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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.

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

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 irrigation, this clone showed low values of stomatal conductance (g s) and of CO 2 assimilation rates (A) suggesting that it had a great efficiency in controlling stomatal closure and transpiration. After water withdrawal, this clone reached a −3.0 MPa after 15 days without irrigation and showed a slow decrease in the pre-dawn leaf water potential. Under drought, the suppression of A was accompanied by maintenance of photochemical quenching (q P) and internal to ambient CO 2 concentration (Ci/Ca) ratios as well as by a decrease of non-photochemical quenching (q N). This is confirmed by the transport rate/CO 2 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 observed suggesting the involvement of the ABA signaling pathway 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 mechanisms amongst the drought-tolerant coffee clones regarding water deficit. Keywords Candidate gene. Coffea canephora. Drought. Gene expression. Real-time quantitative PCR. Water potential Abbreviations CG Candidate gene qPCR Quantitative polymerase chain reaction

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