FRET-SLiM on native autofluorescence: A fast and reliable method to study interactions between fluorescent probes and lignin in plant cell wall

Abstract

Background: Lignocellulosic biomass is a complex network of polymers making the cell walls of plants. It represents a feedstock of sustainable resources to be converted into fuels, chemicals and materials. Because of its complex architecture, lignocellulose is a recalcitrant material that necessitates some pretreatments and several types of catalysts to be transformed efficiently. In particular, enzymes degrading lignocellulose can become inactivated due to their binding to lignin through non-specific interactions, leading to a loss in catalytic efficiency of industrial processes. Gaining more knowledge in the strength of interactions would allow optimizing enzymes and selecting appropriate pretreatments. Results: Measuring interactions directly in plant cell wall can theoretically be performed using confocal fluorescence techniques by evaluating fluorescence resonance energy transfer (FRET) between compatible fluorophores. In this study, autofluorescence of plant cell wall, mainly originating from lignin, was considered as a donor fluorophore while the acceptor was a common rhodamine-based fluorescent probe. To overcome complex plant cell wall fluorescence, which limits FRET analysis by standard techniques, we have developed an original approach, combining spectral and lifetime measurements. It consists in (1) dissecting autofluorescence signal in each spectral channel, (2) optimizing spectral channel choice for lifetime measurements and (3) achieving an unambiguous FRET signature with an autofluorescent donor fluorophore. Interactions between rhodamine-based probes of various sizes and untreated or pretreated wheat sample were evaluated, showing it was possible to discriminate interactions at the nano-scale, revealing some accessibility differences and the effect of pretreatment. Conclusions: SLiM measurement allows precise estimation of the optimal spectral range for FRET measurement. SLiM response allows for the first time doubtless FRET measurements between lignin as a donor, and an acceptor fluorophore with high accuracy and sensitivity related to lifetime decrease studies. As demonstrated, it thus becomes possible to measure interactions of fluorescent probes directly inside plant cell wall samples. This approach can thus be applied to various fields such as lignocellulose deconstruction to optimize the action of enzymes or plant cell wall development to assay in situ the biosynthesis of lignin.