Dual localization of arbuscular mycorrhizal structures and polyphosphate accumulation in soybean roots

【Objectives】 Polyphosphates are major phosphorus forms existing in the structural bodies of arbuscular mycorrhizae. Qualitative and quantitative analyses of polyphosphates are important to understand the phosphorus metabolism in arbuscular mycorrhizae. Many genes have been identified to involve in nutrient exchanges between mycorrhizal fungi and host plants. Therefore, the technology for the staining and localization of polyphosphates in arbuscular mycorrhizae is urgently required. 【Methods】 Fresh roots from Glomus mosseae colonized soybean plants were sampled and made into thin slices. Some of them were stained with low concentration of wheat germ agglutinin for 30 min at room temperature, and photographed using a fluorescence microscope at blue light excitation with wavelength of 488 nm; the others were stained with the fluorescent dye 4, 6-diamidino-2-phenylindole dihydrochloride, and photographed at ultraviolet excitation with wavelength of 405 nm. Thepolyphosphates were thus localized by both the methods. 【Results】1) Using fluorescent active staining method (stained with wheat germ agglutinin), all arbuscular mycorrhizal structures could be clearly detected, including arbuscules, vesicles and intercellular hyphae. 2) In the infected roots, polyphosphates in arbuscular mycorrhizal structures showed yellow fluorescence under excitation of ultraviolet light. DNA in nucleus interacting with 4, 6-diamidino-2-phenylindole dihydrochloride showed blue-white fluorescent dots, and arbuscular membrance of mature arbuscules in the cells exhibited blue-white fluorescence. So, using fluorescent dye 4, 6-diamidino-2-phenylindole dihydrochloride to position the polyphosphates, the pigmentation of polyphosphates, DNA and membrane structure were obviously distinguished. 3) Based on the above results, dual localization of mycorrhizal structures and polyphosphate accumulation was realized by changing the fluorescent channels. Furthermore, polyphosphate accumulation in immature arbuscules showed light yellow fluorescence, and those in mature arbuscules could only partly be observed since highly developed membrane structure affected the staining and appearance of polyphosphates.【Conclusions】The dual staining method using fluorescent dyes wheat germ agglutinin and 4, 6-diamidino-2-phenylindole dihydrochloride is able to localize the mycorrhizal structures and polyphosphate accumulation in soybean roots. Using the method, the nutrient exchanges between arbuscular mycorrhizal fungi and plant roots can be visually observed. Therefore, it is prospective technology for the study of mycorrhizal symbiosis at tissue and cellular level.