Para se avaliar a influência da adubação orgânica, a que foi submetida cana-de-açúcar, na fisiologia de bactérias diazotróficas endofíticas foram identificados e estudados 36 isolados de plantas submetidas a tratamentos orgânico e convencional ou sem tratamento (controle). Foi evidenciado predomínio da família Enterobacteriaceae (75%). Essas bactérias foram avaliadas quanto à excreção de substâncias promotoras de crescimento vegetal (aminoácidos, poliaminas, AIA e etileno), escolhidas como indicadoras da ação do tratamento. Os resultados mostraram que foram significativamente maiores: a excreção de aminoácidos por bactérias provenientes do tratamento orgânico assim como a produção de etileno por bactérias isoladas de plantas controle. Além das 36 linhagens acima citadas, foram identificadas sete linhagens de Rhizobium por sequenciamento dos genes 16S rRNA, recA e gapA. Três dessas linhagens foram identificadas como Rhizobium trifolii, Rhizobium etli e Rhizobium hainanense. O gênero Rhizobium ainda não havia sido descrito como endofítico de cana-de-açúcar. / Aiming to evaluate the influence of the organic fertilization, applied to sugarcane, on the physiology of endophytic nitrogen-fixing bacteria, 36 isolates of plants submitted to organic and conventional fertilization or not fertilized (control) were identified and studied. Results showed that the major part of the strains belong to the Enterobacteriaceae family (75%). The excretions of amino acids, polyamines, IAA and ethylene substances chosen as indicators of the action of the treatment were evaluated in the 36 strains. The release of amino acids was significantly higher in cultures of bacteria isolated from organic treated plants and the production of ethylene was produced in greater amounts by strains isolated from control plants. Seven isolates, different from the 36 tested for the release of plant growth promoters, were identified as Rhizobium strains through 16S rRNA, recA and gapA sequences. Three strains were identified as Rhizobium trifolii, Rhizobium etli and Rhizobium hainanense. This genus was not described as endophytic of sugarcane previously.

Para se avaliar a influência da adubação orgânica, a que foi submetida cana-de-açúcar, na fisiologia de bactérias diazotróficas endofíticas foram identificados e estudados 36 isolados de plantas submetidas a tratamentos orgânico e convencional ou sem tratamento (controle). Foi evidenciado predomínio da família Enterobacteriaceae (75%). Essas bactérias foram avaliadas quanto à excreção de substâncias promotoras de crescimento vegetal (aminoácidos, poliaminas, AIA e etileno), escolhidas como indicadoras da ação do tratamento. Os resultados mostraram que foram significativamente maiores: a excreção de aminoácidos por bactérias provenientes do tratamento orgânico assim como a produção de etileno por bactérias isoladas de plantas controle. Além das 36 linhagens acima citadas, foram identificadas sete linhagens de Rhizobium por sequenciamento dos genes 16S rRNA, recA e gapA. Três dessas linhagens foram identificadas como Rhizobium trifolii, Rhizobium etli e Rhizobium hainanense. O gênero Rhizobium ainda não havia sido descrito como endofítico de cana-de-açúcar. / Aiming to evaluate the influence of the organic fertilization, applied to sugarcane, on the physiology of endophytic nitrogen-fixing bacteria, 36 isolates of plants submitted to organic and conventional fertilization or not fertilized (control) were identified and studied. Results showed that the major part of the strains belong to the Enterobacteriaceae family (75%). The excretions of amino acids, polyamines, IAA and ethylene substances chosen as indicators of the action of the treatment were evaluated in the 36 strains. The release of amino acids was significantly higher in cultures of bacteria isolated from organic treated plants and the production of ethylene was produced in greater amounts by strains isolated from control plants. Seven isolates, different from the 36 tested for the release of plant growth promoters, were identified as Rhizobium strains through 16S rRNA, recA and gapA sequences. Three strains were identified as Rhizobium trifolii, Rhizobium etli and Rhizobium hainanense. This genus was not described as endophytic of sugarcane previously.

Les plantes de la famille des légumineuses ont acquis la capacité d’accueillir au sein d’organes symbiotiques, les nodosités, des bactéries fixatrices d’azote appelées bactéroïdes. Cette symbiose permet aux plantes hôtes de satisfaire leurs besoins en azote. Certaines légumineuses produisent au sein des nodosités une grande famille de peptides antimicrobiens particuliers, les NCR (Nodule-specific Cysteine-Rich). Ils permettent à l’hôte de contrôler la population bactérienne intracellulaire via leurs activités antimicrobiennes, mais aussi d’imposer aux rhizobia une différenciation terminale des bactéroïdes. Durant ma thèse, j’ai participé à l’identification et à la caractérisation de la protéine bactérienne BclA. Ce transporteur ABC est nécessaire pour la formation de bactéroïdes différenciés lors de la symbiose Aeschynomene-Bradyrhizobium. Pour mieux comprendre sa fonction symbiotique, j’ai étudié la relation entre BclA et une enzyme de modification du peptidoglycane, la DD-carboxypeptidase 1. J’ai pu montrer que ces deux facteurs agissent de manière indépendante dans la mise en place de bactéroïdes différenciés. Une analyse fonctionnelle de BclA et une expérience d’évolution expérimentale avec le mutant bacA de Sinorhizobium, un orthologue de bclA, apportent une meilleure compréhension du rôle de ces transporteurs in vivo. L’ensemble des résultats obtenus pendant ma thèse suggère que BclA et BacA assurent la résistance bactérienne face aux NCR in planta, comme un prérequis pour la suite du processus, mais ne sont pas nécessaires à la différenciation per se. De plus, leurs activités d’import des NCR ne semblent pas être le mécanisme sous-jacent du processus de résistance. / Plants of the legume family have acquired the ability to host in specific symbiotic organs, the roots nodules, nitrogen fixing bacteria, called bacteroids. This symbiosis allows plants to fulfill all their nitrogen requirements. Some legume plants produce in their nodules a large family of antimicrobials peptides called the NCRs (Nodule-specific Cysteine-Rich). Their antimicrobial activities allow the host plant to control the intracellular bacterial population. NCRs peptides also govern terminal differentiation of the bacteroids. During my PhD work, I participated in the identification and characterization of BclA. This bacterial ABC transporter is involved in bacteroid differentiation during the Aeschynomene- Bradyrhizobium symbiosis. In order to better understand its symbiotic function, I studied the link between BclA and a peptidoglycan-modifying enzyme, the DD-carboxypeptidase 1. I was able to show that these two factors act in an independent manner in the establishment of bacteroid differentiation. A functional analysis of BclA and an experimental evolution on Sinorhizobium bacA mutant, an orthologue of bclA, conferred a better understanding of the in vivo role of these transporters. The results obtained during my thesis suggest that the BclA and BacA function is to ensure bacterial resistance to NCRs, as a prerequisite for the bacterial differentiation process, but is not needed for differentiation per se. Furthermore, their NCR uptake activities do not seem to be the mechanism underlying the resistance.

L’architecture du système racinaire des légumineuses est déterminée par la croissance des racines et leur capacité à former des racines latérales et des nodosités symbiotiques fixatrices d’azote en fonction des conditions environnementales. Chez la légumineuse modèle Medicago truncatula, nous avons mis en évidence que les phytohormones gibbérellines (GAs) et leur voie de signalisation – médiée par trois protéines MtDELLAs présentant des profils d’expression redondants – jouent un rôle clé dans la régulation du développement du système racinaire. En conditions non-symbiotiques, les GAs régulent négativement la croissance racinaire en réprimant l’activité méristématique et l’élongation cellulaire, et inhibent la formation des racines latérales. En conditions symbiotiques, le rôle des GAs et de leur voie de signalisation est double : au niveau de l’épiderme, les protéines MtDELLAs régulent positivement l’infection par les bactéries symbiotiques Rhizobia en interagissant de façon directe avec la voie de signalisation des facteurs Nod bactériens. Au niveau du cortex, les GAs régulent négativement l’organogénèse des nodosités. Les phytohormones cytokinines (CKs) et leur récepteur MtCRE1 étant essentiels pour initier la nodulation, l’interaction entre les voies de signalisation GA et CK a été analysée en parallèle. Les CKs régulent le niveau de GAs bioactives en modulant l’expression des gènes de leur métabolisme de manière dépendante du récepteur aux CKs MtCRE1. Réciproquement, les GAs régulent les gènes du métabolisme CK et le niveau de CKs bioactives de manière dépendante des MtDELLAs. Par ailleurs, une forme activée de MtDELLA complémente partiellement le phénotype de nodulation du mutant cre1 et lie le promoteur de MtCRE1, indiquant que les voies de signalisation GA et CK régulent la nodulation en étroite association. / Legume root system architecture is determined by both root growth and their ability to form lateral roots and nitrogen-fixing symbiotic nodules, depending on environmental soil conditions. In the model legume Medicago truncatula, we have shown that the phytohormones gibberellins (GAs) and their signaling – involving three MtDELLA proteins with redundant expression patterns – play a crucial role in the regulation of the root system development. In non-symbiotic conditions, GAs negatively regulate root growth through the repression of meristematic activity and cell elongation, and inhibit lateral root formation. In symbiotic conditions, GA and their signaling pathway play a dual role: in the root epidermis, MtDELLA proteins positively regulate infection by symbiotic bacteria Rhizobia, by directly interacting with the bacterial Nod factor signaling pathway. In the root cortex, GAs negatively regulate nodule organogenesis. The phytohormones cytokinins (CKs) and their receptor MtCRE1 being essential for the initiation of nodulation, we additionally analyzed the crosstalk between GA and CK signaling. CKs regulate the bioactive GA pool, controlling the expression of GA-metabolic genes, depending on the MtCRE1 receptor. In turn, GAs regulate CK-metabolic genes and the bioactive CK pool, depending on MtDELLAs. In addition, a dominant version of a MtDELLA partially complements the nodulation phenotype of the cre1 mutant and binds to the MtCRE1 promoter. These results indicate that GA and CK signaling pathways closely interact to regulate nodulation.

Das Bakterium Bradyrhizobium japonicum ist wie andere Rhizobien in der Lage mit Pflanzen der Familie Fabales (Leguminosen) eine Symbiose einzugehen. Im symbiontischen Zustand fixieren die Mikrosymbionten atmosphärischen, molekularen Stickstoff und stellen diesen den Pflanzen in verwertbarer Form zur Verfügung. Im Gegenzug erhalten die Bakterien von den Pflanzen verschiedene Verbindungen als Kohlenstoff- und Energiequelle. Dem geht ein komplexer Signalaustausch voraus um die gegenseitige Erkennung der Partner und die Spezies-spezifische Symbiose zu ermöglichen. Auf Seite der Bakterien erfolgt die Reaktion auf die Gegenwart pflanzlicher Signalmoleküle. In B. japonicum induziert das Flavonoid Genistein die Transkription einer Reihe von Genen. Durch die Expression der nod-Gene erfolgt eine Synthese von Lipochitooligosacchariden. Diese sogenannten Nodulationsfaktoren rufen wiederum eine Reaktion in der Wirtspflanze hervor. Um die zugrunde liegende Genexpression und deren Regulationsmechanismen zu erforschen, standen bis dato nur Methoden für die Untersuchung einzelner Gene zur Verfügung. Die erstmals 1995 publizierte Microarraytechnologie eröffnete die Möglichkeit zu einem Zeitpunkt die Gentranskription eines gesamten Organismus zu untersuchen (Schena et al. 1995). Um mit dieser Technologie die Gentranskription bei B. japonicum zu untersuchen, wurde ein auf PCR-Produkten basierender Microarray hergestellt. Grundlage für die Synthese genspezifischer PCR-Produkte war die symbiontische Region von B. japonicum und andere zu diesem Zeitpunkt bekannte Gensequenzen (Göttfert et al. 2001). Nach der 2002 erfolgten Veröffentlichung des Genoms von B. japonicum erfolgte ein Abgleich der bisher verwendeten Sequenzen mit der vollständigen Sequenzinformation (Kaneko et al. 2002a). Nach Synthese der PCR-Produkte und deren Kontrolle durch Sequenzierung erfolgte die Herstellung des Microarrays unter Einsatz eines Microarrayspotters. Geeignete Techniken der cDNA-Markierung und die Microarray-Hybridisierung wurden mit Total-RNA von B. japonicum-Kulturen ausgetestet und etabliert. Eine differentielle Expremierung war eine Stunde nach Genistein- bzw. Methanolgabe zum Kulturmedium nicht nachweisbar. Zum darauffolgenden Zeitpunkt konnte die bekannte Induktion der nod-Gene durch dass Flavonoid Genistein beobachtet werden. Diese Induktion fiel in den verbleibenden zwei Zeitpunkten stark ab. Dieser Abfall der Induktionsrate ließ sich nicht mit dem Genisteingehalt im Kulturmedium erklären, da dieser Zeitraum konstant blieb. Vier Stunden nach Genisteingabe war die Induktion der Gene nolA und nodD2, deren Produkte sind an der negativen Regulation der nod-Gene beteiligt, nachweisbar. Dies wäre eine mögliche Erklärung für den Abfall der Induktion der nod-Gene. Im gleichen untersuchten Zeitraum wurde die Transkription verschiedener Gene der Stickstofffixierung und Denitrifikation durch das Flavonoid Genistein induziert. Auch für die bekannten Regulatoren dieser Gene war eine Induktion nachweisbar. Nach bisherigen Arbeiten war die Expression dieser Gene auf mikroaerobe und anaerobe Zustände, wie z.B. dem symbiontischen Stadium, beschränkt. Eine Induktion durch Flavonoide ist bisher nicht beobachtet worden. Um die Verwendbarkeit des Microarrays auch für den symbiontischen Zustand von B. japonicum zu testen, wurden Versuche mit Wurzelknöllchen von Sojabohne (Glycine max) durchgeführt. Dafür wurden Keimlinge der Sojabohne (G. max) mit B. japonicum inokuliert und die Total-RNA der entstehenden Wurzelknöllchen isoliert. Jedoch war eine Kreuzhybridisierung mit pflanzlicher Total-RNA zu beobachten. Der auf PCR-Produkten basierende Microarray ist für die Untersuchung des symbiontischen Stadiums von B. japonicum nicht einsetzbar. Im Vergleich dazu wurde ein auf Oligomeren basierender, kommerzieller Microarray für diesen Versuch getestet. Die Kreuzhybridisierung mit pflanzlicher Total-RNA war auf die Oligomere für die 16S rDNA und 23S rDNA reduziert.

Les plantes invasives constituent un phénomène très répandu sur la planète qui entraîne des problèmes environnementaux majeurs aboutissant à des perturbations significatives dans les processus régissant la conservation de la diversité végétale et microbienne des sols. Acacia mearnsii (De Wild) est l'une des espèces d'Acacia ayant un potentiel invasif important. Cette légumineuse d'origine australienne a été introduite dans les subéraies du Parc National d'El-Kala (PNEK), au Nord-Est de l'Algérie, où elle induit des dégradations drastiques sur l'écosystème forestier de chêne-liège Quercus suber (L.) et la diversité végétale et microbienne de cette formation forestière. L'objectif de cette étude a été d'évaluer les transformations induites par A. mearnsii sur les caractéristiques et microbienne du sol (particulièrement les communautés de symbiotes microbiens) et d'en déterminer les conséquences sur le développement de chêne-liège. Une série d'expérimentations a été réalisée sur les sols de trois sites dans le PNEK : forêt naturelle de Q. suber (site non envahi), peuplement mixte Q. suber et A. mearnsii (site envahi récemment par A. mearnsii) et peuplement d'A. mearnsii (site anciennement envahi par A. mearnsii). Les analyses de sol, de la diversité fonctionnelle et structurelle des microorganismes telluriques ont révélé l'existence de modifications liées à la présence de l'espèce envahissante. D'autre part, il a été déterminé que A. mearnsii avait un effet inhibiteur sur le développement du chêne-liège et sur son cortège ectomycorhizien associé. De plus, cette essence semble avoir la capacité de s'adapter facilement au milieu d'introduction, du fait de sa forte mycotrophie vis à vis des champignons mycorhiziens arbusculaires, et de sa facilité de nodulation spontanée avec des bactéries symbiotiques principalement du genre Bradyrhizobium. La présence de symbiotes compatibles avec A. mearnsii dans les habitats envahis représente probablement un des facteurs susceptibles de faciliter le processus d'envahissement de l'espèce. Nos résultats suggèrent que la régression du développement du chêne-liège peut être liée aux modifications induites par A. mearnsii sur le fonctionnement du sol et la structure des microorganismes telluriques. Cet effet est probablement lié à d'autres éléments biotiques et abiotiques du milieu envahi influencé par cet arbre. Il sera donc important d'affiner cette étude, et d'analyser plus précisément les paramètres pouvant être à l'origine du succès de l'invasion par A. mearnsii afin de définir un cadre de lutte contre cette espèce invasive et ainsi sauvegarder la subéraie . / Invasive plants are a global phenomenon causing major environmental problems leading to significant disruptions in the processes governing the conservation of plant diversity and soil microbial communities. Acacia mearnsii (De Wild) is one of the Acacia species with a significant invasive potential. This Australian native legume was introduced in the cork forests of the National Park of El Kala (PNEK), North-eastern Algeria, and induced drastic degradation of the cork oak (Quercus suber (L.)) forest ecosystem, on understorey plant species and soil microbial diversity. The objective of this study was to evaluate changes resulting from the A. mearnsii invasion on chemical characteristics and soil microbiota (especially symbiotic microbial communities) and to determine their impact on cork oak development. Experiments were conducted on soils collected from three sites in the PNEK: Q. suber natural stand (non-invaded site), mixed Q. suber and A. mearnsii stand (recently invaded site) and A. mearnsii stand (fully invaded site). Both chemical and microbiological soil characteristics were affected by the presence of the invasive species. On the other hand, it was determined that A. mearnsii had an inhibitory effect on the development of the cork oak tree and its associated ectomycorrhizal community. In addition, this tree species seems to easily interact with arbuscular mycorrhizal fungi in the introduction area, being abundantly infected. It is also spontaneously nodulated by local symbiotic bacteria, mainly of the genus Bradyrhizobium. The presence of symbionts, compatible with A. mearnsii in invaded habitats are probably one of the factors that facilitate the process of invasion. Our results suggest that the development of the cork oak can be linked to changes induced by A. mearnsii with soil functioning and with the composition of soil microorganism communities. This effect is probably related to other biotic and abiotic components of the environment influenced by this invasive tree species. It seems thus important to analyze more precisely the parameters that cause the success of the invasion by A. mearnsii in order to better control this invasive species and save the cork oak forest in Algeria.

A soja, [Glycine max (L.) Merrill] é uma das espécies com maior teor protéico, contendo cerca de 40% de proteína nos grãos. Em conseqüência disso demanda alta quantidade de nitrogênio (N), o qual pode ser suprido pelo processo de fixação biológica de nitrogênio (FBN), através da simbiose com as bactérias do gênero Bradyrhizobium. No Brasil, a FBN é capaz de suprir toda a demanda de N da cultura da soja, dispensando a aplicação de fertilizantes nitrogenados. No entanto, os caracteres relacionados à FBN não têm sido diretamente considerados em programas de melhoramento genético, em função das dificuldades inerentes às avaliações dos mesmos, que requerem a destruição das plantas. O objetivo deste trabalho foi mapear os locos de caracteres quantitativos (Quantitative Trait Loci: QTLs) dos caracteres relacionados à FBN, visando identificar associações úteis para a seleção assistida por marcadores, bem como obter outras informações sobre a base genética destes caracteres em soja. Uma população composta de 157 F2:7 linhagens endogâmicas recombinantes (Recombinant Inbred Lines RILs), derivada de um cruzamento biparental, foi genotipada com 105 marcadores microssatélites, bem como avaliada para os seguintes caracteres relacionados com a FBN: número de nódulos (NN); peso seco dos nódulos (MNS); peso médio dos nódulos secos (MNS/NN) e peso seco da parte aérea (MPAS). Utilizando o método de mapeamento por intervalo composto para múltiplas características (mCIM) foram mapeados os QTLs para os quatro caracteres. Um mapa genético foi construído com um tamanho estimado em 1.263,2 cM, correspondendo a uma cobertura de 50% do genoma. Oito regiões genômicas foram associadas com os caracteres de FBN. Quatro dessas regiões, localizadas nos grupos de ligação (GL) C1, C2, E e I, foram associadas a mais de um caráter: no GL C1 (Satt190-Satt136) foram mapeados QTLs para MNS, MNS/NN e MPAS; no GL C2 (Satt460-Satt307) foram mapeados QTLs para NN e MNS; no GL E (Satt573-SAtt185) foram mapeados QTLs para MPAS e NN; e no GL I (Satt239-Satt354) foram mapeados QTLs para MNS/NN e NN. A associação dos QTLs nos GL C1, C2 e E foi atribuída à pleiotropia, enquanto que no GL I foi atribuída à ligação gênica. Os QTLs influenciando apenas um caráter foram mapeados nos GL A2, B1, G e L: no GL A2 (Sct067-Satt589) foi mapeado um QTL para MNS/NN; no GL B1 (Satt509-Satt251) foi mapeado um QTL para NN; no GL L (Satt232-Satt418) foi mapeado um QTL para MPAS; e no GL G (Satt394-Satt288) foi mapeado um QTL para MPAS. Os QTLs explicaram, individualmente, muito pouco da variação fenotípica (R2 = 1,2% a 10,0%), sendo o QTL mais significativo mapeado no GL L para MPAS, e explicou 10,0% da variação do caráter, com um efeito aditivo de 0,57 g planta-1. Portanto, foram detectados QTLs em quatro regiões para MPAS (C1, E, G e L), em cinco regiões para NN (B1, C1, C2, E, G); em duas regiões para MNS (C1, C2) e em três regiões para MNS/NN (A2, C1, I) que explicaram 23,0%, 20,0%, 11,8% e 16,0% da variação fenotípica total, respectivamente. Estes resultados estão de acordo com as herdabilidade relativamente baixas dos caracteres (28% a 49%) e refletem a natureza complexa da FBN, que está sob influência ambiental. / Soybeans [Glycine max (L.) Merrill] is a crop with high protein content (about 40%) in the seeds. As a result, the crop demands high nitrogen (N) inputs, which can be supplied by the process of biological nitrogen fixation (BNF), through the symbiosis with bacteria of the genus Bradyrhizobium. In Brazil the BNF allows to fulfill all the demand for N; therefore N fertilizers are not required. However, the traits associated with BNF have not been directly considered in breeding programs due to the difficulties to its evaluation, which require, generally, the plant destruction. The objective of this study was to map Quantitative Trait Loci (QTLs) of traits related to BNF and to identify useful associations for marker-assisted selection, as well as to obtain other information about the genetic basis of these traits in soybeans. A population of 157 F2:7 recombinant inbred lines (RILs), derived from a two-way cross, were genotyped with 105 microsatellite markers as well as evaluated for the following traits related with BNF: number of nodes (NN); nodule dry weight (NDW); mean nodule dry weight (NDW/NN); and shoot dry weight (SDW). Using the composite interval mapping for multiple traits (mCIM) method, the QTLs were mapped for all the traits. A genetic map was constructed with an estimated size of 1,263.2 cM, covering about 50% of the genome. Eight genomic regions were associated with the four traits and four of these regions, located on linkage groups (LG) C1, C2, E and I, were associated with more than one trait: in LG C1 (Satt190-Satt136), QTLs for NDW, NDW/NN and SDW were mapped; in LG C2 (Satt460-Satt307), QTLs for NN and NDW were mapped; in GL E (Satt573-SAtt185), QTLs for SDW and NN were mapped; and in GL I (Satt239-Satt354) QTLs for NDW/NN and NN were mapped. The pleiotropy was attributed to QTL association in the LG C1, C2, and E, whereas genetic linkage was attributed to QTL association in LG I. QTLs affecting only one trait were mapped in LG A2, B1, G and L: in LG A2 (Sct067-Satt589) a QTL was mapped, for NDW/NN; in LG B1 (Satt509-Satt251) a QTL was mapped for NN; in LG L (Satt232-Satt418) a QTL was mapped for SDW; and in LG G (Satt394- Satt288) a QTL was mapped for SDW. The QTLs individually explained very little of the phenotypic variation (R2 = 1.2% to 10.0%), and the most significant QTL was mapped in the LG L, explaining 10.0% of the variation for SDW, with an additive effect of 0.57 g plant-1. Therefore, QTLs in four regions were detected for SDW (C1, E, G, and L), in five regions for NN (B1, C1, C2, E, and G), in two regions for NDW (C1, and C2) and in three regions for SDW/NN (A2, C1, and I), which explained 23.0%, 20.0%, 11.8% and 16.0% of phenotypic variation, respectively. These results are in agreement with the relatively low heritability of the traits (28% to 49%) and reflect the complex nature of BNF traits, which are influenced by the environmental effects.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2018-09-19T12:12:01Z No. of bitstreams: 1 2010 - Rafael Sanches Pacheco.pdf: 669093 bytes, checksum: 3ca2e98e7a5ac781dba791f2e1bfda70 (MD5) / Made available in DSpace on 2018-09-19T12:12:01Z (GMT). No. of bitstreams: 1 2010 - Rafael Sanches Pacheco.pdf: 669093 bytes, checksum: 3ca2e98e7a5ac781dba791f2e1bfda70 (MD5) Previous issue date: 2010-08-10 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico, CNPq, Brasil. / Nutrient concentration in the seeds can affect the initial development, biological nitrogen fixation (BNF) and yield of plants. Common bean (Phaseolus vulgaris L.) has been considered as a species with low capacity of BNF as compared to other grain legumes, but results demonstrate the potential of BNF to supply the N demand of the crop and to achieve adequate yields. The objective of this study was to evaluate the effect of seed concentrations of Mo and P on biomass production, nodulation and grain yield of common bean at different sources of N supply. The field experiment was conducted between April and July 2009, in the Santa Monica Farm of Embrapa Gado de Leite in the municipality of Valen?a ? Rio de Janeiro State. The experimental design was a split-plot 4x4 factorial randomized block with 5 replicates. The main plots consisted of four N sources: control without N, mineral N fertilization with 20 kg N ha-1 at sowing + 40 kg N ha-1 in cover, inoculation with the commercial strain of rhizobia, rhizobia inoculation + 40 kg N ha-1 in cover. The subplots consisted of four combinations between concentrations of P and Mo in the seeds (low P low Mo, low P high Mo, Mo high P low, high-P high-Mo). The seeds of cultivar Carioca were originating from a field experiment when 5 kg P ha-1 and 120 g Mo ha-1 were foliar sprayed at 52 and 71 days after emergence (DAE). In each subplot, plant biomass were sampled at 30, 42 and 57 DAE, and root, shoot and nodules dry mass, the number of nodules and the accumulation of N in the shoot were determined. Grains were harvested at 90 DAE, and grain yield, yield components, N and Mo concentratios in the grains were measured. Interactions between the N sources and the concentrations of P and Mo in the seeds were not significant in the three sampling times, except for shoot dry mass and number of nodules at 57 DAE. Seeds with low P and high Mo increased shoot dry matter and grain yield of beans. Mineral N fertilizer increased root mass in the early growth stages. The rhizobia inoculation increased bean nodulation. There was a small effect of the seeds with high P concentration in the accumulation of N and mass of shoots at 57 DAE. Mineral N fertilizer increased N accumulation in the shoots at 57 DAE. The inoculation + 40 kg N ha-1 in cover gave similar grain yield and N accumulation as the application of 60 kg N ha-1, both treatments superior to the control. The use of seeds enriched with P and Mo, associated with the inoculation of seeds and the application of N in cover, could maximize the process of biological nitrogen fixation in the common bean crop. / A concentra??o de nutrientes nas sementes pode afetar o desenvolvimento inicial, a fixa??o biol?gica de nitrog?nio (FBN) e a produ??o de gr?os das plantas por elas geradas. O feijoeiro (Phaseolus vulgaris L.) tem sido considerado uma esp?cie de baixa capacidade de FBN em compara??o com outras leguminosas de gr?o, mas resultados demonstram o potencial da FBN em suprir a demanda de N do feijoeiro e obter boas produtividades. O objetivo deste estudo foi avaliar o efeito dos teores de P e Mo em sementes na produ??o de biomassa, nodula??o e no rendimento de gr?os de feijoeiro sob diferentes fontes de fornecimento de N. O experimento foi conduzido em condi??es de campo entre abril e julho de 2009, na Fazenda Santa M?nica da Embrapa Gado de Leite, no munic?pio de Valen?a ? RJ, em delineamento em blocos ao acaso em fatorial 4x4 com 5 repeti??es em parcelas subdivididas. As parcelas foram compostas por quatro fontes de N: testemunha absoluta sem N; aduba??o com N mineral com 20 kg N ha-1 no plantio + 40 kg N ha-1 em cobertura; inocula??o com a estirpe comercial de riz?bio recomendada para a cultura; inocula??o com estirpe comercial + 40 kg N ha-1 em cobertura. As subparcelas foram compostas por quatro combina??es entre teores de P e de Mo na semente (baixo P baixo Mo, baixo P alto Mo, alto P baixo Mo, alto P alto Mo). As sementes da cultivar Carioca utilizadas foram oriundas de um experimento de campo onde foram efetuadas aplica??es foliares com 5 kg P ha-1 e 120 g Mo ha-1 aos 52 e 71 dias ap?s emerg?ncia (DAE). Em cada subparcela foram efetuadas tr?s amostragens de biomassa, aos 30, 42 e 57 DAE, determinando-se a massa seca de raiz, parte a?rea e n?dulos, o n?mero de n?dulos e a acumula??o de N na parte a?rea. A colheita de gr?os foi efetuada aos 90 DAE, determinando-se o rendimento de gr?os, os componentes de produ??o e o teor de N e de Mo nos gr?os. As intera??es entre as fontes de N e os teores de P e Mo das sementes, nas tr?s ?pocas de coleta, n?o foram significativas, exceto para massa seca de parte a?rea e n?mero de n?dulos aos 57 DAE. As sementes com baixo P e alto Mo foram as que promoveram maior massa de parte a?rea e tamb?m a maior produ??o de gr?os de feij?o. A aduba??o com N mineral promoveu maior massa radicular nos est?gios iniciais de crescimento. A inocula??o das sementes com riz?bio aumentou a nodula??o do feijoeiro. Houve um pequeno efeito dos altos teores de P nas sementes na acumula??o de N e de biomassa da parte a?rea aos 57 DAE. A aplica??o de N mineral promoveu maior ac?mulo da N na parte a?rea aos 57 DAE. A inocula??o combinada com 40 kg N ha-1 em cobertura forneceu rendimento de gr?os e acumula??o de N nos gr?os similar ? aplica??o de 60 kg N ha-1, por sua vez superiores ? testemunha. O uso de sementes enriquecidas com P e Mo, associada com a inocula??o de sementes e a aplica??o de N em cobertura, podem maximizar o processo de fixa??o biol?gica de nitrog?nio na cultura do feijoeiro.

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Many bacteria accumulate carbon stores as poly-3-hydroxybutyrate (PHB) when growth is limited but carbon availability is not. This stored carbon can then be utilized during conditions of limited carbon availability. The net PHB accumulation in the cell is dependent on the balance between PHB synthesis and degradation. Sinorhizobium meliloti accumulates PHB in the free-living stage but not in the symbiotic stage. The physiological role of the PHB cycle in S. meliloti is unknown. As a first step to understand the genetics of PHB degradation, transposon-generated mutants that were not able to use PHB degradation intermediates, such as 3-hydroxybutyrate and acetoacetate, as a sole carbon source, were isolated. Genetic mapping revealed that there were at least three chromosomal loci involved in acetoacetate metabolism. Identification of these three loci determined that in S. meliloti: (1) acetoacetyl-CoA synthetase (AcsA), encoded by acsA2 gene, rather than the enzyme acetoacetate:succinyl-CoA transferase, is the enzyme that catalyzes activation of acetoacetate to acetoacetyl-CoA; (2) PHB synthase, encoded by phbC, is required for acetoacetate utilization; (3) a putative transporter protein encoding gene, aau-3, may also be involved in acetoacetate metabolism. acsA2 and aau-3 were 78% linked in co-transduction, while phbC was mapped to somewhere else on the chromosome. Biochemical analysis revealed that acsA2::Tn5 mutants lacked AcsA activity but not acetoacetate:succinyl-CoA transferase activity, while phbC::Tn5 maintained similar level of AcsA activity as wild type in vitro. PHB was absent in the phbC mutant. / One transposon-generated mutant, age-1, showed enhanced growth rate on acetoacetate medium. Genetic mapping and transductional analysis indicated that the location of the mutation in age-1 is tightly linked to acsA2. Fine mapping with PCR and DNA sequence techniques showed that Tn5 in age-1 was located at 132 by upstream of the putative translation start site of acsA2. Gene expression analysis indicated that age-1 insertion results in elevated transcription of acsA2. Thus enhanced growth rate on acetoacetate was due to the increased gene expression. acsA2 transcription was induced by acetoacetate and 3-hydroxybutyrate, and repressed by glucose and acetate. / All mutants formed root nodules that fixed nitrogen with varying decrease of impairment. Acetoacetate metabolism and the PHB degradation are not essential for symbiosis.