, Volume 248, Issue 1, pp 171–182 | Cite as

Genome-wide identification of hexokinase gene family in Brassica napus: structure, phylogenetic analysis, expression, and functional characterization

  • Jingxue Wang
  • Xiaomin Wang
  • Siyu Geng
  • Sanjay K. Singh
  • Yaohui Wang
  • Sitakanta Pattanaik
  • Ling Yuan
Original Article


Main conclusion

Genome-wide identification, expression analysis, and functional characterization of previously uncharacterized hexokinase family of oil crop, Brassica napus, underscore the importance of this gene family in plant growth and development.

In plants, the multi-gene family of dual-function hexokinases (HXKs) plays important roles in sugar metabolism and sensing that affect growth and development. Rapeseed (Brassica napus L.) is an important oil crop; however, little is known about the B. napus HXK gene family. We identified 19 putative HXKs in B. napus genome. B. rapa and B. oleracea, the two diploid progenitors of B. napus, contributed almost equally to the BnHXK genes. Phylogenetic analysis divided the 19 BnHXKs into four groups. The exon–intron structures of BnHXKs share high similarity to those of HXKs in Arabidopsis and rice. The group III and IV BnHXKs are highly expressed in roots, whereas group I members preferentially express in leaves. Analysis of seed transcriptomes at different developmental stages showed that most of group I and IV HXKs are highly expressed 2-weeks after pollination (2WAP), compared to 4WAP for group III. BnHKXs are differentially expressed in susceptible and tolerant B. napus cultivars after fungal infection, suggesting the possible involvement in defense response. We generated rapeseed RNAi lines for BnHXK9, a member of relatively less characterized group IV, by pollen-mediated gene transformation. The seedlings of BnHXK9-RNAi lines showed delayed growth compared to the wild type. The RNAi plants were dwarf with curly leaves, suggesting the involvement of BnHXK9 in plant development. Collectively, our findings provides a comprehensive account of BnHXK gene family in an important crop and a starting point for further elucidation of their roles in sugar metabolism and sensing, as well as plant growth and development.


Brassica napus L. Hexokinase Transcriptome analysis RNAi 



Fatty acid








Murashige and Skoog



The authors thank Chunfeng Du and Shuanshi Xian, Cotton Research Institute of Shanxi Academy of Agricultural Sciences, for providing the plant material. This project is supported by Shanxi Provincial Science and Technology Research Grant (20140311010-4), China. The work is supported in part by the National Science Foundation under Cooperative Agreement no. 1355438 to L.Y.

Compliance with ethical standards

Conflict of interest

The authors declare no conflicts of interest.

Supplementary material

425_2018_2888_MOESM1_ESM.pdf (1.2 mb)
Supplementary material 1 (PDF 1266 kb)


  1. Aders K, Larsson B, von Heijne G, Sonnahammer ELL (2001) Predictingtransmembrane protein topology with a hidden markov model: application to complete genomes. J Mol Biol 305:567–580CrossRefGoogle Scholar
  2. Aguilera-Alvarado GP, Sanchez-Nieto S (2017) Plant hexokinases are multifaceted proteins. Plant Cell Physiol 58:1151–1160CrossRefPubMedGoogle Scholar
  3. Ahuatzi D, Herrero P, de la Cera T, Moreno F (2004) The glucose-regulated nuclear localization of hexokinase 2 in Saccharomyces cerevisiae is Mig1-dependent. J Biol Chem 279:14440–14446CrossRefPubMedGoogle Scholar
  4. Allen GC, Flores-Vergara MA, Krasynanski S, Kumar S, Thompson WF (2006) A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nat Protoc 1:2320–2325CrossRefPubMedGoogle Scholar
  5. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL (2009) BLAST+: architecture and applications. BMC Bioinform 10:421CrossRefGoogle Scholar
  6. Chalhoub B, Denoeud F, Liu S, Parkin IA, Tang H, Wang X, Chiquet J, Belcram H, Tong C, Samans B, Correa M, Da Silva C, Just J, Falentin C, Koh CS, Le Clainche I, Bernard M, Bento P, Noel B, Labadie K, Alberti A, Charles M, Arnaud D, Guo H, Daviaud C, Alamery S, Jabbari K, Zhao M, Edger PP, Chelaifa H, Tack D, Lassalle G, Mestiri I, Schnel N, Le Paslier MC, Fan G, Renault V, Bayer PE, Golicz AA, Manoli S, Lee TH, Thi VH, Chalabi S, Hu Q, Fan C, Tollenaere R, Lu Y, Battail C, Shen J, Sidebottom CH, Wang X, Canaguier A, Chauveau A, Berard A, Deniot G, Guan M, Liu Z, Sun F, Lim YP, Lyons E, Town CD, Bancroft I, Wang X, Meng J, Ma J, Pires JC, King GJ, Brunel D, Delourme R, Renard M, Aury JM, Adams KL, Batley J, Snowdon RJ, Tost J, Edwards D, Zhou Y, Hua W, Sharpe AG, Paterson AH, Guan C, Wincker P (2014) Plant genetics. Early allopolyploid evolution in the post-Neolithic Brassica napus oilseed genome. Science 345:950–953CrossRefPubMedGoogle Scholar
  7. Cheng W, Zhang H, Zhou X, Liu H, Liu Y, Li J, Han S, Wang Y (2011) Subcellular localization of rice hexokinase (OsHXK) family members in the mesophyll protoplasts of tobacco. Biol Plant 55:173–177CrossRefGoogle Scholar
  8. Cho JI, Ryoo N, Ko S, Lee SK, Lee J, Jung KH, Lee YH, Bhoo SH, Winderickx J, An G, Hahn TR, Jeon JS (2006a) Structure, expression, and functional analysis of the hexokinase gene family in rice (Oryza sativa L.). Planta 224:598–611CrossRefPubMedGoogle Scholar
  9. Cho YH, Yoo SD, Sheen J (2006b) Regulatory functions of nuclear hexokinase1 complex in glucose signaling. Cell 127:579–589CrossRefPubMedGoogle Scholar
  10. Claeyssen E, Rivoal J (2007) Isozymes of plant hexokinase: occurrence, properties and functions. Phytochemistry 68:709–731CrossRefPubMedGoogle Scholar
  11. da Silva WS, Rezende GL, Galina A (2001) Subcellular distribution and kinetic properties of cytosolic and non-cytosolic hexokinases in maize seedling roots: implications for hexose phosphorylation. J Exp Bot 52:1191–1201CrossRefPubMedGoogle Scholar
  12. Dai N, Schaffer A, Petreikov M, Shahak Y, Giller Y, Ratner K, Levine A, Granot D (1999) Overexpression of Arabidopsis hexokinase in tomato plants inhibits growth, reduces photosynthesis, and induces rapid senescence. Plant Cell 11:1253–1266CrossRefPubMedPubMedCentralGoogle Scholar
  13. Emanuelsson O, Brunak S, von Heijne G, Nielsen H (2007) Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc 2:953–971CrossRefPubMedGoogle Scholar
  14. Finn RD, Coggill P, Eberhardt RY, Eddy SR, Mistry J, Mitchell AL, Potter SC, Punta M, Qureshi M, Sangrador-Vegas A, Salazar GA, Tate J, Bateman A (2016) The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res 44:D279–D285CrossRefPubMedGoogle Scholar
  15. Geng MT, Yao Y, Wang YL, Wu XH, Sun C, Li RM, Fu SP, Duan RJ, Liu J, Hu XW, Guo JC (2017) Structure, expression, and functional analysis of the hexokinase gene family in cassava. Int J Mol Sci 18:E1041CrossRefPubMedGoogle Scholar
  16. Girard IJ, Tong C, Becker MG, Mao X, Huang J, de Kievit T, Fernando WGD, Liu S, Belmonte MF (2017) RNA sequencing of Brassica napus reveals cellular redox control of Sclerotinia infection. J Exp Bot 68:5079–5091CrossRefPubMedPubMedCentralGoogle Scholar
  17. Granot D, Kelly G, Stein O, David-Schwartz R (2014) Substantial roles of hexokinase and fructokinase in the effects of sugars on plant physiology and development. J Exp Bot 65:809–819CrossRefPubMedGoogle Scholar
  18. Gu Z, Eils R, Schlesner M (2016) Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics 32:2847–2849CrossRefPubMedGoogle Scholar
  19. Herbers K, Meuwly P, Frommer WB, Metraux JP, Sonnewald U (1996) Systemic acquired resistance mediated by the ectopic expression of invertase: possible hexose sensing in the secretory pathway. Plant Cell 8:793–803CrossRefPubMedPubMedCentralGoogle Scholar
  20. Hill LM, Morley-Smith ER, Rawsthorne S (2003) Metabolism of sugars in the endosperm of developing seeds of oilseed rape. Plant Physiol 131:228–236CrossRefPubMedPubMedCentralGoogle Scholar
  21. Hu B, Jin J, Guo AY, Zhang H, Luo J, Gao G (2015) GSDS 2.0: an upgraded gene feature visualization server. Bioinformatics 31:1296–1297CrossRefPubMedGoogle Scholar
  22. Jang JC, Leon P, Zhou L, Sheen J (1997) Hexokinase as a sugar sensor in higher plants. Plant Cell 9:5–19CrossRefPubMedPubMedCentralGoogle Scholar
  23. Kandel-Kfir M, Damari-Weissler H, German MA, Gidoni D, Mett A, Belausov E, Petreikov M, Adir N, Granot D (2006) Two newly identified membrane-associated and plastidic tomato HXKs: characteristics, predicted structure and intracellular localization. Planta 224:1341–1352CrossRefPubMedGoogle Scholar
  24. Karve A, Moore BD (2009) Function of Arabidopsis hexokinase-like1 as a negative regulator of plant growth. J Exp Bot 60:4137–4149CrossRefPubMedPubMedCentralGoogle Scholar
  25. Karve A, Rauh BL, Xia X, Kandasamy M, Meagher RB, Sheen J, Moore BD (2008) Expression and evolutionary features of the hexokinase gene family in Arabidopsis. Planta 228:411–425CrossRefPubMedPubMedCentralGoogle Scholar
  26. Karve R, Lauria M, Virnig A, Xia X, Rauh BL, Moore B (2010) Evolutionary lineages and functional diversification of plant hexokinases. Mol Plant 3:334–346CrossRefPubMedGoogle Scholar
  27. Kelly G, Moshelion M, David-Schwartz R, Halperin O, Wallach R, Attia Z, Belausov E, Granot D (2013) Hexokinase mediates stomatal closure. Plant J 75:977–988CrossRefPubMedGoogle Scholar
  28. Kersey PJ, Allen JE, Allot A, Barba M, Boddu S, Bolt BJ, Carvalho-Silva D, Christensen M, Davis P, Grabmueller C, Kumar N, Liu Z, Maurel T, Moore B, McDowall MD, Maheswari U, Naamati G, Newman V, Ong CK, Paulini M, Pedro H, Perry E, Russell M, Sparrow H, Tapanari E, Taylor K, Vullo A, Williams G, Zadissia A, Olson A, Stein J, Wei S, Tello-Ruiz M, Ware D, Luciani A, Potter S, Finn RD, Urban M, Hammond-Kosack KE, Bolser DM, De Silva N, Howe KL, Langridge N, Maslen G, Staines DM, Yates A (2018) Ensembl Genomes 2018: an integrated omics infrastructure for non-vertebrate species. Nucleic Acids Res 46:D802–D808CrossRefPubMedGoogle Scholar
  29. Kim M, Lim JH, Ahn CS, Park K, Kim GT, Kim WT, Pai HS (2006) Mitochondria-associated hexokinases play a role in the control of programmed cell death in Nicotiana benthamiana. Plant Cell 18:2341–2355CrossRefPubMedPubMedCentralGoogle Scholar
  30. Kim YM, Heinzel N, Giese JO, Koeber J, Melzer M, Rutten T, Von Wiren N, Sonnewald U, Hajirezaei MR (2013) A dual role of tobacco hexokinase 1 in primary metabolism and sugar sensing. Plant Cell Environ 36:1311–1327CrossRefPubMedGoogle Scholar
  31. Kodama Y, Shumway M, Leinonen R, International Nucleotide Sequence Database Consortium (2012) The Sequence Read Archive: explosive growth of sequencing data. Nucleic Acids Res 40:D54–D56CrossRefPubMedGoogle Scholar
  32. Kuser PR, Krauchenco S, Antunes OA, Polikarpov I (2000) The high resolution crystal structure of yeast hexokinase PII with the correct primary sequence provides new insights into its mechanism of action. J Biol Chem 275:20814–20821CrossRefPubMedGoogle Scholar
  33. Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359CrossRefPubMedPubMedCentralGoogle Scholar
  34. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948CrossRefPubMedGoogle Scholar
  35. Le BH, Cheng C, Bui AQ, Wagmaister JA, Henry KF, Pelletier J, Kwong L, Belmonte M, Kirkbride R, Horvath S, Drews GN, Fischer RL, Okamuro JK, Harada JJ, Goldberg RB (2010) Global analysis of gene activity during Arabidopsis seed development and identification of seed-specific transcription factors. Proc Natl Acad Sci USA 107:8063–8070CrossRefPubMedPubMedCentralGoogle Scholar
  36. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25:402–408CrossRefPubMedPubMedCentralGoogle Scholar
  37. Lugassi N, Kelly G, Fidel L, Yaniv Y, Attia Z, Levi A, Alchanatis V, Moshelion M, Raveh E, Carmi N, Granot D (2015) Expression of Arabidopsis hexokinase in citrus guard cells controls stomatal aperture and reduces transpiration. Front Plant Sci 6:1114CrossRefPubMedPubMedCentralGoogle Scholar
  38. Mistry J, Finn RD, Eddy SR, Bateman A, Punta M (2013) Challenges in homology search: HMMER3 and convergent evolution of coiled-coil regions. Nucleic Acids Res 41:e121CrossRefPubMedPubMedCentralGoogle Scholar
  39. Moore B, Zhou L, Rolland F, Hall Q, Cheng WH, Liu YX, Hwang I, Jones T, Sheen J (2003) Role of the Arabidopsis glucose sensor HXK1 in nutrient, light, and hormonal signaling. Science 300:332–336CrossRefPubMedGoogle Scholar
  40. Nguyen-Quoc B, Foyer CH (2001) A role for ‘futile cycles’ involving invertase and sucrose synthase in sucrose metabolism of tomato fruit. J Exp Bot 52:881–889CrossRefPubMedGoogle Scholar
  41. Niu Y, Wu GZ, Ye R, Lin WH, Shi QM, Xue LJ, Xu XD, Li Y, Du YG, Xue HW (2009) Global analysis of gene expression profiles in Brassica napus developing seeds reveals a conserved lipid metabolism regulation with Arabidopsis thaliana. Mol Plant 2:1107–1122CrossRefPubMedGoogle Scholar
  42. Olsson T, Thelander M, Ronne H (2003) A novel type of chloroplast stromal hexokinase is the major glucose-phosphorylating enzyme in the moss Physcomitrella patens. J Biol Chem 278:44439–44447CrossRefPubMedGoogle Scholar
  43. Pattanaik S, Kong Q, Zaitlin D, Werkman JR, Xie CH, Patra B, Yuan L (2010) Isolation and functional characterization of a floral tissue-specific R2R3 MYB regulator from tobacco. Planta 231:1061–1076CrossRefPubMedGoogle Scholar
  44. Pelaez R, Herrero P, Moreno F (2010) Functional domains of yeast hexokinase 2. Biochem J 432:181–190CrossRefPubMedPubMedCentralGoogle Scholar
  45. Rojas CM, Senthil-Kumar M, Tzin V, Mysore KS (2014) Regulation of primary plant metabolism during plant–pathogen interactions and its contribution to plant defense. Front Plant Sci 5:17CrossRefPubMedPubMedCentralGoogle Scholar
  46. Rolland F, Moore B, Sheen J (2002) Sugar sensing and signaling in plants. Plant Cell 14(Suppl):S185–S205CrossRefPubMedPubMedCentralGoogle Scholar
  47. Schmieder R, Edwards R (2011) Quality control and preprocessing of metagenomic datasets. Bioinformatics 27:863–864CrossRefPubMedPubMedCentralGoogle Scholar
  48. Sheen J, Zhou L, Jang JC (1999) Sugars as signaling molecules. Curr Opin Plant Biol 2:410–418CrossRefPubMedGoogle Scholar
  49. Singh SK, Wu Y, Ghosh JS, Pattanaik S, Fisher C, Wang Y, Lawson D, Yuan L (2015) RNA-sequencing reveals global transcriptomic changes in Nicotiana tabacum responding to topping and treatment of axillary-shoot control chemicals. Sci Rep 5:18148CrossRefPubMedPubMedCentralGoogle Scholar
  50. Tadege M, Bucher M, Stahli W, Suter M, Dupuis I, Kuhlemeier C (1998) Activation of plant defense responses and sugar efflux by expression of pyruvate decarboxylase in potato leaves. Plant J 16:661–671CrossRefGoogle Scholar
  51. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729CrossRefPubMedPubMedCentralGoogle Scholar
  52. Troncoso-Ponce MA, Rivoal J, Dorion S, Moisan MC, Garces R, Martinez-Force E (2011) Cloning, biochemical characterization and expression of a sunflower (Helianthus annuus L.) hexokinase associated with seed storage compounds accumulation. J Plant Physiol 168:299–308CrossRefPubMedGoogle Scholar
  53. Wang JX, Sun Y, Cui GM, Hu JJ (2001) Transgenic maize plants obtained by pollen-mediated transformation. Acta Bot Sin 43:275–279Google Scholar
  54. Wang J, Singh SK, Du C, Li C, Fan J, Pattanaik S, Yuan L (2016) Comparative transcriptomic analysis of two Brassica napus near-isogenic lines reveals a network of genes that influences seed oil accumulation. Front Plant Sci 7:1498PubMedPubMedCentralGoogle Scholar
  55. Wiese A, Groner F, Sonnewald U, Deppner H, Lerchl J, Hebbeker U, Flugge U, Weber A (1999) Spinach hexokinase I is located in the outer envelope membrane of plastids. FEBS Lett 461:13–18CrossRefPubMedGoogle Scholar
  56. Xiao W, Sheen J, Jang JC (2000) The role of hexokinase in plant sugar signal transduction and growth and development. Plant Mol Biol 44:451–461CrossRefPubMedGoogle Scholar
  57. Xu FQ, Li XR, Ruan YL (2008) RNAi-mediated suppression of hexokinase gene OsHXK10 in rice leads to non-dehiscent anther and reduction of pollen germination. Plant Sci 175:674–684CrossRefGoogle Scholar
  58. Zhang ZW, Yuan S, Xu F, Yang H, Zhang NH, Cheng J, Lin HH (2010) The plastid hexokinase pHXK: a node of convergence for sugar and plastid signals in Arabidopsis. FEBS Lett 584:3573–3579CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018
corrected ​publication April 2018

Authors and Affiliations

  • Jingxue Wang
    • 1
  • Xiaomin Wang
    • 1
  • Siyu Geng
    • 1
  • Sanjay K. Singh
    • 2
  • Yaohui Wang
    • 1
  • Sitakanta Pattanaik
    • 2
  • Ling Yuan
    • 1
    • 2
  1. 1.School of Life SciencesShanxi UniversityTaiyuanChina
  2. 2.Department of Plant and Soil Sciences, Kentucky Tobacco Research and Development CenterUniversity of KentuckyLexingtonUSA

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