Skip to main content
SpringerLink
Log in

Phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) and catechins (flavan-3-ols) accumulation in tea

  • Original Paper
  • Published:
Functional & Integrative Genomics Aims and scope Submit manuscript

Abstract

Phenylalanine ammonia-lyase and cinnamate 4-hydroxylase are important enzymes in allocating significant amounts of carbon from phenylalanine into the biosynthesis of several important secondary metabolites. Tea is an important crop of commerce known for its beverage and medicinally important flavonoid compounds, mainly catechins. As metabolic flux for the operation of the flavonoid pathway is maintained through the activities of PAL and C4H, thus, catechins biosynthesis in tea is critically dependent on the products of these enzymes. We examined the expression of PAL and C4H. Sequence encoding CsPAL was isolated from tea by polymerase chain reaction using sequence information available at the NCBI GenBank. Sequence encoding C4H was isolated from tea by using differential display of mRNA and rapid amplification of cDNA ends technology. CsC4H (AY641731) comprised of 1,352 bp full-length cDNA with open reading frame of 1,173 bp encoding 390 amino acids. Catechin contents decreased in response to drought stress (DS), abscisic acid (ABA), and gibberellic acid (GA3) treatments but increased in response to wounding. The expression of CsPAL and CsC4H showed the same behavior under the above treatments and was also in accordance with the catechin contents. A positive correlation between catechin contents and gene expression suggested a critical role of the enzymes in catechins biosynthesis and a crosstalk between phenylpropanoid and flavonoid pathways.

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
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

ABA:

abscisic acid

C4H:

cinnamate 4-hydroxylase

CsC4H:

Camellia sinensis C4H

CsPAL:

Camellia sinensis PAL

DD:

differential display

DS:

drought stress

FL:

flavonoid

GA3 :

gibberellic acid

PAL:

phenylalanine ammonia-lyase

PP:

phenylpropanoid

RACE:

rapid amplification of cDNA ends

References

  • Appert C, Logemann E, Hahlbrock K, Schmid J, Amrhein N (1994) Structural and catalytic properties of the four phenylalanine ammonia-lyase isozymes from parsley (Petroselinum crispum Nym.). Eur J Biochem 225:491–499

    Article  PubMed  CAS  Google Scholar 

  • Bate NJ, Orrt J, Nit W, Meromit A, Nadler-hassar T, Doerner PW, Dixon RA, Lamb CJ, Elkind Y (1994) Quantitative relationship between phenylalanine ammonia-lyase levels and phenylpropanoid accumulation in transgenic tobacco identifies a rate-determining step in natural product synthesis. Proc Natl Acad Sci USA 91:7608–7612

    Article  PubMed  CAS  Google Scholar 

  • 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 

  • Brignolas F, Lacroix B, Lieutier F, Sauvard D, Drouet A, Claudot AC, Yart A, Berryman AA, Christiansen E (1995) Induced responses in phenolic metabolism in two Norway spruce clones after wounding and inoculations with Ophiostoma polonicum, a bark beetle-associated fungus. Plant Physiol 109:821–827

    PubMed  CAS  Google Scholar 

  • Campos PS, Thi ATP (1997) Effects of an abscisic acid pretreatment on membrane leakage and lipid composition of Vigna unguiculate leaf discs subjected to osmotic stress. Plant Sci 130:11–18

    Article  CAS  Google Scholar 

  • Chapple C (1998) Molecular-genetic analysis of plant cytochrome P450-dependent monooxygenases. Annu Rev Plant Physiol Plant Mol Biol 49:311–343

    Article  PubMed  CAS  Google Scholar 

  • Chen M (2002) Tea and health—an overview. In: Zhen Y, Chen Z, Cheng S, Chen M (eds) Tea. Bioactivity and therapeutic potential. Taylor and Francis, London, pp 1–16

    Google Scholar 

  • Combet C, Blanchet C, Geourjon C, Deléage G (2000) NPS@: network protein sequence analysis. Trends Biochem Sci 25:147–150

    Article  PubMed  CAS  Google Scholar 

  • Davies ME (1972) Effects of auxin on polyphenol accumulation and the development of phenylalanine ammonia-lyase activity in dark grown suspension cultures of Paul’s Scarlet rose. Planta 104:66–77

    Article  CAS  Google Scholar 

  • Dixon R, Paiva NL (1995) Stress-induced phenylpropanoid metabolism. Plant Cell 7:1085–1097

    Article  PubMed  CAS  Google Scholar 

  • Gadallah MAA (1995) Effect of water stress, abscisic acid and proline on cotton plants. J Arid Environ 30:315–325

    Article  Google Scholar 

  • Ghawana S, Singh K, Raizada J, Rani A, Bhardwaj PK, Kumar S (2004) A method for rapid isolation of RNA and a kit thereof. A joint patent between CSIR and DBT 0344 NF 2004/IN, 30 March 2006 (filed).

  • Graham TL, Graham MY (1996) Signaling in soybean phenylpropanoid responses. Dissection of primary, secondary, and conditioning effects of light, wounding, and elicitor treatments. Plant Physiol 110:1123–1133

    PubMed  CAS  Google Scholar 

  • 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 

  • Harborne JB, Williams C (2000) Advances in flavonoid research since 1992. Phytochemistry 55:481–504

    Article  PubMed  CAS  Google Scholar 

  • Hinderer W, Peterson M, Seitz HU (1983) Inhibition of flavonoid biosynthesis by gibberellic acid in cell suspension cultures of Daucus carota. Planta 160:544–549

    Article  Google Scholar 

  • Jaakola L, Maatta K, Pirttila AM, Torronen R, Karenlampi S, Hahtola A (2002) Expression of genes involved in anthocyanin biosynthesis in relation to anthocyanin, proanthocyanidin, and flavonols levels during bilberry fruit development. Plant Physiol 130:729–739

    Article  PubMed  CAS  Google Scholar 

  • Jain NK (1999) Global advances in tea science. Aravali Books International, New Delhi

    Google Scholar 

  • Jeyaramraja PR, Pius PK, Kumar RR, Jayakumar D (2003) Soil moisture stress-induced alterationsin bioconstituents determining tea quality. J Sci Food Agric 83:1187–1191

    Article  CAS  Google Scholar 

  • Jorrin J, Dixon RA (1990) Stress responses in alfalfa (Medicago sativa L.). Purification, characterization, and induction of phenylalanine ammonia-lyase isoforms from elicitor-treated cell suspension cultures. Plant Physiol 92:447–455

    Article  PubMed  CAS  Google Scholar 

  • Joshi CP, Kumar S, Nguyen HT (1995) Application of modified differential display technique for cloning and sequencing of the 3′ region from three putative members of wheat HSP70 gene family. Plant Mol Biol 30:641–646

    Article  Google Scholar 

  • Koes RE, Quattrocchio F, Mol JNM (1994) The flavonoid biosynthetic pathway in plants: function and evolution. BioEssays 16:123–132

    Article  CAS  Google Scholar 

  • Koopmann E, Logemann E, Hahlbrock K (1999) Regulation and functional expression of cinnamate 4-hydroxylase from parsley. Plant Physiol 119:49–56

    Article  PubMed  CAS  Google Scholar 

  • Kumar A, Ellis BE (2001) The phenylalanine ammonialyase gene family in raspberry. Structure, expression, and evolution. Plant Physiol 127:230–239

    Article  PubMed  CAS  Google Scholar 

  • Lal L, Sahoo R, Gupta RK, Sharma P, Kumar S (2001) RNA isolation from high-phenolic tea leaves and apical buds. Plant Mol Biol Report 19:181a–181f

    Article  Google Scholar 

  • Lambert JD, Hong J, Yang G, Liao J, Yang CS (2005) Dietary polyphenols and health: evidence from laboratory investigations. Am J Clin Nutr 81:284S–291S

    PubMed  CAS  Google Scholar 

  • Li X, Li S, Lin J (2003) Effect of GA3 spraying on lignin and auxin contents and the correlated enzyme activities in bayberry (Myrica rubra Bieb.) during flower bud induction. Plant Sci 164:549–556

    Article  CAS  Google Scholar 

  • Li L, Lu S, Chiang V (2006) A genomic and molecular view of wood formation. Crit Rev Plant Sci 25:215–233

    Article  CAS  Google Scholar 

  • Liu R, Xu S, Li J, Hu Y, Lin Z (2006) Expression profile of a PAL gene from Astragalus membranaceus var. Mongholicus and its crucial role in flux into flavonoid biosynthesis. Plant Cell Rep 25:705–710

    Article  PubMed  CAS  Google Scholar 

  • Logemann E, Parniske M, Hahlbrook K (1995) Modes of expression and common structural features of the complete phenylalanine ammonia-lyase gene family in parsley. Proc Nat Acad Sci USA 92:5905–5909

    Article  PubMed  CAS  Google Scholar 

  • Mavandad M, Edwards R, Liang X, Lamb CJ, Dixon RA (1990) Effects of trans-cinnamic acid on expression of the bean phenylalanine ammonia-lyase gene family. Plant Physiol 94:671–680

    Article  PubMed  CAS  Google Scholar 

  • 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 

  • Raes J, Rohde A, Christensen JH, Peer YV, Boerjan W (2003) Genome-wide characterization of the lignification toolbox in Arabidopsis. Plant Physiol 133:1051–1071

    Article  PubMed  CAS  Google Scholar 

  • Rasmussen S, Dixon RA (1999) Transgene-mediated and elicitor-induced perturbation of metabolic channeling at the entry point into the phenylpropanoid pathway. Plant Cell 11:1537–1552

    Article  PubMed  CAS  Google Scholar 

  • Russel DW, Galston AW (1969) Blockage by gibberellic acid on phytochrome effects on growth, auxin responses, and flavonoid biosynthesis in etiolated pea internodes. Plant Physiol 44:1211–1216

    Article  Google Scholar 

  • Schulz W, Eiben HG, Hahlbrock K (1989) Expression in Escherichia coli of catalytically active phenylalanine ammonia-lyase from parsley. FEBS Lett 258:335–338

    Article  PubMed  CAS  Google Scholar 

  • Sharma P, Kumar S (2005) Differential display-mediated identification of three drought-responsive expressed sequence tags in tea (Camellia sinensis L. (O.) Kuntze). J Biosci 30:101–105

    Article  Google Scholar 

  • Shinozaki K, Yamaguchi-Shinozaki K (1997) Gene expression and signal transduction in water stress response. Plant Physiol 115:327–334

    Article  PubMed  CAS  Google Scholar 

  • Singh HP, Ravindranath SD, Singh C (1999) Analysis of tea shoot catechins: spectrophotometric quantitation and selective visualization of two-dimensional paper chromatograms using diazotized sulfanilamide. J Agric Food Chem 47:1041–1045

    Article  PubMed  CAS  Google Scholar 

  • Singh K, Raizada J, Bhardwaj P, Ghawana S, Rani A, Singh H, Kaul K, Kumar S (2004) 26S rRNA-based internal control gene primer pair for reverse transcription-polymerase chain reaction-based quantitative expression studies in diverse plant species. Anal Biochem 335:330–333

    Article  PubMed  CAS  Google Scholar 

  • Singh K, Kumar S, Ahuja PS (2008) Differential expression of Histone H3 gene in tea (Camellia sinensis (L.) O. Kuntze) suggests its role in growing tissue. Mol Biol Rep. doi:10.1007/s11033-008-9211-x.

  • Stafford HA (1990) Flavonoid metabolism. CRC, Boca Raton

    Google Scholar 

  • Teutsch HG, Hasenfratz M-P, Lesot A, Stoltz C, Garnier J-M, Jeltsch J-M, Durst F, Werck-Reichhart D (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 90:4102–4107

    Article  PubMed  CAS  Google Scholar 

  • Thiagarajan G, Chandani S, Sundari CS, Rao SH, Kulkarni AV, Balasubramanian D (2001) Antioxidant properties of green and black tea and their potential ability to retard the progression of eye lens cataract. Exp Eye Res 73:393–401

    Article  PubMed  CAS  Google Scholar 

  • Ward EWB, Cahill DM, Bhattacharayya MK (1989) Abscisic acid suppression of phenylalanaine ammonia-lyase activity and messenger RNA, and resistance of soybeans to Phytopythora megasperma f. sp. Glycinea. Plant Physiol 91:23–27

    Article  PubMed  CAS  Google Scholar 

  • Wink M (1988) Plant breeding: importance of plant secondary metabolites for protection against pathogens and herbivores. Theor Appl Genet 75:225–233

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors are thankful to the Council of Scientific and Industrial Research (CSIR), India for funding the present research work under the New Millennium Indian Technology Leadership Initiative (NMITLI) program entitled “Using functional genomics in plants: development and use of technologies for gene discovery and expression modulation—niche pathway engineering in tea.” KS is thankful to CSIR for awarding the junior and senior research fellowships. The technical help provided by Mr. Digvijay Singh for gene sequencing is duly acknowledged.

Author information

Author notes

  1. Kashmir Singh

    Present address: Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Miedzychodzka 5, 60 371, Poznań, Poland

Authors and Affiliations

  1. Biotechnology Division, Institute of Himalayan Bioresource Technology, P.O. Box No. 6, Palampur, 176061, India

    Kashmir Singh, Sanjay Kumar, Arti Rani & Paramvir Singh Ahuja

  2. Hill Area Tea Science, Institute of Himalayan Bioresource Technology, P.O. Box No. 6, Palampur, 176061, India

    Ashu Gulati

Authors
  1. Kashmir Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sanjay Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Arti Rani
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ashu Gulati
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Paramvir Singh Ahuja
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sanjay Kumar.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Singh, K., Kumar, S., Rani, A. et al. Phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) and catechins (flavan-3-ols) accumulation in tea. Funct Integr Genomics 9, 125–134 (2009). https://doi.org/10.1007/s10142-008-0092-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10142-008-0092-9

Keywords

Search

Navigation