Ultrabroadband Multiplex CARS Imaging Reveals Distinct Protein- and Lipid-Rich Domains in Arbuscules

Arbuscular mycorrhizal (AM) fungi form mutualistic symbioses with a wide range of terrestrial plants, in which they exchange inorganic nutrients taken up from the soil for plant-derived photosynthates such as sugars and lipids. This nutrient exchange occurs exclusively at arbusculeshighly branched hyphal structures that develop in the plant root cortex. Although the molecular mechanisms underlying arbuscule development and function have been extensively studied using targeted approaches based on transgenic plants and fluorescence microscopy, directly visualizing the various molecular components crucial for arbuscule function using nontargeted approaches remains a major challenge. Here, we demonstrate that ultrabroadband multiplex coherent anti-Stokes Raman scattering (CARS) microspectroscopy enables two- and three-dimensional imaging of arbuscule-containing regions in plant roots. Using genetically transformed Lotus japonicus roots colonized by the AM fungus Rhizophagus irregularis, we identify Raman spectroscopic signatures of arbuscules indicative of protein-rich domains. These domains are associated with periarbuscular membrane-embedded transporters that are involved in AM nutrient exchange, including phosphate transporters, as well as other proteins present in fungal and plant cells adjacent to the arbuscules. Additionally, we detect adjacent lipid-rich regions corresponding to arbuscule trunks that mainly contain unsaturated triacylglycerols transferred from the host plant. Our findings highlight the potential of ultrabroadband multiplex CARS imaging as a label-free, in situ imaging tool for studying arbuscules, providing deeper chemical insights into AM symbiosis.