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

Abstract

Background and Aims 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 60s 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. Methods We discuss popular histochemical microscopy techniques for visualization of cell walls, such as autofluorescence or staining with calcofluor, Congo red (CR), fluorol yellow (FY), safranin, and provide detailed descriptions of our own approaches and protocols. Key Results 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 imaging (3D) of all cell types together with chemical information by wide-field fluorescence or CLSM was achieved.

Distribution and Chemical Composition of Lignin in Secondary Xylem of Cactaceae

“…Some authors reported that polysaccharides were compounds without autofluorescence [16,31] . However, with other stains such as Congo red, cellulose could fluoresce between 570–620 nm; [14,32] for holocellulose, Donaldson et al [33] . detected its fluorescence emission in red tones at 600 nm.…”

“…The procedure for preparing plant tissue samples for analysis with epifluorescence microscopy is in Supporting Information , Methods S1. The fixation technique was based on Ruzin [54] with modifications based on Kitin et al [14] . The procedure to stain the samples was based in Loza‐Cornejo and Terrazas [55] .…”

Combining cross‐section images and modeling tools to create high‐resolution root system hydraulic atlases in Zea mays

“…This could be due to the presence of perforation plates (Brodersen et al, 2018;Shane et al, 2000) or persistent xylem cross-walls (Sanderson et al, 1988). In this study, we did not divide 81-37-8) and PEG 400 (PEG 400:glycerol:water = 10:9:1, v:v) allow intense staining of lipids and suberin lamellae (Kitin et al, 2020). We also assume that xylem sap has the same viscosity as water.…”

Improving dimensional stability of Populus cathayana wood by suberin monomers with heat treatment

“…The results of the CLSM were consistent with previous studies. Kitin et al (2020) also reported the fluorescence of lignin and suberin in plant cells under CLSM. Heinämäki et al (2017) confirmed the melting behavior of suberin monomers.…”

“…Suberin and lignin are naturally fluorescent substances in plant cells for its pheno-lic structure (Hutzler et al 1998, Kitin et al 2020. The microscopic distribution of SMs in specimens was detected by a Leica TCS SP8 confocal microscope (Leica Microsystems Inc., Wetzlar, Germany) with a krypton/argon laser emitting at wavelengths of 488 nm.…”