Isolation, characterization and functional analysis of a novel 3-hydroxy-3-methylglutaryl-coenzyme A synthase gene (GbHMGS2) from Ginkgo biloba

  • Xiangxiang Meng
  • Feng Xu
  • Qiling Song
  • Jiabao Ye
  • Yongling Liao
  • Weiwei Zhang
Original Article


Ginkgo biloba, an industrial plant of high medicinal value, is resistant to pest, disease infections, and environmental stresses. 3-Hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS) is a key mevalonic acid pathway enzyme that plays crucial roles in plant defense mechanism. In the present study, a 1407-bp HMGS gene, GbHMGS2, encoding 468 amino acid residues was cloned from G. biloba. A promoter sequence (1479-bp long) of the gene was screened from G. biloba genome data. The promoter contained the putative light-responsive (Box 4, G-Box, GT1-motif, I-box, and Sp1), cold-responsive (LTR), hormone-responsive (auxin and methyl jasmonate), and defense-related (W-box and MBS) regulatory elements. Putative GbHMGS2 protein shared high similarities to other plant HMGSs containing the conserved motif and all conserved active sites possessed by the plant HMGS protein family. Functional complementation of GbHMGS2 in an hmgs-deficient Saccharomyces cerevisiae strain confirmed the catalytic activity of GbHMGS2 protein. GbHMGS2 was preferentially expressed in the roots of G. biloba among the plant organs. The GbHMGS2 transcription was upregulated in response to cold, dark, methyl jasmonate, salicylic acid, and abscisic acid treatments, in agreement with the regulatory elements predicted in promoter region. The present work on GbHMGS2 could help ensuing research on its function, especially in the signal transduction pathways in G. biloba.


Ginkgo biloba 3-Hydroxy-3-methylglutaryl-coenzyme A synthase Functional complementation Promoter Elicitor treatments Expression pattern 



This work was funded by the National Science Foundation of China (No. 31370680).

Compliance with ethical standards

Conflict of interest

The authors state no conflict of interest.

Supplementary material

11738_2018_2650_MOESM1_ESM.docx (521 kb)
Supplementary material 1 (DOCX 520 kb)


  1. Abe H, Urao T, Ito T et al (2003) Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell 15:63–78CrossRefPubMedPubMedCentralGoogle Scholar
  2. Almasia NI, Narhirñak V, Hopp HE et al (2010) Isolation and characterization of the tissue and development-specific potato snakin-1 promoter inducible by temperature and wounding. Electron J Biotechn 13:8–9Google Scholar
  3. Aoyagi K, Beyou A, Moon K et al (1993) Isolation and characterization of cDNAs encoding wheat 3-hydroxy-3-methylglutaryl coenzyme A reductase. Plant Physiol 102:623–628CrossRefPubMedPubMedCentralGoogle Scholar
  4. Argout X, Fouet O, Wincker P et al (2008) Towards the understanding of the cocoa transcriptome: production and analysis of an exhaustive dataset of ESTs of Theobroma cacao L. generated from various tissues and under various conditions. BMC Genom 9:512CrossRefGoogle Scholar
  5. Bari R, Jones JG (2009) Role of plant hormones in plant defence responses. Plant Mol Biol 69:473–488CrossRefPubMedGoogle Scholar
  6. Campobasso N, Patel M, Wilding IE et al (2004) Staphylococcus aureus 3-hydroxy-3-methylglutaryl-CoA synthase: crystal structure and mechanism. J Biol Chem 279:44883–44888CrossRefPubMedGoogle Scholar
  7. Chang J, Ning Y, Xu F et al (2015) Research advance of 3-hydroxy-3-methylglutaryl-coenzyme a synthase in plant isoprenoid biosynthesis. J Anim Plant Sci 25:1441–1450Google Scholar
  8. Cheng H, Li L, Cheng S et al (2013a) Molecular cloning and characterization of three genes encoding dihydroflavonol-4-reductase from ginkgo biloba in anthocyanin biosynthetic pathway. PLoS ONE 8:e72017CrossRefGoogle Scholar
  9. Cheng H, Li L, Xu F et al (2013b) Expression patterns of a cinnamyl alcohol dehydrogenase gene involved in lignin biosynthesis and environmental stress in ginkgo biloba. Mol Biol Rep 40:707–721CrossRefPubMedGoogle Scholar
  10. Cheng S, Wang X, Xu F et al (2016) Cloning, expression profiling and functional analysis of CnHMGS, a gene encoding 3-hydroxy-3-methylglutaryl coenzyme a synthase from chamaemelum nobile. Molecules 21:316CrossRefPubMedGoogle Scholar
  11. Choi D, Bostock RM, Avdiushko S et al (1994) Lipid-derived signals that discriminate wound-and pathogen-responsive isoprenoid pathways in plants: methyl jasmonate and the fungal elicitor arachidonic acid induce different 3-hydroxy-3-methylglutaryl-coenzyme A reductase genes and antimicrobial isoprenoids in Solanum tuberosum L. Proc Natl Acad Sci USA 91:2329–2333CrossRefPubMedPubMedCentralGoogle Scholar
  12. Cutler SR, Rodriguez PL, Finkelstein RR et al (2010) Abscisic acid: emergence of a core signaling network. Annu Rev Plant Biol 61:651–679CrossRefPubMedGoogle Scholar
  13. Diamond BJ, Bailey MR (2013) Ginkgo biloba: indications, mechanisms, and safety. Psychiatr Clin N Am 36:73–83CrossRefGoogle Scholar
  14. Dunn MA, White AJ, Vural S et al (1998) Identification of promoter elements in a low-temperature-responsive gene (blt4.9) from barley (Hordeum vulgare L.). Plant Mol Biol 38:551–564CrossRefPubMedGoogle Scholar
  15. Edwards D, Murray JAH, Smith AG (1998) Multiple genes encoding the conserved CCAAT-Box transcription factor complex are expressed in Arabidopsis. Plant Physiol 117:1015–1022CrossRefPubMedPubMedCentralGoogle Scholar
  16. Eulgem T, Rushton PJ, Robatzek S et al (2000) The WRKY superfamily of plant transcription factors. Trends Plant Sci 5:199–206CrossRefPubMedGoogle Scholar
  17. Eyidogan F, Oz MT, Yucel M et al (2012) Signal transduction of phytohormones under abiotic stresses. In: Khan NA, Nazar R, Iqbal N, Anjum N (eds) Phytohormones and abiotic stress tolerance in plants. Springer, Berlin, pp 1–48Google Scholar
  18. Fujita Y, Fujita M, Shinozaki K et al (2011) ABA-mediated transcriptional regulation in response to osmotic stress in plants. J Plant Res 124:509–525CrossRefPubMedGoogle Scholar
  19. Gaffney T, Friedrich L, Vernooij B et al (1993) Requirement of salicylic acid for the induction of systemic acquired resistance. Science 261:754–756CrossRefPubMedGoogle Scholar
  20. Gao S, Lin J, Liu X et al (2006) Molecular cloning, characterization and functional analysis of a 2C-methyl-d-erythritol 2, 4-cyclodiphosphate synthase gene from Ginkgo biloba. J Biochem Mol Biol 39:502–510PubMedGoogle Scholar
  21. Guan R, Zhao Y, Zhang H et al (2016) Draft genome of the living fossil Ginkgo biloba. Gigascience 5:49CrossRefPubMedPubMedCentralGoogle Scholar
  22. Hemmerlin A, Hoeffler JF, Meyer O et al (2003) Cross-talk between the cytosolic mevalonate and the plastidial alonate and the plastidial methylerythritol phosphate pathways in tobacco bright yellow-2 cells. J Biol Chem 278:26666–26676CrossRefPubMedGoogle Scholar
  23. Heyworth CJ, Iason GR, Temperton V et al (1998) The effect of elevated CO2 concentration and nutrient supply on carbon-based plant secondary metabolites in Pinus sylvestris L. Oecologia 115:344–350CrossRefPubMedGoogle Scholar
  24. Joshi CP (1987) An inspection of the domain between putative TATA box and translation start site in 79 plant genes. Nucleic Acids Res 15:6643–6653CrossRefPubMedPubMedCentralGoogle Scholar
  25. Kai G, Miao Z, Zhang L et al (2006) Molecular cloning and expression analyses of a new gene encoding 3-hydroxy-3-methylglutaryl-coa synthase from Taxus × media. Biol Plantarum 50:359–366CrossRefGoogle Scholar
  26. Kai GY, Li SS, Wang W et al (2013) Molecular cloning and expression analysis of a gene encoding 3-hydroxy-3-methylglutaryl-coa synthase from Camptotheca acuminata. Russ J Plant Physl 60:131–138CrossRefGoogle Scholar
  27. Kang MK, Nargis S, Kim SM et al (2013) Distinct expression patterns of two Ginkgo biloba, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase/isopentenyl diphosphate synthase (HDR/IDS) promoters in Arabidopsis model. Plant Physiol Bioch 62:47–53CrossRefGoogle Scholar
  28. Kawoosa T, Singh H, Kumar A et al (2010) Light and temperature regulated terpene biosynthesis: hepatoprotective monoterpene picroside accumulation in Picrorhiza kurrooa. Funct Integr Genomic 10:393–404CrossRefGoogle Scholar
  29. Kim SM, Kim SU (2010) Characterization of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS) gene from Ginkgo biloba. Mol Biol Rep 37:973–979CrossRefPubMedGoogle Scholar
  30. Kim SM, Kuzuyama T, Chang YJ et al (2006) Identification of class 2 1-deoxy-d-xylulose 5-phosphate synthase and 1-deoxy-d-xylulose 5-phosphate reductoisomerase genes from Ginkgo biloba and their transcription in embryo culture with respect to ginkgolide biosynthesis. Planta Med 72:234–240CrossRefPubMedGoogle Scholar
  31. Kim SM, Kim YB, Kuzuyama T et al (2008) Tw copies of 4-(cytidine 5-diphospho)-2-C-methyl-d-erythritol kinase (CMK) gene in Ginkgo biloba: molecular cloning and functional characterization. Planta 228:941–950CrossRefPubMedGoogle Scholar
  32. Kim JH, Lee KI, Chang YJ et al (2012) Developmental pattern of Ginkgo biloba levopimaradiene synthase (GbLPS) as probed by promoter analysis in Arabidopsis thaliana. Plant Cell Rep 31:1119–1127CrossRefPubMedGoogle Scholar
  33. Korth KL, Jaggard DAW, Dixon RA (2000) Developmental and lightregulated post-translational control of 3-hydroxy-3-methylglutaryl-CoA reductase levels in potato. Plant J 23:507–516CrossRefPubMedGoogle Scholar
  34. Liao HJ, Zheng YF, Li HY et al (2011) Two new ginkgolides from the leaves of Ginkgo biloba. Planta Med 77:1818–1821CrossRefPubMedGoogle Scholar
  35. Liao YL, Xu F, Huang XH et al (2015a) Characterization and transcriptional profiling of Ginkgo biloba mevalonate diphosphate decarboxylase gene (GbMVD) promoter towards light and exogenous hormone treatments. Plant Mol Biol Rep 34:566–581CrossRefGoogle Scholar
  36. Liao YL, Xu F, Huang XH et al (2015b) Promoter analysis and transcriptional profiling of Ginkgo biloba 3-hydroxy-3-methylglutaryl coenzyme a reductase (GbHMGR) gene in abiotic stress responses. Not Bot Horti Agrobo 43:25–34Google Scholar
  37. Lichtenthaler HK, Rohmer M, Schwender J (1997) Two independent biochemical pathways for isopentenyl diphosphate and isoprenoid biosynthesis in higher plants. Physiol Plantarum 101:643–652CrossRefGoogle Scholar
  38. Liu X, Jiang J, Shao J et al (2010) Gene transcription profiling of Fusarium graminearum treated with an azole fungicide tebuconazole. Appl Microbiol Biot 85:1105–1114CrossRefGoogle Scholar
  39. Liu YJ, Zhao YJ, Zhang M et al (2014) Cloning and characterisation of the gene encoding 3-hydroxy-3-methylglutaryl-CoA synthase in Tripterygium wilfordii. Molecules 19:19696–19707CrossRefPubMedGoogle Scholar
  40. Lu J, Wu WS, Cao SW et al (2008) Molecular cloning and characterization of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase gene from Ginkgo biloba. Mol Biol Rep 35:413–420CrossRefPubMedGoogle Scholar
  41. Mantyla E, Lang V, Palva ET (1995) Role of abscisic acid in drought-induced freezing tolerance, cold acclimation, and accumulation of LT178 and RAB18 proteins in Arabidopsis thaliana. Plant Physiol 107:141–148CrossRefPubMedPubMedCentralGoogle Scholar
  42. Martin DM, Gershenzon J, Bohlmann J (2003) Induction of volatile terpene biosynthesis and diurnal emission by methyl jasmonate in foliage of Norway spruce. Plant Physiol 132:1586–1599CrossRefPubMedPubMedCentralGoogle Scholar
  43. Meng XX, Song QL, Ye JB et al (2017) Characterization, function, and transcriptional profiling analysis of 3-hydroxy-3-methylglutaryl-CoA synthase gene (GbHMGS1) towards stresses and exogenous hormone treatments in Ginkgo biloba. Molecules 22:1706CrossRefGoogle Scholar
  44. Menkens AE, Al E (1995) The g-box: a ubiquitous regulatory DNA element in plants bound by the GBF family of bZIP proteins. Trends Biochem Sci 20:506–510CrossRefPubMedGoogle Scholar
  45. Misra I, Wang CZ, Miziorko HM (2003) The influence of conserved aromatic residues in 3-hydroxy-3-methylglutaryl-CoA synthase. J Biol Chem 278:26443–26449CrossRefPubMedGoogle Scholar
  46. Miziorko HM (2011) Enzymes of the mevalonate pathway of isoprenoid biosynthesis. Arch Biochem Biophys 505:131–143CrossRefPubMedPubMedCentralGoogle Scholar
  47. Mohanta TK, Tamboli Y, Zubaidha PK (2014) Phytochemical and medicinal importance of Ginkgo biloba l. Nat Prod Res 28:746–752CrossRefPubMedGoogle Scholar
  48. Mongkolsiriwatana C, Pongtongkam P, Peyachoknagul S (2009) In silico promoter analysis of photoperiod-responsive genes identified by DNA microarray in rice (Oryza sativa L.). Kasetsart J (Nat Sci) 43:544–555Google Scholar
  49. Mosaleeyanon K, Zobayed SMA, Afreen F et al (2005) Relationships between net photosynthetic rate and secondary metabolite contents in St. John’s wort. Plant Sci 169:523–531CrossRefGoogle Scholar
  50. Parveen I, Wang M, Zhao JP et al (2015) Investigating sesquiterpene biosynthesis in Ginkgo biloba: molecular cloning and functional characterization of (E,E)-farnesol and α-bisabolene synthases. Plant Mol Biol 89:451–462CrossRefPubMedGoogle Scholar
  51. Pu GB, Ma DM, Chen JL et al (2009) Salicylic acid activates artemisinin biosynthesis in Artemisia annua L. Plant Cell Rep 28:1127–1135CrossRefPubMedGoogle Scholar
  52. Ren A, Ouyang X, Shi L et al (2013) Molecular characterization and expression analysis of GlHMGS, a gene encoding hydroxymethylglutaryl-CoA synthase from Ganoderma lucidum (Ling-zhi) in ganoderic acid biosynthesis pathway. World J Microb Biot 29:523–531CrossRefGoogle Scholar
  53. Rouster J, Leah R, Mundy J et al (1997) Identification of a methyl jasmonate-responsive region in the promoter of a lipoxygenase 1 gene expressed in barley grain. Plant J 11:513–523CrossRefPubMedGoogle Scholar
  54. Royer DL, Hickey LJ, Wing SL (2003) Ecological conservatism in the “living fossil” Ginkgo. Paleobiology 29:84–104CrossRefGoogle Scholar
  55. Schilmiller AL, Schauvinhold I, Larson M (2009) Monoterpenes in the glandular trichomes of tomato are synthesized from a neryl diphosphate precursor rather than geranyl diphosphate. Proc Natl Acad Sci USA 106:10865–10870CrossRefPubMedPubMedCentralGoogle Scholar
  56. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101–1108CrossRefPubMedGoogle Scholar
  57. Schnable PS, Ware D, Fulton RS et al (2009) The B73 maize genome: complexity, diversity, and dynamics. Science 326:1112–1115CrossRefPubMedGoogle Scholar
  58. Schwarz M, Arigoni D (1999) Ginkgolide biosynthesis. Compr Nat Prod Chem 2:367–399CrossRefGoogle Scholar
  59. Schwede T, Kopp J, Guex N et al (2003) SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res 31:3381–3385CrossRefPubMedPubMedCentralGoogle Scholar
  60. Shen G, Pang Y, Wu W et al (2005) Molecular cloning, characterization and expression of a novel Asr gene from Ginkgo biloba. Plant Physiol Bioch 43:836–843CrossRefGoogle Scholar
  61. Shen GA, Pang YZ, Wu WS et al (2006) Cloning and characterization of a root-specific expressing gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from Ginkgo biloba. Mol Biol Rep 33:117–127CrossRefPubMedGoogle Scholar
  62. Skriver K, Mundy J (1990) Gene expression in response to abscisic acid and osmotic stress. Plant Cell 2:503–512CrossRefPubMedPubMedCentralGoogle Scholar
  63. Smale ST, Kadonaga JT (2002) The RNA polymerase II core promoter. Annu Rev Biochem 72:449–479CrossRefGoogle Scholar
  64. Soitamo AJ, Piippo M, Allahverdiyeva Y et al (2008) Light has a specific role in modulating Arabidopsis gene expression at low temperature. BMC Plant Biol 8:13CrossRefPubMedPubMedCentralGoogle Scholar
  65. Suvachittanont W, Wititsuwannakul R (1995) 3-Hydroxy 3-methylglutaryl-CoA synthase in Hevea brasiliensis. Phytochemistry 40:757–761CrossRefGoogle Scholar
  66. Tao TT, Chen QW, Meng XX et al (2016) Molecular cloning, characterization, and functional analysis of a gene encoding 3-hydroxy-3-methylglutaryl-coenzyme a synthase from matricaria chamomilla. Genes Genom 38:1–9CrossRefGoogle Scholar
  67. Theisen MJ, Misra I, Saadat D et al (2004) 3-Hydroxy-3-methylglutaryl-CoA synthase intermediate complex observed in “real-time”. Proc Natl Acad Sci USA 101:16442–16447CrossRefPubMedPubMedCentralGoogle Scholar
  68. Tholl D (2006) Terpene synthases and the regulation, diversity and biological roles of terpene metabolism. Curr Opin Plant Biol 9:297–304CrossRefPubMedGoogle Scholar
  69. Wang W, Chen M, Yang CX et al (2009) The geranylgeranyl pyrophosphate synthase gene from Ginkgo biloba: cloning, characterization and functional identification. Afr J Biotechnol 8:1203–1210Google Scholar
  70. Wang H, Nagegowda DA, Rawat R et al (2012) Overexpression of Brassica juncea wild type and mutant HMG-CoA synthase 1 in Arabidopsis up-regulates genes in sterol biosynthesis and enhances sterol production and stress tolerance. Plant Biotechnol J 10:31–42CrossRefPubMedGoogle Scholar
  71. Wu CY, Suzuki A, Washida H et al (1998) The GCN4 motif in a rice glutelin gene is essential for endosperm-specific gene expression and is activated by Opaque-2 in transgenic rice plants. Plant J 14:673–684CrossRefPubMedGoogle Scholar
  72. Xu F (2008) Cloning and expression of GbPAL and GbANS genes in Ginkgo biloba and the effect of ALA on flavonoid content. Dissertation, Shandong Agricultural UniversityGoogle Scholar
  73. Xu F, Cai R, Cheng S et al (2008a) Molecular cloning, characterization and expression of phenylalanine ammonia-lyase gene from Ginkgo biloba. Afr J Biotechnol 7:721–729Google Scholar
  74. Xu F, Cheng H, Cai R et al (2008b) Molecular cloning and function analysis of an anthocyanidin synthase gene from Ginkgo biloba, and its expression in abiotic stress responses. Mol Cells 26:536–547PubMedGoogle Scholar
  75. Xu F, Li L, Zhang W et al (2012) Isolation, characterization, and function analysis of a flavonol synthase gene from Ginkgo biloba. Mol Biol Rep 39:2285–2296CrossRefPubMedGoogle Scholar
  76. Xu F, Huang XH, Li LL et al (2013) Molecular cloning and characterization of GbDXS and GbGGPPS gene promoters, from ginkgo biloba. Genet Mol Res 12:293–301CrossRefPubMedGoogle Scholar
  77. Zhang L, Yan X, Wang J et al (2011) Molecular cloning and expression analysis of a new putative gene encoding 3-hydroxy-3-methylglutaryl-CoA synthase from Salvia miltiorrhiza. Acta Physiol Plant 33:953–961CrossRefGoogle Scholar
  78. Zhang CL, Chen P, Zhong YM et al (2012) Full length cDNA cloning and expression analysis of UFGT gene from Ginkgo biloba. Acta Hortic Sinica 60:131–138Google Scholar
  79. Zhang LF, Li WF, Han SY et al (2013) cDNA cloning, genomic organization and expression analysis during somatic embryogenesis of the translationally controlled tumor protein (TCTP) gene from Japanese larch (Larix leptolepis). Gene 529:150–158CrossRefPubMedGoogle Scholar
  80. Zhou DX (1999) Regulatory mechanism of plant gene transcription by GT-elements and GT-factors. Trends Plant Sci 4:210–216CrossRefPubMedGoogle Scholar

Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2018

Authors and Affiliations

  1. 1.College of Horticulture and GardeningYangtze UniversityJingzhouChina

Personalised recommendations