coniferyl-alcohol and 1-phenylpropanol

Verticillium longisporum is a soil-borne vascular pathogen causing economic loss in rape. Using the model plant Arabidopsis this study analyzed metabolic changes upon fungal infection in order to identify possible defense strategies of Brassicaceae against this fungus. Metabolite fingerprinting identified infection-induced metabolites derived from the phenylpropanoid pathway. Targeted analysis confirmed the accumulation of sinapoyl glucosides, coniferin, syringin and lignans in leaves from early stages of infection on. At later stages, the amounts of amino acids increased. To test the contribution of the phenylpropanoid pathway, mutants in the pathway were analyzed. The sinapate-deficient mutant fah1-2 showed stronger infection symptoms than wild-type plants, which is most likely due to the lack of sinapoyl esters. Moreover, the coniferin accumulating transgenic plant UGT72E2-OE was less susceptible. Consistently, sinapoyl glucose, coniferyl alcohol and coniferin inhibited fungal growth and melanization in vitro, whereas sinapyl alcohol and syringin did not. The amount of lignin was not significantly altered supporting the notion that soluble derivatives of the phenylpropanoid pathway contribute to defense. These data show that soluble phenylpropanoids are important for the defense response of Arabidopsis against V. longisporum and that metabolite fingerprinting is a valuable tool to identify infection-relevant metabolic markers.

Topics: Arabidopsis; Biomarkers; Biosynthetic Pathways; Cinnamates; Coumaric Acids; Disease Resistance; Gene Expression Regulation, Plant; Genes, Plant; Glucosides; Lignans; Lignin; Metabolomics; Mutation; Phenols; Plant Diseases; Plant Leaves; Plant Vascular Bundle; Propanols; Solubility; Verticillium

Although the practice of protein engineering is industrially fruitful in creating biocatalysts and therapeutic proteins, applications of analogous techniques in the field of plant metabolic engineering are still in their infancy. Lignins are aromatic natural polymers derived from the oxidative polymerization of primarily three different hydroxycinnamyl alcohols, the monolignols. Polymerization of lignin starts with the oxidation of monolignols, followed by endwise cross-coupling of (radicals of) a monolignol and the growing oligomer/polymer. The para-hydroxyl of each monolignol is crucial for radical generation and subsequent coupling. Here, we describe the structure-function analysis and catalytic improvement of an artificial monolignol 4-O-methyltransferase created by iterative saturation mutagenesis and its use in modulating lignin and phenylpropanoid biosynthesis. We show that expressing the created enzyme in planta, thus etherifying the para-hydroxyls of lignin monomeric precursors, denies the derived monolignols any participation in the subsequent coupling process, substantially reducing lignification and, ultimately, lignin content. Concomitantly, the transgenic plants accumulated de novo synthesized 4-O-methylated soluble phenolics and wall-bound esters. The lower lignin levels of transgenic plants resulted in higher saccharification yields. Our study, through a structure-based protein engineering approach, offers a novel strategy for modulating phenylpropanoid/lignin biosynthesis to improve cell wall digestibility and diversify the repertories of biologically active compounds.

Topics: Arabidopsis; Biocatalysis; Cell Wall; Crystallization; Gene Expression; Genetic Engineering; Lignin; Methylation; Methyltransferases; Models, Molecular; Mutant Proteins; Phenols; Phenotype; Plant Proteins; Plants, Genetically Modified; Propanols; Recombinant Proteins; Structure-Activity Relationship; Substrate Specificity

4-Hydroxycinnamoyl-CoA hydratase/lyase (HCHL), a crotonase homologue of phenylpropanoid catabolism from Pseudomonas fluorescens strain AN103, led to the formation of 4-hydroxybenzaldehyde metabolites when expressed in hairy root cultures of Datura stramonium L. established by transformation with Agrobacterium rhizogenes. The principal new compounds observed were the glucoside and glucose ester of 4-hydroxybenzoic acid, together with 4-hydroxybenzyl alcohol- O-beta- D-glucoside. In lines actively expressing HCHL, these together amounted to around 0.5% of tissue fresh mass. No protocatechuic derivatives were found, although a trace of vanillic acid-beta- D-glucoside was detected. There was no accumulation of 4-hydroxybenzaldehydes, whether free or in the form of their glucose conjugates. There was some evidence suggesting a diminished availability of feruloyl-CoA for the production of feruloyl putrescine and coniferyl alcohol. The findings are discussed in the context of a diversion of phenylpropanoid metabolism, and the ability of plants and plant cultures to conjugate phenolic compounds.

Topics: Benzaldehydes; Datura stramonium; Enoyl-CoA Hydratase; Gene Expression Regulation, Enzymologic; Glucose; Hydro-Lyases; Lignin; Parabens; Phenols; Plant Roots; Plants, Genetically Modified; Propanols; Pseudomonas fluorescens; Vanillic Acid