European oak chemical diversity - from ecotypes to herbivore resistance.

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 nontarget 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, an oak pest that causes severe forest defoliation. Among the detected metabolites, we identified reliable metabolic biomarkers to distinguish S- and 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.

Automatization of metabolite extraction for high-throughput metabolomics: case study on transgenic isoprene-emitting birch.

Metabolomics studies are becoming increasingly common for understanding how plant metabolism responds to changes in environmental conditions, genetic manipulations and treatments. Despite the recent advances in metabolomics workflow, the sample preparation process still limits the high-throughput analysis in large-scale studies. Here, we present a highly flexible robotic system that integrates liquid handling, sonication, centrifugation, solvent evaporation and sample transfer processed in 96-well plates to automatize the metabolite extraction from leaf samples. We transferred an established manual extraction protocol performed to a robotic system, and with this, we show the optimization steps required to improve reproducibility and obtain comparable results in terms of extraction efficiency and accuracy. We then tested the robotic system to analyze the metabolomes of wild-type and four transgenic silver birch (Betula pendula Roth) lines under unstressed conditions. Birch trees were engineered to overexpress the poplar (Populus × canescens) isoprene synthase and to emit various amounts of isoprene. By fitting the different isoprene emission capacities of the transgenic trees with their leaf metabolomes, we observed an isoprene-dependent upregulation of some flavonoids and other secondary metabolites as well as carbohydrates, amino acid and lipid metabolites. By contrast, the disaccharide sucrose was found to be strongly negatively correlated to isoprene emission. The presented study illustrates the power of integrating robotics to increase the sample throughput, reduce human errors and labor time, and to ensure a fully controlled, monitored and standardized sample preparation procedure. Due to its modular and flexible structure, the robotic system can be easily adapted to other extraction protocols for the analysis of various tissues or plant species to achieve high-throughput metabolomics in plant research.

Blue-green fluorescence during hypersensitive cell death arises from phenylpropanoid deydrodimers.

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, localization, and functions during the HCD. Compared with 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-hydroxyferulic acid, and 5-hydroxyferuloylmalate during the HCD. FAH1-dependent BG fluorescence appeared exclusively within dying cells of the upper epidermis as detected by microscopy. Saponification released dehydrodimers from cell wall polymers of WT but not fah1 plants. Collectively, our data suggest that HCD induction leads to the formation of free BG fluorescent dehydrodimers from monomeric sinapates and 5-hydroxyferulates. The formed dehydrodimers move from upper epidermis cells into the apoplast where they esterify cell wall polymers. Possible functions of phenylpropanoid dehydrodimers are discussed.

Application of biodegradable cholinium ionic liquids for the extraction of 5-hydroxymethylfurfural (HMF) from honey.

5-Hydroxymethylfurfural (HMF), a Maillard reaction product, can be formed when honey is subjected to heat treatment or a long storage time, becoming volatile and toxic depending on its concentration. The fact that, until today, there is no literature data on the extraction of 5-hydroxymethylfurfural (HMF) from honey using ionic liquids directed the investigation of the influence of biodegradable cholinium ionic liquids on the formation of aqueous biphasic systems and the application of these systems for the extraction of HMF from honey. The influence of anions of synthesised ionic liquids on the construction of biphasic systems in which an inorganic salt was used as a salting agent was investigated. Then, the extraction of HMF in these systems was examined, and the mechanisms of HMF extraction using ionic liquids were explained using computer simulations. Examining the effect of cholinium ionic liquids (choline chloride ([Ch][Cl]), cholinium nicotinate ([Ch][Nic]), cholinium propionate ([Ch][Prop]), and cholinium butyrate ([Ch][But])) on the formation of aqueous biphasic systems by comparing the phase diagrams, it was concluded that the ability of ionic liquids to form an aqueous biphasic system with tripotassium phosphate (K3PO4) decreases in the following order: [Ch][But] ≈ [Ch][Prop] > [Ch][Nic] > [Ch][Cl]. By applying all tested aqueous biphasic systems for the extraction of HMF from honey, an extraction efficiency of more than 89% was achieved. Complete extraction was achieved using the extraction system with [Ch][But], while the weakest ability to extract HMF was exhibited by the system with [Ch][Cl]. The mechanisms of HMF extraction using ionic liquids are explained on the basis of the optimised structures of the ionic liquid systems with HMF, together with the visualisation of non-covalent interactions, and on the basis of the calculated binding energies ΔGbin, which can be used as a good predictor of the extraction potential of newly synthesised ionic liquids.