The glyoxylate cycle in an arbuscular mycorrhizal fungus. Carbon flux and gene expression

Abstract

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.

Figure 1

¹³C NMR spectra of extracts of germinating spores (A and C) and extraradical mycelium (B and D) of Glomus intraradices after incubation with ¹³C-labeled substrates. ¹³C₂ glycerol (A and B) or ¹³C₂ acetate (C and D) was used. The signals from the six different positions of trehalose are labeled T1 through T6. Splitting of signals in C and D are due to the spectroscopic coupling in multiply labeled molecules. Insets in A and C are subsections of the ¹H spectra of the same samples. These insets show the ¹H signals from the anomeric (C1 and C1′) hydrogens of trehalose, including the ¹H-¹³C satellite peaks whose areas relative to the central ¹H-¹²C signals give the absolute percentage of ¹³C levels in trehalose.

Figure 2

Multiple alignments of known ICL amino acid sequences from several fungi that were used for designing PCR primers and for comparison with the deduced full-length sequence for ICL from G. intraradices. Sequences are shown for: Coprinus cinereus, Eremothecium gossypii, Emericella nidulans, Neurospora crassa, Saccharomyces cerevisiae, and G. intraradices. The degree of homology among the different sequences at each residue is indicated as complete conservation (*), high homology/conserved substitutions (:), moderate conservation (.), or little or no homology (unmarked). Residues shown in bold and underlined are the motifs RRGT and KKFT that contain Tyr phosphorylation sites in S. cerevisiae, shown in bold are the C-terminal tripeptide glyoxysomal-targeting sequences Ser-Lys-Leu and Ala-Lys-Leu in two of the fungal sequences, and another glyoxysomal targeting sequence RDFIAQEQA in the G. intraradices sequence. Also underlined in bold is the decapeptide sequence KTKRNYSARD region that has been shown in S. cerevisiae to be involved in Glc-induced enzyme deactivation.

Figure 3

Multiple alignments of known MS amino acid sequences from several fungi that were used for designing PCR primers and for comparison with the deduced full-length sequence for MS from G. intraradices. Sequences are shown for Hansenula polymorpha, Candida tropicalis, E. nidulans, N. crassa, S. cerevisiae, and G. intraradices. The degree of homology among the different sequences at each residue is indicated as complete conservation (*), high homology/conserved substitutions (:), moderate conservation (.), or little or no homology (unmarked). Shown in bold are the C-terminal tripeptide glyoxysomal-targeting sequences Ser-Lys-Leu and Ala-Lys-Leu that are present in G. intraradices and other fungal sequences.

Figure 4

Electron micrographs of G. intraradices extraradical hyphae grown in AM monoxenic cultures. Black solid arrows indicate glyoxysome-like structures, which usually contained electron-dense cores. a, Section near the apex of a branched absorbing structure (BAS). b, Transverse section at the BAS trunk level. c, Longitudinal section of a runner hypha showing a large number of glyoxysome-like organelles. Gly, Glycogen deposits; V, vacuole; M, mitochondrion; N, nucleus. Scale bars = 500 nm in a and b and 1 μm in c.