Genotyping-by-sequencing provides the first well-resolved phylogeny for coffee (Coffea) and insights into the evolution of caffeine content in its species: GBS coffee phylogeny and the evolution of caffeine content.

A comprehensive and meaningful phylogenetic hypothesis for the commercially important coffee genus (Coffea) has long been a key objective for coffee researchers. For molecular studies, progress has been limited by low levels of sequence divergence, leading to insufficient topological resolution and statistical support in phylogenetic trees, particularly for the major lineages and for the numerous species occurring in Madagascar. We report here the first almost fully resolved, broadly sampled phylogenetic hypothesis for coffee, the result of combining genotyping-by-sequencing (GBS) technology with a newly developed, lab-based workflow to integrate short read next-generation sequencing for low numbers of additional samples. Biogeographic patterns indicate either Africa or Asia (or possibly the Arabian Peninsula) as the most likely ancestral locality for the origin of the coffee genus, with independent radiations across Africa, Asia, and the Western Indian Ocean Islands (including Madagascar and Mauritius). The evolution of caffeine, an important trait for commerce and society, was evaluated in light of our phylogeny. High and consistent caffeine content is found only in species from the equatorial, fully humid environments of West and Central Africa, possibly as an adaptive response to increased levels of pest predation. Moderate caffeine production, however, evolved at least one additional time recently (between 2 and 4Mya) in a Madagascan lineage, which suggests that either the biosynthetic pathway was already in place during the early evolutionary history of coffee, or that caffeine synthesis within the genus is subject to convergent evolution, as is also the case for caffeine synthesis in coffee versus tea and chocolate.

Partial sequencing reveals the transposable element composition of Coffea genomes and provides evidence for distinct evolutionary stories.

The Coffea genus, 124 described species, has a natural distribution spreading from inter-tropical Africa, to Western Indian Ocean Islands, India, Asia and up to Australasia. Two cultivated species, C. arabica and C. canephora, are intensively studied while, the breeding potential and the genome composition of all the wild species remained poorly uncharacterized. Here, we report the characterization and comparison of the highly repeated transposable elements content of 11 Coffea species representatives of the natural biogeographic distribution. A total of 994 Mb from 454 reads were produced with a genome coverage ranging between 3.2 and 15.7 %. The analyses showed that highly repeated transposable elements, mainly LTR retrotransposons (LTR-RT), represent between 32 and 53 % of Coffea genomes depending on their biogeographic location and genome size. Species from West and Central Africa (Eucoffea) contained the highest LTR-RT content but with no strong variation relative to their genome size. At the opposite, for the insular species (Mascarocoffea), a strong variation of LTR-RT was observed suggesting differential dynamics of these elements in this group. Two LTR-RT lineages, SIRE and Del were clearly differentially accumulated between African and insular species, suggesting these lineages were associated to the genome divergence of Coffea species in Africa. Altogether, the information obtained in this study improves our knowledge and brings new data on the composition, the evolution and the divergence of wild Coffea genomes.

Large distribution and high sequence identity of a Copia-type retrotransposon in angiosperm families.

Retrotransposons are the main component of plant genomes. Recent studies have revealed the complexity of their evolutionary dynamics. Here, we have identified Copia25 in Coffea canephora, a new plant retrotransposon belonging to the Ty1-Copia superfamily. In the Coffea genomes analyzed, Copia25 is present in relatively low copy numbers and transcribed. Similarity sequence searches and PCR analyses show that this retrotransposon with LTRs (Long Terminal Repeats) is widely distributed among the Rubiaceae family and that it is also present in other distantly related species belonging to Asterids, Rosids and monocots. A particular situation is the high sequence identity found between the Copia25 sequences of Musa, a monocot, and Ixora, a dicot species (Rubiaceae). Our results reveal the complexity of the evolutionary dynamics of the ancient element Copia25 in angiosperm, involving several processes including sequence conservation, rapid turnover, stochastic losses and horizontal transfer.

Phylogeography and niche modelling of the relict plant Amborella trichopoda (Amborellaceae) reveal multiple Pleistocene refugia in New Caledonia.

Amborella trichopoda Baill. (Amborellaceae, Amborellales), the sole living member of the sister group to all other extant angiosperms, is endemic to New Caledonia. We addressed the intraspecific phylogeography of Amborella by investigating whether its present population genetic structure could be related to its current and past habitats. We found moderate range-wide genetic diversity based on nuclear microsatellite data and detected four well-differentiated, geographically distinct genetic groups using Bayesian clustering analyses. We modelled the ecological niche of Amborella based on the current climatic and environmental conditions. The predictive ability of the model was very good throughout the Central East mainland zone, but Amborella was predicted in the northern part of the island where this plant has not been reported. Furthermore, no significant barrier was detected based on habitat suitability that could explain the genetic differentiation across the area. Conversely, we found that the main genetic clusters could be related to the distribution of the suitable habitat at the last glacial maximum (LGM, c. 21,000 years BP), when Amborella experienced a dramatic 96.5% reduction in suitable area. At least two lineages survived in distinct putative refugia located in the Massif des Lèvres and in the vicinity of Mount Aoupinié. Our findings finally confirmed the importance of LGM rainforest refugia in shaping the current intra- and interspecific diversity in New Caledonian plants and revealed the possibility of an as yet unreported refugium. The combination of niche modelling and population genetics thereby offered novel insight into the biogeographical history of an emblematic taxon.

Genetic structure and diversity of coffee (Coffea) across Africa and the Indian Ocean islands revealed using microsatellites.

BACKGROUND AND AIMS: The coffee genus (Coffea) comprises 124 species, and is indigenous to the Old World Tropics. Due to its immense economic importance, Coffea has been the focus of numerous genetic diversity studies, but despite this effort it remains insufficiently studied. In this study the genetic diversity and genetic structure of Coffea across Africa and the Indian Ocean islands is investigated. METHODS: Genetic data were produced using 13 polymorphic nuclear microsatellite markers (simple sequence repeats, SSRs), including seven expressed sequence tag-SSRs, and the data were analysed using model- and non-model-based methods. The study includes a total of 728 individuals from 60 species. KEY RESULTS: Across Africa and the Indian Ocean islands Coffea comprises a closely related group of species with an overall pattern of genotypes running from west to east. Genetic structure was identified in accordance with pre-determined geographical regions and phylogenetic groups. There is a good relationship between morpho-taxonomic species delimitations and genetic units. Genetic diversity in African and Indian Ocean Coffea is high in terms of number of alleles detected, and Madagascar appears to represent a place of significant diversification in terms of allelic richness and species diversity. CONCLUSIONS: Cross-species SSR transferability in African and Indian Ocean islands Coffea was very efficient. On the basis of the number of private alleles, diversification in East Africa and the Indian Ocean islands appears to be more recent than in West and West-Central Africa, although this general trend is complicated in Africa by the position of species belonging to lineages connecting the main geographical regions. The general pattern of phylogeography is not in agreement with an overall east to west (Mascarene, Madagascar, East Africa, West Africa) increase in genome size, the high proportion of shared alleles between the four regions or the high numbers of exclusive shared alleles between pairs or triplets of regions.

Ancestral synteny shared between distantly-related plant species from the asterid (Coffea canephora and Solanum Sp.) and rosid (Vitis vinifera) clades.

BACKGROUND: Coffee trees (Rubiaceae) and tomato (Solanaceae) belong to the Asterid clade, while grapevine (Vitaceae) belongs to the Rosid clade. Coffee and tomato separated from grapevine 125 million years ago, while coffee and tomato diverged 83-89 million years ago. These long periods of divergent evolution should have permitted the genomes to reorganize significantly. So far, very few comparative mappings have been performed between very distantly related species belonging to different clades. We report the first multiple comparison between species from Asterid and Rosid clades, to examine both macro-and microsynteny relationships. RESULTS: Thanks to a set of 867 COSII markers, macrosynteny was detected between coffee, tomato and grapevine. While coffee and tomato genomes share 318 orthologous markers and 27 conserved syntenic segments (CSSs), coffee and grapevine also share a similar number of syntenic markers and CSSs: 299 and 29 respectively. Despite large genome macrostructure reorganization, several large chromosome segments showed outstanding macrosynteny shedding new insights into chromosome evolution between Asterids and Rosids. We also analyzed a sequence of 174 kb containing the ovate gene, conserved in a syntenic block between coffee, tomato and grapevine that showed a high-level of microstructure conservation. A higher level of conservation was observed between coffee and grapevine, both woody and long life-cycle plants, than between coffee and tomato. Out of 16 coffee genes of this syntenic segment, 7 and 14 showed complete synteny between coffee and tomato or grapevine, respectively. CONCLUSIONS: These results show that significant conservation is found between distantly related species from the Asterid (Coffea canephora and Solanum sp.) and Rosid (Vitis vinifera) clades, at the genome macrostructure and microstructure levels. At the ovate locus, conservation did not decline in relation to increasing phylogenetic distance, suggesting that the time factor alone does not explain divergences. Our results are considerably useful for syntenic studies between supposedly remote species for the isolation of important genes for agronomy.

Microsatellite markers for Amborella (Amborellaceae), a monotypic genus endemic to New Caledonia.

PREMISE OF THE STUDY: Informative markers are required for assessing the diversity of Amborella trichopoda, the only species of its order, endemic to New Caledonia and considered to be the sister species to all flowering plants. Therefore, expressed sequence tag (EST)-based microsatellite markers were developed. • METHODS AND RESULTS: Fifty-five microsatellite loci were characterized in 14896 putative unigenes, which were generated by assembling A. trichopoda ESTs from the public sequence database. Seventeen markers revealed polymorphism in 80 adult shrubs from three populations. The number of alleles per locus ranged from two to 12, with a total of 132 alleles scored. The mean expected heterozygosity per population ranged from 0.336 to 0.567. • CONCLUSIONS: These markers offer an appropriate amount of variation to investigate genetic diversity structure, gene flow, and other conservation issues.

Two novel Ty1-copia retrotransposons isolated from coffee trees can effectively reveal evolutionary relationships in the Coffea genus (Rubiaceae).

In the study, we developed new markers for phylogenetic relationships and intraspecies differentiation in Coffea. Nana and Divo, two novel Ty1-copia LTR-retrotransposon families, were isolated through C. canephora BAC clone sequencing. Nana- and Divo-based markers were used to test their: (1) ability to resolve recent phylogenetic relationships; (2) efficiency in detecting intra-species differentiation. Sequence-specific amplification polymorphism (SSAP), retrotransposon-microsatellite amplified polymorphism (REMAP) and retrotransposon-based insertion polymorphism (RBIP) approaches were applied to 182 accessions (31 Coffea species and one Psilanthus accession). Nana- and Divo-based markers revealed contrasted transpositional histories. At the BAC clone locus, RBIP results on C. canephora demonstrated that Nana insertion took place prior to C. canephora differentiation, while Divo insertion occurred after differentiation. Combined SSAP and REMAP data showed that Nana could resolve Coffea lineages, while Divo was efficient at a lower taxonomic level. The combined results indicated that the retrotransposon-based markers were useful in highlighting Coffea genetic diversity and the chronological pattern of speciation/differentiation events. Ongoing complete sequencing of the C. canephora genome will soon enable exhaustive identification of LTR-RTN families, as well as more precise in-depth analyses on contributions to genome size variation and Coffea evolution.

Current genetic differentiation of Coffea canephora Pierre ex A. Froehn in the Guineo-Congolian African zone: cumulative impact of ancient climatic changes and recent human activities.

BACKGROUND: Among Coffea species, C. canephora has the widest natural distribution area in tropical African forests. It represents a good model for analyzing the geographical distribution of diversity in relation to locations proposed as part of the "refuge theory". In this study, we used both microsatellite (simple sequence repeat, SSR) and restriction fragment length polymorphism (RFLP) markers to investigate the genetic variation pattern of C. canephora in the Guineo-Congolean distribution zone. RESULTS: Both markers were first compared in terms of their informativeness and efficiency in a study of genetic diversity and relationships among wild C. canephora genotypes. As expected, SSR markers were found to have a higher genetic distance detection capacity than RFLP. Nevertheless, similarity matrices showed significant correlations when Mantel's test was carried out (r = 0.66, p < 0.0001). Finally, both markers were equally effective for group discrimination and phylogenetic studies, but SSR markers tended to outperform RFLP markers in discriminating the source of an individual among diversity groups and in putative hybrid detection. Five well defined genetic groups, one in the Upper Guinean forests, the four others in the Lower Guinean forests, were identified, corresponding to geographical patterning in the individuals. CONCLUSION: Our data suggested that the Dahomey Gap, a biogeographical barrier, played a role in wild C. canephora differentiation. Climatic variations during the Pleistocene and/or Holocene probably caused the subgroup differentiation in the Congolese zone through the presence of a mosaic of putative refugia. Recent hybridization between C. canephora diversity groups, both for spontaneous individuals and cultivars, was further characterised according to their geographic dissemination or breeding history as a consequence of human activities.

MoccaDB - an integrative database for functional, comparative and diversity studies in the Rubiaceae family.

BACKGROUND: In the past few years, functional genomics information has been rapidly accumulating on Rubiaceae species and especially on those belonging to the Coffea genus (coffee trees). An increasing number of expressed sequence tag (EST) data and EST- or genomic-derived microsatellite markers have been generated, together with Conserved Ortholog Set (COS) markers. This considerably facilitates comparative genomics or map-based genetic studies through the common use of orthologous loci across different species. Similar genomic information is available for e.g. tomato or potato, members of the Solanaceae family. Since both Rubiaceae and Solanaceae belong to the Euasterids I (lamiids) integration of information on genetic markers would be possible and lead to more efficient analyses and discovery of key loci involved in important traits such as fruit development, quality, and maturation, or adaptation. Our goal was to develop a comprehensive web data source for integrated information on validated orthologous markers in Rubiaceae. DESCRIPTION: MoccaDB is an online MySQL-PHP driven relational database that houses annotated and/or mapped microsatellite markers in Rubiaceae. In its current release, the database stores 638 markers that have been defined on 259 ESTs and 379 genomic sequences. Marker information was retrieved from 11 published works, and completed with original data on 132 microsatellite markers validated in our laboratory. DNA sequences were derived from three Coffea species/hybrids. Microsatellite markers were checked for similarity, in vitro tested for cross-amplification and diversity/polymorphism status in up to 38 Rubiaceae species belonging to the Cinchonoideae and Rubioideae subfamilies. Functional annotation was provided and some markers associated with described metabolic pathways were also integrated. Users can search the database for marker, sequence, map or diversity information through multi-option query forms. The retrieved data can be browsed and downloaded, along with protocols used, using a standard web browser. MoccaDB also integrates bioinformatics tools (CMap viewer and local BLAST) and hyperlinks to related external data sources (NCBI GenBank and PubMed, SOL Genomic Network database). CONCLUSION: We believe that MoccaDB will be extremely useful for all researchers working in the areas of comparative and functional genomics and molecular evolution, in general, and population analysis and association mapping of Rubiaceae and Solanaceae species, in particular.

Microcollinearity in an ethylene receptor coding gene region of the Coffea canephora genome is extensively conserved with Vitis vinifera and other distant dicotyledonous sequenced genomes.

BACKGROUND: Coffea canephora, also called Robusta, belongs to the Rubiaceae, the fourth largest angiosperm family. This diploid species (2x = 2n = 22) has a fairly small genome size of approximately 690 Mb and despite its extreme economic importance, particularly for developing countries, knowledge on the genome composition, structure and evolution remain very limited. Here, we report the 160 kb of the first C. canephora Bacterial Artificial Chromosome (BAC) clone ever sequenced and its fine analysis. RESULTS: This clone contains the CcEIN4 gene, encoding an ethylene receptor, and twenty other predicted genes showing a high gene density of one gene per 7.8 kb. Most of them display perfect matches with C. canephora expressed sequence tags or show transcriptional activities through PCR amplifications on cDNA libraries. Twenty-three transposable elements, mainly Class II transposon derivatives, were identified at this locus. Most of these Class II elements are Miniature Inverted-repeat Transposable Elements (MITE) known to be closely associated with plant genes. This BAC composition gives a pattern similar to those found in gene rich regions of Solanum lycopersicum and Medicago truncatula genomes indicating that the CcEIN4 regions may belong to a gene rich region in the C. canephora genome. Comparative sequence analysis indicated an extensive conservation between C. canephora and most of the reference dicotyledonous genomes studied in this work, such as tomato (S. lycopersicum), grapevine (V. vinifera), barrel medic M. truncatula, black cottonwood (Populus trichocarpa) and Arabidopsis thaliana. The higher degree of microcollinearity was found between C. canephora and V. vinifera, which belong respectively to the Asterids and Rosids, two clades that diverged more than 114 million years ago. CONCLUSION: This study provides a first glimpse of C. canephora genome composition and evolution. Our data revealed a remarkable conservation of the microcollinearity between C. canephora and V. vinifera and a high conservation with other distant dicotyledonous reference genomes. Altogether, these results provide valuable information to identify candidate genes in C. canephora genome and serve as a foundation to establish strategies for whole genome sequencing. Future large-scale sequence comparison between C. canephora and reference sequenced genomes will help in understanding the evolutionary history of dicotyledonous plants.

Shift in precipitation regime promotes interspecific hybridization of introduced Coffea species.

The frequency of plant species introductions has increased in a highly connected world, modifying species distribution patterns to include areas outside their natural ranges. These introductions provide the opportunity to gain new insight into the importance of flowering phenology as a component of adaptation to a new environment. Three Coffea species, C. arabica, C. canephora (Robusta), and C. liberica, native to intertropical Africa have been introduced to New Caledonia. On this archipelago, a secondary contact zone has been characterized where these species coexist, persist, and hybridize spontaneously. We investigated the impact of environmental changes undergone by each species following its introduction in New Caledonia on flowering phenology and overcoming reproductive barriers between sister species. We developed species distribution models and compared both environmental envelopes and climatic niches between native and introduced hybrid zones. Flowering phenology was monitored in a population in the hybrid zone along with temperature and precipitation sequences recorded at a nearby weather station. The extent and nature of hybridization events were characterized using chloroplast and nuclear microsatellite markers. The three Coffea species encountered weak environmental suitability compared to their native ranges when introduced to New Caledonia, especially C. arabica and C. canephora. The niche of the New Caledonia hybrid zone was significantly different from all three species' native niches based on identity tests (I Similarity and D Schoener's Similarity Indexes). This area appeared to exhibit intermediate conditions between the native conditions of the three species for temperature-related variables and divergent conditions for precipitation-related ones. Flowering pattern in these Coffea species was shown to have a strong genetic component that determined the time between the triggering rain and anthesis (flower opening), specific to each species. However, a precipitation regime different from those in Africa was directly involved in generating partial flowering overlap between species and thus in allowing hybridization and interspecific gene flow. Interspecific hybrids accounted for 4% of the mature individuals in the sympatric population and occurred between each pair of species with various level of introgression. Adaptation to new environmental conditions following introduction of Coffea species to New Caledonia has resulted in a secondary contact between three related species, which would not have happened in their native ranges, leading to hybridization and gene flow.

Terminal-repeat retrotransposons with GAG domain in plant genomes: a new testimony on the complex world of transposable elements.

A novel structure of nonautonomous long terminal repeat (LTR) retrotransposons called terminal repeat with GAG domain (TR-GAG) has been described in plants, both in monocotyledonous, dicotyledonous and basal angiosperm genomes. TR-GAGs are relatively short elements in length (<4 kb) showing the typical features of LTR-retrotransposons. However, they carry only one open reading frame coding for the GAG precursor protein involved for instance in transposition, the assembly, and the packaging of the element into the virus-like particle. GAG precursors show similarities with both Copia and Gypsy GAG proteins, suggesting evolutionary relationships of TR-GAG elements with both families. Despite the lack of the enzymatic machinery required for their mobility, strong evidences suggest that TR-GAGs are still active. TR-GAGs represent ubiquitous nonautonomous structures that could be involved in the molecular diversities of plant genomes.

Tolerance of coffee (Coffea spp.) seeds to ultra-low temperature exposure in relation to calorimetric properties of tissue water, lipid composition, and cooling procedure.

The effect of exposure to ultra-low temperature (liquid nitrogen, LN) on viability of seeds desiccated to various water contents was investigated in 9 coffee species. Three groups of species could be distinguished based on seed survival after LN exposure. In group 1 species, no seedling production could be obtained after LN exposure due to endosperm injury. In group 2 species, recovery was very low or nil after rapid cooling, and only moderate after slow cooling. In group 3 species, very high percentages of seedling development were observed after both rapid and slow cooling. A high interspecific variability for the high moisture freezing limit was observed within the species of groups 2 and 3, since it ranged from 0.14 to 0.26 g H2O g-1 dry weight. A very highly significant correlation was found for those species between the unfreezable water content, as determined from DSC analysis, and the high moisture freezing limit of their seeds. No significant correlation was found between seed lipid content, which varied from 9.8 to 34.6% dry weight, and survival after LN exposure. However, a negative relationship was found between seed unfreezable water content and lipid content. Interspecific differences in fatty acid composition of seed lipids resulted in a high variability in the percentage of unsaturated fatty acids, which ranged from 28.7 to 54.4% among the 9 species studied. For all species studied, a highly significant correlation was found between the percentage of unsaturated fatty acids and the percentage of seedling recovery after rapid or slow cooling.

Variability in storage response within a coffee (Coffea spp.) core collection under slow growth conditions.

Anin vitro core collection of African coffee germplasm, structured in 32 diploid diversity groups, was established and conserved under slow growth for 3 years (6 subcultures). The initial objective was to store twenty accessions per group, with four replicates per accession. A statistical model was developed to analyse observations of survival rates within each diversity group. The goodness of fit of the model was shown. Survival analysis indicated a broad variability of the accessions in their response to the storage conditions and confirmed the importance of structuring the coffee complex down to the intraspecific level. Intra- and inter-group differences had consequences on the genetic representativity of thein vitro core collection. For practical purposes, conservation was carried on when the intra-group genetic drift was less than 50%.

Site-Specific Insertion Polymorphism of the MITE Alex-1 in the Genus Coffea Suggests Interspecific Gene Flow.

Miniature Inverted-repeat Transposable Elements (MITEs) are small nonautonomous class-II transposable elements distributed throughout eukaryotic genomes. We identified a novel family of MITEs (named Alex) in the Coffea canephora genome often associated with expressed sequences. The Alex-1 element is inserted in an intron of a gene at the CcEIN4 locus. Its mobility was demonstrated by sequencing the insertion site in C. canephora accessions and Coffea species. Analysis of the insertion polymorphism of Alex-1 at this locus in Coffea species and in C. canephora showed that there was no relationship between the geographical distribution of the species, their phylogenetic relationships, and insertion polymorphism. The intraspecific distribution of C. canephora revealed an original situation within the E diversity group. These results suggest possibly greater gene flow between species than previously thought. This MITE family will enable the study of the C. canephora genome evolution, phylogenetic relationships, and possible gene flows within the Coffea genus.

Development of genomic microsatellite markers in Coffea canephora and their transferability to other coffee species.

Of the 103 accepted Coffea species, 70% are threatened with extinction but only a few of them have been studied. A set of 40 polymorphic microsatellite markers was developed using a GA/GT-enriched Coffea canephora genomic library. Amplification of these markers was tested in accessions of C. heterocalyx (a Critically Endangered species) and C. pseudozanguebariae (a Vulnerable species) belonging to different African geographical clades. All microsatellites were polymorphic in C. canephora, with a mean allele number per polymorphic locus of more than 3 (at least 9 genotypes were tested). Observed and expected heterozygosities calculated for C. canephora and C. pseudozanguebariae varied from 0.10 to 0.91 and from 0.20 to 0.77, respectively. In total, 38 primer pairs (95%) were amplified in C. heterocalyx and C. pseudozanguebariae, indicating their high level of transferability across the genus Coffea. This large marker set will be useful for more extensive genetic studies of threatened Coffea species.

Molecular characterization of an ethylene receptor gene (CcETR1) in coffee trees, its relationship with fruit development and caffeine content.

To understand the importance of ethylene receptor genes in the quality of coffee berries three full-length cDNAs corresponding to a putative ethylene receptor gene (ETR1) were isolated from Coffea canephora cDNA libraries. They differed by their 3'UTR and contained a main ORF and a 5'UTR short ORF putatively encoding a small polypeptide. The CcETR1 gene, present as a single copy in the C. canephora genome, contained five introns in the coding region and one in its 5'UTR. Alternative splicing can occur in C. canephora and C. pseudozanguebariae, leading to a truncated polypeptide. C. pseudozanguebariae ETR1 transcripts showed various forms of splicing alterations. This gene was equally expressed at all stages of fruit development. A segregation study on an inter-specific progeny showed that ETR1 is related to the fructification time, the caffeine content of the green beans, and seed weight. Arabidopsis transformed etiolated seedlings, which over-expressed CcETR1, displayed highly reduced gravitropism, but the triple response was observed in an ethylene enriched environment. These plants behaved like a low-concentration ethylene-insensitive mutant thus confirming the receptor function of the encoded protein. This gene showed no induction during the climacteric crisis but some linkage with traits related to quality.

Functional characterization of two p-coumaroyl ester 3'-hydroxylase genes from coffee tree: evidence of a candidate for chlorogenic acid biosynthesis.

Chlorogenic acid (5-CQA) is one of the major soluble phenolic compounds that is accumulated in coffee green beans. With other hydroxycinnamoyl quinic acids (HQAs), this compound is accumulated in particular in green beans of the cultivated species Coffea canephora. Recent work has indicated that the biosynthesis of 5-CQA can be catalyzed by a cytochrome P450 enzyme, CYP98A3 from Arabidopsis. Two full-length cDNA clones (CYP98A35 and CYP98A36) that encode putative p-coumaroylester 3'-hydroxylases (C3'H) were isolated from C. canephora cDNA libraries. Recombinant protein expression in yeast showed that both metabolized p-coumaroyl shikimate at similar rates, but that only one hydroxylates the chlorogenic acid precursor p-coumaroyl quinate. CYP98A35 appears to be the first C3'H capable of metabolising p-coumaroyl quinate and p-coumaroyl shikimate with the same efficiency. We studied the expression patterns of both genes on 4-month old C. canephora plants and found higher transcript levels in young and in highly vascularized organs for both genes. Gene expression and HQA content seemed to be correlated in these organs. Histolocalization and immunolocalization studies revealed similar tissue localization for caffeoyl quinic acids and p-coumaroylester 3'-hydroxylases. The results indicated that HQA biosynthesis and accumulation occurred mainly in the shoot tip and in the phloem of the vascular bundles. The lack of correlation between gene expression and HQA content observed in some organs is discussed in terms of transport and accumulation mechanisms.