Carbon metabolism in spores of the arbuscular mycorrhizal fungus Glomus intraradices as revealed by nuclear magnetic resonance spectroscopy

Abstract

Arbuscular mycorrhizal (AM) fungi are obligate symbionts that colonize the roots of over 80% of plants in all terrestrial environments. Understanding why AM fungi do not complete their life cycle under free-living conditions has significant implications for the management of one of the world's most important symbioses. We used (13)C-labeled substrates and nuclear magnetic resonance spectroscopy to study carbon fluxes during spore germination and the metabolic pathways by which these fluxes occur in the AM fungus Glomus intraradices. Our results indicate that during asymbiotic growth: (a) sugars are made from stored lipids; (b) trehalose (but not lipid) is synthesized as well as degraded; (c) glucose and fructose, but not mannitol, can be taken up and utilized; (d) dark fixation of CO(2) is substantial; and (e) arginine and other amino acids are synthesized. The labeling patterns are consistent with significant carbon fluxes through gluconeogenesis, the glyoxylate cycle, the tricarboxylic acid cycle, glycolysis, non-photosynthetic one-carbon metabolism, the pentose phosphate pathway, and most or all of the urea cycle. We also report the presence of an unidentified betaine-like compound. Carbon metabolism during asymbiotic growth has features in between those presented by intraradical and extraradical hyphae in the symbiotic state.

Figure 1

¹³C-n.a. signals of trehalose (A) and an isopropyl alcohol extract of unlabeled AM fungal spores (B). Insets, Chemical structure of trehalose and of a triacylglyceride showing the correspondence between the different C positions and their chemical shift in the NMR spectra.

Figure 2

¹³C-NMR spectra of MeOH/H₂O extracts of asymbiotic fungal tissue following different treatments. A, Spores prelabeled but not germinated. Inset, ¹H spectrum of the same sample showing the ¹H resonance of the unidentified betaine-like compound and the upfield ¹³C-¹H satellite used for measuring the ¹³C content in this compound. B, Asymbiotic tissue prelabeled as in A and then germinated for 14 d without external label. C, Unlabeled spores germinated for 14 d in the presence of 25 mm ¹³C₁-Glc. Inset, ¹H spectrum of the same sample showing the ¹H resonance of trehalose and a ¹³C-¹H satellite used for measuring the ¹³C content in the C₁ position. D, Unlabeled spores germinated for 14 d in the presence of ¹³CO₂. E, Same as D, except germinated in the presence of 4 mm ¹³C₁-acetate. F, Same as E, except ¹³C₂-acetate. T₁ to T₆, Trehalose resonances (C₁–C₆); w, choline; v, GAB-betaine; n, unidentified signal.

Figure 3

¹³C-NMR spectra of isopropyl alcohol extracts. A, Spores prelabeled with ¹³C₁-Glc and germinated for 14 d in liquid M medium with no carbon. Inset, ¹H spectrum of the same sample showing the ¹H resonance of the ¹³Glyc_{1, 3} and the downfield ¹³C-¹H satellite used for measuring its ¹³C content. B, Unlabeled spores germinated for 14 d in M minus C liquid medium in the presence of ¹³C₁-Glc. Boxed inset, Spectrum expanded to better detect the signals of interest.

Figure 4

A simplified scheme of the AM fungal metabolic pathways revealed active in the present study. Labeled substrates provided in different experiments are shown in boxes, products detected are shown in capital letters, and certain metabolic intermediates or pools whose presence is inferred but were not detected are shown in italics. 1, Trehalose synthesis from Glc phosphate and UDP-Glc; 2, trehalose breakdown by trehalase; 3, the PPP (also known as the hexose monophosphate pathway); 4, gluconeogenesis, starting with PEP and involving reversal of glycolysis with several differences; 5, glycolysis (the Embden-Meyerhoff-Parnas pathway); 6, non-photosynthetic one-carbon metabolism, typically involving tetrahydrofolate (THF) and S-adenosyl Met as carriers of the methyl groups; 7, lipolysis: storage lipid (tryacylglycerides) breakdown to glycerol and fatty acids, and subsequent glyoxysomal fatty acid β-oxidation to acetyl CoA; 8, Dark fixation of CO₂ by pyruvate carboxylase to oxalocetate (8a) or carbamoyl P synthethase (8d); 9, glyoxylate cycle (or shunt) involving the production of glyoxylate from acetyl-CoA units via part of the TCA cycle; the glyoxylate is condensed with acetyl-CoA (from triacylglyceride degradation) to form triose and CO₂; 10, TCA (also known as the Krebs cycle); 11, Arg synthesis by enzymes of the urea cycle including the incorporation of carbon from carbamoyl P.