Análise de expressão diferencial de proteases em sementes de Coffea arabica

Coffee grains harvested from regions with warmer climates produce distinctly different coffee drinks and simultaneously, present differential levels of protease activity. Based on evidences from our work along this research line, the present study aimed to profile the expression of genes involved in protease production in fruits of a coffee variety along the developmental stages and cultivated under distinct edapho-climatic sites (Adamantina and Mococa). Five genes representing proteases from asparagines and cysteine group were chosen for the expression analyses due to the fact that the corresponding ESTs were fruit-specific in Coffea Genoma databases. Simultaneously, we have investigated four constitutively expressed genes as normalizers for RT-PCR assays in coffee grains and have adopted GPDH C and CYP. The most significant expression differences were observed between the fruit ripening stages (green and “cherry” stage). Among the fruits harvested in Mococa, green fruits had up to 13.93 times higher levels of Cis 5 gene expression than observed in “cherry” stage fruits. In contrast, the expression pattern from fruits harvested from Adamantina did not present the fore mentioned difference. Cis 8 gene was highly expressed in green fruits from both locations. Gene Cis 22 presented lower expression rates in green fruits in comparison to “cherry” fruits in both locations. Taken together, our data demonstrate that the expression rates of these genes is variable along fruit ripening and are likely to be influenced by environmental crop conditions. In order to evaluate the differential expression of proteases in distinct cultivation sites showing contrasting temperature conditions, we have investigated the expression patterns of these genes in fruits harvested at the same developmental stage harvested in distinct locations. Three cysteine genes presented significant differential expression patterns in Adamantina, in comparison to Mococa. At “cherry” stage, the difference between the expression of protease gene Cis 5 was approximately 5-fold higher in Adamantina in comparison to its expression in Mococa. In contrast, in green fruits the expression of Cis 5 was, on average, a 3-fold higher in Mococa, where the mean annual temperature is lower than in Adamantina. A Cis 5-like gene that codes for a papain-type cysteine-endopeptidase from castorbean (Ricinus communis L.) is capable of processing the precursor of glyoxysomal malate dehydrogenase and it has been suggested that this enzyme functions in metabolite transfer from older to newly formed tissues. Although the determination of the influence of proteases on coffee drink needs further biochemical and sensorial analyses, it was possible to establish a close correlation between the expression of this enzyme and environmental factors.