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The compatible solute glycine betaine accumulates in many plants including spinach (Spinacea oleracea) under conditions of water deficit stress. The precursor to glycine betaine is choline, a ubiquitous metabolite in plants as a component of phosphotidylcholine. In spinach choline is synthesized from phosphocholine, a product of three sequential N-methylations of phosphoethanolamine catalysed by the cytosolic enzyme S-adenosyl-L-methionine: phosphoethanolamine-N-methyltransferase (PEAMT). PEAMT activity shows diurnal changes with peak activity at the end of the photoperiod and a decrease overnight. The activity of this enzyme is up-regulated 2 to 3-fold in salt-stressed plants relative to unstressed plants. The objective of this thesis is to determine how PEAMT activity is regulated in vivo. Thus, PEAMT activity, protein and transcript levels were quantified in spinach leaves from plants subjected to different light and salinity conditions. A spinach PEAMT eDNA sequence was used to over-express recombinant PEAMT in the protein expression vector pET30a (+). The presence of a polyhistidine-tag on the overexpressed protein allowed for purification by a cobalt metal affinity column. The affinity purified protein was used to produce polyclonal antibodies for immunoblot hybridization analysis. For these studies, PEAMT protein was first immunoaffinity purified from soluble extracts prepared from leaves and then the protein subjected to electrophoresis by SDS-p AGE. Enzyme assays and immunoblot analysis show PEAMT activity and protein levels increase and become relatively constant in leaves of plants exposed to continuous light. In continuous darkness, PEAMT activity and protein levels decrease and remain low and constant. Thus the pattern of changes in PEAMT activity levels are associated with changes in PEAMT protein levels. In contrast, Northern blot hybridizations show that under conditions of constant light, peamt transcript levels undergo cyclical changes with peak levels at 20 and 40 h and troughs at 28 and 52 h after the continuous light treatment was imposed. These peaks coincide with the dark and light cycles of the normal photoperiod. The same cyclical changes in peamt transcript levels was seen for plants transferred from a normal photoperiod to continuous darkness. Since these changes persist in the absence of a day/night cue we conclude that peamt transcript levels are circadian-regulated. The peamt transcript levels of control unstressed and salt-stressed plants also show circadian rhythms, however the levels found in salt-stressed plants were 0.5 to 2-fold higher than the controls. Therefore, while salinization of plants increases peamt transcript abundance, it does not alter the circadian rhythm that transcripts of this gene display. Changes in PEAMT activity and protein levels are likely controlled by other as yet unknown post-translational mechanisms, processes that override and obscure operation of a circadian rhythm in regulating the level of peamt transcripts. / Thesis / Master of Science (MS)

Made available in DSpace on 2014-06-11T19:26:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-12-17Bitstream added on 2014-06-13T20:53:58Z : No. of bitstreams: 1 martin_lc_me_botib.pdf: 1513851 bytes, checksum: b337e010131dce6a4e203f7b96730b8f (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Estresses abióticos, como a seca, podem reduzir significativamente os rendimentos no cultivo de espécies comercialmente importantes, tais como o eucalipto na produção de celulose e papel. Quando submetidas às condições de déficit hídrico, as plantas desenvolvem alguns mecanismos de defesa. As betaínas participam destes mecanismos, sendo seu soluto mais comum a glicina betaína. Esta é uma amina quaternária distribuída extensamente em diversas espécies de plantas superiores, sintetizada em elevadas taxas, tendo como função, manter a turgescência celular. A identificação e estudo de genes relacionados à tolerância à seca são importantes para os programas de melhoramento florestal. Este estudo teve por objetivo identificar SNPs (Single Nucleotide Polymorphisms) no gene colina monooxigenase relacionado ao metabolismo da glicina betaína em Eucalyptus. A sequência do gene em estudo foi encontrada em genomas de plantas modelos através do banco de dados GenBank, sendo as mesmas, utilizadas para a procura de similaridades no banco de dados de ESTs de eucalipto FORESTs – FAPESP. Após a constatação de homologia no banco de dados de eucalipto, foram confeccionados oito pares de “primers” para flanquear essas regiões, sendo estes, avaliados, amplificando fragmentos únicos. Depois de realizado o seqüenciamento do gene colina monooxigenase, foi utilizado a ferramenta BLAST no GenBank, confirmando com sucesso a identidade da sequência. Em seguida, as sequências foram alinhadas e os SNPs encontrados. No total, foram identificados 49 SNPs, sendo 12 em regiões codificantes e 37 em regiões UTRs e íntrons. Somente os SNPs localizados nas regiões codificantes foram utilizados neste trabalho, sendo 83,3% deles possuindo mutações sinônimas e 16,7% não-sinônimas. Em seguida, foi utilizada uma população mais abrangente composta de E. grandis... / Abiotic stress, such as drought, can reduce significantly the yield in the important commercially species crop, such as the eucalyptus in the cellulose and paper. When submitted to the water stress conditions, the plants develop some defense mechanisms. The betaines are part of these mechanisms , being their solute more common to the glycine betaine. This is a quaternary amine extensively distributed in several species of higher plants synthesized in high taxes with the function of maintaining the cellular turgescence. Identifying and studying the genes related to the drought tolerance are important for the forest improvement programs. This work aimed at identifying SNPs (Single Nucleotide Polymorphisms) in choline monooxygenase related to the glycine betaine in Eucalyptus. The gene sequence was found in model plants genomes through the databank GenBank, being used for searching similarities in the databank ESTs of eucalyptus FORESTs – FAPESP. After noticing homology in the eucalyptus databank, eight pairs of primers were made to amplify these regions, being evaluated, amplifying unique fragments. After the choline monooxygenase sequencing was performed, it was used the BLAST in the GenBank, confirming successfully the sequence identity. Then, the sequences were aligned and the SNPs were found. In the total, 49 SNPs were identified, being 12 in coding regions and 37 in UTRs and intron regions. Only the SNPs located in coding regions were used in this work, being 83.3% of the SNPs with synonymous mutation and 16.7% non-synonymous. Following, it was used a wider population made up of de E. grandis, E. Urophylla and the hybrid “Urograndis” to perform the SNPs genotyping, establishing the formation of 18 haplotypes and 16 possible haplotypical combinations which revealed that some genotypes were exclusive for the species E. grandis and E. urophylla. With the objective of decreasing costs... (Complete abstract click electronic access below)

A novel technique comprising solid phase microextraction and gas chromatography has been developed to analyse the three methylamines (monomethylamine, dimethylamine and trimethylamine) at the concentrations expected in seawater. The volatility of the methylamines was exploited and allowed the headspace pre-concentration and gas chromatographic separation of the analytes. This method achieved limits of detection of 0.43 nmol dm-3, 2.50 nmol dm-3 and 0.37 nmol dm-3 for monomethylamine, dimethylamine and trimethylamine, respectively, which are lower than any previously reported for seawater. This technique was applied to natural samples collected from the Western English Channel (WEC) and the methylamines were successfully quantified. A liquid chromatography-mass spectrometry method was used to analyse glycine betaine in marine particulates. The volume filtered during sample collection was found to be critical in achieving accurate measurements of glycine betaine. Large filter volumes (1 L) significantly decreased observed concentrations compared to smaller volumes, and 10 mL was the optimised amount filtered. The difference in concentrations was attributed to large water volumes causing cell damage and loss of glycine betaine to the dissolved phase. Cell resilience was thought to play a role in the differences observed; for example, diatoms with their rigid silica cell walls were hypothesised to be more resilient to filtration stress. A seasonal study was carried out in the WEC using both 1 L and 10 mL samples and maximum glycine betaine concentrations were 50 nmol dm-3 and 484 nmol dm-3 in the 1 L and 10 mL samples, respectively. The latter concentrations were more than an order of magnitude higher than all previous measurements. Seasonality was observed and a significant correlation was found with chlorophyll a. Statistics were applied to the phytoplankton biomass data and contributions from specific phytoplankton taxa were identified, including from Prorocentrum minimum and Chaetoceros socialis.

Orientador: Celso Luís Marino / Banca: Rinaldo César de Paula / Banca: Ivan de Godoy Maia / Resumo: Estresses abióticos, como a seca, podem reduzir significativamente os rendimentos no cultivo de espécies comercialmente importantes, tais como o eucalipto na produção de celulose e papel. Quando submetidas às condições de déficit hídrico, as plantas desenvolvem alguns mecanismos de defesa. As betaínas participam destes mecanismos, sendo seu soluto mais comum a glicina betaína. Esta é uma amina quaternária distribuída extensamente em diversas espécies de plantas superiores, sintetizada em elevadas taxas, tendo como função, manter a turgescência celular. A identificação e estudo de genes relacionados à tolerância à seca são importantes para os programas de melhoramento florestal. Este estudo teve por objetivo identificar SNPs (Single Nucleotide Polymorphisms) no gene colina monooxigenase relacionado ao metabolismo da glicina betaína em Eucalyptus. A sequência do gene em estudo foi encontrada em genomas de plantas modelos através do banco de dados GenBank, sendo as mesmas, utilizadas para a procura de similaridades no banco de dados de ESTs de eucalipto FORESTs - FAPESP. Após a constatação de homologia no banco de dados de eucalipto, foram confeccionados oito pares de "primers" para flanquear essas regiões, sendo estes, avaliados, amplificando fragmentos únicos. Depois de realizado o seqüenciamento do gene colina monooxigenase, foi utilizado a ferramenta BLAST no GenBank, confirmando com sucesso a identidade da sequência. Em seguida, as sequências foram alinhadas e os SNPs encontrados. No total, foram identificados 49 SNPs, sendo 12 em regiões codificantes e 37 em regiões UTRs e íntrons. Somente os SNPs localizados nas regiões codificantes foram utilizados neste trabalho, sendo 83,3% deles possuindo mutações sinônimas e 16,7% não-sinônimas. Em seguida, foi utilizada uma população mais abrangente composta de E. grandis... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Abiotic stress, such as drought, can reduce significantly the yield in the important commercially species crop, such as the eucalyptus in the cellulose and paper. When submitted to the water stress conditions, the plants develop some defense mechanisms. The betaines are part of these mechanisms , being their solute more common to the glycine betaine. This is a quaternary amine extensively distributed in several species of higher plants synthesized in high taxes with the function of maintaining the cellular turgescence. Identifying and studying the genes related to the drought tolerance are important for the forest improvement programs. This work aimed at identifying SNPs (Single Nucleotide Polymorphisms) in choline monooxygenase related to the glycine betaine in Eucalyptus. The gene sequence was found in model plants genomes through the databank GenBank, being used for searching similarities in the databank ESTs of eucalyptus FORESTs - FAPESP. After noticing homology in the eucalyptus databank, eight pairs of primers were made to amplify these regions, being evaluated, amplifying unique fragments. After the choline monooxygenase sequencing was performed, it was used the BLAST in the GenBank, confirming successfully the sequence identity. Then, the sequences were aligned and the SNPs were found. In the total, 49 SNPs were identified, being 12 in coding regions and 37 in UTRs and intron regions. Only the SNPs located in coding regions were used in this work, being 83.3% of the SNPs with synonymous mutation and 16.7% non-synonymous. Following, it was used a wider population made up of de E. grandis, E. Urophylla and the hybrid "Urograndis" to perform the SNPs genotyping, establishing the formation of 18 haplotypes and 16 possible haplotypical combinations which revealed that some genotypes were exclusive for the species E. grandis and E. urophylla. With the objective of decreasing costs... (Complete abstract click electronic access below) / Mestre

Seasonally high nighttime temperatures (HNT) along the United States Gulf Coast and in regions of similar climate, during the critical stages of development, could reduce rice yield and quality. To study the effects of HNT on plant physiology, a method for applying a controlled heating treatment to plant canopies was developed using overhead infrared heaters, which are relatively inexpensive and are accurate, precise and reliable in rapidly controlling the temperature. The apparatus successfully maintained air temperatures within the set points plus/minus 0.5 degrees C, and was used for all the experiments. Several experiments were conducted to determine the response of various physiological parameters during and following exposure of rice plants to HNT (32 degrees C) or ambient nighttime temperature (ANT) (27 degrees C) starting from 2000 h until 0600 h, and with or without plant growth regulator treatments. The plant growth regulator treatments included alpha-tocopherol (vitamin E), glycine betaine (GB), and salicylic acid (SA), which play different roles in inducing thermo-tolerance in plants. High nighttime temperature had no effect on plant height, number of tillers and panicles, or rice net leaf photosynthetic rates. However, HNT increased leaf respiration (dark respiration in the night) (21%) and decreased membrane thermo-stability (60%), pollen germination (20%), spikelet fertility (18% as a % of total spikelets), grain length (2%), and grain width (2%). The HNT also hastened plant development. The combinations of these effects decreased rice yield by 90%. Moreover, under HNT, there were decreases in leaf chlorophyll concentration (7%) and nitrogen concentration (18%). Application of GB and SA increased total antioxidant capacity of the rice plants by 17%, thereby decreasing the leaf respiration rates, increasing membrane thermo-stability, pollen germination, and spikelet fertility, thus increasing the yield. High nighttime temperature decreased leaf starch concentration (14%), grain total nonstructural carbohydrate (TNC) concentration (9%), and grain extractable invertase activity (20%). Vitamin E- or GB-treated plants had greater grain soluble-sugar concentrations, whereas SA-treated plants had greater leaf soluble-sugar concentrations and lower grain TNC concentrations. Invertase activity was shown to be not rate limiting or required for sucrose degradation for starch synthesis in grain of 'Cocodrie' rice under short-term high nighttime temperatures exposures during grain filling. In conclusion, HNT decreased rice yield by increasing plant respiration, rate of crop development, and decreasing membrane thermo-stability, pollen germination, spikelet fertility and grain dimensions. Exogenous application of GB and SA increased yields under HNT, possibly acting through increased antioxidant levels, which might have protected the membranes and enzymes against heat-induced ROS-mediated degradation.

Arthrobacter sp. yra dirvoje paplitusios bakterijos, kurios gali prisitaikyti prie nepalankių aplinkos sąlygų, tokių kaip osmosinis šokas ar maisto medžiagų trūkumas. Osmoreguliacijoje daugelis organizmų naudoja glicino betainą. Yra žinoma, kad Arthrobacter globiformis bakterijos gali panaudoti glicino betainą kaip anglies ir azoto šaltinį, tačiau nebuvo aišku, ar Arthrobacter globiformis gali šią medžiagą naudoti kaip osmoprotektorių. Iš Arthrobacter globiformis buvo klonuotas DNR fragmentas, kuriame buvo identifikuoti genai, kurie tiesiogiai ir netiesiogiai dalyvauja glicino betaino katabolizme. Struktūrinė genų organizacija leidžia manyti, kad šios bakterijos gali panaudoti glicino betainą kaip osmoprotektorių, Taip pat buvo įdomu patikrinti, ar tokią funkciją glicino betainas gali vykdyti ir kitose A. globiformis giminingose bakterijose. Tyrimui buvo pasirinkti tipiniai Arthrobacter genties bakterijų kamienai (A. atrocyaneus, A. citreus, A. crystalopoietes, A. globiformis, A. ramosus, A. sulfureus bei Arthrobacter spp. (KA3, P3, KA2V2, PY22, KA2, GAZ21, P2G, KA4, KA2V3, GAZ3, PRH1, PY21, VM22, VP23, RD1, VM22, VP22, VP3, VPW7, VPS4, 96, 94, 85, 68M, 83 68B, BL-3 ir 1-IN). Darbo metu paaiškėjo, kad šį junginį apsaugai nuo osmosinio streso naudoja tik dalis Arthrobacter genties bakterijų. Glicino betainas artrobakterėse dažniau naudojamas kaip anglies šaltinis. / Arthrobacter spp. are wide-spread soil bacteria, which are adapted to grow under osmotic stress and shortage in available carbon (energy) sources. Many organisms utilise glycine betaine as an osmoprotectant. It is known, that Arthrobacter globiformis can use glycine betaine as a sole carbon and nitrogen source, however, it is unclear whether this bacterium use this substrate as osmoprotectant. A DNA fragment harbouring genes, which directly and indirectly are involved in glycine betaine degradation, was cloned from A. globiformis. The structural organization of these genes suggested, that this bacterium could use glycine betaine as an osmoprotectant. So, it was tested if other Arthrobacter spp. strains use glycine betaine to protect from osmotic stress. Typical Arthrobacter sp. strains (A. atrocyaneus, A. citreus, A. crystalopoietes, A. globiformis, A. ramosus, A. sulfureus and Arthrobacter spp. (KA3, P3, KA2V2, PY22, KA2, GAZ21, P2G, KA4, KA2V3, GAZ3, PRH1, PY21, VM22, VP23, RD1, VM22, VP22, VP3, VPW7, VPS4, 96, 94, 85, 68M, 83 68B, BL-3 and 1-IN) were chosen in this study. It turned out, that less than a half of studied strains use glycine betaine as osmoprotectant. The arthrobacters used glycine betaine more as a sole carbon source than as an osmoprotectant.

Made available in DSpace on 2015-04-01T14:16:04Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1756619 bytes, checksum: d052f1be18e53f2beddaa57cda5b0a22 (MD5) Previous issue date: 2012-12-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Substrate-binding subunits are very important components of the solute importation system, known as the osmoprotectant system, which consists of a membrane protein belonging to the ABC superfamily. These molecules recognize specific substrates that have different physiological roles in prokaryotes, i.e. roles that contribute to the survival of these organisms in environments with high concentrations of salt. Using MEGA 5.05 software, this study performed a phylogenetic analysis of 431 nucleotide sequences of these subunits, orthologous to each other, collected from the database contained on the website http://www.genome.jp/kegg/. As a result of this analysis, phylogenetic trees were generated that clearly demonstrated that there was a horizontal transfer of some genes due to the sharing by different organisms. Also, two probable ancestral sequences were generated that showed homology with permeases that transport choline, glycine betaine and carnitine, which are trimethylamines currently present in various prokaryotes. Therefore, this system probably arose in prokaryotic organisms with the basic function of capturing nutrients, and by performing this basal function of being shared with other organisms, was fixed to the genome. However, because of the diversification of habitats by the prokaryotes, this system contributed decisively to the adaptation of these organisms to different environments, especially environments that had a high salt concentration; thus, acting and being currently characterized as a system of osmoprotection. / As subunidades ligadoras de substrato são componentes muito importantes do sistema de importação de soluto conhecido, como sistema de osmoproteção, que consiste em uma proteína de membrana pertencente à superfamlía ABC. Estas moléculas reconhecem substratos específicos que apresentam papéis fisiológicos diversos em procariotos, incluindo a colaboração para a sobrevivência destes organismos em ambientes com elevada concentração de sal. Utilizando o software MEGA 5.05, foi realizada uma análise filogenética de 431 sequências nucleotídicas destas subunidades, ortólogas entre si, coletadas a partir do banco de dados contido no site ttp://www.genome.jp/kegg/. Como resultado desta análise, foram geradas árvores filogenéticas que demonstraram claramente que houve a transferência horizontal de alguns dos genes devido ao ompartilhamento por organismos diferentes. Foram geradas também duas prováveis sequências ancestrais que apresentam homologia com permeases que transportam colina, glicina betaína e carnitina, que são aminas trimetiladas, presentes atualmente em diversos procariotos. Portanto, este sistema provavelmente surgiu em organismos procarióticos com a função básica de captura de nutrientes e por desempenhar esta função basal, ao ser compartilhado com outros organismos, foi fixado aos genomas. No entanto, a partir da diversificação de habitats, por parte dos procariotos, este sistema colaborou de forma decisiva para a adaptação destes organismos aos mais diversos ambientes, incluindo, especialmente os ambientes que apresentavam uma elevada concentração de sal, atuando e sendo caracterizado atualmente como um sistema de osmoproteção.

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