Mozambican Coffea accessions from Ibo and Quirimba Islands: identification and geographical distribution.

Mozambique does not have a tradition of farming Coffea arabica or Coffea canephora, the two species that dominate the worldwide coffee market. However, native coffee plants have been growing spontaneously and in some cases cultivated in the Ibo and Quirimba islands in the north of the country and Inhambane province in the south. Historically there has been confusion over the precise taxonomic classification of these indigenous coffee plants, with different botanists identifying the species as C. racemosa, C. zanguebariae or various synonyms of both. The present research aims to clarify the subject and provide new information on these little-described coffee species which may prove valuable as new breeding material for future cultivars, something that is sorely needed to face the present and future challenges of coffee production. Leaf samples were collected from 40 accessions from Ibo Island, Quirimba Island and Inhambane province. The samples were sequenced by whole-genome technology and WGS reads were filtered to identify relevant SNP variants. Diversity among the samples was assessed by PCA, and a phylogenetic tree including several Coffea species was built using additional data available in public databases. Experimental data confirm the presence of C. zanguebariae as the only coffee species present in both Ibo and Quirimba Islands, while it appears that C. racemosa is exclusive to the southern Inhambane province. The present research provides the most detailed analysis so far on the genetic identity of the traditional Mozambican coffee crops. This is the prerequisite for undertaking further scientific studies on these almost unknown coffee species and for starting agronomic development programs for the economic revival of Ibo and Quirimba islands based on coffee cultivation. Furthermore, these species could provide much-needed genetic material for the breeding of new hybrids with the two main commercial coffee species.

A chromosome-scale assembly reveals chromosomal aberrations and exchanges generating genetic diversity in Coffea arabica germplasm.

In order to better understand the mechanisms generating genetic diversity in the recent allotetraploid species Coffea arabica, here we present a chromosome-level assembly obtained with long read technology. Two genomic compartments with different structural and functional properties are identified in the two homoeologous genomes. The resequencing data from a large set of accessions reveals low intraspecific diversity in the center of origin of the species. Across a limited number of genomic regions, diversity increases in some cultivated genotypes to levels similar to those observed within one of the progenitor species, Coffea canephora, presumably as a consequence of introgressions deriving from the so-called Timor hybrid. It also reveals that, in addition to few, early-occurring exchanges between homoeologous chromosomes, there are numerous recent chromosomal aberrations including aneuploidies, deletions, duplications and exchanges. These events are still polymorphic in the germplasm and could represent a fundamental source of genetic variation in such a lowly variable species.

A single polyploidization event at the origin of the tetraploid genome of Coffea arabica is responsible for the extremely low genetic variation in wild and cultivated germplasm.

The genome of the allotetraploid species Coffea arabica L. was sequenced to assemble independently the two component subgenomes (putatively deriving from C. canephora and C. eugenioides) and to perform a genome-wide analysis of the genetic diversity in cultivated coffee germplasm and in wild populations growing in the center of origin of the species. We assembled a total length of 1.536 Gbp, 444 Mb and 527 Mb of which were assigned to the canephora and eugenioides subgenomes, respectively, and predicted 46,562 gene models, 21,254 and 22,888 of which were assigned to the canephora and to the eugeniodes subgenome, respectively. Through a genome-wide SNP genotyping of 736 C. arabica accessions, we analyzed the genetic diversity in the species and its relationship with geographic distribution and historical records. We observed a weak population structure due to low-frequency derived alleles and highly negative values of Taijma's D, suggesting a recent and severe bottleneck, most likely resulting from a single event of polyploidization, not only for the cultivated germplasm but also for the entire species. This conclusion is strongly supported by forward simulations of mutation accumulation. However, PCA revealed a cline of genetic diversity reflecting a west-to-east geographical distribution from the center of origin in East Africa to the Arabian Peninsula. The extremely low levels of variation observed in the species, as a consequence of the polyploidization event, make the exploitation of diversity within the species for breeding purposes less interesting than in most crop species and stress the need for introgression of new variability from the diploid progenitors.

Aquaporins in Coffea arabica L.: Identification, expression, and impacts on plant water relations and hydraulics.

Plant aquaporins (AQPs) are involved in the transport of water and other small solutes across cell membranes, and thus play major roles in the regulation of plant water balance, as well as in growth regulation and response to abiotic stress factors. Limited information is currently available about the presence and role of AQPs in Coffea arabica L., despite the economic importance of the species and its vulnerability to drought stress. We identified candidate AQP genes by screening a proprietary C. arabica transcriptome database, resulting in the identification of nine putative aquaporins. A phylogenetic analysis based on previously characterized AQPs from Arabidopsis thaliana and Solanum tuberosum allowed to assign the putative coffee AQP sequences to the Tonoplast (TIP) and Plasma membrane (PIP) subfamilies. The possible functional role of coffee AQPs was explored by measuring hydraulic conductance and aquaporin gene expression on leaf and root tissues of two-year-old plants (C. arabica cv. Pacamara) subjected to different experimental conditions. In a first experiment, we tested plants for root and leaf hydraulic conductance both before dawn and at mid-day, to check the eventual impact of light on AQP activity and plant hydraulics. In a second experiment, we measured plant hydraulic responses to different water stress levels as eventually affected by changes in AQPs expression levels. Our results shed light on the possible roles of AQPs in the regulation of C. arabica hydraulics and water balance, opening promising research lines to improve the sustainability of coffee cultivation under global climate change scenarios.

Lipase activity and antioxidant capacity in coffee (Coffea arabica L.) seeds during germination.

In this paper, lipase activity was characterized in coffee (Coffea arabica L.) seeds to determine its involvement in lipid degradation during germination. The lipase activity, evaluated by a colorimetric method, was already present before imbibition of seeds and was further induced during the germination process. The activity showed a biphasic behaviour, which was similar in seeds either with or without endocarp (parchment), even though the phenomenon showed a delay in the former. The enzymatic activity was inhibited by tetrahydrolipstatin (THL), a selective and irreversible inhibitor of lipases, and by a polyclonal antibody raised against purified alkaline lipase from castor bean. The immunochemical analysis evidenced a protein of ca. 60 kDa, cross-reacting with an anti-lipase antibody, in coffee samples obtained from seeds of both types. Gas chromatographic analyses of free fatty acid (FFA) content confirmed the differences shown in the lipolytic activity of the samples with or without parchment, since FFA levels increased more rapidly in samples without parchment. Finally, the analyses of the antioxidant capacity showed that the presence of parchment was crucial for lowering the oxidation of the lipophylic fraction, being the seeds with parchment less prone to oxidation processes.

Functional characterization of three Coffea arabica L. monoterpene synthases: insights into the enzymatic machinery of coffee aroma.

The chemical composition of the coffee beverage is extremely complex, being made up of hundreds of volatile and non-volatile compounds, many of which are generated in the thermal reactions that occur during the roasting process. However, in the raw coffee bean there are also compounds that survive roasting and are therefore extracted into the beverage. Monoterpenes are an example of this category, as their presence has been reported in the coffee flower, fruit, seed, roasted bean and in the beverage aroma. The present work describes the isolation, heterologous expression and functional characterization of three Coffea arabica cDNAs coding for monoterpene synthases. RNA was purified from C. arabica (cv. Catuai Red) flowers, seeds and fruits at 4 successive ripening stages. Degenerate primers were designed on the most conserved regions of the monoterpene synthase gene family, and then used to isolate monoterpene synthase-like sequences from the cDNA libraries. After 5'- and 3'-RACE, the complete transcripts of 4 putative C. arabica monoterpene synthases (CofarTPS) were obtained. Gene expression in different tissues and developmental stages was analysed. After heterologous expression in Escherichia coli, enzyme activity and substrate specificity were evaluated in vitro by incubation of the recombinant proteins with geranyl pyrophosphate (GPP), geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP), precursors respectively of mono-, di- and sesquiterpenes. The reaction products were characterized by HS-SPME GC-MS. CofarTPS1 was classified as a limonene synthase gene, while CofarTPS2 and 3 showed lower activity with the production of linalool and ß-myrcene.

Differential responses of Coffea arabica L. leaves and roots to chemically induced systemic acquired resistance.

Coffea arabica is susceptible to several pests and diseases, some of which affect the leaves and roots. Systemic acquired resistance (SAR) is the main defence mechanism activated in plants in response to pathogen attack. Here, we report the effects of benzo(1,2,3)thiadiazole-7-carbothioic acid-S-methyl ester (BTH), a SAR chemical inducer, on the expression profile of C. arabica. Two cDNA libraries were constructed from the mRNA isolated from leaves and embryonic roots to create 1587 nonredundant expressed sequence tags (ESTs). We developed a cDNA microarray containing 1506 ESTs from the leaves and embryonic roots, and 48 NBS-LRR (nucleotide-binding site leucine-rich repeat) gene fragments derived from 2 specific genomic libraries. Competitive hybridization between untreated and BTH-treated leaves resulted in 55 genes that were significantly overexpressed and 16 genes that were significantly underexpressed. In the roots, 37 and 42 genes were over and underexpressed, respectively. A general shift in metabolism from housekeeping to defence occurred in the leaves and roots after BTH treatment. We observed a systemic increase in pathogenesis-related protein synthesis, in the oxidative burst, and in the cell wall strengthening processes. Moreover, responses in the roots and leaves varied significantly.