PEPC [PEP(phosphoenolpyruvate) carboxylase] is an essential and tightly controlled enzyme located at the core of plant C-metabolism. It fulfils a broad spectrum of non-photosynthetic functions, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates consumed during biosynthesis and N-assimilation. In plants, a small multigene family encodes several closely related plant-type PEPC (PTPC) isozymes along with a distantly related bacterial-type PEPC (BTPC) isozyme. The PTPCs are well studied ~110-kDa subunits that typically exist as a homotetramer (Class-1 PEPC). By contrast, little is known about the larger ~118-kDa BTPC isozyme except that it occurs in developing castor (Ricinus communis) endosperm in tight association with PTPC subunits as a ~900-kDa hetero-octameric complex (Class-2 PEPC) that is greatly desensitized to metabolic effectors compared to Class-1 PEPC. This thesis elucidates the physiological purpose of the BTPC subunits by examining their structure/function relationship within Class-2 PEPC and identifying mechanisms of post-translational control. Recombinant expression and purification of the castor bean BTPC revealed unusual physical and kinetic properties including a remarkable insensitivity to metabolic effectors and a dependence upon PTPC subunits for structural stability. The first purification of a non-proteolyzed plant Class-2 PEPC complex was performed, and the kinetic analysis determined that the BTPC and PTPC subunits have complimentary catalytic properties. The BTPC subunits’ high Km(PEP) and desensitization to metabolic effectors may function as a metabolic overflow mechanism for sustaining flux from PEP to malate when PTPC subunits become feedback inhibited. An anti-PTPC co-immunopurification strategy was utilized to highly enrich non-proteolyzed BTPC from developing castor endosperm for downstream immunological and mass spectrometric analysis. BTPC was in vivo phosphorylated at multiple novel sites, identified by mass spectrometry as Thr4 or 5, Ser425 and Ser451. Phosphosite-specific antibodies towards Ser425 and Ser451 confirmed the existence of these sites in vivo and comparisons of Ser425 phosphorylation patterns established that the castor BTPC and PTPC phosphorytation sites are regulated independently. Phosphomimetic mutants of Ser425 caused BTPC inhibition by increasing its Km(PEP) and sensitivity to feedback inhibition. These results establish a novel mechanism of PEPC control whose implications within plant carbon metabolism are discussed. / Thesis (Ph.D, Biology) -- Queen's University, 2011-09-04 16:46:22.024

Our laboratory is interested in studying the junction of glycolysis and the tricarboxylic acid (TCA) cycle, specifically the enzymes phosphoenolpyruvate carboxykinase, pyruvate carboxylase and phosphoenolpyruvate carboxylase. All produce oxaloacetate (OAA) for the cell. OAA production is critical for cell carbon synthesis in the methanogenic archaea. Therefore OAA-generating enzymes are essential for the survival of methanogens. In part of this study we investigated archaeal-type phosphoenolpyruvate carboxylase (PpcA), a new type of phosphoenolpyruvate carboxylase, which is widespread in the archaea and is found in three bacterial species. The form of phosphoenolpyruvate carboxylase (Ppc) that is prevalent in bacteria and plants is not found in the archaea. Due to complications expressing PpcA in the soluble form and difficulty purifying this enzyme from methanogens, an in-depth investigation of this enzyme's biochemical properties has yet to occur. In this study we demonstrate the successful expression of a PpcA homolog in the soluble fraction of Escherichia coli. We purified the recombinant protein to homogeneity. This development provides the means to study the enzyme's biochemical properties and manipulate the primary sequence in order to identify residues critical to the enzyme's function. We also show that this PpcA homolog does have the postulated activity and investigate its biochemical properties. The data show that PpcA has unique properties in regard to the enzyme's substrate and its regulation by metabolites. Our data also reveal that PpcA is a membrane associated protein, unlike Ppc, which is a soluble protein. We also show that pyruvate carboxylase (Pyc) can be expressed recombinantly in Pseudomonas aeruginosa at levels sufficient for structure-function studies. This is a major step forward in the expression in Pyc because it cannot be expressed at high levels in Escherichia coli. These are important developments in studying the enzymes that connect glycolysis and the TCA cycle. / Master of Science

Phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPC) is one of the carbon dioxide- fixing enzymes, which yields oxaloacetate from phosphoenolpyruvate and bicarbonate. Regulation of PEPC activity occurs at many levels. In addition to pH and concentration of activators and inhibitors, it is phosphorylation as well. Phosphorylation of PEPC causes a change of kinetic parameters, such as maximal reaction rate, sensitivity to activation or inhibition. Considering that, there is still little information like this about C3 plants and that regulation is in various plant species different, I have dealt with monitoring of the kinetic parameters and regulation possibilities of PEPC isolated from C3 plant sources (Cannabis sativa L., Chenopodium quinoa, Pisum sativum L., Lens culinaris). While the activity of PEPC from leaves of Cannabis sativa L. was decreased by alkaline phosphatase, the activity of PEPC from seeds of Chenopodium quinoa, Pisum sativum L., Lens culinaris was not affected by alkaline phosphatase. The affinity of PEPC from seeds Chenopodium quinoa, Pisum sativum L., Lens culinaris to the substrate PEP was higher than in the case of PEPC from leaves of Cannabis sativa L.. For PEPC from Cannabis sativa L. was found that the apparent dephosphorylation leads to decrease of sensitivity to the...

Las especies vegetales están continuamente desafiadas por un extenso número de microorganismos patógenos en su ambiente natural, principalmente bacterias, hongos y virus. Las plantas cuentan con un amplio arsenal de mecanismos de defensa tanto constitutivos como inducibles frente a estos patógenos. Los mecanismos inducibles incluyen el estadillo oxidativo, muerte celular rápida y localizada, acumulación de fitoalexinas y la síntesis de proteínas relacionadas con la patogénesis (Proteínas PR). Centrándose en las fitoalexinas, estas son compuestos antimicrobianos de bajo peso molecular producidos por las plantas en respuesta a estrés biótico y abiótico. Las fitoalexinas poseen un amplio rango de propiedades antifúngicas en varias especies de plantas. Debido a esto, estos compuestos han sido objeto de numerosos estudios en los últimos 20 años. En la Vid, Vitis vinífera L., la respuesta mejor caracterizada y más frecuente frente a la infección fúngica es la extensa acumulación de fitoalexinas y de proteínas relacionadas con la patogénesis. Entre la gran diversidad de compuestos naturales bioactivos producidos por las especies de Vitis sp. las fitoalexinas están formadas por un grupo reducido de moléculas que pertenecen a la familia de los estilbenos. Las células de vid son capaces de acumular estilnenos en respuesta a estrés biótico y abiótico. Los estilbenos de la vid derivan primariamente del trans-resveratrol (tR) (3,4`,5 -trihidroxiestilbeno), el cual puede sufrir modificaciones químicas, como glicosilación, metilación, hidroxilación y oligomerización, dando lugar a varios derivados bioactivos. tR es producido como producto final de la ruta fenilpropanoide mediante la condensación de tres moléculas de malonil-CoA con una molécula de 4-cumaroil-CoA en una reacción catalizada por la Estilbeno Sintasa (STS). Los estilbenos de la vid han sido ampliamente estudiados ya que poseen unas propiedades en la salud humana sorprendentes. Se han observado beneficios del tR en las principales enfermedades humanas, como agente cardioprotector, como inhibidor de la carcinogénesis, neuro protector y antienvejecimiento entre otros, ya que tR posee un alto efecto antioxidante. Por lo tanto, el interés farmacéutico, nutracéutico y biotecnológico del tR y por tanto de la vid, han ido en aumento desde los últimos 20 años. De la misma manera, la respuesta de defensa ha sido estudiada en cultivos celulares de vid utilizando inductores naturales de esta respuesta. En este sentido, la 2,6 dimetil-ꞵ-ciclodextrina (MBCD) ha demostrado poseer un gran poder elicitor, promoviendo la producción de tR y su acumulación extracelular. Estos estudios han permitido profundizar en los diferentes mecanismos de regulación de la producción de estos compuestos a través de análisis transcriptómicos y proteómicos. Sin embargo, estos análisis revelaron algunos genes y proteínas cuyo papel en esta respuesta es desconocida hasta hoy. Este es el caso de dos parálogos de Fosfoenolpiruvato carboxilasa quinasa (PPCK). La presente tesis doctoral está centrada en el estudio del posible papel regulador de la enzima fosfoenolpiruvato carboxilasa quinasa (PPCK) en la producción de resveratrol y otros metabolitos secundarios en cultivos celulares de vid. La hipótesis principal de esta tesis es que dicha enzima podría estar regulando el flujo del carbono entre el metabolismo primario y secundario, con la finalidad de potenciar este último en condiciones de estrés. Para demostrar esta hipótesis, el contenido de la tesis se divide en 4 capítulos principales. El primer capítulo consiste en el desarrollo de un método analítico basado en espectrometría de masas de triple cuadrupolo para la detección precisa y la cuantificación exacta de resveratrol y sus derivados bioactivos, producidos por la vid. El segundo capítulo es un estudio transcripcional de 2 isoformas de la enzima PPCK, así como el sustrato de esta última (la enzima fosfoenolpiruvato carboxilsa, PPC), en condiciones de estrés, además de estudiar la coexpression con enzimas y factores de transcripción cuyo papel en la producción de resveratrol se conoce con anterioridad. En este capítulo se demuestra tanto en cultivos celulares de vid, como en hojas intactas, que los genes PPCK son fuertemente reprimidos en condiciones de estrés, mientras que los genes PPC permanecen invariables. En cambio se produce un descenso en torno al 20-30% en la actividad específica de la enzima PPC en tales condiciones. Por otro lado, en tales condiciones se observa la correlación entre la represión de los genes PPCK y el incremento en la producción de resveratrol y otros estilbenos. Es conocido que los cambios de actividad en la enzima PPC son debidos a cambios en la fosforilación de la misma, los cuales a su vez son regulados por la PPCK. Por lo tanto el capítulo 3 consiste en el desarrollo de un método para detectar y cuantificar cambios en el patrón de fosforilación de la enzima PPC, adoptando una estrategia de proteómica dirigida. El desarrollo de esté método para la proteína PPC ha permitido observar como el estrés inducido por MBCD provoca un descenso en el grado de fosforilación de la enzima PPC, lo cual correlaciona directamente con el descenso en su actividad específica, y ambos efectos a su vez están correlacionados con la represión génica de su proteína reguladora PPCK. Por último el capítulo 4 es el análisis funcional de los genes PPCK, a través de transformación genética estable de cultivos celulares de vid, con la finalidad de sobreexpresar los genes PPCK, y del silenciamiento genético de los mismos. Los cultivos celulares transformates que sobreexpresaban alguno de los dos parálogos de PPCK resultaron acumular menos resveratrol en condiciones de estrés en comparación con la estirpe silvestre (WT) avalando de esta manera nuestra hipótesis inicial. Además, este efecto no afectaba únicamente a la producción de resveratrol, si no a una gran variedad de metabolitos secundarios producidos en la ruta fenilpropanoide (Flavonoides, taninos condensados, ácidos hidroxicinámicos etc.) sugiriendo que el papel de la enzima PPCK está implicado en la regulación del suministro de carbono hacia esa vía en condiciones de estrés. Por último, el silenciamiento genético tuvo una eficiencia inadecuada, sugiriendo que los genes PPCK pueden ser esenciales, y difícilmente manipulables por en la región metabólica en la que participan. / La presente tesis doctoral ha sido financiada gracias a la subvención de Ministerio de economía y competitividad (BIO2014-51861-R) y (BIO2017- 82374-R). Así mismo, las dos estancias de investigación realizadas en el Integrative Biology Institute, Liverpool University, (Liverpool) han sido financiadas por la Escuela de Doctorado de la Universidad de Alicante, a través del programa de subvenciones para facilitar la obtención de la mención de Doctorado internacional en el título de Doctor y por la European Molecular Biology Organization (EMBO) a través de su programa Short-term Fellowships (ASTF No 7754).

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.

Estudos anteriores realizados no Laboratório de Fisiologia Vegetal do IBUSP com Guzmania monostachia demonstraram que quando essas plantas são submetidas ao déficit hídrico ocorre a indução do CAM, com maior expressão desse metabolismo na porção foliar apical. Para outra espécie (Vriesea gigantea), foi verificada a maior atividade da enzima nitrato redutase (NR) na porção basal durante o período diurno. Em uma bromélia terrestre (Ananas comosus) foi observada a sinalização por citocininas tanto na indução da expressão gênica, quanto na ativação da NR. Outros laboratórios evidenciaram que plantas de Mesembryanthemum crystallinum induzidas ao CAM apresentaram uma provável regulação negativa da fosfoenolpiruvato carboxilase (PEPC) por citocininas. Em decorrência desses conhecimentos acumulados, surgiram novos questionamentos: haveria variações diuturnas da atividade das enzimas PEPC e NR nas diferentes porções das folhas de G. monostachia induzidas ao CAM? A maior disponibilidade de esqueletos carbônicos à noite (acúmulo de acidez) influenciaria positivamente a atividade da NR, deslocando seu pico de atividade para o período noturno? As variações dos teores endógenos de citocininas acompanhariam as possíveis mudanças da atividade da PEPC e da NR, indicando, assim, a participação dessa classe hormonal na regulação dessas enzimas? O presente trabalho teve por objetivo principal investigar uma possível regulação da atividade das enzimas PEPC e NR por citocininas em folhas destacadas da bromélia epífita com tanque, Guzmania monostachia (Bromeliaceae) induzidas ao CAM. Foi esperado com esta pesquisa aprofundar os estudos sobre a inter-relação entre o comportamento fotossintético, a capacidade de assimilação de nitrogênio e a possível regulação das atividades da PEPC e da NR por citocininas endógenas. Análises de acidez titulável, ácidos orgânicos, amido endógeno e da atividade da enzima malato desidrogenase (MDH) foram realizadas, confirmando a indução do CAM nas folhas isoladas de G. monostachia mantidas em polietilenoglicol (PEG) a uma concentração de 30%. O uso desse composto foi eficiente na redução do conteúdo relativo de água e na imposição da deficiência hídrica foliar. Além disso, pôde-se verificar a maior expressão do CAM na porção apical das folhas mantidas em PEG 30%, quando comparada à porção basal. Análises da atividade da PEPC e da NR permitiram verificar a separação espacial dessas enzimas. A primeira apresentou maior atividade no ápice foliar, enquanto a segunda mostrou a maior atividade na porção basal. Apesar disso, não foi observada a separação temporal dessas enzimas, uma vez que ambas apresentaram picos de atividade noturna. A maior atividade da NR durante o período escuro (01 hora) foi verificada nas folhas-controle ou sob deficiência hídrica. Esse resultado sugere que outros fatores, diferentes do metabolismo CAM, influenciaram para a ocorrência da maior atividade dessa enzima durante o período noturno. Os resultados obtidos ainda sugerem que as citocininas possivelmente atuaram como um regulador negativo para a atividade da PEPC durante o dia, uma vez que os maiores níveis endógenos desse hormônio foram observados durante esse período, enquanto a maior atividade dessa enzima foi verificada durante a noite, quando os teores de Z+iP decaíram significativamente. A aplicação de Z ou iP resultou também num decréscimo da atividade dessa enzima. Por outro lado, as citocininas atuaram como um provável regulador positivo para a atividade da NR, uma vez que a maior atividade noturna dessa enzima foi antecedida em 3 ou 6 horas pelos maiores níveis endógenos de citocininas na porção basal das folhas mantidas em água ou PEG 30%, respectivamente. A aplicação de citocininas-livres aumentou significativamente a atividade da NR na base das folhas destacadas mantidas em água ou PEG 30% / Prior studies undertaken in the Laboratory of Plant Physiology on IBUSP with Guzmania monostachia have shown that during water shortage, CAM induction occurs with greater expression in the apical portion of the leaf. In the case of another species (Vriesea gigantean), more intense nitrate reductase (NR) enzyme activity was observed in the basal portion during the daytime. In a certain terrestrial bromeliad (Ananas comosus), signaling by cytokinins, both in the induction of gene expression as well as NR activation, was observed. According to other laboratories, the cytokinins seem to play a negative regulation of phosphoenolpyruvate carboxylase (PEPC) in CAM induced Mesembryanthemum crystallinum plants. As a result of accumulated knowledge, new questions have arisen, such as: Are there daily variations in PEPC and NR enzymes activity in the different portions of CAM induced leaves of G. monostachia? Would the more pronounced nocturnal availability of carbon skeletons (accumulation of acidity) positively influence NR activity, with consequential displacement of its peak of activity to this period? Would variations in endogenous cytokinins concentration accompany possible changes in PEPC and NR activity, thereby indicating the participation of this hormonal class in their regulation? The main aim in the present study was to investigate the possible regulation of PEPC and NR activity by cytokinins in detached CAM-induced leaves of the epiphyte tank bromeliad Guzmania monostachia (Bromeliaceae). The expectations with this research were to study more deeply the inter-relationship between photosynthetic behavior, the capacity for nitrogen assimilation and the possible regulation of PEPC and NR activity by endogenous cytokinins. Analyses of titratable acidity, organic acids, endogenous starch and malate dehydrogenase (MDH) enzyme activity confirmed CAM induction in isolated leaves of G. monostachia kept in polyethylene glycol (PEG) at a concentration of 30%. The use of this compound was efficient in reducing relative water content and imposing leaf water deficiency. Furthermore, compared to the basal portion, greater CAM expression could be observed in the apical portion of leaves kept in PEG 30%. Analyses of PEPC and NR activity allowed detecting their mutual spatial separation, seeing that, in the first greater activity was concentrated in the leaf apex, while in the second this was more pronounced in the basal portion. Even so, no temporal separation could be observed, since peak of activity for both occurred at night. The peak of nocturnal NR activity (1 hour) was observed in control leaves or those undergoing water deficiency, thereby implying that factors, other than CAM metabolism, exerted an influence on the occurrence of more intense activity of this enzyme at this time. Furthermore, there were indications that cytokinins possibly act as a negative regulator of PEPC activity during the daytime, when the highest endogenous levels of this hormone were observed, whereas it was apparent that the most intense activity of this enzyme actually occurred at night, when Z+iP rates decreased significantly. Z or iP application also induced a decrease in the activity of this enzyme. On the other hand, the cytokinins acted as a positive regulator of NR activity, since the nocturnal peak of activity of this enzyme was preceded by 3 or 6 hours by higher endogenous levels of cytokinins in the basal portion of leaves maintained in water or PEG 30%, respectively. The application of free cytokinins induced a significant increase in NR activity in the base of detached leaves kept in water or PEG 30%

Estudos anteriores realizados no Laboratório de Fisiologia Vegetal do IBUSP com Guzmania monostachia demonstraram que quando essas plantas são submetidas ao déficit hídrico ocorre a indução do CAM, com maior expressão desse metabolismo na porção foliar apical. Para outra espécie (Vriesea gigantea), foi verificada a maior atividade da enzima nitrato redutase (NR) na porção basal durante o período diurno. Em uma bromélia terrestre (Ananas comosus) foi observada a sinalização por citocininas tanto na indução da expressão gênica, quanto na ativação da NR. Outros laboratórios evidenciaram que plantas de Mesembryanthemum crystallinum induzidas ao CAM apresentaram uma provável regulação negativa da fosfoenolpiruvato carboxilase (PEPC) por citocininas. Em decorrência desses conhecimentos acumulados, surgiram novos questionamentos: haveria variações diuturnas da atividade das enzimas PEPC e NR nas diferentes porções das folhas de G. monostachia induzidas ao CAM? A maior disponibilidade de esqueletos carbônicos à noite (acúmulo de acidez) influenciaria positivamente a atividade da NR, deslocando seu pico de atividade para o período noturno? As variações dos teores endógenos de citocininas acompanhariam as possíveis mudanças da atividade da PEPC e da NR, indicando, assim, a participação dessa classe hormonal na regulação dessas enzimas? O presente trabalho teve por objetivo principal investigar uma possível regulação da atividade das enzimas PEPC e NR por citocininas em folhas destacadas da bromélia epífita com tanque, Guzmania monostachia (Bromeliaceae) induzidas ao CAM. Foi esperado com esta pesquisa aprofundar os estudos sobre a inter-relação entre o comportamento fotossintético, a capacidade de assimilação de nitrogênio e a possível regulação das atividades da PEPC e da NR por citocininas endógenas. Análises de acidez titulável, ácidos orgânicos, amido endógeno e da atividade da enzima malato desidrogenase (MDH) foram realizadas, confirmando a indução do CAM nas folhas isoladas de G. monostachia mantidas em polietilenoglicol (PEG) a uma concentração de 30%. O uso desse composto foi eficiente na redução do conteúdo relativo de água e na imposição da deficiência hídrica foliar. Além disso, pôde-se verificar a maior expressão do CAM na porção apical das folhas mantidas em PEG 30%, quando comparada à porção basal. Análises da atividade da PEPC e da NR permitiram verificar a separação espacial dessas enzimas. A primeira apresentou maior atividade no ápice foliar, enquanto a segunda mostrou a maior atividade na porção basal. Apesar disso, não foi observada a separação temporal dessas enzimas, uma vez que ambas apresentaram picos de atividade noturna. A maior atividade da NR durante o período escuro (01 hora) foi verificada nas folhas-controle ou sob deficiência hídrica. Esse resultado sugere que outros fatores, diferentes do metabolismo CAM, influenciaram para a ocorrência da maior atividade dessa enzima durante o período noturno. Os resultados obtidos ainda sugerem que as citocininas possivelmente atuaram como um regulador negativo para a atividade da PEPC durante o dia, uma vez que os maiores níveis endógenos desse hormônio foram observados durante esse período, enquanto a maior atividade dessa enzima foi verificada durante a noite, quando os teores de Z+iP decaíram significativamente. A aplicação de Z ou iP resultou também num decréscimo da atividade dessa enzima. Por outro lado, as citocininas atuaram como um provável regulador positivo para a atividade da NR, uma vez que a maior atividade noturna dessa enzima foi antecedida em 3 ou 6 horas pelos maiores níveis endógenos de citocininas na porção basal das folhas mantidas em água ou PEG 30%, respectivamente. A aplicação de citocininas-livres aumentou significativamente a atividade da NR na base das folhas destacadas mantidas em água ou PEG 30% / Prior studies undertaken in the Laboratory of Plant Physiology on IBUSP with Guzmania monostachia have shown that during water shortage, CAM induction occurs with greater expression in the apical portion of the leaf. In the case of another species (Vriesea gigantean), more intense nitrate reductase (NR) enzyme activity was observed in the basal portion during the daytime. In a certain terrestrial bromeliad (Ananas comosus), signaling by cytokinins, both in the induction of gene expression as well as NR activation, was observed. According to other laboratories, the cytokinins seem to play a negative regulation of phosphoenolpyruvate carboxylase (PEPC) in CAM induced Mesembryanthemum crystallinum plants. As a result of accumulated knowledge, new questions have arisen, such as: Are there daily variations in PEPC and NR enzymes activity in the different portions of CAM induced leaves of G. monostachia? Would the more pronounced nocturnal availability of carbon skeletons (accumulation of acidity) positively influence NR activity, with consequential displacement of its peak of activity to this period? Would variations in endogenous cytokinins concentration accompany possible changes in PEPC and NR activity, thereby indicating the participation of this hormonal class in their regulation? The main aim in the present study was to investigate the possible regulation of PEPC and NR activity by cytokinins in detached CAM-induced leaves of the epiphyte tank bromeliad Guzmania monostachia (Bromeliaceae). The expectations with this research were to study more deeply the inter-relationship between photosynthetic behavior, the capacity for nitrogen assimilation and the possible regulation of PEPC and NR activity by endogenous cytokinins. Analyses of titratable acidity, organic acids, endogenous starch and malate dehydrogenase (MDH) enzyme activity confirmed CAM induction in isolated leaves of G. monostachia kept in polyethylene glycol (PEG) at a concentration of 30%. The use of this compound was efficient in reducing relative water content and imposing leaf water deficiency. Furthermore, compared to the basal portion, greater CAM expression could be observed in the apical portion of leaves kept in PEG 30%. Analyses of PEPC and NR activity allowed detecting their mutual spatial separation, seeing that, in the first greater activity was concentrated in the leaf apex, while in the second this was more pronounced in the basal portion. Even so, no temporal separation could be observed, since peak of activity for both occurred at night. The peak of nocturnal NR activity (1 hour) was observed in control leaves or those undergoing water deficiency, thereby implying that factors, other than CAM metabolism, exerted an influence on the occurrence of more intense activity of this enzyme at this time. Furthermore, there were indications that cytokinins possibly act as a negative regulator of PEPC activity during the daytime, when the highest endogenous levels of this hormone were observed, whereas it was apparent that the most intense activity of this enzyme actually occurred at night, when Z+iP rates decreased significantly. Z or iP application also induced a decrease in the activity of this enzyme. On the other hand, the cytokinins acted as a positive regulator of NR activity, since the nocturnal peak of activity of this enzyme was preceded by 3 or 6 hours by higher endogenous levels of cytokinins in the basal portion of leaves maintained in water or PEG 30%, respectively. The application of free cytokinins induced a significant increase in NR activity in the base of detached leaves kept in water or PEG 30%

Thesis (PhD (Plant biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: In soils, the concentration of available phosphate (P) for plants is normally very low (ca. 1 µM in the soil solution), because most of the P combines with iron, aluminium and calcium to form relatively insoluble compounds. Inorganic P (Pi)-deficiency is thought to be one of the limiting factors of nitrogen fixation due to the high energy requirement for nitrogenase function of plants taking part in nitrogen fixation. Pideficiency has important implications for the metabolic Pi and adenylate pools of plants, which influence respiration and nitrogen fixation. An alternative route of pyruvate supply during Pi stress has been proposed involving the combined activities of phosphoenolpyruvate carboxylase (PEPc), malate dehydrogenase (MDH) and NAD-malic enzyme (ME) supplying pyruvate to the mitochondrion during Pi stress. Previously, three isoforms of PEPc were isolated from lupin nodules and roots, with two forms being nodule specific. The aim of this project was to determine the effect of Pi stress on these PEPc isoforms in Lupinus luteus at transcript and protein expression level with a view to produce genetically modified crops for nutrient-poor soils. Cytosolic P levels were measured over a time course to give an indication of temporal development of P stress in nodules. The changes in enzyme activities of PEPc, MDH and PK (pyruvate kinase) under P stress were measured and the downstream effect on amino and organic acid pools were analysed. Two novel PEPc isoforms, LUP1 (AM235211) and LUP2 (AM237200) were isolated from nodules, followed by transcriptional and protein expression analyses. Nodules under P stress had lower amounts of metabolically available Pi and as P stressed developed, the amount of Pi decreased. This decline in Pi levels was associated with lower growth, but higher biological nitrogen fixation (BNF). A greater proportion of root-nodule respiration was devoted to nutrient acquisition than to new growth. A typical P-stress response is higher anaplerotic carbon fixation via PEPc. However, in this study, no significant differences were found for PEPc, MDH or PK in P-stressed plants compared to P-sufficient plants which would lead to an increase in organic acids. An increase in key amino acids was reported along with unchanged levels of organic acids. These levels of organic and amino acid are in congruence with the increases in BNF under P-starvation. No significant differences were found in expression of PEPC1 or PEPC2 at 12 and 20 days for both P-sufficient and P-stressed plants which further supported the lack of engagement of the PEPc-MDH-ME bypass. PEPc activity appeared not to be regulated by gene expression or phosphorylation indicating that other posttranslational modifications such as a decrease in protein degradation may be of importance. / AFRIKAANSE OPSOMMING: Die konsentrasie van fosfaat (P) beskikbaar vir opname deur plante vanuit die grond is gewoonlik baie laag (in die omgewing van 1 µM) aangesien die P onoplosbare komplekse vorm met katione soos yster, aluminium en kalsium. ‘n Tekort aan anorganiese P (Pi) word gereken as een van die beperkende faktore van stikstofbinding as gevolg van die hoë energie behoefte wat nitrogenase plaas op plante wat van gefikseerde stikstof gebruik maak. Hierdie P-tekort het ook belangrike betrekking op die metaboliese fosfaat- en adenilaatpoele wat weer op hul beurt respirasie en stikstofbinding beÏnvloed. ‘n Alternatiewe roete van pirovaatvoorsiening aan mitochondria tydens fosfaatstres is voorgestel wat bestaan uit die aktiwiteite van fosfoenolpirovaat karboksilase (PEPc), malaat dehidrogenase en NAD-malaat ensiem. Vantevore is drie isovorme van PEPc uit Lupinus luteus wortelknoppies en wortels geïsoleer, met twee van die isovorme wat wortelknoppie-spesifiek was. The doel van hierdie projek was om die invloed van P-tekort op die transkripsie en proteien uitdrukkingsvlak van hierdie PEPc isovorme te bepaal met die doel van gemodifiseerde gewasse vir arm gronde ingedagte. Sitoplasmiese P konsentrasies is gemeet oor tyd om ‘n aanduiding te gee van die ontwikkeling van P-tekort oor tyd. Veranderinge in ensiemaktiwiteite van PEPc, MDH en pirovaatkinase (PK) is gemeet gedurende P-tekort as ook die moontlike effek van hierdie ensiemaktiwiteite op aminosuur en organiese suur poele. Twee nuwe PEPc isovorme, LUP1 (AM235211) en LUP2 (AM237200) is uit wortelknoppies geïsoleer en gekarakteriseer. Transkripsie en proteïenuitdrukking is geanaliseer. Wortelknoppies wat P-tekort behandeling ontvang het, het laer vlakke van metabolise beskikbare Pi gehad en soos die P-tekort ontwikkel het oor tyd, het die Pi vlakke gedaal. Hierdie afname in vlakke van Pi was geassosieer met laer groei, maar met ‘n toename in biologiese stikstofbinding. ‘n Groter proporsie van respirasie is toegestaan aan minerale opname as aan nuwe groei. ‘n Tipiese reaksie op P-tekort is hoër anaplerotiese koolstofbinding via PEPc. Alhoewel, in hierdie studie is geen gevind betekenisvolle verandering gevind in die aktiwiteite van PEPc, MDH en PK nie in plante wat P-tekort ervaar het nie. Verhoogde aktiwiteit van hierdie ensieme sou verhoogde organise suur konsentrasies tot gevolg hê. ‘n Toename in aminosuur konsentrasies is gevind tesame met onveranderde vlakke van organiese sure. Hierdie toename in aminosure word onderskryf deur die verhoogde biologiese stikstofbinding tydens P-tekort. Geen betekenisvolle verskille is gevind in die geenuitdrukking van pepc1 en pepc2 by beide 12 en 20 dae van P-tekort nie, wat verder die afwesigheid van die PEPc- MDH-ME alternatiewe roete beaam het. Dit blyk dat PEPc aktiwiteit nie deur geenuitdrukking of proteïenfosforilering beheer word nie, maar eerder dat ander posttranslasie modifikasies soos ‘n verlaagde afbraak van proteïen ‘n rol speel.

Stomata are small pores that are located on the surface of epidermal leaves, and they can regulate the uptake of CO2 and prevent water lose by opening and closing the pores. Stomata of plants can be regulated by external condition such as CO2, biotic and abiotic stresses and internal factors. CAM (crassulacean acid metabolism) plants adapt to hot and dry environments by closing stomata during the day and opening stomata during the cool night. However, it is still unclear how CAM plants open their stomata during the night and close them during the day. In this study, a number of factors were evaluated for their potential roles in promoting stomatal opening in the model CAM plant Kalanchoe laxiflora. Citrate is an important organic acid and it accumulates during the night in CAM plants. It is shown in this study that citrate promoted stomatal opening in detached leaf epidermis of Kalanchoe laxiflora. Further, the cytokinin zeatin is also shown to stimulate stomatal opening in detached leave of Kalanchoe laxiflora. Melatonin is an important regulator of circadian rhythms in mammals and has been implicated in regulation of plant abiotic stress responses. Melatonin was detected in the leaves of Kalanchoe laxiflora. It promoted stomatal opening in detached epidermis of Kalanchoe laxiflora. Together, these results suggest that stomata of Kalanchoe laxiflora respond to citrate and malate which are the main organic acids accumulate during nighttime and also to some signaling molecules (zeatin, melatonin, and serotonin) by opening stomata during dark period.

No description available.