High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Summary

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Abstract

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

Introduction

The plant cell wall is a dynamic structure. Growing cells are surrounded by a primary cell wall, the organization of which allows cells to expand. Cells that cease to grow deposit a more rigid secondary wall that enhances the mechanical support of the plant. Both cell walls are composed of cellulose microfibrils embedded in a matrix of polysaccharides of different structures (e.g., hemicellulose and pectin) that vary across the different developmental stage and tissues^1,2. Cellulose is synthesized as chains of (1,4)-β-D-glucan that are tightly aligned to form the microfibril of a crystalline structure. Amorphous cellulose refers to the regions....

Protocol

1. Plant Growth

1. Surface sterilize seeds.
   1. Sterilize seeds (up to 30 mg) by wet or dry method. As an example, for dry sterilization method, add 1 ml glacial HCl 37% in 50 ml bleach, in a glass beaker in a closed desiccator for at least 4 hr and up to overnight. Perform this step in a chemical hood.
2. Spread the sterile seeds on plates with 0.8% plant agar in 0.5x Murashige & Skoog (MS) medium. Wrap plates with Parafilm or porous tape and cover with aluminum foil.
3. Store the plates at 4 °C for 1 - 4 days to promote uniform germination.
4. Transfer the plates to a growth chamber suitable for growing

Results

We study the impact of cell type-specific responses to brassinosteroids (BRs), using the Arabidopsis root as a model organ^15-17. When the BR receptor BRI1 is targeted, in the background of bri1 mutant, to a subset of epidermal cells called non-hair cells (Figure 2A,B), it inhibits unidirectional cell expansion of neighboring cells, and whole-root growth¹⁵. Polscope analysis was performed to reveal the mechanism underlying this inhib.......

Discussion

Here, we present a method for determination of the accumulation of crystalline cellulose in the different tissues composing the Arabidopsis roots, while maintaining anatomical information. As such, it provides an additional step towards understanding growth processes in plants at a cellular resolution. This method can be also applied for the study of additional plant species and organs.

A number of points must be considered when applying the method. First, crystalline cellulose accumu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Murashige & Skoog (MS)	Duchefa	M0221
Borax	LOBA CHEMIE	6038
Methylene blue	Sigma	M-9140
Azure	Sigma	861065
Syringe Driven 0.22 mm PVDF Filter	MILLEX-GV
Glutaraldehyde	EMS	16220
Sodium Cacodylate	Sigma	C-0250
Leica kit historesin	Leica	7022 18 500
embedding molds	Agar Scientific	AGG3530
film 100 µ P.P.C.	www.Jolybar.com		overhead film
Knivemaker	LKB	7800
Ultratome III	LKB	8800	Ultratome
Shandon immumount	Thermo	9990402	mounting medium
light microscope	Nikon	Eclipse 80i
Abrio imaging system	CRI	Abrio imaging system
Abrio V2.2 software	CRI	Abrio V2.2 software
Open access polarized light image analysis software	OPS	OpenPolScope	http://www.openpolscope.org/