Skip to main content
SpringerLink
Log in

Cinnamate 4-Hydroxylase (C4H) genes from Leucaena leucocephala: a pulp yielding leguminous tree

  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Leucaena leucocephala is a leguminous tree species accounting for one-fourth of raw material supplied to paper and pulp industry in India. Cinnamate 4-Hydroxylase (C4H, EC 1.14.13.11) is the second gene of phenylpropanoid pathway and a member of cytochrome P450 family. There is currently intense interest to alter or modify lignin content of L. leucocephala. Three highly similar C4H alleles of LlC4H1 gene were isolated and characterized. The alleles shared more than 98 % sequence identity at amino acid level to each other. Binding of partial promoter of another C4H gene LlC4H2, to varying amounts of crude nuclear proteins isolated from leaf and stem tissues of L. leucocephala formed two loose and one strong complex, respectively, suggesting that the abundance of proteins that bind with the partial C4H promoter is higher in stem tissue than in leaf tissue. Quantitative Real Time PCR study suggested that among tissues of same age, root tissues had highest level of C4H transcripts. Maximum transcript level was observed in 30 day old root tissue. Among the tissues investigated, C4H activity was highest in 60 day old root tissues. Tissue specific quantitative comparison of lignin from developing seedling stage to 1 year old tree stage indicated that Klason lignin increased in tissues with age.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. FAOSTAT (2011) Highlights on paper and paperboard: 1999–2009. In: FAO Forestry Department. http://faostat.fao.org/Portals/_Faostat/documents/pdf/Paper and paperboard.pdf. Accessed 1 Oct 2011

  2. Shelton HM, Brewbaker JL (1994) Leucaena leucocephala—the most widely used forage tree legume. http://www.fao.org/ag/AGP/AGPC/doc/Publicat/Gutt-shel/x5556e06.htm. Accessed 1 Oct 2011

  3. Srivastava S, Gupta RK, Arha M et al (2011) Expression analysis of cinnamoyl-CoA reductase (CCR) gene in developing seedlings of Leucaena leucocephala: a pulp yielding tree species. Plant Physiol Biochem 49:138–145. doi:10.1016/j.plaphy.2010.11.001

    Article  PubMed  CAS  Google Scholar 

  4. Dutt D, Tyagi CH, Malik RS (2007) Studies on effect of growth factor on morphological, chemical and pulp and paper making characteristics and its impact on fluff generation. Indian J Chem Technol 14:626–634

    CAS  Google Scholar 

  5. Vanholme R, Demedts B, Morreel K et al (2010) Lignin biosynthesis and structure. Plant Physiol 153:895–905. doi:10.1104/pp.110.155119

    Article  PubMed  CAS  Google Scholar 

  6. Vogt T (2010) Phenylpropanoid biosynthesis. Mol Plant 3:2–20

    Article  PubMed  CAS  Google Scholar 

  7. Boerjan W, Ralph J, Baucher M (2003) Lignin biosynthesis. Annu Rev Plant Biol 54:519–546. doi:10.1146/annurev.arplant.54.031902.134938

    Article  PubMed  CAS  Google Scholar 

  8. Hahlbrock K, Scheel D (1989) Physiology and molecular biology of phenylpropanoid metabolism. Annu Rev Plant Physiol Plant Mol Biol 40:347–369

    Article  CAS  Google Scholar 

  9. Russell DW (1971) The metabolism of aromatic compounds in higher plants X. Properties of the cinnamic acid 4-hydroxylase of pea seedlings and some aspects of its metabolic and developmental control. J Biol Chem 246:3870–3878

    PubMed  CAS  Google Scholar 

  10. Chapple C (1998) Molecular-genetic analysis of plant cytochrome P450-dependent monooxygenases. Annu Rev Plant Physiol Plant Mol Biol 49:311–343. doi:10.1146/annurev.arplant.49.1.311

    Article  PubMed  CAS  Google Scholar 

  11. Ehlting J, Hamberger B, Million-Rousseau R, Werck-Reichhart D (2006) Cytochromes P450 in phenolic metabolism. Phytochem Rev 5:239–270. doi:10.1007/s11101-006-9025-1

    Article  CAS  Google Scholar 

  12. Sakaguchi M, Mihara K, Sato R (1987) A short amino-terminal segment of microsomal cytochrome P-450 functions both as an insertion signal and as a stop-transfer sequence. EMBO J 6(8):2425–2431

    PubMed  CAS  Google Scholar 

  13. Achnine L, Blancaflor EB, Rasmussen S, Dixon RA (2004) Colocalization of l-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase for metabolic channeling in phenylpropanoid biosynthesis. Plant Cell 16:3098–3109

    Article  PubMed  CAS  Google Scholar 

  14. Rasmussen S, Dixon RA (1999) Transgene-mediated and elicitor-induced perturbation of metabolic channeling at the entry point into the phenylpropanoid pathway. Plant Cell 11:1537–1551

    PubMed  CAS  Google Scholar 

  15. Rastogi S, Dwivedi UN (2006) Down-regulation of lignin biosynthesis in transgenic Leucaena leucocephala harboring O-methyltransferase gene. Biotechnol Prog 22:609–616. doi:10.1021/bp050206+

    Article  PubMed  CAS  Google Scholar 

  16. Shaik NM, Arha M, Nookaraju A et al (2009) Improved method of in vitro regeneration in Leucaena leucocephala—a leguminous pulpwood tree species. Physiol Mol Biol Plants 15:311–318. doi:10.1007/s12298-009-0035-5

    Article  Google Scholar 

  17. Chen BJ, Wang Y, Hu YL et al (2005) Cloning and characterization of a drought-inducible MYB gene from Boea crassifolia. Plant Sci 168:493–500. doi:10.1016/j.plantsci.2004.09.013

    Article  CAS  Google Scholar 

  18. Pillai MM, Venkataraman GM, Kosak S, Torok-Storb B (2008) Integration site analysis in transgenic mice by thermal asymmetric interlaced (TAIL)-PCR: segregating multiple- integrant founder lines and determining zygosity. Transgenic Res 17:749–754. doi:10.1007/s11248-007-9161-4

    Article  PubMed  CAS  Google Scholar 

  19. Hartman TPV, Jones J, Blackhall NW, Power JB, Cocking EC, Davey MR (2000) Cytogenetics, molecular cytogenetics and genome size in Leucaena (Leguminosae, Mimosideae). In: Guttenberger H, Borzan Z, Schlarbaum SC, Hartman TPV (eds) Cytogenetic studies of forest trees and shrubs: Review, present status, and outlook of the future. Arora Publishers, Zvolen, pp 57–70

    Google Scholar 

  20. Radonic A, Thulke S, Mackay IM et al (2004) Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun 313:856–862. doi:10.1016/j.bbrc.2003.11.177

    Article  PubMed  CAS  Google Scholar 

  21. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:2002–2007

    Article  Google Scholar 

  22. Lamb CJ, Rubery PH (1975) A spectrophotometric assay for trans-cinnamic acid 4-hydroxylase activity. Anal Biochem 68:554–561

    Article  PubMed  CAS  Google Scholar 

  23. Chen JY, Wen PF, Kong WF et al (2006) Changes and subcellular localizations of the enzymes involved in phenylpropanoid metabolism during grape berry development. J Plant Physiol 163:115–127. doi:10.1016/j.jplph.2005.07.006

    Article  PubMed  CAS  Google Scholar 

  24. Yeh T-F, Yamada T, Capanema E et al (2005) Rapid screening of wood chemical component variations using transmittance near-infrared spectroscopy. J Agric Food Chem 53:3328–3332. doi:10.1021/jf0480647

    Article  PubMed  CAS  Google Scholar 

  25. Yamazaki S, Sato K, Sakaguchi M et al (1993) Importance of the proline-rich region following signal-anchor sequence in the formation of correct conformation of microsomal cytochrome P-450 s. J Biochem 114:652–657

    PubMed  CAS  Google Scholar 

  26. Durst F, Nelson DR (1995) Diversity and evolution of plant P450 and P450-reductases. Drug Metabol Drug Interact 12:189–206

    PubMed  CAS  Google Scholar 

  27. Schoch GA, Attias R, Le Ret M, Werck-Reichhart D (2003) Key substrate recognition residues in the active site of a plant cytochrome P450, CYP73A1. Homology model guided site-directed mutagenesis. Eur J Biochem 270:3684–3695. doi:10.1046/j.1432-1033.2003.03739.x

    Article  PubMed  CAS  Google Scholar 

  28. Nelson DR, Koymans L, Kamataki T et al (1996) P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. DNA Cell Biol 6:1–51

    CAS  Google Scholar 

  29. Lu S, Zhou Y, Li L, Chiang VL (2006) Distinct roles of cinnamate 4-hydroxylase genes in Populus. Plant Cell Physiol 47:905–914. doi:10.1093/pcp/pcj063

    Article  PubMed  CAS  Google Scholar 

  30. Whitbred JM, Schuler MA (2000) Molecular characterization of CYP73A9 and CYP82A1 P450 genes involved in plant defense in pea. Plant Physiol 124:47–58

    Article  PubMed  CAS  Google Scholar 

  31. Bell-lelong DA, Cusumano JC, Meyer K, Chapple C (1997) Cinnamate-4-hydroxylase expression in Arabidopsis regulation in response to development and the environment. Plant Physiol 113:729–738

    Article  PubMed  CAS  Google Scholar 

  32. Kawai S, Mori A, Shiokawa T et al (1996) Isolation and analysis of cinnamic acid 4-hydroxylase homologous genes from a hybrid aspen, Poplus kitakamiensis. Biosci Biotechnol Biochem 60:1586–1597

    Article  PubMed  CAS  Google Scholar 

  33. 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 36:1605–1610. doi:10.1007/s11033-008-9357-6

    Article  PubMed  CAS  Google Scholar 

  34. 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 113:755–763

    Article  PubMed  CAS  Google Scholar 

  35. Pechter P (2001) Characterization of transgenic Aspen (Poplus tremuloides Michx.) harboring a homologous cinnamate 4-hydroxylase (C4H) transgene and analysis of two aspen 4-coumarate: CoA ligase (4CL) gene promoters. PhD Dissertation, Michigan Technological University

  36. 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 374:345–350

    Article  PubMed  CAS  Google Scholar 

  37. Fahrendorf T, Dixon RA (1993) Stress responses in alfalfa (Medicago sativa L.). XVIII: molecular cloning and expression of the elicitor-inducible cinnamic acid 4-hydroxylase cytochrome P450. Arch Biochem Biophys 305:509–515

    Article  PubMed  CAS  Google Scholar 

  38. Betz C, Mccollum TG, Mayer RT (2001) Differential expression of two cinnamate 4-hydroxylase genes in “Valencia” orange (Citrus sinensis Osbeck). Plant Mol Biol 46:741–748

    Article  PubMed  CAS  Google Scholar 

  39. Frank MR, Deyneka JM, Schuler MA (1996) Cloning of wound-induced cytochrome P450 monooxygenases expressed in pea. Plant Physiol 110:1035–1046

    Article  PubMed  CAS  Google Scholar 

  40. Park JH, Park NI, Xu H, Park SU (2010) Cloning and characterization of phenylalanine ammonia-lyase and cinnamate 4-hydroxylase and pyranocoumarin biosynthesis in Angelica gigas. J Nat Prod 73:1394–1397

    Article  PubMed  CAS  Google Scholar 

  41. Ro DK, Mah N, Ellis BE, Douglas CJ (2001) Functional characterization and subcellular localization of poplar (Populus trichocarpa x Populus deltoides) cinnamate 4-hydroxylase. Plant Physiol 126:317–329

    Article  PubMed  CAS  Google Scholar 

  42. Sewalt VJH, Ni W, Blount JW et al (1997) Reduced lignin content and altered lignin composition in transgenic tobacco down-regulated in expression of l-phenylalanine ammonia-lyase or cinnamate 4-hydroxylase. Plant Physiol 115:41–50

    PubMed  CAS  Google Scholar 

  43. Kumar S, Omer S, Chitransh S, Khan BM (2012) Cinnamate 4-Hydroxylase downregulation in transgenic tobacco alters transcript level of other phenylpropanoid pathway genes. Int J Adv Biotechnol Res 3:545–557

    CAS  Google Scholar 

Download references

Acknowledgments

The authors thank Council of Scientific and Industrial Research (CSIR, Govt. of India) for funding the project. SK and KP thankfully acknowledge CSIR; and SO thanks Department of Biotechnology (Govt. of India) for giving assistance in the form of Junior and Senior Research Fellowship. We also thank Dr. David Nelson, University of Tennessee for classifying and assigning names to LlC4H proteins.

Author information

Author notes

  1. Santosh Kumar

    Present address: Division of Plant Biology, Centenary Campus, Bose Institute, Kolkata, 700054, India

Authors and Affiliations

  1. Plant Tissue Culture Division, CSIR-National Chemical Laboratory, Pune, 411008, India

    Santosh Kumar, Sumita Omer, Krunal Patel & Bashir M. Khan

Authors
  1. Santosh Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sumita Omer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Krunal Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bashir M. Khan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bashir M. Khan.

Additional information

Santosh Kumar and Sumita Omer contributed equally to this study.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 17 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kumar, S., Omer, S., Patel, K. et al. Cinnamate 4-Hydroxylase (C4H) genes from Leucaena leucocephala: a pulp yielding leguminous tree. Mol Biol Rep 40, 1265–1274 (2013). https://doi.org/10.1007/s11033-012-2169-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-012-2169-8

Keywords

Search

Navigation