Characterization and expression patterns of a cinnamate-4-hydroxylase gene involved in lignin biosynthesis and in response to various stresses and hormonal treatments in Ginkgo biloba

  • Shuiyuan Cheng
  • Jiaping Yan
  • Xiangxiang Meng
  • Weiwei Zhang
  • Yongling Liao
  • Jiabao Ye
  • Feng XuEmail author
Original Article


Plant cell walls primarily comprise lignin, which performs functions of mechanical support, water transport, and stress responses. Lignin biosynthesis pathway proceeds through metabolic grid featuring complexity and diversity in enzymatic reaction. Cinnamate-4-hydroxylase (C4H, EC is the gene encoding enzyme that catalyzes the second step of phenylpropanoid pathway responsible for biosynthesis of lignin. A full-length cDNA of C4H (designated as GbC4H), which spanned 1816-bp with a 1518-bp open reading frame encoding a 505-amino-acid protein, was cloned from Ginkgo biloba. A GbC4H genomic DNA fragment, spanning 3249-bp, was cloned and found to contain two exons and one intron. GbC4H protein showed high similarities with other plant C4Hs to include conserved domains of cytochrome P450 family. GT-1, W-box, and Myb/Myc recognition sites involved in stress response were detected in a 1265-bp upstream promoter region of GbC4H. Phylogenetic analysis suggested the common evolutionary ancestor shared by plant C4Hs including the gymnosperm enzyme. pET-28a-GbC4H plasmid was constructed and expressed in Escherichia coli strain BL21. Enzymatic assay revealed that recombinant GbC4H protein catalyzes conversion of trans-cinnamic acid to p-coumaric acid. Expression analyses in different organs showed high expression of GbC4H in stems and roots, whereas low expressions was found in fruits, carpopodium, and petioles. Further analysis indicated linear correlation of lignin contents with transcript levels of GbC4H among different tissues. GbC4H transcription was increased by treatments with UV-B, cold, salicylic acid, and abscisic acid, indicating the possible role of GbC4H in response to stresses and hormonal signal. Understanding of GbC4H function could benefit molecular breeding and reinforcement of defense mechanisms in Ginkgo.


Abiotic stresses Cinnamate-4-hydroxylase Ginkgo biloba Hormone Lignin Phenylpropanoid pathway 



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

Supplementary material

11738_2017_2585_MOESM1_ESM.docx (29 kb)
Supplementary material 1 (DOCX 28 kb)


  1. Achnine L, Blancaflor EB, Rasmussen S et al (2004) Colocalization of l-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase for metabolic channeling in phenylpropanoid biosynthesis. Plant Cell 16:3098–3109PubMedPubMedCentralGoogle Scholar
  2. Anterola AM, Lewis NG (2002) Trends in lignin modification: a comprehensive analysis of the effects of genetic manipulations/mutations on lignification and vascular integrity. Phytochemistry 61:221–294PubMedGoogle Scholar
  3. Bhuiyan NH, Selvaraj G, Wei Y et al (2009) Role of lignification in plant defense. Plant Signal Behav 4:158–159PubMedPubMedCentralGoogle Scholar
  4. Boerjan W, Ralph J, Baucher M (2003) Lignin biosynthesis. Annu Rev Plant Biol 54:519–546Google Scholar
  5. Boudet AM, Kajita S, Grima-Pettenati J et al (2003) Lignins and lignocellulosics: a better control of synthesis for new and improved uses. Trends Plant Sci 8:576–581PubMedGoogle Scholar
  6. Buchel AS, Brederode F, Bol JF et al (1999) Mutation of GT-1 binding sites in the Pr-1A promoter influences the level of inducible gene expression in vivo. Plant Mol Biol 40:387–396PubMedGoogle Scholar
  7. Chapple C (1998) Molecular-genetic analysis of plant cytochrome P450-dependent monooxygenases. Annu Rev Plant Biol 49:311–343Google Scholar
  8. Chen AH, Chai YR, Li JN et al (2007) Molecular cloning of two genes encoding cinnamate 4-hydroxylase (C4H) from oilseed rape (Brassica napus). J Biochem Mol Biol 40:247–260PubMedGoogle Scholar
  9. Cheng H, Li LL, Xu F et al (2013a) Expression patterns of an isoflavone reductase-like gene and its possible roles in secondary metabolism in Ginkgo biloba. Plant Cell Rep 32:637–650Google Scholar
  10. Cheng H, Li LL, 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 40:707–721PubMedGoogle Scholar
  11. Chiang VL (2006) Monolignol biosynthesis and genetic engineering of lignin in trees, a review. Environ Chem Lett 4:143–146Google Scholar
  12. Cutler SR, Rodriguez PL, Finkelstein RR et al (2010) Abscisic acid: emergence of a core signaling network. Annu Rev Plant Biol 61:651–679PubMedGoogle Scholar
  13. Desender S, Andrivon D, Val F (2007) Activation of defence reactions in Solanaceae: where is the specificity. Cell Microbiol 9:21–30PubMedGoogle Scholar
  14. Dixon RA, Paiva NL (1995) Stress-induced phenylpropanoid metabolism. Plant Cell 7:1085PubMedPubMedCentralGoogle Scholar
  15. Dixon RA, Lamb CJ, Masoud S et al (1996) Metabolic engineering: prospects for crop improvement through the genetic manipulation of phenylpropanoid biosynthesis and defense responses—a review. Gene 179:61–71PubMedGoogle Scholar
  16. Durst F, Nelson DR (1995) Diversity and evolution of plant P450 and P450-reductases. Drug Metab Drug Interact 12:189–206Google Scholar
  17. Ehlting J, Hamberger B, Million-Rousseau R et al (2006) Cytochromes P450 in phenolic metabolism. Phytochem Rev 5:239–270Google Scholar
  18. Ferrer JL, Austin MB, Stewart C et al (2008) Structure and function of enzymes involved in the biosynthesis of phenylpropanoids. Plant Physiol Biochem 46:356–370PubMedGoogle Scholar
  19. Fujita K, Komatsu K, Tanaka K et al (2006) An in vitro model for studying vascular injury after laser microdissection. Histochem Cell Biol 125:509–514PubMedGoogle Scholar
  20. Gross GG (1981) The biochemistry of lignification. Adv Bot Res 8:25–63Google Scholar
  21. Hahlbrock K, Scheel D (1989) Physiology and molecular biology of phenylpropanoid metabolism. Annu Rev Plant Biol 40:347–369Google Scholar
  22. Hamann T, Bennett M, Mansfield J et al (2009) Identification of cell-wall stress as a hexose-dependent and osmosensitive regulator of plant responses. Plant J 57:1015–1026PubMedGoogle Scholar
  23. Hano C, Addi M, Bensaddek L, Crônier D et al (2006) Differential accumulation of monolignol-derived compounds in elicited flax (Linum usitatissimum) cell suspension cultures. Planta 223:975–989PubMedGoogle Scholar
  24. Himmel ME (2008) Biomass recalcitrance: deconstructing the plant cell wall for bioenergy. Blackwell, Oxford, pp 1–6Google Scholar
  25. Hotze M, Schröder G, Schröder J (1995) Cinnamate 4-hydroxylase from Catharanthus roseus and a strategy for the functional expression of plant cytochrome P450 proteins as translational fusions with P450 reductase in Escherichia coli. FEBS Lett 374:345–350PubMedGoogle Scholar
  26. Huang B, Duan Y, Yi B et al (2008) Characterization and expression profiling of cinnamate 4-hydroxylase gene from Salvia miltiorrhiza in rosmarinic acid biosynthesis pathway. Russ J Plant Physiol 55:390–399Google Scholar
  27. Jaakola L, Määttä-Riihinen K, Kärenlampi S et al (2004) Activation of flavonoid biosynthesis by solar radiation in bilberry (Vaccinium myrtillus L.) leaves. Planta 218:721–728PubMedGoogle Scholar
  28. Janská A, Aprile A, Zámečník J et al (2011) Transcriptional responses of winter barley to cold indicate nucleosome remodelling as a specific feature of crown tissues. Funct Integr Genom 11:307–325Google Scholar
  29. Kadioglu A, Saruhan N, Sağlam A et al (2011) Exogenous salicylic acid alleviates effects of long term drought stress and delays leaf rolling by inducing antioxidant system. Plant Growth Regul 64:27–37Google Scholar
  30. Khan W, Prithiviraj B, Smith DL (2003) Photosynthetic responses of corn and soybean to foliar application of salicylates. J Plant Physiol 160:485–492PubMedGoogle Scholar
  31. Khan MIR, Fatma M, Per TS et al (2015) Salicylic acid-induced abiotic stress tolerance and underlying mechanisms in plants. Front Plant Sci 6:462PubMedPubMedCentralGoogle Scholar
  32. Kim YH, Bae JM, Huh GH (2010) Transcriptional regulation of the cinnamyl alcohol dehydrogenase gene from sweetpotato in response to plant developmental stage and environmental stress. Plant Cell Rep 29:779–791PubMedPubMedCentralGoogle Scholar
  33. Kim J, Choi B, Natarajan S et al (2013) Expression analysis of kenaf cinnamate 4-hydroxylase (C4H) ortholog during developmental and stress responses. Plant Omics 6:65–72Google Scholar
  34. Kirk TK, Obst JR (1988) Lignin determination. Method Enzymol 161:87–101Google Scholar
  35. Kochs G, Grisebach H (1989) Phytoalexin synthesis in soybean: purification and reconstitution of cytochrome P450 3, 9-dihydroxypterocarpan 6a-hydroxylase and separation from cytochrome P450 cinnamate 4-hydroxylase. Arch Biochem Biophys 273:543–553PubMedGoogle Scholar
  36. Kong JQ, Lu D, Wang ZB (2014) Molecular cloning and yeast expression of cinnamate 4-hydroxylase from Ornithogalum saundersiae baker. Molecules 19:1608–1621PubMedPubMedCentralGoogle Scholar
  37. Kumar S, Omer S, Chitransh S et al (2012) Cinnamate 4-hydroxylase downregulation in transgenic tobacco alters transcript level of other phenylpropanoid pathway genes. Int J Adv Biotechnol Res 3:545–557Google Scholar
  38. Kumar S, Omer S, Patel K et al (2013) Cinnamate 4-hydroxylase (C4H) genes from Leucaena leucocephala: a pulp yielding leguminous tree. Mol Biol Rep 40:1265–1274PubMedGoogle Scholar
  39. Lee SC, Luan S (2012) ABA signal transduction at the crossroad of biotic and abiotic stress responses. Plant Cell Environ 35:53–60PubMedGoogle Scholar
  40. Lewis NG, Yamamoto E (1990) Lignin: occurrence, biogenesis and biodegradation. Annu Rev Plant Biol 41:455–496Google Scholar
  41. Liu S, Hu Y, Wang X et al (2009) Isolation and characterization of a gene encoding cinnamate 4-hydroxylase from Parthenocissus henryana. Mol Biol Rep 36:1605–1610PubMedGoogle Scholar
  42. Lu S, Zhou Y, Li L et al (2006) Distinct roles of cinnamate 4-hydroxylase genes in Populus. Plant Cell Physiol 47:905–914PubMedGoogle Scholar
  43. Mahajan S, Tuteja N (2005) Cold, salinity and drought stresses: an overview. Arch Biochem Biophys 444:139–158PubMedGoogle Scholar
  44. Mizutani M, Ohta D, Sato R (1997) Isolation of a cDNA and a genomic clone encoding cinnamate 4-hydroxylase from Arabidopsis and its expression manner in planta. Plant Physiol 113:755–763PubMedPubMedCentralGoogle Scholar
  45. Moura JCMS, Bonine CAV, De Oliveira Fernandes Viana J (2010) Abiotic and biotic stresses and changes in the lignin content and composition in plants. J Integr Plant Biol 52:360–376PubMedGoogle Scholar
  46. Naoumkina MA, Zhao Q, Gallegogiraldo L et al (2010) Genome-wide analysis of phenylpropanoid defence pathways. Mol Plant Pathol 11:829–846PubMedPubMedCentralGoogle Scholar
  47. Nedelkina S, Jupe SC, Blee KA et al (1999) Novel characteristics and regulation of a divergent cinnamate 4-hydroxylase (CYP73A15) from French bean: engineering expression in yeast. Plant Mol Biol 39:1079–1090PubMedGoogle Scholar
  48. Nelson DR, Schuler MA, Paquette SM et al (2004) Comparative genomics of rice and Arabidopsis. Analysis of 727 cytochrome P450 genes and pseudogenes from a monocot and a dicot. Plant Physiol 135:756–772PubMedPubMedCentralGoogle Scholar
  49. Peirats-Llobet M, Han SK, Gonzalez-Guzman M et al (2016) A direct link between abscisic acid sensing and the chromatin-remodeling ATPase BRAHMA via core ABA signaling pathway components. Mol Plant 9:136–147PubMedGoogle Scholar
  50. Plomion C, Leprovost G, Stokes A (2001) Wood formation in trees. Plant Physiol 127:1513–1523PubMedPubMedCentralGoogle Scholar
  51. Pomar F, Novo M, Bernal MA et al (2004) Changes in stem lignins (monomer composition and crosslinking) and peroxidase are related with the maintenance of leaf photosynthetic integrity during Verticillium wilt in Capsicum annuum. New Phytol 163:111–123Google Scholar
  52. Popova LP, Maslenkova LT, Yordanova RY et al (2009) Exogenous treatment with salicylic acid attenuates cadmium toxicity in pea seedlings. Plant Physiol Biochem 47:224–231PubMedGoogle Scholar
  53. Ralph J, Lundquist K, Brunow G et al (2004) Lignins: natural polymers from oxidative coupling of 4-hydroxyphenyl-propanoids. Phytochem Rev 3:29–60Google Scholar
  54. Rani A, Singh K, Ahuja PS (2012) Molecular regulation of catechins biosynthesis in tea [Camellia sinensis (L.) O. Kuntze]. Gene 495:205–210PubMedPubMedCentralGoogle Scholar
  55. Redman J, Whitcraft J, Johnson C et al (2002) Abiotic and biotic stress differentially stimulate as-1 element activity in Arabidopsis. Plant Cell Rep 21:180–185Google Scholar
  56. Reyes JC, Muro-Pastor MI, Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice. Plant Physiol 134:1718–1732PubMedPubMedCentralGoogle Scholar
  57. Ro DK, Douglas CJ (2004) Reconstitution of the entry point of plant phenylpropanoid metabolism in yeast (Saccharomyces cerevisiae) implications for control of metabolic flux into the phenylpropanoid pathway. J Biol Chem 279:2600–2607PubMedGoogle Scholar
  58. Ro DK, Mah N, Ellis BE et al (2001) Functional characterization and subcellular localization of poplar (Populus trichocarpa × Populus deltoides) cinnamate 4-hydroxylase. Plant Physiol 126:317–329PubMedPubMedCentralGoogle Scholar
  59. Saharkhiz MJ, Mohammadi S, Javanmardi J (2011) Salicylic acid changes physio-morphological traits and essential oil content of catnip (Nepeta cataria L.). J Med Spice Plants 16:75–77Google Scholar
  60. Salvador VH, Lima RB, dos Santos WD et al (2013) Cinnamic acid increases lignin production and inhibits soybean root growth. PLoS One 8:e69105PubMedPubMedCentralGoogle Scholar
  61. Sarkanen KV, Ludwig CH (1971) Lignins. Occurrence, formation, structure, and reactions. Wiley, New YorkGoogle Scholar
  62. Schilmiller AL, Stout J, Weng JK (2009) Mutations in the cinnamate 4-hydroxylase gene impact metabolism, growth and development in Arabidopsis. Plant J 60:771–782PubMedGoogle Scholar
  63. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101PubMedGoogle Scholar
  64. Schoch GA, Attias R, Le Ret M et al (2003) Key substrate recognition residues in the active site of a plant cytochrome P450, CYP73A1. FEBS J 270:3684–3695Google Scholar
  65. Shao HB, Guo QJ, Chu LY et al (2007) Understanding molecular mechanism of higher plant plasticity under abiotic stress. Colloid Surface B 54:37–45Google Scholar
  66. Singh K, Kumar S, Rani A et al (2009) Phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) and catechins (flavan-3-ols) accumulation in tea. Funct Integr Genom 9:125–134Google Scholar
  67. Smith JV, Luo Y (2004) Studies on molecular mechanisms of Ginkgo biloba extract. Appl Microbiol Biotechnol 64:465–472PubMedGoogle Scholar
  68. Sykes RW, Gjersing EL, Foutz K et al (2015) Down-regulation of p-coumaroyl quinate/shikimate 3′-hydroxylase (C3′H) and cinnamate 4-hydroxylase (C4H) genes in the lignin biosynthetic pathway of Eucalyptus urophylla × E. grandis leads to improved sugar release. Biotechnol Biofuels 8:128PubMedPubMedCentralGoogle Scholar
  69. Szczesnaskorupa E, Straub P, Kemper B (1993) Deletion of a conserved tetrapeptide, PPGP, in P450 2C2 results in loss of enzymatic activity without a change in its cellular location. Arch Biochem Biophys 304:170–175Google Scholar
  70. Tabata M (1996) The mechanism of shikonin biosynthesis in Lithospermum cell cultures. Plant Tissue Culture Lett 13:117–125Google Scholar
  71. Tao S, Khanizadeh S, Zhang H et al (2009) Anatomy, ultrastructure and lignin distribution of stone cells in two Pyrus species. Plant Sci 176:413–419Google Scholar
  72. Terashima N, Kitano K, Kojima M et al (2009) Nanostructural assembly of cellulose, hemicellulose, and lignin in the middle layer of secondary wall of ginkgo tracheid. J Wood Sci 55:409–416Google Scholar
  73. Teutsch HG, Hasenfratz MP, Lesot A et al (1993) Isolation and sequence of a cDNA encoding the Jerusalem artichoke cinnamate 4-hydroxylase, a major plant cytochrome P450 involved in the general phenylpropanoid pathway. Proc Natl Acad Sci USA 90:4102–4106PubMedGoogle Scholar
  74. Tohge T, Watanabe M, Hoefgen R et al (2013) The evolution of phenylpropanoid metabolism in the green lineage. Crit Rev Biochem Mol 48:123–152Google Scholar
  75. Van Beek TA (2002) Chemical analysis of Ginkgo biloba leaves and extracts. J Chromatogr A 967:21–55PubMedGoogle Scholar
  76. Vanholme R, Morreel K, Ralph J et al (2008) Lignin engineering. Curr Opin Plant Biol 11:278–285PubMedGoogle Scholar
  77. Vogt T (2010) Phenylpropanoid biosynthesis. Mol Plant 3:2–20Google Scholar
  78. Wei HUI, Dhanaraj AL, Arora R et al (2006) Identification of cold acclimation-responsive Rhododendron genes for lipid metabolism, membrane transport and lignin biosynthesis: importance of moderately abundant ESTs in genomic studies. Plant Cell Environ 29:558–570PubMedGoogle Scholar
  79. Weisshaar B, Jenkins GI (1998) Phenylpropanoid biosynthesis and its regulation. Curr Opin Plant Biol 1:251–257PubMedGoogle Scholar
  80. Weng JK, Chapple C (2010) The origin and evolution of lignin biosynthesis. New Phytol 187:273–285PubMedGoogle Scholar
  81. Weng JK, Li X, Bonawitz ND (2008) Emerging strategies of lignin engineering and degradation for cellulosic biofuel production. Curr Opin Biotechnol 19:166–172PubMedGoogle Scholar
  82. Werck-Reichhart D, Batard Y, Kochs G (1993) Monospecific polyclonal antibodies directed against purified cinnamate 4-hydroxylase from Helianthus tuberosus (immunopurification, immunoquantitation, and interspecies cross-reactivity). Plant Physiol 102:1291–1298PubMedPubMedCentralGoogle Scholar
  83. Xu F, Cheng H, Cai R (2008) Molecular cloning and function analysis of an anthocyanidin synthase gene from Ginkgo biloba, and its expression in abiotic stress responses. Mol Cell 26:536–547Google Scholar
  84. Xu Z, Zhang D, Hu J (2009) Comparative genome analysis of lignin biosynthesis gene families across the plant kingdom. BMC Bioinform 10:S3Google Scholar
  85. Xu H, Park NI, Li X et al (2010) Molecular cloning and characterization of phenylalanine ammonia-lyase, cinnamate 4-hydroxylase and genes involved in flavone biosynthesis in Scutellaria baicalensis. Bioresour Technol 101:9715–9722PubMedGoogle Scholar
  86. Xu F, Ning YJ, Zhang WW et al (2014) An R2R3-MYB transcription factor as a negative regulator of the flavonoid biosynthesis pathway in Ginkgo biloba. Funct Integr Genom 14:177–189Google Scholar
  87. Yamazaki S, Sato K, Suhara K (1993) Importance of the proline-rich region following signal-anchor sequence in the formation of correct conformation of microsomal cytochrome P-450s. J Biochem 114:652–657PubMedGoogle Scholar
  88. Yang DH, Chung BY, Kim JS (2005) cDNA cloning and sequence analysis of the rice cinnamate-4-hydroxylase gene, a cytochrome P450-dependent monooxygenase involved in the general phenylpropanoid pathway. J Plant Biol 48:311–318Google Scholar
  89. Yeh TF, Yamada T, Capanema E (2005) Rapid screening of wood chemical component variations using transmittance near-infrared spectroscopy. J Agric Food Chem 53:3328–3332PubMedGoogle Scholar
  90. Zeng Y, Zhao S, Yang S (2014) Lignin plays a negative role in the biochemical process for producing lignocellulosic biofuels. Curr Opin Biotechnol 27:38–45PubMedGoogle Scholar
  91. Zhu JK (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Biol 53:247–273PubMedPubMedCentralGoogle Scholar

Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2017

Authors and Affiliations

  1. 1.School of Biology and Pharmaceutical EngineeringWuhan Polytechnic UniversityWuhanChina
  2. 2.College of Horticulture and GardeningYangtze UniversityJingzhouChina

Personalised recommendations