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Summary [ABSTRACT FROM AUTHOR]

Presents molecular method to examine plasma membrane H[sup +]-adenosine triphosphatases in arbuscular mycorrhizas (AM). Translocation of cations, anions, amino acids and sugars; Electrochemical gradient of H[sup +] across the cell plasma membrane; Nutrient exchange processes taking place between the plant and the fungus in AM symbiosis.

To analyze the effect of arbuscular mycorrhizal (AM) colonization on tomato gene expression, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) patterns of crude extracts, soluble and membrane proteins of tomato roots, either mycorrhizal and the AM fungus Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe or non-mycorrhizal, have been compared. In the three fractions analyzed. AM colonization induced up-regulation with down-regulation of the synthesis of polypeptides already present in tomato roots and induction of some new polypeptides. Separation of root extracts into soluble and membrane fractions allowed us to identify two soluble, and five membrane-bound, newly induced polypeptides in AM roots. Comparison of the protein patterns of AM roots with those of the external mycelium of G. mosseae showed that one of the newly induced polypeptides might correspond to a fungal polypeptide. By using this experimental approach, we have been able to detect 44 polypeptides tbat arc differentially displayed in tomato roots as a consequence of the establishment of the AM symbiosis. [ABSTRACT FROM AUTHOR]

Optimizing agroecosystems and crops for micronutrient uptake while reducing issues with inorganic contaminants (metal(loid)s) is a challenging task. One promising approach is to use arbuscular mycorrhizal fungi (AMF) and investigate the physiological, molecular and epigenetic changes that occur in their presence and that lead to changes in plant metal(loid) concentration (biofortification of micronutrients or mitigation of contaminants). Moreover, it is important to understand these mechanisms in the context of the soil microbiome, particularly those interactions of AMF with other soil microbes that can further shape crop nutrition. To address these challenges, a two-pronged approach is recommended: exploring molecular mechanisms and investigating microbiome management and engineering. Combining both approaches can lead to benefits in human health by balancing nutrition and contamination caused by metal(loid)s in the agro-ecosystem., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

Variations in arbuscular mycorrhizae (AM) effects on plant growth (MGR) are commonly assumed to result from cost : benefit balances, with C as the cost and, most frequently, P as the benefit. The trade-balance model (TBM) adopts these assumptions and hypothesizes that mycorrhizal benefit depends on C : N : P stoichiometry. Although widely accepted, the TBM has not been experimentally tested. We isolated the parameters included in the TBM and tested these assumptions using it as framework. Oryza sativa plants were supplied with different N : P ratios at low light level, establishing different C : P and C : N exchange rates, and C, N or P limitation. MGR and effects on nutrient uptake, %M, ERM, photosynthesis and shoot starch were measured. C distribution to AM fungi played no role in MGR, and N was essential for all AM effects, including on P nutrition. C distribution to AM and MGR varied with the limiting nutrient (N or P), and evidence of extensive interplay between N and P was observed. The TBM was not confirmed. The results agreed with the exchange of surplus resources and source-sink regulation of resource distribution among plants and AMF. Rather than depending on exchange rates, resource exchange may simply obey both symbiont needs, not requiring further regulation., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

Aluminum (Al) toxicity and phosphorus (P) deficiency are widely recognized as major constraints to agricultural productivity in acidic soils. Under this scenario, the development of ryegrass plants with enhanced P use efficiency and Al resistance is a promising approach by which to maintain pasture production. In this study, we assessed the contribution of growth traits, P efficiency, organic acid anion (OA) exudation, and the expression of Al-responsive genes in improving tolerance to concurrent low-P and Al stress in ryegrass ( Lolium perenne L.). Ryegrass plants were hydroponically grown under optimal (0.1 mM) or low-P (0.01 mM) conditions for 21 days, and further supplied with Al (0 and 0.2 mM) for 3 h, 24 h and 7 days. Accordingly, higher Al accumulation in the roots and lower Al translocation to the shoots were found in ryegrass exposed to both stresses. Aluminum toxicity and P limitation did not change the OA exudation pattern exhibited by roots. However, an improvement in the root growth traits and P accumulation was found, suggesting an enhancement in Al tolerance and P efficiency under combined Al and low-P stress. Al-responsive genes were highly upregulated by Al stress and P limitation, and also closely related to P utilization efficiency. Overall, our results provide evidence of the specific strategies used by ryegrass to co-adapt to multiple stresses in acid soils.

Arbuscular mycorrhizal (AM) fungi establish a mutualistic symbiosis with most land plants. AM fungi regulate plant copper (Cu) acquisition both in Cu deficient and polluted soils. Here, we report characterization of RiCRD1, a Rhizophagus irregularis gene putatively encoding a Cu transporting ATPase. Based on its sequence analysis, RiCRD1 was identified as a plasma membrane Cu  +  efflux protein of the P 1B1 -ATPase subfamily. As revealed by heterologous complementation assays in yeast, RiCRD1 encodes a functional protein capable of conferring increased tolerance against Cu. In the extraradical mycelium, RiCRD1 expression was highly up-regulated in response to high concentrations of Cu in the medium. Comparison of the expression patterns of different players of metal tolerance in R. irregularis under high Cu levels suggests that this fungus could mainly use a metal efflux based-strategy to cope with Cu toxicity. RiCRD1 was also expressed in the intraradical fungal structures and, more specifically, in the arbuscules, which suggests a role for RiCRD1 in Cu release from the fungus to the symbiotic interface. Overall, our results show that RiCRD1 encodes a protein which could have a pivotal dual role in Cu homeostasis in R. irregularis, playing a role in Cu detoxification in the extraradical mycelium and in Cu transfer to the apoplast of the symbiotic interface in the arbuscules., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Transporters of the NRAMP family are ubiquitous metal-transition transporters, playing a key role in metal homeostasis, especially in Mn and Fe homeostasis. In this work, we report the characterization of the NRAMP family members ( RiSMF1 , RiSMF2 , RiSMF3.1 and RiSMF3.2 ) of the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis. Phylogenetic analysis of the NRAMP sequences of different AM fungi showed that they are classified in two groups, which probably diverged early in their evolution. Functional analyses in yeast revealed that RiSMF3.2 encodes a protein mediating Mn and Fe transport from the environment. Gene-expression analyses by RT-qPCR showed that the RiSMF genes are differentially expressed in the extraradical (ERM) and intraradical (IRM) mycelium and differentially regulated by Mn and Fe availability. Mn starvation decreased RiSMF1 transcript levels in the ERM but increased RiSMF3.1 expression in the IRM. In the ERM, RiSMF1 expression was up-regulated by Fe deficiency, suggesting a role for its encoded protein in Fe-deficiency alleviation. Expression of RiSMF3.2 in the ERM was up-regulated at the early stages of Fe toxicity but down-regulated at later stages. These data suggest a role for RiSMF3.2 not only in Fe transport but also as a sensor of high external-Fe concentrations. Both Mn- and Fe-deficient conditions affected ERM development. While Mn deficiency increased hyphal length, Fe deficiency reduced sporulation.

Arbuscular mycorrhizal (AM) fungi, symbionts of most land plants, increase plant fitness in metal contaminated soils. To further understand the mechanisms of metal tolerance in the AM symbiosis, the expression patterns of the maize Heavy Metal ATPase (HMA) family members and the ionomes of non-mycorrhizal and mycorrhizal plants grown under different Cu supplies were examined. Expression of ZmHMA5a and ZmHMA5b, whose encoded proteins were predicted to be localized at the plasma membrane, was up-regulated by Cu in non-mycorrhizal roots and to a lower extent in mycorrhizal roots. Gene expression of the tonoplast ZmHMA3a and ZmHMA4 isoforms was up-regulated by Cu-toxicity in shoots and roots of mycorrhizal plants. AM mitigates the changes induced by Cu toxicity on the maize ionome, specially at the highest Cu soil concentration. Altogether these data suggest that in Cu-contaminated soils, AM increases expression of the HMA genes putatively encoding proteins involved in Cu detoxification and balances mineral nutrient uptake improving the nutritional status of the maize plants., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Cadmium is a heavy metal (HM) that inhibits plant growth and leads to death, causing great losses in yields, especially in Cd hyperaccumulator crops such as Glycine max (L.) Merr. (soybean), a worldwide economically important legume. Furthermore, Cd incorporation into the food chain is a health hazard. Oxidative stress (OS) is a plant response to abiotic and biotic stresses with an intracellular burst of reactive oxygen species (ROS) that causes damage to lipids, proteins, and DNA. The arbuscular mycorrhizal fungal (AMF) association is a plant strategy to cope with HM and to alleviate OS. Our aim was to evaluate the mitigation effects of mycorrhization with AMF Rhizophagus intraradices on soybean growth, nutrients, Cd accumulation, lipid peroxidation, and the activity of different antioxidant agents under Cd (0.7-1.2 mg kg -1 bioavailable Cd) induced OS. Our results suggest that glutathione may act as a signal molecule in a defense response to Cd-induced OS, and mycorrhization may avoid Cd-induced growth inhibition and reduce Cd accumulation in roots. It is discussed that R. intraradices mycorrhization would act as a signal, promoting the generation of a soybean cross tolerance response to Cd pollution, therefore evidencing the potential of this AMF association for bioremediation and encouragement of crop development, particularly because it is an interaction between a worldwide cultivated Cd hyperaccumulator plant and an AMF-HM-accumulator commonly present in soils., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

The obligate symbiotic nature of arbuscular mycorrhizal (AM) fungi makes extremely difficult their genetic manipulation or transformation. For this reason, a heterologous system has been traditionally used for functional analysis of AM fungal genes, being the budding yeast Saccharomyces cerevisiae an organism suitable for this purpose. Here we present the yeast methods required for the functional analysis of AM fungal genes, including protocols for yeast transformation, heterologous gene expression, functional complementation assays, preparation of yeast extracts, and subcellular localization of the encoded protein.

An in vivo whole-plant bi-dimensional experimental system has been devised and tested with different host plants, in order to obtain extraradical mycelium (ERM) produced by different arbuscular mycorrhizal fungi (AMF). In this system, a host plant germling is inoculated with AMF to establish mycorrhizal symbiosis, and, after colonization, newly formed extraradical hyphae and spores are removed. Then the mycorrhizal root system is wrapped in a nylon net and placed between membranes in a Petri dish, allowing ERM to grow on the membrane surface. Such extraradical hyphae may be used for in situ morphometric analyses or collected for molecular or biochemical assays: in the latter case, the plant with its root sandwich may be reassembled to renew mycelium production. In this experimental system, which was tested with diverse host plant species and lines, values of explored membrane surface areas and densities of ERM showed wide ranges of variation, and its length ranged from 9.7 ± 2.0 to 48.8 ± 9.9 m per plant, depending on host and AMF identity. Across the different plant-AMF combinations tested, the whole-plant system produced 2.0 ± 0.6 to 5.3 ± 0.3 mg of ERM fresh biomass per plant per harvest. This experimental system can be used for a wide range of AMF and host plants species, either establishing arbuscular mycorrhizas or other mycorrhizal interactions. ERM produced and collected in the whole-plant system is suitable for morphological, physiological, and molecular analyses, facilitating studies on the different aspects of mycorrhizal symbiotic interactions.

Main Conclusion: The phosphate transporters LpPHT1;1 and LpPHT1;4 have different roles in phosphate uptake and translocation in ryegrass under P stress condition. The phosphate transporter 1 (PHT1) family are integral membrane proteins that operate in phosphate uptake, distribution and remobilization within plants. In this study, we report on the functional characterization and expression of two PHT1 family members from ryegrass plants (Lolium perenne L.) and determine their roles in the specificity of Pi transport. The expression level of LpPHT1;4 was strongly influenced by phosphorus (P) status, being higher under P-starvation condition. In contrast, the expression level of LpPHT1;1 was not correlated with P supply. Yeast mutant complementation assays showed that LpPHT1;4 can complement the growth defect of the yeast mutant Δpho84 under Pi-deficient conditions, whereas the yeast mutant expressing LpPHT1;1 was not able to restore growth. Phylogenetic and molecular analyses indicated high sequence similarity to previously identified PHT1s from other species in the Poaceae. These results suggest that LpPHT1;1 may function as a low-affinity Pi transporter, whereas LpPHT1;4 could acts as a high-affinity Pi transporter to maintain Pi homeostasis under stress conditions in ryegrass plants. This study will form the basis for the long-term goal of improving the phosphate use efficiency of ryegrass plants.

Arbuscular mycorrhizal fungi increase fitness of their host plants under Cu deficient and toxic conditions. In this study, we have characterized two Cu transporters of the CTR family (RiCTR1 and RiCTR2) and a CTR-like protein (RiCTR3A) of Rhizophagus irregularis . Functional analyses in yeast revealed that RiCTR1 encodes a plasma membrane Cu transporter, RiCTR2 a vacuolar Cu transporter and RiCTR3A a plasma membrane protein involved in Cu tolerance. RiCTR1 was more highly expressed in the extraradical mycelia (ERM) and RiCTR2 in the intraradical mycelia (IRM). In the ERM, RiCTR1 expression was up-regulated by Cu deficiency and down-regulated by Cu toxicity. RiCTR2 expression increased only in the ERM grown under severe Cu-deficient conditions. These data suggest that RiCTR1 is involved in Cu uptake by the ERM and RiCTR2 in mobilization of vacuolar Cu stores. Cu deficiency decreased mycorrhizal colonization and arbuscule frequency, but increased RiCTR1 and RiCTR2 expression in the IRM, which suggest that the IRM has a high Cu demand. The two alternatively spliced products of RiCTR3, RiCTR3A and RiCTR3B , were more highly expressed in the ERM. Up-regulation of RiCTR3A by Cu toxicity and the yeast complementation assays suggest that RiCTR3A might function as a Cu receptor involved in Cu tolerance.

Phosphorus (P) is a poorly available macronutrient essential for plant growth and development and consequently for successful crop yield and ecosystem productivity. To cope with P limitations plants have evolved strategies for enhancing P uptake and/or improving P efficiency use. The universal 450-million-yr-old arbuscular mycorrhizal (AM) (fungus-root) symbioses are one of the most successful and widespread strategies to maximize access of plants to available P. AM fungi biotrophically colonize the root cortex of most plant species and develop an extraradical mycelium which overgrows the nutrient depletion zone of the soil surrounding plant roots. This hyphal network is specialized in the acquisition of low mobility nutrients from soil, particularly P. During the last years, molecular biology techniques coupled to novel physiological approaches have provided fascinating contributions to our understanding of the mechanisms of symbiotic P transport. Mycorrhiza-specific plant phosphate transporters, which are required not only for symbiotic P transfer but also for maintenance of the symbiosis, have been identified. The present review provides an overview of the contribution of AM fungi to plant P acquisition and an update of recent findings on the physiological, molecular and regulatory mechanisms of P transport in the AM symbiosis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Soil acidity, associated with aluminium (Al) toxicity and low phosphorus (P) availability, is considered the most important problem for agricultural production. Even though the Al-P interaction has been widely investigated, the impact of P-nutrition on Al-toxicity still remains controversial and poorly understood. To elucidate further insights into the underlying mechanisms of this interaction in ryegrass (Lolium perenne L.), P uptake, antioxidant responses and the gene expression of phosphate transporters were determined. Two ryegrass cultivars with different Al resistances, the Al-tolerant Nui cultivar and the Al-sensitive Expo cultivar were hydroponically grown under low (16 μM) and optimal (100 μM) P doses for 16 days. After P treatments, plants were exposed to Al doses (0 and 200 μM) under acidic conditions (pH 4.8) for 24 h. Al and P accumulation were higher in the roots of Nui than that of Expo. Moreover, lower Al accumulation was found in shoots of Nui independent of P supplies. Oxidative stress induced by Al-toxicity and P-deficiency was more severe in the Al-sensitive Expo. Expression levels of L. perenne phosphate transporters were higher in Nui than they were in Expo. While LpPHT1 expression was up-regulated by P deficiency and Al toxicity in both cultivars, LpPHT4 expression only increased in the Al-tolerant cultivar. This report shows that the higher Al-tolerance of Nui can be attributed to a greater antioxidant system under both P conditions. The observation of higher P and Al accumulation in roots of Nui might indicate that the Al-tolerance of Nui is a consequence of Al immobilization by P mediated by the high expression of phosphate transporters., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Arbuscular mycorrhizal (AM) fungi can improve iron (Fe) acquisition of their host plants. Here, we report a characterization of two components of the high-affinity reductive Fe uptake system of Rhizophagus irregularis, the ferric reductase (RiFRE1) and the high affinity Fe permeases (RiFTR1-2). In the extraradical mycelia (ERM), Fe deficiency induced activation of a plasma membrane-localized ferric reductase, an enzyme that reduces Fe(III) sources to the more soluble Fe(II). Yeast mutant complementation assays showed that RiFRE1 encodes a functional ferric reductase and RiFTR1 an iron permease. In the heterologous system, RiFTR1 was expressed in the plasma membrane while RiFTR2 was expressed in the endomembranes. In the ERM, the highest expression levels of RiFTR1 were found in mycelia grown in media with 0.045 mM Fe, while RiFTR2 was upregulated under Fe-deficient conditions. RiFTR2 expression also increased in the intraradical mycelia (IRM) of maize plants grown without Fe. These data indicate that the Fe permease RiFTR1 plays a key role in Fe acquisition and that RiFTR2 is involved in Fe homeostasis under Fe-limiting conditions. RiFTR1 was highly expressed in the (IRM), which suggests that the maintenance of Fe homeostasis in the IRM might be essential for a successful symbiosis., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)