A H+-ATPase That Energizes Nutrient Uptake during Mycorrhizal Symbioses in Rice and Medicago truncatula

Most plant species form symbioses with arbuscular mycorrhizal (AM) fungi, which facilitate the uptake of mineral nutrients such as phosphate from the soil. Several transporters, particularly proton-coupled phosphate transporters, have been identified on both the plant and fungal membranes and contribute to delivering phosphate from fungi to plants. The mechanism of nutrient exchange has been studied in plants during mycorrhizal colonization, but the source of the electrochemical proton gradient that drives nutrient exchange is not known. Here, we show that plasma membrane H⁺-ATPases that are specifically induced in arbuscule-containing cells are required for enhanced proton pumping activity in membrane vesicles from AM-colonized roots of rice (Oryza sativa) and Medicago truncatula. Mutation of the H⁺-ATPases reduced arbuscule size and impaired nutrient uptake by the host plant through the mycorrhizal symbiosis. Overexpression of the H⁺-ATPase Os-HA1 increased both phosphate uptake and the plasma membrane potential, suggesting that this H⁺-ATPase plays a key role in energizing the periarbuscular membrane, thereby facilitating nutrient exchange in arbusculated plant cells.     

The sucrose transporter SlSUT2 from tomato interacts with brassinosteroid functioning and affects arbuscular mycorrhiza formation

Mycorrhizal plants benefit from the fungal partners by getting better access to soil nutrients. In exchange, the plant supplies carbohydrates to the fungus. The additional carbohydrate demand in mycorrhizal plants was shown to be balanced partially by higher CO₂ assimilation and increased C metabolism in shoots and roots. In order to test the role of sucrose transport for fungal development in arbuscular mycorrhizal (AM) tomato, transgenic plants with down‐regulated expression of three sucrose transporter genes were analysed. Plants that carried an antisense construct of SlSUT2 (SlSUT2as) repeatedly exhibited increased mycorrhizal colonization and the positive effect of plants to mycorrhiza was abolished. Grafting experiments between transgenic and wild‐type rootstocks and scions indicated that mainly the root‐specific function of SlSUT2 has an impact on colonization of tomato roots with the AM fungus. Localization of SISUT2 to the periarbuscular membrane indicates a role in back transport of sucrose from the periarbuscular matrix into the plant cell thereby affecting hyphal development. Screening of an expression library for SlSUT2‐interacting proteins revealed interactions with candidates involved in brassinosteroid (BR) signaling or biosynthesis. Interaction of these candidates with SlSUT2 was confirmed by bimolecular fluorescence complementation. Tomato mutants defective in BR biosynthesis were analysed with respect to mycorrhizal symbiosis and showed indeed decreased mycorrhization. This finding suggests that BRs affect mycorrhizal infection and colonization. If the inhibitory effect of SlSUT2 on mycorrhizal growth involves components of BR synthesis and of the BR signaling pathway is discussed.     

A set of fluorescent protein‐based markers expressed from constitutive and arbuscular mycorrhiza‐inducible promoters to label organelles,membranes and cytoskeletal elements in Medicago truncatula

Medicago truncatula is widely used for analyses of arbuscular mycorrhizal (AM) symbiosis and nodulation. To complement the genetic and genomic resources that exist for this species, we generated fluorescent protein fusions that label the nucleus, endoplasmic reticulum, Golgi apparatus, trans‐Golgi network, plasma membrane, apoplast, late endosome/multivesicular bodies (MVB), transitory late endosome/ tonoplast, tonoplast, plastids, mitochondria, peroxisomes, autophagosomes, plasmodesmata, actin, microtubules, periarbuscular membrane (PAM) and periarbuscular apoplastic space (PAS) and expressed them from the constitutive AtUBQ10 promoter and the AM symbiosis‐specific MtBCP1 promoter. All marker constructs showed the expected expression patterns and sub‐cellular locations in M. truncatula root cells. As a demonstration of their utility, we used several markers to investigate AM symbiosis where root cells undergo major cellular alterations to accommodate their fungal endosymbiont. We demonstrate that changes in the position and size of the nuclei occur prior to hyphal entry into the cortical cells and do not require DELLA signaling. Changes in the cytoskeleton, tonoplast and plastids also occur in the colonized cells and in contrast to previous studies, we show that stromulated plastids are abundant in cells with developing and mature arbuscules, while lens‐shaped plastids occur in cells with degenerating arbuscules. Arbuscule development and secretion of the PAM creates a periarbuscular apoplastic compartment which has been assumed to be continuous with apoplast of the cell. However, fluorescent markers secreted to the periarbuscular apoplast challenge this assumption. This marker resource will facilitate cell biology studies of AM symbiosis, as well as other aspects of legume biology.     

Arbuscular mycorrhizal symbiosis and plant aquaporin expression

Almost all land plants have developed a symbiosis with arbuscular mycorrhizal fungi. Establishment of the association is accompanied by structural changes in the plant root. During arbuscule formation fungal hyphae penetrate the root apoplast and install highly specialized interfaces for solute transport between plant and fungus. The periarbuscular membrane which is part of the plant plasma membrane surrounding arbuscular structures was shown to harbour a high density of different transport systems. Among these also expression of aquaporins was described, which potentially can act as a low affinity transport system for ammonia or ammonium. The present study provides data for expression, localization and function of plant aquaporins in the periarbuscular membrane of mycorrhizal Medicago truncatula plants.     

Differential activation of H+-ATPase genes by an arbuscular mycorrhizal fungus in root cells of transgenic tobacco

In arbuscular mycorrhizas, H⁺-ATPase is active in the plant membrane around arbuscules but absent from plant mutants defective in arbuscule development (Gianinazzi-Pearson et al. 1995, Can J Bot 73: S526–S532). The proton-pumping H⁺-ATPase is encoded by a family of genes in plants. Immunocytochemical studies and promoter-gusA fusion assays were performed in transgenic tobacco (Nicotiana tabacum L.) to determine whether the periarbuscular enzyme activity results from de-novo activation of plant genes by an arbuscular mycorrhizal fungus. The H⁺-ATPase protein was localized in the plant membrane around arbuscule hyphae. The enzyme was absent from non-colonized cortical cells. Regulation of seven H⁺-ATPase genes (pma) was compared in non-mycorrhizal and mycorrhizal roots by histochemical detection of β-glucuronidase (GUS) activity. Two genes (pma2, pma4) were induced in arbuscule-containing cells of mycorrhizal roots but not in non-mycorrhizal cortical tissues or senescent mycorrhiza. It is concluded that de-novo H⁺-ATPase activity in the periarbuscular membrane results from selective induction of two H⁺-ATPase genes, which can have diverse roles in plant-fungal interactions at the symbiotic interface. Received: 23 October 1999 / Accepted: 7 February 2000     

Medicago truncatula Mtha1-2 mutants loose metabolic responses to mycorrhizal colonization

Bidirectional nutrient transfer is one of the key features of the arbuscular mycorrhizal symbiosis. Recently we were able to identify a Medicago truncatula mutant (mtha1-2) that is defective in the uptake of phosphate from the periarbuscular space due to a lack of the energy providing proton gradient provided by the symbiosis specific proton ATPase MtHA1¹ In order to further characterize the impact of fungal colonization on the plant metabolic status, without the beneficial aspect of improved mineral nutrition, we performed leaf ion analyses in mutant and wildtype plants with and without fungal colonization. Although frequency of fungal colonization was unaltered, the mutant did not show a positive growth response to mycorrhizal colonization. This indicates that nutrient transfer into the plant cell fails in the truncated arbuscules due to lacking expression of a functional MtHA1 protein. The leaves of wildtype plants showed clear metabolic responses to root mycorrhizal colonization, whereas no changes of leaf metabolite levels of mycorrhizal mtha1-2 plants were detected, even though they were colonized. These results show that MtHa1 is indispensable for a functional mycorrhizal symbiosis and, moreover, suggest that fungal root colonization per se does not depend on nutrient transfer to the plant host.     

A mass spectrometric approach to identify arbuscular mycorrhiza-related proteins in root plasma membrane fractions

One of the most important morphological changes occurring in arbuscular mycorrhizal (AM) roots takes place when the plant plasma membrane (PM) invaginates around the fungal arbuscular structures resulting in the periarbuscular membrane formation. To investigate whether AM symbiosis-specific proteins accumulate at this stage, two complementary MS approaches targeting the root PM from the model legume Medicago truncatula were designed. Membrane extracts were first enriched in PM using a discontinuous sucrose gradient method. The resulting PM fractions were further analysed with (i) an automated 2-D LC-MS/MS using a strong cation exchange and RP chromatography, and (ii) SDS-PAGE combined with a systematic LC-MS/MS analysis. Seventy-eight proteins, including hydrophobic ones, were reproducibly identified in the PM fraction from non-inoculated roots, representing the first survey of the M. truncatula root PM proteome. Comparison between non-inoculated and Glomus intraradices-inoculated roots revealed two proteins that differed in the mycorrhizal root PM fraction. They corresponded to an H(+)-ATPase (Mtha1) and a predicted glycosylphosphatidylinositol-anchored blue copper-binding protein (MtBcp1), both potentially located on the periarbuscular membrane. The exact role of MtBcp1 in AM symbiosis remains to be investigated.     

The H+-ATPase HA1 of Medicago truncatula Is Essential for Phosphate Transport and Plant Growth during Arbuscular Mycorrhizal Symbiosis

A key feature of arbuscular mycorrhizal symbiosis is improved phosphorus nutrition of the host plant via the mycorrhizal pathway, i.e., the fungal uptake of Pi from the soil and its release from arbuscules within root cells. Efficient transport of Pi from the fungus to plant cells is thought to require a proton gradient across the periarbuscular membrane (PAM) that separates fungal arbuscules from the host cell cytoplasm. Previous studies showed that the H⁺-ATPase gene HA1 is expressed specifically in arbuscule-containing root cells of Medicago truncatula. We isolated a ha1-2 mutant of M. truncatula and found it to be impaired in the development of arbuscules but not in root colonization by Rhizophagus irregularis hyphae. Artificial microRNA silencing of HA1 recapitulated this phenotype, resulting in small and truncated arbuscules. Unlike the wild type, the ha1-2 mutant failed to show a positive growth response to mycorrhizal colonization under Pi-limiting conditions. Uptake experiments confirmed that ha1-2 mutants are unable to take up phosphate via the mycorrhizal pathway. Increased pH in the apoplast of abnormal arbuscule-containing cells of the ha1-2 mutant compared with the wild type suggests that HA1 is crucial for building a proton gradient across the PAM and therefore is indispensible for the transfer of Pi from the fungus to the plant.     

Pathways of Phosphorus Absorption and Early Signaling between the Mycorrhizal Fungi and Plants

This review highlights the key role that mycorrhizal fungi play in making phosphorus (Pi) more available to plants, including pathways of phosphorus absorption, phosphate transporters and plant-mycorrhizal fungus symbiosis, especially in conditions where the level of inorganic phosphorus (Pi) in the soil is low. Mycorrhizal fungi colonization involves a series of signaling where the plant root exudates strigolactones, while the mycorrhizal fungi release a mixture of chito-oligosaccharides and liposaccharides, that activate the symbiosis process through gene signaling pathways, and contact between the hyphae and the root. Once the symbiosis is established, the extraradical mycelium acts as an extension of the roots and increases the absorption of nutrients, particularly phosphorus by the phosphate transporters. Pi then moves along the hyphae to the plant root/fungus interface. The transfer of Pi occurs in the apoplectic space; in the case of arbuscular mycorrhizal fungi, Pi is discharged from the arbuscular to the plant’s root symplasm, in the membrane that surrounds the arbuscule. Pi is then absorbed through the plant periarbuscular membrane by plant phosphate transporters. Furthermore, plants can acquire Pi from soil as a direct absorption pathway. As a result of this review, several genes that codify for high-affinity Pi transporters were identified. In plants, the main family is Pht1 although it is possible to find others such as Pht2, Pht3, Pho1 and Pho2. As in plants, mycorrhizal fungi have genes belonging to the Pht1 subfamily. In arbuscular mycorrhizal fungi we found L1PT1, GiPT, MtPT1, MtPT2, MtPT4, HvPT8, ZmPht1, TaPTH1.2, GmosPT and LYCes. HcPT1, HcPT2 and BePT have been characterized in ectomycorrhizal fungi. Each gene has a different way of expressing itself. In this review, we present diagrams of the symbiotic relationship between mycorrhizal fungi and the plant. This knowledge allows us to design solutions to regional problems such as food production in soils with low levels of Pi.

     

A phosphate transporter from Medicago truncatula involved in the acquisition of phosphate released by arbuscular mycorrhizal fungi

Many plants have the capacity to obtain phosphate via a symbiotic association with arbuscular mycorrhizal (AM) fungi. In AM associations, the fungi release phosphate from differentiated hyphae called arbuscules, that develop within the cortical cells, and the plant transports the phosphate across a symbiotic membrane, called the periarbuscular membrane, into the cortical cell. In Medicago truncatula, a model legume used widely for studies of root symbioses, it is apparent that the phosphate transporters known to operate at the root-soil interface do not participate in symbiotic phosphate transport. EST database searches with short sequence motifs shared by known phosphate transporters enabled the identification of a novel phosphate transporter from M. truncatula, MtPT4. MtPT4 is significantly different from the plant root phosphate transporters cloned to date. Complementation of yeast phosphate transport mutants indicated that MtPT4 functions as a phosphate transporter, and estimates of the K(m) suggest a relatively low affinity for phosphate. MtPT4 is expressed only in mycorrhizal roots, and the MtPT4 promoter directs expression exclusively in cells containing arbuscules. MtPT4 is located in the membrane fraction of mycorrhizal roots, and immunolocalization revealed that MtPT4 colocalizes with the arbuscules, consistent with a location on the periarbuscular membrane. The transport properties and spatial expression patterns of MtPT4 are consistent with a role in the acquisition of phosphate released by the fungus in the AM symbiosis.     

Medicago truncatula mtpt4 mutants reveal a role for nitrogen in the regulation of arbuscule degeneration in arbuscular mycorrhizal symbiosis

Plants acquire essential mineral nutrients such as phosphorus (P) and nitrogen (N) directly from the soil, but the majority of the vascular plants also gain access to these mineral nutrients through endosymbiotic associations with arbuscular mycorrhizal (AM) fungi. In AM symbiosis, the fungi deliver P and N to the root through branched hyphae called arbuscules. Previously we identified MtPT4, a Medicago truncatula phosphate transporter located in the periarbuscular membrane that is essential for symbiotic phosphate transport and for maintenance of the symbiosis. In mtpt4 mutants arbuscule degeneration occurs prematurely and symbiosis fails. Here, we show that premature arbuscule degeneration occurs in mtpt4 mutants even when the fungus has access to carbon from a nurse plant. Thus, carbon limitation is unlikely to be the primary cause of fungal death. Surprisingly, premature arbuscule degeneration is suppressed if mtpt4 mutants are deprived of nitrogen. In mtpt4 mutants with a low N status, arbuscule lifespan does not differ from that of the wild type, colonization of the mtpt4 root system occurs as in the wild type and the fungus completes its life cycle. Sulphur is another essential macronutrient delivered to the plant by the AM fungus; however, suppression of premature arbuscule degeneration does not occur in sulphur-deprived mtpt4 plants. The mtpt4 arbuscule phenotype is strongly correlated with shoot N levels. Analyses of an mtpt4-2 sunn-1 double mutant indicates that SUNN, required for N-mediated autoregulation of nodulation, is not involved. Together, the data reveal an unexpected role for N in the regulation of arbuscule lifespan in AM symbiosis.     

Ultrastructural localization of acid phosphatase in arbusculate coils of mycorrhizal Phoenix canariensis roots

Acid phosphatase (ACP) activity has been detected in roots of mycorrhizal and non-mycorrhizal Phoenix canariensis. This enzyme was ultrastructurally localized in arbusculate coils for the first time. This localization was carried out using a cerium-based method, which minimizes non-specific precipitation. The ACP was localized in inter- and intracellular hyphae, in the fungal cytoplasm as well as at the interface and the fungal cell wall and the periarbuscular membrane limiting it. The novel localization of an ACP in the arbuscular mycorrhizal (AM) interface of arbusculate coils suggests that this enzyme may be involved in the phosphorus efflux from the mycorrhizal fungus to the host. The results presented in this article indicate that the role played by ACP in AM symbiosis may be more important than was previously thought and that arbusculate coils are highly relevant when considering nutrient transfer through AM symbiosis.     

Apoplastic plant subtilases support arbuscular mycorrhiza development in Lotus japonicus

In the arbuscular mycorrhiza (AM) symbiosis, plant roots accommodate Glomeromycota fungi within an intracellular compartment, the arbuscule. At this symbiotic interface, fungal hyphae are surrounded by a plant membrane, which creates an apoplastic compartment, the periarbuscular space (PAS) between fungal and plant cell. Despite the importance of the PAS for symbiotic signal and metabolite exchange, only few of its components have been identified. Here we show that two apoplastic plant proteases of the subtilase family are required for AM development. SbtM1 is the founder member of a family of arbuscular mycorrhiza-induced subtilase genes that occur in at least two clusters in the genome of the legume Lotus japonicus . A detailed expression analysis by RT-PCR revealed that SbtM1 , SbtM3 , SbtM4 and the more distantly related SbtS are all rapidly induced during development of arbuscular mycorrhiza, but only SbtS and SbtM4 are also up-regulated during root nodule symbiosis. Promoter–reporter fusions indicated specific activation in cells that are adjacent to intra-radical fungal hyphae or in cells that harbour them. Venus fluorescent protein was observed in the apoplast and the PAS when expressed from a fusion construct with the SbtM1 signal peptide or the full-length subtilase. Suppression of SbtM1 or SbtM3 by RNAi caused a decrease in intra-radical hyphae and arbuscule colonization, but had no effect on nodule formation. Our data indicate a role for these subtilases during the fungal infection process in particular arbuscule development.