RESUMEN
In most studies, amounts of protein complexes of the oxidative phosphorylation (OXPHOS) system in different organs or tissues are quantified on the basis of isolated mitochondrial fractions. However, yield of mitochondrial isolations might differ with respect to tissue type due to varying efficiencies of cell disruption during organelle isolation procedures or due to tissue-specific properties of organelles. Here we report an immunological investigation on the ratio of the OXPHOS complexes in different tissues of Arabidopsis thaliana which is based on total protein fractions isolated from five Arabidopsis organs (leaves, stems, flowers, roots and seeds) and from callus. Antibodies were generated against one surface exposed subunit of each of the five OXPHOS complexes and used for systematic immunoblotting experiments. Amounts of all complexes are highest in flowers (likewise with respect to organ fresh weight or total protein content of the flower fraction). Relative amounts of protein complexes in all other fractions were determined with respect to their amounts in flowers. Our investigation reveals high relative amounts of complex I in green organs (leaves and stems) but much lower amounts in non-green organs (roots, callus tissue). In contrast, complex II only is represented by low relative amounts in green organs but by significantly higher amounts in non-green organs, especially in seeds. In fact, the complex I-complex II ratio differs by factor 37 between callus and leaf, indicating drastic differences in electron entry into the respiratory chain in these two fractions. Variation in amounts concerning complexes III, IV and V was less pronounced in different Arabidopsis tissues (quantification of complex V in leaves was not meaningful due to a cross-reaction of the antibody with the chloroplast form of this enzyme). Analyses were complemented by in gel activity measurements for the protein complexes of the OXPHOS system and comparative 2D blue native/SDS PAGE analyses using isolated mitochondria. We suggest that complex I has an especially important role in the context of photosynthesis which might be due to its indirect involvement in photorespiration and its numerous enzymatic side activities in plants.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Proteínas Mitocondriales/metabolismo , Transporte de Electrón , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Fosforilación Oxidativa , Hojas de la Planta/metabolismo , Raíces de Plantas/metabolismo , Tallos de la Planta/metabolismoRESUMEN
Processing of 5'-ends is a frequently observed step during maturation of plant mitochondrial mRNAs. Up to now, very little is known about the biochemistry of this process and the proteins involved in the removal of 5' leader sequences. Based on natural genetic variation we have used linkage mapping and complementation studies to identify a nuclear gene required for the efficient generation of a 5'-end 228 nucleotides upstream of the mitochondrial nad4 gene encoding subunit 4 of the NADH dehydrogenase complex. This nuclear gene, At1g12700, that we designate RNA PROCESSING FACTOR 1 (RPF1), encodes a pentatricopeptide repeat (PPR) protein of the P-class containing canonical PPR-repeats. RPF1 belongs to a subgroup of PPR proteins, which includes the RESTORER OF FERTILITY (RF) gene products restoring cytoplasmic male sterility (CMS) in various plant species. CMS is a mitochondrially inherited trait caused by the expression of aberrant, chimeric genes, which has not been observed in the predominantly inbreeding species Arabidopsis thaliana. The here reported results are a further step towards the characterization of the plant mitochondrial RNA processing machinery and provide additional insights into the function of RF-like PPR proteins.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , ARN de Planta/metabolismo , Proteínas de Unión al ARN/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Mapeo Cromosómico , Codón de Terminación , Prueba de Complementación Genética , ARN Mitocondrial , Proteínas de Unión al ARN/genéticaRESUMEN
In mitochondria of higher plants, the majority of 5' termini of mature mRNAs are generated posttranscriptionally. To gain insight into this process, we analyzed a natural 5' end polymorphism in the species Arabidopsis thaliana. This genetic approach identified the nuclear gene At1g62670, encoding a pentatricopeptide repeat protein. The functional importance of this mitochondrial restorer of fertility-like protein, designated RNA PROCESSING FACTOR2 (RPF2), is confirmed by the analysis of a respective T-DNA knockout mutant and its functional restoration by in vivo complementation. RPF2 fulfills two functions: it is required for the generation of a distinct 5' terminus of transcripts of subunit 9 of the NADH DEHYDROGENASE complex (nad9) and it determines the efficiency of 5' end formation of the mRNAs for subunit 3 of the CYTOCHROME C OXIDASE (cox3), the latter also being influenced by mitochondrial DNA sequences. Accordingly, recombinant RPF2 protein directly binds to a nad9 mRNA fragment in vitro. Two-dimensional gel electrophoresis and immunodetection analyses reveal that altered 5' processing does not influence accumulation of the nad9 and cox3 polypeptides. In accessions C24, Oystese-1, and Yosemite-0, different inactive RPF2 alleles exist, demonstrating the variability of this gene in Arabidopsis. The identification of RPF2 is a major step toward the characterization of 5' mRNA processing in mitochondria of higher plants.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Mitocondrias/metabolismo , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , ARN de Planta/genética , Proteínas de Unión al ARN/genética , AlelosRESUMEN
In our analysis of 5' and 3' end formation in plant mitochondria, we compared the major transcript ends of all mitochondrial protein-coding genes between the three Arabidopsis (Arabidopsis thaliana) accessions Columbia (Col), C24, and Landsberg erecta (Ler). Differences between transcript patterns were found for seven genes. For atp6-2, no transcripts at all were detected in Ler. This and further analyses suggest that the atp6-2 gene arrangement is absent from the mitochondrial DNA of this accession. All other transcript polymorphisms are attributed to variations at the 5' termini and were consistently observed in all tissues investigated. mRNA phenotyping of reciprocal Col/Ler, Col/C24, and Ler/C24 F(1) hybrids revealed the differing transcript patterns of ccmC to be inherited maternally, suggesting these to arise from differences in the mitochondrial DNA. Biparental inheritance was observed for the polymorphic transcripts of nad4, nad9, ccmB, and rpl5, indicating these differences to be caused by nuclear-encoded trans-factors. Deviant transcript patterns were tested in further accessions and were found in at least three additional accessions. Detailed examination of the nad4 and the nad9 transcripts demonstrates that the respective polymorphisms affect the major mRNAs of these genes. This study shows that natural genetic variation in Arabidopsis can also affect mitochondrial mRNA end processing. These variations can now be used to identify the nuclear genes responsible, as well as the mitochondrial cis-elements required, for 5' end generation of mitochondrial transcripts.
Asunto(s)
Arabidopsis/genética , Polimorfismo Genético , ARN Mensajero/genética , ARN de Planta/genética , Células Cultivadas , Cruzamientos Genéticos , ADN Mitocondrial/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genotipo , Patrón de Herencia , Mitocondrias/genética , ARN Mitocondrial , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADNRESUMEN
EosFP is a fluorescent protein from the coral Lobophyllia hemprichii that changes its fluorescence emission from green to red upon irradiation with near-UV light. Here we present the spectroscopic properties of wild-type EosFP and a variety of monomeric and dimeric mutants and provide a structural interpretation of its oligomerization and photoconversion, which is based on X-ray structure analysis of the green and red species that we reported recently. Because functional expression of the monomeric EosFP variant is limited to temperatures of 30 degrees C, we have developed a tandem dimer. This construct, in which two EosFP subunits are connected by a flexible 12 amino acid linker, expresses well after fusion with the androgen and endothelin A receptors at 37 degrees C. A variety of applications in cellular imaging, developmental biology and automated high-content screening applications are presented, which demonstrate that EosFP is a powerful tool for in vivo monitoring of cellular processes.