Structural requirements of strigolactones for hyphal branching in AM fungi

Abstract

Strigolactones are a group of terpenoid lactones that act as a host-derived signal in the rhizosphere communication of plants with arbuscular mycorrhizal (AM) fungi and root parasitic weeds as well as an endogenous plant hormone regulating shoot branching in plants. Strigolactones induce hyphal branching in AM fungi at very low concentrations, suggesting a highly sensitive perception system for strigolactones present in AM fungi. However, little is known about the structural requirements of strigolactones for hyphal branching in AM fungi. Here, we tested a series of natural and synthetically modified strigolactones as well as non-strigolactone-type germination stimulants for hyphal branching-inducing activity in germinating spores of the AM fungus Gigaspora margarita. All tested compounds with a tricyclic lactone coupled to a methylbutenolide via an enol ether bond showed activity, but differed in the active concentration and in the branching pattern of hyphae. Truncation of the A- and AB-rings in the tricyclic ABC lactone of strigolactones resulted in a drastic reduction in hyphal branching activity. Although the connection of the C-ring in the tricyclic lactone to the methylbutenolide D-ring was shown to be essential for hyphal branching, the bridge structure in the C-D part was found not necessarily to be enol ether, being replaceable with either alkoxy or imino ethers. These structural requirements in AM fungi are very similar but not identical to those observed in root parasitic weeds, especially with respect to the enol ether bridge in the C-D part.

Fig. 1

Chemical structures of natural and chemically modified natural strigolactones. Hyphal branching-inducing activities in Gigaspora margarita are indicated by a colored background to each chemical structure.

Fig. 2

Chemical structures of synthetic GR (‘germination releaser’) analogs and non-strigolactone-type germination stimulants. Formyl tricyclic lactone and hydroxymethylbutenolide identified as inactive water degradation products of GR24 are also included. Hyphal branching-inducing activities in Gigaspora margarita are indicated by a colored background to each chemical structure.

Fig. 3

Hyphal branching of Gigaspora margarita induced by natural and synthetically modified strigolactones. (A) Control (70% ethanol in water). (B) (+)-5DS, (3 pg per disc). (C) (+)-Orobanchol (1 pg per disc). (D) (+)-2′-Epiorobancol (10 pg per disc). (E) (+)-7-Oxoorobanchyl acetate (100 pg per disc). (F) (−)-Fabacyl acetate (10 pg per disc). (G) (±)-2′,5-Bisepistrigol (100 pg per disc). (H) (±)-2′-epi-5-Oxo-5DS (10 pg per disc). (I) (+)-ent-2′-epi-3,6′-dihydro-GR24 (10 ng per disc). (J) Imino GR24F (1 μg per disc). Arrows indicate the direction of growth of primary hyphae. Control hyphae, which were treated with 70% ethanol–water, formed no hyphal branches, as shown in A. Scale bars, 1 mm.

Fig. 4

Spectroscopic analysis of water degradation products of GR24 and 5DS. EI-MS spectra of formyl tricyclic lactone (A) and hydroxymethylbutenolide (B) isolated as degradation products of GR24 in water. (C) EI-MS spectrum of formyl tricyclic lactone isolated in the water degradation experiment of 5DS. (D) Time course of 5DS levels in water (filled circle) or acetone (open circle) incubated at 32°C. (E) Time course of GR24 levels in water incubated at 32°C.