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International audience; The rust fungi Melampsora larici-populina (Mlp) and Melampsora allii-populina (Map) are the main phytosanitary constraints for commercial poplar cultivation in Europe. Although Mlp is more aggressive and prevalent than Map, the two species may co-infect the same poplar tree or even the same poplar leaf, making the epidemiological surveys of each species difficult to achieve. In this study, a new duplex real-time PCR assay targeting each species was developed, based on single-copy genes. This test proved to be specific, inclusive and was successfully used to detect and quantify each species, starting from urediniospore samples or directly from infected poplar leaves, with or without visible symptoms. This new molecular tool was also assessed for comparative studies of time-course infection experiments on artificially inoculated poplar leaf discs. These studies showed that the growth dynamics of Map were significantly slower when the two species were co-inoculated on the same leaf disc, confirming that Map is less aggressive than Mlp.

Nickel-hyperaccumulator plants are of interest due to their potential use in agromining. We aimed to characterize leaf traits and Ni concentration variabilities occurring between individual plants, leaves of differing age or between various leaf tissues, in a single Greek population of the Ni-hyperaccumulator Leptoplax emarginata (Boiss.) O.E. Schulz. We linked these results to ecophysiological characteristics and other element concentrations at leaf and leaf tissue scales. We measured leaf gas exchanges, stomatal density, and we carried out rapid freezing and freeze-drying processes on leaf sections before microanalysis with scanning electron microscopy and energy-dispersive spectrometry. Leaf or leaf-tissue Ni concentrations were influenced by a combination of individual plant and leaf age factors. The greatest Ni concentrations were found in the highly transpiring young and thin leaves with the greatest stomatal densities. Indeed, Ni was statistically seven times more concentrated in both epidermis layers than in their bulk neighbour leaf counterparts, whatever the leaf age. In both epidermis layers, increases in the Ni-S and Mg-S correlations from the oldest leaves to the youngest ones were observed. The Mg:Ni, Ca:Ni and P:Ni mass ratios decreased from the oldest leaves to the mature leaves. We would recommend time-series characterization of leaf traits belonging to at least three plant replicates in order to take into account the allogamous character of many Ni-hyperaccumulator plants. Long-distance Ni transport via the xylem is predominant in the Ni-hyperaccumulator L. emarginata whereas a complementary redistribution via the phloem should also occur. The perspectives of this study are the validation and refinement of this process over shorter periods using relevant tracers.

The glycosylation of therapeutic monoclonal antibodies (mAbs), a known critical quality attribute, is often greatly modified during the production process by animal cells. It is essential for biopharmaceutical industries to monitor and control this glycosylation. However, current glycosylation characterization techniques involve time- and labor-intensive analyses, often carried out at the end of the culture when the product is already synthesized. This study proposes a novel methodology for real-time monitoring of antibody glycosylation site occupancy using Raman spectroscopy. It was first observed in CHO cell batch culture that when low nutrient concentrations were reached, a decrease in mAb glycosylation was induced, which made it essential to rapidly detect this loss of product quality. By combining in situ Raman spectroscopy with chemometric tools, efficient prediction models were then developed for both glycosylated and nonglycosylated mAbs. By comparing variable importance in projection profiles of the prediction models, it was confirmed that Raman spectroscopy is a powerful method to distinguish extremely similar molecules, despite the high complexity of the culture medium. Finally, the Raman prediction models were used to monitor batch and feed-harvest cultures in situ. For the first time, it was demonstrated that the concentrations of glycosylated and nonglycosylated mAbs could be successfully and simultaneously estimated in real time with high accuracy, including their sudden variations due to medium exchanges. Raman spectroscopy can thus be considered as a promising PAT tool for feedback process control dedicated to on-line optimization of mAb quality. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:486-493, 2018.

International audience; Fungi are among the principal causal agents of emerging plant diseases, which are a matter of worldwide concern. Changes in land use, such as the expansion of cultivated areas, are implicated in the emergence of fungal diseases, but have been less often reported for native species plantations. In the maritime pine (Pinus pinaster) forest of the Landes de Gascogne (south-western France), pine mortality due to the root rot fungus Armillaria ostoyae (Basidiomycete) has been increasing over the last 30 years. The first cases of this disease occurred in 1920 only few years after a period of rapid major change to the landscape. During the second half of the 19th century the landscape was transformed from marshes to the largest monospecific maritime pine plantation forest in Europe. We carried out two surveys (0.24 and 1 million hectares) of Armillaria root rot disease in the Landes area, to investigate the spatial distribution of pathogen damage and to determine the role of historical factors in the establishment of this pattern. For the two surveys, spatial analyses and generalised linear models revealed a significant effect on the current geographical distribution of A. ostoyae disease of the proportion of pre-existing forest in the vicinity of afforested areas and a significant effect of the proximity of the first forests planted in the coastal dunes. These results suggest that A. ostoyae was commonly distributed in pre-existing forest areas, and that most of these fragments acted as source for the colonisation of newly planted forests. Better predictions on the risk of establishment of new disease foci in this forest area can be achieved from these results.

International audience; Ozone is an air pollutant that causes oxidative stress by generation of reactive oxygen species (ROS) within the leaf. The capacity to detoxify ROS and repair ROS-induced damage may contribute to ozone tolerance. Ascorbate and glutathione are known to be key players in detoxification. Ozone effects on their biosynthesis and on amino acid metabolism were investigated in three Euramerican poplar genotypes (Populus deltoides Bartr. x Populus nigra L.) differing in ozone sensitivity. Total ascorbate and glutathione contents were increased in response to ozone in all genotypes, with the most resistant genotype (Carpaccio) showing an increase of up to 70%. Reduced ascorbate (ASA) concentration at least doubled in the two most resistant genotypes (Carpaccio and Cima), whereas the most sensitive genotype (Robusta) seemed unable to regenerate ASA from oxidized ascorbate (DHA), leading to an increase of 80% of the oxidized form. Increased ascorbate (ASA + DHA) content correlated with the increase in gene expression in its biosynthetic pathway, especially the putative gene of GDP-L-galactose phosphorylase VTC2. Increased cysteine availability combined with increased expression of gamma-glutamylcysteine synthetase (GSH1) and glutathione synthetase (GSH2) genes allows higher glutathione biosynthesis in response to ozone, particularly in Carpaccio. In addition, ozone caused a remobilization of amino acids with a decreased pool of total amino acids and an increase of Cys and putrescine, especially in Carpaccio. In addition, the expression of genes encoding threonine aldolase was strongly induced only in the most tolerant genotype, Carpaccio. Reduced ascorbate levels could partly explain the sensitivity to ozone for Robusta but not for Cima. Reduced ascorbate level alone is not sufficient to account for ozone tolerance in poplar, and it is necessary to consider several other factors including glutathione content.

Multiple factors, including small sulfur-containing molecules and oxidoreductases, are involved in the control of intracellular redox homeostasis. In this chapter, we first review properties and functions of the small sulfur-containing molecules glutathione, mycothiol, bacillithiol and trypanothione. These low molecular weight thiols, which cycle between a reduced and oxidized form, are present at high intracellular concentrations and function as redox buffers to protect cells against oxidative stress conditions. In the second part of this chapter, we focus on the two oxidoreductases, thioredoxin and glutaredoxin. These enzymes are key players in pathways aimed to reduce disulfide bonds in intracellular proteins and to maintain cellular redox homeostasis. We review the general properties of these enzymes and highlight their significant diversity. Finally, we discuss the recent discovery that monothiol glutaredoxins coordinate an iron sulfur cluster, which suggests a novel link between redox and iron homeostasis.

Photosynthetic organisms and especially terrestrial plants contain expanded redox regulatory pathways. Proteins involved in these redox chains and detoxification reactions often evolved on a thioredoxin fold structural basis. We discuss in this review the proteins associated with this superfamily, their modes of reduction/regeneration and their known protein targets, focusing in particular on the protein disulfide isomerase and glutathione transferase families. We also discuss the evolution of the redoxin and target families along the plant kingdom and provide information on the systems biology of the redox circuitry.

NADPH is a pivotal molecule in oxidative stress, during which it is potentially produced by several cytosolic NADP-linked dehydrogenases. This study investigated the response and functional importance of the major leaf cytosolic NADP-malic enzyme in Arabidopsis (NADP-ME2) during oxidative stress. Data from both microarray and targeted quantitative PCR analyses showed that NADP-ME2 transcripts accumulated in response to ozone or in mutants undergoing intracellular oxidative stress. To test the functional importance of this response, loss-of-function nadp-me2 mutants were obtained and the effects of oxidative stress of intracellular and extracellular origin were tested. Despite much decreased leaf NADP-ME activity, nadp-me2 showed a wild-type phenotype when exposed to ozone. Introduction of the nadp-me2 mutations into the catalase-deficient cat2 background did not alter growth inhibition or lesions triggered by intracellular oxidative stress. Similarly, loss of NADP-ME2 function had little effect on cat2-triggered changes in glutathione or NADPH. While single nadp-me2 mutations produced slight effects on basal resistance to one type of bacteria, they did not affect resistance induced by the cat2 mutation. Taken together, the results suggest that, although NADP-ME2 induction is part of the response to oxidative stress, the enzyme is not an essential determinant of the outcome of such stress.

Melampsora medusae is a quarantine fungus in the European Union (EU) that causes a damaging leaf rust disease on poplars. Two formae speciales of the pathogen can be distinguished, M. medusae f. sp. deltoidae and M. medusae f. sp. tremuloidae, but the EU plant health directive 2000/29/EC currently in force does not make the distinction between them. EU countries must have the ability to detect and identify rapidly the introduction of these quarantine fungi and to conduct extensive surveys in case of outbreaks. Efficient detection tools are thus needed. In this study, a sensitive real-time PCR assay was developed to detect the presence of M. medusae in poplar leaf samples. A unique primer/hydrolysis probe combination targeting both formae speciales was designed using species-specific polymorphisms observed within the internal transcribed spacer region. An additional primer/hydrolysis probe combination was designed from a region of the 28S rDNA that is highly conserved in the genus Melampsora and used in a separate real-time PCR assay in order to check the quality of the DNA extracted from Melampsora urediniospores. The test developed demonstrated a high sensitivity since it enables the reproducible detection of two M. medusae urediniospore in a mixture of 2 mg of urediniospores (ca 800 000 urediniospores) of other Melampsora species. This new real-time PCR tool should be useful for laboratories in charge of official analyses since it has many advantages over the techniques currently used to monitor this quarantine pathogen in Europe.

The dead wood and forest soils are sources of diversity and under-explored fungal strains with biotechnological potential, which require to be studied. Numerous enzymatic tests have been proposed to investigate the functional potential of the soil microbial communities or to test the functional abilities of fungal strains. Nevertheless, the diversity of these functional markers and their relevance in environmental studies or biotechnological screening does still have not been demonstrated. In this work, we assessed ten different extracellular enzymatic activities involved in the wood decaying process including beta-etherase that specifically cleaves the beta-aryl ether linkages in the lignin polymer. For this purpose, a collection of 26 fungal strains, distributed within three ecological groups (white, brown and soft rot fungi), has been used. Among the ten potential functional markers, the combinatorial use of only six of them allowed separation between the group of white and soft rot fungi from the brown rot fungi. Moreover, our results suggest that extracellular beta-etherase is a rare and dispensable activity among the wood decay fungi. Finally, we propose that this set of markers could be useful for the analysis of fungal communities in functional and environmental studies, and for the selection of strains with biotechnological interests. (c) 2012 Elsevier B.V. All rights reserved.

Cell capacity for cytosolic NADPH regeneration by NADP-dehydrogenases was investigated in the leaves of two hybrid poplar (Populus deltoides × Populus nigra) genotypes in response to ozone (O3 ) treatment (120 ppb for 17 days). Two genotypes with differential O3 sensitivity were selected, based on visual symptoms and fallen leaves: Robusta (sensitive) and Carpaccio (tolerant). The estimated O3 flux (POD0 ), that entered the leaves, was similar for the two genotypes throughout the treatment. In response to that foliar O3 flux, CO2 assimilation was inhibited to the same extent for the two genotypes, which could be explained by a decrease in Rubisco (EC 4.1.1.39) activity. Conversely, an increase in PEPC (EC 4.1.1.31) activity was observed, together with the activation of certain cytosolic NADP-dehydrogenases above their constitutive level, i.e. NADP-G6PDH (EC 1.1.1.49), NADP-ME (malic enzyme) (EC 1.1.1.40) and NADP-ICDH (NADP-isocitrate dehydrogenase) (EC1.1.1.42). However, the activity of non-phosphorylating NADP-GAPDH (EC 1.2.1.9) remained unchanged. From the 11th fumigation day, NADP-G6PDH and NADP-ME profiles made it possible to differentiate between the two genotypes, with a higher activity in Carpaccio than in Robusta. At the same time, Carpaccio was able to maintain high levels of NADPH in the cells, while NADPH levels decreased in Robusta O3 -treated leaves. All these results support the hypothesis that the capacity for cells to regenerate the reducing power, especially the cytosolic NADPH pool, contributes to improve tolerance to high ozone exposure.

International audience; Melampsora medusae (Mm), one of the causal agents of poplar rust, is classified as an A2 quarantine pest for European Plant Protection Organization (EPPO) and its presence in Europe is strictly controlled. Two formae speciales have been described within Mm, Melampsora medusae f. sp. deltoidae (Mmd), and Melampsora medusae f. sp. tremuloidae (Mmt) on the basis of their pathogenicity on Populus species from the section Aigeiros (e.g. Populus deltoides) or Populus (e.g. Populus tremuloides), respectively. In this study, a real-time polymerase chain reaction (PCR) assay was developed allowing the detection of Mmd, the forma specialis that is economically harmful. A set of primers and hydrolysis probe were designed based on sequence polymorphisms in the large ribosomal RNA subunit (28S). The real-time PCR assay was optimized and performance criteria of the detection method, i.e. sensitivity, specificity, repeatability, reproducibility, and robustness, were assessed. The real-time PCR method was highly specific and sensitive and allowed the detection of one single urediniospore of Mmd in a mixture of 2 mg of urediniospores of other Melampsora species. This test offers improved specificity over currently existing conventional PCR tests and can be used for specific surveys in European nurseries and phytosanitary controls, in order to avoid introduction and spread of this pathogen in Europe.

International audience; The recent release of several basidiomycete genome sequences allows an improvement of the classification of fungal glutathione S-transferases (GSTs). GSTs are well-known detoxification enzymes which can catalyze the conjugation of glutathione to non-polar compounds that contain an electrophilic carbon, nitrogen, or sulfur atom. Following this mechanism, they are able to metabolize drugs, pesticides, and many other xenobiotics and peroxides. A genomic and phylogenetic analysis of GST classes in various sequenced fungi—zygomycetes, ascomycetes, and basidiomycetes—revealed some particularities in GST distribution, in comparison with previous analyses with ascomycetes only. By focusing essentially on the wood-degrading basidiomycete Phanerochaete chrysosporium, this analysis highlighted a new fungal GST class named GTE, which is related to bacterial etherases, and two new subclasses of the omega class GSTs. Moreover, our phylogenetic analysis suggests a relationship between the saprophytic behavior of some fungi and the number and distribution of some GST isoforms within specific classes.