Characterization of the Promoter of the Homeobox Gene CaHB12 in Coffea arabica

Abiotic stresses, especially drought, are major bottlenecks for crop production. The CaHB12 gene codes for a transcription factor belonging to the HD-Zip family, and this gene was found to be induced by drought conditions in Coffea arabica. The overexpression of CaHB12 confers drought tolerance to transgenic Arabidopsis thaliana plants. In this study, the promoter of CaHB12 was isolated and characterized. Two different promoter sequences were identified in C. arabica, a tetraploid species. Cloning and sequencing of the HB12 promoter regions of the ancestral species C. canephora and C. eugenioides suggest that the C. arabica sequences originated from the diploid parental ancestors. In silico promoter analysis showed typical cis-elements for responses to drought, salinity and abscisic acid in both sequences identified in the C. arabica genome. The expression activity of two fragments from each promoter, referred to as pCc_CaHB12-979, pCc_CaHB12-356, pCe_CaHB12-749 and pCe_CaHB12-356, were investigated using uidA as the reporter gene in transgenic A. thaliana plants. In plants bearing the pCc_CaHB12-979::GUS construct, GUS activity was observed in leaves during drought stress and in roots after treatment with polyethylene glycol or mannitol. The pCc_CaHB12 (356 bp) promoter fragment showed activity after treatment with ABA. Real-time qPCR analysis of the uidA gene was used to quantify expression of the promoter fragments. Interestingly, the two pCe_CaHB12 promoter fragments showed no activity under the experimental conditions tested. These results suggest that the CaHB12 gene is stress-inducible and regulated by the pCc_CaHB12 promoter, a sequence putatively inherited from the drought-tolerant coffee species C. canephora.