Get Complete Project Material File(s) Now! »

Mitochondrion and chloroplast: two essential organelles in plant

Mitochondria and chloroplasts are often defined as the “powerhouses” of photosynthetic cells. This definition, although simplistic, has the merit of highlighting the primordial function of these organelles in plants. Indeed, both organelles generate reducing power and ATP, which provide energy for many reactions occurring in organisms, including biosynthesis, transport and degradation pathways of RNA, proteins and metabolites.
Photosynthesis and respiration are two processes that balance each other: one produces substrates that can be broken down by the other process. However, the functions of the mitochondrion and chloroplast and their interactions are much more complex, as these organelles constitute major metabolic and signalling hubs (Figure 1.10, Araújo et al., 2014).
The mitochondrial energy metabolism has generally been considered to be a dedicated pathway that uses breakdown products of glycolysis to produce reductants through the TCA cycle (Fernie et al., 2004). By oxidizing NADH and FADH2, the electron transport chain (ETC) then builds up the proton gradient necessary for the operation of ATP synthase. However, this simple vision of the mitochondrial metabolism has evolved to a more complex one, in particular concerning the TCA cycle (Figure 1.10). The beta-oxidation of fatty acids and the degradation of amino acids (especially branched-chain amino acids) were found to be other important sources of TCA cycle metabolites (Sweetlove et al., 2010). Moreover, most of the reactions of the TCA cycle can be bypassed by cytosolic enzymes and thus, the mitochondrial TCA cycle does not operate independently, but rather interacts with cytosolic and plastidial metabolites via metabolic shuttles present at the mitochondrial membrane (Sweetlove et al., 2010; Millar et al., 2011). Another adaptive trait of plant mitochondria is the presence of additional complexes in the ETC: an alternative oxidase (AOX), several NAD(P)H dehydrogenases and uncoupling proteins (UCPs) (reviewed in Møller, 2001; Millenaar & Lambers, 2003; Rasmusson et al., 2008). They play a significant role in response to many stresses such as drought, salt and cold, by controlling the mitochondrial ROS production and maintaining photosynthetic activity throughout the operation of the malate valve (Begcy et al., 2011; Dinakar et al., 2016; Wanniarachchi et al., 2018). AOX was also shown to contribute to the reoxidation of mitochondrial NADH during photorespiration (Vanlerberghe, 2013). These supplementary machineries increase the flexibility of energy metabolism, and thus, play an important role in the rapid adaptation to environmental changes, which is crucial for sessile organisms like plants.
Concerning photosynthesis, it has been one of the most extensively characterized physiological process in plants, as it is a major component influencing plant growth and yield. By the 1970s, a broad overview of the light reactions, the Calvin cycle and photorespiration had already been obtained (Halliwell, 1978). Besides energy transduction, plastids are involved in the biosynthesis of carbohydrates, amino acids, lipids, nucleotides, pigments, hormones and vitamins. For example, they are able to produce 17 out of the 20 proteinaceous amino acids, among which ten are exclusively synthetized in the chloroplast, which makes plastids essential components of nitrogen metabolism (Rolland et al., 2018). Lipid synthesis necessitates the coordination of chloroplasts with the ER: C16 and C18 fatty acids are produced in the chloroplast and can then be used to produce lipids in chloroplast (said “prokaryotic pathway ») or in the ER (« eukaryotic pathway ») (Li-Beisson et al., 2013).
Metabolite exchanges between the two compartments might occur through unidentified transporters or by direct contact between their membranes (Bobik & Burch-Smith, 2015).
The chloroplast metabolism is also connected to peroxisomes because they participate in photorespiration, fatty acid degradation, ROS detoxification but also in the synthesis of hormones (JAs, auxin, SA) (Kaur et al., 2009; Bobik & Burch-Smith, 2015).
The chloroplast and mitochondrion thus constitute two essential metabolic hubs connected with each other and with the rest of the cell and their various activities must be finely controlled to ensure the proper functioning of plant metabolism.

Communicating organelles: anterograde and retrograde signalling

The mitochondrion and chloroplast are organelles originating from two endosymbiotic events: the first would have derived from an alpha-proteobacterium–like ancestor entering an Archea-type host around 1.5 billion years ago, while the second would have derived from a cyanobacterium entering a mitochondriate eukaryote between 1.2 and 1.5 billion years ago (Dyall et al., 2004; Martin et al., 2015). After these events, the original genomes of the endosymbionts were considerably reduced, many genes being transferred to the nucleus and others lost. Thus, these previously autonomous endosymbionts changed into organelles. In Arabidopsis thaliana, the mitochondrial genome currently encodes around 122 protein coding genes, while the chloroplast genome encodes 88 protein coding genes as defined by the TAIR 10 database (, while their original endosymbionts had several thousand genes. According to the subcellular localization database for Arabidopsis proteins (SUBA 4; consensus, 3060 proteins are predicted to be localized in mitochondria and 3597 proteins in plastids, which means that respectively only 4 % and 2.4 % of the mitochondrial and plastidial proteins are encoded by the organellar genomes (Hooper et al., 2017). Moreover, many components of the organellar transcriptional and translational machinery are encoded by the nucleus, even if the genomes of the
organelles comprise some ribosomal subunits and ribosomal RNA encoding genes (Liere et al., 2011). All known organelle transcription factors including RNA polymerases are encoded by the nucleus, except one plastid-encoded RNA polymerase. Organellar post-transcriptional regulation also involves nuclear-encoded proteins for RNA splicing, processing, editing and translation, such as the pentatricopeptide-repeat (PPR) proteins, which constitute a large family of proteins conserved in eukaryotes, but with highly variable targets (Manna, 2015; Guillaumot et al., 2017). This complex partnership implies a tight coordination in the regulation of the expression of organelle and nuclear genes through anterograde (nucleus-to-organelles) and retrograde (organelles-to-nucleus) signalling pathways (Woodson & Chory, 2008). Via retrograde signalling, mitochondria and chloroplasts can transmit developmental or stress signals to the nucleus (Figure 1.11). In mitochondria, a dysfunction of the electron transfer chain affects the transcription of several genes (e.g. AOX in plants or genes involved in ageing control or programmed cell death in yeast or animals), via an increased concentration in ROS or Ca2+ which can activate kinases and protein phosphatases for signal transduction (Amuthan et al., 2001; Borghouts et al., 2004; Lai et al., 2006; Rhoads & Subbaiah, 2007). In yeast, aerobic or hypoxic conditions are perceived through the oxygenic-dependent haem synthesis, which can thereafter interact with Hap1 in the nucleus to regulate the transcription of genes involved in respiration and ergosterol synthesis (Hickman & Winston, 2007). ROS also seem to play a significant role in the oxidation of mitochondrial proteins, which would trigger their degradation. Some oxidized peptides would then be exported to the cytosol and addressed to the nucleus, where they could activate the expression of specific sets of genes (Møller & Sweetlove, 2010). However, whether such a process occurs in plants has not been clearly demonstrated.
The retrograde signalling of the chloroplast has been more extensively studied than that of the plant
mitochondrion. Six Arabidopsis mutants, called genome uncoupled (gun), which exhibited deregulation of nuclear encoded plastid proteins led to the identification of key regulators of the chloroplast-nuclear communication.
GUN1 encodes a PPR-containing protein that would interact with many plastidial components, such as transcriptional, translational or protein import machineries (Colombo et al., 2016). GUN1 was shown to induce the transcription of ABI4 (ABA-insensitive 4), a TF which represses several nuclear genes encoding photosynthetic proteins. The activation of ABI4 would occur through the translocation to the nucleus of PTM, a plant homeodomain transcription factor otherwise localized at the chloroplast envelope (Chan et al., 2016). GUN1 was also proposed to interact with the synthesis of tetrapyrrole derivatives such as heme or Mg-protoporphyrin IX (a precursor involved in chlorophyll biosynthesis), in which the other five gun mutants are involved (de Souza et al., 2017). An increased level of β-cyclocitral, a byproduct of carotenoid oxidation, would also activate the transcription of the singlet oxygen responsive genes, while methylerythritol cyclodiphosphate (MEcPP), an isoprenoid precursor, would modify chromatin remodeling and thus gene expression (Chi et al., 2015). As major metabolites in chloroplasts, fatty acids, and especially their oxidized derivatives, are also important actors of retrograde signaling (de Souza et al., 2017).

READ  Stable water isotopes: a major climate proxy

Oxygen measurements

Measurements of O2 exchange were performed using an Oxylab electrode system with a white light source LED1/W (Hansatech, King’s Lynn, UK) at 25°C, in 1 mL Evian natural mineral water containing around 20 mg germinating seeds, 50 mg seedlings or vacuum-infiltrated fragments of mature leaves from four-week-old plants.
For net photosynthesis assay, light was calibrated at 150 (growth room conditions) or 700 μmol photons.m-2.s-1 (saturating light) using a QRT1 Quantitherm (Hansatech). For light response assay, different light intensities (from 0 to 100 μmol photons.m-2.s-1) were applied. Rates were standardized using dry weight or chlorophyll amount. Total chlorophyll was extracted in 1 mL dimethyl-N-formamide overnight in the dark and chlorophyll amount was determined by spectrophotometry according to the formula (Total chlorophyll (μg) = 7.04 A664 + 20.27 A647) of Moran (1982), using a microplate reader FLUOstar Omega (BMG Labtech, Ortenberg, Germany).

Western blot

For the immunodetection of glycine decarboxylase H protein (GDC-H), alternative oxidase (AOX1 and AOX2 isoforms) and isocitrate lyase (ICL), total proteins were extracted in 50 mM NaPO4 pH 8, 10 mM Ethylenediaminetetraacetic acid (EDTA), 0.1 % (v/v) Triton-X 100, 0.1 % (m/v) Sarcosyl, 10 mM dithiothreitol (DTT), 1 mM phenylmethane sulfonyl fluoride (PMSF) and antiproteases (cOmplete EDTA-free, Roche, Basel, Switzerland). Protein concentration was determined using the Bradford protein assay (Bio-rad, Marnes-La- Coquette, France) with bovine serum albumin (BSA) as protein standard. Proteins separated on 12 % Stain Free Gels (Bio-Rad) were blotted to PVDF (Merck KGaA, Darmstadt, Germany) using tank transfer (10 mM Ncyclohexyl-3-aminopropane sulfonic acid (CAPS) pH 11, 10 % EtOH). Blots were blocked in TBST buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.1 % (v/v) Tween 20) containing 3 % (m/v) defatted dry milk, then incubated in the same buffer containing primary antibodies for 1.5 h, at 4°C, under shaking. Anti-GDC-H (Cat# AS05074, RRID:AB_1031689), anti-AOX1/2 (Cat# AS04054, RRID:AB_1031839) and anti-ICL (Cat# AS09500, RRID:AB_1832014) rabbit polyclonal antibodies from Agrisera (Vännäs, Sweden) were respectively diluted to 1:3000, 1:1000 and 1:1000 and secondary antibody (goat anti-rabbit IgG horse radish peroxidase conjugated (Cat# AS09602, RRID:AB_1966902), Agrisera) diluted to 1:75,000. Blots were developed with Western Clarity ECL (Bio-Rad). Oleosin immunodetection was performed according to D’Andréa et al. (2007). Protein loading on membrane was estimated by UV detection according to Stain-free technology (Bio-Rad). For semi-quantitative analysis, pixels of chemiluminescence were quantified in every lane and were normalized based on the intensity of the RbcL band detected by UV on the membranes. For each protein, the normalized intensities were then expressed as the percentage of the signal detected in d28 sample on the same membrane.

Table of contents :

1. Impact of heat stress on plant development
1.1. Impact of climate change on crop productivity
1.1.1. Climate change and yield projections
1.1.2. Heat stress impact on phenology
1.2. Heat stress impacts on plant physiology
1.2.1. Heat damage on photosynthesis and respiration
1.2.2. Heat perturbation on cell organization
2. The heat stress response
2.1. Early signalling cascade
2.2. Transcriptional factors
2.2.1. Heat shock factors
2.2.2. Other transcriptional factors involved in the HSR
2.3. Implication of hormones in thermotolerance
2.4. Protective mechanisms
2.4.1. Heat shock proteins
2.4.2. ROS balance
2.4.3. Metabolic adaptation
3. Mitochondrion and chloroplast: two essential organelles in plant
3.1. Metabolic hubs
3.2. Communicating organelles: anterograde and retrograde signalling
3.3. Dynamic organelles
4. Thesis aims
1. Introduction
2. Publication
2.1. Introduction
2.2. Materials and methods
2.2.1. Plant material
2.2.2. Biomass measurements
2.2.3. Oxygen measurements
2.2.4. Western blot
2.2.5. Metabolic profiling
2.2.6. Optical and transmission electron microscopy
2.2.7. Enzyme assays
2.2.8. Statistical analyses
2.3. Results
2.3.1. Prolonging early seedling life
2.3.2. Energy metabolism maintenance under developmental arrest
2.3.3. Metabolic adaptation to mineral nutrient starvation
2.3.4. Carbon mobilization and storage during germination and seedling life
2.4. Discussion
2.5. Acknowledgements
2.6. References
2.7. Supporting Information
1. Introduction
2. Results and discussion
2.1. Heat stress regimes
2.2. Respiratory and photosynthetic acclimation
2.2.1. Measurements during stress at 38°C or 43°C
2.2.2. Measurements at 38°C or 43°C at the end of stress application
2.2.3. Measurements at 25°C after heat stress and during recovery
2.2.4. Chlorophyll fluorescence after heat-shock
2.2.5. Overview of energy metabolism responses
2.3. Heat stress response of organelle dynamics
1. Introduction
2. Transcriptomics
2.1. Strategy
2.2. Results and discussion
2.2.1. General heat stress response
2.2.2. Specific responses of priming
2.2.3. Specific responses in heat-shocked samples
2.2.4. Mitochondria and chloroplast heat stress response
3. Proteomics
3.1. Methods
3.2. Results and discussion
3.2.1. Core response
3.2.2. Specific responses of primed samples
3.2.3. Specific responses of heat-shocked samples
3.2.4. Data integration between transcriptomic and proteomic analyses
4. Metabolic profiling
4.1. Methods
4.2. Results and discussion
4.2.1. Profiling of polar primary metabolites
4.2.2. Fatty acid profiling
5. Conclusion
6. Supporting Information
1. Introduction
2. Results and discussion
1. High resilience of seedlings
2. Effect of priming on heat shock response
3. Critical step of the recovery phase in thermotolerance
4. Perspectives
1. Plant material and growth conditions
2. Heat stress regimes
3. Green pixel detection and viability assay
4. Measurements of respiration and photosynthesis
4.1. Oxygraphy
4.2. Chlorophyll fluorescence
5. Organelle dynamics
5.1. Live cell imaging
5.2. Quantification of organelle dynamics
6. Transcriptomic analysis
6.1. RNA extraction and sequencing
7. Proteome analysis
8. Metabolic profiling
9. Western blot


Related Posts