IJOEAR-MAY-2018-12

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-4 Issue-5 May- 2018 Page | 58 Chloroplasts and Mitochondria: Similarities and Differences Firoozeh Chamandoosti Iranian Research Institute of Plant Protection Agricultural Research Education and Extension Organization AREEO Tehran Iran PhD of Cellular and Developmental Biology Assistant Professor of Iranian Research Institute of Plant Protection Department of Plant Diseases Abstract — Eukaryotic cells contain two organelles originally derived from endosymbiotic bacteria: mitochondria and plastids only plants. In eukaryotes owner mitochondria and chloroplast ATP synthase complex is located in the inner membrane of mitochondria and thylakoids membrane of chloroplast. ATP synthesis utilization and provision of both ADP and Pi need to be fine – tuned for optimal ATP synthase activity. Mitochondria and chloroplast have their DNA. The vast majority of mitochondrial and plastid proteins are encoded in the nucleus synthesized by cytosolic ribosomes and subsequently imported into the organelles via active protein transport systems. Keywords — ATP synthesis Chlororplast Mitochondria Protein targeting. I. INTRODUCTION Several proposals have been made to explain the rise of multicellular life forms. An internal environment can be created and controlled germ cells can be protected in novel structures and increased organismal size allows a „„division of labor‟‟ among cell types. These proposals describe advantages of multicellular versus unicellular organisms at levels of organization at or above the individual cell. It have been focused on a subsequent phase of evolution when multicellular organisms initiated the process of development that later became the more complex embryonic development found in animals and plants. The advantage here is realized at the level of the mitochondria and chloroplast 20. Eukaryotic cells have chloroplast and mitochondria that both are membrane bound organelles. Prokaryotic cells for example bacteria have not chloroplast and mitochondria. Mitochondria occur in the cells of animals and plants but chloroplast only occur in the photosynthesising tissues of plants. These two organelles are best known for their roles in energy metabolism notably respiration and photosynthesis 85. Respiration occurs in mitochondria. Mitochondria were originally identified as the site of oxidative energy metabolism 13. Mitochondria are also the host for enzymes of the Krebs cycle and β – oxidation of fatty acids. In today‟s world mitochondria are known not only as the “power station” of the cell but also for playing a vital role in the transmission of extra – and intracellular signals that activate reaction cascades leading to cellular senescence and programmed cell death PCD 104. The discovery of a number of human diseases associated with mitochondrial dysfunctions once again brought mitochondria into the spotlight of biological research. Chloroplasts are members of a class of plant cell organelles known as plastids that all originate from protoplastids. During plant development the protoplastids differentiate to form three major groups of plastids the green chloroplasts the colored chromoplasts and the colorless leucoplasts. The most abundant and important plastids are the chloroplasts. Chloroplasts harvest energy from sunlight to split water and fix carbon dioxide to produce sugars. This process called photosynthesis also converts harvested solar energy into a conserved form of energy: ATP and NADPH through a complex set of processes. II. SYNTHESIS OF ATP IN MITOCHONDRIA AND CHLOROPLAST As it has been mentioned earlier mitochondria and chloroplasts are best known for their roles in energy metabolism notably respiration and photosynthesis 85. It is clear that ATP synthesis is the central bioenergetic engine of all organisms and represents the smallest molecular motor which was optimized in the course of evolution 17. In eukaryotes the ATP synthase complex is located in the inner membrane of mitochondria with ATP synthesis reaction occurring on the membrane side toward matrix compartment. In plants the enzyme is in addition localized in the thylakoid membrane of chloroplasts with the ATP – forming – moiety facing the stroma. These topological differences between the mitochondrial and chloroplastic ATP synthases bring about two very distinct metabolic environments for ATP synthesis where ATP utilization and provision of both ADP and Pi need to be fine–for optimal ATP synthase activity. In chloroplasts ATP synthase receives protons from thylakoid lumen which volume is small as compared to the mitochondrial

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-4 Issue-5 May- 2018 Page | 59 intermembrane space IMS and which pH value can drop to the values below 5 102 while in the mitochondrial IMS it drops only slightly below 7 11 12. In mitochondria adenylates are transported through the membrane whereas their stromal pool is self – sufficient to support chloroplastic ATP synthase the activity of adenylate transport between chloroplast and cytosol is very low representing _1 of activity of the triose phosphate translocator 14. While generation of proton electrochemical potential became the central theory in the chemiosmotic concept of ATP synthase operation 84 the optimal conditions of delivery of ADP and phosphate were analyzed in the concept of thermodynamic buffering 59 60 underlying the importance of auxiliary buffering enzymes such as adenylate kinase AK and creatine kinase in provision of the stable flux of ADP to ATP synthase. This theory was extended in relation to operation of AK in the IMS of mitochondria 15. The energy balance of photosynthetic cells is provided by equilibration of adenylate levels by chloroplasts and mitochondria and the cytosol and the role of AK in this equilibration appears to be important. III. MAGNESIUM AND THE ROLE OF ITS IN ATP SYNTHESIS Before any more explanation and commentary about mitochondria and chloroplast and their differences and similarities also ATP synthase it is better to have a statement about the role of magnesium in ATP synthase by these two important and crucial organelle. The role of magnesium in ATP synthesis is underlined not only by the fact that MgATP is the actual product of the reaction but also as we show below that Mg 2+ acts as a separate substrate in the ATP synthas reaction under physiological conditions ADP can exist both in a free and Mg - bound state and this dual chemical capacity determines a way that magnesium becomes a part of “energy charge”. The Mg 2+ pool is not less important than protons and it is generated kept stable by the AK reaction which determines the equilibrium value of Mg 2+ in cellular compartments 16. This results in efficient regulation of Mg - dependent enzymes and one such enzymeis ATP synthase. The rotation mechanism of ATP synthase was suggested by Boyer 1989 86 and then it was demonstrated empirically 51. The role of proton translocation consists in deforing an open catalytic site to increase the affinity for ADP and Pi which then bind and pass through the transition state yielding tightly bound ATP in one binding change. ADP binding appears to be a key parameter controlling rotation during synthesis while MgADP is inhibiting. The essential role of Mg 2+ in ATP synthase catalysis was recently established in works of scientists. Previously it was assumed that the substrate of ATP synthase was MgADP 87. Later studies however have indicated that it is free ADP in the presence of magnesium which represents the real substrate. It was shown 105 that inhibition of catalysis by vanadate in the presence of MgADP could be substituted by the Mg - vanadate complex indicating that Mg 2+ plays a pivotal role in transition state formation during ATP synthesis. This state involves the preferential coordination with Pi and the repositioning of the P – loop to bring the nonpolar alanine 158 into the catalytic pocket which is achieved in the presence of Mg 2+ 35. According to these more recent data it is correct to consider ADP free rather than MgADP as a true substrate rather than MgADP as a true substrate while Mg 2+ acts independently. Therefore the substrates of ATP synthase are ADP free Pi free Mg 2+ free and H + while the product is MgATP. The reaction can be presented by the following equation one proton is the substrate whereas other protons have catalytic function: ADP 3- + HPO 4 2- + Mg 2+ + H + → MgATP 2- + H 2 O The difference in pH between matrix and IMS results in deprotonation of phosphate and of ADP in the matrix side facilitating the Mg - dependent mechanism. Magnesium participating in ATP synthase catalysis exhibits a profound catalytic effect as shown by 10. The activity with 25 Mg which has magnetic isotopic nucleus is two to three times higher than with 24 Mg or 25 Mg isotopes having spinless non – magnetic isotopic nuclei. This suggests that the ATP synthesis is a spin – dependent ion – radical process. It implies a reversible electron transfer from the terminal phosphate of ADP3 - to Mg +2 generating ion – radical pair with singlet and triplet spin states. The yields of ATP along the singlet and triplet channels are controlled by hyperfine coupling of unpaired electron in 25 Mg +2 ion with magnetic nucleus. The magnesium bivalent cation transforms the protein molecule mechanics into a chemical reaction 10. Although this mechanism was suggested for the mitochondrial ATP synthase potentially it can be generalized for all ATP sythases including the chloroplast and even for other Mg - dependent enzymes. Mg 2+ uptake by mitochondria and its efflux are mediated by a channel or transporter responding to changes in membrane potential in particular in pH gradient 40 68. The concentration of Mg 2+ in the mitochondrial matrix depends on Pi which interacts strongly with Mg +2 to decrease its concentration and in the absence of external Mg +2 promotes respiration – dependent Mg +2 efflux and its decrease in the matrix to very low levels 41. The uptake of Pi by respiring mitochondria converts ∆ pH to ∆  and provides additional Mg - binding sites permitting its large accumulations. This means that Pi in addition to AK buffers Mg +2 concentration and this buffering is important in the matrix of plant mitochondria where AK is absent. While Mg 2+ is an important catalyst and substrate of ATP synthesis and

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-4 Issue-5 May- 2018 Page | 60 many other processes the changes of its content result in significant shifts in bioenergetic state of the cell. These Mg 2+ dependent shifts strongly affect Ca 2+ concentration in the IMS 89 4. Ca 2+ uptake by mitochondria is inhibited by Mg 2+ via a mixed type inhibition in the process of multistate catalytic binding and interconversion in which phosphate is also involved as a regulator 90. A frequently observed increase in Ca 2+ under stress conditions is therefore mediated by fluctuations in Mg 2+ and results in activation of Ca 2+ dependent stress induced enzymes. Thus the signaling and metabolic roles of Ca 2+ are under control of magnesium phosphate and adenylate energy charge that establishes equilibrium Mg 2+ concentration IV. DNA IN MICHONDRIA AND CHLOROPLAST The DNA in both mitochondria and chloroplasts can be extremely unstable as illustrated by the following examples. i The half – life of rat mitochondrial DNA mtDNA in days is 6.7 for heart 9.4 for liver 10.4 for kidney and 31 for brain 73 ii In the single – celled alga Euglena the half – lives for chloroplast DNA cpDNA and mtDNA are 1.6 and 1.8 cell doublings respectively but nuclear DNA is so stable that turnover could not be detected 56 80. iii Two days after sowing mung bean seeds the mtDNA in dark – grown seedlings turns over entirely in 24 hours 50. iv The half – life of mtDNA in yeast is 4 hours for a mutant defective in the mtDNA polymerase gamma 106 v Light triggers the degradation of DNA in maize chloroplasts 10. Four hours after exposing 10 – day – old dark grown seedlings to light the leaf begins to green and the average DNA content per chloroplast decreases to 54 by hour 6 and 9 by hour 24 . vi During 6 stages of development of maize leaf tissue the size and structural integrity of cpDNA decreases progressively from branched molecules of multigenomic size in the basal meristem of seedlings to fragments of subgenomic size in adult plants as observed in moving pictures of individual ethidium – stained DNA molecules 36. A similar degradative progression of individual cpDNA molecules is observed during leaf development for tobacco and the legume Medicago truncatula 57 and Arabidopsis 18. vii In fully expanded leaves of adult plants of Arabidopsis 18 19 and maize 36 more than half the chloroplasts contain no detectable DNA. How can we explain this remarkable instability of organellar DNA It is suggested that the ROS generated during electron transport that accompanies oxidative phosphorylation and photosynthesis leads to oxidative stress and extensive damage to the DNA 20. For Euglena repair of the mtDNA and cpDNA is the only option because it is a unicellular organism. For dark – grown mung bean seedlings repair again is the only option for mtDNA since respiration must provide the energy for this aerobic organism. The mtDNA is so extensively damaged that it turns over completely in one day. For a light – grown plant however there is another option. If some of the organellar DNA can be sequestered in quiescent germ line cells the highly damaged organellar DNA in somatic cells can be left unrepaired it is eventually degraded and its nucleotides are recycled for their nutritive value 23. Similarly oxidatively damaged mtDNA in active somatic cells can either be repaired or abandoned as long as undamaged mtDNA is retained in quiet germ line cells. For the mesozoan Dicyema japonicum mtDNA is retained in "stem" mitochondria of germ cells but mtDNA is undetectable in most somatic cells of mature larvae and adults a result of either dilution without replication 49 or it is suggested that abandonment and degradation of mtDNA 20. 4.1 DNA damage and repair in mitochondria and chloroplasts From an evolutionary perspective the only objective for an organism is to replicate its DNA and pass it on to the next generation. Unintended alterations in chromosomal DNA molecules can arise in various ways including DNA polymerase errors and changes to the DNA template from internal ROS for example and external radiation for example sources. Only internal sources because these can be modulated during development. Changes in DNA can be perceived and acted upon as needed during development. Changes in DNA can occur as nucleotide alterations insertions/deletions inversions and DNA strand breaks. Those lesions recognized as "damage" can be either repaired or removed by degrading the DNA 42. Most information on the repair of mtDNA comes from yeast and somatic cells of mammals 57 72 63 96 54 whereas very little is known about mtDNA repair in plants or about cpDNA repair 24 82 25 5. A detected change in mtDNA is the result of both the rate of damage and the efficiency of correcting the damage. The power of genetics can sometimes be used to study each of these parameters separately in yeast. Overall two conclusions seem generally supported. First most DNA damage in mitochondria is due to oxidative damage as may be expected for the site of respiration and base excision repair

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-4 Issue-5 May- 2018 Page | 61 BER is the main way to rectify oxidative damage 72 71 61 . If BER fails human mtDNA molecules containing the damage are usually degraded and base substitution point mutation is thus avoided 28. Degradation of damaged DNA molecules to avoid mutation is feasible for the high – genome – copy cytoplasmic organelles but not for the diploid nucleus. Such degradation would mask a higher rate of damage in the organelles than in the nucleus. The second conclusion is that the capacity to repair stress – induced DNA damage is lower in mitochondria than the nucleus because mitochondria are the principal site of ROS production employ fewer repair processes than do nuclei or lack protective histones on their mtDNA molecules 75 72 61 45 53 83. Damage to organellar DNA is indicated by a rapidly increasing mutation rate point mutations per kb of mtDNA as mouse tissues age 71 an accumulation of mtDNA deletions with age in humans monkeys and rodents 75 62 and a decline in structural integrity of cpDNA molecules as leaves develop. Thus it would be advantageous to shelter organellar DNA before tissues mature in the adult. V. GENES IN MITOCHONDRIA AND CHLOROPLAST INHERITANCE: LAWS AND MECHANISMS The literature on the inheritance of genes in mitochondria and chloroplasts hereafter organelle genes has changed grown and advanced tremendously 30. Some of the most exciting advances since the previous reviews have been made in understanding the molecular and cellular mechanisms of organelle division and distribution between daughter cells partitioning in yeast animals and plants genetic studies of segregation and within generation selection of mitochondrial genes in mammals and Drosophila and the controversial subject of mitochondrial bottlenecks in mammals. Other exciting discoveries dealt with the mechanisms of uniparental inheritance in Chlamydomonas and mammals and a controversy over whether there is a low level of biparental inheritance and recombination in humans. The growing excitement about mitochondrial genetics in humans and mammals has been driven in large part by their application to human diseases caused by mitochondrial mutations and by the widespread use of mitochondrial genes to study the population genetics and evolution of humans and other animals. These subjects have also been reviewed in the past three years but mainly as separate subjects and not in the context of organelle heredity in general. 5.1 Chlamydomonas Chloroplasts 5.1.1 Vegetative Segregation is rapid in Chlamydomonas chloroplasts Chlamydomonas reinhardtii has been used extensively for chloroplast genetics since the pioneering studies of Sager 93 94 and Gillham 77 78. In contrast to the plants discussed above Chlamydomonas cells have one chloroplast which divides into two equal parts just before the cell divides consequently vegetative segregation cannot be explained by the partitioning of chloroplasts. Most data come from crosses of antibiotic – resistant by sensitive clones. Vegetative segregation can be studied in vegetative zygotes which divide by mitosis instead of meiosis or in the meiotic and early mitotic divisions of the small percentage of zygospores that show biparental inheritance. In either case segregation is complete within a few cell generations. This is much too fast to be accounted for by random partitioning of the approximately 50 – 100 genomes. 5.1.2 Genome partitioning is probably stochastic but not strictly random One possible explanation for rapid segregation is that when the two gamete chloroplasts fuse in the zygote the plastid genomes from the parents tend to remain in different parts of the chloroplast and consequently tend to segregate together rather than strictly randomly 67. The chloroplast genomes are grouped in about 5 – 15 nucleoids and it is possible that the 10 or more genomes in each nucleoid tend to be replication products of one genome. In other words genome partitioning is stochastic but not strictly random like molecules tend to segregate together because they are joined in nucleoids and/or the nucleoids from the gametes are not completely mixed in the zygote. 5.1.3 Genome replication is stochastic Different Chlamydomonas zygotes from the same mating give rise to clones with very different frequencies of alleles from the two parents. Some zygote clones are uniparental with organelle genomes from only one parent or the other. Frequency distributions of gene frequencies in a large number of zygote clones bear a striking resemblance to the gene frequency distributions of Mendelian populations undergoing random genetic drift 33. When the mitotic division of vegetative zygotes 66 or the meiotic divisions of zygospores 22 was delayed for a time by starvation the variance in gene frequencies increased and more uniparental zygote clones were produced. These data suggested that plastid genomes continue to replicate during starvation and that replication is stochastic with some genomes replicating more often than others by chance. The result is that gene frequencies within cells undergo stochastic changes which is called intracellular

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-4 Issue-5 May- 2018 Page | 62 random drift by analogy to random drift of nuclear gene frequencies in populations of organisms 30. Stochastic replication by itself will not completely eliminate an allele from a cell or clone but may reduce it to a frequency too low to detect. Alternatively there may be some degradation of organelle DNA molecules which will then be replaced by additional replications of other molecules turnover. Note that the stochastic replication of genomes and the stochastic partitioning of genomes into daughter organelles when an organelle divides can also explain how a mutant genome becomes homoplasmic in plant plastids. 5.2 Yeast Mitochondria Much has been learned about organelle heredity from the study of another model genetic system mitochondrial genes in budding or baker‟s yeast Saccharomyces cerevisiae. The best markers are mutant genes conferring antibiotic resistance respiration – deficient mutants petites are also used but their inheritance is strongly affected by selection. When heteroplasmic zygotes are produced by mating yeast strains that differ in one or more mitochondrial alleles the majority of diploid progeny are homoplasmic after no more than 20 cell generations. Strictly random partitioning could only explain this rate of segregation if there were no more than 2 to 5 segregating units 30. This is much smaller than the number of mtDNA molecules in diploid cells approximately 100 and slightly smaller than the number of nucleoids. Mitochondria from the two parents cannot be the segregating units because they fuse in the zygote. Consequently vegetative segregation in yeast must be explained by some combinations of the same factors that were invoked above for chloroplast genes in Chlamydomonas: a partitioning of genes that is stochastic but not strictly random with similar molecules tending to remain together b stochastic replication or c turnover. There is experimental evidence only for the first two processes but it is likely that all three are involved. 5.3 Mitochondrial fusion and fission A yeast cell may contain a single large mitochondrial network or a network plus a few small separate mitochondria or many small discrete mitochondria depending on its physiological state. Yeast mitochondrial genomes undergo multiple pairings with recombination in zygotes showing that genomes from the two parents can interact extensively. Considerable progress has been made in understanding mitochondrial fusion and fission in yeast. Fission is accomplished by the dynamin system in yeast and animals 74. The dynamin Dnmp1p localizes to mitochondria at division sites and tips and is required for normal mitochondrial morphology. Mitochondrial fusion requires the fzo1 fuzzy onion gene a homologue of the fuzzy onion gene that is required for mitochondrial fusion in Drosophila. In yeast normal mitochondrial morphology requires a balance between the activities of Dnm1p and Fzo1 52. 5.4 Bud position effects: non random partitioning Early models of mitochondrial gene inheritance in yeast assumed that fusion was so frequent that a cell is effectively a single population of freely interacting genomes. That this could not be strictly true was demonstrated by pedigree studies of zygotes 30 34 91 which showed that a when the first bud comes from one end of the zygote the majority of its mitochondrial genes come from the parent which formed that end of the zygote and b buds that arise from the neck of the zygote receive markers from both parents as well as a higher frequency of recombinant genotypes. This indicates that the mixing of mitochondrial genomes from the two parents is incomplete when the first bud is formed later buds usually include markers from both parents indicating more complete mixing. This interpretation was verified by showing that labeled mtDNA from one parent failed to enter the opposite side of the zygote until sometime after the first bud was formed although it did enter first center buds 58. The mitochondrial membranes from the two parents fused quickly so delayed mixing of mtDNA was not due to delayed mitochondrial fusion evidently the movement of mtDNA across the zygote involves a different mechanism from the movement of mitochondria. Mitochondrial proteins also move more quickly through the mitochondrial network than does mtDNA 88 58 69. 5.5 Mitochondrial movment from mother to bud Because Saccharomyces cells bud rather than undergoing binary fission a mechanism is required to move mitochondria and their genes from the mother into the growing bud. The experimental studies of this process have been reviewed 74. Mitochondria are actively transported from the mother cell into the bud where they are immobilized at the tip of the bud until cytokinesis is complete. Mitochondria probably move along actin filaments by a motor that depends on actin polymerization 103 and movement also requires intermediate filaments encoded by the MDM gene 99. It is not surprising that a mechanism evolved which ensures that buds receive at least some mitochondria which are required for survival and mitochondrial genomes which are required for respiratory competence.

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-4 Issue-5 May- 2018 Page | 63 5.6 Stochastic replication As was the case for Chlamydomonas chloroplast genes yeast cells can become homoplasmic for mitochondrial genes without dividing owing to random genetic drift of gene frequencies within the cell 31. This was demonstrated using delayed division experiments with both budding and fission yeast 64 analogous to those in Chlamydomonas. Birky and colleagues 32 reported that many first central buds are uniparental producing clones with mitochondrial genes from only one parent however when wild – type cells were mated with ½ mutants that have mitochondria but no mtDNA all first central buds receive mtDNA. They suggested that all first central buds probably receive mtDNA from both parents but that stochastic replication possibly combined with turnover eliminates genes from one parent or the other. Stochastic replication is almost certainly a major contributor to the production of homoplasmic cells during asexual reproduction in yeast i.e. to vegetative segregation. 5.6.1 Nucleoid structure affects mitochondrial gene inheritance It was suggested that the segregating units in yeast mitochondria might be nucleoids 30 and recent studies suggest that nucleoid structure does affect the inheritance of mitochondrial genes. The mtDNA molecules in a nucleoid appear to be held together by Holliday structures 100 37 38 81 perhaps because mtDNA replication is initiated by recombination 7 99 44 as it is in T – even phage 46. Mutations that affect the resolution of the Holliday structures also modify the inheritance of neutral ρ - genomes in ρ _ × ρ + crosses 38 97. VI. PROTEIN TARGETING TO MITOCHONDRIA AND CHLOROPLASTS One of the most interesting subjects about mitochondria and chloroplasts which should be considered earlier is the origin of these organells. As all of us know these organells originally derive from endosymbiotic bacteria: The closest bacterial organisms to the endosymbiotic ancestors of these organelles have nearly a thousand genes Rickettsia 98 or several thousands cyanobacteria 95. Since the endosymbiosis many of the genes of the endosymbiotic bacteria have been lost leaving the organelle genomes with less than a hundred proteins – coding genes each 101 70. The vast majority of mitochondrial and plastid proteins are encoded in the nucleus synthesized by cytosolic ribosomes and subsequently imported into the organelles via active protein transport systems. The total number of proteins present in mitochondria and chloroplasts is thought to be about 2000 – 3000 for each of them 3. Mitochondria originated much earlier than plastids and thus the first plastids arose in cells that already contained an efficient system for targeting cytosolically synthesized proteins to mitochondria. One might have expected evolution to have seized this opportunity to reuse the same machinery for targeting proteins to plastids but in fact this seems not to be the case the two protein import systems have clearly been derived independently and do not share homology. In this situation it is thus easy to understand why protein targeting is usually highly specific. Nevertheless it is becoming increasingly clear that despite the profound differences in the two import machineries a certain number of proteins are efficiently recognized by both systems and are imported into both organelles 85. 6.1 Targeting protein to mitochondria Mitochondria have two complexes of proteins called TOM proteins and TIM proteins respectively located in the outer membrane and the inner membrane which together form the protein import channel. The proteins that will be imported generally have a mitochondrial targeting sequence located at the N – terminus although there are proteins that have internal or even C – terminal targeting signals. This latter case has been found only once for a yeast mitochondrial helicase 27. The N – terminal presequence cannot be described as a consensus sequence but contains conserved features that can be identified with more or less confidence. In plants mitochondrial targeting sequences are generally longer than in other organisms 40 amino acids on average 9 they have a net positive charge rich in arginine and poor in acidic amino acids and contain many aliphatic residues mainly leucine and alanine. The structure adopted by the presequence is generally an amphiphilic K helix 47. It can be noted also that plant mitochondrial targeting sequences are particularly rich in serine residues. How the translated protein is actually targeted to the mitochondria is not well understood yet. In the case of a protein targeted to the matrix of mitochondria and possessing an N – terminal presequence cytosolic protein factors interact with the presequence. These factors are generally chaperones and can require ATP. The presequence is then transferred to the mitochondrial TOM complex proteins. These proteins namely TOM70 TOM20 and TOM22 are generally negatively charged and can thus form

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-4 Issue-5 May- 2018 Page | 64 electrostatic interactions with the presequence. Once the presequence is engaged in the outer membrane channel negative charges present on the inner membrane protein TIM23 along with the electrochemical gradient across the inner membrane gradient created by the electron transport along the mitochondrial respiratory chain allow the presequence to tow the protein through both the outer and the inner membrane. The last steps of protein import are carried out by mitochondrial chaperones which literally pull the protein inside the matrix. The imported protein can then be cleaved from its import signal by specific proteases and be refolded to carry out its function inside the mitochondria. 6.2 Targeting protein to chloroplast Chloroplasts also possess an outer envelope protein complex called TOC and an inner envelope protein complex TIC which differ in many ways from the equivalent mitochondrial complexes. Chloroplast proteins can be located in even more compartments than mitochondrial proteins. In addition to the envelope membranes and the inter membrane space and the stroma many important chloroplast proteins photosynthesis – related proteins are located in the membrane and the lumen of the thylakoids. In this study will be focused only on the presequence needed for the protein to cross the double membrane envelope of the chloroplast. These targeting sequences are different from their mitochondrial counterparts but do present some similarities. They are about 50 amino acids long rich in the hydroxylated residue serine and unlike mitochondrial presequences they do not contain many positively charged residues especially in the first ten amino acids and do not contain many leucine residues. However like mitochondrial targeting sequences they contain very few acidic amino. The structure of the presequence is somewhat less well defined than for mitochondria 48. Proteins targeted to the chloroplast are probably also recognized in the cytosol by chaperone proteins 48 before interacting with the components of the import machinery. The major difference with protein import into mitochondria is that there is no comparable electrical gradient in chloroplasts. None of the proteins from the TOC and TIC complexes have homologues in the TOM or TIM machinery 2. Protein import into chloroplasts largely depends on the subsequent action of different protein chaperones the process requiring GTP and ATP. A large GTPase protein TOC160 is one of the most cytosolic – accessible TOC proteins and is involved in recognition of the presequence. The TOC and TIC protein complexes are in close contact with each other. TIC22 is the first protein from the inner membrane complex to interact with the presequence 9. TIC110 is believed to form the canal through which the proteins are eventually imported into the stroma. It seems that TIC110 is also in close interaction with stromal chaperones which could be the final motor for the import of the chloroplast – targeted protein. As in mitochondria specific proteases can remove the presequence from the mature protein. 6.3 Dual targeting proteins to chloroplast and mitochondria There are approximately 50 proteins in different species reported up to date to be encoded by a single gene synthesized as one gene product but imported into both mitochondria and chloroplasts using an ambiguous dual targeting peptide dTP 29 6 69. The first protein shown to be dually targeted to mitochondria and chloroplasts was glutathione reductase GR from Pisum sativum. Since then other proteins involved in many essential organellar functions such as DNA and RNA synthesis and processing protein folding and fate energy metabolism and stress response have been shown to be dually targeted. Eighteen of the dually targeted proteins are aminoacyl – tRNA synthetases identified in A. thaliana 6 76. The overall properties of dTPs resemble standard characteristics of mTPs and cTPs but there are quantitative and distributional differences. Continuous identification of new dually targeted proteins gives opportunity for reinvestigating the overall properties. Determinants for dual targeting are not fully understood. It has been proposed that the information for organellar targeting can be organized in domains as for example in dTPs of GR 29 RNA polymerase RpoT:2 21 and Presequence Protease or spread all through the targeting peptide 79 or associated with the occurrence of arginine and the properties of the second amino acid of the N – terminal sequence 28. Moreover expression of the 5 untranslated region UTR upstream of the ATG start codon has also been shown to be involved in generating a dTP 1. Here we have analyzed the amino acid content and distribution of 43 dual targeted proteins in A. thaliana using SequenceLogos and statistical methods. We have investigated targeting determinants of the dual targeting peptide of Thr – tRNA synthetase ThrRS – dTP studying organellar import of N and C – terminal deletion constructs coupled to GFP. Furthermore it have been produced chemical quantities of the shortest peptide of ThrRS – dTP that was capable of conferring dual targeting capacity ThrRS – dTP 2 – 60 and it has been studied its biochemical and biophysical properties.

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-4 Issue-5 May- 2018 Page | 65 VII. CONCLUSION Chloroplasts and mitochondria are both membrane bound organelles of eukaryotic cells. They do not occur in prokaryotic cells for example bacteria. Mitochondria occur in the cells of animals and plants but chloroplasts only occur in the photosynthesising tissues of plants. Chloroplasts are concerned with the process of photosynthesis whereas mitochondria are concerned with aerobic respiration. It is clear that one of the most important products of these two povital process is ATP. ATP synthesis is the central bioenergetic engine of all organisms and represents the smallest molecular motor which was optimized in the course of evolution. In eukaryotes the ATP synthase complex is located in the inner membrane of mitochondria with ATP synthesis reaction occurring on the membrane side toward matrix compartment. In plants the enzyme is in addition localized in the thylakoid membrane of chloroplasts with the ATP – forming – moiety facing the stroma. So we observe that there are topological differences between the mitochondrial and chloroplastic ATP synthases. Also magnesium is a important element in ATP synthesis. The role of its is to form of Mg 2+ which acts as separate substrate in the ATP synthas. It has also been shown that DNA mitochondria chloroplast has its own and that the DNA in both mitochondria and chloroplasts can be extremely unstable. Most information on the repair of mtDNA comes from yeast and somatic cells of mammals whereas very little is known about mtDNA repair in plants or about cpDNA repair. An ambiguous dual targeting peptide is a tool for importing one gene product into both mitochondria and chloplast. The first protein shown to be dually targeted to mitochondria and chloroplasts was glutathione reductase GR from Pisum sativum. It has been proposed that the information for organellar targeting can be organized in domains. A protein domain is a conserved part of a given protein sequence and tertiary structure that can evolve function and exist independently of the rest of the protein chain. REFERENCES 1 A.C. Christensen et al. „„Dual – domain dual – targeting organellar protein presequences in Arabidopsis can use non – AUG start codons‟‟ Plant Cell vol. 17 pp. 2805 – 2816 2005. 2 A. Caliebe J. Soill „‟News in chloroplast protein import ‟‟ Plant Mol Biol vol. 39 641 – 645 1999. 3 AGI „„Analysis of the genome sequence of the flowering plant Arabidopsis thaliana‟‟ Nature vol. 408 pp. 796 – 815. 2000. Doi: 10.1038/35048692 4 A. Malmendal S. Linse J. Evenas S. Forsen and T. Drakenberg „„Battle for EF – hands: magnesium – calcium interference in calmodulin‟‟ Biochemistry vol. 38 pp. 11844 – 11850 1999. 5 A. Marechal N. Brisson „„Recombination and the maintenance of plant organelle genome stability‟‟ New Phytologist vol. 186 pp. 299 – 317 2010. 6 A.M. Duchene et al. „„Dual targeting is the rule for organellar aminoacyl–tRNA synthetases in Arabidopsis thaliana‟‟ Proc Natl Acad Sc. U S A vol. 102 pp. 16484 – 16489 2005. 7 A.J. Bendich „„Structural analysis of mitochondrial DNA molecules from fungi and plants using moving pictures and pulsed-field gel electrophoresis‟‟ J Mol Biol vol. 255 pp. 564 – 588 1996. 8 J. Bendich „„Mitochondrial DNA chloroplast DNA and the origins of development in eukaryotic organisms‟‟vol. 42 pp. 1 – 8 May 2010 9 A. Kouranov X. Chen B. Fuks D.J. Schnell „„Tic20 and Tic22 Are New Components of the Protein Import Apparatus at the Chloroplast Inner Envelope Membrane‟‟ J Cell Biol Vol. 143 pp. 991 – 1002 1998. 10 A.L. Buchachenko D.A. Kouznetsov N.N. Breslavskaya and M.A. Orlova „„Magnesium isotope effects in enzymatic phosphorylation‟‟ J Phys Chem B 112 pp. 2548 – 2556 2008. 11 A.L. Moore and P.R. Rich “Organization of the respiratory chain and oxidative phosphorylation in Encyclopedia of Plant Physiology‟‟ Higher Plant Cell Respiration Vol. 18 eds R. Douce and D.A. Day Berlin: Springer pp. 134 – 172 1985. 12 A.M. Porcelli A. Ghelli C. Zanna P. Pinton R. Rizzuto and M. Rugolo „„pH difference across the outer mitochondrial membrane measured with a green fluorescent protein mutant‟‟ Biochem. Biophys. Res. Commun vol. 326 pp. 799 – 804 2005. 13 A.P. Kennedy and A.L. Lehninger „„The products of oxidation of fatty acids by isolated rat liver mitochondria‟‟ J Biol Chem vol. 185 pp. 275 – 285 1950. 14 A.P.M. Weber and K. Fischer „„Making the connections – the crucial role of metabolite transporters at the interface between chloroplast and cytosol ‟‟ FEBS Lett vol. 581 pp. 2215 – 2222 2007. 15 A.U. Igamberdiev L.A. Kleczkowski „„Metabolic systems maintain stablenon – equilibrium via thermodynamic buffering ‟‟ Bioessays vol. 31 pp. 1091 – 1099 2009.

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