Photosynthetica 2005, 43(4):509-517 | DOI: 10.1007/s11099-005-0083-1

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

H. Synkova1,*, R. Schnablova1,2, M. Husak3, P. Siffel4, L. Bumba4,5, I. Hunalova4, N. Cerovska1, F. Vacha3,4
1 Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Praha 6, Czech Republic
2 Department of Plant Anatomy and Physiology, Faculty of Sciences, Charles University, Praha 2, Czech Republic
3 Institute of Physical Biology, University of South Bohemia, Nove Hrady, Czech Republic
4 Institute of Plant Molecular Biology, Academy of Sciences of the Czech Republic, Ceske Budejovice, Czech Republic
5 Faculty of Biological Sciences, University of South Bohemia, Ceske Budejovice, Czech Republic

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.

Additional key words: aggregates; chloroplast ultrastructure; cytokinins; fluorescence emission spectra; light-harvesting complex proteins; Nicotiana; three-dimensional reconstruction

Received: July 29, 2004; Accepted: March 7, 2005; Published: December 1, 2005  Show citation

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Synkova, H., Schnablova, R., Husak, M., Siffel, P., Bumba, L., Hunalova, I., Cerovska, N., & Vacha, F. (2005). Isolation and characterization of paracrystalline structures from transgenic Pssu-ipt tobacco. Photosynthetica, 43(4), 509-517. doi: 10.1007/s11099-005-0083-1
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