Heterologous expression of coffee HB12 confers tolerance to water deficit in transgenic plants through an ABA-independent route

Drought is one of the major abiotic stresses affecting plant growth, with serious negative consequences for crop yields worldwide. Among these crops, coffee is severely injured by water deficiency. Despite its economic importance, very little is known about the molecular mechanisms governing coffee responses to water deficit. In the present work, a total of 288 members of the homeobox (HB) gene family were identified in the genome of the Coffea arabica Brazilian Coffee Genome Project database. In silico analysis allowed to determine the expression pattern of 33 HD genes. Among them, three genes (CaZHD4, CaHB1-like2 and CaHB12) were found to be up-regulated by osmotic stress in the database. Expression analyses revealed that CaHB12 is highly up-regulated in the leaves and lateral roots of Coffea arabica plants under moderate and severe water deficit conditions even after 10 days of drought induction. Functional characterization of transgenic Arabidopsis plants constitutively expressing CaHB12 resulted in increased tolerance to water deficit at different developmental stages and increased tolerance to salt stress during seed germination. To gain further insights into genes modulated by the ectopic expression of CaHB12, a RNA-Seq was performed revealing that classical drought-responsive genes were mostly repressed, suggesting that other mechanisms likely contribute to the tolerant phenotype exhibited by CaHB12-expressing plants, such as the pathway signalled by heat shock proteins, reactive oxygen species and heat shock transcription factor signalling pathways. Moreover, to provide further support for the involvement of CaHB12 in drought stress tolerance, three independent soybean transgenic lines overexpressing CaHB12 were employed in this study. Accordingly, at a physiological level, the constitutive expression of CaHB12 promotes the regulation of stomatal conductance and antioxidant activity under drought conditions, suggesting that this gene plays a key role in plant responses to water deprivation and can confer tolerance to drought stress. Our data suggest that CaHB12 is a positive regulator of the stress response in coffee plants and indicate that this gene is a potential candidate for biotechnological approaches.