Background: Quantitative data from gene expression experiments are often normalized by transcription levels of reference or housekeeping genes. An inherent assumption for their use is that the expression of these genes is highly uniform in living organisms during various phases of development, in different cell types and under diverse environmental conditions. To date, the validation of reference genes in plants has received very little attention and suitable reference genes have not been defined for a great number of crop species including Coffea arabica. The aim of the research reported herein was to compare the relative expression of a set of potential reference genes across different types of tissue/organ samples of coffee. We also validated the expression profiles of the selected reference genes at various stages of development and under a specific biotic stress.

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The regulation of the CaWRKY1 homoeologous genes were analyzed through the characterization of their promoters. The pW1a promoter is proposed as a new tool for coffee plant biotechnologies. WRKY transcription factors are important elements of the plant immune response. The CaWRKY1 gene from Coffea arabica is induced by several biotic and abiotic stresses, including challenge by the rust fungus Hemileia vastatrix. Two homoeologous CaWRKY1 genes, named CaWRKY1a and CaWRKY1b, were previously identified in the C. arabica allotetraploid genome. To gain insight into the transcriptional regulation of these genes, their promoter sequences, named pW1a and pW1b, respectively, were cloned and characterized in this study. In silico analysis revealed some important defense-associated regulatory elements, including W-boxes and as-1 elements. Promoter activities were analyzed in transient assays conducted by agroinfiltration of tobacco leaves. Exogenous salicylic acid (SA) treatments increased promoter activities corroborating the presence of as-1 regulatory elements. Transactivation assays with the CaWRKY1 protein showed the reduction of both pW1a and pW1b promoter activities, indicating that the CaWRKY1 protein may negatively regulate its own promoters. Stable transgenic C. arabica lines expressing a pW1a::GUS construct were obtained by Agrobacterium-mediated transformation and high GUS activity was observed in leaves subjected to mechanical wounding. Hence, the ability of pW1a to drive transgene expression in coffee plants as well as to enhance expression in response to stresses opens possibilities for using this promoter as a new tool for biotechnological approaches in coffee plants.

In the present work, we report the use of bacterial colonies to optimize macroarray technique. The devised system is significantly cheaper than other methods available to detect large-scale differential gene expression. Recombinant Escherichia coli clones containing plasmid-encoded copies of 4,608 individual expressed sequence tag (ESTs) were robotically spotted onto nylon membranes that were incubated for 6 and 12 h to allow the bacteria to grow and, consequently, amplify the cloned ESTs. The membranes were then hybridized with a beta-lactamase gene specific probe from the recombinant plasmid and, subsequently, phosphorimaged to quantify the microbial cells. Variance analysis demonstrated that the spot hybridization signal intensity was similar for 3,954 ESTs (85.8%) after 6 h of bacterial growth. Membranes spotted with bacteria colonies grown for 12 h had 4,017 ESTs (87.2%) with comparable signal intensity but the signal to noise ratio was fivefold higher. Taken together, the results of this study indicate that it is possible to investigate large-scale gene expression using macroarrays based on bacterial colonies grown for 6 h onto membranes.

A seed maturation protein gene (CaSMP) from Coffea arabica is expressed in the endosperm of yellow/green fruits. The CaSMP promoter drives reporter expression in the seeds of immature tomato fruits. In this report, an expressed sequence tag-based approach was used to identify a seed-specific candidate gene for promoter isolation in Coffea arabica. The tissue-specific expression of the cognate gene (CaSMP), which encodes a yet uncharacterized coffee seed maturation protein, was validated by RT-qPCR. Additional expression analysis during coffee fruit development revealed higher levels of CaSMP transcript accumulation in the yellow/green phenological stage. Moreover, CaSMP was preferentially expressed in the endosperm and was down-regulated during water imbibition of the seeds. The presence of regulatory cis-elements known to be involved in seed- and endosperm-specific expression was observed in the CaSMP 5'-upstream region amplified by genome walking (GW). Additional histochemical analysis of transgenic tomato (cv. Micro-Tom) lines harboring the GW-amplified fragment (~ 1.4 kb) fused to uidA reporter gene confirmed promoter activity in the ovule of immature tomato fruits, while no activity was observed in the seeds of ripening fruits and in the other organs/tissues examined. These results indicate that the CaSMP promoter can be used to drive transgene expression in coffee beans and tomato seeds, thus representing a promising biotechnological tool.