Philip E. Pfeffer

Nitrogen transfer in the arbuscular mycorrhizal symbiosis

Most land plants are symbiotic with arbuscular mycorrhizal fungi (AMF), which take up mineral nutrients from the soil and exchange them with plants for photosynthetically fixed carbon. This exchange is a significant factor in global nutrient cycles as well as in the ecology, evolution and physiology of plants. Despite its importance as a nutrient, very little is known about how AMF take up nitrogen and transfer it to their host plants. Here we report the results of stable isotope labelling experiments showing that inorganic nitrogen taken up by the fungus outside the roots is incorporated into amino acids, translocated from the extraradical to the intraradical mycelium as arginine, but transferred to the plant without carbon. Consistent with this mechanism, the genes of primary nitrogen assimilation are preferentially expressed in the extraradical tissues, whereas genes associated with arginine breakdown are more highly expressed in the intraradical mycelium. Strong changes in the expression of these genes in response to nitrogen availability and form also support the operation of this novel metabolic pathway in the arbuscular mycorrhizal symbiosis.

Partitioning of Intermediary Carbon Metabolism in Vesicular-Arbuscular Mycorrhizal Leek.

Vesicular-arbuscular mycorrhizal fungi are symbionts for a large variety of crop plants; however, the form in which they take up carbon from the host is not established. To trace the course of carbon metabolism, we have used nuclear magnetic resonance spectroscopy with [13C]glucose labeling in vivo and in extracts to examine leek (Allium porrum) roots colonized by Glomus etunicatum (and uncolonized controls) as well as germinating spores. These studies implicate glucose as a likely substrate for vesicular-arbuscular mycorrhizal fungi in the symbiotic state. Root feeding of 0.6 mM 1-[13C]glucose labeled only the fungal metabolites trehalose and glycogen. The time course of this labeling was dependent on the status of the host. Incubation with 50 mM 1-[13C]glucose caused labeling of sucrose (in addition to fungal metabolites) with twice as much labeling in uncolonized plants. There was no detectable scrambling of the label from C1 glucose to the C6 position of glucose moieties in trehalose or glycogen. Labeling of mannitol C1,6 in the colonized root tissue was much less than in axenically germinating spores. Thus, carbohydrate metabolism of host and fungus are significantly altered in the symbiotic state.

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.

Fungal nutrient allocation in common mycorrhizal networks is regulated by the carbon source strength of individual host plants

Common mycorrhizal networks (CMNs) of arbuscular mycorrhizal (AM) fungi in the soil simultaneously provide multiple host plants with nutrients, but the mechanisms by which the nutrient transport to individual host plants within one CMN is controlled are unknown. Using radioactive and stable isotopes, we followed the transport of phosphorus (P) and nitrogen (N) in the CMNs of two fungal species to plants that differed in their carbon (C) source strength, and correlated the transport to the expression of mycorrhiza-inducible plant P (MtPt4) and ammonium (1723.m00046) transporters in mycorrhizal roots. AM fungi discriminated between host plants that shared a CMN and preferentially allocated nutrients to high-quality (nonshaded) hosts. However, the fungus also supplied low-quality (shaded) hosts with nutrients and maintained a high colonization rate in these plants. Fungal P transport was correlated to the expression of MtPt4. The expression of the putative ammonium transporter 1723.m00046 was dependent on the fungal nutrient supply and was induced when the CMN had access to N. Biological market theory has emerged as a tool with which the strategic investment of competing partners in trading networks can be studied. Our work demonstrates how fungal partners are able to retain bargaining power, despite being obligately dependent on their hosts.

Carbon Partitioning, Cost, and Metabolism of Arbuscular Mycorrhizas

Colonization of roots by arbuscular mycorrhizal [AM] fungi results in many changes in the carbon partitioning and metabolism of the host plant. The rate of carbon assimilation, the export of photosynthates from leaves, and the sink strength of roots may be increased relative to that in uncolonized plants. Hexose is taken up by the obligately symbiotic fungus for its growth, maintenance, and reproduction. This can represent a significant cost to the host, most notably under conditions in which the fungus offers little nutritive benefit. The components of the carbon partitioning and cost of arbuscular mycorrhizas, as well as current knowledge of the carbon metabolism of germinating spores and infra- and extraradical hyphae of AM fungi are reviewed in this chapter.

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.

Structural studies of a phosphocholine substituted β-(1,3); (1,6) macrocyclic glucan from Bradyrhizobium japonicum USDA 110

In our previous in vivo 31P study of intact nitrogen-fixing nodules (Rolin, D.B., Boswell, R.T., Sloger, C., Tu, S.I. and Pfeffer, P.E., 1989 Plant Physiol. 89, 1238-1246), we observed an unknown phosphodiester. The compound was also observed in the spectra of isolated bacteroids as well as extracts of the colonizing Bradyrhizobium japonicum USDA 110. In order to characterize the phosphodiester in the present study, we took advantage of the relatively hydrophobic nature of the material and purified it by elution from a C-18 silica reverse-phase chromatography column followed by final separation on an aminopropyl silica HPLC column. Structural characterization of this compound with a molecular weight of 2271 (FAB mass spectrometry), using 13C-1H and 31P-1H heteronuclear 2D COSY and double quantum 2D phase sensitive homonuclear 1H COSY NMR spectra, demonstrated that the molecule contained beta-(1,3); beta-(1,6); beta-(1,3,6) and beta-linked non-reducing terminal glucose units in the ratio of 5:6:1:1, respectively, as well as one C-6 substituted phosphocholine (PC) moiety associated with one group of (1,3) beta-glucose residues. Carbohydrate degradation analysis indicated that this material was a macrocyclic glucan, (absence of a reducing end group) with two separated units containing three consecutively linked beta-(1,3) glucose residues and 6 beta-(1,6) glucose residues. The sequences of beta-(1,3)-linked glucose units contained a single non-reducing, terminal, unsubstituted glucose linked at the C-6 position and a PC group attached primarily to an unsubstituted C-6 position of a beta-(1,3)-linked glucose.

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.

Complex formation in sonicated mixtures of β-lactoglobulin and phosphatidylcholine

Abstractβ-Lactoglobulin, the major whey protein of bovine milk, is secreted via the endomembrane system of the mammary gland. The primary structure of β-lactoglobulin shares certain characteristics with membrane proteins, although the soluble protein assumes a globular conformation. We have prepared complexes of β-lactoglobulin and phosphatidylcholines by dissolving both in a helix-forming solvent (chloroform methanol). The complex is stable when transferred to aqueous solutions and sonicated to form vesicles. Both ionic and hydrophobic interactions appear to be involved in complex formation. We have used spectroscopy (circular dichroism, fluorescence, and nuclear magnetic resonance) and electron microscopy to study these complexes. At pH 3.7, the small, single bilayer vesicles produced by sonication are protected against aggregation by the presence of the protein. As determined by circular dichroism, the proportion of α-helix in β-lactoglobulin is increased by complexation with phosphatidylcholine. Circular dichroism and fluorescence spectra show the involvement of at least 1 tryptophan residue in the conformational change. At pH 7.2, β-lactoglobulin-phosphatidylcholine vesicles form aggregates as observed by electron microscopy and31P nuclear magnetic resonance spectroscopy. These aggregated vesicles could be resuspended by raising the pH. The ability of the partially unfolded β-lactoglobulin to interact with lipids is believed to be important to its transport through the endomembrane system.