Untargeted metabolomics reveals novel metabolites in Lotus japonicus roots during arbuscular mycorrhiza symbiosis

Abstract

Introduction

Arbuscular mycorrhiza (AM), a symbiosis between c. 80% of land plant species and fungi of the Glomeromycotina (Spatafora et al., 2016), increases mineral nutrient uptake and stress tolerance of plants (Smith & Smith, 2011; Zhang et al., 2023; Zou et al., 2023), improving overall fitness (Liu et al., 2007), boosting photosynthetic rates (Zhu et al., 2012), growth, and yield (Ramírez-Flores et al., 2020; Di Tomassi et al., 2021; Igiehon et al., 2021; Sheteiwy et al., 2021).

The fungi collect mineral nutrients from the soil via their extraradical mycelium and release them to the plant inside the root. The plant, in return, nourishes the fungi with photoassimilates, mainly hexoses and lipids (Keymer & Gutjahr, 2018; Wipf et al., 2019). This nutrient exchange requires the formation of intracellular, highly branched fungal structures called arbuscules, which are surrounded by a plant membrane called the peri-arbuscular membrane, across which nutrient exchange takes place (Gutjahr & Parniske, 2013). The environment affects plant development and physiology, and thus considerably influences the state of the symbiosis. Accordingly, the host plant dynamically regulates the formation of intracellular fungal structures such as hyphae and arbuscules and the extent of root colonization to keep the symbiotic advantages at an optimum (Koide & Schreiner, 1992; Gutjahr & Parniske, 2017).

Symbiosis is initiated through an interchange of molecular signals between plant and fungus. Plants grown under phosphate or nitrogen limitation release strigolactones, which activate fungal germination and hyphal branching. In turn, the fungi release chito-oligosaccharides and lipochito-oligosaccharides, which activate LysM receptor-like kinases on the plant side (summarized in Delaux & Gutjahr, 2024). This triggers a symbiotic signalling cascade, including ion channels in the nuclear membrane evoking nuclear calcium oscillations, which are thought to be interpreted by a Ca/calmodulin-dependent protein kinase (CCamK, Charpentier et al., 2016; Miller et al., 2013). Activated CCaMK binds and phosphorylates the protein CYCLOPS, a transcription factor that regulates the expression of crucial symbiosis genes (Singh et al., 2014). Additionally, CYCLOPS forms a complex with DELLA proteins to activate the transcription factor REQUIRED FOR ARBUSCULAR MYCORRHIZATION 1 (RAM1; Pimprikar et al., 2016). The GIBBERELLIC-ACID INSENSITIVE (GAI), REPRESSOR of GAI (RGA) and SCARECROW (SCR) (GRAS; Pysh et al., 1999)) transcription factor RAM1 is involved in activating RAM2 (Gobbato et al., 2012; Park et al., 2015; Pimprikar et al., 2016), which encodes a glycerol-3-phosphate acyltransferase 6 (GPAT6) involved in providing lipids for transfer to the fungus in arbuscule-containing cells (summarized in Keymer & Gutjahr, 2018). Mutants in ccamk block colonization at the hyphopodium stage (Demchenko et al., 2004; Pimprikar et al., 2016), whereas cyclops mutants allow rare events of root entry but no formation of arbuscules (Yano et al., 2008). ram1 and ram2 allow arbuscule formation, but the arbuscules remain stunted and underdeveloped (Pimprikar et al., 2016; Keymer et al., 2017).

Colonization of plant roots by AM fungi is accompanied by drastic changes in the transcriptome (reviewed in Pimprikar & Gutjahr, 2018) including many genes involved in secondary metabolism. In Medicago truncatula and some other plant species, metabolomics of symbiotic roots, as well as leaves of symbiotic plants, revealed strong AM-induced changes in the metabolome, but as usual in untargeted metabolomics, many compounds could not be identified (Schliemann et al., 2007; Schweiger & Müller, 2015; Kaur et al., 2022). In general, the knowledge on alterations of the secondary metabolome in response to AM symbiosis is rather poor (Ranner et al., 2023).

Therefore, we analysed extracts from roots of the model legume L. japonicus colonized by the model AM fungus Rhizophagus irregularis compared to control roots, using untargeted metabolomics by ultraperformance liquid chromatography-electrospray ionization-ion mobility-time-of-flight-mass spectrometry (UPLC-ESI-IM-ToF-MS). We identified AM-enhanced compounds via co-chromatography with authentic standards or isolation followed by spectroscopic and spectrometric analyses. Multi-omics correlation analysis with metabolomics and transcriptomics data was used to visualize AM-induced trends. Finally, we evaluated the activity of identified and highly induced compounds on fungal spore germination. Root colonization by AM fungi induced massive metabolome changes. We found novel compounds induced by AM and provided a basis for the elucidation of further novel compounds, metabolic pathways, and their functions in plants and AM symbiosis.