Fernanda A Carneiro

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.

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

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.

Functional analysis of different promoter haplotypes of the coffee ( Coffea canephora ) CcDREB1D gene through genetic transformation of Nicotiana tabacum

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.