Dissertations / Theses on the topic 'Ammonium assimilation'

Saccharomyces cerevisiae can assimilate ammonium by NADP-GDH or by GS-GOGAT. The aim of this project was to improve the efficiency of ammonium assimilation, and therefore substrate utilisation, of S. cerevisiae by elimination of the energy inefficient pathway (GS-GOGAT). GOGAT- mutants were isolated from a GDH- parent strain by their inability to use ammonium as sole nitrogen source. Two structural gene mutants were identified, one in each of the two structural genes encoding GOGAT. Constructs with different combinations of GDH- and GOGAT- mutations and corresponding wild type alleles were made, and their growth studied in medium supplemented with different levels of ammonium. The growth properties (as final culture density and growth rate) of GOGAT- and GOGAT+ strains transformed with the GDH1 gene, and grown with excess ammonium were very similar. It was concluded that, under the conditions used in this study, the loss of GOGAT does not improve the growth properties of the strain. Non-transformed constructs were grown with excess and limiting ammonium. Growth properties of the GDH- and GOGAT- strains suggest that GS-GOGAT functions in ammonium assimilation at very low ammonium levels. This conclusion needs further investigation because the GDH+ GOGAT- construct had lower NADP-GDH activity than the wild type. The physiology of ammonium assimilation by two industrial strains was compared to that of a laboratory wild type at different ammonium concentrations using shake-flask culture. All three strains possessed the three activities in MM+20mM NH4+, and the profiles of appearance/disappearance of activity were very similar. At lower ammonium concentrations, important differences between the strains became apparent. It is unclear if it is due to simple strain heterogeneity or represents significant differences between industrial and laboratory strains. On the basis of the enzyme data, GS-GOGAT appears to be important in ammonium assimilation by DCL1 at limiting concentrations.

Bibliography: pages 98-112.
A review of the recent literature concerning the assimilation and utilisation of nitrate and ammonium nitrogen within plants has been presented. Barley plants (Hordeum vulgare L.cv. Clipper) were grown hydroponically under controlled environmental conditions. The aerated nutrient solutions contained 2mM inorganic ¹⁴N supplied as either nitrate alone, ammonium alone, a 1:1 nitrate plus ammonium mixture or a 3:1 nitrate plus ammonium mixture. After 20 days of growth ¹⁵N nutrient solutions were substituted. The plant material was harvested four and eight hours after the commencement of the ¹⁵N feeding experiment and prepared for analysis. Xylem sap was also collected for a period of one hour beginning half an hour before each harvest and continuing for half an hour after harvesting. Separate batches of plants were used for harvesting and sap collection. In nitrate-fed plants the shoot was shown to be the main organ of nitrate assimilation. Xylem sap analysis indicated that 66% of the ¹⁵N supply to the shoot was in the form of nitrate and the majority of the absorbed and assimilated ¹⁵N was located in this region. In ammonium-fed plants, however, ¹⁵N-ammonium accumulated in the root with only a very small amount detectable in the xylem sap. Some 93% of the ¹⁵N exported from root to shoot in the xylem stream was in the form of organic nitrogen (mainly glutamine). This indicated that the root was the major organ of ammonium assimilation and that the shoot was the main destination of root assimilated nitrogen.

L’activité de croissance des plantes se trouve souvent limitée par les conditions contraignantes de l’environnement. La salinité du sol est l’une des majeures contraintes abiotiques qui ne cesse d’envahir les surfaces cultivés chaque année. Elle entraine chez les espèces glycophytes des perturbations d’ordre osmotique, nutritionnel et métaboliques. La nutrition et le métabolisme de l’azote minéral constituent des étapes primordiales dans la synthèse des acides aminés et des composés azotés indispensables chez les plantes. Par conséquent, l’étude de l’expression des enzymes impliquées dans l’assimilation d’azote telle que l’asparagine synthétase (AS, EC 6.3.5.4) chez l’arabette des dames (Arabidopsis thaliana) permet d’avancer nos connaissances sur la régulation transcriptionnelle du métabolisme azoté sous stress salin. Au cours des travaux de recherche entamés dans le cadre de cette thèse, un intérêt particulier est accordé au gène ASN2 chez Arabidopsis. Les résultats obtenus ont montré que la mutation ASN2 a accentué les effets du NaCl sur l’assimilation de l’ammonium. Le mutant asn2-1 se montre plus sensible au stress salin que le sauvage malgré que l’absence des transcrits du gène ASN2 est associé à une expression importante du gène ASN1. L’inhibition de l’activité glutamine synthétase (GS, EC 6.3.1.2), la faible activité aminatrice de la GDH (NADH-GDH, EC 1.4.1.2) sous stress salin ainsi que l’absence des transcrits ASN2 seraient à l’origine de l’accumulation de l’ammonium chez le mutant asn2-1. Toutefois, l’application exogène de l’ammonium nous a montré que l’action du NaCl sur l’expression de l’asparagine synthétase n’est pas directement liée à l’accumulation endogène d’ammonium. L’accumulation d’autres métabolites tels que l’asparagine, la glutamine et la glutamate pourrait être à l’origine des effets du sel sur l’expression des gènes ASN
Plant growth activity is often limited by constraint environment conditions. Soil salinity is one of major abiotic stress which is becoming more problematic every year. In glycophytes species, it induced osmotic, nutritional and metabolic disturbances. The nitrogen nutrition and metabolism constitute an essential step in amino acid and nitrogen compounds synthesis in plants. Therefore, studying the expression of enzymes involved in nitrogen assimilation such as asparagine synthetase (AS, EC 6.3.5.4) in Arabidopsis thaliana will improve our knowledge on the transcriptional regulation of nitrogen metabolism under salt stress. In the present work of this thesis, a special attention was taken on AS gene (ASN2) wild type and mutants. Obtained results showed that ASN2 mutation accentuated the salt-induced effects on ammonium assimilation. The asn2-1 mutant was more sensitive to salt stress than the wild type, while the ASN2 transcript absence was associated with an important ASN1expression. The observed inhibition of glutamine synthetase (GS, EC 6.3.1.2) activity, the low aminatrice GDH (NADH-GDH, EC 1.4.1.2) activity under salt stress as well as the ASN2 transcript loss brought to an ammonium accumulation in asn2-1mutant. However, exogenous ammonium application showed that NaCl effect on asparagine synthetase expression was not directly related to the endogenous ammonium accumulation. Other metabolites accumulation such as asparagine, glutamine and glutamate could be involved in the obtained salt-effects on ASN expression in Arabidopsis

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In this work, tobacco plants with a high proline accumulation due to the insertion of the VaP5CSF129A mutant gene on the control of the promoter constitutive CaMV 35S were used as a model to verify the effect of transgene overexpression on the uptake and utilization of N. The experiment was conducted in a greenhouse for a period of 30 days. The arrangement was completely randomized in the 2x5 factorial scheme, consisting of two genotypes (one transgenic event and one unprocessed control) and five nitrogen fertilization doses (ammonium nitrate (NO3NH4): 25, 50, 100, 200 and 400 kgN ha-¹), totaling 10 treatments with 4 replicates. The plants were evaluated for height, leaf number, dry mass, root/shoot ratio, chlorophyll content, free proline content, nitrogen content, total protein foliar content, carbon/nitrogen ratio and nutritional efficiency for N (Absorption efficiency - EAbs, leaf utilization - EUtil and plant use - EUN). The plants of the transgenic event with high proline accumulation were less efficient than the control plants in terms of nitrogen absorption (lower levels of N in root, leaves and role plant), which resulted in lower EAbs in these plants. For EUtil (given by the relation between leaf dry mass and N content in the plant) there was no difference between the transgenic and control genotypes. However, the transgenic plants presented lower EUN in comparison to the control plants. It was concluded that the use of tobacco plants with overexpression of the VaP5CS129A gene on control of the constitutive promoter CaMV 35S for a high proline accumulation does not present potential of direct use in agricultural systems, since the alteration of the biochemical balance between the metabolisms of nitrogen and carbon did not result in plants with higher EUN.
O aumento da eficiência de uso do nitrogênio (EUN) nas plantas cultivadas é resultado da melhoria da absorção e/ou da utilização do N. Nesse trabalho, plantas de tabaco com alto acúmulo de prolina devido à inserção do gene mutante VaP5CSF129A sobre controle do promotor constitutivo CaMV 35S foram utilizadas como modelo para verificar o efeito da super-expressão do transgene na absorção e utilização de N. O experimento foi conduzido em casa de vegetação por um período de 30 dias. O arranjo foi inteiramente casualizado, no esquema fatorial 2x5, formado por dois genótipos (um evento transgênico e um controle não transformado) e cinco doses de adubação nitrogenada (nitrato de amônio (NO3NH4): 25, 50, 100, 200 e 400 kgN ha-¹), totalizando 10 tratamentos com 4 repetições. As plantas foram avaliadas quanto à altura, número de folhas, massa seca, razão raiz/parte aérea, teor de clorofila, teor de prolina livre, teor de nitrogênio, teor foliar de proteína total e eficiência nutricional quanto ao N (eficiências de absorção - EAbs, utilização na folha - EUtil e uso na planta - EUN). As plantas controle e transgênicas responderam de forma positiva ao incremento de N no solo, tanto em relação às variáveis de crescimento quanto aos parâmetros bioquímicos. As plantas transgênicas apresentaram menor biomassa e maiores teores de compostos nitrogenados nas folhas (na forma de prolina e proteínas) quando comparadas às plantas controle. Quanto à eficiência nutricional, as plantas transgênicas foram menos eficientes que as plantas controle quanto à absorção de nitrogênio (menores teores de N na raiz, nas folhas e na planta); o que resultou portanto em uma menor EAbs nestas plantas. Para a EUtil (dada pela relação entre a massa seca de folhas e o teor de N na planta) não houve diferença entre os genótipos transgênico e controle. Porém, as plantas transgênicas apresentaram menor EUN em comparação às plantas controle. Concluiu-se que o uso de plantas de tabaco com super-expressão do gene VaP5CS129A sobre controle do promotor constitutivo CaMV 35S para um alto acúmulo de prolina não apresenta potencial de utilização direta em sistemas agrícolas, desde que a alteração do equilíbrio bioquímico entre os metabolismos de nitrogênio e do carbono não resultou em plantas com maior EUN.

Étude des mouvements d'azote entre les racines et les organes photosynthétiques. La fonction d'assimilation de NO::(3-) et de NH::(4+) est assurée par la racine mais la compartimentation cellulaire et le devenir des produits de l'assimilation primaire différents. En nutrition ammoniacale il existe un pool racinaire d'exportation de l'azote constitué essentiellement d'asparagine, arginine et glutamine, distinct du pool d'aminoacides de la racine. Au contraire, la nutrition nitrique entraîne une forte dépendance de la sève vis-à-vis d'une source d'azote réduit, préalablement stocké dans la racine

In order to investigate the effect of the elimination of GOGAT activity in S. cerevisiae, the pool sizes of ammonia, glutamate and glutamine plus the specific activities of the enzymes involved in ammonia assimilation were determined for two genetically engineered strains (AR2 and AR5) and an haploid wild type ( Sigma 1278b). AR2 and AR5 strains carry the plasmid pCYG4 which directs about 5 fold more NADPH-GDH activity than wild type cells. AR5 strain is a double mutant, which lacks GOGAT activity. The studies were carried out using a microprocessor-controlled fermenter (PCS) which has the following features: 3 Main Boards (Central Processor Board, Memory Board and Analog/Digital - ON/OFF Switch Board). 4 Auxiliary Boards (pH, Oxygen, Temperature and Biomass Interface Boards). A connection block to link the PCS with the video terminal, with sensors from the fermenter, with a control box and with other microcomputer. AR2 and AR5 showed lower values of maximum specific growth rates than the wild type, determined either by batch mode or by washout kinetics. The reduction in the growth rate for AR2 and AR5 can be related to the added metabolic loads due to the plasmid encoded genes. Under carbon limitation there were no remarkable differences between the NADPH-GDH activities of AR2 (GOGAT+) and AR5 (GOGAT-). However, the concentrations of glutamate and glutamine for AR2 were higher (from 20 to 40 %) than those of AR5. The lack of the GOGAT activity also resulted in a decrease in the biomass concentration for AR5 compared to the GOGAT+ strains. Under nitrogen limitation NADPH-GDH activities were higher and intracellular ammonia concentrations lower than under carbon limited conditions. The intracellular concentrations of glutamate and glutamine were higher for the GOGAT+ strain than for the GOGAT- one. Although the biomass level was the same for the three strains, AR5 (GOGAT") cells changed from rounded to ellipsoidal form under nitrogen limited conditions. Oscillations were present in the NADPH-GDH activities of AR2 and AR5 strains growing under carbon and nitrogen limited media. They are probably due to segregational instability of the plasmid pCYG4 in these microorganisms.

The structural gene (g1nA) encoding the ammonia assimilation enzyme glutamine synthetase (GS) has been cloned from the obligate methanotroph Methylococcus capsulatus (Bath). Complementation of Escherichia coli and Klebsiella pneumoniae g1nA mutants was demonstrated. In vitro and in vivo expression analysis revealed the cloned g1nA gene to encode a polypeptide with an apparent M_r of 60,000 as determined by PAGE. Expression of the M. capsulatus (Bath) g1nA gene in E. coli was found to be regulated by nitrogen levels in an Ntr+ but not an Ntr− background. This regulation was not observed when the cloned H. capsulatus (Bath) g1nA gene was under the influence of the chloramphenicol acetyl transferase gene of the vector. The nucleotide sequence of the M. capsulatus (Bath) g1nA gene and flanking sequences has also been determined. The gene comprises 1407 bp encoding a polypeptide of M_r 51,717 containing 468 amino acids. The 5' leader region contains three putative promoters. Promoters P1 and P3 resemble the canonical -10 -35 E. coli type promoter. Promoter P2 which is located between P1 and P3, resembles the NtrA dependent promoters of enteric organisms. A potential NtrC-binding site was also determined. The 3' flanking region contained a small putative open reading frame (ORF) encoding a polypeptide of M_r 7022. The identity of this polypeptide remains to be elucidated. Comparisons of g1nA structural genes and GS enzymes at the nucleotide and amino acid levels between M. capsulatus (Bath) and both prokaryotes and eukaryotes have been determined. The presence of ntrA, ntrB, ntrC, g1nB and rpoD homologues in the M. capsulatus (Bath) genome was determined by heterologous hybridization studies. Type I and Type II obligate methanotrophs were also screened for g1nA, ntrC and ntrA homologues. Both Type I and Type II organisms were found to have homologues to each of these gene probes. A portion of a M. capsulatus (Bath) putative ntrC gene has been cloned on a cosmid, pCOS1 and was found to be unlinked to g1nA and lies some 8.5 kb downstream of g1nA. The development of a plasmid transformation and gene transfer system for M. capsulatus (Bath) based on previously published methods has also been assessed.

Abstract Investigation of a Commercial Product (BiOWiSH­­TM) for Nitrogen Management Eva Lee BiOWiSH–Aqua, which has the capability of treating nitrogen from wastewater through bioaugmentation, is a commercial product consisting of a blend of microorganisms developed by BiOWiSH Technologies. A study of the treatment of nitrogen compounds (i.e. , , and ) using Biowish–Aqua was conducted using small scale experiments (flask experiments) and large scale experiments (column reactor experiments). In this work, column reactors were created to test Biowish–Aqua’s nitrogen treating capabilities by providing enough depth to simulate the dissolved oxygen gradient that can be observed in a pond. The results show that the optimal growth conditions for both ammonia assimilating and denitrifying bacteria are an anoxic environment with a carbon-to-nitrogen ratio of 2:1. Under this optimal growth environment, Biowish–Aqua was able to assimilate ammonia with a zero order k value of 3.06 ppm/day. Also, under the same conditions, Biowish–Aqua was able to eliminate nitrate ( ) and nitrite ( ) at a rate of 9.58 ppm/day and 5.64 ppm/day respectively. The experiments also suggested that with a C:N ratio of 2:1, Biowish–Aqua did not have an effect in slowing the hydrolysis of urea. Overall, this research suggests that the application of Biowish–Aqua is a feasible nitrogen removing strategy for wastewater with initial presence of ammonia and nitrate between 10 to 20 ppm. Keywords: Ammonia assimilation, Bioaugmentation, BiOWiSH, Denitrification

Biological nitrogen fixation (BNF) is the nitrogenase-catalyzed conversion of dinitrogen to ammonia by a select group of Bacteria and Archaea called diazotrophs. In turn, plants and other microbes assimilate ammonia during the synthesis of nucleic acids, proteins and other biomolecules. BNF is of special interest in agriculture where it replenishes soil nitrogen lost during repetitive farming. Basic knowledge of BNF might eventually lead to less dependence on expensive and polluting chemical fertilizers. For the studies presented here, two model diazotrophs, the free-living Azotobacter vinelandii , and the alfafa symbiont, Sinorhizobium meliloti, were used to investigate mechanisms controlling nitrogen fixation and nitrogen metabolism. In A. vinelandii, ammonia inhibits nitrogenase expression by limiting activity of the two-component activator, NifA; this involves the negatively acting sensor protein, NifL. Groundwork indicated that a global nitrogen-sensing system, present in many bacteria might control NifA activity since glnD mutants were unable to fix nitrogen. In other organisms, nitrogen limitation signals GlnD-mediated uridylylation of PII-like signal transduction proteins, which signals activation of a suite of genes involved in nitrogen source utilization. The goals of the current study were to characterize the operon encoding a PII-like protein in A. vinelandii, named GlnK, and determine its influence on NifA and nitrogen metabolism. The results indicated that glnK is an essential gene and that uridylylation of GlnK is required for activation of glutamine synthetase and NifA. Also presented here is evidence that GlnK interacts with NifL to stimulate its inhibitory properties. These results are consistent with a model in which uridylylation of GlnK in response to nitrogen limitation signals relief of NifL inhibition. In the last section of this dissertation, glnD of Sinorhizobium meliloti was cloned and sequenced because a PII-like protein had been previously implicated in control of nodule development and symbiosis. Unfortunately, S. meliloti glnD mutants could not be isolated unless glnD and flanking genes were provided in trans, indicating that the glnD operon is indispensable. These studies provide new insight into the global mechanisms controlling nitrogen fixation and metabolism and suggest that GlnD and PII-like proteins may regulate other targets, some of which are essential.

Ce travail contribue a l'etude de l'assimilation des acides gras a courte chaine (agcc) et de l'azote ammoniacal chez les levures en vue d'evaluer leur interet vis-a-vis de l'epuration carbonee et azotee d'effluents agro-industriels. L'assimilation des acides acetique (c2), propionique (c3) et butyrique (c4) etudiee separement et en melange chez quatre especes: candida ingens, candida tropicalis, candida utilis et yarrowia lipolytica, montre que l'acide propionique n'est jamais degrade en premier et que des mecanismes de regulation de la -oxydation et de la voie du propionyl-coa regissent differemment l'ordre de degradation des acides selon que les especes sont oxydatives ou fermentatives. L'etude cinetique des cultures de c. Utilis sur acides butyrique et propionique a permis de determiner les parametres cinetiques de croissance et de quantifier les effets inhibiteurs lies aux formes non dissociees des acides (1,2 g/l et 0,81 g/l respectivement pour c3 et c4) et de l'azote (nh3). Plusieurs modeles cinetiques issus de la litterature ont ete examines mais aucun n'a donne entiere satisfaction pour l'ensemble des phenomenes presents dans notre etude. Un nouveau modele mathematique integrant les divers effets inhibiteurs est donc propose. Par ailleurs, la tres bonne maitrise des cultures de levures s'est pretee a l'investigation d'une nouvelle methodologie de modelisation: les reseaux de neurones. Pour en demontrer tout le potentiel, cette approche est comparee avec les modeles classiques par bilan matiere. Finalement, apres l'etude des cultures de levures en milieu synthetique, l'experimentation des cultures sequentielles: (i. E. , bacteries anaerobies produisant les agcc et nh3, et levures aerobies) a ete engagee sur des milieux complexes (effluents agro-industriels) en vue d'envisager un nouveau concept de traitement

Un appareillage simple et efficace a été mis au point pour la culture de bactéries méthanogènes. Grâce à ce système, la nutrition soufrée et azotée de Methanobacterium (Mb. ) ivanovi et de Mb. Thermoautotrophicum souche delta H a été étudiée. En présence de mercapto-2-éthanol, agent réducteur non métabolisé, il a été montré que, en plus du sulfure, Mb. Ivanovi utilise le soufre élémentaire, la cystéine, et la méthionine comme source de soufre et que Mb. Thermoautotrophicum utilise le soufre élémentaire et la cystéine. Les autres composés testés n'ont aucun effet (sulfate) ou inhibent (sulfite, thiosulfate et dithionite) la croissance et la méthanogénèse des deux souches. En ce qui concerne la nutrition azotée, outre l'ammoniaque, (i) seule la glutamine est utilisée comme source d'azote chez Mb. Ivanovi, (ii) la glutamine et l'urée sont utilisées comme sources Irazote chez Mb. Thermoautotrophicum. Les autres composés testés n'ont aucun effet (nitrate chez la souche delta H) ou inhibent (nitrite) la croissance et la méthanogénèse des deux souches. La mesure des activités glutamine synthétase (GS), glutamate synthase (GOGAT) et alanine déshydrogénase (ADH) chez les deux souches suggèrent l'existence d'une voie GS-GOGAT pour l'assimilation de l'ammoniaque. A des concentrations élevées d'ammoniaque l'ADH pourrait jouer un rôle. Par contre, la glutamate déshydrogénase n'a pas été détectée chez ces méthanogènes. La GS de Mb. Ivanovi a été purifiée à homogénéité. C'est un dodécamère d'environ 600. 000 daltons composé d'un seul type de sous-unité, thermostable et insensible à l'oxygène. Son pI est de 4,6. Son activité n'est pas régulée par un système d'adénylation mais elle est sensible à une inhibition allostérique. Aucune réaction croisée n'a été détectée entre la GS de Mb. Ivanovi et des anticorps contre la GS d'Escherichia coli, d'Anabaena 7120 ou de Bacillus megaterium. Des mutants de 141 ivanovi auxotrophes pour la glutamine ont été caractérisés. Ces mutants ont une activité GS fortement diminuée par rapport à celle de la souche sauvage. En utilisant la technique de Southern, de l'homologie a été mise en évidence entre les gènes de structure de la nitrogénase (nifHDK) de K. Pneumoniae et d'Anabaena et l'ADN de Mb. Ivanovi et Mb. Thermoautotrophicum. De plus, de l'homologie avec le gène de structure de la GS (glnA) d'Anabaena a été détectée dans l'ADN de Mb. Ivanovi.

Le métabolisme azoté a été étudié chez deux champignons ectomycorhiziens : l'ascomycète Cenococcum geophilum et le basidiomycète Scleroderma verrucosum ainsi que chez les ectomycorhizes constituées par S. Verrucosum et Eucalyptus diversicolor. Chez C. Geophilum, la méthionine sulfoximine (MSX) inhibe fortement l'activité de la glutamine synthétase (GS) et de la glutamate déshydrogénase à NADP (GDH à NADP). L'albizzine inhibe l'accumulation du glutamate ce qui indique une implication de la glutamate synthase (GOGAT). C. Geophilum assimile donc l'azote par la voie GDH à NADP et le cycle GS/GOGAT. S. Verrucosum utilise bien l'azote nitrique et la nitrate réductase est réprimé par l'ammonium. Les dosages enzymatiques montrent que le champignon assimile l'azote via le cycle GS/GOGAT et l'activité de la GDH à NADP est faible. Chez S. Verrucosum associé à E. Diversicolor, le 14C-glutamate est rapidement métabolisé en glutamine par la GS. Cependant, l'accumulation de l'aspartate et de l'alanine marque au 14C est importante, même en présence de MSX, indiquant la grande efficacité des aminotransférases de la plante-hôte pour fournir des acides aminés. Les champignons étudiés appartiennent par conséquent à deux groupes: l'un représenté par C. Geophilum qui possède la GDH à NADP et le cycle GS/GOGAT et l'autre représenté par S. Verrucosum qui ne possède que le cycle GS/GOGAT. L’utilisation de l'électrophorèse capillaire a permis la séparation des acides organiques, en particulier ceux du cycle de Krebs et la détermination de l'ammonium, du nitrate, d'acides aminés particuliers (desmosine, isodesmosine), ainsi que de nombreux autres anions et cations inorganiques

The initiation, persistence, and termination of harmful algal blooms (HABs) can all be influenced by nutrient availability. Recent studies have highlighted the role of both organic and inorganic nitrogen sources in HAB dynamics. The pelagophyte Aureoumbra lagunensis causes ecosystem disruptive algal blooms and is responsible for the longest recorded harmful algal bloom (1989-1997). Because of Aureoumbra's small size and its inability to use nitrate, it has been hypothesized that its ability to use ammonium and organic nitrogen, especially at low concentrations, contributed to the unusual persistence of this bloom. This project aimed to assess the response of Aureoumbra to inorganic and organic nitrogen sources by examining the expression of genes responsible for nitrogen assimilation, with an eventual intent of developing expression assays that are indicative of nitrogen source use and/or sufficiency in Aureoumbra. Large volume batch cultures of Aureoumbra were grown with either ammonium or urea as a nitrogen source. Physiological characteristics (C:N, chlorophyll [alpha] cell⁻¹, and Fv/Fm) were monitored throughout the growth period, and the expression of the AMT-1, AMT-2 and UREC genes was assayed at early-, mid- and late-exponential phases. The results show that Aureoumbra can use both ammonium and urea, and that it is well adapted to low-nutrient environments. Only one gene, AMT-1, appeared to be transcriptionally regulated in response to changing nitrogen concentration, and only to ammonium. The results of this study contribute to our understanding of how algae in general cope with low nutrient availability and should ultimately help to define the dynamics of these HAB events.
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碩士
國立臺南大學
生物科技學系碩士班
107
Plasticizers are a kind of compounds that often use as a shaping agents and stabilizer in various types of products. With the development of industry, plasticizers are widely used in the production of various industrial and consumer goods. However, phthalates, the most prevalent plasticizers, are very easy to release from the product into the environment because they combine with substances by non-chemical bond. Duckweed is a common aquatic herb that is found in freshwater from temperate to tropical regions. Because of its high sensitivity to many pollutants, duckweed is often used as an indicator of the water environment. Our laboratory has conducted a number of studies to investigate the toxic effects of diethyl phthalate (DEP), a type of short-chain phthalate, on Greater duckweed (S. polyrhiza). We found that DEP causes chloroplast electron transport chain disorder and decreased photosynthesis efficiency, accumulation of reactive oxidants species (ROS) and malondialdehyde (MDA), increased ammonia nitrogen content, and a decrease in growth rate. In this study, we found that in the treatment of 0.25, 0.5, 1 mM DEP, the water-soaked appeared; in 2 mM DEP treatment, the yellowing phenomenon appeared, the cause of water-soked greater duckweed increased endogenous esterase as a detoxification means of DEP, the cell structure is destroyed by DEP treatment, or the carbon regulation is changed in DEP stress. The relative growth rate (RGR) showed a negative effect under DEP treatment, indicating that DEP caused cell division and reproductive toxicity to greater duckweed. DEP caused the change of nitrogen distribution, and nitrogen concentrate to chlorophyll at 0.25, 0.5 mM DEP concentration. Nitrogen is released from chlorophyll by 1,2 mM DEP treatment. The increase of low concentration of chlorophyll may be caused by the increase of glutamine synthetase (GS) activity, and the high concentration of chlorophyll may be caused by chlorophyll procedurality degradation. High concentration of DEP treatment caused GS-GOGAT cycle disability, NADH-GDH activity increased, which means that glutamate dehydrogenase (GDH) may be used as a substitute for ammonia in the DEP stress and the product of GS-GOGAT cycle and GDH pathway tends to different route of utilization. DEP treatment increases the activity of succinate dehydrogenase (SDH), indicating that the rate of TCA cycle rised under DEP stress, and GDH acts as a provider of TCA cycle substrates NAD-GDH activity also increased along with SDH activity. Correlation analysis result indicated that GDH 1 and SDH 1 gene expression showed a highly positive correlation; GDH 2 and GS total activity showed a highly negative correlation, which revealed the possible regulation mechanism of GDH gene under DEP stress.