RESUMO
N-fixing nodules are new organs formed on legume roots as a result of the beneficial interaction with soil bacteria, rhizobia. The nodule functioning is still a poorly characterized step of the symbiotic interaction, as only a few of the genes induced in N-fixing nodules have been functionally characterized. We present here the characterization of a member of the Lotus japonicus nitrate transporter1/peptide transporter family, LjNPF8.6 The phenotypic characterization carried out in independent L. japonicus LORE1 insertion lines indicates a positive role of LjNPF8.6 on nodule functioning, as knockout mutants display N-fixation deficiency (25%) and increased nodular superoxide content. The partially compromised nodule functioning induces two striking phenotypes: anthocyanin accumulation already displayed 4 weeks after inoculation and shoot biomass deficiency, which is detected by long-term phenotyping. LjNPF8.6 achieves nitrate uptake in Xenopus laevis oocytes at both 0.5 and 30 mm external concentrations, and a possible role as a nitrate transporter in the control of N-fixing nodule activity is discussed.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Lotus/metabolismo , Família Multigênica , Fixação de Nitrogênio , Proteínas de Plantas/metabolismo , Nódulos Radiculares de Plantas/metabolismo , Animais , Antocianinas/metabolismo , Biomassa , Éxons/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Íntrons/genética , Lotus/efeitos dos fármacos , Lotus/genética , Mutagênese Insercional/genética , Mutação/genética , Transportadores de Nitrato , Nitratos/farmacologia , Fixação de Nitrogênio/efeitos dos fármacos , Fixação de Nitrogênio/genética , Nitrogenase/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Especificidade de Órgãos/efeitos dos fármacos , Fenótipo , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/metabolismo , Nódulos Radiculares de Plantas/efeitos dos fármacos , Nódulos Radiculares de Plantas/genética , Superóxidos/metabolismo , Transcrição Gênica/efeitos dos fármacos , Xenopus laevisRESUMO
The endosomal-lysosomal system is a series of organelles in the endocytic pathway that executes trafficking and degradation of proteins and lipids and mediates the internalization of nutrients and growth factors to ensure cell survival, growth, and differentiation. Here, we reveal regulatory, non-proteolytic ubiquitin signals in this complex system that are controlled by the enigmatic deubiquitinase USP32. Knockout (KO) of USP32 in primary hTERT-RPE1 cells results among others in hyperubiquitination of the Ragulator complex subunit LAMTOR1. Accumulation of LAMTOR1 ubiquitination impairs its interaction with the vacuolar H+-ATPase, reduces Ragulator function, and ultimately limits mTORC1 recruitment. Consistently, in USP32 KO cells, less mTOR kinase localizes to lysosomes, mTORC1 activity is decreased, and autophagy is induced. Furthermore, we demonstrate that depletion of USP32 homolog CYK-3 in Caenorhabditis elegans results in mTOR inhibition and autophagy induction. In summary, we identify a control mechanism of the mTORC1 activation cascade at lysosomes via USP32-regulated LAMTOR1 ubiquitination.
Assuntos
Autofagia , Alvo Mecanístico do Complexo 1 de RapamicinaRESUMO
G Protein Coupled Receptor (GPCRs) are integral membrane proteins involved in various signalling pathways by perceiving many extracellular signals and transducing them to heterotrimeric G proteins, which further transduce these signals to intracellular downstream effectors. GCR1 is the only reliable plant candidate as a member of the GPCRs superfamily. In the legume/rhizobia symbiotic interaction, G proteins are involved in signalling pathways controlling different steps of the nodulation program. In order to investigate the putative hierarchic role played by GCR1 in these symbiotic pathways we identified and characterized the Lotus japonicus gene encoding the seven transmembrane GCR1 protein. The detailed molecular and topological analyses of LjGCR1 expression patterns that are presented suggest a possible involvement in the early steps of nodule organogenesis. Furthermore, phenotypic analyses of independent transgenic RNAi lines, showing a significant LjGCR1 expression down regulation, suggest an epistatic action in the control of molecular markers of nodulation pathways, although no macroscopic symbiotic phenotypes could be revealed.