Purification and Analysis of Signaling Molecules in Plant-Arbuscular Mycorrhizal Fungal Communication

The arbuscular mycorrhizal (AM) symbiosis is the most widespread mutualistic association between plants and fungi. The most widely recognized benefit of AM symbiosis for the host plant is a significant enhancement in the acquisition of mineral nutrients from the soil, particularly phosphorus. In addition to improved nutrition, AM symbiosis confers enhanced tolerance to a wide range of environmental stresses. Strigolactones (SLs) are a family of natural products produced by plants as shoot branching factors and are responsible for the induction of hyphal branching in arbuscular mycorrhizal fungi (AMF). On the other hand, flavonoids also play crucial roles in various signaling processes, such as legume-rhizobia symbiosis or in the AM symbiosis. Being able to accurately analyze strigolactones and flavonoids is essential for unraveling the basic mechanisms of AM symbiosis. However, the low amount of them produced by plants and their rapid degradability make it crucial to develop fast analytical methods with very low limits of quantification to study the interaction and communication between plants and AMF. Herein, a protocol is described for the development of a LC-MS/MS method for the quantification of SLs and flavonoids, using GR24 and chrysin as internal standards, respectively, in roots, exudates, and extracts.