Direct fluorescence imaging of lignocellulosic and suberized cell walls in roots and stems.

Peter Kitin,Satoshi Nakaba,Sierin Lim,Ryo Funada,Christopher G Hunt,Daniel Johnson

doi:10.1093/aobpla/plaa032

Abstract

Investigating plant structure is fundamental in botanical science and provides crucial knowledge for the theories of plant evolution, ecophysiology and for the biotechnological practices. Modern plant anatomy often targets the formation, localization and characterization of cellulosic, lignified or suberized cell walls. While classical methods developed in the 1960s are still popular, recent innovations in tissue preparation, fluorescence staining and microscopy equipment offer advantages to the traditional practices for investigation of the complex lignocellulosic walls. Our goal is to enhance the productivity and quality of microscopy work by focusing on quick and cost-effective preparation of thick sections or plant specimen surfaces and efficient use of direct fluorescent stains. We discuss popular histochemical microscopy techniques for visualization of cell walls, such as autofluorescence or staining with calcofluor, Congo red (CR), fluorol yellow (FY) and safranin, and provide detailed descriptions of our own approaches and protocols. Autofluorescence of lignin in combination with CR and FY staining can clearly differentiate between lignified, suberized and unlignified cell walls in root and stem tissues. Glycerol can serve as an effective clearing medium as well as the carrier of FY for staining of suberin and lipids allowing for observation of thick histological preparations. Three-dimensional (3D) imaging of all cell types together with chemical information by wide-field fluorescence or confocal laser scanning microscopy (CLSM) was achieved.

Highlights

A reignited interest in studies of plant structure is evident in the last two–three decades triggered by the crucial economic and ecological importance of plants as renewable sources of energy and biomaterials (Van de Wouwer et al 2018; Ralph et al 2019)
Depending on the purpose of observation, different chemical fixatives have traditionally been used in plant anatomy, such as FAA; GA (4 % glutaraldehyde solution in 0.1 M phosphate buffer of pH 7.3); 1–3 % OsO4; or methanol solutions (Talbot and White 2013)
We found that the fluorescence of fluorol yellow (FY)-stained suberized walls in the range of 510–630 nm is considerably stronger than the native fluorescence of lignin and suberin

Summary

Introduction

A reignited interest in studies of plant structure is evident in the last two–three decades triggered by the crucial economic and ecological importance of plants as renewable sources of energy and biomaterials (Van de Wouwer et al 2018; Ralph et al 2019). The formation and characterization of lignocellulosic or suberized cells are frequently targeted The innovations include every main step: plant material preparation, such as the fixation and clearing of plant material, the methods of sectioning and staining, as well as exciting new developments of the microscopy equipment (Kitin et al 2003, 2010; Lux et al 2005; Sano et al 2005; Truernit et al 2008; Liesche et al 2013; Thomas et al 2013; Palmer et al 2015; Hasegawa et al 2016; Ursache et al 2018; Yazaki et al 2019)

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