List of Figures

Context

The application of Green Fluorescent Protein, as an in vivo marker of gene expression, together with development of wide-field and confocal microscopes, their associated detection devices, and image capture, and image analysis software has revolutionised cell biology. Together this new technology enables not just observation and recording of events in living cells, and in real time, but also allows the identification of the objects, the intracellular location of specific proteins in cells, and opportunities to investigate interactions between specific proteins.

This thesis describes research that uses Arabidopsis as a model organism in a live-cell bioimaging investigation into the roles of mitochondrial dynamics and mitophagy in the development of mitochondria to fuel seed germination. The results demonstrate how these processes are tightly interwoven with mitochondrial membrane biogenesis to provide the germinated seedling with mitochondria tailored for the transition from heterotrophy to autotrophy that occurs with seedling establishment.

The first chapter introduces the mitochondrial compartment and genetic components known to be involved in the control of mitochondrial dynamics, in addition to describing current knowledge about the selective degradation of mitochondria, and presents an overview of seed germination and the involvement of mitochondria therein.

The second chapter describes experiments and analyses performed to understand the mechanisms responsible for the development of mitochondria in the dry seed, the promitochondria, firstly into organelles able to fuel germination and then into organelles with the physical attributes and functions typical of mature mitochondria.

The third chapter presents experiments investigating mitochondrial quality control, and mitophagy in particular, and how these processes interface with mitochondrial dynamics during the developmental transitions leading to germination and subsequent seedling

The mitochondrial compartment

Mitochondria are one of the oldest organelles and define eukaryotic organisms, together with the presence of a nucleus, as underlined by the knowledge of only one eukaryote organism, the oxymonad Monocercomonoides sp., a eukaryotic microorganism, that lacks mitochondria or any traces of mitochondria in its lineage [START_REF] Karnkowska | A eukaryote without a mitochondrial organelle[END_REF].

Mitochondria are derived from an ancestral endosymbiosis of an α-proteobacteria by a pro-eukaryote organism, and during evolution mitochondria have retained their own DNA genome (mtDNA) despite a high level of genetic transfer to nucleus: for example, in the model plant Arabidopsis thaliana L. only 57 proteins are encoded by mtDNA relative to the >726 mitochondrial proteins confirmed in proteomic studies as being mitochondrial resident [START_REF] Lee | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy[END_REF][START_REF] Unseld | The mitochondrial genome of Arabidopsis thaliana contains 57 genes in 366,924 nucleotides[END_REF].

Mitochondria perform many vital functions such as the synthesis of carbon skeletons, iron-cluster assembly and as signalling hubs, but are best known as the sites of oxidative phosphorylation. Mitochondria are composed of two distinct phospholipid bilayer membranes, defining an inter membrane space (IMS) and an inner matrix. While the outer mitochondrial membrane (OMM) is characterised by the presence of large proteinaceous pores allowing diffusion of small molecules, the inner mitochondrial membrane (IMM) contains numerous specific channels and carriers leading to a less permissive flow of ions and molecules. Invagination in the IMM leads to the formation of the characteristic cristae observed in "typical" mitochondria of adult tissues that are structures with a tightly linked form and function, such that OXPHOS complex assembly in the IMM helps form the cristae and thus the presence of well-developed cristae is an indicator of enhanced OXPHOS activity. The matrix contains the mtDNA, in the form of a nucleoid composed of DNA and protein, that is, at least in yeast and human, attached to the IMM [START_REF] Hobbs | Mmm1p, a mitochondrial outer membrane protein, is connected to MtDNA nucleoids and required for mtDNA[END_REF][START_REF] Gerhold | Human Mitochondrial DNA-Protein Complexes Attach to a Cholesterol-Rich Membrane Structure[END_REF]. The distribution of mtDNA among the individual members of the chondriome (chondriome refers to all mitochondria in a cell, together), can be heterogeneous as indicated by the differential intensity of staining with fluorescent probes for mtDNA of individual mitochondria observed within plant cells, and lack of detected mtDNA in numerous individual mitochondria [START_REF] Satoh | Organization of heterogeneous mitochondrial DNA molecules in mitochondrial nuclei of cultured tobacco cells[END_REF][START_REF] Arimura | Frequent fusion and fission of plant mitochondria with unequal nucleoid distribution[END_REF][START_REF] Sheahan | Mitochondria as a connected population: Ensuring continuity of the mitochondrial genome during plant cell dedifferentiation through massive mitochondrial fusion[END_REF][START_REF] Takanashi | Different amounts of DNA in each mitochondrion in rice root[END_REF].

The Arabidopsis mtDNA genome can be represented as a master circle following sequence assembly, with a size of 367 Kbp in Arabidopsis and, as in the case of all plant mitochondrial genomes analysed, the genes of known function are very stable, exhibiting very low mutation rates relative to nuclear DNA, such that mtDNA genes are amongst the slowest evolving genes known [START_REF] Palmer | Plant Mitochondrial DNA Evolves Rapidly in Structure, but Slowly in Sequence[END_REF][START_REF] Mower | Extensive variation in synonymous substitution rates in mitochondrial genes of seed plants[END_REF][START_REF] Christensen | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy[END_REF].

Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ............................. 7 However, mtDNA is organised in a complex spectrum of circular, linear or multibranched molecules that are highly recombinogenic, thereby generating a population of subgenomic molecules, at the mitochondrial level, that drive mtDNA evolution [START_REF] Lonsdale | The Plant Mitochondrial Genome : Homologous Recombination as a Mechanism for Generating Heterogeneity[END_REF][START_REF] Bendich | The size and form of chromosomes are constant in the nucleus, but highly variable in bacteria, mitochondria and chloroplasts[END_REF][START_REF] Mower | Extensive variation in synonymous substitution rates in mitochondrial genes of seed plants[END_REF][START_REF] Arrieta-Montiel | Diversity of the arabidopsis mitochondrial genome occurs via nuclearcontrolled recombination activity[END_REF][START_REF] Davila | Double-strand break repair processes drive evolution of the mitochondrial genome in Arabidopsis[END_REF][START_REF] Miller-Messmer | RecA-dependent DNA repair results in increased heteroplasmy of the Arabidopsis mitochondrial genome[END_REF][START_REF] Gualberto | The plant mitochondrial genome: Dynamics and maintenance[END_REF][START_REF] Wallet | The RECG1 DNA Translocase Is a Key Factor in Recombination Surveillance, Repair, and Segregation of the Mitochondrial DNA in Arabidopsis[END_REF]. In vegetative tissues of higher plants, the chondriome is organised as a population of highly dynamic separate units, exhibiting mainly a spherical to ovoid morphology of diameter circa 0.5 to 1 µm, but more elongate morphologies are also found which can reach lengths of tens of microns. The heterogeneous mix of mtDNA molecules both in sequence and structure, generated by recombination, distributed amongst the hundreds of individual mitochondria, led Londsdale to propose that the plant chondriome should be considered as a dynamic syncytium in order to set a recombination equilibrium between mitochondrial subgenomes [START_REF] Lonsdale | The Plant Mitochondrial Genome : Homologous Recombination as a Mechanism for Generating Heterogeneity[END_REF]. However, the dynamic syncytium as defined by Lonsdale is ambiguous since mitochondria of higher plants are mainly present as physically separate units, while syncytium suggests a more connected fused state such as in yeast, leading to the suggestion of the discontinuous whole vision of the plant chondriome by Logan [START_REF] Lonsdale | The Plant Mitochondrial Genome : Homologous Recombination as a Mechanism for Generating Heterogeneity[END_REF][START_REF] Logan | The mitochondrial compartment[END_REF].

Plant mitochondrial undergo frequent fusion and fission events [START_REF] Arimura | Frequent fusion and fission of plant mitochondria with unequal nucleoid distribution[END_REF][START_REF] Sheahan | Mitochondria as a connected population: Ensuring continuity of the mitochondrial genome during plant cell dedifferentiation through massive mitochondrial fusion[END_REF], partly likely to ensure distribution of the multipartite mtDNA genome within the mitochondrial discontinuous whole. The events of mitochondrial fusion and fission are integral activities of the research field of mitochondrial dynamics, which defines the processes and underlying mechanisms leading to changes in mitochondrial shape, size, number, motility and cellular distribution.

Studies into the dynamic nature of mitochondria were first performed by observation of mitochondria after the staining of cells or tissues with dyes such as Janus Green before the development of Green Fluorescent Protein (GFP) as a genetically encoded marker, and the subsequent explosion of GFP-derived fluorescent proteins (FPs) or similar FPs from different organisms (e.g. DsRed). The application of fluorescent protein marker technology revolutionised cell biology in general and specifically facilitated the observation of organelle dynamics in living tissues. Genetically encoded translational fusions of GFP to native targeting sequences provides robust detection based on highcontrast fluorescence and enables real-time visualisation of dynamics without the need for any additional treatment of the cells or tissues. Since the first use of a FP as a specific marker of mitochondria in 1995 by Rizzuto et al. [START_REF] Rizzuto | Chimeric green fluorescent protein as atool for visualizing subcellular organelles in living cells[END_REF], a range of genetic constructs have been engineered and used for the tailored transformation of a range of different eukaryotes with matrix targeted GFP (simply termed mito-GFP), including tobacco and Arabidopsis [START_REF] Kohler | The green fluorescent protein as a marker to visualize plant mitochondria in vivo[END_REF]Zhu et al., 1997b;[START_REF] Logan | Mitochondria-targeted GFP highlights the heterogeneity of mitochondrial shape, size and movement within living plant cells[END_REF]. The availability of stable transformed Arabidopsis lines enabled functional genomics studies using forward and reverse genetics aimed at identifying the genes, proteins and mechanisms involved in the control of mitochondrial shape, size, Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ............................. 8 movement, cellular distribution and inheritance: collectively termed mitochondrial dynamics. For example, mutant screens based on the microscopic identification of individual mutants with altered mitochondrial dynamics were preformed which led to the identification of the NETWORK MITOCHONDRIAL (NMT) fission gene and the FRIENDLY (FMT) gene involved in mitochondrial distribution in Arabidopsis [START_REF] Logan | The genetic control of plant mitochondrial morphology and dynamics[END_REF]. In turn, the identification of subcellular location of proteins then enabled their use as fusion partners to direct the FP to the same location. In this way, matrix-targeted mito-GFP has been joined by FP constructs and stable plant lines with FPs targeted to the IM, the IMS, and the OMM. FP technology is now central to molecular cell biology and in addition to a whole spectrum of different colour variants, new techniques have been developed such as FRET, and Bimolecular Fluorescence Complementation, to investigate interactions between proteins in vivo as well as the identification and application of FPs that can undergo photoconversion in situ to change their emission spectrum (e.g. Kaede and EOS).

Mitochondrial dynamics

Displacement

The motility of plant mitochondria is necessary to enable the individual physically discrete mitochondria to interact and to fuse, and to facilitate redistribution of mitochondria within the cell.

Actin mediated displacement

Mitochondrial movement in plants depends mainly on the F-actin cytoskeleton [START_REF] Olyslaegers | Improved staining of F-actin and colocalization of mitochondria in plant cells[END_REF][START_REF] Van Gestel | Plant mitochondria move on F-actin, but their positioning in the cortical cytoplasm depends on both F-actin and microtubules[END_REF][START_REF] Sheahan | Organelle inheritance in plant cell division: The actin cytoskeleton is required for unbiased inheritance of chloroplasts, mitochondria and endoplasmic reticulum in dividing protoplasts[END_REF][START_REF] Sheahan | Mitochondria as a connected population: Ensuring continuity of the mitochondrial genome during plant cell dedifferentiation through massive mitochondrial fusion[END_REF] (Figure 1.1), which contrasts with the situation in most fungi and animals where mitochondrial movement is microtubule-based. Mitochondrial movement on actin occurs either by the dynamic rearrangement of F-actin, or because of the activity of motor proteins.

Actin dynamics is party controlled by the rate of polymerization and depolymerisation of actin monomers and these processes can also control mitochondrial movement as the actin filaments with bound mitochondria undergo "treadmilling". This process is known to be a key means of mitochondrial movement in Saccharomyces cerevisiae, and is dependent on the protein complex Arp2 / 3 [START_REF] Boldogh | Mitochondria on the move[END_REF]. In Arabidopsis, proteins forming the ARP2 / 3 complexes are also present, and mutation of the ARP2 gene greatly reduces the movement of mitochondria [START_REF] Zhao | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy[END_REF]. The involvement of the ARP2/3 complex was confirmed by an observed perimitochondrial localization of some ARP2 proteins, however the complex is also linked to the dynamics of other organelles [START_REF] Zhao | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy[END_REF][START_REF] Zhang | ARP2/3 localization in Arabidopsis leaf pavement cells: a diversity of intracellular pools and cytoskeletal interactions[END_REF]. Indeed, mitochondria are still able to move, and are seen in association with actin filaments, in arp2 mutant plants, indicating that the ARP2/3 complex is more involved in the formation of actin tracks favourable to the movement of mitochondria and other organelles than more specifically involved in mitochondrial movement per se [START_REF] Zhao | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy[END_REF].

The known motor proteins related to the actin-dependant displacement of organelles are members of the myosin family. Two groups of MYOSIN (groups VIII and XI) are found in

Arabidopsis, but only alteration of myosins in group XI induce disruption of the movement of various organelles [START_REF] Avisar | Myosin XI-K is required for rapid trafficking of Golgi stacks, peroxisomes, and mitochondria in leaf cells of Nicotiana benthamiana[END_REF][START_REF] Avisar | A comparative study of the involvement of 17 Arabidopsis myosin family Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy[END_REF]. Indeed, mutations of only two genes (MYOSIN XI-K and XI-2) are able to significantly interfere with the movement of organelles in plants, including mitochondria [START_REF] Avisar | Myosin XI-K is required for rapid trafficking of Golgi stacks, peroxisomes, and mitochondria in leaf cells of Nicotiana benthamiana[END_REF][START_REF] Avisar | A comparative study of the involvement of 17 Arabidopsis myosin family Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy[END_REF][START_REF] Peremyslov | Two class XI myosins function in organelle trafficking and root hair development in Arabidopsis[END_REF][START_REF] Sparkes | Truncated myosin XI tail fusions inhibit peroxisome, Golgi, and mitochondrial movement in tobacco leaf epidermal cells: A genetic tool for the next generation[END_REF]. However the XI group has 13 members and therefore functional redundancy may have masked the contribution of some myosin genes to organelle dynamics [START_REF] Avisar | Myosin XI-K is required for rapid trafficking of Golgi stacks, peroxisomes, and mitochondria in leaf cells of Nicotiana benthamiana[END_REF][START_REF] Peremyslov | Two class XI myosins function in organelle trafficking and root hair development in Arabidopsis[END_REF]. Indeed, observation of YFP fused to the myosin tail domain, that enables association with organelles, demonstrated the colocalisation of MYOSIN XI-J with mitochondria [START_REF] Reisen | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy[END_REF]. More recently, another study using YFP-myosin tail fusions for each of the 13 group XI myosins identified a perimitochondrial localization for 6 of the class XI myosins (XI-C, XI-E, XI-G, XI-H; XI, XI-K, XI-B) and also confirmed some functional redundancy due to the fact that disruption of multiple MYOSIN XI genes was necessary in order to seriously affect the motility of organelles [START_REF] Sattarzadeh | Arabidopsis myosin XI sub-domains homologous to the yeast myo2p organelle inheritance sub-domain target subcellular structures in plant cells[END_REF]. Little is known about how myosin motors are attached to mitochondria, but recent work suggested that they are connected to either the mitochondria, peroxisome and Golgi by a transient association involving MYOSIN BINDING PROTEIN (MYOB) adaptor proteins and that mutation of MYOB disturbs the dynamics of organelles dependant on class-XI motors [START_REF] Peremyslov | Identification of myosin XI receptors in Arabidopsis defines a distinct class of transport vesicles[END_REF][START_REF] Peremyslov | Myosin-powered membrane compartment drives cytoplasmic streaming, cell expansion and plant development[END_REF] (Figure 1.1).

Tubulin mediated displacement

Plant mitochondrial motility has also been reported to be affected by perturbation of the microtubule cytoskeleton [START_REF] Van Gestel | Plant mitochondria move on F-actin, but their positioning in the cortical cytoplasm depends on both F-actin and microtubules[END_REF][START_REF] Zheng | Actin turnover is required for myosin-dependent mitochondrial movements in Arabidopsis root hairs[END_REF] (Figure 1.1). In most yeast and animals, mitochondria associate with kinesins and dynein, two motor proteins that move along microtubules, by interaction of the adapter protein MILTON with the OMM protein MIRO [START_REF] Fransson | Atypical Rho GTPases have roles in mitochondrial homeostasis and apoptosis[END_REF][START_REF] Frederick | Yeast Miro GTPase, Gem1p, regulates mitochondrial morphology via a novel pathway[END_REF]. Arabidopsis contains three MIRO orthologues: MIRO1 and 2 are expressed ubiquitously, while MIRO3 is seed endosperm specific [START_REF] Yamaoka | EMB2473/MIRO1, an Arabidopsis Miro GTPase, is required for embryogenesis and influences mitochondrial morphology in pollen[END_REF]. MIRO1 and 2 localise to mitochondria and their functional disruption leads to altered mitochondrial morphology [START_REF] Yamaoka | EMB2473/MIRO1, an Arabidopsis Miro GTPase, is required for embryogenesis and influences mitochondrial morphology in pollen[END_REF][START_REF] Yamaoka | MIRO1 Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy[END_REF] MILTON [START_REF] Yamaoka | EMB2473/MIRO1, an Arabidopsis Miro GTPase, is required for embryogenesis and influences mitochondrial morphology in pollen[END_REF][START_REF] Yamaoka | The mitochondrial Ras-related GTPase Miro: views from inside and outside the metazoan kingdom[END_REF]. This fact can perhaps be explained by the observed functional divergence between the various types of MIRO protein present in the eukaryotic kingdom, which is presumably linked to an evolutionary divergence of MIRO-dependant mechanisms (Logan, 2010a;[START_REF] Yamaoka | The mitochondrial Ras-related GTPase Miro: views from inside and outside the metazoan kingdom[END_REF]. Indeed, MIRO1 and 2 are both involved in plant mitochondrial inheritance and are required for embryonic development [START_REF] Sørmo | Arabidopsis thaliana MIRO1 and MIRO2 GTPases are unequally redundant in pollen tube growth and fusion of polar nuclei during female gametogenesis[END_REF][START_REF] Yamaoka | MIRO1 Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy[END_REF][START_REF] Yamaoka | The mitochondrial Ras-related GTPase Miro: views from inside and outside the metazoan kingdom[END_REF], as also observed in yeast [START_REF] Mishra | Mitochondrial dynamics and inheritance during cell division, development and disease[END_REF], despite the lack of evidence of interaction with tubulin in Arabidopsis.

The fission apparatus

Although numerous proteins involved in the mitochondrial fusion and fission mechanisms have been identified using animal and yeast models, few have been identified in plants, and all of those that have been identified are implicated in fission.

Fission apparatus -dynamin dependant

The principle fission apparatus genes identified are members of the dynamin superfamily, and homologues are found in all eukaryotic kingdoms [START_REF] Otsuga | The dynamin-related GTPase, Dnm1p, controls mitochondrial morphology in yeast[END_REF][START_REF] Arimura | A dynamin-like protein (ADL2b), rather than FtsZ, is involved in Arabidopsis mitochondrial division[END_REF]. Dynamins are large GTPases, distinguishing them from the smaller Ras-like GTPases and other GTPases with regulatory functions such as trimeric GTPases, and belong to a superfamily of proteins involved in various processes related to the dynamics and fissions of membranes [START_REF] Antonny | Membrane fission by dynamin : what we know and what we need to know[END_REF]. Different subfamilies of dynamin are involved in the division of several types of organelle in S. cerevisiae, mammals and Arabidopsis, having functionally replaced the bacterial FtsZ-derived fission systems during evolution [START_REF] Leger | An ancestral bacterial division system is widespread in eukaryotic mitochondria[END_REF]. In Arabidopsis, 16 dynamin and dynaminlike proteins have been identified and classified by sequence similarity and putative functions into six subfamilies [START_REF] Hong | A unified nomenclature for Arabidospsis dynamin-related large GTPase based on homology and possible function[END_REF]. Dynamins from the DRP1 and DRP2 families are involved in phragmoplast formation and clathrin vesicle trafficking, but DRP2 sub-family members are specific to the plant kingdom [START_REF] Hong | A unified nomenclature for Arabidospsis dynamin-related large GTPase based on homology and possible function[END_REF][START_REF] Taylor | A role for Arabidopsis dynamin related proteins DRP2A/B in endocytosis; DRP2 function is essential for plant growth[END_REF][START_REF] Huang | Arabidopsis dynamin-related proteins, DRP2A and DRP2B, function coordinately in post-Golgi trafficking[END_REF]. DRP3s have been shown to be involved in mitochondrial and peroxisomal fission [START_REF] Arimura | A dynamin-like protein (ADL2b), rather than FtsZ, is involved in Arabidopsis mitochondrial division[END_REF][START_REF] Logan | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy[END_REF][START_REF] Mano | An Arabidopsis dynamin-related protein, DRP3A, controls both peroxisomal and mitochondrial division[END_REF]Logan, 2010a), while two DRP5 dynamins have been implicated in plastid and peroxisomal fission. The functions of the four dynamins from the DRP4 subfamily, and the single DRP6, are still unknown. The DRP3 subfamily is the main group of dynamins regulating mitochondrial fission in Arabidopsis, and consists of two members: DRP3A and DRP3B, that share 37-47% amino acid similarity with the human mitochondrial fission proteins DRP1 and yeast Dnm1 (Logan, 2010b). Disruption of DRP-dependent fission causes elongation of mitochondria and an increased mitochondrion volume [START_REF] Arimura | A dynamin-like protein (ADL2b), rather than FtsZ, is involved in Arabidopsis mitochondrial division[END_REF][START_REF] Arimura | Frequent fusion and fission of plant mitochondria with unequal nucleoid distribution[END_REF][START_REF] Logan | The genetic control of plant mitochondrial morphology and dynamics[END_REF][START_REF] Mano | An Arabidopsis dynamin-related protein, DRP3A, controls both peroxisomal and mitochondrial division[END_REF]) (Figure 1.2). Visualisation of DRP3A or B location using GFP fusions has demonstrated a primarily cytosolic location but, importantly, DRP3A/B-GFP also localises at the ends and constriction sites of mitochondria [START_REF] Arimura | A dynamin-like protein (ADL2b), rather than FtsZ, is involved in Arabidopsis mitochondrial division[END_REF][START_REF] Fujimoto | Arabidopsis dynaminrelated proteins DRP3A and DRP3B are functionally redundant in mitochondrial fission, but have distinct roles in peroxisomal fission[END_REF] (Figure 1.3). The DRP3s also have the capacity to form homo-or hetero-polymers, as observed for yeast Dnm1 [START_REF] Arimura | Arabidopsis ELONGATED MITOCHONDRIA1 is required for localization of DYNAMIN-RELATED PROTEIN3A to mitochondrial fission sites[END_REF][START_REF] Aung | Differential Roles of Arabidopsis Dynamin-Related Proteins DRP3A, DRP3B, and DRP5B in Organelle Division[END_REF], and are partially redundant in their role in mitochondrial division since the phenotype of drp3a/b double mutants is more severe than a single mutation alone. In yeast, regulation of Dnm1 association to mitochondria is mainly achieved posttranslationally, through phosphorylation, ubiquitination, SUMOylation or S-nitrolysation, and by its interaction with other partners and effectors [START_REF] Elgass | Recent advances into the understanding of mitochondrial fission[END_REF][START_REF] Koppenol-Raab | A targeted mutation identified through p K a measurements indicates a postrecruitment role for Fis1 in yeast mitochondrial fission[END_REF]. Indeed, yeast Dnm1 is present in the form of dimers or trimers in the cytoplasm and mitochondrial recruitment requires partners to stabilize the GTP-bindingdependant assembly into a spiral of several Dnm1 polymers surrounding the constriction sites. Assembly into a helix in turn triggers GTP hydrolysis which then drives a conformational change resulting in eventual membrane scission [START_REF] Elgass | Recent advances into the understanding of mitochondrial fission[END_REF].

Similarly, DRP3s of Arabidopsis are mainly regulated by their interactions with adapter proteins and effectors, but also post-translationally by phosphorylation and ubiquitination [START_REF] Arimura | Arabidopsis ELONGATED MITOCHONDRIA1 is required for localization of DYNAMIN-RELATED PROTEIN3A to mitochondrial fission sites[END_REF][START_REF] Aung | Differential Roles of Arabidopsis Dynamin-Related Proteins DRP3A, DRP3B, and DRP5B in Organelle Division[END_REF][START_REF] Wang | Phosphorylation and ubiquitination of dynamin-related proteins (AtDRP3A/3B) synergically regulate mitochondrial proliferation during mitosis[END_REF]. DRP3-dependant fission is also affected by mitochondrial membrane composition. Thus, for Arabidopsis, the synthesis of cardiolipin (CL) at the mitochondrial inner membrane by the activity of the cardiolipin synthase (CLS) enzyme is required for normal ultrastructure and promotes the stabilization of DRP3 oligomers at constrictions sites [START_REF] Katayama | Identification of an Arabidopsis thaliana gene for cardiolipin synthase located in mitochondria[END_REF][START_REF] Pineau | The importance of cardiolipin synthase for mitochondrial ultrastructure, respiratory function, plant development, and stress responses in Arabidopsis[END_REF][START_REF] Pan | Cardiolipin-mediated mitochondrial dynamics and stress response in Arabidopsis[END_REF].

Forward and reverse genetic screens enabled identification of three genes in addition to DRPs that are involved in the maintenance of mitochondrial morphology: NETWORK (At5g22350, [START_REF] Logan | The genetic control of plant mitochondrial morphology and dynamics[END_REF]), and BIGYIN1 and 2 (At3g57090 and At5g12390; [START_REF] Scott | BIGYIN, an orthologue of human and yeast FIS1 genes functions in the control of mitochondrial size and number in Arabidopsis thaliana[END_REF][START_REF] Zhang | FISSION1A and FISSION1B proteins mediate the fission of peroxisomes and mitochondria in Arabidopsis[END_REF])) (Figure 1.2). The BIGYIN1 and 2 genes, also named FISSION1A and B, are orthologues of the outer mitochondrial membrane protein Fis1 from human and Fis1p from S. cerevisiae. The BIGYIN proteins are anchored to the outer membrane of mitochondria through a C-terminal domain and gene mutation leads to fewer and enlarged mitochondria [START_REF] Scott | BIGYIN, an orthologue of human and yeast FIS1 genes functions in the control of mitochondrial size and number in Arabidopsis thaliana[END_REF][START_REF] Zhang | FISSION1A and FISSION1B proteins mediate the fission of peroxisomes and mitochondria in Arabidopsis[END_REF]) (Figure 1.2). BIGYINs may interact indirectly with the DRP3s to mediate the formation of the constriction ring around the mitochondria since no direct interaction had been observed by Y2H nor BiFC, in comparison to human and yeast studies [START_REF] Arimura | Arabidopsis ELONGATED MITOCHONDRIA1 is required for localization of DYNAMIN-RELATED PROTEIN3A to mitochondrial fission sites[END_REF][START_REF] Lingard | Arabidopsis PEROXIN11c-e, FISSION1b, and DYNAMIN-RELATED PROTEIN3A cooperate in cell cycle-associated replication of peroxisomes[END_REF]Zhang andHu, 2008, 2009). Thus, the functions of BIGYIN proteins in fission are not clear due to the lack of known protein interactors in plants. In yeast, a recent study indicated that in addition to being required for Dnm1 mitochondrial recruitment, Fis1 proteins also function in the assembly of massive and continuous Dnm1 polymers into the form of a constriction ring with the help of adapter proteins complexes [START_REF] Koppenol-Raab | A targeted mutation identified through p K a measurements indicates a postrecruitment role for Fis1 in yeast mitochondrial fission[END_REF].

NETWORK (NMT), also known as ELM1, has one homologue (At5g06180) in Arabidopsis, but no homologue outside the plant kingdom [START_REF] Logan | The genetic control of plant mitochondrial morphology and dynamics[END_REF][START_REF] Arimura | Arabidopsis ELONGATED MITOCHONDRIA1 is required for localization of DYNAMIN-RELATED PROTEIN3A to mitochondrial fission sites[END_REF].

NMT localises to the outer mitochondrial membrane but does not have a transmembrane domain [START_REF] Arimura | Arabidopsis ELONGATED MITOCHONDRIA1 is required for localization of DYNAMIN-RELATED PROTEIN3A to mitochondrial fission sites[END_REF][START_REF] Wagner | The EF-Hand Ca2+ Binding Protein MICU Choreographs Mitochondrial Ca2+ Dynamics in Arabidopsis[END_REF][START_REF] Yamashita | Formation of mitochondrial outer membrane derived protrusions and vesicles in arabidopsis thaliana[END_REF] (Figure 1.4).

It has been suggested that NMT functions to physically mediate the recruitment of cytosolic DRPs to the mitochondrial membrane at subsequent constriction sites [START_REF] Arimura | Arabidopsis ELONGATED MITOCHONDRIA1 is required for localization of DYNAMIN-RELATED PROTEIN3A to mitochondrial fission sites[END_REF] in a manner similar to that performed in S. cerevisiae where recruitment of Dnm1p by Fis1p is mediated by an adapter protein complex involving Mdv1p and Caf4p (Logan, 2010b). In contrast, there is uncertainty about any interactions between NMT1

and BIGYIN in Arabidopsis because no interaction was identified by yeast two-hybrid screening (Y2H) [START_REF] Arimura | Arabidopsis ELONGATED MITOCHONDRIA1 is required for localization of DYNAMIN-RELATED PROTEIN3A to mitochondrial fission sites[END_REF] (Figure 1.4). Nevertheless, an interaction has been reported using BiFC [START_REF] Ruberti | Molecular Characterization of Mitochondria Interactions with other Organelles[END_REF] but not in a peer reviewed scientific journal and no data was presented, so no definitive conclusions can be reached. Thus, other adapter proteins are likely present in plants in order to regulate interaction between BIGYIN and DRP3s and / or for the stabilization at the constriction sites of the DRP3s polymer in a NMT1-dependent manner (Logan, 2010b).

In an attempt to discover the identity of these adaptors, an in silico study identified eight genes in Arabidopsis with a similar domain organisation to the S. cerevisiae proteins Mdv1 and Caf4 [START_REF] Li | YAO is a nucleolar WD40-repeat protein critical for embryogenesis and gametogenesis in Arabidopsis[END_REF][START_REF] Pan | The conserved fission complex on peroxisomes and mitochondria[END_REF], however none of those genes have been reported to affect mitochondria, showing instead involvement in embryo patterning or being identified as component parts of ubiquitination complexes. Members of the dynamin superfamily other than DRP3 have also been implicated in mitochondrial division. For example, a similar mitochondrial phenotype to drp3a/b mutants has been observed in a drp1c dominant negative mutant and in drp1e T-DNA insertion mutants, although the images presented were of generally poor quality and the objects identified as mitochondria had very suspicious morphologies [START_REF] Jin | The Arabidopsis dynamin-like proteins ADL1C and ADL1E play a critical role in mitochondrial morphogenesis[END_REF]. DRP1C and E proteins were claimed by [START_REF] Jin | The Arabidopsis dynamin-like proteins ADL1C and ADL1E play a critical role in mitochondrial morphogenesis[END_REF] to show partial colocalisation with mitochondria, but only data obtained with GFP-DRP1C was presented, with no quantification, and it can be argued that the distribution was simply random resulting in three overlapping dots amongst five times as many not overlapping. A second study presented more convincing evidence of association between GFP-DRP1E and Mitotracker stained structures [START_REF] Tang | A mutation in the GTP hydrolysis site of Arabidopsis dynamin-related protein 1E confers enhanced cell death in response to powdery mildew infection[END_REF] whereas other studies have localised DRP1C and E to clathrin endocytic vesicles and at the phragmoplast of dividing cells [START_REF] Hong | A unified nomenclature for Arabidospsis dynamin-related large GTPase based on homology and possible function[END_REF][START_REF] Jin | The Arabidopsis dynamin-like proteins ADL1C and ADL1E play a critical role in mitochondrial morphogenesis[END_REF][START_REF] Konopka | Dynamics of Arabidopsis dynamin-related protein 1C and a clathrin light chain at the plasma membrane[END_REF]. Given the lack of robust data it must be concluded that the question of the involvement of these two dynamins in mitochondrial division is still open.

DRP3A DRP3B

Additional dynamins have been suggested to be involved in mitochondrial dynamics. A study identified DRP5B involvement in the fission of mitochondria in addition to its role in the division of plastids and peroxisomes [START_REF] Aung | Differential Roles of Arabidopsis Dynamin-Related Proteins DRP3A, DRP3B, and DRP5B in Organelle Division[END_REF]. Indeed, the drp5b mutants have a mitochondrial elongation phenotype similar to Drp3a/b mutants but also show a swollen morphology, and drp3 / drp5 double mutants have a stronger aberrant mitochondrial phenotype [START_REF] Aung | Differential Roles of Arabidopsis Dynamin-Related Proteins DRP3A, DRP3B, and DRP5B in Organelle Division[END_REF] (Figure 1.2). However, the role of DRP5B in mitochondrial fission is not clear since localisation to the exterior of chloroplasts and peroxisomes was detected using YFP fusions but no association with mitochondria was detected [START_REF] Aung | Differential Roles of Arabidopsis Dynamin-Related Proteins DRP3A, DRP3B, and DRP5B in Organelle Division[END_REF]) (Figure 1.3). Moreover, the same authors identified interactions between DRP3A or DRP3B and DRP5B, as well as between DRP5B and BIGYIN1, in vivo by BiFC and in vitro by co-immunoprecipation (co-IP) but not in vitro by Y2H, which suggests the requirement of additional partners (absent in the Y2H experiment) to mediate BIGYIN1 interaction with DRP5B, similarly to DRP3A and DRP3B, or for yeast Dnm1p [START_REF] Zhang | The Arabidopsis chloroplast division protein DYNAMIN-RELATED PROTEIN5B also mediates peroxisome division[END_REF]Aung andHu, 2011, 2012).

Fission apparatus -dynamin independent

The role of DRP5 in the fission of plastids has been investigated in greater detail than its role in mitochondrial fission and shows similarity to the mitochondrial system in the requirement for anchored proteins at the plastid outer membrane and adapters to form stable periplastidic DRP5 polymers [START_REF] Holtsmark | Plastid division control: The PDV proteins regulate DRP5B dynamin activity[END_REF]. Two PLANT MITOCHONDRIA DIVISION proteins (PMD1 and 2) were identified following an in-silico screen searching for proteins containing a transmembrane domain and several protein interaction domains organised similary to the chloroplastic outer membrane PDVs proteins mediating DRP5s assembly [START_REF] Aung | The Arabidopsis tail-anchored protein PEROXISOMAL AND MITOCHONDRIAL DIVISION FACTOR1 is involved in the morphogenesis and proliferation of peroxisomes and mitochondria[END_REF]. Disruption of PMD1 or 2 gene function causes a mitochondrial phenotype similar to drp3 mutations: disruption of PMD2 function disturbs specifically mitochondrial morphology while disruption of PMD1 leads also to elongated peroxisomes [START_REF] Aung | The Arabidopsis tail-anchored protein PEROXISOMAL AND MITOCHONDRIAL DIVISION FACTOR1 is involved in the morphogenesis and proliferation of peroxisomes and mitochondria[END_REF] (Figure 1.2). Analysis of the intracellular location of fluorescent proteins fused to PMD1 or 2 indicates a homogeneous pattern of fluorescence at the outer mitochondrial membrane [START_REF] Aung | The Arabidopsis tail-anchored protein PEROXISOMAL AND MITOCHONDRIAL DIVISION FACTOR1 is involved in the morphogenesis and proliferation of peroxisomes and mitochondria[END_REF][START_REF] Marty | New insights into the targeting of a subset of tailanchored proteins to the outer mitochondrial membrane[END_REF]) (Figure 1.3). However, the mechanism of PMD action is not resolved. Indeed, co-IP and Y2H

experiments have shown that PMD1 and 2 can form homo-or hetero-complexes but no interactions with DRP3s or 5B or BIGIYIN1 have been identified [START_REF] Aung | The Arabidopsis tail-anchored protein PEROXISOMAL AND MITOCHONDRIAL DIVISION FACTOR1 is involved in the morphogenesis and proliferation of peroxisomes and mitochondria[END_REF].

Interaction with NMT has not been tested, but given that mutation of PMDs does not affect the mitochondrial location of DRP3s, the authors suggested that these two proteins may have a role in the mitochondrial fission machinery independent of the currently known actors [START_REF] Aung | The Arabidopsis tail-anchored protein PEROXISOMAL AND MITOCHONDRIAL DIVISION FACTOR1 is involved in the morphogenesis and proliferation of peroxisomes and mitochondria[END_REF]. However, the possible independence of this pathway can be questioned, especially concerning the regulation of DRP3B localisation, since the supplementary data presented by [START_REF] Aung | The Arabidopsis tail-anchored protein PEROXISOMAL AND MITOCHONDRIAL DIVISION FACTOR1 is involved in the morphogenesis and proliferation of peroxisomes and mitochondria[END_REF] clearly show a reduction in mitochondrial constriction sites marked by DRP3B and increased sizes of the fluorescent spots in the pmd2 mutant (sup data [START_REF] Aung | The Arabidopsis tail-anchored protein PEROXISOMAL AND MITOCHONDRIAL DIVISION FACTOR1 is involved in the morphogenesis and proliferation of peroxisomes and mitochondria[END_REF] (Figure 1.4). In addition, the results of co-IP investigating interaction of PMD2 and FIS1A are not convincing since recognition of the FIS1A HA tag was very weak, and interactions with NMT should be tested since NMT could interact directly with DRP3s as an adaptor. Thus, the possibility that PMD2 could be part of the complex promoting the recruitment of DRPs to the subsequent constriction site via NMT1 and BIGYIN1/2 cannot be ruled out.

In summary, studies of mitochondrial fission in plants have revealed eight actors and indicate the importance of post-translational regulatory mechanisms for the activity of these proteins, in addition to a contribution from the lipid composition of the membranes.

However, there are still dark areas, such as the processes leading to formation of the constriction site, the existence of others adaptor proteins to recruit DRP3s, and the involvement of DRP5B or other DRPs (Figure 1.5).

The fusion apparatus

In general, disruption of mitochondrial fission causes an elongate, often reticulated, mitochondrial morphology, as fusion continues in the absence of fission. In contrast, disruption of the fusion apparatus, at least in S. cerevisiae, leads to an increase in the number of individual mitochondria together with a decreased volume of individual organelles [START_REF] Mozdy | Dnm1p Gtpase-Mediated Mitochondrial Fission Is a Multi-Step Process Requiring the Novel Integral Membrane Component Fis1p[END_REF] (Figure 1.6). These clear and distinct phenotypes have enabled the identification of several components of the fusion apparatus in non-plant organisms. In yeast, for example, three key genes are known to be involved in the fusion of outer and inner mitochondrial membranes. Two of these genes, FZO1 (MFN1 in mammals) and UGO1 (similar to members of the mitochondrial solute carrier family in mammals), code for proteins that localise to the outer mitochondrial membrane [START_REF] Herlan | Processing of Mgm1 by the rhomboid-type protease Pcp1 is required for maintenance of mitochondrial morphology and of mitochondrial DNA[END_REF]Sesaki andJensen, 2001, 2004), while the third gene MGM1 (OPA1 in mammals), encodes a protein of the dynamin large GTPase family, with an intermembrane space localisation [START_REF] Wong | The dynamin-related GTPase, Mgm1p, is intermembrane space protein required for maintenance of fusion competent mitochondria[END_REF]. Homologues of some of the fusion genes identified in yeast are also found in other eukaryotes, such as MFN1/2 and OPA1 genes mentioned above, that are mammalian homologues of the FZO1 and MGM1 genes respectively. (Logan, 2010a). Also, the different screens undertaken in Arabidopsis have failed to identify genes actively involved in the fusion of mitochondrial membranes, and leading to a mitochondrial morphology similar to that observed following the shift in fusion/fission balance by overexpression of fission components, nor have mutants been identified with phenotypes similar to the mitochondrial phenotype in yeast fusion mutants (Logan, 2010a) (Figure 1.6). One explanation for the failure to identify genes involved in plant mitochondrial fusion from loss-of-function mutant screens is the fact that plant mitochondria are mainly present as discrete units generated by fission. Given that late steps of mitochondrial fission also require membrane fusion in order to reseal the OMM and IMM to maintain their integrity and avoid leakage of compartment contents, loss of mitochondrial membrane fusion capability may simply be lethal.

Electron cryotomographic microscopy techniques and an in vitro mitochondrial fusion assay have recently enabled observation of the progression of fusion yeast mitochondria and enabled the detection of discrete steps in this process [START_REF] Brandt | A mitofusindependent docking ring complex triggers mitochondrial fusion in vitro[END_REF].

Separate mitochondria are first tethered to each other by a protein aggregate and then the two OMMs dock over an extended area aided by a ring of protein which triggers subsequent fusion of the OMM by GTPase activity, resulting in conformational change of the membrane, and localised fusion. OMM fusion is then followed by inner membrane fusion mediated by proteins localised to the inner membrane space that are in part regulated by membrane lipid composition [START_REF] Brandt | A mitofusindependent docking ring complex triggers mitochondrial fusion in vitro[END_REF]. No such high-resolution observations have been made using plants but careful observations, based on live-cell visualisation of targeted fluorescence proteins, allow to distinguish early and late steps in the course of the mitochondrial fusion process, at least in Arabidopsis (Figure 1.6).

In plants, it is clear from observing mito-GFP, or OMM targeted FP, that fusion or fission events happen very rapidly, within a few seconds, involving fast membrane dynamics and remodelling [START_REF] Logan | Mitochondrial biogenesis during germination in maize embryos[END_REF][START_REF] Arimura | Frequent fusion and fission of plant mitochondria with unequal nucleoid distribution[END_REF]) (Logan, unpublished, personal observations). The most frequent kind of fusion observed occurs between two mitochondria that become physically close and maintain this association until fusion. Less frequently, alternative, albeit putative, fusion mechanisms may be involved. For example, connection and exchange between fusion partners that have their OMMs physically separated by a distance of few micrometers may be facilitated by the formation of relatively thin membranous extensions from the OMM and IMM called matrixules, which infers the involvement of a much smaller area of membrane to make the connection between the fusion partners. However, it is emphasised here that mitochondrial fusion via matrixules has not been proven experimentally [START_REF] Logan | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy[END_REF][START_REF] Logan | The mitochondrial compartment[END_REF]Scott et al., 2007;[START_REF] Mueller | Mitochondrial dynamics and the ER: The plant perspective[END_REF][START_REF] Yamashita | Formation of mitochondrial outer membrane derived protrusions and vesicles in arabidopsis thaliana[END_REF] (Figure 1 It is also possible that matrixules represent a stalled, or failed, attempt to make connections between physically separate mitochondria. In this way, matrixule formation could been indicative of mitochondria that are able to initiate fusion but unable to accomplish the transition from the tethering/docking steps to fusion, and subsequent differential motility of the failed fusion partners may stretch the mitochondrial membranes of one of the two partners (Figure 1.7). In this regard, the increase in matrixule frequency observed in the drp3a mutant [START_REF] Logan | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy[END_REF]) could be indicative of either defective fission or defective fusion.

The fact that mitochondria fuse raises questions about the dynamic nature of the matrix.

Indeed, in-vivo observation of remodelling of the matrix, enabled using matrix targeted mito-GFP, from a round to a tubular morphology within seconds, indicates the high elasticity of mitochondria that allows transient compaction or extension of their membrane without the requirement for membrane degradation or synthesis. The numerous cristae observed in mature mitochondria demonstrate the highly folded nature of the IMM in mature mitochondria which may be extended easily upon release of the exterior restraining force supplied by the OMM. Overcoming a mechanical constraint on the IMM during mitochondrial shape change, such as by stretching of the OMM, with assistance of membrane remodelling proteins, may provide a means of rapid matrix volume regulation. Equally, swelling of the mitochondrial matrix, such as results from a mitochondrial permeability transition, may cause stretching of the OMM enabling rapid shape changes. Such matrix-driven expansion of the OMM could equally explain an increase in matrixule frequency in WT plants at times, or under conditions, were extension of mitochondrion membrane is promoted (Figure 1.7).

Mitochondrial dynamics during development and following modification of cell environment

Plants bearing mutations in genes involved in mitochondrial dynamics have alterered plant growth. For example, drp3a/b double mutants showing a reticulated chondriome phenotype exhibit an overall reduction in growth, while the less reticulated chondriome phenotype of single drp3 mutants have less pronounced growth defects: 25% reduction in hypocotyl length of 5 day-old dark-grown single drp3 mutant seedlings, relative to wild type (WT), versus a 40% reduction for the double mutant [START_REF] Fujimoto | Arabidopsis dynaminrelated proteins DRP3A and DRP3B are functionally redundant in mitochondrial fission, but have distinct roles in peroxisomal fission[END_REF][START_REF] Zhang | Two small protein families, DYNAMIN-RELATED PROTEIN3 and FISSION1, are required for peroxisome fission in Arabidopsis[END_REF][START_REF] Aung | The Arabidopsis tail-anchored protein PEROXISOMAL AND MITOCHONDRIAL DIVISION FACTOR1 is involved in the morphogenesis and proliferation of peroxisomes and mitochondria[END_REF][START_REF] Pan | Cardiolipin-mediated mitochondrial dynamics and stress response in Arabidopsis[END_REF]. Similarly, nmt/elm1 mutants have also been reported to have altered vegetative growth and were reported to exhibit a reduction in pollen production leading to loss in fertility [START_REF] Scott | The control of mitochondrial morphology and dynamics in Arabidopis thaliana[END_REF][START_REF] Arimura | Arabidopsis ELONGATED MITOCHONDRIA1 is required for localization of DYNAMIN-RELATED PROTEIN3A to mitochondrial fission sites[END_REF]. In addition to results from the study of mutants, the importance of mitochondrial dynamics is clear from fluctuations in chondriome structure linked to plant development.

For example, extensive changes have been recorded in mitochondrial structure during protoplast culture and especially during dedifferentiation of protoplasts when the mitochondria undergo a massive fusion event prior to subsequent fission and reformation of a typical discontinuous chondriome [START_REF] Sheahan | Mitochondria as a connected population: Ensuring continuity of the mitochondrial genome during plant cell dedifferentiation through massive mitochondrial fusion[END_REF]. Moreover, Arabidopsis leaf meristematic cells display a reorganisation of the chondriome leading to a perinuclear cage at the time of mitosis and a subsequent fragmentation at the time of cytokinesis, which may help to ensure mitochondrial function in vegetative cells [START_REF] Segui-Simarro | The mitochondrial cycle of Arabidopsis shoot apical meristem and leaf primordium meristematic cells is defined by a perinuclear tentaculate/cage-like mitochondrion[END_REF].

Plant responses to abiotic stress and environmental perturbation also involve modification of mitochondrial morphology. Indeed, mitochondria display an aberrant swollen morphology upon tissue exposure to ROS or heat-induced stress, in addition to blocked cytoplasmic streaming [START_REF] Yoshinaga | Mitochondrial behaviour in the early stages of ROS stress leading to cell death in Arabidopsis thaliana[END_REF]Scott and Logan, 2007), while chilling stress leads to an increase in the number of mitochondria with unusual morphologies, such as ring-shaped, in mesophyll cells [START_REF] Vella | Chilling-induced ultrastructural changes to mesophyll cells of Arabidopsis grown under short days are almost completely reversible by plant re-warming[END_REF]. Exposure of the whole plant to environmental change during development also causes changes to mitochondrial dynamics. In leaf epidermal tissues of two-month-old plants grown under cold conditions, or in plants cold-treated for three weeks, cells were shown to have twice as many mitochondria, of higher mean volume, as an adaptive response to cold to reestablish respiratory flux (Armstrong et al., 2006a(Armstrong et al., , 2006b)). Furthermore, exposure of plants to an elevated flux of ultraviolet radiation led to irregular clustering of mitochondria together with blocked mitochondrial motility [START_REF] Gao | Implication of reactive oxygen species and mitochondrial dysfunction in the early stages of plant programmed cell death induced by ultraviolet-C overexposure[END_REF]. Starvation also affects mitochondrial dynamics: anoxia has been reported to induce a swelling of mitochondria leading to a transient ring shaped mitochondrial morphology [START_REF] Logan | The mitochondrial compartment[END_REF][START_REF] Jaipargas | Mitochondrial pleomorphy in plant cells is driven by contiguous ER dynamics[END_REF], whereas dark starvation caused by growing plants in the dark for seven days led to an increased proportion of mitochondria with a tubular morphology, as did growing plants on zero-sucrose medium in the dark or light [START_REF] Jaipargas | Mitochondrial pleomorphy in plant cells is driven by contiguous ER dynamics[END_REF].

Biotic stresses have also been reported to influence and be influenced by mitochondrial dynamics. Indeed, an Arabidopsis mutant screen for plants that fail to respond to oxylipins, oxidised fatty acid molecules known to activate local plant defences against pathogens, led to the identification of mutant alleles of DRP3A and DRP3B, in addition to a mutant named noxy38 which is allelic to friendly [START_REF] Vellosillo | Defense activated by 9-lipoxygenasederived oxylipins requires specific mitochondrial proteins[END_REF]. These mutants displayed altered mitochondrial dynamics but were identified in the screen by showing an increased susceptibility to the biotrophic pathogen Pseudomonas syringae, thus demonstrating the involvement of mitochondrial dynamics in the response to pathogen attack [START_REF] Vellosillo | Defense activated by 9-lipoxygenasederived oxylipins requires specific mitochondrial proteins[END_REF] [START_REF] Koh | Arabidopsis thaliana subcellular responses to compatible Erysiphe cichoracearum infections[END_REF][START_REF] Fuchs | Immobilized Subpopulations of Leaf Epidermal Mitochondria Mediate PENETRATION2-Dependent Pathogen Entry Control in Arabidopsis[END_REF].

Taken together, these results demonstrate that plant cells are able to constantly adjust chondriome structure in order to respond to a changing environment, notably through homeostatic modification of mitochondrial dynamics, and that modification of mitochondrial dynamics is a common cellular response to perturbation and provides a means to physiological adaptation.

The central role of mitochondrial dynamics in shaping cell and whole organism physiology, and its role in development, reproduction, pathology, disease progression, aging and death in non-plant eukaryotes is well established. Indeed, disruption of fission is connected to cyto-protective effects in a range of cardiovascular and brain cell types, while cytotoxic effects result from preventing normal mitochondrial fission in hyperproliferative cells, such as cancer cells [START_REF] Rosdah | Mitochondrial fission -a drug target for cytoprotection or cytodestruction?[END_REF]. Deregulation of mitochondrial fusion leads to impaired OXPHOS activity together with loss of mtDNA in yeast, and is linked to heart and neurodegenerative disorders such as Alzheimer's and

Parkinson's diseases in mammals [START_REF] Hermann | Mitochondrial fusion in yeast requires the transmembrane GTPase Fzo1p[END_REF][START_REF] Lenaers | Mitochondrial Dynamics and Neurodegeneration[END_REF][START_REF] Mao | The Scaffold Protein Atg11 Recruits Fission Machinery to Drive Selective Mitochondria Degradation by Autophagy[END_REF][START_REF] Trushina | Defects in mitochondrial dynamics and metabolomic signatures of evolving energetic stress in mouse models of familial alzheimer's disease[END_REF][START_REF] Reddy | Dynamin-related protein 1 and mitochondrial fragmentation in neurodegenerative diseases[END_REF]. Additionally, it has been demonstrated in yeast and human that mitochondrial dynamics is linked to aging and cell death phenomena, notably through adaptation of the chondriome to the damage produced by ROS during metabolic activity [START_REF] Braun | Mitochondrial dynamics in yeast cell death and aging[END_REF][START_REF] Breitenbach | Mitochondria in ageing: There is metabolism beyond the ROS[END_REF][START_REF] Birch-Machin | Oxidative stress and ageing[END_REF].

In summary, these studies provide strong evidence of the tight interconnections between mitochondrial dynamics and cell and tissue function, and thus organism health. However, mitochondrial dynamics is also key to the processes maintaining the quality of the mitochondrial themselves, which underpins cell, tissue and whole organism health.

Mitochondrial quality control and regulation of dynamics to enable selective degradation of mitochondria

Mitochondrial quality control (MQC) is the name given to a series of processes and mechanisms that act to maintain a healthy chondriome. Our understanding of these processes and mechanism is rudimentary at present, but it is a rapidly expanding field of study, especially due to the central role mitochondrial function plays in cellular homeostasis, and in the prevention of disease, as outlined in the previous section. The PINK and PARKIN genes found in mammals and insects are the main actors in one of the most studied mitochondrial quality control pathways [START_REF] Narendra | Parkin is recruited selectively to impaired mitochondria and promotes their autophagy[END_REF][START_REF] Kanki | Atg32 is a mitochondrial protein that confers selectivity during mitophagy[END_REF][START_REF] Okamoto | Mitochondria-Anchored Receptor Atg32 Mediates Degradation of Mitochondria via Selective Autophagy[END_REF] [START_REF] Narendra | PINK1 is selectively stabilized on impaired mitochondria to activate Parkin[END_REF][START_REF] Chan | Broad activation of the ubiquitinproteasome system by Parkin is critical for mitophagy[END_REF][START_REF] Matsuda | PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy[END_REF][START_REF] Eiyama | PINK1/Parkin-mediated mitophagy in mammalian cells[END_REF](Firgure 1.8).

The interweaving of mitochondrial dynamics and MQC is complex, and quality control (QC) involves fusion and fission in order to drive biogenesis, while fission is additionally required to isolate damaged mitochondria from the rest of the chondriome. These processes have been highlighted in an elegant study showing that in some cases after the fusion of two mitochondria the subsequent fission event regenerated one mitochondrion with a membrane potential similar to before fission, while the second depolarised without observed recovery. Because fusion is dependent on membrane potential this fission event may contribute to the isolation of defective mitochondria [START_REF] Twig | Fission and selective fusion govern mitochondrial segregation and elimination by autophagy[END_REF].

Events downstream of the activation of PINK1/Parkin ensure isolation of defective mitochondria via modulation of mitochondrial dynamics. Upon depolarisation, the PINK/Parkin pathway results in ubiquitination, and consequent proteasome-degradation, of MITOFUSIN proteins (large GTPases involved in mitochondrial fusion) to promote increased fission and decreased fusion [START_REF] Yang | Pink1 regulates mitochondrial dynamics through interaction with the fission/fusion machinery[END_REF][START_REF] Poole | The Mitochondrial Fusion-Promoting Factor Mitofusin Is a Substrate of the PINK1/Parkin Pathway[END_REF]Tanaka et al., 2010;[START_REF] Ziviani | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy[END_REF][START_REF] Yoshii | Parkin mediates proteasome-dependent protein degradation and rupture of the outer mitochondrial membrane[END_REF]. In addition, PINK1/Parkin-mediated proteasome-dependant degradation of MIRO (an OMM adaptor protein for tubulin motors, see paragraph 2.1.2) has been observed to lead to reduced mitochondrial displacement, and thus may promote immobilisation of defective mitochondria to promote subsequent degradation and avoid spread of damage throughout the chondriome [START_REF] Liu | Mitochondrial outer-membrane protein FUNDC1 mediates hypoxiainduced mitophagy in mammalian cells[END_REF].

In summary, MQC, by modification of mitochondrial dynamics, helps identify and isolate defective mitochondria than can then be removed from the chondriome by selective mitophagy. However, there is a complex interweave of quality control mechanisms, biogenesis, and dynamics and the situation in plants is very poorly understood as shown in the following section.

The mitophagy apparatus

Named by Christian de Duve during his presentation at the CIBA Foundation Symposium in 1963 [START_REF] Feng | The machinery of macroautophagy[END_REF], autophagy is a conserved process that enables cells to degrade or recycle components. Selective autophagy of superfluous or damaged organelles is achieved by the macroautophagy pathway. Macroautophagy is considered a relatively non-specific bulk waste-collection and recycling service, while the specific removal and recycling of mitochondria and mitochondrial components is termed mitophagy (Figure 1.8). In yeast and mammals, genetic screens and related research have identified many actors in the autophagy and mitophagy pathways, and most of them are classified as members of the ATG gene family (AuTophaGy-related gene), with different sub-family members mediating different functions in order to accomplish autophagy [START_REF] Feng | The machinery of macroautophagy[END_REF]. Indeed, during the activation of mitophagy, defective organelles harbouring specific OMM proteins, or else bearing a polyubiquitin tag, are recognized by adaptor proteins and consequently surrounded by a growing doublemembrane structure termed the phagophore. Once growth of the phagophore is complete, the new organelle, composed of phagophore membrane and engulfed material, is called an autophagosome [START_REF] Feng | The machinery of macroautophagy[END_REF]. The autophagosome is next targeted to the lytic vacuole in order to deliver its contents to the vacuole lumen where the degradation and recycling process can continue [START_REF] Reggiori | Autophagic processes in yeast: Mechanism, machinery and regulation[END_REF])(Figure 1.8). Mitophagy has been reported to require from several hours to days to perform the complete clearance of a mitochondrion, whereas an alternative process, at least in human, involving Mitochondrial Derived Vesicles (MDV) formation, by a Parkin/PINK1dependant and DRP1-independant pathway, requires only a few hours to extract oxidised mitochondrial proteins and destroy them in the lysosome [START_REF] Chan | Broad activation of the ubiquitinproteasome system by Parkin is critical for mitophagy[END_REF]Soubannier et al., 2012bSoubannier et al., , 2012a;;[START_REF] Mclelland | Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control[END_REF].

In plants, both the bulk and selective autophagy pathways, each dependent on the formation of ATG8-decorated phagophores, have been observed using ATG8-FP fusions, both during development and in response to various stresses [START_REF] Sláviková | The autophagy-associated Atg8 gene family operates both under favourable growth conditions and under starvation stresses in Arabidopsis plants[END_REF][START_REF] Slavikova | An autophagy-associated Atg8 protein is involved in the responses of Arabidopsis seedlings to hormonal controls and abiotic stresses[END_REF]. However, while the main autophagy actors from non-plant eukaryotes are conserved in Arabidopsis [START_REF] Thompson | Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways[END_REF][START_REF] Bassham | Autophagy in development and stress responses of plants[END_REF], few components of the plant mitophagy process have been identified due to a lack of published functional studies about the assembly of the mitophagy apparatus. In fact, while the assembly of the phagophore surrounding mitochondria is well described in yeast and human studies [START_REF] Youle | Mechanisms of mitophagy[END_REF][START_REF] Feng | The machinery of macroautophagy[END_REF], experimental comparison with the situation in plants has been made only twice, and in only one published study using Arabidopsis [START_REF] Li | AUTOPHAGY-RELATED11 plays a critical role in general autophagy-and senescence-induced mitophagy in Arabidopsis[END_REF][START_REF] Li | AUTOPHAGY-RELATED11 plays a critical role in general autophagy-and senescence-induced mitophagy in Arabidopsis[END_REF], despite numerous studies indicating coordinated disassembly of mitochondria during plant development, or in response to starvation, or supplemented ROS [START_REF] Logan | Mitochondrial biogenesis during germination in maize embryos[END_REF][START_REF] Minibayeva | Oxidative stress-induced autophagy in plants: The role of mitochondria[END_REF][START_REF] Avin-Wittenberg | Global analysis of the role of autophagy in cellular metabolism and energy homeostasis in Arabidopsis seedlings under carbon starvation[END_REF][START_REF] Michaeli | Autophagy in Plants -What's New on the Menu?[END_REF]. There are no Arabidopsis orthologues of genes known to be involved in the recognition of, and subsequent recruitment of, the phagophore to defective mitochondria such as the yeast ATG32 OMM protein, or the mammalian PINK/Parkin complex, but one Arabidopsis gene, NBR1 is orthologous to mammalian OMM proteins with LC3 domains, such as the FUNDC1, NIX and SQSTM1 proteins [START_REF] Youle | Mechanisms of mitophagy[END_REF][START_REF] Svenning | Plant NBR1 is a selective autophagy substrate and a functional hybrid of the mammalian autophagic adapters NBR1 and p62/SQSTM1[END_REF].

Paszkiewicz
In human, NBR1 interacts with ATG8 protein to mediate mitophagy, but in plants, while localisation to the phagophore has been reported, AtNBR1 mitochondrial localisation has not, and thus it remains possible that AtNBR1 is not involved in mitophagy [START_REF] Svenning | Plant NBR1 is a selective autophagy substrate and a functional hybrid of the mammalian autophagic adapters NBR1 and p62/SQSTM1[END_REF]. An Arabidopsis orthologue of the yeast ATG11 / mammalian FIP200 scaffold protein, which is required to recruit tagged mitochondria to the phagophore, shows conserved scaffold and recruitment functions, and atg11 mutants show decreased clearance of mitochondria upon transfer to conditions promoting mitophagy [START_REF] Li | AUTOPHAGY-RELATED11 plays a critical role in general autophagy-and senescence-induced mitophagy in Arabidopsis[END_REF]. In addition, mitochondria have been shown to be surrounded by phagophores decorated with AtATG8a, orthologous to yeast Atg8 (the AtAtg8 sub-family contains eight members), in an AtATG7 dependent-manner, as first demonstrated in yeast, to drive phagophore assembly, autophagosome formation, and subsequent mitochondrial clearance [START_REF] Li | AUTOPHAGY-RELATED11 plays a critical role in general autophagy-and senescence-induced mitophagy in Arabidopsis[END_REF]. Finally, the ATG8/ATG12 conjugation pathway, whose human orthologues are required for the phagophore assembly process, appears conserved in plants and mediates the removal of ubiquitinated proteins contained in extracts of leaves of 14 day-old maize plants following N starvation, in addition to mediating the turnover of mitochondrial proteins (such as VDAC and COX2), and ribosomal or peroxisomal ones, but not the chloroplastic proteins TIC110 and TOC33 [START_REF] Li | AUTOPHAGY-RELATED11 plays a critical role in general autophagy-and senescence-induced mitophagy in Arabidopsis[END_REF].

To conclude for this section, despite a limited number of published studies into the plant mitophagy apparatus, the evidence indicates that key aspects of phagophore-dependant mitophagy are conserved across the yeast, animal and plant kingdoms, but that there is low conservation of genes related to the process of mitochondrial recognition by the phagophore.

FRIENDLY/Clu-type proteins

Clu-type genes have been reported to be involved in mitochondrial dynamics in protists (cluA), yeast (CLU1), flies (Clu), Caenorhabditis elegans, (clu-1), human (CLUH) and plants (FRIENDLY) (Zhu et al., 1997a;[START_REF] Fields | The S. cerevisiae CLU1 and D. discoideum cluA genes are functional homologues that influence mitochondrial morphology and distribution[END_REF][START_REF] Logan | The genetic control of plant mitochondrial morphology and dynamics[END_REF][START_REF] Cox | clueless, a conserved Drosophila gene required for mitochondrial subcellular localization, interacts genetically with parkin[END_REF][START_REF] Gao | CLUH regulates mitochondrial biogenesis by binding mRNAs of nuclear-encoded mitochondrial proteins[END_REF]. Clu-type proteins are not integral components of mitochondrial compartments but, as indicated by localisation of fluorescent protein fusions in Arabidopsis, Drosophila and human cells, show a cytosolic location in addition to the presence of aggregates close to the OMM of some mitochondria [START_REF] El Zawily | FRIENDLY regulates mitochondrial distribution, fusion, and quality control in Arabidopsis[END_REF][START_REF] Gao | CLUH regulates mitochondrial biogenesis by binding mRNAs of nuclear-encoded mitochondrial proteins[END_REF][START_REF] Sen | Clueless is a conserved ribonucleoprotein that binds the ribosome at the mitochondrial outer membrane[END_REF].

Paszkiewicz

FRIENDLY function

In Arabidopsis, the FRIENDLY gene was first identified following a microscopy based screen of EMS mutants for individuals with altered mitochondrial dynamics [START_REF] Logan | The genetic control of plant mitochondrial morphology and dynamics[END_REF]. Thus, friendly plants have mitochondria in clusters of discrete organelles as a result of an increased association time between individual mitochondria that meet through movement on the actin cytoskeleton [START_REF] El Zawily | FRIENDLY regulates mitochondrial distribution, fusion, and quality control in Arabidopsis[END_REF]. Further evidence for a role for FRIENDLY in the plant stress response and mitochondrial quality control is provided by the independent discovery of FRIENDLY in a mutant screen (see section 2.4) for plants with enhanced susceptibility to P. syringae, a pathogen that is known to employ effectors targeted to mitochondria, confirming the involvement of FRIENDLY in adjustment of the chondriome under stressed conditions [START_REF] Vellosillo | Defense activated by 9-lipoxygenasederived oxylipins requires specific mitochondrial proteins[END_REF].

Clu interacts with MQC pathways

Evidence of a role for CLU-type proteins in MQC also comes from studies on FRIENDLY orthologues in human and Drosophila. Drosophila S2R+ cells expressing an RNAi knockdown for the clueless gene display the characteristic clustered mitochondria phenotype, but this phenotype is absent in RNAi cells overexpressing Park, while cells overexpressing PINK1 maintained a mutant clustered phenotype [START_REF] Cox | clueless, a conserved Drosophila gene required for mitochondrial subcellular localization, interacts genetically with parkin[END_REF][START_REF] Sen | Clueless, a protein required for mitochondrial function, interacts with the PINK1-Parkin complex in Drosophila[END_REF]. Similar results were obtained using the corresponding knock-out mutants indicating that disruption of Clu function influences the PINK/Parkin quality control pathway [START_REF] Wang | Drosophila clueless is involved in Parkin-dependent mitophagy by promoting VCP-mediated Marf degradation[END_REF].

Moreover, while in untreated cells Clu protein associates with TOM20 and PINK1, but not or H 2 O 2 ) leads to detection of association between Clu and Parkin protein [START_REF] Sen | Clueless, a protein required for mitochondrial function, interacts with the PINK1-Parkin complex in Drosophila[END_REF]. However, in clu RNAi cells, PINK1 and Parkin are still found associated, while PINK1 abundance is increased, demonstrating that although Clu is involved in mitochondrial quality control it is not required for PINK1 and Parkin interaction [START_REF] Sen | Clueless, a protein required for mitochondrial function, interacts with the PINK1-Parkin complex in Drosophila[END_REF]. CLU involvement with the macro-autophagy pathway has also been suggested by the increased proportion of mitochondria surrounded by Parkin, and association with a phagophore membrane, in flight muscle cells of Drosophila clu mutants, although completion of engulfment by the phagophore and consequent degradation is strongly inhibited in the absence of CLU [START_REF] Wang | Drosophila clueless is involved in Parkin-dependent mitophagy by promoting VCP-mediated Marf degradation[END_REF]. Moreover, a physical association between CLU and valosin-containing protein (VCP/p97), which mediates specific proteasomal degradation of components of the mammalian mitochondrial fusion apparatus [START_REF] Tanaka | Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin[END_REF], has been demonstrated by co-IP, and fly clu mutants display disruption of the PARKIN-dependant VCP-mediated degradation of fusion machinery, thereby suggesting that CLU function may be to prevent unwanted interaction between functional and dysfunctional mitochondria [START_REF] Wang | Drosophila clueless is involved in Parkin-dependent mitophagy by promoting VCP-mediated Marf degradation[END_REF].

Taken together, these studies indicate that key mechanisms linking MQC and mitophagy, such as PINK1/ parkin and VCP proteins, are misregulated upon CLU dysfunction, which triggers mitophagy.

In conclusion, disruption of CLU induces a specific mitochondrial clustering phenotype in all eukaryotic kingdoms. However, no functional orthologues of any protein known to interact with CLU in non-plant eukaryotes has been identified in the Arabidopsis genome.

This is similar to the situation observed for genes involved in recognition of defective mitochondria by the phagophore assembly machinery. Thus, investigation of the extent of conservation of the FRIENDLY-dependent processes may help identify novel mechanisms underpinning the regulation of mitochondrial quality in plants.

Germination

The germination process

Arabidopsis seeds are composed of an embryo stuffed with reserves, surrounded by two additional tissues layers: the endosperm and the testa [START_REF] Bentsink | Seed dormancy and germination[END_REF].

Following production, the seed is in a quiescent state (sometimes dormant), awaiting suitable conditions for germination [START_REF] Baskin | A classification system for seed dormancy[END_REF][START_REF] Finkelstein | Molecular Aspects Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy[END_REF]. The first phase (Phase 1) is brief, involving rehydration of the dry seed, and is named imbibition. The second phase (Phase 2) comprises rupture of the testa, mainly due to cell expansion, and this is followed by the third phase (Phase 3) involving rupture of the endosperm and protrusion of the radicle out of the seed. The third phase is usually associated with a post-germination heterotrophic transition, corresponding to the early establishment of the seedling (Figure 1.9) [START_REF] Bewley | Seed Germination and Dormancy[END_REF].

During imbibition, there is a rapid influx of water into the dry seed leading to a progressive change in the transcriptome, indicating the end of the developmental program implemented during late seed maturation [START_REF] Weitbrecht | First off the mark: Early seed germination[END_REF]. The ABA content of the seed declines sharply and this continues into Phase 2 [START_REF] Chiwocha | The etr1-2 mutation in Arabidopsis thaliana affects the abscisic acid, auxin, cytokinin and gibberellin metabolic pathways during maintenance of seed dormancy, moist-chilling and germination[END_REF]. This ABA catabolism is a prerequisite during early germination in order to break seed dormancy, and also to allow the completion of the last germination step [START_REF] Müller | Endosperm-limited Brassicaceae seed germination: Abscisic acid inhibits embryo-induced endosperm weakening of Lepidium sativum (cress) and endosperm rupture of cress and Arabidopsis thaliana[END_REF][START_REF] Rodríguez-Gacio | Seed dormancy and ABA signaling: the breakthrough goes on[END_REF]. During Phase 2, the amount of water absorbed by the seed is low and stable [START_REF] Bewley | Seed Germination and Dormancy[END_REF]. The enzymes involved in the synthesis of bioactive gibberellins are produced [START_REF] Yamauchi | Activation of gibberellin biosynthesis and response pathways by low temperature during imbibition of Arabidopsis thaliana seeds[END_REF]). Thus, the high ratio of GA / ABA that is generated during this phase, in addition to the perception of environmental conditions such as light and temperature, cause a shift of the transcriptome and proteome to a development program that supports the accomplishment of germination [START_REF] Nonogaki | Germination-still a mystery[END_REF][START_REF] Weitbrecht | First off the mark: Early seed germination[END_REF]. Thereafter, expansion growth of the embryo increases and the cell walls of the envelope cells surrounding the embryo are weakened, allowing fracture of the testa and weakening of the endosperm [START_REF] Müller | Endosperm-limited Brassicaceae seed germination: Abscisic acid inhibits embryo-induced endosperm weakening of Lepidium sativum (cress) and endosperm rupture of cress and Arabidopsis thaliana[END_REF][START_REF] Nonogaki | Germination-still a mystery[END_REF]. In parallel, progression of the cell cycle has been demonstrated, but a blockage in the pre-mitotic phase (G2) is observed until the end of germination [START_REF] Barroco | The Role of the Cell Cycle Machinery in Resumption of Postembryonic Development[END_REF].

Following Phase 2, a second rapid and transient influx of water into the seed marks the beginning of Phase 3. This water influx is associated with the elongation of cells between the hypocotyl and root, which causes the protrusion of the root out of the seed, marking the end of germination [START_REF] Sliwinska | Germination of Arabidopsis thaliana seeds is not completed as a result of elongation of the radicle but of the adjacent transition zone and lower hypocotyl[END_REF].

During Phase 3, but post-germination sensu stricto, cell division is gradually reactivated and a metabolic transition allows the mobilization of the majority of reserves accumulated in seed in order to provide energy for growth and to support seedling establishment [START_REF] Sliwinska | Germination of Arabidopsis thaliana seeds is not completed as a result of elongation of the radicle but of the adjacent transition zone and lower hypocotyl[END_REF][START_REF] Nonogaki | Seed dormancy and germination-emerging mechanisms and new hypotheses[END_REF].

Arabidopsis seeds do not require stratification (imbibition at low, above freezing temperature) to germinate, however, stratification synchronizes germination of treated seed and this facilitates the study of the germination especially during early stages which present no obvious morphological indicators of the physiological state [START_REF] Weitbrecht | First off the mark: Early seed germination[END_REF]. Indeed, during stratification there is a stimulation of the production of bioactive GA as well as a drop in ABA content, which reduces seed dormancy and therefore promotes germination [START_REF] Yamauchi | Activation of gibberellin biosynthesis and response pathways by low temperature during imbibition of Arabidopsis thaliana seeds[END_REF][START_REF] Chiwocha | The etr1-2 mutation in Arabidopsis thaliana affects the abscisic acid, auxin, cytokinin and gibberellin metabolic pathways during maintenance of seed dormancy, moist-chilling and germination[END_REF].

5.2

The chondriome during germination

Energy metabolism during germination

Germination involves reactivation of cellular activity and therefore metabolism. In fact, during drying of seeds at the end of their development, metabolism is slowed and differs from that observed during maturation of the seed, leading to the accumulation of particular metabolites before the developmental stasis observed in the dry seed [START_REF] Baud | An integrated overview of seed development in Arabidopsis thaliana ecotype WS[END_REF][START_REF] Baud | Storage Reserve Accumulation in Arabidopsis: Metabolic and Developmental Control of Seed Filling[END_REF][START_REF] Fait | Arabidopsis seed development and germination is associated with temporally distinct metabolic switches[END_REF]. During seed imbibition there is an increase in oxygen consumption and consequently ATP production, which then stabilizes during Phase 2 of germination [START_REF] Botha | Respiratory metabolism and gene expression during seed germination[END_REF][START_REF] Benamar | Membrane integrity and oxidative properties of mitochondria isolated from imbibing pea seeds after priming or accelerated ageing[END_REF][START_REF] Stupnikova | Pea seed mitochondria are endowed with a remarkable tolerance to extreme physiological temperatures[END_REF][START_REF] Macherel | Function and stress tolerance of seed mitochondria[END_REF]. The respiratory substrates for ATP synthesis during early germination are provided by metabolites accumulated during drying, and provide cytosolic NADH and/or succinate to the mitochondria, since the tricarboxylic acid (TCA) cycle enzyme activities are low [START_REF] Logan | Mitochondrial biogenesis during germination in maize embryos[END_REF][START_REF] Benamar | Membrane integrity and oxidative properties of mitochondria isolated from imbibing pea seeds after priming or accelerated ageing[END_REF][START_REF] Howell | Ordered assembly of mitochondria during rice germination begins with promitochondrial structures rich in components of the protein import apparatus[END_REF][START_REF] Fait | Arabidopsis seed development and germination is associated with temporally distinct metabolic switches[END_REF][START_REF] Weitbrecht | First off the mark: Early seed germination[END_REF]. As germination progresses, mitochondria display increasing activities of the pyruvate dehydrogenase complex (PDC)

and TCA cycle enzymes [START_REF] Logan | Mitochondrial biogenesis during germination in maize embryos[END_REF][START_REF] Howell | Ordered assembly of mitochondria during rice germination begins with promitochondrial structures rich in components of the protein import apparatus[END_REF]. However, the majority of lipid and protein reserves accumulated during seed maturation are used only during Phase 3, in order to fuel cell division, expansion and tissue differentiation during seedling establishment [START_REF] Fait | Arabidopsis seed development and germination is associated with temporally distinct metabolic switches[END_REF][START_REF] Pinfield-Wells | Sucrose rescues seedling establishment but not germination of Arabidopsis mutants disrupted in peroxisomal fatty acid catabolism[END_REF]. This metabolic burst is associated with increased oxygen consumption and ATP production, but requires peroxisomal (glyoxysomal) activity for fatty-acid oxidation and the glyoxylate cycle [START_REF] Hayashi | 2,4-Dichlorophenoxybutyric acid-resistant mutants of Arabidopsis have defects in glyoxysomal fatty acid beta-oxidation[END_REF][START_REF] Pinfield-Wells | Sucrose rescues seedling establishment but not germination of Arabidopsis mutants disrupted in peroxisomal fatty acid catabolism[END_REF][START_REF] Carrera | Seed after-ripening is a discrete developmental pathway associated with specific gene networks in Arabidopsis[END_REF]. Indeed, mutants having a total disruption of peroxisomal-dependant fatty acid catabolism have reduced germination even in the presence of sucrose, whereas the addition of sucrose post-germination, facilitated establishment of those mutants [START_REF] Pinfield-Wells | Sucrose rescues seedling establishment but not germination of Arabidopsis mutants disrupted in peroxisomal fatty acid catabolism[END_REF][START_REF] Nonogaki | Germination-still a mystery[END_REF]. Taken together, these studies indicate the involvement of mitochondria in energy metabolism during germination, and increasing mitochondrial activity following completion of germination sensu stricto.

Mitochondrial biogenesis during germination

Mitochondrial biogenesis defines the processes leading to the establishment of a mitochondrion able to support cellular metabolic activity of adult tissues. Biogenesis is not a ex-nihilo process but instead involves modification and development of pre-existing mitochondria inherited from the mother cell during cell division, as demonstrated for pea and soybean by the detection of mitochondrial respiratory activities in crude extracts of germinated seed [START_REF] Akazawa | Mitochondria in the endosperm of the germinating castor bean; a developmental study[END_REF][START_REF] Howell | Changes in Metabolic Characteristics of Mitochondria from Soyhean Cotyledons During Germination[END_REF]. Mitochondria in dry seeds are in a state of suspended animation and have a rudimentary structure, with few cristae and a matrix of low electron density when tissue is viewed by transmission electron microscopy, relative to mitochondria in adult tissues.

thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 35

In addition, seed mitochondria exhibit low metabolic activity, leading to their definition as promitochondria, in line with the poorly structured mitochondria in yeast under anoxia [START_REF] Logan | Mitochondrial biogenesis during germination in maize embryos[END_REF]. Mitochondrial biogenesis has been observed during the germination of seeds of several families of higher plant, especially in monocots, and in Arabidopsis [START_REF] Yoo | Ultrastructural changes in cells of pea embryo radicles during germination[END_REF][START_REF] Logan | Mitochondrial biogenesis during germination in maize embryos[END_REF][START_REF] Howell | Ordered assembly of mitochondria during rice germination begins with promitochondrial structures rich in components of the protein import apparatus[END_REF][START_REF] Howell | Oxygen initiation of respiration and mitochondrial biogenesis in rice[END_REF][START_REF] Howell | Mapping Metabolic and Transcript Temporal Switches during Germination in Rice Highlights Specific Transcription Factors and the Role of RNA Instability in the Germination Process[END_REF]Law et al., 2012), and these have demonstrated the generality of the transition from promitochondria. One of the earliest detected events associated with mitochondrial biogenesis during germination is the synthesis of components of the protein import machinery prior to synthesis of protein components of the mitochondrial metabolic apparatus, which reach peak abundance and activities post-germination to support altered substrate use to fuel seedling establishment [START_REF] Logan | Mitochondrial biogenesis during germination in maize embryos[END_REF][START_REF] Howell | Ordered assembly of mitochondria during rice germination begins with promitochondrial structures rich in components of the protein import apparatus[END_REF][START_REF] Howell | Mapping Metabolic and Transcript Temporal Switches during Germination in Rice Highlights Specific Transcription Factors and the Role of RNA Instability in the Germination Process[END_REF]. Study of the Arabidopsis mitochondrial transcriptome during germination has complemented previous semi-quantitative studies examining transcripts by RT-PCR or Northern blot and highlights a burst of transcription of genes related to mtDNA metabolism slightly before the burst of gene transcripts from the protein import machinery (Law et al., 2012).

However, in that study, seed expressing mito-GFP did not show detectable GFPfluorescence until after 48 hours of stratification, while FP targeted to plastids or peroxisomes was detected soon after the start of hydration, despite expression being driven by the same constitutive promotor. Law et al concluded that Arabidopsis seed mitochondria required extensive biogenesis during stratification to become metabolically active (Law et al., 2012). Nevertheless, the maize and rice studies concluded that import competent promitochondria were active from the start of imbibition, fuelled by external NADH, raising the possibility that monocots and dicots differed significantly in the early stages of mitochondrial biogenesis during germination [START_REF] Logan | Mitochondrial biogenesis during germination in maize embryos[END_REF][START_REF] Howell | Ordered assembly of mitochondria during rice germination begins with promitochondrial structures rich in components of the protein import apparatus[END_REF][START_REF] Benamar | Membrane integrity and oxidative properties of mitochondria isolated from imbibing pea seeds after priming or accelerated ageing[END_REF].

Most experimental investigations of mitochondrial biogenesis during germination have been performed using methods analysing the state of a population of mitochondria from hundreds or thousands of cell, for example, analyses that start with the purification of organelles from tissue homogenates. As a result, the events at the level of the cell and individual mitochondrial are obscure.

The presence of mitochondrial subpopulations, differing in buoyant density and protein complement, in the dry seed and during germination was highlighted by performing subcellular fractionation of total cell homogenates by sucrose density gradient centrifugation along the germination time-course of maize seeds [START_REF] Logan | Mitochondrial biogenesis during germination in maize embryos[END_REF]. On the basis of protein content and enzyme activity it was demonstrated that the heavy subpopulation underwent biogenesis: this fraction retained a relatively high abundance of mtDNA (detected by Southern blot of the sucrose gradient fractions using a mtDNA probe), and displayed increasing activities of the PDC and TCA cycle enzymes during germination. In contrast, the light subpopulation appeared to be progressively disassembled, perhaps continuing a process started during seed maturation: less mtDNA was detected as germination proceeded and the light mitochondria contained relatively low abundances of mitochondrial proteins including very low or undetectable protein abundances of PDC and TCA cycle enzyme activities [START_REF] Logan | Mitochondrial biogenesis during germination in maize embryos[END_REF]. The apparent disassembly of the light sub-population reported in that study may represent the specific removal by autophagy of mitochondria during seed germination. Another study, this time on soybean seeds, also demonstrated the presence of light and heavy mitochondria, again by density gradient centrifugation, and reported decreasing COX activity of the light fraction as germination progressed [START_REF] Yin | Mitochondrial damage in the soybean seed axis during imbibition at chilling temperatures[END_REF], suggesting that disassembly of the light mitochondrial subclass during germination is a common feature of the germination of monocot and dicot seeds.

The presence of mitochondrial subpopulations has also been reported post-germination, in four day-old etiolated mung bean seedlings. Again, the two populations differed in buoyant density on sucrose gradients with the light fraction containing mitochondrial with a lower mtDNA content and oxygen consumption capacity, indicating a potentially similar process of mitochondrial disassembly to that observed during seed germination [START_REF] Dai | Population heterogeneity of higher-plant mitochondria in structure and function[END_REF]. However, the processes involved in the disassembly of mitochondria during germination, and also subsequently, at the transition to autotrophic growth, have been subject to little study since these cited works.

Introduction

Seed germination is a remarkable transition in the life cycle of spermatophytes, whereby the next generation sprouts from the dead protective tissues of the mother plant. The Greek philosopher Theophrastus (circa 372-287 B.C.) knew that food reserves were stored in seeds [START_REF] Baskin | Ecology, Biogeography, and Evolution of Dormancy and Germination[END_REF], but hundreds of years later, in the 17 th century, many who wondered about the miraculous regeneration they witnessed considered germination to be essentially ex nihilo [START_REF] Preston | Thomas Browne and the Writing of Early Modern Science[END_REF], since not only were dry seeds usually tiny compared with the size of the mature plant, but they lacked signs of life. Humankind, collectively fascinated by mortality, has long been fascinated with the reanimation that results in seed germination.

thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 39

Seeds of the model plant Arabidopsis (Arabidopsis thaliana L. Heynth) are composed of an embryo, charged with protein reserves, surrounded by two envelopes: the endosperm and the multi-layered testa [START_REF] Debeaujon | Influence of the testa on seed dormancy, germination, and longevity in Arabidopsis[END_REF]. Following maturation and afterripening to overcome dormancy the seed is in a quiescent state, awaiting favourable conditions for germination [START_REF] Finkelstein | Molecular Aspects Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy[END_REF][START_REF] Baskin | Seeds : ecology, biogeography, and evolution of dormancy and germination[END_REF]. Upon perception of the appropriate environmental conditions, germination of Arabidopsis proceeds in two sequential steps: the first step involves expansion of the embryonic axis leading to rupture of the testa [START_REF] Liu | Large-scale screening of Arabidopsis enhancer-trap lines for seed germination-associated genes[END_REF], while the second step involves elongation growth of the lower hypocotyl to drive protrusion of the radicle through the endosperm [START_REF] Sliwinska | Germination of Arabidopsis thaliana seeds is not completed as a result of elongation of the radicle but of the adjacent transition zone and lower hypocotyl[END_REF]. The reactivation of growth requires energy and thus reactivation of primary metabolism. However, mitochondria in dry seed are in a state of suspended animation and are structurally and functionally deficient relative to typical mitochondria extracted from mature tissue, lacking defined cristae and internal structure [START_REF] Logan | Mitochondrial biogenesis during germination in maize embryos[END_REF][START_REF] Howell | Ordered assembly of mitochondria during rice germination begins with promitochondrial structures rich in components of the protein import apparatus[END_REF][START_REF] Attucci | Oxidative Phosphorylation by Mitochondria Extracted from Dry Sunflower Seeds[END_REF]. Thus, for many years, researchers have investigated the extent to which these rudimentary mitochondria, termed promitochondria, are functional early during imbibition and the processes, collectively termed biogenesis, that are responsible for their full reactivation [START_REF] Dai | Population heterogeneity of higher-plant mitochondria in structure and function[END_REF][START_REF] Ehrenshaft | Respiration and Mitochondrial Biogenesis in Germinating Embryos of Maize[END_REF][START_REF] Logan | Mitochondrial biogenesis during germination in maize embryos[END_REF][START_REF] Howell | Ordered assembly of mitochondria during rice germination begins with promitochondrial structures rich in components of the protein import apparatus[END_REF]. Many of the results from those studies were obtained from experiments performed in vitro, on cell-free homogenates derived from thousands of cells, originating from a mix of embryonic tissues, and thus interpretation is hampered by dilution and averaging errors.

Nevertheless, those studies demonstrated that although mitochondria isolated from dry embryos had low tricarboxylic acid (TCA) cycle enzyme activities, they were able to oxidise supplied succinate and/or NADH enabling the generation of a membrane potential needed for protein import crucial for biogenesis [START_REF] Benamar | Membrane integrity and oxidative properties of mitochondria isolated from imbibing pea seeds after priming or accelerated ageing[END_REF][START_REF] Ehrenshaft | Respiration and Mitochondrial Biogenesis in Germinating Embryos of Maize[END_REF][START_REF] Logan | Mitochondrial biogenesis during germination in maize embryos[END_REF][START_REF] Howell | Ordered assembly of mitochondria during rice germination begins with promitochondrial structures rich in components of the protein import apparatus[END_REF]. However, because isolation procedures necessarily involved several hours of hydration, questions remained about the true state of promitochondria in vivo. In a recent study, primarily focused on changes in transcript abundance previously published by [START_REF] Narsai | In-depth temporal transcriptome profiling reveals a crucial developmental switch with roles for RNA processing and organelle metabolism that are essential for germination in Arabidopsis[END_REF], Law et al (2012) used a mitochondrial targeted GFP Arabidopsis line [START_REF] Carrie | Nine 3ketoacyl-CoA thiolases (KATs) and acetoacetyl-CoA thiolases (ACATs) encoded by five genes in Arabidopsis thaliana are targeted either to peroxisomes or cytosol but not to mitochondria[END_REF] to visualise mitochondria in germinating Arabidopsis seeds. However, in that study, mitochondria expressing GFP were not detected in the dry seed embryos. Because fluorescent proteins driven by the same promoter but targeted to peroxisomes or plastids were detected it was concluded that the lack of a mitochondrial GFP signal from dry embryos reflected low mitochondrial mass or protein content (Law et al., 2012). GFP was detected in mitochondria after 48 h stratification (imbibition in the dark at 4°C), leading to the conclusion that Arabidopsis seed mitochondria required extensive biogenesis at the end of stratification, marked by a transient increase in abundance of a subset of mitochondrial transcripts and concomitant mitochondrial mass increase, in order to become metabolically active (Law et al., 2012). This conclusion contrasts with the maize and rice studies that concluded import competent promitochondria were active from the start of imbibition, fuelled by external NADH [START_REF] Logan | Mitochondrial biogenesis during germination in maize embryos[END_REF][START_REF] Howell | Ordered assembly of mitochondria during rice germination begins with promitochondrial structures rich in components of the protein import apparatus[END_REF][START_REF] Benamar | Membrane integrity and oxidative properties of mitochondria isolated from imbibing pea seeds after priming or accelerated ageing[END_REF]. Furthermore, changes in mitochondrial mass in maize and rice only occur late during germination, rather than before germination has been triggered, as suggested by the Arabidopsis study. We decided to revisit the role of promitochondria during germination, taking an in vivo bioimaging approach to investigate the reactivation of mitochondria bioenergetics, dynamics and biogenesis during germination of Arabidopsis. We show here that mitochondria expressing mito-GFP [START_REF] Logan | Mitochondria-targeted GFP highlights the heterogeneity of mitochondrial shape, size and movement within living plant cells[END_REF] [START_REF] Logan | The mitochondrial compartment[END_REF], is tailored to the changing role of mitochondria from the primary role of promitochondria as mtDNA-containing molecular scaffolds, to a mixed population of organelles providing for the more complex metabolic needs of autotrophic life. Measurement of oxygen uptake (Figure 2.1C) demonstrated a rapid resumption of respiration upon rehydration of the dry seed at 21°C, with a progressive increase of O 2 consumption during the first hour of imbibition until full rehydration, as described before [START_REF] Sew | Multiplex micro-respiratory measurements of Arabidopsis tissues[END_REF]. Oxygen consumption was also monitored during imbibition and incubation of seeds for 48h in the dark at 4°C, conditions that correspond to the stratification treatment widely used with Arabidopsis. Even after 3h imbibition in the dark a significant level of O 2 consumption at 4°C was detected, revealing that resumption of mitochondrial respiration was also proceeding, albeit more slowly, at low temperature (Figure 2.1C). After 48h at 4°C in the dark oxygen consumption measured at 4°C had almost doubled, but was still lower than if measured after imbibition at 21°C. Transfer of stratified seeds to 21ºC in the light resulted in a >5-fold increase in oxygen consumption (Figure 2.1C), reflecting the thermal dependence of respiration, and the high respiratory activity of the stratified seeds. From these data the Q 10 of respiration can be estimated between 2.46 and 3.22 (using the equation Q10 = (R 2 /R 1 ) [10/T2-T1) ). These relatively high values suggest that respiration of stratified seeds was not limited by substrate availability [START_REF] Atkin | Thermal acclimation and the dynamic response of plant respiration to temperature[END_REF]. Cyanide inhibited O 2 consumption more strongly at 21° than at 4°C, suggesting a higher capacity of the alternative pathway at low temperature, which agrees with the cold induction of alternative oxidase gene expression [START_REF] Wang | Impact of mitochondrial alternative oxidase expression on the response of Nicotiana tabacum to cold temperature[END_REF]. Together, these data show that mitochondria quickly regain membrane potential and metabolic activity upon embryo imbibition, and that incubation at low temperature during stratification maintains a lower rate of metabolism because of the thermal constraint.

Paszkiewicz

Results

Mitochondrial motility increases in response to environmental germination cues

Given that Arabidopsis seed mitochondria regain bioenergetic activity within 15 min of re-hydration (Figure 2. Our experimental procedure included 48 h stratification (4°C, dark), in order to ensure synchronous germination and consistency in the physiological stage of embryos selected at fixed time points. This is the method of choice at early stages of development, when physical descriptors cannot be used, and allows comparison of our results with the two key transcriptomic studies of (Law et al., 2012;[START_REF] Narsai | In-depth temporal transcriptome profiling reveals a crucial developmental switch with roles for RNA processing and organelle metabolism that are essential for germination in Arabidopsis[END_REF]. However, we also Images generated using Imaris to track mitochondria in cotyledon cells, maintained throughout the time course in 100 µM ABA, GA 3 or paclobutrazol, after stratification for 48h at 4ºC and 2 h after transfer to light/21ºC. Detected mitochondria were coloured green and tracks are rainbow coloured according to speed (no movement is purple and movement above 0.032 µm/s is red). Bar = 5 µm.

1 2 h S 2 4 h S 3 6 h S 4 8 h S 2 h L 1 2 h L T R E R L R R H 1 2 h S 2 4 h S 3 6 h S 4 8 h S 2 h L 1 2 h L T R E R L R R H 1 2 h S 2 4 h S 3 6 h S 4 8 h S 2 h L 1 2 h L T R E R L R R H 1 2 h S 2 4 h S 3 6 h S 4 8 h S 2 h L 1 2 h L T R E R L R R H 1 2 h S 2 4 h S 3 6 h S 4 8 h S 2 h L 1 2 h L T R E R L R
4 8 h S 2 h L 2 h D 2 h L 4 C 2 h L G A 3 2 h D G A 3 2 h L A B A 2 h D A B A 2 h L P A C 2 h D P A C 4 8 h S 2 h L 2 h D 2 h L 4 C 2 h L 4 C 2 h L 4 C 2 h L G A 2 h L G A 2 h L G A 3 2 h D G A 2 h D G A 2 h D G A 3 2 h L A B A 2 h L A B A 2 h L A B A 2 h D A B A 2 h D A B A 2 h D A B A 2 h L P A C 2 h L P A C 2 h L P A C 2 h D P A C 2 h D P A C 2 h D P A C 4 8 h S 4 8 h S 2 h L 2 h L 2 h D 2 h D 2 h L 4 C 2 h L 4 C 2 h L G A 2 h L G A 3 2 h D G A 2 h D G A 3 2 h L A B A 2 h L A B A 2 h D A B A 2 h D A B A 2
As in A. The promitochondria reactivated during imbibition have survived challenging conditions during seed drying, the dry state itself, and then strains imposed by rehydration. We therefore tested for the induction of autophagy following the initiation of imbibition and transfer to germination promoting conditions. To visualise and quantify the AUTOPHAGY8-related (ATG8) protein labeled phagophores / autophagosomes (hereafter referred collectively as autophagosomes) we used lines expressing both mRFP1-ATG8F [START_REF] Honig | A New Type of Compartment, Defined by Plant-Specific Atg8-Interacting Proteins, Is Induced upon Exposure of Arabidopsis Plants to Carbon Starvation[END_REF] and mito-GFP [START_REF] Logan | Mitochondria-targeted GFP highlights the heterogeneity of mitochondrial shape, size and movement within living plant cells[END_REF]. Automatic segmentation of the mRFP1 signal from autophagosomes was complicated due to the red autofluorescence of the protein storage vacuoles (PSVs). However, mRFP1-ATG8F could be detected by its brighter fluorescence, association into spheroid bodies of Twelve hours after transfer to light/21ºC (60 h from the start of imbibition) the number of mitochondria was unchanged, but they were more elongate (42% of mitochondria had a sphericity of <0.8, median = 0.83) and individual mitochondria were larger (25% of objects had a volume greater than 0.43 µm 3 , Figure 6E). The changes to mitochondrial morphology observed at 60 h, from spherical to more elongate structures, continued over the next hours till the TR stage (Figure 2.6). At the TR stage, most mitochondria displayed a tubular morphology with frequent branching (75% of objects had OS <0.9; andE). The wide distribution of volumes also reflects the continued presence of small spherical mitochondria (similar as observed during stratification) which do not interact and remain as singletons. By the end of germination, ER stage, the number of reticular/tubular mitochondria had decreased with OS values rising (50% of objects had OS>0.8 and 50%). Mitochondria remained visibly clustered (Figure 2.6A "ER"), but the volumes of the largest objects were reduced which together with the increased sphericity reflects the change from a tubuloreticular chondriome to a more discontinuous population. The disassembly of the chondriome structure led to an increase in number of identifiable discrete mitochondria (Figure 2.6A and B). The disintegration of the mitochondrial reticulum and reduction in mitochondrial tubulation continued during early seedling growth such that by the LR stage over 75% of mitochondria had an OS >0.8, and 50% had an OS> 0.9. This change in morphology was accompanied by a further reduction in the volumes of the largest objects (Figure 2.6E) and associated reduction in morphological heterogeneity. In addition, the number of identifiable discrete mitochondria, which was observed to increase from the TR to the ER stage continued to rise through the LR and RH stages. By the time the seedling had developed root hairs (RH stage) mitochondria were typically round to ovoid although some tubules were observed. The distribution of OS and individual organelle volume values were similar to those measured after 12 h 21ºC/light, although mitochondria were more heterogeneous at the RH stage, particularly due to an increase in number of small mitochondria (Figure 2.6D and E). The increase in proportion of small mitochondria was concomitant with an increase in total mitochondrial number, and consequently there was little change in total mitochondrial volume (Figure 2.6C and E). Our analysis of the 4D structure of the chondriome during germination showed an activation of mitochondrial dynamics that led to the generation of a tubuloreticular structure composed of tubular and/or branched mitochondria at the end of germination (TR/ER stages). To further investigate the subcellular location of this structure we examined its localization with respect to PSVs, taking advantage of their specific autofluorescence [START_REF] Bolte | Distinct lytic vacuolar compartments are embedded inside the protein storage vacuole of dry and germinating arabidopsis thaliana Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy[END_REF][START_REF] Hunter | Fluorescent reporter proteins for the tonoplast and the vacuolar lumen identify a single vacuolar compartment in Arabidopsis cells[END_REF][START_REF] Fuji | Arabidopsis vacuolar sorting mutants (green fluorescent seed) can be identified efficiently by secretion of vacuole-targeted green fluorescent protein in their seeds[END_REF]. In the dry seed, the PSVs were spherical to ovoid structures and filled much of the cell volume (Figure 2.7; as previously described by [START_REF] Bolte | Distinct lytic vacuolar compartments are embedded inside the protein storage vacuole of dry and germinating arabidopsis thaliana Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy[END_REF]). The spherical mitochondria were distributed seemingly randomly in the remaining cortical space and between the PSVs. Following transfer to light, the mitochondria congregated in the space between the PSVs (Figure 2.7A) and from 12h light (60 h time point) the congregated mitochondria formed the tubuloreticular structure in the PSV-free areas (Figure 2.7A). Following germination, when the tubular network disassembled, most mitochondria were redistributed more evenly within the cell (Figure 2.7A; see also Figures 2.2 and 2.6).

Our investigations on the nature of the organelle surrounded by the tubular mitochondria coincided with investigations of the abundance and distribution of mtDNA in vivo, using the DNA-binding fluorescent dye SYBR Green I (see below), which also stains the nucleus (although not uniformly across cells) under longer staining times. We were therefore able to observe both mitochondria and the nucleus simultaneously, which revealed that, at the TR stage, when mitochondrial reticulation is most pronounced, SYBR Green I stained mtDNA nucleoids encircled the nucleus within a PSV-free area (Figure 2.7B, see also

The perinuclear mitochondrial enable interaction and redistribution of mtDNA nucleoids at the end of germination

mtDNA is packaged into compact nucleoid structures composed of variable amounts of DNA and protein. Nucleoids are distributed heterogeneously within the mitochondrial population in tobacco and onion cells [START_REF] Arimura | Frequent fusion and fission of plant mitochondria with unequal nucleoid distribution[END_REF][START_REF] Sheahan | Mitochondria as a connected population: Ensuring continuity of the mitochondrial genome during plant cell dedifferentiation through massive mitochondrial fusion[END_REF] and can be redistributed by mitochondrial fusion and fission [START_REF] Sheahan | Mitochondria as a connected population: Ensuring continuity of the mitochondrial genome during plant cell dedifferentiation through massive mitochondrial fusion[END_REF]. We checked how remodelling of the chondriome during germination affected the distribution of mitochondrial nucleoids. By staining with SYBR Green I in a line expressing mCherry in the mitochondrial matrix [START_REF] Candat | The ubiquitous distribution of late embryogenesis abundant proteins across cell compartments in Arabidopsis offers tailored protection against abiotic stress[END_REF] we were able to both visualise mtDNA and mitochondria in the embryo, from 10 min imbibition till the end of germination. 91% of mitochondria contained detectable mtDNA at the onset of imbibition and during stratification (Figure 2.8). During stratification, there was no measurable movement of nucleoids within the mitochondria beyond the slight wobbling of mitochondria detected previously (Figure 2.1). However, it was clear from time-lapse imaging that the redistribution of nucleoids within the chondriome during late germination was an active process combining mitochondrial movement and movement of the nucleoid within mitochondria, enabling transient association between mtDNA molecules that were previously segregated (Figure 2.8).

mtDNA quantity, quality, and recombination are tightly controlled during germination

Lack of mitochondrial motility inhibits mitochondrial fusion and therefore isolates the mtDNA within each physically discrete mitochondrion. We hypothesised that the lack of mitochondrial motility during early germination coincided with the induction of mtDNA repair, to ensure that subsequent mixing and redistribution of mtDNA did not involve molecules damaged during drying/imbibition of the seed. We further hypothesised that the massive fusion favoured recombination (which may in turn enable recombinationbased repair) between mtDNA molecules before partition of the newly organised genome amongst the newly fragmented mitochondria population. To test these hypotheses, we first quantified total mtDNA during stratification and germination (Figure 2.9). No significant endoreduplication of the nuclear genome occurs in the embryo during stratification, or early germination [START_REF] Sliwinska | Germination of Arabidopsis thaliana seeds is not completed as a result of elongation of the radicle but of the adjacent transition zone and lower hypocotyl[END_REF], that would affect the relative quantification.

However, the increase in mtDNA copy number at the ER, TR and RH stages may be slightly underestimated due to an increase in ploidy, specifically in the hypocotyl-radicle axis during elongation growth [START_REF] Sliwinska | Germination of Arabidopsis thaliana seeds is not completed as a result of elongation of the radicle but of the adjacent transition zone and lower hypocotyl[END_REF]. We next quantified nucDNA and mtDNA quality by calculating the relative abundance of qPCR amplification of short versus long DNA fragments, on the premise that a relative reduction in long fragment abundance is proportional to DNA damage [START_REF] Miller-Messmer | RecA-dependent DNA repair results in increased heteroplasmy of the Arabidopsis mitochondrial genome[END_REF]. No significant change was detected in the relative amplification of long versus short DNA fragments of either nucDNA or mtDNA, although there was a reduction in mtDNA quality after the start of stratification followed by a non-significant rise in quality coincident with the resumption of mitochondrial motility before a second drop in quality at the end of germination (Figure 2.9B). Since mtDNA replication and mtDNA repair are both hypothesised to involve recombination as part of their mechanism, we next chose to investigate recombinational activity of the mtDNA genome. As a first approach, we quantified the relative abundance of recombination-derived products that are known to increase in abundance when control over recombination is relaxed (e.g. in recA3 and recG1 mutants). There was no significant increase in recombination products across repeats L or EE during germination, with only repeat EE showing significant increase in recombination in 10 day-old-seedlings (Figure 2.9C). In comparison, mutants defective in suppressing ectopic recombination show greatly increased fold changes in the copy number of these recombination products [START_REF] Wallet | The RECG1 DNA Translocase Is a Key Factor in Recombination Surveillance, Repair, and Segregation of the Mitochondrial DNA in Arabidopsis[END_REF]. To confirm the unchanged mtDNA recombination during germination we next calculated the relative copy number of different regions of the entire mtDNA genome by qPCR using a set of primer pairs spaced between 5-10 kbp apart [START_REF] Wallet | The RECG1 DNA Translocase Is a Key Factor in Recombination Surveillance, Repair, and Segregation of the Mitochondrial DNA in Arabidopsis[END_REF]. No significant change in sequences stoichiometry was detected. Taken together, our results show that the quantity, quality, recombination/stoichiometry of the mtDNA inherited from the mother, as stored in the seed, is under tight homeostatic control during stratification and germination despite the

Discussion

Promitochondria bioenergetics reactivate immediately upon imbibition but reactivation of dynamics only occurs upon transfer to germination conditions

The term promitochondria has been used as a name for mitochondria in embryos of dry seed [START_REF] Logan | Mitochondrial biogenesis during germination in maize embryos[END_REF][START_REF] Howell | Ordered assembly of mitochondria during rice germination begins with promitochondrial structures rich in components of the protein import apparatus[END_REF] due to their limited internal membrane structure and reduced metabolic activity compared to typical mitochondria seen in most adult plant tissues (cf. promitochondria of yeast, [START_REF] Criddle | Promitochondria of anaerobically grown yeast. I. Isolation and biochemical properties[END_REF][START_REF] Logan | Mitochondrial biogenesis during germination in maize embryos[END_REF]). However, rather than being non-functional scaffolds requiring de novo biogenesis for their activity, promitochondria are specialised for their role in the maintenance of mitochondrial structure and genetic integrity during the challenging periods of maturation drying and seed dormancy. Promitochondria have reduced metabolic activities but are able to generate a membrane potential immediately upon rehydration, and can support energy metabolism throughout germination. As such, the initial activation of mitochondrial activity is a biophysical process, controlled principally by hydration status and temperature, rather than a coordinated genetic programme controlled by the nucleus.

Promitochondria in the dry seed are immobile due to a lack of hydration, and only local disorganised movement occurs during stratification. However, a massive reactivation of mitochondrial dynamics occurred within 2 h of transfer of stratified seeds to germinating conditions leading to a large increase in inter-mitochondria interactions (Figure 2.10). An increase in transcription of nuclear genes encoding proteins involved in import across the outer membrane and in mitochondria transcription and RNA editing accompanies this stage (48 h S / 1 h SL) (Law et al., 2012). This reactivation, representing one of the earliest events recorded in the transition from stasis to germination, also occurred in embryos of seed germinating in the absence of stratification, but at a slower rate. The effect of stratification, during which there is limited transcriptional and translational activity of mitochondrial genes (Law et al., 2012), is therefore to synchronise the reactivation across the chondriome. Given that temperature is a key factor controlling the reactivation of mitochondrial dynamics, stratification also acts through inhibition of mitochondrial dynamics until conditions promoting germination are encountered. This inhibition of dynamics may be simply through lower biological activity of the various proteins responsible for mitochondrial dynamics rather than being due to changes in gene expression or protein activity. Increased mitochondrial motility over the next few hours leads to increased fusion concomitant with an increase in mitochondrial membrane biogenesis, with promitochondria acting as scaffolds for the insertion of new polypeptides and lipids (Figure 2.10). These changes are accompanied with increased transcription at 6 h SL of genes encoding components of the inner membrane import apparatus, protein folding and mtDNA metabolism (Law et al., 2012). The modulation of germination by the hormones ABA and GA had little effect on mitochondrial dynamics, reinforcing the view that their reactivation results from the release of biophysical constraints imposed by low tissue hydration and temperature rather than from specific germination cues. ABA, which inhibits germination at the concentration used, reduced the reactivation of mitochondrial dynamics, but only under the germination-promoting conditions of light and 21ºC. This suggests there are different requirements for mitochondrial dynamics linked to the two independent programmes reported to operate during germination: the ABA-sensitive developmental growth programme and the largely ABA-independent storage lipid mobilisation programme [START_REF] Pritchard Sarah | Germination and storage reserve mobilization are regulated independently in Arabidopsis[END_REF]. While temperature is the key trigger for the activation of mitochondrial dynamics, there is clearly a light-mediated effect, which, as demonstrated by the effect of paclobutrazol, is linked to the role of light as a germination trigger via a GA-dependent pathway. Mitochondrial dynamics is reduced following paclobutrazol treatment in the light to a similar extent as measured in seed imbibing in the dark at 21ºC without stratification. Thus, light triggers the culmination of germination and this is accompanied by a burst in mitochondrial motility and subsequent inter-mitochondrial interaction generating the formation of a tubuloreticular chondriome.

Paszkiewicz

Reactivation of mitochondrial dynamics leads to the formation of a perinuclear tubuloreticular

The formation of the perinuclear mitochondrial coincides with a massive increase in chondriome volume. Therefore, this change is not simply due to a shift in the fissionfusion balance in favour of a fused state, a conclusion supported by the fact that the number of mitochondria is relatively stable between time of transfer to germination promoting conditions and the TR stage. Instead, because there is an increase in chondriome volume while mitochondrial numbers are unchanged, the measured increase in fusion must be balanced by fission and the increase in volume can thus be attributable to a net increase in de novo membrane biogenesis (Figure 2.10). This increase in membrane biogenesis is a rapid yet transient response to germination triggers leading to a doubling of chondriome volume that is coincident with the second burst of oxygen uptake that occurs during germination [START_REF] Sew | Multiplex micro-respiratory measurements of Arabidopsis tissues[END_REF]. While chondriome volume stabilises at the end of germination, the redifferentiation of promitochondria continues with a peak in abundance at 24/48 h SL of transcripts involved in mitochondrial bioenergetics and metabolism (Law et al., 2012), that is initially coincident with a massive fragmentation of the mitochondrial reticulum leading to a doubling of the number of mitochondria per cell relative to the number before germination was initiated In dry seed, and during the first hours of imbibition (the first phase of germination), the mitochondria are static and have low metabolic activity despite an immediate re-establishment of a membrane potential. A high percentage of mitochondria contain at least one mtDNA nucleoid. It is proposed that these attributes define these mitochondria as promitochondria, and provide a structure and organisation that act to minimise damage and thus help ensure mitochondrial quality throughout seed dormancy or quiescence. During the early second phase of germination, initiated upon the transfer of seed to germination promoting conditions, the constraint imposed on mitochondrial dynamics by cold temperature is released leading to a burst of motility, inter-mitochondrial interactions and upregulation of mitophagy. During the middle of the second phase of germination, mitochondria become more dynamic, displaying higher rates of inter-mitochondrial interaction together with a shift to an ovoid morphology. Membrane biogenesis is initiated. At the late second phase of germination, mitochondria are preferentially located around the nucleus and further reticulation and fusion leads to the formation of a perinuclear tubuloreticular mitochondrial structure. This organisation may provide a means to ensure mixing of the chondriome and exchange of molecules, together with facilitating synchronisation of mitochondrial biogenesis through efficient delivery and import of tRNA and polypeptides encoded by nuclear transcripts and assisting the necessary associated cross-talk between the mitochondrial and nuclear genomes. Following the end of germination, the perinuclear tubuloreticular structure fragments, leading to the reformation of a population of physically discrete mitochondria. The distribution of mtDNA nucleoids among the mitochondria is uneven, indicating division of labour amongst the physically discrete members of the chondriome. The reorganisation of the chondriome, generating an organisation typical of more adult tissues, coincides with a burst in mitochondrial metabolic activity as storage reserves are used to fuel postgerminative growth. Blue spots represent mtDNA nucleoids; the nucleus is colored yellow; the matrix is shaded from light to dark grey to indicate increasing bioenergetic activity; increased membrane thickness represents membrane biogenesis; mitochondrial motility is represented by tracks with a scale from blue (slow) to red (fast); nucleation of ATG8 and phagophore formation is indicated in green. Similar transient structures have been reported to occur due to massive mitochondrial fusion prior to division of Arabidopsis, Medicago, or tobacco protoplasts [START_REF] Sheahan | Mitochondria as a connected population: Ensuring continuity of the mitochondrial genome during plant cell dedifferentiation through massive mitochondrial fusion[END_REF], and encircling of the nucleus by mitochondria has been reported to occur during male and female gametogenesis of a number of species [START_REF] Dickinson | Organelle behaviour during gametogenesis[END_REF][START_REF] Nagata | Differentiation of organelles during male gametophyte development in higher plants[END_REF]. A perinuclear location has been suggested to facilitate ATP "funnelling" to the nucleus during G1/G2 phases of the cell cycle and to the growing spindle and then cell plate at later stages of mitosis and cytokinesis [START_REF] Segui-Simarro | The mitochondrial cycle of Arabidopsis shoot apical meristem and leaf primordium meristematic cells is defined by a perinuclear tentaculate/cage-like mitochondrion[END_REF]. However, the tubuloreticular structure at the end of germination is not similarly synchronised with the cell cycle. In the case of the SAM and LP, the cage remained in place during cytokinesis and it was only after cell division was complete that the chondriome fragmented [START_REF] Segui-Simarro | The mitochondrial cycle of Arabidopsis shoot apical meristem and leaf primordium meristematic cells is defined by a perinuclear tentaculate/cage-like mitochondrion[END_REF]. In contrast, in seed cotyledons, formation and fragmentation of the tubuloreticular structure occurred without cell division. Indeed, cell division of Arabidopsis cotyledon cells is initiated after protrusion of the radicle, which signals the end of germination [START_REF] Sliwinska | Germination of Arabidopsis thaliana seeds is not completed as a result of elongation of the radicle but of the adjacent transition zone and lower hypocotyl[END_REF]. Whilst ATP provision to the nucleus to support the transcriptional burst concomitant with the end of germination is likely more efficient as a result of a perinuclear location, this location may also facilitate mitochondrial biogenesis through efficient delivery of nuclear-encoded transcripts to ribosomes in the cytosol around the nucleus/perinuclear mitochondria and also import of nuclear synthesised tRNA molecules for mitochondrial translation. For example, mitochondria with a perinuclear location were shown to be enriched in components of the outer-membrane protein import apparatus, relative to mitochondria in the cell periphery of cultured mammalian cells [START_REF] Wurm | Nanoscale distribution of mitochondrial import receptor Tom20 is adjusted to cellular conditions and exhibits an inner-cellular gradient[END_REF]. In addition, and as suggested by [START_REF] Segui-Simarro | The mitochondrial cycle of Arabidopsis shoot apical meristem and leaf primordium meristematic cells is defined by a perinuclear tentaculate/cage-like mitochondrion[END_REF] in the case of SAM and LP cells, the formation of a cage-like mitochondrial reticulum, provides conditions for intermixing of, and recombination between, the population of mtDNA molecules of different sizes and compositions that comprise the mitochondrial genome. Mitochondrial genome continuity in the new generation is then ensured by redistribution of the homogenised genome throughout the mitochondrial population of the daughter cells following division of the fused chondriome [START_REF] Segui-Simarro | The mitochondrial cycle of Arabidopsis shoot apical meristem and leaf primordium meristematic cells is defined by a perinuclear tentaculate/cage-like mitochondrion[END_REF]. Whilst reorganisation of the chondriome during germination enables homogenisation of the mtDNA, subsequent fragmentation at the end of germination generates a population of discrete mitochondria with heterogeneous, rather than homogeneous mtDNA content, in contrast to the situation with dividing protoplasts [START_REF] Sheahan | Mechanisms of organelle inheritance in dividing plant cells[END_REF][START_REF] Sheahan | Mitochondria as a connected population: Ensuring continuity of the mitochondrial genome during plant cell dedifferentiation through massive mitochondrial fusion[END_REF]; the distribution of nucleoids in SAM and LP cells before and after cage formation has not been reported. A heterogeneous distribution of mtDNA within the physically discrete mitochondria is typical of plant cells [START_REF] Lonsdale | The Plant Mitochondrial Genome : Homologous Recombination as a Mechanism for Generating Heterogeneity[END_REF][START_REF] Satoh | Organization of heterogeneous mitochondrial DNA molecules in mitochondrial nuclei of cultured tobacco cells[END_REF][START_REF] Preuten | Fewer genes than organelles: extremely low and variable gene copy numbers in mitochondria of somatic plant cells[END_REF][START_REF] Sheahan | Mitochondria as a connected population: Ensuring continuity of the mitochondrial genome during plant cell dedifferentiation through massive mitochondrial fusion[END_REF][START_REF] Arimura | Frequent fusion and fission of plant mitochondria with unequal nucleoid distribution[END_REF][START_REF] Logan | The mitochondrial compartment[END_REF] and led to the plant chondriome being termed a discontinuous whole [START_REF] Logan | The mitochondrial compartment[END_REF]. This heterogeneity is evidence of division of labour between members of the chondriome, with some mitochondria probably functioning as "genetic vaults" designed to protect vital genetic material [START_REF] Logan | The mitochondrial compartment[END_REF].

(

Paszkiewicz Gael

thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 63

Promitochondria are specialised transgenerational genetic vaults that redifferentiate during germination for autotrophic growth

All promitochondria are specialised as genetic vaults rather than being degradation products, or structures requiring biogenesis in order to assume function: over 90% contain a visible mtDNA nucleoid, recombination is under tight control in order to maintain structural integrity, mtDNA replication is tightly coupled to that of the nuclear DNA, and they can fuel energy metabolism, relying on metabolites accumulated during maturation [START_REF] Fait | Arabidopsis seed development and germination is associated with temporally distinct metabolic switches[END_REF]. These characteristics enable promitochondria to faithfully fulfil their role in maintaining the integrity of the mitochondrial genome during embryo maturation, imbibition, and germination, that in turn ensures continuation of the species.

The fact that mito-GFP is readily detected in dry seeds provides additional evidence that promitochondria are protected. In addition, the lack of mitochondrial motility even when the actin cytoskeleton is present and dynamic may help ensure safe storage of the mtDNA within each discrete compartment. Such a role for promitochondria is in agreement with the results of (Law et al., 2012) that showed relatively low transcript abundances for genes involved in mitochondrial biogenesis during stratification, before a transient increase in abundance upon transfer to germination promoting conditions which precedes the doubling of mitochondrial volume as promitochondria are used as a scaffold for the incorporation of new membrane lipids and proteins. Thus, promitochondria are apparently specialised for specific roles, but even within the promitochondrial population there is evidence of further division of labour. The reactivation of mitochondria after transfer to germinating-promoting conditions is heterogeneous, with some organelles becoming mobile after only a few minutes of observation. These "first responders" then interact/fuse with nearby partners before the wave of interaction spreads to more distant mitochondria. At the same time, there are other mitochondria that display no organised movement and do not interact with others.

The violin plots of rates of fusion and number of interactors clearly show the existence of such subpopulations. One possibility is that these singleton mitochondria are specialised for tasks that do not require them to fuse, or they may be isolated from the rest of the chondriome due to dysfunction, perhaps an inability to fuse, and instead these mitochondria may be the targets for the measured upregulation of mitophagy following initiation of germination. However, the clearest indicator of division of labour is the increase in the percentage of mitochondria with no visible nucleoid following fragmentation of the perinuclear tubuloreticular structure, a condition maintained in seedling tissue [START_REF] Preuten | Fewer genes than organelles: extremely low and variable gene copy numbers in mitochondria of somatic plant cells[END_REF]. This heterogeneity is generated simultaneously with the doubling of mitochondrial volume and number, and precedes a boost in mitochondrial dynamics in the newly germinated seedling and the coincident increased respiratory activity that fuels seedling growth and development (Figure 2 It is hypothesised that biogenesis is tailored to the generation of mitochondria specialised for energy production alongside mitochondria with other functions (for example, as genetic vaults) rather than a uniform rebuilding of all promitochondria to enable full mitochondrial functionality. Concomitant with this hypothetical phase of specialisation of mitochondrial functions was the uncoupling of mtDNA replication from that of the nuclear genome and a relaxing of control over mtDNA recombination. Taken together, these discoveries indicate that mitochondrial specialisation is a highly orchestrated event in the transition from heterotrophic growth during germination to autotrophy in the developing seedling.

Author Contributions

This chapter section is a reproduction of a paper accepted for publication in The Plant Cell and included work performed, in part, by others as follows: José M. Gualberto performed the qPCR analysis of mitochondrial sequences stoichiometry and analyzed the data. Abdel Benamar and David Macherel performed respiration measurements and analyzed the data. GP and DCL wrote the manuscript with critical additions and revisions by JMG and DM.

1.5

Supplementary bioimaging investigations 1.5.1 Dynamics of mitochondria in mature green cotyledons.

Mitochondria in relatively mature green cotyledons of 4 day-old seedlings move at much greater speed than those in germinating seeds, and faster still than mitochondrial in germinated seedlings at the RH stage, and therefore accurate tracking of each organelle in that tissue necessitated modification of CLSM parameters (faster scan speed) relative to those used for germinating seeds. Images were collected at 1024 pixel resolution, and the PSV 405 nm channel was not used so as to allow an increased frame capture rate of 0.95 fps. The recorded mean speed of mitochondrial movement in 4 days-old cotyledons was over five-times greater, at 360 nm s -1 , than that of mitochondria during germination.

This greater speed was concomitant with longer displacement values compare to that measured at time points during germination. Mitochondrial dynamics in green cotyledons remains heterogeneous with some mitochondrial displaying very low displacement values (< 256 nm) due to wiggling motion which generations short tracks with low straightness values. There is little difference between the numbers of interactors per shared track (cf. Nevertheless, a higher fusion rate is an expected outcome of an increase in mitochondrial speed due to the higher likelihood of faster-moving mitochondria encountering a fusion partner, however, the fact that mitochondria in 4 days-old seedlings are typically round to spherical rather than tubular products of repeated fusion events suggests that fusion is rapidly followed by subsequent division to maintain a fragmented chondriome in mature green cotyledons compared to the uncoupling of fusion from division that occurs during ER/TR stages in germinating seed.

Dynamics with no stratification

Seed were stratified to minimise variation due to variable dormancy between biological replicates thereby helping synchronise germination, and also to enable direct comparison with previous studies (Law et al., 2012;[START_REF] Birky | The inheritance of genes in mitochondria and chloroplasts: laws, mechanisms, and models[END_REF]. Whilst stratification replicates a natural phenomenon, we wanted to rule out possible artefactual effects of the process on the reactivation of mitochondrial dynamics. Seed were surface sterilised and incubated directly under light/21ºC. After 2 h of imbibition, few mitochondria displayed a reactivation of mitochondrial dynamics and instead they behaved like mitochondria under stratification (Figure 2.12A and C, blue = median speed after 48h stratification).

Mitochondrial speed of movement increased during continued imbibition both in the light and dark but it took more than 6 h incubation before the median speed reached that of mitochondria in seed stratified for 48 h and then incubated at light/21ºC for 2 h (Figure 2.12C, red line = median at 50 h L). Following 24 h light/21ºC, seed were close to germinating (but before testa rupture stage), and mitochondrial dynamics had increased relative to 6 h: 37% of mitochondria were interacting and the fusion rate per mitochondrial had increased substantially Figure 2.12). Mitochondrial fusion and the number of interacting partners were highest at the end of germination (ER/TR stages, Figure 2.12 F, G and O) leading to the formation of perinuclear tubuloreticular structure as observed and described at the same developmental stage in stratified seed. Similar to stratified seed, the newly formed tubuloreticular structure is a transient structure and a reduction in mitochondrial speed, fusion rate per mitochondrion, number of interacting partners accompanies the fragmentation of the reticular structure as observed with stratified seed (Figure 2.12 cf. The initially lower dynamics parameters measured in the dark were coincident with a failure to form the perinuclear tubuloreticular structure (Figure 2.12A, B). Even after 48 h, when larger values of mitochondrial speed, displacement and track straightness were measured relative to 24 h L or 24 h D these activities were not translated into a greatly increased fusion rate, number of interactors or percentage of interactions (Figure 2.12) such that clusters of mitochondria formed but these did not coalesce into the higherordered perinuclear cage. Despite an increase in dynamics over the first 48 h in the dark, further incubation up to 96 h D had little effect beyond a slight decrease in fusion rate, number of interactors and percentage interactions. Together these results demonstrate that the reactivation of mitochondrial dynamics occurs similarly in stratified and nonstratified seeds, with stratification acting to reduce the time required for reactivation of mitochondrial dynamics. In addition, it is clear that formation of the mitochondrial cage is tightly linked to the completion of germination and is required to generate the highly dynamic and metabolically active mitochondria of the developing seedling. Introduction

h L 6 h L 2 4 h L T R /E R L R R H time 2 h L 2 h L 6 h L 6 h L 2 4 h L 2 4 h L T R /E R T R /E R L R R H time 2 h L 6 h L 2 4 h L T R /E R L R R H time 2 h L 2 h L 6 h L 6 h L 2 4 h L 2 4 h L T R /E R T R /E R L R
The reactivation of mitochondrial dynamics during germination described in the previous section is a component part of the activation of a programme of mitochondrial biogenesis that is required in order to provide cells with the support required first for germination, and then for the cell division and tissue differentiation required for seedling establishment. The use of seed germination as a model process for the study of mitochondrial dynamics provides the advantages of a clearly defined and relative short developmental time course which can be visibly correlated with defined physiological states and therefore enables use of, and comparison with, pre-existing datasets generated over identical germination time-courses. This section thus describes the results of a transcriptomic meta-analysis investigation of genes involved in mitochondrial dynamics and biogenesis.

Methods

Investigations were performed using three GEO datasets of gene transcript abundances during germination of Arabidopsis seed: GSE41212 [START_REF] Dekkers | Transcriptional dynamics of two seed compartments with opposing roles in Arabidopsis seed germination[END_REF], GSE65394 [START_REF] Silva | A predictive coexpression network identifies novel genes controlling the seed-toseedling phase transition in Arabidopsis thaliana[END_REF], and GSE30223 [START_REF] Narsai | In-depth temporal transcriptome profiling reveals a crucial developmental switch with roles for RNA processing and organelle metabolism that are essential for germination in Arabidopsis[END_REF] In addition, the fact that the datasets were obtained without using stratification in two cases, and with stratification in the third, allows the effect of stratification on transcript abundance to be compared with the results presented in this thesis on mitochondrial dynamics under both stratified and non-stratified conditions.

Result and discussion

Analysis of gene transcripts linked to reactivation of mitochondrial motility

F-actin filaments are the main tracks for movement of plant mitochondria. Transcripts for the ARP2/3 complexes, promoting assembly and organization of the F-actin cytoskeleton, had a complex profile during germination, likely highlighting the massive changes in cell morphology that accompany this process (Figure 2.14). Transcripts of ARP3 and ARPC2A

genes increased in abundance from the middle of Phase 2 of germination (i.e. after 12 h imbibition) and were more abundant at the last step of germination, in agreement with their putative involvement in the reorganisation of the cytoskeleton to support increased mitochondrial dynamics and the perinuclear mitochondrial localisation at that time. In contrast, ARPC5 and ARPC3 transcripts decreased in abundance during early Phase 2 (i.e.

after 3 h imbibition), suggesting that these genes are more important during seed maturation. The transcript for ARP2 showed increased abundance after stratification followed by decreasing abundance as germination progressed, and therefore had a profile consistent with a role in the eventual reactivation of organelle dynamics (upon transfer to conditions promoting germination) that is stimulated by stratification. The datasets also highlight what is perhaps a key discovery -myosin transcripts increased in abundance during stratification such that maximum expression coincided with a time when mitochondrial activities were still cold limited. Stratification may, therefore, provide a means to promote more rapid reactivation of mitochondrial dynamics upon transfer to germination promoting conditions, relative to non-stratified seed.

With regard to the expression of genes encoding for Myo-B adaptors, interpretation is difficult since, for unknown reasons, the transcript profiles do not match well between the different experiments (Figure 2.15). However, it is clear that MYOB7 was the most abundant transcript in dry seed, and as such is a good candidate to be involved in the regulation of organelle motility during seed maturation. Stratification promoted MYOB7 expression until a reduction during mid-germination (i.e. 12 h after transfer) while a lack of stratification led to static MYOB7 abundance until again there was a decrease in expression during the middle of germination (i.e. 12 h after the start of imbibition). As with the expression of MYOSIN XI genes, stratification may act as a priming mechanism to ensure rapid expression of the protein upon transfer from inhibitory cold conditions, and therefore rapid resumption of mitochondrial dynamics.

The MYOB1 and MYOB3 genes showed low transcript abundances relative to MYOB7, but showed a profile consistent with a role in organelle motility during germination by stimulating motility during major developmental transitions such as during Phase 1 (i.e.

1 to 7 h after imbibition), and post-germination (after ER). The MYOB2 gene appears not to be involved in organelle motility during germination, acting instead after the transition to an autotrophic state, as indicated by the increased transcript abundance following ER and a maximal abundance at the open cotyledon stages (i.e. post RH stage).

Analysis of gene transcripts linked to mitochondrial fission

Eight genes are known to encode for components of the mitochondrial fission apparatus (referred to as fission genes subsequently). PMD2 and BIGYIN1 were the most abundantly expressed of all fission genes in dry seed in all three datasets, while PMD1

was the fission gene most induced during germination (16 h imbibition) relative to dry seed (Figure 2.16). The increases in transcript abundances of PMD1 and PMD2 occurred before entry into Phase 2 of germination (1 to 3 h after the start of imbibition) and they both reached maximal abundance mid-way into this phase (i.e. after 12 to 16 h of imbibition). However, the abundance of PMD1 transcript was markedly reduced after ER, while the abundance of PMD2 decreased after 16 h imbibition. Since PMD2 is specific to mitochondria, while PMD1 also affects peroxisome fission, the decreased abundance of BIGYIN genes show the opposite profile to PMDs. BIGYIN1 transcript was more abundant following imbibition relatively to dry seed, when mitochondria are still not moving, and thereafter showed reduced abundance (i.e. high from 3 to 7 h imbibition without stratification, or from 12 h stratification to 1 h after transfer to germination promoting conditions for stratified seed). This profile is consistent with a role in mitochondrial fission at the start of Phase 2. Transcript abundance of BIGYIN2 decreased from early Phase 2 (after 3-7 h of imbibition) and this reduction continued during germination until stabilisation beyond the ER stage. This decreased abundance of BIGYIN2 transcript is consistent with a role in promoting mitochondrial fusion/elongation by limiting assembly of the division apparatus. Stratification induced a decrease in abundance of BIGYIN2 transcript, providing a mechanism to reduce the activity of mitochondrial fission concomitant with the observed stimulation of mitochondrial biogenesis. In addition, the BIGYIN2 transcript profile, and the high abundance of BIGYIN1 transcripts, suggests an important role for these proteins during seed development and maturation in order to ensure correct segregation of mitochondrial units.

Unfortunately, data for the only putative adaptor protein mediating mitochondrial fission in plants, NMT, is available only in the set covering the end stages of germination.

However, the NMT transcript profile is similar to that of BIGYIN2: high abundance during seed development / maturation, but the absence of additional data prevents further analysis.

The abundances of transcripts of members of the dynamin family of large GTPase involved in mitochondrial fission were stable (maximal abundances of DRP3A and DRP3B

during germination are 5.7 and 5.8% greater than in dry seed respectively), throughout germination, but the profile varies depending on the seed tissues examined (Figure 2.16 and 2.17). Stratification induces an increased and earlier accumulation of DRP3A and DRP3B transcripts (1 h after stratification) suggesting a requirement for mitochondrial division, or remodelling under stratification conditions. Transcript abundance of DRP5B, which is involved in plastid and peroxisome division, showed an profile opposite to that of the DRP3s, whether in stratified or non-stratified seed: expression of DRP5B dropped after imbibition, and during germination, until a slight increase at the RH stage; while stratification promoted a small transient increase in transcript abundance (+30%) before a reduction in abundance after transfer to germination promoting conditions (i.e.

reduction between 1 to 6 h after transfer). The relatively small-scale changes in DRP transcript abundance likely reflect, at least in part, the importance of post-translational regulation of the activities of these proteins. However, the results of this analysis highlight the different roles DRP3s and DRP5B play in the division of organelles other than mitochondria. For example, DRP3s are specific to mitochondrial and peroxisomal fission, and show relatively stable expression during germination, whereas DRP5A and B increased in abundance during the late stages of germination, and following completion of germination, concomitant with chloroplast biogenesis which occurs later during germination than mitochondrial biogenesis [START_REF] Pogson | Genetic dissection of chloroplast biogenesis and development: an overview[END_REF][START_REF] Demarsy | Characterization of the plastid-specific germination and seedling establishment transcriptional programme[END_REF]. This differential expression ensures uncoupling of mitochondrial/peroxisomal fission from plastid fission. The measured increase of DRP5A abundance at this stage correlates with reactivation of the cell cycle, and cell division post-germination when DRP5A function has been reported to be necessary for cell plate formation [START_REF] Miyagishima | Evolutionary linkage between eukaryotic cytokinesis and chloroplast division by Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy[END_REF].

Analysis of the transcript abundances of other members of the DRP family indicates that DRP1C and DRP2A transcripts, known to be involved in endocytosis and cytokinesis, are the most abundant and show regulated expression during germination (Figure 2.17).

DRP1E transcript abundance is the highest of any DRP in dry seed and the expression level is maintained during imbibition until a reduction during Phase 2, highlighting a potential involvement of DRP1E in seed development / maturation and/or during imbibition. DRP family members with unknown function, such as DRP4s and DRP6 do not appear to be involved in germination since their absolute abundances are the lowest of all DRPs (for example, absolute transcript levels differ up to 425-fold relative to the more abundant dynamin in the stratification dataset) and their abundances decrease during germination and beyond.

Analysis of gene transcripts linked to mitochondrial membrane biogenesis and import

Mitochondrial membranes define the identity of the mitochondrial compartment and, focusing on the lipid component, are mainly composed of phosphatidycholine, phosphatidylethanolamine and phospatidylserine, in addition to lipids specific to this organelle such as cardiolipins. Genes coding for key enzymes required for the synthesis of mitochondrial membrane lipids displayed a range of transcript abundance profiles during germination (Figure 2.18). CLS transcripts had a maximal abundance in dry seeds, higher in absolute quantity than other enzymes of this class, but abundance fell gradually after seed imbibition and during germination. Given that the transcript profile of CLS obtained using the web-based resource BAR (http://bar.utoronto.ca/efp/cgibin/efpWeb.cgi) indicates that higher abundances are observed during late seed development and seed maturation, cardiolipin synthesis seems to be set, at least at the transcriptional level, during seed development more so than during germination. Other transcripts such as DLAH, encoding for an enzyme in the cardiolipin biosynthetic pathway, and PSD3, which is involved in the biosynthesis of phosphatidylserine, are downregulated following imbibition compared to dry seed, but show an increase in abundance from the TR stage. Membrane biosynthesis genes whose transcripts are upregulated from imbibition until the end of germination show similar trends, reaching maximal abundance at mid Phase 2 (i.e. 16 h imbibition). The transcript abundance profile of PSD1, coding for an IMM enzyme involved in the synthesis of phosphatidylserine, is consistent with a role in mitochondrial membrane biogenesis during germination since abundance is regulated during this developmental program. For example, stratification led to an increase in PSD1 transcript abundance of 761% relative to dry seed 1 h after transfer to germination promoting conditions while there were no changes in the abundances of PGPS2 or PGP1 transcripts. Transcripts of other genes involved in lipid synthesis showed maximal increases in abundance of 43% during germination relative to dry seed and therefore regulation at the transcriptional level appears an unlikely means to regulate the activity of genes involved in membrane biogenesis during germination.

Har-vest Dry 1 h 12 h 48 h 1 h 6 h 12 h 24 h

The mitochondrial import apparatus is required for the transfer of proteins encoded by the nucleus, and synthesised on cytoplasmic ribosomes, into mitochondria. The higher transcript abundance detected at Phase 2 (i.e. 7-21 h after imbibition) of genes encoding components of the mitochondrial import apparatus, together with the high absolute abundances of those transcripts across the three datasets relative to other observed gene classes (Figure 2.19), highlights the previously described early upregulation of this class of transcript (Law et al., 2012[START_REF] Law | Mitochondrial biogenesis in plants during seed germination[END_REF]. Indeed, almost all transcripts for genes of this class increased in abundance during germination and reached maximal abundance at Phase 2 (i.e. 7-20 h imbibition), while transcripts that displayed decreased abundance during germination were already highly abundant at absolute levels in dry seeds, or in early after the start of imbibition (except for TIM17-1; TIM44-1; TOM20-1 and VDAC5 transcripts which had low absolute abundance relative to other components of the import apparatus). The Narsai dataset, which included stratification in the germination protocol, yielded a similar transcript profile to germination without stratification, but additionally highlights the fact that the cold treatment allowed the generation of relatively high transcript abundances while enzyme activities were still inhibited by low temperature.

Transcript abundance of import components localised at the IMM peak earlier than for those localised at the OMM, such that there is lower abundance of OMM import apparatus transcripts from RH to OC stages, relative to in dry seed. While the abundance of transcripts involved in import through the IMM drops at ER through RH stages.

Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis Therefore, based on these transcriptomic data, the increase in expression of genes involved in protein import into mitochondria during Phase 2, a time when there is a large increase in mitochondrial membrane (as shown in Chapter 2.1) may reflect turnover of the already present protein import apparatus, in addition to supplying these protein components of the newly synthesised membrane.

Analysis of gene transcripts linked to organellar DNA maintenance

The majority of genes known to be involved in the maintenance of mtDNA show high 1 Introduction

Autophagy is central to the plant response to starvation, for example, sugar, or nitrogen, and the pathways were first elucidated in plants using mutants with an accelerated senescence phenotype [START_REF] Sláviková | The autophagy-associated Atg8 gene family operates both under favourable growth conditions and under starvation stresses in Arabidopsis plants[END_REF][START_REF] Bassham | Autophagy in development and stress responses of plants[END_REF]. Indeed, mutants of ATI1, that encodes a protein that interacts with ATG8F, display decreased germination on media containing ABA, while ATI1 over-expression increased germination rate [START_REF] Honig | A New Type of Compartment, Defined by Plant-Specific Atg8-Interacting Proteins, Is Induced upon Exposure of Arabidopsis Plants to Carbon Starvation[END_REF]. ATI1 interacts with plastid-localised proteins which are selected for specific autophagy and therefore suggests that selective autophagy of organelles is important for germination [START_REF] Avin-Wittenberg | ATI1, a newly identified atg8-interacting protein, binds two different Atg8 homologs[END_REF][START_REF] Michaeli | Arabidopsis ATG8-INTERACTING PROTEIN1 is involved in autophagy-dependent vesicular trafficking of plastid proteins to the vacuole[END_REF].

Reactivation of mitochondrial dynamics in cotyledon cells of embryos during germination was observed to be concomitant with reactivation of interactions between mitochondria and ATG8F-decorated bodies (ATG8F-bodies) and consequent mitophagy as detailed in Chapter 2. Those results are in line with a previous study investigating mitochondrial biogenesis during maize germination that indicated there was a coordinated disassembly of a subpopulation of mitochondria that could be isolated by isopycnic density gradient fractionation [START_REF] Logan | Mitochondrial biogenesis during germination in maize embryos[END_REF]. However, [START_REF] Logan | Mitochondrial biogenesis during germination in maize embryos[END_REF] could not rule out the possibility that the low density mitochondria were biogenesis intermediates rather than degradation intermediates and, in addition, the specific processes involved, and their extent was not investigated. Therefore, using a double-transgenic line expressing mito-GFP and mRFP1-ATG8F, a fusion that tags the growing phagophore, it was decided to perform a more detailed study of the progression of mitophagy both during germination but also during subsequent seedling growth. In addition, the effect of a disturbed chondriome structure, using the friendly mutant, and the process of autophagy in friendly was investigated in order to learn more about the role of autophagy and MQC during germination. The increased number of ATG8F-bodies described in Chapter 1 occurred 6 h after transfer to germination promoting conditions. However, in this additional study it was decided to use an earlier time point of 4 h in the expectation of recording high numbers. As a result, it is clear that the increase in mitophagy occurred within 4 h after transfer to germination promoting conditions. In addition, more ATG8F-bodies were observed 4 h after transfer compared to after 6 h (mean of 1.38 per cell compared to 0.86), although the percentage of ATG8F-bodies that were associated with mitochondria remained similar (73%) highlighting requirement for mitophagy for germination per se (Figure 3.2), as previously observed (Figure 2.5). Indeed, of the total number of ATG8F-bodies detected 4 h after transfer, 57% were juxtaposed to mitochondria and 16% were seen to be engulfing complete mitochondria, indicating that a part of mitochondrial interaction with ATG8F-bodies may be transient during early germination. It could be also be noted that all seed observed at 4 h L (31 seeds) had ATG8F-bodies associated with mitochondria, providing evidence that mitochondrial quality control, including terminal mitophagy, is reactivated during early germination.

Results

Mitophagy is activated during early germination and during the transition to autotrophy

Twelve hours after the end of stratification there was a 2.5-times reduction in number of

In-depth in vivo observations of ATG8F-mediated mitophagy

There are few published studies of the observation of mitophagy in vivo in plants. [START_REF] Li | AUTOPHAGY-RELATED11 plays a critical role in general autophagy-and senescence-induced mitophagy in Arabidopsis[END_REF] showed mitochondria surrounded by ATG8A-bodies, however little is known about the early process leading to interaction of ATG8-bodies and mitochondria. Therefore, because mitophagy is strongly induced during early-and post-germination, it was reasoned that in vivo observations by CLSM at this particular stage would provide valuable information. The more detailed studies described here confirm the existence of populations of ATG8F-bodies, some of which do not interact with mitochondria, while others have a perimitochondrial localisation. However, perhaps more surprising is the transient aspect of the interactions with mitochondria for a majority of ATG8F-bodies that are mitochondrial-associated. Indeed, once a mitochondrion becomes juxtaposed to an ATG8F-body tethering between the two is suggested by co-directional movement and the duration of the tethering varies from one association to the next. In addition, association of ATG8F-bodies with a mitochondrion within a group can lead to a breakdown in the apparent co-ordinated movement of the associated mitochondria, followed by immobilisation and division of the tethered mitochondrion (Figure 3.3). Together, these observations highlight the possibility that mitochondria undergoing longer interaction with ATG8F-bodies are immobilised, which could be a requirement for successful mitophagy. For example, distinct types of interaction takes place such as tethering of preassembled ATG8F-decorated phagophores to previously immobilized mitochondria, or the assembly of the ATG8F phagophore in in a manner constricting part of mitochondria motile been observed (Figure 3.4). Mito-GFP remained detectable in mitochondria inside ATG8F-autophagosomes during early germination and since it is known that GFP fluorescence is quenched in low pH environments [START_REF] Campbell | The effect of pH on Green Fluorescent Protein: a brief review[END_REF]

The mitophagy transciptome during germination

The coordination of mitophagy with particular stages of germination (increased mitophagy during early germination Phase 2 and early post-germination) permits use of GEO datasets (Dekkers et al., 2013 (GSE41212), [START_REF] Silva | A predictive coexpression network identifies novel genes controlling the seed-toseedling phase transition in Arabidopsis thaliana[END_REF](GSE65394), Narsai et al., 2011 (GSE30223)) of transcript abundance in order to try to identify genes involved in the processes (Figure 3.5).

From the GEO class "mitophagy", only three genes (close to 25% of the genes represented) are upregulated during germination within each dataset, underlining the lack of actors known to be involved in mitophagy. In the transcriptome datasets from experiments that did not include a stratification step (GSE41212, GSE65394

FRIENDLY is preferentially expressed in meristematic tissue

Disruption of FRIENDLY function leads to both unusual mitochondrial dynamics and an increase in indicators of a loss of MQC [START_REF] Logan | The genetic control of plant mitochondrial morphology and dynamics[END_REF][START_REF] El Zawily | FRIENDLY regulates mitochondrial distribution, fusion, and quality control in Arabidopsis[END_REF]. The In seed stratified for 48 h, the chondriome is structured in similar way in both friendly and WT. Indeed, as described in Chapter 1, mitochondria are homogenously distributed within cells, in the form of mostly spherical discrete units in the cytosol as single units, or in small groups of a few mitochondria. However, 12 h after transfer of seed to germination conditions, clustering of mitochondria in friendly was increased relatively to what was observed in WT embryos, and few mitochondria remained isolated from the growing clusters. The clustering phenotype observed was maintained during subsequent steps, with an increase in the numbers of matrixules observed at 22 h L. At the ER stage, mitochondrial clusters remain, but the morphologies of individual mitochondria within the cluster appears more uniform than previously. In addition, clusters appear clearly distributed around the PSV "hole", identified previously as the location of the nucleus. At the LR step, massive mitochondria clusters are progressively split into smaller clusters, but these remain in the perinuclear region throughout the whole of the cotyledon tissue (not shown), and an increase in the number of individual mitochondria was observed, but in a less clear manner than was observed in WT in Chapter 2. However, in cotyledons from 4 day-old seedlings the massive perinuclear mitochondrial cluster was no longer detected, instead the chondriome was organised as a mix of discrete units and small clusters containing no more than a few tens of mitochondria, such as typically observed in friendly seedlings [START_REF] Logan | The genetic control of plant mitochondrial morphology and dynamics[END_REF].

The friendly mutant shows impaired mitophagy during germination and post germination

The consequence of the friendly mutation on mitophagy during germination and seedling establishment was assessed by CSLM observation of cotyledon cells of friendly embryos expressing both mito-GFP and mRFP1-ATG8F compared to an otherwise WT mito-GFP / mRFP1-ATG8F double expressing line. Unfortunately, the double transgenic friendly line displayed very low mito-GFP fluorescence during early germination stages and therefore mitophagy could not be investigated, although the numbers of ATG8F-bodies was determined for those steps.

The numbers of ATG8F-bodies detected in cotyledon cells of friendly embryos following 48 h of stratification were similar to WT, with a median number of 0 ATG8F-bodies and a mean of 0.041 ATG8F-bodies per cell coming from 5 embryos over 39 counted (Figure

Discussion

During early Phase 2, low numbers of promitochondria are subject to mitophagy, but there is a high level of mitochondria-phagophore interactions, relative to later germination phases, indicating that mitochondrial quality is under tight control during early germination stages. As a result, there is no blanket destruction of promitochondria, instead the majority are maintained and mitochondrial fission acts to segregate damaged mitochondria that may be unable to reactivate, or facilitates the removal of damaged mitochondrial components. The temporal correlation between the decrease in mitophagy, and orientation of mitochondrial dynamics to facilitate high rates of mitochondrial interaction, and mitochondrial biogenesis, underlines the interweaving of both processes in order to coordinate re-activation of mitochondrial activities required to support the end of germination and subsequent seedling establishment.

Disruption of FRIENDLY function is known to induce clustering of mitochondria in Arabidopsis, and similarly, disruption of FRIENDLY homologues (CLU-type genes) leads to mitochondrial clustering in a range of species across the eukaryote kingdom. A recent extended characterisation of friendly plants demonstrated that not only were mitochondria in the mutant defective, relative to WT, in their interactions with each other, but also displayed disruption of mitochondrial metabolism (such as respiration) and quality control (indicated by a high level of mitochondrial pulsing) in young seedlings [START_REF] El Zawily | FRIENDLY regulates mitochondrial distribution, fusion, and quality control in Arabidopsis[END_REF]. Analysis of FRIENDLY transcript abundance during development using the BAR resource indicated that Phase 2 of germination is one of the developmental stages when FRIENDLY transcript is at its most abundant. Regulation of Indeed, in Chapter 2, the reactivation of mitochondrial dynamics was shown to lead to increased mitochondrial interaction during germination, coincident with the time when the steady-state abundance of FRIENDLY is relatively high. In addition, friendly cotyledon cells contain mitochondrial clusters upon the reactivation of mitochondrial dynamics, but the size of these clusters increase during germination such that at late stages there is a massive perinuclear accumulation (a location shared with mitochondria in WT), but this location is maintained for longer in friendly, up to early seedling establishment. Mitochondria in clusters have been observed to exchange matrix content at a greater rate, at least for mitochondria in leaf epidermal cells of 14-day-old seedlings, although clusters have reduced mitochondrial motility mitochondria such that mitochondria within the same cluster, or in different clusters, have fewer opportunities for new interaction that mitochondria distributed normally in WT [START_REF] El Zawily | FRIENDLY regulates mitochondrial distribution, fusion, and quality control in Arabidopsis[END_REF].

In this way, extensive clustering at the end of germination in the mutant may promote heterogeneity of mitochondrial content. However, the perinuclear localisation of the clusters might be a means to counteract the effects of FRIENDLY dysfunction on the mixing of the chondriome by promoting an efficient uptake of mitochondrial protein encoded by the nuclear genome such as in cultured mammalian cells were mitochondria with a perinuclear location were shown to be enriched in components of the outermembrane protein import apparatus [START_REF] Wurm | Nanoscale distribution of mitochondrial import receptor Tom20 is adjusted to cellular conditions and exhibits an inner-cellular gradient[END_REF]. The absence of massive clusters in four-day-old seedlings may be related to the active growth of the cotyledon cells as they expand to generate a useful photosynthetic organ, since those changes in cellular architecture involve extensive remodelling of the F-actin cytoskeleton, and this remodeliing has been shown to lead to the break-up of clusters in friendly [START_REF] El Zawily | FRIENDLY regulates mitochondrial distribution, fusion, and quality control in Arabidopsis[END_REF].

MQC is known to be defective in other CLU-type mutants, as indicated, for example, by an increased quantity of PINK/parkin complexes observed in flight muscle of mutant Drosophila, and by an increase in the size of the lytic vacuole compartment in friendly [START_REF] El Zawily | FRIENDLY regulates mitochondrial distribution, fusion, and quality control in Arabidopsis[END_REF][START_REF] Sen | Drosophila Clueless Is Highly Expressed in Larval Neuroblasts, Affects Mitochondrial Localization and Suppresses Mitochondrial Oxidative Damage[END_REF]. Despite the absence of data for mitophagy during early germination (for the technical reasons given above), the number of ATG8F-bodies was higher in friendly than in WT, and since the majority of ATG8F-bodies are interacting with mitochondria at this time in WT, it could be concluded that it is this interaction that is defective in friendly during early germination leading to an upregulation of phagophore formation. Beyond late germination, assessment of ATG8Fbodies confirmed the increase in numbers in friendly at this stage, as was found during germination, and analysis of their subclasses indicated that few mitochondria were observed to undergo disassembly in autophagosomes in friendly. Together, these data suggest that FRIENDLY is also involved in the regulation of mitochondrial association with ATG8F-bodies. Spermatophytes, such as Arabidopsis, are defined as such by the production of seeds.

L 4 h L 1 2 h L 1 2 h L 2 2 h L 2 2 h L E R E R L R L R
Within the most diverse group, the angiosperms, the seeds are enclosed within protective layers provided by the mother plant which help the seed survive as a newly independent organism until conditions are suitable for germination. Within the angiosperms, some species, again such as Arabidopsis have been classified as orthodox if they can be dried to low moisture contents (as low as 5% w/w) and can tolerate freezing (relative to recalcitrant seeds which cannot survive drying below approximately 20-30% w/w, nor can they survive freezing, or intermediate seeds with limited desiccation tolerance) [START_REF] Finkelstein | Molecular Aspects Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy[END_REF][START_REF] Bentsink | Seed dormancy and germination[END_REF]. The low water content of orthodox seeds at maturity, when drying is complete, means the cytoplasm enters a high viscosity glassy state in which metabolism is impossible due to low molecular mobility [START_REF] Buitink | Dehydration-induced redistribution of amphiphilic molecules between cytoplasm and lipids is associated with desiccation tolerance in seeds[END_REF][START_REF] Buitink | Intracellular glasses and seed survival in the dry state[END_REF]. In this state the seed can survive for years, or decades, yet as long as they remain viable many species will germinate within 48 h if they become sufficiently hydrated and other species-specific environmental criteria are met [START_REF] Bentsink | Seed dormancy and germination[END_REF][START_REF] Holdsworth | Tansley review Arabidopsis seed maturation , after[END_REF].

Rehydration and perception of stimulatory environmental signals trigger cellular reanimation that is fuelled by an activation of metabolism which starts to convert stored reserves into useful energy. This energy is required for both for the continued reactivation of the quiescent tissues and cell growth which will in turn lead to completion of germination.

Mitochondria are central to plant cell metabolism and therefore their reactivation from stasis in the dry seed is a vital component part of the germination program. However, mitochondria in seeds are rudimentary, both in structure and function relative to mitochondrial in mature cells in adult tissues such as developed leaves and roots. The relatively low metabolic activity and rudimentary protein composition and structure of dry seed mitochondria led to them being termed promitochondria [START_REF] Logan | Mitochondrial biogenesis during germination in maize embryos[END_REF], and the activities of these mitochondria and the mechanisms underpinning their reactivation during germination have been the focus of several studies on maize, rice, and Arabidopsis [START_REF] Logan | Mitochondrial biogenesis during germination in maize embryos[END_REF][START_REF] Howell | Ordered assembly of mitochondria during rice germination begins with promitochondrial structures rich in components of the protein import apparatus[END_REF][START_REF] Howell | Mapping Metabolic and Transcript Temporal Switches during Germination in Rice Highlights Specific Transcription Factors and the Role of RNA Instability in the Germination Process[END_REF]Law et al., 2012).

The transformation from promitochondria into organelles with the functions typical of mature mitochondria is known to involve mitochondrial biogenesis [START_REF] Logan | Mitochondrial biogenesis during germination in maize embryos[END_REF][START_REF] Howell | Ordered assembly of mitochondria during rice germination begins with promitochondrial structures rich in components of the protein import apparatus[END_REF][START_REF] Law | Mitochondrial biogenesis in plants during seed germination[END_REF], however, many questions remain unanswered about the function of promitochondria during early germination events and the extent to which biogenesis is necessary for these functions.

The objective of the work described in this thesis was to investigate, using a live-cell bioimaging approach and Arabidopsis as a model system, the roles of mitochondrial Despite the rudimentary state of promitochondria, they are protected in orthodox seeds in the dry state as simply demonstrated by continued fluorescence of matrix-targeted fluorescent protein in dry tissues sensu stricto, and the measured stability of mtDNA structure and abundance. Promitochondria in the dry seed are discrete spherical/ovoid structures and once hydrated ten minute each mitochondrion contains a visible mtDNA nucleoid (Chapitre two -1.3.8), which contrasts with the heterogeneous distribution of mtDNA amongst the discrete members of the chondriome of more mature cells in autotrophic seedlings [START_REF] Arimura | Frequent fusion and fission of plant mitochondria with unequal nucleoid distribution[END_REF][START_REF] Takanashi | Different amounts of DNA in each mitochondrion in rice root[END_REF]. Within 10 mins from the start of imbibition, promitochondria are able to generate a membrane potential, which is vital to fuel protein import for maintenance and biogenesis, likely by oxidising succinate and/or NADH as previously determined in vitro [START_REF] Logan | Mitochondrial biogenesis during germination in maize embryos[END_REF][START_REF] Benamar | Membrane integrity and oxidative properties of mitochondria isolated from imbibing pea seeds after priming or accelerated ageing[END_REF][START_REF] Howell | Ordered assembly of mitochondria during rice germination begins with promitochondrial structures rich in components of the protein import apparatus[END_REF][START_REF] Fait | Arabidopsis seed development and germination is associated with temporally distinct metabolic switches[END_REF]. These results indicate that promitochondria are specialised in the maintenance of mitochondria structure and genetic integrity. However, the immediate reactivation of membrane potential is not concomitant with the reactivation of mitochondrial dynamics, since promitochondria remain static, just as in dry seed, up to the time of transition to Phase 2.

From Phase 2, mitochondrial motility is reactivated, in a manner independent of the hormone (ABA/GA) status of seeds, enabling physically discrete mitochondrial to interact and fuse. This is accompanied by activation of autophagy and specially mitochondrial quality control, as demonstrated by an increase in the number of detectable phagophores and number of interactions between phagophores and mitochondria suggesting that interaction between mitochondria is necessary for identification of damaged mitochondria or mitochondrial components as shown in mouse embryonic fibroblasts [START_REF] Twig | Fission and selective fusion govern mitochondrial segregation and elimination by autophagy[END_REF].

The reactivation of mitochondrial dynamics at the start of Phase 2 is coincident with a burst in expression of genes encoding mitochondrial membrane proteins, including components of the import apparatus [START_REF] Logan | Mitochondrial biogenesis during germination in maize embryos[END_REF][START_REF] Howell | Ordered assembly of mitochondria during rice germination begins with promitochondrial structures rich in components of the protein import apparatus[END_REF]Law et al., 2012). This leads to biogenesis of mitochondrial membrane using promitochondria as scaffolds to expand chondriome volume as described in Chapter 2. As germination progresses from Phase 2 to the end stages (i.e. though TR to the ER stage), a further increase in inter-mitochondria interactions, and specifically a higher fusion rate, leads to the transient formation of a tubuloreticular chondriome with a distinct perinuclear location. This burst in mitochondrial interaction and massive fusion brings together, within the same matrix, mtDNA that was previously segregated amongst discrete mitochondria, providing an opportunity to homogenise the different mtDNA molecules that have been stored independently since seed maturity. These changes in chondriome architecture are similar to those seen during cell division of meristematic cells of young Arabidopsis seedlings wherein the mitochondria form what was described as a tentaculate cage-like structure surrounding the nucleus [START_REF] Segui-Simarro | The mitochondrial cycle of Arabidopsis shoot apical meristem and leaf primordium meristematic cells is defined by a perinuclear tentaculate/cage-like mitochondrion[END_REF]. germination stages (TR to ER stages), mitophagy is downregulated, suggesting that the activation of biogenesis improves overall mitochondrial quality. The changes in morphology and location of the chondriome at this time coincide with a transcriptional reprogramming of energy metabolism (Law et al., 2012) consistent with the developing metabolic activities (increased OXPHOS and TCA cycle enzyme activities) measured in previous studies [START_REF] Logan | Mitochondrial biogenesis during germination in maize embryos[END_REF][START_REF] Howell | Ordered assembly of mitochondria during rice germination begins with promitochondrial structures rich in components of the protein import apparatus[END_REF] and thus the perinuclear location of the chondriome in seed at this stage may enable efficient fuelling of transcription and mRNA transport to mitochondria by delivery of ATP to the perinuclear cytosol as suggested by [START_REF] Segui-Simarro | The mitochondrial cycle of Arabidopsis shoot apical meristem and leaf primordium meristematic cells is defined by a perinuclear tentaculate/cage-like mitochondrion[END_REF] in the case of the cage-like chondriome in meristematic cells.

After ER, the tubuloreticular chondriome fragments to regenerate a chondriome consisting of hundreds of physically discrete mitochondria. Despite the previous massive fusion event providing the opportunity for mixing of mtDNA, an event which in meristematic cells leads to the even distribution of mtDNA amongst the newly fragmented chondriome [START_REF] Segui-Simarro | The mitochondrial cycle of Arabidopsis shoot apical meristem and leaf primordium meristematic cells is defined by a perinuclear tentaculate/cage-like mitochondrion[END_REF], fission of the tubuloreticular chondriome in cotyledon cells at the end of Arabidopsis seed germination seed generates a population of mitochondria with a very heterogeneous distribution of nucleoids such that a mean of thirty five percent have no detectable mtDNA (Chapitre two -1.3.8).

This new distribution is coincident with uncoupling of mtDNA replication from that of the nuclear genome reduced control over mtDNA recombination.

The results presented in this thesis show that the development of promitochondria during seed germination involves specific modulation of mitochondrial dynamics to control the level of inter-mitochondrial interactions and to facilitate interaction of mitochondrial with other cell compartments. These physical interactions underpin mitochondrial quality control during early germination and a massive restructuration of the chondriome, through the combined activities of mitochondrial fusion and membrane biogenesis, in the later stages of germination. Formation of the tubuloreticular chondriome facilitates mixing and exchange of mtDNA while subsequent fragmentation of the chondriome at the end of germination leads to an uneven redistribution of the mtDNA amongst the discrete mitochondria. These processes are interwoven with transcriptional reprogramming of mitochondrial function first to support germination and then seedling establishment. The living world is a dynamic world and the results presented in this thesis highlight the importance of intracellular dynamics, and in particular the dynamics of mitochondria, to the life and fate of the mitochondria, cell, tissue and organism.

Perspective

Given that Arabidopsis mitochondria are inherited from the mother, and seed quality is known to be influenced by the environment during seed set, it will be interesting to investigate how the maternal environment influences the development of promitochondria, and to determine how mitochondrial dynamics during the later stages of seed set shape mtDNA genome architecture and distribution within the chondriome. Furthermore, focusing on germination, the results presented highlight the existence of subpopulations of mitochondria (that differ in mtDNA content, in their motility, in their metabolism, and in their frequency of interaction with other mitochondria): future studies should seek to discover the mechanisms that generate and maintain these subpopulations within the whole.

Finally, this study has provided some clues about the role of autophagy in the control of mitochondrial development and turnover. A key challenge for the future will be to discover and investigate, in vivo, the proteins mediating mitochondria-phagophore interactions, to understand precisely how defective mitochondria are identified and

Plant materials and growth conditions

All experiments were performed using the Arabidopsis thaliana Columbia ecotype (Col-0).

Visualisation of mitochondria was performed using transgenic lines expressing mGFP5 [START_REF] Siemering | Mutations that suppress the thermosensitivity of green fluorescent protein[END_REF], or mCherry [START_REF] Shaner | Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein[END_REF] targeted to the mitochondrial matrix (mito-GFP [START_REF] Logan | Mitochondria-targeted GFP highlights the heterogeneity of mitochondrial shape, size and movement within living plant cells[END_REF], or mito-mCherry, [START_REF] Candat | The ubiquitous distribution of late embryogenesis abundant proteins across cell compartments in Arabidopsis offers tailored protection against abiotic stress[END_REF]). In order to visualize actin filaments and mitochondria, a double transgenic line was used expressing mito-GFP and an in-frame fusion of mCherry to the actin binding domain of mouse talin [START_REF] Kost | A GFP-mouse talin fusion protein labels plant actin filaments in vivo and visualizes the actin cytoskeleton in growing pollen tubes[END_REF][START_REF] El Zawily | FRIENDLY regulates mitochondrial distribution, fusion, and quality control in Arabidopsis[END_REF]. The endoplasmic reticulum was observed in a line expressing YFP-HDEL [START_REF] Saint-Jore | Redistribution of membrane proteins between the Golgi apparatus and endoplasmic reticulum in plants is reversible and not dependent on cytoskeletal networks[END_REF][START_REF] Teh | An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells[END_REF] while peroxisomes were observed in a line expressing YFP-SKL [START_REF] Mathur | Simultaneous visualization of peroxisomes and cytoskeletal elements reveals actin and not microtubule-based peroxisome motility in plants[END_REF]. To visualise autophagosomes and mitochondria, the mito-GFP line and friendly mutant (kanamycin resistant) were retransformed with a construct containing mRFP1-ATG8F under the control of the 35S promoter in a binary plasmid conferring resistance to BASTA [START_REF] Honig | A New Type of Compartment, Defined by Plant-Specific Atg8-Interacting Proteins, Is Induced upon Exposure of Arabidopsis Plants to Carbon Starvation[END_REF]. The friendly mutant was identified in a microscope-based screen of a mito-GFP line mutated with ethyl methansulfonate. FRIENDLY was identified by mapbased cloning and DNA sequencing as At3g52140, with the mutant allele bearing a single G-to-A mutation in the first intron/exon boundary [START_REF] Logan | The genetic control of plant mitochondrial morphology and dynamics[END_REF]. Double transgenics were selected by BASTA resistance and by microscopy. All lines were used within six months of harvest and seeds stored over desiccant at -20ºC, except for Atg8 experiments when seed were used within one month of harvest. Seeds were surface sterilized in the dark by washing in 80% (v/v) ethanol and then in 30% (v/v) household bleach for 5 min with continual inversion, followed by three rinses in sterile water.

Sterilized seeds were immediately spread on a nitrocellulose membrane (Sartorius) over two circles of Whatman No1 filter paper in 47mm glass Petri dish. Dishes were wrapped in aluminium foil and transferred to 4°C in the dark for stratification. After 48h the plates were unwrapped and transferred to a growth chamber at 21°C under continuous light (50 µmol m -2 s -1 ). For treatment with 100 µM abscisic acid (ABA, Sigma), 100 µM gibberellic acid 3 (GA3, Sigma), or 100 µM paclobutrazol (Riedel-de Haen), seed were incubated in test or control solutions from the start of imbibition and throughout the germination time course. Drug concentrations were chosen to saturate the responses as determined in previous studies [START_REF] Müller | Endosperm-limited Brassicaceae seed germination: Abscisic acid inhibits embryo-induced endosperm weakening of Lepidium sativum (cress) and endosperm rupture of cress and Arabidopsis thaliana[END_REF][START_REF] Debeaujon | Influence of the testa on seed dormancy, germination, and longevity in Arabidopsis[END_REF].

Live cell imaging

Observation of dry embryos was performed after applying gentle pressure on seeds held between two glass microscope slides in order to break the testa and endosperm tissues and gain visible access to the embryonic tissues. During stratification and germination time courses embryos were carefully removed from the testa and endosperm by the application of gentle pressure to seeds in water between microscope slide and coverslip.

Résumé

La dynamique mitochondriale est impliquée dans la maintenance et la fonction des mitochondries. Dans les graines sèches tout les processus cellulaires sont arrêtés du fait de la faible teneur en eau des tissues, et la transition développementale que représente la germination requiert la réactivation de la dynamique cellulaire. Une approche de bio-imagerie sur la plante modèle Arabidopsis a été utilisée afin d'étudier la réactivation des mitochondries nécessaire à la germination. La réactivation bioénergétique des mitochondries, mesurée par la présence du potentiel membranaire, intervient dès le début de l'hydratation des tissus. Cependant les mitochondries restent statiques et la dynamique mitochondriale ne reprend que plus tardivement. La réactivation des mitochondries provoque une réorganisation du chondriome impliquant la biogenèse de membranes et une fusion massive menant à la formation de structures réticulaires et périnucléaires, qui permet le mélange des nucléoides d'ADNmt précédemment isolés en unités discrètes. La mitophagie, un indicateur de la qualité mitochondriale, est réactivée de manière concomitante à la dynamique, alors qu'elle est réprimée durant la biogenèse des mitochondries. La fin de la germination coïncide avec la fragmentation du chondriome tubulaire, menant au doublement du nombre de mitochondrie et à une redistribution hétérogène des nucléoides dans le chondriome, générant une population de mitochondrie adaptée à la croissance des plantules. Cette thèse met en évidence l'imbrication des processus de dynamique mitochondriale, de biogenèse et de contrôle qualité des mitochondries requis pour la germination et pour la transition vers l'autotrophie.

Mots clés

Arabidopsis thaliana, dynamique mitochondriale, mitophagie, germination des graines, biogenèse mitochondriale, ADNmt, mito-GFP, phagophore.

Abstract

Mitochondrial dynamics underpin their function and maintenance. In dry seeds, all cellular processes are in stasis due to a low water content. Thus, the developmental switch leading to germination necessarily involves a reactivation of cellular dynamics. In order to better understand the role played by mitochondrial dynamics during germination we used Arabidopsis as a model for a bioimaging approach to investigate the rapid reactivation of mitochondria that is required in order to provide ATP to support germination. Bioenergetic reactivation, visualised as the presence of a mitochondrial membrane potential, is almost immediate upon rehydration. However, the reactivation of mitochondrial dynamics only occurs after several hours of rehydration. The reactivation of mitochondrial bioenergetics and dynamics lead to a dramatic reorganisation of the chondriome involving massive fusion and membrane biogenesis to form a perinuclear tubuloreticular structure enabling mixing of previously discrete mtDNA nucleoids. Mitophagy, an indicator of mitochondrial quality, is reactivated concomitant with a reactivation of mitochondrial dynamics, but is repressed at time of mitochondrial biogenesis. The end of germination coincides with fragmentation of the tubular chondriome leading to a doubling of mitochondrial number and heterogeneous redistribution of the nucleoids amongst the mitochondria, generating a population of mitochondria tailored to seedling growth. This thesis provides strong evidence for the tight interweaving of mitochondrial dynamics, mitochondrial biogenesis and mitochondrial quality control that is required to ensure effective germination and the transition to autotrophy.

Key Words

Arabidopsis thaliana, mitochondrial dynamics, mitophagy, seed germination, mitochondrial biogenesis, mtDNA, mito-GFP, phagophore.

Paszkiewicz Gael |

 Gael Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . .............................

Figure 1 . 1

 11 Figure 1.1 Mitochondrial displacement in plants .......................................................

Figure 1 . 2

 12 Figure 1.2 Mitochondrial morphology in Arabidopsis plants homozygous for mutations in genes involved in mitochondrial fission. ..................................................................

Figure 1 . 3

 13 Figure 1.3 Intracellular location of members of the mitochondrial division apparatus. .

Figure 1 . 4

 14 Figure 1.4 Interactions between putative components of the plant mitochondrial fission apparatus. ..........................................................................................................

Figure 1 . 5

 15 Figure 1.5 Proteins involved in plant mitochondrial fission. ......................................

Figure 1 . 6

 16 Figure 1.6 Fission-fusion balance and mitochondrial size, number and morphology. ...

Figure 1 . 7

 17 Figure 1.7 Putative mechanisms for plant mitochondrial fusion. ...............................

Figure 1 . 8

 18 Figure 1.8 Mitochondrial quality control and mitophagy...........................................

Figure 1 . 9 Figure 2 . 1

 1921 Figure 1.9 Germination of Arabidopsis seeds. ........................................................

Figure 2 . 2

 22 Figure 2.2 Mitochondrial motility increases in response to germination cues and leads to increased inter-mitochondrial interaction and fusion. ...............................................

Figure 2 . 3

 23 Figure 2.3 Measurement of dynamic reactivation of F-actin, the endoplasmic reticulum, or peroxisomes. ..................................................................................................

Figure 2 . 4

 24 Figure 2.4 Effects of ABA, GA 3 and paclobutrazol on the reactivation of mitochondrial dynamics. ...........................................................................................................

Figure 2 . 5

 25 Figure 2.5 Transfer of seed to germination promoting conditions induces the formation of ATG8F decorated autophagosomes.....................................................................

Figure 2 . 6

 26 Figure 2.6 Morphological changes affecting the embryonic cotyledon chondriome during stratification and germination................................................................................

Figure 2 . 7

 27 Figure 2.7 Mitochondria localise around the nucleus at the end of germination. .........

Figure 2 . 8

 28 Figure 2.8 Distribution of mtDNA nucleoids within the chondriome of cotyledon cells during stratification and germination. .....................................................................

Figure 2 . 9

 29 Figure 2.9 Embryo mtDNA quantity, quality, recombination and sequences stoichiometry are tightly controlled during germination. ...............................................................

Figure 2 .

 2 Figure 2.10 Schematic representation of mitochondrial dynamics during germination.

Figure 2 .

 2 Figure 2.11 Mitochondrial dynamics in cells of green cotyledon 4-days post-germination. .........................................................................................................................

Figure 2 .

 2 Figure 2.12 Reactivation of dynamics of cotyledon mitochondria is delayed without stratification but displays a similar profile ...............................................................

Figure 2 .

 2 Figure 2.13 Description of tissue collection used for GEO datasets ...........................

Figure 2 .

 2 Figure 2.14 Transcript abundance of genes of the ARP2/3 complex ..........................

Figure 2 .

 2 Figure 2.15 Transcript abundance of genes involved in mitochondrial and organelle displacement ......................................................................................................

Figure 2 .

 2 Figure 2.16 Transcript abundance of genes involved in mitochondrial fission ............

Figure 2 .

 2 Figure 2.17 Transcript abundance of dynamin family genes .....................................

Figure 2 .

 2 Figure 2.18 Transcript abundance of genes involved in synthesis of mitochondrial membrane lipid ...................................................................................................

Figure 2 .

 2 Figure 2.19 Transcript abundance of genes involved in mitochondrial protein import .

Figure 2 .Figure 3 . 1

 231 Figure 2.20 Transcript abundance of genes involved in mitochondrial and organellar DNA maintenance ................................................................................................

Figure 3 . 2

 32 Figure 3.2 Quantification of ATG8F-bodies associated with mitochondria during germination. .......................................................................................................

Figure 3 . 3

 33 Figure 3.3 Immobilisation of mitochondria and interaction with ATG8F-bodies to promote mitophagy. ............................................................................................

Figure 3 . 4

 34 Figure 3.4 Engulfement of previously immobilized mitochondria by ATG8F decorated autophagosomes. ................................................................................................

Figure 3 . 5

 35 Figure 3.5 Abundance of transcripts of ATG family genes during germination ............

Figure 3 . 6

 36 Figure 3.6 Profile of FRIENDLY trancript abundance during plant development ...........

Figure 3 . 7

 37 Figure 3.7 Mitochondrial clusters increase in size in friendly during germination and reduce in size at seedling establishment. ...............................................................

Figure 3 . 8

 38 Figure 3.8 Association of ATG8F-bodies with mitochondria are impaired during germination of friendly ........................................................................................

  Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ............................. 5 Chapter І -Introduction Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ............................. 6

Figure 1 . 1

 11 Figure 1.1 Mitochondrial displacement in plants A. Confocal images of tobacco cells expressing mito-GFP following 2 to 4 h treatments with drugs perturbing the actin (Latrunculin B), or microtubule (Oryzalin) cytoskeletal elements, or both actin filaments and microtubules together (Latrunculin + oryzalin). Bars = 25 μm. Images from Van Gestel 2002. B. Wide-field image of root cells of an Arabidopsis line expressing both mito-GFP and an mCherry protein fusion to visualise F-actin. Bars = 5 μm. C. Proteins known to be involved in the movement of mitochondria in plants. Red explanation marks highlight a lack of knowledge about the underlying mechanisms.

Figure 1 . 2

 12 Figure 1.2 Mitochondrial morphology in Arabidopsis plants homozygous for mutations in genes involved in mitochondrial fission. Green coloured images are from plants expressing the mito-YFP, from Aung 2011 and 2012. Grey-scale images is of mitochondria stained with MitoTracker Orange CMTMRos, from Arimura 2008. Magenta coloured images show mitochondria stained with MitoTracker Orange CMTMRos, from Zhang 2009. Bars = 10 μm.

Figure 1 . 3

 13 Figure 1.3 Intracellular location of members of the mitochondrial division apparatus. The perimitochondrial localisation of DRP3A, DRP3B, NMT, FIS1A, FIS1B, PMD1 and PMD2 have been confirmed in vivo by confocal microscopy of plants expressing translational fusions to fluorescent protein markers and either MitoTracker or mito-FP. Images from[START_REF] Aung | The Arabidopsis tail-anchored protein PEROXISOMAL AND MITOCHONDRIAL DIVISION FACTOR1 is involved in the morphogenesis and proliferation of peroxisomes and mitochondria[END_REF] and 2012[START_REF] Yamashita | Formation of mitochondrial outer membrane derived protrusions and vesicles in arabidopsis thaliana[END_REF][START_REF] Zhang | Two small protein families, DYNAMIN-RELATED PROTEIN3 and FISSION1, are required for peroxisome fission in Arabidopsis[END_REF]

Figure 1 . 5

 15 Figure 1.5 Proteins involved in plant mitochondrial fission. Schematic representation of the known actors in fission, and their locations. Dynamins are depicted by circles, OMM-residents are represented by squares, and proteins that also show a perimitochondrial localization are presented by triangles. Red indicates putative unknown proteins, while red explanation marks highlight a lack of knowledge about the underlying mechanisms (red arrow indicates initiation of constriction ring assembly).

AFromFigure 1 . 8

 18 Figure 1.8 Mitochondrial quality control and mitophagy. A. Models of MQC pathways under control of PINK1 and Parkin. (a) the behaviour of healthy mitochondria and (b) the behaviour of unpolarized mitochondria. B. Representation of non-selective autophagy (left) and selective mitophagy (right).

  Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 29

  Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 30

  The FRIENDLYdependant regulation of association time is affected by acetylation of two lysine residues located in the C-terminal of the FRIENDLY protein, indicating once again the importance of post-translation regulation in the control of mitochondrial dynamics. The mitochondrial cluster phenotype is accompanied by a 10-fold higher frequency of mitochondrial membrane potential pulsing, an increased flux through the alternative respiratory pathway, and a global reprogramming of the transcriptome, indicating broad-spectrum cellular stress resulting from a disrupted chondriome structure[START_REF] El Zawily | FRIENDLY regulates mitochondrial distribution, fusion, and quality control in Arabidopsis[END_REF]. A stress response is also indicated by the increased number and size of acidic vacuoles, as stained by Lysotracker Red, in friendly relative to wild type, highlighting upregulation of macro-autophagy and thus potentially a deregulation of MQC and plant metabolism. To date, friendly is the only plant mutant identified to show defects in mitochondrial dynamics and MQC. At the tissue level, friendly plants have significantly more dead cells, as well as a reduced root cell size leading to a short root phenotype, indicating that FRIENDLY function is required for correct plant development[START_REF] El Zawily | FRIENDLY regulates mitochondrial distribution, fusion, and quality control in Arabidopsis[END_REF].

with

  Parkin as measured by co-IP, treatment with drugs damaging mitochondria (CCCP Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 31

  Germination occurs in three phases and is complete upon protrusion of the root through the endosperm (Figure 1.9). Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 32

FromFigure 1 . 9

 19 Figure 1.9 Germination of Arabidopsis seeds. A. Time course of physical and metabolic events occurring during germination (Phases 1 and 2) and early seedling growth (Phase 3). From Nonogaki et al., 2010. B. Anatomy of mature Arabidopsis seed and representative images of the testa rupture (TR), and endosperm rupture (ER) stages of germination. From Mueller et al., 2006.

  Two clear subpopulations were identified: heavy, banding at 37-42 % (w/v) sucrose and light, banding at 20-28 % (w/v) sucrose and the relative composition of each subpopulation changed as germination progressed. Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 36

  Gael | Mitochondrial biogenesis during seed germination of Arabidopsis Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 40

1. 3 . 1

 31 Figure 2.1 Mitochondria are clearly detectable in the dry embryo and are bioenergetically reactivated within minutes of hydration. A. Images of embryo mitochondria from seed expressing mito-GFP under control of 35S promoter. Seed were either dry or imbibed at 4°C in the dark. Embryos were mounted in PFD to avoid tissue hydration and observed within 5 min. Bar = 5 µm. B. Colocalisation of TMRM, a reporter of membrane potential, and mito-GFP in cotyledon mitochondria. The majority of mitochondria in the merged image display white pixels indicating colocalisation of TMRM (magenta) and GFP (green). Quantification of colocalisation is provided by the Mander's coefficient below images ( S.E., n=9). Bars = 5 µm. C. Measurement of respiration in mito-GFP seed after the first hours of imbibition, at the end of stratification, and upon transfer to germination promoting conditions. Cyanide (500 µM KCN) was used to estimate alternative pathway capacity.

  1) we were interested to determine when during germination mitochondria regain motility. Mitochondria were immobile in the dry embryonic cotyledon, however, challenging imaging conditions (slight tissue movement and general image clarity) prevented accurate quantification. After 12 h stratification (Figure 2.2) mitochondria exhibited disordered localised oscillatory movements, generating low displacement values and track straightness (Figure 2.2D and E), with three-quarters of mitochondria displaying speeds <6.1 nm/s (Figure 2.2C). This pattern of mitochondria motility was maintained until the seeds were transferred, after 48 h imbibition, to the illuminated growth chamber at 21°C (Figure 2.2C, D and E).

  R H Paszkiewicz Gael Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 43 A. Germination protocol and images of seed at the developmental stages used for experiments. Developmental stages: TR = testa rupture, ER = endosperm rupture, LR = long root, RH = root hair. L = after transfer to light; S = stratification. Bar = 200 µm. B. Images generated using Imaris to track mitochondria in cotyledon cells over a period of 5 min. Detected mitochondria were coloured green and tracks are rainbow coloured according to speed (no movement is purple and movement above 0.032 µm/s is red). Each image is re presentative of data captured from six seeds (total number of tracks analysed per time point ranges from 623 to 2180). Bar = 5 µm. C. Violin box plots of the mean speed recorded per track in nm/s. Box plot whiskers indicate 1.5 x IQR and any outliers are represented by an open circle, as described by Tukey. Means are represented by a solid circle. The notch corresponds to the median  1.58 x IQR/ n. D, E, F, G. Violin box plots of (D) track displacement length, (E) track straightness (1= perfectly straight), (F) numbers of interactors per track for tracks having at least one in teraction between detected mitochondrial objects, (G) rate of fusion between mitochondrial objects expressed per mitochondria. Plots design as in C. H . Dot plot of the percentage of mitochondria sharing a track within the 5 min tracking period. Open circles indicate percentage mitochondrial objects sharing a track in each image stack, while the solid circle indicates the mean (n=6). The line represents the Loess regression, while the grey area corresponds to the 95% confidence interval.

Figure 2 . 2

 22 Figure 2.2 Mitochondrial motility increases in response to germination cues and l eads to increased inter-mitochondrial interaction and fusion.

D

  Figure 2.3 Measurement of dynamic reactivation of F-actin, the endoplasmic reticulum, or peroxisomes. A. Visualisation of F-actin in cotyledon cells of a double transgenic line expressing mCherry-mTalin and mito-GFP during stratification, and near the end of germination. Bar = 5 µm. B. Time-lapse series of images showing changes in position of mitochondria (green) as a r esult of limited reorganisation of F-actin (magenta) that occurs during stratification. Bar = 2 µm. C. Images generated from the merging of two single frame optical slices, captured 5 min apart, of embryonic cotyledon cells expressing the ER marker YFP-HDEL after 24h of imbibition in dark at 4°C, or at the TR stage. The first image (t = 0) was false coloured green, the second (t = 5 min), magenta: white pixels indicate colocalisation and therefore relative immobility. Pearson's colocalisation coefficient is the average from the analysis of 9 seeds. Bar = 5 µm. D. Embryonic cotyledon cells expressing the peroxisomal marker YFP-SKL, images were captured as in C. Dynamics was measured by object tracking: speed are in nm/s, displacement in µm. Measurement was performed on at least 7 plants at each time point. Bar = 2 µm.

Figure 2 . 4

 24 Figure 2.4 Effects of ABA, GA 3 and paclobutrazol on the reactivation of mitochondrial dynamics.

  but seed were transferred to dark/21ºC after 48 h stratification. Bar = 5 µm. Violin box plots of the mean speed recorded per track in nm/s. Box plot whiskers indicate 1.5 x IQR and any outliers are represented by an empty circle, as described by Tukey. Means are represented by a full circle. The notch corresponds to the median  1.58 x IQR/ n. The coloured lines indicate the median of the mean speed of mitochondria following 48h of stratification (blue), or 2 h after transfer to 21°C/dark (violet), or 21ºC/light (red). Total number of objects tracked per time point ranges from 636 to 1386 from between 6 and 9 seeds. Violin box plots of track displacement length. The blue, violet and red lines indicate the median of the displacement length after 48h of stratification (blue), or 2 h after transfer to 21ºC/dark (violet), or 21ºC/ light (red). Plot design as in C. Violin box plots of track straightness (1= perfectly straight). Plot design as in C. Violin box plots of numbers of interactors per track for tracks having at least one interaction between detected mitochondrial objects. Plot design as in C. Violin box plots of the rate of fusion between mitochondrial objects expressed per mitochondria. Plot design as in C. Dot plot of the percentage of mitochondria sharing a track within the 5 min tracking period. Empty circles indicate percentage mitochondrial objects sharing a track in each image stack, while the full circle indicates the mean. Paszkiewicz Gael Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 48 Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 49 1.3.5 Transfer of seed to germination-promoting conditions induces formation of ATG8-labeled autophagosomes

  approximately 0.5 to 1 µm in diameter and absence of characteristic PSV fluorescence at 425-475 nm upon excitation at 405 nm (Figure 2.5). Due to the interfering autofluorescence we were not able to automatically quantify colocalisation with confidence and therefore manual counts were made of the combined number of instances of close juxtaposition of mitochondria and mRFP1-ATG8F structures (Figure 2.5) and instances where mitochondria appear enclosed by mRFP1-ATG8F (Figure 2.5). Autophagosome number was very low after 48 h stratification, with only 0.08 bodies visible per 10 cotyledon epidermal pavement cells (Figure 2.5C). However, 6 h after transfer to germinating conditions (6 h L) a large increase in autophagosome number per cell was detected (Figure 2.5C). Concomitant with the increased autophagosome number we observed that the majority of these bodies were closely associated with, or colocalised with mitochondria (Figure 2.5B and C), suggesting their specific participation in mitophagy. 1.3.6 Activation of mitochondrial motility during germination leads to the formation of a tubuloreticular chondriome through a shift in the fusion/fission balance Mitochondrial motility is required in order to affect changes to mitochondrial number per cell and individual mitochondrial volume via the motility dependent processes of mitochondrial fusion and division. Thus, we next quantified these parameters to better understand mitochondrial dynamics during germination (Figure 2.6). Mitochondrial number decreased slightly during stratification concomitant with a reduction in total mitochondrial volume (Figure 2.6A, B and C). The spherical morphology of mitochondria in dry seeds (Figure 2.1) was maintained during stratification, with more than 75% of the population having an object sphericity (OS) >0.8 (Figure 2.6A and D). Despite the relatively homogeneous morphology of mitochondria during stratification, the volume of individually resolvable mitochondria (mito-GFP objects) varied 1000 fold from 0.004 to 3.470 µm 3 (Figure 2.6E).

Figure 2 . 5

 25 Figure 2.5 Transfer of seed to germination promoting conditions induces the formation of ATG8F decorated autophagosomes. Seed expressing both mito-GFP (green) and mRFP1-ATG8F (red) were observed by CLSM. PSVs autofluorescence was coloured blue. Bar = 5 µm. Enlarged micrographs to show putative mitophagy during early germination. Arrows indicate ATG8F bodies, either associated with or surrounding mitochondria (white) or nonassociated (yellow) . Bars = 2 µm. Quantification of the numbers of ATG8F decorated bodies per 10 cells and the percentage of ATG8F bodies close or containing mito-GFP signal. Data from 19 different embryos per time point from th ree independent experiments. Box plot whiskers indicate 1.5 x IQR and outliers are represe nted by an empty circle, as described by Tukey. Means are represented by a full circle. The notch corresponds to the median  1.58 x IQR/ n.

Figure 2 .

 2 Figure 2.6A and D) thereby forming a tubuloreticular structure, with only approximately 25% of the population displaying sphericity values in the range previously observed for 75% of mitochondria during stratification (when 75% of mitochondria had a OS of >0.8 (Figure 2.6D)). The change in mitochondrial shape was accompanied by a shift in the volumes of individual organelles to larger values (over 25% of organelles had a volume between 0.54 and 38.7 µm 3 ) and an increase in total chondriome volume (Figure 2.6C

Figure 2 . 6

 26 Figure 2.6 Morphological changes affecting the embryonic cotyledon chondriome during stratification and germination. Reconstructed images of CSLM z-stacks of cotyledon mitochondria tagged by mito-GFP. Bars = 5 µm. Each image is representative of images captured from at least five seeds at each time point. The bottom image at each time point shows the same z-stack but tilted at -45º relative to the top image. Dot plot of the numbers of mitochondrial objects per stack. Open circles indicate total number for each stack while the solid circle indicates the mean. Total number of objects per stack ranges from 1168 to 3954 from at least five seeds per time point. The line represents the Loess regression, while the grey area corresponds to the 95% confidence interval. Dot plot of the total volume of detected objects per stack, in µm 3 . Open circles indicate the mean chondriome volume per stack, while the solid circle indicates the mean for all stacks. The line represents the Loess regression, while the grey area corresponds to the 95% confidence interval. D, E. Violin box plots of (D) object sphericity (OS, a value of 1 means a perfect sphere), (E) volume per object (in µm 3 ). Box plot whiskers indicate 1.5 x IQR and outliers are represented by an open circle, as described by Tukey. Means are represented by a solid circle. The notch corresponds to the median  1.58 x IQR/ n. Paszkiewicz Gael Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 52

Figure 2 .

 2 Figure 2.8A panel TR).

  Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 54

Figure 2 . 7

 27 Figure 2.7 Mitochondria localise around the nucleus at the end of germination. A. Visualisation of mitochondria (mito-GFP, green) and PSVs (autofluorescence, blue) in cotyledon cells observed by CLSM. Arrows indicate PSV-free area. Bar = 5 µm. B. Visualisation of mtDNA nucleoids and nuclear DNA in Col-0 seed stained with the DNA binding fluorescent dye SYBR Green I. Seed were observed following 1h of imbibition or during TR stage. The PSV-free area within cells correspond to the position of the SYBR Green I stained nucleus. Arrows indicate PSV-free area/nucleus. Bar = 5 µm.

Figure 2 . 8

 28 Figure 2.8 Distribution of mtDNA nucleoids within the chondriome of cotyledon cells during stratification and germination. Images of mtDNA nucleoids and mitochondria in embryonic cotyledon cells of seed expressing mito-mCherry stained with SYBR Green I. Arrows indicate mitochondria with no apparent SYBR Green I fluorescence. Bars= 5 µm. Graph of percentage of mitochondria having a detectable mtDNA nucleoid and of the average nucleoid number per mitochondria (mitochondrial numbers were >1129 and nucleoid numbers >1073, depending on time point, n= 7 seeds). Violin box plot of the mean fluorescence intensity measured per mtDNA nucleoid in relative units. Box plot whiskers indicate 1.5 x IQR and any outliers are represented by an open circle, as described by Tukey. Means are represented by a solid circle. The notch corresponds to the median  1.58 x IQR/ n. Violin and box plot of the variation of the fluorescence within each mtDNA nucleoid in relative units. Plot design as in C.

Figure 2 . 9

 29 Figure 2.9 Embryo mtDNA quantity, quality, recombination and sequences stoichiometry are tightly controlled during germination. A. Quantification of the variation in total mtDNA and cpDNA relative copy numbers during stratification, germination and in 10 day-old seedlings, relative to the dry seed. Values are normalised to nucDNA and presented as means S.D. of biological duplicates each with three technical replicates. B. Quality of mtDNA during germination as detected by qPCR assay. Values represent long PCR fragment abundance relative to that in dry seeds and normalised by the short fragment abundance, and relative to amplification of nucDNA targets. Replication as in A. C. Quantification by qPCR of the accumulation of recombination products across two repeats within the mitochondrial genome. Values indicate the quantity of parental sequence (L-1, L-2, EE-1, EE-2) or crossover product (L-1/2, L-2/1, EE-1/2, EE-2/1) relative to in dry seeds, for pairs of repeats L and EE. Replication as in A. D. Mitochondrial genome-wide scan for changes in sequences stoichiometry. Sequences spaced 5 to 10 kbp apart were quantified by qPCR. Values are relative to the dry seed time point for each PCR product. Replication as in A.

  Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 60

  Figure 2.10 Schematic representation of mitochondrial dynamics during germination.In dry seed, and during the first hours of imbibition (the first phase of germination), the mitochondria are static and have low metabolic activity despite an immediate re-establishment of a membrane potential. A high percentage of mitochondria contain at least one mtDNA nucleoid. It is proposed that these attributes define these mitochondria as promitochondria, and provide a structure and organisation that act to minimise damage and thus help ensure mitochondrial quality throughout seed dormancy or quiescence. During the early second phase of germination, initiated upon the transfer of seed to germination promoting conditions, the constraint imposed on mitochondrial dynamics by cold temperature is released leading to a burst of motility, inter-mitochondrial interactions and upregulation of mitophagy. During the middle of the second phase of germination, mitochondria become more dynamic, displaying higher rates of inter-mitochondrial interaction together with a shift to an ovoid morphology. Membrane biogenesis is initiated. At the late second phase of germination, mitochondria are preferentially located around the nucleus and further reticulation and fusion leads to the formation of a perinuclear tubuloreticular mitochondrial structure. This organisation may provide a means to ensure mixing of the chondriome and exchange of molecules, together with facilitating synchronisation of mitochondrial biogenesis through efficient delivery and import of tRNA and polypeptides encoded by nuclear transcripts and assisting the necessary associated cross-talk between the mitochondrial and nuclear genomes. Following the end of germination, the perinuclear tubuloreticular structure fragments, leading to the reformation of a population of physically discrete mitochondria. The distribution of mtDNA nucleoids among the mitochondria is uneven, indicating division of labour amongst the physically discrete members of the chondriome. The reorganisation of the chondriome, generating an organisation typical of more adult tissues, coincides with a burst in mitochondrial metabolic activity as storage reserves are used to fuel postgerminative growth. Blue spots represent mtDNA nucleoids; the nucleus is colored yellow; the matrix is shaded from light to dark grey to indicate increasing bioenergetic activity; increased membrane thickness represents membrane biogenesis; mitochondrial motility is represented by tracks with a scale from blue (slow) to red (fast); nucleation of ATG8 and phagophore formation is indicated in green.

  Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 61 Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 62

  .10). Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 64

Figure 2 .

 2 Figure 2.2F and Figure 2.11), nor in the percentage of mitochondria that interact at least once (cf.Figure 2.2H and Figure 2.11G), however, the fusion rate in 4 days-old seedlings is approximately 10-times greater than during or immediately after germination (cf.

Figure 2 .Figure

 2 Figure 2.2G and Figure 2.11F, note scales are not identical); although part of this measured increase in the relative rate of fusion may result from the necessary image capture resolution.

  Figure 2.2). During early seedling growth mitochondrial dynamics changes dramatically with mitochondria moving at much greater speeds, following straighter tracks over greater distances resulting in a reduction in the fusion rate per mitochondrion. Reactivation of dynamics also occurred in the dark, although mitochondria were slower to reactivate relative to incubation in the light, showing slower speeds, along less-straight tracks, resulting in lower rates of fusion per mito (Figure 2.12B L & N), and lower numbers of interacting partners early during imbibition (Figure 2.12M), such that 24 h incubation in the dark was required for the dynamics parameters to reach levels similar to those measured after only 6 h in the light. Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis

  2

Figure 2 .

 2 Figure2.12 Reactivation of dynamics of cotyledon mitochondria is delayed without stratification but displays a similar profile A, B. Tracking images of time lapse of cotyledon mitochondria tagged by mito-GFP. Detected mitochondria were coloured green and movement was followed over 5 min in order to reconstruct tracks. Seeds were imbibed under light (A) or dark (B) conditions, without stratification. Tracks are rainbow coloured according to speed (no movement is purple and movement above 0.032 µm/s is red). Each image is representative of data captured from at least five seeds. Bar = 5 µm. Bars = 5 µm. C to N. Quantification of the dynamic parameters of tracked mitochondria from seed germinated under light (C to G and M) or dark (H to L and N) conditions, without stratification. The total numbers of tracks analysed per time point range from 659 to 1775. Replication as in A and B. C, H. Violin and box plot of the mean speed recorded per track in nm/s. Box plot whiskers indicate 1.5xIQR and outliers are represented with empty open circle, as described by Tukey. Means are represented by a full circle. The notch corresponds to the mean 1.58 xIQR/ n. The blue and red lines indicate the median of the mean speed of mitochondria after 48h of stratification or 2 h after transfer to light/21°C, respectively. D, I. Violin box plots of track displacement length. Plot design as in C. E, J. Violin box plots of track straightness (1= perfectly straight). Plot design as in C. F, K. Violin box plots of numbers of interactors per track for tracks having at least one interaction between detected mitochondrial objects. Plot design as in C. G, L. Violin box plots of the rate of fusion between mitochondrial objects expressed per mitochondria. Plot design as in C. M, N. Dot plot of the percentage of mitochondria sharing a track within the 5 min tracking period. Open circles indicate percentage mitochondrial objects sharing a track in each image stack, while the solid circle indicates the mean. The line represents the Loess regression, while the grey area corresponds to the 95% confidence interval.

  (Figure 2.13). The Dekkers dataset covers germination and are the most detailed, being subdivided into separate transcript profiles of "root + hypocotyls" and "cotyledon". In addition, Dekkers et al used a variety of physiological parameters to select tissues, such as water content, and calculated T 50 values for TR and ER stages for the seed population in order that sampling was truly representative of the seed germination phases (i.e. Phase 1 from 0-3 h; Phase 2 from 7-25 h; Phase 3 from 25-38 h). Furthermore, for tissues collected at the T 50 for TR or ER stages, the physiological descriptor of ruptured layers (R) or not ruptured layers (NR) was used. The Silva dataset has less coverage of germination per se and instead is oriented to seedling establishment, using mainly physical descriptors to select tissues from TR until the open cotyledon step. The Narsai dataset covers germination and differs from the other datasets by inclusion of a 48 h stratification step and data from mature dry seed (i.e. Phase 2 from 12 h S to "around" 24 h L, and 48 h L correspond to seedling establishment). Because early germination steps are represented in each dataset, combining all datasets allows coverage of the whole period of germination and early seedling establishment (Figure2.13).

Figure 2 .

 2 Figure 2.13 Description of tissue collection used for GEO datasets A. Sampling protocol for dataset from Dekkers et al., 2013 (GSE41212). B. Sampling protocol for dataset from Silva et al., 2016 (GSE65394). C. Sampling protocol for dataset from Narsai et al., 2011 (GSE30223). D. Colour scale used for change in transcript abundance relative to dry seed.

FFigure 2 .

 2 Figure 2.14 Transcript abundance of genes of the ARP2/3 complex A. Data from Dekkers et al., 2013. B. Data from Silvia et al., 2015. C. Data from Narsai et al., 2011. Change in transcript abundance during germination, relative to abundance measured in dry seeds are represented by a colour scale. Numbers indicate the absolute abundance in dry seed and maximal or minimal variation during germination. Black dot indicates maximal abundance.

PMD2

  could be part of a mechanism leading to a specific burst of mitochondrial fusion towards the end of germination. On the basis of transcript profiles alone, PMDs seem particularly involved in the control of mitochondrial dynamics during germination. Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 74 Figure 2.15 Transcript abundance of genes involved in mitochondrial and organelle displacement A. Data from Dekkers et al., 2013. B. Data from Silvia et al., 2015. C. Data from Narsai et al., 2011. Plots designs as in 2.14. Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 75

Figure 2 .

 2 Figure 2.16 Transcript abundance of genes involved in mitochondrial fission A. Data from Dekkers et al., 2013. B. Data from Silvia et al., 2015.C. Data from[START_REF] Narsai | In-depth temporal transcriptome profiling reveals a crucial developmental switch with roles for RNA processing and organelle metabolism that are essential for germination in Arabidopsis[END_REF] Plots designs as in 2.14.

  Figure 2.17 Transcript abundance of dynamin family genes A. Data from Dekkers et al., 2013. B. Data from Silvia et al., 2015. C. Data from Narsai et al., 2011. Plots designs as in 2.14. (no probe for DRP4D).

Figure 2 .

 2 Figure 2.18 Transcript abundance of genes involved in synthesis of mitochondrial membrane lipid A. Data from Dekkers et al., 2013. B. Data from Silvia et al., 2015.C. Data from[START_REF] Narsai | In-depth temporal transcriptome profiling reveals a crucial developmental switch with roles for RNA processing and organelle metabolism that are essential for germination in Arabidopsis[END_REF] Plots designs as in 2.14.

Figure 2 .

 2 Figure 2.19 Transcript abundance of genes involved in mitochondrial protein import A. Data from Dekkers et al., 2013. B. Data from Silvia et al., 2015. C. Data from Narsai et al., 2011. Plots designs as in 2.14.

FollowingFigure 3 . 1

 31 Figure 3.1 Reduction in number of ATG8F-bodies correlated with the timing of mitochondrial biogenesis during seed germination. Seed expressing both mito-GFP (green) and mRFP1-ATG8F (red) were observed by CLSM during germination and seedling establishment. PSVs autofluorescence is coloured blue. Arrows indicate ATG8F bodies, either juxtaposed (yellow) or surrounding mitochondria (white). Bars = 5 µm. Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 88

  ATG8F-bodies, to an average of 0.55 per cell, reducing to 0.16 per cell at 22 h L, after which the numbers remained low, up to and including ER (mean of 0.15 per cell). This reduction in number was concomitant with a decreased percentage of structures associated with mitochondria: a reduction of both ATG8F-bodies/mitochondrial juxtaposition and parallel mitophagy events. Despite the almost complete absence of detectable ATG8F-bodies at the ER stage, mRFP1 fluorescence was detected in the cytosol with preferentially accumulation around the nucleus (Figure3.1).At the LR stage, the number of detectable ATG8F-bodies had increased to a mean of 0.49 per cell, and continued to increase up to a mean of 2.01 bodies per cell in green cotyledon of four days-old seedlings. Despite this increase in the number of detectable structures, the percentage of bodies associated with mitochondria at these two postgermination time points are similar (46% at LR and 43% at 4 days), despite an increased percentage of mitochondria being engulfed by ATG8F-bodies at the LR stage (15% at LR and 10% at 4 days), suggesting a constant basal level of mitochondrial and ATG8Fbodies interaction. Post-germination, a stronger mRFP1 fluorescence was detected in distinct strand-like structures in the cytoplasm and in structures appearing to encircle mitochondria

  Figure 3.3 Immobilisation of mitochondria and interaction with ATG8F-bodies to promote mitophagy. CLSM images putatively showing the early steps of mitophagy during early germination of seed expressing both mito-GFP (green) and mRFP1-ATG8F (red). PSV autofluorescence is coloured blue. Arrows indicate phagophore undergoing tethering to mitochondria. Bar = 2 µm. Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis

  Figure 3.4 Engulfement of previously immobilized mitochondria by ATG8F decorated autophagosomes. Enlarged CLSM micrographs to show putative early mitophagy process during early germination of seed expressing both mito-GFP (green) and mRFP1-ATG8F (red). PSVs aut ofluorescence was coloured blue. Arrows indicate mitochondria undergoing tethering to phagophores and subsequent division. Bar = 2 µm. Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis

  Figure 3.5 Abundance of transcripts of ATG family genes during germination A, B, C Variation of transcript abundance during germination observed in three different data-set covering germination time course. (A) Data coming from Dekkers et al., 2013 (GSE41212), (B) Data coming from Silvia et al., 2015 (GSE65394), (C) Data coming from Narsai et al., 2011 (GSE30223) E. Colour scales used to show variation of transcript abundance during germination, relatively to abundance measured in dry seeds. Numbers indicate the absolute abundance in dry seed, and maximal or minimal variation during germination. Black dot indicates maximal abundance.

Figure 3 . 6

 36 Figure 3.6 Profile of FRIENDLY trancript abundance during plant development A. Abundance of FRIENDLY transcript depicted by the developmental map option of the BAR expression brower B. Variation of transcript abundance during germination observed in three different dataset covering germination time course. Dataset are equivalent as in 2.13. Abundance relative to abundance in dry seed is indicated by a colour scale (blue to red for increasing abundance) and the maximal abundance is marked by black circle. The absolute abundance in dry seed are, moreover to the maximal variation and lower variation in abundance relative to dry seeds observed during germination are indicates below each dataset.

FRIENDLY

  function has been shown to involve posttranslational modification, whereby acetylation of lysine residues towards the C-terminus of FRIENDLY modulates the clustering behaviour (El Zawily et al., 2014). However, variation of FRIENDLY transcript abundance correlated with the microscopy-based observations presented in this study and is consistent with the proposed role for FRIENDLY as a regulator of intermitochondrial association time. Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ......................... 100

Figure 3 . 7

 37 Figure 3.7 Mitochondrial clusters increase in size in friendly during germination and reduce in size at seedling establishment. Seed of friendly expressing mito-GFP (green) were observed by CLSM during germination and seedling establishment. PSVs autofluorescence was coloured blue to helps visualisation of cells. Bars = 5 µm.

Figure 3 . 8

 38 Figure 3.8 Association of ATG8F-bodies with mitochondria are impaired during germination of friendly A. Quantification of the numbers of ATG8F-bodies per cells and the percentage of ATG8F-bodies close to mito-GFP signal or percentage of ATG8F-bodies containing mito-GFP signal in friendly. Data from at least 31 different embryos per time point from three independent experiments. Box plot whiskers indicate 1.5 x IQR and outliers are represented by an empty circle, as described by Tukey. Means are represented by a full circle. The notch corresponds to the median 1.58 x IQR/ n. B. Enlarged sections of CLSM images of friendly seed expressing both mito-GFP (green) and mRFP1-ATG8F (red), as used to count mitophagy events during post-germinative growth. Bars = 5 µm.

  dynamics and mitophagy in the development of promitochondria and the provision of the germinated seedling with mitochondria tailored for autotrophic growth. Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ......................... 106

  Despite the increased frequency of inter-mitochondrial interaction during these late Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ......................... 107

  Dynamics influence mitochondrial function from biogenesis to destruction, and through its effects on mtDNA maintenance, energy metabolism and cell health mitochondrial dynamics has a profound influence on organism physiology. Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ......................... 108

  targeted for selective destruction. Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ......................... 109 Materials and methods Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ......................... 110

  seed germination of Arabidopsis thal iana is dependent on mitochondrial dynamics and mitophagy La biogenèse mitochondriale durant la germination d'Arabidopsis thaliana est dépendante de la dynamique mitochondriale et de la mitophagy L'Université Bretagne Loire

  3

	Abbreviations
	ABA	Abscisic acid
	ATG	AuTophaGy
	ATP	Adenosine triphosphate
	BiFC	bimolecular fluorescence complementation
	CFP	Cyan Fluorescent Protein
	CLSM	Confocal laser scanning microscopy
	Co-IP	Co-immunoprecipation
	DNA	Deoxyribonucleic acid
	DRP	Dynamin related protein
	ER	Endosperm rupture
	g	gram
	GA	Gibberellins
	GEO	Gene expression omnibus
	GFP	Green Fluorescent Protein
	GO	Gene ontology
	GTP	Guanosine triphosphate
	h	Hours
	IMS	Intermembrane space
	IMM	Inner mitochondrial membrane
	m	meter
	mCherry	monomeric Cherry fluorescent protein
	mtDNA	Mitochondrial DNA
	mRFP	monomeric Red Fluorescent Protein
	MQC	Mitochondrial quality control
	NADH	Nicotinamide adenine dinucleotide
	O 2	Molecular oxygen
	OMM	Outer mitochondrial membrane
	OS	Object sphericity
	OXPHOS	Oxidative phosphorylation
	PACLO	Paclobutrazol
	PCR	Polymerase chain reaction
	PDC	Pyruvate dehydrogenase complex
	PFD	Perfluorodecalin
	PSV	Protein storage vacuole
	RFP	Red Fluorescent Protein
	RNA	Ribonucleic acid
	ROS	Reactive oxygen species
	s	Second
	S	Stratification
	TCA	Tricarboxylic acid
	TMRM	Tetramethylrhodamine methyl ester
	TR	Testa rupture
	Y2H	Yeast two hybrid
	YFP	Yellow Fluorescent Protein

Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ............................. 4

20 Figure 1.6 Fission-fusion balance and mitochondrial size, number and morphology

	Unfortunately, there is little conservation in Arabidopsis of genes involved in
	mitochondrial fusion identified from other organisms, indicating a potentially divergent
	machinery in plants
	.
	A. In yeast, deletion of Dnm1 leads to an over-connected chondriome while deletion of a
	gene involved in fusion leads to a fragmented chondriome. Images from Mozdi 2000.
	B. In plants, deletion of a gene involved in fission (PMD1 or DRP3A or B) leads to a
	phenotype similar to that in the corresponding mutant in yeast, while overexpression of
	PMD1 (in WT), or DRP3B in drp3a/b background leads to a mitochondrial morphology
	similar to that caused by impairment of fusion in yeast. Images from Aung 2011 and
	Fujimoto 2009; Bars = 10 µm.
	C. Schematic representation of mitochondrial fusion and fission.

Paszkiewicz Gael Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 21

22 Figure 1.7 Putative mechanisms for plant mitochondrial fusion. A,B. Mechanism of mitochondrial fusion. Mitochondrial fusion involves docking of

  .7).

	A
	B	?
	C
	D	?
		closely
	juxtaposed mitochondrial units (A), or potentially could involve more distant units
	hypothetically performing fusion by means of a narrow area of membrane that forms an
	extension (termed a matrixule) from the mitochondria (B).
	C. Formation of matrixules could be indicators of mitochondria units defective in either a
	membrane tethering process or in membrane fission.
	D. Hypothetical model for the formation of a matrixule as a result of pressure from within
	the matrix and IMM being exerted on the OMM, together with localised membrane
	remodeliing.

Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 23

Arabidopsis seed mitochondria are genetic vaults tailored to fuel germination before specialization for autotrophic growth

Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 37 Chapter ІІ -Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 38 1

Arabidopsis seed mitochondria are bioenergetically active immediately upon imbibition and specialize via biogenesis in preparation for autotrophic growth 1.1 Abstract

	Seed germination is a vital developmental transition in the production of progeny by
	sexual reproduction in spermatophytes. Quiescent cells in non-dormant dry embryos are
	reawakened first by imbibition and then by perception of additional germination triggers
	that vary depending on species. The reanimated tissues then enter into a germination
	programme that requires energy for expansion growth. However, germination is also a
	time when embryonic tissues must develop to support the more energy-demanding
	processes of cell division and organogenesis of the newly germinated seedling.

Reactivation of mitochondria, from their state of suspended animation in the dry seed, to supply the required energy, is thus a key process underpinning germination and subsequent seedling survival. Using a live imaging approach, we investigated reactivation of mitochondrial bioenergetics and dynamics using Arabidopsis thaliana L. Heynth, as a model. Bioenergetic reactivation, visualised by the presence of a membrane potential, is almost immediate upon re-hydration. However, reactivation of mitochondrial dynamics only occurs after transfer to germination conditions. Reactivation of mitochondrial bioenergetics and dynamics is followed by dramatic reorganisation of the chondriome (all mitochondrial in a cell, collectively) involving transient massive fusion and membrane biogenesis to form a perinuclear tubuloreticular structure enabling mixing of previously discrete mtDNA nucleoids. The end of germination coincides with fragmentation of the tubular chondriome leading to a doubling of mitochondria number and heterogeneous redistribution of the nucleoids amongst the mitochondria, generating a population of mitochondria tailored to seedling growth.

Total vol me o o ects er stack m Mitochon rial o ects er stack

		2 h S 1 2 h S 2 4 h S 3 6 h S 4 8 h S 1 2 h L	T R	E R	L R	R H	2 h S 1 2 h S 2 4 h S 3 6 h S 4 8 h S 1 2 h L	T R	E R	L R	R H	2 h S 1 2 h S 2 4 h S 3 6 h S 4 8 h S 1 2 h L	T R	E R	L R	R H
		32 00
	Vol me er o ects m	0 008 0 032 0 125 0 50 2 00 8 00				time
		0 002
		2 h S 1 2 h S 2 4 h S 3 6 h S 4 8 h S 1 2 h L	T R	E R	L R	R H

B

  Paszkiewicz Gael Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 90 Quantification of the numbers of ATG8F-bodies per cell and the percentage of ATG8Fbodiesclose to mitochondria, or those containing mito-GFP. Data from at least 31 different embryos per time point from three independent experiments. Box plot whiskers indicate 1.5 x IQR and outliers are represented by an empty circle, as described by Tukey. Means are rep resented by a full circle. The notch corresponds to the median 1.58 x IQR/ n.

	A		mito-GFP			mRFP1-ATG8F		PSV		Merge
		Not associated
	Associated	uxtaposed Engul ed
		ATG8F-bodies (per cell)									ATG8F-bodies associated to	mitochondria (per cell)
			4 8 h S	4 h L	1 2 h L	2 2 h L	E R	L R	4 a y s 4 a y s 4 a y s		4 8 h S	4 h L 4 h L	1 2 h L 1 2 h L	2 2 h L 2 2 h L	E R	L R	4 4 a y s a y s
		Percentage o ATG8F-bodies	juxtaposed to mitochondria								Percentage o ATG8F-bodies	engul ing mitochondria
			4 8 h S 4 8 h S	4 h L 4 h L	1 2 h L 1 2 h L	2 2 h L 2 2 h L	E R	L R	4 a y s 4 a y s 4 a y s		4 8 h S 4 8 h S	4 h L 4 h L	1 2 h L 1 2 h L	2 2 h L 2 2 h L	E R	L R	4 4 4 a y s a y s a y s
	Figure 3.2 Quantification of ATG8F-bodies associated with mitochondria during
	germination.
	A. Enlarged sections of CLSM images, as used to count mitophagy events during germination,
	shown to indicate classes of ATG8-decoracted structures . Bars = 2 µm.
	B.

Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ........................... 91

ies er cell ATG8F-o ies associate to mitochon ria er cell Percentage o ATG8F-o ies ta ose to mitochon ria Percentage o ATG8F-o ies eng l ing mitochon ria t Percentage o ATG8F-o ies eng l ing mitochon ria t ercentage o ATG8F-o ies ta ose to mitochon ria t ATG8F-o ies associate to mitochon ria er cell

	ATG8F-o 4 8 h S 4 8 h S 4 8 h S 4 8 h S 4 h L 4 h L 1 2 h L 1 2 h L 2 2 h L 2 2 h L E R E R L R L R 4 a y s 4 a y s 4 8 h S 4 h L 1 2 h L 4 2 2 h L E R L R a y s	4 h L 4 h L	1 2 h L 1 2 h L 2 2 h L 2 2 h L 2 2 h L 2 2 h L	E R E R	L R L R	4 a y s 4 a y s			a y s a y s
	4 8 h S 4 8 h S	4 h L 4 h L	1 2 h L 1 2 h L	2 2 h L 2 2 h L	E R	L R	4 4 4 4 a y s a y s a y s a y s	2 2 h L	E R	L R	4 a y s	2 2 h L	E R	L R	4 a y s	2 2 h L	E R	L R	a y s

friendly WT Paszkiewicz Gael Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ......................... 102

thaliana is dependent on mitochondrial dynamics and mitophagy . ......................... 111

All tissue was mounted in perfluorodecalin (octadecafluorodecahydro-naphtalene, Fluka) [START_REF] Littlejohn | Perfluorodecalin enhances in vivo confocal microscopy resolution of Arabidopsis thaliana mesophyll[END_REF]. For all experiments each image stack corresponds to a different embryo. Live cell confocal laser scanning microscopy (CLSM) was performed using a Nikon A1 microscope driven by NIS Elements software (Nikon) using Nikon 40x CFI Plan Apochromat oil-immersion objective (NA 1.3), or 60x CFI Plan Apochromat oil-immersion objective (NA 1.4) for experiments using SYBR Green I. Excitation and emission wavelengths for the different probes were: mito-GFP/SYBR Green I/YFP, 488/500-550; mCherry/mRFP1/TMRM, 561/570-620; and PSV autofluorescence, 405/425-475. Dichroic mirrors of 405/488 or 405/488/561 were used as appropriate. Live imaging, using line switching between channels, was performed on the same area of embryonic cotyledons of between 5 to 11 different seeds from at least two independent experiments per time point and/or per condition in Chapter 2. Observations detailed in Chapter 3 were performed on the same area of embryonic cotyledons of at least 31 different seed from three independent experiments per time point.

Quantification of mitochondria morphologies

Z-stacks were captured first at 2h from the start of imbibition (stratification) and then at 12h intervals up to 60h. In addition, stacks were captured of the four late-germination and post-germination stages of testa rupture (TR), endosperm rupture, long root (LR) and root hair (RH). Stacks of slices 37.29 x 37.29 µm with a z-step of 0.15 µm were captured at an image size of 1024x1024 pixels with a pixel size of 0.04 µm and pixel dwell time of 1.2 µs. The z-stacks were then reconstructed using IMARIS v7 (Bitplane).

Object reconstructions were performed by automatic thresholding using background subtraction (rolling ball of 0.273 µm). The parameters measured to describe the 3-D reconstruction are: objects numbers (object per stacks) or volume (in relative unit, per object or total) and sphericity (0 to 1 unit, per object). Data were then exported to R (R- [START_REF]R: A language and environment for statistical computing[END_REF] to perform statistical analysis. To facilitate visualisation, the distribution of object volume is presented on logarithmic scale (base 2) while object numbers and sphericity are on linear scales.

Quantification of organelle dynamics

Stacks of images, 37.29 x 37.29, were captured at 15.47s intervals for 5 min 15s, with an image size of 2048 x 2048 pixels, giving a pixel size of 0.02 µm. Stacks were processed using IMARIS v7 to enable object identification, tracking and the calculation of various dynamics parameters. Object reconstructions were performed by automatic thresholding using background subtraction (rolling ball of 0.237 µm). Tracking over time was performed using the connected component algorithm. Parameters measured were mean track speed, track displacement, track straightness (relative unit from 0 to 1), total number of interactions with other mitochondria, fusion rate per mitochondria for Paszkiewicz Gael | Mitochondrial biogenesis during seed germination of Arabidopsis thaliana is dependent on mitochondrial dynamics and mitophagy . ......................... 112 mitochondria interacting at least once, and the percentage of mitochondria interacting at least once. Peroxisome movement was also quantified by ImarisTrack. Endoplasmic reticulum time stacks were processed using Fiji [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF] to filter noise and to superimpose two images (25 x 25 µm) of the same field-of-view captured 309 second apart. Colocalisation analysis of these two images was performed with the Coloc 2 plug-in using the Costes method [START_REF] Costes | Automatic and quantitative measurement of protein-protein colocalization in live cells[END_REF]. Following stratification for 48h, plates were transferred to light/21ºC or dark/21ºC for 2h. Image capture was performed within 30min of opening the plate. Statistical analysis was performed using R. Trackderived parameter measurements were weighted to account for number of mitochondria per track to avoid underestimation resulting from track sharing.

TMRM staining

The ability of mitochondria to generate a membrane potential was determined in vivo by the observation of the fluorescent dye TMRM which accumulates in mitochondria in a potentiometric manner [START_REF] Brand | Assessing mitochondrial dysfunction in cells[END_REF]. Dry mito-GFP seed were incubated under gentle vacuum in freshly prepared 50 nM TMRM for 10 min before dissection.

Dissected embryos were mounted in 50 nM TMRM and image capture (image size 2048 x 2048 pixels covering an area of 125.88 x 125.88 µm with a pixel size of 0.06 µm) by CSLM was achieved within a further 5 min. Colocalisation analysis of the TMRM and GFP fluorescence signal was performed on ROIs of 50 x 50 µm in Fiji using the Coloc2 plugin. The Mander's coefficient was chosen in order to calculate the percentage of pixels registering mito-GFP that also register TMRM fluorescence. Statistical analysis was performed using R.

Respiration measurement

Oxygen consumption of seeds was measured in 1 mL of deionized water with a liquidphase Oxytherm oxygen electrode system (Hansatech Ltd, Norfolk, UK) calibrated at the measurement temperature. Dry seeds (20-40 mg dry weight) were directly imbibed in the electrode chamber. For experiments with stratified seeds (48 h, 4°C in the dark), seeds (20-40 mg dry weight) were transferred to the electrode chamber for measurements at 4°C, or after 2h of incubation at 21°C in the light (50 µmol.m -2 .s -1 ) for measurements at 21°C. To estimate capacity of the alternative pathway, O 2 consumption rate was measured after addition of 500 µM KCN.

Actin imaging

Image capture of actin dynamics was performed at an image size of 2048 x 2048 pixels (line averaging of 4), with a pixel size of 0.04 µm covering an area of 81.6 x 81.6 µm.

Movies were captured at a frame rate of 1 per 92s over a recording period of 369s. Images were imported to Fiji where they were cropped to 20 x 20 µm and processed to reduce noise by applying Gaussian blur.

SYBR Green I staining

SYBR Green I (Thermo Scientific) is a cell permeable dye that binds preferentially to double-stranded DNA and then emits green fluorescence under blue light excitation. To enable visualisation of mtDNA and mitochondria, we used seed from a transgenic Arabidopsis line expressing matrix targeted mCherry [START_REF] Candat | The ubiquitous distribution of late embryogenesis abundant proteins across cell compartments in Arabidopsis offers tailored protection against abiotic stress[END_REF] stained with SYBR Green I at a dilution of 1:10000. For the 10 min time point, dry seed was incubated in SYBR Green I for 10 min with gentle vacuum before the embryo was carefully dissected from the surrounding tissues. For later time-points, embryos were first dissected from seed imbibed for the appropriate time before. Each image, 66.88 x 66.88 µm, was captured from a different embryo (n=7) and was captured at 2048px, with a pixel size of 0.03 µm. Stacks were processed using IMARIS v7 using the same thresholding for all images. Object reconstructions were performed by automatic absolute thresholding. Parameters measured were number of mitochondria, number of mitochondria with mtDNA, the numbers of mtDNA nucleoid, the nucleoid plan area (µm²), the nucleoid relative fluorescence (relative unit) and the spatial variation of fluorescence within nucleoid. Statistical significance testing was performed by analysis of variance (ANOVA). The second derivative maximum method was used to determine Cp values, and PCR efficiencies were determined from DNA serial dilution curves or using LinRegPCR software (http://LinRegPCR.nl). Three technical replicates were performed for each experiment.

Measurement of interaction between mitochondria and ATG8F-bodies

The numbers of biological replicates are indicated in the figures. For quantification of mtDNA and cpDNA copy numbers, a set of primers located along the organellar genomes was used, and results were normalized against the UBQ10 (At4g05320) and ACT1 (At2g37620) nuclear genes. The same method was used to record mtDNA stoichiometry [START_REF] Wallet | The RECG1 DNA Translocase Is a Key Factor in Recombination Surveillance, Repair, and Segregation of the Mitochondrial DNA in Arabidopsis[END_REF]. To measure the accumulation of ectopic recombination in mtDNA, primers flanking repeat R15 and R26 were used [START_REF] Wallet | The RECG1 DNA Translocase Is a Key Factor in Recombination Surveillance, Repair, and Segregation of the Mitochondrial DNA in Arabidopsis[END_REF]. The COX2 (AtMG00160) and 18S rRNA (AtMG01390) mitochondrial genes were used for normalization. DNA quality assays were performed in a LightCycler CFX96 (Bio-Rad), in 10 µL of reaction containing 1 X SSoAdvanced Universal SYBR Green SuperMix (Bio-Rad) and 0.5 μM of each primer. The thermocycling program was as follows: 7 min denaturing step at 95°C, then 40 cycles of 10s at 95°C and 45s at 60°C. To quantify DNA quality, sets of primers were used to amplify either long fragments, or short fragments of the mitochondrial or nuclear genomes as described by [START_REF] Miller-Messmer | RecA-dependent DNA repair results in increased heteroplasmy of the Arabidopsis mitochondrial genome[END_REF] .

Meta-analysis of transciptomic data

Investigations were performed using three GEO datasets of gene transcript abundances during germination of Arabidopsis seed: GSE41212 [START_REF] Dekkers | Transcriptional dynamics of two seed compartments with opposing roles in Arabidopsis seed germination[END_REF], GSE65394 [START_REF] Silva | A predictive coexpression network identifies novel genes controlling the seed-toseedling phase transition in Arabidopsis thaliana[END_REF], and GSE30223 [START_REF] Narsai | In-depth temporal transcriptome profiling reveals a crucial developmental switch with roles for RNA processing and organelle metabolism that are essential for germination in Arabidopsis[END_REF]. Gene classes were determined