Stereochemical identification of glucans by oligothiophenes enables cellulose anatomical mapping in plant tissues

Agneta Richter-Dahlfors,Marcus Bäck,Ulrica Edlund,Ferdinand X Choong,K Peter R Nilsson,Anette Schulz

doi:10.1038/s41598-018-21466-y

Abstract

Efficient use of plant-derived materials requires enabling technologies for non-disruptive composition analysis. The ability to identify and spatially locate polysaccharides in native plant tissues is difficult but essential. Here, we develop an optical method for cellulose identification using the structure-responsive, heptameric oligothiophene h-FTAA as molecular fluorophore. Spectrophotometric analysis of h-FTAA interacting with closely related glucans revealed an exceptional specificity for β-linked glucans. This optical, non-disruptive method for stereochemical differentiation of glycosidic linkages was next used for in situ composition analysis in plants. Multi-laser/multi-detector analysis developed herein revealed spatial localization of cellulose and structural cell wall features such as plasmodesmata and perforated sieve plates of the phloem. Simultaneous imaging of intrinsically fluorescent components revealed the spatial relationship between cell walls and other organelles, such as chloroplasts and lignified annular thickenings of the trachea, with precision at the sub-cellular scale. Our non-destructive method for cellulose identification lays the foundation for the emergence of anatomical maps of the chemical constituents in plant tissues. This rapid and versatile method will likely benefit the plant science research fields and may serve the biorefinery industry as reporter for feedstock optimization as well as in-line monitoring of cellulose reactions during standard operations.

Highlights

Composition analysis of polysaccharide rich materials addresses the need for non-destructive, accurate and efficient techniques, preferentially at low cost
To define the optical changes h-FTAA undergoes when binding to cellulose, we first defined the spectra of unbound probe in phosphate-buffered saline (PBS) using spectrophotometric recordings between [400–750] nm
The Spec-Plot showed that binding of h-FTAA to M. cellulose brings a red shift in the excitation λmax to 497.5 ± 17.5 nm and the appearance of characteristic shoulders at 460 nm and 525 nm

Summary

Introduction

Composition analysis of polysaccharide rich materials addresses the need for non-destructive, accurate and efficient techniques, preferentially at low cost. Whereas most polysaccharides are invisible to standard immunofluorescence techniques[9] as they lack unique surface epitopes, the newly reported oligothiophene-based method enables optical analysis by spectrophotometry as well as spectral imaging[1,2]. This technique is highly advantageous for visualization of repetitive polysaccharide structures. The standard techniques for carbohydrate determination are mainly disruptive in the sense that the biomass is degraded prior to, or during analysis[13,14] Mainstay methods, such as ion exchange chromatography, identify carbohydrates based on their constituent monosaccharides. This demonstrates a novel non-destructive method to visualize the anatomical location of cellulose in plant tissues by standard fluorescence microscopy

Methods

Results

Conclusion