Ectomycorrhizae symbiosis in Castanea mollissima improves phosphate acquisition through activating gene expression and H⁺ efflux

Chestnuts are important ecological forest tree species. They can form mycorrhizal connections with a variety of ectotrophic mycorrhizal fungi. Mycorrhizal hyphae can expand the absorbing area to facilitate the intake of nutrients, thereby promoting plant growth and development. In this study, a chestnut-fungus symbiosis system was established in vitro, and typical mycorrhizal structures (root tips bifurcated and apical enlargement) were found after three-month inoculation. Phosphorus content and H⁺ efflux rate of chestnut roots increased significantly after being inoculated with Scleroderma citrinum compared to the non-inoculated control. The increased H⁺ efflux rate provides indirect evidence for the role of ecto-mycorrhizae in increased phosphate uptake. Finally, phylogenetic tree was clustered by MEGA5.0 based on amino acid sequences of five phosphate transporters of Castanea mollissima and known phosphate transporters of other plants. The results showed that CmPT4 was in a clade with known mycorrhizal phosphate transporters, and thus this gene was preliminarily a predicted mycorrhizal phosphate transporter. Further real-time quantitative PCR showed that five phosphate transporter genes (CmPT1, CmPT2, CmPT3, CmPT4, and CmPT5) were upregulated under phosphorus deficiency treatment, and only CmPT4 was induced in the mycorrhizal roots of chestnut. Therefore, It can be concluded CmPT4 is a mycorrhizal phosphate transporter gene in Castanea mollissima. This study provides a foundation for further analysis of chestnut mycorrhizal formation and phosphate uptake improvement by phosphate transporters.