4.2.2—
Freeze-Etching

The freeze-etch technique is a different approach to the study of the substructure of the thylakoid membrane, because chemical fixatives are not used. Leaf material or isolated chloroplasts are frozen and fractured (Moor et al., 1961). The fracture plane in the frozen sample occurs along hydrophobic regions within the thylakoid membrane exposing complementary faces. Platinum-carbon replicas of the fracture planes are made and examined in the electron microscope.

Two major sizes of particles are seen when mature plant chloroplasts are freeze-fractured; large particles of average diameter, 17.5 nm and small particles of average diameter, 11 nm (Fig. 4.1a). The small and large particles occur on different fracture faces (Fig. 4.1b). Fracture face C, which is towards the stroma

Figure 4.1a
Freeze-fracture electron micrograph of a spinach grana thylakoid, showing
the large and small particle fracture faces, B and C, and the surfaces, A' and D.
(By courtesy of Dr. D.J. Goodchild.)

of the chloroplast contains tightly packed small particles, and the complementary fracture face B contains the large particles at about half the density per µm² . The large particles are only observed within grana stacks, where the adjacent thylakoid membranes are in close contact, while the small particles are observed in grana and stroma thylakoids. In the unstacked stroma thylakoids the complementary fracture face (designated B_u ) is relatively smooth and shows only a few small particles (Park & Sane, 1971).

Figure 4.1b
Model of the thylakoid membrane, showing grana and stroma regions.
The particles on the A' surface represent the coupling factor (CF₁ ).
(By courtesy of Dr. D. J. Goodchild.)

The exterior thylakoid surface, A', and the interior surface, D, are observable by the deep etch technique, in which the ice covering the surfaces is sublimed off before the replica is made. In well-washed thylakoids the A' surface shows little surface relief, but is covered with proteins in unwashed lamellae. The interior surface is covered with particles about 18.5 × 15.5 nm in size which show slight surface relief. These particles on the D surface contain 3 or 4 subunits and they may correspond to the quantasomes as seen by metal shadowing.

The freeze-etch data suggest that the particles within the thylakoid membrane are arranged somewhat asymmetrically. The larger particles are located towards the inner surface and partly protrude into the intrathylakoid space, and the smaller particles are near the outer surface. The model of the thylakoid membrane shown in Fig. 4.2 appears to be consistent with the freeze-etching results and also with X-ray diffraction studies of thylakoid membranes (Sadler et al., 1973). The membrane is viewed as a lipid bilayer in which the particles are embedded, in accordance with the fluid mosaic model for biological membranes (Singer & Nicolson, 1972; see chapter 2).

Figure 4.2
Structural model of the thylakoid membrane showing an asymmetric
distribution of large and small particles. The two membranes belong to
one thylakoid. The lipid molecules are indicated as a bilayer, the circles
representing the polar head groups. Lipid asymmetry is indicated by the
shading of the head groups. The model appears to be consistent with freeze
-etch electron microscopy and X-ray diffraction of thylakoid membranes.
(From Sadler et al.,  1973.)