FMO and CYP monooxygenase families determine the metabolic flux of hydroxylated tryptamine derivatives in barley ( Hordum vulgare ) following pathogen infection

To counteract pathogenic microorganisms, plants execute a complex resistance response that includes major metabolic reprogramming and production of bioactive defensive compounds. Barley ( Hordeum vulgare ) is a major cereal crop, but suffers significant yield losses due to pathogen attack every year. Here we use an untargeted metabolomic approach to assess the diversity and shifts in key barley metabolites produced in response to Pyrenophora teres f. teres infection, a hemibiotrophic fungal pathogen and causal agent of net blotch disease. Tryptophan-derived compounds, including tryptamine, serotonin, and a novel indole alkaloid – 2-oxo-tryptamine (2OT) – were among the most significantly induced and abundant compounds, with mass spectrometry imaging revealing that these metabolites accumulate at the site of infection. A transcriptomic approach identified a flavin-containing monooxygenase (FMO), which was functionally characterized as a 2-oxo-tryptamine synthase (2OTS). In addition, a cytochrome P450 (CYP71P10) was characterized as a tryptamine-5-hydroxylase, responsible for serotonin biosynthesis. These characterized genes are tightly co-expressed with genes involved in tryptophan biosynthesis and signify a major metabolic flux towards indolic compounds after infection, potentially serving as bioactive phytoalexins. Additional microbial interactions using a biotrophic fungus ( Blumeria hordei ; powdery mildew), a hemibiotrophic bacterium ( Pseudomonas syringae ), and alternative cultivars suggest that these pathways represent a generally activated resistance response in barley. These results provide new insights within the barley defense response relevant for the development of disease resistant traits in cereal crops.