Arctic and alpine plants like Oxyria digyna have to face
enhanced environmental stress. This study compared leaves
from Oxyria digyna collected in the Arctic at Svalbard (788N)
and in the Austrian Alps (478N) at cellular, subcellular, and ultra-
structural levels. Oxyria digyna plants collected in Svalbard had
significantly thicker leaves than the samples collected in the
Austrian Alps. This difference was generated by increased thick-
ness of the palisade and spongy mesophyll layers in the arctic
plants, while epidermal cells had no significant size differences
between the two habitats. A characteristic feature of arctic, al-
pine, and cultivated samples was the occurrence of broad stro-
ma-filled chloroplast protrusions, 2 – 5 μm broad and up to 5 μm
long. Chloroplast protrusions were in close spatial contact with
other organelles including mitochondria and microbodies. Mito-
chondria were also present in invaginations of the chloroplasts.
A dense network of cortical microtubules found in the meso-
phyll cells suggested a potential role for microtubules in the for-
mation and function of chloroplast protrusions. No direct inter-
actions between microtubules and chloroplasts, however, were
observed and disruption of the microtubule arrays with the anti-
microtubule agent oryzalin at 5 – 10 μM did not alter the appear-
ance or dynamics of chloroplast protrusions. These observations
suggest that, in contrast to studies on stromule formation in
morphology of chloroplast protrusions in Oxyria digyna. The ac-
tin microfilament-disrupting drug latrunculin B (5 – 10 μM for
2 h) arrested cytoplasmic streaming and altered the cytoplas-
mic integrity of mesophyll cells. However, at the ultrastructural
level, stroma-containing, thylakoid-free areas were still visible,
mostly at the concave sides of the chloroplasts. As chloroplast
protrusions were frequently found to be mitochondria-associat-
ed in Oxyria digyna, a role in metabolite exchange is possible,
which may contribute to an adaptation to alpine and arctic con-
Actin, chloroplast, chloroplast protrusion, high Alps,
high arctic, latrunculin B, microfilaments, microtubules, oryza-
Arctic and alpine plants have to face enhanced environmen-
tal stress in their natural habitats. They have developed multi-
ple strategies to cope with this situation (for a summary see
Körner, 2003). Among physiological adaptations, temperature
tolerance and enhanced photosynthetic performance have
Oxyria digyna is a well studied arctic- and alpine-specialized
plant, with data available on numerous characters in different
ecotypes (Billings et al., 1961; Mooney and Billings, 1961; Bill-
ings et al., 1971; Chrtek and Sourková, 1992; Heide, 2005). Oxy-
by respiration inhibition and other adaptation mechanisms
(Semikhatova et al., 1985; Tolvanen and Henry, 2001). Protec-
tion against cold temperatures has been described (Engel et al.,
1986 a, b; Koroleva et al., 1994; Pyankov and Vaskorskii, 1994).
The plant has been investigated concerning oxalate metab-
olism (Bornkamm, 1969), unusual chemical compounds (Zhou
et al., 2001), the occurrence of epicuticular waxes (Lütz and
Gülz, 1985), and photosynthetic pigments (Gerasimenko et
al., 1993; Lütz and Holzinger, 2004). Non- and cold-acclimated
photoinhibition, zeaxanthin formation, and chlorophyll fluo-
rescence quenching (Koroleva et al., 1994).
Despite its adaptability to withstand arctic and alpine environ-
mental conditions, Oxyria digyna can also be cultivated under
standard laboratory conditions. The maximum total photosyn-
thesis was found to range between 21 8C and 28 8C (Engel et al.,
1986 a; Koroleva et al., 1994; Kurets et al., 2002), a feature that
may explain why cultivation is relatively easy. This is impor-
Investigating Cytoskeletal Function in Chloroplast Protrusion
Formation in the Arctic-Alpine Plant Oxyria digyna
, G. O. Wasteneys
, and C. Lütz
Department of Physiology and Cell Physiology of Alpine Plants, Institute of Botany, University of Innsbruck, Sternwartestraße 15,
Department of Botany, University of British Columbia, 6270 Univ. Blvd., Vancouver BC, V6T 14Z, Canada
Plant Biol. 9 (2007): 400 – 410
© Georg Thieme Verlag KG Stuttgart · New York
DOI 10.1055/s-2006-924727 · Published online January 19, 2007
imaging, cryo-preservation or immunolabelling.
The present study focuses on chloroplast behaviour and chlo-
roplast interactions with the cytoskeleton and with other or-
ganelles. The latter are likely to play important roles in phys-
iological responses. Therefore, in situ fixation for the transmis-
sion electron microscope (TEM) has been performed at the
growth site to document adaptation of the plants in their nat-
ural environment. The first examination at the ultrastructural
level of alpine-adapted plants involved Ranunculus glacialis, a
plant growing up to 4270 m a.s.l. in the European Alps (Lütz
and Moser, 1977; Lütz, 1987). A large number of microbodies
with high catalase activity were described. These microbodies,
as well as mitochondria, were found to be in close contact with
long chloroplast protrusions (described as “proliferations” in
these publications). Chloroplast protrusions consist of broad
thylakoid-free stroma portions, occurring mostly on the lati-
tudinal ends of chloroplasts. A possible interaction between
these organelles to enhance photorespiration and protect
against photoinhibition was discussed, but not studied. Chlo-
roplast protrusions have also been described for the two high-
er plant species found in Antarctica (Gielwanowska and Szczu-
ka, 2005; Lütz et al., 2006), suggesting that this phenomenon
is common to plants growing in both alpine and polar environ-
ments. A recent analysis of cell structures in leaves from six
high-alpine plants has shown that chloroplast protrusions are
very abundant and well-developed in Oxyria digyna (Lütz and
Are these chloroplast protrusions of alpine and arctic plants
analogous to the recently defined stromules? Stromules are
thin (less than 800 nm wide) stroma-filled projections of plas-
tids, and most prominent in non-green plastids, e.g., leuco-
plasts (Köhler and Hanson, 2000; Gray et al., 2001; Kwok and
Hanson, 2004 a). In contrast, the chloroplast protrusions of
alpine and arctic plants are significantly larger and appear in
photosynthetic tissues. Despite these differences, they may,
like the speculated function of stomules, facilitate organelle
contact, increase chloroplast surface area and stromal content.
In this study, we compared structural features of leaves of Oxy-
ria digyna from arctic and high alpine growth sites. Morpho-
logical differences were conspicuous at the tissue level, while
prominent chloroplast protrusions were found in mesophyll
cells from both ecotypes. The cytoskeleton plays a role in chlo-
roplast organization (Kandasamy and Meagher, 1999), and
there is some evidence that stromule formation depends on
the activities of actin microfilaments (MFs) and microtubules
(MTs) (Kwok and Hanson 2003, 2004 b). We therefore exam-
ined cytoskeletal involvement in the formation and behaviour
of chloroplast protrusions through the use of MT and MF dis-
Materials and Methods
Plant material and study sites
Specimens of Oxyria digyna were collected in Svalbard (Spitz-
bergen) in Ny-Ålesund, Norway, 78855
′N, 11856′E, 10 m a.s.l.
and in the Austrian Alps in the Pitztal 47834
2600 m a.s.l. near Innsbruck, Austria. Additionally, plant mate-
rial was raised from seeds collected in the Austrian Alps and
cultivated in a growth chamber at 20 8C, 14 h light (approx.
400 μmol photons m
Cryo scanning electron microscopy was performed with a
Hitachi S 4700 Cryo SEM. Oxyria digyna leaves were rapidly
frozen in sub-cooled liquid nitrogen at approx. – 210 8C in an
Emitech K 1250 freezing device. Samples were then kept at
about – 170 8C and examined with or without gold coating
in a vacuum of 1 – 5 × 10
mbar. Excess water was sublimed
vacuum chamber for 20 min.
Leaf sections were treated with 5 μM and 10 μM oryzalin
(Supleco, Bellefonte, USA) or 5 μM and 10 μM latrunculin B
(Sigma) for 2 h, with DMSO used as a solvent in all treatments
and controls at 0.2 %. Control and drug-treated cells were
examined with a Zeiss 200 M microscope using a 63 × 1.4 NA
objective lens. Images were collected with a Zeiss Axoicam
MCR5. Inhibitor-treated samples were fixed for TEM as de-
MT staining was carried out by the freeze shattering method
(Wasteneys et al., 1997). Leaf samples were fixed for 40 min
in 0.5 % glutaraldehyde and 1.5 % formaldehyde in buffer con-
taining 50 mM PIPES, pH = 7.2, 2 mM EGTA, 2 mM MgSO
ton X-100. Samples were then frozen in liquid nitrogen and
freeze-shattered between two glass slides by compressing
them with a pair of pliers. Samples were transferred to perme-
abilization buffer containing PBS and 1 % Triton X-100 for 1 h.
Subsequently, samples were transferred to PBS (pH = 7.4) for
10 min, followed by a 20-min incubation in PBS containing
1 mg · ml
. Primary antibody incubation (Sigma B 512
anti alpha tubulin, 1 : 1000) was carried out overnight at 4 8C,
secondary antibody (Alexa conjugated goat anti-mouse IgG,
1 : 200) was applied for 1 h at 37 8C. Samples were mounted in
Citifluor AF1 antifade agent and examined with a Biorad Multi-
photon microscope using Lasersharp 2000 software or a Zeiss
Pascal CLSM, 63 × 1.4 NA objective lens. Excitation was gener-
ated with an argon laser at 488 nm. Long pass (LP) 560 nm fil-
tered emission and band pass (BP) 505 – 530 nm filtered emis-
sion were recorded simultaneously. Z stacks were captured
and projections generated with ImageJ software (freeware).
Approximately 2 × 2 mm pieces of freshly harvested leaves
were cut with a razor blade and fixed with 2.5 % glutaraldeyde
for 2 h in 50 mM sodium cacodylate buffer, pH 7.0 at 20 8C,
rinsed and post-fixed in 1 % OsO
in the same buffer for 12 h
of ethanol (10%, 20 %, 40 %, 60 %, 80 %, 90 %, 95 %, 100%), each for
20 min, infiltrated with Spurr’s resin (Serva, Heidelberg, Ger-
many), and polymerized for 8 h at 70 8C. Ultrathin sections
were post-stained and examined with a Zeiss EM 902 trans-
mission electron microscope or a Zeiss Libra 120 EFTEM at
80 kV or 120 kV. Images were captured with a ProScan Slow
Cytoskeleton-Independent Chloroplast Protrusions
Plant Biology 9 (2007)
ing System GmbH).
In addition, semithin sections (0.7 μm) were toluidine blue-
stained and examined on a Zeiss Axiovert 200 light microscope
with a 63 × 1.4 NA objective lens for measurements of leaf, epi-
dermis, palisade, and spongy parenchyma thickness.
Thickness of the leaves, epidermis, palisade, and spongy pa-
renchyma were compared between samples collected in the
Austrian Alps and samples collected in Svalbard (n = 10). The
comparison was undertaken on semithin sections prepared
from the different samples. Mean value ± standard deviation
(SD) calculations and t-test comparisons were undertaken us-
ing SPSS software (SPSS Inc. Chicago, Ill.).
of embedded samples (Figs. 1 a, b) and by cryo SEM (Figs. 1 c, d).
The adaxial epidermis contained irregularly shaped pavement
cells, the abaxial epidermis had additional secretory trichomes
and stomatal guard cells (Figs. 1 a – c). The leaves had a bifa-
cial architecture, with palisade and spongy parenchyma cells
(Figs. 1 a – c). The adaxial epidermal cells were significantly (t-
test, p < 0.001) larger (61.4 ± 8.7 μm, given as mean value ± SD,
the same for all subsequent values) than the abaxial epider-
mal cells (31.5 ± 7.3 μm) but there were no significant differ-
ences in the size of either adaxial or abaxial epidermal cells be-
tween the two sampling sites. Measurements of the leaf thick-
ness performed on semithin sections of embedded material
revealed a statistically significant (t-test, p < 0.001) differ-
ence between samples (each n = 10) collected in the Austrian
Alps (314.3 ± 33.5 μm) and Svalbard (538.2 ± 72.7 μm). The in-
creased leaf thickness in the arctic ecotype was correlated with
greater thickness of both the palisade parenchyma (Austrian
Alps: 99.0 ± 17.3 μm, Svalbard: 232.8 ± 51.6 μm) and spongy
parenchyma (Austrian Alps: 125.6 ± 35.4 μm, Svalbard: 230.2 ±
37.4 μm) layers (t-test, p < 0.001). Samples raised in growth
chambers from seed collected in the Austrian Alps had approx-
imately the same sizes for all measured parameters as samples
collected in the Austrian Alps (data not shown).
Chloroplast protrusions were visible in live leaf sections from
structures were found in most mesophyll cells; no difference
was found between the palisade and spongy parenchyma cells.
Chloroplast protrusions were, however, more abundant when
chloroplasts were not as densely packed. These structures
were polymorphic and dynamic, with size and shape changing
continuously. The chloroplast protrusions were from 2 to 5 μm
broad and up to 5 μm long. They frequently established close
spatial contact with smaller spherical organelles. When sam-
ples were examined by TEM, this close contact was confirmed.
Mesophyll cells of Oxyria digyna exhibited a typical arrange-
ment of nuclei, mitochondria, golgi stacks (Fig. 2 b), starch-
containing chloroplasts, and microbodies (Fig. 2 c). Chloroplast
protrusions were evident as stroma-containing regions emerg-
ing from the chloroplast body, either at the lateral edges or the
concave side (Figs. 2 c – f). In TEM micrographs mostly longitu-
dinal sections through chloroplasts were observed, exhibiting
the lens-shaped organization of this organelle, with the con-
cave side pointing towards the cell cortex, and the convex side
facing the centre of the cell. These structures were observed in
samples collected in the Austrian Alps (Figs. 2 c, f) as well as in
samples collected in Svalbard (Figs. 2 d, e). The shape of chloro-
plast protrusions was irregular, with invaginations frequently
observed (Figs. 2 d – f). In most cases, a close association be-
tween chloroplasts, microbodies, and mitochondria was ob-
served (Figs. 2 c – e). In addition, golgi stacks were often found
in the vicinity of other organelles, such as within invaginations
of the nucleus (Fig. 2 b). In cells connected to the vascular tis-
sue, styloid crystals were found in vacuoles, likely to be com-
prised of oxalate (Fig. 2 g).
Immunofluorescence labelling by a freeze-shattering method
revealed that mesophyll cells of Oxyria digyna contain a dense
array of cortical MTs (Figs. 3 a – e). In palisade parenchyma
cells, the cortical MTs appear to be arranged in a net-like pat-
tern (Fig. 3 a). In epidermal cells, vast networks of cortical
MTs were observed (Fig. 3 b). MTs showed no preferential ori-
entation, and their lengths appeared to be quite variable. In
spongy parenchyma cells, MT arrays formed apparent focal
points, which appeared as areas with bright fluorescence
(Fig. 3 c). MTs were predominantly found in the cell cortex,
and although merged images of fluorescently labelled MTs
(green) and chlorophyll autofluorescence (red) demonstrated
that MTs are often in close proximity to chloroplasts, there
was no clear MT pattern to suggest a direct association with
chloroplast distribution (Fig. 3 d). Instead, the dense cortical
MT arrays extended far beyond the location of chloroplasts
(Fig. 3 e).
To investigate whether MTs might play a role in regulating the
formation and activity of chloroplast protrusions, leaves were
treated with the MT depolymerizing agent, oryzalin. A 10-μM
concentration of oryzalin depolymerized all MTs in mesophyll
cells (Fig. 3 f). Examining live cells under DIC revealed that
treatment with 10 μM oryzalin did not abolish chloroplast pro-
trusions or their interactions with smaller organelles (Fig. 4 a).
In specimens prepared for TEM, chloroplast protrusions were
still evident after treatments with 10 μM oryzalin for 2 h
(Figs. 4 b – f). Invaginations in the stroma-filled parts were fre-
quently observed (Figs. 4 c, d). Cytoplasmic organization did
not appear to be altered upon treatment with oryzalin and
close contact of chloroplasts with other organelles, like mito-
chondria, continued (Figs. 4 b, f). ER tubules, however, were
sometimes observed in close vicinity to the chloroplast protru-
sions (Fig. 4 e) or the organelle contact area (Fig. 4 f).
Latrunculin B treatment affects cytoplasmic integrity, but
does not prevent chloroplast protrusion formation. Treatment
with 5 μM or 10 μM latrunculin B arrested cytoplasmic stream-
ing. Chloroplasts became spherical and protrusions were
hardly visible. Numerous spherical structures became visible
around the chloroplasts (Fig. 5 a). Transmission electron mi-
Plant Biology 9 (2007)
A. Holzinger, G. O. Wasteneys, and C. Lütz
mesophyll cells when leaf sections were treated with 5 μM
(Figs. 5 b – d) or 10 μM (Fig. 5 e) latrunculin B. For example, ER
accumulations were found in the vicinity of the nucleus
(Fig. 5 b) and disconnected membranes and unusual mem-
brane vesicles were sometimes observed (Fig. 5 c). Neverthe-
less, massive thylakoid-free chloroplast protrusions were still
frequently present (Figs. 5 c – e), located mostly at the concave
sides of the chloroplasts (Fig. 5 e). After latrunculin B treat-
ments, the thylakoid-free chloroplast protrusions still con-
tained invaginations, where organelles like mitochondria re-
mained in close contact with the chloroplast surface (Fig. 5 d).
Comparative anatomy of alpine and arctic Oxyria digyna leaves.
(a, b) Toluidine blue-stained semithin sections observed with the light
microscope. a Sample collected in Svalbard; b sample collected in the
Austrian Alps. Note the substantial difference in leaf thickness be-
tween the two sampling sites. Especially the palisade parenchyma
(pp) and the spongy parenchyma (sp) differ in size. The epidermis
(ep) has significantly different cell sizes on the adaxial (top) and abaxial
(bottom) sides; on the abaxial sides secretory trichomes (arrow) and
stomatal guard cells are found. (c, d) 4-week-old cultivated leaf ob-
served by cryo SEM. c Cross fracture exhibiting the bifacial arrange-
ment; d abaxial epidermis, stomata (st), and secretory trichomes (ar-
rows). Bars: 100 μm.
Plant Biology 9 (2007)
Leaves of Oxyria digyna control samples from different habi-
tats. a DIC image of mesophyll cell from a cultivated plant containing
a large number of chloroplast protrusions (arrows). (b – g) Details of
the ultrastructure of samples collected in the Austrian Alps (b, c, f, g)
and Svalbard (d, e). b Dictyosome (arrow) in invagination of the nu-
cleus (N), mitochondrion (M); c tangential section of leaf mesophyll
cell, chloroplast containing starch grains (S), transversely sectioned
chloroplast with broad stroma portion (str), mitochondrion (M), micro-
body (MB); d mitochondrion (M) almost covered by a chloroplast pro-
trusion; e chloroplast protrusion (arrow) in close contact with mito-
chondrion (M) in the vicinity of the cell wall (CW); f stroma (str)-filled
chloroplast protrusions; g mesophyll cell in close vicinity to vascular tis-
sue containing oxalate crystal (arrow) in the vacuole (V). Bars: a 5 μm,
A. Holzinger, G. O. Wasteneys, and C. Lütz
Microtubule arrangement in cultivated Oxyria digyna leaves.
cal microtubules. b Epidermal cells with dense cortical arrays of micro-
tubules. c Spongy parenchyma cell with net-like arrangement of corti-
cal microtubules, and apparent focal points with very bright staining
(arrows). d Merged chlorophyll autofluorescence (red) representing
chloroplasts (Chl) and microtubules (green) in a mesophyll cell. e High-
er magnification of dense microtubules in mesophyll cells. f Mesophyll
cell treated for 2 h with 10 μM oryzalin showing destruction of micro-
tubules. Only diffuse fluorescence remains (arrows). Bars: 20 μm.
Plant Biology 9 (2007)
Effects of treatment with 10 μM oryzalin for 2 h on mesophyll
cells of cultivated Oxyria digyna plants. a DIC image exhibiting chlo-
roplast protrusions (arrows), some have close contact to smaller or-
ganelles. (b – f) Details of the ultrastructure. b Chloroplast protrusion
containing stroma (str) in close vicinity to mitochondrion (M); c chlo-
roplasts with large areas exclusively containing stroma (str) and an in-
vagination in this area (arrow); d tangentially sectioned chloroplasts
with chloroplast protrusions and invaginations (arrows); e ER cisternae
(arrows) next to chloroplast with protrusion; f vicinity of chloroplast
protrusion containing stroma (str), ER (arrow), and mitochondrion (M).
Bars: a 5 μm, b – f 1 μm.
Plant Biology 9 (2007)
A. Holzinger, G. O. Wasteneys, and C. Lütz
Our investigations compared leaves, from the gross anatomical
to the ultrastructural level, in the arctic-alpine species Oxyria
digyna collected from different habitats. In all samples, mas-
sive chloroplast protrusions containing only stromal material
and free of thylakoid membranes were observed. Chloroplast
protrusions similar to those found in plants collected in the
field were identified in cultivated samples. In this study, we
explored the possible involvement of cytoskeletal elements in
the regulation of chloroplast protrusion formation and be-
haviour. Our results suggest that MTs, and possibly MFs, are
not directly involved in the formation of these structures.
Several reports have described the ability of plastids to form
stroma-containing compartments (for a summary see Gray et
al., 2001). Recently, the term stromule (Köhler and Hanson,
2000) has been used to describe tubular projections emerging
from plastids. These stromules are highly dynamic as they
grow and retract from chloroplasts along MF tracks (Kwok
and Hanson, 2004 b; Gunning, 2005). Also, MTs have been
found to be crucial for their formation (Kwok and Hanson,
2003). Stromules have been defined as being up to 800 nm
wide, and may reach a length of several tens of micrometers
(for a summary see Kwok and Hanson, 2004 a). The structures
presented herein are likely to be distinct from stromules on
account of their size and their MT-independent formation and
behaviour. Instead, these protrusions are most likely analo-
Effects of treatment with 5 μM (b – d) or 10 μM (a, e) latruncu-
lin B on mesophyll cells of cultivated Oxyria digyna plants. a DIC image.
No cytoplasmic streaming is detected, chloroplasts appear spherical
and numerous small, spherical organelles (arrows) are present. (b – e)
Details of the ultrastructure. b Nucleus (N) and massive amount of ER;
roplast with protrusion and invagination (arrow); d mitochondrion (M)
in chloroplast invagination, surrounded by area exclusively containing
stroma (str); e chloroplast with stroma (str) containing protrusion in
vicinity of the nucleus (N). Bars: a 5 μm, b – e 1 μm.
Cytoskeleton-Independent Chloroplast Protrusions
alpine plant Ranunculus glacialis (Lütz, 1987; Lütz and Moser,
1977; Larcher et al., 1997). A distinction between stromules
and chloroplast protrusions is given by the establishment of
a “shape index”, a ratio between length and radius of these
structures in Arabidopsis thaliana (Holzinger et al., 2006).
Speculations on the function of stromules and chloroplast pro-
trusions exist. Most point towards the increased surface to
volume ratio of chloroplasts in response to physiological de-
mands. One possibility is that the formation of chloroplast
protrusions would increase RUBISCO concentrations and chlo-
roplast ribosome levels. RUBISCO, whose regulation by activity
levels is likely more important than concentration, has never
been found to be a limiting factor for photosynthesis in Oxyria
al., 1972). A more likely advantage is that chloroplast protru-
sions establish close contact with other organelles. This can
also be concluded from the present study, in which mitochon-
dria were frequently found in the invaginations of chloro-
plasts, thus being in close contact to the stroma-rich areas.
However, we were unable to document membrane fusion
these chloroplast protrusions in Oxyria digyna. Using immuno-
fluorescence microscopy, we observed a dense network of cor-
tical MTs in mesophyll cells, and showed that this network was
effectively destroyed by the anti-MT drug oryzalin. Although
MTs were found in the vicinity of chloroplasts, we detected
no direct interactions between MTs and the chloroplast pro-
trusions. MT disruption also failed to abolish chloroplast pro-
trusions. Kandasamy and Meagher (1999) similarly concluded
that MTs did not influence the position or movement of chlo-
roplasts in Arabidopsis thaliana leaf cells. The finding that
chloroplast protrusions in Oxyria digyna are not disrupted
when MTs are depolymerized, however, is particularly impor-
tant because MTs have been described as being a critical factor
in the establishment of stromules in Nicotiana tabacum (Kwok
and Hanson, 2003). Our analysis indicates that stromule for-
mation and the development of the more massive chloroplast
protrusions in Oxyria digyna are likely to be controlled by dis-
Our study also failed to demonstrate any clear function for MFs
in the occurrence of chloroplast protrusions. Treatment with
latrunculin B, a potent disruptor of MFs (Spector et al., 1989),
at concentrations sufficient to fully destroy the MF system (cf.
Kandasamy and Meagher, 1999) failed to abolish the occur-
rence of stroma-filled chloroplast areas interacting with other
organelles. This treatment, however, did cause severe changes
to the cytoplasmic integrity, so some role for MFs in the es-
tablishment of chloroplast protrusions cannot be ruled out.
Latrunculin B arrested cytoplasmic streaming and altered cy-
toplasmic integrity, including the generation of membrane
anomalies and massive ER accumulation. The latter can be re-
garded as a general defence mechanism, also observed upon
applying various MF-disrupting agents (e.g., Holzinger and
Meindl, 1997; Holzinger and Lütz-Meindl, 2001). Despite the
clear signs of MF disruption and DIC observations that protru-
sions were totally lost, thylakoid-free areas of the chloroplasts
were clearly detected at the TEM level. These areas, however,
were mainly at the concave side of the chloroplast, suggesting
that expansion of chloroplast protrusions may depend on an
intact MF cytoskeleton, as suggested for stromules (Kwok and
Hanson, 2004 b; Gunning, 2005). However, in Oxyria digyna
the proportion of thylakoid-free stroma area in chloroplasts
remained constant after MF disruption when compared to the
controls. Thus, the destruction of MFs may influence the shape
of chloroplast protrusions, but not their occurrence. This find-
ing accords with observations in Nicotiana tabacum, where dif-
ferent MF inhibitors were found to alter chloroplast stromules
in a way that they became shorter and thicker (Kwok and Han-
son, 2003). In a study employing GFP-hTalin to visualize MFs,
a close correlation of MFs with stromules was observed in Ara-
would suggest that MFs are responsible for establishing the
narrow stromules, but are not causally involved in the occur-
rence of thylakoid-free stroma-filled areas in chloroplasts seen
in our study. While it would be interesting to visualize the MF
cytoskeleton in Oxyria digyna, only thick MF bundles are ob-
served after phalloidin staining (not shown). If there is a mesh-
work of individual MFs surrounding chloroplasts, GFP-talin
would be better for revealing it. However, it has not yet been
possible to introduce GFP-talin constructs into Oxyria digyna.
The close spatial contacts between chloroplasts and organelles
like mitochondria are retained in the presence of latrunculin B,
which confirms that these processes are also not dependent on
an intact MF cytoskeleton.
What then can explain the formation and activity of chloro-
plast protrusions? A highly active chloroplast “mobile jacket”
has been described in isolated tobacco and spinach chloro-
plasts (Spencer and Wildman, 1964; Spencer and Unt, 1965),
suggesting that an intrinsic mechanism for chloroplast flexi-
bility exists. It has also been claimed that osmotic phenomena
in salt- or water-stressed plants are responsible for “amoe-
boid” plastids (Huang and Steveninck, 1990) or the develop-
ment of chloroplast extensions (Freeman and Duysen, 1975).
The structures described by these authors are very similar in
appearance to the chloroplast protrusions found in our study.
The detection of chloroplast protrusions in the Antarctic spe-
cies Deschampsia antarctica (Gielwanowska and Szczuka,
2005) was recently confirmed and extended to a second Ant-
arctic phanerogam, Colobanthus quitensis (Lütz et al., 2006).
This points to chloroplast protrusions being an adaptation to
special environmental conditions. Recent observations have
demonstrated that temperature might be a critical factor in
chloroplast protrusion formation in Arabidopsis thaliana (Hol-
zinger et al., 2006) and alpine plants (Buchner et al., 2007).
Chloroplast swelling, and the appearance of thylakoid-free
areas, have been described in association with chilling effects
ˇ iamporová and Trginová, 1999). MTs, in turn, are cold-sensi-
2000). Some indirect signalling mechanisms upon MT disrup-
tion may be responsible for cold-induced chloroplast swelling.
The significant difference in leaf thickness between samples
collected from Svalbard and the Austrian Alps is an interesting
observation but difficult to interpret. It could be related to the
24 h daylight situation Oxyria digyna plants have to face in
Svalbard. Distinct ecotypes of Oxyria digyna with substantial
anatomical differences have been described (Billings et al.,
1961, 1971; Heide, 2005). For a detailed understanding of this
observation, extended studies will need to be performed.
A. Holzinger, G. O. Wasteneys, and C. Lütz
al., 1971) and structural (Pyankov and Vaskorskii, 1994; Koro-
leva, 1996) adaptations of the photosynthetic apparatus to arc-
tic and alpine conditions have been described for the different
ecotypes of Oxyria digyna. In particular, rearrangements of
grana in Oxyria digyna chloroplasts have been detected (Miro-
slavov et al., 1996). Our study extends these findings to the ob-
servation of thylakoid-free, stroma-filled chloroplast protru-
sions (Lütz and Engel, 2007) with close associations to other
organelles. These protrusions are likely to be an additional
adaptation to harsh environments. In future, a detailed inves-
tigation of temperature and light conditions that favour the
occurrence of chloroplast protrusions may elucidate their role
in plant adaptation to challenging environmental conditions.
We would like to thank the EU Large-Scale Facility for Arctic
Environmental Research, Norwegian Polar Board, Longyearby-
en for financial support and Alfred Wegener Institute (AWI) for
their cooperation in Ny Alesund, Svalbard. Technical assistance
in TEM section preparation by L. Di Piazza and B. Defregger is
acknowledged. We thank S. Marcante and Prof. Dr. J. Wagner
for help in seed collection in the Austrian Alps. TEM observa-
tions were performed in the Department of Zoology at the Uni-
versity of Innsbruck, Prof. Dr. R. Rieger is to be acknowledged
for providing access to this instrument. We would also like to
thank E. Kawamura for practical instructions in the MT stain-
ing procedure and the UBC Bioimaging Facility of the Univer-
sity of British Columbia, Vancouver, BC for access to the cryo
SEM and confocal microscope. The scientific visit to Vancouver,
BC was supported by a “Habilitation” stipend from the LFU
Innsbruck (Dept. for Academic Mobility) to A. H. The study
has been supported by a FWF (Austrian Science Foundation)
grant P 17184 to C. L. and an NSERC Discovery grant 298264-
04 to G. O. W.
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Department of Physiology and Cell Physiology of Alpine Plants
Institute of Botany
University of Innsbruck
Editor: S. M. Wick