Skip to main content
SpringerLink
Log in

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

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

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.

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
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • 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 30:241–246

    Article  CAS  Google Scholar 

  • Ashihara H, Crozier A (1999) Biosynthesis and metabolism of caffeine and related purine alkaloids in plants. Adv Bot Res 30:118–205

    Google Scholar 

  • Ashihara H, Crozier A (2001) Caffeine: a well known but little mentioned compound in plant science. Trends Plant Sci 6:407–413

    Article  PubMed  CAS  Google Scholar 

  • Ashihara H, Sano H, Crozier A (2008) Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. Phytochemistry 69:841–856

    Article  PubMed  CAS  Google Scholar 

  • Bhattacharya A, Saini U, Ahuja PS (2006) Transgenic tea. Inter J Tea Sci 5:39–52

    Google Scholar 

  • 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 79:419–424

    Article  CAS  Google Scholar 

  • Channel Check (2008) No movement at the top for tea. Bev Spect 6:14–15

    Google Scholar 

  • Chen X, Whitford GM (1999) Effects of caffeine on fluoride, calcium and phosphorus metabolism and calcified tissues in the rat. Arch Oral Biol 44:33–39

    Article  PubMed  CAS  Google Scholar 

  • Chou TM, Benowitz NL (1994) Caffeine and coffee: effects on health and cardiovascular disease. Comp Biochem Physiol 109C:173–189

    CAS  Google Scholar 

  • FDA [Food and Drug Administration] (2003) Affirmation of generally recognized as safe (GRAS) status. #21CFR-170.35. 4-1-08 edition

  • Fujiki H (1999) Two stages of cancer prevention with green tea. J Cancer Res Clin Oncol 125:589–597

    Article  PubMed  CAS  Google Scholar 

  • 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 21:51–58

    Article  CAS  Google Scholar 

  • Graham HN (1992) Green tea composition, consumption, and polyphenol chemistry. Prev Med 21:334–350

    Article  PubMed  CAS  Google Scholar 

  • 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–R87

    CAS  Google Scholar 

  • Higdon JV, Frei B (2006) Coffee and health: a review of recent human research. Crit Rev Food Sci Nutr 46:101–123

    Article  PubMed  CAS  Google Scholar 

  • Jain SM, Newton RJ (1990) Prospects of biotechnology for tea improvement. Proc Indian Nat Sci Acad 6:441–448

    Google Scholar 

  • Jankun J, Selman SH, Swiercz R et al (1997) Why drinking green tea could prevent cancer. Nature 387:561

    Article  PubMed  CAS  Google Scholar 

  • 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–299i

    Article  Google Scholar 

  • 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 98:629–636

    Article  CAS  Google Scholar 

  • Kato A, Crozier A, Ashihara H (1998) Subcellular localization of the N-3 methyltransferase involved in caffeine biosynthesis in tea. Phytochemistry 48:777–779

    Article  CAS  Google Scholar 

  • Kato M, Mizuno K, Fujimura T et al (1999) Purification and characterization of caffeine synthase from tea leaves. Plant Physiol 120:586–597

    Article  Google Scholar 

  • Kato M, Mizuno K, Crozier A et al (2000) Caffeine synthase gene from tea leaves. Nature 406:956–957

    Article  PubMed  CAS  Google Scholar 

  • Koshiishi C, Crozier A, Ashihara H (2001) Profiles of purine and pyrimidine nucleotides in fresh and manufactured tea leaves. J Agri Food Chem 49:4378–4382

    Article  CAS  Google Scholar 

  • Li Y, Ogita S, Keya CA, Ashihara H (2008) Expression of caffeine biosynthesis genes in tea (Camellia sinensis). Zeitschrift fur Naturforschung C 63:267–270

    CAS  Google Scholar 

  • 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–201j

    Article  Google Scholar 

  • Lopez-Garcia E, Rodriguez-Artalejo F, Rexrode KM et al (2009) Coffee consumption and risk of stroke in women. Circulation 119:1116–1123

    Article  PubMed  CAS  Google Scholar 

  • Marimuthu S, Muraleedharan N (2004) Tea quality: present status of research in India. J Plant Crops 32:1–12

    Google Scholar 

  • Massey LK (2001) Is caffeine a risk factor for bone loss in the elderly. Am J Clin Nutr 74:569–570

    PubMed  CAS  Google Scholar 

  • 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 547:56–60

    Article  PubMed  CAS  Google Scholar 

  • 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 534:75–81

    Article  PubMed  CAS  Google Scholar 

  • 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 43:104–111

    Article  PubMed  CAS  Google Scholar 

  • Mohanpuria P, Kumar V, Yadav SK (2010) Tea caffeine: metabolism, functions, and reduction strategies. Food Sci Biotechnol 19:275–287

    Article  CAS  Google Scholar 

  • 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 20:712–720

    Article  CAS  Google Scholar 

  • Mondal TK, Bhattacharya A, Ahuja PS (2001b) Induction of synchronous secondary somatic embryogenesis in Camellia sinensis (L.) O. Kuntze. J Plant Physiol 158:945–951

    Article  CAS  Google Scholar 

  • Mondal TK, Bhattacharya A, Ahuja PS (2004) Recent advances of tea (Camellia sinensis) biotechnology. Plant Cell Tiss Org Cult 76:194–254

    Article  Google Scholar 

  • Nawrot P, Jordan S, Eastwood J, Rotstein J, Hugenholtz A, Feely M (2003) Effects of caffeine on human health. Food Addit Contam 20:1–30

    PubMed  CAS  Google Scholar 

  • Nurminen ML, Niittynen L, Korpela R et al (1999) Coffee, caffeine and blood pressure. Eur J Clin Nut 53:831–839

    Article  CAS  Google Scholar 

  • Ogawa H, Ueki N (2007) Clinical importance of caffeine dependence and abuse. Psych Clin Neurosci 61:263–268

    Article  CAS  Google Scholar 

  • Ogita S, Uefuji H, Yamaguchi Y et al (2003) Production of decaffeinated coffee plants by genetic engineering. Nature 423:823

    Article  PubMed  CAS  Google Scholar 

  • 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 54:931–941

    Article  PubMed  CAS  Google Scholar 

  • Pan X, Guaguang N, Liu H (2003) Microwave-assisted extraction of tea polyphenols and tea caffeine from green tea leaves. Chem Eng Process 42:129–133

    Article  CAS  Google Scholar 

  • Riksen NP, Rongen GA, Smits P (2009) Acute and long-term cardiovascular effects of coffee: implications for coronary heart disease. Pharmacol Ther 121:185–191

    Article  PubMed  CAS  Google Scholar 

  • 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 26:169–176

    Article  PubMed  CAS  Google Scholar 

  • Sharma V, Rao LJ (2009) A thought on the biological activities of black tea. Crit Rev Food Sci Nutr 49:379–404

    Article  PubMed  CAS  Google Scholar 

  • 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 18:583–594

    Article  CAS  Google Scholar 

  • 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 335:330–333

    Article  PubMed  CAS  Google Scholar 

  • Smith A (2002) Effects of caffeine on human behavior. Food Chem Tox 40:1243–1255

    Article  CAS  Google Scholar 

  • Smith NA, Singh SP, Wang MB et al (2000) Total silencing by intron-spliced hairpin RNAs. Nature 407:319–320

    Article  PubMed  CAS  Google Scholar 

  • Tomita R, Hamada T, Horiguchi G et al (2004) Transgene overexpression with cognate small interfering RNA in tobacco. FEBS Lett 573:117–120

    Article  PubMed  CAS  Google Scholar 

  • 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 132:372–380

    Article  PubMed  CAS  Google Scholar 

  • Van Dieren S, Uiterwaal CSPM, Van der Schouw YT et al (2009) Coffee and tea consumption and risk of type 2 diabetes. Diabetologia 52:2561–2569

    Article  PubMed  Google Scholar 

  • Wang H, Provan GJ, Helliwell K (2000) Tea flavonoids: their functions, utilization and analysis. Trends Food Sci Tech 11:152–160

    Article  CAS  Google Scholar 

  • Yadav SK, Ahuja PS (2007) Towards generating caffeine-free tea by metabolic engineering. Plant Food Humn Nutr 62:185–191

    Article  CAS  Google Scholar 

  • 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 55:3498–3502

    Article  PubMed  CAS  Google Scholar 

  • 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 275:125–135

    Article  CAS  Google Scholar 

  • Yukiaki K, Yukihiko H (1999) Antimutagenic and anticarcinogenic activity of tea polyphenols. Mut Res 436:69–97

    Google Scholar 

Download references

Acknowledgments

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.

Author information

Authors and Affiliations

  1. Biotechnology Division, Institute of Himalayan Bioresource Technology, CSIR, Palampur, 176061, HP, India

    Prashant Mohanpuria, Vinay Kumar, Paramvir Singh Ahuja & Sudesh Kumar Yadav

Authors
  1. Prashant Mohanpuria
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Vinay Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Paramvir Singh Ahuja
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sudesh Kumar Yadav
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sudesh Kumar Yadav.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mohanpuria, P., Kumar, V., Ahuja, P.S. et al. Producing low-caffeine tea through post-transcriptional silencing of caffeine synthase mRNA. Plant Mol Biol 76, 523–534 (2011). https://doi.org/10.1007/s11103-011-9785-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11103-011-9785-x

Keywords

Search

Navigation