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

  • Kashmir Singh
  • Sanjay KumarEmail author
  • Arti Rani
  • Ashu Gulati
  • Paramvir Singh Ahuja
Original Paper

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.

Keywords

Phenylalanine ammonia-lyase Cinnamate 4-hydroxylase Differential display Expression analysis Catechins Tea 

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

Notes

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.

References

  1. 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 225:491–499PubMedCrossRefGoogle Scholar
  2. 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 91:7608–7612PubMedCrossRefGoogle Scholar
  3. 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 113:729–738PubMedCrossRefGoogle Scholar
  4. 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 109:821–827PubMedGoogle Scholar
  5. 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 130:11–18CrossRefGoogle Scholar
  6. Chapple C (1998) Molecular-genetic analysis of plant cytochrome P450-dependent monooxygenases. Annu Rev Plant Physiol Plant Mol Biol 49:311–343PubMedCrossRefGoogle Scholar
  7. 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 1–16Google Scholar
  8. Combet C, Blanchet C, Geourjon C, Deléage G (2000) NPS@: network protein sequence analysis. Trends Biochem Sci 25:147–150PubMedCrossRefGoogle Scholar
  9. 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 104:66–77CrossRefGoogle Scholar
  10. Dixon R, Paiva NL (1995) Stress-induced phenylpropanoid metabolism. Plant Cell 7:1085–1097PubMedCrossRefGoogle Scholar
  11. Gadallah MAA (1995) Effect of water stress, abscisic acid and proline on cotton plants. J Arid Environ 30:315–325CrossRefGoogle Scholar
  12. 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).Google Scholar
  13. 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 110:1123–1133PubMedGoogle Scholar
  14. Hahlbrock K, Scheel D (1989) Physiology and molecular biology of phenylpropanoid metabolism. Annu Rev Plant Physiol Plant Mol Biol 40:347–369CrossRefGoogle Scholar
  15. Harborne JB, Williams C (2000) Advances in flavonoid research since 1992. Phytochemistry 55:481–504PubMedCrossRefGoogle Scholar
  16. Hinderer W, Peterson M, Seitz HU (1983) Inhibition of flavonoid biosynthesis by gibberellic acid in cell suspension cultures of Daucus carota. Planta 160:544–549CrossRefGoogle Scholar
  17. 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 130:729–739PubMedCrossRefGoogle Scholar
  18. Jain NK (1999) Global advances in tea science. Aravali Books International, New DelhiGoogle Scholar
  19. Jeyaramraja PR, Pius PK, Kumar RR, Jayakumar D (2003) Soil moisture stress-induced alterationsin bioconstituents determining tea quality. J Sci Food Agric 83:1187–1191CrossRefGoogle Scholar
  20. 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 92:447–455PubMedCrossRefGoogle Scholar
  21. 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 30:641–646CrossRefGoogle Scholar
  22. Koes RE, Quattrocchio F, Mol JNM (1994) The flavonoid biosynthetic pathway in plants: function and evolution. BioEssays 16:123–132CrossRefGoogle Scholar
  23. Koopmann E, Logemann E, Hahlbrock K (1999) Regulation and functional expression of cinnamate 4-hydroxylase from parsley. Plant Physiol 119:49–56PubMedCrossRefGoogle Scholar
  24. Kumar A, Ellis BE (2001) The phenylalanine ammonialyase gene family in raspberry. Structure, expression, and evolution. Plant Physiol 127:230–239PubMedCrossRefGoogle Scholar
  25. 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–181fCrossRefGoogle Scholar
  26. 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–291SPubMedGoogle Scholar
  27. 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 164:549–556CrossRefGoogle Scholar
  28. Li L, Lu S, Chiang V (2006) A genomic and molecular view of wood formation. Crit Rev Plant Sci 25:215–233CrossRefGoogle Scholar
  29. 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 25:705–710PubMedCrossRefGoogle Scholar
  30. 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 92:5905–5909PubMedCrossRefGoogle Scholar
  31. 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 94:671–680PubMedCrossRefGoogle Scholar
  32. 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 113:755–763PubMedCrossRefGoogle Scholar
  33. Raes J, Rohde A, Christensen JH, Peer YV, Boerjan W (2003) Genome-wide characterization of the lignification toolbox in Arabidopsis. Plant Physiol 133:1051–1071PubMedCrossRefGoogle Scholar
  34. Rasmussen S, Dixon RA (1999) Transgene-mediated and elicitor-induced perturbation of metabolic channeling at the entry point into the phenylpropanoid pathway. Plant Cell 11:1537–1552PubMedCrossRefGoogle Scholar
  35. 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 44:1211–1216CrossRefGoogle Scholar
  36. Schulz W, Eiben HG, Hahlbrock K (1989) Expression in Escherichia coli of catalytically active phenylalanine ammonia-lyase from parsley. FEBS Lett 258:335–338PubMedCrossRefGoogle Scholar
  37. 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 30:101–105CrossRefGoogle Scholar
  38. Shinozaki K, Yamaguchi-Shinozaki K (1997) Gene expression and signal transduction in water stress response. Plant Physiol 115:327–334PubMedCrossRefGoogle Scholar
  39. 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 47:1041–1045PubMedCrossRefGoogle Scholar
  40. 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 335:330–333PubMedCrossRefGoogle Scholar
  41. 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.
  42. Stafford HA (1990) Flavonoid metabolism. CRC, Boca RatonGoogle Scholar
  43. 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 90:4102–4107PubMedCrossRefGoogle Scholar
  44. 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 73:393–401PubMedCrossRefGoogle Scholar
  45. 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 91:23–27PubMedCrossRefGoogle Scholar
  46. Wink M (1988) Plant breeding: importance of plant secondary metabolites for protection against pathogens and herbivores. Theor Appl Genet 75:225–233CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Kashmir Singh
    • 1
    • 3
  • Sanjay Kumar
    • 1
    Email author
  • Arti Rani
    • 1
  • Ashu Gulati
    • 2
  • Paramvir Singh Ahuja
    • 1
  1. 1.Biotechnology DivisionInstitute of Himalayan Bioresource TechnologyPalampurIndia
  2. 2.Hill Area Tea ScienceInstitute of Himalayan Bioresource TechnologyPalampurIndia
  3. 3.Department of Gene Expression, Institute of Molecular Biology and BiotechnologyAdam Mickiewicz UniversityPoznańPoland

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