Inner structure of isolated intact chloroplasts was observed for the first time by a method of laser scanning microscopy at the temperature of liquid nitrogen at 77 K. The microscope, based on gradient index optics, has a maximum resolution of 440 nm at the wavelength of 650 nm. Chloroplasts were excited into the Q-band of chlorophyll b by a krypton laser line at 647.6 nm and fluorescence was detected using two different interference filters. The 680 nm interference filter detects the regions where photosystem (PS) 2 mainly occurs, the 730 nm interference filter detects domains with predominant location of PS1. Since PS1 occurs mainly in stroma lamellae, whereas PS2 occurs mainly in grana regions we were able to view the structure of thylakoid membrane in isolated intact chloroplast that is the closest to in vivo state. and F. Vácha ... [et al.].
Two types of photosystem 2-light-harvesting complex 2 (PS2-LHC2) supercomplexes with similar pigment and protein composition were isolated directly from thylakoid membranes by sucrose density gradient centrifugation. Electron microscopy and single particle image analysis revealed the first Type as single unpaired PS2-LHC2 supercomplexes, whereas the second Type was characterized as pairs of two PS2-LHC2 supercomplexes attached together by their stromal sides. Unstacking of thylakoid membranes resulted in a spontaneous disintegration of the paired supercomplexes into single unpaired particles. A model of the organisation of the pigment-protein complexes in grana region is proposed. and L. Bumba, M. Hušák, F. Vácha.
Distinct crystalloids were found in chloroplasts of transgenic Pssu-ipt tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) overproducing endogenous cytokinins. They were present both in rooted (T) and grafted (TC) transgenic plants contrary to control tobacco (C). The fractions enriched by crystalloids were isolated from chloroplasts using a continuous or a discontinuous Percoll gradient. Chlorophyll (Chl) fluorescence emission spectra at 77 K indicated the presence of aggregates of light-harvesting complex proteins (LHC2) that was not connected to reaction centres of photosystem 2 both in isolated chloroplasts and in the fraction of 80 % Percoll gradient from both types of transgenic tobacco. Further analyses, i.e. pigment contents, polypeptide composition by SDS-PAGE, and immunoblotting support our hypothesis that crystalloids inside chloroplasts of transgenic tobacco are formed by LHC2 aggregates. Treatment with two distinct detergents, chosen with respect to their effects (i.e. β-dodecyl maltoside or Triton X-100), resulted in different degree of disintegration of Chl a/b proteins in transgenic plants compared to the control. Electron microscopic observations and immunogold labelling with specific LHC2 antibodies carried on the resin embedded leaf sections or free suspensions of chloroplasts showed that gold particles were bound preferentially on the outer surface of crystalloids. Three-dimensional reconstruction of chloroplasts and crystalloids proved that paracrystalline structures varied moderately in their size and took up a significant portion of total chloroplast volume. and H. Synková ... [et al.].