Analyse des mécanismes moléculaires responsables des échanges de solutés nutritifs entre le champignon et l'arbre dans la symbiose ectomycorhizienne

La symbiose mycorhizienne entre les champignons du sol et les racines de la plupart des plantes constitue une relation à bénéfice réciproque et joue un rôle majeur dans la productivité des écosystèmes. Les récentes avancées dans le domaine ont abouties à l'identification et à la caractérisation fonctionnelle de nombreux systèmes de transport du partenaire fongique. Le travail présenté s'inscrit dans le cadre de développement d'outils permettant la localisation de gènes d'intérêts du champignon ectomycorhizien Hebeloma cylindrosporum et de leur caractérisation fonctionnelle. Les systèmes de transport candidats ont été identifiés au sein d'une banque EST du champignon et semblent impliqués dans les échanges de phosphate (Pi) et de potassium (K+) entre Hebeloma et la plante hôte Pinus pinaster. Une stratégie de fusion transcriptionnelle utilisant l'EGFP comme gène rapporteur a été développée pour permettre la localisation de deux transporteurs de phosphate, HcPT1 et HcPT2, d'un transporteur de potassium, HcTrk1, et d'un canal potassique de type Shaker, HcSKC1, dans les hyphes en culture pure et au sein de l'ectomycorhize. Les Agrotransformations de la souche h7 d'Hebeloma avec des vecteurs de fusion transcriptionnelle ont montré une expression mycélienne de l'EGFP sous contrôle des promoteurs de nos gènes d'intérêts. Sous contrôle des différents promoteurs, l'expression de l'EGFP apparait comme étant site-spécifique dans les hyphes différenciés des ectomycorhizes. Le promoteur du transporteur de Pi HcPT1 induit l'expression du gène rapporteur au niveau des hyphes extramatriciels et du manteau mycélien entourant la racine. De plus, son expression est stimulée en cas de carence en Pi, indiquant ainsi l'implication de ce transporteur dans la récupération du Pi du sol lorsque celui-ci devient limitant. Pour ce qui est du promoteur de HcTrk1, il permet l'expression de l'EGFP dans les hyphes extraracinaires et dans le manteau, tandis que celui de HcSKC1 permet son expression au niveau du réseau de Hartig et du manteau. Ceci indique, qu'ils semblent respectivement participer à la récupération du K+ du sol et à son excrétion vers la plante. Pour poursuivre la caractérisation fonctionnelle de nos systèmes de transport candidats, un second canal potassique, HcSKC2, a été isolé à partir de la souche h1 et exprimé dans des ovocytes de xénope. Tout comme HcSKC1, HcSKC2 n'a pas été actif en système d'expression hétérologue. Cependant, des fusions traductionnelles avec l'EGFP ont montré que la protéine HcSKC2 est bien dirigée à la membrane. En perspective, la caractérisation fonctionnelle de ce canal issue de la souche h7 récemment séquencée sera tentée.The mycorrhizal symbiosis made it possible the first plants to conquest emerged lands and is a major biological phenomenon of terrestrial ecosystems. The fungal partner efficiently takes up nutritive ions from the soil solution and transfers them to the host plants in exchange for photosynthetates. However, despite the importance of this symbiosis on ecosystem productivity, our knowledge about molecular processes controlling this symbiotic interaction and solute transports at the membrane level is very scarce. The objective of the project aims at dissecting part of the molecular mechanisms required for a functional ectomycorrhizal symbiosis associated with most of the woody species from boreal and temperate forests, by focusing on K+ exchanges occurring through the continuum soil-hyphae-plant. The general aim of the project is to gain new insights into the molecular mechanisms responsible for the polarization and differentiation of the plasma membrane between the site of nutrient uptake and the site of efflux into the apoplastic space in the ectomycorrhizal root. The team "Canaux Ioniques – Ion channels" has obtained an EST library of the fungus Hebeloma cylindrosporum (1) and has identified and characterized a potassium transporter of the Trk family (2). Also a Shaker-type potassium cannel was identified within the EST library but it is not yet functionally characterized. A second transcript was found from this channel with a longer N-terminus compared to the first transcript isolated in the beginning. Also, a sugar transporter was identified among the ESTs that could participate in the absorption of sugars, coming from the host plant, by the fungus. The objective of the PhD thesis is the functional characterization of these fungal transport systems as well as their localization. The functional characterization of these candidate genes will be accomplished using heterologous expression systems (Xenopus oocytes, COS cells, complementation of yeast mutants) and by the means of electrophysiology. Localization of genes within the fungus being in symbiotic interaction with the host plant, the tree Pinus pinaster, will help to better understand the role of the transport systems. The differentiation of the fungus, when establishing symbiosis, into the specialized interfaces soil-fungus and fungal cell- host plant cell within the ectomycorrhiza (Hartig net) is probably accompanied by a specific expression of transport proteins and ion channel

Bentonite and Biochar Mitigate Pb Toxicity in Pisum sativum by Reducing Plant Oxidative Stress and Pb Translocation

Lead (Pb)-polluted soils pose a serious threat to human health, particularly by transmitting this heavy metal to the food chain via the crops grown on them. The application of novel amendments in Pb-polluted soils can significantly reduce this problem. In this research, we report the effects of various organic and inorganic amendments i.e., bentonite (BN), biochar (BR), lignin (LN), magnesium potassium phosphate cement (CM) and iron hydroxyl phosphate (FeHP), on the Pb bioavailability in Pb-polluted soil, upon Pb distribution in shoots, roots, grain, the translocation factor (TF) and the bioconcentration factor (BCF) of Pb in pea (Pisum sativum L.) grain. Furthermore, effects of the said amendments on the plant parameters, as well as grain biochemistry and nutritional quality, were also assessed. Lead pollution significantly elevated Pb concentrations in roots, shoots and grain, as well as the grain TF and BCF of Pb, while reducing the nutritional quality and biochemistry of grain, plant height, relative water content (RWC), chlorophyll contents (chl a and chl b) and the dry weight (DW) of shoot, root and grain. The lowest Pb distribution in shoots, roots and grain were found with BN, FeHP and CM, compared to our control. Likewise, the BN, FeHP and CM significantly lowered the TF and BCF values of Pb in the order FeHP > CM > BN. Similarly, the highest increase in plant height, shoot, root and grain DW, RWC, chl a and chl b contents, grain biochemistry and the micronutrient concentrations, were recorded with BR amendment. Biochar also reduced grain polyphenols as well as plant oxidative stress. Given that the BR and BN amendments gave the best results, we propose to explore their potential synergistic effect to reduce Pb toxicity by using them together in future research

Drought Stress Amelioration in Maize (Zea mays L.) by Inoculation of Bacillus spp. Strains under Sterile Soil Conditions

The aim of the present study was to promote plant growth characteristics including mineral uptake and various phytohormone production by indigenously isolated Bacillus spp. strains. Plants subjected to normal and water stress conditions were collected after 21 days to measure physiological parameters, photosynthetic pigment estimation, biochemical attributes, lipid peroxidation and antioxidant enzyme response modulation. Our results correlated with drought stress amelioration with the inoculation of Bacillus spp. strains BEB1, BEB2, BEB3 and BEB4 under sterile soil condition. Inoculated plants of both maize cultivars showed increases in fresh (56.12%) and dry (103.5%) biomass, plant length (42.48%), photosynthetic pigments (32.76%), and biochemical attributes with enhanced nutrient uptake. The overall maize antioxidant response to bacterial inoculation lowered the malonaldehyde level (59.14%), generation of hydrogen peroxide (45.75%) and accumulation of flavonoid contents in both control and water stress condition. Activity of antioxidant enzymes, catalase (62.96%), peroxidase (23.46%), ascorbate peroxidase (24.44%), and superoxide dismutase (55.69%) were also decreased with the application of bacterial treatment. Stress amelioration is dependent on a specific plant–strain interaction evident in the differences in the evaluated biochemical attributes, lipid peroxidation and antioxidant responses. Such bacteria could be used for enhancing the crop productivity and plant protection under biotic and abiotic stresses for sustainable agriculture

Drought Stress Amelioration in Maize (<i>Zea mays</i> L.) by Inoculation of <i>Bacillus</i> spp. Strains under Sterile Soil Conditions

The aim of the present study was to promote plant growth characteristics including mineral uptake and various phytohormone production by indigenously isolated Bacillus spp. strains. Plants subjected to normal and water stress conditions were collected after 21 days to measure physiological parameters, photosynthetic pigment estimation, biochemical attributes, lipid peroxidation and antioxidant enzyme response modulation. Our results correlated with drought stress amelioration with the inoculation of Bacillus spp. strains BEB1, BEB2, BEB3 and BEB4 under sterile soil condition. Inoculated plants of both maize cultivars showed increases in fresh (56.12%) and dry (103.5%) biomass, plant length (42.48%), photosynthetic pigments (32.76%), and biochemical attributes with enhanced nutrient uptake. The overall maize antioxidant response to bacterial inoculation lowered the malonaldehyde level (59.14%), generation of hydrogen peroxide (45.75%) and accumulation of flavonoid contents in both control and water stress condition. Activity of antioxidant enzymes, catalase (62.96%), peroxidase (23.46%), ascorbate peroxidase (24.44%), and superoxide dismutase (55.69%) were also decreased with the application of bacterial treatment. Stress amelioration is dependent on a specific plant–strain interaction evident in the differences in the evaluated biochemical attributes, lipid peroxidation and antioxidant responses. Such bacteria could be used for enhancing the crop productivity and plant protection under biotic and abiotic stresses for sustainable agriculture

Harnessing the Phytase Production Potential of Soil-Borne Fungi from Wastewater Irrigated Fields Based on Eco-Cultural Optimization under Shake Flask Method

Indigenous fungi present in agricultural soils could have synchronized their inherent potentials to the local climatic conditions. Therefore, the fungi resident in the untreated wastewater irrigated agricultural field might develop their potential for producing various enzymes to handle the induced full organic load from domestic wastewater and toxic chemicals from the textile industry. Around 53 various fungal isolates were grown and separated from the soil samples from these sites through soil dilution, soil-culture plate, and soil-culture plate methods. All the purified fungi were subjected to a phosphatase production test, and only 13 fungal strains were selected as phosphatase producers. Among them, only five fungi identified as Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, Penicillium purourogenum, and Mucor rouxii based on morphological similarities, showing higher phosphate solubilizing indices, were utilized for eco-cultural fine-tuning to harness their full production potential under shake flask (SF) method. Among various media, orchestral tuning, 200 µM sodium phytate as substrate with 1.5 mL of inoculum size of the fungi, pH 7, temperature 30 °C, glucose, and ammonium nitrate as carbon and nitrogen additive with seven days of incubation were found to be the most appropriate cultural conditions to harness the phytase production potential of the selected fungi. Aspergillus niger and Aspergillus flavus showed initial phytase activity (5.2 Units/mL, 4.8 Units/mL) and phytase specific activity (2.85, 2.65 Units/mL per mg protein) during screening to be enhanced up to 17 ± 0.033 (Units/mL), 16 ± 0.033 (Units/mL) and (13 ± 0.012), 10 ± 0.066 (Units/mL per mg protein), respectively, with the above-mentioned conditions. The phytase enzyme produced from these fungi were found to be almost stable for a wide range of pH (4–8); temperature (20–60 °C); insensitive to Ca2+ and Mg2+ ions, and EDTA, Ni2+, and Ba2+ inhibitors but highly sensitive to Mn2+, Cu2+, and Zn2+ ions, and Co2+, Cr3+, Al3+, Fe2+ and Ag1+ inhibitors. It was suggested that both phytase-producing strains of A. niger and A. flavus or their crude phytase enzymes might be good candidates for application in soils to release phosphates from phytate and a possible valuable substitute of phosphate fertilizers

Harnessing the Phytase Production Potential of Soil-Borne Fungi from Wastewater Irrigated Fields Based on Eco-Cultural Optimization under Shake Flask Method

Indigenous fungi present in agricultural soils could have synchronized their inherent potentials to the local climatic conditions. Therefore, the fungi resident in the untreated wastewater irrigated agricultural field might develop their potential for producing various enzymes to handle the induced full organic load from domestic wastewater and toxic chemicals from the textile industry. Around 53 various fungal isolates were grown and separated from the soil samples from these sites through soil dilution, soil-culture plate, and soil-culture plate methods. All the purified fungi were subjected to a phosphatase production test, and only 13 fungal strains were selected as phosphatase producers. Among them, only five fungi identified as Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, Penicillium purourogenum, and Mucor rouxii based on morphological similarities, showing higher phosphate solubilizing indices, were utilized for eco-cultural fine-tuning to harness their full production potential under shake flask (SF) method. Among various media, orchestral tuning, 200 &micro;M sodium phytate as substrate with 1.5 mL of inoculum size of the fungi, pH 7, temperature 30 &deg;C, glucose, and ammonium nitrate as carbon and nitrogen additive with seven days of incubation were found to be the most appropriate cultural conditions to harness the phytase production potential of the selected fungi. Aspergillus niger and Aspergillus flavus showed initial phytase activity (5.2 Units/mL, 4.8 Units/mL) and phytase specific activity (2.85, 2.65 Units/mL per mg protein) during screening to be enhanced up to 17 &plusmn; 0.033 (Units/mL), 16 &plusmn; 0.033 (Units/mL) and (13 &plusmn; 0.012), 10 &plusmn; 0.066 (Units/mL per mg protein), respectively, with the above-mentioned conditions. The phytase enzyme produced from these fungi were found to be almost stable for a wide range of pH (4&ndash;8); temperature (20&ndash;60 &deg;C); insensitive to Ca2+ and Mg2+ ions, and EDTA, Ni2+, and Ba2+ inhibitors but highly sensitive to Mn2+, Cu2+, and Zn2+ ions, and Co2+, Cr3+, Al3+, Fe2+ and Ag1+ inhibitors. It was suggested that both phytase-producing strains of A. niger and A. flavus or their crude phytase enzymes might be good candidates for application in soils to release phosphates from phytate and a possible valuable substitute of phosphate fertilizers

Isolation of natural herbicidal compound from Lantana camara

A flavone glucoside 'vitexin' (C21H20O10) was isolated from the leaves of Lantana camara through bioassay-guided isolation. Kupchan method of solvent-solvent partitioning of crude methanol extract from leaves of L. camara gave ethyl acetate, chloroform, hexane and aqueous fraction. Bio-assay with four fractions at 500 ppm, 1000 ppm and 10,000 ppm indicated chloroform fraction to be most potent phytotoxic against weeds (monocot: Phalaris minor, Avena fatua; Dicot: Chenopodium album, Rumex dentatus). Column chromatography of chloroform fraction with hexane: ethyl acetate (60:40) based on TLC profiling with vanillin visualisation stain and subsequent bioassays indicated sub-fraction (iii) of fraction 23 as most growth inhibitory. The chemical structure of the isolated compound was identified by gas chromatography-mass spectrometry (Shimadzu GC-MS-QP2010 ultra). The natural herbicidal activity of the isolated compound against various weeds in laboratory and field conditions is suggested

α-Tocopherol Foliar Spray and Translocation Mediates Growth, Photosynthetic Pigments, Nutrient Uptake, and Oxidative Defense in Maize (Zea mays L.) under Drought Stress

A pot experiment was conducted to assess the induction of drought tolerance in maize by foliar-applied &alpha;-tocopherol at early growth stage. Experiment was comprised two maize cultivars (Agaiti-2002 and EV-1098), two water stress levels (70% and 100% field capacity), and two &alpha;-tocopherol levels (0 mmol and 50 mmol) as foliar spray. Experiment was arranged in a completely randomized design in factorial arrangement with three replications of each treatment. &alpha;-tocopherol was applied foliary at the early vegetative stage. Water stress reduced the growth of maize plants with an increase in lipid peroxidation in both maize cultivars. Contents of non-enzymatic antioxidants and activities of antioxidant enzymes increased in studied plant parts under drought, while the nutrient uptake was decreased. Foliary-applied &alpha;-tocopherol improved the growth of both maize cultivars, associated with improvements in photosynthetic pigment, water relations, antioxidative mechanism, and better nutrient acquisition in root and shoot along with tocopherol contents and a decrease in lipid peroxidation. Furthermore, the increase of tocopherol levels in roots after &alpha;-Toc foliar application confers its basipetal translocation. In conclusion, the findings confer the role of foliar-applied &alpha;-tocopherol in the induction of drought tolerance of maize associated with tissue specific improvements in antioxidative defense mechanism through its translocation

Promoter-dependent expression of the fungal transporter HcPT1.1 under Pi shortage and its spatial localization in ectomycorrhiza.

International audienceMycorrhizal exchange of nutrients between fungi and host plants involves a specialization and polarization of the fungal plasma membrane adapted for the uptake from the soil and for secretion of nutrient ions towards root cells. In addition to the current progress in identification of membrane transport systems of both symbiotic partners, data concerning the transcriptional and translational regulation of these proteins are needed to elucidate their role for symbiotic functions. To answer whether the formerly described Pi-dependent expression of the phosphate transporter HcPT1.1 from Hebeloma cylindrosporum is the result of its promoter activity, we introduced promoter-EGFP fusion constructs in the fungus by Agrotransformation. Indeed, HcPT1.1 expression in pure fungal cultures quantified and visualized by EGFP under control of the HcPT1.1 promoter was dependent on external Pi concentrations, low Pi stimulating the expression. Furthermore, to study expression and localization of the phosphate transporter HcPT1.1 in symbiotic conditions, presence of transcripts and proteins was analyzed by the in situ hybridization technique as well as by immunostaining of proteins. In ectomycorrhiza, expression of the phosphate transporter was clearly enhanced by Pi-shortage indicating its role in Pi nutrition in the symbiotic association. Transcripts were detected in external hyphae and in the hyphal mantle, proteins in addition also within the Hartig net. Exploiting the transformable fungus H. cylindrosporum, Pi-dependent expression of the fungal transporter HcPT1.1 as result from its promoter activity as well as transcript and protein localization in ectomycorrhizal symbiosis are shown

Fungal Shaker-like channels beyond cellular K+ homeostasis: a role in ectomycorrhizal symbiosis between Hebeloma cylindrosporum and Pinus pinaster

International audiencePotassium (K +) acquisition, translocation and cellular homeostasis are mediated by various membrane transport systems in all organisms. We identified and described an ion channel in the ectomycorrhizal fungus Hebeloma cylindrosporum (HcSKC) that harbors features of animal voltage-dependent Shaker-like K + channels, and investigated its role in both free-living hyphae and symbiotic conditions. RNAi lines affected in the expression of HcSKC were produced and used for in vitro mycorrhizal assays with the maritime pine as host plant, under standard or low K + conditions. The adaptation of H. cylindrosporum to the downregulation of HcSKC was analyzed by qRT-PCR analyses for other K +-related transport proteins: the transporters HcTrk1, HcTrk2, and HcHAK, and the ion channels HcTOK1, HcTOK2.1, and HcTOK2.2. Downregulated HcSKC transformants displayed greater K + contents at standard K + only. In such conditions, plants inoculated with these transgenic lines were impaired in K + nutrition. Taken together, these results support the hypothesis that the reduced expression of HcSKC modifies the pool of fungal K + available for the plant and/or affects its symbiotic transfer to the roots. Our study reveals that the maintenance of K + transport in H. cylindrosporum, through the regulation of HcSKC expression, is required for the K + nutrition of the host plant