Caffeine biosynthesis and adenine metabolism in transgenic Coffea canephora plants with reduced expression of N-methyltransferase genes

In anti-sense and RNA interference transgenic plants of Coffea canephora in which the expression of CaMXMT1 was suppressed, caffeine biosynthesis from [8-(14)C]adenine was investigated, together with the overall metabolism of [8-(14)C]adenine. Compared with wild type control plants, total purine alkaloid biosynthesis from adenine and conversion of theobromine to caffeine were both reduced in the transgenic plants. As found previously, [8-(14)C]adenine was metabolised to salvage products (nucleotides and RNA), to degradation products (ureides and CO(2)) and to purine alkaloids (theobromine and caffeine). In the transgenic plants, metabolism of [8-(14)C]adenine shifted from purine alkaloid synthesis to purine catabolism or salvage for nucleotides. HPLC analysis revealed a significantly reduced caffeine content in the transgenic plants. A small quantity (less than 20 nmol g(-1) fresh weight) of xanthosine had accumulated in at least one of the transgenic plants.     

Theobromine, theophylline, and caffeine in 42 samples and products of Guaraná (Paullinia cupana, Sapindaceae)

Guaranás, the carbonated beverages (sodas) of choice throughout much of Brazil, are mandated by Brazilian law to contain at least 300 mg guara’a (Paullinia cupana, Sapindaceae) seed per 100 ml soda. Were all the soda manufacturers adhering to the law, they would be consuming almost three times the annual production of seed. Guaraná seeds contain unusually high levels of caffeine, along with smaller amounts of the related purine alkaloids theobromine and theophylline. We investigated the purine alkaloid content of three ethnobotanical guaraná collections and 39 commercial products using HPLC/UV. Many of the products did contain caffeine as the major alkaloid, with traces of theobromine and theophylline. Numerous sodas and syrups contained up to ten times more theobromine than caffeine, and we suspect that these products were adulterated with cacao (Theobroma cacao, Sterculiaceae), the major source of theobromine.     

Metabolism of purine bases, nucleosides and alkaloids in theobromine-forming Theobroma cacao leaves

We examined the purine alkaloid content and purine metabolism in cacao (Theobroma cacao L.) plant leaves at various ages: young small leaves (stage I), developing intermediate size leaves (stage II), fully developed leaves (stage III) from flush shoots, and aged leaves (stage IV) from 1-year-old shoots. The major purine alkaloid in stage I leaves was theobromine (4.5 μmol g^–1 fresh weight), followed by caffeine (0.75 μmol g^–1 fresh weight). More than 75% of purine alkaloids disappeared with subsequent leaf development (stages II–IV). In stage I leaves, ¹⁴C-labelled adenine, adenosine, guanine, guanosine, hypoxanthine and inosine were converted to salvage products (nucleotides and nucleic acids), to degradation products (ureides and CO₂) and to purine alkaloids (3- and 7-methylxanthine, 7-methylxanthosine and theobromine). In contrast, ¹⁴C-labelled xanthine and xanthosine were not used for nucleotide synthesis. They were completely degraded, but nearly 20% of [8-¹⁴C]Xanthosine was converted in stage I leaves to purine alkaloids. These observations are consistent with the following biosynthetic pathways for theobromine: (a) AMP → IMP → 5′-xanthosine monophosphate → xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine; (b) GMP → guanosine → xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine; (c) xanthine → 3-methylxanthine → theobromine. Although no caffeine biosynthesis from ¹⁴C-labelled purine bases and nucleosides was observed during 18 h incubations, exogenously supplied [8-¹⁴C]Theobromine was converted to caffeine in young leaves. Conversion of theobromine to caffeine may, therefore, be slow in cacao leaves. No purine alkaloid synthesis was observed in the subsequent growth stages (stages II–IV). Significant degradation of purine alkaloids was found in leaves of stages II and III, in which [8-¹⁴C]Theobromine was degraded to CO₂ via 3-methylxanthine, xanthine and allantoic acid. [8-¹⁴C]Caffeine was catabolised to CO₂ via theophylline (1,3-dimethylxanthine) or theobromine.     

Biosynthesis and metabolism of purine alkaloids in leaves of cocoa tea (Camellia ptilophylla)

The biosynthesis and metabolism of purine alkaloids in leaves ofCamellia ptilophylla (cocoa tea), a new tea resource in China, have been investigated. The major purine alkaloid was theobromine, with theophylline also being present as a minor component. Caffeine was not accumulated in detectable quantities. Theobromine was synthesized from [8-¹⁴C] adenine and the rate of its biosynthesis in the segments from young and mature leaves from flush shoots was approximately 10 times higher than that from aged leaves from 1-year old shoots. Neither cellfree extracts nor segments fromC. ptilophylla leaves could convert theobromine to caffeine. A large quantity of [2-¹⁴C] xanthine taken up by the leaf segments was degraded to¹⁴CO₂ via the conventional purine catabolic pathway that includes allantoin as an intermediate. However, small amounts of [2-¹⁴C] xanthine were also converted to theobromine. Considerable amounts of [8-¹⁴C] caffeine exogenously supplied to the leaf segments ofC. ptilophylla was changed to theobromine. These results indicate that leaves ofC. ptilophylla exhibit unusual purine alkaloid metabolism as i) they have the capacity to synthesize theobromine from adenine nucleotides, but they lack adequate methyltransferase activity to convert of theobromine to caffeine in detectable quantities, ii) the leaves have a capacity to convert xanthine to theobromine, probably via 3-methylxanthine.     

Theacrine (1,3,7,9-tetramethyluric acid) synthesis in leaves of a Chinese tea,kucha (Camellia assamica var. kucha)

Theacrine (1,3,7,9-tetramethyluric acid) and caffeine were the major purine alkaloids in the leaves of an unusual Chinese tea known as kucha (Camellia assamica var. kucha). Endogenous levels of theacrine and caffeine in expanding buds and young leaves were ca. 2.8 and 0.6-2.7% of the dry wt, respectively, but the concentrations were lower in the mature leaves. Radioactivity from S-adenosyl-L-[methyl-14C]methionine was incorporated into theacrine as well as theobromine and caffeine by leaf disks of kucha, indicating that S-adenosyl-L-methionine acts as the methyl donor not only for caffeine biosynthesis but also for theacrine production. [8-14C]Caffeine was converted to theacrine by kucha leaves with highest incorporation occurring in expanding buds. When [8-14C]adenosine, the most effective purine precursor for caffeine biosynthesis in tea (Camellia sinensis), was incubated with young kucha leaves for 24 h, up to 1% of total radioactivity was recovered in theacrine. However, pulse-chase experiments with [8-14C]adenosine demonstrated much more extensive incorporation of label into caffeine than theacrine, possibly because of dilution of [14C]caffeine produced by the large endogenous caffeine pool. These results indicate that in kucha leaves theacrine is synthesized from caffeine in what is probably a three-step pathway with 1,3,7-methyluric acid acting an intermediate. This is a first demonstration that theacrine is synthesized from adenosine via caffeine.     

Occurrence of theobromine synthase genes in purine alkaloid-free species of <Emphasis Type="Italic">Camellia</Emphasis> 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 [8-¹⁴C]adenine and [8-¹⁴C]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. The nucleotide sequence data reported here have been deposited in the GenBank database under the accession numbers AB297451 (CjCS1), AB362882 (CgCS1), AB362883 (CgCS2), AB362884 (CkCS1), AB362885 (ClCS1), and AB362886 (CcCS2).     

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.     

可可茶经栽培后化学成分的变化及其与传统茶的比较分析

野生茶树可可茶(Camellia Ptilophylla Chang)由于其芽叶中的嘌呤生物碱主要为可可碱,因而不同于传统茶叶。通过化学筛选,在纯种无性苗建立的可可茶基地上,随机进行单株取样,对可可茶的水浸出物、游离氨基酸、水溶性糖、茶多酚、儿茶素类、花青素、嘌呤生物碱等成分进行了检测。将这些结果与野生可可茶相关成份进行比较,发现可可茶经人工栽培后,含优势可可碱的特点保持不变,游离氨基酸总量和儿茶素类含量得到了明显的提高。进一步与传统茶叶比较后,得出两者之间的最大差异是可可茶含可可碱,不含咖啡碱;传统茶叶含咖啡碱为主,同时伴生相当于0．5～1％咖啡碱量的可可碱。     

Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering

Details of the recently elucidated biosynthetic pathways of caffeine and related purine alkaloids are reviewed. The main caffeine biosynthetic pathway is a sequence consisting of xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine. Genes encoding N-methyltransferases involved in three of these four reactions have been isolated and the molecular structure of N-methyltransferases investigated. Pathways for the catabolism of caffeine have also been studied, although there are currently no reports of enzymatic and genetic studies having been successfully carried out. Metabolism of purine alkaloids in species including Camellia, Coffea, Theobroma and Ilex plants is summarised, and evidence for the involvement of caffeine in chemical defense and allelopathy is discussed. Finally, information is presented on metabolic engineering that has produced coffee seedlings with reduced caffeine content, and transgenic caffeine-producing tobacco plants with enhanced disease resistance.     

Caffeine biosynthesis in Camellia sinensis

The metabolism of adenine and guanine, relating to the biosynthesis of caffeine, in excised shoot tips of tea was studied with micromolar amounts of adenine-[8-¹⁴C] or guanine-[8-¹⁴C]. Among the presumed precursors of caffeine biosynthesis, adenine was the most effective, whereas guanine was the least effective. After administration of a ‘pulse’ of adenine-[8-¹⁴C], almost all of the adenine-[¹⁴C] supplied disappeared by 30 hr, and ¹⁴C-labelled caffeine and RNA purine nucleotide (AMP and GMP) synthesis increased throughout the experimental period, whereas the radioactivities of free purine nucleotides, 7-methylxanthine and theobromine increased during the first 10 hr incubation period, followed by a steady decrease. By contrast, more than 45% of the guanine-[8-¹⁴C] supplied remained unchanged even after a 120 hr period. The main products of guanine-[8-¹⁴C] metabolism in tea shoot tips were guanine nucleotides, theobromine, caffeine and the GMP of RNA. The results support the hypothesis that the purine nucleotides are synthesized from adenine and guanine via the pathway of purine salvage. Adenylate is readily converted into other purine nucleotides, whereas the conversion rate of guanylate into other purine nucleotides is very low.The results also support the view that 7-methylxanthine and theobromine are precursors of caffeine. For the origin of the purine ring in caffeine, purine nucleotides in the nucleotide pool rather than in nucleic acids are suggested.     

Pathogen resistance of transgenic tobacco plants producing caffeine

Caffeine (1,3,7-trimethylxanthine) is a typical purine alkaloid, and produced by a variety of plants such as coffee and tea. Its physiological function, however, is not completely understood, but chemical defense against pathogens and herbivores, and allelopathic effects against competing plant species have been proposed. Previously, we constructed transgenic tobacco plants, which produced caffeine up to 5 microg per gram fresh weight of leaves, and showed them to repel caterpillars of tobacco cutworms (Spodoptera litura). In the present study, we found that these transgenic plants constitutively expressed defense-related genes encoding pathogenesis-related (PR)-1a and proteinase inhibitor II under non-stressed conditions. We also found that they were highly resistant against pathogens, tobacco mosaic virus and Pseudomonas syringae. Expression of PR-1a and PR-2 was higher in transgenic plants than in wild-type plants during infection. Exogenously applied caffeine to wild-type tobacco leaves exhibited the similar resistant activity. These results suggested that caffeine stimulated endogenous defense system of host plants through directly or indirectly activating gene expression. This assumption is essentially consistent with the idea of chemical defense, in which caffeine may act as one of signaling molecules to activate defense response. It is thus conceivable that the effect of caffeine is bifunctional; direct interference with pest metabolic pathways, and activation of host defense systems.     

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.     

Catabolism of caffeine and related purine alkaloids in leaves ofCoffea arabica L.

In a study of purine alkaloid catabolism pathways in coffee,¹⁴C-labelled theobromine, caffeine, theophylline and xanthine were incubated with leaves ofCoffea arabica. Incorporation of label into¹⁴CO₂ was determined and methanol-soluble metabolites were analysed by high-performance liquid chromatography-radiocounting. The data obtained demonstrate catabolism of caffeine theophylline 3-methylxanthine xanthine. Xanthine is degraded further by the conventional purine catabolism pathway to CO₂ and NH₃ via uric acid, allantoin and allantoic acid. The conversion of caffeine to theophylline is the rate-limiting step in purine alkaloid catabolism and provides a ready explanation for the high concentration of endogenous caffeine found inC. arabica leaves. Although theobromine is converted primarily to caffeine, a small portion of the theobromine pool appears to be degraded to xanthine by a caffeine-independent pathway. In addition to being broken down to CO₂, via the purine catabolism pathway, xanthine is metabolised to 7-methylxanthine. Metabolism of [2-¹⁴C]xanthine byC. arabica leaves in the presence of 5 mM allopurinol results in very large increases in incorporation of radioactivity into 7-methylxanthine as degradation of the substrate via the purine catabolism pathway is blocked. The identity of 7-methylxanthine in these studies was confirmed by gas chromatography-mass spectrometry analysis.Abbreviations HPLC-RC high-performance liquid chromatography-radiocounting This work was supported by the British Council which provided H.A. with Japan-UK travel grants. F.M.G. was supported by a Biotechnology and Biological Sciences Research Council grant to A.C.     

Metabolism of Caffeine and Related Purine Alkaloids in Leaves of Tea (Camellia sinensis L.)

Purine alkaloid catabolism pathways in young, mature and agedleaves of tea (Camellia sinensis L.) were investigated by incubatingleaf sections with ¹⁴C-labelled theobromine, caffeine, theophyllineand xanthine. Incorporation of label into CO₂ was determinedand methanol-soluble metabolites were analysed by high-performanceliquid chromatography-radiocounting and thin layer chro-matography.The data obtained demonstrate that theobromine is the immediateprecursor of caffeine, which accumulates in tea leaves becauseits conversion to theophylline is the rate limiting step inthe purine alkaloid catabolism pathway. The main fate of [8-¹⁴C]theophyllineincubated with mature and aged leaves, and to a lesser extentyoung leaves, is conversion to 3-methylxanthine and onto xanthinewhich is degraded to ¹⁴CO₂ via the purine catabolism pathway.However, with young leaves, sizable amounts of [8-¹⁴C]-theophyllinewere salvaged for the synthesis of caffeine via a 3-methylxanthine     

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.     

Cultivable bacterial composition and BIOLOG catabolic diversity of biofilm communities developed on Phragmites australis

Biofilm samples formed on submerged young and old stems of reed, Phragmites australis (Cav.) Trin ex Steudel were taken during summer at different sites of Lake Velencei, Hungary. BIOLOG GN microplates were used to analyze the patterns of sole carbon source utilizations by microbial communities. From the carbon sources, carbohydrates and amino acids were preferred by all microbial communities. In the case of the old reed stem samples, higher number of carbohydrates, carboxylic acids and polymers were used than in young samples. Biofilm bacterial communities from the old reed samples of the nature conservation area of the lake used the highest number of (≥50% of the available) substrates. In principal component analysis (PCA), the metabolic potential of the microbial communities from the middle open water region of the lake showed the smallest variability. The variability within metabolic potential of the reed stem microbial communities from a given sampling site was the largest in the case of samples originating from the western, reed-covered nature conservation area. A total of 251 bacterial isolates obtained after serial dilutions and plating onto different media were characterized by traditional phenotypic tests. The strains showed high activities mainly in the hydrolysis of certain biopolymers (gelatine and casein). PCA was used to evaluate the phenotypic variability of strain groups of different sampling sites. The two open water regions were similar to each other, and separated from the western reed covered part of the lake. Similarly to the BIOLOG community-level physiological profiles, strain groups of the young and old reed stem samples originating from the nature conservation area had the largest metabolic potential. On the basis of 16S rDNA sequence analysis, 23 representative strains with different ARDRA patterns were identified. The cultivation-based investigations of bacterial diversity showed characteristic differences in the number of identified taxa in connection with the sampling sites. No characteristic differences could be observed according to medium or sample type (young, first year and more than 1-year old stems) among the identified species. 16S rDNA sequence comparisons resulted in the identification of the genera Aureobacterium, Arthrobacter, Kocuria, Microbacterium, Micrococcus, Rhodococcus, Bacillus, Marinibacillus, Rhodobacter, Defluvibacter, Pseudomonas, Klebsiella, Serratia and Aeromonas. The results of the cultivation-based and BIOLOG investigations revealed characteristic differences in the bacterial community composition and activities of the open water region and the reed covered nature conservation part of the lake.     

CaXMT和TCS1突变体基因串联共表达及体外酶活检测分析

咖啡碱和可可碱是茶叶生物碱的主要组分,且咖啡碱是茶叶重要的滋味物质,随着咖啡碱在食品和药物领域的应用愈发广泛,咖啡碱的生物合成成为新的研究热点.目前市场上的咖啡碱主要靠化学合成,为了探索其生物合成途径,该研究将咖啡黄嘌呤核苷甲基转移酶(coffee xanthosine methyltransferase,CaXMT)基因和茶树咖啡碱合成酶(tea caffeine synthase,TCS1)基因的4个突变体分别串联至同一大肠杆菌表达载体pMAL-c5X,诱导融合蛋白共表达,并进行SDS-PAGE凝胶电泳分析.结果表明:目的蛋白成功表达后,应用超声破碎法制备含有目的蛋白的粗酶液,添加底物黄嘌呤核苷(xanthosine,XR)和甲基供体S-腺苷甲硫氨酸(S-adenosyl-L-methionine,SAM)进行体外酶促反应,将反应产物进行高效液相色谱检测.检测结果显示,pMAL-CaXMT-TM2/3/4的体外酶促反应产物仅有可可碱生成,均未见咖啡碱生成.该研究结果为构建生物合成咖啡碱和可可碱的串联共表达载体奠定了基础,也为进一步研究生物合成咖啡碱和可可碱提供了新思路.     

Metabolic Relations between Methylxanthines and Methyluric Acids in Coffea L

Metabolism of purine alkaloids in the leaves of Coffea dewevrei De Wild et Durand var excelsa Chev, Coffea liberica Bull ex Hiern and Coffea abeokutae Cramer was studied by analyzing leaf discs collected during vegetative development and by feeding the following radioactive tracers: [¹⁴C]theobromine, [¹⁴C]caffeine, and [¹⁴C]theacrine (1,3,7,9-tetramethyluric acid). Their principal metabolites were quantitatively and qualitatively determined. All three species convert the precursors to the same radioactive products, and proceed through the same four maturity stages characterized by the alkaloid accumulation pattern and by a particular transformation potency: (stage 1) young plant accumulating caffeine, transforms theobromine to caffeine; (stage 2) caffeine is gradually replaced by theacrine, theobromine and caffeine are converted to theacrine; (stage 3) theacrine disappears whereas liberine (O(2), 1,9-thrimethyluric acid) accumulates, theacrine is metabolized to liberine; (stage 4) branched-out plant containing liberine but no theacrine, caffeine is converted rapidly to liberine via theacrine. Methylliberine (O(2),1,7,9-tetramethyluric acid), presumably the direct precursor of liberine, is occasionally found in low concentrations at stage 3 and 4.

The collective term `liberio-excelsoid' introduced by geneticists for the numerous races or species of Pachycoffea is in accordance with the phytochemical equality found in this work.

     

Purine Alkaloid Production and Accumulation in Cocoa Callus and Suspension Cultures

Cocoa callus and suspension cultures were found to produce caffeine,theobromine, and theophylline which are typical of the purinealkaloids found in cocoa beans. Production of these purine alkaloidswas monitored in callus cultures for over 2 years and shownto stabilize at concentrations of about 10% those found in vivo.Caffeine and theobromine were produced concomitant with logphase growth of the cultures whilst theophylline productionreached a maximum during stationary phase, reflecting the possiblerole of the latter as a catabolite of caffeine. The effectsof choice of cytokinin, explant tissue, cocoa type, light conditionsand time in culture on purine alkaloid production by callushave been examined. Purine alkaloid production by cocoa suspensioncultures has also been examined and these cultures were shownto be less productive and more variable than callus cultures.The results demonstrate that cocoa tissue cultures can be usefulfor studying secondary metabolism in vitro. Key words: Theobroma cacao, caffeine, theobromine, tissue culture, secondary metabolism     

Unification of the genera Nonlabens, Persicivirga, Sandarakinotalea and Stenothermobacter into a single emended genus, Nonlabens, and description of Nonlabens agnitus sp. nov

Phylogenetic analyses based on 16S rRNA gene sequences showed that a bacterial isolate, designated JC2678(T), represents a distinct phyletic line within the suprageneric monophyletic clade containing the genera Nonlabens, Persicivirga, Stenothermobacter and Sandarakinotalea. The polyphasic data presented in this study demonstrated that the members belonging to the Nonlabens-like clade overall constitute a single genus. Therefore, it is proposed to transfer the members of genera Persicivirga O'Sullivan et al. 2006, Stenothermobacter Lau et al. 2006 and Sandarakinotalea Khan et al. 2006 to the genus Nonlabens Lau et al. 2005. Thus, P. dokdonensis (Yoon et al. 2006) Nedashkovskaya et al. 2009, P. ulvanivorans Barbeyron et al. 2010, P. xylanidelens O'Sullivan et al. 2006, Sandarakinotalea sediminis Khan et al. 2006 and Stenothermobacter spongiae Lau et al. 2006 should be transferred to Nonlabens dokdonensis comb. nov., Nonlabens ulvanivorans comb. nov., Nonlabens xylanidelens comb. nov., Nonlabens sediminis comb. nov. and Nonlabens spongiae comb. nov., respectively. In addition, strain JC2678(T) (=KACC 14155(T)=JCM 17109(T)) is proposed to constitute a novel species belonging to the genus Nonlabens with the name of Nonlabens agnitus sp. nov.