RESUMEN
Frataxin plays a key role in eukaryotic cellular iron metabolism, particularly in mitochondrial heme and iron-sulfur (Fe-S) cluster biosynthesis. However, its precise role has yet to be elucidated. In this work, we studied the subcellular localization of Arabidopsis frataxin, AtFH, using confocal microscopy, and found a novel dual localization for this protein. We demonstrate that plant frataxin is targeted to both the mitochondria and the chloroplast, where it may play a role in Fe-S cluster metabolism as suggested by functional studies on nitrite reductase (NIR) and ferredoxin (Fd), two Fe-S containing chloroplast proteins, in AtFH deficient plants. Our results indicate that frataxin deficiency alters the normal functioning of chloroplasts by affecting the levels of Fe, chlorophyll, and the photosynthetic electron transport chain in this organelle.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Proteínas de Unión a Hierro/fisiología , Proteínas Hierro-Azufre/metabolismo , Mitocondrias/metabolismo , Arabidopsis/genética , Arabidopsis/ultraestructura , Proteínas de Arabidopsis/análisis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Clorofila/análisis , Cloroplastos/química , Ferredoxinas/genética , Ferredoxinas/metabolismo , Eliminación de Gen , Proteínas de Unión a Hierro/análisis , Proteínas de Unión a Hierro/genética , Microscopía Confocal , Mitocondrias/química , Proteínas Mitocondriales/fisiología , Nitrito Reductasas/genética , Nitrito Reductasas/metabolismo , Plantas Modificadas Genéticamente , Protoplastos/metabolismo , Protoplastos/ultraestructura , ARN Mensajero/genética , ARN de Planta/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
A simple and reproducible method for the treatment of Arabidopsis thaliana leaves with rotenone is presented. Rosette leaves were incubated with rotenone and Triton X-100 for at least 15 h. Treated leaves showed increased expression of COX19 and BCS1a, 2 genes known to be induced in Arabidopsis cell cultures after rotenone treatment. Moreover, rotenone/Triton X-100 incubated leaves presented an inhibition of oxygen uptake. The simplicity of the procedure shows this methodology is useful for studying the effect of the addition of rotenone to a photosynthetic tissue in situ.
Asunto(s)
Arabidopsis/efectos de los fármacos , Biología Celular , Mitocondrias/efectos de los fármacos , Proteínas Mitocondriales/metabolismo , Oxígeno/metabolismo , Hojas de la Planta/efectos de los fármacos , Rotenona/farmacología , Arabidopsis/citología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Botánica , Técnicas de Cultivo de Célula , Complejo I de Transporte de Electrón/metabolismo , Genes de Plantas , Mitocondrias/metabolismo , Octoxinol , Fotosíntesis/efectos de los fármacos , Hojas de la Planta/metabolismoRESUMEN
Frataxin, a conserved mitochondrial protein implicated in cellular iron homeostasis, has been involved as the iron chaperone that delivers iron for the Fe-S cluster and heme biosynthesis. However, its role in iron metabolism remains unclear, especially in photosynthetic organisms. In previous work, we found that frataxin deficiency in Arabidopsis results in decreased activity of the mitochondrial Fe-S proteins aconitase and succinate dehydrogenase, despite the increased expression of the respective genes, indicating an important role for Arabidopsis thaliana frataxin homolog (AtFH). In this work, we explore the hypothesis that AtFH can participate in heme formation in plants. For this purpose, we used two Arabidopsis lines, atfh-1 and as-AtFH, with deficiency in the expression of AtFH. Both lines present alteration in several transcripts from the heme biosynthetic route with a decrease in total heme content and a deficiency in catalase activity that was rescued with the addition of exogenous hemin. Our data substantiate the hypothesis that AtFH, apart from its role in protecting bioavailable iron within mitochondria and the biogenesis of Fe-S groups, also plays a role in the biosynthesis of heme groups in plants.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/metabolismo , Hemo/biosíntesis , Proteínas Mitocondriales/fisiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Hierro/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Fenotipo , Plantas Modificadas Genéticamente/metabolismo , ARN Mensajero/metabolismoRESUMEN
Frataxin, a nuclear-encoded mitochondrial protein, has been proposed to participate in Fe-S cluster assembly, mitochondrial energy metabolism, respiration, and iron homeostasis. However, its precise function remains elusive. Frataxin is highly conserved in living organisms with no major structural changes, in particular at the C-terminal protein domain, suggesting that it plays a key function in all organisms. Recently, a plant gene, AtFH, with significant homology to other members of the frataxin family has been described. To gain insight on the frataxin role in plants, the frataxin domain was expressed in Escherichia coli BL21-codonPlus (DE3)-RIL cells and purified using a Ni-chelating column. The purified protein, added to a mixture containing Fe(II) and H2O2, attenuates the Fenton reaction indicating that the recombinant plant frataxin is functional. The procedure described here produced high yield of 99% pure protein through only one chromatographic step, suitable for further structure-function studies.
Asunto(s)
Proteínas de Arabidopsis/biosíntesis , Proteínas de Arabidopsis/aislamiento & purificación , Proteínas de Unión a Hierro/biosíntesis , Proteínas de Unión a Hierro/aislamiento & purificación , Proteínas Mitocondriales/biosíntesis , Proteínas Mitocondriales/aislamiento & purificación , Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Clonación Molecular , Escherichia coli/metabolismo , Peróxido de Hidrógeno/química , Hierro/química , Proteínas de Unión a Hierro/metabolismo , Proteínas Mitocondriales/metabolismo , FrataxinaRESUMEN
Frataxin, a protein crucial for the biogenesis of mitochondria in different organisms, was recently identified in Arabidopsis thaliana. To investigate the role of frataxin in higher plants, we analyze two knock-out and one knock-down T-DNA insertion mutants. The knock-out mutants present an embryo-lethal phenotype, indicating an essential role for frataxin. The knock-down mutant has reduced frataxin mRNA and protein levels. This mutant also presents retarded growth, reduced fresh weight of fruits and reduced number of seeds per fruit. Surprisingly, transcription of aconitase and the Fe-S subunit of succinate dehydrogenase (SDH2-1) are increased in mutant plants; however, the activity of these proteins is reduced, indicating a role for frataxin in Fe-S cluster assembly or insertion of Fe-S clusters into proteins. Mutant plants also have increased CO(2) assimilation rates, exhibit increased formation of reactive oxygen species (ROS) and have increased levels of transcripts for proteins known to be involved in the ROS stress responses. These results indicate that frataxin is an essential protein in plants, required for full activity of mitochondrial Fe-S proteins and playing a protective role against oxidative damage.