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1. Details of Module and its Structure

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Module Name/Title Chloroplast and mitochondria > Module Id <10a>

Pre-requisites Basic knowledge about plant cells, their contents and basic idea about cell organelles

Objectives To make the students aware of the structure and molecular organization of chloroplast and mitochondria.

Keywords Chloroplast, mitochondria, stroma, grana, ATP synthase, thylakoids, organelle genome

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2. 2. Development Team

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National Coordinator

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Content Reviewer (CR) < Dr. Nutan Malpathak> Language Editor (LE) < Dr. Nutan Malpathak>

TABLE OF CONTENTS 1. Introduction 2. Structure and molecular organization of chloroplast 2.1 Outer membrane 2.2 Intermembrane space 2.3 Inner membrane 2.4 Stroma 2.5 Thylakoid system 2.6 Antenna systems 2.7 Chloroplast genome 3. Structure of mitochondria 3.1 Outer membrane 3.2 Inner membrane 3.3 ATP synthase 3.4 Mitochondrial genome

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1. Introduction:

A typical plant cell has two energy-producing organelles: mitochondria and chloroplasts. Chloroplasts belong to a group of double membrane–enclosed organelles called plastids. They are rich in chlorophylls that give them green colour. Plastids containing high concentrations of carotenoid pigments instead of chlorophyll are called chromoplasts. They cause colouration of leaves and fruits other than green. Non pigmented plastids are called leucoplasts. Amyloplast is starch storing plastid and is most important type of leucoplast. They are abundant in storage tissues of the shoot and root, and in seeds. Specialized amyloplasts in the root cap also serve as gravity sensors which direct root growth into the soil. The chloroplasts are vital and unique organelles of green plants and some eukaryotic organisms. They carry out photosynthesis, whereby they convert solar energy into chemical energy and also produce free energy stored in the form of ATP and NADPH. The chloroplasts and ER are the key organelles of pathogen defense.

2. Structure and molecular organization of chloroplast –

Higher plant chloroplasts are generally biconvex or planoconvex. They have different shapes in different plants like discoid, ovoid, spheroid, filamentous, saucer- shaped etc. They are vesicular and have a colorless center. Some chloroplasts are in shape of club, they have a thin middle zone and the ends are filled with chlorophyll. In algae a single huge chloroplast is seen that appears either as a network, a spiral band or a stellate plate. The size of the chloroplast also varies from species to species.

The chloroplasts are bound by double membranes. They show a system of three membranes: the outer membrane, the inner membrane and the thylakoid membrane system (Fig 1).

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Fig 1: Schematic picture of the overall organization of the membranes in the chloroplast. The chloroplast of higher plants is surrounded by the inner and outer membranes (envelope). The region of the chloroplast that is inside the inner membrane and surrounds the thylakoid membranes is known as the stroma. The thylakoid membranes form one or a few large interconnected membrane systems, with a well-defined interior and exterior with respect to the stroma. The inner space within a thylakoid is known as the lumen (Taiz & Zeiger, Plant Physiology, 2010).

The outer and the inner membrane of the chloroplast enclose a semi-gel-like fluid known as the stroma. This stroma makes up much of the volume of the chloroplast. The thylakoid system floats in the stroma (Fig 2).

Fig 2: (A) Electron micrograph of a chloroplast from a leaf of timothy grass, Phleum pratense. (18,000×) (B) The same preparation at higher magnification. (52,000×) (Taiz & Zeiger, Plant Physiology, 2010).

2.1 Outer membrane – It is a semi-porous membrane and is permeable to small molecules and ions, which diffuses easily. The outer membrane is not permeable to larger proteins.

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2.2 Intermembrane Space –

It is usually a thin intermembrane space about 10-20 nm and it is present between the outer and the inner membrane of the chloroplast.

2.3 Inner membrane –

The inner membrane of the chloroplast regulates passage of materials in and out of the chloroplast. In addition, the fatty acids, lipids and carotenoids are synthesized in the inner chloroplast membrane. The ATP is synthesized by a large (400 kDa) enzyme complex called as ATP synthase or ATPase or CFo–CF1. It consists of two parts: a hydrophobic membrane-bound portion called CFo and a portion that sticks out into the stroma called CF1 (Fig 3). CFo forms a channel across the membrane through which protons pass. CF1 is made up of three copies each of α and β polypeptides arranged alternately. The catalytic sites are present on β polypeptide. CF1 synthesizes ATP. The chloroplast and mitochondrial ATP synthases have the same overall architecture and probably nearly identical catalytic sites.

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Fig 3: Structure of ATP synthase. This enzyme consists of a large multisubunit complex, CF1, attached on the stromal side of the membrane to an integral membrane portion, known as CFo. CF1 consists of five different polypeptides, with a stoichiometry of α3, β3, γ, δ, ε. CFo contains probably four different polypeptides, with a stoichiometry of a, b, b′, c12. (Taiz & Zeiger, Plant Physiology, 2010).

2.4 Stroma –

Stroma is a protein rich, alkaline, aqueous fluid. It is present within the inner membrane of the chloroplast. The carbon reduction reactions take place in the stroma.

2.5 Thylakoid System –

The thylakoid system is suspended in the stroma. Thylakoids are membranous sacks that contain chlorophylls and associated proteins. The thylakoids are about 300-600 nm in diameter and are arranged in stacks known as grana. Each granum contains around 10-20 thylakoids. A typical granum from Arabidopsis thaliana shows average 4.0 nm thick membrane bilayers, lumen thickness is 4.7 nm and discs are separated by a 3.6 nm gap (Kirchhoff et al, 2011). The fluid compartment surrounding the thylakoids is called the stroma. Adjacent grana are connected by unstacked membranes called stroma lamellae. The stromal thylakoids are in the form of helicoid sheets. The different components of the photosynthetic apparatus are localized in different areas of the grana and the stroma lamellae. The ATP synthases of the chloroplast are located on the thylakoid membranes.

Along with chlorophylls, a wide variety of proteins essential for photosynthesis are embedded in the thylakoid membranes. Many of these proteins are integral membrane proteins. They contain a large amount of hydrophobic amino acids and so are much more stable in a non-aqueous medium. The reaction centers, antenna pigment–protein complexes, and most of the electron transport enzymes are all integral membrane proteins. They have a unique orientation within the membrane - one region pointing toward the stromal side of membrane and the other oriented towards the lumen (Fig 4).

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Fig 4: Predicted folding pattern of the D1 protein of the PSII reaction center. The hydrophobic portion of the membrane is traversed five times by the peptide chain rich in hydrophobic amino acid residues. The protein is asymmetrically arranged in the thylakoid membrane, with the amino (NH2) terminus on the stromal side of the membrane and the carboxyl (COOH) terminus on the lumen side (Taiz & Zeiger, Plant Physiology, 2010).

The chlorophylls and accessory light-harvesting pigments in the thylakoid membrane are always associated with proteins in a specific non-covalent way. Photosystems I and II are spatially separated in the thylakoid membrane (Fig 5).

Fig 5: Organization of the protein complexes of the thylakoid membrane. PS II is located predominantly in the stacked regions of the thylakoid membrane; PS I and ATP synthase are found in the unstacked regions protruding into the stroma. Cytochrome b6/f complexes are evenly distributed. This lateral separation of the two photosystems takes care that electrons and protons produced by PS II are moved to a considerable distance before they are acted on by PS I and the ATP-coupling enzyme (Allen and Forsberg, Trends Plant Sci. 6: 317–326, 2001).

The PSII reaction center, its antenna chlorophylls and associated proteins are primarily located in the grana lamellae (Allen & Forsberg, 2001). The PSI reaction center

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along with its associated antenna pigments, proteins and the coupling-factor enzymes are found almost exclusively in the stroma lamellae and at the edges of the grana lamellae. The cytochrome b6/f complex of the electron transport chain is evenly distributed between stroma and grana. If we see the relative quantities of PS I and PS II, photosystem II is always in excess in chloroplasts. Usually the ratio of PSII to PSI is about 1.5:1, but it can change when plants are grown in different light conditions. The spatial separation between photosystems I and II indicates that a strict equal amounts of two photosystems is not required. PSII provides reducing equivalents into a common intermediate pool of soluble electron carriers (plastoquinone). PSI removes the reducing equivalents from the common pool.

Two different types of models have been proposed for the 3-dimensional structure of grana –

a) Helical model – The model postulates that thylakoids comprise a network of stroma lamellae. These lamellae wind around grana stacks as a right-handed helix and connect individual grana disks via narrow membrane protrusions (Fig 6A). The grana are connected to each other solely by the stroma lamella helices. The helices are tilted at an angle ranging from 10 to 25°, with respect to the grana stacks (Austin & Staehelin, 2011) and they make multiple contacts with successive layers in the grana through slits located in the rims of the stacked discs.

Fork models

A B C

Fig 6: Models of thylakoid architecture. A) The helical model. In fork models, B) stroma lamellae bifurcate to generate grana discs, suggest that grana consist of repetitive units, each containing three grana discs formed by symmetrical invaginations of a thylakoid pair. C) Later, it was proposed that grana are composed of paired layers formed by bifurcations of stroma lamellar sheets. These layers are interconnected by membrane bridges (dotted lines) that emerge from one layer and fuse to the next (Pribil et al, J Exp Bot, 65(8):1955 – 1972, 2014).

b) Fork / bifurcation model – The model was given by Arvidsson and Sundby in 1999. It proposes that the grana themselves are formed by bifurcations of stroma

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lamellae. According to this model, granum is composed of piles of repeat units, each containing three grana discs (Fig 6B). They are formed by symmetrical invaginations of a thylakoid pair caused by bifurcation of the thylakoid membrane. The model explains unstacking and restacking of grana under changing light conditions.

c) Fork / bifurcation model – Recently Shimoni et al. in 2005 presented another model. Here, the granum–stroma assembly is formed by bifurcations of the stroma lamellar membranes into multiple parallel discs. The stromal membranes form wide, slightly undulating, lamellar sheets that intersect the granum body roughly perpendicular to the long axis of the granum cylinder. Instead of winding around the grana and fusing to form multiple granum layers at various levels, each stroma lamellar sheet enters and exits the granum body in approximately the same plane (Fig 6C). Adjacent granum layers are joined through the stroma lamellae and also through the bifurcations and direct membrane bridges. Direct membrane bridges are formed by bending of the granum discs, leading to fusion with their neighbours at the edges. This model also explains the rearrangements in thylakoids during state transitions.

2.6 Antenna systems –

The antenna systems vary greatly in different photosynthetic organisms, but reaction centers are quite similar in even distantly related organisms. The variation in antenna complexes plausibly is due to diverse environments in which the organisms live. This also balances the energy input in the two photosystems that is essential in some organisms (Grossman et al. 1995; Green and Durnford 1996).

Antenna systems are associated with certain reaction centres. They deliver energy very efficiently to the reaction centers with which they are associated (Pullerits and Sundström 1996). There is large variation in size of the antenna systems. They can be small (about 20 to 30 bacteriochlorophylls per reaction center) as in some photosynthetic bacteria, or may be of the size of 200 to 300 chlorophylls per reaction center in higher plants, or can be very large having few thousand pigments per reaction center in some types of algae and bacteria. All antenna pigments are linked to the photosynthetic membranes. There is much diversity in their molecular structures. The excitation energy from the antenna molecule is transferred to the reaction center by resonance transfer.

Antenna complexes transfer energy very efficiently. Approximately 95 to 99% of the energy is delivered to the reaction center by the antenna pigments. These antenna molecules channelize the energy to the reaction center. The pigments in the antenna that

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transfer the absorbed energy to reaction centre have a particular sequence. These pigments have absorption maxima progressively shifting towards longer red wavelengths (carotenoids to chlorophyll b to chlorophyll a). This red shift in absorption maxima signifies that energy of the excited state is somewhat lower near reaction center than in peripheral portions of the antenna system. So when there is transfer of excitation e.g. from a chlorophyll b molecule (absorption maxima at 650 nm) to a chlorophyll a molecule (absorption maxima at 670 nm), the difference in energy between these two excited chlorophylls is lost to the environment as heat. The probability of reverse transfer is small just because thermal energy is not sufficient for such transfers. The energy trapping process and delivery of excitation to the reaction center goes in one direction or it becomes irreversible.

Many photosynthetic eukaryotic organisms contain both chlorophyll a and chlorophyll b. These are the most abundant antenna proteins. They belong to a large family of structurally related proteins. Some of them are primarily associated with PS II and are called light-harvesting complex II (LHCII) proteins. Some others are associated with PS I and are called LHCI proteins. The antenna complexes are also known as chlorophyll a/b antenna proteins (Paulsen 1995; Green and Durnford 1996). The structure of one of the LHCII proteins has been shown in Fig 7 (Kühlbrandt et al. 1994).

Fig 7: Two-dimensional view of the structure of the LHCII antenna complex from higher plants. The antenna complex is a transmembrane pigment protein, with three helical regions that cross the nonpolar part of the membrane. Approximately 15 chlorophyll a and b molecules are associated with the complex, as well as

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several carotenoids. In the membrane, the complex is trimeric and aggregates around the periphery of the PSII reaction center complex. (Kühlbrandt et al, Nature 367: 614–621, 1994).

The protein contains three α-helical regions and binds about 15 chlorophyll a and b molecules, as well as a few carotenoids. The structure of the LHCI proteins is thought to be similar to that of the LHCII proteins. All these proteins have significant sequence similarity and probably they originate from a common ancestral protein (Green and Durnford 1996). Light absorbed by carotenoids or chlorophyll b in the LHC proteins is rapidly transferred to chlorophyll a and then to other antenna pigments that are intimately associated with the reaction center.

Photosystem II is a multisubunit pigment – protein supercomplex (Fig 8) (Barber et al, 1999). It has two complete reaction centers and some antenna complexes In higher plants. The core consists of two membrane proteins – D1 and D2, as well as other proteins. The primary donor chlorophyll (P680), additional chlorophylls, carotenoids, pheophytins, and plastoquinones are bound to D1 and D2. Other proteins serve as antenna complexes or are involved in oxygen evolution.

Fig 8: Structure of dimeric multisubunit protein supercomplex of photosystem II from higher plants. Figure shows two complete reaction centers, each of which is a dimeric complex. (A) Helical arrangement of the D1 and D2 (red) and CP43 and CP47 (green) core subunits. (B) View from the lumenal side of the supercomplex, including additional antenna complexes, LHCII, CP26 and CP29, and extrinsic oxygen- evolving complex, shown as orange and yellow circles. Unassigned helices are shown in gray. (Barber et al, Trends Biochem. Sci. 24: 43–45, 1999)

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2.7 Chloroplast genome –

Chloroplasts reproduce by division rather than by de novo synthesis. During cell division, chloroplasts are divided between the two daughter cells. In most sexual plants, zygote receives chloroplasts only from the maternal plant. In these plants the normal Mendelian pattern of inheritance does not apply to chloroplast-encoded genes. So plastids show non-Mendelian or maternal inheritance. Many important traits like herbicide resistance are inherited from chloroplasts.

Chloroplasts contain their own DNA and protein-synthesizing machinery. They are considered to have evolved from endosymbiotic bacteria. The chloroplast genome of higher plants is smaller than the mitochondrial genome and ranges between 120 – 160 kilobase pairs and encodes approximately 120 proteins (Jensen & Leister, 2014). The genome of green algal chloroplasts typically ranges between 100 and 200 kbp. Some Acetabularia species have chloroplast genomes up to 1.5 Mbp in size (Simpson & Stern 2002) while the smallest conventional chloroplast genome of Ostreococcus tauri, shows only 86 genes closely packed into 72 kbp chromosome (Robbens et al. 2007).The DNA is in the form of circular chromosome and is localized in specific regions of the plastid stroma. DNA replication in chloroplasts is independent of DNA replication in the nucleus. The average amount of DNA per chloroplast in plants is much greater than that of the mitochondria. The total amount of DNA from the mitochondria and plastids together amounts for about one-third of the nuclear genome. Chloroplast DNA encodes rRNA; tRNA, the large subunit of the enzyme ribulose-1,5-bisphosphate carboxylase / oxygenase (RUBISCO) and several of the proteins that participate in photosynthesis. Most of chloroplast proteins are encoded by nuclear genes. They are synthesized in cytoplasm and are delivered to the organelle. Chloroplasts are considered as semi-autonomous organelles because they depend on the nucleus for the majority of their proteins.

A single mesophyll chloroplast is able contain up to 300 chromosomes organized into nucleoids. These are complex structures each consisting of 10-20 copies of the plastid genome along with RNA and various proteins (Krupinska et al, 2013). Nucleoids display unique composition and organization. Certain typical features of prokaryotic nucleoids along with characteristics of eukaryotic chromatin are observed in them. New DNA-binding proteins have been identified in nucleoid using proteomic analysis. Some of these proteins were not inherited from the prokaryotic ancestors (Melonek et al, 2012). One group of proteins in particular includes a SWIB domain. This domain was shown to be part of chromatin remodelling complexes in yeast. This domain is present in 20 proteins in Arabidopsis, of which, at least four are located in the chloroplast. The SWIB-domain proteins in chloroplasts are small proteins with a high isoelectric point and high lysine

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content. They possibly serve as functional replacements for the bacterial histone-like, DNA-binding HU proteins known from E. coli (Melonek et al, 2012).

It was observed in spinach that plastid chromosome is organized in a folded form around a central body which is composed of proteins. These proteins are either firmly bound to DNA in a central region or are more loosely bound to the peripheral DNA fibrils. These observations proposed a layered structure of plastid nucleoids (Fig 9) (Pribil et al, 2014). About 30–50 % of the plastid DNA is included in the central body. Surprisingly, the layered structure is not static but is under developmental control.

Fig 9: Model for the layered structure of plastid nucleoids. Plastid nucleoids consist of a dense layer, or nucleoid core, where transcriptional activity is particularly high. Many membrane anchor proteins in this layer mediate the attachment of the DNA to chloroplast membranes. The nucleoid core is surrounded by a second layer where DNA–protein interactions and DNA compaction are less tight. Switching between the ‘‘core DNA’’ conformation and the ‘‘surrounding DNA’’ conformation could be mediated by chromatin remodeling proteins like SWIB-4 (Pribil et al, J Exp Bot 65(8): 1955 – 1972, 2014).

3. Structure of mitochondria –

Mitochondria are the sites of respiration. They show variation in shape from spherical to tubular. In higher plants, mitochondria are typically discrete, spherical to sausage shaped organelles (Fig 10) but all have a smooth outer membrane and a highly

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intricate inner membrane. The inner membrane in folded and the invaginations are called cristae. In electron micrographs, plant mitochondria usually look spherical or rodlike, ranging from 0.5 – 1.0 μm in diameter and up to 3 μm in length. Most of the plant cells have a substantially lower number of mitochondria than that in a typical animal cell. The number of mitochondria per plant cell is variable. Generally it is related to the metabolic activity of the tissue. A typical Arabidopsis mesophyll cell contains approximately 200–300 discrete mitochondria, while tobacco mesophyll protoplasts show 500–600 (Scott & Logan, 2007).

Fig 10: Electron micrograph of mitochondria in a mesophyll cell of Vicia faba (Taiz & Zeiger, Plant Physiology, 2010).

It is made up of 70% of proteins and some phospholipids. The inner membrane encloses the mitochondrial matrix. The matrix contains enzymes of the Krebs cycle. The inner membrane serves as a barrier to the movement of protons. This allows the formation of electro-chemical gradients. Dissipation of such gradients is linked with production of ATP.

3.1 Mitochondrial outer membrane (MOM) –

The outer membrane typically contains 8–10% of the total proteins of the organelle. The two mitochondrial membranes differ considerably in their structure and composition. The region between the two mitochondrial membranes is known as the intermembrane space. Intact mitochondria are osmotically active. Most inorganic ions and charged organic molecules are able to diffuse freely into the matrix space. The outer membrane is permeable to solutes having a molecular mass of less than approximately 10,000 Da. The lipid fraction of both membranes is primarily made up of phospholipids. About 80% of lipids

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in the membrane are either phosphatidylcholine or phosphatidylethanolamine. MOM is more homogenous in structure and contains much less amount of cardiolipin. Proportion of proteins to phospholipids is much lower. All known proteins in the MOM are encoded by the nuclear genome. None of the MOM proteins contains a typical N-terminal signal sequence that would be cleaved. The outer membrane plays a crucial role in the biogenesis, inheritance and morphology of the mitochondria.

Integral proteins of the MOM are designated to different categories (Fig 11) based on their structure and conformation (Walther & Rapaport, 2009).

a) Signal anchored proteins - Many of these proteins are attached to membrane via a single membrane spanning helix present at the N- terminus of the polypeptide chain. The known signal-anchored proteins in the yeast MOM (Tom20, Tom70, OM45, Mcr1) are primary import receptors. They have single N-terminally located membrane spanning helix that is moderately hydrophobic and contains approximately 20 amino acids. Very often positively charged residues are present on either side of this membrane spanning helices. The helix anchors the protein to the membrane and represents the sorting signal, thus assuming a “signal-anchor” double function.

b) Tail anchored proteins – They are anchored to membrane by a single membrane spanning helix located at the C-terminus of the polypeptide chain.

In both cases, the bulk of the protein is exposed to the cytosol and only a short segment is located within the intermembrane space (IMS).

c) Certain proteins like Tom22 and Mim1have a single transmembrane segment. They take up an orientation where the N-terminal domain is in the cytosol and the C- terminal part is facing the IMS.

d) Proteins like Fzo1 in yeast are anchored in the membrane via multiple α-helices. Here majority of the protein is facing the cytosol and the two membrane embedded helices are connected via a short loop in the IMS.

e) Ugo1 is a multipass membrane protein and possess at least three transmembrane helices.

f) MOM also contains distinct class of β-barrel proteins. They span the membrane via multiple anti-parallel amphiphatic β-strands arranged in a cylindrical shape.

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Fig 11: Topologies of proteins in the mitochondrial outer membrane. a) They contain a single trans- membrane segment at the N-terminus. E.g. Tom20, Tom70, OM45, Mcr1. For these proteins, the bulk of the polypeptide is exposed to the cytosol and only a small N-terminal segment crosses the outer membrane. b) Fis1, Tom5, Tom6, Gem1, Bcl-2, VAMP-1B form another distinct class. These proteins have a single membrane spanning sequence at their C-terminus and display their large N-terminal portion to the cytosol. C) A third type of proteins, like Mim1 and Tom22, traverse the outer membrane once with an N-terminal domain pointing to the cytosol and a soluble C-terminal domain to the intermembrane space. D) Other proteins span the membrane twice e.g. Fzo1/Mfn1,2, e) or several times e.g. Ugo1 or PBR. f) Many proteins (porin, Tom40, Tob55, Mdm10) cross the outer membrane as a series of antiparallel β-strands, forming a β- barrel structure.

3.2 Mitochondrial Inner membrane (MIM) –

Due to invaginations, inner membrane shows much enlarged surface area and can contain more than 50% of the total mitochondrial protein. Mitochondrial matrix is present inside the inner membrane. The inner membrane is the osmotic barrier. MIM contains much high amounts of cardiolipin as compared to MOM and the ratio between proteins and phospholipids in this membrane is also unusually high.

MIM is divided in 2 sub types of membranes - the inner boundary membrane (IBM) and the cristae membrane (CM). The IBM is present adjoining the outer membrane. CM represents invaginations of the IBM that protrude into the matrix space. We now know that mitochondria exhibit great variation in ultra structures depending on the tissue, the

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physiological state, and the developmental stage. Tubular, lamellar and even triangle shaped cristae membranes (Fig 12) have been observed (Zick et al, 2009).

Fig 12: Diversity of mitochondrial ultrastructures. Different types of cristae are shown (Zick et al, Biochem Biophys Acta 1793:5–19, 2009).

Cristae are attached to the IBM by narrow, tubular openings called crista junctions (CJs) (Frey et al, 2002). Although, CJs are present in mitochondria of many different tissues and organism in animals as well as fungi and yeasts, they demonstrate uniform sizes and shapes. These are narrow tubular, ring or slot-like structures with 12 - 40 nm diameters. Usually they are 30 - 50 nm in length, but N. crassa shows longer CJs – about 150–200 nm (Nicastro et al, 2000).

There are 20 inner membrane proteins and they belong to seven major processes viz. oxidative phosphorylation, protein translocation, metabolite exchange, mitochondrial morphology, protein translation, iron/sulfur cluster biogenesis, and protein degradation. The CM shows large amounts of proteins involved in oxidative phosphorylation, iron/sulfur cluster biogenesis, protein synthesis and transport of mtDNA-encoded proteins, whereas the IBM was enriched in proteins involved in mitochondrial fusion and protein transport of nuclear-encoded proteins. If there is any change in the physiological state of plant,

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proteins present in these 2 compartments of the inner membrane can be redistributed (Vogel et al, 2006).

3.3 ATP synthase –

ATP synthase (F1–F0 complex) utilizes the energy of the proton gradient produced during electron transport for ATP synthesis. The ATP synthase consists of the hydrophilic F1 ATPase attached to a hydrophobic unit F0 present in the MIM (Fig 13). F1 and F0 are physically connected by means of a central stalk and a peripheral stalk called as stator (Rak et al, 2009). F1 is composed of five subunits – three α subunits, three β subunits, one each of γ, δ and ɛ. The protein contains three catalytic sites of the enzyme. The core

of α3β3 hexamer is occupied by an elongated α-helical coiled-coil domain of the γ subunit. Another portion of the γ subunit protrudes from the basal part of the F1 sphere and interacts with the small δ and ɛ subunits to form the central stalk. The stalk has a broad

base that makes contact with subunit 9 of F0. F0 has no intrinsic ATPase activity. Its primary function is to use the electrochemical energy into a rotational movement of polymeric subunit 9. During this process protons are recycled from the intermembrane space to the matrix. Proton translocation occurs at an interface between subunit 9 (subunit c) and subunit 6 (subunit a). Subunit 9 is a low-molecular weight proteolipid with a hairpin structure. It contains two transmembrane α-helices separated by a small loop of polar residues extending into the matrix. The yeast ATP synthases have ten copies of subunit 9 arranged in a ring.

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Fig 13: The subunit organization of the mitochondrial ATP synthase. F1 is composed of subunits α, β and the three central stalk subunits γ, δ and ɛ. The F0 sector contains the subunit 9 oligomer, subunit 6, subunits 8, f and i/j. The peripheral stalk consists of subunits 4, d, h and OSCP. The rotor is made up of the central stalk and the subunit 9 ring (Rak et al, Biochem Biophys Acta 1793:108–116, 2009).

3.4 Mitochondrial genome –

Both mitochondria and chloroplasts contain their own DNA and protein-synthesizing machinery. They are supposed to have evolved from endosymbiotic bacteria. Mitochondria divide by fission and also are able to undergo extensive fusion to form elongated structures or networks. Its DNA is in the form of circular chromosomes, similar to those of bacteria. It is confined to specific regions of the mitochondrial matrix called nucleoids. DNA replication in mitochondria is independent of nuclear DNA replication. Number of mitochondria in a given cell type remain approximately constant.

Plant mitochondrial genome ranges between 200 – 2400 kilobase pairs in size. It is considerably larger than that of most animal mitochondria. Much larger variations in size are observed even in closely related species. The mitochondria of meristematic cells typically contain multiple copies of the circular chromosome. But as cells mature, the number of copies per mitochondrion gradually decreases. This happens due to a unique

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feature – mitochondria continue to divide in the absence of DNA synthesis. Mitochondrial genome mostly encodes prokaryotic-type 70S ribosomal proteins and also components of the electron transfer system. Large numbers of mitochondrial proteins, including Krebs cycle enzymes, are encoded by nuclear genes and are transported to mitochondria. .

There is a difference between RNA processing of plant mitochondria and of other organisms. Many mitochondrial genes contain introns, and some genes also show splicing. Plant mtDNA also lacks strict complementarity to translated mRNA. Mitochondrial genome strictly observes the universal genetic code and does not show any deviations that are commonly observed in mtDNA in all other kingdoms. Plant mtDNA shows large amount of non-coding sequences, including introns. Arabidopsis mtDNA codes for 35 known proteins in contrast to just 13 proteins encoded by mammalian mtDNA.

The genes of the mtDNA can be divided into two main classes –

a) tRNA, rRNA, and ribosome proteins encoding genes, and

b) Encoding for oxidative phosphorylation complexes.

Plant mtDNA encodes nine subunits for complex I, one for complex III, three for complex IV, three for ATP synthase, and five proteins for biogenesis of cytochromes (Marienfeld et al. 1999). The subunits encoded by mtDNA are essential for the activity of the respiratory complexes. Remaining proteins are encoded by nuclear genome. So, oxidative phosphorylation depends on expression of nuclear as well as mtDNA genes. The circular plant mtDNA is normally split into several smaller sub-genomic segments. By decreasing the copy number of a particular segment of mtDNA, one can down regulate the genes (Leon et al. 1998). There are several different gene promoters in mtDNA and their transcriptional activity also differs. Major control of mitochondrial gene expression probably occurs by degradation of excess polypeptides at the post-translational level (McCabe et al. 2000).

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References:

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