Plant Molecular Biology

, Volume 76, Issue 6, pp 523–534 | Cite as

Producing low-caffeine tea through post-transcriptional silencing of caffeine synthase mRNA

  • Prashant Mohanpuria
  • Vinay Kumar
  • Paramvir Singh Ahuja
  • Sudesh Kumar Yadav


In this study, attempt has been made to produce a selected cultivar of tea with low-caffeine content using RNAi technology. The caffeine biosynthetic pathway in tea has been proposed to involve three N-methyltransferases such as xanthosine methyltransferase, 7-N-methylxanthine methyltransferase and 3, 7-dimethylxanthine methyltransferase. Last two steps of caffeine biosynthesis in tea have been known to be catalyzed by a bifunctional enzyme known as caffeine synthase. To suppress the caffeine synthesis in the selected tea [Camellia sinensis (L.) O. Kuntze] cv. Kangra jat, we isolated a partial fragment of caffeine synthase (CS) from the same cultivar and used to design RNAi construct (pFGC1008-CS). Somatic embryos were transformed with the developed construct using biolistic method. Transformed somatic embryos showed reduction in the levels of CS transcript expression as well as in caffeine content. Plants were regenerated from the transformed somatic embryos. Transgenic plants showed a significant suppression of CS transcript expression and also showed a reduction of 44–61% in caffeine and 46–67% in theobromine contents as compared to the controls. These results suggest that the RNAi construct developed here using a single partial fragment of CS gene reduced the expression of the targeted endogenous gene significantly. However, the reduction in theobromine content in addition to caffeine documented the involvement of this single CS in the catalysis of last two methyl transfer steps in caffeine biosynthesis of tea.


Caffeine Caffeine synthase Camellia sinensis Gene silencing Somatic embryo Transformation 



The authors thank Dr. Ashwani Pareek, Jawahar Lal Nehru University (New Delhi) for his generous help in making RNAi construct. This work was supported by the research grants from Department of Science and Technology (DST; Grant No GAP095) and Council of Scientific and Industrial Research (CSIR; Grant No SIP003), Govt. of India, New Delhi. Prashant Mohanpuria is also thankful to CSIR for providing research fellowship in the form of SRF. The IHBT communication number for this article is 2202.


  1. Akula A, Akula C, Bateson M (2000) Betaine a novel candidate for rapid induction of somatic embryogenesis in tea (Camellia sinensis (L.) O. Kuntze). Plant Growth Regul 30:241–246CrossRefGoogle Scholar
  2. Ashihara H, Crozier A (1999) Biosynthesis and metabolism of caffeine and related purine alkaloids in plants. Adv Bot Res 30:118–205Google Scholar
  3. Ashihara H, Crozier A (2001) Caffeine: a well known but little mentioned compound in plant science. Trends Plant Sci 6:407–413PubMedCrossRefGoogle Scholar
  4. Ashihara H, Sano H, Crozier A (2008) Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. Phytochemistry 69:841–856PubMedCrossRefGoogle Scholar
  5. Bhattacharya A, Saini U, Ahuja PS (2006) Transgenic tea. Inter J Tea Sci 5:39–52Google Scholar
  6. Borse BB, Jagan Mohan Rao L, Nagalakshmi S et al (2002) Fingerprint of black teas from India: identification of the regio-specific characteristics. Food Chem 79:419–424CrossRefGoogle Scholar
  7. Channel Check (2008) No movement at the top for tea. Bev Spect 6:14–15Google Scholar
  8. Chen X, Whitford GM (1999) Effects of caffeine on fluoride, calcium and phosphorus metabolism and calcified tissues in the rat. Arch Oral Biol 44:33–39PubMedCrossRefGoogle Scholar
  9. Chou TM, Benowitz NL (1994) Caffeine and coffee: effects on health and cardiovascular disease. Comp Biochem Physiol 109C:173–189Google Scholar
  10. FDA [Food and Drug Administration] (2003) Affirmation of generally recognized as safe (GRAS) status. #21CFR-170.35. 4-1-08 editionGoogle Scholar
  11. Fujiki H (1999) Two stages of cancer prevention with green tea. J Cancer Res Clin Oncol 125:589–597PubMedCrossRefGoogle Scholar
  12. Goto K, Kanazawa A, Kusaba M et al (2003) A simple and rapid method to detect plant siRNAs using nonradioactive probes. Plant Mol Biol Rep 21:51–58CrossRefGoogle Scholar
  13. Graham HN (1992) Green tea composition, consumption, and polyphenol chemistry. Prev Med 21:334–350PubMedCrossRefGoogle Scholar
  14. Heckman MA, Weil J, De Mejia EG (2010) Caffeine (1, 3, 7-trimethylxanthine) in foods: a comprehensive review on consumption, functionality, safety, and regulatory matters. JFS R Con Rev Hypo Food Sci 75:R77–R87Google Scholar
  15. Higdon JV, Frei B (2006) Coffee and health: a review of recent human research. Crit Rev Food Sci Nutr 46:101–123PubMedCrossRefGoogle Scholar
  16. Jain SM, Newton RJ (1990) Prospects of biotechnology for tea improvement. Proc Indian Nat Sci Acad 6:441–448Google Scholar
  17. Jankun J, Selman SH, Swiercz R et al (1997) Why drinking green tea could prevent cancer. Nature 387:561PubMedCrossRefGoogle Scholar
  18. Jeyaramraja PR, Meenakshi S (2005) Agrobacterium tumefaciens-mediated transformation of embryogenic tissues of tea (Camellia sinensis (L.) O. Kuntze). Plant Mol Biol Rep 23:299a–299iCrossRefGoogle Scholar
  19. Kato M, Kanehara T, Shimizu H et al (1996) Caffeine biosynthesis in young leaves of Camellia sinensis: in vitro studies on N-methyltransferase activity involved in the conversion of xanthosine to caffeine. Physiol Plant 98:629–636CrossRefGoogle Scholar
  20. Kato A, Crozier A, Ashihara H (1998) Subcellular localization of the N-3 methyltransferase involved in caffeine biosynthesis in tea. Phytochemistry 48:777–779CrossRefGoogle Scholar
  21. Kato M, Mizuno K, Fujimura T et al (1999) Purification and characterization of caffeine synthase from tea leaves. Plant Physiol 120:586–597CrossRefGoogle Scholar
  22. Kato M, Mizuno K, Crozier A et al (2000) Caffeine synthase gene from tea leaves. Nature 406:956–957PubMedCrossRefGoogle Scholar
  23. Koshiishi C, Crozier A, Ashihara H (2001) Profiles of purine and pyrimidine nucleotides in fresh and manufactured tea leaves. J Agri Food Chem 49:4378–4382CrossRefGoogle Scholar
  24. Li Y, Ogita S, Keya CA, Ashihara H (2008) Expression of caffeine biosynthesis genes in tea (Camellia sinensis). Zeitschrift fur Naturforschung C 63:267–270Google Scholar
  25. Lopez SJ, Rajkumar R, Pius PK et al (2004) Agrobacterium tumefaciens–mediated genetic transformation in tea (Camellia sinensis (L.) O. Kuntze). Plant Mol Biol Rep 22:201a–201jCrossRefGoogle Scholar
  26. Lopez-Garcia E, Rodriguez-Artalejo F, Rexrode KM et al (2009) Coffee consumption and risk of stroke in women. Circulation 119:1116–1123PubMedCrossRefGoogle Scholar
  27. Marimuthu S, Muraleedharan N (2004) Tea quality: present status of research in India. J Plant Crops 32:1–12Google Scholar
  28. Massey LK (2001) Is caffeine a risk factor for bone loss in the elderly. Am J Clin Nutr 74:569–570PubMedGoogle Scholar
  29. Mizuno K, Kato M, Irino F et al (2003a) The first committed step reaction of caffeine biosynthesis: 7-methylxanthosine synthase is closely homologous to caffeine synthases in coffee (Coffea arabica L.). FEBS Lett 547:56–60PubMedCrossRefGoogle Scholar
  30. Mizuno K, Okuda A, Kato M et al (2003b) Isolation of a new dual-functional caffeine synthase gene encoding an enzyme for the conversion of 7-methylxanthine to caffeine from coffee (Coffea arabica L.). FEBS Lett 534:75–81PubMedCrossRefGoogle Scholar
  31. Mohanpuria P, Kumar V, Joshi R et al (2009) Caffeine biosynthesis and degradation in tea [Camellia sinensis (L.) O. Kuntze] is under developmental and seasonal regulation. Mol Biotechnol 43:104–111PubMedCrossRefGoogle Scholar
  32. Mohanpuria P, Kumar V, Yadav SK (2010) Tea caffeine: metabolism, functions, and reduction strategies. Food Sci Biotechnol 19:275–287CrossRefGoogle Scholar
  33. Mondal TK, Bhattacharya A, Ahuja PS et al (2001a) Transgenic tea [Camellia sinensis (L.) O. Kuntze cv. Kangra Jat] plants obtained by Agrobacterium-mediated transformation of somatic embryos. Plant Cell Rep 20:712–720CrossRefGoogle Scholar
  34. Mondal TK, Bhattacharya A, Ahuja PS (2001b) Induction of synchronous secondary somatic embryogenesis in Camellia sinensis (L.) O. Kuntze. J Plant Physiol 158:945–951CrossRefGoogle Scholar
  35. Mondal TK, Bhattacharya A, Ahuja PS (2004) Recent advances of tea (Camellia sinensis) biotechnology. Plant Cell Tiss Org Cult 76:194–254CrossRefGoogle Scholar
  36. Nawrot P, Jordan S, Eastwood J, Rotstein J, Hugenholtz A, Feely M (2003) Effects of caffeine on human health. Food Addit Contam 20:1–30PubMedGoogle Scholar
  37. Nurminen ML, Niittynen L, Korpela R et al (1999) Coffee, caffeine and blood pressure. Eur J Clin Nut 53:831–839CrossRefGoogle Scholar
  38. Ogawa H, Ueki N (2007) Clinical importance of caffeine dependence and abuse. Psych Clin Neurosci 61:263–268CrossRefGoogle Scholar
  39. Ogita S, Uefuji H, Yamaguchi Y et al (2003) Production of decaffeinated coffee plants by genetic engineering. Nature 423:823PubMedCrossRefGoogle Scholar
  40. Ogita S, Uefuji H, Morimoto M et al (2004) Application of RNAi to confirm theobromine as the major intermediate for caffeine biosynthesis in coffee plants with potential for construction of decaffeinated varieties. Plant Mol Biol 54:931–941PubMedCrossRefGoogle Scholar
  41. Pan X, Guaguang N, Liu H (2003) Microwave-assisted extraction of tea polyphenols and tea caffeine from green tea leaves. Chem Eng Process 42:129–133CrossRefGoogle Scholar
  42. Riksen NP, Rongen GA, Smits P (2009) Acute and long-term cardiovascular effects of coffee: implications for coronary heart disease. Pharmacol Ther 121:185–191PubMedCrossRefGoogle Scholar
  43. Sandal I, Saini U, Lacroix B et al (2007) Agrobacterium-mediated genetic transformation of tea leaf explants: effects of counteracting bactericidity of leaf polyphenols without loss of bacterial virulence. Plant Cell Rep 26:169–176PubMedCrossRefGoogle Scholar
  44. Sharma V, Rao LJ (2009) A thought on the biological activities of black tea. Crit Rev Food Sci Nutr 49:379–404PubMedCrossRefGoogle Scholar
  45. Sharma V, Gulati A, Ravindranath SD et al (2005) A simple and convenient method for analysis of tea biochemicals by reverse phase HPLC. J Food Comp Anal 18:583–594CrossRefGoogle Scholar
  46. Singh K, Raizada J, Bhardwaj P et al (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
  47. Smith A (2002) Effects of caffeine on human behavior. Food Chem Tox 40:1243–1255CrossRefGoogle Scholar
  48. Smith NA, Singh SP, Wang MB et al (2000) Total silencing by intron-spliced hairpin RNAs. Nature 407:319–320PubMedCrossRefGoogle Scholar
  49. Tomita R, Hamada T, Horiguchi G et al (2004) Transgene overexpression with cognate small interfering RNA in tobacco. FEBS Lett 573:117–120PubMedCrossRefGoogle Scholar
  50. Uefuji H, Shinjiro O, Yamaguchi Y et al (2003) Molecular cloning and functional characterization of three distinct N-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. Plant Physiol 132:372–380PubMedCrossRefGoogle Scholar
  51. Van Dieren S, Uiterwaal CSPM, Van der Schouw YT et al (2009) Coffee and tea consumption and risk of type 2 diabetes. Diabetologia 52:2561–2569PubMedCrossRefGoogle Scholar
  52. Wang H, Provan GJ, Helliwell K (2000) Tea flavonoids: their functions, utilization and analysis. Trends Food Sci Tech 11:152–160CrossRefGoogle Scholar
  53. Yadav SK, Ahuja PS (2007) Towards generating caffeine-free tea by metabolic engineering. Plant Food Humn Nutr 62:185–191CrossRefGoogle Scholar
  54. Ye JH, Liang YR, Jin J, Liang HL, Du YY, Lu JL, Ye Q, Lin C (2007) Preparation of partially decaffeinated instant green tea. J Agric Food Chem 55:3498–3502PubMedCrossRefGoogle Scholar
  55. Yoneyama N, Morimoto H, Ye CX et al (2006) Substrate specificity of N-methyltransferase involved in purine alkaloids synthesis is dependent upon one amino acid residue of the enzyme. Mol Gen Genom 275:125–135CrossRefGoogle Scholar
  56. Yukiaki K, Yukihiko H (1999) Antimutagenic and anticarcinogenic activity of tea polyphenols. Mut Res 436:69–97Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Prashant Mohanpuria
    • 1
  • Vinay Kumar
    • 1
  • Paramvir Singh Ahuja
    • 1
  • Sudesh Kumar Yadav
    • 1
  1. 1.Biotechnology DivisionInstitute of Himalayan Bioresource Technology, CSIRPalampurIndia

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