Carbon export from arbuscular mycorrhizal roots involves the translocation of carbohydrate as well as lipid

Abstract

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 (13)C(1) 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 (13)C-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.

Figure 1

13C NMR spectra of trehalose (A), Glc from glycogen (B), and NAG from chitin (C). The carbohydrates were obtained by sequential extraction and hydrolysis from ERM that was harvested 2 months after providing ¹³C₁ Glc to the mycorrhizal roots. Spectroscopic conditions (see “Materials and Methods”) were such that the different intensities of the signals from different carbons accurately reflect the different labeling levels in those positions of the carbohydrates. The C1 and C6 signals are indicated for each compound. Greek symbols refer to the two anomeric forms in which Glc and NAG exist in solution.

Figure 2

The relative labeling in the C1 and C6 positions of carbohydrates from the ERM as a function of time after providing ¹³C₁ Glc to the mycorrhizal roots. Data are derived from the intensities of C1 and C6 signals in spectra such as those shown in Figure 1. Error bars for are ± sem for at least three replicates per experiment.

Figure 3

Multiple alignments of glycogen synthase enzymes. The organisms are G. intraradices, Neurospora crassa, Saccharomyces cerevisiae, and human (Homo sapiens). Conservation of phosphorylation sites at the C terminus are shown in bold: Ser(650), Ser(654), and Thr(667) (numbering is according to the S. cerevisiae protein). Thr 667 is conserved between S. cerevisiae and N. crassa, but substituted by Ser in G. intraradices and human. The region defining the Glc-6-P allosteric binding site known in yeast (Pedersen et al., 2000) is also highlighted.

Figure 4

Multiple alignments of BE sequences. The organisms are G. intraradices, A. oryzae, Emericella nidulans, N. crassa, and humans. Two Arg residues and a Tyr in the human protein (Y329, R515, and R524) whose mutation is associated with glycogen storage disease (Ziemssen et al., 2000; Lossos et al., 1998) are also highlighted.

Figure 5

Illustration of the labeling patterns in the hexose units of carbohydrates in the ERM that result from the activity of different pathways after providing ¹³C₁ Glc to the mycorrhizal roots. A, The direct export of carbohydrate from the IRM; Glc uptake in the IRM results in labeling in C1 of carbohydrate in the IRM that is then exported to the ERM. B, Hexose triose cycling: Hexose initially labeled in C1 is broken down to dihydroxyacetone phosphate labeled in the C3 position, this then labels the C3 of glyceraldehyde-3-P through the action of triose phosphate isomerase, subsequently, gluconeogenic flux results in the labeling of C6 of carbohydrates. C, Synthesis of carbohydrates from lipids made in the IRM: The fatty acids (mainly C16) of lipid made in the IRM from ¹³C₁ Glc are labeled in the even-numbered positions, export of this lipid to the ERM followed by β-oxidation produces acetyl-CoA units labeled in C2, the glyoxylate cycle produces malate labeled in C2 and C3, which after decarboxylation labels triose in C2 and C3, this triose labels carbohydrates in C1, C2, C5, and C6. D, The oxidative pentose phosphate pathway operating in cyclic mode: Three hexose molecules are converted to two hexose molecules, three molecules of CO₂, and one of triose. The fate of each of the six carbons of hexose is shown. The light and dark shading of C1 and C6 carbons in C is used to emphasize the loss of label from the C1 positions and the retention of label in the C6 positions. This acts to lower the ratio of ¹³C1:¹³C₆ in carbohydrates.

Figure 6

Illustration of the two routes by which we propose that carbon is moved from the IRM to the ERM in the AM symbiosis. Carbon taken up from the host in the form of hexose is converted to carbohydrates and storage lipids in the IRM. Lipid and glycogen are translocated from IRM to ERM. Storage (glycogen and trehalose) and structural (chitin) carbohydrates are synthesized in the ERM from hexose that is derived from exported carbohydrate as well as from lipid.