Background Arbuscular mycorrhizal fungi (AMF) have a positive effect on drought tolerance of plants after establishing reciprocal resymbiosis with roots, while the underlying mechanism is not deciphered. Metabolomics can explain the mechanism of plant response to environmental stress by analyzing the changes of all small molecular weight metabolites. The purpose of this study was to use Ultra High Performance Liquid Chromatography Q Exactive Mass Spectrometer to analyze changes in root metabolites of walnut (Juglans regia) after inoculation with an arbuscular mycorrhizal fungus Diversispora spurca under well-watered (WW) and drought stress (DS). Results Sixty days of soil drought significantly inhibited root mycorrhizal colonization rate, shoot and root biomass production, and leaf water potential in walnut, while AMF inoculation significantly increased biomass production and leaf water potential, accompanied by a higher increase magnitude under DS versus under WW. A total of 3278 metabolites were identified. Under WW, AMF inoculation up-regulated 172 metabolites and down-regulated 61 metabolites, along with no changes in 1104 metabolites. However, under DS, AMF inoculation up-regulated 49 metabolites and down-regulated 116 metabolites, coupled with no changes in 1172 metabolites. Among them, juglone (a quinone found in walnuts) as the first ranked differential metabolite was up-regulated by AMF under WW but not under DS; 2,3,5-trihydroxy-5–7-dimethoxyflavanone as the first ranked differential metabolite was increased by AMF under DS but not under WW. The KEGG annotation showed a large number of metabolic pathways triggered by AMF, accompanied by different metabolic pathways under WW and DS. Among them, oxidative phosphorylation and phenylalanine metabolism and biosynthesis were triggered by AMF in response to WW and DS, where N-acetyl-L-phenylalanine was induced by AMF to increase under DS, while decreasing under WW. Conclusion This study provides new insights into the metabolic mechanisms of mycorrhiza-enhanced drought tolerance in walnuts.

Arbuscular mycorrhizal fungi (AMF) have important roles in enhancing drought tolerance of host plants, but it is not clear whether and how AMF increase drought tolerance in walnut (Juglans regia). We hypothesized that AMF could activate antioxidant defense systems and heat shock transcription factors (Hsfs) transcription levels to alleviate oxidative damage caused by drought. The walnut variety ‘Liaohe No. 1’ was inoculated with Diversispora spurca and exposed to well-watered (WW, 75% of the maximum soil water capacity) and drought stress (DS, 50% of the maximum soil water capacity) for 6 weeks. Plant growth, antioxidant defense systems, and expressions of five JrHsfs in leaves were studied. Such drought treatment inhibited root mycorrhizal colonization, while plant growth performance was still improved by AMF inoculation. Mycorrhizal fungal inoculation triggered the increase in soluble protein, glutathione (GSH), ascorbic acid (ASC), and total ASC contents and ascorbic peroxidase and glutathione reductase activities, along with lower hydrogen peroxide (H2O2), superoxide anion radical (O2•−), and malondialdehyde (MDA) levels, compared with non-inoculation under drought. Mycorrhizal plants also recorded higher peroxidase, catalase, and superoxide dismutase activities than non-mycorrhizal plants under drought. The expression of JrHsf03, JrHsf05, JrHsf20, JrHsf22, and JrHsf24 was up-regulated under WW by AMF, while the expression of JrHsf03, JrHsf22, and JrHsf24 were up-regulated only under drought by AMF. It is concluded that D. spurca induced low oxidative burst in drought-stressed walnut through activating antioxidant defense systems and part Hsfs expressions.