The benzoxazinoid breakdown product 5-Methoxy-2-Nitrophenol has a dual function in maize defense against herbivory

Catabolism of plant secondary metabolites can yield bioactive breakdown products. However, these compounds often remain poorly characterized. Here, we report on the discovery, biosynthesis, and biological relevance of 5-Methoxy-2-Nitrophenol (5M2NP), a secondary metabolite breakdown product which accumulates in damaged maize tissues. We used mutant plants, biochemical complementation, and metabolomic analyses to determine the biosynthetic origin of 5M2NP. Additionally, we assessed 5M2NP’s role in plant defense against herbivores. For this, we surveyed defense-associated responses (gene expression, phytohormones, volatile organic compounds) in wounded, 5M2NP-supplemented maize leaves, and performed bioassays with herbivorous insects. We found that 5M2NP accumulation upon tissue disruption is contingent upon a functional benzoxazinoid biosynthesis pathway. Labeling experiments demonstrated that 5M2NP is derived from DIMBOA. Physiological doses of exogenous 5M2NP increased the wound-induced expression of defense genes and emission of terpenoids. Additionally, 5M2NP exhibited antibiotic and antixenotic activities towards both generalist and specialist herbivores in nano-to micromolar quantities. We conclude that 5M2NP, which represents a novel class of plant-derived compounds, can act as a direct defense and a defense modulator. 5M2NP thus expands the functional repertoire of benzoxazinoids and likely contributes to their protective function against insect herbivores. The discovery of 5M2NP highlights the multifunctionality of plant secondary metabolites and their breakdown products. ### Competing Interest Statement The authors have declared no competing interest. * DIMBOA : 2,4-Dihydroxy-7-Methoxy-2H-1,4-Benzoxazin-3( 4H )-One DIM2BOA-Glc : ( 2R )-β-D-Glucopyranosyl-2,4-Dihydroxy-7,8-Dimethoxy-2H-1,4-Benzoxazin-3( 4H )-One DIMBOA-Glc : ( 2R )-β-D-Glucopyranosyl-2,4-Dihydroxy-7-Methoxy-2H-1,4-Benzoxazin-3( 4H )-One DMNT : ( 3E )-4,8-Dimethyl-1,3,7-Nonatriene HDM2BOA-Glc : ( 2R )-β-D-Glucopyranosyl-2-Hydroxy-4,7,8-Trimethoxy-2H-1,4-Benzoxazin-3( 4H )-One HDMBOA-Glc : ( 2R )-β-D-Glucopyranosyl-2-Hydroxy-4,7-Dimethoxy-2H-1,4-Benzoxazin-3( 4H )-One HM2BOA-Glc : ( 2R )-β-D-Glucopyranosyl-2-Hydroxy-7,8-Dimethoxy-2H-1,4-Benzoxazin-3( 4H )-One MBOA : 6-Methoxy-Benzoxazolin-2( 3H )-One TMTT : ( 3E,7E )-4,8,12-Trimethyl-1,3,7,11-Tridecatetraene