γ-Hydroxybutyrate (GHB) is an intermediate of γ-aminobutyrate (GABA) catabolism in plants subjected to abiotic stress and its formation is catalyzed by two NADPH-dependent glyoxylate/succinic semialdehyde reductases (GLYRs). GABA and/or GHB accumulation in ‘Honeycrisp’ and ‘Empire’ apple fruit stored under controlled atmosphere (CA) conditions (i.e., low temperature, low O2, elevated CO2) in the presence or absence of the ethylene-antagonist 1-methylcyclopropene, coincided with the onset of physiological injury, suggesting an association with cellular disruption. Salinity and chilling stresses differentially influenced the expression of GABA pathway genes and the levels of GHB among various GABA pathway mutants of Arabidopsis. Furthermore, the occurrence of GHB in glyr1/glyr2 double knockout mutants indicates the presence of an additional pathway for GHB production. Evidence for GHB oxidation was not detectable in cell-free leaf extracts, suggesting the existence of a novel enzyme for GHB turnover. / NSERC Alexander Graham Bell Canada Graduate Scholarship (CGS-M), Ontario Graduate Scholarship (OGS), NSERC, Ontario Apple Growers, Rohm & Haas, MITACS

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.

O nitrogênio é o nutriente mineral essencial mais limitante ao crescimento das plantas. Apenas alguns microorganismos procariotos desenvolveram um mecanismo bioquímico que permite reduzir N2, abundante na atmosfera, a amônia, que pode ser assimilada pelas plantas. Muitos destes microorganismos conseguem associar-se simbioticamente com outros organismos, sendo a associação de bactérias do grupo Rhizobium com plantas do grupo das leguminosas a mais importante a nível econômico. Por outro lado, quando existe disponibilidade de nitrato no meio ambiente, a leguminosa abandona a fixação biológica absorvendo nitrato, que é reduzido a amônia pelas enzimas nitrato redutase (NR) e nitrito redutase (NiR) e, ao final, assimilada pelo sistema GS/GOGAT. Apesar de as duas vias terem amônia como produto final, em leguminosas tropicais, principalmente as pertencentes à tribo Phaseolae, quando a amônia provém de associação simbiótica a planta exporta, via xilema, preferencialmente ureídeos, enquanto quando a amônia provém da redução de nitrato, a planta exporta amidas, principalmente asparagina e glutamina. O objetivo do presente trabalho foi identificar, em Canavalia ensiformes (L.), as alterações metabólicas que ocorriam em função da concentração de nitrato fornecida à planta, através da quantificação localizada dos principais compostos nitrogenados do metabolismo de nitrogênio e dos principais sítios de redução de nitrato; bem como estudar o metabolismo de asparagina na leguminosa em questão desde o estádio de germinação até a fase reprodutiva, identificando os sítios preferenciais de metabolismo desta amida. Observou-se que o metabolismo de aminoácidos foi profundamente alterado em função da concentração de nitrato fornecida. Os níveis de aminoácidos solúveis totais não se alteraram drasticamente, o mesmo ocorrendo com os níveis de proteína e ureídeos, considerando-se os diferentes tratamentos em um mesmo estádio de desenvolvimento. Com a alteração do estádio de desenvolvimento, especialmente com o início da fase reprodutiva, o metabolismo de nitrogênio foi profundamente alterado, observando-se inversão no sítio de redução de nitrato, alterações nas concentrações totais de ureídeos e aminoácidos e alterações profundas no metabolismo de aminoácidos, quando se relaciona com o estádio vegetativo. A atividade de nitrato redutase, em Canavalia ensiformes (L.) mostrou-se regulada pela concentração de glutamina presente nos tecidos, tendo se mostrado mais elevada nos tecidos onde os níveis de glutamina eram menores. A alteração no sítio de redução de nitrato em função da mudança no estádio de desenvolvimento foi acompanhada pelo aumento simultâneo na concentração de glutamina solúvel nos tecidos onde a atividade da enzima foi menor. Em função das mudanças no estádio de desenvolvimento, percebeu-se também uma redução no número de sítios de possível atividade da enzima asparaginase, o que foi inferido pela redução no número de tecidos onde a enzima esteve presente, sugerindo então um possível aumento no catabolismo desta amida por ação da enzima asparagina-aminotransferase. Para maior entendimento dos processos que levam às alterações no metabolismo de nitrato, asparagina e aminoácidos de uma forma geral, os mecanismos de síntese e utilização de aminoácidos devem ser analisados para melhor compreensão dos processos envolvidos, através da análise da atividade das principais enzimas envolvidas nestes processos, bem como de estudos da transcrição dos respectivos genes. / Nitrogen is the most limiting essential nutrient for plant growth. Some prokaryotic microorganisms have developed a biochemical mechanism, which allows the reduction of N2, which is abundantly present in the atmosphere, to ammonium that can be assimilated by the plants. Many of these microorganisms form symbiotic associations with other organisms. This is especially true for leguminous plants that form symbiotic associations with bacteria belonging to the Bradyrhizbium, Rhizobium, and Sinorhizobium groups. Bacterial nitrogen fixation from these interactions are extremely important for the global nitrogen balance and plays a major economically role in agriculture. On the other hand, when nitrate is available in the environment, leguminous plants interrupt the symbiotic fixation process to directly use of the nitrate, which is reduced to ammonium by the enzymes nitrate reductase (NR) and nitrite reductase (NiR), and is finally assimilated by the GS/GOGAST system. Although both will result in ammonium as the end-product, in tropical leguminous plants species, mainly those of the Phaseoleae tribe, when ammonium is produced by the symbiotic association the plant translocates mainly ureides via xylem, whereas the plant translocate mainly amides such as asparagine and glutamine, when the ammonium is produced by nitrate reduction. The objective of this study was to identify in Canavalia ensiformes (L.), metabolic alterations dependent upon the concentration of nitrate supplied to the plant. Specific attention was given to the quantity of nitrogen compounds from nitrogen metabolism and asparagine metabolism from the early stage of germination to the reproductive stage, with the identification of the main locations of metabolism for this amide. Amino acids metabolism was significantly altered when nitrate was supplied at different concentrations. Total soluble amino acids, total protein and ureide contents were not dramatically altered when considering the different treatments at the same developmental stage. However, nitrogen metabolism was shown to be drastically altered when different development stages were compared, particularly at the beginning of the reproductive stage, at which time a switch in the location of nitrate reduction, alterations in the total concentration of ureides and amino acids were observed, when compared to the vegetative stage. Nitrate reductase activity of Canavalia ensiformes was shown to be regulated by the concentration of glutamine present in the tissues, exhibiting higher activity in tissues containing lower concentrations of glutamine, which coincided with the shift of the site of nitrate reduction with the changing developmental stage. The understanding of the processes leading to the alterations in the metabolism of nitrate, asparagine, amino acids, and the mechanism related to the synthesis and utilization of amino acids requires further studies.

A produção de banana no Vale do Ribeira, em São Paulo, está inserida numa região que abriga um fragmento remanescente da Mata Atlântica, considerada um dos principais repositórios de biodiversidade e a floresta mais devastada entre os biomas brasileiros. Medidas sustentáveis, como a adoção de práticas agrícolas alternativas, tem sido o caminho para a conservação da fauna e flora, como também para a redução do uso inapropriado de terras que consequentemente pode aumentar o índice de doenças que atacam as plantas. Apesar, do razoável conhecimento científico sobre a fisiologia pós-colheita da banana, ainda não foram elucidadas quais são as respostas fisiológicas do fruto diante das alterações do meio ambiente decorrentes das práticas agroecológicas. Principalmente quais são os mecanismos de ação de compostos específicos, relacionados com a resistência da planta contra os estresses bióticos e abióticos. Também, não há uma abordagem analítica integrada que identifique os reguladores das vias metabólicas e possibilite um estudo holístico a nível molecular. Neste trabalho, foi avaliado o efeito da proximidade da biodiversidade nativa da Mata Atlântica sobre o perfil de metabólitos da banana (Musa acuminata AAA, Cavendish, cv. Nanicão). Foram comparados os frutos da parcela Biodiversidade, o qual apresenta 60% de seu perímetro limítrofe à floresta com a parcela Controle, inserida em uma área de produção de banana convencional. Neste estudo, foram determinados os perfis de etileno, vida verde, amido, açúcares, textura, cor, metabólitos semi-voláteis e não-voláteis e poliaminas. Como resultado, ambas as parcelas avaliadas apresentaram diferenças significativas no metabolismo primário e secundário, nos frutos verdes e maduros, respectivamente. Os metabólitos com diferença significativa entre as parcelas experimentais apresentaram maior envolvimento no metabolismo primário, sobretudo na via de biossíntese de aminoácidos, compostos precursores de uma ampla faixa de metabólitos secundários. As alterações quanto à abundância destes compostos são uma referência de que as mudanças do meio modificam as respostas da planta a estresses e estímulos. / Banana production in the Ribeira Valley, in São Paulo, is set in a region that embraces a remaining fragment of the Atlantic Coastal Forest (Mata Atlântica), considered one of the main biodiversity repositories and the most devastated forest among the Brazilians´ biomes. Sustainable measures such as the adoption of alternative farming practices has been the way for the conservation of fauna and flora, but also to reduce the inappropriate use of land which in turn may increase the disease index that attack plants. Although, reasonable scientific knowledge about banana postharvest physiology is available, the fruit´s physiologic response given the environment changes as a result agroecological practices has not been elucidated yet. Primarily what are the mechanisms of action of specific compounds, related to plant resistance against biotic and abiotic stresses. In addition, there is not an integrated analytical approach to identify regulators of metabolic pathways and enable a holistic study at the molecular level. In this study, we evaluated the effect of proximity to the native biodiversity of the Atlantic Forest on the banana metabolites profile (Musa acuminata AAA, Cavendish, cv. Nanicão). The fruits of Biodiversity group were compared, which has 60% of its perimeter adjacent to the forest with the Control group set in a conventional banana production area. In this study, we determined the ethylene profiles, green life, starch, sugars, texture, colour, semi- volatile and non- volatile metabolites and polyamines. As a result, both evaluated groups showed significant differences in primary and secondary metabolism, green and ripen fruits respectively. The metabolites with significant differences between treatments showed greater involvement in primary metabolism, especially in amino acid biosynthesis, precursors of secondary metabolites. Changes in the abundance of these compounds are indicators that alterations in the environment modify the plant responses to stresses and stimuli.

A produção de banana no Vale do Ribeira, em São Paulo, está inserida numa região que abriga um fragmento remanescente da Mata Atlântica, considerada um dos principais repositórios de biodiversidade e a floresta mais devastada entre os biomas brasileiros. Medidas sustentáveis, como a adoção de práticas agrícolas alternativas, tem sido o caminho para a conservação da fauna e flora, como também para a redução do uso inapropriado de terras que consequentemente pode aumentar o índice de doenças que atacam as plantas. Apesar, do razoável conhecimento científico sobre a fisiologia pós-colheita da banana, ainda não foram elucidadas quais são as respostas fisiológicas do fruto diante das alterações do meio ambiente decorrentes das práticas agroecológicas. Principalmente quais são os mecanismos de ação de compostos específicos, relacionados com a resistência da planta contra os estresses bióticos e abióticos. Também, não há uma abordagem analítica integrada que identifique os reguladores das vias metabólicas e possibilite um estudo holístico a nível molecular. Neste trabalho, foi avaliado o efeito da proximidade da biodiversidade nativa da Mata Atlântica sobre o perfil de metabólitos da banana (Musa acuminata AAA, Cavendish, cv. Nanicão). Foram comparados os frutos da parcela Biodiversidade, o qual apresenta 60% de seu perímetro limítrofe à floresta com a parcela Controle, inserida em uma área de produção de banana convencional. Neste estudo, foram determinados os perfis de etileno, vida verde, amido, açúcares, textura, cor, metabólitos semi-voláteis e não-voláteis e poliaminas. Como resultado, ambas as parcelas avaliadas apresentaram diferenças significativas no metabolismo primário e secundário, nos frutos verdes e maduros, respectivamente. Os metabólitos com diferença significativa entre as parcelas experimentais apresentaram maior envolvimento no metabolismo primário, sobretudo na via de biossíntese de aminoácidos, compostos precursores de uma ampla faixa de metabólitos secundários. As alterações quanto à abundância destes compostos são uma referência de que as mudanças do meio modificam as respostas da planta a estresses e estímulos. / Banana production in the Ribeira Valley, in São Paulo, is set in a region that embraces a remaining fragment of the Atlantic Coastal Forest (Mata Atlântica), considered one of the main biodiversity repositories and the most devastated forest among the Brazilians´ biomes. Sustainable measures such as the adoption of alternative farming practices has been the way for the conservation of fauna and flora, but also to reduce the inappropriate use of land which in turn may increase the disease index that attack plants. Although, reasonable scientific knowledge about banana postharvest physiology is available, the fruit´s physiologic response given the environment changes as a result agroecological practices has not been elucidated yet. Primarily what are the mechanisms of action of specific compounds, related to plant resistance against biotic and abiotic stresses. In addition, there is not an integrated analytical approach to identify regulators of metabolic pathways and enable a holistic study at the molecular level. In this study, we evaluated the effect of proximity to the native biodiversity of the Atlantic Forest on the banana metabolites profile (Musa acuminata AAA, Cavendish, cv. Nanicão). The fruits of Biodiversity group were compared, which has 60% of its perimeter adjacent to the forest with the Control group set in a conventional banana production area. In this study, we determined the ethylene profiles, green life, starch, sugars, texture, colour, semi- volatile and non- volatile metabolites and polyamines. As a result, both evaluated groups showed significant differences in primary and secondary metabolism, green and ripen fruits respectively. The metabolites with significant differences between treatments showed greater involvement in primary metabolism, especially in amino acid biosynthesis, precursors of secondary metabolites. Changes in the abundance of these compounds are indicators that alterations in the environment modify the plant responses to stresses and stimuli.

O nitrogênio é o nutriente mineral essencial mais limitante ao crescimento das plantas. Apenas alguns microorganismos procariotos desenvolveram um mecanismo bioquímico que permite reduzir N2, abundante na atmosfera, a amônia, que pode ser assimilada pelas plantas. Muitos destes microorganismos conseguem associar-se simbioticamente com outros organismos, sendo a associação de bactérias do grupo Rhizobium com plantas do grupo das leguminosas a mais importante a nível econômico. Por outro lado, quando existe disponibilidade de nitrato no meio ambiente, a leguminosa abandona a fixação biológica absorvendo nitrato, que é reduzido a amônia pelas enzimas nitrato redutase (NR) e nitrito redutase (NiR) e, ao final, assimilada pelo sistema GS/GOGAT. Apesar de as duas vias terem amônia como produto final, em leguminosas tropicais, principalmente as pertencentes à tribo Phaseolae, quando a amônia provém de associação simbiótica a planta exporta, via xilema, preferencialmente ureídeos, enquanto quando a amônia provém da redução de nitrato, a planta exporta amidas, principalmente asparagina e glutamina. O objetivo do presente trabalho foi identificar, em Canavalia ensiformes (L.), as alterações metabólicas que ocorriam em função da concentração de nitrato fornecida à planta, através da quantificação localizada dos principais compostos nitrogenados do metabolismo de nitrogênio e dos principais sítios de redução de nitrato; bem como estudar o metabolismo de asparagina na leguminosa em questão desde o estádio de germinação até a fase reprodutiva, identificando os sítios preferenciais de metabolismo desta amida. Observou-se que o metabolismo de aminoácidos foi profundamente alterado em função da concentração de nitrato fornecida. Os níveis de aminoácidos solúveis totais não se alteraram drasticamente, o mesmo ocorrendo com os níveis de proteína e ureídeos, considerando-se os diferentes tratamentos em um mesmo estádio de desenvolvimento. Com a alteração do estádio de desenvolvimento, especialmente com o início da fase reprodutiva, o metabolismo de nitrogênio foi profundamente alterado, observando-se inversão no sítio de redução de nitrato, alterações nas concentrações totais de ureídeos e aminoácidos e alterações profundas no metabolismo de aminoácidos, quando se relaciona com o estádio vegetativo. A atividade de nitrato redutase, em Canavalia ensiformes (L.) mostrou-se regulada pela concentração de glutamina presente nos tecidos, tendo se mostrado mais elevada nos tecidos onde os níveis de glutamina eram menores. A alteração no sítio de redução de nitrato em função da mudança no estádio de desenvolvimento foi acompanhada pelo aumento simultâneo na concentração de glutamina solúvel nos tecidos onde a atividade da enzima foi menor. Em função das mudanças no estádio de desenvolvimento, percebeu-se também uma redução no número de sítios de possível atividade da enzima asparaginase, o que foi inferido pela redução no número de tecidos onde a enzima esteve presente, sugerindo então um possível aumento no catabolismo desta amida por ação da enzima asparagina-aminotransferase. Para maior entendimento dos processos que levam às alterações no metabolismo de nitrato, asparagina e aminoácidos de uma forma geral, os mecanismos de síntese e utilização de aminoácidos devem ser analisados para melhor compreensão dos processos envolvidos, através da análise da atividade das principais enzimas envolvidas nestes processos, bem como de estudos da transcrição dos respectivos genes. / Nitrogen is the most limiting essential nutrient for plant growth. Some prokaryotic microorganisms have developed a biochemical mechanism, which allows the reduction of N2, which is abundantly present in the atmosphere, to ammonium that can be assimilated by the plants. Many of these microorganisms form symbiotic associations with other organisms. This is especially true for leguminous plants that form symbiotic associations with bacteria belonging to the Bradyrhizbium, Rhizobium, and Sinorhizobium groups. Bacterial nitrogen fixation from these interactions are extremely important for the global nitrogen balance and plays a major economically role in agriculture. On the other hand, when nitrate is available in the environment, leguminous plants interrupt the symbiotic fixation process to directly use of the nitrate, which is reduced to ammonium by the enzymes nitrate reductase (NR) and nitrite reductase (NiR), and is finally assimilated by the GS/GOGAST system. Although both will result in ammonium as the end-product, in tropical leguminous plants species, mainly those of the Phaseoleae tribe, when ammonium is produced by the symbiotic association the plant translocates mainly ureides via xylem, whereas the plant translocate mainly amides such as asparagine and glutamine, when the ammonium is produced by nitrate reduction. The objective of this study was to identify in Canavalia ensiformes (L.), metabolic alterations dependent upon the concentration of nitrate supplied to the plant. Specific attention was given to the quantity of nitrogen compounds from nitrogen metabolism and asparagine metabolism from the early stage of germination to the reproductive stage, with the identification of the main locations of metabolism for this amide. Amino acids metabolism was significantly altered when nitrate was supplied at different concentrations. Total soluble amino acids, total protein and ureide contents were not dramatically altered when considering the different treatments at the same developmental stage. However, nitrogen metabolism was shown to be drastically altered when different development stages were compared, particularly at the beginning of the reproductive stage, at which time a switch in the location of nitrate reduction, alterations in the total concentration of ureides and amino acids were observed, when compared to the vegetative stage. Nitrate reductase activity of Canavalia ensiformes was shown to be regulated by the concentration of glutamine present in the tissues, exhibiting higher activity in tissues containing lower concentrations of glutamine, which coincided with the shift of the site of nitrate reduction with the changing developmental stage. The understanding of the processes leading to the alterations in the metabolism of nitrate, asparagine, amino acids, and the mechanism related to the synthesis and utilization of amino acids requires further studies.

<p>Juglone is a 1,4-naphthoquinone (1,4-NQ) and the allelochemical responsible for the well-known toxic effects of black walnut (<i>Juglans nigra</i>)<i> </i>and other members of the Juglandaceae. Juglone affects a variety of weed species via a mode of action unlike any commercially available herbicides, and thus has the potential to be used as a new natural product-based herbicide. However, lack of knowledge about its metabolism precludes introducing juglone biosynthesis traits into resistant crops through biotechnology. Herein, we established that juglone is derived from the phylloquinone pathway at the level of the intermediate 1,4-dihydroxy-2-naphthoic acid (DHNA). Phylloquinone is a primary 1,4-NQ made by all plants for photosynthetic electron transport. Despite the fundamental importance of phylloquinone, there are still unanswered questions about the subcellular architecture of the phylloquinone pathway. In chapter 3, we show that <i>o</i>-succinylbenzoate CoA-ligase is localized to both chloroplasts and peroxisomes and that its activity is vital in both organelles. The required dual localization of CoA ligase activity is a theme common to other plant pathways with CoA metabolic steps occurring in peroxisomes and thus leads us to propose a revised model of the phylloquinone pathway. Lastly, given the potential of introducing juglone biosynthesis as part of novel weed management strategies, we investigated the circumstances, costs, and benefits of producing allelochemicals in crops using an evolutionary game theory model. Together, this work (i) shows that the phylloquinone pathway provides crops with the biosynthetic framework to produce juglone, (ii) sheds new light on the phylloquinone pathway architecture, and (iii) reveals the circumstances in which producing an allelochemical will be an evolutionarily stable strategy. We envision these results will assist biotechnological efforts to utilize juglone as a novel, natural product-based herbicide.</p>

A principal fonte de proteínas para alimentação humana e animal é fornecida pelas sementes de cereais e leguminosas. O conteúdo de aminoácidos solúveis em endospermas de milho normal e mutantes opaco-2 e floury foram determinadas por HPLC. A análise indicou que a concentração total de aminoácidos solúveis variou entre os mutantes e seus tipos selvagens. Nos mutantes o10, o11 e o13, as concentrações foram aumentadas significativamente quando comparadas ao tipo selvagem W22, enquanto os mutantes o1, o2, o13, fl1 e fl2 exibiram baixas concentrações em relação ao seu respectivo tipo selvagem Oh43. Resultados similares foram obtidos para os mutantes o5, o7 e fl3 em relação aos seus tipos selvagens (B79, B37 e WT3, respectivamente). Para metionina, o mutante o2 e o tipo selvagem Oh43 apresentaram as mais altas concentrações deste aminoácido. Diferenças significativas não foram observadas para os outros aminoácidos analisados, tais como lisina e treonina. Os resultados sugerem que as altas concentrações sugeridas originalmente para estes mutantes devem ser devidas aos níveis destes aminoácidos incorporados nas proteínas de reserva, mas não na forma solúvel. / For human nutrition the main source of vegetable proteins are cereal and legume seeds. The content of total soluble amino acids in mature endosperms of wildtype and maize opaque and floury mutants have been determined by HPLC. The total absolute concentration of soluble amino acids among the mutants and their wild-type counterparts varied depending on the mutant. In the o10, o11 and o13 mutants the concentrations were significantly increased when compared to their wild-type counterpart W22, whereas the mutants o1, o2, o13, fl1 and fl2 exhibited lower concentrations when compared to the wild-type Oh43, Similar results were observed for o5, o7 and fl3 in relation to their specific wild-type counterparts (B79, B37 and WT3, respectively). For soluble methionine content, o2 and Oh43 exhibited the highest concentrations. Significant differences were not observed for other amino acids such as lysine and threonine. The results suggest that the high-lysine concentrations indicated originally for these mutants must be due to the amino acids incorporated into storage proteins, but not in the soluble form.

Thesis (M.Sc. (Microbiology)) --University of Limpopo, 2011 / Pterocarpus angolensis seed lectin (PAL), a 28 kDa non glycosylated protein, was initially successfully cloned and expressed in E. coli for ease of high protein production. It was discovered, however, as in similar studies that the recombinant PAL yield in E. coli is low and localized intracellularly. This makes extraction even more difficult because most of the protein is lost either when the cell undergoes lysing or when there is incomplete extraction. As a result of the low yields in E. coli, expression vectors were constructed for pal expression in S. cerevisiae, Y. lipolytica and A. niger. Colony PCR of S. cerevisiae transformants confirmed the presence of pal gene whilst sequencing revealed a 66% homology to native PAL. Expression of recombinant PAL in S. cerevisiae, which was expected to be intracellular, was doubtfully unsuccessful since no signal was detected following Western blot analysis. A pBARMTE1-pal expression vector was successfully constructed and could be used for expression studies in Aspergillus niger, however, it was not used in this study. A pal gene whose codons were optimized for Y. lipolytica was synthesized and successfully cloned and expressed in Y. lipolytica. Gene sequence alignment of native pal and the codon optimized pal showed 81% homology whilst the amino acid alignment showed 100% homology. A 31 kDa, recombinant PAL was successfully expressed in Y. lipolytica. The recombinant PAL was approximately 3 kDa larger than native PAL. It was established that this is due to glycosylation of the recombinant PAL. This recombinant protein was found to be more thermostable than native PAL since it demonstrated haemagglutination activity after 10 minutes of exposure in a boiling water bath and only lost activity after 2 hours of exposure to boiling. This study succeeded in producing a more stable extracellular recombinant PAL which demonstrated biochemical activity that was largely similar to that of native PAL but only differed in carbohydrate specificity and haemagglutinating strengths. / Flemish Interuniversity Council (VLIR-UOS)-Own Initiative Project,the SARBIO- South African Regional Co-operation in Biochemistry, Molecular Biology and Biotechnology, the (CSIR) Council for Scientific and Industrial Research,the (NRF) National Research Foundation,(TBI) The Biovac Institute Foundation, and the (SIDA) Swedish International Agency

A principal fonte de proteínas para alimentação humana e animal é fornecida pelas sementes de cereais e leguminosas. O conteúdo de aminoácidos solúveis em endospermas de milho normal e mutantes opaco-2 e floury foram determinadas por HPLC. A análise indicou que a concentração total de aminoácidos solúveis variou entre os mutantes e seus tipos selvagens. Nos mutantes o10, o11 e o13, as concentrações foram aumentadas significativamente quando comparadas ao tipo selvagem W22, enquanto os mutantes o1, o2, o13, fl1 e fl2 exibiram baixas concentrações em relação ao seu respectivo tipo selvagem Oh43. Resultados similares foram obtidos para os mutantes o5, o7 e fl3 em relação aos seus tipos selvagens (B79, B37 e WT3, respectivamente). Para metionina, o mutante o2 e o tipo selvagem Oh43 apresentaram as mais altas concentrações deste aminoácido. Diferenças significativas não foram observadas para os outros aminoácidos analisados, tais como lisina e treonina. Os resultados sugerem que as altas concentrações sugeridas originalmente para estes mutantes devem ser devidas aos níveis destes aminoácidos incorporados nas proteínas de reserva, mas não na forma solúvel. / For human nutrition the main source of vegetable proteins are cereal and legume seeds. The content of total soluble amino acids in mature endosperms of wildtype and maize opaque and floury mutants have been determined by HPLC. The total absolute concentration of soluble amino acids among the mutants and their wild-type counterparts varied depending on the mutant. In the o10, o11 and o13 mutants the concentrations were significantly increased when compared to their wild-type counterpart W22, whereas the mutants o1, o2, o13, fl1 and fl2 exhibited lower concentrations when compared to the wild-type Oh43, Similar results were observed for o5, o7 and fl3 in relation to their specific wild-type counterparts (B79, B37 and WT3, respectively). For soluble methionine content, o2 and Oh43 exhibited the highest concentrations. Significant differences were not observed for other amino acids such as lysine and threonine. The results suggest that the high-lysine concentrations indicated originally for these mutants must be due to the amino acids incorporated into storage proteins, but not in the soluble form.