Le colza d’hiver (Brassica napus) est cultivé pour l’huile contenue dans ses graines ainsi que pour les tourteaux qui sont une source de protéines pour l’alimentation animale. La culture de colza demande de forts apports d’azote et cette espèce est caractérisée par sa faible efficacité d’utilisation de l’azote. Une forte proportion de l’azote absorbé est restituée au sol lors de la chute précoce des feuilles au stade végétatif. L’amélioration de la remobilisation de l’azote est donc de première importance pour améliorer le rendement de cette culture tout en satisfaisant le besoin de réduction des intrants. La glutamine et l’asparagine jouent un rôle important dans le transport de l’azote au sein de la plante, notamment au cours de la sénescence foliaire. Les deux familles multigéniques des glutamine synthétases cytosoliques (GLN1) et des asparagine synthétases (ASN) assurent leur synthèse. Ce travail de thèse s’est intéressé à ces enzymes chez deux Brassicacées : le colza et Arabidopsis thaliana. Dans un premier temps, l’expression des gènes GLN1 a été étudiée chez Arabidopsis par une combinaison d’approches de biologie moléculaire, cellulaire et de cytologie. Les spécificités d’expression de chacun des cinq gènes d’Arabidopsis ont été mises en évidence. L’identification des gènes BnaGLN1 chez Brassica napus a permis une analyse de leur expression en fonction de l’âge des feuilles et de la disponibilité en azote. Les profils d’expression observés chez le colza se sont révélés similaires à ceux des gènes homologues d’Arabidopsis, amenant l’hypothèse d’une conservation des fonctions chez les deux espèces. Le rôle des gènes GLN1 d’Arabidopsis dans la remobilisation de l’azote vers les graines a été étudié grâce à un marquage ¹⁵N effectué sur des mutants simples. Le rôle des gènes GLN1 dans la remobilisation de l’azote des tissus végétatifs vers les tissus reproducteurs a été mis en évidence sans toutefois cibler spécifiquement une isoforme. L’étude de la famille ASN chez Arabidopsis a permis de mettre en évidence des profils d’expression spécifiques en fonction des organes, de l’âge des tissus et de la disponibilité en azote pour chacun des trois gènes. Le marquage ¹⁵N a également révélé une implication des gènes ASN1 et ASN2 dans la remobilisation de l’azote de la rosette vers les tissus reproducteurs. Les travaux présentés dans ce manuscrit sont une base pour de futures approches translationnelles vers le colza. / Winter oilseed rape (Brassica napus) is grown for its oil-rich seeds and for proteins, used in animal feed cake. It requires high nitrogen inputs due to the low efficiency of nitrogen utilization that characterizes this species. A large proportion of absorbed nitrogen is indeed returned to the soil when leaves fall. Improving nitrogen remobilization to promote seed filling is then required to improve yield and limit fertilizer use. Asparagine and glutamine are important amino acids for phloem translocation. This thesis focuses on the two multigenic families in charge of asparagine and glutamine synthesis: cytosolic glutamine synthetase (GLN1) and asparagine synthetase (ASN). Studies were performed on the two Brassicaceae, rapeseed and Arabidopsis thaliana. The GLN1 gene expressions were investigated in Arabidopsis by a combination of molecular biology and cytology. The five GLN1 genes are differentially expressed in Arabidopsis depending on ageing and nitrogen availability. The identified BnaGLN1 genes in Brassica napus also showed age and nitrogen dependent expressions. Interestingly, expression profiles were similar between homologous genes in Arabidopsis and rapeseed, suggesting that homologous genes share similar function in the two species. The role of Arabidopsis GLN1 genes for nitrogen remobilization to the seeds was monitored using ¹⁵N tracing experiments on individual mutants. The GLN1 genes play a role in the remobilization of nitrogen from the rosette leaves to the reproductive organs. However, their effect is weak and non-specific of one GS1 isoform. ASN genes also presented specific expression profiles depending on organs, age and nitrogen availability. The ¹⁵N tracing revealed that ASN1 and ASN2 are both involved in nitrogen remobilization from the rosette to the seeds. Our studies provide a basis for future translational approaches to improve oilseed rape.

Chagas disease, is caused by the protozoan Trypanosoma cruzi. The available chemotherapy is restricted to two compounds: Nifurtimox and Benzonidazol. The T. cruzi life cycle occurs among invertebrate and vertebrate hosts and its survival strongly depends on its ability to catabolise both, carbohydrates and amino acids to obtain energy. The amino acids have an important role in several biological processes in this parasite. In this context, we studied the L-asparagine (Asn) relevance in the metabolism, differentiation and stress resistance of the T cruzi. Our results have shown that epimastigotes use Asn as carbon source, and contributes with the resistance to nutritional and oxidative stress. The Asn also showed a positive influence in metacyclogenesis and supported the ATP synthesis. Finally, the absence of Asn interfered with the proliferation of the replicative forms of T. cruzi. Taken together, our results suggest the involvement of the Asn metabolism in the protection of T. cruzi when submitted to different stress conditions. / Chagas disease, is caused by the protozoan Trypanosoma cruzi. The available chemotherapy is restricted to two compounds: Nifurtimox and Benzonidazol. The T. cruzi life cycle occurs among invertebrate and vertebrate hosts and its survival strongly depends on its ability to catabolise both, carbohydrates and amino acids to obtain energy. The amino acids have an important role in several biological processes in this parasite. In this context, we studied the L-asparagine (Asn) relevance in the metabolism, differentiation and stress resistance of the T cruzi. Our results have shown that epimastigotes use Asn as carbon source, and contributes with the resistance to nutritional and oxidative stress. The Asn also showed a positive influence in metacyclogenesis and supported the ATP synthesis. Finally, the absence of Asn interfered with the proliferation of the replicative forms of T. cruzi. Taken together, our results suggest the involvement of the Asn metabolism in the protection of T. cruzi when submitted to different stress conditions.

Com o objetivo de estudar a potencialidade do emprego de termolisina como catalisador nas reações de incorporação de N-acil-asparagina a ésteres de aminoácidos e peptídeos, foram sintetizados os seguintes di- e tripeptídeos: Boc-Asn-Ile-OBzl, Z-Asn-Ile-OBzl, Moz-Asn-Ile-OBzl, Boc-Asn-Leu- OBzl, Z-Asn-Leu-OBzl, Moz-Asn-Leu-OBzl, Z-Asn-Leu-OEt, Boc-Asn-Phe-OBzl, Z-Asn-Phe-OBzl, Moz-Asn-Phe-OBzl, Z-Asn-Phe- OEt, Z-Asn-Val-OBzl, Moz-Asp-Val-OBzl, Moz-Asn-Ile-Gly-OBzl, Moz-Asn-Ile-Ala-OBzl, Moz-Asn-Ile-Leu-OBzl e Moz-Asn- Ile-Phe-OBzl. Todos os peptídeos foram obtidos na forma pura, com bom rendimento e foram analisados e caracterizados por cromatografia em camada delgada, ponto de fusão, análise elementar, análise de aminoácidos e ressonância magnética protônica. Entre os grupos protetores de asparagina, benziloxicarbonil e p-metoxibenziloxicarbonil permitiram a obtenção dos dipeptídeos com excelentes rendimentos. Foi observado que os tripeptídeos requerem para a sua síntese menores concentração de enzima e tempo de reação em relação aos dipeptídeos. Não foi possível estabelecer a especificidade secundária da termolisina para o resíduo P\'2 pois os rendimentos dos tripeptídeos sintetizados não apresentaram diferença significativa. Foi também realizado um estudo metodológico para determinar as condições ótimas de síntese de Boc-Asn-Ile-OBzl, que consistiu em analisar a influência do pH, concentração de enzima, concentraçao e volume da solução de acetato de sódio, proporção entre os componentes carboxílico e amínico, temperatura e adição de solvente orgânico ao meio de reação. / With de objective of studying the potential for the use of thermolysin as a catalyst in reactions of incorporation of N-acyl-asparagine into esters of aminoacids and peptides, the following di- and tripeptides were synthesized: Boc-Asn-Ile-OBzl, Z-Asn-Ile-OBzl, Moz-Asn-Ile-OBzl, Boc-Asn-Leu-OBzl, Z-Asn-Leu-OBzI, Moz-Asn-Leu-OBzl, Z-Asn-Leu-OEt, Boc-Asn-Phe-OBzl,Z-Asn-Phe-OBzl, Moz-Asn-Phe-OBzl, Z-Z-Asn-Phe-OEt, Z-Asn-Val-OEt, Moz-Asn-Val-OBzl, Moz-Asn-Ile-Gly-OBzl, Moz-Asn-Ile-Ala-OBzl, Moz-Asn-Ile-Leu-OBzl e Moz-Asn-Ile-Phe-OBzl. All of these peptides were obtained in pure form in good yield and analyzed and characterized by thin layer chromatography, melting point, elemental analysis, aminoacid analysis and proton magnetic resonance. Among the protecting groups of asparagine, benzyloxycarbonyl and p-methoxybenzyloxycarbonyl gave excellent yields of the dipeptides. Relative to the dipeptides, the synthesis of the tripeptides was found to require lower enzyme concentrations and temperatures. Since the yields of the tripeptides failed to exhibit significant differences, it was not possible to establish the existence of a secondary specificity of thermolysin for the residue P\' 2 . A methodological study was also performed to determine the optimum conditions for synthesis of Boc-Asn-Ile-OBzl. This study consisted of an analysis of the influence of pH, enzyme concentration, concentration and volume of the solution of sodium acetate, relative proportions of the carboxyl and amino components, temperature, and addition of organic solvent to the reaction medium.

Chagas disease, is caused by the protozoan Trypanosoma cruzi. The available chemotherapy is restricted to two compounds: Nifurtimox and Benzonidazol. The T. cruzi life cycle occurs among invertebrate and vertebrate hosts and its survival strongly depends on its ability to catabolise both, carbohydrates and amino acids to obtain energy. The amino acids have an important role in several biological processes in this parasite. In this context, we studied the L-asparagine (Asn) relevance in the metabolism, differentiation and stress resistance of the T cruzi. Our results have shown that epimastigotes use Asn as carbon source, and contributes with the resistance to nutritional and oxidative stress. The Asn also showed a positive influence in metacyclogenesis and supported the ATP synthesis. Finally, the absence of Asn interfered with the proliferation of the replicative forms of T. cruzi. Taken together, our results suggest the involvement of the Asn metabolism in the protection of T. cruzi when submitted to different stress conditions. / Chagas disease, is caused by the protozoan Trypanosoma cruzi. The available chemotherapy is restricted to two compounds: Nifurtimox and Benzonidazol. The T. cruzi life cycle occurs among invertebrate and vertebrate hosts and its survival strongly depends on its ability to catabolise both, carbohydrates and amino acids to obtain energy. The amino acids have an important role in several biological processes in this parasite. In this context, we studied the L-asparagine (Asn) relevance in the metabolism, differentiation and stress resistance of the T cruzi. Our results have shown that epimastigotes use Asn as carbon source, and contributes with the resistance to nutritional and oxidative stress. The Asn also showed a positive influence in metacyclogenesis and supported the ATP synthesis. Finally, the absence of Asn interfered with the proliferation of the replicative forms of T. cruzi. Taken together, our results suggest the involvement of the Asn metabolism in the protection of T. cruzi when submitted to different stress conditions.

Visando dar continuidade ao estudo de incorporação de N-acil-asparagina a derivados de aminoácidos e a peptídeos mediante catálise por termolisina, foi estudada a viabilidade de síntese dos peptídeos Z-Asn-Cys(S-Bzl)-OBut e Moz-Asn-Cys(S-Bzl)-Pro-Leu-Gly-NH2. Durante a síntese do pentapeptídeo Moz-Asn-Cys(S-Bzl)-Pro-Leu-Gly-NH2 foi constatada a formação de um subproduto cuja estrutura foi elucidada a partir da comparação ao padrão autêntico Moz-Asn-Leu-Gly-NH2. Para a obtenção deste padrão, sintetizamos o tripeptídeo Moz-Asn-Leu-Gly-OEt a partir de Moz-Asn-OH e H-Leu-Gly-OEt na presença de termolisina. A recristalização deste peptídeo em MeOH/H2 O levou à transformação do mesmo em Moz-Asn-Leu-Gly-OMe, confirmada pelo isolamento e caracterização do produto por espectrometria de massa e ressonância magnética protônica, análise elementar e de aminoácidos e por determinação do tempo de retenção por HPLC. Estudos posteriores demonstraram o envolvimento do íon Ca(II) no processo e nos permitiram sugerir um modelo da ligação deste íon ao peptídeo e da sequência de reação da transesterificação estudada. Também foi investigada a possibilidade de diversos ésteres de peptídeos e de peptidilresinas de Merrifield sofrerem transesterificação quando incubados em metanol na presença de Ca(II). Para tanto, os tripeptídeos Z-Asn-Leu-Gly-OEt, Boc-Asn-Leu-Gly-OEt, Moz-Asn-Leu-Gly-OBzl, Moz-Asn-Leu-Gly-OBu Moz-Gln-Leu-Gly-OEt, Moz-Asn-Ile-Gly-OEt e Moz-Asn-Leu-Ala-OEt foram sintetizados mediante catálise por termolisina. Também foram testados vários outros peptídeos disponíveis no laboratório e Boc-Leu-Gly-Res e Moz-Asn-Leu-Gly-Res. Com exceção dos ésteres terc-butílicos, todos sofreram transesterificação em soluções metanólicas de acetato de cálcio. / The main obJective was the study of thermolysin catalyzed incorporation of N-acyl-asparagine into amino acid and peptide derivatives. Both Z-Asn-Cys(S-Bzl)-OBut and Moz-Asn-Cys(S-Bzl)-Pro-Leu-Gly-NH2 syntheses have been studied. The formation of a by-product occurred during the synthesis of the later pentapeptide. By comparison with an authentic standard this by-product was identified as Moz-Asn-Leu-Gly-NH2. The standard Moz-Asn-Leu-Gly-NH2 was obtained by aminolYSlS of Moz-Asn-Leu-Gly-OEt. This peptide was synthesized by coupling Moz-Asn-OH with H-Leu-Gly-OEt using thermolysin as catalyst. During the recrystallization in MeOH/H2O, the transformation of Moz-Asn-Leu-Gly-OEt to its methyl-ester was obtained. This observation has been confirmed by purification and caracterization of the peptide by: Mass Spectroscopy, Nuclear Magnetic Ressonance, Amino Acid and Elemental Analysis and HPLC. Later studies have shown that the Ca(II) ion participates in the process. A model for the binding of this ion to the peptide and for the transesterification reaction has been suggested. Further studies have been performed with (1) the newly synthesized peptides Z-Asn-Leu-Gly-OEt, Boc-Asn-Leu-Gly-OEt, Moz-Asn-Leu-Gly-OBzl, Moz-Asn-Leu-Gly-OBut, Moz-Gln-Leu-Gly-OEt, Moz- Asn-Ile-Gly-OEt and Moz-Asn-Leu-Ala-OEt; (2) several other peptides available in our laboratory; and (3) Boc-Leu-Gly-Res and Moz-Asn-Leu-Gly-Res. With the exception of t-butyl-esters, all peptides tested have transesterified in methanolic calcium acetate solutions.

Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 203-215). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

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Com o objetivo de estudar a potencialidade do emprego de termolisina como catalisador nas reações de incorporação de N-acil-asparagina a ésteres de aminoácidos e peptídeos, foram sintetizados os seguintes di- e tripeptídeos: Boc-Asn-Ile-OBzl, Z-Asn-Ile-OBzl, Moz-Asn-Ile-OBzl, Boc-Asn-Leu- OBzl, Z-Asn-Leu-OBzl, Moz-Asn-Leu-OBzl, Z-Asn-Leu-OEt, Boc-Asn-Phe-OBzl, Z-Asn-Phe-OBzl, Moz-Asn-Phe-OBzl, Z-Asn-Phe- OEt, Z-Asn-Val-OBzl, Moz-Asp-Val-OBzl, Moz-Asn-Ile-Gly-OBzl, Moz-Asn-Ile-Ala-OBzl, Moz-Asn-Ile-Leu-OBzl e Moz-Asn- Ile-Phe-OBzl. Todos os peptídeos foram obtidos na forma pura, com bom rendimento e foram analisados e caracterizados por cromatografia em camada delgada, ponto de fusão, análise elementar, análise de aminoácidos e ressonância magnética protônica. Entre os grupos protetores de asparagina, benziloxicarbonil e p-metoxibenziloxicarbonil permitiram a obtenção dos dipeptídeos com excelentes rendimentos. Foi observado que os tripeptídeos requerem para a sua síntese menores concentração de enzima e tempo de reação em relação aos dipeptídeos. Não foi possível estabelecer a especificidade secundária da termolisina para o resíduo P\'2 pois os rendimentos dos tripeptídeos sintetizados não apresentaram diferença significativa. Foi também realizado um estudo metodológico para determinar as condições ótimas de síntese de Boc-Asn-Ile-OBzl, que consistiu em analisar a influência do pH, concentração de enzima, concentraçao e volume da solução de acetato de sódio, proporção entre os componentes carboxílico e amínico, temperatura e adição de solvente orgânico ao meio de reação. / With de objective of studying the potential for the use of thermolysin as a catalyst in reactions of incorporation of N-acyl-asparagine into esters of aminoacids and peptides, the following di- and tripeptides were synthesized: Boc-Asn-Ile-OBzl, Z-Asn-Ile-OBzl, Moz-Asn-Ile-OBzl, Boc-Asn-Leu-OBzl, Z-Asn-Leu-OBzI, Moz-Asn-Leu-OBzl, Z-Asn-Leu-OEt, Boc-Asn-Phe-OBzl,Z-Asn-Phe-OBzl, Moz-Asn-Phe-OBzl, Z-Z-Asn-Phe-OEt, Z-Asn-Val-OEt, Moz-Asn-Val-OBzl, Moz-Asn-Ile-Gly-OBzl, Moz-Asn-Ile-Ala-OBzl, Moz-Asn-Ile-Leu-OBzl e Moz-Asn-Ile-Phe-OBzl. All of these peptides were obtained in pure form in good yield and analyzed and characterized by thin layer chromatography, melting point, elemental analysis, aminoacid analysis and proton magnetic resonance. Among the protecting groups of asparagine, benzyloxycarbonyl and p-methoxybenzyloxycarbonyl gave excellent yields of the dipeptides. Relative to the dipeptides, the synthesis of the tripeptides was found to require lower enzyme concentrations and temperatures. Since the yields of the tripeptides failed to exhibit significant differences, it was not possible to establish the existence of a secondary specificity of thermolysin for the residue P\' 2 . A methodological study was also performed to determine the optimum conditions for synthesis of Boc-Asn-Ile-OBzl. This study consisted of an analysis of the influence of pH, enzyme concentration, concentration and volume of the solution of sodium acetate, relative proportions of the carboxyl and amino components, temperature, and addition of organic solvent to the reaction medium.

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Orientador: Ladaslav Sodek / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T05:22:48Z (GMT). No. of bitstreams: 1 Souza_SarahCarolineRibeiro_D.pdf: 2548021 bytes, checksum: 61437f1032c9e4fcc9ee2131a8f6dabe (MD5) Previous issue date: 2014 / Resumo: A soja é uma leguminosa amplamente utilizada para estudos envolvendo a fixação biológica de Nitrogênio (N), seja por sua grande importância econômica, seja por sua elaborada e bem sucedida relação simbiótica com rizóbios do gênero Bradyrhizobium, sendo capaz de obter todo N necessário para seu desenvolvimento através da fixação do N2 atmosférico. Todavia, o metabolismo de N em plantas de soja noduladas é sensível à hipóxia provocada pelo alagamento do sistema radicular. Dessa forma, este trabalho teve por objetivo, avaliar os efeitos do alagamento sobre o metabolismo de N em nódulos de soja em diferentes períodos de inundação e recuperação após a drenagem. Para isto, plantas de soja noduladas com o B. elkanii foram submetidas aos experimentos de inundação e recuperação, sendo os períodos de inundação/recuperação variáveis de acordo com o experimento. As alterações no metabolismo foram avaliadas através da análise da composição de aminoácidos por HPLC e avaliação da incorporação de 15N2 nos aminoácidos dos nódulos. Também foi avaliada a atividade da nitrogenase e a expressão dos genes nifH e nifD (nitrogenase), e dos genes que codificam as enzimas glutamato descarboxilase (GAD) e asparagina sintetase (AS) em soja. Verificamos que asparagina (ASN) é o aminoácido mais abundante em nódulos de soja (50%), seguido por glutamato (GLU), serina (SER) e ácido gama-aminobutírico (GABA). Com a inundação observou-se principalmente, uma redução acentuada de ASN, e aumento de GABA, após 4 dias (quando ASN reduziu a quase 1%), além de pequenas alterações na composição de outros aminoácidos. Com os tratamentos de recuperação ASN recuperou-se lentamente e quanto maior o período de exposição ao estresse mais lento o período de recuperação. Aparentemente a redução de ASN nos nódulos foi compensada pelo aumento de GABA. A atividade da nitrogenase foi fortemente inibida pela inundação, mas se recuperou totalmente. Quanto à incorporação de 15N2, verificamos que GLN foi o aminoácido marcado em grau mais elevado, seguido respectivamente por GLU, ASP, ALA e SER. A marcação dos aminoácidos ASN e GABA foi baixa, e isso pode ser devido ao grande "pool" destes aminoácidos no nódulo, ou pela entrada destes aminoácidos a partir de uma fonte não-marcada como o floema. Com relação à inundação, observou-se uma redução na incorporação de 15N2 em ASN, e a recuperação também foi lenta, também houve redução na incorporação em outros aminoácidos como ASP e GLN. A hipóxia afetou a expressão dos genes avaliados nos nódulos. Houve uma redução na expressão dos genes AS1 e AS2, o que condiz com a redução nos teores de ASN. O gene que codifica a GAD também foi menos expresso em nódulos submetidos à inundação o que não explica o aumento de GABA no nódulo durante o estresse. A expressão dos genes nifH e nifD também diminuiram com a inundação, mas se recuperam, e condizem com o observado para atividade da nitrogenase. Dessa forma, verificamos que a inundação afeta o metabolismo de N nos nódulos de soja, em diversos aspectos, como a composição de aminoácidos, atividade da nitrogenase e na expressão de genes envolvidos na assimilação do N em aminoácidos nos nódulos / Abstract: Soybean has been widely used in studies of biological nitrogen fixation, not only because of its economic importance, but in view of its highly efficient symbiotic relationship with rhizobia of the genus Bradyrhizobium, which can supply all the N needed for full development of the plant. However, the process is highly sensitive to oxygen deficiency provoked by waterlogging of the root system, resulting in a rapid and strong inhibition of nitrogen fixation since the availability of oxygen for nitrogenase activity is tightly controlled in the nodule and close to limiting under normal conditions. Thus, this study aimed to evaluate the effects of flooding on the N metabolism in nodules of soybean in different periods of flooding and recovery after drainage. For this, soybean plants nodulated with B. elkanii were subjected to flooding and recovery experiments at stages V7/V8 The flooding/recovery duration was where the flooding/recovery periods were variable according to the experiment. Changes in metabolism were evaluated by analyzing the amino acid composition by HPLC and by assessing the amino acid incorporation of 15N2 of the nodules. Nitrogenase activity and expression of nifH and nifD (nitrogenase) genes, and genes encoding GAD and AS in soybean were also evaluated. The most abundant amino acid in soybean nodules was asparagine (ASN) (50%), followed by glutamate (GLU), serine (SER) and gama-aminobutyric acid (GABA). On flooding, there was a marked decrease of ASN, and increased GABA, mainly after 4 days when ASN dropped to near 1%, as well as smaller alterations in the composition of other amino acids. With the recovery treatments, ASN recovered slowly and the longer the period of exposure to the stress the longer the recovery period. It appears that the reduction of the ASN in nodules is compensated by the increase of GABA. The nitrogenase activity was strongly inhibited by flooding, but full recovery was possible. Regarding the incorporation of 15N2, it was found that GLN is the amino acid labelled to the highest degree, followed respectively by GLU, ASP, ALA and SER. The labelling of the amino acids GABA and ASN was low, which may be due either to the large pool of these amino acids in the nodule, or to the entry of these amino acids from a non-labelled source such as the phloem. Flooding resulted in a reduction of the incorporation of 15N2 in ASN, and recovery was also slow. There was also reduction in the incorporation of 15N2 in other amino acids, such as Asp and GLN. Hypoxia affected the expression of all genes evaluated in nodules. There was a reduction in the expression of the AS1 and AS2 genes, which is consistent with the fall in levels of ASN. Recovery of expression was slow and gradual. Expression of the gene encoding the enzyme GAD was also strongly suppressed in nodules under flooding which does not therefore explain the increase of GABA in the nodule during stress. The expression of nifH and nifD genes were also strongly decreased on flooding, but recovered fully, consistent with the observed data for nitrogenase activity. In conclusion, it was found that flooding affects the metabolism of N in soybean nodules, in diverse ways, such as the amino acid composition, nitrogenase activity, and the expression of genes involved in N assimilation of nodule amino acids / Doutorado / Biologia Vegetal / Doutora em Biologia Vegetal