RESUMO
Abscisic acid (ABA) is a phytohormone that promotes leaf senescence in response to environmental stress. We previously identified methyl CpG-binding domain 10 (MBD10) as a phosphoprotein that becomes differentially phosphorylated after ABA treatment in Arabidopsis. ABA-induced leaf senescence was delayed in mbd10 knockout plants but accelerated in MBD10-overexpressing plants, suggesting that MBD10 positively regulates ABA-induced leaf senescence. ABA-induced phosphorylation of MBD10 occurs in planta on Thr-89, and our results demonstrated that Thr-89 phosphorylation is essential for MBD10's function in leaf senescence. The in vivo phosphorylation of Thr-89 in MBD10 was significantly downregulated in a quadruple mutant of group C MAPKs (mpk1/2/7/14), and group C MAPKs directly phosphorylated MBD10 in vitro. Furthermore, mpk1/2/7/14 showed a similar phenotype as seen in mbd10 for ABA-induced leaf senescence, suggesting that group C MAPKs are the cognate kinases of MBD10 for Thr-89. Because group C MAPKs have been reported to function downstream of SnRK2s, our results indicate that group C MAPKs and MBD10 constitute a regulatory pathway for ABA-induced leaf senescence.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas Quinases Ativadas por Mitógeno , Folhas de Planta , Senescência Vegetal , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Fosforilação , Reguladores de Crescimento de Plantas/metabolismo , Folhas de Planta/genética , Folhas de Planta/fisiologia , Folhas de Planta/metabolismo , Senescência Vegetal/genética , Plantas Geneticamente ModificadasRESUMO
PURPOSE: In this study, we compared two triarylmethyl (TAM) spin probes, Ox071 and Ox063 for their efficacy in measuring tissue oxygen levels under hypoxic and normoxic conditions by R2 *-based EPR oximetry. METHODS: The R2 * dependencies on the spin probe concentration and oxygen level were calibrated using deoxygenated 1, 2, 5, and 10 mM standard solutions and 2 mM solutions saturated at 0%, 2%, 5%, 10%, and 21% of oxygen. For the hypoxic model, in vivo imaging of a MIA PaCa-2 tumor implanted in the hind leg of a mouse was performed on successive days by R2 *-based EPR oximetry using either Ox071 or Ox063. For the normoxic model, renal imaging of healthy athymic mice was performed using both spin probes. The 3D images were reconstructed by single point imaging and multi-gradient technique was used to determine R2 * maps. RESULTS: The signal intensities of Ox071 were approximately three times greater than that of Ox063 in the entire partial pressure of oxygen (pO2 ) range investigated. The histograms of the tumor pO2 images were skewed for both spin probes, and Ox071 showed more frequency counts at pO2 > 32 mm Hg. In the normoxic kidney model, there was a clear delineation between the high pO2 cortex and the low pO2 medulla regions. The histogram of high-resolution kidney oximetry image using Ox071 was nearly symmetrical and frequency counts were seen up to 55 mm Hg, which were missed in Ox063 imaging. CONCLUSION: As an oximetric probe, Ox071 has clear advantages over Ox063 in terms of sensitivity and the pO2 dynamic range.