The regulation of arbuscular mycorrhizal symbiosis by phosphate in pea involves early and systemic signalling events

Abstract

Most plants form root symbioses with arbuscular mycorrhizal (AM) fungi, which provide them with phosphate and other nutrients. High soil phosphate levels are known to affect AM symbiosis negatively, but the underlying mechanisms are not understood. This report describes experimental conditions which triggered a novel mycorrhizal phenotype under high phosphate supply: the interaction between pea and two different AM fungi was almost completely abolished at a very early stage, prior to the formation of hyphopodia. As demonstrated by split-root experiments, down-regulation of AM symbiosis occurred at least partly in response to plant-derived signals. Early signalling events were examined with a focus on strigolactones, compounds which stimulate pre-symbiotic fungal growth and metabolism. Strigolactones were also recently identified as novel plant hormones contributing to the control of shoot branching. Root exudates of plants grown under high phosphate lost their ability to stimulate AM fungi and lacked strigolactones. In addition, a systemic down-regulation of strigolactone release by high phosphate supply was demonstrated using split-root systems. Nevertheless, supplementation with exogenous strigolactones failed to restore root colonization under high phosphate. This observation does not exclude a contribution of strigolactones to the regulation of AM symbiosis by phosphate, but indicates that they are not the only factor involved. Together, the results suggest the existence of additional early signals that may control the differentiation of hyphopodia.

Fig. 1.

Effect of phosphate fertilization on mycorrhizal root colonization. Plants inoculated with 600 spores of Gl. intraradices (grey bars) or 100 spores of Gi. rosea (white bars) were grown under low (LP) or high (HP) phosphate fertilization. The extent of root colonization was determined after observation of stained root samples as the fraction of root length showing arbuscules, vesicles, or both in the case of Gl. intraradices, and arbuscules in the case of Gi. rosea. Error bars show the SEM; n=5–6 plants when inoculated with Gl. intraradices and n=7–8 plants when inoculated with Gi. rosea. Different letters indicate statistically significant differences according to Student's t-test (P <0.05).

Fig. 2.

Gigaspora rosea hyphal branching in response to GR24, LP or HP root exudates. Germinated spores of Gi. rosea were treated with GR24 and/or root exudates of low (LP) or high (HP) phosphate-grown plants, or with the solvent alone as negative control (10% acetonitrile; AcN). Newly formed hyphal apices were counted 48 h after treatment. White bars, controls; grey bars, root exudates alone; black bars, root exudates+GR24. Error bars show the SEM; n=24–26 treated spores for each condition. Different letters indicate statistically significant differences according to one-way ANOVA followed by Tukey's test (P <0.05).

Fig. 3.

Mycorrhizal root colonization in split-root systems. (A) Experimental design. Each root system was divided into two parts placed in different pots to allow differential phosphate fertilization. Both sides were inoculated with 90 spores of Gl. intraradices and plants were grown for 6 weeks. Control plants were fertilized with the same solution on both sides. Results in B, C, and D correspond to the same plants. (B) Root colonization levels determined by observation of stained root samples. For control plants, colonization levels measured on both sides were averaged. (C, D) Inorganic orthophosphate (Pi) content in leaves (C) and roots (D). Error bars show the SEM; n=5–7 plants for each condition. Different letters indicate statistically significant differences according to one-way ANOVA followed by Tukey's test (P <0.05).

Fig. 4.

Systemic control of strigolactone production. Split-root plants were fertilized with low phosphorus (LP) on one side and high phosphorus (HP) on the other (LP/HP plants), or with the same solution on both sides (LP/LP and HP/HP plants). Root exudate extracts were analysed by LC-MS/MS in the MRM mode. The synthetic strigolactone analogue GR24 was added in equal quantity to all samples as an external standard. Chromatograms show the most abundant mass transition for each of the two major pea strigolactones, fabacyl acetate and orobanchyl acetate. Insets show the mass transition corresponding to the external standard GR24 (299>202 m/z).

Fig. 5.

Effect of strigolactone supplementation on root colonization. Plants inoculated with 150 spores of Gl. intraradices were fertilized daily with LP or HP nutrient solution, supplemented or not with 10 nM GR24. The extent of root colonization was determined by observation of stained root samples. Error bars show the SEM; n=7–8 plants for each condition. Different letters indicate statistically significant differences according to one-way ANOVA followed by Tukey's test (P <0.05).