L’azote (N) est un facteur majeur limitant la croissance des plantes. Sa disponibilité peut également avoir un impact sur la résistance des plantes aux pathogènes en régulant leur immunité. Afin de mieux comprendre les liens entre la nutrition azotée et les défenses de la plante, nous avons analysé l’impact de la disponibilité en N sur la résistance de Medicago truncatula à un pathogène racinaire, Aphanomyces euteiches, en prenant en compte la variabilité génétique de la plante. Cet oomycète est considéré comme un des facteurs limitant le plus la production des légumineuses. Deux conditions de nutrition azotée, non limitante ou carencée en N, et dix génotypes de M. truncatula ont été testés in vitro. Les résultats ont montré que la résistance est modulée par les conditions nutritionnelles, dépendament du génotype. Les analyses d’expression de gènes impliqués dans le métabolisme azoté et dans les réponses de défense ainsi que la quantification des teneurs en acides aminés et des composés métaboliques secondaires ont montré des réponses différentes selon les génotypes et la condition nutritive. Elles ont souligné en particulier le rôle potentiellement important de la glutamine dans ce pathosystème. De plus, nous avons mis en évidence l’importance de l’homéostasie du monoxyde d’azote (NO) dans la résistance de M. truncatula à A. euteiches et que la disponibilité en azote impactait l’homéostasie du NO en affectant les niveaux de S-nitrosothiols et l’activité de la S-nitrosoglutathion réductase dans les racines. Ces résultats soulignent l’importance du métabolisme azoté et de son interaction avec le génotype de la plante dans les réactions de défense chez M. truncatula. / Nitrogen (N) is a major limiting factor for plant growth. N availability can also impact plant resistance to pathogens by regulating plant immunity. To better understand the links between N nutrition and plant defense, we analyzed the impact of N availability of plant on Medicago truncatula resistance to the root pathogen, Aphanomyces euteiches, taking into account plant genetic variability. This oomycete is considered as the most limiting factor for legume production. Two conditions of N nutrition, non-limiting or deprived in N, and ten plant genotypes were tested in vitro. The results showed that the resistance is modulated by nutritional conditions, depending on plant genotype. Analysis of the expression of genes involved in N metabolism and defense and quantification of different amino-acids contents and secondary metabolic compounds showed different responses of the genotypes and highlighted a potential role of glutamine in this pathosystem. Furthermore, our work underlined the importance of nitric oxide (NO) homeostasis for M. truncatula resistance to A. euteiches and that N availability impacts NO homeostasis by affecting S-nitrosothiol levels and S-nitrosoglutathione reductase activity in roots. These studies highlight, therefore, the importance of N metabolism and its interaction with plant genotype in defense responses in M. truncatula.

A fase de desenvolvimento é um importante fator a ser considerado em pesquisas sobre nutrição de bromélias. O hábito de vida dessas plantas pode mudar de: atmosférica (com folhas sem formar um tanque) para o com tanque ao longo do seu desenvolvimento. Algumas pesquisas mostraram que o conteúdo de nitrogênio foliar ou capacidade fotossintética são significantemente influenciados pela fase de desenvolvimento, porém não há registros de que a nutrição e o metabolismo do nitrogênio diferem entre bromélias jovens ou adultas. O objetivo principal deste projeto foi verificar se existem diferenças na dinâmica do metabolismo do nitrogênio (absorção, transporte e assimilação), decorrente da utilização de fontes de distintas (amônio, nitrato ou ureia), entre bromélias nas fases atmosférica ou adultas com tanque desenvolvido. Para tanto, plantas de Vriesea gigantea foram regadas com uma solução nutritiva que conteve 5mM de N total, disponível nas formas: 15NH4+ ou 15NO3- ou 15N-ureia. Foram feitas coletas temporais das raízes e de duas diferentes porções da folha (ápice e base) das bromélias jovens e de três regiões foliares (ápice, mediana e base) das folhas das bromélias adultas com tanque. Todas as amostras vegetais foram utilizadas na avaliação das atividades da: urease, redutase do nitrato, sintetase da glutamina e desidrogenase do glutamato; e da quantificação da abundância isotópica do 15N. Segundo os resultados, o nitrato foi considerado a fonte de nitrogênio absorvida em concentrações menores quando comparada com a ureia e o amônio pelas bromélias de ambas as fases de desenvolvimento. Entretanto, as bromélias atmosféricas mostraram ser capazes de capturar essa fonte inorgânica de nitrogênio mais eficientemente do que as bromélias com tanque, uma vez que o nitrato foi absorvido, transportado e assimilado rapidamente na 1ª hora após o fornecimento dessa fonte. Já para as bromélias adultas, a absorção do nitrato foi lenta e ocorreu, principalmente, no final do experimento (12ª e 24ª hora). O amônio e a ureia foram as fontes absorvidas em maiores concentrações tanto pelas bromélias jovens quanto pelas adultas. Entretanto, as bromélias atmosféricas foram capazes de captar e metabolizar maiores concentrações de nitrogênio proveniente do amônio, enquanto que as da fase adulta com tanque foram mais aptas a absorver e assimilar maiores concentrações de ureia em seus tecidos. A bromélia V. gigantea pode mudar a sua morfologia e fisiologia ao longo de seu desenvolvimento, tornando-se apta a captar as fontes de nitrogênio que, talvez, sejam mais abundantes em cada fase de seu desenvolvimento. A água da chuva que contém, principalmente, fontes inorgânicas de nitrogênio diluídas, pode ser o principal meio por onde as bromélias jovens captam o nitrogênio. Ao desenvolverem um tanque, as bromélias podem mudar a sua fisiologia, capturando preferencialmente fontes de nitrogênio provenientes de matéria orgânica decomposta que se acumula no interior da cisterna. As raízes das bromélias atmosféricas também mostraram cumprir um papel fundamental na nutrição dessas plantas durante a fase juvenil, pois aumentaram a capacidade de absorção e assimilação de fontes de nitrogênio. Quando as bromélias iniciam o desenvolvimento de um tanque, as bases das folhas passaram a assumir a função do sistema radicular, enquanto que as raízes, talvez, começassem a diminuir sua capacidade de captar os nutrientes do meio ambiente. Os resultados bioquímicos demonstraram que existe uma forte sincronização de todas as etapas do metabolismo do nitrogênio (absorção, transporte e assimilação) envolvendo diferentes partes do corpo das bromélias (raízes, porções foliares da base, mediana ou ápice) de ambas as fases de desenvolvimento, sugerindo que nos tecidos vegetais dessas plantas, existe uma fina regulação de todos os processos fisiológicos e metabólicos que compreendem o metabolismo do nitrogênio. Essa regulação controlada seria necessária para que as bromélias atmosféricas ou com tanque desenvolvido consigam absorver, transportar e assimilar as fontes de nitrogênio rapidamente e com grande eficiência. Para finalizar, o novo termo \"bromélia epífita jovem sem tanque\" foi sugerido para se referir à bromélia V. gigantea na fase juvenil ao invés de \"bromélia epífita atmosférica\". As raízes dessa bromélia jovem demonstraram ter um papel fundamental nos processos de absorção e assimilação das fontes de nitrogênio, uma característica que geralmente não é atribuída para as raízes das bromélias com o hábito de vida atmosférico / The stages of ontogenetic development of bromeliad can be an important feature to be considered in the physiology studies because the young plants can be classified as atmospheric bromeliads, while the adult plants have a special structure formed by leaves called tank. Some studies showed that some physiological characteristics can be influenced by the stages of ontogenetic development in bromeliads as photosynthetic taxes or the total nitrogen (N) content in leaves. However, there are no records that nutrition and nitrogen metabolism differ between young and adult epiphytic bromeliads. The objective of this project was to verify the existence of differences in the dynamics of nitrogen metabolism (absorption, transportation and assimilation) arising from the use of distinct nitrogen sources (NH4+, NO3- or urea) in epiphytic bromeliad Vriesea gigantea with different stages of ontogenetic development (atmospheric or tank). A nutrient solution, consisting 5mM of total N, was offered to bromeliads. Three different forms of N sources were used: NH4+, NO3- or urea, enriched with 15N isotopes. Three distinct portions of leaf (apex, middle and base) of adult tank bromeliad and two different regions of leaf (apex and base) and roots of young bromeliads were harvested in six different times. All samples were used in enzymatic assays of urease, nitrate reductase, glutamate sinthetase and glutamate dehydrogenase and in the 15N isotope quantification. According to the results, the nitrate was considered the nitrogen source absorbed at lower concentration by young and adult bromeliads. The atmospheric bromeliads were able to capture nitrate more efficiently than the tank plants, since this inorganic nitrogen source was absorbed and assimilated quickly in the 1st hour of the experimental time while the tank bromeliads absorbed nitrate slowly at the end of the experiment (12th and 24th hour). Ammonium and urea sources were absorbed in higher concentrations by atmospheric and tank bromeliads. The young bromeliads were able to absorb and assimilate higher concentrations of nitrogen from ammonium, while tank bromeliad absorbed and assimilated higher concentrations of urea. In each development stage, the epiphytic bromeliad V. gigantea can absorb and assimilate the nitrogen sources which are more available in the environment. The atmospheric bromeliads get to absorb diluted nutrients as inorganic nitrogen sources mainly from rainwater. After the tank structure developed in the rosette, the morphology and/or physiology features changes in the adult bromeliads. The tank bromeliads get to absorb mainly organic nitrogen sources from decomposed organic matter which accumulates inside the tank. The roots of atmospheric bromeliads also showed an important role in the nutrition of the young plants since the atmospheric bromeliads get to improve the nitrogen sources uptake and nitrogen assimilation. When the bromeliads developed a tank, the bases of the leaves might assume the absorption function, whereas the roots, perhaps, might decrease its capacity to capture the nutrients from the environment. The biochemical results showed that there is a strong synchronization of all stages of nitrogen metabolism (uptake, transport and assimilation) involving different body parts of bromeliads (roots, leaf portions of the base, middle or apex) of both development stages, suggesting that there might have a thin regulation of all physiological and metabolic processes of nitrogen metabolism in the bromeliad\'s tissues. This controlled regulation might be important to the atmospheric or tank bromeliads are able to absorb, allocate and assimilate nitrogen sources quickly and with great efficiency. Finally, the terminology “atmospheric epiphytic bromeliad” might not be appropriated to refer to young plants since their roots showed an important role in the absorption and assimilation of nitrogen sources. This feature is not usually attributed to the roots of atmospheric bromeliads. Then, the new terminology “young epiphytic bromeliad without tank” was suggested to refer the bromeliad V. gigantea in the juvenile phase

Les communautés microbiennes du sol peuvent améliorer la croissance de la plante en augmentant la disponibilité en nutriments du sol, favorisant ainsi leur prélèvement par la plante. Dans le contexte d’une production agricole à bas niveau d’intrants, la nutrition de la plante est susceptible de reposer de plus en plus sur les interactions plante-communautés microbiennes de la rhizosphère, qui peuvent être modulées par le génotype de la plante. Pourtant, très peu d’études se sont intéressées aux modifications des communautés microbiennes de la rhizosphère dans leur globalité et ce en relation avec à la fois le génotype et le phénotype de la plante. Ces travaux de thèse ont été consacrés à étudier l’effet du génotype de la plante sur la structure génétique des communautés microbiennes de la rhizosphère en relation avec les stratégies nutritionnelles de la plante.L’interaction plante-communautés microbiennes de la rhizosphère a été évaluée par une approche multidisciplinaire alliant écophysiologie et écologie microbienne. L’effet du génotype de la plante sur la structure génétique des communautés microbiennes de la rhizosphère qui lui sont associées a été analysé par DNA fingerprint. Les différentes stratégies nutritionnelles de la plante ont été analysées par une approche de type structure/fonction prenant en compte la mise en place des structures (feuilles, racines) et leur fonctionnement (photosynthèse, rhizodéposition, prélèvement spécifique d’azote).Dans une première expérimentation réalisée sur sept génotypes de Medicago truncatula, nous avons montré qu’à un stade précoce du développement de la plante, le génotype de Medicago truncatula affectait la structure génétique des communautés bactériennes du sol. En revanche, à ce stade précoce, peu de différences de croissance ont été observées entre les différents génotypes étudiés. Ces derniers ont par contre présenté des stratégies nutritionnelles contrastées. Les descripteurs fonctionnels sont donc plus efficaces que les descripteurs structurels pour discriminer les génotypes de plantes à un stade précoce du développement de la plante. De plus, nous avons montré un lien entre les stratégies nutritionnelles de la plante et la sélection des communautés bactériennes associées. Cette étude nous a également permis de développer un cadre d’analyse écophysiologique appliqué à l’étude des interactions plante-communautés microbiennes de la rhizosphère.Outre l’effet majeur du génotype de la plante dans les interactions plante-communautés bactériennes de la rhizosphère, nous avons également montré qu’il y avait un effet important de l’environnement, comme la disponibilité en azote minéral du sol. En effet, la disponibilité en azote minéral du sol a affecté la structure génétique des communautés bactériennes rhizosphériques via un effet indirect de la plante dépendant du génotype considéré. Les effets des différents génotypes de Medicago truncatula et de leurs stratégies de réponses à des contraintes environnementales, comme la disponibilité de l’azote du sol, se sont révélées être des composantes majeures de la sélection des communautés microbiennes. [...] / The soil microbial communities can improve plant growth by increasing soil nutrient availability, thereby promoting their uptake by the plant. In an overall context of input reduction, the plant nutrition should be increasingly based on plant- rhizosphere microbial communities’ interactions. Yet, very few studies have examined the entire rhizosphere microbial communities in relationship with both plant genotype and phenotype. The aim of this thesis was to study the plant genotype effect on the rhizosphere microbial communities in relationship with the plant nutritional strategies.To do so, the plant-rhizosphere microbial communities’ interaction was assessed by a multidisciplinary approach combining ecophysiology and microbial ecology. The plant genotype effect on the genetic structure of the associated rhizosphere microbial communities was analyzed by DNA fingerprinting. The different plant nutritional strategies were analyzed by a structural/functional approach taking into account both structure establishment e.g. leaves and functions e.g. photosynthesis.In a first experiment carried out on seven genotypes of Medicago truncatula, we showed that the Medicago truncatula genotype affected the genetic structure of the rhizosphere bacterial communities very early relatively to the plant development stages. However, at this early stage, few growth differences could be observed among the different genotypes. Yet, those genotypes presented contrasted nutritional strategies. Therefore, the functional descriptors were more efficient than the structural ones to discriminate plant genotypes at an early developmental stage. In addition, we showed that a link existed between the plant nutritional strategies and the rhizosphere bacterial communities selection. Finally, this study enabled to develop a multidisciplinary framework applied to the study of the plant- rhizosphere microbial communities’ interactions.In addition to the plant genotype effect, we showed that there is an environmental effect e.g. soil mineral nitrogen availability on the rhizosphere bacterial communities. Indeed, the soil mineral nitrogen availability affected the genetic structure of the rhizosphere bacterial communities via an indirect effect of the plant depending on its genotype. The effects of the different Medicago truncatula genotypes and their response strategies to environmental constraints (soil mineral nitrogen availability), proved to be a major component of the selection of the rhizosphere microbial communities.In order to identify the genetic determinisms of the interaction between the plant and the rhizosphere microbial communities, a second experiment was conducted on a core collection of 184 genotypes of Medicago truncatula. Initial results enabled to identify and characterize four groups of genotypes with contrasted phenotypes for their growth and their specific nitrogen uptake. Thanks to high-throughput sequencing, we will analyze the rhizosphere microbial communities’ diversity associated with the different Medicago truncatula genotypes. These results should determine if the plant genotype influences the selection of beneficial rhizosphere microbial communities. Moreover, when the whole genome sequencing data would be available for the 184 genotypes of the Medicago truncatula core collection, a genome-wide association study will be proceed. The creation of plant ideotypes, which will promote beneficial interactions with rhizosphere microbial communities, will be possible. Plant growth and yield will be improved without the concomitant increase of agricultural inputs.

Σκοπός της παρούσας διδακτορικής διατριβής ήταν η μελέτη της δυναμικής του αζώτου σε καλλιέργεια γλυκού σόργου. Χρησιμοποιήθηκαν φυτά γλυκού σόργου [Sorghum bicolor (L) Moench] ποικιλίας Keller και πραγματοποιήθηκαν αφ’ ενός πειράματα στο πεδίο αφ’ ετέρου πειράματα σε πλαστικά δοχεία. Τα πειράματα πεδίου πραγματοποιήθηκαν σε αγρόκτημα του Πανεπιστημίου Πατρών, τις καλλιεργητικές περιόδους του 2004 και 2005, και περιλάμβαναν δύο χειρισμούς (βιολογική και συμβατική διαχείριση της γονιμότητας του εδάφους), με τρεις επαναλήψεις ο καθένας σε πλήρη τυχαιοποιημένη διάταξη. Τα αποτελέσματα των πειραμάτων πεδίου, έδειξαν ότι η βιολογική διαχείριση της γονιμότητας του εδάφους ευνόησε τη διαθεσιμότητα του εδαφικού νιτρικού αζώτου. Το 2004, μέχρι και την ολοκλήρωση της άνθησης, τα φυτά του βιολογικού χειρισμού συσσώρευσαν περισσότερο άζωτο από ό,τι τα φυτά του συμβατικού χειρισμού, αν και οι διαφορές δεν ήταν στατιστικά σημαντικές, εκτός από τη δεύτερη δειγματοληψία, όπου τα φυτά του συμβατικού χειρισμού συσσώρευσαν 2,69 N gm-2, ενώ για τα φυτά του βιολογικού χειρισμού η αντίστοιχη τιμή ήταν 5,12 N gm-2. Το 2005, η συσσώρευση αζώτου ακολούθησε παρόμοια πορεία μέχρι τα μέσα της άνθησης και οι διαφορές δεν ήταν στατιστικά σημαντικές, εκτός από τη τρίτη δειγματοληψία, όπου τα φυτά του συμβατικού χειρισμού συσσώρευσαν 3,61 N gm-2, ενώ για τα φυτά του βιολογικού χειρισμού η αντίστοιχη τιμή ήταν 4,61 N gm-2. H επίδραση εμβολίων Azospirillum brasilense, σε παραμέτρους αύξησης φυτών γλυκού σόργου και στην πρόσληψη αζώτου από τα φυτά, υπό διαφορετικά επίπεδα αζωτούχου λίπανσης μελετήθηκε σε πειράματα σε πλαστικά δοχεία. Το κάθε πείραμα περιλάμβανε πέντε χειρισμούς με τριάντα επαναλήψεις, έκαστος. Τα φυτά που εμβολιάστηκαν με Azospirillum brasilense και έλαβαν τη μισή ποσότητα αζώτου, έδωσαν 7,69 g ξηρής βιομάζας φυτό-1 στο τέλος του πρώτου πειράματος και 4,89 g ξηρής βιομάζας φυτό-1 στο τέλος του δεύτερου πειράματος, ενώ τα φυτά που δέχθηκαν πλήρη λίπανση αζώτου χωρίς εμβόλιο έδωσαν 2,39 και 2,04 g ξηρής βιομάζας φυτό-1 στο τέλος του πρώτου και δεύτερου πειράματος αντιστοίχως. Οι διαφορές των χειρισμών ανά πείραμα ήταν στατιστικά σημαντικές. Το συνολικό προσλαμβανόμενο άζωτο από τα φυτά που εμβολιάστηκαν με Azospirillum brasilense και έλαβαν τη μισή ποσότητα αζώτου ήταν 153,23 και 99,96 mg φυτό-1 στο τέλος του πρώτου και του δεύτερου πειράματος αντιστοίχως. Οι τιμές ήταν υψηλότερες σε σχέση με τις τιμές του συνολικού προσλαμβανόμενου αζώτου των φυτών των λοιπών και οι διαφορές ήταν στατιστικά σημαντικές. Τα αποτελέσματα των πειραμάτων έδειξαν υψηλή αξιοποίηση του εφαρμοζόμενου αζώτου των φυτών που εμβολιάστηκαν με Azospirillum. Τα αποτελέσματα υποδεικνύουν την βιωσιμότητα ενός βιολογικού συστήματος καλλιέργειας, όσον αφορά την κάλυψη των αναγκών των φυτών σε άζωτο. Η υψηλή αξιοποίηση του αζώτου από τα φυτά γλυκού σόργου, οδηγεί στη μείωση εφαρμογής αζωτούχων λιπασμάτων και συμβάλλει στη μείωση του κόστους καλλιέργειας, στη μείωση εκπομπών αερίων του θερμοκηπίου και στην μείωση διήθησης νιτρικών στο υπόγειο υδροφόρο ορίζοντα. / The aim of the present thesis was to study the nitrogen dynamics in sweet sorghum crop. Sweet sorghum plants [Sorghum bicolor (L) Moench] variety Keller were used, and field and experiments were conducted. Field experiments were conducted during 2004 and 2005 growing seasons at the experimental station of the University of Patras, Greece and there were two treatments (biological and conventional soil fertility treatment), in a randomized complete block experimental design with three replications. The results showed that biological soil fertility treatment favour soil nitrate nitrogen concentration. In 2004, until completion of blooming, nitrogen uptake was higher in plants cultivated on biological fertility treated soil than in them cultivated on conventional but the differences were not statistically significant, except in second sampling, which nitrogen uptake was 2,69 N gm-2 in plants cultivated on conventional fertility treated soil and it was 5,12 N gm-2 in plants cultivated on conventional fertility treated soil. In 2005, until half of blooming, nitrogen uptake was similarly ranged for both treatments but the differences were not statistically significant, except in third sampling, which nitrogen uptake was 3,61 N gm-2 in plants cultivated on conventional fertility treated soil and it was 4,61 N gm-2 in plants cultivated on conventional fertility treated soil. Τhe effect of Azospirillum brasilense inoculation on growth parameters and nitrogen uptake in sweet sorghum plants fertilized with various nitrogen levels, was studied during pot experiments. Each experiment was including five treatments and thirty replications of each. Plants inoculated with Azospirillum brasilense and receiving half amount of nitrogen fertilizer produced 7,69 g dry biomass plant-1 at the end of the first experiment and they produced 4,89 at the end of the second one, while plants receiving full amount of nitrogen fertilizer and no inoculation produced 2,39 and 2,04 g dry biomass plant-1 at the end of the first and second experiment, respectively. Differences among treatments in each experiment, were statistically significant. Total nitrogen uptake in Plants inoculated with Azospirillum brasilense and receiving half amount of nitrogen fertilizer was 153,23 and it was 99,96 mg plant-1 at the end of first and second experiment, respectively. Those plants showed higher nitrogen uptake than plants from each other treatment and the differences were statistically significant. Results showed that treatments which inoculated with Azospirillum brasilense were the most efficient in terms of nitrogen uptake. These results indicate that biological management provides an adequate nitrogen nutrition to sweet sorghum crop. High nitrogen efficiency in sweet sorghum plants leads in reduced applying nitrogen fertilization and contribute in reduced crop cost, in reduced emission of the greenhouse gas and in reduced leaching of NO3-N to ground water.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A busca por estratÃgias de cultivo que possam contribuir para a aclimataÃÃo de plantas à salinidade à de fundamental importÃncia, pois, alÃm de possibilitar a identificaÃÃo de genes potenciais para guiar ensaios de modificaÃÃo genÃtica, permite selecionar cultivares com maior capacidade de crescer em solos com excesso de sais. A fim de testar a hipÃtese que a nutriÃÃo nitrogenada com NH4+ aumenta a tolerÃncia de plantas de Sorghum bicolor à salinidade, atravÃs da ativaÃÃo de mecanismos voltados ao controle da homeostase iÃnica, estabeleceram-se trÃs etapas experimentais. Na primeira delas, que objetivou definir a relaÃÃo entre as fontes de nitrogÃnio (N), NO3- e NH4+, que proporcionasse melhor crescimento das plantas sob salinidade, observou-se claramente que a nutriÃÃo somente com NH4+ (proporÃÃo NO3-/NH4+ de 0:100) foi mais vantajosa para o crescimento de S. bicolor sob salinidade que a nutriÃÃo apenas com NO3- ou com o regime misto desses dois Ãons, dado os maiores Ãndices de Ãrea foliar e massa seca da parte aÃrea. Verificou-se tambÃm que, sob estresse, as plantas nutridas somente com NH4+ acumularam menos Na+ nas folhas e nas raÃzes, influenciando positivamente a relaÃÃo K+/Na+, e apresentaram maiores teores de aminoÃcidos solÃveis, principalmente aqueles ricos em N (glutamina e asparagina), que contribuÃram para evitar a toxicidade do NH4+ e provavelmente para o ajustamento osmÃtico. AlÃm disso, enquanto plantas nutridas com proporÃÃes NO3-/NH4+ de 100:0, 75:25, 50:50 e 25:75 apresentaram taxas de assimilaÃÃo lÃquida de CO2 inalteradas ou reduzidas pela salinidade, plantas cultivadas somente com NH4+ (proporÃÃo 0:100) apresentaram incrementos nessa variÃvel, em reposta ao estresse. A segunda etapa teve como objetivo principal investigar se a tolerÃncia à salinidade mediada pelo NH4+ era resultante da regulaÃÃo efetiva dos processos relacionados à fotossÃntese. Nessa ocasiÃo, esse argumento foi refutado, pois a melhor eficiÃncia do fotossistema II sob estresse salino foi observada nas plantas cultivadas com a mesma proporÃÃo de NO3- e NH4+ (proporÃÃo 50:50). Nesse grupo de plantas, a reduÃÃo no quenching nÃo fotoquÃmico (NPQ) confirmou a maior eficiÃncia fotoquÃmica, dado o aumento na eficiÃncia quÃntica potencial (Fv/Fm) e efetiva (ΦPSII) do fotossistema II e a elevada taxa de transporte de elÃtrons (ETR). Esse fenÃmeno foi diretamente relacionado com os incrementos nos teores de clorofila b e de antocianinas. Por fim, na terceira etapa, objetivou-se elucidar os mecanismos envolvidos no controle do acÃmulo de Na+, sob salinidade, na cÃlula e na planta inteira, bem como identificar o papel da nutriÃÃo com NH4+ nesses processos. Em estudos com vesÃculas de membrana de raÃzes, verificou-se que plantas estressadas cultivadas somente com NH4+ apresentaram maior ativaÃÃo dos transportadores do tipo antiporte Na+/H+ (SOS1) de membrana plasmÃtica e, em menor proporÃÃo, do antiporte Na+/H+ (NHX) de tonoplasto, ao passo que o oposto foi observado nas plantas nutridas com NO3-. Esses dados sugerem que o cultivo somente com NO3- induziu o mecanismo de compartimentaÃÃo de Na+ no vacÃolo, como evidenciado pela anÃlise dos transcritos da famÃlia NHX, em que a expressÃo do gene SbNHX2 (principal isoforma expressa) nas raÃzes das plantas foi aumentada em quase todos os tempos analisados (24, 48, 120 e 240 horas apÃs exposiÃÃo ao NaCl). Mesmo assim, essa resposta nÃo foi suficiente para o controle do Na+, jà que a entrada contÃnua desse Ãon no xilema radicular afetou o influxo de K+ na seiva e limitou o acÃmulo de K+ nas folhas. Por outro lado, a nutriÃÃo somente com NH4+ ativou potencialmente mecanismos de controle do acÃmulo de Na+, uma vez que houve acionamento efetivo do efluxo de Na+ para o apoplasto via SOS1, que restringiu o carregamento desse Ãon no xilema e, consequentemente, limitou a acumulaÃÃo dele nos tecidos aÃreos. A formaÃÃo do gradiente de potencial eletroquÃmico, essencial para a atividade dos transportadores Na+/H+, foi modulada diferencialmente pela fonte de N. A atividade de bombeamento de prÃtons da H+-ATPase de membrana plasmÃtica (P-ATPase) foi estimulada em maior proporÃÃo pela presenÃa de NH4+, sem haver, contudo, aumento na atividade de hidrÃlise de ATP. Jà o aumento da translocaÃÃo de H+ pela P-ATPase em plantas estressadas cultivadas com NO3- foi diretamente relacionado ao incremento na hidrÃlise de ATP. Esses resultados sugerem que a disponibilidade de NH4+ aumentou a afinidade da P-ATPase por H+, pois houve melhor eficiÃncia de acoplamento H+/ATP, e isso tornou a enzima mais efetiva para transportar H+ com menos gasto de energia. AlÃm disso, esse aumento no bombeamento de prÃtons resultou em um maior potencial eletroquÃmico, e favoreceu diretamente a atividade do antiporte SOS1 de membrana plasmÃtica. Os nÃveis de transcritos dos genes SbPHA3 e SbPHA5 (principais isoformas expressas da famÃlia) foram aumentados nas plantas cultivadas somente com NO3-, nos tempos iniciais de exposiÃÃo ao estresse salino (12 e 24 h), enquanto que, nos cultivos somente com NH4+, essa resposta sà foi detectada apÃs 24 h. No vacÃolo, a principal bomba responsÃvel pela formaÃÃo do gradiente de H+ durante a exposiÃÃo ao estresse salino foi a H+-ATPase (V-ATPase), em comparaÃÃo à H+-PPiase. Nas plantas cultivadas somente com NO3-, observou-se uma melhor regulaÃÃo da V-ATPase, em associaÃÃo à atividade aumentada do antiporte NHX, enquanto que no cultivo com NH4+, a ativaÃÃo do transporte de H+ sob salinidade foi diretamente relacionada a incrementos na atividade de hidrÃlise de ATP da V-ATPase, bem como ao aumento da expressÃo dos transcritos do gene SbVHA2, ao longo de todo o perÃodo experimental. Essas observaÃÃes revelam que o NH4+, como fonte Ãnica de N, ativa mecanismos que envolvem uma regulaÃÃo coordenada, nas raÃzes, da atividade e da expressÃo gÃnica de bombas de H+ e transportadores Na+/H+ de membrana plasmÃtica e de tonoplasto, que culminam no controle do acÃmulo de Na+ na planta inteira e aumentam a tolerÃncia de S. bicolor ao estresse salino. / Over the last decades, several researchers have focused the development of cultivation strategies in order to improve the plantâs ability to withstand salinity. Understanding the plant salt tolerance is one of important trait to enhance productivity of crops in saline soils because it provides molecular basis for plant breeding, as well as allows identify plant species with a greater ability to grown in salinized areas. In order to test the hypothesis that nitrogen nutrition with NH4+ improves the salt tolerance in Sorghum bicolor plants, through the restrict control of ionic homeostasis, three experimental steps were established. In the first one, we investigated what would nitrogen regime, as NO3-:NH4+ ratio, contribute to the better growth of plants under salinity. Our data clearly showed that the nutrition with only NH4+ (NO3-/NH4+ at 0:100) was more advantageous for the growth of S. bicolor under salinity than the supply with solely NO3- or the mixed regimes, as evidenced by the higher leaf area and shoot dry mass. Under salinity, Na+ accumulation was severely limited in presence of NH4+ (0:100), which positively influenced on K+/Na+ homeostasis. In parallel, NH4+-fed plants displayed a substantial accumulation of N-rich amino acids (mainly glutamine and asparagine) in both tissues, which seems to be fundamental in alleviating the NH4+ toxicity. Furthermore, whereas plants treated with NO3-:NH4+ ratio of 100:0, 75:25, 50:50 and 25:75 ratios had their photosynthetic rates (A) unaltered or reduced by salinity, plants supplied with only NH4+ showed an increased CO2 assimilation in response to stress. During the second step, we evaluated if the better salt tolerance in NH4+ cultivated plants was due to an effective regulation of photosynthesis-related processes. This idea was rejected because of the most striking effects of nitrogen regime were observed in plants supplied with equal amounts of NO3-: NH4+ (50:50). Under salt stress, plants from 50:50 NO3-:NH4+ treatments displayed a lower non-photochemical quenching (NPQ) and an improved photosystem II maximum efficiency (Fv/Fm). Their superior performance was also indicated by a higher effective quantum yield of PSII (ΦPSII) and electron transport rate (ETR), as well as increased chlorophyll b and anthocyanins. Finally, at the third step, we supply S. bicolor plants with NO3- or NH4+ to investigate changes in pathways for control of Na+ accumulation, at cell and whole plant level, in response to 75 mM NaCl-stress. By using root membrane-enriched vesicles, it was found that a more pronounced plasma membrane Na+/H+ antiporter (SOS1) activity and low loading of Na+ in the xylem in the NH4+ treated plants, whereas a largest vacuolar Na+/H+ exchanger (NHX) activity was noticed by NO3- grown plants. These data suggest that the NO3- availability induced the compartmentalization of Na+ into the vacuole, as supported by the upregulation of SbNHX2 gene expression over time of NaCl exposure (12, 24, 48, 120 and 240 h). Nonetheless, it composed an inefficient pathway of Na+ control, since the incessant entrance of Na+ in the xylem sap impaired the K+ loading and limited the K+ accumulation in the shoot. On the other hand, the NH4+ supply potentially activated the mechanisms for control of Na+ accumulation, driving an effective efflux of Na+ out of the cell, via SOS1, restricting its loading in the xylem and thus limiting Na+ reach and accumulation in the aerial tissues. Surprisingly, we found that the generation of electrochemical potential gradient for Na+/H+ exchange activity is differentially modulated by the nitrogen source. The H+-pumping activity driven by plasma membrane H+-ATPase (P-ATPase) was greatly stimulated by the presence of NH4+ in growth medium, however, without an increase in ATP hydrolysis activity. Conversely, the improvement of P-ATPase-generated H+-pumping of NO3- fed stressed plants was directly related to the increase of ATP hydrolysis. These data show that the NH4+ availability enhances the H+/ATP coupling efficiency of P-ATPase, i. e. the enzyme displayed a high capacity of transport H+ across plasma membrane with low ATP consumption. Moreover, the bigger H+ translocation resulted in a greater electrochemical potential which in turn favored the SOS1 activity. The expression of SbPHA3 and SbPHA5 genes was upregulated in NO3- grown stressed plants at the beginning of salt exposure (12 and 24 h), whereas it was enhanced in NH4+ supplied stressed plants only after 24 h. At vacuole level, the H+-ATPase (V-ATPase) was the main proton pump responsive to salinity, as compared do H+-Pyrophosphatase (PPase). A better regulation between V-ATPase and NHX antiporter activities was noticed by plants from NO3- treatments. Under NH4+ supply, the increase of H+ pumping was directly associated to the improvement of ATP hydrolysis by V-ATPase, coupled to upregulation of SbVHA2 gene expression over time of salinity exposure. Taken together, our data reveal that the NH4+, as the only nitrogen source, activates an intricate regulation of Na+ control pathways, involving the existence of a robust regulation and systematic mechanism firstly on root cell and subsequently on whole plant in sorghum upon salinity. In conclusion, the NH4+ stimulated salt tolerance is resulted from a more active SOS1 protein and high efficiency of P- and V-ATPase in the roots, which help to efficient Na+ exclusion and counteract net Na+ accumulation in the cytosol, thus preventing the loading of Na+ in the xylem sap and its reach in the photosynthetic tissues.

Submitted by Johnny Rodrigues (johnnyrodrigues@ufcg.edu.br) on 2018-05-09T21:25:32Z No. of bitstreams: 1 EWERTON GONÇALVES DE ABRANTES - DISSERTAÇÃO PPGHT 2014..pdf: 742061 bytes, checksum: fb0ea15cd43e74db00440e44ab2d3d33 (MD5) / Made available in DSpace on 2018-05-09T21:25:32Z (GMT). No. of bitstreams: 1 EWERTON GONÇALVES DE ABRANTES - DISSERTAÇÃO PPGHT 2014..pdf: 742061 bytes, checksum: fb0ea15cd43e74db00440e44ab2d3d33 (MD5) Previous issue date: 2014-02-26 / Capes / A berinjela (Solanum melongena L.) é uma hortaliça de fruto pertencente à família solanácea considerada exigente em nitrogênio para uma produção adequada. Dessa forma, o fornecimento de silício (Si) pode aumentar a eficiência da adubação nitrogenada e reduzir as doses a serem aplicadas. O trabalho teve por objetivo avaliar o efeito da adubação com N e Si e da interação N x Si sobre o crescimento, aspectos fisiológicos, nutrição nitrogenada e silicatada, visando ao aumento da eficiência da adubação nitrogenada, o aumento da produtividade e da qualidade dos frutos da berinjela. Dois experimentos, em delineamento inteiramente casualizado, foram conduzidos no Centro de Ciências e Tecnologia Agroalimentar (CCTA/UFCG), Campus de Pombal-PB, com plantas de berinjela, cultivar “Embú”. No primeiro, os tratamentos foram constituídos por um arranjo fatorial 5 x 4, compreendendo 5 doses de N (25; 125; 250; 350 e 500 mg dm-3) e 4 doses de Si (0; 75; 150 e 200 mg dm-3) aplicados via radicular com quatro repetições. No segundo, os tratamentos foram constituídos por um arranjo fatorial 5 x 2, sendo 5 doses N (25; 125; 250; 350 e 500 mg dm-3) e 2 doses de Si (sem silício e aplicação foliar de uma solução 10 mmol L-1 de Si) e seis repetições. Na fase de pré-florescimento foram avaliados o crescimento (produção de matéria seca das folhas, caule e de raízes e o índice de área foliar); as trocas gasosas, quais sejam taxa fotossintética, condutância estomática, taxa de transpiração e concentração intercelular de CO2; os teores e acúmulos das frações de nitrogênio (NO3-, NH4+, total e orgânico), a estimativa da eficiência de utilização do N (EUN) e o teor de Si nas folhas. No segundo experimento, além das variáveis citadas, foram avaliados também o número de frutos por planta, produção por planta, o peso médio dos frutos, o diâmetro longitudinal e transversal, a firmeza, °Brix, o pH da polpa, o teor de vitamina C e a acidez titulável. A adubação nitrogenada proporcionou aumentos na produção de matéria seca, nas trocas gasosas, nos teores das frações de N assim como seus respectivos acúmulos, elevou os atributos de qualidade dos frutos, porém diminuiu o teor de Si nas folhas e a eficiência de utilização de N (EUN). A adubação silicatada aplicada via radicular elevou o teor e acúmulo de N-NH4+ nas folhas, o acúmulo de N-NH4+ nas raízes, e o teor de Si nas folhas, o teor e o acúmulo de N-NO3- nas raízes, e diminuiu o teor e o acúmulo de N-NO3- nas folhas, sem influenciar no crescimento e nas trocas gasosas. A adubação silicatada aplicada via foliar influenciou negativamente nas taxas de transpiração e fotossintética; diminuiu os teores de N-NH4+ nas folhas, N-NO3-, Norgânico e N-total no caule, e aumentou o teor de N-NO3- nas raízes; influenciou positivamente na EUN; proporcionou aumentos no tamanho dos frutos, no °Brix, e diminuiu a acidez titulável dos frutos. Concluiu-se que a berinjela respondeu positivamente a adubação nitrogenada, proporcionando aumentos no crescimento, nas trocas gasosas, nos teores das frações de N, porém com efeito negativo na EUN e no teor foliar de Si. O silício exerceu influência nos teores foliares das frações de N e nitrato nas raízes, e na qualidade dos frutos. / The eggplant (Solanum melongena L.) is a vegetable fruit that belongs to the solanaceous family,considered demanding in nitrogen for adequate production. So, the supply of silicon (Si) may increase the efficiency of nitrogen fertilization and reduces the doses to be applied. The study aimed to evaluate the fertilization effect with N and Si, and Si x N interaction on growth, physiological aspect, silicon and nitrogen nutrition, aiming at increasing the nitrogen efficiency fertilizer, and also increasing of productivity and eggplant fruit quality. Two experiments in completely randomized design were conducted at the Centro de Ciências e Tecnologia Agroalimentar (CCTA/UFCG), Campus Pombal - PB, with eggplants cultivate "Embu". In the first experiment, treatments consisting of a 5 x 4 factorial arrangement, comprising 5 N rates (25, 125, 250, 350 and 500 mg dm-3) and 4 Si rates (0, 75, 150 and 200 mg dm-3) applied via root with four replications. In the second experiment, treatments consisting of a 5 x 2 factorial arrangement, with 5 N rates (25, 125, 250, 350 and 500 mg dm-3) and 2 Si rates (silicon and without foliar application of a 10 mmol L-1 Si) and six replications. During pre-flowering, were evaluated growth (dry matter production of leaves, stems and roots, and leaf area index); gas exchanges, which are photosynthetic rate, stomatal conductance, transpiration rate and intercellular CO2 concentration; the contents and accumulation of fractional nitrogen (NO3-, NH4+, and total and organic), the estimated N use efficiency (NUE) and Si content in the leaves. In the second experiment, in addition to the aforementioned variables were also evaluated the number of fruits per plant, yield per plant, average fruit weight, the longitudinal and transverse diameter, firmness, °Brix, the pH of the pulp, the vitamin content C and titratable acidity. Nitrogen fertilization increased the yield in dry matter production, gas exchange, the levels of N fractions as well as their accumulation, increased the quality attributes of the fruit, but decreased the Si content in leaves and N utilization efficiency (EUN). Silicon fertilization applied via roots elevated the content and accumulation of N-NH4+ in the leaves , the accumulation of N-NH4+ in the roots , and the Si content in the leaves , the concentration and accumulation of N-NO3- in roots and decreased and N-NO3- accumulation in leaves without influence on growth and gas exchange. Silicon fertilization foliar applied negatively influenced the rates of transpiration and photosynthesis; decreased levels of N-NH4+ in leaves , N-NO3-, N-organic and N-total in the stem, and increased the content of N-NO3- in roots; positively influenced the NUE; yielded increases in fruit size, in °Brix, and decreased acidity of the fruits. It was concluded that eggplant responded positively to nitrogen fertilization, providing increases in growth, gas exchange, the levels of N fractions, but with a negative effect on NUE and leaf content in Si. Silicon exerted influence on foliar concentrations of fractions N and nitrate in roots, and fruit quality.