Philippe Chétrit

Regeneration of cytoplasmic male sterile protoclones of Nicotiana sylvestris with mitochondrial variations

SummaryOne fertile and two male-sterile diploid plants were regenerated from the same callus after two cycles of protoplast culture from fertile Nicotiana sylvestris. Genetic studies indicated that the male-sterile trait was under cytoplasmic control. Progenies of each regenerated plant possessed different mitochondrial (mt) DNA restriction patterns. Both cms protoclone types were characterized by specific mtDNA deletions. In addition, a 40 kD mitochondrially encoded polypeptide is lacking in the cms plants.

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

SummaryANicotiana 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 by 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 ofms4 in the appearance of the U mt DNA organization was investigated by introducing this gene into normalN. sylvestris cytoplasm. No mt DNA change could be found in homozygousms4/ms4 plants of the F2 generation.

The mitochondrial respiratory chain and ATP synthase complexes: Composition, structure and mutational studies

Abstract The oxidative phosphorylation process is dependent on the assembly of both the respiratory chain that generates the electrochemical potential of the mitochondrial inner membrane and the ATP synthase complex which uses this membrane potential to drive ATP synthesis. The five respiratory enzymes involved in this process, complexes I to V, are composed of multiple subunits, some of which are synthesized on mitochondrial ribosomes, whereas others are a product of the nucleocytoplasmic genetic system. The mitochondrial genome has a limited coding capacity and the co-ordinate expression of all the subunits forming these complexes has been shown to be under nuclear control. Present knowledge of complexes I to V mainly comes from studies of bovine and fungal mitochondria. If beef heart mitochondria represent a choice material for studying the composition and structure of these complexes, Saccharomyces cerevisiae and Neurospora crassa and their numerous respiratory mutants, are ideal organisms for investigating the co-ordination of nuclear and mitochondrial genomes in their assembly. The major reason for the interest in respiratory complexes and ATP synthase from the mitochondrial inner membrane in Homo sapiens and in higher plants is the relationship between enzyme deficiencies and human diseases and ageing on one hand, and such plant phenotypic abnormalities as cytoplasmic male sterility on the other.

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

SummaryMale 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 ofNicotiana 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 andms3, while no changes can be seen in the mitochondrial genome. However, differences were found between thein 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.

Changes in antioxidant expression and harpin-induced hypersensitive response in a Nicotiana sylvestris mitochondrial mutant

Abstract The present study was designed to investigate the role of plant mitochondria in redox sensing and hypersensitive response in a Nicotiana sylvestris mitochondrial mutant (CMSII mutant) that lacks a functional respiratory complex I. The abundance of compartment-specific transcripts encoding isoforms of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6) and ascorbate peroxidase (APX; EC 1.11.1.11) was higher in the CMSII mutant. This suggests the presence of redox signalling, originating in the mitochondria, that affects the rest of the cell. In order to explore this further, the hypersensitive response induced in tobacco by harpin—a bacterial elicitor from Erwinia amylovora —was exploited as a model system for programmed cell death. Although the time course of harpin-induced necrosis was similar in the N. sylvestris wild-type and CMSII, several aspects of the hypersensitive response were found to be different in the mutant. For example, the accumulation of autofluorescent compounds, as observed under UV light, was lower in the mutant. In addition, presymptomatic transpiration was absent while cytosolic APX and PAL transcripts were enhanced. These results strongly suggest that mitochondrial functions (possibly mediated by redox changes) participate in the hypersensitive response to bacterial pathogens.

A mitochondrial sub-stoichiometric orf87-nad3-nad1 exonA co-transcription unit present in solanaceae was amplified in the genus Nicotiana.

Abstract Unlike other plant species, two copies of nad3 are present in Nicotiana sylvestris mitochondria. Both are localized downstream from an open reading frame (orf87 ), and are associated with either rps12 or the first exon of the nad1 gene. The orf87-nad3-nad1/A cluster is present in normal stoichiometry in Nicotiana tomentosiformis and is sub-stoichiometric in other Solanaceae, revealing recent amplification in the genus Nicotiana. It is suggested from sequence analysis that this cluster originated in an homologous recombination event that involved the nad3-rps12 intergenic region and the upstream region of an ancestral nad1 gene. Transcription patterns and RT-PCR showed that orf87-nad3-rps12 and orf87-nad3-nad1/A clusters are both co-transcription units.

Pollen Mitochondrial Proteins in Fertile and Male Sterile Nicotiana sylvestris

Rower development is a complex process, and little is known concerning molecular events related to gamete formation (Mascarenhas, 1990). Male sterility, i.e. the inability of plants to produce viable pollen, may be controlled by the nuclear genome alone, or by interactions between mitochondrial and nuclear genes (Hanson aConde, 1985). Mitochondria are semi-autonomous organelles, the genome of which encodes less than 10% of the several hundred mt polypeptides. The remaining proteins encoded by nuclear genes are then imported (Tzagoloff aMyers, 1986). Mt systems in plants can be distinguished from those of other organisms by larger heterogeneous genomes, with complex gene expression (QuAtier aVedel, 1977; Palmer aShield, 1984; Levings aBrown, 1989).

Cytoplasmic or Nuclear Control of Male Sterility in Protoplast-Derived Plants of Nicotiana Sylvestris

Different lines of Nicotiana sylvestris, a diploid autogamous species (2n =24), were used to initiate protoplast culture from leaf mesophyll cells: The original line (T line) is a pure line from the Institut des Tabacs, Bergerac, France, morphologically very homogeneous (“flat” phenotype). Diploid androgenetic lines were obtained by consecutive pollen cultures from the T line. These lines, theoretically completely homozygous (Doubled-Haploid or DH) show typical alterations of leaf morphology that we previously called “crumpling” (FR: fripe) (De Paepe et al, 1977, De Paepe et Pernes, 1978).