Arbuscular mycorrhizal symbiosis regulates plasma membrane H+-ATPase gene expression in tomato plants

Regulation by arbuscular mycorrhizal symbiosis of three tomato plasma membrane H+-ATPase genes (LHA1, LHA2 and LHA4) has been analysed in wild-type and mycorrhiza-defective tomato plants. Expression of these genes was differentially regulated in leaves and roots of both tomato phenotypes after inoculation with Glomus mosseae.     

Mechanisms of nutrient transport across interfaces in arbuscular mycorrhizas

Bidirectional nutrient transfer between the plant and the fungus is a key feature of arbuscular mycorrhizal symbiosis. The major nutrients exchanged between the symbiotic partners are reduced carbon, assimilated through the plant photosynthesis and phosphate, taken up by the fungal hyphae exploring soil microhabitats. This nutrient exchange takes place across the symbiotic interfaces which are bordered by the plant and fungal plasma membranes. This review provides an overview of the current knowledge of the mechanisms underlying nutrient transport processes in the symbiosis, with special emphasis on recent developments in the molecular biology of the plant and fungal primary (H⁺-ATPases) and secondary transporters.     

Cloning, sequencing and functional expression in Escherichia coli of the gene for a P-type Na(+)-ATPase of a facultatively anaerobic alkaliphile, Exiguobacterium aurantiacum

Cloning and sequencing of the gene encoding a P-type Na(+)-ATPase of a facultatively anaerobic alkaliphile, Exiguobacterium aurantiacum, were conducted. The structural gene was composed of 2628 nucleotides. The deduced amino acid sequence (876 amino acid residues; Mr, 96,664) suggested that the enzyme possesses 10 membrane-spanning regions. When the amino acid sequences of the four putative membrane regions, M4, M5, M6 and M8, of BL77/1 ATPase were aligned with those of fungal Na(+)-ATPase, Na(+)/K(+)-ATPase, H(+)-ATPases and sarcoplasmic reticulum Ca(2+)-ATPase, it exhibited the highest homology with Ca(2+)-ATPase except M5 region. By the transformation of Escherichia coli with the expression vector (pQE30) containing the ATPase gene, the enzyme was functionally expressed in E. coli membranes.     

The plant plasma membrane H(+)-ATPase: structure, function and regulation

The proton-pumping ATPase (H(+)-ATPase) of the plant plasma membrane generates the proton motive force across the plasma membrane that is necessary to activate most of the ion and metabolite transport. In recent years, important progress has been made concerning the identification and organization of H(+)-ATPase genes, their expression, and also the kinetics and regulation of individual H(+)-ATPase isoforms. At the gene level, it is now clear that H(+)-ATPase is encoded by a family of approximately 10 genes. Expression, monitored by in situ techniques, has revealed a specific distribution pattern for each gene; however, this seems to differ between species. In the near future, we can expect regulatory aspects of gene expression to be elucidated. Already the expression of individual plant H(+)-ATPases in yeast has shown them to have distinct enzymatic properties. It has also allowed regulatory aspects of this enzyme to be studied through random and site-directed mutagenesis, notably its carboxy-terminal region. Studies performed with both plant and yeast material have converged towards deciphering the way phosphorylation and binding of regulatory 14-3-3 proteins intervene in the modification of H(+)-ATPase activity. The production of high quantities of individual functional H(+)-ATPases in yeast constitutes an important step towards crystallization studies to derive structural information. Understanding the specific roles of H(+)-ATPase isoforms in whole plant physiology is another challenge that has been approached recently through the phenotypic analysis of the first transgenic plants in which the expression of single H(+)-ATPases has been up- or down-regulated. In conclusion, the progress made recently concerning the H(+)-ATPase family, at both the gene and protein level, has come to a point where we can now expect a more integrated investigation of the expression, function and regulation of individual H(+)-ATPases in the whole plant context.     

Chemotropism in the arbuscular mycorrhizal fungus Glomus mosseae

In this work, we report the occurrence of chemotropism in the arbuscular mycorrhizal (AM) fungus Glomus mosseae. Fungal hyphae were able to respond to host-derived signals by reorienting their growth towards roots and to perceive chemotropic signals at a distance of at least 910 microm from roots. In order to reach the source of chemotropic signals, hyphal tips crossed interposed membranes emerging within 1 mm from roots, eventually establishing mycorrhizal symbiosis. The specificity of chemotropic growth was evidenced by hyphal growth reorientation and membrane penetration occurring only in experimental systems set up with host plants. Since pre-symbiotic growth is a critical stage in the life cycle of obligate AM fungal symbionts, chemotropic guidance may represent an important mechanism functional to host root location, appressorium formation and symbiosis establishment.     

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.     

Novel genes induced during an arbuscular mycorrhizal (AM) symbiosis formed between Medicago truncatula and Glomus versiforme

Many terrestrial plant species are able to form symbiotic associations with arbuscular mycorrhizal fungi. Here we have identified three cDNA clones representing genes whose expression is induced during the arbuscular mycorrhizal symbiosis formed between Medicago truncatula and an arbuscular mycorrhizal fungus, Glomus versiforme. The three clones represent M. truncatula genes and encode novel proteins: a xyloglucan endotransglycosylase-related protein, a putative arabinogalactan protein (AGP), and a putative homologue of the mammalian p110 subunit of initiation factor 3 (eIF3). These genes show little or no expression in M. truncatula roots prior to formation of the symbiosis and are significantly induced following colonization by G. versiforme. The genes are not induced in roots in response to increases in phosphate. This suggests that induction of expression during the symbiosis is due to the interaction with the fungus and is not a secondary effect of improved phosphate nutrition. In situ hybridization revealed that the putative AGP is expressed specifically in cortical cells containing arbuscules. The identification of two mycorrhiza-induced genes encoding proteins predicted to be involved in cell wall structure is consistent with previous electron microscopy data that indicated major alterations in the extracellular matrix of the cortical cells following colonization by mycorrhizal fungi.     

Is a fully established arbuscular mycorrhizal symbiosis required for a bioprotection of Pisum sativum roots against Aphanomyces euteiches?

Bioprotection of pea roots against Aphanomyces euteiches by the arbuscular mycorrhizal fungus G. mosseae was demonstrated to depend on a fully established symbiosis. This was related with induction of mycorrrhiza-related chitinolytic enzymes. Possible mechanisms implicated in bioprotection are discussed.     

Soluble and membrane symbiosis-related polypeptides associated with the development of arbuscular mycorrhizas in tomato (Lycopersicon esculentum)

To analyse 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 analysed, 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 that are differentially displayed in tomato roots as a consequence of the establishment of the AM symbiosis.     

Molecular approaches to study plasma membrane H+-ATPases in arbuscular mycorrhizas

The activity of H⁺-ATPases of plant and fungi generates an electrochemical gradient of H⁺ across the cell plasma membrane that drives a number of secondary transport systems, including those responsible for the translocation of cations, anions, amino acids and sugars. During the last years, several studies have been aimed at elucidating the role of plasma membrane H⁺-ATPases in the nutrient exchange processes taking place between the plant and the fungus in arbuscular mycorrhizal (AM) symbiosis. This paper reviews present knowledge about plasma membrane H⁺-ATPases and experimental evidence supporting the involvement of H⁺-ATPases of both organisms in the bidirectional transport of nutrients between partners. Molecular strategies that will provide further information on the function and regulation of plasma membrane H⁺-ATPases in AM symbiosis are presented and discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Further root colonization by arbuscular mycorrhizal fungi in already mycorrhizal plants is suppressed after a critical level of root colonization

An established arbuscular mycorrhizal symbiosis suppresses further mycorrhization. It is not clear whether the observed suppressional effect is linked with the level of root colonization or not. In the present work we studied the effect of the degree of root colonization by the arbuscular mycorrhizal fungus Glomus mosseae on further root colonization by G. mosseae. At different time points barley plants grown in split-root compartments were pre-inoculated on one half of the split-root system with G. mosseae. Sequential inoculation resulted in different colonization levels. Thereafter, the second half of the split root system was inoculated. The results indicate an enhanced suppression of root colonization on the second side of the split-root system when colonization levels increased on the first side.     

Impact of temperature on the arbuscular mycorrhizal (AM) symbiosis: growth responses of the host plant and its AM fungal partner

The growth response of the hyphae of mycorrhizal fungi has been determined, both when plant and fungus together and when only the fungus was exposed to a temperature change. Two host plant species, Plantago lanceolata and Holcus lanatus, were grown separately in pots inoculated with the mycorrhizal fungus Glomus mosseae at 20/18 degrees C (day/night); half of the pots were then transferred to 12/10 degrees C. Plant and fungal growth were determined at six sequential destructive harvests. A second experiment investigated the direct effect of temperature on the length of the extra-radical mycelium (ERM) of three mycorrhizal fungal species. Growth boxes were divided in two equal compartments by a 20 micro m mesh, allowing only the ERM and not roots to grow into a fungal compartment, which was either heated (+8 degrees C) or kept at ambient temperature. ERM length (LERM) was determined on five sampling dates. Growth of H. lanatus was little affected by temperature, whereas growth of P. lanceolata increased with temperature, and both specific leaf area (SLA) and specific root length (SRL) increased independently of plant size. Percentage of colonized root (LRC) and LERM were positively correlated with temperature when in symbiosis with P. lanceolata, but differences in LRC were a function of plant biomass. Colonization was very low in H. lanatus roots and there was no significant temperature effect. In the fungal compartment LERM increased over time and was greatest for Glomus mosseae. Heating the fungal compartment significantly increased LERM in two of the three species but did not affect LRC. However, it significantly increased SRL of roots in the plant compartment, suggesting that the fungus plays a regulatory role in the growth dynamics of the symbiosis. These temperature responses have implications for modelling carbon dynamics under global climate change.     

Proton (H+) flux signature for the presymbiotic development of the arbuscular mycorrhizal fungi

Ion dynamics are important for cell nutrition and growth in fungi and plants. Here, the focus is on the relationship between the hyphal H(+) fluxes and the control of presymbiotic growth and host recognition by arbuscular mycorrhizal (AM) fungi. Fluxes of H(+) around azygopores and along lateral hyphae of Gigaspora margarita during presymbiotic growth, and their regulation by phosphate (P) and sucrose (Suc), were analyzed with an H(+)-specific vibrating probe. Changes in hyphal H(+) fluxes were followed after induction by root exudates (RE) or by the presence Trifolium repens roots. Differential sensitivity to P-type ATPase inhibitors (orthovanadate or erythrosin B) suggests an asymmetric distribution or activation of H(+)-pump isoforms along the hyphae of the AM fungi. Concentration of P and Suc affected the hyphal H(+) fluxes and growth rate. However, further increases in H+ efflux and growth rate were observed when the fungus was growing close to clover roots or pretreated with RE. The H(+) flux data correlate with those from polarized hyphal growth analyses, suggesting that spatial and temporal alterations of the hyphal H(+)fluxes are regulated by nutrient availability and might underlie a pH signaling elicitation by host RE during the early events of the AM symbiosis.     

Flavonoid levels in roots ofMedicago sativa are modulated by the developmental stage of the symbiosis and the root colonizing arbuscular mycorrhizal fungus

Abundant data on the effect of flavonoids on spore germination, hyphal growth and root colonization by AMF are available. Moreover, the flavonoid pattern in mycorrhizal roots changes, thus flavonoids have been suggested as arbuscular mycorrhizal signalling compounds. In our work we studied the accumulation of flavonoids in roots of Medicago sativa i) after the exposure of uncolonized roots to sterile solutions containing Glomus intraradices tissue, ii) at three different stages of colonization by G. mosseae, iii) colonized by G. mosseae, G. intraradices or Gigaspora rosea.

We could show that flavonoid accumulation in M. sativa roots i) is induced before root colonization, pointing towards the presence of a fungal-derived signal, ii) depends on the developmental stage of the symbiosis and iii) depends on the root-colonizing arbuscular mycorrhizal fungus. The data presented indicate not only a time-specificity of the flavonoid accumulation during the mycorrhizal association, but also an arbuscular mycorrhizal fungal-specificity. The possible functions of the flavonoid pattern changes are discussed.     

Fungal genes related to calcium homeostasis and signalling are upregulated in symbiotic arbuscular mycorrhiza interactions

Fluctuations in intracellular calcium levels generate signalling events and regulate different cellular processes. Whilst the implication of Ca²⁺ in plant responses during arbuscular mycorrhiza (AM) interactions is well documented, nothing is known about the regulation or role of this secondary messenger in the fungal symbiont. The spatio-temporal expression pattern of putatively Ca²⁺-related genes of Glomus intraradices BEG141 encoding five proteins involved in membrane transport and one nuclear protein kinase, was investigated during the AM symbiosis. Expression profiles related to successful colonization of host roots were observed in interactions of G. intraradices with roots of wild-type Medicago truncatula (line J5) compared to the mycorrhiza-defective mutant dmi3/Mtsym13. Symbiotic fungal activity was monitored using stearoyl-CoA desaturase and phosphate transporter genes. Laser microdissection based-mapping of fungal gene expression in mycorrhizal root tissues indicated that the Ca²⁺-related genes were differentially upregulated in arbuscules and/or in intercellular hyphae. The spatio-temporal variations in gene expression suggest that the encoded proteins may have different functions in fungal development or function during symbiosis development. Full-length cDNA obtained for two genes with interesting expression profiles confirmed a close similarity with an endoplasmic reticulum P-type ATPase and a Vcx1-like vacuolar Ca²⁺ ion transporter functionally characterized in other fungi and involved in the regulation of cell calcium pools. Possible mechanisms are discussed in which Ca²⁺-related proteins G. intraradices BEG141 may play a role in mobilization and perception of the intracellular messenger by the AM fungus during symbiotic interactions with host roots.