Investigating the simultaneous effect of chitosan and arbuscular mycorrhizal fungi on growth, phenolic compounds, PAL enzyme activity and lipid peroxidation in Salvia nemorosa L.

Considering the importance of Salvia nemorosa L. in the pharmaceutical and food industries, and also beneficial approaches of arbuscular mycorrhizal fungi (AMF) symbiosis and the use of bioelicitors such as chitosan to improve secondary metabolites, the aim of this study was to evaluate the performance of chitosan on the symbiosis of AMF and the effect of both on the biochemical and phytochemical performance of this plant and finally introduced the best treatment. Two factors were considered for the factorial experiment: AMF with four levels (non-inoculated plants, Funneliformis mosseae, Rhizophagus intraradices and the combination of both), and chitosan with six levels (0, 50, 100, 200, 400 mg L-1 and 1% acetic acid). Four months after treatments, the aerial part and root length, the levels of lipid peroxidation, H2O2, phenylalanine ammonia lyase (PAL) activity, total phenol and flavonoid contents and the main secondary metabolites (rosmarinic acid and quercetin) in the leaves and roots were determined. The flowering stage was observed in R. intraradices treatments and the highest percentage of colonization (78.87%) was observed in the treatment of F. mosseae × 400 mg L-1 chitosan. Furthermore, simultaneous application of chitosan and AMF were more effective than their separate application to induce phenolic compounds accumulation, PAL activity and reduce oxidative compounds. The cluster and principal component analysis based on the measured variables indicated that the treatments could be classified into three clusters. It seems that different treatments in different tissues have different effects. However, in an overview, it can be concluded that 400 mg L-1 chitosan and F. mosseae × R. intraradices showed better results in single and simultaneous applications. The results of this research can be considered in the optimization of this medicinal plant under normal conditions and experiments related to abiotic stresses in the future.

Gibberellin Promotes Fungal Entry and Colonization during Paris-Type Arbuscular Mycorrhizal Symbiosis in Eustoma grandiflorum.

Arbuscular mycorrhizas (AMs) are divided into two types according to morphology: Arum- and Paris-type AMs. Gibberellins (GAs) mainly inhibit the establishment of Arum-type AM symbiosis in most model plants, whereas the effects of GAs on Paris-type AM symbiosis are unclear. To provide insight into the mechanism underlying this type of symbiosis, the roles of GAs were investigated in Eustoma grandiflorum when used as the host plant for Paris-type AM establishment. Eustoma grandiflorum seedlings were inoculated with the model AM fungus, Rhizophagus irregularis, and the effects of GA and the GA biosynthesis inhibitor uniconazole-P on the symbiosis were quantitatively evaluated. Exogenous GA significantly increased hyphopodium formation at the epidermis, thus leading to the promotion of fungal colonization and arbuscule formation in the root cortex. By contrast, the suppression of GA biosynthesis and signaling attenuated fungal entry to E. grandiflorum roots. Moreover, the exudates from GA-treated roots strongly induced the hyphal branching of R. irregularis. Our results show that GA has an contrasting effect on Paris-type AM symbiosis in E. grandiflorum compared with Arum-type AM symbiosis. This finding could be explained by the differential regulation of the early colonization stage, where fungal hyphae make contact with and penetrate the epidermis.

Understanding interaction effect of arbuscular mycorrhizal fungi in rice under elevated carbon dioxide conditions.

Arbuscular mycorrhizal fungi (AMF), particularly the Glomerales group, play a paramount role in plant nutrient uptake, and abiotic and biotic stress management in rice, but recent evidence revealed that elevated CO2 concentration considerably reduces the Glomerales group in soil. In view of this, the present study was initiated to understand the interaction effect of native Glomerales species application in rice plants (cv. Naveen) under elevated CO2 concentrations (400 ± 10, 550 ± 20, and 700 ± 20 ppm) in open-top chambers. Three different modes of application of the AMF inoculum were evaluated, of which, combined application of AMF at the seedling production and transplanting stages showed increased AMF colonization, which significantly improved grain yield by 25.08% and also increased uptake of phosphorus by 18.2% and nitrogen by 49.5%, as observed at 700-ppm CO2 concentration. Organic acids secretion in rice root increased in AMF-inoculated plants exposed to 700-ppm CO2 concentration. To understand the overall effect of CO2 elevation on AMF interaction with the rice plant, principal component and partial least square regression analysis were performed, which found both positive and negative responses under elevated CO2 concentration.

Stimulation of asymbiotic sporulation in arbuscular mycorrhizal fungi by fatty acids.

Arbuscular mycorrhizal (AM) fungi are obligate symbionts that depend on living host plants to complete their life cycle1,2. This feature, which leads to their unculturability in the absence of plants, strongly hinders basic research and agricultural application of AM fungi. However, at least one AM fungus can grow and develop fertile spores independently of a host plant in co-culture with the bacterium Paenibacillus validus3. The bacteria-derived substances are thought to act as stimulants or nutrients for fungal sporulation, but these molecules have not been identified. Here, we show that (S)-12-methyltetradecanoic acid4,5, a methyl branched-chain fatty acid isolated from bacterial cultures, stimulates the branching of hyphae germinated from mother spores and the formation of secondary spores in axenic culture of the AM fungus Rhizophagus irregularis. Extensive testing of fatty acids revealed that palmitoleic acid induces more secondary spores than the bacterial fatty acid in R. irregularis. These induced spores have the ability to infect host plant roots and to generate daughter spores. Our work shows that, in addition to a major source of organic carbon6-9, fatty acids act as stimulants to induce infection-competent secondary spores in the asymbiotic stage and could provide the key to developing the axenic production of AM inoculum.

Two herbicides, two fungicides and spore-associated bacteria affect Funneliformis mosseae extraradical mycelium structural traits and viability.

The extraradical mycelium (ERM) produced by arbuscular mycorrhizal fungi is fundamental for the maintenance of biological fertility in agricultural soils, representing an important inoculum source, together with spores and mycorrhizal root fragments. Its viability and structural traits, such as density, extent and interconnectedness, which are positively correlated with the growth and nutrition of host plants, may be affected by different agronomic practices, including the use of pesticides and by different mycorrhizospheric communities. This work, carried out using a whole-plant experimental model system, showed that structural traits of ERM, such as length and density, were strongly decreased by the herbicides dicamba and glufosinolate and the fungicides benomyl and fenhexamid, while anastomosis frequency and hyphal branching were differentially modulated by singly inoculated mycorrhizospheric bacteria, depending on their identity.

Biogeochemical distribution of Pb and Zn forms in two calcareous soils affected by mycorrhizal symbiosis and alfalfa rhizosphere.

Using of arbuscular mycorrhizal fungi (AMF) has emerged as a new technique to alleviate the toxic metals stress through changing their chemical behavior. The present work was conducted as a factorial arrangement based on a completely randomized design to study the inoculation effects of Glomus intraradices, Glomus mosseae and Glomus etunicatum, on Pb and Zn fractions in the rhizosphere of alfalfa by using rhizobox technique in two agricultural soils with different Zn and Pb concentrations [with low (LH) and high (HH) concentration levels]. The results showed that AMF colonization promoted plant growth and lowered the shoot and root Pb and shoot Zn concentrations in the studied soils compared to uninoculated treatments. Mycorrhizal colonization significantly increased the Ca(NO3)2- extractable Zn and ORG-Zn (respectively 500 and 59.6% more than the uninoculated treatment) and decreased the OXI-Zn (20.32% less than the none inoculated treatment) in the HH soil. By contrast, mycorrhizae slightly increased the CARB, OXI and ORG-Zn forms in the LH soil compared to the uninoculation condition. In the AMF- treated HH soil, an increase was recorded in the Ca(NO3)2- extractable Pb, EXCH-Pb and CARB-Pb (respectively, 17.65, 3.09 and 14.22% compared to the none inoculated treatment) and a decrease in the OXI and ORG-Pb forms (respectively, 28.79 and 13.51% compared to the uninoculated treatment). A reverse status was observed for Pb changes in the LH soil. Depending on the contamination level, the mycorrhizal inoculation differentially affected the Pb and Zn fractions at different distances from the root surface. In the LH soil, at <5 mm distance (i.e. rhizospheric soil), the mycorrhizal inoculation decreased the CARB (about 17.99%) and OXI -Zn (about 29.63%) forms compared to bulk soil (i.e. > 5 mm distance) while ORG-Zn was increased up to 48.63%. However, Ca(NO3)2- extractable, CARB and ORG-Pb was increased in rhizosphere soil (respectively, 89.33, 3.84 and 6.14%) and OXI-Pb was decreased up to 10.36% compared to the bulk soil. In the HH soil, mycorrhizal inoculation increased the CARB and OXI-Zn (respectively, 1.76 and 5.71%) and OXI-Pb fractions (11.56%) compared to the <5 mm distances. Whereas, it reduced the Ca(NO3)2- extractable, EXCH, and ORG-Zn (Respectively, 52.70, 19.19 and 30.16%) and Ca(NO3)2- extractable, CARB and ORG-Pb (respectively, 47.18, 3.70 and 5.79%). These results revealed that depending on the soil contamination level and nature of the element, AMF colonization affects biogeochemical fractions of the metals and their accumulation in the plant tissues.

Foliar Roundup application has minor effects on the compositional and functional diversity of soil microorganisms in a short-term greenhouse experiment.

The herbicide Roundup (and glyphosate, its active ingredient) is extensively used for weed control on a worldwide scale. It is absorbed after foliar application and quickly translocated inside the plant. In this study, we investigated the effects of Roundup speed, a commercial glyphosate formulation, on the structural composition (dominance of microbial groups, phospholipid fatty acid analysis - PLFA) and functional diversity (use of carbon sources, Multiple Substrate Induced Respiration - MSIR) of soil microorganisms. We specifically aimed at understanding the potential impact of biotic interactions on herbicide effects and included plants, earthworms, and endomycorrhizal fungi in the experimental setup. For this, we grew clover (Trifolium repens) in the greenhouse and added mycorrhizal inoculum (Glomus mosseae) and earthworms (Lumbricus terrestris) to the pots. Two weeks after foliar Roundup application and subsequent plant death, the pots were destructively sampled. The application resulted in a significant increase of microbial respiration (SIR) by approximately 30%. A multivariate analysis of the MSIR data exhibited small but significant differences between the microbial communities of treated and untreated pots, while no significant difference was apparent for the PLFA data. Bacterial PLFAs generally decreased following herbicide application, while mycorrhizal and fungal PLFAs were not affected. We did not find a consistent difference between the fatty acid markers of gram negative and gram positive bacteria. For all investigated parameters, there were highly significant differences between the upper (0-5 cm depth) and lower (5-10 cm) soil layers. The fact that rooting density differed by a factor of 3.5 between the two layers indicated that herbicide effects were especially pronounced in the clover rhizosphere and were likely due to changes in root exudate composition. We found significant, though very small, interactions between Roundup and other experimental factors (especially mycorrhizal inoculum).

Proteomic analysis of the response of Funnelifor mismosseae/Medicago sativa to atrazine stress.

BACKGROUND: Arbuscular mycorrhizal (AM) fungi form symbiotic associations with host plants can protect host plants against diverse biotic and abiotic stresses, and promote biodegradation of various contaminants. However, the molecular mechanisms of how the arbuscular mycorrhizal fungi and host plant association on atrazine stress were still poorly understood. To better characterize how arbuscular mycorrhizal fungi and host plant interactions increase atrazine stress, we performed physiological and proteomic analysis of Funneliformis mosseae (mycorrhizal fungi) and Medicago sativa (alfalfa) association under atrazine stress. RESULTS: The results showed that in the Arbuscular mycorrhizal, protective enzymes were up regulated and the malondialdehyde content increased relative to those of non-mycorrhizal M.sativa. We also examined the atrazine degradation rates within the nutrient solution, and a 44.43% reduction was observed with the mycorrhizal M.sativa, with 30.83% of the reduction attributed to F. mosseae. The accumulation content in root and stem of mycorrhizal M.sativa were obviously increased 11.89% and 16.33% than those of non- mycorrhizal M.sativa. The activity of PPO, POD, CAT and SOD in mycorrhizal M.sativa were obviously higher than non mycorrhizal M.sativa under atrazine stess. We identified differential root proteins using isobaric tags for relative and absolute quantization coupled with liquid chromatography-mass spectrometry, with 533 proteins identified (276 unregulated and 257 downregulated). The differentially expressed proteins were further examined using GO, BLAST comparisons, and a literature inquiry and were classified into the categories of atrazine degradation (37.1%); atrazine stress response (28.6%); plant immune responses (14.3%); translation, synthesis, and processing (10%); and signal transduction and biological processes (10%). Furthermore, we identified glycosyl transferase, glutathione S-transferase, laccase, cytochrome P450 monooxygenase, peroxidase, and other proteins closely related to the degradation process. CONCLUSIONS: Mycorrhizal Medicago showed improved atrazine degradation within the culturing medium and increased atrazine enrichment in the roots and stems. Additionally, AMF increased the plant root response to atrazine, with relevant enzymes up regulated and toxic effects alleviated. Overall, the findings of this study show that AMF played an important role in easing atrazine stress in plants and contributed to atrazine remediation and further contributed to the understanding of the molecular mechanism associated with atrazine stresses and potential mycorrhizal contributions in M.sativa.

Placing arbuscular mycorrhizal fungi on the risk assessment test battery of plant protection products (PPPs).

Arbuscular mycorrhizal fungi (AMF) are mutualistic symbionts considered a key group in soil systems involved in the provision of several ecosystem services. Recently they have been listed by EFSA as organisms to be included in the test battery for the risk assessment of plant protection product (PPPs). This study aimed to contribute to improve the ISO Protocol (ISO 10832: 2009) by assessing the feasibility of using other AMF species under different test conditions. Overall, results showed that AMF species Gigaspora albida and Rhizophagus clarus (selected out of five AMF species) are suitable to be used in spore germination tests using the ISO protocol (14 days incubation with sand or artificial soil as substrate) to test PPPs. However, several modifications to the protocol were made in order to accommodate the use of the tested isolates, namely the incubation temperature (28 °C instead of 24 °C) and the change of reference substance (boric acid instead of cadmium nitrate). The need for these changes, plus the results obtained with the three fungicides tested (chlorothalonil, mancozeb and metalaxyl-M) and comparisons made with literature on the relevance of the origin of AMF isolates in dictating the adequate test conditions, emphasize the importance of adjusting test conditions (AMF species/isolates and test temperature) when assessing effects for prospective risk assessment targeting different climatic zones. So, further studies should be conducted with different AMF species and isolates from different climatic regions, in order to better define which species/isolate and test conditions should be used to assess effects of a particular PPP targeting a given climatic zone.

Can Cd translocation in Oryza sativa L. be attenuated by arbuscular mycorrhizal fungi in the presence of EDTA?

Arbuscular mycorrhizal (AM) fungi play an important role in plant tolerance of heavy metal contamination. In this study, a pot experiment was conducted to illustrate the effects of the two AM fungi species Funneliformis mosseae (Fm) and Rhizophagus irregularis (Ri) on plant growth of Oryza sativa L. either with or without ethylenediamine tetraacetate (EDTA) addition and during exposure to five Cd concentrations (in the range of 0-5 mg kg-1). The results showed that Fm inoculation achieved greater mycorrhizal colonization and mycorrhizal dependency indexes than Ri inoculation. In addition, the effects of AM fungi on Cd biosorption and translocation in rice were also investigated in the presence of EDTA. Despite cooperative adsorption, the Freundlich isotherm could describe the biosorption effects of Cd on rice roots regardless of AM fungi inoculation or EDTA addition. Cd concentrations in mycorrhizal roots increased but decreased in mycorrhizal shoots in contrast to the control treatment. Although EDTA addition negatively inhibited the uptake of Cd to mycorrhizal shoots, lower translocation factor (TF) and bioconcentration factor (BCF) were still observed in treatments with EDTA compared to control treatment. Our findings suggest that Ri and Fm inoculation enhanced Cd immobilization in the roots, thus preventing Cd entry into the food chain during exposure to low and high Cd stress, respectively.

Arbuscular mycorrhizal fungi enhance the copper tolerance of Tagetes patula through the sorption and barrier mechanisms of intraradical hyphae.

Arbuscular mycorrhizal fungi (AMF) are widespread soil fungi that can form endosymbiotic structures with the root systems of most plants and can improve the tolerance of host plants to heavy metals. In the present study, we investigated the effects of AMF (Glomus coronatum) inoculation on the tolerance of Tagetes patula L. to Cu. Almost all of the non-mycorrhizal plants exposed to 100 µM Cu died after 3 d, whereas phytotoxicity was only observed in mycorrhizal plants that were exposed to Cu concentrations greater than 100 µM. Analysing the dynamic accumulation of Cu indicated that, after 7 d of Cu exposure, less Cu was absorbed or accumulated by mycorrhizal plants than by control plants, and significantly less Cu was translocated to the shoots. Meanwhile, analysing the root morphology, the integrity of the root plasma membranes, the photosynthesis rate, and the content of essential elements of plants growing in cultures with 50 µM Cu revealed that AMF inoculation markedly alleviated the toxic effects of Cu stress on root system activity, photosynthesis rate, and mineral nutrient accumulation. In addition, to understand the Cu allocation, an energy spectrum analysis of Cu content at the transverse section of root tips was conducted and subsequently provided direct evidence that intraradical hyphae at the root endodermis could selectively immobilise large amounts of Cu. Indeed, the sorption and barrier mechanisms of AMF hyphae reduce Cu toxicity in the roots of T. patula and eventually enhance the plants' Cu tolerance.

Glomeromycota communities survive extreme levels of metal toxicity in an orphan mining site.

Abandoned tailing basins and waste heaps of orphan mining sites are of great concern since extreme metal contamination makes soil improper for any human activity and is a permanent threat for nearby surroundings. Although spontaneous revegetation can occur, the process is slow or unsuccessful and rhizostabilisation strategies to reduce dispersal of contaminated dust represent an option to rehabilitate such sites. This requires selection of plants tolerant to such conditions, and optimization of their fitness and growth. Arbuscular mycorrhizal fungi (AMF) can enhance metal tolerance in moderately polluted soils, but their ability to survive extreme levels of metal contamination has not been reported. This question was addressed in the tailing basin and nearby waste heaps of an orphan mining site in southern France, reaching in the tailing basin exceptionally high contents of zinc (ppm: 97,333 total) and lead (ppm: 31,333 total). In order to contribute to a better understanding of AMF ecology under severe abiotic stress and to identify AMF associated with plants growing under such conditions, that may be considered in future revegetation and rhizostabilisation of highly polluted areas, nine plant species were sampled at different growing seasons and AMF root colonization was determined. Glomeromycota diversity was monitored in mycorrhizal roots by sequencing of the ribosomal LSU. This first survey of AMF in such highly contaminated soils revealed the presence of several AMF ribotypes, belonging mainly to the Glomerales, with some examples from the Paraglomerales and Diversisporales. AMF diversity and root colonization in the tailing basin were lower than in the less-contaminated waste heaps. A Paraglomus species previously identified in a polish mining site was common in roots of different plants. Presence of active AMF in such an environment is an outstanding finding, which should be clearly considered for the design of efficient rhizostabilisation processes.

Transcriptional profiling of arbuscular mycorrhizal roots exposed to high levels of phosphate reveals the repression of cell cycle-related genes and secreted protein genes in Rhizophagus irregularis.

The development of arbuscular mycorrhiza (AM) is strongly suppressed under high-phosphate (Pi) conditions. To investigate AM fungal responses during the suppression of AM by high Pi, we performed an RNA-seq analysis of Rhizophagus irregularis colonizing Lotus japonicus roots at different levels of Pi (20, 100, 300, and 500 µM). AM fungal colonization decreased markedly under high-Pi conditions. In total, 163 fungal genes were differentially expressed among the four Pi treatments. Among these genes, a cell cycle-regulatory gene, cyclin-dependent kinase CDK1, and several DNA replication- and mitosis-related genes were repressed under high-Pi conditions. More than 20 genes encoding secreted proteins were also downregulated by high-Pi conditions, including the strigolactone-induced putative secreted protein 1 gene that enhances AM fungal colonization. In contrast, the expression of genes related to aerobic respiration and transport in R. irregularis were largely unaffected. Our data suggest that high Pi suppresses the expression of genes associated with fungal cell cycle progression or that encode secreted proteins that may be required for intercellular hyphal growth and arbuscule formation. However, high Pi has little effect on the transcriptional regulation of the primary metabolism or transport in preformed fungal structures.

Role of Rhizophagus irregularis in alleviating cadmium toxicity via improving the growth, micro- and macroelements uptake in Phragmites australis.

Arbuscular mycorrhizal (AM) fungi have been used to alleviate heavy metal stress on plant growth and uptake of micro- and macroelements. A greenhouse pot experiment was conducted to verify the effects of AM fungus Rhizophagus irregularis on the growth, physiological characteristics, total Cd, and element uptake of Phragmites australis under different Cd stress (in the range of 0-20 mg L-1). The results showed that the symbiosis could effectively alleviate Cd toxicity with greater root biomass, higher photosynthesis rate, and lower levels of malonaldehyde (MDA) and proline than non-mycorrhizal plants could. However, reduced transpiration rate (Tr) and stomatal conductance (g s) indicated R. irregularis protected host plants from Cd stress (≥5 mg L-1) via the stomatal closure. Although micro- and macroelements displayed differently in the presence of Cd, higher concentrations were still detected in mycorrhizal plants in contrast to non-mycorrhizal plants. Moreover, step multiple regression significantly demonstrated Pnmax, stem diameter (Sd), and g s were the important factors with regard to total Cd uptake in the symbiosis, but Mn affected to non-mycorrhizal plants. These results suggested R. irregularis could alleviate the competition between Mn and Cd by altering plant physiology. This work clearly demonstrated that R. irregularis can be able to support P. australis growth better even though under high Cd stress (>1 mg L-1), suggesting its good potential for practical use in high Cd-contaminated areas.

Cd-induced production of glomalin by arbuscular mycorrhizal fungus (Rhizophagus irregularis) as estimated by monoclonal antibody assay.

Glomalin is a specific fungal glycoprotein produced by arbuscular mycorrhizal (AM) fungi belonging to the Glomerales which could efficiently sequestrate heavy metals. The glomalin has been introduced as a heat shock protein and there are evidences that increasing levels of heavy metals could enhance its production. We examined the influence of Cd concentrations on glomalin production by AM fungus, as well as its contribution to the sequestration of Cd in both pot and in vitro culture conditions. Pot experiment was carried out using pure sand with Trifolium repens L. as host plant, mycorrhized by Rhizophagus irregularis and treated with Cd levels of 0, 15, 30, and 45 µM. In vitro experiment was performed in two-compartment plates containing the transformed carrot roots mycorrhized with the same fungus and treated with Cd levels of 0, 0.001, 0.01, and 0.1 mM. The immunoreactive and Bradford reactive glomalin contents in both experiments increased as so raising Cd concentration. Total Cd sequestrated by hyphal glomalin in both cultures was significantly increased as the levels of Cd increased. The highest contents of Cd sequestration in pot (75.78 µg Cd/mg glomalin) and in vitro (11.44 µg Cd/mg glomalin) cultures were recorded at the uppermost levels of Cd, which significantly differed with other levels. Our results suggested that under Cd-induced stress, stimulated production of glomalin by AM fungus may be a protective mechanism against the toxic effect of Cd.

Sex-specific responses of Populus deltoides to Glomus intraradices colonization and Cd pollution.

The positive effects of arbuscular mycorrhizal fungi (AM) on the survival, growth and physiology of plants under various stress conditions have been widely recognized. However, whether sex-dependent susceptibility to AM colonization exists, which can induce a differential tolerance between the sexes to stress conditions, is still unclear. In this study, we investigated the effects of Glomus intraradices on Cd-stressed males and females of Populus deltoides (spiked with 10 mg Cd per kg dry substrate) in terms of morphology, physiology, biochemistry, ultrastructure, and toxin storage and translocation. Exposure to Cd promoted the colonization by G. intraradices in males, but not in females. Generally, females suffered more impairments than males in response to Cd stress, reflected by leaf symptoms, the extent of lipid peroxidation, and integrity of the cellular ultrastructure, whether they were inoculated or not. Inoculation with G. intraradices alleviated the phytotoxic effects of Cd in females by stimulating antioxidant enzymes, decreasing levels of reactive oxygen species (ROS) and restricting Cd transfer to the shoots. In contrast, these beneficial effects induced by AM were not detected in mycorrhizal males compared to non-mycorrhizal males, based on thiobarbituric acid-reactive substances (TBARS) and cellular ultrastructure. Inoculation with AM promoted Cd accumulation in males but not in females, and caused the sequestration of more toxic Cd in the root systems in both sexes. Therefore, our results suggest that inoculated males of P. deltoides are suitable candidates for phytostabilization in Cd-polluted soils, due to their higher accumulation ability and greater tolerance relative to inoculated females.

Characterization of Three New Glutaredoxin Genes in the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis: Putative Role of RiGRX4 and RiGRX5 in Iron Homeostasis.

Glutaredoxins (GRXs) are small ubiquitous oxidoreductases involved in the regulation of the redox state in living cells. In an attempt to identify the full complement of GRXs in the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis, three additional GRX homologs, besides the formerly characterized GintGRX1 (renamed here as RiGRX1), were identified. The three new GRXs (RiGRX4, RiGRX5 and RiGRX6) contain the CXXS domain of monothiol GRXs, but whereas RiGRX4 and RiGRX5 belong to class II GRXs, RiGRX6 belongs to class I together with RiGRX1. By using a yeast expression system, we observed that the newly identified homologs partially reverted sensitivity of the GRX deletion yeast strains to external oxidants. Furthermore, our results indicated that RiGRX4 and RiGRX5 play a role in iron homeostasis in yeast. Gene expression analyses revealed that RiGRX1 and RiGRX6 were more highly expressed in the intraradical (IRM) than in the extraradical mycelium (ERM). Exposure of the ERM to hydrogen peroxide induced up-regulation of RiGRX1, RiGRX4 and RiGRX5 gene expression. RiGRX4 expression was also up-regulated in the ERM when the fungus was grown in media supplemented with a high iron concentration. These data indicate the two monothiol class II GRXs, RiGRX4 and RiGRX5, might be involved in oxidative stress protection and in the regulation of fungal iron homeostasis. Increased expression of RiGRX1 and RiGRX6 in the IRM suggests that these GRXs should play a key role in oxidative stress protection of R. irregularis during its in planta phase.

Functional analysis of liverworts in dual symbiosis with Glomeromycota and Mucoromycotina fungi under a simulated Palaeozoic CO2 decline.

Most land plants form mutualistic associations with arbuscular mycorrhizal fungi of the Glomeromycota, but recent studies have found that ancient plant lineages form mutualisms with Mucoromycotina fungi. Simultaneous associations with both fungal lineages have now been found in some plants, necessitating studies to understand the functional and evolutionary significance of these tripartite associations for the first time. We investigate the physiology and cytology of dual fungal symbioses in the early-diverging liverworts Allisonia and Neohodgsonia at modern and Palaeozoic-like elevated atmospheric CO2 concentrations under which they are thought to have evolved. We found enhanced carbon cost to liverworts with simultaneous Mucoromycotina and Glomeromycota associations, greater nutrient gain compared with those symbiotic with only one fungal group in previous experiments and contrasting responses to atmospheric CO2 among liverwort-fungal symbioses. In liverwort-Mucoromycotina symbioses, there is increased P-for-C and N-for-C exchange efficiency at 440 p.p.m. compared with 1500 p.p.m. CO2. In liverwort-Glomeromycota symbioses, P-for-C exchange is lower at ambient CO2 compared with elevated CO2. No characteristic cytologies of dual symbiosis were identified. We provide evidence of a distinct physiological niche for plant symbioses with Mucoromycotina fungi, giving novel insight into why dual symbioses with Mucoromycotina and Glomeromycota fungi persist to the present day.

Integrated multi-omics analysis supports role of lysophosphatidylcholine and related glycerophospholipids in the Lotus japonicus-Glomus intraradices mycorrhizal symbiosis.

Interaction of plant roots with arbuscular mycorrhizal fungi (AMF) is a complex trait resulting in cooperative interactions among the two symbionts including bidirectional exchange of resources. To study arbuscular mycorrhizal symbiosis (AMS) trait variation in the model plant Lotus japonicus, we performed an integrated multi-omics analysis with a focus on plant and fungal phospholipid (PL) metabolism and biological significance of lysophosphatidylcholine (LPC). Our results support the role of LPC as a bioactive compound eliciting cellular and molecular response mechanisms in Lotus. Evidence is provided for large interspecific chemical diversity of LPC species among mycorrhizae with related AMF species. Lipid, gene expression and elemental profiling emphasize the Lotus-Glomus intraradices interaction as distinct from other arbuscular mycorrhizal (AM) interactions. In G. intraradices, genes involved in fatty acid (FA) elongation and biosynthesis of unsaturated FAs were enhanced, while in Lotus, FA synthesis genes were up-regulated during AMS. Furthermore, FAS protein localization to mitochondria suggests FA biosynthesis and elongation may also occur in AMF. Our results suggest the existence of interspecific partitioning of PL resources for generation of LPC and novel candidate bioactive PLs in the Lotus-G. intraradices symbiosis. Moreover, the data advocate research with phylogenetically diverse Glomeromycota species for a broader understanding of the molecular underpinnings of AMS.

Arbuscular mycorrhiza improve growth, nitrogen uptake, and nitrogen use efficiency in wheat grown under elevated CO2.

Effects of the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis on plant growth, carbon (C) and nitrogen (N) accumulation, and partitioning was investigated in Triticum aestivum L. plants grown under elevated CO2 in a pot experiment. Wheat plants inoculated or not inoculated with the AM fungus were grown in two glasshouse cells with different CO2 concentrations (400 and 700 ppm) for 10 weeks. A (15)N isotope labeling technique was used to trace plant N uptake. Results showed that elevated CO2 increased AM fungal colonization. Under CO2 elevation, AM plants had higher C concentration and higher plant biomass than the non-AM plants. CO2 elevation did not affect C and N partitioning in plant organs, while AM symbiosis increased C and N allocation into the roots. In addition, plant C and N accumulation, (15)N recovery rate, and N use efficiency (NUE) were significantly higher in AM plants than in non-AM controls under CO2 enrichment. It is concluded that AM symbiosis favors C and N partitioning in roots, increases C accumulation and N uptake, and leads to greater NUE in wheat plants grown at elevated CO2.