Translocation and utilization of fungal storage lipid in the arbuscular mycorrhizal symbiosis

Abstract

The arbuscular mycorrhizal (AM) symbiosis is responsible for huge fluxes of photosynthetically fixed carbon from plants to the soil. Carbon is transferred from the plant to the fungus as hexose, but the main form of carbon stored by the mycobiont at all stages of its life cycle is triacylglycerol. Previous isotopic labeling experiments showed that the fungus exports this storage lipid from the intraradical mycelium (IRM) to the extraradical mycelium (ERM). Here, in vivo multiphoton microscopy was used to observe the movement of lipid bodies through the fungal colony and to determine their sizes, distribution, and velocities. The distribution of lipid bodies along fungal hyphae suggests that they are progressively consumed as they move toward growing tips. We report the isolation and measurements of expression of an AM fungal expressed sequence tag that encodes a putative acyl-coenzyme A dehydrogenase; its deduced amino acid sequence suggests that it may function in the anabolic flux of carbon from lipid to carbohydrate. Time-lapse image sequences show lipid bodies moving in both directions along hyphae and nuclear magnetic resonance analysis of labeling patterns after supplying 13C-labeled glycerol to either extraradical hyphae or colonized roots shows that there is indeed significant bidirectional translocation between IRM and ERM. We conclude that large amounts of lipid are translocated within the AM fungal colony and that, whereas net movement is from the IRM to the ERM, there is also substantial recirculation throughout the fungus.

Figure 1

Two-photon microscopy of storage lipids in the AM fungus G. intraradices growing asymbiotically. a, Storage lipid deposits within a mature spore; b, lipid bodies in germ tubes are most abundant in the close proximity of the spore; c, further along the germ-tube apex less lipid bodies are visualized; d, close to the hyphal tip almost no lipid bodies are observed; e, projection of a z-series of a G. intraradices germ-tube (left) and visualization of the same image after applying the software for lipid globules volume measurements (right).

Figure 2

Two-photon microscopy of storage lipids in the AM fungus Gi. rosea growing in the absence of a host root. Fewer lipid globules are observed as compared with G. intraradices germ-tubes. Lipid globules preferentially accumulate at hyphal branching zones (a and b) and are almost absent at germ-tube zones close to the apex (d). Note the presence of black vacuoles in zones closer to the apex (c and d).

Figure 3

Two-photon microscopy of storage lipids in the ERM of G. intraradices during symbiosis. Lipid globules are most abundant in hyphal zones closer to the root (a–c), then its number progressively decrease as we move to zones closer to the hyphal growing front (d–f). Storage lipid globules are least abundant in zones of high C consumption, as BAS (c). Percentages represent the fraction of total hyphal volume occupied by lipid globules ± se. An estimate of total TAGs volume present in each image is also provided.

Figure 4

Storage lipid bodies in a BAS (a) and a developing group of spores (b) of symbiotic G. intraradices.

Figure 5

Two-photon microscopy of storage lipids in symbiotic Gi. margarita extraradical hyphae. The same gradient of lipids than in the case of G. intraradices (most abundant in zones closer to the host root [a–c], less abundant approaching hyphal growing front [d–f]) is observed for this AM fungus. Percentages represent the fraction of total hyphal volume occupied by lipid globules ± se. An estimate of total TAGs volume present in each image is also provided.

Figure 6

The gradient of storage lipid globules along symbiotic hyphae of G. intraradices (a) and Gi. margarita (b) as a result of plotting total storage lipid volume versus distance to the hyphal tip. The dashed line in b represents the expected curve we should obtain for Gi. margarita if applying the same equation curve as for G. intraradices.

Figure 7

¹³C-NMR spectra of neutral lipids extracted from the ERM of G. intraradices (a and c) and from transformed carrot roots colonized by G. intraradices (b and d) after incubation with [¹³C_1,3]Glyc, which was added to make a final concentration of 10 mm either to the ERM in the fungal compartment (a and b) or to the mycorrhizal root compartment (c and d). Peaks from ¹³C at different molecular positions of TAG are labeled: glyc1,3 the spectroscopically equivalent C₁ and C₃ carbons of the glyceryl moiety of TAG, glyc2 the C₂ carbon of the glyceryl moiety, CH₃ the terminal carbon of the FA moieties, C(ω-1) the penultimate carbon of the FA moieties. The fungal TAG contains almost entirely a shorter chain length FA, C16:1c_11, whereas the host TAG is C18:2c_11. Natural abundance signals from unlabeled positions give integrals that are proportional to the number of carbons of each type. Thus CH₃= C(ω-1) when FAs are unlabeled and C_1,3 = 2 × C₂ when the glyceryl is unlabeled. Incorporation of label from [¹³C_1,3]Glyc results in increased intensity at C_1,3 and at CH₃, but not at the (ω-1) position since labeling is almost entirely at even-numbered carbons.

Figure 8

Multiple alignments of acyl-CoA dehydrogenase sequences from G. intraradices, Neurospora crassa, Pseudomonas aeruginosa, and Homo sapiens. Noteworthy are the substantial N-terminal extension and the C-terminal PTS-1 type peroxisomal/glyoxysomal tripeptide targeting sequence (in bold) that G. intraradices shares with the Neurospora sp. sequence but not with the bacterial or human sequences.