Degradação de aminoácidos e sua associação com o metabolismo energético em Arabidopsis thaliana

Plant mitochondrion are involved in several key cellular processes that goesbeyond energy production being also associated with programmed cell death, fruit ripening and even light- associate process including phorespiration and photosynthesis. In this context, mitochondria acquisition by host cell brought evolutionary advances for the existing plant cell by the preservation of diverse metabolic pathways including both those related to energy metabolism as well as those associated with lipids, nucleotides and vitamin biosynthesis. The most notorious heritage is related to the tricarboxylic acid (TCA) cycle. The TCA cycle is an essential pathway which is related to reducing power (NADH and FADH2) generation, nitrogen assimilation and photosynthesis optimization. It has been suggested that the TCA cycle operated as isolated steps prior endosymbiosis events and that only after mitochondria acquisition it was possible for it to be organized and function as a cycle. The TCA cycle is composed by a set of eight enzymes. However, each enzyme is encoded by several genes which are targeting not just to mitochondria, but that are also imported into others subcellular compartments. These TCA enzymes located in other subcellular compartiments result in likely a broader connection between mitochondria and other organelles (e.g. peroxissome and chloroplast) allowing a bypass of the intermediates of the cycle switching his operation to an unusual in non-cyclic modes flux. It is also currently accepted that under stress conditions, which leads to decreases in carbohydrate levels, the TCA cycle can function in non-cyclic flux mode due to diminishing of carbon skeleton the enter it making required that be fed by anauplerotic reactions. Therefore, amino acids become essential to support respiration and ATP synthesis under such situations. Compelling evidence have demonstrated that branched chain amino acids (BCAA) and lysine can supply electrons to the mitochondrial electron transport chain (mETC) by the action of the electron transfer flavoprotein (ETF)-ETF: ubiquinone oxidoreductase (ETF/ETFQO) system and associated dehydrogenases. In plants, only isovaleryl- CoA dehydrogenase (IVDH) and (D)-2-hydroxyglutarate dehydrogenase (D2HGDH) have been characterized as electron donnor to the ubiquinol pool via this system so far by the degradation of BCAA and lysine, respectively. In fact, BCAA catabolism is of pivotal importance to provide intermediates to TCA cycle, particularly under stress situations, whereas lysine shows a strict association with the TCA cycle being required to couple amino acid degradation and energy generation. The electron transfer through the mETC is tightly coupled to ATP synthesis and use electron donates by NADH and FADH 2 to phosphorylate ADP to ATP. However, our knowledged regarding the organization of the mitochondrial oxidative phosphorylation (OXPHOS) system and its alternatives pathways under energy limitation remains elusive. Thus, this thesis, which is focused on the function of respiration within the context of the role of the TCA cycle as well as the function of alternative electron donors to the mETC, iscomprised by three independent stand-alone chapters focusing on energy metabolism and alternative respiration in Arabidopsis thaliana. Hence to obtain a compreenhesive picture of how the TCA cycle evolved and to which extend its alternative pathways interact to adjust to different cellular and metabolic requirements, three experimental approaches were used: (i) by using bioinformatic approaches we investigated the evolutionary history of TCA cycle genes allowing the generation of a model for the origin of the TCA cycle genes in plants and connected its evolution with TCA cycle behavior under a range of stress; (ii) the importance of lysine deficiency were investigated by using an Arabidopsis mutant with reduced activity of the lysine biosynthesis enzyme L,L-diaminopimelate aminotransferase (dapat), and (iii) the metabolic reprograming associated with the OXPHOS system were investigated following carbon limitation.. In brief, the results presented here provided several novel findings and allowed, at least preliminarly, mechanistic interpretation thereof. First, it facilitate the elucidation of the evolutionary origem of the TCA cycle in land plants providing support to the contention that the origin of isoforms present in different subcellular compartments might be associated either with gene-transfer events which did not result in correct targeting or with new gene copys generated by genome duplication and horizontal transfer gene. Additionally, coexpression analyses of TCA cycle genes following different stress conditions in both shoot and root tissues demonstrated the presence of a large molecular plasticity and provided an explanation for the modular operation of the TCA cycle in land plants. Secondly, by using an Arabidopsis mutant with reduced activity of the Lys biosynthesis enzyme L,L-diaminopimelate aminotransferase (dapat) it was demonstrated that lysine biosynthesis deficiency mimics stress situation and impacts both plant growth and leaf metabolism.Thirdly, by evaluating OXPHOS system behavior following carbon starvation and how a range of amino acids can impact respiratory complexes it was possible to further demonstrate that OXPHOS is affected in function of the carbon source and that alternative pathways are induced under this condition.In addition, immunoblotting assays revealed that OXPHOS system is most likely regulated by posttranslational modification. When considered together these results highlight the complexity and specificity of plant respiration during evolution and that it is differently affected following energy limitation by the usage of alternative substrates. The results discussed here support the contention that ETF/ETFQO is an essential pathway able to donate electrons to the mETC and that amino acids are alternative substrates maintaining respiration under carbon starvation.The results obtained are discussed in the context of current models of metabolic evolution showing the strict association of energy metabolism with amino acids metabolism, and where possible, mechanistic insights are properly discussed. Key-words: alternative substrate respiration; energy deprivation; mitochondria evolution; mitochondria metabolism; neofunctionalization; OXPHOS; paralogous genes; stress response; TCA cycle;Mitocondrias vegetais estão envolvidas em vários processos chaves da célula, vão além da produção de energica, tais como morte celular programada, amadurecimento de frutos, ou mesmo aqueles processos dependente de luz como fotossíntese e fotorrespiração. Dessa forma, aquisição mitocondrial pela célula hospedeira trouxe avanços para as atuais células vegetais: desde a manutenção de diversas vias metabólicas que incluem o metabolismo energético bem como processos de bissíntese de lipidios, nucleotidios e vitaminas. No tocante ao metabolismo energ- etico, destaca-se a herança do ciclo do ácido tricarboxílico. Este ciclo é uma via essencial relacionada com a produção de poder redutor (NADH e FADH 2 ), assimilação de nitrogênio e otimização da fotssíntese. Acredita-se que o ciclo do ácido tricarboxílico oprerasse como passos isoladados antes do processo endossimbiótico e somente após a aquisição da mitocôndria resultou que aquele organizar-se e atuasse como uma via cíclica. O cíclo do ácido tricarboxílico é composto por oito enzimas. Contudo, cada enzima é codificada por vários genes os quais são endereçados para diversos compartimetos celulares e, não somente, mitocôndrias. Essas enziimas locallizadas em diferentes comparimentos subcelulares acarretaram em uma possível ampla conecção entre mitocôndrias e outras organelas (peroxissomos e cloroplastos) permitindo fluxos alternativos dos intermediários do ciclo cujo resultado alterou seu funcionamento para um não convencional modo não cíclico. É bastante aceito que sob estresses, no quais reduzem os níveis de carboidratos. O ciclo do ácido tricarboxílico pode funcionar no modo não cíclico, devido a perda de esquelos carbônico que entram se fazendo necessário ser alimentado por reações anapleuróticas. Portanto, aminoácidos tornam-se fundamentais para suprir a respiração e síntese de ATP sob tais situações. Fortes evidencias demonstraram que aminoácidos de cadeia ramificada (BCAA) e lisina podem fornecer elétrons para o sistema a cadeia de transporte de elétrons mitocondrial pela ação do sistema flavoproteína de transferência de elétrons (ETF)- ETF: ubiquinona oxidorredutase (ETF/ETFQO). Em plantas, duas enzimas: Isovaleril-CoA desidorgenase (IVDH) e (D)-2-hidroxidoglutarato desidrogenase (D2HGDH) foram caracterizadas como doadores de elétrons para o pool de ubiquinone através do sistema ETF/ETFQO a partir da degradação de BCAA e lisina, respectivamente. Na verdade, o catabolismo de BCAA mostra-se de uma importância fundamental para nutrir o ciclo do ácido tricarboxílico, principalmente, em situações de estresse enquanto lisina mostra uma estreia associação com o ciclo do ácido tricarboxílico sendo importante para fazer um elo da degradação de aminoácido com a geração de energia. A transferência de elétrons através da fdoacadeia transportadora de elétrons mitocondrial acopla a síntese de ATP a partir da regeneração de NADH e FADH 2 para fosforilar ADP a ATP. Contudo, o conhecimento com relação a organização do sistema de fosforilação oxidativa (OXPHOS) e sua via alternativa sob limitação energética permanece escasso. Assim, essa tese, a qual se concentra no funcionamento da respiração em um contexto que o ciclo do ácido tricarboxílico e via alternativa como doador de elétrons para cadeia transportadora de elétrons mitocondrial, é composta por três independentes capítulos centrados no metabolismo energético e respiração alternativa em Arabidopsis thaliana. Por isso, para se obter uma visão global de como ocorre o envolvimento e interação do ciclo do ácido tricarboxílico juntamente da via alternativa para coordenar o ajustamento das necessidades metabólicas e celulares, três abordagens experimentais foram usadas (i) uma abordagem in silico, nós investigamos a história evolucionária dos genes do ciclo do ácido tricarboxílico gerando um modelo para a origem dos genes do ciclo em plantas bem como seu comportamento submetido a uma série de estresse; (ii) a importância da biossíntese de lisina foi investigado usando mutante de Arabidopsis com reduzida atividade da enzima L,L-diaminopimelato aminotransferase (dapat) da via biossintética de lisina; (iii) reprogramação metabólica do sistema OXPHOS associado a limitação de carbono foi investigado. Rapidamente, os resultados apresentados aqui forneceram resultados que permitiu, no mínimo um prévio, a elaboração de mecanismo do metabolismo energético junto a vias alternativas. Primeiramente, permitiu a elucidação da origem evolutiva dos constituintes do ciclo do ácido tricarboxílico em plantas fornecendo elemento para a origem das isoformas presentes nos diferentes compartimentos subcelulares os quais que devem ser associados com eventos de transferência gênica ou com novas cópias geradas por duplicação genômica. Ademais, análises de co-expressão dos genes do ciclo em diferentes condições estressantes em ambos tecidos parte aérea e raiz demonstrou a presença de plasticidade molecular e forneceu uma explicação para o funcionamento do ciclo do ácido tricarboxílico em plantas. Após isso, o uso de Arabidopsis mutante com reduzida atividade para biossíntesi de lisina L,L-diaminopimelato aminotransferase (dapat) foi demonstrada que biossíntese de lisina simula condições de estresse e impacta no crescimento e metabolismo foliar. Por fim, uma avaliação de como o comportamento do sistema OXPHOS sob limitação de carbono e como vários aminoácidos podem impactar os complexos respiratórios foi possível demonstrar que o sistema OXPHOS tem sua função afetada por diferentes fontes de carbono e que vias alternativas são induzidas sob essas condições. Ademais, imunoensaios revelaram que é mais provável ser regulado por modificações pós traducionais. Juntos, esses resultados realçam a complexidade e especificidade da respiração vegetal durante evolução e que é differentemente afetado por linitações energéticas e pelo uso de substratos alternativos. Os resultados discutidos aqui suportam que ETF/ETFQo é uma via essencial capaz de doa elétrons para a cadeia transportadora de elétrons e que amioácidos são substratos alternativos para manter a respiração sob limitação de carbono. Os resultados obtidos são discutidos em um contexto de evolução metabólica mostrando estreia associação da metabolismo energético com metabolismo de aminoácidos e onde possível mcanísticos são devidamente discutidos. Palavras chaves: ciclo do ácido tricarboxílico; escassez de energia; evolução mitochondrial; fosforilação oxidativa; genes parálogos, metabolismo mitocondrial; neofuncionalização; respiração; resposta a estresse; substratos alternativosFundação de Amparo à Pesquisa do Estado de Minas Gerai

Cyanobacterial nitrogenases: phylogenetic diversity, regulation and functional predictions

Abstract Cyanobacteria is a remarkable group of prokaryotic photosynthetic microorganisms, with several genera capable of fixing atmospheric nitrogen (N2) and presenting a wide range of morphologies. Although the nitrogenase complex is not present in all cyanobacterial taxa, it is spread across several cyanobacterial strains. The nitrogenase complex has also a high theoretical potential for biofuel production, since H2 is a by-product produced during N2 fixation. In this review we discuss the significance of a relatively wide variety of cell morphologies and metabolic strategies that allow spatial and temporal separation of N2 fixation from photosynthesis in cyanobacteria. Phylogenetic reconstructions based on 16S rRNA and nifD gene sequences shed light on the evolutionary history of the two genes. Our results demonstrated that (i) sequences of genes involved in nitrogen fixation (nifD) from several morphologically distinct strains of cyanobacteria are grouped in similarity with their morphology classification and phylogeny, and (ii) nifD genes from heterocytous strains share a common ancestor. By using this data we also discuss the evolutionary importance of processes such as horizontal gene transfer and genetic duplication for nitrogenase evolution and diversification. Finally, we discuss the importance of H2 synthesis in cyanobacteria, as well as strategies and challenges to improve cyanobacterial H2 production

Evolution and functional implications of the tricarboxylic acid cycle as revealed by phylogenetic analysis

The tricarboxylic acid (TCA) cycle, a crucial component of respiratory metabolism, is composed of a set of eight enzymes present in the mitochondrial matrix. However, most of the TCA cycle enzymes are encoded in the nucleus in higher eukaryotes. In addition, evidence has accumulated demonstrating that nuclear genes were acquired from the mitochondrial genome during the course of evolution. For this reason, we here analyzed the evolutionary history of all TCA cycle enzymes in attempt to better understand the origin of these nuclear-encoded proteins. Our results indicate that prior to endosymbiotic events the TCA cycle seemed to operate only as isolated steps in both the host (eubacterial cell) and mitochondria (alphaproteobacteria). The origin of isoforms present in different cell compartments might be associated either with gene-transfer events which did not result in proper targeting of the protein to mitochondrion or with duplication events. Further in silico analyses allow us to suggest new insights into the possible roles of TCA cycle enzymes in different tissues. Finally, we performed coexpression analysis using mitochondrial TCA cycle genes revealing close connections among these genes most likely related to the higher efficiency of oxidative phosphorylation in this specialized organelle. Moreover, these analyses allowed us to identify further candidate genes which might be used for metabolic engineering purposes given the importance of the TCA cycle during development and/or stress situations

Identification of duplicated and stress-inducible Aox2b gene co-expressed with Aox1 in species of the Medicago genus reveals a regulation linked to gene rearrangement in leguminous genomes

In flowering plants, alternative oxidase (Aox) is encoded by 3-5 genes distributed in 2 subfamilies (Aox1 and Aox2). In several species only Aox1 is reported as a stress-responsive gene, but in the leguminous Vigna unguiculata Aox2b is also induced by stress. In this work we investigated the Aox genes from two leguminous species of the Medicago genus (Medicago sativa and Medicago truncatula) which present one Aox1, one Aox2a and an Aox2b duplication (named here Aox2b1 and Aox2b2). Expression analyses by semi-quantitative RT-PCR in M. sativa revealed that Aox1, Aox2b1 and Aox2b2 transcripts increased during seed germination. Similar analyses in leaves and roots under different treatments (SA, PEG, H2O2 and cysteine) revealed that these genes are also induced by stress, but with peculiar spatio-temporal differences. Aox1 and Aox2b1 showed basal levels of expression under control conditions and were induced by stress in leaves and roots. Aox2b2 presented a dual behavior, i.e., it was expressed only under stress conditions in leaves, and showed basal expression levels in roots that were induced by stress. Moreover, Aox2a was expressed at higher levels in leaves and during seed germination than in roots and appeared to be not responsive to stress. The Aox expression profiles obtained from a M. truncatula microarray dataset also revealed a stress-induced co-expression of Aox1, Aox2b1 and Aox2b2 in leaves and roots. These results reinforce the stress-inducible co-expression of Aox1/Aox2b in some leguminous plants. Comparative genomic analysis indicates that this regulation is linked to Aox1/Aox2b proximity in the genome as a result of the gene rearrangement that occurred in some leguminous plants during evolution. The differential expression of Aox2b1/2b2 suggests that a second gene has been originated by recent gene duplication with neofunctionalization. © 2013 Elsevier GmbH. All rights reserved