Čeština
EnglishIsolation and characterization of paracrystalline structures from transgenic Pssu-ipt tobacco
Synková, Helena; Schnablová, R.; Hušák, M.; Šiffel, Pavel; Bumba, Ladislav; Hunalová, Ivana; Čeřovská, Noemi; Vácha, František
PHOTOSYNTHETICA 43: 509-517, 2005
Klíčová slova: chloroplast ultrastructure; cytokinins; light-harvesting complex proteins
Abstrakt: 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. The results obtained from chlorophyll (Chl) fluorescence emission spectra at 77 K indicated the presence of aggregates of light-harvesting complex proteins (LHC II) that was not connected to reaction centres of Photosystem II 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 LHC II 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 chlorophyll a/b proteins in transgenic plants compared to the control. Electron microscopic observations and immunogold labelling with specific LHC II 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.
DOI:
Autoři z ÚEB: Noemi Čeřovská, Helena Synková
PHOTOSYNTHETICA 43: 509-517, 2005
Klíčová slova: chloroplast ultrastructure; cytokinins; light-harvesting complex proteins
Abstrakt: 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. The results obtained from chlorophyll (Chl) fluorescence emission spectra at 77 K indicated the presence of aggregates of light-harvesting complex proteins (LHC II) that was not connected to reaction centres of Photosystem II 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 LHC II 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 chlorophyll a/b proteins in transgenic plants compared to the control. Electron microscopic observations and immunogold labelling with specific LHC II 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.
DOI: