Biomedical subjects

R De Paepe

Publications and source records attributed to R De Paepe.

14 recordsLinked to original sources

PubMed2001

Infra-red thermography revealed a role for mitochondria in pre-symptomatic cooling during harpin-induced hypersensitive response.

The establishment of Erwinia amylovora harpin-induced hypersensitive response (HR) in Nicotiana sylvestris was followed by infra-red thermography (IRT). Three to four hours after elicitation, the temperature decreased in the harpin-infiltrated zone associated to stomatal opening. The marked drop in temperature which reached 2 degrees C and preceded necrosis symptoms for several hours, is thus likely caused by higher transpiration. Neither of these effects was observed in a respiratory mutant, affected in complex I structure and function and over-expressing alternative oxidase, indicating that they are directly or indirectly mediated by mitochondrial function. However, as the HR establishment was similar in both wild type and mutant, cell death was either uncorrelated with the observed epidermal changes or occurred by a different signalling pathway in the two genotypes. IRT revealed a novel aspect of plant-pathogen interactions and could be applied to screen for mutants affected in elicitor signalling and/or for respiratory mutants.

Bacterial Outer Membrane Proteins↗

PubMed2000

Defective splicing of the first nad4 intron is associated with lack of several complex I subunits in the Nicotiana sylvestris NMS1 nuclear mutant.

In this work, we provide evidence for the existence of a nuclear factor involved in the splicing of a specific mitochondrial intron in higher plants. In the Nicotiana sylvestris nuclear NMS1 mutant, defective in both vegetative and reproductive development, the first intron of the nad4 transcript encoding the complex I NAD4 subunit is not removed, whatever the tissue analysed. Transcript patterns of other standard mitochondrial genes are not affected in NMS1. However, numerous polypeptides are missing in two-dimensional in organelle mitochondrial protein synthesis patterns and several nuclear and mitochondrial complex I subunits are present in trace amounts. This indicates that translational or post-translational steps in the synthesis of other mitochondrial proteins are affected. All of these defects co-segregated with the abnormal phenotype in the offspring of a NMS1 x wild-type cross, showing that they are controlled by the same nuclear gene (MS1) or tightly linked loci. Such a complex situation has been described in chloroplasts and mitochondria of fungi, but never in higher plant mitochondria.

Alternative Splicing↗

PubMed2000

Complex I impairment, respiratory compensations, and photosynthetic decrease in nuclear and mitochondrial male sterile mutants of Nicotiana sylvestris.

We have previously shown that in Nicotiana sylvestris cytoplasmic male-sterile (CMS) mutants where the mtDNA lacks the nad7 gene coding for a subunit of respiratory Complex I (NADH:ubiquinone oxidoreductase, EC 1.6.5.3), glycine (Gly) oxidation was lower than in the wild type and insensitive to rotenone, suggesting Complex I dysfunction. In contrast, the oxidation rate of exogenous NADH and the capacity of the cyanide-resistant respiration (AOX) were enhanced. Here we report that, in contrast to Gly, the rate of malate oxidation was not affected, but proceeded totally in a rotenone-insensitive pathway, strongly suggesting that survival of CMS plants depends on the activation of internal and external alternative NAD(P) H dehydrogenases and that Gly decarboxylase activity depends on Complex I functioning. A similar defect in Complex I activity and Gly oxidation was found in the NMS1 nuclear mutant, defective in the processing of the nad4 transcript, but alternative NAD(P) H dehydrogenases were less activated. In CMS and NMS1, the fraction of the AOX pathway was increased, as compared to wild type, associated with higher amounts of aox transcripts, AOX protein, and plant resistance to cyanide. Non-phosphorylating respiratory enzymes maintained normal in vivo respiration levels in both mutants, but photosynthesis was decreased, in correlation with lower leaf conductance, emphasizing mitochondrial control on photosynthesis.

Cell Nucleus↗

PubMed1999

In the Nicotiana sylvestris CMSII mutant, a recombination-mediated change 5' to the first exon of the mitochondrial nad1 gene is associated with lack of the NADH:ubiquinone oxidoreductase (complex I) NAD1 subunit.

We previously reported that the Nicotiana sylvestris CMSII mutant mitochondrial DNA carried a large deletion. Several expressed sequences, most of which are duplicated, and the unique copy of the nad7 gene encoding the NAD7 subunit of the NADH:ubiquinone oxidoreductase complex (complex I) are found in the deletion. Here, we show that the orf87-nad3-nad1/A cotranscription unit transcribed from a unique promoter element in the wild-type, is disrupted in CMSII. Nad3, orf87 and the promoter element are part of the deleted sequence, whilst the nad1/A sequence is present and transcribed from a new promoter brought by the recombination event, as indicated by Northern and primer extension experiments. However, Western analyses of mitochondrial protein fractions and of complex I purified using anti-NAD9 affinity columns, revealed that NAD1 is lacking in CMSII mitochondria. Our results suggest that translation of nad1 transcripts rather than transcription itself could be altered in the mutant. Consequences of lack of this submit belonging the membrane arm of complex I and thought to contain the ubiquinone-binding site, are discussed.

Amino Acid Sequence↗

PubMed1998

Analysis of gentamicin by liquid chromatography with pulsed electrochemical detection.

The analysis of gentamicin by liquid chromatography using a column packed with poly(styrene-divinylbenzene) and pulsed electrochemical detection on a gold electrode is described. The mobile phase consists of an aqueous solution containing sodium sulfate, tetrahydrofuran, sodium 1-octanesulfonate and a phosphate buffer of pH 3.0. In contradistinction to methods previously published, this method not only allows a better separation of gentamicins C1, C1a, C2, C2a and C2b, but also the separation of several other, minor components, most of which were not identified. The effects of the different chromatographic parameters on the separation were also investigated. A number of commercial samples was analysed using this method, allowing sensitive detection of gentamicin without derivatization, and the results were compared with the results obtained with the European Pharmacopoeia method, prescribing pre-column derivatization.

Carbohydrate Sequence↗

PubMed1998

Tissue-specific expression of genes encoding isoforms of the mitochondrial ATPase beta subunit in Nicotiana sylvestris.

We report here that the catalytic beta subunit of the mitochondrial ATPase/ATP synthase is encoded by a small multigenic family in the diploid tobacco Nicotiana sylvestris (nsatp2 genes). cDNAs and genes corresponding to the beta1, beta2 and beta3 (pollen specific) isoforms previously detected by 2D-SDS PAGE were isolated. Nsatp2.1 and nsatp2.2 transcripts were found in all vegetative and reproductive tissues analysed. In contrast, nsatp2.3 transcripts were found exclusively in bicellular pollen. As a whole, steady-state transcript levels of nuclear nsatp2 and mitochondrial atp1 genes were found to be closely correlated.

Amino Acid Sequence↗

PubMed1998

Organization and expression of the mitochondrial genome in the Nicotiana sylvestris CMSII mutant.

Previous analyses suggested that the Nicotiana sylvestris CMSII mutant carried a large deletion in its mitochondrial genome. Here, we show by cosmid mapping that the deletion is 60 kb in length and contains several mitochondrial genes or ORFs, including the complex I nad7 gene. However, due to the presence of large duplications in the progenitor mitochondrial genome, the only unique gene that appears to be deleted is nad7. RNA gel blot data confirm the absence of nad7 expression, strongly suggesting that the molecular basis for the CMSII abnormal phenotype, poor growth and male sterility, is the altered complex I structure. The CMSII mitochondrial genome appears to consist essentially of one of two subgenomes resulting from recombination between direct short repeats. In the progenitor mitochondrial genome both recombination products are detected by PCR and, reciprocally, the parental fragments are detected at the substoichiometric level in the mutant. The CMSII mtDNA organization has been maintained through six sexual generations.

Cosmids↗

PubMed1997

Lack of mitochondrial and nuclear-encoded subunits of complex I and alteration of the respiratory chain in Nicotiana sylvestris mitochondrial deletion mutants.

We previously have shown that Nicotiana sylvestris cytoplasmic male sterile (CMS) mutants I and II present large mtDNA deletions and that the NAD7 subunit of complex I (the main dehydrogenase of the mitochondrial respiratory chain) is absent in CMS I. Here, we show that, despite a large difference in size in the mtDNA deletion, CMS I and II display similar alterations. Both have an impaired development from germination to flowering, with partial male sterility that becomes complete under low light. Besides NAD7, two other complex I subunits are missing (NAD9 and the nucleus-encoded, 38-kDa subunit), identified on two-dimensional patterns of mitochondrial proteins. Mitochondria isolated from CMS leaves showed altered respiration. Although their succinate oxidation through complex II was close to that of the wild type, oxidation of glycine, a priority substrate of plant mitochondria, was significantly reduced. The remaining activity was much less sensitive to rotenone, indicating the breakdown of Complex I activity. Oxidation of exogenous NADH (coupled to proton gradient generation and partly sensitive to rotenone) was strongly increased. These results suggest respiratory compensation mechanisms involving additional NADH dehydrogenases to complex I. Finally, the capacity of the cyanide-resistant alternative oxidase pathway was enhanced in CMS, and higher amounts of enzyme were evidenced by immunodetection.

Amino Acid Sequence↗

PubMed1996

Dynamics of plant mitochondrial genome: model of a three-level selection process.

The plant mitochondrial genome is composed of a set of molecules of various sizes that generate each other through recombination between repeated sequences. Molecular observations indicate that these different molecules are present in an equilibrium state. Different compositions of molecules have been observed within species. Recombination could produce deleted molecules with a high replication rate but bearing little useful information for the cell (such as "petite" mutants in yeast). In this paper we use a multilevel model to examine selection among rapidly replicating incomplete molecules and relatively slowly replicating complete molecules. Our model simulates the evolution of mitochondrial information through a three-level selection process including intermolecular, intermitochondrial, and intercellular selection. The model demonstrates that maintenance of the mitochondrial genome can result from multilevel selection, but maintenance is difficult to explain without the existence of selection at the intermitochondrial level. This study shows that compartmentation into mitochondria is useful for maintenance of the mitochondrial information. Our examination of evolutionary equilibria shows that different equilibria (with different combinations of molecules) can be obtained when recombination rates are lower than a threshold value. This may be interpreted as a drift-mutation balance.

Genome, Plant↗

PubMed1995

Deletion of the last two exons of the mitochondrial nad7 gene results in lack of the NAD7 polypeptide in a Nicotiana sylvestris CMS mutant.

In Nicotiana sylvestris, two cytoplasmic male sterile (CMS) mutants obtained by protoplast culture show abnormal developmental features of both vegetative and reproductive organs, and mitochondrial gene reorganization following homologous recombination between 65 bp repeated sequences. A mitochondrial region of 16.2 kb deleted from both CMS mutants was found to contain the last two exons of the nad7 gene coding for a subunit of the mitochondrial respiratory chain complex I, which is encoded in the nucleus in fungi and animals but was recently found to be encoded by the mitochondrial genome in wheat. Although the N. sylvestris nad7 gene shows strong homology with its wheat counterpart, it contains only three introns instead of four. Polymerase chain reaction (PCR) experiments indicated that the parental gene organization, including the complete nad7 gene, is probably maintained at a substoichiometric level in the CMS mutants, but this proportion is too low to have a significant physiological role, as confirmed by expression studies showing the lack of detectable amounts of the NAD7 polypeptide. Consequently, absence of NAD7 is not lethal to plant cells but a deficiency of complex I could be involved in the abnormal CMS phenotype.

Amino Acid Sequence↗

PubMed1993

Specific mitochondrial proteins in pollen: presence of an additional ATP synthase beta subunit.

A protocol was designed to obtain a pure fraction of pollen mitochondria from the diploid species Nicotiana sylvestris, the female parent of the allotetraploid Nicotiana tabacum. Most organelles were morphologically intact and able to perform in organello mitochondrial (mt) protein synthesis. As revealed by two-dimensional protein electrophoresis, numerous quantitative differences exist between leaf and pollen mt proteins. Moreover, additional mt polypeptides, named R (for reproductive), encoded by either nuclear or mitochondrial genes, are found in pollen. The most abundant R polypeptide, R1 (M(r) 53,000, pI 5.6), is nuclearly encoded, is membrane bound, and cross-reacts with an antibody directed against the beta subunit of the mt ATP synthase (ATPase). N-terminal microsequence analysis showed that the two ATPase beta subunits present in leaves (beta 1 and beta 2) and the R1 pollen-specific subunit are encoded by distinct genes. A similar additional ATPase beta subunit was observed in pollen mitochondria from Petunia, suggesting that this polypeptide is of general importance for male gametophytic development in Solanaceaes.

Amino Acid Sequence↗

PubMed1992

Cytoplasmic male sterility is associated with large deletions in the mitochondrial DNA of two Nicotiana sylvestris protoclones.

Two cytoplasmic male-sterile plants (CMSI and CMSII) were obtained by protoplast culture in Nicotiana sylvestris. Both plants showed large deletions (up to 50 kb) in their mitochondrial DNA. Restriction maps of the reorganized regions suggested that the deletions occurred via two homologous recombination events (rec. 1 and rec. 2) in the parental mitochondrial genome. With the exception of nad5, no mitochondrial DNA polymorphism could be detected between parental and CMS lines using different heterologous genes probes. A sequence homologous to the Oenothera nad5 mitochondrial gene was located close to the CMSI-specific rec. 2 region. Moreover, a cDNA probe corresponding to total mitochondrial RNA from the parent line was found to hybridize to mitochondrial DNA fragments involved in the rec. 1 event common to both CMS lines, suggesting that rec. 1 lies in a transcribed region. Cytoplasmic male sterility in the Nicotiana sylvestris CMS mutants could be due either to gene deletion or to a regulatory effect of such a deletion on mitochondrial gene expression, rather than to the presence of specific polypeptides as has been shown in the T cytoplasm of maize, or in CMS Petunia.

Blotting, Southern↗

PubMed1992

Amplification of substoichiometric recombinant mitochondrial DNA sequences in a nuclear, male sterile mutant regenerated from protoplast culture in Nicotiana sylvestris.

A Nicotiana sylvestris plant regenerated from protoplast culture was found to be mutated in both the mitochondrial (mt) and nuclear genomes. The novel mt DNA organization, called U, is due to the amplification of recombinant substoichiometric DNA sequences that preexist in the parent line. The recombination event involves two 404 bp repeats, which hybridize to a 2.1 kb transcript. Although the sequence of both repeats was not altered by the recombination, an additional transcript of 2.5 kb was detected in U mitochondria. In addition to this mitochondrial reorganization, the protoclone carried a recessive nuclear mutation conferring male sterility (ms4). A possible role of ms4 in the appearance of the U mt DNA organization was investigated by introducing this gene into normal N. sylvestris cytoplasm. No mt DNA change could be found in homozygous ms4/ms4 plants of the F2 generation.

Amino Acid Sequence↗

PubMed1990

Several nuclear genes control both male sterility and mitochondrial protein synthesis in Nicotiana sylvestris protoclones.

Male sterile plants appeared in the progeny of three fertile plants obtained after one cycle of protoplast culture from a fertile botanical line and two androgenetic lines of Nicotiana sylvestris. These plants showed the same foliar and floral abnormalities as the cytoplasmic male sterile (cms) mitochondrial variants obtained after two cycles of culture. We show that male sterility in these plants is controlled by three independent nuclear genes, ms1, ms2 and ms3, while no changes can be seen in the mitochondrial genome. However, differences were found between the in organello mitochondrial protein synthesis patterns of male sterile and parent plants. Two reproducible changes were observed: the presence of a new 20 kDa polypeptide and the absence of a 40 kDa one. Such variations were described previously in mitochondrial protein synthesis patterns of the cms lines. Fertile hybrids of male sterile plants showed normal synthesis patterns. The male sterile plants are thus mutated in nuclear genes involved in changes observed in mitochondrial protein synthesis patterns.

Cloning, Molecular↗