Gabriel Sergio Costa Alves

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.

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

Fine-tuning of DREB1D expression in stomatal guard cells under water deficit is mediated differentially by promoter haplotypes from sensitive and tolerant coffee genotypes.

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.

Nucleotide Diversity of the Coding and Promoter Regions of DREB1D, a Candidate Gene for Drought Tolerance in Coffea Species

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.