Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled anaplerotic enzyme situated at a pivotal branchpoint of plant C-metabolism. Plant genomes encode several closely related plant-type PEPC (PTPC) isozymes, and a distantly related bacterial-type PEPC (BTPC). Two physically and kinetically distinct oligomeric classes of PEPC occur in the endosperm of developing castor oil seeds (COS). Class-1 PEPC is a typical homotetramer composed of 107-kDa PTPC subunits, whereas the novel 910-kDa Class-2 PEPC hetero-octameric complex arises from a tight interaction between Class-1 PEPC and 118-kDa bacterial-type PEPC (BTPC) subunits. BTPC functions as a catalytic and regulatory subunit of the allosterically-desensitized Class-2 PEPC, hypothesized to support PEP-flux to malate for leucoplast fatty acid synthesis. Previous studies established that BTPC: (i) subunits of COS Class-2 PEPC are phosphorylated at multiple sites in vivo and (ii) phosphorylation at Ser425 provides a new tier of enzyme control in developing COS. LC MS/MS and LTQ-FT MS identified Thr4 and Ser451 as additional in vivo phosphorylation sites of immunopurified COS BTPC (corresponding to acidophilic and basophilic protein kinase consensus sequences, respectively). Immunoblots probed with a phosphorylation-site specific antibody raised against a synthetic phosphopeptide indicated that Ser451 phosphorylation is promoted during seed development, becoming maximal in stage VII (full cotyledon) COS. Although several pThr4 containing BTPC peptides were non-immunogenic, the collective results indicate that Thr4 is also phosphorylated in vivo. Kinetic effects of each phosphorylation site were examined using phospho-mimetic mutants of heterologously expressed COS BTPC. BTPC’s phosphorylation at Ser451 appears to be inhibitory, as reflected by significantly increased Km(PEP) values, and reduced I50(malate) and I50(Asp) values of a S451D mutant. By contrast, kinetic characterization of a T4D phosphomimetic mutant indicated that Thr4 phosphorylation is not regulatory in nature. However, Thr4 exists in a conserved forkhead-associated (FHA) binding domain (pTXXD) that has received considerable prominence as a phospho-Thr dependent protein interaction module. These results further our understanding of multisite phosphorylation of BTPC in developing COS and its possible contribution to the control of Class-2 PEPC activity. / Thesis (Master, Biology) -- Queen's University, 2011-08-22 19:05:03.077

Strategic procurement items, including complex engineered equipment and systems essential for project performance, are frequently designed, manufactured, and delivered by suppliers who are outside the circle of cooperation between owner, engineer, and contractor. When suppliers are excluded from the design and planning stages of a project, much of the knowledge needed for successful design and integration is lost or underutilized. This research was done as part of a Construction Industry Institute sponsored project to develop and quantify a step change to the EPC process that will bring the supplier into the circle of cooperation between the owner, engineer, and contractor. The result was a step change entitled PEpC (Procure, Engineer, procure, and Construct). This research also sought to provide implementation guidelines for the recommended step change. Through an examination of the literature, a survey of industry experts, and the review of four case studies, this research found that PEpC, the step change recommended by the Construction Industry Institute research team, may reduce both the time and cost required to complete a project. The anticipated savings in project duration is expected to fall between 10 and 15 percent, while the anticipated reduction in project direct labor cost is expected to fall between 4 and 8 percent. / Master of Science

Co-immunoprecipitation (co-IP) followed by proteomic analysis was employed to examine the phosphoenolpyruvate carboxylase (PEPC) interactome of developing castor oil seed (COS) endosperm. Earlier studies suggested that immunologically unrelated 107-kDa plant-type and 118-kDa bacterial-type PEPCs (p107/PTPC and p118/BTPC, respectively) are subunits of an unusual ~910-kDa hetero-octameric Class-2 PEPC complex of developing COS. The current results confirm that a tight physical interaction occurs between p118 and p107 since p118 quantitatively co-IP’d with p107 following elution of COS extracts through an anti-p107-IgG immunoaffinity column. No PEPC activity or immunoreactive PTPC or BTPC polypeptides were detected in the corresponding flow-through fractions. Although BTPCs lack the N-terminal phosphorylation site characteristic of PTPCs, Pro-Q Diamond Phosphoprotein staining, immunoblotting with phospho-(Ser/Thr) Akt substrate IgG, and phosphate-affinity PAGE demonstrated that the co-IP’d p118 was significantly phosphorylated at unique Ser and/or Thr residue(s). The co-IP of p118 and p107 was not influenced by their phosphorylation status. As p118 phosphorylation appeared unchanged 48 h following elimination of photosynthate supply due to COS depodding, the signaling mechanisms responsible for photosynthate-dependent p107 phosphorylation differ from those controlling p118’s in vivo phosphorylation. A third PEPC polypeptide of ~110-kDa (p110; RcPPC1) co-IP’d with p118 and p107 when depodded COS was used. Analysis of RcPpc1’s full-length cDNA sequence revealed p110’s identity with PTPCs, but that a pair of unique amino-acid substitutions occurs in its N-terminal sequence that may render p110 non-phosphorylatable in vivo. The plastidial pyruvate dehydrogenase complex (PDCpl) was identified as a novel PEPC interactor. Subcellular fractionation indicated that p118 and p107 are strictly cytosolic, but that PDCpl is targeted to both the cytosol and leucoplast of developing COS. Thus, a putative cytosolic metabolon involving PEPC and PDCpl could function to channel carbon from phosphoenolpyruvate to acetyl-CoA and/or to recycle CO2 from PDCpl to PEPC. / Thesis (Master, Biology) -- Queen's University, 2007-09-26 15:57:52.216

Phosphoenolpyruvate (PEP) carboxylase (PEPC) is a tightly controlled enzyme situated at a pivotal branchpoint of plant C-metabolism. Two physically and kinetically distinct oligomeric classes of PEPC exist in the endosperm of developing castor oil seeds (COS). Class-1 PEPC is a typical homotetramer composed of 107-kDa plant-type PEPC (PTPC) subunits, whereas the 910-kDa Class-2 PEPC hetero-octameric complex arises from a tight interaction between Class-1 PEPC and distantly related 118-kDa bacterial-type PEPC (BTPC) subunits. BTPC functions as both a catalytic and regulatory subunit of the allosterically-desensitized Class-2 PEPC, which has been hypothesized to support massive PEP-flux to malate for leucoplast fatty acid synthesis. Previous studies established that BTPC: (i) subunits of COS Class-2 PEPC are subject to inhibitory phosphorylation in vivo, and (ii) at Ser425 and Ser451 within an intrinsically disordered region. This study focuses on characterization of the COS protein kinase (BTPC-K) that phosphorylates BTPC at Ser451. BTPC-K, having a native molecular mass of 63 kDa, was purified ~500-fold from developing COS endosperm. Its activity was absolutely dependent upon the presence of Ca2+ (Ka= 2.7 μM) and millimolar Mg2+. BTPC-K phosphorylated BTPC subunits of Class-2 PEPC strictly at Ser451 (Km= 1.1 μM), as well as histone type III-S (Km= 1.7 μM), but did not phosphorylate a BTPC S451D phosphomimetic mutant, native COS PTPC or sucrose synthase, or α-casein. BTPC-K displayed a broad pH-activity optima of pH 7.3, a Km for Mg2+-ATP of 6.6 μM, and marked inhibition by 3-P-glycerate and PEP. The possible control of BTPC-K by disulfide-dithiol interconversion was suggested by its rapid inactivation and subsequent reactivation when incubated with oxidized glutathione and then dithiothreitol. BTPC-K activity was insensitive to exogenous calmodulin, but potently inhibited by 100 µM trifluoperazine (a calmodulin antagonist). BTPC-K-mediated Ser451 phosphorylation of BTPC subunits of Class-2 PEPC inhibited BTPC activity by ~50% when assayed under suboptimal conditions (pH 7.3, 1 mM PEP with 10 mM L-malate). Overall the results of this study have led to the hypothesis that in vivo phosphorylation of COS BTPC at Ser451 is mediated by a dedicated calcium-dependent protein kinase (CDPK). / Thesis (Master, Biology) -- Queen's University, 2013-08-30 14:23:39.648

Submitted by Secretaria Pós de Produção (tpp@vm.uff.br) on 2017-07-27T19:41:59Z No. of bitstreams: 1 D2014 - Carlos Eduardo Ribeiro de Barros Barateiro .pdf: 6039808 bytes, checksum: 44d3013c2830bb2b96f353a17b0bf002 (MD5) / Made available in DSpace on 2017-07-27T19:41:59Z (GMT). No. of bitstreams: 1 D2014 - Carlos Eduardo Ribeiro de Barros Barateiro .pdf: 6039808 bytes, checksum: 44d3013c2830bb2b96f353a17b0bf002 (MD5) / O objetivo principal deste trabalho é o desenvolvimento de um modelo de implementação da automação destinada ao controle de processos industriais em plantas petroquímicas, cujos projetos são enquadrados como complexos pelas suas particularidades. O modelo criado aplica-se no conceito do Main Automation Contractor (MAC – Contratista Principal da Automação) onde uma única empresa é responsável pela automação de todo o empreendimento. Este trabalho é uma continuação da dissertação de mestrado do autor onde foram encontrados os construtos que justificam a elaboração do modelo proposto. O levantamento de dados foi baseado em pesquisa bibliográfica e entrevistas semi-estruturadas com o corpo gerencial de mais alto nível das empresas envolvidas na construção de três grandes plantas petroquimicas. Os resultados foram obtidos a partir de uma abordagem qualitativa com aplicação de uma metodologia de análise de conteúdo e serviram para levantamento das condições de aplicação e a validação do modelo. A pesquisa bibliográfica exploratória teve por finalidade obter maior conhecimento sobre os temas principais incluindo uma análise sobre a evolução da automação e dos modelos de contratação praticados pela indústria, além da análise dos impactos da automação no ciclo de vida desses projetos e das metodologias de seleção de fornecedores. A pesquisa concluiu sobre a viabilidade de utilização do modelo proposto que é composto de três partes: a) a escolha do fornecedor do MAC; b) sua participação nas fases iniciais do projeto (FEED - Front End Engineering and Design) e; c) o processo de implementação do projeto de automação. A automação para o controle de processos industriais, principalmente destinado a grandes plantas petroquimicas é de fundamental importância para a otimização dos resultados desses investimentos e o modelo apresentado é suficientemente robusto, permitindo uma padronização das atividades e a minimização de subjetividades que podem ocorrer sem apoio de uma metodologia adequada. / The main objective of this work is the development of an automation implementation model designed to industrial process control in petrochemical plants, whose projects are classified as complex by its peculiarities. The created model applies the concept of Main Automation Contractor (MAC) which one company is responsible for automation of the entire project. This work is a continuation of the master science program of author that has found the constructs that justify the proposed model. The survey was based on a literature review and semi-structured interviews with the top management team of companies involved in construction of three large petrochemical plants. The results were obtained by a qualitative approach with application of a content analysis methodology and were used to identify the application conditions and model validation. The exploratory literature search aimed to gain more knowledge about key topics including a discussion on the automation evolution and contract models practiced by the industry, besides studying the automation impacts in the life cycle of these projects and methods of supplier selection. The research concluded on the feasibility of the proposed model, which is composed of three parts: a) the choice of the MAC vendor; b) their participation in the early stages of design (FEED - Front End Engineering and Design) and; c) the process of implementation of the automation project. Automation for industrial process control, mainly to large petrochemical plants, is crucial for optimizing the results of these investments and the model is sufficiently robust, allowing standardization activities and to minimize subjectivity that can occur without the support of an appropriate methodolog.

In this project the effect of viral infection on the metabolism of nitrogenous compounds in tobacco plants (Nicotiana tabacum L. cv. Petit Havana SR1) was studied. The tobacco plants were infected with Potato virus Y, strain NTN, ELISA confirmed the presence of the virus. Enzymes that participate in C4 plants in Hatch-Slack cycle fosfoenolpyruvate carboxylase (EC 4.1.1.31, PEPC), NADP-dependent malic enzyme (EC 1.1.1.40, NADP-ME), pyruvate, phosphate dikinase (EC 2.7.9.1, PPDK) are present also in C3 plants and are related to plant responses to stress conditions. As a result of viral infection, the activities of all these enzymes were increased. Infection caused by PVYNTN decreased activity of nitrate reductase (EC 1.7.1.1, NR), an enzyme catalyzing reduction of nitrates to nitrites. The activity of enzymes catalyzing the synthesis of glutamine from glutamate and ammonium ions: glutamine synthetase (EC 6.3.2.1, GS) and glutamate synthase (EC 1.4.1.14, GOGAT) was enhanced. In addition to this main route of nitrogen fixation the plant can still use glutamate dehydrogenase (EC 1.4.1.2, GDH). This enzyme can also catalyze the opposite reaction, deamination of glutamate. The direction of response depends on environmental conditions. In this case a significant increase of oxidative-deaminating activity...

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Plants exhibit a flexible array of morphological, physiological and biochemical adaptations during phosphorous limitation. Legumes are vulnerable to P deficiency, because it affects their ability to fix atmospheric nitrogen (N2). In particular, legumes from nutrient-poor ecosystems, such as the Fynbos in the Cape Floristic Region (CFR) evolved on P deficient soils and may therefore display unique adaptations to soil P stress. In general, very few studies on legumes have focussed on the belowground structures of nodules as a plant organ. Moreover, even less is known about the P stressed responses in nodules from legumes in nutrient-poor ecosystems. The aim of this research was to investigate the metabolic flexibility of organic acid and amino acid metabolism in the nodulated root system of the Fynbos legume Virgilia divaricata, during low P stress. Virgilia divaricata, which grows in the Cape Floristic Region, was used in this study to enhance our knowledge regarding the vital role that the cytosolic enzyme, phosphoenol pyruvate carboxylase (PEPC) plays in phosphoenol pyruvate (PEP) metabolism, in roots and nodules of this legume during phosphate stress. V. divaricata was grown under glasshouse conditions (20 - 25°C) in sterilized quartz sand for 2-3 months whilst being inoculated with the nitrogen fixation bacteria, Burkholderia phytofirmans, which was isolated from V. divaricata nodules grown in fynbos soil. Two phosphate treatments, 5 μM and 500 μM, were applied simulating low-phosphate and high phosphate conditions respectively using a modified Long Ashton Nutrient Solution to simulate a low nutrient ecosystem such as the Cape Floristic Region. Roots and nodules were then analysed for growth kinetics, nutrient acquisition and distribution, enzyme activity and genetic responses. It was shown that during phosphate deficiency, V. divaricata nodules experienced less Pi stress than roots, due to increased metabolic phosphate conservation reactions during organic acid synthesis via an increased PEPC activity. The increased PEPC activity resulted in an increase in downstream metabolic products such as organic acids, (malic acid and citric acid), and amino acids (glutamate, aspartate and asparagine). Although the biological nitrogen fixation (BNF) declined, the high efficiency of BNF may be underpinned by these altered phosphate conservation pathways and enhanced resource allocation during growth particularly under low phosphate (LP) conditions. Therefore, it can be concluded that the efficiency of the nodules via an increased allocation of resources and P acquiring mechanisms in V. divaricata may be the key to the plant’s ability to adapt to poor P environments and thus sustaining its reliance on BNF. From the data obtained as well as previous findings, it has been established that the phosphate conservation mechanisms in roots and nodules, involve the non-adenylate requiring PEPC-bypass route. 13C Nuclear magnetic resonance (NMR) gave us a better understanding regarding the incorporation rates of the PEPCderived C into malate, α-ketoglutarate and asparagine. It therefore is suggested that V. divaricata nodules may use their large PEPC-derived malate pool to prevent large declines in BNF under low phosphate conditions. The nodules of V. divaricata were able to offset an excessive drop in BNF, despite a decline in inorganic phophosphate (Pi) levels. It therefore appears that nodules have evolved to acquire different mechanisms than roots to adapt to phosphate deficiency in order to maintain their function. This was achieved via increased regulation of nodule PEPC and its downstream products. This implies that compared to roots under low P, nodules alter the metabolism of PEPC derived C, in order to maintain nodule respiration and amino acid synthesis. This trait could be observed in the synthesis of larger 13C malate pools of nodules compared to roots, from PEPC, which was underpinned by their different regulation mechanisms of enzyme activity, of the same protein isoform. Since malate is a potent inhibitor of PEPC activity, roots appear to have invested in more PEPC protein compared to nodules. In contrast, nodules with lower PEPC protein, achieved greater enzyme activity than roots, possibly due to higher phosphorylation in order to reduce the malate effect. The subsequent metabolism of this PEPCderived malate, caused roots and nodules to synthesise asparagine via different pathways. These findings imply that roots and nodules under P stress, synthesise their major export amino acid, asparagine, via different routes. This research has generated new knowledge regarding the physiological impact of the organic and amino acid metabolism, derived from PEPC-C in the roots and nodules of legumes growing in nutrient poor ecosystems. It has demonstrated for the first time that the nodules of legume from a nutrient-poor ecosystem rely on improved resource allocation, Pi distribution, and PEPC-derived organic acids to maintain the efficient functioning of N assimilation under P stress. This may be a consequence of having evolved in a nutrient-poor ecosystem, so that nodule-bacteroid respiration and N metabolism can be maintained in P-poor soils such as the Fynbos. / AFRIKAANSE OPSOMMING: Tydens fosfaat stremming maak plante gebruik van buigsame kombinasies van morfologiese, fisiologiese en biochemiese aanpassings. Peulplante is sensitief vir fosfaat tekorte omdat dit die vermoë om atmosferiese stikstof te kan fikseer, grootliks beïnvloed. Peulplante vanuit ekosisteme met mineraal-arme gronde, soos Fynbos binne die Kaapse Blommeryk, het ontwikkel in grond met lae fosfaatvlakke en mag dus unieke aanpassings tot fosfaat tekorte toon. Oor die algemeen is daar baie min peulplant studies wat fokus op die ondergrondse strukture van wortelknoppies as ‘n plant orgaan. Nog minder inligting is beskikbaar oor wortelknoppies, van peulplante, vanuit mineraalarme ekosisteme, se reaksie teenoor ‘n fosfaat tekort. Die doel van hierdie navorsing was om die metaboliese buigsaamheid van organiese- en aminosuur metabolisme in die (nodulated) wortelknoppie-wortelstelsel van die Fynbos peulplant Virgilia divaricata, tydens fosfaat tekort te ondersoek. Virgilia divaricata wat voorkom in die Kaapse Blommeryk, was gebruik in hierdie studie om die huidige kennis te verbeter van die essensiële rol wat die sitisoliese ensiem, fosfo-enol piruvaat karboksilase (PEPC) in fosfo-enol piruvaat metabolisme tydens ‘n fosfaat tekort speel binne die wortels en wortelknoppies van hierdie peulplant. V. divaricata was gegroei onder glashuis toestande (20 - 25°C) in gesteriliseerde kwartssand vir 2-3 maande. Die plante was geïnokuleer met die stikstoffikserende bakterie, Burkholderia phytofirmans, wat geïsoleer is vanaf V. divaricata wortelknoppies wat in Fynbos grond gegroei is. Twee fosfaatbehandelings, 5μM and 500μM, was toegedien om lae en hoë fosfaat toestande, onderskeidelik, na te boots deur gebruik te maak van ‘n aangepasde Long Ashton voedingstofmengsel om ‘n ekosisteem, soos die Kaapse Blommeryk, met lae voedingstofvlakke na te boots. Die wortels en knoppies was geanaliseer ten opsigte van die groeikinetika, opname en verspreiding van voedingstowwe, ensiemaktiwiteit en genetiese aanpassings. Dis is bewys dat tydens fosfaat tekort V. divaricata wortelknoppies minder fosfaat stres ervaar as die wortels, as gevolg van die verhoogde metaboliese fosfaat bewaringsreaksies tydens organise suur sintese via die styging in PEPC aktiwiteit. Die styging in PEPC aktiwiteit lei tot ‘n verhoging in stroomaf metaboliese produkte soos organiese- (appel- en sitroënsuur) en aminosure (glutamaat, aspartaat en asparagien). Alhoewel biologiese stikstoffiksering verlaag het, kan die hoë doeltreffendheid daarvan ondersteun word deur díe aangepasde fosfaat bewarings weë asook verhoogde hulpbron toekenning tydens groei onder lae fosfaat omstandighede. Dit kan dus afgelei word dat die doeltreffendheid van die wortelknoppies via die verhoging in belegging van hulpbronne en fosfaat opname meganismes in V. divaricata moontlik die sleutel is tot die plant se vermoë om aan te pas tot omgewings met lae fosfaatvlakke en sodoende die afhanklikheid van biologiese stikstofbinding te kan onderhou. Data in hierdie as ook vorige studies, wys dat die fosfaat bewaringsmeganismes in wortels en wortelknoppies die PEPC-ompad roete, wat nie adenilaat benodig nie, gebruik. 13C NMR het meer lig gewerp aangaande die vaslegging van koolstof vanaf PEPC na malaat, α-ketoglutaraat en asparagien. Dit word voorgestel dat V. divaricata knoppies ‘n groot hoeveelheid malaat, afkomstig van PEPC-werking, gebruik om groot dalings in biologiese stikstofbinding tydens fosfaat tekort, te verhoed. Die wortelknoppies van V. divaricata kon ‘n oormatige verlaging in biologiese stikstofbinding voorkom ten spyte van die verlaging in fosfaatvlakke. Dit wil voorkom dat wortelknoppies ander meganismes as die wortels ontwikkel het om aan te pas tot fosfaat tekort en sodoende dus hul funksie behou. Dit word bereik deur ‘n verhoging in die regulering van PEPC en die stroomaf produkte in die wortelknoppies. Dit blyk dat wortelknoppies tydens fosfaat te kort, in vergelyking met wortels, die metabolisme van die koolstof vanaf PEPC verander om sodoende respirasie en aminosuursintese te onderhou. Dit wil voorkom dat hierdie meganismes verskil van die van wortel meganismes. Hierdie eienskap kan toegeskryf word aan die produksie van ‘n groter hoeveelheid van 13C malaat vanaf PEPC in die wortelknoppies teenoor die wortels, wat ondersteun word die verskillende reguleringsmeganismes van ensiemaktiwiteit van dieselfde proteïen isoform. Malaat is ‘n kragtige inhibeerder van PEPC-aktiwiteit, dus blyk dit dat die wortels belê in meer PEPC proteïene as die wortelknoppies. In teenstelling, toon die wortelknoppies met laer PEPC proteïene, ‘n hoër ensiem aktiwiteit as die wortels. Dit kan wees as gevolg van hoër fosforilasie om die effek van malaat te verlaag. Die metabolisme van die malaat vanaf PEPC het die sintese van asparagien in die wortels en wortelknoppies via verskillende roetes tot gevolg gehad. Dit impliseer dat tydens ‘n tekort aan fosfaat, wortels en wortelknoppies hul hoof uitvoer aminosuur, asparagien, deur verskillende roetes sintetiseer. Hierdie studie het nuwe kennis aangaande die fisiologiese impak van organiese- en aminosuur metabolisme met koolstof vanaf PEPC in die wortels en wortelknoppies van peulplante wat voorkom in ekosisteme met lae voedingstofvlakke, voortgebring. Vir die eerste keer is dit bewys dat die wortelknoppies vanaf peulplante wat voorkom in mineraal-arme ekosisteme, staatmaak op verbeterde hulpbron beleggings, fosfaat verspreiding en organiese sure vanaf PEPC om die doeltreffendheid van funksionele stikstofassimilasie tydens fosfaat tekort, te onderhou. Dit mag die gevolg wees van, om in ‘n voedingstof arme ekosisteem te ontwikkel sodat die wortelknoppiebakteroïed respirasie en stikstofmetabolisme onderhou kan word in fosfaat arme grond soos die Fynbos.

The metabolism of plants grown in the cultivation medium is influenced by its composition and availability of CO2. In this project the effect of cultivation medium on the activity phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), NADP-malic enzyme (NADP-ME, EC 1.1.1.40), pyruvate, phosphate dikinase (PPDK, EC 2.7.9.1) and enzymes of nitrogen metabolism: nitrate reductase (NR, EC 1.7.1.1), glutamine synthetase (GS, EC 6.3.1.2), glutamate synthase (GOGAT, EC 1.4.1.13) and glutamate dehydrogenase (GDH, EC 1.4.1.2) was studied. The tobacco plant Nicotiana tabacum L., cv. Petit Havana SR1 were grown in vitro in containers fitted with a filter, which caused limited access of CO2. The cultivation medium was modified Murashige-Skoog agar with decreased amounts of phosphate, nitrate, ammonium, or with casein as source of nitrogen and with or without of 1.5% sucrose as additional carbon source Activity of PEPC was higher in plants grown in medium in the presence of sucrose. Reduced concentrations of phosphates, nitrates or NH4 + ions or when casein was the only source of N in medium caused decreased activity of PEPC. Under these conditions, also activity of NADP-ME and of enzymes of nitrogen metabolism: NR, GS decreased. Activity of all enzymes was also negatively affected by limited CO2. On the other...

L'ozone (O3), polluant atmosphérique et gaz à effet de serre, est responsable des pertes de production pour plusieurs espèces végétales. Actuellement, les seuils de risques ne prennent pas en considération la capacité de détoxication intrinsèque des cellules, qui dépend en partie de la régénération du NADPH. Dans cette dernière perspective, l'étude des différentes NADP-déshydrogénases cytosoliques dans les feuilles de deux génotypes de peuplier euraméricain (Populus deltoides x Populus nigra), montre que les activités de la cG6PDH (glucose-6-phosphate déshydrogénase) et de la ME (enzyme malique) sont plus élevées chez le génotype tolérant Carpaccio en réponse à un traitement O3 (120 ppb pendant 17 jours). Dans ces conditions, le maintien des teneurs en NADPH chez Carpaccio, nous a permis de conclure que la capacité des cellules à régénérer ce nucléotide, contribue à améliorer la tolérance face à l'O3. Par une approche de génétique inverse chez Arabidospis, nous avons étudié l'importance de l'activité isocitrate déshydrogénase (ICDH) et de la glutathion réductase 1 (GR1) sous O3, dans un contexte de modulation de la durée du jour. Nous montrons ainsi pour les différents génotypes une plus grande sensibilité à l'O3 lorsque les plantes croissent sous un régime de jours longs, nous soulignons l'interconnexion entre déshydrogénases NADPdépendantes chez le mutant icdh et nous mettons en évidence un possible rôle du glutathion dans le contrôle des interactions entre les fonctions de signalisation oxydante et antioxydante chez le mutant gr1. Enfin, nous avons montré que le monoxyde d'azote (NO) pouvait être en partie impliqué dans la régulation post-traductionnelle de la PEPC sous O3. En effet, nous avons montré que le SNP (donneur de NO) mime les effets de l'O3 alors que le prétraitement de rcd1, mutant surproducteur de NO sous O3, avec du cPTIO + LNNA prévient partiellement ces effets. De plus, l'incubation in vitro d'extraits protéiques foliaires avec du GSNO stimule fortement l'activité PEPC. Tous ces résultats corroborent l'hypothèse que sous O3, l'activité PEPC pourrait être modulée via S-nitrosylation. Cette hypothèse est confortée par la prédiction dans la séquence d'acides aminés de la PEPC de trois cystéines S-nitrosylées, très conservées chez les plantes / Ozone (O3), both an air pollutant and a greenhouse gas, is responsible for yield production losses. Current flux based threshold indices do not take in consideration intrinsic cell capacity for detoxification, which partly depends on NADPH regeneration. In this optic, the study of the NADP-dehydrogenases within leaves of two hybrid poplar (Populus deltoides x Populus nigra) genotypes showed that the activation of cG6PDH (glucose-6-phosphate dehydrogenase) and of ME (malic enzyme) was higher in the tolerant genotype (Carpaccio) in response to O3 treatment (120 ppb for 17 days). The maintenance of NADPH levels in Carpaccio, supported the hypothesis that the capacity for cells to regenerate this nucleotide, contributes to improve tolerance to O3.To investigate the importance of a specific cytosolic NADP?dehydrogenase, we used a reverse genetic approach to test isocitrate dehydrogenase (icdh) and glutathione reductase 1 (gr1) mutants to O3 in a context of day?length modulation. Our study reveals an important role for day length conditions in influencing O3 responses with extended damage for all the genotypes in long days, we pointed to overlapping functions of NADP-dependent dehydrogenases in icdh mutants, and highlights novel roles for the glutathione system in controlling the interactions between antioxidative and oxidative signaling functions in gr1 mutant. Finally, we showed that nitric oxide (NO) could be partly implied in the posttranslational regulation of PEPC under ozone. Treatment of the wild ecotype Col?0 with the NO donor SNP mimicked the O3 effects concerning development of visible injuries and the stimulation of PEPC activity. The pre-treatment of the NO-overproducer mutant rcd1, with cPTIO+L-NNA partially prevented both of these O3 effects. Additionally, in vitro incubation of a protein extract with GSNO strongly enhanced PEPC activity. All together, these results corroborated the hypothesis of the modulation of PEPC activity via Snitrosylation under O3, which is reinforced by the presence of three predicted and highly preserved Snitrosylated cysteines in the protein sequence

EEffffeecctt ooff ddrroouugghhtt oonn tthhee mmeettaabboolliissmm ooff ttaabbaaccccoo ppllaannttss (Nicotiana tabacum L.) Diploma thesis - abstract Lucia Miedzińska, Bc. Work Supervisor: Doc. RNDr. Helena Ryšlavá, CSc. Consultant: RNDr. Veronika Doubnerová, PhD. Drought stress is one of the most frequent form of plant stress, which occurs not only under condition of limited water availability, but also under reduced ability to accept water by roots, for example in salted or cold soils. In this thesis the changes in enzyme activities of: NADP-malic enzyme (EC 1.1.1.40; NADP-ME), phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPC) and pyruvate, phosphate dikinase (EC 2.7.9.1; PPDK) in tobacco plants (Nicotiana tabacum L., cv. W38) after drought were investigated. Enzyme activities in tobacco leaves were significantly increased during 11 days of stress, PEPC 2-fold, PPDK 3,3- fold and NADP-ME 4-fold compared to control plants. The regulation of NADP-ME and PEPC activities were studied on transcriptional level - by the real-time PCR method and on translational level - immunochemically. The expression of NADP-ME protein and transcription of mRNA for chloroplast NADP-ME isoform were increased, but mRNA for cytosolic isoform was not affected. The protein expression of PEPC was slightly increased, transcription of...