylate glucose in Arabidopsis, and AtHXK3 present in the cytoplasm may have only catalytic functions (Karve et al., 2008). The overexpression of AtHXK1 not only inhibits seedling growth but also decreases the expression levels of photosynthetic genes, the transpiration rate, hydraulic conductivities of the root and stem and the CO2 conductance of leaf mesophyll in Arabidopsis (Dai et al., 1999). Seedlings are hypersensitive to increasing concentrations of exogenous glucose, root growth is inhibited, and the greening of cotyledons is reduced in the AtHXK1-overexpressing Arabidopsis seedlings. However, the transgenic Arabidopsis plants overexpressing YHXK2, encoding a heterologous hexokinase from yeast, showed sugar hyposensitivity to glucose, with normal growth and a significantly greater HXK activity level (Gonzali et al., 2002). In Solanum tuberosum, the antisense repression of StHK1 led to an over accumulation of starch. Additionally, the HXK activity varied 22-fold in leaves and 7-fold in developing tubers, but nonsignificant changes were found in fresh weight yield, starch, sugar and metabolite levels (Veramendi et al., 1999). Overexpressing AtHXK1 and NtAQP1 simultaneously in tobacco significantly improved growth and increased the transpiration rate, photosynthesis rate and leaf mesophyll CO2 conductance (Kelly et al., 2012, 2013, 2014). Furthermore, the roles of HXK have been studied in signal transduction and hormone regulation. For example, HXK activates the signaling cascade through the HXKinteracting proteins (Yim et al., 2012). Additionally, genetic and chromatin immunoprecipitation analyses suggested that the nuclear specific complex generated by HXK1, VHA-B1 and RPT5B formed a glucose signaling complex core by directly modulating specific target gene transcription independent of glucose metabolism (Jung-Il et al., 2006). Godbole et al. (2013) also demonstrated that the voltage-dependent anion channel interacts with mitochondrial HXK in regulating plant programmed cell death, which is mediated by myo-inositol accumulation through the HXK (Bruggeman et al., 2015). Genetic and physiological analyses have demonstrated that HXK-mediated glucose signaling interacts with abscisic acid and ethylene signaling (Cho et al., 2010; Karve et al., 2012). Pear (Pyrus × bretschneideri) is a commercially important crop that is cultivated in temperate regions worldwide. The fruit are popular because the pulp is refreshing and sweet. The fruit HXK activity is positively correlated with the sugar content, and the sugar content significantly influences the fruit's quality (Ramon et al., 2008; Cheng et al., 2018). However, HXK family members have not yet been identified in pear, and the functions of HXK-related features in pear are still poorly understood. In this study, we employed bioinformatics and publicly available data to identify the pear HXK genes on a genome-wide scale, and we cloned and functionally characterized a Type B HXK gene from P. bretschneideri. A sequence analysis indicated the conserved domains of the putative HXK, PbHXK1, which are specific to HXKs and essential for its function as a catalyst of glycolysis. The overexpression of PbHXK1 in tomatoes significantly increased the HXK activity and decreased the sugar content. Furthermore, the growth of transgenic

tomatoes overexpressing PbHXK1 was inhibited, resulting in shortened internodes and smaller leaves. Thus, the data indicated that PbHXK1 played a negatively role in the sugar content, and it has great potential in the bioengineering of the quality in perennial fruit. 2. Materials and methods 2.1. Plant materials Fruit of P. bretschneideri. ‘Yali’ and Pyrus pyrifolia Nakai ‘Aikansui’, grown in the Jiangpu Orchard of the National Center of Pear Breeding, Nanjing Agricultural University, Nanjing, Jiangsu, China were used in this study. The orchard was managed according to normal commercial practices, and the pear samples were collected in 2015. ‘Yali’ and ‘Aikansui’ were collected randomly at 20 d intervals from 10 d after full bloom (DAFB) until fruit ripening from three 12-year-old pear trees from each cultivar. The fresh weights of fruit were measured using an electronic analytical balance (FA, 2014), and repeated three times for each sample. Flesh tissue was diced into small pieces, sampled at the designated time points, immediately frozen with liquid nitrogen, and placed at −80 °C until analyzed. 2.2. Identification of the HXK gene family members in pear To identify the HXK genes in pear, multiple database searches were performed. The Arabidopsis HXK protein sequences were downloaded from the Arabidopsis Information Resource (TAIR; http://www. arabidopsis.org/) and the tomato HXK protein sequences were downloaded from Phytozome (https://phytozome.jgi.doe.gov/pz/portal). These sequences were used as queries to perform BLAST algorithmbased searches against pear genome databases. Furthermore, the seed alignment file for the HXK gene family domains (PF00349.21 and PF03727.16) were downloaded from the Pfam database (http://pfam. xfam.org/) and used to build a Hidden Markov Model (HMM) file with the HMMER3 software package (Eddy, 2011). Then, HMM searches were performed against the local protein databases of pear using HMMER3. Finally, all the obtained sequences were checked for the presence of HXK protein domains using SMART (http://smart.emblheidelberg.de/) and Pfam, and then the redundant sequences or those lacking the HXK protein domains were removed. 2.3. Phylogenetic and structural analysis of the HXK gene family The HXK amino acid sequences were aligned using ClustalW2 (Larkin et al., 2007), and a Neighbor-Joining-based phylogenetic tree was constructed using MEGA6.0 (http://www.megasoftware.net/). Bootstrapping was performed with 1000 replications. The p-distance and pairwise deletion option parameters were selected (Tamura et al., 2013). The structures of the HXK genes were analyzed using Gene Structure Display Server (GSDS 2.0; http://gsds.cbi.pku.edu.cn/) by aligning the cDNA sequences with their corresponding genomic DNA sequences. In addition, the N-terminal signal sequences of the putative proteins were predicted using SignalP 4.1 Server (Petersen et al., 2011). 2.4. Total RNA extraction and cDNA synthesis Total genomic RNA was extracted according to the CTAB protocol found in Gasic et al. (2004), treated with DNaseI (Invitrogen, CA, USA) and then reverse transcribed into cDNA using ReverTra Ace-α FirstStrand cDNA Synthesis Kit (TOYOBO, TOYOBO Biotech Co. Ltd., Japan) following the manufacturer's instructions. All cDNA samples were stored at −20 °C before being used as templates in cloning and quantitative real-time reverse transcriptase PCR (qRT-PCR). 2

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2.5. Gene isolation

integrated peak areas of external standards (Sigma-Aldrich, UK).

The full-length coding sequence of the PbHXK1 gene was amplified from pear fruit using a nested PCR-system. The specific primers of the PbHXK1 gene of pear were designed using Primer Premier 5.0 software (Supplementary Table S1). To verify the specificity of these primers, we used the program “primer search-paired” against the pear genome. The first nested-PCR reaction contained 100 ng cDNA of P. bretschneideri. ‘Yali’, 1× PCR buffer (TaKaRa, Daliang, China), 0.25-mM dNTPs, 0.4 μM of each primer (F1 and R1; Supplementary Table S1) and 1 Unit Taq DNA polymerase (TaKaRa). The first nested-PCR was carried out as follows: initial denaturation at 94 °C for 3 min, then 35 cycles of 94 °C for 30 s, 58 °C for 40 s and 72 °C for 1.5 min, and a final extension at 72 °C for 10 min. The second nested-PCR reaction contained 1 μL first nested-PCR product, 1× PCR buffer (TaKaRa), 0.25-mM dNTPs, 0.4 μM each primer (F2 and R2; Supplementary Table S1) and 1 unit Taq DNA polymerase (TaKaRa). The second nested-PCR was performed as follows: initial denaturation at 94 °C for 3 min, then 35 cycles of 94 °C for 30 s, 56 °C for 40 s and 72 °C for 1.5 min, and a final extension at 72 °C for 10 min. The second nested-PCR products were separated by 1.2% agarose gel electrophoresis, and the target fragment was recycled and purified using the QiagenII Gel Extraction Kit (Qiagen, Valencia, CA, USA). The purified products were added to the enzyme site (Nco I and Kpn I) using primer P4 (F4 and R4; Supplementary Table S1) and inserted into the pMD19-T vector (TaKaRa) and transformed into Escherichia coli DH5α cells. The primer P2 was used for identifying transformed clones. Then, five independent colonies were sequenced by Invitrogen (Shanghai, China).

2.8. Plant transformation and generation of transgenic plants Full-length PbHXK1 cDNA was amplified using PCR with forward and reverse (F4 and R4; Supplementary Table 1) primers, which have NcoI and BstEII restriction sites, respectively, on their 5′ ends. Then, PbHXK1 was inserted as an NcoI/BstEII fragment into the NcoI/BstEII sites within the pCAMBIA1301 vector driven by the CaMV 35S promoter. The recombinant plasmid was verified and designated pCAMBIA1301-PbHXK1, which was introduced into Agrobacterium tumefaciens strain GV3101 using the freeze-thaw method. Tomato (Solanum lycopersicum ‘Micro-Tom’) transformations were carried out using leaf disks and epicotyls as explants according to Sun et al. (Sun et al., 2006). Transgenic plants were cultured on selective medium containing hygromycin as the selective marker, and were verified as containing the plasmid by PCR using specific primers (Supplementary Table S1). Hygromycin resistant seedlings were transferred to rooting medium. The PbHXK1 expression levels in leaf, young fruit and mature fruit of transgenic and wild-type (WT) plants was determined by RTPCR using primer pair P3 (Supplementary Table S1). The actin (FJ532351) gene was used as the internal control (Supplementary Table S1). 2.9. Enzyme extraction and assay Fruit HXKs were extracted according to Teo et al. (2006). In brief, fruit were collected and frozen in liquid nitrogen. Then, fruit were ground to fine powders using a mortar and pestle in liquid nitrogen containing 0.5 g polyvinyl-pyrrolidone. Then, the fruit was homogenized in an extraction buffer that contained 50-mM HEPES/NaOH (pH 7.5), 1-mM EDTA, 10-mM MgCl2, 2.5-mM DTT, 1% Tween 20, and 5% (wt/v) glycerol. Finally, the slurry was filtered through Miracloth and, after centrifugation at 5000 ×g for 15 min, the supernatant was used for enzyme assays. Fruit HXK activity levels were assayed according to Kanayama et al. (1997) with modifications. The reaction contained 30-mM HEPES/ NaOH (pH 7.5), 0.6 mM EDTA, 9 mM KCl, 1 mM MgCl2, 1 mM NAD, 1 mM ATP, 2 units of G6P dehydrogenase, 30 mM glucose and enzyme solution. Enzyme activities were determined by changes in absorbance at 340 nm at 25 °C.

2.6. Quantitative real-time RT-PCR analysis (qRT-PCR) The qRT-PCR was performed using SYBR Green Master Mix (SYBR Premix EX Taq™, TaKaRa) according to the manufacturer's instructions. Reactions were performed in triplicate using 10 μL of SYBR-Green PCR Master Mix, 0.4 μL of each primer, 200 ng cDNA and nuclease-free water for a total volume of 20 μL. The qRT-PCR was performed in a LightCycler 480 (Roche, USA). The PCR reaction conditions were as follows: preincubation at 95 °C for 5 min, then 45 cycles of 95 °C for 3 s, 60 °C for 10 s and 72 °C for 30 s, and a final extension at 72 °C for 3 min. P3 (Supplementary Table S1) was the specific primer pair for the qRTPCR analysis of PbHXK1. Tubulin (AB239681) and Actin (FJ532351) genes were used as the internal controls to normalize the relative expression levels of PbHXK1 in pear and tomato, respectively. The relative expression levels were calculated using the 2−ΔΔCt method (Livak and Schmittgen, 2001). Data were analyzed using Office 2010 software, and statistical analyses were conducted with SPSS 17.0 software using Duncan's multiple range test at the P < 0.05 level of significance.

2.10. Statistical analyses The SPSS 17.0 software package (IBM Software Group) and Microsoft Excel 2010 were used for statistical analyses. Data were presented as means ± SDs of three replicates. Significant differences were identified using Fisher's least significant difference test and were considered significant at P < 0.05 or highly significant at P < 0.01.

2.7. Soluble sugar determinations

3. Results

Sugar extractions and concentration measurements were performed according to Xin et al. (2009) with some modifications. Flesh and leaves of frozen tissues (2 g) were homogenized in 80% (v/v) ethanol, incubated for 30 min at 37 °C and centrifuged at 12,000 ×g for 15 min. The residues were extracted twice in 80% (v/v) ethanol, and the supernatants were combined. The combined supernatants were diluted to 25 mL with double-distilled water. The sugar supernatants (2 mL) were evaporated and dissolved in 1 mL sterile water. The aliquots were passed through a 0.45-μm membrane filter and injected into a Waters 1525 high-performance liquid chromatography (HPLC) system (Waters, USA). The HPLC was equipped with a 6.5 × 300-mm Sugar-Pak™ Column (Waters, USA) and a Waters 2414 refractive index detector (Waters, USA). The column and reference cell were maintained at 85 °C and 35 °C, respectively. Samples were eluted by redistilled water at a constant flow of 0.6 mL·min−1. Sucrose, glucose and fructose were identified and quantified by comparisons with retention times and

3.1. Genome-wide identification and classification of the HXK genes in pear A total of 14 HXK genes were identified in pear based on BLAST and HMM searches using Arabidopsis and tomato annotated protein sequences as query. Subsequently, we removed the incomplete gene sequences, redundant sequences and transcripts of the same genes, and the remaining sequences were analyzed for the presence of the HXK domain by SMART and Pfam. Finally, 10 nonredundant HXK genes were identified in pear (Supplementary Table S2). To classify the HXK identified in pear, Arabidopsis and tomato, and investigate their evolutionary relationships, a phylogenetic tree was constructed based on the amino acid sequences of each protein, showed that the HXK from pear could be separated into two major groups, Type A and Type B (Fig. 1A). In addition, the phylogenetic analysis also indicated that the 3

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Fig. 1. The phylogenetic relationships and schematic diagrams of HXK genes intron/exon structures in pear, tomato and Arabidopsis thaliana. A. The phylogenetic tree was constructed using the full-length protein sequences of HXK genes with the Neighbor-Joining method and 1000 bootstrap replicates. Different colors in the subgroups represent the Type-A (red) and Type-B (yellow), respectively. B. The gene structural diagram. The cyan boxes, black lines and pink boxes represent exons, introns and UTRs, respectively. Gene models are drawn to scale as indicated at the bottom. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. Multiple alignment of PbHXK1, LeHXK3, AtHXK1 and AtHXK2 amino acid sequences. Identical and similar amino acid residues are highlighted with black and pink shades, respectively. Conserved domains specific to HXKs are framed and indicated using A–D, which refer to phosphate 1, sugar binding, phosphate 2 and adenosine, respectively. Phosphate 1 and 2 and adenosine are involved in ATP binding; the sugar-binding domain corresponds to the hexose-binding site. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

putative protein PbHXK1 is a Type B HXK (Fig. 1A). To explore and compare the structural diversity of HXK genes in pear, Arabidopsis and tomato, the exons and introns were analyzed by aligning genomic sequences with their corresponding cDNAs (Fig. 1B). Most members of the HXK gene family have nine exons, except for AtHXK1, which had seven, and AtHKL3, which has eight. This was

consistent with previous results (Karve et al., 2008). All the tomato HXKs also had nine exons. In pear, most HXKs had nine exons, except for PbHXK4, which had 11 (Fig. 1B). Thus, the HXK genes had similar exon numbers in different species, which indicated that their gene structures have the same characteristics, which drove the phosphorylation function of the HXK. 4

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PbHXK8, PbHXK9 and PbHXK10 were also highly expressed in sepal, with expression levels in petal being secondary. 3.4. Sugar content, HXK activity and expression of PbHXK1 in pear fruit The sugar contents in pear fruit were measured during fruit development, and the growth curves of pear fruit are presented in Fig. 4. The fruit growth of ‘Yali’ showed a typical sigmoidal curve from 10 to 164 DAFB, while that of ‘Aikansui’ formed a more logarithmic curve from 10 to 110 DAFB. In the fruit of both ‘Yali’ and ‘Aikansui’, the glucose and fructose contents increased slightly at the early stage, rapidly during fruit enlargement and gradually at the maturation stage (Fig. 4A and B). The sucrose accumulation pattern in ‘Yali’ differed from that of ‘Aikansui’. The sucrose content of Yali was maintained at a low level, 0.12–5.66 mg·g−1 fresh weight (FW), with a slight increase during fruit development (Fig. 4A). However, the content of sucrose in ‘Aikansui’ was maintained at a very low level (0.34–0.92 mg·g−1 FW) before 70 DAFB and increased sharply during fruit maturation to an extremely high level (23.66 mg·g−1 FW) (Fig. 4B). The expression of PbHXK1 in ‘Yali’ and ‘Aikansui’ during fruit development was analyzed by qRT-PCR. PbHXK1 was differentially expressed at different stages throughout fruit development (Fig. 4). The transcript level in ‘Yali’ increased rapidly during the early stages, peaked on 50 DAFB, decreased sharply on 70 DAFB and then remained roughly constant (Fig. 4C). For ‘Aikansui’, the transcript level of PbHXK1 reached similar peaks at 30 and 70 DAFB (Fig. 4D). Therefore, ‘Yali’ and ‘Aikansui’ had different expression patterns in which the transcript level of PbHXK1 increased during fruit development. “Aikansui” has two PbHXK1 expression peaks (on 30 and 70 DAFB), while “Yali” has one PbHXK1 expression peak (on 50 DAFB). As a key enzyme in fruit glycolysis, HXK levels were measured to provide an insight to sugar metabolism. The HXK activity in ‘Yali’ increased rapidly during the early stages, peaked at 50 DAFB, decreased gradually and was maintained at a roughly constant level until fruit maturation (Fig. 4E). A similar trend was found in ‘Aikansui’, but with a peak at 30 DAFB (Fig. 5F). Independent of the cultivar, a comprehensive analysis showed that the sucrose, glucose and fructose contents were nonsignificantly negatively correlated with HXK activity and the relative expression level of PbHXK1 (Table 1). However, the latter was positively correlated with HXK activity.

Fig. 3. Analysis of the HXK gene expression levels in different pear tissues. A heat map depicting the overall trend of the differential expression profiles of the HXK genes in different pear tissues as constructed by MeV. The rows in the heat map represent genes, and the columns represent tissues. The colors of heat map cells indicate scaled expression levels of genes across different tissues. The color gradient from blue to red corresponds to transcript levels from low to high, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.2. Protein sequence analyses of PbHXK1 The HXK sequences structures have been well studied in varies species, such as Arabidopsis (Karve et al., 2008) and tomato (Jung-Il et al., 2006). HXK proteins contain a large and a small domain, which are located in the sugar-binding site, and four additional peptide segments (Loops 1–4) that are induced to move upon binding the sugar ligand. The sugar-binding sites and the nucleotide-binding site are completed or “pre-formed” by these loops (Katz et al., 2000). To better study the structural difference between PbHXK1 and Arabidopsis or tomato and the possible functional differences, we aligned and analyzed their amino acid sequences (Fig. 2). The multiple alignments indicated that the deduced amino acid sequence of PbHXK1 shared roughly 76.51%, 76.49% and 75.4% identity levels with homologous sequences in AtHXK1, AtHXK2 and LeHXK3, respectively. PbHXK1 contains four conserved domains, phosphate 1 and 2, sugar-binding and adenosine, that are specific to plant HXKs and essential for their enzymatic functions (Bork et al., 1993).

3.5. PbHXK1 overexpression and HXK activity in transgenic tomato regenerants To further investigate the function of PbHXK1, transgenic tomato plants over-expressing PbHXK1 were generated by Agrobacteriummediated transformation using tomato leaf disks and epicotyls as explants. DNA of independent tomato regenerants was extracted and used as template in PCR to determine the presence of PbHXK1. Then, the expression levels of PbHXK1 in leaves and young and mature fruit of transgenic lines (#93, #95 and #98) were analyzed by qRT-PCR. Transgenic tomato plants over-expressing PbHXK1 showed extremely significant increases in the relative expression levels of PbHXK1 by 13to 54-fold in leaf (Fig. 5A), 25- to 75-fold in young fruit (Fig. 5B) and 6to 22-fold in mature fruit (Fig. 5C), when compared with WT. In addition, the HXK activity levels in the transgenic tomato plants significantly increased by 1.5- to 3.4-fold in leaf (Fig. 5D), 3- to 4.6-fold in young fruit (Fig. 5E) and 1.1- to 2-fold in mature fruit (Fig. 5F), when compared with WT. Thus, the HXK activity required to phosphorylate hexose correlated directly with the expression level of heterologous PbHXK1.

3.3. Expression patterns of PbHXKs To study the accurate expression patterns of PbHXKs in different pear tissues, a heat map depicting the overall expression patterns of the HXK genes in different tissues (root, stem, leaf, fruit, petal, sepal, ovary, bud, pollen, pollen tube and stop-growth pollen tube) was constructed using Mev software, which was based on the transcriptome (Chen et al., 2018; Li et al., 2019). PbHXKs were expressed in various pear tissues (Fig. 3). For example, PbHXK1 showed preferential expression levels in fruit, while the expression levels in petal, sepal, ovary and bud were secondary. PbHXK2 and PbHXK3 were mainly expressed in sepal and petal, respectively. PbHXK4 and PbHXK5 showed similar expression patterns that were significantly greater in ovary, with the expression levels in bud and sepal being secondary. In contrast, PbHXK6 and PbHXK7 also showed similar expression patterns, but they were greatly expressed in bud, and were also detected in root, petal and ovary.

3.6. PbHXK1 overexpression decreased the sugar content and inhibited transgenic tomato-regenerant growth PbHXK1 was a candidate gene to encode HXK, which catalyzes 5

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Fig. 4. Sucrose, glucose and fructose contents, fresh weights (FWs), PbHXK1 expression and HXK activity were measured during pear fruit development. A. Sucrose, glucose and fructose contents, and FWs were measured during fruit development in Pyrus bretschneideri. ‘Yali.’ B. Sucrose, glucose and fructose contents, and FWs were measured during fruit development in P. bretschneideri. ‘Aikansui.’ C. PbHXK1 expression was analyzed by qRT-PCR during fruit development in P. bretschneideri. ‘Yali.’ D. PbHXK1 expression was analyzed by qRT-PCR during fruit development in P. bretschneideri. ‘Aikansui.’ E. HXK activity was measured during fruit development in P. bretschneideri. ‘Yali.’ 1 U = ΔA340 min−1·mg−1 protein. F. HXK activity was measured during pear fruit development in P. bretschneideri. ‘Aikansui’. 1 U = ΔA340 min−1·mg−1 protein. Fruit samples were collected once every 20 d since 10 d after full bloom (DAFB). Furthermore, in ‘Yali’, the early stage was 10–50 DAFB, the fruit enlargement stage was 50–150 DAFB, and the fruit maturation stage was 50–164 DAFB; In ‘Aikansui’, the early stage was 10–50 DAFB, the fruit enlargement stage was 50–100 DAFB, and the fruit maturation stage was 100–110 DAFB. The data is presented as the means ± SDs of three replicates. Different superscript letters indicate significant differences (p < 0.05).

hexose phosphorylation, the first step of glycolysis. The overexpression of heterologous PbHXK1 and increased HXK activity were expected to decrease the sugar contents in transgenic lines. As shown in Fig. 6A, B and C, the contents of sucrose, glucose and fructose, respectively, in two tissues (leaves, and young and mature fruit) of the transgenic lines (#93, #95 and #98) decreased significantly, compared with those in WT. Glucose was barely detectable in the leaves of the transgenic lines. The overexpression of heterologous PbHXK1 in transgenic lines resulted in several characteristic phenotypes. For example, the most prominent phenotype was the inhibition of plant growth (Fig. 7A). The growth inhibition of transgenic regenerants was accompanied by a decrease in

the amount of sugar, which is essential for plant growth and development. The transgenic lines (#93, #95 and #98) were stunted, with decreased stem lengths and smaller leaves, compared with WT (Fig. 7B and C). Thus, the plants growth was inhibited, which correlated directly with the overexpression of heterologous PbHXK1 and the increased HXK activity level. 4. Discussion HXK, the rate-limiting enzyme in glycolysis, controls cell survival by promoting metabolism and/or inhibiting apoptosis. HXK not only plays 6

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Fig. 5. PbHXK1 expression and HXK activity were measured in wild-type (WT) tomato (‘Micro-Tom’) and transgenic tomato overexpressing PbHXK1. Total mRNA was extracted from tomato leaves (A), young fruit (B) and mature fruit (C) and used for qRT-PCR analysis. HXK activity was measured in crude protein extracts prepared from tomato leaves (D), young fruit (E) and mature fruit (F). The three independent regenerants, #93, #95 and #98, represent PbHXK1-overexpression lines. 1 U = 0.001 ΔA340 min−1·g−1 FW. ⁎⁎ indicates that the expression level of PbHXK1 in the transgenic regenerant was significantly greater than that in WT tomato (P ≤ 0.01); ⁎ indicates statistical significance (P ≤ 0.05). The data are presented as the means ± SDs of three replicates.

key roles in sugar signaling, but it also acts as a sugar sensor (Moore et al., 2003; Kim et al., 2013). Moore et al. (2003) also concluded that the glucose-6-phosphate metabolism is uncoupled from HKX-dependent signaling. HXK also plays critically important roles in plant growth and development (Xiao et al., 2000). Thus, the characterization of more HXK genes is crucial to fully understand sugar signaling and provide functional gene candidates for genetic manipulation. However, to date, no HXK gene family members of pear had been cloned and characterized. To identify and study the HXK genes in pear, Arabidopsis and tomato HXK protein sequences were used as query to perform BLAST algorithm-based searches against pear genome databases. A total of 10 nonredundant HXK genes were identified in pear, which was more than in any other species. For example, there are six members in Arabidopsis (Karve et al., 2008), six members in tomato (Damari-Weissler et al., 2006) and 10 members in rice (Jung-Il et al., 2006). In addition, to

Table 1 Correlation analysis of the sugar content, HXK activity and relative expression level of PbHXK1 during pear fruit development. Cultivar

Sugar content/HXK activity

cv. Yali

Sucrose Glucose Fructose HXK activity Sucrose Glucose Fructose HXK activity

cv. Aikansui

HXK activity −0.146 −0.367 −0.345 – −0.538 −0.556 −0.564 –

Relative expression level −0.257 −0.29 −0.278 0.682⁎ −0.438 −0.285 −0.4 0.850⁎

Note: ⁎ Significant at P ≤ 0.05.

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LeHXK3 in Arabidopsis and tomato, respectively, using multiple amino acid sequence alignments. We found that PbHXK1 contains found conserved domains, phosphate 1 and 2, sugar binding and adenosine, which are specific to plant HXKs and essential for enzymatic functions (Bork et al., 1993). These domains shared significant degrees of identity with those from other plant species, indicating that the protein structure of PbHXK1 was similar to those of other species and has the basic functional domains of an HXK. In our study, to classify the HXK genes in pear, Arabidopsis and tomato, and to investigate their evolutionary relationships, a phylogenetic tree incorporating the amino acid sequences of each protein was constructed and analyzed. The result is consistent with previous studies, which was divided into two major groups, Types A and B (Moore et al., 2003). Furthermore, the phylogenetic analysis indicated that the putative protein PbHXK1 was a Type B HXK, which is highly expressed in pear fruit and highly homologous to AtHXK1 and LeHXK3 in Arabidopsis and tomato, respectively. So, PbHXK1 was chosen to study on the functional characteristics of HXK family members in pear fruit. To further explore the roles of the HXK in sugar accumulation and sugar metabolism in pear, we studied the expression patterns of the HXK gene family in different pear tissues, and the transcriptome data were analyzed using a heat map. The different HXK family members had different expression levels. In particular, PbHXK1 was mainly expressed in fruit, which was similar to previous results in tomato (Dai et al., 2002). Thus, we speculated that PbHXK1 had important functions in the fruit related to the catalyzing glucose and phosphorylating fructose. Then, the sugar contents were measured in pear fruit during fruit development, and the growth curves of pear fruit weights were determined. ‘Yali’ and ‘Aikansui’ had different fruit growth curves, and their sucrose contents also showed different trends during fruit growth. However, the fructose and glucose contents of ‘Yali’ and ‘Aikansui’ showed similar changes during fruit growth. Furthermore, the expression levels of PbHXK1 in ‘Yali’ and ‘Aikansui’ were analyzed during fruit development using qRT-PCR. The PbHXK1 transcript level in ‘Yali’ increased rapidly at early stages, peaked at 50 DAFB, decreased sharply at 70 DAFB and then remained roughly constant. The PbHXK1 transcript level in ‘Aikansui’ peaked to similar levels at 30 and 70 DAFB. This indicated that in different varieties of pear, the PbHXK1 expression pattern was also different during fruit growth, which is most likely caused by their different maturity periods. The HXK enzyme's activity level was also measured to provide an insight into sugar metabolism, and a similar trend was shown in ‘Yali’ and ‘Aikansui’, with activity peaking at 50 and 30 DAFB, respectively. Thus, the sucrose, glucose and fructose contents were nonsignificantly negatively correlated with HXK enzymes activity and the relative expression level of PbHXK1. However, the relative expression level of PbHXK1 was positively correlated with the HXK enzyme's activity level. With the development and improvement of plant transgenic technologies, more heterologous expression experiments have verified the specific functions of homologous genes, making them increasingly effective and efficient. In the present study, PbHXK1 was transformed into tomato under the control of CaMV 35S promoter using Agrobacterium to give an insight to its function. The expression levels of PbHXK1 increased significantly in the transgenic lines from 6- to 75-fold compared with WT plants. At the same time, the HXK enzyme activity also significantly increased in transgenic tomato lines from 1.5- to 4.6-fold compared with WT plants. A high HXK enzyme activity is necessary and sufficient for limiting photosynthesis and growth, and the sugar-related regulation of plant photosynthesis and growth is entirely mediated through HXK (Dai et al., 1999). Most of the known HXK isoforms have been characterized as having dual functions, acting as sugar-phosphorylating enzymes and as mediators of signaling pathways (Jang et al., 1997; Dai et al., 1999; Moore et al., 2003; Olsson et al., 2003). The data presented by Kim et al. (2013) clearly demonstrated that NtHXK1 was not only essential for the glycolytic pathway but also for regulating starch turnover, especially during the night. OsHXK7 functions in a glycolysis-

Fig. 6. Sugar contents were measured in wild-type (WT) tomato (‘Micro-Tom’) and transgenic tomato overexpressing PbHXK1. Soluble sugar extracts prepared from tomato leaves, and young and mature fruit were used to measure sucrose (A), glucose (B) and fructose (C) contents. The three independent regenerants, #93, #95 and #98, represent PbHXK1-overexpression lines. ⁎⁎ indicates that sugar content in the transgenic regenerant was significantly greater than that in WT tomato (P ≤ 0.01); ⁎ indicates statistical significance (P ≤ 0.05); ns indicates nonsignificance (P > 0.05). The data are presented as the means ± SDs of three replicates.

further study the functional characteristics of HXK family members in pear fruit and to explore the commonalities and differences with other species' HXK members, we screened a sequence PbHXK1, which is highly expressed in pear fruit and highly homologous to AtHXK1 and 8

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Fig. 7. PbHXK1 overexpression inhibited transgenic tomato plant growth. A. The overexpression of PbHXK1 inhibited transgenic tomato plant growth. The wild-type (WT) tomato and transgenic regenerant lines #93, #95 and #98 grown in regular soil under normal growth conditions. B. The plant height was analyzed in WT tomato and transgenic regenerant lines #93, #95 and #98. C. Leaf area was analyzed in WT tomato and transgenic regenerant lines #93, #95 and #98. ⁎⁎ indicates that the plant height or leaf area of the transgenic regenerant was significantly greater than that of WT tomato (P ≤ 0.01); ⁎ indicates statistical significance (P ≤ 0.05). The data are presented as the means ± SDs of three replicates.

correlated with the sugar content but significantly positively correlated with HXK activity during fruit development in pear. Furthermore, the overexpression of PbHXK1 in tomato significantly enhanced the HXK activity and decreased the sugar content. In addition, the growth of transgenic tomato plants overexpressing PbHXK1 was inhibited, having shortened internodes and smaller leaves. Thus, in pear, PbHXK1 appeared to be a gene encoding HXK, which regulated the sugar content in fruit and affected the growth and development of plants. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.gene.2019.06.022.

dependent manner during sugar signaling under normal conditions and plays a role in sugar metabolism to enforce glycolysis-mediated fermentation in rice when O2 is deficient (Kim et al., 2016). Moore et al. (2003) also showed that HKX1 is a glucose sensor and glucose signal that interacts with additional signaling pathways controlling plant growth and development. In this study, when the PbHXK1 gene was overexpressed in tomato, the plant heights and leaf areas decreased, perhaps because the sugar content was significantly reduced and the HXK enzyme activity was also decreased in leaves. This indicated that PbHXK1 primarily fulfils a catalytic function. Thus, PbHXK1 might play a key role in regulating plant growth, which occurs mainly through the participation of HXK enzymes. However, the molecular mechanism and regulatory pathways behind PbHXK1's role in regulating plant growth and development require further research. Furthermore, the sugar (sucrose, glucose and fructose) contents were significantly decreased in the transgenic lines. Glucose was barely detected in the leaves of transgenic lines, indicating that PbHXK1 has a greater affinity for glucose. These results are similar to those of a previous study in which the functional complementation of a HXK-deficient yeast strain YSH7.4-3C (hxk1, hxk2 and glk1) suggested that HXK2 was an enzyme catalyzing hexose phosphorylation (Geng et al., 2017).

Author contributions Zhang SL designed the research. Zhao BY and Qi KJ performed the experiments. Yi XR, Chen GD, Liu X, Qi XX, and Zhao BY proofread this manuscript. Zhao BY wrote the manuscript. All authors read and approved the final manuscript. Acknowledgments This work was funded by the National Key Research and Development Program of China (2018YFD1000107), the National Natural Science Foundation of China (31830081), China Agriculture Research System, Guangxi deciduous fruit tree innovation team (nycytxgxcxtd-13-06), Science and Technology Support Program of Jiangsu Province (BE2018389), Taishan Scholar Project of Shandong Province.

5. Conclusion Here, we report the functional characterization of a glucose sensor gene (PbHXK1) from P. bretschneideri. In total, 10 HXK genes were identified from pear. A multiple sequence alignment and phylogenetic analysis showed that PbHXK1 was a Type B HXK and contained four conserved domains, phosphate 1 and 2, sugar binding and adenosine, which were specific to plant HXKs and essential for enzymatic functions. A heat map depicting the overall expression patterns of the HXK genes in different tissues was constructed using Mev software, and the PbHXKs were expressed in different pear tissues. A qRT-PCR analysis revealed that the relative expression level of PbHXK1 was negatively

Data archiving statement The authors declare that all the work described in this manuscript followed the standard Gene policy. 9

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