 Climate change is increasing insect pressure and forcing plants to adapt. Although chemotypic differentiation and phenotypic plasticity in spatially separated tree populations are known for decades, understanding their importance in herbivory resistance across forests remains challenging. We studied four oak forest stands in Germany using non-target metabolomics, elemental analysis, and chemometrics and mapped the leaf metabolome of herbivore-resistant (T-) and herbivore-susceptible (S-) European oaks (Quercus robur) to Tortrix viridana, a oak pest that causes severe forest defoliation. Among the detected metabolites, we identified reliable metabolic biomarkers to distinguish Sand T-oak trees. Chemotypic differentiation resulted in metabolic shifts of primary and secondary leaf metabolism. Across forests, T-oaks allocate resources towards constitutive chemical defense enriched of polyphenolic compounds, e.g. the flavonoids kaempferol, kaempferol and quercetin glucosides, while S-oaks towards growth-promoting substances such as carbohydrates and amino-acid derivatives. This extensive work across natural forests shows that oaks' resistance and susceptibility to herbivory are linked to growth-defense trade-offs of leaf metabolism. The discovery of biomarkers and the developed predictive model pave the way to understand Quercus robur's susceptibility to herbivore attack and to support forest management, contributing to the preservation of oak forests in Europe.

1. Plants harbour a wide range of leaf-feeding insects whose survival and fitness are influenced by both energy-rich molecules and phytochemicals in the host foliage.Yet, how leaf host chemical diversity and insect microbiota-key factors in ecological and physiological processes-impact insect nutrition and fitness are still poorly understood.2. To study the effects of leaf metabolic composition on insect herbivory resistance and performance, we fed the larvae of the specialist herbivory Tortrix viridana with leaves of susceptible and resistant Quercus robur trees that are characterized by contrasting metabolomes. We analysed the larval performance and mortality, the metabolomes in plant leaves, and in the insects' saliva and faeces by nontargeted metabolomics. Using chemometrics, mass difference network analysis and metabarcoding, we show the metabolome changes and chemical reactions associated with the different diets as well as their impact on insect fitness and gut microbiota.3. In the saliva and faeces of larvae, plant secondary metabolites (e.g. flavonoids) persisted more the insect digestion while compounds from primary metabolism were more depleted. In addition, metabolic reactions within the larvae indicated different degradation pathways used on the two plant metabolic types (syn. metabotypes), including sulfation and sulfonation. We show that feeding insects with resistant oak leaves, enriched in secondary metabolites and depleted in primary metabolites, impaired insect performance and mortality. Although the insects' gut microbiota was slightly different upon the contrasting diets, overall, it was fairly stable. Despite the impact of host chemicals on herbivores, larvae were generally highly efficient in nutrient assimilation (feed conversion ratios of 3.3-3.6) and able to minimize plant defences (78% of secondary metabolites were converted, broken down or sequestrated).

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Infection of Arabidopsis with avirulent Pseudomonas syringae and exposure to nitrogen dioxide (NO2) both trigger hypersensitive cell death (HCD) that is characterized by the emission of bright blue-green (BG) autofluorescence under UV illumination. The aim of our current work was to identify the BG fluorescent molecules and scrutinize their biosynthesis and functions during the HCD. Compared to wild-type (WT) plants, the phenylpropanoid-deficient mutant fah1 developed normal HCD except for the absence of BG fluorescence. Ultrahigh resolution metabolomics combined with mass difference network analysis revealed that WT but not fah1 plants rapidly accumulate dehydrodimers of sinapic acid, sinapoylmalate, 5-OH-ferulic acid, and 5-OH-feruloylmalate during the HCD. FAH1-dependent BG fluorescence appeared exclusively within dying cells of the upper epidermis as detected by microscopy. Saponification released dehydrodimers from extracted cell wall material. Collectively, our data suggest that HCD induction leads to the formation of free BG fluorescent dehydrodimers from monomeric sinapates and 5-hydroxyferulates. Reactive oxygen species from de-regulated photosynthesis likely contribute to the radical-radical coupling. The formed dehydrodimers move from upper epidermis cells into the apoplast where they esterify and thereby cross-link cell wall polymers. Both, free as well as wall-bound phenylpropanoid dehydrodimers are defense-related compounds in Arabidopsis. We propose that other plants also employ dehydrodimers of highly abundant phenylpropanoids for rapid defense against pathogen attack.

Plants harbor a wide range of leaf-feeding insects. Insect survival and fitness are influenced by both energy-rich molecules and phytochemicals in the host foliage. Yet, how leaf chemical diversity and insect microbiota - key factors in ecological and physiological processes – shape insect nutrition and impact insect performance is still poorly understood. Here we forced Tortrix viridana larvae, an oak-specialized herbivore, to feed on two Quercus robur susceptible and resistant metabolic phenotypes (metabotypes) and examined leaf, salivary, and fecal metabolomes associated with larval performance, mortality, and fecal microbiota. We show that host chemical diversity affects larval development and that the distinct signatures of oak metabotypes are maintained in the insect digestive system. Larvae were highly efficient in nutrient assimilation and able to minimize plant chemical defenses, thanks in part to the adaptation of the gut microbiota to the different food qualities.