Ultrastructure of the thylakoid membrane in tomato leaf chloroplast revealed by liquid helium rapid-freezing and substitution-fixation method

The ultrastructure of the chloroplast in tomato leaves was studied by a liquid helium rapid-freezing and substitution-fixation method. Distinct morphological differences were observed between the ultrastructure of the thylakoid membrane fixed by this method and the one fixed by the conventional chemical fixation method. The membrane was asymmetrical and consisted of triple-layered leaflets. While the leaflet facing the stroma (outer leaflet) was very electron-dense, the one facing the thylakoid lumen (inner leaflet) was less electron-dense. The triple-layered leaflets showed a structural difference between the grana thylakoid and the stroma thylakoid. In the grana region, where thylakoid membranes are appressed, the part of outer leaflets were looked like fused or adhered to each other making one highly electron-dense layer composed of linings of particles (about 4 nm in diameter). The inner leaflet of the grana thylakoid membrane showed discontinuous structure. This discontinuous structure was composed of a cluster of irregular particles (about 2–3 nm in diameter). The size of cluster varied from about 10nm to 16nm. The discontinuity of the inner leaflet, i.e. linings of the clusters at rather constant intervals, was not observed clearly in the stroma thylakoid. The liquid helium rapid-freezing and substitution-fixation method can be used to observe the details of protein complexes in the thylakoid membrane organization.