Journal of Experimental Botany, Vol. 53, No. 371, pp. 1215-1218,
May 2002
© 2002 Oxford University Press
Short Communication |
Giant mitochondria are a response to low oxygen pressure in cells of tobacco (Nicotiana tabacum L.)
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 µm 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 (Bereiter-Hahn 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 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 µm). 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 µM 3,3'-dihexyloxa-carbocyanine iodide (DiOC6(3)) (Molecular Probes).
Cells were treated with KCN 4 mM, dinitrophenol 40 µM, CCCP 5 µM, H2O2 0.2 mM, CuSO4 10–250 µM, paraquat 100 µM, or menadion 100 µM for periods ranging from 30 min to 16 h. Treatments with latrunculin B 1.25 µM, or oryzalin 10 µM 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 µm and 5 µm. However, when elongated 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 µm 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.
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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 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 (Bereiter-Hahn 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 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 plant-specific 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).
Footnotes
1 To whom correspondence should be addressed. Fax: +32 3 8202271. E-mail: verbelen{at}uia.ua.ac.be 
References
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