Plant Cell Advance Publication. Published on March 21, 2016, doi:10.1105/tpc.16.00230
IN BRIEF
3D Visualization of Thylakoid Membrane Development
Proper biogenesis of the chloroplast is
essential for all photosynthetic plant cells. As
germinating seedlings are exposed to light,
etioplasts in young mesophyll cells become
chloroplasts in the developing seedling. The
chloroplast thylakoids develop from a
paracrystalline tubular structure that is
transformed when illuminated into a series of
flattened membranes that become the grana
thylakoids. Traditional transmission electron
microscopy (TEM) shows subcellular fine
structure in two-dimensional thin slices, but a
comprehensive study of chloroplast biogenesis
requires three-dimensional (3D) visualization
of membrane transformations.
Electron tomography (ET) creates 3D
reconstructions of subcellular objects by taking
a series of traditional TEMs collected at
different angles and using computer algorithms
to generate detailed views of the surfaces of
structures (Daum and Kühlbrandt, 2011;
Shimoni, et al., 2005). Kowalewska et al.
(2016) have used ET and confocal laser
scanning microscopy to follow the
development of stacked grana thylakoids as
etiolated runner bean seedlings are exposed to
light. They characterize membrane structure
during the first three days of illumination (under
a 16-h light/8-h dark photoperiod) of darkadapted seedlings.
After 8 days of etiolation, etioplasts in
dark-adapted seedlings showed a regular
arrangement of branched membrane tubules
These tubular units are joined as a
paracrystalline network in the prolamellar body
(PLB), much like the cells in a honeycomb (see
figure, A and B). As soon as one hour after
illumination, the symmetry of the PLB was lost
as the internal membranes rearranged (figure,
C). The tetrahedral network was no longer
visible and the margins of the tubules
developed flattened stromal slats that would
develop into prothylakoids.
After 2 h of illumination, only a remnant of
the PLB was present and membranes became
organized parallel to the long axis of the
chloroplast (figure, D). These PLB remnants
were continuous, as opposed to the porous
membranes seen in prothylakoids (PTs). By 4
h, the PLB was transformed into a parallel
arrangement of PTs with local connections,
often split dichotomously into two adjacent
Thylakoid development in three dimensions. As dark-adapted runner bean seedlings
are illuminated, the paracrystalline prolamellar body of the etioplast (A and B) becomes
disorganized (C and D), as membranes flatten (E and F), and are transformed into stacked
grana (G and H) . (Reprinted from Kowalewska, et al. [2016]
branches (figure, E). By 8 h, the first stacked
membranes had appeared (figure, F). The
appressed thylakoids were no longer porous
and membranes were seen to be continuous in
stacked regions, with each stacked membrane
connected to a single split PT.
By the beginning of the second day, small
grana stacks were observed, and both stroma
thylakoids and grana thylakoids were evident
(figure, G). Each stroma thylakoid was
connected to two adjacent grana thylakoids at
an angle of approximately 20°, as seen by
Austin and Staehelin (2011). By day three,
stroma thylakoids had split dichotomously,
connecting with two grana thylakoids, except at
the top and bottom of the grana stack, where
the stroma thylakoid connected to only one
grana thylakoid (figure, H).
The entire transformation of the
paracrystalline tubular membrane network of
the PLB to the organized, stacked grana
thylakoid membranes is shown in a
supplemental movie created by the authors in
2D and 3D (this wonderful video should not be
missed!). Every stage of thylakoid
development is shown in a traditional TEM
view, outlining the membranes to be visualized
in 3D. Then, the 3D structures of the outlined
areas are presented and rotated to show
membrane surfaces from many angles during
the membrane transformations.
The authors also use confocal laser
scanning microscopy to monitor red
chlorophyll fluorescence, as it helps to show
the distribution of appressed thylakoids. And,
because chlorophyll-protein complexes affect
membrane stacking, they use low temperature
fluorescence emission and excitation
spectroscopy
to
characterize
these
complexes. The authors complement this work
with mild-denaturing electrophoresis and
immunodetection of the chlorophyll-protein
complexes. In summary, the authors propose
a theoretical model of the membrane changes
during grana development and greatly
contribute to our understanding of chloroplast
and thylakoid biogenesis.
Gregory Bertoni
Science Editor
[email protected]
ORCID ID: 0000-0001-7977-3724
REFERENCES
Austin, J.R. and Staehelin, L.A. (2011).
Three-dimensional architecture of grana and
stroma thylakoids of higher plants as
determined by electron tomography. Plant
Physiol. 155: 1601–1611.
Daum, B. and Kühlbrandt, W. (2011).
Electron tomography of plant thylakoid
membranes. J. Exp. Bot. 62: 2393–2402.
Kowalewska, L., Mazur, R., Suski, S.,
Garstka, M., and Mostowska, A. (2016).
Three-dimensional visualization of the
tubular-lamellar transformation of the
internal plastid membrane network during
runner bean chloroplast biogenesis.. Plant
Cell 10.1105/tpc.15.01053.
Shimoni, E., Rav-Hon, O., Ohad, I.,
Brumfeld, V., and Reich, Z. (2005). Threedimensional organization of higher-plant
chloroplast thylakoid membranes revealed
by electron tomography. Plant Cell 17:
2580–2586.
©2016 American Society of Plant Biologists. All Rights Reserved.
3D Visualization of Thylakoid Membrane Development
Gregory Bertoni
Plant Cell; originally published online March 21, 2016;
DOI 10.1105/tpc.16.00230
This information is current as of March 21, 2016
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ADVANCING THE SCIENCE OF PLANT BIOLOGY
3D Visualization of Thylakoid Membrane Development
Gregory Bertoni
Plant Cell; originally published online March 21, 2016;
DOI 10.1105/tpc.16.00230
This information is current as of June 18, 2017
Supplemental Data
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