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The aim of this study was to identify the correlation between photochemical efficiency and candidate genes expression to elucidate the drought tolerance mechanisms in coffee progenies (Icatu Vermelho IAC 3851-2 x Catimor UFV 1602-215) previously identified as tolerant in field conditions. Four progenies (2, 5, 12 and 15) were evaluated under water-deficit conditions (water deficit imposed 8 months after transplanting seedlings to the pots) and under irrigated system. Evaluations of physiological parameters and expression of candidate genes for drought tolerance were performed. Progeny 5 showed capacity to maintain water potential, which contributed to lower qP variation between irrigated and deficit conditions. However, the increases of qN and NPQ in response to stress indicate that this progeny is photochemically responsive to small variations of am protecting the photosystem and maintaining qP. Data obtained for progeny 12 indicated a lower water status maintenance capacity, but with increased qN and NPQ providing maintenance of the PSII and ETR parameters. A PCA analysis revealed that the genes coding regulatory proteins, ABA-synthesis, cellular protectors, isoforms of ascorbate peroxidase clearly displayed a major response to drought stress and discriminated the progenies 5 and 12 which showed a better photochemical response. The genes CaMYB1, CaERF017, CaEDR2, CaNCED, CaAPX1, CaAPX5, CaGolS3, CaDHN1 and CaPYL8a were up-regulated in the arabica coffee progenies with greater photochemical efficiency under deficit and therefore contributing to efficiency of the photosynthesis in drought tolerant progenies Made available in DSpace on 2021-07-20T15:01:13Z (GMT). No. of bitstreams: 1 Photochemical-efciency-correlated.pdf: 1599792 bytes, checksum: 8fcd09d5ca7177dd144b3a900116a490 (MD5) Previous issue date: 2021

International audience; Climate change is posing a major challenge to coffee production worldwide leading to a need for the development of coffee cultivars with increased drought tolerance. In several plant species, the use of DREB genes in crop improvement has achieved promising results to desiccation tolerance engineering. Recent studies reported CcDREB1D specific patterns of expression in Coffea canephora and functional evidence of this gene involvement in drought stress responses. However, knowledge on natural diversity of this gene is largely unknown. In this context, this study aimed at evaluating the sequence variability of the DREB1D gene in several Coffea genotypes. Nucleotide variation in promoters and coding regions of this gene were evaluated in a population consisting of 38 genotypes of C. canephora, C. arabica and C. eugenioides, most of them characterized by different phenotypes (tolerance vs. susceptibility) in relation to drought. The genetic diversity of the loci revealed different haplotypes for the promoter and coding regions. In particular, our findings suggest association between drought tolerance and the genetic variations on DREB1D promoter regions, but not with those from its corresponding coding regions. Gene expression studies revealed up-regulated expression of DREB1D gene upon drought mainly in leaves of drought-tolerant clones of C. canephora, and in response to drought, high, and low temperatures in leaves of C. arabica, suggesting a key role of this gene in coffee responses to abiotic stress.

International audience; Previous results showed that the three promoter haplotypes (HP15, HP16 and HP17) of the CcDREB1D gene (encoding the dehydration responsive element binding transcription factor) found in the drought-tolerant (HP15/HP16) and drought-sensitive (HP15/HP17) clones of Coffea canephora, diverged by several single nucleotide polymorphisms and insertions/deletions. In order to compare the activities and regulation of these haplotypes in response to abiotic stresses, these sequences were cloned in front of the uidA and analyzed in transgenic tobacco (Nicotiana tabacum) for their ability to regulate the expression of this reporter gene by monitoring GUS histochemical activity under drought (mimicked by dehydration), heat shock and cold treatments. Under unstressed condition, GUS staining was mainly observed in leaf and root vascular tissues of young tobacco plants transformed by the longest sequences of CcDREB1D promoter haplotypes. These GUS activities were not observed in the same tissues of older plants as well as in plants transformed by shorter proximal regions, suggesting a developmentally-regulated activity of CcDREB1D promoters in tobacco and the existence of cis-regulatory elements essential for their regulation in distal regions. Under dehydration and heat shock conditions, GUS staining detected in leaf midribs and secondary veins of pHP17L-transformed plants was correlated with up-regulated expression of uidA reporter gene while no GUS activities were observed in pHP16L-transformed plants. However, all CcDREB1D promoter haplotypes were positively regulated by cold stress in transgenic tobacco. These results showed that these coffee promoters were recognized by the tobacco transcriptional machinery but were regulated in different manners in response to abiotic stress.

The effects of water deficit on photochemical parameters and expression of several candidate genes were investigated in drought-tolerant clone 73 of Coffea canephora submitted to slowly imposed water limitation. Under irriga- tion, this clone showed low values of stomatal conductance (gs) and of CO2 assimilation rates (A )s uggesting that it had a great efficiency in controlling stomatal closure and transpira- tion. After water withdrawal, this clone reached a −3.0 MPa after 15days without irrigation and showeda slow decrease in the pre-dawn leaf water potential. Under drought, the suppres- sion of Awas accompanied by maintenance of photochemical quenching (qP) and internal to ambient CO2 concentration (Ci/Ca) ratios as well as by a decrease of non-photochemical quenching (qN). This is confirmed by the transport rate/CO2 assimilation (ETR/A) rates that suggested the participation of an alternative electron sink protecting the photosynthetic apparatus against photoinhibition. At the transcriptomic level, high up-regulation of genes encoding for a dehydrin (CcDH3), an ascorbate peroxidase (CcAPX1), a prephenate-dehydrogenase like protein (CcPDH1) and a non-symbiotic haemoglobin (CcNSH1) was also observed upon drought suggesting a strong induction of antioxidant and osmoprotection systems in this clone. High expression levels of gene-encoding ABA receptors (CcPYL3 and CcPYL7) under water limitation were also ob- served suggesting the involvement of the ABA signaling path- way in response to drought. All these results where compared to those previously obtained for drought-tolerant clones 14 and 120. Our results demonstrated the existence of different mecha- nisms amongst the drought-tolerant coffee clones regarding water deficit.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12870-016-0777-5) contains supplementary material, which is available to authorized users.

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.

MADS-box genes comprise a family of transcription factors that act as key regulators in many cellular development processes of several organisms. Members of this family have highly conserved regions and play important roles as transcription factors, activating target genes. The present work aimed at analyzing the MADS-box gene family present in a database generated by the Brazilian Coffee Genome Project (CAFEST) as well as of observing their respective sites of expression within these data. Through the use of bioinformatics tools, it was possible to identify and classify 26 expressed sequence tag contigs, 13 of them were expressed exclusively in vegetative tissues, 11 in reproductive tissues, and two in both. Later, quantitative analysis by quantitative reverse transcriptase PCR was carried out for three of them belonging to the groups of genes APETALA3 (B genes), AGAMOUS (C genes), and SEPALLATAS (E genes), which could be compared with the expression profile of in silico analysis and of its putative orthologous genes. Therefore, this study aims at a better understanding of the development processes performed by this family of genes in Coffea arabica, mainly in reproductive organs, as well as to compare functions of MADS-box orthologous studied in other species.

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.

Coffee, tea, and chocolate converge Caffeine has evolved multiple times among plant species, but no one knows whether these events involved similar genes. Denoeud et al. sequenced the Coffea canephora (coffee) genome and identified a conserved gene order (see the Perspective by Zamir). Although this species underwent fewer genome duplications than related species, the relevant caffeine genes experienced tandem duplications that expanded their numbers within this species. Scientists have seen similar but independent expansions in distantly related species of tea and cacao, suggesting that caffeine might have played an adaptive role in coffee evolution. Science , this issue p. 1181 ; see also p. 1124

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 beta-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.

Made available in DSpace on 2014-12-03T13:11:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-11-11Bitstream added on 2014-12-03T13:22:47Z : No. of bitstreams: 1 WOS000327221600081.pdf: 1259635 bytes, checksum: 94a7f854e23462a4f12622699cd436d4 (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Centre de Cooperation Internationale en Recherche Agronomique pour le Developpement 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. Univ Estadual Paulista, Univ Estadual Paulista Julio de Mesquita Filho, Dept Biol, Sao Jose do Rio Preto, Brazil Georgia Inst Technol, Sch Biol, Atlanta, GA 30332 USA Univ Estadual Londrina, Dept Biol Geral, Londrina, Brazil Empresa Brasileira Pesquisa Agr Recursos Genet &, Brasilia, DF, Brazil Ctr Cooperat Int Rech Agron Dev, UMR AGAP, Montpellier, France Univ Estadual Campinas, Dept Genet Evolucao & Bioagentes, Campinas, SP, Brazil Empresa Brasileira Pesquisa Agr Cafe, Brasilia, DF, Brazil PanAmer Bioinformat Inst, Santa Marta, Magdalena, Colombia Univ Estadual Paulista, Univ Estadual Paulista Julio de Mesquita Filho, Dept Biol, Sao Jose do Rio Preto, Brazil FAPESP: 08/05894-1 FAPESP: 07/55985-0

Polyploidization constitutes a common mode of evolution in flowering plants. This event provides the raw material for the divergence of function in homeologous genes, leading to phenotypic novelty that can contribute to the success of polyploids in nature or their selection for use in agriculture. Mounting evidence underlined the existence of homeologous expression biases in polyploid genomes; however, strategies to analyze such transcriptome regulation remained scarce. Important factors regarding homeologous expression biases remain to be explored, such as whether this phenomenon influences specific genes, how paralogs are affected by genome doubling, and what is the importance of the variability of homeologous expression bias to genotype differences. This study reports the expressed sequence tag assembly of the allopolyploid Coffea arabica and one of its direct ancestors, Coffea canephora. The assembly was used for the discovery of single nucleotide polymorphisms through the identification of high-quality discrepancies in overlapped expressed sequence tags and for gene expression information indirectly estimated by the transcript redundancy. Sequence diversity profiles were evaluated within C. arabica (Ca) and C. canephora (Cc) and used to deduce the transcript contribution of the Coffea eugenioides (Ce) ancestor. The assignment of the C. arabica haplotypes to the C. canephora (CaCc) or C. eugenioides (CaCe) ancestral genomes allowed us to analyze gene expression contributions of each subgenome in C. arabica. In silico data were validated by the quantitative polymerase chain reaction and allele-specific combination TaqMAMA-based method. The presence of differential expression of C. arabica homeologous genes and its implications in coffee gene expression, ontology, and physiology are discussed.

Coffee is one of the world's most valuable agricultural export commodities. In particular, coffee is a key export and cash crop in numerous tropical and subtropical countries having a generally favorable impact on the social and physical environment.While coffee species belong to the Rubiaceae family, one of the largest tropical angiosperm families, commercial production relies mainly on two species, Coffea arabica L. and Coffea canephora Pierre, known as Robusta. Although a considerable genetic diversity is potentially available, coffee breeding is still a long and difficult process. Nevertheless, genomic approaches offer feasible strategies to decipher the genetic and molecular bases of important biological traits in coffee tree species that are relevant to the growers, processors, and consumers. This knowledge is fundamental to allow efficient use and preservation of coffee genetic resources for the development of improved cultivars in terms of quality and reduced economic and environmental costs. This review focuses on the recent progress of coffee genomics in relation to crop improvement.

Corresponding author: E-mail: thierry.leroy@cirad.fr; International audience; The first bacterial artificial chromosome (BAC) library of Robusta coffee (Coffea canephora) was constructed, with the aim of developing molecular resources to study the genome structure and evolution of this perennial crop. Clone 126, which is highly productiveand confers good technological and organoleptic qualities of beverage, was chosen for development of this library. The BAC library contains 55,296 clones, with an average insert size of 135 Kb per plasmid, therefore representing theoretically nine haploid genome equivalents of C. canephora. Its validation was achieved with a set of 13 genetically anchored single-copy and 4 duplicated RFLP probes and yielded on average 9 BAC clones per probe. Screening of this BAC library was also carried out with partial cDNA probes coding for enzymes of sugar metabolism like invertases and sucrose synthase, with the aim of characterizing the organization and promoter structure of this important class of genes. It was shown that genes for both cell wall and vacuolar forms of invertases were probably unique in the Robusta genome whereas sucrose synthase was encoded by at least two genes. One of them (CcSUS1) was cloned and sequenced, showing that our BAC library is a valuable tool to rapidly identify genes of agronomic interest or linked to cup quality in C. canephora.

Two single-copy genes, designated atrA and atrB (ATP-binding cassette transporter A and B), were cloned from the filamentous fungus Aspergillus nidulans and sequenced. Based on the presence of conserved motifs and on hydropathy analysis, the products encoded by atrA and atrB can be regarded as novel members of the ATP-binding cassette (ABC) superfamily of membrane transporters. Both products share the same topology as the ABC transporters PDR5 and SNQ2 from Saccharomyces cerevisiae and CDR1 from Candida albicans, which are involved in multidrug resistance of these yeasts. Significant homology also occurs between the ATP-binding cassettes of AtrA and AtrB, and those of mammalian ABC transporters (P-glycoproteins). The transcription of atrA and, in particular, atrB in mycelium of A. nidulans is strongly enhanced by treatment with several drugs, including antibiotics, azole fungicides and plant defense toxins. The enhanced transcription is detectable within a few minutes after drug treatment and coincides with the beginning of energy-dependent drug efflux activity, reported previously in the fungus for azole fungicides. Transcription of the atr genes has been studied in a wild-type and in a series of isogenic strains carrying the imaA and/or imaB genes, which confer multidrug resistance to various toxic compounds such as the azole fungicide imazalil. atrB is constitutively transcribed at a low level in the wild-type and in strains carrying imaA or imaB. Imazalil treatment enhances transcription of atrB to a similar extent in all strains tested. atrA, unlike atrB, displays a relatively high level of constitutive expression in mutants carrying imaB. Imazalil enhances transcription of atrA more strongly in imaB mutants, suggesting that the imaB locus regulates atrA. Functional analysis demonstrated that cDNA of atrB can complement the drug hypersensitivity associated with PDR5 deficiency in S. cerevisiae.

The aim of this work was to study the regulation of coffee DREB-like genes in leaves of C. arabica subjected to cold, heat, low relative humidity, exogenous abscisic acid and high light stress, as well as in leaves and roots of drought-tolerant and drought-susceptible clones of Coffea canephora subjected to water limitation. In C. arabica, CaERF017 was the most expressed gene under low temperatures and relative humidity, while low humidity and high temperatures up-regulated the expression of CaERF053 and CaERF014, respectively. Under water limitation, CcDREB1B, CcRAP2.4, CcERF027, CcDREB1D and CcTINY were the most expressed genes mainly in leaves of drought-tolerant C. canephora. On the other hand, expression of the CcERF016, CcRAP2.4 and CcDREB2F genes was highly up-regulated under water limitation in the roots of drought-susceptible C. canephora clone 22. We previously reported fine-tuned regulation of CcDREB1D promoter haplotypes (HP15, HP16 and HP17) in transgenic C. arabica subjected to low humidity. Here, we investigated the regulation of these haplotypes under high light, cold, heat, and abscisic acid (ABA) stress. In apical buds and leaf guard cells, GUS-stained percentages were higher in pHP16L-transformed plants subjected to low humidity, high light and ABA stress than in pHP17L- and pHP15L-transformed plants. We also reported up-regulated expression of the endogenous CaDREB1D gene for both the cold and low humidity in leaves of pHP16L-transformed C. arabica suggesting a key role of this gene in controlling the responses of coffee plants to abiotic stress probably through an ABA-dependent pathway.