RESUMEN
Plant mutants for genes encoding subunits of mitochondrial complex I (CI; NADH:ubiquinone oxidoreductase), the first enzyme of the respiratory chain, display various phenotypes depending on growth conditions. Here, we examined the impact of photoperiod, a major environmental factor controlling plant development, on two Arabidopsis (Arabidopsis thaliana) CI mutants: a new insertion mutant interrupted in both ndufs8.1 and ndufs8.2 genes encoding the NDUFS8 subunit and the previously characterized ndufs4 CI mutant. In the long day (LD) condition, both ndufs8.1 and ndufs8.2 single mutants were indistinguishable from Columbia-0 at phenotypic and biochemical levels, whereas the ndufs8.1 ndufs8.2 double mutant was devoid of detectable holo-CI assembly/activity, showed higher alternative oxidase content/activity, and displayed a growth retardation phenotype similar to that of the ndufs4 mutant. Although growth was more affected in ndufs4 than in ndufs8.1 ndufs8.2 under the short day (SD) condition, both mutants displayed a similar impairment of growth acceleration after transfer to LD compared with the wild type. Untargeted and targeted metabolomics showed that overall metabolism was less responsive to the SD-to-LD transition in mutants than in the wild type. The typical LD acclimation of carbon and nitrogen assimilation as well as redox-related parameters was not observed in ndufs8.1 ndufs8 Similarly, NAD(H) content, which was higher in the SD condition in both mutants than in Columbia-0, did not adjust under LD We propose that altered redox homeostasis and NAD(H) content/redox state control the phenotype of CI mutants and photoperiod acclimation in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/fisiología , Complejo I de Transporte de Electrón/genética , Fotoperiodo , Antioxidantes/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Carbono/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Regulación de la Expresión Génica de las Plantas , Luz , Mutación , Nitrógeno/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/metabolismoRESUMEN
Cardiolipin (CL) is the signature phospholipid of the mitochondrial inner membrane. In animals and yeast (Saccharomyces cerevisiae), CL depletion affects the stability of respiratory supercomplexes and is thus crucial to the energy metabolism of obligate aerobes. In eukaryotes, the last step of CL synthesis is catalyzed by CARDIOLIPIN SYNTHASE (CLS), encoded by a single-copy gene. Here, we characterize a cls mutant in Arabidopsis thaliana, which is devoid of CL. In contrast to yeast cls, where development is little affected, Arabidopsis cls seedlings are slow developing under short-day conditions in vitro and die if they are transferred to long-day (LD) conditions. However, when transferred to soil under LD conditions under low light, cls plants can reach the flowering stage, but they are not fertile. The cls mitochondria display abnormal ultrastructure and reduced content of respiratory complex I/complex III supercomplexes. The marked accumulation of tricarboxylic acid cycle derivatives and amino acids demonstrates mitochondrial dysfunction. Mitochondrial and chloroplastic antioxidant transcripts are overexpressed in cls leaves, and cls protoplasts are more sensitive to programmed cell death effectors, UV light, and heat shock. Our results show that CLS is crucial for correct mitochondrial function and development in Arabidopsis under both optimal and stress conditions.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/enzimología , Proteínas de la Membrana/fisiología , Mitocondrias/ultraestructura , Transferasas (Grupos de Otros Fosfatos Sustitutos)/fisiología , Antioxidantes/metabolismo , Apoptosis , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Cardiolipinas/química , ADN Bacteriano , Luz , Proteínas de la Membrana/genética , Membranas Mitocondriales/química , Mutagénesis Insercional , Protoplastos/enzimología , Plantones/crecimiento & desarrollo , Estrés Fisiológico , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genéticaRESUMEN
Mitochondrial NADH-ubiquinone oxidoreductase (complex I) is the largest enzyme of the oxidative phosphorylation system, with subunits located at the matrix and membrane domains. In plants, holocomplex I is composed of more than 40 subunits, 9 of which are encoded by the mitochondrial genome (NAD subunits). In Nicotiana sylvestris, a minor 800-kDa subcomplex containing subunits of both domains and displaying NADH dehydrogenase activity is detectable. The NMS1 mutant lacking the membrane arm NAD4 subunit and the CMSII mutant lacking the peripheral NAD7 subunit are both devoid of the holoenzyme. In contrast to CMSII, the 800-kDa subcomplex is present in NMS1 mitochondria, indicating that it could represent an assembly intermediate lacking the distal part of the membrane arm. L-galactono-1,4-lactone dehydrogenase (GLDH), the last enzyme in the plant ascorbate biosynthesis pathway, is associated with the 800-kDa subcomplex but not with the holocomplex. To investigate possible relationships between GLDH and complex I assembly, we characterized an Arabidopsis thaliana gldh insertion mutant. Homozygous gldh mutant plants were not viable in the absence of ascorbate supplementation. Analysis of crude membrane extracts by blue native and two-dimensional SDS-PAGE showed that complex I accumulation was strongly prevented in leaves and roots of Atgldh plants, whereas other respiratory complexes were found in normal amounts. Our results demonstrate the role of plant GLDH in both ascorbate biosynthesis and complex I accumulation.
Asunto(s)
Complejo I de Transporte de Electrón , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/fisiología , Arabidopsis/genética , Membrana Celular/enzimología , Genes de Plantas , Modelos Biológicos , Mutación , Fosforilación Oxidativa , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Oxígeno/metabolismo , Fosforilación , Extractos Vegetales/metabolismo , Hojas de la Planta/metabolismo , Nicotiana/genéticaRESUMEN
The mitochondrial DNA of the Nicotiana sylvestris CMSII mutant carries a 72-kb deletion comprising the single copy nad7 gene that encodes the NAD7 subunit of the respiratory complex I (NADH-ubiquinone oxidoreductase). CMSII plants lack rotenone-sensitive complex I activity and are impaired in physiological and phenotypical traits. To check whether these changes directly result from the deletion of nad7, we constructed CMS transgenic plants (termed as CMSnad7) carrying an edited nad7 cDNA fused to the CAMV 35S promoter and to a mitochondrial targeting sequence. The nad7 sequence was transcribed and translated and the NAD7 protein directed to mitochondria in CMSnad7 transgenic plants, which recovered both wild type morphology and growth features. Blue-native/SDS gel electrophoresis and enzymatic assays showed that, whereas fully assembled complex I was absent from CMSII mitochondria, a functional complex was present in CMSnad7 mitochondria. Furthermore, a supercomplex involving complex I and complex III was present in CMSnad7 as in the wild type. Taken together, these data demonstrate that lack of complex I in CMSII was indeed the direct consequence of the absence of nad7. Hence, NAD7 is a key element for complex assembly in plants. These results also show that allotopic expression from the nucleus can fully complement the lack of a mitochondrial-encoded complex I gene.
Asunto(s)
Complejo I de Transporte de Electrón/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/fisiología , Proteínas de Plantas/genética , Proteínas de Plantas/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Núcleo Celular/metabolismo , Clonación Molecular , ADN Complementario/metabolismo , ADN Mitocondrial/metabolismo , Electroforesis en Gel de Poliacrilamida , Formiato Deshidrogenasas/metabolismo , Eliminación de Gen , Membranas Intracelulares/metabolismo , Espectrometría de Masas , Datos de Secuencia Molecular , Mutación , NAD/metabolismo , Péptidos/química , Fenotipo , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Unión Proteica , ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rotenona/farmacología , Solanum tuberosum/metabolismo , Nicotiana/metabolismoRESUMEN
Two mutants of Chlamydomonas reinhardtii, mf1 and mf2, characterized by a marked reduction in their phosphatidylglycerol content together with a complete loss in its Delta3-trans hexadecenoic acid-containing form, also lost photosystem II (PSII) activity. Genetic analysis of crosses between mf2 and wild-type strains shows a strict cosegregation of the PSII and lipid deficiencies, while phenotypic analysis of phototrophic revertant strains suggests that one single nuclear mutation is responsible for the pleiotropic phenotype of the mutants. The nearly complete absence of PSII core is due to a severely decreased synthesis of two subunits, D1 and apoCP47, which is not due to a decrease in translation initiation. Trace amounts of PSII cores that were detected in the mutants did not associate with the light-harvesting chlorophyll a/b-binding protein antenna (LHCII). We discuss the possible role of phosphatidylglycerol in the coupled process of cotranslational insertion and assembly of PSII core subunits.