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IN BRIEF
Observe Them in Their Native Habitat: Atomic Force Microscopy of
Photosynthetic Complexes in Thylakoid Membranes
The distribution of photosynthetic complexes within heterogeneous thylakoid
membranes has been a subject of study
for many years (reviewed in Nevo et al.,
2012). Thylakoids of vascular plants include
granal stacks and nonstacked regions called
stromal lamellae; the small amount of space
between the stacked grana limits which
protein complexes can fit in that region.
Photosystem I (PSI) and ATP synthase, both
of which have large domains that protrude
into the stroma (outside the thylakoid), are
found in the nonappressed stromal lamellae.
Photosystem II (PSII) has smaller stromal
protrusions and is present mainly in the
appressed granal membranes. The distribution of the fourth complex associated with
photosynthesis, cytochrome b6f (cytb6f ),
remains a matter of debate. Cytb6f carries
out electron transfer reactions linking PSII
and PSI: It oxidizes plastoquinone (the electron acceptor for PSII) and reduces plastocyanin (the electron donor for PSI). Thus, its
distance from PSI and PSII is mechanistically
important, determining how far the electron
carriers must diffuse. Whereas some studies
have suggested an even distribution of cytb6f
throughout the thylakoid membrane, other
approaches have supported its exclusive
localization in the stromal lamellae region.
Now, Johnson et al. (2014) report direct
evidence that a large proportion of cytb6f
complexes are found in the grana stacks.
Johnson and coworkers prepared granal
membranes from spinach (Spinacia oleracea)
thylakoids using three different methods,
one of which led to loss of cytb6f from the
samples. Since the PSII and cytb6f complexes possess a similar lateral size and
shape, the authors instead used the exquisite z-resolution of atomic force microscopy (AFM) to differentiate them by their
heights. They found two populations of
topographical features on the lumenal side
of the granal membranes. One of ;4 nm
was found in all preparations, and another
of ;3 nm was absent in the preparation
lacking cytb6f. Together with spectroscopic
www.plantcell.org/cgi/doi/10.1105/tpc.114.130161
complex, the interaction between plastocyanin and cytb 6 f increased the force
needed to pull the probe back (see figure).
This mapped the areas of interaction between plastocyanin and cytb6f, revealing
the location of cytb6f within the samples. In
addition, it quantified the force needed to
pull plastocyanin from cytb6f at the singlemolecule level. Furthermore, the topography of the membrane could be analyzed
simultaneously, such that areas of interaction could be correlated with specific height
features. Together, these analyses supported
the assignment of the 4- and 3-nm features
as PSII and cytb6f, respectively.
Overall, Johnson et al. performed a compelling analysis of large protein complexes
in native membranes with only minimal
disruption. In doing so, they provide strong
evidence that cytb6f is surrounded by PSII
in nanodomains within granal stacks (see
figure), an arrangement that would limit the
distance plastoquinone is required to travel
during electron transport in the extremely
protein-crowded granal membranes.
Nanodomains of cytb6f and PSII in thylakoid
membranes. Top: Interaction between AFM
probe loaded with plastocyanin (Pc) and cytb6f in
the sample increases the force required to
pull the probe off the sample. Bottom: This
affinity-mapping AFM allowed the positions
of cytb 6 f and PSII to be precisely assigned
in AFM images of the thylakoid granal membrane.
(Adapted from Johnson et al. [2014], Figures 2B
and 5D.)
data, these findings suggested that the 4-nm
feature represented PSII and the 3-nm
feature was cytb6f.
Johnson et al. used affinity-mapping
AFM (Vasilev et al., 2014) to confirm the
identity of the 3-nm features. Granal membrane samples were queried with an AFM
probe to which plastocyanin proteins
were attached. When this plastocyaninfunctionalized probe encountered a cytb6f
Nancy R. Hofmann
Science Editor
[email protected]
ORCID ID: 0000-0001-9504-1152
REFERENCES
Johnson, M.P., Vasilev, C., Olsen, J.D., and
Hunter, C.N. (2014). Nanodomains of cytochrome b6f and photosystem II complexes in
spinach grana thylakoid membranes. Plant
Cell 26: 10.1105/tpc.114.127233.
Nevo, R., Charuvi, D., Tsabari, O., and Reich,
Z. (2012). Composition, architecture and dynamics of the photosynthetic apparatus in higher
plants. Plant J. 70: 157–176.
Vasilev, C., Brindley, A.A., Olsen, J.D., Saer,
R.G., Beatty, J.T., and Hunter, C.N. (2014).
Nano-mechanical mapping of the interactions
between surface-bound RC-LH1-PufX core
complexes and cytochrome c 2 attached to
an AFM probe. Photosynth. Res. 120: 169–
180.
The Plant Cell Preview, www.aspb.org ã 2014 American Society of Plant Biologists. All rights reserved.
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Observe Them in Their Native Habitat: Atomic Force Microscopy of Photosynthetic Complexes in
Thylakoid Membranes
Nancy R. Hofmann
Plant Cell; originally published online July 28, 2014;
DOI 10.1105/tpc.114.130161
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