Made available in DSpace on 2016-08-17T17:11:04Z (GMT). No. of bitstreams: 1 Dissertacao-AnagilaJanenisCardosoSilva.pdf: 387241 bytes, checksum: 6333a1930e36328efecb8d1915cc5248 (MD5) Previous issue date: 2016-06-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In the humid tropical soils, the efficiency of nutrient use is very low, especially those required in larger quantities by plants, such as nitrogen. The objectives of this study were to evaluate the effect of using leucena legume biomass coverage associated with mineral fertilizer and humic acid on the availability and efficiency of use of N; evaluate the agronomic performance of the variety of corn QPM BR 473 associated with the following treatments: 133 kg ha-1 of urea as a source of nitrogen (N); 15 t ha-1 leucena (L); 133 kg ha-1 of urea + 15 t ha-1 leucena (N + L); 500 L ha-1 humic acid + 15 t ha-1 leucena (AH + L); 500 L ha-1 humic acid + 133 kg ha-1 of urea (AH + N) and 500 L ha-1 humic acid + 133 kg ha-1 of urea + 15 t ha-1 leucena (AH + N + U). All treatments received 120 kg ha-1 of P2O5, 60 kg ha-1 of K2O and 25 kg ha-1 ZnSO4. The N + L treatment was the highest productivity (5385 kg ha-1), nitrogen remobilization (35.20 kg ha-1), agronomic efficiency (32.61 kg kg-1) and higher recovery efficiency (52.44%). For this investigation, with the data efficiencies mainly shows the agronomic and nitrogen recovery efficiency and nitrogen remobilized and grain production, it can be inferred that the combination of biomass leucena with urea showed the highest productivity , noting that the use of these two sources of nitrogen together in tillage system is an important alternative for small producers in the humid tropics. Therefore, the effect of the existing synchronization between the release of nitrogen by organic and mineral sources was well demonstrated by the data presented in this study, however, the synergy of these sources with the humic acid was not fully clarified. Thus, increasing the efficiency of nutrients by plants through novel mechanisms as the application of humic acid may be a means of improving productivity. However, it is still necessary to better understand the mechanisms involved in this process to improve them. / Nos solos do trópico úmido, a eficiência do uso de nutrientes é muito baixa, principalmente daqueles exigidos em maiores quantidades pelas plantas, como o nitrogênio. Os objetivos com esse estudo foram avaliar o efeito da utilização de cobertura com biomassa da leguminosa leucena associada ao adubo mineral e o ácido húmico sobre a disponibilidade e eficiência do uso de N; avaliar o desempenho agronômico da variedade de milho QPM BR 473 associada aos seguintes tratamentos: 133 kg ha-1 de uréia como fonte de Nitrogênio (N); 15 t ha-1 de leucena (L); 133 kg ha-1 de uréia + 15 t ha-1 de leucena (N + L); 500 L ha-1 de ácido húmico + 15 t ha-1 de leucena (AH + L); 500 L ha-1 de ácido húmico + 133 kg ha-1 de uréia (AH + N) e 500 L ha-1 de ácido húmico + 133 kg ha-1 de uréia + 15 t ha-1 de leucena (AH + N + L). Todos os tratamentos receberam 120 kg ha-1 de P2O5, 60 kg ha-1 de K2O e 25 kg ha-1 de ZnSO4. O tratamento N + L foi o que proporcionou maior produtividade (5385 kg ha-1), remobilização do nitrogênio (35,20 kg ha-1), eficiência agronômica (32,61 kg kg-1) e maior eficiência de recuperação (52,44%). Para esta investigação, com os dados das eficiências apresentadas principalmente as eficiências agronômica e de recuperação do nitrogênio, bem como o nitrogênio remobilizado e a produção de grãos, pode-se inferir que a combinação da biomassa de leucena com uréia foi a que apresentou maior produtividade, constatando que o uso dessas duas fontes de nitrogênio juntas em sistema de plantio direto é uma importante alternativa para os pequenos produtores do trópico úmido. Portanto, o efeito da sincronia existente entre a liberação de nitrogênio pelas fontes orgânica e mineral foi perfeitamente comprovado pelos dados apresentados neste estudo, no entanto, a sinergia dessas fontes com o ácido húmico não ficou perfeitamente esclarecida. Sendo assim, aumentar a eficiência de nutrientes pelas plantas através de novos mecanismos como a aplicação de ácidos húmicos pode ser um meio de melhorar a produtividade. Entretanto, ainda é necessário conhecer melhor os mecanismos envolvidos neste processo para aperfeiçoá-los.

Orientador: Eduardo Caruso Machado / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T03:39:07Z (GMT). No. of bitstreams: 1 Dovis_VeronicaLorena_D.pdf: 1810872 bytes, checksum: 889e3b63cede2d73563ccbfb91cdd3e1 (MD5) Previous issue date: 2012 / Resumo: As hipóteses deste estudo foram: a presença de frutos nas variedades de laranja tardia e as variações sazonais no ambiente afetam o desenvolvimento reprodutivo, o metabolismo de carbono e nitrogênio e o acúmulo e remobilização de reservas nos órgãos da planta; cada estádio fenológico tem necessidades nutritivas específicas, que afetariam a assimilação de CO2 e o metabolismo de carboidratos e nitrogênio. Estas hipóteses foram testadas em plantas que sofreram ou não desbaste total de frutos com o intuito de induzir variações na demanda de assimilados. O experimento foi executado em Cordeirópolis (SP) em plantas de laranjeira "Valência? de 2,5 anos de idade, crescidas em vasos de 100 L. Em maio de 2009, a metade das plantas foi completamente desbastada de frutos. A partir de julho e ao longo do ciclo produtivo avaliaram-se a dinâmica de assimilação de CO2, carboidratos e compostos nitrogenados, e a atividade de enzimas da assimilação de nitrogênio. O experimento foi inteiramente casualizado com três ou quatro repetições, segundo o parâmetro avaliado, o tratamento estatístico dos dados foi como parcelas subdivididas no tempo. Em relação à primavera a fotossíntese é reduzida no inverno e no verão, como consequência da ocorrência de temperaturas mais baixas e mais altas, respectivamente. Porém, a condição fisiológica da planta também afeta a assimilação de CO2, a qual é incrementada pela alta demanda de carboidratos antes e durante a floração, mesmo sob baixa temperatura. A presença de frutos inibe parcialmente a brotação e floração visto que plantas desbastadas têm sete vezes mais flores; fixam 31% mais frutos e eles são 6% maiores. A floração é o processo de maior consumo de assimilados, e quanto mais intensa, maior o consumo de amido. Floração e frutificação são os únicos estádios nos quais se observaram diferenças entre os tratamentos; plantas com maior quantidade de flores têm maior consumo de assimilados. O amido continua a ser consumido até o fim da queda fisiológica dos frutos no verão, quando atinge o teor mínimo. Ramos e raízes maiores que 1 mm de diâmetro aportam amido durante o desenvolvimento das brotações e dos frutos. Entre o desbaste e a frutificação há aumento no teor de nitrogênio total nas folhas do último fluxo, ramos e raízes, ainda com baixa temperatura, resultado da maior demanda. O aumento no metabolismo induz maior síntese de proteína nas folhas e raízes menores que 1 mm de diâmetro. A enzima redutase do nitrato (RN) tem a menor atividade quando comparada com o sistema glutamina sintetase/glutamato sintase, sugerindo ser a que limita a taxa de assimilação de NO3-. Nas folhas a atividade in vitro da RN incrementa até a frutificação, e segue o padrão de variação sazonal observado na fotossíntese; nas raízes sua atividade é constante ao longo do ano / Abstract: The hypotheses of the study are that seasonal variations in environmental conditions affect the assimilation rate of resources while there is accumulation and remobilization of reserves in all the organs of plant to attend variations in nutrients demand. Each phenological stage has different needs, which would affect CO2 assimilation rate and metabolism of carbohydrates and nitrogen. To analyzer these hypotheses the objective was to evaluate the photosynthesis, flowering and fruit production, accumulation and remobilization of carbohydrates during of production cycle, in plants with and without fruit. Also it was objective to evaluate the annual dynamics of nitrogenous compounds and activity of enzymes of nitrogen assimilation. The study was conducted in Cordeirópolis (SP) in plants 'Valencia' orange of 2.5 years old, grown in pots of 100 L. In may 2009, at the start of the trial, half of plants was completely defruit, and starting from july and throughout the production cycle were evaluated photosynthesis, budding, flowering and fruit production, status of reserves of sugars, nitrogen compounds and enzymatic activities. The experimental design was completely randomized with three or four replications, with three or four repetitions, depending on the variable assessed, with split plots in time. It is observed that photosynthesis decreased in winter and summer, consequence of low and high temperature and radiation, respectively. However, the physiological condition of plant also affects the uptake, inducing an increase in photosynthesis prior to flowering, even at low temperatures. The largest demand of plants with fruits results in an increase on efficiency of energy use which comes of the assimilation and reserves. The presence of fruits partially inhibits the sprouting and blooming, even with high leaves starch content, defruited plants have seven times more flowers, fruit set is 31% higher and they are 6% greater. The flowering has the greater consumption of all phenological stages, when it is more intense, largest starch consumption. The flowering and fruiting are the only stages with differences between the treatments; plants with the more fruits and demand have more assimilates consumption. Starch is consumed until the end of june drop, in summer, when it reaches minimum content. Starch for development of flowers and fruits can be contributed by younger shoots, shoots older than one year and roots larger than 1 mm across. From the defruited plants until fruiting the total nitrogen content increases in mature leaves, shoots and roots, even at low temperatures, as result of the higher demand. Soluble protein is accumulated in mature leaves and roots smaller than 1 mm across as a result of increased metabolism. The nitrate reductase enzyme has the lowest activity when compared with the system glutamine synthetase/glutamate synthase, which would indicate that nitrate reductase activity controls the assimilation rate of NO3-. Nitrate reductase in vitro activity in leaves is increased until fruiting, and has seasonal variations similar like was observed in photosynthesis. Nitrate reductase in vitro activity in roots is constitutive throughout the year / Doutorado / Biologia Vegetal / Doutor em Biologia Vegetal

Master of Science / Department of Agronomy / Ignacio Ciampitti / Soybean [Glycine max (L.) Merr.] yields have continuously increased over time. Seed yields are determined by the genotype, environment, and management practices (G × E × M) interaction. Closing yield gaps require a continuous improvement in the use of the available resources, which must be attained via implementation of better management decisions. Linear relationships between seed yield and nitrogen (N) demand are reported in the scientific literature. Main sources of N to the plant are the biological N fixation (BNF) and the soil mineralization processes. On overall, only 50-60% of soybean N demand is met by the BNF process. An unanswered scientific knowledge is still related to the ability of the BNF to satisfy soybean N demand at varying yield levels. Seed N demand not met by N fixation plus soil mineral N, is then fulfilled by the remobilization of N from vegetative organs during the seed filling period. An early remobilization process reduces the photosynthetic activity (leaves) and can limit seed yield. The objectives of this project were to: i) study yield improvements and contribution of N via utilization of contrasting N conditions under historical and modern soybean genotypes, and ii) quantify main seed N sources during the seed filling period. For objective one, four field experiments were conducted during the 2016 and 2017 growing seasons in Kansas, United States (US) and Santa Fe Province, Argentina (ARG). Those experiments investigated twenty-one historical and modern soybean genotypes with release decades from 1980s to 2010s. As for objective two, three field experiments were conducted during the 2015 and 2016 growing seasons in Kansas, US, studying three soybean genotypes: non-roundup ready (RR), released in 1997; RR-1, released in 2009; and RR-2, released in 2014. Across all studies, seeds were inoculated and tested under three N management strategies: i) control without N application (Zero-N); ii) 56 kg N ha-1 applied at reproductive growth stages (Late-N); and iii) 670 kg ha-1 equally split at three timings (Full-N). As for yield improvements and N limitation, soybean yield improvements from the 1980s to 2010s were documented, representing 29% increases in the US and 21% in ARG. Regarding N management, the Full-N fertilization produced a 12% increase in seed yields in the US and 4% in ARG. As for main seed N sources in objective two, remobilization accounted for 59% of seed N demand, and was negatively related to new N uptake occurring during the seed filling period. Seed N demand for greater yields was dependent on both, N remobilization and new N uptake, while for lower yields, seed N demand was mainly supported by the N remobilization process. These results suggest that: a) high seed yields are somehow limited by the availability of N to express their potential, although the question about N application still remains to be fully investigated, as related to the timing and the environment by plant interactions that could promote a N limitation in soybeans; b) remobilization accounts for majority (59%) of N sourced to the seed, and c) high yielding soybean (modern genotypes) rely on diverse N sources: the N remobilization process plus new uptake of N.

Les plantes sont des organismes statiques et tributaires des ressources minérales présentes dans leur rhizosphère. La remobilisation des nutriments est un processus qui permet une économie nutritionnelle et un recyclage de macro- et micro - nutriments qui sont le plus souvent limitants. Le rôle du démantèlement des chloroplastes au cours de ce processus est très important pour le recyclage de l’azote, puisque ceux-ci contiennent la majeure partie des protéines foliaires. Bien que les protéines chloroplastiques soient une source essentielle pour le recyclage de l’azote foliaire, leur mécanisme de dégradation est mal connu. L’autophagie, a été proposée comme mécanisme participant au recyclage des nutriments, notamment en situation de carence ou de limitation en azote. L’autophagie, processus cellulaire de dégradation, représente un mécanisme de survie et d’adaptation, par le recyclage et l’élimination des protéines et organelles altérés.La détermination des flux d’azote, entre la rosette et les graines par l’utilisation du marquage à l’isotope stable 15N chez des mutants d’autophagie, nous a permis de montrer que l’autophagie est nécessaire à la remobilisation de l’azote. L’analyse fonctionnelle des mutants d’autophagie a permis de mettre en évidence de profondes perturbations métaboliques résultant dans l’élévation du rapport C/N. Les modifications métaboliques observées montre que les mutants d’autophagie ne présentent pas les signatures métaboliques habituellement retrouvées chez les plantes adaptées à la limitation en azote minéral, qu’ils accumulent au contraire les composés azotés et sont pauvres en ressources carbonées. Les investigations ont également révélé que l’autophagie est sélective envers certaines protéines. L’activité autophagique a été évaluée en fonction de différents niveaux d’expression d’AtTOR et à la suite de l’inhibition de son activité kinase. Ces résultats ont montré qu’AtTOR, senseur du statut nutritionnel, est un régulateur négatif de l’autophagie. L’autophagie est une étape clef du recyclage nutritionnel en réponse à une situation de stress telle que la limitation en azote. / Plants are static organisms dependent on minerals resources available in the rhizosphere. Nutrient recycling is a process allowing a nutritional economy and recycling of macro- and micro- nutrients, which are often limiting. The role of chloroplast dismantling during this process is very important for nitrogen recycling because chloroplasts contain the major part of foliar proteins. Albeit chloroplastic proteins are an essential source for foliar nitrogen recycling, their degradation process is not well understood. Autophagy has been proposed to participate in nutrients recycling, notably in nitrogen starvation or limitation. Autophagy, a cellular degradation process, represents a survival and an adaptation mechanism by recycling and eliminating defectives proteins and organelles.Based on nitrogen fluxes determination between the rosette and the seeds by using 15N labeling in autophagy (atg) mutants, the study has shown that autophagy is necessary for nitrogen remobilization. The functional analysis of atg mutants revealed deep metabolic perturbations resulting in elevated C/N ratio, marker of plant physiology status. The observed metabolic modifications are not the hallmarks of an adaptation to nitrogen limitation. Autophagy mutants indeed accumulate nitrogen compounds and present low carbohydrate contents. The investigations also revealed that autophagy is selective towards some proteins. Autophagic ativity has been evaluated function of different AtTOR expression levels and following AtTOR activity inhibition. Results have shown that AtTOR, a sensor of the nutritional status, is a negative regulator of autophagy. Autophagy is a key step for nitrogen recycling in response to stress situation like nitrogen limitation.

Le phosphore (P) est un élément indispensable pour la croissance des plantes. De nombreux travaux montrent des réponses très précoces à une limitation de la disponibilité en P. Pendant la germination et la croissance juvénile, la demande en P des plantules peut être satisfaite par la remobilisation des réserves en P des graines et le prélèvement racinaire. Les objectifs de la thèse sont d’étudier la contribution respective de la remobilisation des réserves en P des graines et du prélèvement racinaire de P à l’alimentation en P des plantules de maïs, et les interactions entre ces deux processus. Différentes expériences ont été conduites pour i) étudier les cinétiques de la remobilisation des réserves en P des graines, ii) identifier précisément le début du prélèvement de P exogène par les racines, iii) quantifier la contribution relative de ces flux à l’alimentation en P de la plantule, iv) comprendre les interactions entre ces flux. Des graines riches et des graines pauvres en P on été cultivées à différents niveaux de disponibilités P exogènes pendant quatre semaines. Le traçage isotopique du P exogène (32P) a été utilisé pour quantifier le flux de prélèvement et calculer le flux de remobilisation du P des graines. Initialement, 86% du P sous forme phytate et 13% du C de la graine est localisé dans le scutellum indépendamment du niveau de richesse en P de la graine. 4 jours après le semis, 98% des phytates des graines sont hydrolysés. La cinétique d’hydrolyse des phytates est indépendante de la richesse en P des graines et de la disponibilité en P dans le milieu. Le P issu de l’hydrolyse des phytates est stocké temporairement dans la graine avant d’être transporté vers les organes en croissance de la plantule. Le prélèvement de P exogène commence dès l’émergence de la radicule (4ième jour) et dépend de la disponibilité en P dans le milieu. L’initiation du prélèvement et son intensité ne dépend pas du flux de remobilisation des réserves en P de la graine. Le P issu de la remobilisation et du prélèvement est distribué dans les mêmes proportions entre les parties ariennes et racinaires. Un bilan de P à l’échelle de la plantule entière et de la graine a permis de mettre en évidence un efflux de P depuis la graine vers l’extérieur pendant la phase d’hydrolyse des phytates. La modélisation des flux de P pendant la germination et la croissance précoce permet de rendre compte des observations sous l’hypothèse d’absence d’interaction entre les flux de remobilisation et de prélèvement de P bien que ces deux processus se chevauchent dans le temps. Nos résultats démontrent l’importance de la disponibilité locale en P dans le milieu pendant les stades précoces indépendamment du niveau de richesse en P des graines. / Phosphorus (P) is an essential element for plant growth. Many studies have shown a very early seedling response to the limitation on the availability of P. During germination and early growth, the seedling P demand may be satisfied by the remobilization of seed P reserves and exogenous P uptake by developing roots. The objective of the thesis was to study the relative contribution of remobilization of seed P reserves, the exogenous P uptake by seedling roots and the interaction between these two processes. Various experiments were conducted to i) study the kinetics of the remobilization of seed P reserves, ii) identify precisely the beginning of exogenous P uptake by seedling roots, iii) quantify the relative contribution P fluxes in developing seedlings and iv) the interaction between these two P fluxes. Seeds with low and high P reserves were cultivated at different levels of exogenous P availability for the growth period of four weeks. The exogenous P was labelled with radioactive P (32P) to identify and quantify the P flux in young seedlings coming from exogenous P uptake and seed P reserves remobilization. Initially, 86% of P in the form of phytate and 13% C of seed reserves is localised in scutellum regardless of P initial seed P reserves. Four days after germination, 98% of seed phytate reserves are hydrolyzed. The kinetics of seed phytate hydrolysis was independent of seed P reserves and exogenous P availability. The hydrolyzed forms of phytate were temporarily stored in the seed before being translocated towards newly growing seedling compartments. The exogenous P uptake started soon after the radicle emergence (4th day) and depend mainly on the availability of exogenous P in the growth medium. The beginning of exogenous P uptake and its intensity was not influenced by the seed P reserves remobilization. The proportion of distribution of remobilized seed P reserves and the exogenous P uptake was similar among seedling shoot and roots. The whole seed and seedling P budget showed the significant P losses from germinating seeds by P efflux with the beginning of phytate hydrolysis in seeds. We proposed a model for the seed P remobilization and exogenous P uptake during germination and early growth. Assuming no interaction between seed P reserves remobilization and exogenous P uptake, the simulations were found to be in close agreement with experimental data. Our results showed the importance of exogenous P availability in growth medium during early growth stages regardless of seed P reserves.

Chromium exists largely in two oxidation states, namely hexavalent chromium (Cr(VI)) which is carcinogenic, mutagenic to living organisms including humans and trivalent chromium (Cr(III)) which is known to be 1000 times less toxic than Cr(VI). It is therefore desirable in most cases to reduce Cr(VI) to Cr(III). Various studies have been conducted on the Cr(VI) reduction process either in situ or ex situ. However in situ bioremediation using permeable reactive barrier system appears as a potential and attractive technology compared to other in situ technologies. This study was conducted to evaluate the reduction of Cr(VI) to Cr(III) in the short term and regeneration of the biological reactive barrier to achieve continuous long term operation. It was observed from the study that the chromium hydroxide Cr(OH)3(s) precipitated and thus affected the porosity and hydraulic conductivity of the barrier system. It was therefore proposed to implement a regeneration process involving remobilization of precipitated Cr(OH)3 using a dilute acid (0.1% HCl) and recover Cr(III) by electrokinetics. Lowering the pH in the reactor introduced harsh conditions which necessitated the evaluation of a possible culture shift during the regeneration phase. Microbial culture composition during bioremediation and after soil washing was evaluated using a 16S rRNA finger printing method. The microbial barrier was initially inoculated with indigenous bacterial species from dried sludge. The results presented in the phylogenic tree diagrams confirm that, after microbial barrier system operation, the well-known Cr(VI) reducers Bacillus mycoides, Lysinibacillus fusiformis and Micrococcus lylae were the predominant species in the microbial community of the barrier. The microbial barrier system successfully achieved near complete removal of Cr(VI), whereby approximately 75% Cr(VI) removal was achieved within 63 days of operation. The formation of Cr(OH)3(s) was observed in the second week of operation. After 4 weeks of operating the mesocosm under soil washing with 0.1% HCl and electrokinetics remediation with a DC voltage of 50-150 V an increase in total chromium (73%) was observed suggesting that the trapped chromium species in the mesocosm was effectively remobilized with the assumption that Cr(III) had attached to the cathode forming a white-yellow precipitate layer around the cathode. Additionally more than 95% Cr(VI) was transformed to lower toxicity Cr(III) during electrokinetics and soil washing remediation. However, one of the limitations of electrokinetics is near anode focusing effect whereby a layer of precipitate is formed around the anode that lead to the reduction of efficiency of the technology. / Dissertation (MSc)--University of Pretoria, 2014. / lk2014 / Chemical Engineering / MSc / Unrestricted

L’autophagie est un processus vésiculaire des organismes eucaryotes permettant de véhiculer au sein d’autophagosomes des protéines dysfonctionnelles et/ou des organites défectueux qui sont apportés à la vacuole pour y être dégradés. Les acides aminés et les squelettes carbonés ainsi générés pourront ensuite être exportés vers le cytosol et recyclés. Ce travail de thèse a consisté à identifier les rôles que joue l’autophagie au cours du développement et de la germination de la graine d’Arabidopsis thaliana. Dans une première partie, les graines du mutant d’autophagie atg5 ont été caractérisées d’un point de vue morphologique et leur remplissage en molécules de réserve a été étudié. Il a notamment été montré que la graine mutante présente une maturation accélérée et accumule plus de protéines que la graine sauvage. Dans une seconde partie, l’expression de gènes ATG8 a été mise en évidence au cours du développement de l’embryon, dans le phloème de la silique et du funicule, dans les téguments externes et internes, ainsi que dans l’endosperme de la graine. L’activité autophagique a été visualisée par l’observation en microscopie de structures autophagiques dans l’embryon en développement. Enfin, dans une dernière partie, les rôles de l’autophagie au cours de la germination ont été étudiés via le suivi de la mobilisation des molécules de réserve chez le mutant atg5, comparativement à des graines sauvages. Il a ainsi été montré que la graine mutante présente un défaut de mobilisation des protéines. Les résultats obtenus montrent donc que l’autophagie jouerait différents rôles dans la graine, notamment dans sa maturation et son vieillissement, dans l’apport des nutriments depuis la plante mère jusqu’à l’embryon, et encore dans la constitution des réserves au cours du développement, puis leur mobilisation après la germination. / Autophagy is a vesicular process of eukaryotic organisms, which consists of the transport of dysfunctional proteins and/or defective organelles within auto phagosomes toward the vacuole in order to be degraded. The generated amino acids and carbon skeletons are transported to the cytosol and recycled. The aim of this thesis work was to identify the roles of autophagy during seed development and germination in Arabidopsis thaliana. In the first part, seeds of the atg5 autophagy mutant have been morphologically characterized in order to study the accumulation of storage molecules. We demonstrated that atg5 mutant seeds are affected by an accelerated maturation and accumulate more proteins than wild type seeds. In a second part, the expression of ATG8 genes has been exhibited during the embryo development, into the phloem of silique and funiculus, in the outer and the inner integument, and in the seed endosperm. Autophagic activity has been visualized by microscopy observation of autophagic structures in the developing embryo. Finally, in the last part, the roles of autophagy during germination have been studied by monitoring the mobilization of storage molecules in the atg5 mutant seeds and compared with the wild type. We thus established that mutant seeds are affected by a defect in protein mobilization. These results show that autophagy may play several roles in seeds, for instance in the ageing and maturation processes, in the transport of nutrients from the mother plant to the embryo, or in the constitution of storage compounds during seed development and their mobilization after germination.

Le colza (Brassica napus L.) est une plante de grande culture particulièrement exigeante en azote (N) et caractérisée par une faible Efficience d’Usage de l’Azote (EUA), principalement due à une mauvaise Efficience de Remobilisation de l’Azote (ERA) au cours de la sénescence foliaire. L’optimisation de l’ERA est donc un enjeu majeur pour améliorer le bilan agro-environnemental de cette culture. La dégradation des protéines étant l’un des processus clés de la remobilisation de l’N associée à la sénescence, l’objectif de ce travail est d’identifier et caractériser les principaux acteurs de la protéolyse lors de la sénescence naturelle ou induite par une limitation en N. Chez la plante modèle Arabidopsis thaliana cultivée en condition de forte disponibilité en N (HN), nous avons montré que SAG12 est une protéase majeure lors de la sénescence foliaire afin d’assurer la remobilisation de l’N essentielle à l’élaboration du rendement et au remplissage en N des graines. En effet, nos travaux montrent que le rôle de SAG12 est central et qu’en absence de son activité, d’autres protéases appartenant au CPs, mais aussi à d’autres classes (protéases à aspartate ; APs), sont sollicitées pour soutenir la remobilisation de l’N foliaire. Dans ce contexte, AED1 (une APs CND41-like) est particulièrement intéressante puisqu’elle semble collaborer étroitement avec SAG12. Par ailleurs, cette étude met en évidence pour la première fois une localisation racinaire de SAG12. Dans cet organe, le rôle de SAG12 est crucial pour remobiliser l’N des racines pour maintenir le rendement et la teneur en N des graines lorsque les plantes sont soumises à une limitation en N. Un autre volet de ce travail a consisté à étudier d’autres moyens de réguler l’activité protéolytique en se focalisant notamment sur des systèmes anti-protéolytiques. Cette étude suggère qu’une Water Soluble Chlorophyll binding Protein (WSCP), la protéine WSCP1, porte effectivement une double fonction de protection des chlorophylles et d’inhibiteur de protéases à sérine, ce qui en fait un potentiel candidat pour prolonger la durée de vie des feuilles et ainsi réduire l’asynchronisme entre la période de vidage des feuilles et la période de remplissage en N des graines.L’ensemble de ces travaux permet de proposer de nouveaux candidats pertinents pour la sélection de variétés de colza présentant une remobilisation efficiente de l’N dans un contexte de limitation des intrants azotés. / Rapeseed (Brassica napus L.) is a field crop plant that is particularly requiring nitrogen (N) and characterized by a low Nitrogen Use Efficiency (NUE), mainly due to a poor Nitrogen Remobilization Efficiency (NRE) during foliar senescence. The optimization of NRE is therefore a major challenge to improve the agro-environmental balance of this crop. Since protein degradation is one of the key processes in the remobilization of N associated with senescence, the objective of this work is to identify and characterize the main actors of proteolysis during natural or induced by N limitation senescence. In the plant model Arabidopsis thaliana grown under high N conditions (HN), we have shown that SAG12 is a major protease during leaf senescence to ensure the remobilization of N essential for yield and seed N filling. Indeed, our work shows that the role of SAG12 is pivotal and that in the absence of its activity, other proteases belonging to the CPs but also to other classes (aspartate proteases; APs) are requested to support the remobilization of foliar N. In this context, AED1 (a CND41-like APs) is particularly interesting since it seems to collaborate closely with SAG12. In addition, this study shows for the first time a root localization of SAG12. In this organ, the role of SAG12 is crucial, in particular to remobilize N from the roots to sustain yield and N content of the seeds when plants face an N limitation. Another aspect of this work was to study other ways of regulating proteolytic activity, focusing in particular on anti-proteolytic systems. This study suggests that a Water Soluble Chlorophyll binding Protein (WSCP), the WSCP1 protein, has effectively a dual function of chlorophyll protection and serine proteases inhibition, which make it a potential candidate to extend leaf lifespan and thus, reduce asynchronism between leaf emptying time and the N seed filling time.Altogether, these results allow to suggest new relevant candidates for the selection of rapeseed varieties with an efficient N remobilization in a context of nitrogen input limitation.

L’orge (Hordeum vulgare L.) est l'une des céréales les plus importantes du monde et l’une des premières cultures domestiquées. Elle a été utilisée pendant des siècles pour l'alimentation humaine. Comme toutes les autres plantes, l'orge est dépendante de l'azote inorganique. L’efficacité de remobilisation de l'azote est donc très importante pour le remplissage des grains et pour la teneur en protéines du grain. L'objectif de ce travail est de donner une image du métabolisme des feuilles sénescence chez l'orge lorsque les plantes sont cultivées dans des conditions limitantes ou non en nitrates. Les analyses biochimiques, physiologiques et moléculaires de la sénescence des feuilles d'orge ont été réalisées. La gestion de l'azote pendant la sénescence des feuilles a été suivie par l'évolution des différents composés azotés au cours du vieillissement de la feuille. Une étude de profilage métabolique a été effectuée afin de déterminer les caractéristiques métaboliques de la sénescence des feuilles dans l'orge. En parallèle, les enzymes impliquées dans la remobilisation de l'azote ont été étudiées. Leurs activités et les niveaux de leurs transcripts ont été mesurés. Une attention particulière a été portée aux glutamine synthétases et asparagine synthétases et aux protéines de la machinerie de l'autophagie, processus connus pour jouer un rôle dans la remobilisation de l'azote pendant la sénescence des feuilles. A partir de toutes les données de séquences disponibles, ADNc, EST et séquences génomiques, cinq gènes codant pour les isoformes de glutamine synthétase cytosoliques (GS1), cinq gènes codant pour les isoformes d’asparagine synthétase (AS) isoformes et 19 gènes codant pour des protéines de la machinerie de l'autophagie ont été identifiés. Les expressions de tous les gènes identifiés ont été suivies au cours de la sénescence des feuilles et en fonction de l'alimentation en nitrates. La plupart de ces gènes sont sur-exprimés dans les feuilles sénescentes et de façon différentielle en fonction des conditions de nutrition. Toutes les données de séquences fournies par ce travail seront utiles à d'autres études translationelles et d'association génétique. / Barley (Hordeum vulgare L.) is one of the most important cereals in the world. It was one of the first domesticated crops and was used for centuries for human food. As all plants, barley has a fundamental dependence of inorganic nitrogen and nitrogen remobilization efficiency is very important for grain filling and grain protein content. The aim of this work was then to give a picture of the leaf-senescence metabolism in barley leaves when plants are grown under low or high nitrate conditions. Biochemical, physiological and molecular analyses of barley leaf senescence were performed. Nitrogen management during leaf senescence was monitored measuring changes in the different nitrogen pools during leaf ageing. In addition a large metabolite profiling study was performed in order to determine the metabolic hallmarks of leaf senescence in barley. In parallel enzymes involved in nitrogen remobilization were studied measuring their activity and the transcript levels of their coding genes. There was a special focus on glutamine synthetase and asparagine synthetase enzymes and for autophagy machinery that are known to play a role in nitrogen remobilisation during leaf senescence.From all the sequences data available, cDNA, EST and genomic sequences, we could identified five genes coding for cytosolic glutamine synthetase (GS1), five genes coding for asparagine synthetase (AS) and 19 genes coding for autophagy machinery proteins. Transcript levels of all the genes identified were monitored during leaf senescence and depending on nitrate nutrition. Most of these genes were over-expressed in senescing leaves and differentially expressed depending on nitrate conditions. In addition to the characterization of autophagy, GS1 and ASN genes, phylogenic and gene structures were analysed. All the sequences data provided by this work will be helpful to further translational and genetic association studies.

A cana-de-açúcar (Saccharum spp.) desempenha um papel de grande importância no cenário socioeconômico brasileiro, e representa 42% da matriz energética renovável do país. A expansão da área de cultivo da cana-de-açúcar para solos marginais e a necessidade de manutenção da alta produtividade dessa cultura tem levado à maior aplicação de fertilizantes a base de nitrogênio (N). Tal fato aliado à baixa responsividade da cana-de-açúcar a fertilizantes nitrogenados acarreta altos custos econômicos e ambientais. O amônio é a fonte preferencial de N para essa gramínea, sendo que pouco se conhece sobre a funcionalidade dos transportadores de NH4+ pertencentes à família gênica AMT (AMMONIUM TRANSPORTER). Neste contexto, é relevante esclarecer os mecanismos que influenciam na eficiência do uso de N (NUE), visando reduzir o impacto econômico e ambiental da aplicação dos fertilizantes nitrogenados nos sistemas agrícolas. Dessa forma, esse trabalho teve como objetivo a caracterização molecular e funcional de membros da subfamília AMT2 de cana-de-açúcar através de expressão heteróloga em mutantes de Saccharomyces cerevisiae (cepa 31019b) e Arabidopsis thaliana (qko), defectivos no transporte de amônio. As sequências gênicas e promotoras de ScAMT2;1 e ScAMT3;3A foram identificadas em biblioteca de BAC (bacterial artificial chromosome) de cana-de-açúcar (cultivar \'R570\'). Análises de expressão gênica de ScAMT2;1 e ScAMT3;3A em cana-de-açúcar demonstraram uma expressão preferencial em raízes e em folhas maduras, respectivamente, e que estes genes são regulados de maneira distinta entre si e entre os órgãos, de acordo com o desenvolvimento e com o status de N da planta. A complementação de levedura com os AMT2 de cana-de-açúcar demonstrou que estes genes restauram o crescimento do mutante, sendo que ScAMT2;1 permite maior absorção de amônio; porém o experimento não indicou sensibilidade dessas proteínas ao metilamônio (análogo tóxico ao amônio). Experimentos de localização da expressão órgão/tecido específico em arabidopsis selvagem \'Col-0\', utilizando os promotores de ScAMT2;1 ou ScAMT3;3A fusionados a GUS ou GFP, demonstraram que esses AMTs são preferencialmente expressos na região da endoderme/periciclo e vascular das células das raízes e região vascular da parte aérea, sendo regulados pela disponibilidade e fonte de N. Plantas de arabidopsis qko superexpressando ScAMT2;1, ScAMT3;3A ou transformadas com ScAMT2;1 dirigido por seu promotor endógeno, crescidas in vitro com amônio como fonte exclusiva de N, apresentaram um aumento significativo na produção de biomassa em relação a qko não transformada, principalmente para ScAMT2;1, indicando que essas proteínas são capazes de transportar amônio e complementar o mutante. Dados de influxo e acúmulo de 15N-amônio in vivo em raízes e parte aérea de plantas qko superexpressando ScAMT2;1 ou ScAMT3;3A demonstraram que ScAMT2;1 atua na absorção de amônio pelas raízes e provavelmente do carregamento do xilema, enquanto ScAMT3;3A está possivelmente envolvida na remobilização de amônio na parte aérea, podendo atuar aditivamente na absorção de NH4+ em raízes sob alto amônio. Esses resultados indicam que os transportadores ScAMT2;1 e ScAMT3;3A de cana-de-açúcar são funcionais, atuando com propriedades e funções distintas no transporte de amônio nessa gramínea e de acordo com a disponibilidade de N. / Sugarcane (Saccharum spp.) plays a major role in the Brazilian socio-economic scenario, and represents 42% of renewable energy sources in the country. The expansion of sugarcane cultivation to marginal lands and the requirement to maintain high yields have led to increased application of nitrogen (N) fertilizer. This fact, coupled with the low response of sugarcane to N fertilization, entails high economic and environmental costs. Ammonium is the preferred source of N by this grass; however, little is known about the functionality of NH4+ transporters belonging to the AMT gene family (AMMONIUM TRANSPORTER). In this context, it is important to clarify the mechanisms that affect the nitrogen use efficiency (NUE) in order to reduce the economic and environmental impact of the application of N fertilizers in agricultural systems. Therefore, this study aimed to conduct the molecular and functional characterization of members of the AMT2 subfamily from sugarcane by heterologous expression in mutants of Saccharomyces cerevisiae (strain 31019b) and Arabidopsis thaliana (qko), both defective in ammonium transport. Gene and regulatory region sequences of ScAMT2;1 and ScAMT3;3A were identified in a bacterial artificial chromosome (BAC) library of sugarcane (cultivar \'R570\'). Expression analysis of ScAMT2;1 and ScAMT3;3A in sugarcane showed a preferential expression in roots and mature leaves, respectively, and indicated a distinct expression pattern between genes and organs according to the ontogeny and the N status of the plant. The yeast complementation with AMT2 of sugarcane demonstrated that these genes restore the mutant growth, with ScAMT2;1 enabling higher ammonium absorption; however, the experiment did not indicate sensitivity to methylammonium (toxic ammonium analog). Arabidopsis wild type \'Col-0\' transformed with the promoter region of ScAMT2;1 or ScAMT3;3A directing the expression of GUS or GFP, demonstrated preferential expression in the endodermis/pericycle regions of roots and vascular region in shoots, being regulated by the availability and source of N. Arabidopsis qko overexpressing ScAMT2;1, ScAMT3;3A or transformed with ScAMT2;1 driven by its endogenous promoter, grown in vitro with ammonium as the sole source of nitrogen, showed a significant increase in biomass production compared to untransformed qko, especially for ScAMT2;1, indicating that these proteins are capable of transporting ammonium and complementing the mutant. Data of 15N-ammonium influx and accumulation in vivo in roots and shoots of qko plants overexpressing ScAMT2;1 or ScAMT3;3A showed that ScAMT2;1 acts in ammonium uptake by roots and probably in the xylem loading, while ScAMT3;3A is possibly involved in ammonium remobilization in shoots, and may act additively in the absorption of NH4+ in roots under high ammonium. These results indicate that ScAMT2;1 and ScAMT3;3A from sugarcane are functional, working with distinct properties and functions in ammonium transport according to the availability of N