Characterization of a candidate gene for drought tolerance in Coffea: the CcDREB1D gene, in contrasting genotypes of Coffea canephora and related species

Abstract

Drought is the key factor affecting plant development, flowering, productivity, fruits development and their quality. In several plant species, the DREBs genes play a key role in plant responses to abiotic stress. In coffee, several candidate genes for drought tolerance have been identified, like CcDREB1D that showed increased gene expression upon drought stress mainly in leaves of Coffea canephora drought-tolerant clones. Based on these information, an in depth characterization of DREB1D was initiated by (i) studying the genetic diversity present in DREB1D loci in C. canephora and C. arabica, (ii) identifying DREB coffee subfamily members and evaluating phylogenetic relations between CcDREB1D and homologs from other plant species, and (iii) performing a functional characterization of CcDREB1D promoters by homologous genetic transformation of C. arabica. Our findings show several evidences of association between drought tolerance and the genetic variations on DREB1D promoter region. Further analyses indicated that these promoters are evolving by the rearrangement of cis-regulatory elements, and could influence DREB1D expression. The recent release of the C. canephora genome sequence allowed us to identify the DREB gene family in this species composed of at least 31 canonical DREB genes. Compared to other plants, no specific amplification of the DREB gene family was observed. A reconciled phylogenetic tree constructed with homologs from other species allowed us to identify the coffee members of the DREB subgroups I, II, III and IV, in reference to their classification previously established in Arabidopsis. In addition, CcDREB1D is the nearest C. canephora orthologue of AtDREB1/CBF group, but any functional inference could not be done due to a recent duplication that lead to DREB1/CBF group subfunctionalization in Arabidopsis. These results reinforced the necessity of CcDREB1D functional characterization in coffee. The study of genetic variability revealed a major source of nucleic diversity in the promoter region of DREB1D genes from C. canephora, with potential implication in local adaptation to drought tolerance. This is for example the case for the promoters of droughttolerant clone 14 and drought-sensitive clone 22 of C. canephora known to present different CcDREB1D expression profiles regarding drought. In order to perform the functional analysis of these promoters, full length and truncated version of allelic D14-hp15pro and D14-hp16pro, and homolog D22-hp17pro forms of CcDREB1D promoters were fused to the GUS reporter gene. Transgenic plants of C. arabica harboring these constructions were regenerated. The spatiotemporal, tissue, cell and allelic-specific regulation of CcDREB1D promoters was analyzed by detecting GUS enzymatic activity. In response to different (PEG and relative humidity) treatments mimicking drought stress, the full-length CcDREB1D promoter drives leaf-specific GUS activity only in epidermic, parenchyma and guard-cells. By comparing the different constructs, the most upstream 700 bp of allelic and homolog CcDREB1D promoters were shown to be necessary for the leaf-specific expression. In addition, allelic-specific expression observed showed that nucleic variations present between CcDREB1D promoter allelic forms affect the spatiotemporal regulation of the GUS reporter gene.