Plant hormones as signals in arbuscular mycorrhizal symbiosis

Abstract

Arbuscular mycorrhizal (AM) fungi are non-specific symbionts developing mutual and beneficial symbiosis with most terrestrial plants. Because of the obligatory nature of the symbiosis, the presence of the host plant during the onset and proceeding of symbiosis is necessary. However, AM fungal spores are able to germinate in the absence of the host plant. The fungi detect the presence of the host plant through some signal communications. Among the signal molecules, which can affect mycorrhizal symbiosis are plant hormones, which may positively or adversely affect the symbiosis. In this review article, some of the most recent findings regarding the signaling effects of plant hormones, on mycorrhizal fungal symbiosis are reviewed. This may be useful for the production of plants, which are more responsive to mycorrhizal symbiosis under stress.     

Arbuscular mycorrhizal hyphopodia and germinated spore exudates trigger Ca2+ spiking in the legume and nonlegume root epidermis

? The aim of this study was to investigate Ca(2+) responses to endosymbiotic arbuscular mycorrhizal (AM) fungi in the host root epidermis following pre-infection hyphopodium formation in both legumes and nonlegumes, and to determine to what extent these responses could be mimicked by germinated fungal spore exudate. ? Root organ cultures of both Medicago truncatula and Daucus carota, expressing the nuclear-localized cameleon reporter NupYC2.1, were used to monitor AM-elicited Ca(2+) responses in host root tissues. ? Ca(2+) spiking was observed in cells contacted by AM hyphopodia for both hosts, with highest frequencies correlating with the epidermal nucleus positioned facing the fungal contact site. Treatment with AM spore exudate also elicited Ca(2+) spiking within the AM-responsive zone of the root and, in both cases, spiking was dependent on the M. truncatula common SYM genes DMI1/2, but not on the rhizobial Nod factor perception gene NFP. ? These findings support the conclusion that AM fungal root penetration is preceded by a SYM pathway-dependent oscillatory Ca(2+) response, whose evolutionary origin predates the divergence between asterid and rosid clades. Our results further show that fungal symbiotic signals are already generated during spore germination, and that cameleon-expressing root organ cultures represent a novel AM-specific bio-assay for such signals.     

Carbon export from arbuscular mycorrhizal roots involves the translocation of carbohydrate as well as lipid

Arbuscular mycorrhizal (AM) fungi take up photosynthetically fixed carbon from plant roots and translocate it to their external mycelium. Previous experiments have shown that fungal lipid synthesized from carbohydrate in the root is one form of exported carbon. In this study, an analysis of the labeling in storage and structural carbohydrates after (13)C(1) glucose was provided to AM roots shows that this is not the only pathway for the flow of carbon from the intraradical to the extraradical mycelium (ERM). Labeling patterns in glycogen, chitin, and trehalose during the development of the symbiosis are consistent with a significant flux of exported glycogen. The identification, among expressed genes, of putative sequences for glycogen synthase, glycogen branching enzyme, chitin synthase, and for the first enzyme in chitin synthesis (glutamine fructose-6-phosphate aminotransferase) is reported. The results of quantifying glycogen synthase gene expression within mycorrhizal roots, germinating spores, and ERM are consistent with labeling observations using (13)C-labeled acetate and glycerol, both of which indicate that glycogen is synthesized by the fungus in germinating spores and during symbiosis. Implications of the labeling analyses and gene sequences for the regulation of carbohydrate metabolism are discussed, and a 4-fold role for glycogen in the AM symbiosis is proposed: sequestration of hexose taken from the host, long-term storage in spores, translocation from intraradical mycelium to ERM, and buffering of intracellular hexose levels throughout the life cycle.     

The exudate from an arbuscular mycorrhizal fungus induces nitric oxide accumulation in Medicago truncatula roots

Nitric oxide (NO) is a signaling molecule involved in plant responses to abiotic and biotic stresses. While there is evidence for NO accumulation during legume nodulation, almost no information exists for arbuscular mycorrhizas (AM). Here, we investigated the occurrence of NO in the early stages of Medicago truncatula–Gigaspora margarita interaction, focusing on the plant response to fungal diffusible molecules. NO was visualized in root organ cultures and seedlings by confocal microscopy using the specific probe 4,5-diaminofluorescein diacetate. Five-minute treatment with the fungal exudate was sufficient to induce significant NO accumulation. The specificity of this response to AM fungi was confirmed by the lack of response in the AM nonhost Arabidopsis thaliana and by analyzing mutants impaired in mycorrhizal capacities. NO buildup resulted to be partially dependent on DMI1, DMI2, and DMI3 functions within the so-called common symbiotic signaling pathway which is shared between AM and nodulation. Significantly, NO accumulation was not induced by the application of purified Nod factor, while lipopolysaccharides from Escherichia coli, known to elicit defense-related NO production in plants, induced a significantly different response pattern. A slight upregulation of a nitrate reductase (NR) gene and the reduction of NO accumulation when the enzyme is inhibited by tungstate suggest NR as a possible source of NO. Genetic and cellular evidence, therefore, suggests that NO accumulation is a novel component in the signaling pathway that leads to AM symbiosis.     

The Glyoxylate Cycle in an Arbuscular Mycorrhizal Fungus. Carbon Flux and Gene Expression

The arbuscular mycorrhizal (AM) symbiosis is responsible for huge fluxes of photosynthetically fixed carbon from plants to the soil. Lipid, which is the dominant form of stored carbon in the fungal partner and which fuels spore germination, is made by the fungus within the root and is exported to the extraradical mycelium. We tested the hypothesis that the glyoxylate cycle is central to the flow of carbon in the AM symbiosis. The results of (13)C labeling of germinating spores and extraradical mycelium with (13)C(2)-acetate and (13)C(2)-glycerol and analysis by nuclear magnetic resonance spectroscopy indicate that there are very substantial fluxes through the glyoxylate cycle in the fungal partner. Full-length sequences obtained by polymerase chain reaction from a cDNA library from germinating spores of the AM fungus Glomus intraradices showed strong homology to gene sequences for isocitrate lyase and malate synthase from plants and other fungal species. Quantitative real-time polymerase chain reaction measurements show that these genes are expressed at significant levels during the symbiosis. Glyoxysome-like bodies were observed by electron microscopy in fungal structures where the glyoxylate cycle is expected to be active, which is consistent with the presence in both enzyme sequences of motifs associated with glyoxysomal targeting. We also identified among several hundred expressed sequence tags several enzymes of primary metabolism whose expression during spore germination is consistent with previous labeling studies and with fluxes into and out of the glyoxylate cycle.     

Fungal symbiosis in rice requires an ortholog of a legume common symbiosis gene encoding a Ca2+/calmodulin-dependent protein kinase

In natural ecosystems, many plants are able to establish mutually beneficial symbioses with microorganisms. Of critical importance to sustainable agriculture are the symbioses formed between more than 80% of terrestrial plants and arbuscular mycorrhizal (AM) fungi and between legumes and nitrogen-fixing rhizobial bacteria. Interestingly, the two symbioses share overlapping signaling pathways in legumes, suggesting that the evolutionarily recent root nodule symbiosis may have acquired functions from the ancient AM symbiosis. The Medicago truncatula DMI3 (DOESN'T MAKE INFECTIONS3) gene (MtDMI3) and its orthologs in legumes are required for both bacterial and fungal symbioses. MtDMI3 encodes a Ca(2+)/calmodulin-dependent protein kinase (CCaMK) essential for the transduction of the Ca(2+) signal induced by the perception of Nod factors. Putative orthologs of MtDMI3 are also present in non-legumes, but their function in AM symbiosis has not been demonstrated in any non-legume species. Here, we combine reverse genetic approaches and a cross-species complementation test to characterize the function of the rice (Oryza sativa) ortholog of MtDMI3, namely, OsDMI3, in AM symbiosis. We demonstrate that OsDMI3 is not only required for AM symbiosis in rice but also is able to complement a M. truncatula dmi3 mutant, indicating an equivalent role of MtDMI3 orthologs in non-legumes.     

Transformed soybean (Glycine max) roots as a tool for the study of the arbuscular mycorrhizal symbiosis

Ri T-DNA transformed roots have been used effectively in studying the interaction between various plant hosts and arbuscular mycorrhizal (AM) fungi. We investigated the in vitro monoxenic symbiosis between the AM fungus Glomus intraradices and transformed soybean roots (TSRs). Comparisons were made between TSR system and plants of the same genotype. The extraradical fungal structures generated in vitro culture showed normal development. Straight runner hyphae branched into short simple branched absorbing structures and spores were initiated. AM symbiosis was confirmed by the presence of arbuscules and vesicles in cortical cells of the TSRs. The frequency of intraradical colonization in TSRs was higher than in plants grown in soil, whereas the intensity values of intraradical colonization in TSR cultures were similar to those in whole plants. These results show that TSR cultures were able to support the growth and characteristic development of G. intraradices.     

GR24, a synthetic analog of strigolactones, stimulates the mitosis and growth of the arbuscular mycorrhizal fungus Gigaspora rosea by boosting its energy metabolism

Arbuscular mycorrhizal (AM) fungi are obligate biotrophs that participate in a highly beneficial root symbiosis with 80% of land plants. Strigolactones are trace molecules in plant root exudates that are perceived by AM fungi at subnanomolar concentrations. Within just a few hours, they were shown to stimulate fungal mitochondria, spore germination, and branching of germinating hyphae. In this study we show that treatment of Gigaspora rosea with a strigolactone analog (GR24) causes a rapid increase in the NADH concentration, the NADH dehydrogenase activity, and the ATP content of the fungal cell. This fully and rapidly (within minutes) activated oxidative metabolism does not require new gene expression. Up-regulation of the genes involved in mitochondrial metabolism and hyphal growth, and stimulation of the fungal mitotic activity, take place several days after this initial boost to the cellular energy of the fungus. Such a rapid and powerful action of GR24 on G. rosea cells suggests that strigolactones are important plant signals involved in switching AM fungi toward full germination and a presymbiotic state.     

The pre-symbiotic growth of arbuscular mycorrhizal fungi is induced by a branching factor partially purified from plant root exudates

Arbuscular mycorrhizal (AM) symbiosis is an association between obligate biotrophic fungi and more than 80% of land plants. During the pre-symbiotic phase, the host plant releases critical metabolites necessary to trigger fungal growth and root colonization. We describe the isolation of a semipurified fraction from exudates of carrot hairy roots, highly active on germinating spores of Gigaspora gigantea, G. rosea, and G. margarita. This fraction, isolated on the basis of its activity on hyphal branching, contains a root factor (one or several molecules) that stimulates, directly or indirectly, G. gigantea nuclear division. We demonstrate the presence of this active factor in root exudates of all mycotrophic plant species tested (eight species) but not in those of nonhost plant species (four species). We negatively tested the hypothesis that it was a flavonoid or a compound synthesized via the flavonoid pathway. We propose that this root factor, yet to be chemically characterized, is a key plant signal for the development of AM fungi.     

Soil nutritional status, not inoculum identity, primarily determines the effect of arbuscular mycorrhizal fungi on the growth of Knautia arvensis plants

Arbuscular mycorrhizal (AM) symbiosis is among the factors contributing to plant survival in serpentine soils characterised by unfavourable physicochemical properties. However, AM fungi show a considerable functional diversity, which is further modified by host plant identity and edaphic conditions. To determine the variability among serpentine AM fungal isolates in their effects on plant growth and nutrition, a greenhouse experiment was conducted involving two serpentine and two non-serpentine populations of Knautia arvensis plants grown in their native substrates. The plants were inoculated with one of the four serpentine AM fungal isolates or with a complex AM fungal community native to the respective plant population. At harvest after 6-month cultivation, intraradical fungal development was assessed, AM fungal taxa established from native fungal communities were determined and plant growth and element uptake evaluated. AM symbiosis significantly improved the performance of all the K. arvensis populations. The extent of mycorrhizal growth promotion was mainly governed by nutritional status of the substrate, while the effect of AM fungal identity was negligible. Inoculation with the native AM fungal communities was not more efficient than inoculation with single AM fungal isolates in any plant population. Contrary to the growth effects, a certain variation among AM fungal isolates was revealed in terms of their effects on plant nutrient uptake, especially P, Mg and Ca, with none of the AM fungi being generally superior in this respect. Regardless of AM symbiosis, K. arvensis populations significantly differed in their relative nutrient accumulation ratios, clearly showing the plant’s ability to adapt to nutrient deficiency/excess.     

A novel nuclear protein interacts with the symbiotic DMI3 calcium- and calmodulin-dependent protein kinase of Medicago truncatula

Many higher plants establish symbiotic relationships with arbuscular mycorrhizal (AM) fungi that improve their ability to acquire nutrients from the soil. In addition to establishing AM symbiosis, legumes also enter into a nitrogen-fixing symbiosis with bacteria known as rhizobia that results in the formation of root nodules. Several genes involved in the perception and transduction of bacterial symbiotic signals named "Nod factors" have been cloned recently in model legumes through forward genetic approaches. Among them, DMI3 (Doesn't Make Infections 3) is a calcium- and calmodulin-dependent kinase required for the establishment of both nodulation and AM symbiosis. We have identified, by a yeast two-hybrid system, a novel protein interacting with DMI3 named IPD3 (Interacting Protein of DMI3). IPD3 is predicted to interact with DMI3 through a C-terminal coiled-coil domain. Chimeric IPD3::GFP is localized to the nucleus of transformed Medicago truncatula root cells, in which split yellow fluorescent protein assays suggest that IPD3 and DMI3 physically interact in Nicotiana benthamiana. Like DMI3, IPD3 is extremely well conserved among the angiosperms and is absent from Arabidopsis. Despite this high level of conservation, none of the homologous proteins have a demonstrated biological or biochemical function. This work provides the first evidence of the involvement of IPD3 in a nuclear interaction with DMI3.     

Medicago truncatula Vapyrin is a novel protein required for arbuscular mycorrhizal symbiosis

Arbuscular mycorrhizal (AM) symbiosis is a widespread mutualism formed between vascular plants and fungi of the Glomeromycota. In this endosymbiosis, fungal hyphae enter the roots, growing through epidermal cells to the cortex where they establish differentiated hyphae called arbuscules in the cortical cells. Reprogramming of the plant epidermal and cortical cells occurs to enable intracellular growth of the fungal symbiont; however, the plant genes underlying this process are largely unknown. Here, through the use of RNAi, we demonstrate that the expression of a Medicago truncatula gene named Vapyrin is essential for arbuscule formation, and also for efficient epidermal penetration by AM fungi. Vapyrin is induced transiently in the epidermis coincident with hyphal penetration, and then in the cortex during arbuscule formation. The Vapyrin protein is cytoplasmic, and in cells containing AM fungal hyphae, the protein accumulates in small puncta that move through the cytoplasm. Vapyrin is a novel protein composed of two domains that mediate protein–protein interactions: an N‐terminal VAMP‐associated protein (VAP)/major sperm protein (MSP) domain and a C‐terminal ankyrin‐repeat domain. Putative Vapyrin orthologs exist widely in the plant kingdom, but not in Arabidopsis, or in non‐plant species. The data suggest a role for Vapyrin in cellular remodeling to support the intracellular development of fungal hyphae during AM symbiosis.     

Mycorrhiza of plants in different vegetation types in tropical ecosystems of Xishuangbanna,southwest China

We examined plants growing in four tropical vegetation types (primary forest, secondary forest, limestone forest and a slash and burn field) in Xishuangbanna, southwest China for mycorrhizal associations. Of the 103 plant species examined (belonging to 47 families), 81 had arbuscular mycorrhizal (AM) associations, while three species possessed orchid mycorrhiza. AM colonization levels ranged between 6% and 91% and spore numbers ranged between 1.36 spores and 25.71 spores per 10 g soil. Mean AM colonization level was higher in primary and secondary forest species than in plant species from limestone forests and a slash and burn field. In contrast, mean AM fungal spore numbers of the primary and limestone forest were lower than in the secondary forest or the slash and burn field. AM fungal spores belonging to Glomus and Acaulospora were the most frequent in soils of Xishuangbanna. AM fungal colonization and spore numbers were significantly correlated to each other and were significantly influenced by vegetation type.     

丛枝菌根共生体中碳、氮代谢及其相互关系

丛枝菌根共生体(arbuscular mycorrhiza, AM)是丛枝菌根真菌(arbuscular mycorrhizal fungi, AMF)与宿主植物之间形成的互惠共生形式.共生体中的碳、氮交换和代谢影响着宿主植物和共生真菌之间的营养平衡和资源重新分配,在物质和能量循环中发挥着重要作用.宿主植物光合固定的碳输送到真菌内,并且分解和释放真菌所需的生命物质和能量,包括促进孢子萌发、菌丝生长和提高氮等营养元素的吸收;而菌根真菌利用宿主植物提供的碳骨架和能量,发生氮的转化和运输,最终传递给宿主植物供其利用.本文综述了丛枝菌根共生体中碳、氮传输和代谢的主要模式,碳、氮的交互影响和调控机制,以促进丛枝菌根在可持续农业和生态系统中的应用.     

植物菌根共生磷酸盐转运蛋白

大多数植物能和丛枝菌根（arbuscular mycorrhiza, AM）真菌形成菌根共生体。AM能够促进植物对土壤中矿质营养的吸收,尤其是磷的吸收。磷的吸收和转运由磷酸盐转运蛋白介导。总结了植物AM磷酸盐转运蛋白及其结构特征,分析其分类及系统进化,并综述了AM磷酸盐转运蛋白介导的磷的吸收和转运过程及其基因的表达调控。植物AM磷酸盐转运蛋白属于Pht1家族成员,它不仅对磷的吸收和转运是必需的,而且对AM共生也至关重要,为进一步了解菌根形成的分子机理及信号转导途径提供了理论基础。     

Widely distributed native and alien plant species differ in arbuscular mycorrhizal associations and related functional trait interactions

It is debated whether alien plants in new environments benefit from being mycorrhizal and whether widely distributed natives and aliens differ in their associations with mycorrhizal fungi. Here, we compared whether species differing in their origin status, i.e. natives, archaeophytes (alien species introduced before the year 1500) and neophytes (introduced after the year 1500), and arbuscular mycorrhizal (AM) status (obligate, facultative, non‐mycorrhizal) differ in their area of occupancy in Germany (i.e. number of occupied grid cells, each ~130 km²). We used generalized linear models, incorporating main effects and up to three‐way interactions combining AM status, origin status and plant functional traits. The latter were chosen to describe the possible trade‐off in carbon allocation either towards the symbiosis or to other plant structures, such as storage organs (significant interactions involving traits were assumed to indicate the existence of such trade‐offs). AM status significantly explained the area of occupancy of natives and neophytes – with facultative mycorrhizal species occupying the largest area in both groups – but was less pronounced among archaeophytes. Archaeophytes may have reduced dependency on AM fungi, as they are generally agricultural weeds and the symbiosis potentially becomes obsolete for plants growing in habitats providing a steady provision of nutrients. Trait interactions between AM status and other functional traits were almost exclusively detected for neophytes. While facultative mycorrhizal neophytes benefit from trade‐offs with other traits related to high C cost in terms of area of occupancy, such trade‐offs were almost absent among natives. This indicates that natives and neophytes benefit differently from the symbiosis and suggests that native AM fungal partners might be less important for neophytic than for native plant species or that more time is required to establish similar relationships between neophytes and native fungal symbionts.     

Arbuscular mycorrhizal symbiosis on serpentine soils: the effect of native fungal communities on different Knautia arvensis ecotypes

Serpentine soils represent a unique environment that imposes multiple stresses on vegetation (low Ca/Mg ratios, macronutrient deficiencies, elevated heavy metal concentrations and drought). Under these conditions, a substantial role of arbuscular mycorrhizal (AM) symbiosis can be anticipated due to its importance for plant nutrition and stress alleviation. We tested whether serpentine and non-serpentine populations of Knautia arvensis (Dipsacaceae) differ in the benefits derived from native AM fungal communities. Four serpentine and four non-serpentine populations were characterised in terms of mycorrhizal colonisation and soil characteristics. The serpentine populations showed significantly lower mycorrhizal colonisation than their non-serpentine counterparts. The mycorrhizal colonisation positively correlated with soil pH, Ca and K concentrations and Ca/Mg ratio. Seedlings from each population were then grown for 3 months in their sterilised native substrates, either uninoculated or reinoculated with native AM fungi. Two serpentine and two non-serpentine populations responded positively to mycorrhizal inoculation, while no significant change in plant growth was observed in the remaining populations. Contrary to our hypothesis, serpentine populations of K. arvensis did not show higher mycorrhizal growth dependence than non-serpentine populations when grown in their native soils and inoculated with native AM fungi.