Isolation and characterization of paracrystalline structures from transgenic Pssu-ipt tobacco

Abstract

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. Threedimensional reconstruction of chloroplasts and crystalloids proved that paracrystalline structures varied moderately in their size and took up a significant portion of total chloroplast volume.

Three-dimensional reconstruction of anomalous chloroplasts in transgenic ipt tobacco

“…We can only speculate on the fact that, if any imbalance in LHC apoprotein and Chl a and b synthesis caused by CKs exists, then it could probably be the mechanism used to balance the optimal functioning of the photosynthetic apparatus again. This is supported by the fact that those aggregates are not connected to photosystems and photosynthetic complexes in normal thylakoid membranes, which was proved by spectrofluorometric analysis (Synkova´et al 2005).…”

Chlorophyll a fluorescence as a tool for a study of the Potato virus Y effects on photosynthesis of nontransgenic and transgenic Pssu-ipt tobacco

“…non-Q Breducing or silent reaction centres), which may act as dissipative sinks, thereby decreasing F 0 (Öquist et al 1992). In our previous study, Chl fluorescence emission spectra at 77 K indicated the presence of aggregates of light-harvesting complex proteins (LHCII) that were not connected to reaction centres of PSII in Pssu-ipt transgenic tobacco (Synková et al 2005). Lower F m is again an indication of the energy dissipation via nonphotochemical pathways.…”