Inheritance of caffeine and heteroside contents in an interspecific cross between a cultivated coffee species Coffea liberica var dewevrei and a wild species caffeine-free C. pseudozanguebariae

 Coffee species originating from Africa, in particular the two major cultivated species C. arabica and C. canephora, usually contain caffeine in their beans, whereas almost all Malagasy coffee species are caffeine-free. However, one wild coffee species C. pseudozanguebariae, collected near the coast in south Kenya, is also caffeine-free. Beans of this species contain a specific heteroside diterpene (hereinafter referred to simply as heteroside) and give a bitter coffee beverage. We have investigated the inheritance of the caffeine and heteroside contents in the first and second generations of an interspecific cross between C. pseudozanguebariae and C. liberica var. dewevrei, for which the caffeine content is about 1% dmb (dry matter basis). The caffeine content of F₁ hybrids (0.2% dmb) was lower than the parental average (0.47% dmb). Caffeine and heteroside contents appeared to be under polygenic control with a strong genetic effect. Nevertheless, one major gene with two alleles seemed to be involved in the control of both compounds. Absence of caffeine was apparently controlled by one recessive gene. Heteroside content seemed to be controlled by one co-dominant gene, heterozygotes being intermediate between the two different groups of homozygotes. Received: 15 September 1997 / Accepted: 6 October 1997     

Genetic investigations on the caffeine and chlorogenic acid relationship in an interspecific cross between Coffea liberica dewevrei and C. pseudozanguebariae

The objective of the paper was to identify the number of major loci explaining caffeine content in coffee seeds. Investigations were based on previously published results: (1) Caffeine binds to chlorogenic acids in a 1:1 molecular ratio; (2) Between species, the caffeine content is correlated to the chlorogenic acid content; (3) Only a part of chlorogenic acids is bound to caffeine. Especially, the content ratio between caffeine and chlorogenic acids varied between species. For identifying the number of major loci, a quantitative trait locus (QTL) approach was carried out using an interspecific cross between two highly differentiated species—Coffea liberica dewevrei and Coffea pseudozanguebariae, the latter being a caffeine-free species. As main finding, two QTLs, i.e., RCQ1 and CQA1, were identified allowing us to explain up to 97 % of the caffeine content variance. RCQ1 explained variation of the caffeine/chlorogenic acid ratio and was genetically independent of the second QTL. The latter explained the part of the caffeine content which was dependent on the chlorogenic acid content. The findings also confirmed that only a part of chlorogenic acids were trapped by caffeine, as in wild species.     

Morphological and histological impacts of the laurina mutation on fructification and seed characteristics in Coffea arabica L.

Key message

The comparison between the cultivar Bourbon and its mutant, the Bourbon pointu, of Coffea arabica led to five novel findings on fruit development and three main impacts of the mutation.

Abstract

Coffea arabica ‘Laurina’ (Bourbon pointu) is a natural mutant of Coffea arabica ‘Bourbon’. Relative to the ‘Bourbon’ cultivar, it is characterized by internode dwarfism, a Christmas tree shape, and lower caffeine content. The effects of the laurina mutation on fructification over time, the fruit structure and seed characteristics were studied here. Fruits of ‘Bourbon’ and ‘Bourbon pointu’ were monitored. The trees were grown in the same plot and flowered on the same day. Harvesting was done every 2 weeks from the 6th to the 26th week after flowering. Histological observations were carried out using multiphoton and conventional microscopes. The measurements concerned the fruit, parchment and seed. Five novel findings on fructification development were obtained: (1) a sigmoid model and non-linear regression efficiently described the phenomenon; (2) a precise relationship was defined between the qualitative stages of fructification and quantitative observations, thus revealing key weeks in this process; (3) the parchment had a mesocarpic origin; (4) a meristematic zone was present close to the parchment; and (5) an endocarp with three cell layers was visible in young fruits. Three effects of the laurina mutation were highlighted: (1) fruit growth ended 1 week earlier in ‘Bourbon’, but without difference in fruit length. In contrast, fruits were wider on average in ‘Bourbon’; (2) the parchment of narrow seeds in ‘Bourbon pointu’ was thicker than in other ‘Bourbon pointu’ and ‘Bourbon’ seeds; and (3) the narrow seed frequency in ‘Bourbon pointu’ depended on environmental conditions.     

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.

     

Improving the quality of African robustas: QTLs for yield- and quality-related traits in Coffea canephora

Coffea canephora breeding requires combining sustainable productivity with improved technological and cup quality characteristics. Beverage quality is a complex and subjective trait, and breeding for this trait is time consuming and depends on knowledge of the genetics of its components. A highly variable C. canephora progeny resulting from an intraspecific cross was assessed for 63 traits over 5?years. To identify quantitative trait loci (QTLs) controlling agronomic, technological, and quality-related traits, a genetic map comprising 236 molecular markers was constructed, and composite interval mapping was performed. Beverage quality was evaluated in relation to biochemical and cup tasting traits. QTLs were identified for almost half of the traits evaluated, with effects ranging from 6% to 80% of phenotypic variation. Most of them present a consistent detection over years. The strongest QTLs explained a high percentage of the variation for yield in 2006 (34% to 57%), bean size (25% to 35%), content of chlorogenic acids (22% to 35%), sucrose and trigonelline content (29% to 81%), and acidity and bitterness of coffee beverages (30% to 55%). Regions of the C. canephora genome influencing beverage quality were identified. Five QTL zones were co-localized with candidate genes related to the biosynthesis of the analyzed traits: two genes coding for caffeine biosynthesis, one gene implicated in the biosynthesis of chlorogenic acids, and two genes implicated in sugar metabolism. This is one of the first studies on the identification of QTLs combining agronomic and quality traits in coffee. The high variability of quality traits within C. canephora and the presence of consistent QTLs offer breeders a promising tool to improve coffee cup quality.     

Cloning of caf1 +, caf2 + and caf4 + from Schizosaccharomyces pombe : their involvement in multidrug resistance, UV and pH sensitivity

We previously identified four nuclear genes (caf1 ⁺ to caf4 ⁺) in Schizosaccharomyces pombe, mutations in which can confer caffeine resistance. Here we report the cloning and sequencing of caf1 ⁺, caf2 ⁺ and caf4 ⁺. All three genes are allelic to genes (hba1 ⁺ , crm1 ⁺ and trr1 ⁺ , respectively) involved in multidrug resistance mechanisms or in stress response systems. In agreement with this the caffeine-resistant mutants caf1(hba1)-21, caf2(crm1)-3 and caf4(trr1)-83 are also resistant to brefeldin. Disruption of caf1(hba1) ⁺ and caf4(trr1) ⁺ makes cells sensitive to high pH. The overlapping ranges of pleiotropic effects and the genetic interaction detected between caf1(hba1) ⁺ and caf2(crm1) ⁺ suggest that the three genes function in interlinked systems.     

The biosynthesis of caffeine in the coffee plant

Leaf disks of Coffea arabica were infiltrated simultaneously with L-methionine-(methyl-¹⁴C) and with various possible precursors of caffeine biosynthesis. The results permit the identification of theobromine, 7-methylxanthine and 7-methylxanthosine as precursors of caffeine. 7-methylguanosine seems not to be an intermediate in caffeine formation.     

Metabolic Engineering of Saccharomyces cerevisiae for Caffeine and Theobromine Production

Caffeine (1, 3, 7-trimethylxanthine) and theobromine (3, 7-dimethylxanthine) are the major purine alkaloids in plants, e.g. tea (Camellia sinensis) and coffee (Coffea arabica). Caffeine is a major component of coffee and is used widely in food and beverage industries. Most of the enzymes involved in the caffeine biosynthetic pathway have been reported previously. Here, we demonstrated the biosynthesis of caffeine (0.38 mg/L) by co-expression of Coffea arabica xanthosine methyltransferase (CaXMT) and Camellia sinensis caffeine synthase (TCS) in Saccharomyces cerevisiae. Furthermore, we endeavored to develop this production platform for making other purine-based alkaloids. To increase the catalytic activity of TCS in an effort to increase theobromine production, we identified four amino acid residues based on structural analyses of 3D-model of TCS. Two TCS1 mutants (Val317Met and Phe217Trp) slightly increased in theobromine accumulation and simultaneously decreased in caffeine production. The application and further optimization of this biosynthetic platform are discussed.     

Comparison between <Emphasis Type="Italic">Coffea arabica</Emphasis> L. ‘Laurina’ and <Emphasis Type="Italic">C. arabica</Emphasis> ‘Bourbon’ seedlings grown in daylight or darkness for their polysaccharidic cell wall composition and caffeine and chlorogenic acid contents

Key message

Cell wall polysaccharidic composition changed between cotyledons, hypocotyls and roots. Neither the laurina mutation nor the presence of light had an impact on this composition.

Abstract

Coffea arabica ‘Laurina’, a natural mutant of Coffea arabica ‘Bourbon’ (B), is also known as ‘Bourbon Pointu’ (BP). In seedlings under daylight, the laurina mutation leads to semi-dwarf hypocotyls, but this effect disappears in darkness conditions. The first step of our work was to analyze the impact of the mutation on the monosaccharide cell wall composition in cotyledons, hypocotyls and roots in relation to growth conditions (daylight vs darkness). Secondly, the same type of comparison was carried out for caffeine and chlorogenic acid (CQA) contents. Cell wall polysaccharides (CWP) present in cotyledons, hypocotyls and roots were identified. Neither the laurina mutation nor the growth conditions had an impact on the CWP composition. By contrast, there were marked differences between cotyledons, hypocotyls and roots regarding their CWP composition, CQA and caffeine contents. Lastly, the mutation and the light did not modify the CQA content in the three organs, whereas the mutation, but the light, lowered the caffeine (CAF) content.

     

QTL and gene expression analyses identify genes affecting carcass weight and marbling on BTA14 in Hanwoo (Korean Cattle)

Causal mutations affecting quantitative trait variation can be good targets for marker-assisted selection for carcass traits in beef cattle. In this study, linkage and linkage disequilibrium analysis (LDLA) for four carcass traits was undertaken using 19 markers on bovine chromosome 14. The LDLA analysis detected quantitative trait loci (QTL) for carcass weight (CWT) and eye muscle area (EMA) at the same position at around 50?cM and surrounded by the markers FABP4SNP2774C>G and FABP4_??sat3237. The QTL for marbling (MAR) was identified at the midpoint of markers BMS4513 and RM137 in a 3.5-cM marker interval. The most likely position for a second QTL for CWT was found at the midpoint of tenth marker bracket (FABP4SNP2774C>G and FABP4_??sat3237). For this marker bracket, the total number of haplotypes was 34 with a most common frequency of 0.118. Effects of haplotypes on CWT varied from a ?5-kg deviation for haplotype 6 to +8?kg for haplotype 23. To determine which genes contribute to the QTL effect, gene expression analysis was performed in muscle for a wide range of phenotypes. The results demonstrate that two genes, LOC781182 (p?=?0.002) and TRPS1 (p?=?0.006) were upregulated with increasing CWT and EMA, whereas only LOC614744 (p?=?0.04) has a significant effect on intramuscular fat (IMF) content. Two genetic markers detected in FABP4 were the most likely QTL position in this QTL study, but FABP4 did not show a significant effect on both traits (CWT and EMA) in gene expression analysis. We conclude that three genes could be potential causal genes affecting carcass traits CWT, EMA, and IMF in Hanwoo.     

Sequence diversity, haplotype analysis, association mapping and functional marker development in the waxy and starch synthase IIa genes for grain-yield-related traits in hexaploid wheat (Triticum aestivum L.)

Development of high-yielding cereal crops could meet increasing global demands for food, feed and bio-fuels. Wheat is one of the world??s most important cereal crops. The biosynthesis of starch is the major determinant of yield in wheat. Two starch biosynthesis genes, the waxy (Wx) genes and the starch synthase IIa (SSIIa) genes, were amplified and sequenced in 92 diverse wheat genotypes using genome-specific primers. Nucleotide diversity, haplotype analysis and association mapping were performed. The first exon (5??-UTR) and the first intron of the three homoeologous Wx genes were isolated using expressed sequence tag sequences. The Wx genes contained 12 exons separated by 11 introns. SNP (single nucleotide polymorphism) frequency ranged from 1 SNP/3,648?bp for Wx-D1 to 1 SNP/135?bp for SSIIa-A1, with an average of 1 SNP/230?bp. The average SNP frequencies in exon and intron regions were 1 SNP/322?bp and 1 SNP/228?bp, respectively. Thirty, 23 and 5 SNPs were identified and formed five, six and five haplotypes for SSIIa-A1, SSIIa-B1 and SSIIa-D1, respectively. However, no association was found between these SNPs and seven yield-related traits. Twenty-two, 15 and 1 SNPs were detected and formed nine, five and two haplotypes for Wx-A1, Wx-B1 and Wx-D1, respectively. Three unique nucleotides C+A+T at SNP5, SNP6 and SNP12 formed Wx-B1-H3, which was significantly associated with increased grain weight, thousand kernel weight, and total starch content in three spring wheat genotypes and five winter wheat genotypes. Cost-effective and co-dominant SNP markers were developed using temperature-switch (TS)-PCR and are being used for marker-assisted selection of doubled haploid lines with enhanced grain yield and starch content in winter wheat breeding programs.     

Establishment of somaclonal variants of Robusta coffee with reduced levels of cafestol and kahweol

Cafestol (caf) and kahweol (kah) are two diterpenes uniquely associated with the unsaponified lipid fraction of coffee brew and are reported to be responsible for an increase in serum cholesterol and triglyceride levels. The plant growth regulators (PGRs) indole-3-acetic acid (IAA), N ⁶-benzyladenine (BA), and thidiazuron (TDZ); the plant growth-promoting agents silver nitrate, triacontanol (TRIA), and coconut water; and some PGR antagonists such as lovastatin, paclobutrazol, and 2,3,5-triiodobenzoic acid (TIBA) were used to determine the variation of caf and kah in somatic embryos of Robusta coffee (Coffea canephora, CxR variety). Embryogenic (EG) medium was comprised of half-strength Murashige and Skoog basal components (½MS) supplemented with 2.85 μM IAA and 1.11 μM BA. After an 8-wk culture, somatic embryos were subjected to diterpene extraction and HPLC analysis of caf and kah profiles. TRIA-supplemented (5 μg L^?1) EG medium devoid of IAA reduced the levels of caf and kah by 18–24 and 48–55%, respectively, in coffee somatic embryos. Similarly, the combination of 2.85 μM IAA, 2.27 μM TDZ, and 5–10% coconut water in ½MS basal medium drastically reduced the caf and kah levels in somatic embryos. There was 60–75% reduction in both caf and kah in the presence of 5 μM TIBA, followed by 56–62% reduction in the presence of 10 μM silver nitrate. In contrast, there was 25–32% elevation of caf and kah in EG medium supplemented with 5 μM paclobutrazol. In this study, for the first time, somaclonal variants of C. canephora with reduced levels of diterpenes caf and kah were established. Furthermore, these lines exhibited consistency in their metabolite profiles when cultivated under greenhouse conditions. In-depth investigations at physiological level are warranted in order to elucidate the actual mechanism of these PGR inhibitors on alterations in endogenous pools of diterpenes in coffee somatic embryos.     

Purine Alkaloids of the Fruits of Camellia sinensis L. and Coffea arabica L. during Fruit Development

The amounts of two purine alkaloids, caffeine and theobromine,in the fruit of tea (Camellia sinensis L.) increased markedlyduring the growing season until the fruit was full-ripened anddried. In the dry fruit, the pericarp contained the most alkaloids,but there were also considerable amounts in the seed coat and,to a lesser extent, the fruit stalk and the seed. The shed seedsalso contained significant amounts of the alkaloids, especiallyin the seed coats. In contrast with the dry fruit of tea, seedsand pericarp of coffee (Coffea arabica L.) fruit contained aconsiderable amount of caffeine and a small amount of theobromide.A small amount of theophylline was also present in the pericarpof the ripened fruit. Relationships between growth and purinealkaloid content in tea and coffee fruits and their roles duringseed formation are discussed. Camellia sinensis L., tea, Coffea arabica L., coffee, purine alkaloids, fruit development, seed, seed coat, caffeine, theobromine, theophylline     

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.     

Construction of a genetic map for arabica coffee

We have used AFLPs to construct a genetic linkage map on a pseudo-F₂ population of arabica coffee (Coffea arabica L.) derived from a cross between the cultivars Mokka hybrid and Catimor. Sixty trees from this population were selected on the basis of plant height distribution to construct a linkage map. A total of 456 dominant markers and eight co-dominant markers were generated from 288 AFLP primer combinations. Of the total number of markers generated, 68% were from cv. Catimor, 30% from cv. Mokka hybrid, and 2% were co-dominant. This distribution suggests that the heterozygosity within the cv. Catimor sub-genomes was twice that within the cv. Mokka hybrid sub-genomes. Linkage groups were constructed using MAPMAKER version 3.0, resulting in 16 major linkage groups containing 4–21 markers, and 15 small linkage groups consisting of 2–3 linked markers each. The total length of the map was 1,802.8 cM, with an average distance of 10.2 cM between adjacent markers. This genetic map will serve as the framework for mapping QTL controlling source-sink traits in the same population.Communicated by H.F. Linskens     

Functional coordination between branch hydraulic properties and leaf functional traits in miombo woodlands: implications for water stress management and species habitat preference

We investigated functional coordination between branch hydraulic properties and leaf functional traits among nine miombo woodlands canopy tree species differing in habitat preference and phenology. Specifically, we were seeking to answer the question: are branch hydraulic properties coordinated with leaf functional traits linked to plant drought tolerance in seasonally dry tropical forests and what are the implications for species habitat preference? The hydraulic properties investigated in this study were stem area specific hydraulic conductivity (K _S), Huber value (H _v), and xylem cavitation vulnerability (??₅₀). The leaf functional traits measured were specific leaf area (SLA), leaf dry matter content (LDMC), and mean leaf area (MLA). Generalists displayed significantly (P?<?0.05) higher cavitation resistance (??₅₀) and SLA, but lower sapwood specific hydraulic conductivity (K _S), leaf specific conductivity (K _L), MLA, and LDMC than mesic specialists. Although MLA was uncorrelated with ??₅₀, we found significant (P?<?0.05) positive and negative correlations between plant hydraulic properties and leaf functional traits linked to plant drought tolerance ability, indicating that the interactions between branch hydraulics and leaf functional traits related to plant drought tolerance ability may influence tree species habitat preference in water-limited ecosystems.     

Long term culture and caffeine production of immobilized coffee (Coffea arabica) L. cells in polyurethane foam

Coffee (Coffea arabica L.) cells could be immobilized in polyurethane foam and subcultured repeatedly for a long time. Four phases were observed for cell growth and caffeine production, I; immobilization, II; growth, III; caffeine production, IV; regrowth. Their periods were influenced by the number of foam particles. Especially in the phase III, the immobilized cells produced a relatively large amount of caffeine in the subculture numbers 5–8 (34 cubes) when the fresh weight of the immobilized cells decreased despite culture in growth medium (DK medium). Caffeine production appeared to have a negative correlation with the growth of the immobilized cells throughout the subcultures.This paper is Part 71 in the series of Studies on Plant Tissue Cultures. For Part 70, see Orihara Y, Furuya T. submitted for publication.     

Decaf and the Steeplechase Towards Decaffito—the Coffee from Caffeine-Free Arabica Plants

Unquestionably, the popularity of the coffee beverage relies on its alerting attribute caffeine. However, susceptibilities to this purine alkaloid, quite frequently associated with health concerns, encouraged a significant market for decaffeinated coffee. The beans of Coffea arabica render the best beverage and a decaffeinated coffee has to preserve the desired organoleptic characteristics of this species. Consequently, besides technical removal of caffeine, the endeavors to attain a decaffeinated Arabica coffee range from traditional studies on genetic variability to advanced techniques to produce genetic modified coffee. The aim of this review is to recover part of this subject focusing mainly on the natural genetic variation for caffeine content in Arabica. We also present historical information about caffeine discovery and briefly discuss molecular approaches to reduce caffeine. We introduce here the term decaffito for coffee derived from Arabica plants with beans naturally low in or almost devoid of caffeine. In the near future, coffee drinkers avoiding caffeine will have the choice between basically three Arabica coffees, namely decaffeinated by (a) selection and breeding, (b) genetic modification and (c) industrial extraction. Although only the last decaf coffee is available for the consumers, we believe that the size of the market of each type will occupy in the future depend on the price and health aspects related to the way the decaffeinated coffee beans are obtained.     

Purine alkaloid formation and CO2 gas exchange in dependence of development and of environmental factors in leaves of Coffea arabica L.

In the leaves of Coffea arabica L., purine alkaloid formation was estimated by analyzing the theobromine and caffeine content and by measuring the methylation rate of [2-¹⁴C]theobromine to [2-¹⁴C]caffeine in short-term experiments (6–24 h). At the same time, growth (in terms of dry weight and area), net photosynthesis (NPS), and dark respiration were determined. During leaf development, which was considered to be terminated when NPS was at a maximum (60–80 mol g^-1 s^-1) and dark respiration at a minimum (5–7.5 mol g^-1 s^-1), the content of theobromine and the velocity of caffeine formation were both found to decrease by a factor of more than 100. The close correlation between the theobromine content and the methylation rate is suspended when purine alkaloid formation is influenced by factors other than leaf development. Among these factors, temperature is the most effective: the velocity of caffeine biosynthesis is increased by raising the temperature and vice versa. Although the plants were well irrigated, a drastic decrease of NPS in the afternoon was observed under all environmental conditions tested. Light saturation was reached between 170–360 mol m^-2 s^-1. The temperature optimum of NPS was shown to be very broad (24–33°C)m provided the adaptation time was sufficiently long.Abbreviations MR methylation rate - NPS net photosynthesis - RMC relative methylation coefficient Dedicated to Professor Hans Wanner, as promoter of these investigations, on occasion of his 65th birthday