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
Budějovice
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 Spurr’‘s
resin. Ultrathin sections were stained by uranyl acetate and Reynold’‘s
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.