Molecular And Biochemical Characterization Of Caffeine Synthase And Purine Alkaloid Concentration In Guarana Fruit

Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083 bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein. © 2014 Elsevier Ltd. All rights reserved.1052536Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., Basic local alignment search tool (1990) J. Mol. Biol., 215, pp. 403-410Angelo, P.C.S., Nunes-Silva, C.G., Brigido, M.M., Azevedo, J.S.N., Assuncao, E.N., Sousa, A.R.B., Patricio, F.J.B., Oliveira Jr., W.P., Guarana (Paullinia cupana var. Sorbilis), an anciently consumed stimulant from the Amazon rain forest: The seeded-fruit transcriptome (2008) Plant Cell Reports, 27 (1), pp. 117-124. , DOI 10.1007/s00299-007-0456-yAraújo, P., Cesarino, I., Carmello-Guerreiro, S.M., Dornelas, M.C., Citrus sinensis L. Osbeck orthologs of FRUITFULL and SHATTERPROOF are differentially expressed during fruit development (2012) Plant Growth Regul., 70, pp. 1-13Ashihara, H., Metabolism of alkaloids in coffee plants (2006) Brazilian Journal of Plant Physiology, 18 (1), pp. 1-8. , http://www.scielo.br/pdf/bjpp/v18n1/a01v18n1.pdf, DOI 10.1590/S1677-04202006000100001Ashihara, H., Suzuki, T., Distribution and biosynthesis of caffeine in plants (2004) Frontiers in Bioscience, 9, pp. 1864-1876. , d1500-1993Ashihara, H., Sano, H., Crozier, A., Caffeine and related purine alkaloids: Biosynthesis, catabolism, function and genetic engineering (2008) Phytochemistry, 69, pp. 841-856Ashihara, H., Ogita, S., Crozier, A., Purine alkaloid metabolism (2011) Plant Metabolism and Biotechnology, pp. 163-189. , H. Ashihara, A. Crozier, A. Komamine, John Wiley & Sons Ltd ChichesterBaumann, T., Frischknecht, P., Caffeine: Production by plant (Coffea spp.) cell cultures (1988) Medicinal and Aromatic Plants i, 4 VOL., pp. 264-281. , Y. Bajaj, Springer Verlag, Berlin, HeidelbergBaumann, T.W., Schulthess, B.H., Hanni, K., Guarana (Paullinia cupana) rewards seed dispersers without intoxicating them by caffeine (1995) Phytochemistry, 39, pp. 1063-1070Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding (1976) Anal. Biochem., 72, pp. 248-254Deng, W.-W., Ashihara, H., Profiles of purine metabolism in leaves and roots of Camellia sinensis seedlings (2010) Plant Cell Physiol., 51, pp. 2105-2118Figueiredo, L.C., Faria-Campos, A.C., Astolfi-Filho, S., Azevedo, J.L., Identification and isolation of full-length cDNA sequences by sequencing and analysis of expressed sequence tags from guarana (Paullinia cupana) (2011) Genet. Mol. Res., 10, pp. 1188-1199Fujimori, N., Ashihara, H., Biosynthesis of caffeine in flower buds of Camellia sinensis (1993) Ann. Bot., 71, pp. 279-284Gouet, P., Courcelle, E., Stuart, D.I., Metoz, F., ESPript: Analysis of multiple sequence alignments in PostScript (1999) Bioinformatics, 15 (4), pp. 305-308. , DOI 10.1093/bioinformatics/15.4.305Henman, A.R., (1986) Vida Natural, O Guaraná: Sua Cultura, Propriedades, Formas de Preparação e Uso, , Global/Ground São PauloIshida, M., Kitao, N., Mizuno, K., Tanikawa, N., Kato, M., Occurrence of theobromine synthase genes in purine alkaloid-free species of Camellia plants (2009) Planta, 229, pp. 559-568Joshi, C.P., Chiang, V.L., Conserved sequence motifs in plant S-adenosyl-L-methionine-dependent methyltransferases (1998) Plant Molecular Biology, 37 (4), pp. 663-674. , DOI 10.1023/A:1006035210889Kato, M., Mizuno, K., Caffeine synthase and related methyltransferases in plants (2004) Frontiers in Bioscience, 9, pp. 1833-1842. , d1500-1993Kato, M., Kaneharu, T., Shimizu, H., Suzuki, T., Gillies, F.M., Crozier, A., Ashihara, H., Caffeine biosynthesis in young leaves of Camellia sinensis: In vitro studies on N-methyltransferase activity involved in the conversion of xanthosine to caffeine (1996) Physiol. Plant., 98, pp. 629-636Kato, M., Mizuno, K., Crozier, A., Fujimura, T., Ashihara, H., Caffeine synthase gene from tea leaves (2000) Nature, 406, pp. 956-957Kretschmar, J.A., Baumann, T.W., Caffeine in Citrus flowers (1999) Phytochemistry, 52 (1), pp. 19-23. , DOI 10.1016/S0031-9422(99)00119-3, PII S0031942299001193Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4 (1970) Nature, 227, pp. 680-685Livak, K.J., Schmittgen, T.D., Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method (2001) Methods, 25 (4), pp. 402-408. , DOI 10.1006/meth.2001.1262Maravalhas, N., (1965) Estudos Sobre O Guaraná e Outras Plantas Produtoras de Cafeína, p. 25. , Instituto Nacional de Pesquisas da Amazônia (INPA) ManausMazzafera, P., Catabolism of caffeine in plants and microorganisms (2004) Frontiers in Bioscience, 9, pp. 1348-1359. , d1000-1499Mazzafera, P., Carvalho, A., Breeding for low seed caffeine content of coffee (Coffea L.) by interspecific hybridization (1991) Euphytica, 59, pp. 55-60Mazzafera, P., Crozier, A., Magalhães, A.C., Caffeine metabolism in Coffea arabica and other species of coffee (1991) Phytochemistry, 30, pp. 3913-3916Mazzafera, P., Crozier, A., Sandberg, G., Studies on the metabolic control of caffeine turnover in developing endosperms and leaves of Coffea arabica and Coffea dewevrei (1994) J. Agric. Food Chem., 42, pp. 1423-1427Mazzafera, P., Wingsle, G., Olsson, O., Sandberg, G., S adenosyl-l-methionine:theobromine 1-N-methyltransferase, an enzyme catalyzing the synthesis of caffeine in coffee (1994) Phytochemistry, 37, pp. 1577-1584McCarthy, A.A., McCarthy, J.G., The structure of two N-methyltransferases from the caffeine biosynthetic pathway (2007) Plant Physiology, 144 (2), pp. 879-889. , http://www.plantphysiol.org/cgi/reprint/144/2/879.pdf, DOI 10.1104/pp.106.094854(2012) Tropicos, , http://www.tropicos.org/, Missouri Botanical Garden (accessed in Feb., 2012)Mithöfer, A., Boland, W., Plant defense against herbivores: Chemical aspects (2012) Annu. Rev. Plant Biol., 63, pp. 431-450Mizuno, K., Kato, M., Irino, F., Yoneyama, N., Fujimura, T., Ashihara, H., The first committed step reaction of caffeine biosynthesis: 7-methylxanthosine synthase is closely homologous to caffeine synthases in coffee (Coffea arabica L.) (2003) FEBS Letters, 547 (1-3), pp. 56-60. , DOI 10.1016/S0014-5793(03)00670-7Mizuno, K., Okuda, A., Kato, M., Yoneyama, N., Tanaka, H., Ashihara, H., Fujimura, T., 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.) (2003) FEBS Letters, 534 (1-3), pp. 75-81. , DOI 10.1016/S0014-5793(02)03781-XNagata, T., Sakai, S., Purine base pattern of Camellia irrawadiensis (1985) Phytochemistry, 24, pp. 2271-2272Oliveira, E.R.N., (2010) Características Morfofisiológicas e Bioquímicas de Clones de Guaraná Paullinia Cupana Kunt. Var. Sorbilis (Mart.) Paullinia Cupana Ducke Cultivados Sob Plantio Comercial. Botânica, , Instituto Nacional de Pesquisas da Amazônia - INPA ManauPires, J.M., Guaraná e cupana (1949) Sociedade de Agrônomos e Veterinários Do Pará, 1, pp. 9-20Raju, K.I., Gopal, N.H., Distribution of caffeine in arabica and robusta coffee plants (1979) J. Coffee Res., 9, pp. 83-90Rezaian, M., Krake, L., Nucleic acid extraction and virus detection in grapevine (1987) J. Virol. Methods, 17Saitou, N., Nei, M., The neighbor-joining method: A new method for reconstructing phylogenetic trees (1987) Mol. Biol. Evol., 4, pp. 406-425Sartor, R.M., Mazzafera, P., Caffeine formation by suspension cultures of Coffea dewevrei (2000) Braz. Arch. Biol. Technol., 43, pp. 61-69Schimpl, F.C., (2013) Teores de Metilxantinas e Metabolismo de Cafeína em Frutos de Guaraná (Paullinia Cupana Var. Sorbilis Kunth.), p. 72. , Instituto de Biologia. Universidade Estadual de Campinas CampinasSchimpl, F.C., Da Silva, J.F., Gonçalves, D.F.J.C., Mazzafera, P., Guarana: Revisiting a highly caffeinated plant from the Amazon (2013) J. Ethnopharmacol., 150, pp. 14-31Schmidt, F., (1941) O Guaraná, Sua Cultura e Indústria, , Serviço de Informação Agrícola, Ministério da Agricultura Rio de JaneiroSenanayake, U., Wijesekera, R., Theobromine and caffeine content of the cocoa bean during its growth (1971) J. Sci. Food Agric., 22, pp. 262-263Silvarolla, M.B., Mazzafera, P., Fazuoli, L.C., A naturally decaffeinated arabica coffee (2004) Nature, 429 (6994), p. 826. , DOI 10.1038/429826aSpoladore, D.S.B., Antonio Milan, M., Sáes, Alberto, L., Teor de cafeína em sementes matrizes do guaranazeiro (1987) Bragantia, 46, pp. 425-429Suzuki, T., Participation of S-adenozylmethionine in biosynthesis of caffeine in tea plant (1972) FEBS Lett., 24, pp. 18-20Suzuki, T., Waller, G.R., Biodegradation of caffeine - Formation of theophylline and theobromine from caffeine in mature Coffea arabica fruits (1984) J. Sci. Food Agric., 35, pp. 66-70Suzuki, T., Waller, G.R., Biosynthesis and biodegradation of caffeine, theobromine, and theophylline in Coffea arabica L. Fruits (1984) J. Agric. Food Chem., 32, pp. 845-848Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods (2011) Mol. Biol. Evol., 28, pp. 2731-2739Thompson, J.D., Higgins, D.G., Gibson, T.J., CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice (1994) Nucleic Acids Research, 22 (22), pp. 4673-4680Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., Sano, H., Molecular cloning and functional characterization of three distinct N-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants (2003) Plant Physiology, 132 (1), pp. 372-380. , DOI 10.1104/pp.102.019679Weckerle, C.S., Stutz, M.A., Baumann, T.W., Purine alkaloids in Paullinia (2003) Phytochemistry, 64 (3), pp. 735-742. , DOI 10.1016/S0031-9422(03)00372-8Ye, C., Lin, Y., Zhou, H., Cheng, F., Li, X., Isolation and analysis of purine alkaloids from Camellia ptilophylla Chang (1997) Acta Sci. Nat. Univ. Sunyatseni, 36, pp. 30-33Yoneyama, N., Morimoto, H., Ye, C.-X., Ashihara, H., Mizuno, K., Kato, M., Substrate specificity of N-methyltransferase involved in purine alkaloids synthesis is dependent upon one amino acid residue of the enzyme (2006) Molecular Genetics and Genomics, 275 (2), pp. 125-135. , DOI 10.1007/s00438-005-0070-