Haitian coffee agroforestry systems harbor complex arabica variety mixtures and under-recognized genetic diversity.

Though facing significant challenges, coffee (Coffea arabica) grown in Haitian agroforestry systems are important contributors to rural livelihoods and provide several ecosystem services. However, little is known about their genetic diversity and the variety mixtures used. In light of this, there is a need to characterize Haitian coffee diversity to help inform revitalization of this sector. We sampled 28 diverse farms in historically important coffee growing regions of northern and southern Haiti. We performed KASP-genotyping of SNP markers and HiPlex multiplex amplicon sequencing for haplotype calling on our samples, as well as several Ethiopian and commercial accessions from international collections. This allowed us to assign Haitian samples to varietal groups. Our analyses revealed considerable genetic diversity in Haitian farms, higher in fact than many farmers realized. Notably, genetic structure analyses revealed the presence of clusters related to Typica, Bourbon, and Catimor groups, another group that was not represented in our reference accession panel, and several admixed individuals. Across the study areas, we found both mixed-variety farms and monovarietal farms with the historical and traditional Typica variety. This study is, to our knowledge, the first to genetically characterize Haitian C. arabica variety mixtures, and report the limited cultivation of C. canephora (Robusta coffee) in the study area. Our results show that some coffee farms are repositories of historical, widely-abandoned varieties while others are generators of new diversity through genetic mixing.

A voltammetric peptide biosensor for Cu2+ metal ion quantification in coffee seeds.

A prion-derived copper(II)-binding peptide was assembled onto a gold electrode for the building of a voltammetric biosensor for measuring the Cu2+ metal ion in biological samples. The chosen sequence was H-CVNITKQHTVTTTT-NH2, with an appended cysteine residue for binding to the gold surface as a self-assembled monolayer and a histidine residue as the anchorage point for copper(II) complexation. The biosensor showed a linear range of 10-7 to 10-6 M with an 8.0 × 10-8 M detection limit and a 1.0 × 10-7 M quantification limit, with good precision, trueness, and absence of matrix effect. The quantification of Cu2+ was performed in the presence of other transition metal ions, such as Zn2+, Cd2+, Fe2+, or Ni2+, which indicates the excellent selectivity of the biosensor. When the modified electrode was applied for measuring copper(II) in calcined coffee seeds, a difference in copper amount was observed between two Coffea arabica cultivars that were submitted to a treatment with a copper-based antifungal, showing the applicability of the biosensor in the agricultural field.

Genome-Wide Admixture Mapping Identifies Wild Ancestry-of-Origin Segments in Cultivated Robusta Coffee.

Humans have had a major influence on the dissemination of crops beyond their native range, thereby offering new hybridization opportunities. Characterizing admixed genomes with mosaic origins generates valuable insight into the adaptive history of crops and the impact on current varietal diversity. We applied the ELAI tool-an efficient local ancestry inference method based on a two-layer hidden Markov model to track segments of wild origin in cultivated accessions in the case of multiway admixtures. Source populations-which may actually be limited and partially admixed-must be generally specified when using such inference models. We thus developed a framework to identify local ancestry with admixed source populations. Using sequencing data for wild and cultivated Coffea canephora (commonly called Robusta), our approach was found to be highly efficient and accurate on simulated hybrids. Application of the method to assess elite Robusta varieties from Vietnam led to the identification of an accession derived from a likely backcross between two genetic groups from the Congo Basin and the western coastal region of Central Africa. Admixtures resulting from crop hybridization and diffusion could thus lead to the generation of elite high-yielding varieties. Our methods should be widely applicable to gain insight into the role of hybridization during plant and animal evolutionary history.

Genetic-environment interactions and climatic variables effect on bean physical characteristics and chemical composition of Coffea arabica.

BACKGROUND: The effects of the environment and genotype in the coffee bean chemical composition were studied using nine trials covering an altitudinal gradient [600-1100 m above sea level (a.s.l.)] with three genotypes of Coffea arabica in the northwest mountainous region of Vietnam. The impacts of the climatic conditions on bean physical characteristics and chemical composition were assessed. RESULTS: We showed that the environment had a significant effect on the bean density and on all bean chemical compounds. The environment effect was stronger than the genotype and genotype-environment interaction effects for cafestol, kahweol, arachidic (C20:0), behenic acid (C22:0), 2,3-butanediol, 2-methyl-2-buten-1-ol, benzaldehyde, benzene ethanol, butyrolactone, decane, dodecane, ethanol, pentanoic acid, and phenylacetaldehyde bean content. A 2 °C increase in temperature had more influence on bean chemical compounds than a 100 mm increase in soil water content. Temperature was positively correlated with lipids and volatile compounds. With an innovative method using iterative moving averages, we showed that correlation of temperature, vapour pressure deficit (VPD) and rainfall with lipids and volatiles was higher between the 10th and 20th weeks after flowering highlighting this period as crucial for the synthesis of these chemicals. Genotype specific responses were evidenced and could be considered in future breeding programmes to maintain coffee beverage quality in the midst of climate change. CONCLUSION: This first study of the effect of the genotype-environment interactions on chemical compounds enhances our understanding of the sensitivity of coffee quality to genotype environment interactions during bean development. This work addresses the growing concern of the effect of climate change on speciality crops and more specifically coffee. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Multiplex CRISPR/Cas9-mediated knockout of the phytoene desaturase gene in Coffea canephora.

Coffea canephora (2n = 2x = 22 chromosomes) is a species with extensive genetic diversity and desirable agronomic traits for coffee breeding programs. However, obtaining a new coffee cultivar through conventional breeding techniques may require more than 30 years of crossing cycles and selection, which hampers the effort of keeping up with market demands and rapidly proposing more resilient to climate change varieties. Although, the application of modern biotechnology tools such as precision genetic engineering technologies may enable a faster cultivar development process. Therefore, we aimed to validate the CRISPR/Cas9 system to generate mutations on a selected genotype of C. canephora, the clone 14. Embryogenic calli and a multiplex binary vector containing two sgRNAs targeting different exons of the CcPDS gene were used. The sgRNAs were under the C. canephora U6 promoter regulation. The target gene encodes phytoene desaturase, an enzyme essential for photosynthesis involved in ß-carotene biosynthesis. Somatic seedlings and embryos with albino, variegated and green phenotypes regenerated after Agrobacterium tumefaciens-mediated genetic transformation were analyzed by verifying the insertion of the Cas9 gene and later by sequencing the sgRNAs target regions in the genome of Robusta modified seedlings. Among them, 77% had the expected mutations, and of which, 50% of them had at least one target with a homozygous mutation. The genotype, temperature of co-cultivation with the bacteria, and light intensity used for subsequent embryo regeneration appeared to strongly influence the successful regeneration of plants with a mutated CcPDS gene in the Coffea genus.

Adaptive potential of Coffea canephora from Uganda in response to climate change.

Understanding vulnerabilities of plant populations to climate change could help preserve their biodiversity and reveal new elite parents for future breeding programmes. To this end, landscape genomics is a useful approach for assessing putative adaptations to future climatic conditions, especially in long-lived species such as trees. We conducted a population genomics study of 207 Coffea canephora trees from seven forests along different climate gradients in Uganda. For this, we sequenced 323 candidate genes involved in key metabolic and defence pathways in coffee. Seventy-one single nucleotide polymorphisms (SNPs) were found to be significantly associated with bioclimatic variables, and were thereby considered as putatively adaptive loci. These SNPs were linked to key candidate genes, including transcription factors, like DREB-like and MYB family genes controlling plant responses to abiotic stresses, as well as other genes of organoleptic interest, such as the DXMT gene involved in caffeine biosynthesis and a putative pest repellent. These climate-associated genetic markers were used to compute genetic offsets, predicting population responses to future climatic conditions based on local climate change forecasts. Using these measures of maladaptation to future conditions, substantial levels of genetic differentiation between present and future diversity were estimated for all populations and scenarios considered. The populations from the forests Zoka and Budongo, in the northernmost zone of Uganda, appeared to have the lowest genetic offsets under all predicted climate change patterns, while populations from Kalangala and Mabira, in the Lake Victoria region, exhibited the highest genetic offsets. The potential of these findings in terms of ex situ conservation strategies are discussed.

Genetic diversity of native and cultivated Ugandan Robusta coffee (Coffea canephora Pierre ex A. Froehner): Climate influences, breeding potential and diversity conservation.

Wild genetic resources and their ability to adapt to environmental change are critically important in light of the projected climate change, while constituting the foundation of agricultural sustainability. To address the expected negative effects of climate change on Robusta coffee trees (Coffea canephora), collecting missions were conducted to explore its current native distribution in Uganda over a broad climatic range. Wild material from seven forests could thus be collected. We used 19 microsatellite (SSR) markers to assess genetic diversity and structure of this material as well as material from two ex-situ collections and a feral population. The Ugandan C. canephora diversity was then positioned relative to the species' global diversity structure. Twenty-two climatic variables were used to explore variations in climatic zones across the sampled forests. Overall, Uganda's native C. canephora diversity differs from other known genetic groups of this species. In northwestern (NW) Uganda, four distinct genetic clusters were distinguished being from Zoka, Budongo, Itwara and Kibale forests A large southern-central (SC) cluster included Malabigambo, Mabira, and Kalangala forest accessions, as well as feral and cultivated accessions, suggesting similarity in genetic origin and strong gene flow between wild and cultivated compartments. We also confirmed the introduction of Congolese varieties into the SC region where most Robusta coffee production takes place. Identified populations occurred in divergent environmental conditions and 12 environmental variables significantly explained 16.3% of the total allelic variation across populations. The substantial genetic variation within and between Ugandan populations with different climatic envelopes might contain adaptive diversity to cope with climate change. The accessions that we collected have substantially enriched the diversity hosted in the Ugandan collections and thus contribute to ex situ conservation of this vital genetic resource. However, there is an urgent need to develop strategies to enhance complementary in-situ conservation of Coffea canephora in native forests in northwestern Uganda.

Identification and characterization of Vietnamese coffee bacterial endophytes displaying in vitro antifungal and nematicidal activities.

The endophytic bacteria were isolated from coffee roots and seeds in Vietnam and identified with 16S rDNA sequencing as belonging to the Actinobacteria, Firmicutes and Proteobacteria phyla with the Nocardia, Bacillus and Burkholderia as dominant genera, respectively. Out of the thirty genera recovered from Coffea canephora and Coffea liberica, twelve were reported for the first time in endophytic association with coffee including members of the genera Brachybacterium, Caballeronia, Kitasatospora, Lechevalieria, Leifsonia, Luteibacter, Lysinibacillus, Mycolicibacterium, Nakamurella, Paracoccus, Sinomonas and Sphingobium. A total of eighty bacterial endophytes were characterized in vitro for several plant growth promoting and biocontrol traits including: the phosphate solubilization, the indolic compounds, siderophores, HCN, esterase, lipase, gelatinase and chitinase production. A subset of fifty selected bacteria were tested for their potential as biocontrol agents with in vitro confrontations with the fungal pathogen Fusarium oxysporum as well as the coffee parasitic nematodes Radopholus duriophilus and Pratylenchus coffeae. The three most efficient isolates on F. oxysporum belonging to the Bacillus, Burkholderia, and Streptomyces genera displayed a growth inhibition rate higher than 40%. Finally, five isolates from the Bacillus genus were able to lead to 100% of mortality in 24 h on both R. duriophilus and P. coffeae.

Starmaya: The First Arabica F1 Coffee Hybrid Produced Using Genetic Male Sterility.

In the present paper, we evaluated the implementation of a seed production system based on the exploitation of male sterility on coffee. We studied specifically the combination between CIR-SM01 and Marsellesa® (a Sarchimor line), which provides a hybrid population called Starmaya. We demonstrated that the establishment of seed garden under natural pollination is possible and produces a sufficient amount of hybrid seeds to be multiplied efficiently and economically. As expected for F1 hybrid, the performances of Starmaya are highly superior to conventional cultivars. However, we observed some heterogeneity on Starmaya cultivar in the field. We confirmed by genetic marker analysis that the off-types were partly related to the heterozygosity of the CIR-SM01 clone and could not be modified. Regarding the level of rust resistance of Starmaya cv., we saw that it could be improved if Marsellesa was more fully fixed genetically. If so, we should be able to decrease significantly the percentage of rust incidence of Starmaya from 15 to 5%, which would be quite acceptable at a commercial level. Starmaya represents the proof of concept for the mass propagation of Arabica F1 hybrid seeds using male sterility. Finally, we discuss the possibility to increase the number of hybrid varieties produced by seed, exploring some initiatives to identify male sterility markers to induce male sterility on any conventional cultivar. This would definitively open up the universe of known Arabica cultivars to be used in breeding new F1 hybrids.

Identification and characterization of core abscisic acid (ABA) signaling components and their gene expression profile in response to abiotic stresses in Setaria viridis.

Abscisic acid (ABA) is an essential phytohormone that regulates growth, development and adaptation of plants to environmental stresses. In Arabidopsis and other higher plants, ABA signal transduction involves three core components namely PYR/PYL/RCAR ABA receptors (PYLs), type 2C protein phosphatases (PP2Cs) and class III SNF-1-related protein kinase 2 (SnRK2s). In the present study, we reported the identification and characterization of the core ABA signaling components in Setaria viridis, an emerging model plant for cereals and feedstock crops presenting C4 metabolism, leading to the identification of eight PYL (SvPYL1 to 8), twelve PP2C (SvPP2C1 to 12) and eleven SnRK2 (SvSnRK2.1 through SvSnRK2.11) genes. In order to study the expression profiles of these genes, two different S. viridis accessions (A10.1 and Ast-1) were submitted to drought, salinity and cold stresses, in addition to application of exogenous ABA. Differential gene expression profiles were observed in each treatment and plant genotype, demonstrating variations of ABA stress responses within the same species. These differential responses to stresses were also assessed by physiological measurements such as photosynthesis, stomatal conductance and transpiration rate. This study allows a detailed analysis of gene expression of the core ABA signaling components in Setaria viridis submitted to different treatments and provides suitable targets for genetic engineering of C4 plants aiming tolerance to abiotic stresses.

Coffee Somatic Embryogenesis: How Did Research, Experience Gained and Innovations Promote the Commercial Propagation of Elite Clones From the Two Cultivated Species?

Since the 1990s, somatic embryogenesis (SE) has enabled the propagation of selected varieties, Arabica F1 hybrid and Robusta clones, originating from the two cultivated coffee species, Coffea arabica and Coffea canephora, respectively. This paper shows how mostly empirical research has led to successful industrial transfers launched in the 2000s in Latin America, Africa, and Asia. Coffee SE can be considered as a model for other woody perennial crops for the following reasons: (i) a high biological efficiency has been demonstrated for propagated varieties at all developmental stages, and (ii) somaclonal variation is understood and mastered thanks to intensive research combining molecular markers and field observations. Coffee SE is also a useful model given the strong economic constraints that are specific to this species. In brief, SE faced four difficulties: (i) the high cost of SE derived plants compared to the cost of seedlings of conventional varieties, (ii) the logistic problems involved in reaching small-scale coffee growers, (iii) the need for certification, and (iv) the lack of solvency among small-scale producers. Nursery activities were professionalized by introducing varietal certification, quality control with regard to horticultural problems and somaclonal variation, and sanitary control for Xylella fastidiosa. In addition, different technology transfers were made to ensure worldwide dissemination of improved F1 Arabica hybrids and Robusta clones. Innovations have been decisive for successful scaling-up and reduction of production costs, such as the development of temporary immersion bioreactors for the mass production of pre-germinated embryos, their direct sowing on horticultural soil, and the propagation of rejuvenated SE plants by rooted mini-cuttings. Today, SE is a powerful tool that is widely used in coffee for biotechnological applications including propagation and genetic transformation. Basic research has recently started taking advantage of optimized SE protocols. Based on -omics methodologies, research aims to decipher the molecular events involved in the key developmental switches of coffee SE. In parallel, a high-throughput screening of active molecules on SE appears to be a promising tool to speed-up the optimization of SE protocols.

Differential fine-tuning of gene expression regulation in coffee leaves by CcDREB1D promoter haplotypes under water deficit.

Despite the importance of the DREB1D gene (also known as CBF4) in plant responses to water deficit and cold stress, studies analysing its regulation by transgenic approaches are lacking. In the current work, a functional study of three CcDREB1D promoter haplotypes (named HP15, HP16 and HP17) isolated from drought-tolerant and drought-sensitive clones of Coffea canephora was carried out in plants of C. arabica stably transformed by Agrobacterium tumefaciens by analysing their ability to regulate the expression of the uidA reporter gene in response to water deficit mimicked by polyethylene glycol (-2.0 MPa) and low relative humidity treatments. A deletion analysis of their corresponding 5'-upstream regions revealed increased specificity of ß-glucuronidase activity in the polyethylene glycol and low relative humidity treatments, with high expression in leaf mesophyll and guard cells in full-length constructs. RT-qPCR assays also revealed that the HP16 haplotype (specific to clone tolerant to water deficit) had stronger and earlier activity compared with the HP15 and HP17 haplotypes. As most of the cis-regulatory elements involved in ABA-dependent and -independent networks, tissue specificity and light regulation are common to these haplotypes, we propose that their organization, as well as the nucleic acid polymorphisms present outside these boxes, may play a role in modulating activities of DREB1D promoters in guard cells.

Identification of candidate genes for drought tolerance in coffee by high-throughput sequencing in the shoot apex of different Coffea arabica cultivars.

BACKGROUND: Drought is a widespread limiting factor in coffee plants. It affects plant development, fruit production, bean development and consequently beverage quality. Genetic diversity for drought tolerance exists within the coffee genus. However, the molecular mechanisms underlying the adaptation of coffee plants to drought are largely unknown. In this study, we compared the molecular responses to drought in two commercial cultivars (IAPAR59, drought-tolerant and Rubi, drought-susceptible) of Coffea arabica grown in the field under control (irrigation) and drought conditions using the pyrosequencing of RNA extracted from shoot apices and analysing the expression of 38 candidate genes. RESULTS: Pyrosequencing from shoot apices generated a total of 34.7 Mbp and 535,544 reads enabling the identification of 43,087 clusters (41,512 contigs and 1,575 singletons). These data included 17,719 clusters (16,238 contigs and 1,575 singletons) exclusively from 454 sequencing reads, along with 25,368 hybrid clusters assembled with 454 sequences. The comparison of DNA libraries identified new candidate genes (n = 20) presenting differential expression between IAPAR59 and Rubi and/or drought conditions. Their expression was monitored in plagiotropic buds, together with those of other (n = 18) candidates genes. Under drought conditions, up-regulated expression was observed in IAPAR59 but not in Rubi for CaSTK1 (protein kinase), CaSAMT1 (SAM-dependent methyltransferase), CaSLP1 (plant development) and CaMAS1 (ABA biosynthesis). Interestingly, the expression of lipid-transfer protein (nsLTP) genes was also highly up-regulated under drought conditions in IAPAR59. This may have been related to the thicker cuticle observed on the abaxial leaf surface in IAPAR59 compared to Rubi. CONCLUSIONS: The full transcriptome assembly of C. arabica, followed by functional annotation, enabled us to identify differentially expressed genes related to drought conditions. Using these data, candidate genes were selected and their differential expression profiles were confirmed by qPCR experiments in plagiotropic buds of IAPAR59 and Rubi under drought conditions. As regards the genes up-regulated under drought conditions, specifically in the drought-tolerant IAPAR59, several corresponded to orphan genes but also to genes coding proteins involved in signal transduction pathways, as well as ABA and lipid metabolism, for example. The identification of these genes should help advance our understanding of the genetic determinism of drought tolerance in coffee.

Lipid transfer proteins in coffee: isolation of Coffea orthologs, Coffea arabica homeologs, expression during coffee fruit development and promoter analysis in transgenic tobacco plants.

The aim of the present study was to perform a genomic analysis of non-specific lipid-transfer proteins (nsLTPs) in coffee. Several nsLTPs-encoding cDNA and gene sequences were cloned from Coffea arabica and Coffea canephora species. In this work, their analyses revealed that coffee nsLTPs belong to Type II LTP characterized under their mature forms by a molecular weight of around 7.3 kDa, a basic isoelectric points of 8.5 and the presence of typical CXC pattern, with X being an hydrophobic residue facing towards the hydrophobic cavity. Even if several single nucleotide polymorphisms were identified in these nsLTP-coding sequences, 3D predictions showed that they do not have a significant impact on protein functions. Northern blot and RT-qPCR experiments revealed specific expression of Type II nsLTPs-encoding genes in coffee fruits, mainly during the early development of endosperm of both C. arabica and C. canephora. As part of our search for tissue-specific promoters in coffee, an nsLTP promoter region of around 1.2 kb was isolated. It contained several DNA repeats including boxes identified as essential for grain specific expression in other plants. The whole fragment, and a series of 5' deletions, were fused to the reporter gene ß-glucuronidase (uidA) and analyzed in transgenic Nicotiana tabacum plants. Histochemical and fluorimetric GUS assays showed that the shorter (345 bp) and medium (827 bp) fragments of nsLTP promoter function as grain-specific promoters in transgenic tobacco plants.

Transcriptional activity, chromosomal distribution and expression effects of transposable elements in Coffea genomes.

Plant genomes are massively invaded by transposable elements (TEs), many of which are located near host genes and can thus impact gene expression. In flowering plants, TE expression can be activated (de-repressed) under certain stressful conditions, both biotic and abiotic, as well as by genome stress caused by hybridization. In this study, we examined the effects of these stress agents on TE expression in two diploid species of coffee, Coffea canephora and C. eugenioides, and their allotetraploid hybrid C. arabica. We also explored the relationship of TE repression mechanisms to host gene regulation via the effects of exonized TE sequences. Similar to what has been seen for other plants, overall TE expression levels are low in Coffea plant cultivars, consistent with the existence of effective TE repression mechanisms. TE expression patterns are highly dynamic across the species and conditions assayed here are unrelated to their classification at the level of TE class or family. In contrast to previous results, cell culture conditions per se do not lead to the de-repression of TE expression in C. arabica. Results obtained here indicate that differing plant drought stress levels relate strongly to TE repression mechanisms. TEs tend to be expressed at significantly higher levels in non-irrigated samples for the drought tolerant cultivars but in drought sensitive cultivars the opposite pattern was shown with irrigated samples showing significantly higher TE expression. Thus, TE genome repression mechanisms may be finely tuned to the ideal growth and/or regulatory conditions of the specific plant cultivars in which they are active. Analysis of TE expression levels in cell culture conditions underscored the importance of nonsense-mediated mRNA decay (NMD) pathways in the repression of Coffea TEs. These same NMD mechanisms can also regulate plant host gene expression via the repression of genes that bear exonized TE sequences.

Differentially expressed genes and proteins upon drought acclimation in tolerant and sensitive genotypes of Coffea canephora.

The aim of this study was to investigate the molecular mechanisms underlying drought acclimation in coffee plants by the identification of candidate genes (CGs) using different approaches. The first approach used the data generated during the Brazilian Coffee expressed sequence tag (EST) project to select 13 CGs by an in silico analysis (electronic northern). The second approach was based on screening macroarrays spotted with plasmid DNA (coffee ESTs) with separate hybridizations using leaf cDNA probes from drought-tolerant and susceptible clones of Coffea canephora var. Conilon, grown under different water regimes. This allowed the isolation of seven additional CGs. The third approach used two-dimensional gel electrophoresis to identify proteins displaying differential accumulation in leaves of drought-tolerant and susceptible clones of C. canephora. Six of them were characterized by MALDI-TOF-MS/MS (matrix-assisted laser desorption-time of flight-tandem mass spectrometry) and the corresponding proteins were identified. Finally, additional CGs were selected from the literature, and quantitative real-time polymerase chain reaction (qPCR) was performed to analyse the expression of all identified CGs. Altogether, >40 genes presenting differential gene expression during drought acclimation were identified, some of them showing different expression profiles between drought-tolerant and susceptible clones. Based on the obtained results, it can be concluded that factors involved a complex network of responses probably involving the abscisic signalling pathway and nitric oxide are major molecular determinants that might explain the better efficiency in controlling stomata closure and transpiration displayed by drought-tolerant clones of C. canephora.

Characterization and expression of two cDNA encoding 3-Hydroxy-3-methylglutaryl coenzyme A reductase isoforms in coffee (Coffea arabica L.).

In higher plants there are two independent pathways for isoprenoid biosynthesis, located in the cytosol (mevalonic acid or MVA pathway) or in the plastids [methylerythritol phosphate (MEP) pathway]. The 3-hydroxy-3-methyglutaryl-CoA reductase (HMGR) is the first committed step in the MVA pathway. Using the information available from the Brazilian Coffee Genome Project, we found 13 ESTs that originated two isoforms, CaHMGR1 and CaHMGR2, for the enzyme HMGR of Coffea arabica. A complementary DNA encoding the isoform CaHMGR1 was cloned, and its complete nucleotide sequence determined. The full-length cDNA of CaHMGR1 was 2,242 bp containing a 1,812-bp ORF encoding 604 amino acids. Bioinformatic analyses revealed that the deduced CaHMGR1 had extensive homology with other plant HMGRs and contained two transmembrane domains and two putative HMGR binding sites and two NADP(H)-binding sites. Under normal growth conditions, transcripts of isoform CaHMRG1 were detected in fruit tissues (pulp, perisperm, and endosperm) only at the initial stages of development, flower buds and leaves. CaHMRG2 was expressed in all tissues and during all fruit development stages examined. These results suggest a constitutive expression of isoform CaHMGR2, while the isoform CaHMGR1 shows temporal and tissue-specific transcriptional activation.

RBCS1 expression in coffee: Coffea orthologs, Coffea arabica homeologs, and expression variability between genotypes and under drought stress.

BACKGROUND: In higher plants, the inhibition of photosynthetic capacity under drought is attributable to stomatal and non-stomatal (i.e., photochemical and biochemical) effects. In particular, a disruption of photosynthetic metabolism and Rubisco regulation can be observed. Several studies reported reduced expression of the RBCS genes, which encode the Rubisco small subunit, under water stress. RESULTS: Expression of the RBCS1 gene was analysed in the allopolyploid context of C. arabica, which originates from a natural cross between the C. canephora and C. eugenioides species. Our study revealed the existence of two homeologous RBCS1 genes in C. arabica: one carried by the C. canephora sub-genome (called CaCc) and the other carried by the C. eugenioides sub-genome (called CaCe). Using specific primer pairs for each homeolog, expression studies revealed that CaCe was expressed in C. eugenioides and C. arabica but was undetectable in C. canephora. On the other hand, CaCc was expressed in C. canephora but almost completely silenced in non-introgressed ("pure") genotypes of C. arabica. However, enhanced CaCc expression was observed in most C. arabica cultivars with introgressed C. canephora genome. In addition, total RBCS1 expression was higher for C. arabica cultivars that had recently introgressed C. canephora genome than for "pure" cultivars. For both species, water stress led to an important decrease in the abundance of RBCS1 transcripts. This was observed for plants grown in either greenhouse or field conditions under severe or moderate drought. However, this reduction of RBCS1 gene expression was not accompanied by a decrease in the corresponding protein in the leaves of C. canephora subjected to water withdrawal. In that case, the amount of RBCS1 was even higher under drought than under unstressed (irrigated) conditions, which suggests great stability of RBCS1 under adverse water conditions. On the other hand, for C. arabica, high nocturnal expression of RBCS1 could also explain the accumulation of the RBCS1 protein under water stress. Altogether, the results presented here suggest that the content of RBCS was not responsible for the loss of photosynthetic capacity that is commonly observed in water-stressed coffee plants. CONCLUSION: We showed that the CaCe homeolog was expressed in C. eugenioides and non-introgressed ("pure") genotypes of C. arabica but that it was undetectable in C. canephora. On the other hand, the CaCc homeolog was expressed in C. canephora but highly repressed in C. arabica. Expression of the CaCc homeolog was enhanced in C. arabica cultivars that experienced recent introgression with C. canephora. For both C. canephora and C. arabica species, total RBCS1 gene expression was highly reduced with WS. Unexpectedly, the accumulation of RBCS1 protein was observed in the leaves of C. canephora under WS, possibly coming from nocturnal RBCS1 expression. These results suggest that the increase in the amount of RBCS1 protein could contribute to the antioxidative function of photorespiration in water-stressed coffee plants.

Evaluation of kahweol and cafestol in coffee tissues and roasted coffee by a new high-performance liquid chromatography methodology.

A reverse phase high-performance liquid chromatography (HPLC) method was developed for the simultaneous quantification of kahweol and cafestol in tissues of fresh fruits, leaves, and roasted coffee beans. The best resolution was obtained with isocratic elution of acetonitrile/water (55/45% v/v) and UV detection. A single sample preparation method carried out by direct saponification and extraction with organic solvent was standardized for all matrices. Good recovery (average of 99% for kahweol and 94% for cafestol), repeatability, and linearity were obtained. Detection limits of 2.3 and 3.0 mg/100 g were observed for kahweol and cafestol. The HPLC method was effective in quantifying these diterpenes in the different coffee matrices. The endosperm and perisperm of Coffea arabica cv. IAPAR 59 showed elevated amounts of kahweol as compared to the pericarp and leaves. On the other hand, cafestol was detected in all samples except in leaves from Coffea canephora cv. Apoata.

Effects of shade on the development and sugar metabolism of coffee (Coffea arabica L.) fruits.

Coffee fruits grown in shade are characterized by larger bean size than those grown under full-sun conditions. The present study assessed the effects of shade on bean characteristics and sugar metabolism by analyzing tissue development, sugar contents, activities of sucrose metabolizing enzymes and expression of sucrose synthase-encoding genes in fruits of coffee (Coffea arabica L.) plants submitted to full-sun (FS) and shade (SH) conditions. Evolution of tissue fresh weights measured in fruits collected regularly from flowering to maturation indicated that this increase is due to greater development of the perisperm tissue in the shade. The effects of light regime on sucrose and reducing sugar (glucose and fructose) contents were studied in fresh and dry coffee beans. Shade led to a significant reduction in sucrose content and to an increase in reducing sugars. In pericarp and perisperm tissues, higher activities of sucrose synthase (EC 2.4.1.13) and sucrose-phosphate synthase (SPS: EC 2.4.1.14) were detected at maturation in the shade compared with full sun. These two enzymes also had higher peaks of activities in developing endosperm under shade than in full sun. It was also noted that shade modified the expression of SUS-encoding genes in coffee beans; CaSUS2 gene transcripts levels were higher in SH than in FS. As no sucrose increase accompanied these changes, this suggests that sucrose metabolism was redirected to other metabolic pathways that need to be identified.