Produção recombinante e caracterização funcional de uma legumaína de cana-de-açúcar
Silva, Ludier Kesser Santos
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Plant legumains, also termed vacuolar processing enzymes (VPEs), are cysteine peptidases that play key roles in seed maturation, germination, senescence, stress response, programmed cell death during development and defense against pathogens. Despite the increasing number of reports on VPEs, most of studies are on dicotyledonous legumain and their role in seeds. In this study, it was performed the characterization of sugarcane legumain, named CaneLEG. Kinetic characterization of the recombinant CaneLEG expressed in Pichia pastoris revealed that this enzyme has the main characteristics of VPEs, such as self-activation and activity under acidic pH. CaneLEG activity was strongly inhibited by the sugarcane cystatin named CaneCPI-3. In vivo interaction between legumain and cystatin was observed to barley proteins, indicating that legumains might be inhibited by cystatins in the plant. Furthermore, the CaneLEG and CaneCPI-3 gene expression was analyzed throughout sugarcane development and in plantlets treated with phytohormones. From these results, it was observed that these genes showed a tissue-specific expression pattern, with the strong accumulation of CaneLEG transcripts throughout the internode development. The up-regulation of CaneLEG and CaneCPI-3 genes in plantlets treated with ABA suggests that these proteins encoded by these genes may be involved in stress response and CaneCPI-3 may act on endogenous cysteine peptidase regulation. Our results suggest the CaneCPI-3 as the endogenous inhibitor of CaneLEG and the involvement of these proteins on the ABA-regulated stress response. Further, the gradual increase on CaneLEG expression in internode suggests the action of this enzyme on the development of this storage tissue. The results obtained in this work will serve as an initial tool to understand the roles played by CaneLEG and CaneCPI-3 on sugarcane.