Academic literature on the topic 'Eukaryotic mitochondria'

Recombinase enzymes promote DNA repair by homologous recombination. The genes that encode them are ancestral to life, occurring in all known dominions: viruses, Eubacteria, Archaea and Eukaryota. Bacterial recombinases are also present in viruses and eukaryotic groups (supergroups), presumably via ancestral events of lateral gene transfer. The eukaryotic recA genes have two distinct origins (mitochondrial and plastidial), whose acquisition by eukaryotes was possible via primary (bacteria–eukaryote) and/or secondary (eukaryote–eukaryote) endosymbiotic gene transfers (EGTs). Here we present a comprehensive phylogenetic analysis of the recA genealogy, with substantially increased taxonomic sampling in the bacteria, viruses, eukaryotes and a special focus on the key eukaryotic supergroup Amoebozoa, earlier represented only by Dictyostelium . We demonstrate that several major eukaryotic lineages have lost the bacterial recombinases (including Opisthokonta and Excavata), whereas others have retained them (Amoebozoa, Archaeplastida and the SAR-supergroups). When absent, the bacterial recA homologues may have been lost entirely (secondary loss of canonical mitochondria) or replaced by other eukaryotic recombinases. RecA proteins have a transit peptide for organellar import, where they act. The reconstruction of the RecA phylogeny with its EGT events presented here retells the intertwined evolutionary history of eukaryotes and bacteria, while further illuminating the events of endosymbiosis in eukaryotes by expanding the collection of widespread genes that provide insight to this deep history.

Classical ideas for early eukaryotic evolution often posited a period of anaerobic evolution producing a nucleated phagocytic cell to engulf the mitochondrial endosymbiont, whose presence allowed the host to colonize emerging aerobic environments. This idea was given credence by the existence of contemporary anaerobic eukaryotes that were thought to primitively lack mitochondria, thus providing examples of the type of host cell needed. However, the groups key to this hypothesis have now been shown to contain previously overlooked mitochondrial homologues called hydrogenosomes or mitosomes; organelles that share common ancestry with mitochondria but which do not carry out aerobic respiration. Mapping these data on the unfolding eukaryotic tree reveals that secondary adaptation to anaerobic habitats is a reoccurring theme among eukaryotes. The apparent ubiquity of mitochondrial homologues bears testament to the importance of the mitochondrial endosymbiosis, perhaps as a founding event, in eukaryotic evolution. Comparative study of different mitochondrial homologues is needed to determine their fundamental importance for contemporary eukaryotic cells.

For over 100 years, endosymbiotic theories have figured in thoughts about the differences between prokaryotic and eukaryotic cells. More than 20 different versions of endosymbiotic theory have been presented in the literature to explain the origin of eukaryotes and their mitochondria. Very few of those models account for eukaryotic anaerobes. The role of energy and the energetic constraints that prokaryotic cell organization placed on evolutionary innovation in cell history has recently come to bear on endosymbiotic theory. Only cells that possessed mitochondria had the bioenergetic means to attain eukaryotic cell complexity, which is why there are no true intermediates in the prokaryote-to-eukaryote transition. Current versions of endosymbiotic theory have it that the host was an archaeon (an archaebacterium), not a eukaryote. Hence the evolutionary history and biology of archaea increasingly comes to bear on eukaryotic origins, more than ever before. Here, we have compiled a survey of endosymbiotic theories for the origin of eukaryotes and mitochondria, and for the origin of the eukaryotic nucleus, summarizing the essentials of each and contrasting some of their predictions to the observations. A new aspect of endosymbiosis in eukaryote evolution comes into focus from these considerations: the host for the origin of plastids was a facultative anaerobe.

All extant eukaryotes are now considered to possess mitochondria in one form or another. Many parasites or anaerobic protists have highly reduced versions of mitochondria, which have generally lost their genome and the capacity to generate ATP through oxidative phosphorylation. These organelles have been called hydrogenosomes, when they make hydrogen, or remnant mitochondria or mitosomes when their functions were cryptic. More recently, organelles with features blurring the distinction between mitochondria, hydrogenosomes and mitosomes have been identified. These organelles have retained a mitochondrial genome and include the mitochondrial-like organelle of Blastocystis and the hydrogenosome of the anaerobic ciliate Nyctotherus . Studying eukaryotic diversity from the perspective of their mitochondrial variants has yielded important insights into eukaryote molecular cell biology and evolution. These investigations are contributing to understanding the essential functions of mitochondria, defined in the broadest sense, and the limits to which reductive evolution can proceed while maintaining a viable organelle.

Bacterial division initiates at the site of a contractile Z-ring composed of polymerized FtsZ. The location of the Z-ring in the cell is controlled by a system of three mutually antagonistic proteins, MinC, MinD, and MinE. Plastid division is also known to be dependent on homologs of these proteins, derived from the ancestral cyanobacterial endosymbiont that gave rise to plastids. In contrast, the mitochondria of model systems such asSaccharomyces cerevisiae, mammals, andArabidopsis thalianaseem to have replaced the ancestral α-proteobacterial Min-based division machinery with host-derived dynamin-related proteins that form outer contractile rings. Here, we show that the mitochondrial division system of these model organisms is the exception, rather than the rule, for eukaryotes. We describe endosymbiont-derived, bacterial-like division systems comprising FtsZ and Min proteins in diverse less-studied eukaryote protistan lineages, including jakobid and heterolobosean excavates, a malawimonad, stramenopiles, amoebozoans, a breviate, and an apusomonad. For two of these taxa, the amoebozoanDictyostelium purpureumand the jakobidAndalucia incarcerata, we confirm a mitochondrial localization of these proteins by their heterologous expression inSaccharomyces cerevisiae. The discovery of a proteobacterial-like division system in mitochondria of diverse eukaryotic lineages suggests that it was the ancestral feature of all eukaryotic mitochondria and has been supplanted by a host-derived system multiple times in distinct eukaryote lineages.

AbstractThe discovery that the protist Monocercomonoides exilis completely lacks mitochondria demonstrates that these organelles are not absolutely essential to eukaryotic cells. However, the degree to which the metabolism and cellular systems of this organism have adapted to the loss of mitochondria is unknown. Here, we report an extensive analysis of the M. exilis genome to address this question. Unexpectedly, we find that M. exilis genome structure and content is similar in complexity to other eukaryotes and less “reduced” than genomes of some other protists from the Metamonada group to which it belongs. Furthermore, the predicted cytoskeletal systems, the organization of endomembrane systems, and biosynthetic pathways also display canonical eukaryotic complexity. The only apparent preadaptation that permitted the loss of mitochondria was the acquisition of the SUF system for Fe–S cluster assembly and the loss of glycine cleavage system. Changes in other systems, including in amino acid metabolism and oxidative stress response, were coincident with the loss of mitochondria but are likely adaptations to the microaerophilic and endobiotic niche rather than the mitochondrial loss per se. Apart from the lack of mitochondria and peroxisomes, we show that M. exilis is a fully elaborated eukaryotic cell that is a promising model system in which eukaryotic cell biology can be investigated in the absence of mitochondria.

Published data suggest that hydrogenosomes, organelles found in diverse anaerobic eukaryotes that make energy and hydrogen, were once mitochondria. As hydrogenosomes generally lack a genome, the conversion is probably one way. The sources of the key hydrogenosomal enzymes, pyruvate : ferredoxin oxidoreductase (PFO) and hydrogenase, are not resolved by current phylogenetic analyses, but it is likely that both were present at an early stage of eukaryotic evolution. Once thought to be restricted to a few unusual anaerobic eukaryotes, the proteins are intimately integrated into the fabric of diverse eukaryotic cells, where they are targeted to different cell compartments, and not just hydrogenosomes. There is no evidence supporting the view that PFO and hydrogenase originated from the mitochondrial endosymbiont, as posited by the hydrogen hypothesis for eukaryogenesis. Other organelles derived from mitochondria have now been described in anaerobic and parasitic microbial eukaryotes, including species that were once thought to have diverged before the mitochondrial symbiosis. It thus seems possible that all eukaryotes may eventually be shown to contain an organelle of mitochondrial ancestry, to which different types of biochemistry can be targeted. It remains to be seen if, despite their obvious differences, this family of organelles shares a common function of importance for the eukaryotic cell, other than energy production, that might provide the underlying selection pressure for organelle retention.

Phagocytosis, or ‘cell eating’, is a eukaryote-specific process where particulate matter is engulfed via invaginations of the plasma membrane. The origin of phagocytosis has been central to discussions on eukaryogenesis for decades­, where it is argued as being either a prerequisite for, or consequence of, the acquisition of the ancestral mitochondrion. Recently, genomic and cytological evidence has increasingly supported the view that the pre-mitochondrial host cell—a bona fide archaeon branching within the ‘Asgard’ archaea—was incapable of phagocytosis and used alternative mechanisms to incorporate the alphaproteobacterial ancestor of mitochondria. Indeed, the diversity and variability of proteins associated with phagosomes across the eukaryotic tree suggest that phagocytosis, as seen in a variety of extant eukaryotes, may have evolved independently several times within the eukaryotic crown-group. Since phagocytosis is critical to the functioning of modern marine food webs (without it, there would be no microbial loop or animal life), multiple late origins of phagocytosis could help explain why many of the ecological and evolutionary innovations of the Neoproterozoic Era (e.g. the advent of eukaryotic biomineralization, the ‘Rise of Algae’ and the origin of animals) happened when they did.

The availability of complete genome sequence data from both bacteria and eukaryotes provides information about the contribution of bacterial genes to the origin and evolution of mitochondria. Phylogenetic analyses based on genes located in the mitochondrial genome indicate that these genes originated from within the α–proteobacteria. A number of ancestral bacterial genes have also been transferred from the mitochondrial to the nuclear genome, as evidenced by the presence of orthologous genes in the mitochondrial genome in some species and in the nuclear genome of other species. However, a multitude of mitochondrial proteins encoded in the nucleus display no homology to bacterial proteins, indicating that these originated within the eukaryotic cell subsequent to the acquisition of the endosymbiont. An analysis of the expression patterns of yeast nuclear genes coding for mitochondrial proteins has shown that genes predicted to be of eukaryotic origin are mainly translated on polysomes that are free in the cytosol whereas those of putative bacterial origin are translated on polysomes attached to the mitochondrion. The strong relationship with α–proteobacterial genes observed for some mitochondrial genes, combined with the lack of such a relationship for others, indicates that the modern mitochondrial proteome is the product of both reductive and expansive processes.

It had been believed that when individual eukaryotic cells engulfed aerobic and photosynthetic bacteria, the bacteria became endosymbionts with the phagocyte and evolved into mitochondria and chloroplasts respectively. The key objective of this study is to prove that the Endosymbiotic Theqory about the origins of mitochondria and chloroplasts is a misleading lie, being ridiculously laughable. Each of the micrographs or videos in the Figures, and each of the concrete evidences listed were designed to have targetful aims. The micrographs, videos, and concrete evidences were devised to serve as addressive findings (results). The human mitochondrial DNA is a transcript obtained when two separate spliced or mature RNAs (of DNA type) transcribed from the nuclear Genome of human oocyte have annealed into double-stranded transcript and exported into the cytoplasmic organelle called mitochondrion (plural, mitochondria) that mainly performs the task of producing energy (ATP) to power the genomic reactions of the cell. Lynn Margulis’s Endosymbiotic Theory which stated that mitochondrion & chloroplast found in eukaryotic cells had evolved from bacteria when they were engulfed by eukaryotic cells, was a misleading lie that would had been ridiculously laughable!!!!

Mitochondria are eukaryotic cellular organelles responsible for power-generation, believed to have come into existence by an endo-symbiontic event where a bacterial cell was incorporated by an un-specified "proto-eukaryote". Phylogenetic analysis have shown that the mitochondrial ancestor was most related to present-day alpha-proteobacteria, although the exact nature of the mitochondrial progenitor remains disputed. In this work, I have used phylogenetic and other methods to investigate the identity of the organism giving rise to mitochondria, by analysing the evolutionary history of select proteins, the events where they have been transfered to the eukaryotic nucleus, and the time-point of mitochondrial establishment. In addition, a search for mitochondrially related organisms in the ocean metagenome was performed, in the hope that something more related to the mitochondrial progenitor than anything previously identified could be found. Previous analysis have shown that a large fraction of mitochondrial proteins does indeed trace their descent to the alpha-proteobacteria, but I found that the amino-acyl tRNA-synthetases display more general bacterial descent, making it likely that these proteins are of a different origin from the mitochondria themselves. While the synthetases are encoded on the nuclear genome, most mitochondria still posses most of the tRNA on their own genomes. In the cases where the tRNA has been lost from the mitochondrial genome, I found that the probability of loss correspond to the evolutionary history of their synthetase. The ocean metagenome represents an order of magnitude more data than previously available, making it suitable for improving the analyses dealing with mitochondrial placement. This large of amount of data was utilised to improve the phylogenetic analyses, showing that previous works might have suffered from artefacts inflating the support for placement of mitochondria with a specific alpha-proteobacterial group. Eukaryotic/mitochondrial radiation was shown to be as old, or older, than radiation of extant alpha-proteobacteria, casting doubt on previous analysis identifying a specific alpha-proteobacterial group as the mitochondrial ancestor.

Tutti gli eucarioti presentano sistemi di percezione per le fonti di carbonio e azoto tramite la quale la crescita cellulare è coordinata con lo stato nutrizionale. In particolare, I metaboliti della parte alta della glicolisi influenzano lo stato di attivazione della via di PKA e di Snf1/AMPK/SnRK1 in lievito, mammifero e piante. Mentre, la via del complesso di TORC1 è al centro di un sistema di segnalazione per la disponibilità di amminoacidi. Di particolare interesse è il meccanismo di interazione tra la via di Snf1/AMPK e Ras/PKA con il metabolismo del glucosio la cui regolazione non è tuttora compresa nel dettaglio. In questa tesi, si dimostra che le vie di Snf1/AMPK e Ras/PKA sono indipendentemente connesse al metabolismo del glucosio tramite la sintesi di glucosio-6 fosfato e fruttosio 1,6 bisfosfato. Inoltre, l’attività della chinasi Snf1/AMPK è risultata essere regolata dalla velocità di importo del glucosio nelle cellule, piuttosto che dalla sua disponibilità nel terreno di coltura. Esclusi meccanismi alternativi, il glucosio-6 fosfato potrebbe influenzare lo stato di fosforilazione e attivazione di Snf1 tramite un’interazione diretta e causando una maggiore accessibilità alle fosfatasi della treonina regolatoria T210. I nutrienti hanno inoltre un forte impatto sull’invecchiamento cellulare e microorganismi eucariotici e gli organismi pluricellulari a bassa complessità possono essere utilizzati come organismi modello per lo studio di tali processi. In collaborazione con altri gruppi di ricerca, sono state studiate le proprietà del fagiolo Vigna unguicolata nel prevenire l’invecchiamento e la neuro-degenerazione. Gli estratti di fagiolo hanno aumentato le aspettative di vita di Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans e di cellule di mammifero. Inoltre, gli stessi estratti hanno mostrato proprietà neuroprotettive riducendo l’aggregazione dell’α-sinucleina in vitro e la morte di neuroni dopaminergici in Caenorhabditis elegans. Nella seconda parte della tesi vengono investigati nuovi approcci per il trattamento del carcinoma epatocellulare. Uno studio preliminare ha infatti dimostrato che, in lievito, l’abbinamento della delezione di SNF1 e l’aggiunta di metionina determinano un riarrangiamento metabolico ed una riduzione della crescita cellulare. Essendo il fegato l’organo in cui prende luogo la maggior parte del metabolismo della metionina e della S-adenosilmetionina (SAM), abbiamo investigato l’effetto dell’inibizione di AMPK e l’aggiunta di metionina sul fenotipo tumorale di linee cellulari derivate dal carcinoma epatocellulare. Le condizioni analizzate hanno mostrato di essere in grado di aumentare l’attività del ciclo di Krebs e la quantità di ATP derivata dalla respirazione mitocondriale. Questo, in associazione ad una riduzione dell’aggressività delle linee di carcinoma epatocellulare HepG2 e Huh7. La S-adenosilmetionina è un’importante molecola per il trattamento dell’alcolismo e della depressione, inoltre è utilizzata nella sintesi di melatonina, antibiotici e flavonoidi. Nell’ultima parte di questa tesi viene presentato lo stato di avanzamento di un progetto di ingegnerizzazione del batterio del suolo Pseudomonas putida per la produzione di SAM. Il disegno sperimentale prevede la duplicazione della sua via di sintesi con una via sintesi resistente ai controlli endogeni ed accoppiata al ciclo di Krebs. Questo ha implicato lo studio delle vie di anaplerosi del ciclo di Krebs ed ha evidenziato come le informazioni ottenute in Escherichia coli non siano sempre traslabili su altri tipi di batteri.
Receptors and signal transduction pathways have been studied for decades depicting the mechanism responsible for the perception of nutrients and growth factors. Nevertheless, an increasing amount of evidence suggest that signal transduction is inherently connected also to intracellular metabolism through protein-metabolite interactions (PMIs) between metabolites and proteins of the signal transduction pathways. All the eukaryotes present conserved pathway for the sensing of carbon and nitrogen sources responsible for the coordination of cell growth with its nutritional state. Metabolites belonging to the upper glycolysis strongly influence PKA and Snf1/AMPK/SnRK1 activation state in yeast and mammalian and plants cells. In the meanwhile, components of the TORC1 pathway result to be the center of interaction for the sensing of amino acids availability. Interestingly, the crosstalk between Snf1/AMPK and Ras/PKA pathways, as well as glucose regulation of Snf1/AMPK activity in yeast is not completely understood yet. In the present thesis, we demonstrate that Snf1/AMPK and Ras/PKA pathway are independently controlled by glucose metabolism through the synthesis of glucose 6-phosphate and fructose 1,6-bisphosphate, respectively. Hence, we proved that Snf1/AMPK activation state is controlled by glucose transport rate and not by glucose availability, providing evidence suggesting that glucose 6-phosphate may directly interact with Snf1 complex and enhance the exposure to phosphatases of the phosphorylated regulatory threonine (T210). Nutrients also have a strong impact on cellular aging and eukaryotic microorganisms or simple pluricellular organisms can be useful model organisms for the study of the aging process. In a collaborative study, we evaluated the properties of the bean Vigna unguicolata as functional food ameliorating aging and neurodegeneration. Bean extracts extend the life span of Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans and mammalian cells. Furthermore, bean extracts also showed neuroprotective properties, reducing the in vitro aggregation of α-synuclein and decreasing the age-related degeneration of cephalic dopaminergic neurons in Caenorhabditis elegans. In the second part of the thesis, we investigate new putative approaches for the treatment of hepatocellular carcinoma (HCC). A preliminary study showed that the coupling of SNF1 deletion with methionine supplementation rewires yeast metabolism and reduces its proliferation. Being methionine and S-adenosylmethionine metabolism mainly active in the liver, we investigated whether AMKP inhibition coupled with a high methionine dosage can ameliorate the phenotype of hepatocellular carcinoma cell lines. These conditions increased the activity of the TCA cycle and the amount of ATP derived from respiration. Furthermore, this reduction of the Warburg phenotype was associated with a reduction of the aggressiveness of the hepatocellular carcinoma cell lines HepG2 and Huh7. S-adenosylmethionine is also an important fine chemical used in the treatment of alcoholism and depression or for the synthesis of melatonin, antibiotics and flavonoids. In the last part of this thesis, I present the advancement of the engineering of the environmental bacteria Pseudomonas putida for the overproduction of SAM. To pursue this goal, we designed a feedback-free inducible pathway to duplicate SAM production pathways in P. putida and coupling it with the TCA cycle. The building of this engineered strain forced us to deal with the robustness of P. putida central carbon metabolism and to investigate possible anaplerotic reaction replenishing the TCA cycle. This allowed us to gain useful details on the regulation of the TCA metabolism in P. putida and highlighted that information acquire in enterobacteria Escherichia coli are not always translatable to other type of bacteria.

There are ~12 supergroups of complex-celled organisms (eukaryotes), but relationships among them (including the root) remain elusive. For Paper I, I developed a dataset of 37 eukaryotic proteins of bacterial origin (euBac), representing the conservative protein core of the proto-mitochondrion. This gives a relatively short distance between ingroup (eukaryotes) and outgroup (mitochondrial progenitor), which is important for accurate rooting. The resulting phylogeny reconstructs three eukaryote megagroups and places one, Discoba (Excavata), as sister group to the other two (neozoa). This rejects the reigning “Unikont-Bikont” root and highlights the evolutionary importance of Excavata. For Paper II, I developed a 150-gene dataset to test relationships in supergroup SAR (Stramenopila, Alveolata, Rhizaria). Analyses of all 150-genes give different trees with different methods, but also reveal artifactual signal due to extremely long rhizarian branches and illegitimate sequences due to horizontal gene transfer (HGT) or contamination. Removing these artifacts leads to strong consistent support for Rhizaria+Alveolata. This breaks up the core of the chromalveolate hypothesis (Stramenopila+Alveolata), adding support to theories of multiple secondary endosymbiosis of chloroplasts. For Paper III, I studied the evolution of cox15, which encodes the essential mitochondrial protein Heme A synthase (HAS). HAS is nuclear encoded (nc-cox15) in all aerobic eukaryotes except Andalucia godoyi (Jakobida, Excavata), which encodes it in mitochondrial DNA (mtDNA) (mt-cox15). Thus the jakobid gene was postulated to represent the ancestral gene, which gave rise to nc-cox15 by endosymbiotic gene transfer. However, our phylogenetic and structure analyses demonstrate an independent origin of mt-cox15, providing the first strong evidence of bacteria to mtDNA HGT. Rickettsiales or SAR11 often appear as sister group to modern mitochondria. However these bacteria and mitochondria also have independently evolved AT-rich genomes. For Paper IV, I assembled a dataset of 55 mitochondrial proteins of clear α-proteobacterial origin (including 30 euBacs). Phylogenies from these data support mitochondria+Rickettsiales but disagree on the placement of SAR11. Reducing amino-acid compositional heterogeneity (resulting from AT-bias) stabilizes SAR11 but moves mitochondria to the base of α-proteobacteria. Signal heterogeneity supporting other alternative hypotheses is also detected using real and simulated data. This suggests a complex scenario for the origin of mitochondria.

Mitochondria are eukaryotic organelles derived in evolution from within the ? subdivision of Proteobacteria. Although mitochondria are structurally and metabolically complex, modern-day mitochondrial genomes (mtDNA) encode only a small number of RNAs and proteins predominantly involved in adenosine triphosphate (ATP) formation through electron transport coupled to oxidative phosphorylation, as well as translation of mtDNA-encoded proteins. In humans, only 13 of the >1000 polypeptides that constitute the complete mitochondrial protein complement (proteome) are encoded in mtDNA; the remainder is encoded by nuclear DNA (nuDNA). It is therefore imperative to comprehensively catalog nuDNA-encoded mitochondrial proteins in order to understand holistically the evolution of mitochondria. Mitochondrial proteome investigations of animals, fungi and land plants have dramatically altered our conception of mitochondrial evolution: in contrast to mtDNA-encoded proteins, few nuDNA-encoded mitochondrial proteins are demonstrably derived from the eubacterial progenitor of mitochondria, and many are found only in eukaryotes. Notably, however, little is known about the mitochondria of eukaryotic microbes (protists), which constitute the bulk of biochemical and genetic diversity within the domain Eucarya. The proteomic characterization of protist mitochondria is therefore crucial to fully elucidating mitochondrial function and evolution. Employing tandem mass spectrometry (MS/MS), I have analyzed highly purified mitochondria from Acanthamoeba castellanii (Amoebozoa). In combination, nearly 750 nuDNA- and mtDNA-encoded proteins were identified. These data were used to catalog metabolic pathways and protein complexes, and to infer functional and evolutionary profiles of A. castellanii mitochondria. My analyses suggest that while A. castellanii mitochondria have many features in common with other eukaryotes, they possess several novel attributes and pronounced metabolic versatility. An analysis of the A. castellanii electron transport chain (ETC) was also performed, utilizing a combination of blue native polyacrylamide gel electrophoresis (BN-PAGE), MS/MS and bioinformatic queries. A significant proportion of A. castellanii ETC proteins was identified, yielding several insights into ETC evolution in eukaryotes. Lastly, I present two unusual cases of ‘split’ mitochondrial proteins: the iron-sulfur subunit SdhB of succinate:ubiquinone oxidoreductase (Complex II), in the phylum Euglenozoa and Cox1 of cytochrome c:O2 oxidoreductase (Complex IV) in various eukaryotes, including A. castellanii. Functional and evolutionary implications of these findings are discussed.

The mitochondrion is a double membrane-bound organelle found in all eukaryotic organisms. Mitochondria are endosymbiotic, autogenous organelles referred to as “Powerhouse” of cells due to their ability to synthesize ATP from oxidative phosphorylation. The number of mitochondria varies from cell to cell in an organism, depending on the function of the cells. For example, liver and muscle cells are rich in mitochondria, on the other hand, RBC cells are devoid of mitochondria. In healthy cells, mitochondrial homeostasis is mainly due to Biogenesis (fission and fusion), maintenance (recycling) and clearance. Mitochondria also act as a molecular sink to regulate the activities of many proteins. In Eukaryotes mitochondria are the major hub for the synthesis of Fe-S clusters. The Fe-S cluster biogenesis process is essentially constituted of two major events; first, the assembly of Fe-S clusters on a scaffold protein. Second, the transfer of the assembled Fe-S clusters to a recipient apo-protein. In Eukaryotic mitochondria, the sulfur transfer was accomplished by cysteine desulfurase protein NFS1, which was stabilized by ISD11 protein. ISD11 protein exclusively presents only in the eukaryotic system and absent in bacteria. On the other hand, the iron-binding protein frataxin donates Iron. The electrons required for the process of Fe-S cluster biogenesis was provided by ferredoxin reductase and ferredoxin 2. A highly conserved matrix protein ISCU acts as the scaffold where the assembly of Fe-S cluster occurs. The transfer of Fe-S cluster process is mediated by chaperone machinery comprising the mtHsp70 namely HSPA9, the J-protein cochaperone, HSCB along GLRX5. However, transfer of Fe-S clusters to apoprotein was not clearly understood. Dysfunction of Mitochondrial proteins involved in Fe-S cluster biogenesis and transfer leads to a pathological condition in humans. For example, deficiency or loss of function of scaffold protein ISCU, iron donor protein frataxin, sulfur donor protein NFS1, sulfur transfer assisting protein, ISD11 and electron transfer protein FDX2 results in mitochondrial disorders ISCU myopathy, Friedreich’s ataxia, Infantile Mitochondrial Complex II/III Deficiency (IMC23D), Combined Oxidative Phosphorylation Deficiency19 (COXPD19) and FDX2 myopathy respectively.

in English Formation of mitochondria by the conversion of a bacterial endosymbiont is the fundamental moment in the evolution of eukaryotes. An integral part of the organelle genesis was the displacement of the endosymbiont genes to host nucleus and simultaneous creation of new pathways for delivery of proteins synthesized now in the host cytoplasm. Resulting protein translocases are complexes combining original bacterial components and eukaryote-specific proteins. In addition to these novel protein import machines, some components of the original bacterial secretory pathways have remained in the organelle. While the function of a widely distributed mitochondrial homolog of YidC, Oxa1, is well understood, the role of infrequent components of Sec or Tat translocases has not yet been elucidated. So far, more attention has been paid to their abundant plastid homologs, which assemble photosynthetic complexes in the thylakoid membrane. In the thesis, the structure and function of prokaryotic YidC, Sec and Tat machineries and their eukaryotic homologs are described. By comparing both organelles of the endosymbiotic origin, the hypothesis is drawn on why these translocases have been more "evolutionary successful" in plastids than in mitochondria.

碩士
國立清華大學
動力機械工程學系
101
Cytochrome c oxidase is a mitochondrial membrane bounded enzyme which is the fourth complex of the respiratory electron transport chain. Cytochrome c oxidase catalyzes the respiratory reduction reaction of O2 to water. Reduction of O2 takes places at the metallic center of the cytochrome c oxidase. This thesis intensively studies the oxygen reduction reaction using the first principles calculations based on the time-independent density functional theory (TI-DFT) with the B3LYP /6-31G (d, p) method in the Gaussian09 program. It is generally agreed that DFT methods give accurate results for the geometries and vibrational frequencies of transition metals. In this study, the functional model of the metallic active site in the respiratory enzyme cytochrome c oxidase is simulated and the output data are used to analyze the bond length, band gaps, molecular orbitals, IR spectra, the structure energy and the reaction energy of the oxygen reduction reaction (ORR). The metallic active center was calculated with three different multiplicities, which are singlet, triplet, and quintet. According to the results of geometric energy of different multiplicities, we can sum up the reaction center of cytochrome c oxidase to be quintet. Finally, the total energy of the reaction product is calculated and the reaction energy of the ORR is discussed in this thesis.

Mitochondria are essential eukaryotic organelles, acting as the sites for numerous crucial metabolic and signalling pathways. The biogenesis of mitochondria requires efficient targeting of several hundreds of proteins from the cytosol, to their varied functional locations within the organelle. The translocation of localized proteins across the inner membrane, and their subsequent folding is achieved by the ATP-dependent function of mitochondrial Hsp70 (mtHsp70). It is a bonafide member of the Hsp70 chaperone family, which are involved in a multitude of functions, together aimed at protein quality control and maintenance of cellular homeostasis. These varied functions of Hsp70 proteins require binding to exposed hydrophobic patches in substrate polypeptides thus preventing non-productive associations. The interaction with substrates occurs through the substrate-binding domain (SBD) and is regulated by the ATPase activity of the nucleotide-binding domain (NBD), through a series of conformational changes. Conversely, substrate binding to the SBD also stimulates ATP hydrolysis, and thereby the core activities of the two domains are regulated by mutual allosteric signalling. This mechanism of bidirectional inter-domain communication is indispensable for Hsp70 function, which is characterized by cycles of substrate binding and release, coupled to cycles of ATP binding and hydrolysis. The process of allosteric regulation in Hsp70 proteins has been comprehensively investigated, especially in the bacterial homolog, DnaK. However, the in vivo functional significance of inter-domain communication in the eukaryotic mtHsp70 system and the mechanism of its regulation remain unexplored. Furthermore, the complex physiological implications of impairment in allosteric communication and their correlation with diverse disease conditions, including Myelodysplastic syndrome (MDS), and Parkinson’s disease (PD), are yet to be elucidated. Based on this brief introduction, the primary research objectives set out in the present thesis were to: 1. uncover the regulation of ligand-modulated allosteric communication between the two domains of mtHsp70; and its in vivo significance in the context of protein import into the organelle. (Chapter 2) 2. understand the role of mtHsp70 in progression of Parkinson’s disease; and to study the modulation of α-synuclein toxicity by the protein quality control function of the mtHsp70 chaperone network. (Chapters 3 and 4) We have employed a battery of genetic and biochemical approaches to investigate the above questions using the Saccharomyces cerevisiae mtHsp70 protein, Ssc1; an essential protein that is involved in a plethora of critical functions in this eukaryotic model system. Objective 1: Structural studies, primarily in bacterial DnaK, have yielded mechanistic insights into its interactions with ligands and cochaperones, as well as conformational transitions in different ligand-bound states. In recent years, the availability of crystal structures of full-length DnaK and detailed information from NMR studies and single-molecule resolution spectroscopic analyses (both DnaK and eukaryotic Hsp70s), have significantly contributed to our understanding of the inter-domain interface, critical residues and contacts, and the energetics of the entire process of ligand-modulated conformational changes. Although eukaryotic mtHsp70s have a high degree of conservation with DnaK, they possess significant differences in their conformational and biochemical properties. They are essential for a vast repertoire of physiological functions, which are distinctly different from their bacterial counterpart. Using a combined in vivo and in vitro approach, we have uncovered specific structural elements within mtHsp70s, which are required for allosteric modulation of the chaperone cycle and maintenance of in vivo functions of the protein. Foremost, we demonstrate that a conserved SBD loop, L4,5 plays a critical role in inter-domain communication, and multiple mutations in this loop result in significant growth and protein translocation defects. The mutants are associated with a specific set of altered biochemical properties, which are indicative of impaired inter-domain communication. Using the loop L4,5 mutant, E467A as a template for genetic screening, we report a series of intragenic suppressor mutations, which are capable of correcting a distinct subset of the altered properties, and thereby leading to restoration of in vivo functions, including growth, preprotein import and mitochondria biogenesis. The suppressors modify the altered conformational landscape associated with E467A, and also provide us with information regarding unique aspects governing the regulation of allosteric communication, especially in physiological contexts. Strikingly, they reveal that restoration of communication in the NBD to SBD direction is sufficient for function, when the protein is primed in a high ATPase activity state. In this unique scenario, the requirement for ATPase stimulation upon substrate binding is rendered unnecessary, thereby making conformational changes in the SBD to NBD direction, dispensable for function. Further, we provide evidence to show that loop L4,5 functions synergistically with the linker region, working in tandem for organization of the inter-domain interface and propagation of communication. Together, our analyses provide the first insights into regulation of allosteric inter-domain communication in vivo and their implications in mitochondrial protein translocation and organelle biogenesis. Objective 2: Point mutations in the loop L4,5 have been associated with Myelodysplastic syndrome. Additionally, a mutation isolated in clinical cases of Parkinson’s disease was found to be impaired in allosteric communication. These observations further highlight the importance of efficient inter-domain communication in mtHsp70 in the complex physiological scenario of eukaryotic cells. Independent clinical screens of PD patients have revealed unique point mutations in the mtHsp70 and a strong association of the gene locus with the disease progression. This is also correlated with decreased mtHsp70 levels in affected neurons and the interactions of this protein with established PD-candidate proteins like α-synuclein and Dj-1. Further, mitochondrial dysfunction is a common phenomenon associated with neurodegenerative disorders. To understand the specific role of mtHsp70 in PD, we have developed a yeast model for studying the disease variants in isolation from other players of the multifactorial disease, and in complete absence of the wild type protein. We generated two analogous PD-mutations in Ssc1, R103W and P486S; which recapitulated the symptoms of mitochondrial dysfunction in affected neurons, including cell death, inner membrane depolarization, increased generation of ROS, and respiratory incompetence. At the molecular level, we observed an increased aggregation propensity of R103W, while P486S exhibited futile enhanced interaction with J-protein cochaperone partners thereby resulting in loss of chaperoning activity and impaired mitochondrial protein quality control. Remarkably, these altered biochemical properties mimicked similar defects in the human mtHsp70 variants, therefore, affirming the involvement of mtHsp70 in PD progression. To further investigate the relevance of impaired mitochondrial protein quality control in PD, we have explored whether mtHsp70 can act as a genetic modifier of α-synuclein toxicity. It is known that α-synuclein can act as an unfolded substrate for the Hsp70 chaperone system and also deposits as intracellular aggregates in PD-affected brains. Intriguingly, it is known to translocate into mitochondria under conditions of neuronal stress in spite of lacking a canonical mitochondrial signal sequence. Utilizing our yeast-PD model, we find that targeting of α-synuclein A30P disease variant into mitochondria leads to a severe mitochondrial dysfunction phenotype in the wild type Ssc1 background, but not the P486S mutant background. This results in multiple cellular manifestations, which are reversed upon overexpression of the Ssc1 chaperone. Significantly, increasing the J-protein cochaperone availability also leads to reversal of the mutant-associated defects. However, the simultaneous overexpression of both together does not additively improve the protective effects; highlighting the importance of the relative availability of chaperone and cochaperone proteins in preventing aggregation. Our analyses further reveal that while both the wild type and P486S Ssc1 proteins are equally capable of delaying aggregation of α-synuclein, only the wild-type chaperone is better able to prevent aggregation in the presence of its J-protein cochaperone, leading to accumulation of soluble oligomeric species. These observations raised the intriguing possibility, that the reduced chaperoning ability of the proline to serine PD-mutant is, in fact, a compensatory adaptation, favoring the aggregation of α-synuclein over its more toxic soluble oligomeric form. We verify this hypothesis with the aggregation kinetics of A30P α-synuclein, whose intrinsically lower aggregation tendency results in a pronounced delay in aggregation with the wild-type chaperone, thereby strongly favoring the toxic oligomeric species and correlating with the observed lethality in yeast cells. In conclusion, our study provides a model of α-synuclein aggregation-related toxicity and its modulation by the extent of protein quality control within the mitochondrial matrix, through the action of the mtHsp70 chaperone network.

Mitochondria are essential eukaryotic organelles, acting as the sites for numerous crucial metabolic and signalling pathways. The biogenesis of mitochondria requires efficient targeting of several hundreds of proteins from the cytosol, to their varied functional locations within the organelle. The translocation of localized proteins across the inner membrane, and their subsequent folding is achieved by the ATP-dependent function of mitochondrial Hsp70 (mtHsp70). It is a bonafide member of the Hsp70 chaperone family, which are involved in a multitude of functions, together aimed at protein quality control and maintenance of cellular homeostasis. These varied functions of Hsp70 proteins require binding to exposed hydrophobic patches in substrate polypeptides thus preventing non-productive associations. The interaction with substrates occurs through the substrate-binding domain (SBD) and is regulated by the ATPase activity of the nucleotide-binding domain (NBD), through a series of conformational changes. Conversely, substrate binding to the SBD also stimulates ATP hydrolysis, and thereby the core activities of the two domains are regulated by mutual allosteric signalling. This mechanism of bidirectional inter-domain communication is indispensable for Hsp70 function, which is characterized by cycles of substrate binding and release, coupled to cycles of ATP binding and hydrolysis. The process of allosteric regulation in Hsp70 proteins has been comprehensively investigated, especially in the bacterial homolog, DnaK. However, the in vivo functional significance of inter-domain communication in the eukaryotic mtHsp70 system and the mechanism of its regulation remain unexplored. Furthermore, the complex physiological implications of impairment in allosteric communication and their correlation with diverse disease conditions, including Myelodysplastic syndrome (MDS), and Parkinson’s disease (PD), are yet to be elucidated. Based on this brief introduction, the primary research objectives set out in the present thesis were to: 1. uncover the regulation of ligand-modulated allosteric communication between the two domains of mtHsp70; and its in vivo significance in the context of protein import into the organelle. (Chapter 2) 2. understand the role of mtHsp70 in progression of Parkinson’s disease; and to study the modulation of α-synuclein toxicity by the protein quality control function of the mtHsp70 chaperone network. (Chapters 3 and 4) We have employed a battery of genetic and biochemical approaches to investigate the above questions using the Saccharomyces cerevisiae mtHsp70 protein, Ssc1; an essential protein that is involved in a plethora of critical functions in this eukaryotic model system. Objective 1: Structural studies, primarily in bacterial DnaK, have yielded mechanistic insights into its interactions with ligands and cochaperones, as well as conformational transitions in different ligand-bound states. In recent years, the availability of crystal structures of full-length DnaK and detailed information from NMR studies and single-molecule resolution spectroscopic analyses (both DnaK and eukaryotic Hsp70s), have significantly contributed to our understanding of the inter-domain interface, critical residues and contacts, and the energetics of the entire process of ligand-modulated conformational changes. Although eukaryotic mtHsp70s have a high degree of conservation with DnaK, they possess significant differences in their conformational and biochemical properties. They are essential for a vast repertoire of physiological functions, which are distinctly different from their bacterial counterpart. Using a combined in vivo and in vitro approach, we have uncovered specific structural elements within mtHsp70s, which are required for allosteric modulation of the chaperone cycle and maintenance of in vivo functions of the protein. Foremost, we demonstrate that a conserved SBD loop, L4,5 plays a critical role in inter-domain communication, and multiple mutations in this loop result in significant growth and protein translocation defects. The mutants are associated with a specific set of altered biochemical properties, which are indicative of impaired inter-domain communication. Using the loop L4,5 mutant, E467A as a template for genetic screening, we report a series of intragenic suppressor mutations, which are capable of correcting a distinct subset of the altered properties, and thereby leading to restoration of in vivo functions, including growth, preprotein import and mitochondria biogenesis. The suppressors modify the altered conformational landscape associated with E467A, and also provide us with information regarding unique aspects governing the regulation of allosteric communication, especially in physiological contexts. Strikingly, they reveal that restoration of communication in the NBD to SBD direction is sufficient for function, when the protein is primed in a high ATPase activity state. In this unique scenario, the requirement for ATPase stimulation upon substrate binding is rendered unnecessary, thereby making conformational changes in the SBD to NBD direction, dispensable for function. Further, we provide evidence to show that loop L4,5 functions synergistically with the linker region, working in tandem for organization of the inter-domain interface and propagation of communication. Together, our analyses provide the first insights into regulation of allosteric inter-domain communication in vivo and their implications in mitochondrial protein translocation and organelle biogenesis. Objective 2: Point mutations in the loop L4,5 have been associated with Myelodysplastic syndrome. Additionally, a mutation isolated in clinical cases of Parkinson’s disease was found to be impaired in allosteric communication. These observations further highlight the importance of efficient inter-domain communication in mtHsp70 in the complex physiological scenario of eukaryotic cells. Independent clinical screens of PD patients have revealed unique point mutations in the mtHsp70 and a strong association of the gene locus with the disease progression. This is also correlated with decreased mtHsp70 levels in affected neurons and the interactions of this protein with established PD-candidate proteins like α-synuclein and Dj-1. Further, mitochondrial dysfunction is a common phenomenon associated with neurodegenerative disorders. To understand the specific role of mtHsp70 in PD, we have developed a yeast model for studying the disease variants in isolation from other players of the multifactorial disease, and in complete absence of the wild type protein. We generated two analogous PD-mutations in Ssc1, R103W and P486S; which recapitulated the symptoms of mitochondrial dysfunction in affected neurons, including cell death, inner membrane depolarization, increased generation of ROS, and respiratory incompetence. At the molecular level, we observed an increased aggregation propensity of R103W, while P486S exhibited futile enhanced interaction with J-protein cochaperone partners thereby resulting in loss of chaperoning activity and impaired mitochondrial protein quality control. Remarkably, these altered biochemical properties mimicked similar defects in the human mtHsp70 variants, therefore, affirming the involvement of mtHsp70 in PD progression. To further investigate the relevance of impaired mitochondrial protein quality control in PD, we have explored whether mtHsp70 can act as a genetic modifier of α-synuclein toxicity. It is known that α-synuclein can act as an unfolded substrate for the Hsp70 chaperone system and also deposits as intracellular aggregates in PD-affected brains. Intriguingly, it is known to translocate into mitochondria under conditions of neuronal stress in spite of lacking a canonical mitochondrial signal sequence. Utilizing our yeast-PD model, we find that targeting of α-synuclein A30P disease variant into mitochondria leads to a severe mitochondrial dysfunction phenotype in the wild type Ssc1 background, but not the P486S mutant background. This results in multiple cellular manifestations, which are reversed upon overexpression of the Ssc1 chaperone. Significantly, increasing the J-protein cochaperone availability also leads to reversal of the mutant-associated defects. However, the simultaneous overexpression of both together does not additively improve the protective effects; highlighting the importance of the relative availability of chaperone and cochaperone proteins in preventing aggregation. Our analyses further reveal that while both the wild type and P486S Ssc1 proteins are equally capable of delaying aggregation of α-synuclein, only the wild-type chaperone is better able to prevent aggregation in the presence of its J-protein cochaperone, leading to accumulation of soluble oligomeric species. These observations raised the intriguing possibility, that the reduced chaperoning ability of the proline to serine PD-mutant is, in fact, a compensatory adaptation, favoring the aggregation of α-synuclein over its more toxic soluble oligomeric form. We verify this hypothesis with the aggregation kinetics of A30P α-synuclein, whose intrinsically lower aggregation tendency results in a pronounced delay in aggregation with the wild-type chaperone, thereby strongly favoring the toxic oligomeric species and correlating with the observed lethality in yeast cells. In conclusion, our study provides a model of α-synuclein aggregation-related toxicity and its modulation by the extent of protein quality control within the mitochondrial matrix, through the action of the mtHsp70 chaperone network.

Lipid droplets (LDs) are small mobile organelles conserved in all eukaryotic cells. We wanted to test if the LD movement can be muffled by an incomplete inhibition of mitochondrial respiration, induced by treating hyphae of filamentous fungus Phycomyces blakesleeanus with 0.5 mM sodium azide. Nitrogen starved hyphae were used, in order to obtain LDs in larger sizes and numbers. The data obtained unequivocally showed: 1. Sodium azide treatment dramatically reduces the LD velocity and the distances LDs travel; 2. LDs in both controls and in azide-treated hyphae oscillate in a small confined space instead of travelling through the cell; 3. Azide-treated LDs oscillate less frequently and in smaller confinement than controls.

Abstract Mitochondria are the site of respiration and numerous other metabolic processes required for plant growth and development. Increased demands for metabolic energy are observed during different stages in the plants life cycle, but are particularly ample during germination and reproductive organ development. These activities are dependent upon the tight regulation of the expression and accumulation of various organellar proteins. Plant mitochondria contain their own genomes (mtDNA), which encode for rRNAs, tRNAs and some mitochondrial proteins. Although all mitochondria have probably evolved from a common alpha-proteobacterial ancestor, notable genomic reorganizations have occurred in the mtDNAs of different eukaryotic lineages. Plant mtDNAs are notably larger and more variable in size (ranging from 70~11,000 kbp in size) than the mrDNAs in higher animals (16~19 kbp). Another unique feature of plant mitochondria includes the presence of both circular and linear DNA fragments, which undergo intra- and intermolecular recombination. DNA-seq data indicate that such recombination events result with diverged mitochondrial genome configurations, even within a single plant species. One common plant phenotype that emerges as a consequence of altered mtDNA configuration is cytoplasmic male sterility CMS (i.e. reduced production of functional pollen). The maternally-inherited male sterility phenotype is highly valuable agriculturally. CMS forces the production of F1 hybrids, particularly in predominantly self-pollinating crops, resulting in enhanced crop growth and productivity through heterosis (i.e. hybrid vigor or outbreeding enhancement). CMS lines have been implemented in some cereal and vegetables, but most crops still lack a CMS system. This work focuses on the analysis of the molecular basis of CMS. We also aim to induce nuclear or organellar induced male-sterility in plants, and to develop a novel approach for fertility restoration. Our work focuses on Brassicaceae, a large family of flowering plants that includes Arabidopsis thaliana, a key model organism in plant sciences, as well as many crops of major economic importance (e.g., broccoli, cauliflower, cabbage, and various seeds for oil production). In spite of the genomic rearrangements in the mtDNAs of plants, the number of genes and the coding sequences are conserved among different mtDNAs in angiosperms (i.e. ~60 genes encoding different tRNAs, rRNAs, ribosomal proteins and subunits of the respiratory system). Yet, in addition to the known genes, plant mtDNAs also harbor numerous ORFs, most of which are not conserved among species and are currently of unknown function. Remarkably, and relevant to our study, CMS in plants is primarily associated with the expression of novel chimericORFs, which likely derive from recombination events within the mtDNAs. Whereas the CMS loci are localized to the mtDNAs, the factors that restore fertility (Rfs) are identified as nuclear-encoded RNA-binding proteins. Interestingly, nearly all of the Rf’s are identified as pentatricopeptide repeat (PPR) proteins, a large family of modular RNA-binding proteins that mediate several aspects of gene expression primarily in plant organelles. In this project we proposed to develop a system to test the ability of mtORFs in plants, which are closely related to known CMS factors. We will induce male fertility in various species of Brassicaceae, and test whether a down-relation in the expression of the recombinantCMS-genes restores fertility, using synthetically designed PPR proteins.

New potential for engineering chloroplasts to express novel traits has stimulated research into relevant techniques and genetic processes, including plastid transformation and gene regulation. This proposal continued our long time BARD-funded collaboration research into mechanisms that influence chloroplast RNA accumulation, and thus gene expression. Previous work on cpRNA catabolism has elucidated a pathway initiated by endonucleolytic cleavage, followed by polyadenylation and exonucleolytic degradation. A major player in this process is the nucleus-encoded exoribonuclease/polymerasepolynucleotidephoshorylase (PNPase). Biochemical characterization of PNPase has revealed a modular structure that controls its RNA synthesis and degradation activities, which in turn are responsive to the phosphate (P) concentration. However, the in vivo roles and regulation of these opposing activities are poorly understood. The objectives of this project were to define how PNPase is controlled by P and nucleotides, using in vitro assays; To make use of both null and site-directed mutations in the PNPgene to study why PNPase appears to be required for photosynthesis; and to analyze plants defective in P sensing for effects on chloroplast gene expression, to address one aspect of how adaptation is integrated throughout the organism. Our new data show that P deprivation reduces cpRNA decay rates in vivo in a PNPasedependent manner, suggesting that PNPase is part of an organismal P limitation response chain that includes the chloroplast. As an essential component of macromolecules, P availability often limits plant growth, and particularly impacts photosynthesis. Although plants have evolved sophisticated scavenging mechanisms these have yet to be exploited, hence P is the most important fertilizer input for crop plants. cpRNA metabolism was found to be regulated by P concentrations through a global sensing pathway in which PNPase is a central player. In addition several additional discoveries were revealed during the course of this research program. The human mitochondria PNPase was explored and a possible role in maintaining mitochondria homeostasis was outlined. As polyadenylation was found to be a common mechanism that is present in almost all organisms, the few examples of organisms that metabolize RNA with no polyadenylation were analyzed and described. Our experiment shaded new insights into how nutrient stress signals affect yield by influencing photosynthesis and other chloroplast processes, suggesting strategies for improving agriculturally-important plants or plants with novel introduced traits. Our studies illuminated the poorly understood linkage of chloroplast gene expression to environmental influences other than light quality and quantity. Finely, our finding significantly advanced the knowledge about polyadenylation of RNA, the evolution of this process and its function in different organisms including bacteria, archaea, chloroplasts, mitochondria and the eukaryotic cell. These new insights into chloroplast gene regulation will ultimately support plant improvement for agriculture