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.
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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