Promoter analysis of the WRKY transcription factors CaWRKY1a and CaWRKY1b homoeologous genes in coffee (Coffea arabica).

CONCLUSIONS

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.