Membrane pseudo-crystal structures in Pssu-ipt tobacco chloroplasts


Helena Synková1, Renáta Pechová3, Michal Hušák2, František Vácha 2, Pavel Šiffel2


1 Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Na Karlovce 1a,

  CZ-160 00 Praha 6

2 Insitute of Physical Biology, University of South BohemiaLaboratory of Biomembranes, South Bohemian University, Branišovská 31, 370 05 České


3 Department of Plant Physiology, Faculty of Sciences, Charles University, Viničná 5, CZ-128 44   Praha 2, Czech Republic


Our study is focused on  native pseudo-crystalline structures, which were observed in chloroplasts of transgenic tobacco overproducing plant hormones cytokinins. The structures were not positively identified until now. We suppose that they are formed by light harvesting protein (LHC) aggregating in a form of 2D crystal, which then constitute membrane stacks. Our hypothesis is supported by fluorescence emission spectra, which showed certain bands corresponding to LHC aggregates and higher emission of chlorophyll b in chloroplasts izolated from transgenic plants.

The aim of this experiment was the estimation of relative size of pseudo-crystals compared to chloroplast and the size of basic cell unit, which can be determined from analysis of TEM images from ultrathin sections of leaves and isolated chloroplast suspensions.

Transgenic tobacco containing a supplementary ipt-gene under a control of the promoter for the small subunit of RuBPCO (Pssu-ipt) was grown as grafts on non-transgenic tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) rootstock as described by Synková et al. (J. Plant Physiol.155 : 173-182, 1999) or as rooted plants (kanamycin resistant progeny of the transgenic grafts). Samples for TEM were taken from the central part of the young fully developed leaf or isolated chloroplast suspension and after overnight fixation in 3 % glutaraldehyde were embedded in Spurrs resin. Ultrathin sections were stained by uranyl acetate and Reynolds lead citrate and examined in JEM 1010 (Jeol, Japan). Analysis of serial sections by program IMOD 2.42 enabled three dimensional (3D) reconstructions of chloroplasts and pseudo-crystals. The size of basic structural unit was calculated using MRC Cambridge Image Processing System (1994).

3D reconstruction showed that pseudo-crystalline structures occupy up to 20 % of chloroplast volume (at least in that part of chloroplast which was studied).

The average size of basic cell unit was calculated as: a = 11 nm, b = 12 nm, γ = 100o.This size parameters support our hypothesis, although dehydration preceedingpreceding embedding in epoxide resin cause a shrinkage of natural structures. Therefore further experiments are needed to prove it.




The authors thank to Dr.J. Nebesářová and the staff of her laboratory (IP, Lab.of EM, České Budějovice) for the assistenceassistance  in electron microscopy. The work was supported by grant of the Grant Agency of the Czech Republic No. 206/01/1061.