Patricia Roeckel-Drevet

Seleno-independent glutathione peroxidases. More than simple antioxidant scavengers.

Glutathione peroxidases (GPXs, EC 1.11.1.9) were first discovered in mammals as key enzymes involved in scavenging of activated oxygen species (AOS). Their efficient antioxidant activity depends on the presence of the rare amino‐acid residue selenocysteine (SeCys) at the catalytic site. Nonselenium GPX‐like proteins (NS‐GPXs) with a Cys residue instead of SeCys have also been found in most organisms. As SeCys is important for GPX activity, the function of the NS‐GPX can be questioned. Here, we highlight the evolutionary link between NS‐GPX and seleno‐GPX, particularly the evolution of the SeCys incorporation system. We then discuss what is known about the enzymatic activity and physiological functions of NS‐GPX. Biochemical studies have shown that NS‐GPXs are not true GPXs; notably they reduce AOS using reducing substrates other than glutathione, such as thioredoxin. We provide evidence that, in addition to their inefficient scavenging action, NS‐GPXs act as AOS sensors in various signal‐transduction pathways.

Genotypic variability and phenotypic plasticity of cavitation resistance in Fagus sylvatica L. across Europe

Xylem cavitation resistance is a key physiological trait correlated with species tolerance to extreme drought stresses. Little is known about the genetic variability and phenotypic plasticity of this trait in natural tree populations. Here we measured the cavitation resistance of 17 Fagus sylvatica populations representative of the full range of the species in Europe. The trees were grown in three field trials under contrasting climatic conditions. Our findings suggest that the genotypic variability of cavitation resistance is high between genotypes of a given population. By contrast, no significant differences were found for this trait across populations, the mean population cavitation resistance being remarkably constant in each trial. We found a significant site effect and a significant site × population interaction, suggesting that cavitation resistance has a high phenotypic plasticity and that this plasticity is under genetic control. The implications of our findings for beech forest management in a context of climate change are discussed.

The genetics of resistance to five races of downy mildew (Plasmopara halstedii) in sunflower (Helianthus annuus L.)

Abstract These studies were undertaken to determine whether downy mildew resistance genes in sunflower were independent as first reported, or linked as suggested by more recent hypotheses. The segregations for downy mildew reaction of 111 F3 progenies from a cross between a susceptible line and a line with Pl2 were used to locate this gene on the sunflower consensus RFLP linkage map. It was shown that Pl2 was linked to the same RFLP markers on linkage group 1 as Pl1 and Pl6, mapped earlier, and at a very similar distance. The F3 progenies showed exactly the same segregation patterns when tested with race 1 and race D. One hundred and fifty four progenies from a cross between a susceptible line and HA335, containing Pl6 (considered as giving resistance to all Plasmopara halstedii races), were tested with the five French downy mildew races, 1, A, B, C and D. Two progenies were observed to show segregation for races 1 and D, while appearing homozygous-resistant to races A , B and C. Tests on F4 progenies confirmed this separation of resistances with fixation of susceptibility to races 1 and D and resistance to races A, B and C. It is concluded that the Pl6 gene is not a “strong” gene, giving resistance to all downy mildew races, but rather a cluster of genes, each providing resistance to one, or a few, downy mildew races. The genes giving resistance to races 1 and D, on one hand, and to races A, B and C, on the other hand, must be very closely linked, with about 0.6 cM between the two groups.

Study of the variability and evolution of Orobanche cumana populations infesting sunflower in different European countries

Abstract The parasitic plant Orobanche cumana Wallr. has become a limiting factor for sunflower crops in infested countries. Over the past few years the progression of this parasitic plant, its introduction into new countries, and the development of new and more virulent races have all been observed. Consequently, the survey and understanding of broomrape population evolution is now crucial for the establishment of efficient breeding programmes. With this in prospect, the genetic variability of O. cumana populations from infested European countries, Bulgaria, Romania, Turkey and Spain, was studied using RAPD markers. Eight populations with a total of 180 plants were analysed. Twenty three primers were used to obtain 133 reproducible bands which led to a binary matrix. This matrix was subjected to various complementary analyses including pairwise distances computed with the Nei and Li coefficient, AMOVA, Nei’s genetic diversity statistics, and an estimation of gene flow among populations with the infinite-island formula. The results gave consistent conclusions whatever the method used for data treatment. We show that this parasitic plant is probably self-pollinated, that there is little intra-population variability, and very little gene exchange appears to occur between different geographic regions. Populations were well structured and organized into two distinct groups (one group corresponding to the East European countries, Bulgaria, Romania and Turkey, and the other group corresponding to Spanish populations) and could have a monophyletic origin. These results are discussed in relation to the applied uses of RAPD markers in the determination of true O. cumana races instead of populations.

Colocation of downy mildew (Plasmopara halstedii) resistance genes in sunflower (Helianthus annuus L.)

SummaryThe Pl6 locus in the inbred sunflower (Helianthus annuus L.) line HA335 giving resistance to French races of downy mildew (Plasmopara halstedii (Farl.) Berl. & de Toni. was localized by molecular techniques. A bulked segregant analysis was made on the F2 progeny from a cross between this line and H52, a downy mildew susceptible line. The resistance gene in HA335 was found to have the same linked RFLP marker loci as those determined for Pl1 (resistance to race 1 in the line RHA266) on linkage group 1 of the consensus RFLP map of the cultivated sunflower. Pl1 and Pl6 thus appear either to be allelic or closely linked. The implications for sunflower breeding are discussed.

Glutathione Peroxidase Regulation of Reactive Oxygen Species Level is Crucial for In Vitro Plant Differentiation

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is overexpressed in plants under abiotic and biotic stress conditions that mediate oxidative stress. To study its biological role and its ability to confer stress resistance in plants, we tried to obtain transgenic plants overexpressing citrus (Citrus sinensis) PHGPx (cit-PHGPx). All attempts to obtain regenerated plants expressing this enzyme constitutively failed. However, when the enzymes catalytic activity was abolished by active site-directed mutagenesis, transgenic plants constitutively expressing inactive cit-PHGPx were successfully regenerated. Constitutive expression of enzymatically active cit-PHGPx could only be obtained when transformation was based on non-regenerative processes. These results indicate that overexpression of the antioxidant enzyme PHGPx interferes with shoot organogenesis and suggests the involvement of reactive oxygen species (ROS) in this process. Using transgenic tobacco (Nicotiana tabacum) leaves obtained from plants transformed with a beta-estradiol-inducible promoter, time-dependent induction of cit-PHGPx expression was employed. A pronounced inhibitory effect of cit-PHGPx on shoot formation was found to be limited to the early stage of the regeneration process. Monitoring the ROS level during regeneration revealed that upon cit-PHGPx induction, the lowest level of ROS correlated with the maximal level of shoot inhibition. Our results clearly demonstrate the essential role of ROS in the early stages of in vitro shoot organogenesis and the possible involvement of PHGPx in maintaining ROS homeostasis at this point.

Accumulation of defense related transcripts in sunflower hypocotyls (Helianthus annuus L.) infected with Plasmopara halstedii

A cDNA clone encoding a sunflower chitinase was obtained using degenerated primers in PCR amplifications and RACE procedures. This clone, a phenylalanine ammonia-lyase (PAL) clone and ubiquitin clone were used to analyse the resistance of sunflower (Helianthus annuus) to downy mildew. The differential regulation of amounts of PAL (involved in the general pathway of phenylpropanoid synthesis), chitinase (a pathogenesis-related protein) and ubiquitin (involved in proteolytic pathways) mRNA was studied in hypocotyls during the early stages after an aerial infection of sunflower inbred line RHA274 with zoospores from either race 1 (incompatible, host resistant) or race B (compatible, host susceptible) of Plasmopara halstedii. Northern analyses showed that transcript levels of PAL, chitinase and ubiquitin were rapidly and strongly increased after infection in incompatible interactions but not in the compatible ones, suggesting that regulation of these mRNAs is an important component of the resistance mechanisms in sunflower.

Proteome analysis of apical and basal regions of poplar stems under gravitropic stimulation

Gravity is a constant force guiding the direction of plant growth. In young poplar stem, reorientation of the apical region is mainly obtained by differential growth of elongating primary tissues. At the base, where elongation is achieved but where the cambium is active, reorientation is due to asymmetrical formation of reaction wood. After 45 min of gravistimulation, the stem showed no reorientation, but 1 week later, reaction wood was observed at the base of the stem. To determine the molecular mechanisms taking place at the top and base of the stem, after 45 min or 1 week of inclination, the changes induced in protein accumulation were studied by two-dimensional polyacrylamide gel electrophoresis and quantitatively analyzed using image analysis software. Around 300 protein spots were reproducibly detected and analyzed. Forty percent of these proteins showed significant changes after inclination. Mass spectrometry analysis of 135 spots led to the identification of 60 proteins involved in a wide range of activities and metabolisms. Very different patterns of protein expression were obtained according to conditions tested, highlighting the complexity of gravitropic responses. Our results suggest that primary and secondary tissues present specific mechanisms to sense reorientation and to respond to inclination. Some selected proteins are discussed.

Transgenic tomatoes showing higher glutathione peroxydase antioxidant activity are more resistant to an abiotic stress but more susceptible to biotic stresses

The function of selenium independent glutathione peroxidase (GPx) in response to biotic and abiotic stresses was investigated in transgenic tomato plants overexpressing an exogenous GPx and exhibiting a 50% increase in total GPx activity. GPx-overexpressing and control plants were challenged either by a mechanical stress or by infection with the biotrophic parasite Oidium neolycopersici or the necrotrophic parasite Botrytis cinerea. In mechanically stressed plants, internode growth was significantly less modified in GPx-overexpressing plants compared to controls. This stress resistant phenotype was not accompanied with any change in the global antioxidant response of the plants other than their increased GPx activity. Following infection by O. neolycopersici or by B. cinerea, lesion extension was increased in GPx-overexpressing plants compared with controls. These results showed that GPx overexpression provoked opposite effects in situations of biotic and abiotic challenges, suggesting a key role for this scavenger enzyme in controlling biotic and abiotic stress responses.

Molecular Variability Within Diaporthe/Phomopsis helianthi from France.

ABSTRACT Diaporthe/Phomopsis helianthi causes brown stem canker of sunflower (Helianthus annuus) and is responsible for considerable yield loss. This species shows considerable variation for morphological characters, growth, and pathogenicity. Molecular variability of two sample groups was assessed with amplified fragment length polymorphism (AFLP) markers. Isolates of the first sample were collected from infected sunflower tissues from the main regions in France where the crop is grown, whereas isolates from the second sample came from stems within a single field of sunflower. A soybean strain was taken as an outgroup for AFLP analyses. Within sample one, the greatest genetic distance among isolates was 0.97, whereas it was 0.44 within sample two isolates. For the whole of France, the average genetic distance was 0.68, whereas in the one field it was 0.12. Neis genetic diversity indices were 0.20 and 0.06 for France and for one field, respectively. The greatest genetic distance was found between isolates from the most northern crops. The greatest genetic distance between D. helianthi isolates and the strain isolated from soybean was similar to that observed for D. helianthi isolates from different geographical areas. The problems in defining the genus Phomopsis are discussed. It is shown that internal transcribed spacer sequencing could be a useful criteria for Diaporthe/Phomopsis species determination. The considerable genetic variability of the pathogen could lead to the occurrence of new strains that could be more aggressive or more resistant to chemical control.