Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

Abstract

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by Förster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

Fluorescence Microscopy Methods for the Analysis and Characterization of Lignin

“…This method consists of measuring the molecular interaction between two fluorophores by superimposing the emission and excitation spectra [ 103 ]. The process occurs when a donor fluorophore and an acceptor fluorophore are within 10 nm, making it possible for a non-radiative transfer of excitation energy from donor to acceptor to occur [ 31 ].…”

“…FRET has been used to determine the rate of saccharification of species with potential use [ 25 ]; in addition to that, FRET has allowed to determine the route of transport and polymerization of lignin in the cell wall [ 44 ] and the inhibition of peroxidase and oxidase enzymes due to their association with lignin nanoparticles [ 66 ]. On the other hand, FRET has been used as a basis for other methods, such as FRAP, TPM, and the SFLiM variant for the analysis of enzymatic interaction with lignin [ 103 ], or in conjunction with CLSM for the detection of nanostructural interactions between additives, such as polyethylene glycol (PEG), for the detection of recalcitrant lignin [ 67 ].…”