Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Summary

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

Abstract

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40–50%), hemicellulose (25–30%), and lignin (20–30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas Mäule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Introduction

The plant cell wall holds a plethora of information within its various components: lignin, cellulose, hemicelluloses (xylan, glucuronoxylan, xyloglucan, arabinoxylan, mixed linkage glucan, or glucomannan), and pectin. Histological techniques provide important visual cues in studying differences within the secondary cell walls at organizational and cellular levels. Various histological techniques were developed and can be found in the literature, but these techniques can be challenging and time-consuming for beginners because very detailed protocols with simple visual instructions are seldom if ever available. The goal of this study is to provide simple guidelines for ....

Protocol

1. Stem Embedding

1. Make a 7% agarose solution in water (7 g of electrophoresis-grade agarose in 100 ml distilled water). Dissolve the agarose by autoclaving for 20 min or by microwaving for 20 min at lowest intensity (e.g., 10% intensity of a 1,250 watt microwave).
2. Prepare a homemade mold for embedding the stems using plastic vials.
   1. Using a razor blade, cut off the conical bottom of a 2 ml screw-cap microcentrifuge tube (Part A). With a syringe needle, puncture a hole in the tube cap slightly larger than the diameter of the stem to be embedded. Next, cut 0.5 cm off the bottom of a 0.6 ml microcentrifuge tube (Part B).

Results

Stem Embedding and Sectioning:
The use of the homemade plastic mold to embed the stems in 7% agarose proved to be fast and easy (Figure 1). The two parts (A and B; Figure 1) of the embedded vial system make it simple to easily release the stem embedded in agarose as the agarose does not stick to the vial parts that are inert, keeping the system clean. The vials can be reused multiple times for years. The convenience of storing the embedded stem also makes the next steps easier. In or.......

Discussion

A. thaliana stem sections are widely used to study the organization of the cells in the secondary cell wall and to qualitatively examine the differences between wild type and transgenic plants. The commonly used techniques for sectioning the specimens are direct hand cutting; or when specimens are embedded in agarose or fixative, sectioning can be done with a vibratome or microtome. In contrast to hand cutting, the last two allow reducing the risk of producing inaccurate differences between samples that would be.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agarose	EMD	MERC2125	CAS Number: 9012-36-6
Phloroglucinol	Sigma	P 3502	1,3,5-trihydroxybenzene [CAS Number: 108-73-6]
Hydrochloric acid	EMD	HX0603-75	CAS Number: 7647-01-0
Ammonium hydroxide	EMD	AX1303-6	CAS Number: 1336-21-6
Toulidine Blue O	Sigma	T3260	Blutene chloride, Tolonium Chloride [CAS Number 92-31-9]
Potassium permanganate	Sigma	223468	CAS Number 7722-64-7
Ethanol 190 proof	KOPTEC	V1401	CAS Number: 64-17-5
Congo Red	Sigma	C6277	Disodium 3,3'-[[1,1'-biphenyl]-4,4'-diylbis(azo)]bis(4-aminonaphthalene 1-sulphonate) [CAS Number 573-58-0]
Fluorescent Brightener 28/ Calcofluor White Stain	Sigma	F3543	4,4'-Bis[[4-[bis(2-hydroxyethyl)amino]-6-anilino-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonic acid [CAS Number 4404-43-7]
Vibratome	Leica	Leica Vibrating blade microtome VT1000S	http://www.leicabiosystems.com/products/sectioning/vibrating-blade-microtomes/details/product/leica-vt1000-s/
Razor	American Safety razor company	Item # 60-0139-0000	Stainless Steel Double Edge Blade (Personna Super)
Screw Cap Microcentrifuge Tubes (2 ml)	VWR	16466-044
Microcentrifuge Tubes (0.6 ml)	Axygen Scientific	MCT-060-C
Mitt	Bel-Art	380000000	SCIENCEWARE  Hot Hand Protector Mitt
Tissue adhesive	Ted Pella Inc	10033	Store at 4 °C or 20 °C for 3 months or longer storage
Microwave	Panasonic	NN-SD762S	PELCO Pro CA 44 Instant tissue adhesive
Camera with CCD chip with no mechanical shutter	Hamamatsu	C4742-95
High speed color camera	QImaging	MicroPublisher 5.0 RTV
Camera software	Molecular Devices	MetaMorph version 7.7.0.0
Imagining anaylsis	Adobe	Photoshop CS4
Micro Cover Glasses, Square, No. 1	VWR	48366-067	22 x 22 mm (7/8 x 7/8")-Cover glasses are corrosion-resistant and uniformly thick and flat. No. 1 thickness is 0.13 to 0.17 mm.
Frosted Micro Slides, 1 mm	VWR	48312-003	75 x 25 mm - 1 mm
Parafilm M	Alcan packaging	BRNDPM998
TX2 Filter cube	Leica	11513851/11513885	Filter used for Congo red analysis with a band-pass of 560/40.