N-methyltransferases and 7-methyl-N9-nucleoside hydrolase activity in Coffea arabica and the biosynthesis of caffeine

The incorporation of radioactivity from L-[¹⁴CH₃]-methionine into caffeine by coffee fruits was enhanced by additions of theobromine and paraxanthine but was reduced by additions of theophylline and caffeine. Cell-free extracts prepared from seedlings, partially ripe and unripe coffee fruits showed that only the unripe green fruits contained significant methyltransferase and 7-methyl-N⁹-nucleoside hydrolase activity. The cell-free extracts catalysed the transfer of methyl groups fromS-adenosyl-L-[¹⁴CH₃]-methionine to 7-methylxanthine, and 7-methylxanthosine, producing theobromine and to theobromine producing caffeine. The two enzymic methylations exhibited a sharp pH max at 8.5 and a similar pattern of effects with metal chelators, thiol reagents and Mg²⁺ ions, which were slightly stimulating though not essential to enzyme activity. Paraxanthine (1,7-dimethylxanthine) was sh own to be the most active among methylxanthines as methyl acceptors; however its formation from 1-methylxanthine and 7-methylxanthine was not detectable, and biosynthesis from paraxanthine in the intact plant would therefore appear not to occur. The apparent K_m values are as follows: 7-methylxanthine 0.2 mM, theobromine 0.2 mM, paraxanthine 0.07 mM and S-adenosyl-L-methionine with each substrate 0.01 mM. The results suggest the pathway for caffeine biosynthesis in Coffea arabica is: 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine.     

7-methylxanthosine—An intermediate in caffeine biosynthesis

Ring-labelled 7-methylxanthosine was synthesized and fed to leaves of Coffea arabica. Up to 26% of the tracer was converted into caffeine within 8 days.     

Changes in the composition of coffee leaf wax with development

Coffee leaf wax contains alkanes, free primary alcohols and free acids, together with unidentified substances. The relative amounts of each fraction varied with age: alkanes from 22–35% alcohols from 28-25%, and free acids from 22-14%. The major homologues of the alkane fraction were C₂₉ and C₃₁, of the alcohol fraction C₃₀ and C₃₂ and of the acid fraction C₂₈, C₃₀ and C₃₂. The ratio of both C₂₉:C₃₁ alkanes and C_3O:C₃₂ alcohols changed from 1:1 to 1:2 during development, although their combined sum in each case remained constant at 90% (for alkanes) and 73% (for alcohols) of the weight of the respective fractions.     

Stress induced formation of purine alkaloids in plant tissue culture of Coffea arabica

Reports that environmental stress may enhance the accumulation of secondary substances in plants led to the idea of introducing stress into tissue cultures with the aim of improving the in vitro production of pharmaceutically active compounds. The test was made with low- and high-producing cell suspension cultures of Coffea arabica. The production of the purine alkaloid caffeine was shown to be stimulated by stressors such as high light intensity and—depending on the culture type—high NACl concentration.     

3-O-feruloyl-4-O-caffeoylquinic acid from coffee beans

A new phenolic ester was isolated from unroasted robusta coffee beans (Coffea canephora) by HPLC. The isolated compound was identified as an ester of caffeic acid and ferulic acid with quinic acid (3-O-feruloyl-4-O-caffeoylquinic acid) using ¹H NMR and mass spectroscopy.     

Caffeine adsorption of montmorillonite in coffee extracts

The growth in health-conscious consumers continues to drive the demand for a wide variety of decaffeinated beverages. We previously developed a new technology using montmorillonite (MMT) in selective decaffeination of tea extract. This study evaluated and compared decaffeination of coffee extract using MMT and activated carbon (AC). MMT adsorbed caffeine without significant adsorption of caffeoylquinic acids (CQAs), feruloylquinic acids (FQAs), dicaffeoylquinic acids (di-CQAs), or caffeoylquinic lactones (CQLs). AC adsorbed caffeine, chlorogenic acids (CGAs) and CQLs simultaneously. The results suggested that the adsorption selectivity for caffeine in coffee extract is higher in MMT than AC. The caffeine adsorption isotherms of MMT in coffee extract fitted well to the Langmuir adsorption model. The adsorption properties in coffee extracts from the same species were comparable, regardless of roasting level and locality of growth. Our findings suggest that MMT is a useful adsorbent in the decaffeination of a wide range of coffee extracts.     

Identification of a coffee berry borer‐associated yeast: does it break down caffeine?

Two yeasts isolated from laboratory reared adult coffee berry borers [Hypothenemus hampei (Ferrari) (Coleoptera: Scolytidae)] and from insects collected in the field in Colombia were identified as Pichia burtonii Boidin and Candida fermentati (Saito) Bai, based on sequencing of the nuclear large subunit 26S rDNA variable D1/D2 domain. Liquid culture experiments using P. burtonii in media containing different caffeine levels indicated that caffeine levels in a range found within coffee seeds can retard yeast growth. HPLC analysis shows that P. burtonii does not break down caffeine.     

Intermediacy of iridodial in the biosynthesis of some iridoid glucosides

Administration of MVA-[2-¹⁴C] to Lamium applexicaule and Deutzia crenata as well as of 11-hydroxyiridodial glucoside-[10-³H] and 7-deoxyloganic acid-[10-³H] to the former plant suggested that, in contrast to secoiridoid-indole alkaloids, iridoid glucosides such as ipolamiide, lamiide, lamioside, deutzioside and scabroside in these plants are biosynthesized via iridodial. Iridodial as a precursor for the biosynthesis of asperuloside was also suggested from the results of the administration of MVA-[2-¹⁴C] to Galium spurium var. echinospermon.     

Conflicting phylogenetic signals in genomic data of the coffee family (Rubiaceae)

Reconstructions of phylogenetic relationships in the flowering plant family Rubiaceae have up until now relied heavily on single‐ or multi‐gene data, primarily from the plastid compartment. With the availability of cost‐ and time‐efficient techniques for generating complete genome sequences, the opportunity arises to resolve some of the relationships that, up until now, have proven problematic. Here, we contribute new data from complete 58 plastid genome sequences, representing 55 of the currently 65 recognized tribes of the Rubiaceae. Also contributed are new data from the nuclear rDNA cistrons for corresponding taxa. Phylogenetic analyses are conducted on two plastid data sets, one including data from the protein coding genes only, and a second where protein coding data are combined with non‐coding regions, and on a nuclear rDNA data set. Our results clearly show that simply adopting a “more characters” approach does not resolve the relationships in the Rubiaceae. More importantly, we identify conflicting phylogenetic signals in the data. Analyses of the same plastid data, treated as nucleotides or as codon‐degenerated data, resolve and support conflicting topologies in the subfamily Cinchonoideae. As these analyses use the same data, we interpret the conflict to result from erroneous assumptions in the models used to reconstruct our phylogenies. Conflicting signals are also identified in the analyses of the plastid versus the nuclear rDNA data sets. These analyses use data from different genomic compartments, with different inheritance patterns, and we interpret the conflicts as representing “real” conflicts, reflecting biological processes of the past.     

Sterol biosynthesis in heterotrophic plant parasites

Cycloartenol derivatives are present in the non-photosynthetic parasitic plants Cuscuta europaea (dodder), Cuscuta epithymum and Orobanche lutea (broomrape). C.europaea and O.lutea are capable of biosynthesizing their own sterols. There is therefore no direct link, in a chlorophyll-containing phylum, between the cycloartenol pathway to sterols and photosynthesis.     

The complete nucleotide sequence of the coffee (Coffea arabica L.) chloroplast genome: organization and implications for biotechnology and phylogenetic relationships amongst angiosperms

The chloroplast genome sequence of Coffea arabica L., the first sequenced member of the fourth largest family of angiosperms, Rubiaceae, is reported. The genome is 155 189 bp in length, including a pair of inverted repeats of 25 943 bp. Of the 130 genes present, 112 are distinct and 18 are duplicated in the inverted repeat. The coding region comprises 79 protein genes, 29 transfer RNA genes, four ribosomal RNA genes and 18 genes containing introns (three with three exons). Repeat analysis revealed five direct and three inverted repeats of 30 bp or longer with a sequence identity of 90% or more. Comparisons of the coffee chloroplast genome with sequenced genomes of the closely related family Solanaceae indicated that coffee has a portion of rps19 duplicated in the inverted repeat and an intact copy of infA . Furthermore, whole-genome comparisons identified large indels (> 500 bp) in several intergenic spacer regions and introns in the Solanaceae, including trnE (UUC)– trnT (GGU) spacer, ycf4 – cemA spacer, trnI (GAU) intron and rrn5 – trnR (ACG) spacer. Phylogenetic analyses based on the DNA sequences of 61 protein-coding genes for 35 taxa, performed using both maximum parsimony and maximum likelihood methods, strongly supported the monophyly of several major clades of angiosperms, including monocots, eudicots, rosids, asterids, eurosids II, and euasterids I and II. Coffea (Rubiaceae, Gentianales) is only the second order sampled from the euasterid I clade. The availability of the complete chloroplast genome of coffee provides regulatory and intergenic spacer sequences for utilization in chloroplast genetic engineering to improve this important crop.     

Caffeine from green beans of Mascarocoffea

Unequivocal evidence is presented for the presence of 0.55–0.81% caffeine in the beans of population A213 of Coffea kianjavatensis, one taxon of the Mascarocoffea, which traditionally have been viewed as caffeine-free.     

Caffeine metabolism in Coffea arabica and other species of coffee

High performance liquid chromatography has been used to measure the quantities of caffeine, theobromine, and theophylline in aqueous extracts of endosperm from immature and mature fruits of Coffea arabica and six other species of Coffea. Caffeine was the alkaloid present in largest amounts and, with one exception, in concentrations that were broadly similar in immature and mature fruit. The highest concentrations of caffeine were found in C. canephora at 35.1 and 24.5 mg g⁻¹, respectively, in immature and mature endosperm. The lowest concentrations were in C. bengalensis, where caffeine was not detected in extracts from mature fruit. [8-³H]Caffeine was metabolised relatively slowly by immature endosperm of C. arabica and C. canephora. In contrast, C. dewevrei, C. eugenioides, C. stenophylla, C. salvatrix and C. bengalensis all appeared to metabolise [8-³H]caffeine much more rapidly, as the percentage recovery of the applied label was much lower and there was more extensive incorporation of radioactivity into theobromine, theophylline, 3-methylxanthine and two unidentified polar metabolites. The endogenous caffeine concentrations and the metabolism data indicate that there may be marked differences in the rate of turnover of caffeine in the various species of Coffea. Potential sources of material for the production of naturally decaffeinated coffee are discussed.     

Dimerization of N-methyltransferases involved in caffeine biosynthesis

Caffeine is synthesized from the precursor xanthosine through three methylation and one nucleoside removal steps. Methylation is catalyzed by N-methyltransferases, designated as CaXMT1, CaMXMT1 and CaDXMT1, which, respectively, convert xanthosine into 7-methylxanthosine, 7-methylxanthine into 3,7-dimethylxanthine, and 3,7-dimethylxanthine into 1,3,7-trimethylxanthine (caffeine). In the present study, we examined their cytological and biochemical properties using fusion proteins with fluorescent proteins. All three enzymes were found to localize in cytosol as visualized by green fluorescence protein fusions. The possibility of dimer formation among these enzyme proteins was examined in vivo by transient expression of bimolecular fluorescence complementation of yellow fluorescent protein (YFP) using onion epidermal cell layers. Results showed that each enzyme protein formed a homo-dimer in cytosol as seen by a clear reconstituted YFP fluorescence. In addition, each enzyme also formed a hetero-dimer with each of the other two enzymes in cytosol. The biological significance of dimerization among structurally resembling methyltransferases involved in caffeine biosynthesis is discussed.     

The methyl migration in rosenonolactone biosynthesis

The C-10 to C-9 methyl group migration in rosane biosynthesis proceeded without loss of mevalonoid hydrogen from C-20 thus excluding a cyclopropane intermediate from the biosynthesis sequence. 10β,19-Dihydroxyrosa-15-ene was comparatively efficiently incorporated into rosenonolactone but it could not be detected in the fermentation.     

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.     

Wild coffee management and plant diversity in the montane rainforest of southwestern Ethiopia

Coffea arabica occurs naturally in the montane rainforests of Ethiopia, but large areas of these unique forests have been converted to other land-uses. In the remaining forest, wild coffee is managed and harvested with increasing intensity because of rising coffee prices in the world market. This study evaluated the impact of coffee management on wild coffee populations and the forest vegetation as a basis for conservation planning in southwestern Ethiopia. Vegetation surveys and yield assessments were carried out in unmanaged natural forest and in managed semi-forest coffee (SFC) systems. Analyses show that wild coffee density and coffee yields were low in natural forest (max. 15 kg ha⁻¹ year⁻¹). In SFC systems, 30% of the canopy trees and most undergrowth vegetation were removed. This stimulated wild coffee growth and strongly enhanced yields (max. 54 kg ha⁻¹ year⁻¹), but severely disturbed forest structure. Species richness increased by 26% because of an increase in species of ruderal and secondary vegetation; however, species richness and abundance of typical forest species declined. Conservation of the natural forest therefore requires the control of wild coffee management. Wild coffee certification is discussed as one tool to reconcile conservation measures and the interests of local farmers.