4.2—
Thylakoid Structure

4.2.1—
Thin Sectioning and Heavy Metal Shadowing

When thin sections of fixed and stained leaf material are examined in the electron microscope, the chloroplast thylakoid membrane appears as a single electron dense line or as a tripartite structure (two dense lines with an intermediate light line) characteristic of the unit membrane (chapter 2). In high resolution electron micrographs of thin sections, fixed in permanganate, the thylakoid membranes give the appearance of containing globular subunits, 9 nm in diameter. It is possible, however, that these are an artefact since it is difficult to understand how several layers of overlapping 9 nm particles can be clearly resolved in a section thicker than 40 nm (Branton, 1968).

Some of the earliest electron micrographs of thylakoid membranes were obtained simply by drying isolated chloroplast fragments of Spirogyra and Mougeotia on an electron microscope grid and shadowing the preparations with chromium (Steinmann, 1952). The membranes showed a granular or particulate structure. Similar observations were made subsequently with spinach thylakoid membranes (Park & Pon, 1961). The particles usually existed in a random array but sometimes they were seen in highly ordered arrays. Their dimensions were 15 × 18 × 10 nm and they appeared to contain subunits. Park (1962) suggested

that the particle, which he termed quantasome, might be the morphological expression of the so-called photosynthetic unit, defined as the minimum number of chlorophyll molecules needed to fix one molecule of carbon dioxide per intense flash of light (Emerson & Arnold, 1932). The quantasomes were seen on the inner surface of the thylakoid membrane, suggesting that there was some distortion of the thylakoid membrane during drying.

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.)

4.2.3—
Negative Staining

Two sizes of particles are observed on the surface of unwashed thylakoids by negative staining with phosphotungstic acid. The larger particles (11 nm in diameter) are removed by water washing the thylakoids and correspond to the CO₂ -fixing enzyme, ribulose bisphosphate carboxylase. The smaller particles (9 nm) are removed by washing with the chelating agent, ethylenediamine tetraacetate, and are identical with the coupling factor (CF₁ ) which catalyses the terminal steps in the energy coupling process of ATP formation. These surface particles are not related to the membrane particles seen by freeze-etching (Park & Sane, 1971).