The Mitochondrion

by David Logan

School of Biology, University of St Andrews, St Andrews, Scotland, UK.

Mitochondria in epidermal cells of an Arabidopsis plant (6X real time)
The Mitochondrion is a vital organelle that performs a variety of fundamental functions ranging from the synthesis of much of the cell’s energy currency, adenosine triphosphate (ATP), through to its involvement in the initiation and promulgation of cell death.
Mitochondria therefore have a dual role as key players in the provision of the energy for life, and in providing a trigger for death.
Comprised of at least six compartments: outer membrane, inner boundary membrane, inter-membrane space, cristal membranes, inter-cristal space, and matrix, mitochondria have a complex, dynamic internal structure. This internal dynamic structure is reflected in mitochondrial pleomorphy and motility. Targeted fluorescent proteins have enabled the study of mitochondrial morphology and dynamics, led to many exciting new discoveries, and provide excellent tools for further study of these fascinating organelles in living tissue and in real time.
The first reported use of GFP in the context of plant mitochondrial research was a study by Zhu and colleagues who used GFP to analyse the intracellular location of a gernylgeranyl pyrophosphate synthase (GGPS6) [1]. The signal sequence of GGPS6 targeted GFP to mitochondria. At around the same time a number of stably transformed plant lines were produced in which GFP was targeted to the mitochondrial matrix. All these constructs make use of different targeting signals that are present on the N-terminus of proteins, synthesised in the cytosol, but destined for mitochondria. Kohler and colleagues [2] produced a mitochondrial-GFP (mito-GFP) construct comprising mgfp4 (a GFP with plant optimised codon usage that removes a cryptic intron [3]) downstream of the signal sequence from a yeast mitochondrial protein (COXIV) under the control of the CaMV 35S promoter, to produce stable transgenic tobacco plants and cell suspension cultures with GFP labelled mitochondria. Stable transgenic Arabidopsis lines were generated independently by two groups. One group [4] used the gamma subunit of the Arabidopsis mitochondrial F1-ATPase fused to sGFP(S65T) (a synthetic GFP with humanized codon usage) and under control of a 35S promoter with the omega translation enhancer from tobacco mosaic virus [5]. Logan and Leaver [6] generated two Arabidopsis transgenic lines using either of two different constructs comprised of the signal sequence from either the beta subunit of the F1-ATPase of Nictotiana plumbaginifolia, or from Arabidopsis CPN-60, followed by mgfp5 (as mgfp4 but with improved thermostability and greatly improved excitation maxima at 473 nm) and under the control of the 35S promoter.
The availability of mito-GFP lines and constructs has had a profound effect on our knowledge of plant mitochondria both by enabling studies aimed at understanding mitochondria from a cell biological perspective, and by providing the proof-of-concept and controls for researchers simply using GFP as a reporter molecule for subcellular localisation of their protein of interest.
The chondriome (all mitochondria in a cell collectively) can vary from cell-type to cell-type and from organism to organism and is also controlled by environmental and bioenergetic conditions [7, 8]. However, even within this variation it is clear that the steady-state structure of the plant chondriome visualised unambiguously and in living tissue, as enabled by mito-GFP, differs from that of other organisms [9]. The plant chondriome is composed of numerous, physically discrete mitochondria [2, 6], rather than forming a reticular structure more typical of yeast and animal chondriomes, although the plant chondriome functions, at least genetically, as a discontinuous whole [10].
Researchers using yeast as a model system (and mito-GFP) first demonstrated that chondriome structure is maintained by a balance between inter-mitochondrial fusion, and intra-mitochondrial fission: fission leads to the formation of a greater number of smaller mitochondria, fusion leads to a smaller number of larger mitochondria [11, 12]. Prior to the development of plant mito-GFP nothing was known about the genes and processes controlling plant mitochondrial division, but once researchers were able to visualise mitochondria reliably by fluorescence microscopy, components of the mitochondrial division apparatus were identified [13-15]. Nothing is known about the genes and mechanisms controlling mitochondrial fusion in plants, but an elegant use of a photoconvertible fluorescent protein called Kaede [16], which can be converted by laser from fluorescing green to fluorescing red has confirmed by cell biology what was known to occur from studying mitochondria DNA following the production of plant cybrids [17], namely that mitochondria fuse [18]. The cybrid results demonstrated, using tradition geneome analysis techniques, that mixing, exchange, and possibly complementation, of mtDNA requires mitochondrial fusion. Intra-mitochondrial fusion has also been shown to occur during cell cycle progression, at least in de-differentiating protoplast cultures, a result enabled by the use of mito-GFP [19].
Mitochondria are highly pleomorphic and motile structures [6]. Indeed, mitochondrial motility has been shown to require actin filaments, an important component of the cytoskeleton, and the use of mito-GFP has once again been instrumental in this discovery [20, 21] and in research aimed at identifying the motors that move mitochondria on the cytoskeleton [22].
As endosymbiosis-derived organelles containing their own genome [23, 24] mitochondria cannot be synthesised de novo, instead mitochondria are inherited from one generation to the next. As with many areas of mitochondrial cell biology in plants, little is known about the mechanics of this process. Yet again, the use of mito-GFP has enabled studies of mitochondrial inheritance during cell division, and demonstrated the importance of the cytoskeleton in mediating this process [25].
Much remains unknown about the genes and mechanisms controlling fundamental aspects of plant mitochondrial cell biology and one way to help us learn more is the use of mutants. As a means to learn more about the genes and mechanisms controlling mitochondrial morphology, division, fusion and distribution in the cell, a mutant screen was performed using microscopy of an Arabidopsis line expressing mito-GFP [26]. A suite of mutants was identified with a range of aberrant mitochondrial morphologies [26]. This type of screen, based on the visualisation of organelles, or organelle interactions could also be easily applied to other organelles.
Finally to come full circle in the life cycle of a cell, GFP has recently been used to investigate mitochondrial morphological changes during cell death (Scott and Logan, in press). Upon induction of cell death by means of chemical (strong oxidants), or physical (heat) shock mitochondria in Arabidopsis undergo a morphology transition involving a swelling of the mitochondrial matrix. This is likely synonymous with the mitochondrial permeability transition, affecting the permeability of the inner boundary membrane a phenomenon which had previously only been measured by indirect in vitro methods using isolated mitochondria. Use of mito-GFP allows a direct measure of mitochondrial swelling in living cells and allows a per-cell quantification of any subsequent cell death (Scott and Logan, in press). Interestingly, lanthanum chloride, an inhibitor of calcium channels, inhibited the morphological transition, a result that could be investigated further by the use of mitochondrial targeted aequorin (a calcium sensitive photoprotein [27]), which has been targeted to mitochondria downstream of mito-GFP, which allows confirmation of the subcellular location of the aequorin [28].
Despite the usefulness of mito-GFPs, which to date have all been targeted to the matrix, no constructs that target fluorescent proteins to the other mitochondrial compartments have been described in the literature, at least as far as I am aware. An outer-membrane targeting construct has been made and works well (Sparkes and Hawes unpublished), but also localises to peroxisomes. Recently an array of organelle targeted constructs and Arabidopsis lines have been generated in green, cyan, yellow and red (mCherry) variants, mostly using previously published targeting information, and are available conferring either kanamycin or BASTA resistance [29]. These lines and constructs, like others before them will continue to enable advances in our understanding of plant mitochondrial biology.

References

1.    Zhu, X.F., Suzuki, K., Saito, T., Okada, K., Tanaka, K., Nakagawa, T., Matsuda, H., and Kawamukai, M. 1997. Geranylgeranyl pyrophosphate synthase encoded by the newly isolated gene GGPS6 from Arabidopsis thaliana is localized in mitochondria. Plant Mol Biol 35, 331-341.

2.    Kohler, R.H., Zipfel, W.R., Webb, W.W., and Hanson, M.R. 1997. The green fluorescent protein as a marker to visualize plant mitochondria in vivo. Plant Journal 11, 613-621.

3.    Haseloff, J., Siemering, K.R., Prasher, D.C., and Hodge, S. 1997. Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proceedings of the National Academy of Sciences of the United States of America 94, 2122-2127.

4.    Niwa, Y., Hirano, T., Yoshimoto, K., Shimizu, M., and Kobayashi, H. 1999. Non-invasive quantitative detection and applications of non-toxic, S65T-type green fluorescent protein in living plants. Plant Journal 18, 455-463.

5.    Luehrsen, K.R., de Wet, J.R., and Walbot, V. 1992. Transient expression analysis in plants using firefly luciferase reporter gene. Methods Enzymol 216, 397-414.

6.    Logan, D.C., and Leaver, C.J. 2000. Mitochondria-targeted GFP highlights the heterogeneity of mitochondrial shape, size and movement within living plant cells. Journal of Experimental Botany 51, 865-871.

7.    Benard, G., Bellance, N., James, D., Parrone, P., Fernandez, H., Letellier, T., and Rossignol, R. 2007. Mitochondrial bioenergetics and structural network organization. Journal of Cell Science 120, 838-848.

8.    Logan, D.C. 2003. Mitochondrial dynamics. New Phytologist 160, 463-478.

9.    Logan, D.C. 2006. Plant mitochondrial dynamics. Biochim Biophys Acta 1763, 430-441.

10.    Logan, D.C. 2006. The mitochondrial compartment. J Exp Bot 57, 1225-1243.

11.    Okamoto, K., and Shaw, J.M. 2005. Mitochondrial morphology and dynamics in yeast and multicellular eukaryotes. Annual Review of Genetics 39, 503-536.

12.    Sesaki, H., and Jensen, R.E. 1999. Division versus fusion: Dnm1p and Fzo1p antagonistically regulate mitochondrial shape. Journal of Cell Biology 147, 699-706.

13.    Arimura, S., and Tsutsumi, N. 2002. A dynamin-like protein (ADL2b), rather than FtsZ, is involved in Arabidopsis mitochondrial division. Proceedings of the National Academy of Sciences of the United States of America 99, 5727-5731.

14.    Scott, I., Tobin, A.K., and Logan, D.C. 2006. BIGYIN, an orthologue of human and yeast FIS1 genes functions in the control of mitochondrial size and number in Arabidopsis thaliana. J Exp Bot 57, 1275-1280.

15.    Logan, D.C., Scott, I., and Tobin, A.K. 2004. ADL2a, like ADL2b, is involved in the control of higher plant mitochondrial morphology. Journal of Experimental Botany 55, 783-785.

16.    Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H., and Miyawaki, A. 2002. An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc Natl Acad Sci U S A 99, 12651-12656.

17.    Belliard, G., Vedel, F., and Pelletier, G. 1979. Mitochondrial recombination in cytoplasmic hybrids of Nicotiana tabacum by protoplast fusion. Nature 281, 401-403.

18.    Arimura, S., Yamamoto, J., Aida, G.P., Nakazono, M., and Tsutsumi, N. 2004. Frequent fusion and fission of plant mitochondria with unequal nucleoid distribution. Proceedings of the National Academy of Sciences of the United States of America 101, 7805-7808.

19.    Sheahan, M.B., McCurdy, D.W., and Rose, R.J. 2005. Mitochondria as a connected population: ensuring continuity of the mitochondrial genome during plant cell dedifferentiation through massive mitochondrial fusion. The Plant Journal 44, 744-755.

20.    Mathur, J., Mathur, N., and Hulskamp, M. 2002. Simultaneous visualization of peroxisomes and cytoskeletal elements reveals actin and not microtubule-based peroxisome motility in plants. Plant Physiol 128, 1031-1045.

21.    Van Gestel, K., Kohler, R.H., and Verbelen, J.P. 2002. Plant mitochondria move on F-actin, but their positioning in the cortical cytoplasm depends on both F-actin and microtubules. Journal of Experimental Botany 53, 659-667.

22.    Reisen, D., and Hanson, M.R. 2007. Association of six YFP-myosin XI-tail fusions with mobile plant cell organelles. BMC Plant Biol 7, 6.

23.    Unseld, M., Marienfeld, J.R., Brandt, P., and Brennicke, A. 1997. The mitochondrial genome of Arabidopsis thaliana contains 57 genes in 366,924 nucleotides. Nature Genetics 15, 57-61.

24.    Gray, M.W., Burger, G., and Lang, B.F. 1999. Mitochondrial evolution. Science 283, 1476-1481.

25.    Sheahan, M.B., Rose, R.J., and McCurdy, D.W. 2004. Organelle inheritance in plant cell division: the actin cytoskeleton is required for unbiased inheritance of chloroplasts, mitochondria and endoplasmic reticulum in dividing protoplasts. Plant Journal 37, 379-390.

26.    Logan, D.C., Scott, I., and Tobin, A.K. 2003. The genetic control of plant mitochondrial morphology and dynamics. Plant Journal 36, 500-509.

27.    Shimomura, O., and Johnson, F. 1970. Calcium Binding, Quantum Yield, and Emitting Molecule in Aequorin Bioluminescence. Nature 227, 1356-&.

28.    Logan, D.C., and Knight, M.R. 2003. Mitochondrial and cytosolic calcium dynamics are differentially regulated in plants. Plant Physiology 133, 21-24.

29.    Nelson, B., Cai, X., and Nebenfuhr, A. 2007. A multicolored set of in vivo organelle markers for co-localisation studies in Arabidopsis and other plants. The Plant Journal 51, 1126-1136.

30.    Forner, J., and Binder, S. 2007. The red fluorescent protein eqFP611: application in subcellular localization studies in higher plants. Bmc Plant Biology

FP probes - Mitochondria
FP probes - Mitochondria

References for the Table


1. Zhu, X.F., Suzuki, K., Saito, T., Okada, K., Tanaka, K., Nakagawa, T., Matsuda, H., and Kawamukai, M. (1997). Geranylgeranyl pyrophosphate synthase encoded by the newly isolated gene GGPS6 from Arabidopsis thaliana is localized in mitochondria. Plant Mol Biol 35, 331-341.

2. Kohler, R.H., Zipfel, W.R., Webb, W.W., and Hanson, M.R. (1997). The green fluorescent protein as a marker to visualize plant mitochondria in vivo. Plant Journal 11, 613-621.

3. Logan, D.C., and Leaver, C.J. (2000). Mitochondria-targeted GFP highlights the heterogeneity of mitochondrial shape, size and movement within living plant cells. Journal of Experimental Botany 51, 865-871.

4. Logan, D.C., Scott, I., and Tobin, A.K. (2004). ADL2a, like ADL2b, is involved in the control of higher plant mitochondrial morphology. Journal of Experimental Botany 55, 783-785.

5. Nelson, B., Cai, X., and Nenenfuhr, A. (2007). A multicolored set of in vivo organelle markers for co-localisation studies in Arabidopsis and other plants. The Plant Journal 51, 1126-1136.

6. Forner, J., and Binder, S. (2007). The red fluorescent protein eqFP611: application in subcellular localization studies in higher plants. Bmc Plant Biology

 

External Links: Wikipedia • Mitochondrial Inner Membrane Proteins • Mitochondrial Outer Membrane Proteins • Mitochondrion Atlas