GR24, a synthetic analog of strigolactones, stimulates the mitosis and growth of the arbuscular mycorrhizal fungus Gigaspora rosea by boosting its energy metabolism

Abstract

Arbuscular mycorrhizal (AM) fungi are obligate biotrophs that participate in a highly beneficial root symbiosis with 80% of land plants. Strigolactones are trace molecules in plant root exudates that are perceived by AM fungi at subnanomolar concentrations. Within just a few hours, they were shown to stimulate fungal mitochondria, spore germination, and branching of germinating hyphae. In this study we show that treatment of Gigaspora rosea with a strigolactone analog (GR24) causes a rapid increase in the NADH concentration, the NADH dehydrogenase activity, and the ATP content of the fungal cell. This fully and rapidly (within minutes) activated oxidative metabolism does not require new gene expression. Up-regulation of the genes involved in mitochondrial metabolism and hyphal growth, and stimulation of the fungal mitotic activity, take place several days after this initial boost to the cellular energy of the fungus. Such a rapid and powerful action of GR24 on G. rosea cells suggests that strigolactones are important plant signals involved in switching AM fungi toward full germination and a presymbiotic state.

Figure 1.

NADPH autofluorescence in hyphae of G. rosea. A, Autofluorescence emission spectra of a G. rosea hypha treated with 10⁻⁸ m GR24. Emission spectra were obtained at 405-nm excitation and recorded 0 and 15 min after treatment (black triangles and circles, respectively). They are compared with the emission spectrum of a standard NADH solution (white squares). B, Time course measurements of emission (450 nm) intensities of NADPH autofluorescence in a G. rosea hypha, treated (black square) or not (white squares) with 10⁻⁸ m GR24 and excited at 405 nm. Values are means of fluorescence signals measured in four regions (15 μm² each) of three hyphal tips treated in three independent experiments. Polynomial fitted curves (solid lines) are shown. Error bars are ses of the means.

Figure 2.

Time lapse sequence of NADPH autofluorescence changes after GR24 treatment. A and G, Bright-field micrographs of control (A) and GR24-treated (G) hyphae, respectively. Autofluorescence of NADPH was monitored after 0, 5, 10, 15, and 20 min, respectively, in control hyphae (B–F) and in hyphae treated by 10⁻⁸ m GR24 (H–L). In all pseudocolor images, the color follows convention, with red indicating high and blue indicating low levels of fluorescence signal (color intensity scale on F and L). Bars = 10 μm.

Figure 3.

NADH dehydrogenase activity. A to D, Hyphae were treated 1 h with acetone (A and B) or with 10⁻⁸ m GR24 (C and D). They were incubated for 20 min with NBT reaction mix alone (A–C) or with NBT reaction mix supplemented with 28 mm NADH (B–D). NBT precipitates (in blue) were observed in hyphal tips (eight per treatment). Images were similar within each treatment. Bars = 5 μm. A quantitative analysis of NADH dehydrogenase activity in control (white bar) and GR24-treated hyphae (black bar), is presented (E). The conditions of incubation were as in B and D. Statistical analysis was performed with Student's t test (P < 0.01; n = 15). Error bars are ses of mean values. Different letters above histograms indicate that mean values from control and treated samples are statistically different.

Figure 4.

Effect of GR24 on gene regulation. Real-time RT-PCR was carried out on total mRNA extracted from GR24-treated hyphae after 5 and 10 d of treatment. Total RNA yield was measured and cDNA synthesis performed as described in the experimental procedures section. Relative nuclear gene expression in GR24-treated hyphae was compared to acetone-treated controls. Expression of each gene was obtained using the 2^−ΔΔCT method with NOC4 as the reference gene (see details in text). Data are the means of two independent experiments.

Figure 5.

Effect of GR24 on the number of nuclei in G. rosea hyphal tips. A and B, Fungal cells treated with 0.0001% (A) or 10⁻⁸ M GR24 (B) were stained with AO after 5 d. Bars = 10 μm. C, After each treatment, nuclei were counted in the first 100-μm segment of 18 hyphal tips. Different letters above histograms indicate a significant difference between the mean values (Student's t test, P < 0.001; n = 18). Error bars are ses of mean values. [See online article for color version of this figure.]

Figure 6.

Effect of GR24 on spore nucleus number. Spores were incubated in the presence of 10⁻⁸ M GR24 (black bars) or 0.0001% acetone (white bars) for 5 d. A, Using five spores (germinated hyphae removed) per DNA extraction, real-time PCR absolute quantification was carried out on putative α-tubulin and NOC4 homolog genes. The copy number per spore was determined as described in “Materials and Methods.” Bars are means ± se of seven replicates. For both genes, there was no significant difference between control and treated spores (P = 0.57). B, Direct quantification of the nucleus number in crushed spores (10 spores per treatment) and mithramycin staining, showing no significant difference between control and treated spores (Student's t test, P = 0.24; n = 10). Error bars are se of mean values.