Fungal X-Intrinsic Protein Aquaporin from Trichoderma atroviride: Structural and Functional Considerations

The major intrinsic protein (MIP) superfamily is a key part of the fungal transmembrane transport network. It facilitates the transport of water and low molecular weight solutes across biomembranes. The fungal uncharacterized X-Intrinsic Protein (XIP) subfamily includes the full protein diversity of MIP. Their biological functions still remain fully hypothetical. The aim of this study is still to deepen the diversity and the structure of the XIP subfamily in light of the MIP counterparts-the aquaporins (AQPs) and aquaglyceroporins (AQGPs)-and to describe for the first time their function in the development, biomass accumulation, and mycoparasitic aptitudes of the fungal bioagent Trichoderma atroviride. The fungus-XIP Glade, with one member (TriatXIP), is one of the three clades of MIPs that make up the diversity of T. atroviride MIPs, along with the AQPs (three members) and the AQGPs (three members). TriatXIP resembles those of strict aquaporins, predicting water diffusion and possibly other small polar solutes due to particularly wider ar/R constriction with a Lysine substitution at the LE2 position. The XIP loss of function in Delta TriatXIP mutants slightly delays biomass accumulation but does not impact mycoparasitic activities. Delta TriatMIP forms colonies similar to wild type; however, the hyphae are slightly thinner and colonies produce rare chlamydospores in PDA and specific media, most of which are relatively small and exhibit abnormal morphologies. To better understand the molecular causes of these deviant phenotypes, a wide-metabolic survey of the ATriatXIPs demonstrates that the delayed growth kinetic, correlated to a decrease in respiration rate, is caused by perturbations in the pentose phosphate pathway. Furthermore, the null expression of the XIP gene strongly impacts the expression of four expressed MIP-encoding genes of T. atroviride, a plausible compensating effect which safeguards the physiological integrity and life cycle of the fungus. This paper offers an overview of the fungal XIP family in the biocontrol agent T. atroviride which will be useful for further functional analysis of this particular MIP subfamily in vegetative growth and the environmental stress response in fungi. Ultimately, these findings have implications for the ecophysiology of Trichoderma spp. in natural, agronomic, and industrial systems

Dynamique de recolonisation d’une carrière de pouzzolane : étude du fonctionnement hydrique du pin sylvestre (Pinus sylvestris L.)

National audienceSur plusieurs sites naturels ayant un intérêt patrimonial, la colonisation par des ligneux est observée et pose des questions sur l’évolution des communautés végétales en place. Mieux prévoir ces évolutions et prendre les décisions appropriées implique de mieux connaître les raisons de ces évolutions, et notamment les capacités de colonisation des ligneux. Le site d’étude est une carrière de pouzzolane abandonnée depuis 1980 situé au pied du Puy de la Vache, dans la chaîne des Puys. Dans cette carrière, comme au sommet du Puy, c’est une végétation pionnière herbacée des substrats meubles (scories) qui est installée. On observe aussi une colonisation par le Pin sylvestre (Pinus sylvestris L.), et dans une moindre mesure, par l’épicea (Picea abies (L.) H. Karst) et le bouleau (Betula pendula Roth), de faciès secs et très filtrants, car en pente forte et sur scories. Nous avons analysé les particularités morphologiques et physiologiques des Pins sylvestres colonisant ces faciès, en les comparant à des individus installés à proximité sur des faciès ayant une réserve en eau. Les paramètres physiologiques suivis portent sur l’état hydrique de l’arbre, les échanges gazeux et le fonctionnement hydraulique. Pour ce dernier, nous avons mesuré la résistance à la cavitation qui est un paramètre de tolérance à la sécheresse

Protective effects of EPA and deleterious effects of DHA on eNOS activity in Ea hy 926 cultured with lysophosphatidylcholine

International audienceOxidized low density lipoprotein (Ox-LDL) is a well-established risk factor in atherosclerosis and lysophosphatidylcholine (LysoPtdCho) is considered to be one of the major atherogenic component of Ox-LDL. The purpose of this work was to investigate the effects of two membrane n-3 long chain polyunsaturated fatty acids (n-3 PUFAs), EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) compared to n-6 PUFA, ARA (arachidonic acid), on the activation of endothelial NO synthase (eNOS) by histamine in Ea hy 926 endothelial cells incubated during 24 h in the presence or the absence of LysoPtdCho. DHA (50 mu M) produced a ROS induction in cells and aggravated the LysoPtdCho-induced oxidative stress. It did not modify the basal eNOS activity but impaired the stimulation of eNOS induced by histamine and was unable to correct the deleterious effect of LysoPtdCho on histamine-stimulated eNOS activity or phosphorylation of Ser 1177. In contrast, EPA (90 mu M) did not modify the ROS level produced in the presence or absence of LysoPtdCho or basal eNOS activity and the stimulating effect of histamine on eNOS. However, it diminished the deleterious effect of LysoPtdCho as well as on the histamine-stimulated eNOS activity on the phosphorylation on Ser 1177 of eNOS. The beneficial effect of EPA but not DHA on endothelial eNOS activity in Ea hy 926 could be also partially due to a slight decrease in membrane DHA content in EPA-treated cells. Consequently, the equilibrium between NO generated by eNOS and ROS due to oxidative stress could explain, in part, the beneficial effect of EPA on the development of cardiovascular diseases. By contrast ARA an n-6 PUFA was devoid of any effect on ROS generation or eNOS activity in the basal state or after histamine-induced stimulation. In vivo experiments should be undertaken to confirm these results

Transcriptome and ecophysiological data of Populus nigra genotypes during drought stress

National audienc

The impact of PEPC phosphorylation on growth and development of Arabidopsis thaliana: Molecular and physiological characterization of PEPC kinase mutants

Two phosphoenolpyruvate carboxylase (PEPC) kinase genes (PPCk1 and PPCk2) are present in the Arabidopsis genome; only PPCk1 is expressed in rosette leaves. Homozygous lines of two independent PPCk1 T-DNA-insertional mutants showed very little (dln1), or no (csi8) light-induced PEPC phosphorylation and a clear retard in growth under our greenhouse conditions. A mass-spectrometry-based analysis revealed significant changes in metabolite profiles. However, the anaplerotic pathway initiated by PEPC was only moderately altered. These data establish the PPCk1 gene product as responsible for leaf PEPC phosphorylation in planta and show that the absence of PEPC phosphorylation has pleiotropic consequences on plant metabolism. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved

High-throuput workflow for RNAseq data treatment linking laboratory data server and remote parallel calculation platform

National audienc

Whole transcriptome sequencing and ecophysiological traits to qualify drought responses in Populus nigra genotypes

PosterClimate evolution will tends to an increase of frequency and severity of droughts. These changes may alter the distribution and potential survival of plant species. Drought treatment observed on trees induces diverse responses in function of ecological strategies which depend on genotype x environment interaction. Earlier modifications of ecophysiological traits (stomata closure, photosynthesis decrease, and hydraulic tension increase) in response to drought stress can be qualified as isohydric as opposed to anisohydric behaviour. Then our objective was to test if these contrasted phenotypes had a specific transcriptional regulation. Production and hydraulic traits were monitored during severe stress on six Populus nigra genotypes in order to bring out ecophysiological traits which are genotypespecific and to characterise droughtresponse strategies. Multivariate analysis on ecophysiological traits allowed us to choose the most indicative ones of hydraulic and physiological plant status and to define iso- or anisohydric genotypes. Total RNA sequencing was performed by RNAseq, on new leaves formed during stress, sampled at the maximum drought stress intensity indicated by predawn leaf water potential. A statistical analysis of transcripts expression levels was carried out to clean out the data and to reveal trends. Afterwards, these informations were analysed along with genes groups involved in leaf blade water regulation to estimate relationships between expression level and ecophysiological responses

Phenotypic and transcriptional plasticities in response to drought in black poplar

International audienceDrought severity and frequency are increasing with climate changes threatening plant survival. Perennials species such as poplar trees are concerned through their water dependence and economical impact. Phenotypic plasticity is considered as a key lever in plant response to environmental fluctuation but little is known concerning transcriptional plasticity in water fluctuating conditions. This project explored relationships between ecophysiological and transcriptional responses to identify genes involved in drought tolerance. Six Populus nigra genotypes, contrasted by their geographical origin, were submitted to water withholding up to a severe drought fixed thanks to predawn leaf water potential measurements (-2 MPa). Hydraulic, physiological and architectural leaf measurements revealed genotype-specific drought responses and plasticity. One genotype was particularly interesting because of its low sensibility to drought while keeping biomass production. RNAseq was used to analyze the transcriptome of leaf grown during the stress. At least 30% of total leaf transcripts were differentially expressed. Among them, genes families related to water flux regulation (aquaporins) and transcription factors were identified. Along with ecophysiological plasticity, transcriptional plasticity was genotype specific and showed particular transcripts involved in plasticity maximization. Such transcripts were also correlated to ecophysiological traits within networks. These results highlighted the importance to integrate plasticity in improvement of tree breeding

Calcium et contrôle de la photosynthèse C4

absen

Aquaporins and water control in drought-stressed poplar leaves: A glimpse into the extraxylem vascular territories

Leaf hydraulic conductance (Kleaf) and capacitance (Cleaf) are among the key parameters in plant-water regulation. Understanding the responses of these hydraulic traits to drought conditions remains a challenge for describing comprehensive plant-water relationships. The ability of an organism to resist and/or tolerate embolism events, which may occur at high negative pressure caused by hydric stress, relies on how well it can sustain a hydraulic system in a dynamic equilibrium. Populus deltoides is a water-saving tree species with a stomatal conductance that declines rapidly with reduced water availability. Under unfavorable conditions, the stomatal control of transpiration is known to be closely coordinated with a loss of plant hydraulic functioning that can ultimately result in hydraulic failure through xylem embolism, notably in leaves. The effects of drought on leaf hydraulics are also related to regulation in water permeases such as the aquaporins. To describe the responses linked to leaf hydraulics under severe drought and rewatering conditions, water-stressed poplars were monitored daily on an ecophysiological and a molecular scale. A structural and expression analysis on a set of aquaporins was carried out in parallel by in situ hybridization analysis and quantitative PCR. In complement, water distribution in water-challenged leaves was investigated using X-ray microtomography. A general depression of leaf hydraulic conductance and relative water content occurred under drought, but was reversed when plants were rewatered. More interestingly, (i) extreme leaf water deficiency led to marked xylem and lamina embolism, but a degree of hydric integrity in the midrib extraxylem territories and the bundle sheath of the minor veins was maintained, and (ii) the sub-tissue water allocation correlated well with an over-accumulation of several PIP and TIP aquaporins. Our multi-facet molecular ecophysiological approach revealed that leaves were able to secure a certain level of hydric status, in particular in cell territories near the "living ribs", which provided rapid hydric adjustment responses once favorable conditions were restored. These findings contribute to an integrated approach to leaf hydraulics, thus favoring a better understanding of the cell mechanisms involved in tree vulnerability to climate changes