Berta Bago

Nitrate depletion and pH changes induced by the extraradical mycelium of the arbuscular mycorrhizal fungus Glomus intraradices grown in monoxenic culture

The effect of the extraradical mycelium of the arbuscular mycorrhizal (AM) fungus Glomus intraradices Smith & Schenck on nitrate uptake and on the pH of the medium was studied in a monoxenic culture with tomato (Lycopersicon esculentum Mill. var. Vendor) roots obtained from root organ culture. The symbiosis was established in compartmented Petri dishes containing agar media amended with the pH indicator bromocresol purple. A pattern of pH changes was revealed as the symbiosis progressed in the media of the Petri dish compartments containing the dual, arbuscular-mycorrhizal fungi/root, culture as well as in the media of the hyphae, root-free compartments, in which the extraradical hyphae developed extensively, coming from the compartment containing the symbiosis. The colour changes in the media were measured spectrophotometrically, whilst maintaining the monoxenic conditions. The extraradical hyphae of G. intraradices strongly increased the pH of nutrient-free medium when supplied with nitrate, whereas the pH decreased m the absence of this N source. The hyphae developing from germinated spores and growing in axenic, nitrate-amended media did not induce any increase in pH. Nitrogen analysis revealed that a depletion of nitrate in the media accompanied increased pH. These results point towards an active uptake of nitrate by the extraradical mycelium of G. intraradices, probably coupled to a H+ -symport mechanism. The pH changes induced by AM fungal hyphae and the possible influence of the establishment of a functional symbiosis on these pH changes are discussed.

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 13C1 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 13C-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.

Architecture and developmental dynamics of the external mycelium of the arbuscular mycorrhizal fungus Glomus intraradices grown under monoxenic conditions

The structural development of arbuscular mycorrhiza extraradical mycelium is difficult to fol- low in soil-based systems. The use of dual arbuscular mycorrhizal fungi/in vitro root organ cultures (mon- oxenic AM cultures) allowed the nondestructive study of hyphal development following establishment of the symbiosis. The present study shows that the extraradical spreading of the arbuscular mycorrhizal fungus Glomus intraradices grown monoxenically with tomato roots can be divided into three stages: (i) pro- liferation of runner hyphae acting as conducting channels, which divide dichotomously and extend the fungal colony radially; (ii) development of arbus- cule-like structures, which are formed at regular in- tervals along the runner hyphae and which might play a preferential role in nutrient uptake; and (iii) formation of spores in zones already colonized by runner hyphae and arbuscule-like structures. The de- velopment of the mycorrhiza is accompanied by changes in the pH of the medium. In particular, pH decreases in zones of the medium in which a high number of arbuscular mycorrhizal fungal spores are formed. The intricate architecture shown by the ex- traradical mycelium highlights the potential for en- hanced nutrient uptake by mycorrhizal roots, and their role in the maintainance and amelioration of soil structure.

FLAVONOIDS AND ARBUSCULAR-MYCORRHIZAL FUNGI

Arbuscular mycorrhizal fungi (AMF) are ancient Zygomycetes forming the most widespread plant-fungus symbiosis. The regulation of this association is still poorly understood in terms of the communication between the two partners. Compounds inside the root and released by the root, such as flavonoids, are hypothesized to play a role in this plant-fungus communication, as already demonstrated in other symbiotic associations (e.g. Rhizobium-leguminoseae). Here we give a general overview of the research concerning this question.

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.

Tracking metabolism and imaging transport in arbuscular mycorrhizal fungi

In the last few years the application of modern techniques to the study of arbuscular mycorrhizas has greatly increased our understanding of the mechanisms underlying carbon metabolism in these mutualistic symbioses. Arbuscular mycorrhizal (AM) monoxenic cultures, nuclear magnetic resonance spectroscopy together with isotopic labeling, and analyses of expressed sequence tags (ESTs) have shed light on the metabolic processes taking place in these interactions, particularly in the case of the mycobiont. More recently, in vivo multiphoton microscopy has provided us with some new insights in the allocation and translocation processes which play crucial roles in the distribution of host plant-derived C throughout the fungal colony. In this mini-review we highlight recent advances in these fields, with special attention to the visualization of oleosomes (i.e., lipid bodies) as they move along the long, coenocytic AM fungal hyphae. Volumetric measurements of such oleosomes have allowed us to estimate the flux of triacylglycerides from the intraradical to the extraradical phase of the AM fungal colony. We raise questions and postulate regulatory mechanisms for C metabolism and translocation within the arbuscular mycorrhizal fungal colony.

Tracking metabolism and imaging transport in arbuscular mycorrhizal fungi. Metabolism and transport in AM fungi

In the last few years the application of modern techniques to the study of arbuscular mycorrhizas has greatly increased our understanding of the mechanisms underlying carbon metabolism in these mutualistic symbioses. Arbuscular mycorrhizal (AM) monoxenic cultures, nuclear magnetic resonance spectroscopy together with isotopic labeling, and analyses of expressed sequence tags (ESTs) have shed light on the metabolic processes taking place in these interactions, particularly in the case of the mycobiont. More recently, in vivo multiphoton microscopy has provided us with some new insights in the allocation and translocation processes which play crucial roles in the distribution of host plant-derived C throughout the fungal colony. In this mini-review we highlight recent advances in these fields, with special attention to the visualization of oleosomes (i.e., lipid bodies) as they move along the long, coenocytic AM fungal hyphae. Volumetric measurements of such oleosomes have allowed us to estimate the flux of triacylglycerides from the intraradical to the extraradical phase of the AM fungal colony. We raise questions and postulate regulatory mechanisms for C metabolism and translocation within the arbuscular mycorrhizal fungal colony.

In vivo studies on the nuclear behavior of the arbuscular mycorrhizal fungusGigaspora rosea grown under axenic conditions

SummaryThe distribution and fate of nuclei of the arbuscular-my-corrhizal fungusGigaspora rosea during late stages of axenic cultures were studied in fixed cultures by transmitted light, conventional and confocal laser scanning microscopy, and in live cultures with two-photon fluorescence microscopy. Mature specimens not yet showing apical septation displayed oval-shaped nuclei localized in lateral positions of the hypha all along the germ-tube length. Beside these, round-shaped nuclei were found to migrate along the central germ-tube core. Some (rare) germ-tube areas, delimited by septa and containing irregularly shaped, much brighter fluorescent nuclei were also found. Specimens that had just initiated the septation process after germ-tube growth arrest displayed round or oval-shaped nuclei in several portions of the germ tubes. These hyphal areas often alternated with other septa-delimited cytoplasmic clusters which contained distorted, brightly fluorescent nuclei. Completely septated specimens mostly lacked nuclei along their germ tubes. However, highly fluorescent chromatin masses appeared within remnants of cytoplasmic material, often compressed between close septa. Our results provide a first clear picture of the in vivo distribution of nuclei along arbuscular mycorrhizal fungal germ tubes issued from resting spores, and suggest that selective areas of their coenocytic hyphae are under specific, single nuclear control. They indicate as well that random autolytic processes occur along senescingG. rosea germ tubes, probably as a consequence of the absence of a host root signal for mycorrhizal formation. Finally, the data presented here allow us to envisage the fate of nuclei released by the germinating spore after nonsymbiotic fungal growth arrest.

Putative sites for nutrient uptake in arbuscular mycorrhizal fungi

Nutrition of the arbuscular mycorrhiza (AM) is addressed from a fungal point of view. Intraradical and extraradical structures proposed as preferential sites for nutrient acquisition in arbuscular mycorrhizal (AM) fungi are considered, and their main features compared. This comparison includes the formation and function of branched structures (either intra- or extraradical) as putative nutrient uptake sites with unique morphological and physiological features in the AM fungal colony. The morphology and functioning of these structures are further affected by intra- or extraradical environmental factors. A model is presented which portrays the intrinsic developmental and physiological duality of the AM fungus.

Nuclei of symbiotic arbuscular mycorrhizal fungi as revealed by in vivo two-photon microscopy.

SummaryThe present work reports the results obtained from in vivo studies on the distribution and behavior of nuclei of two arbuscular mycorrhizal (AM) fungi growing in symbiosis with tomato root organ cultures (AM monoxenic cultures). Upon staining with 4′,6-diamidino-2-phenylindole and two-photon microscopy (2PM) observations, symbiotic thick runner hyphae appeared mostly opaque to 2PM and did not reveal nuclei within them; thin runner hyphae showed dimly stained nuclei along them, whereas nuclei were clearly visible within the branches of the so-called branched absorbing structures. When visible, nuclei appeared anchored laterally at regular intervals along the symbiotic AM extraradical hyphae. Other nuclei migrate through the hyphal central core; this migration occurs in pulses. Simultaneous observations on different areas of extraradical AM mycelium revealed the existence of lysed compartments along the fungal hyphae, containing nuclei remnants and/or chromatin masses. All these results give new insights in (i) the differential permeability of AM hyphae in the symbiotic versus the asymbiotic state; (ii) the behavior and distribution of nuclei along the symbiotic extraradical mycelium; (iii) the occurrence of ageing events within the AM fungal colony; and (iv) the existence of “healing” mechanisms aiming to restrict the damage induced by such ageing or lytic events. An AM fungal strategy for hyphal survival under adverse conditions is also suggested.