Fluorescence Lifetime Imaging as an In Situ and Label-Free Readout for the Chemical Composition of Lignin (original) (raw)
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bioRxiv (Cold Spring Harbor Laboratory), 2021
Important structures and functions within living organisms rely on naturally fluorescent polymeric molecules such as collagen, keratin, elastin, resilin, or lignin. Theoretical physics predict that fluorescence lifetime of these polymers is related to their chemical composition. We verified this prediction for lignin, a major structural element in plant cell walls and one of the most abundant components of wood. Lignin is composed of different types of phenylpropanoid units, and its composition affects its properties, biological functions, and the utilization of wood biomass. We carried out fluorescence lifetime imaging microscopy (FLIM) measurements of wood cell wall lignin in a population of 90 hybrid aspen trees genetically engineered to display differences in cell wall chemistry and structure. We also measured wood cell wall composition by classical analytical methods in the wood cell walls of these trees. Using statistical modelling and machine learning algorithms, we identified parameters of fluorescence lifetime that predict the content of S-type and G-type lignin units, the two main types of units in the lignin of angiosperm plants. Finally, we show how quantitative measurements of lignin chemical composition by FLIM can reveal the dynamics of lignin biosynthesis in two different biological contexts, including in vivo while lignin is being synthesized in the walls of living cells.
Journal of Photochemistry and Photobiology A-chemistry, 2001
Four fluorescent probes (biphenyl, naphthalene, pyrene and phenanthrene) were used to map the energy distribution of the structural units present in lignin fragments from Eucalyptus grandis wood. This distribution shows that these fragments present two regions with a high concentration of chromophores, one between 418 and 385 kJ/mol, and the other below 322 kJ/mol. When this lignin was treated with NaBH 4 , the two more intense regions occurs between 418 and 346 kJ/mol, followed by a significant increase in the concentration of chromophores in almost all the studied energy range. Lifetime distributions present a bimodal pattern, with two typical peak lifetime values, the first of 1.36 ± 0.17 ns, with a relative amplitude above 80%, and the second of 8.48 ± 2.32 ns, for both species, with some fluctuation for different λ em . The synchronous spectra indicates, for this lignin, at least three broad spectral envelopes, with a large superposition of the emission maxima. The results indicate the existence of at least three most representative fluorophores, most probably due to biphenyl, coniferyl alcohol and stilbene structures, with varying substituents. The majority of the fluorescence complexity of this lignin seems to be associated with the inhomogeneous emission decay kinetics associated with ground state heterogeneity, due to the complex mixture of the different fluorophores, on which are superimposed different distributions of environments.
The origin of lignin fluorescence
Journal of Molecular Structure, 1999
Spruce lignin exhibits fluorescence emission spectra that peaks at Ϸ 360 nm on excitation at wavelengths ranging from 240 to 320 nm. This can be explained by non-radiative energy transfer from lignin chromophores, that are excited in the wavelength range 240-320 nm, to an acceptor that emits fluorescent light at Ϸ 360 nm. Examinations of lignin samples and model compounds suggest that small amounts of phenylcoumarone structures in the lignin is a conceivable acceptor. Such structures and stilbene structures are formed from structural elements in lignin of the phenylcoumaran type on various treatments. The photophysical properties of models for phenylcoumarone structures [2-(3,4-dimethoxyphenyl)-7-methoxy-3-methylbenzo[b]furan, 2-(3,4-dimethoxyphenyl)-3-hydroxymethyl-7-methoxybenzo[b]furan] and stilbene structures (the E and Z forms of 2-hydroxy-3,3 H ,4 H-trimethoxystilbene) have been examined and are discussed on the basis of crystal structure determinations.
ACS Sustainable Chemistry & Engineering
A novel analytical approach based on fluorescence labeling was developed in the effort to increase the understanding of phenolic group distribution in technical lignins. Selective derivatization with a fluorophore (dansyl chloride) of lignin phenolic functionalities was quantitatively achieved under mild reaction conditions. Reference acetylated lignin and labeled lignin were analyzed by gel permeation chromatography (GPC) coupled to a UV−vis detector (set at 280 nm) and a fluorescence detector (λ excitation: 390 nm, λ emission: 550 nm) to discern the dansyl-linked phenol response from the lignin aromatic skeleton input. After data elaboration, valuable information about the phenolic group distribution as a function of molecular weight for different technical lignins was gathered. This novel analytical approach is applied to model lignin polymer thermal protection properties, a useful parameter in lignin valorization strategies.
Biotechnology for Biofuels, 2013
Background: Delignification pretreatments of biomass and methods to assess their efficacy are crucial for biomassto-biofuels research and technology. Here, we applied confocal and fluorescence lifetime imaging microscopy (FLIM) using one-and two-photon excitation to map the lignin distribution within bagasse fibers pretreated with acid and alkali. The evaluated spectra and decay times are correlated with previously calculated lignin fractions. We have also investigated the influence of the pretreatment on the lignin distribution in the cell wall by analyzing the changes in the fluorescence characteristics using two-photon excitation. Eucalyptus fibers were also analyzed for comparison.
Component analysis of the fluorescence spectra of a lignin model compound
Journal of Photochemistry and Photobiology B: Biology, 2006
In order to test whether lignin fluorescence originates from discrete fluorophores, fluorescence emission spectra of the lignin model dehydrogenative polymer (DHP) were analyzed by the band deconvolution method and time-resolved analysis of both the excitation and emission spectra. Two series of 22 fluorescence emission spectra of DHP in chloroform/methanol (3:1, v/v) solution, and as a solid suspension in water, were deconvoluted into three fluorescence and one Raman Gaussian components. Emission spectra were obtained by stepwise variation of the excitation wavelength from 360 to 465 nm. Deconvolution was performed by nonlinear fitting of all three Gaussian parameters: area, width and position. Position of all components in a series was treated as a random variable and its approximate probability distribution (APD) calculated from a series of histograms with increasing number of abscissa intervals. A five peak multimodal APD profile was obtained for both series of DHP emission spectra. The mean fluorescence lifetime varied with wavelength both in the emission and the excitation decay-associated spectra (DAS), where four kinetic components were resolved. The shapes of the excitation spectra of the four components were quite different and gradually shifted bathochromically. The multicomponent nature of the DHP emission spectra along with the changes in the mean fluorescence lifetime and the form of the excitation DAS of the four components give evidence of the heterogeneous origin of fluorescent species emitting in the visible.
Visualization of plant cell wall lignification using fluorescence-tagged monolignols
The Plant Journal, 2013
Lignin is an abundant phenylpropanoid polymer produced by the oxidative polymerization of p-hydroxycinnamyl alcohols (monolignols). Lignification, i.e., deposition of lignin, is a defining feature of secondary cell wall formation in vascular plants, and provides an important mechanism for their disease resistance; however, many aspects of the cell wall lignification process remain unclear partly because of a lack of suitable imaging methods to monitor the process in vivo. In this study, a set of monolignol analogs c-linked to fluorogenic aminocoumarin and nitrobenzofuran dyes were synthesized and tested as imaging probes to visualize the cell wall lignification process in Arabidopsis thaliana and Pinus radiata under various feeding regimens. In particular, we demonstrate that the fluorescence-tagged monolignol analogs can penetrate into live plant tissues and cells, and appear to be metabolically incorporated into lignifying cell walls in a highly specific manner. The localization of the fluorogenic lignins synthesized during the feeding period can be readily visualized by fluorescence microscopy and is distinguishable from the other wall components such as polysaccharides as well as the pre-existing lignin that was deposited earlier in development.
New Insights into the Structural Organization of the Plant Polymer Lignin
Annals of the New York Academy of Sciences, 2005
The organizational features of lignin structure and the mechanism of its synthesis have significant implications for the response of the plant to stress. It was unknown whether the enzymic formation of lignin in the cell wall is an uncontrolled process or finely regulated in time and space. In vitro scanning tunneling microscopy (STM), atomic force microscopies (AFM), nearfield scanning optical microscopy (NSOM). and the novel environmental scanning electron microscopy (ESEM) imaging studies of the lignin model compounds have directly shown its highly ordered structure and elucidated its modular and fractal organization. Direct evidence was presented for the existence of strong intermolecular forces responsible for holding lignin globules together in highly ordered structures. Fractal analysis was applied as a theoretical approach, to show regularity and modular organization of lignin. Surface chemistry studies of the lignin monolayer reveal intrinsic properties that may be a key to osmotic pressure and cell size control mechanism in the higher plant cells. The obtained data contribute to the explanation of the mechanisms of cell wall synthesis in vivo.