Elsevier

Gene

Volume 711, 30 August 2019, 143932
Gene

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

https://doi.org/10.1016/j.gene.2019.06.022Get rights and content

Highlights

  • PbHXK1 were positively correlated with HXK activity during pear fruit development

  • PbHXK1 can significantly enhance the HXK activity and decreased the sugar content in transgenic plants

  • The growth of transgenic tomato plants was inhibited, which over-expressed the PbHXK1 gene

Abstract

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.

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 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, Kelly et al., 2013, Kelly et al., 2014). Furthermore, the roles of HXK have been studied in signal transduction and hormone regulation. For example, HXK activates the signaling cascade through the HXK-interacting 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.

Section snippets

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

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

Discussion

HXK, the rate-limiting enzyme in glycolysis, controls cell survival by promoting metabolism and/or inhibiting apoptosis. HXK not only plays 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

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

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.

Data archiving statement

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

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