RESUMO
Plants respond to oxygen deprivation by activating the expression of a set of hypoxia-responsive genes (HRGs). The master regulator of this process is a small group of transcription factors belonging to group VII of the ethylene response factors (ERF-VIIs). ERF-VIIs are highly unstable under aerobic conditions due to the continuous oxidation of their characteristic Cys residue at the N terminus by plant cysteine oxidases (PCOs). Under hypoxia, PCOs are inactive and the ERF-VIIs activate transcription of the HRGs required for surviving hypoxia. However, if the plant exposed to hypoxia has limited sugar reserves, the activity of ERF-VIIs is severely dampened. This suggests that oxygen sensing by PCO/ERF-VII is fine-tuned by another sensing pathway, related to sugar or energy availability. Here, we show that oxygen sensing by PCO/ERF-VII is controlled by the energy sensor target of rapamycin (TOR). Inhibition of TOR by genetic or pharmacological approaches leads to a much lower induction of HRGs. We show that two serine residues at the C terminus of RAP2.12, a major ERF-VII, are phosphorylated by TOR and are needed for TOR-dependent activation of transcriptional activity of RAP2.12. Our results demonstrate that oxygen and energy sensing converge in plants to ensure an appropriate transcription of genes, which is essential for surviving hypoxia. When carbohydrate metabolism is inefficient in producing ATP because of hypoxia, the lower ATP content reduces TOR activity, thus attenuating the efficiency of induction of HRGs by the ERF-VIIs. This homeostatic control of the hypoxia-response is required for the plant to survive submergence.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Oxigênio , Fosfatidilinositol 3-Quinases , Trifosfato de Adenosina/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Carboidratos , Cisteína Dioxigenase/metabolismo , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Hipóxia , Oxigênio/metabolismo , Açúcares/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
Thrombomodulin is a membrane-bound protein that plays an active role in the blood coagulation system by binding thrombin and initiating the protein C anticoagulant pathway. Solulin is a recombinant soluble derivative of human thrombomodulin. It is used for the treatment of thrombotic disorders. To evaluate the production of this pharmaceutical protein in plants, expression vectors were generated using four different N-terminal signal peptides. Immunoblot analysis of transiently transformed tobacco leaves showed that intact Solulin could be detected using three of these signal peptides. Furthermore transgenic tobacco plants and BY2 cells producing Solulin were generated. Immunoblot experiments showed that Solulin accumulated to maximum levels of 115 and 27 microg g(-1) plant material in tobacco plants and BY2 cells, respectively. Activity tests performed on the culture supernatant of transformed BY2 cells showed that the secreted Solulin was functional. In contrast, thrombomodulin activity was not detected in total soluble protein extracts from BY2 cells, probably due to inhibitory effects of substances in the cell extract. N-terminal sequencing was carried out on partially purified Solulin from the BY2 culture supernatant. The sequence was identical to that of Solulin produced in Chinese hamster ovary cells, confirming correct processing of the N-terminal signal peptide. We have demonstrated that plants and plant cell cultures can be used as alternative systems for the production of an active recombinant thrombomodulin derivative.