In this chapter, I describe how AFM can be applied to measure
the mechanical properties of plant cell walls. The interdigitated
epidermal pavement cells from Arabidopsis thaliana leaf are used
as a model in this study [33]. Epidermal cells are surrounded by two
types of walls: thick and long periclinal walls (parallel to the leaf
surface), and very short and thin anticlinal walls (perpendicular to
the leaf surface). I focus on the anticlinal cell walls, which are
hidden in the cleft between periclinal walls and are covered by a
thick layer of wax. Measuring the mechanical properties in anticlinal
pavement cell walls is very challenging due to several factors that
influence the mechanical measurement such as the cell wall thickness; direction and distribution of wall stiffnesses; cell, tissue, and
organ geometry; and turgor pressure [34]. Anticlinal walls are also
very short (less than 1 μm long) and the leaf surface is irregular,
making it difficult to capture many anticlinal walls on the same
plane. To overcome all the abovementioned difficulties, the leaf
pieces are embedded in resin and cut into ultrathin paradermal
sections. This allows to perform high-resolution AFM indentation
and to measure local stiffness within different regions of anticlinal
cell walls (including the mix of cellulose and different matrix polysaccharides) [33, 35].
2 Materials
2.1 Plant Cultivation
1. Arabidopsis thaliana wild type and selected transgenic lines.
2. Ethanol–Tween-20 (70%; 0.1%).
3. Ethanol (95%).
4. Square Petri dishes (120 mm).
5. Plant agar.
6. Murashige and Skoog (MS) basal medium.
7. Sucrose.
8. Analytical balance.
9. Growth chamber.
2.2 Sample
Preparation
The preparation of resin-embedded sections described here is
adapted from the sample preparation protocol for transmission
electron microscopy [36].
1. Disodium phosphate (Na 2 HPO 4 ) (0.1 M).
2. Monosodium phosphate (NaH 2 PO 4 ) (0.1 M).
3. Phosphate buffer solution (0.1 M, pH 7.2) [37].
4. Tweezers with thin tips.
5. Paraformaldehyde (PFA) for histology.
354
Mateusz Majda
the mechanical properties of plant cell walls. The interdigitated
epidermal pavement cells from Arabidopsis thaliana leaf are used
as a model in this study [33]. Epidermal cells are surrounded by two
types of walls: thick and long periclinal walls (parallel to the leaf
surface), and very short and thin anticlinal walls (perpendicular to
the leaf surface). I focus on the anticlinal cell walls, which are
hidden in the cleft between periclinal walls and are covered by a
thick layer of wax. Measuring the mechanical properties in anticlinal
pavement cell walls is very challenging due to several factors that
influence the mechanical measurement such as the cell wall thickness; direction and distribution of wall stiffnesses; cell, tissue, and
organ geometry; and turgor pressure [34]. Anticlinal walls are also
very short (less than 1 μm long) and the leaf surface is irregular,
making it difficult to capture many anticlinal walls on the same
plane. To overcome all the abovementioned difficulties, the leaf
pieces are embedded in resin and cut into ultrathin paradermal
sections. This allows to perform high-resolution AFM indentation
and to measure local stiffness within different regions of anticlinal
cell walls (including the mix of cellulose and different matrix polysaccharides) [33, 35].
2 Materials
2.1 Plant Cultivation
1. Arabidopsis thaliana wild type and selected transgenic lines.
2. Ethanol–Tween-20 (70%; 0.1%).
3. Ethanol (95%).
4. Square Petri dishes (120 mm).
5. Plant agar.
6. Murashige and Skoog (MS) basal medium.
7. Sucrose.
8. Analytical balance.
9. Growth chamber.
2.2 Sample
Preparation
The preparation of resin-embedded sections described here is
adapted from the sample preparation protocol for transmission
electron microscopy [36].
1. Disodium phosphate (Na 2 HPO 4 ) (0.1 M).
2. Monosodium phosphate (NaH 2 PO 4 ) (0.1 M).
3. Phosphate buffer solution (0.1 M, pH 7.2) [37].
4. Tweezers with thin tips.
5. Paraformaldehyde (PFA) for histology.
354
Mateusz Majda