Nutritional composition, bioactive compounds and volatile profile of cocoa beans from different regions of Cameroon.

Analysis of the complex composition of cocoa beans provides fundamental information for evaluating the quality and nutritional aspects of cocoa-based food products, nutraceuticals and supplements. Cameroon, the world's fourth largest producer of cocoa, has been defined as "Africa in miniature" because of the variety it habitats. In order to evaluate the nutritional characteristics of cocoa beans from five different regions of Cameroon, we studied their polyphenolic content, volatile compounds and fatty acids composition. The High Performance Thin Layer Chromatography (HPTLC) analysis showed that the Mbalmayo sample had the highest content of theobromine (11.6 mg/g) and caffeic acid (2.1 mg/g), while the Sanchou sample had the highest level of (-)-epicatechin (142.9 mg/g). Concerning fatty acids, the lowest level of stearic acid was found in the Mbalmayo sample while the Bertoua sample showed the highest content of oleic acid. Thus, we confirmed that geographical origin influences the quality and nutritional characteristics of cocoa from these regions of Cameroon.

Transcriptomic analysis of Camellia ptilophylla and identification of genes associated with flavonoid and caffeine biosynthesis.

Camellia ptilophylla, or cocoa tea, is naturally decaffeinated and its predominant catechins and purine alkaloids are trans-catechins and theobromine Regular tea [Camellia sinensis (L.) O. Ktze.] is evolutionarily close to cocoa tea and produces cis-catechins and caffeine. Here, the transcriptome of C. ptilophylla was sequenced using the 101-bp paired-end technique. The quality of the raw data was assessed to yield 70,227,953 cleaned reads totaling 7.09 Gbp, which were assembled de novo into 56,695 unique transcripts and then clustered into 44,749 unigenes. In catechin biosynthesis, leucoanthocyanidin reductase (LAR) catalyzes the transition of leucoanthocyanidin to trans-catechins, while anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR) catalyze cis-catechin production. Our data demonstrate that two LAR genes (CpLAR1 and CpLAR2) by C. ptilophylla may be advantageous due to the combined effects of this quantitative trait, permitting increased leucoanthocyanidin consumption for the synthesis of trans-catechins. In contrast, the only ANS gene observed in C. sinensis (CsANS) shared high identity (99.2%) to one homolog from C. ptilophylla (CpANS1), but lower identity (~80%) to another (CpANS2). We hypothesized that the diverged CpANS2 might have lost its ability to synthesize cis-catechins. C. ptilophylla and C. sinensis each contain two copies of ANR, which share high identity and may share the same function. Transcriptomic sequencing captured two N-methyl nucleosidase genes named NMT1 and NMT2. NMT2 was highly identical to three orthologous genes TCS2, PCS2, and ICS2, which did not undergo methylation in vitro; in contrast, NMT1 was less identical to TCS, PCS and ICS, indicating that NMT1 may undergo neofunctionalization.

Involvement of a novel intronic microRNA in cross regulation of N-methyltransferase genes involved in caffeine biosynthesis in Coffea canephora.

There are numerous reports on intronic miRNAs from plants, most of which are involved in the regulation of unrelated genes. Some of the target genes are antagonistic to the host genes. Intronic miRNAs in animal systems, however, are known to have synergistic effects. This article is the first to report a similar regulatory effect of a miRNA originating from an intron in plants. NMT genes involved in caffeine biosynthesis were silenced to obtain transformants with reduced caffeine. Transcript analysis revealed the accumulation of transcripts for a related NMT gene (CaMTL1) in transformants bearing either antisense or RNAi constructs. The altered expression was assumed to relate to the silencing of the NMT genes. Bioinformatics analysis of the genes involved in biosynthesis revealed the presence of an intronic miRNA originating from the intron of the theobromine synthase gene targeting CaMTL1. The putative miRNA was cloned and sequenced. Modified 5'-RLM-RACE mapping of the cleavage site and subsequent Northern blotting experimentally demonstrated the presence and activity of such a miRNA in Coffea canephora. This novel regulatory mechanism previously unreported in plants will shed more light onto the evolution of multigene families and the role of introns in this process.

Agrobacterium-mediated silencing of caffeine synthesis through root transformation in Camellia sinensis L.

Tea [Camellia sinensis (L.) O. Kuntze] is a perennial and most popular non-alcoholic caffeine-containing beverage crop. Tea has several constraints for its genetic improvement such as its high polyphenolic content and woody perennial nature. The development of transgenic tea is very difficult, laborious, and time taking process. In tea, regeneration requires minimum 8-12 months. In view of this, attempt has been made in this article to develop a rapid, efficient, and quite economical Agrobacterium-mediated root transformation system for tea. The feasibility of the developed protocol has been documented through silencing caffeine biosynthesis. For this, one-month-old tea seedlings were exposed to fresh wounding at the elongation zone of roots and were inoculated with Agrobacterium tumefaciens cultures carrying a RNAi construct (pFGC1008-CS). The pFGC1008-CS contained 376 bp of caffeine synthase (CS) cDNA fragment in sense and antisense direction with an intron in between. This has made the RNAi construct to produce a hairpin RNA (ihpRNA). The suppressed expression of CS gene and a marked reduction in caffeine and theobromine contents in young shoots of tea seedlings were obtained after root transformation through Agrobacterium infiltration. Such transformation system could be useful for functional analysis of genes in tea like woody and perennial plants.

Occurrence of theobromine synthase genes in purine alkaloid-free species of Camellia plants.

Caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine) are purine alkaloids that are present in high concentrations in plants of some species of Camellia. However, most members of the genus Camellia contain no purine alkaloids. Tracer experiments using [8-(14)C]adenine and [8-(14)C]theobromine showed that the purine alkaloid pathway is not fully functional in leaves of purine alkaloid-free species. In five species of purine alkaloid-free Camellia plants, sufficient evidence was obtained to show the occurrence of genes that are homologous to caffeine synthase. Recombinant enzymes derived from purine alkaloid-free species showed only theobromine synthase activity. Unlike the caffeine synthase gene, these genes were expressed more strongly in mature tissue than in young tissue.

Substrate specificity of N-methyltransferase involved in purine alkaloids synthesis is dependent upon one amino acid residue of the enzyme.

Caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine) are the major purine alkaloids in plants. To investigate the diversity of N-methyltransferases involved in purine alkaloid biosynthesis, we isolated the genes homologous for caffeine synthase from theobromine-accumulating plants. The predicted amino acid sequences of N-methyltransferases in theobromine-accumulating species in Camellia were more than 80% identical to caffeine synthase in C. sinensis. However, there was a little homology among the N-methyltransferases between Camellia and Theobroma. The recombinant enzymes derived from theobromine-accumulating plants had only 3-N-methyltransferase activity. The accumulation of purine alkaloids was, therefore, dependent on the substrate specificity of N-methyltransferase determined by one amino acid residue in the central part of the protein.

Biosynthesis, accumulation and degradation of theobromine in developing Theobroma cacao fruits.

We have studied the purine alkaloid content and purine metabolism in Theobroma cacao fruits at differing growth stages: Stage A (young small fruit, fresh weight, ca. 2 g); stage B (medium size fruit, fresh weight, ca. 100 g) and stage C (large size, fresh weight, ca. 500 g). The major purine alkaloid in stage A fruits (mainly pericarp) was theobromine (0.7 micromol g(-1) fresh weight), followed by caffeine (0.09 micromol g(-1) fresh weight). The theobromine content of the pericarp decreased sharply with tissue age, and the caffeine content decreased gradually. A large amount of theobromine (22 micromol g(-1) fresh weight) had accumulated in seeds (mainly cotyledons) of stage C fruits. Theobromine was found also in the seed coat and placenta. Tracer experiments with [8-(14)C]adenine show that the major sites of theobromine synthesis are the young pericarp and cotyledons of T. cacao fruits. Limited amounts of purine alkaloids may be transported from the pericarp to seed tissue, but most purine alkaloids that accumulated in seeds appeared to be synthesised in cotyledons. Degradation of [8-(14)C]theobromine and [8-(14)C]caffeine to CO2 via 3-methylxanthine and ureides (allantoin and allantoic acid) was detected only in the pericarp of stage C fruits.

Biosynthesis of caffeine by tea-leaf extracts. Enzymic formation of theobromine from 7-methylxanthine and of caffeine from theobromine.

1. Extracts prepared from tea leaves with Polyclar AT (insoluble polyvinylpyrrolidine) contained two methyltransferase activities catalysing the transfer of methyl groups from S-adenosylmethionine to 7-methylxanthine, producing theobromine, and to theobromine, producing caffeine. 2. The methyltransferases exhibited the same pH optimum (8.4) and a similar pattern of effects by metal ions, thiol inhibitors and metal-chelating reagents, both for theobromine and caffeine synthesis. Mg2+, Mn2+ and Ca2+ slightly stimulated enzyme activity but they were not essential. Paraxanthine was shown to be most active among methylxanthines, as the methyl acceptor. However, the formation of paraxanthine from 1-methylxanthine was very low and that from 7-methylxanthine was nil, suggesting that the synthesis of caffeine from paraxanthine is of little importance in intact plants. Xanthine, xanthosine, XMP and hypoxanthine were all inactive as methyl acceptors, whereas [2(-14)C]xanthine and [8(-14)C]hypoxanthine were catabolized to allantoin and urea by tea-leaf extracts. The apparent Km values are as follows: 7-methylxanthine, 1.0 times 10(-14)M; theobromine, 1.0 times 10(-3)M; paraxanthine, 0.2 times 10(-3)M; S-adenosylmethionine, 0.25 times 10(-4)M (with each of the three substrates). 3. The results suggest that the pathway for caffeine biosynthesis is as follows: 7-methylxanthine leads to theobromine leads to caffeine. In contrast, it is suggested that theophylline is synthesized from 1-methylxanthine. The methyl groups of the purine ring of caffeine are all derived directly from the methyl group of S-adenosylmethionine. Little is known about the pathways leading to the formation of 7-methylxanthine. 4. A good correlation between caffeine synthesis and shoot formation or growth of tea seedlings was shown, suggesting that the methylating systems in caffeine synthesis are closely associated with purine nucleotide and nucleic acid metabolism in tea plants.

Biosynthesis of caffeine in tea callus tissue.

1. A study of caffeine biosynthesis has been made by following the incorporation of radioactive carbon dioxide and methionine into the methylated purines produced by tea callus tissue. 2. The uptake of the radioactive labels into nucleic acid and caffeine was followed over a period of approximately 9h. 3. The distribution of the radioactive labels in both nucleic acid and caffeine was determined after incorporation and subsequent incubation of the tissue in a non-radioactive medium. 4. The results of the experiments indicated that the caffeine arose from purines released from the breakdown of nucleic acids rather than that it was formed directly from a purine pool. 5. A metabolic scheme to show the production of caffeine from the nucleotides of the nucleic acid is discussed.