David C Logan, UMR 1345, Institut de Recherche en Horticulture et Semences, Université d’Angers, Angers, France.

(November, 2012)

Introduction

Mitochondria are vitally important eukaryotic organelles. It is not possible to attribute the discovery of mitochondria to a single person since between 1850 and 1890 many cytologists observed granular bodies within cells, and some of these bodies may have been mitochondria (Lehninger 1964). It is believed mitochondria were first named cytomikrosomen by Adolf Freiherr von La Valette St. George in 1886 but the name that stuck, mitochondria, was coined by Carl Benda, a German zoologist in 1898 (Cavers 1914). The occurrence of mitochondria in plant cells was first described by Meves in 1904 (see Cavers 1914), although it was many years until the common role of these organelles in all eukaryotes was established.

Mitochondria were recognised, over sixty years ago, as the site of oxidative energy metabolism (Kennedy and Lehninger 1949), and are now known to synthesize the majority of respiratory ATP in plants, animals, and fungi. In addition to this crucial role, mitochondria are involved in the de novo synthesis of many compounds, such as iron-sulphur clusters, phospholipids, nucleotides, and several amino acids; this ensures that mitochondria are essential to eukaryotic life—even organisms able to respire anaerobically can only survive when mitochondria are present to manufacture these unique metabolites.

Mitochondria cannot be created de novo, meaning that any new mitochondrion must be formed from the division of an existing organelle. In addition to division, mitochondria also undergo fusion, where two or more individual organelles join to produce a single mitochondrion. Mitochondrial fission and fusion are the primary processes controlling mitochondrial form and together they control mitochondrial size and number (Logan 2006, 2010). Traditionally, the mitochondrion has been portrayed as an immobile, oval-shaped body. In reality, mitochondria are very dynamic organelles, capable of changing size and shape in a matter of seconds (Bereiter-Hahn and Voth 1994; see Movie 1). Additionally, they undergo short- and long-distance vectorial transport mediated by association with the cytoskeleton. Advances in bioimaging have allowed scientists to re-evaluate the behavior of mitochondria in vivo, stimulating a surge of interest in determining the genes, proteins, and mechanisms that control mitochondrial shape, size, number, and distribution (collectively termed mitochondrial dynamics) (Logan 2010).

Movie 1: The movie shows highly dynamic wild-type mitochondria (sped up 6 times) in the epidermal cell layer of one of the first true leaves of a 7-day-old Arabidopsis seedling. The arrow points to a mitochondrion that will become constricted and then divide. Note that the constricted part of the mitochondrion remains associated with one daughter organelle.

One of the primary goals of this research is the identification of genes and mechanisms controlling mitochondrial division and fusion—two processes that underpin mitochondrial form. In budding yeast (Saccharomyces cerevisiae), several genes have been identified that play a role in mitochondrial division and fusion. One main protagonist in yeast mitochondrial division is the dynamin-related protein Dnm1p. Dynamin is a GTPase mechano-enzyme involved in vesicle membrane constriction and severance during endocytosis. Dnm1p is believed to function similarly to dynamin but instead acting on mitochondrial membranes. Fusion of mitochondria in S. cerevisiae is controlled by a second GTPase, encoded by FZO1, homologous to the fuzzy onions gene from Drosophila. Fzo1p belongs to a functional class of proteins called mitofusins that are involved in inter-mitochondrial tethering (Escobar-Henriques and Anton 2012).

Genes Involved in Higher Plant Mitochondrial Dynamics

Two main approaches have been taken to identify genes involved in the control of higher plant mitochondrial dynamics. The first approach relies on the fact that mitochondria in all eukaryotes have a shared ancestry. This means that plant genes with significant similarity to genes involved in yeast mitochondrial dynamics are likely to have analogous functions. This gene homology provides researchers with a variety of “reverse-genetics” approaches (called reverse genetics because the gene is identified before the phenotype, cf. forward genetics, see below) to analyze the effect of gene knock-outs or knock-downs on mitochondrial dynamics and cell function. Such an approach has provided researchers with several successes to date: (i) two plant dynamin-like homologues, DRP3A and DRP3B, have been shown to be involved in mitochondrial division (Arimura et al. 2004; Logan et al. 2004); and (ii) an Arabidopsis protein called BIGYIN, orthologous to FIS1 in humans and yeast, has also been identified as a likely component of the plant mitochondrial division apparatus (Scott et al. 2006). However, this reverse-genetics method of identifying genes involved in plant mitochondrial dynamics is ultimately of limited use. The wild-type morphology of the plant chondriome (all mitochondria in a cell, collectively) is considerably different to the yeast chondriome, as are the mechanisms controlling mitochondrial inheritance. In a typical yeast cell, 5–10 mitochondria form a cortical network, and mitochondrial inheritance is an active process involving movement of parental mitochondria into the developing bud. In plants, there may be several hundred discrete mitochondria per cell, usually seen as small spherical or sausage-shaped organelles, and it is thought that mitochondrial partitioning (inheritance) into daughter cells during mitosis is, at least in part, due to the stochastic distribution of mitochondria in the parental cell (Sheahan et al. 2004). These morphological and organizational differences suggest differences in the mechanisms and proteins involved in the processes. Indeed, interrogation of the Arabidopsis thaliana genome database shows that there are no sequence homologues of many genes crucial to yeast mitochondrial morphology and dynamics. For example, while we know that plant mitochondria fuse, there is no Arabidopsis homologue of the important yeast fusion protein Fzo1p.

These facts suggest that the genes, proteins, and mechanisms controlling plant mitochondrial dynamics, while exhibiting some similarities to yeast, are predominantly distinct. With this in mind, a mutant screening approach was used to try to identify plant-specific genes involved in the control of plant mitochondrial morphology and dynamics (Logan et al. 2003). Arabidopsis plants expressing green fluorescent protein (GFP) targeted to the mitochondria (Logan and Leaver 2000) were mutagenized using ethyl methanosulfonate (EMS) and the second (M2) generation were then screened for altered mitochondrial shape, size, number, and distribution using a fluorescence microscope. Six viable mutants with distinct mitochondrial phenotypes were identified from a population of approximately 9500 individuals. Forward genetics (i.e., mutant phenotype identified prior to identification of the mutated gene, cf. reverse genetics above) is being used in the form of positional cloning and genome sequencing to identify the mutant genes.

The six mutants identified have distinct aberrant mitochondrial phenotypes such as changes in the sizes of individual mitochondria (mmt1, mmt2, and bmt), the presence of a mitochondrial reticulum rather than discrete organelles (nmt), and the presence of large clusters of mitochondria instead of the normal apparently random distribution (fmt) (Figure 1).

Figure 1 Images captured using either epifluorescence microscopy (left hand panels) or transmission electron microscopy (right hand panels) of Arabidopsis leaf mitochondria in the wild type and five mutants. Epifluorescent micrographs are false-coloured for GFP (green) and chlorophyll (red) fluorescence (from Logan et al. 2003).

a & b, wild type, arrows = mitochondria, * = chloroplast.

c & d, mmt1 mutant, * = chloroplast.

e & f, mmt2 mutant, plain arrows = large mitochondria, arrows with circle = small mitochondria, the boxes indicate an area magnified to highlight the heterogeneity of mitochondrial size within a single cell, * = chloroplast.

g & h, bmt mutant, arrow = mitochondrion.

i & j, nmt mutant, arrows = small mitochondria, * = chloroplast.

k & l, fmt mutant, arrow = large mitochondrial cluster, the boxes indicate a region enlarged to highlight a cluster of mitochondria.

Scale bars in epifluorescent images = 5 µm; in TEMs = 1 µm, except in d where bar = 5 µm. 

Plant Mitochondrial Dynamics and Human Disease

The friendly mitochondria mutant (fmt) was identified in Arabidopsis by the presence of discrete clusters of tens of mitochondria (see Figure 1, k & l; Figure 2). While only a proportion of the cell’s mitochondrial population form clusters (many maintain a wild-type distribution), the phenotype of this mutant is striking. Forward and reverse genetics were used to reveal that the mitochondrial phenotype of the friendly mutant was due to a single point mutation and we named the Arabidopsis gene FMT (FRIENDLY, Logan et al. 2003). We have since demonstrated that FRIENDLY is a cytosolic protein that associates with mitochondria and regulates mitochondrial fusion through mediating the length of time mitochondria remain in close association during “kiss and run” fusion (El Zawily et al. 2014).

Figure 2 Arabidopsis mesophyll protoplasts from wild type (left) or friendly mutant (right) plants expressing GFP targeted to mitochondria (Logan et al. 2003). The large clusters of mitochondria (green) are clearly evident in the mutant, interspersed between the autofluorescing chloroplasts (red) (from Scott and Logan 2007).

A recent study identified a Drosophila orthologue of FRIENDLY, named clueless, and implicated this gene in a mitochondrial quality control pathway (Cox and Spradling 2009). The clueless gene is, like FRENDLY, required for the correct subcellular distribution of mitochondria (see Logan 2010). Clueless was shown to interact genetically with parkin, the Drosophila orthologue of a human gene, PARK2 (Cox and Spradling 2009). PARK2 is mutated in many cases of early-onset Parkinson’s disease and it is believed that mitochondrial dysfunction in dopamine-producing nerve cells may be important in causing Parkinson's symptoms. 

Research is underway to exploit the genetic differences between Arabidopsis and Homo sapiens that have arisen over the past 1.6 ´ 109 years in order to identify novel components of a conserved mitochondrial quality control pathway involving FRIENDLY and to uncover the exact role FRIENDLY and its orthologues play in maintaining normal mitochondrial dynamics. It is possible, indeed likely, that there are metazoan-specific and plant-specific components of the mitochondrial quality control pathway, but screening an evolutionarily distant eukaryote (Arabidopsis) for candidate proteins in this pathway may identify additional mechanistically important components. Nevertheless, identification of plant-specific components will also inform research on metazoan species. There is potential for both conserved and plant-specific genes to be used as tools to identify conserved ligands that could be the focus of drug development studies.

Conclusion

Observations of mitochondrial behavior have been made for over a century (see Lehninger 1964). However, recently developed techniques using green fluorescent protein, enabling the unambiguous visualization of mitochondria in living tissue, have revolutionized research in this area. Such advances in cell biology have been complemented by advances in molecular genetics, supported by the Arabidopsis Genome Initiative; together, these advances have enabled a new era of functional genomics research into mitochondrial dynamics. One of the main conclusions that can be reached from recent research on mitochondrial dynamics is the clear interplay between mitochondrial form and function. For example, studies into plant cell death and apoptosis (a morphologically-distinct type of programmed cell death occurring in yeast and animals, see Web Essay 12.5) have uncovered the vital role played by mitochondrial dynamics in these processes. As we learn more about plant mitochondrial dynamics we can expect to discover many more instances where mitochondrial form and function are integrated and where plants can be useful eukaryotic models for the cell and for the molecular biology of human disease.

References

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 USA 99: 5727–5731.

Bereiter–Hahn, J., and Voth, M. (1994) Dynamics of mitochondria in living cells: shape changes, dislocations, fusion, and fission of mitochondria. Microscopy Research and Technique 27: 198–219.

Cavers, F. (1914) Chondriosomes (mitochondria) and their significance. New Phytologist 13: 96–106.

Cox, R. T., and Spradling, A. C. (2009) clueless, a conserved Drosophila gene required for mitochondrial subcellular localization, interacts genetically with parkin. Disease Models & Mechanisms 2: 490–499.

El Zawily, A.M., Schwarzländer, M., Finkemeier, I., Johnston, I.G., Benamar, A., Cao, Y., Gissot, C., Meyer, A.J., Wilson, K., Datla, R., Macherel, D., Jones, N.S., Logan, D.C. (2014) FRIENDLY regulates mitochondrial distribution, fusion, and quality control in Arabidopsis.  Plant Physiology 166 :808-828.

Escobar-Henriques, M., and Anton, F. (2012) Mechanistic perspective of mitochondrial fusion: Tubulation vs. fragmentation. Biochimica et Biophysica Acta – Molecular Cell Research http://dx.doi.org/10.1016/j.bbamcr.2012.07.016

Kennedy, E. P., and Lehninger, A. L. (1949) Oxidation of fatty acids and tricarboxylic acid cycle intermediates by isolated rat liver mitochondria. Journal of Biological Chemistry 179: 957–972.

Lehninger, A. L. (1964) The Mitochondrion. W. A. Benjamin, Inc., New York.

Logan, D. C. (2006) The mitochondrial compartment. Journal of Biological Chemistry 57: 1225–1243.

Logan, D. C. (2010) Mitochondrial fusion, division, and positioning in plants. Biochemical Society Transactions 38: 789–795.

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.

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

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.

Scott, I., and Logan, D. C. (2007) Mitochondrial dynamics: the control of mitochondrial shape, size, number, motility, and cellular inheritance. In Plant Mitochondria, David C. Logan, ed., Blackwell Publishing Ltd., Oxford, UK, pp. 1–35.

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. Journal of Experimental Botany 57: 1275–1280.

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.