Download Giant mitochondria are a response to low oxygen pressure in cells of

Journal of Experimental Botany, Vol. 53, No. 371, pp. 1215–1218, May 2002
SHORT COMMUNICATION
Giant mitochondria are a response to low oxygen pressure
in cells of tobacco (Nicotiana tabacum L.)
K. Van Gestel and J-P. Verbelen1
Department of Biology, University of Antwerp UIA, Universiteitsplein 1, 2610 Wilrijk, Belgium
Received 24 September 2001; Accepted 18 January 2002
Abstract
Low oxygen pressure induces fast and reversible
formation of giant mitochondria in cells of Nicotiana
tabacum. These can have unusual shapes, attain a
length of 80 mm and even form a reticulum. In contrast to animal cells, there is no such response to
chemically induced oxidative stress.
Key words: GFP, giant mitochondria, Nicotiana, oxygen,
respiration.
Introduction
In animal cells, the shape and size of mitochondria are
controlled by the metabolic state of the cells. Disc-shaped
or ring-like mitochondria are induced by oxygen deprivation and by uncouplers and inhibitors of respiration
and phosphorylation in a range of animal cells (BereiterHahn and Vöth, 1983; Markova et al., 1990). Extremely
enlarged and elongated mitochondria, called megamitochondria, have been induced by free-radical inducing
chemicals (Karbowski et al., 1999). Giant mitochondria
were also observed in chloroplast-deprived Euglena gracilis
two years after a treatment with N-succinimidylofloxacin
(Polónyi et al., 1998).
Reports on extremely long or abnormally shaped
mitochondria in plants are scarce. In Arabidopsis thaliana,
variation in the size of mitochondria has been related
to their motility (Logan and Leaver, 2000). Giant mitochondria were reported in egg cells of Pelargonium zonale,
where they appear just before double fertilization and
persist until early embryogenesis (Kuroiwa and Kuroiwa,
1992). Recently, a causal link was found between the
formation of long mitochondria in leaves of Arabidopsis
thaliana and long-term exposure to low oxygen pressure
(Ramonell et al., 2001).
In cultured plant cells, mitochondria can also vary in
size and shape (Stickens and Verbelen, 1996), but giant
1
mitochondria have not been previously reported. Here,
the fast and reversible induction of giant mitochondria in
cells of Nicotiana tabacum is reported and their general
features are described.
Materials and methods
Elongating and dividing tobacco cells were cultured in Petri
dishes on an agarose layer, starting from mesophyl protoplasts
(Vissenberg et al., 2000). These were isolated from leaves
of sterile-grown wild-type or transgenic plants of Nicotiana
tabacum L. cv. Petite Havana, the latter expressing mitochondrion targeted GFP (Köhler et al., 1997). This targeting is
mediated by the yeast cytochrome oxidase subunit IV transit
peptide, which is removed upon import of the GFP molecule
into the mitochondrion. The specificity and the non-toxicity of
the GFP targeting have been demonstrated previously (Köhler
et al., 1997). After several days of culture, regenerated cells were
harvested from the agarose layer and collected on a nylon filter
(mesh size 30 mm). Cells were mounted at different densities
between slide and coverslip or first kept on the filter for some
hours, submerged in culture medium. In cells obtained from
wild-type plants, mitochondria were stained with 0.4 mM
3,39-dihexyloxa-carbocyanine iodide (DiOC6(3)) (Molecular
Probes).
Cells were treated with KCN 4 mM, dinitrophenol 40 mM,
CCCP 5 mM, H2O2 0.2 mM, CuSO4 10–250 mM, paraquat
100 mM, or menadion 100 mM for periods ranging from 30 min
to 16 h. Treatments with latrunculin B 1.25 mM, or oryzalin
10 mM lasted 2 h. To create hypoxic conditions, a sealed Petri
dish was equipped with needle inlets and outlets. The culture
medium covering the cells was purged with pure nitrogen gas
for 4 h.
Fluorescence of GFP and DiOC6(3), was detected using
the 488 nm laser line of a Bio-Rad MRC 600 confocal system
mounted on a Zeiss Axioskop microscope. Phase-contrast
micrographs were made with a Nikon DXM 1200 digital
camera mounted on a Leitz Orthoplan microscope.
Results
Throughout cell culture, the size of the mitochondria is
between 0.5 mm and 5 mm. However, when elongated
To whom correspondence should be addressed. Fax: [32 3 8202271. E-mail: [email protected]
ß Society for Experimental Biology 2002
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Van Gestel and Verbelen
cells are mounted at high density for microscopy, giant
mitochondria develop. The transformation of the mitochondria into giant mitochondria is clearly illustrated by
images of four successive stages recorded in the same cell
(Fig. 1). Immediately after mounting, the mitochondria
still have their normal size and shape (Fig. 1A). Fusion or
aggregation of mitochondria is visible after 1 h (Fig. 1B),
and proceeds further (Fig. 1C). After 3 h, the cell contains
fewer but very long or circular mitochondria (Fig. 1D).
Once formed, many long mitochondria transform into
flat discs with thick margins (Fig. 2A). The speed of this
process varies with cell density, but after 4 h the majority
of cells have generally developed giant mitochondria. In
a transvacuolar strand a single long mitochondrion of
80 mm was recorded. The elongated and disc-shaped
mitochondria are often branched and connected with
each other, forming a mitochondrial reticulum (Fig. 2B).
FRAP (fluorescence recovery after photobleaching)
applied to the GFP indicated that the stroma inside
giant mitochondria is forming a continuum. Figure 3A,
B, and C show part of a cell before, immediately after,
and 30 s after strong photobleaching of the GFP in a
small area (indicated with a rectangle). In wild-type cells,
using calibrated conditions, the membrane potential
sensitive dye DiOC6(3) gives the same fluorescence
intensity for both giant mitochondria and normal
mitochondria (data not shown), indicating that both
types of mitochondria have a similar membrane potential. Giant mitochondria are less mobile than normal
mitochondria. An intact cytoskeleton is, however, not
a prerequisite for their formation. Cells in which the
actin filaments or the microtubules were disturbed with
latrunculin B and oryzalin, respectively, were still capable
of forming giant mitochondria (data not shown).
Dividing cells react in the same way as elongating cells
and also form giant mitochondria.
The formation of giant mitochondria is certainly not
caused by the GFP-tagging, as wild-type tobacco cells
show exactly the same phenomenon. In these cells the
mitochondria can be observed by phase-contrast microscopy (Fig. 2C) or by staining with the dye DiOC6(3).
However, there is a relationship between cell density
and the formation of giant mitochondria. Densely packing the cells on a nylon filter in culture medium, had the
same effect as mounting the cells between slide and
coverslip. Low density mounting of cells, either on a filter
or on a microscope slide, never led to giant mitochondria
in the time range used (1–16 h). Also cells adjacent to
air bubbles never formed giant mitochondria, suggesting
oxygen deprivation may be the causal factor. Indeed,
giant mitochondria formation was induced in cells by
purging the culture dish with pure nitrogen for 4 h
(Fig. 2D). The process is fully reversible just by diluting
the cells or by increasing the oxygen concentration. One
population of cells can even be subjected to multiple
cycles of giant mitochondria formation and subsequent
fission.
However, treating the cells with various concentrations
of KCN, dinitrophenol or CCCP had no effect on the size
of mitochondria. Also oxidative stress conditions induced
Fig. 1. Four stages of the formation of giant mitochondria in an elongated tobacco cell, densely packed for microscopy. (A) Immediately after
mounting, the mitochondria are normal. (B) After 1 h a slight increase in size is visible. (C) After 2 h both circular and very long mitochondria are
formed. (D) After 3 h typical giant mitochondria are predominant. All pictures are Z-series projections of confocal images displaying GFP-tagged
mitochondria of one cell. Scale bar \ 50 mm.
Giant mitochondria in tobacco
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Fig. 2. Z-series projections of confocal images of GFP-tagged mitochondria in tobacco cells (A, B, D), and a phase-contrast image of mitochondria
in a wild-type tobacco cell (C). (A) Plate-like giant mitochondria after densely mounting of the cells between microscopy glasses for 4 h. (B) A
mitochondrial reticulum as it occurs after 4 h. (C) Giant mitochondria (tubular and plate-like) in wild-type cells after 4 h. (D) Giant mitochondria
generated in the culture dish after 4 h of nitrogen purging. Scale bars (A, B, D) \ 50 mm, scale bar (C) \ 10 mm.
Fig. 3. Three stages of a FRAP (fluorescence recovery after photobleaching) experiment on GFP-labelled mitochondria in a tobacco cell. (A) Plate-like
giant mitochondria before photobleaching. (B) Immediately after thorough photobleaching of a small area (rectangle). (C) Thirty s after the
photobleaching, the lower part is filled up again by GFP molecules flowing from the upper part of the plate-like mitochondrion to the lower.
The pictures represent single plane confocal images. Scale bar \ 25 mm.
by applying H2O2, CuSO4, menadion or paraquat did not
result in giant mitochondria formation.
Discussion
Recently, abnormally long mitochondria in leaves of
Arabidopsis thaliana were linked to long-term hypoxia
(Ramonell et al., 2001). In cultured cells of tobacco,
the effect of low oxygen pressure is impressive as it
induces giant mitochondria, leading eventually to an
extensive mitochondrial reticulum including large plates.
As the number of mitochondria decreases when their size
increases, the size increase is most probably due to fusion.
FRAP experiments on the GFP labelled giant mitochondria indicate a continuity of the stroma. The size
ultimately reached exceeds by far that reported for the
so-called megamitochondria in animal cells (Karbowski
et al., 1999).
In animal cells, hypoxia and anoxia cause elongation
and disc-shaped swelling of mitochondria (Bereiter-Hahn
and Vöth, 1983), but also respiratory inhibitors and
uncouplers provoke disc-shaped mitochondria (BereiterHahn and Vöth, 1983; Markova et al., 1990). However,
KCN, dinitrophenol and CCCP did not induce abnormal
mitochondria in the tobacco cells. Recent research has
focused on the association between megamitochondria
and apoptosis in cultured rat cells (Karbowski et al.,
1999), whereby free radical generating agents induce large
mitochondria. This was suggested to be an adaptation
to high oxidative stress. However, neither H2O2 nor
other oxidative stress inducers (CuSO4, menadion and
paraquat) induced enlarged mitochondria in tobacco
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Van Gestel and Verbelen
cells. The different reaction of the tobacco mitochondria
to respiratory inhibitors and to oxidative stress could
relate with the fact that plant mitochondria differ from
their animal counterparts: they possess an alternative
(cyanide-resistant) respiratory pathway to meet plantspecific demands. This alternative pathway is upregulated
by a range of stress conditions and is suggested to
mitigate reactive oxygen species (ROS) damage in plant
cells (Mackenzie and McIntosh, 1999).
From a practical standpoint, the very specific state of
giant mitochondria reported here is most readily induced
in large populations of cells by densely packing of the
cells. This offers opportunities for further research.
Acknowledgements
We thank Dr Rainer Köhler for the transgenic tobacco plants
and Dr Robbie Wilson for critical reading of the manuscript.
This work is supported by the Research program of the
Fund for Scientific Research, Flanders (grants 3.0028.90
and G.0034.97).
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