Chantal Mathieu

Leaf Mitochondria Modulate Whole Cell Redox Homeostasis, Set Antioxidant Capacity, and Determine Stress Resistance through Altered Signaling and Diurnal Regulation

To explore the role of plant mitochondria in the regulation of cellular redox homeostasis and stress resistance, we exploited a Nicotiana sylvestris mitochondrial mutant. The cytoplasmic male-sterile mutant (CMSII) is impaired in complex I function and displays enhanced nonphosphorylating rotenone-insensitive [NAD(P)H dehydrogenases] and cyanide-insensitive (alternative oxidase) respiration. Loss of complex I function is not associated with increased oxidative stress, as shown by decreased leaf H2O2 and the maintenance of glutathione and ascorbate content and redox state. However, the expression and activity of several antioxidant enzymes are modified in CMSII. In particular, diurnal patterns of alternative oxidase expression are lost, the relative importance of the different catalase isoforms is modified, and the transcripts, protein, and activity of cytosolic ascorbate peroxidase are enhanced markedly. Thus, loss of complex I function reveals effective antioxidant crosstalk and acclimation between the mitochondria and other organelles to maintain whole cell redox balance. This reorchestration of the cellular antioxidative system is associated with higher tolerance to ozone and Tobacco mosaic virus.

Lack of Respiratory Chain Complex I Impairs Alternative Oxidase Engagement and Modulates Redox Signaling during Elicitor-Induced Cell Death in Tobacco

Alternative oxidase (AOX) functions in stress resistance by preventing accumulation of reactive oxygen species (ROS), but little is known about in vivo partitioning of electron flow between AOX and the cytochrome pathway. We investigated the relationships between AOX expression and in vivo activity in Nicotiana sylvestris and the complex I–deficient CMSII mutant in response to a cell death elicitor. While a specific AOX1 isoform in the active reduced state was constitutively overexpressed in CMSII, partitioning through the alternative pathway was similar to the wild type. Lack of correlation between AOX content and activity indicates severe metabolic constraints in nonstressed mutant leaves. The bacterial elicitor harpin NEa induced similar timing and extent of cell death and a twofold respiratory burst in both genotypes with little change in AOX amounts. However, partitioning to AOX was increased twofold in the wild type but remained unchanged in CMSII. Oxidative phosphorylation modeling indicated a twofold ATP increase in both genotypes. By contrast, mitochondrial superoxide dismutase activity and reduced forms of ascorbate and glutathione were higher in CMSII than in the wild type. These results demonstrate genetically programmed flexibility of plant respiratory routes and antioxidants in response to elicitors and suggest that sustained ATP production, rather than AOX activity by itself or mitochondrial ROS, might be important for in planta cell death.

Mitochondrial DNA polymorphism induced by protoplast fusion in Cruciferae

SummaryThe mitochondrial genomes of five rapeseed somatic hybrid plants, which combine in a first experimentBrassica napus chloroplasts and a cytoplasmic male sterility trait coming fromRaphanus sativus, and in a second experiment chloroplasts of a triazine resistantB. compestris and a cytoplasmic male sterility trait fromR. sativus, were analyzed by restriction endonucleases. Restriction fragment patterns indicate that these genomes differ from each other and from both parents. The presence of new bands in the somatic hybrid mitochondrial DNA restriction patterns is evidence of mitochondrial recombination in somatic hybrid cells. In both parental and somatic hybrid plants large quantitative variations in a mitochondrial plasmid-like DNA have been observed. Our results suggest that the cytoplasmic support for male sterility is located in the chromosomal mitochondrial DNA instead of the plasmid-like DNA.

Interspecific somatic hybridization between Brassica napus and Brassica hirta (Sinapis alba L.)

SummarySomatic hybridization between Brassica napus and B. hirta (or Sinapis alba) is described. No cybrid plant with B. napus nucleus exhibiting cytoplasmic male sterility was recovered. Somatic hybrids were identified morphologically and, for some of them, by cytological observations. They were also characterised by Southern hybridization of nuclear rDNA. Chloroplast and mitochondrial DNA restriction analysis showed that 2 plants out of 14 have B. hirta ctDNA, one the B. napus mtDNA and the other a hybrid. Nine possess B. napus ctDNA with a hybrid mtDNA. For six of them, mtDNA patterns present novel bands, suggesting intergenomic recombination during fusion. These hybrids will be included in the breeding program.

Several different mitochondrial DNA regions are involved in intergenomic recombination in Brassica napus cybrid plants

SummaryAn important mitochondrial (mt) DNA polymorphism was detected by SalI restriction enzyme analysis in five Brassica napus cybrids plants which combine B. napus chloroplasts and a cytoplasmic male sterility (cms) trait from Raphanus sativus. Novel restriction fragments observed in these cybrids were analysed. One of them was found to be constituted by fragments of both parent mt genomes. Sites involved in rut recombination in cybrids were compared by molecular hybridization to sites supposedly implicated in intragenomic mt recombination in B. oleracea The results indicate that mt recombination events arising through protoplast fusion involve several different rut DNA regions. Some of these regions appeared homologous to regions presumably involved in intragenomic mt recombination in B. oleracea.

Phosphorylation of p27KIP1 homologs KRP6 and 7 by SNF1‐related protein kinase–1 links plant energy homeostasis and cell proliferation

SNF1-related protein kinase-1 (SnRK1), the plant kinase homolog of mammalian AMP-activated protein kinase (AMPK), is a sensor that maintains cellular energy homeostasis via control of anabolism/catabolism balance. AMPK-dependent phosphorylation of p27(KIP1) affects cell-cycle progression, autophagy and apoptosis. Here, we show that SnRK1 phosphorylates the Arabidopsis thaliana cyclin-dependent kinase inhibitor p27(KIP1) homologs AtKRP6 and AtKRP7, thus extending the role of this kinase to regulation of cell-cycle progression. AtKRP6 and 7 were phosphorylated in vitro by a recombinant activated catalytic subunit of SnRK1 (AtSnRK1α1). Tandem mass spectrometry and site-specific mutagenesis identified Thr152 and Thr151 as the phosphorylated residues on AtKRP6- and AtKRP7, respectively. AtSnRK1 physically interacts with AtKRP6 in the nucleus of transformed BY-2 tobacco protoplasts, but, in contrast to mammals, the AtKRP6 Thr152 phosphorylation state alone did not modify its nuclear localization. Using a heterologous yeast system, consisting of a cdc28 yeast mutant complemented by A. thaliana CDKA;1, cell proliferation was shown to be abolished by AtKRP6(WT) and by the non-phosphorylatable form AtKRP6(T152A) , but not by the phosphorylation-mimetic form AtKRP6(T152D). Moreover, A. thaliana SnRK1α1/KRP6 double over-expressor plants showed an attenuated AtKRP6-associated phenotype (strongly serrated leaves and inability to undergo callogenesis). Furthermore, this severe phenotype was not observed in AtKRP6(T152D) over-expressor plants. Overall, these results establish that the energy sensor AtSnRK1 plays a cardinal role in the control of cell proliferation in A. thaliana plants through inhibition of AtKRP6 biological function by phosphorylation.

Physical and gene mapping of chloroplast DNA from normal and cytoplasmic male sterile (radish cytoplasm) lines of Brassica napus.

SummaryUsing the restriction endonucleases SaII, SmaI, BgII and KpnI, physical maps of chloroplast DNA isolated from normal and cytoplasmic male sterile (radish cytoplasm) lines of B. napus were constructed and compared. In this study, a rapid and simple procedure was developed for the isolation of chloroplast DNA restriction fragments from low gelling temperature agarose gels.The overall structural organization of N and cms B. napus appears to be rather similar to that of cpDNAs of other higher plants. It is composed of two identical sequences (each about 15 Md) arranged as an inverted repeat separated by two single copy-regions of different sizes (about 54 and 15 Md). In both genomes the ribosomal RNAs are encoded by duplicate genes situated at one end of the inverted repeat. The two chloroplast genomes are distinguished by a point mutation in the rRNA locus. Genes for the large subunit of ribulose-1.5-biphosphate carboxylase and a 32 kilodalton photosystem II polypeptide are separated by a minimum of 30 Md of DNA within the large single copy region.

Variation of plastid and mitochondrial DNAs in the genus Hedysarum

SummaryPlastid and mitochondrial DNAs from Hedysarum species of the western Mediterranean basin, H. spinosissimum ssp eu-spinosissimum, H. spinosissimum ssp capitatum, H. carnosum, H. coronarium and H. flexuosum, were compared by restriction endonuclease fragment analysis. ctDNA fragment patterns for ssp eu-spinosissimum and ssp capitatum were indistinguishable in different enzyme digests. An identical ctDNA variation was found in Hpa II digests with two Sardinian populations of ssp capitatum. Each of the two subspecies was characterized by specific mt DNA patterns with Pst I, Bam HI, Sma I and EcoRI. No variation was detected in populations of different geographical origins for a given subspecies. H. carnosum, H. coronarium and H. flexuosum generated specific ct and mt DNA patterns. Comparison of mitochondrial fragments indicated: — a strong homology between the two subspecies, — a closer homology among the three other diploids, each being closer to the other two than to H. spinosissimum subspecies — as was also the case for the plastid genomes.

Isolation of purified mitochondrial DNA from Brassicae.

Abstract The ability to isolate purified mitochondrial DNAs from higher plants is particularly useful in the study of mitochondrial genome organization through cloning and gene mapping. Mitochondrial DNAs from cauliflower, turnip, and rape have been isolated either by cesium chloride-ethidium bromide or cesium chloride-diamidinophenylindole density gradient centrifugation. After removal of the fluorescent dyes and extensive dialysis, mitochondrial DNAs were analyzed by analytical centrifugation and gel electrophoresis of restriction fragments. It was found that (i) mitochondrial DNAs prepared with ethidium bromide were contaminated both by nuclear and chloroplast DNAs and (ii) cesium chloride-diamidinophenylindole gradients led to pure mitochondrial DNAs.

Arabidopsis monomeric G-proteins, markers of early and late events in cell differentiation

In Schizosaccharomyces pombe, septum formation is intricately controlled by proteins which constitute the SIN (Septum Initiation Network) signalling cascade. The SIN ensures the coordination between mitotic exit and cytokinesis. Yeast spg1p is a core component of the SIN pathway and we have previously characterized the two orthologs of this G-protein in Arabidopsis thaliana (named AtSGP1 and 2). In this work, the cell and tissue expression of AtSGP genes during plant development has been analysed using AtSGP promoter::GUS fusions in stably transformed A. thaliana lines. AtSGP1 promoter activity was restricted to the quiescent centre, collumella cells, stomata guard cells and the stele while AtSGP2 promoter activity was detected in atrichoblasts, trichomes and pollen. The observed promoter activities are in accordance with publicly available pollen, stomata guard cell and root transcriptome data. Two-hybrid experiments previously evidenced an interaction between AtMAP3Kepsilon1 and AtSGP1. The AtMAP3Kepsilon1 promoter activity was detected in root apices, trichomes and ovule integuments. A genetic approach involving both markers of these specialized cells and mutant backgrounds was used to reinforce our hypothesis. It appears that, although highly conserved between plants and fungi, the spg1p G-protein has evolved in plants to perform a function different from the SIN pathway. Interestingly, cells expressing AtSGPs possessed limited or null mitotic activity. Our data suggests that AtSGP are crucial signalling components involved either in early cell fate specification, or in the final steps of cell differentiation. This is an interesting starting point for a wider study devoted to functional experiments designed to test these hypotheses.