Strigolactones stimulate arbuscular mycorrhizal fungi by activating mitochondria

Abstract

The association of arbuscular mycorrhizal (AM) fungi with plant roots is the oldest and ecologically most important symbiotic relationship between higher plants and microorganisms, yet the mechanism by which these fungi detect the presence of a plant host is poorly understood. Previous studies have shown that roots secrete a branching factor (BF) that strongly stimulates branching of hyphae during germination of the spores of AM fungi. In the BF of Lotus, a strigolactone was found to be the active molecule. Strigolactones are known as germination stimulants of the parasitic plants Striga and Orobanche. In this paper, we show that the BF of a monocotyledonous plant, Sorghum, also contains a strigolactone. Strigolactones strongly and rapidly stimulated cell proliferation of the AM fungus Gigaspora rosea at concentrations as low as 10(-13) M. This effect was not found with other sesquiterperne lactones known as germination stimulants of parasitic weeds. Within 1 h of treatment, the density of mitochondria in the fungal cells increased, and their shape and movement changed dramatically. Strigolactones stimulated spore germination of two other phylogenetically distant AM fungi, Glomus intraradices and Gl. claroideum. This was also associated with a rapid increase of mitochondrial density and respiration as shown with Gl. intraradices. We conclude that strigolactones are important rhizospheric plant signals involved in stimulating both the pre-symbiotic growth of AM fungi and the germination of parasitic plants.

Figure 1. ESI-MS/MS Analysis, in the Positive EPI Scan Mode, of a Purified Sorghum Extract Active in a Gi. rosea Hyphal Branching Assay

The parent ion m/z = 339 shows the characteristic daughter ion of sorgolactone [M + Na] ⁺ at m/z = 242.

Figure 2. Time-Course Analyses of Gi. rosea Hyphal Branching (A) and Growth (B) Treated with 10 ⁻⁷ M GR7 in Methanol:Water (v/v) or with Methanol:Water (v/v) (Control)

Number of hyphal branches and total hyphal length (including hyphal branches) were recorded and cumulated from day 5 to 14. Twenty spores were analysed per treatment. This experiment was repeated three times with similar results. Error bars show SEM.

Figure 3. Cellular Response of Germinating Spores of Gi. rosea to GR24 after 1 h or 5 h of Treatment with GR24

Hyphae were stained with MitoTracker Green and treated with 0.001% acetone (control) or 30 nM GR24. Mitochondrial density is significantly higher in treated samples than in control at 1 h and 5 h ( p < 0.001, n = 26; and p < 0.001, n = 42, respectively). For porin and COX immunolabelling, hyphae were treated with acetone or GR24 (as above) and incubated with antibodies as described in Materials and Methods. The fluorescence density of porin and COX immunolabelling is significantly higher in treated hyphae than in controls both at 1 h ( p < 0.001, n = 52; and p < 0.05, n = 60, respectively) and at 5 h ( p < 0.05, n = 28; and p < 0.001, n = 29, respectively). Error bars show SEM.

Figure 4. Effect of GR24 on Mitochondrial Shape and Density in Hyphae of Gi. rosea Stained with Mitotracker Green

Hyphae were treated with 30 nM GR24 (up) or with 0.001% acetone (bottom) for 1 h. Bar = 10 μm.

Figure 5. Effect of GR24 on Size of Mitochondria in Hyphae of Gi. rosea Stained with Mitotracker Green

Hyphae were treated with 30 nM GR24 or with 0.001% acetone for 1 h (A) or 5 h (B). Results are expressed in frequency (%) distribution of length/width ratios ( n = 140–150 mitochondria).

Figure 6. Effect of GR24 on Size of Mitochondria in Hyphae of Gl. intraradices Stained with Mitotracker Green

Hyphae were treated with 30 nM GR24 or with 0.001% acetone for 1 h. Results are expressed in frequency (%) distribution of length/width ratios ( n = 200–220 mitochondria).