Summary: In monocotyledonous plants, 1,4‐benzoxazin‐3‐ones, also referred to as benzoxazinoids or hydroxamic acids, are one of the most important chemical barriers against herbivores. However, knowledge about their behavior after attack, mode of action and potential detoxification by specialized insects remains limited. We chose an innovative analytical approach to understand the role of maize 1,4‐benzoxazin‐3‐ones in plant–insect interactions. By combining unbiased metabolomics screening and simultaneous measurements of living and digested plant tissue, we created a quantitative dynamic map of 1,4‐benzoxazin‐3‐ones at the plant–insect interface. Hypotheses derived from this map were tested by specifically developed in vitro assays using purified 1,4‐benzoxazin‐3‐ones and active extracts from mutant plants lacking 1,4‐benzoxazin‐3‐ones. Our data show that maize plants possess a two‐step defensive system that effectively fends off both the generalist Spodoptera littoralis and the specialist Spodoptera frugiperda. In the first step, upon insect attack, large quantities of 2‐β‐d‐glucopyranosyloxy‐4,7‐dimethoxy‐1,4‐benzoxazin‐3‐one (HDMBOA‐Glc) are formed. In the second step, after tissue disruption by the herbivores, highly unstable 2‐hydroxy‐4,7‐dimethoxy‐1,4‐benzoxazin‐3‐one (HDMBOA) is released by plant‐derived β‐glucosidases. HDMBOA acts as a strong deterrent to both S. littoralis and S. frugiperda. Although constitutively produced 1,4‐benzoxazin‐3‐ones such as 2,4‐dihydroxy‐7‐methoxy‐1,4‐benzoxazin‐3‐one (DIMBOA) are detoxified via glycosylation by the insects, no conjugation of HDMBOA in the insect gut was found, which may explain why even the specialist S. frugiperda has not evolved immunity against this plant defense. Taken together, our results show the benefit of using a plant–insect interface approach to elucidate plant defensive processes and unravel a potent resistance mechanism in maize.

Plants respond to herbivory by reprogramming their metabolism. Most research in this context has focused on locally induced compounds that function as toxins or feeding deterrents. We developed an ultra‐high‐pressure liquid chromatography time‐of‐flight mass spectrometry (UHPLC‐TOF‐MS)‐based metabolomics approach to evaluate local and systemic herbivore‐induced changes in maize leaves, sap, roots and root exudates without any prior assumptions about their function. Thirty‐two differentially regulated compounds were identified fromSpodoptera littoralis‐infested maize seedlings and isolated for structure assignment by microflow nuclear magnetic resonance (CapNMR). Nine compounds were quantified by a high throughput direct nano‐infusion tandem mass spectrometry/mass spectrometry (MS/MS) method. Leaf infestation led to a marked local increase of 1,3‐benzoxazin‐4‐ones, phospholipids,N‐hydroxycinnamoyltyramines, azealic acid and tryptophan. Only few changes were found in the root metabolome, but 1,3‐benzoxazin‐4‐ones increased in the vascular sap and root exudates. The role ofN‐hydroxycinnamoyltyramines in plant–herbivore interactions is unknown, and we therefore tested the effect of the dominatingp‐coumaroyltyramine onS. littoralis. Unexpectedly,p‐coumaroyltyramine was metabolized by the larvae and increased larval growth, possibly by providing additional nitrogen to the insect. Taken together, this study illustrates that herbivore attack leads to the induction of metabolites that can have contrasting effects on herbivore resistance in the leaves and roots.