En raison de leur rôle central dans la physiologie et le développement des plantes, les phythormones ont depuis longtemps été considérées comme des médiateurs chimiques déterminants dans la capacité des insectes à contrôler leur environnement végétal. Les mécanismes permettant aux insectes de manipuler la balance phytohormonale permettant ainsi la régulation des apports nutritifs et la modulation des défenses végétales demeurent néanmoins pour la plupart inconnus, en particulier pour les insectes galligènes et mineurs de feuilles. L’objectif de ma thèse visait à caractériser les capacités de modulation phytohormonale par les insectes manipulateurs de plantes avec un accent particulier sur le lépidoptère mineur de feuille Phyllonorycter blancardella. Nous avons ainsi développés une caractérisation spatio-temporelle de la réponse des plantes aux attaques des larves mineuses au niveau moléculaire et biochimique. Une comparaison entre différents systèmes biologiques nous a permis d’évaluer les similitudes entre les stratégies adoptées par les les insectes galligènes et les insectes mineurs de feuilles, d’identifier l’origine possible des phytohormones impliquées dans la manipulation de la plante et le rôle des bactéries endosymbiotiques d’insectes dans ces interactions. / Because phytohormones lie at the very core of molecular mechanisms controlling the plant physiology and development, they have long been hypothesized to be involved in insect-induced plant manipulations. Insects are using phytohormones to manipulate their host plants for their own benefit, regulating nutrient provisioning and plant defenses. However, a mechanistic understanding of how phytohormones operate in plant reconfigurations by plant-manipulating insects, especially by gall-inducing and leaf-minging insects, is lacking. The objective of my Ph.D. was to provide an extensive characterization of how plant-manipulating insects modulate the plant global hormonal balance with a specific focus on the leaf-mining moth Phyllonorycter blancardella. We thus developed a time course characterization of plant transcriptomic and biochemical responses following attack by leaf-mining larvae. A comparative analysis between different biological systems allowed us to estimate similarities in strategies developed leaf-mining and gall-inducing insects, to identify the possible origin of phytohormones involved in the plant manipulation and to estimate the role of insect endosymbiotic bacteria in these interactions.

Les phytoplasmes sont des bactéries phytopathogènes, sans paroi, qui appartenant à la classe des Mollicutes. Ils ne peuvent pas etre cultivés in vitro et sont limités à des tubes du phloème. Ils provoquent des centaines de maladies chez de nombreuses espèces végétales dans le monde entier, ce qui conduit à des pertes de récolte importantes. Les phytoplasmes sont transmis naturellement par des insectes suceurs de sève dans laquelle ils se multiplient. Ils induisent des symptômes graves, notamment le jaunissement, la croissance limitée, déclin, ainsi que des anomalies des fleurs et des fruits. L'infection par le phytoplasme du stolbur, en particulier, affect fortement la morphologie florale. Dans la tomate, deux isolats différents du phytoplasme du stolbur, nommé C et PO, induisent des symptômes différents. La tomate infectée par le phytoplasme du stolbur PO montrent des malformations florale telles que les sépales hypertrophiés, les pétales et les étamines avortées ce qui conduit à la stérilité. En revanche, la tomate infecté par le phytoplasme du stolbur C ont de petites feuilles de tomate en retrait, mais les fleurs presque normale, et produisent des fruits. Nous avons précédemment montré que SlDEF, un gène impliqué dans la formation des pétales est réprimé dans des plantes de tomate infectée par le stolbur phytoplasme PO. Toutefois, l'expression de son facteur de transcription, codée par le gène FA, est resté stable ou voir légèrement augmentée. Nous avons donc émis l'hypothèse que la répression de SlDEF pourrait être dû à une méthylation de l'ADN. Pour tester cette hypothèse, nous avons étudié l'expression des gènes de méthylases et de déméthylases. Ils étaient en général réprimés dans les tomates infectées par le phytoplasme du stolbur PO, ce qui était en accord avec l'hypothèse De plus, nous avons étudié les voies de défense activée chez les tomates infectées par le phytoplasme du stolbur. Pour se défendre, les plantes utilisées des molécules de signalisation comme l'acide salicylique (SA), l'acide jasmonique (JA) et d'éthylène (ET). Nous avons étudié l'expression de 21 gènes de défense dépendants SA / JA / ET, des gènes de biosynthèse et les facteurs de transcription chez les tomates infectées par les phytoplasmes du stolbur C et PO. Nous avons également étudié l'effet de la pré-activation des voies de SA et JA sur la production des symptômes. Nos résultats montrent clairement que les voies de défense ont été activées différemment dans les tomates infectés par le phytoplasme du stolbur C et PO. En effet, les voies de défense dépendantes de SA, ET et JA ont été activées chez les tomates infectées par le phytoplasme du stolbur C alors que seulement les voies dépendantes SA et ET ont été activés dans les tomates infectées par stolbur PO . En outre, la pré-activation de la voie de défense dépendante SA par l'application de BTH modifie légèrement l'évolution des symptômes de maladies causées par le phytoplasme du stolbur PO / Phytoplasma are cell wall-less, phytopathogenic bacteria belonging to the class Mollicutes. They have not been cultured in vitro and are restricted to the phloem sieve tubes. They cause hundreds of diseases in many plant species worldwide, resulting in important crop losses. Phytoplasmas are naturally transmitted by sap-sucking insects in which they multiply. They induce severe symptoms including yellowing, restricted growth, decline, as well as major flowers and fruits abnormalities.The stolbur phytoplasma infection, in particular, has been reported to strongly affect floral morphology. In tomato, two different isolates of stolbur phytoplasma, named C and PO, induce different symptoms. The stolbur PO phytoplasma-infected plants show abnormal flower development such as hypertrophied sepals, and aborted petals and stamens leading to sterility. In contrast, stolbur C phytoplasma-infected tomato have small indented leaves but nearly normal flowers, and produce fruits. We have previously shown that SlDEF, one gene involved in petal formation, was repressed in stolbur PO phytoplasma-infected tomato. However, the expression of its transcription factor, encoded by the gene FA, was unchanged or slightly up-regulated. So we hypothesized that SlDEF repression could be due to DNA methylation. To test this hypothesis, we studied the expression of DNA methylases and demethylases genes. They were in general down-regulated in stolbur PO infected tomato, which was in agreement with the hypothesis. However, the regulation of SlDEF expression could not be firmly correlated to the DNA methylation status of its promoter region. In addition, we studied the plant defense pathways activated in stolbur phytoplasma-infected tomato. To defend themselves, plants used signalling molecules like Salicylic acid (SA), Jasmonic acid (JA) and Ethylene (ET). We studied the expression of 21 SA/JA/ET regulated defense and biosynthesis genes including transcription factors in stolbur C and PO phytoplasma-infected tomato as compared to healthy ones. We also studied the effect of pre-activation of SA and JA mediated defense pathways on symptom production. Our results clearly showed that defense pathways were activated differently in stolbur C and PO phytoplasma-infected tomato. Indeed, SA ET and JA dependant pathways were activated in stolbur C-infected tomato while only SA and ET dependant pathways were activated in stolbur PO-infected plants. In addition, pre-activation of SA-dependent defense pathway by application of BTH slightly modify the evolution of disease symptoms caused by stolbur PO phytoplasma whereas no effect was observed after treatment with an analogue of JA.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The use of cowdung as an organic fertilizer in Asian and African agriculture is an ancient practice. This explains its renewed interest, partly due to the financial inability of most farmers to purchase agrochemicals but also the ever increasing need to adopt greener technologies that do not adversely affect soil health, water quality, biodiversity and promote sustained or even increased food production. In this context, many innovative farmers have developed their own novel technologies based on the use of local resources. One such innovation is Sanjeevak (a mix of cow dung, cow urine, water and a handful of sugar); which showed very promising boosting effect on crop productivity. However, very little scientific work has so far been conducted to evaluate its effect as an organic product for soil amendments. The present study was subdivided into three main objectives. (i) To assess the fertilizing value, human health and ecological risk profiles of Sanjeevak; (ii) To screen Sanjeevak for phytohormones content using Salkowski colorimetric method and liquid chromatography – mass spectrometry (LC-MS) (iii) To evaluate Sanjeevak application at various rates on growth parameters and yield of various crops cultivated in glasshouse and field conditions. Sanjeevak was assessed for its micro and macro nutrients contents. The analysis showed the presence of micronutrients such as Mg, Na, Ca and Zn at variable concentrations and phosphorus (P) (0.007%) and potassium (K) (0.063%). However, Sanjeevak content in total nitrogen (TN) (0.11%), and total organic carbon (TOC) (0.71%) was very low; suggesting that it may be a viable source of nutrients only if applied at higher and consistent rates or alternatively by improving its formulation. Also, Sanjeevak was analysed for its microbiological characteristics and level of heavy metals content in comparison to the strictest legislations that regulate the use and application of wastewater sludge to agricultural land in South Africa. The findings showed that heavy metals, which averaged from 0.03±0.01 for Arsenic (As) to 4.74±0.92 mg/kg for Zinc (Zn) and feacal coliform was estimated at 1.2×102 CFU/g dry matter measured were considerably below the threshold (for Arsenic between 40 to 75 mg/kg dry weight; for Zinc between 2800 to 7500 mg/kg dry weight) and faecal coliform bacteria between 1000 to 1×107 CFU/g dry weight for application as a source of soil amendments. Studies investigating the detection and concentration of phytohormones in Sanjeevak were carried out. In using the Salkowski colorimetric method to detect and quantify auxins from Sanjeevak and its composites (cow urine and dung), the results showed the presence of indole-3-acetic acid (IAA) at variable concentrations ranging from 20.38±2.1 ppm in cow urine, 20.1±6.6 ppm in cow dung, Sanjeevak 17.90±1.1 ppm to up to 138.31±12.6 ppm when LTRP was added to Sanjeevak bacterial cultures and by varying parameters such as incubaton time and temperature. Screening of the above mentioned samples for IAA using LC-MS analysis validated earlier findings. Further analysis of these results strongly emphasized the influence of bacteria in Sanjeevak in producing IAA. Trials were carried out both in the glasshouse and the field. In the greenhouse, different Sanjeevak application rates consistently confirmed its root promoting effect on crops such as tomato, cucumber and grapevine and increased wheat yield independent of the nutrients it contains. Marginal increases were recorded between treatments under field conditions; for example compost and compost + Sanjeevak 20.35 and 20.61 t/ha; and 2.46 and 2.60 t/ha compared to the control 11.67 t/ha and 1.29 t/ha respectively for tomato and maize. However, statistical analysis of the results obtained, revealed that there was no difference between treatments (control, compost, Sanjeevak and compost + Sanjeevak) for the same crop tested due to the high coefficient of variation of the data. Therefore, the use of Sanjeevak as an organic source of soil amendments may be considered as a cheaper alternative to effective microorganisms (EM) technology made up of local and natural resources. As observed in the study, it may be best used in combination with a reliable source of plant nutrients. / RESUMEE: L'utilisation des excréments de vaches comme engrais organique dans l'agriculture asiatique et africaine est une pratique très ancienne. Ceci explique son intérêt renouvelé, en partie due à aux restrictions monétaires de la plupart des agriculteurs d'acheter des produits agrochimiques, mais aussi la nécessité croissante d'adopter des technologies vertes qui ne nuissent pas à la qualité des sols, des eaux, la biodiversité et qui permettent d’améliorer la production agricole. C’est dans ce contexte que de nombreux paysans ont développé des techniques nouvelles dépendant des ressources naturelles et locales. L’utilisation de Sanjeevak (un mélange d’excréments de vaches, d’urine de vaches, l'eau et du sucre), a démontré sa capacité à accroître la productivité des plantes vivrières. Cependant, très peu de travaux scientifiques ont jusqu'à présent été menée pour étudier ce produit organique. La présente étude a été subdivisée en trois objectifs principaux. (i) Evaluation de la valeur fertilisante, et les profils de risques écologiques et sanitaires de Sanjeevak, (ii) Etudes de détection des phytohormones et leurs concentrations en utilisant une méthode colorimétrique adaptée de celle de Salkowski et la chromatographie liquide - spectrométrie de masse (LC-MS) (iii) Etude des effets de l’utilisation de Sanjeevak a différents taux d’applications sur la croissance et le développement des plants. Sanjeevak a été évaluée pour son contenu en micro et macro nutriments. L'analyse a montré la présence d'oligo-éléments tels que Mg, Na, Ca et Zn à des concentrations variables. De plus, son contenu en éléments majors tels que le phosphore (P) (0.007%), le potassium (K) (0.063%), l’azote (N) (0.11%), et carbone (C) (0.71%) est très faible; suggérant qu'il pourrait être une source viable de nutriments que si elle est appliquée à des taux plus élevés et répétés ou alternativement en améliorant sa formulation. En outre, Sanjeevak a été analysé pour ses caractéristiques microbiologiques et sa concentration en métaux lourds en comparaison a la législation qui réglemente l'utilisation et l'application de déchets liquide d’origines domestiques sur les terres agricoles en Afrique du Sud. Les résultats ont révélé que des métaux lourds et le niveau de coliformes fécaux mesuré était inférieur aux seuils d'application en tant que source d'amendements de sols agricoles. Les études portant sur la détection et la concentration d’hormones végétales ont été effectuées. En utilisant la méthode colorimétrique de Salkowski pour détecter et quantifier les auxines de Sanjeevak, les urines et les excréments de vaches; les résultats ont révélé la présence d'acide indole-3-acétique (AIA) à des concentrations variables dans les urines, les excréments et Sanjeevak. Une autre analyse des échantillons mentionnés ci-dessus pour les AIA en utilisant LC-MS a validé les résultats obtenus au préalable. L’étude détaillée de ces résultats confirme l'influence des micro-organismes dans la production des hormones végétales. Concernant les expériences sous serre, les différents taux d'application de Sanjeevak ont confirmé son effet stimulant à la croissance accélérée des racines des plantes telles que la tomate et les raisins et augmenté le rendement du blé indépendamment des nutriments qu'il contient. L'analyse statistique des résultats obtenus dans des conditions de terrain, a révélé qu'il n'y avait pas de différence entre les traitements (contrôle, compost, compost + Sanjeevak et Sanjeevak) pour la même plantes testées. Par conséquent, l'utilisation de Sanjeevak comme un produit organique qui améliore la qualité des sols et le rendement des cultures vivrières; peu être considéré comme un inoculum contenant des microorganismes constitué de ressources locales et naturelles. Comme l'a observé dans l'étude, il pourrait être mieux utilisé en combinaison avec une source fiable de nutriments végétaux. / AFRIKAANSE OPSOMMING: Die gebruik van beesmis as 'n organiese kunsmis in Asië en Afrika is' n eeu-oue landbou praktyk. Dit verklaar die hernude belangstelling, deels vanweë die finansiële onvermoë van meeste boere om landbouchemikalieë aan te koop, maar ook as gevolg van die toenemende behoefte vir groener tegnologie wat nie nadelig is vir grond gesondheid, waterkwaliteit, biodiversiteit en wat volhoubaarheid of selfs verhoogde voedselproduksie bevorder. In hierdie konteks het baie vindingryke boere hul eie nuwe tegnologie, gebaseer op die gebruik van plaaslik verkrygde hulpbronne, ontwikkel. `n Voorbeeld hiervan is Sanjeevak ('n mengsel van beesmis, beesurine, water en die handvol melasse), wat belowende bevorderende effekte op gewas produktiwiteit en grond mikroflora getoon het. Tot dusver was daar egter baie min wetenskaplike werk gedoen om die effek daarvan as 'n organiese produkte vir grond wysigings te evalueer. Hierdie studie was verdeel in vier belangrike doelwitte. (i) Om die bemestingswaarde, menslike gesondheid en ekologiese risiko-profiele van Sanjeevak te evalueer; (ii) Om Sanjeevak vir fitohormone inhoud en vlakke met behulp van 'n kolorimetriese metode afgelei van dié van Salkowski en vloeistofchromatografie – massaspektrometrie (LC-MS) te besigtig; (iii) Glashuis en veld waarneming reaksies met betrekking tot groei parameters en opbrengs van verskeie gewasse na die toediening van Sanjeevak by verskillende tempos; (iv) Laastens, om die effek van die Sanjeevak voorbehandeling op saad ontkieming en voortplanting te toets in vergelyking met die metodes en tegnieke wat gereeld gebruik word. Sanjeevak is geassesseer vir die mikro-en makro voedingstowwe inhoud. Die analise het die teenwoordigheid van mikrovoedingstowwe soos Mg, Na, Ca en Zn by wisselende konsentrasies, asook fosfor (P) (0.007%) en kalium (K) (0.063%), getoon. Sanjeevak inhoud van totale stikstof (TN) (0.11%), en die totale organiese koolstof (TOC) (0.71%) was egter baie laag, wat daarop dui dat dit slegs 'n lewensvatbare bron van voedingstowwe is indien dit by hoër en konsekwente tempos toegedien word of alternatiewelik wanneer formulering daarvan verbeter word. Sanjeevak was ook ontleed vir die mikrobiologiese eienskappe en die vlakke van swaar metale in vergelyking met die streng wetgewing wat die gebruik en toediening van afvalwater slyk op landbougrond in Suid-Afrika reguleer. Die bevindinge het getoon dat swaar metale en fekalieë kolivorm vlakke hier gemeet, aan die drumpel vereistes voldoen vir die toediening as ‘n grondverbeteringsmiddel. Studies wat die opsporing en die konsentrasie van fitohormone in Sanjeevak ondersoek is uitgevoer. In die gebruik van die Salkowski kolorimetrise metode om die ouksiene op te spoor en te kwantifiseer uit Sanjeevak en sy mengsel (beesurine en mis), het die resultate die teenwoordigheid van indol-3-asynsuur (IAA) by wisselende konsentrasies wat wissel van 20 tot 140 ppm in beesurine, beesmis en Sanjeevak getoon. Evaluering van die bogenoemde monsters vir IAA met behulp van LC-MSanalise bevestig vroeër bevindings. Verdere ontleding van hierdie resultate beklemtoon sterk die invloed van Sanjeevak mikrobiota in fitohormone produksie. Proewe is uitgevoer in die glashuis en die veld. In die glashuis eksperimente, is het die verskeie toedieningstempo van Sanjeevak herhalend die wortelbevorderende effekte bevestig op gewasse soos tamaties, komkommer en wingerdstok en dit het opbrengs van koring verhoog, onafhanklik van die voedingstowwe wat dit bevat. Statistiese analise van die resultate verkry onder veldtoestande, het getoon dat daar geen verskil tussen die behandelings (kontrole, kompos, Sanjeevak en kompos + Sanjeevak) was nie, gegee dat dieselfde gewas getoets was. Ten slotte, laboratorium-eksperimente op Sanjeevak as voor-behandeling om die beworteling te verbeter van die wingerdstok (Ramsey) onderstok steggies, het baie belowende resultate getoon in vergelyking met naftaleen asynsuur (NAA) voorbehandeling en die kontrole. Dit beklemtoon die feit dat Sanjeevak 'n alternatief kan wees en wat verdere studie verdien, hoofsaaklik as gevolg van sy lae-koste en omgewingsvriendelike prosedures. Die gebruik van Sanjeevak as 'n organiese grondverbeteringsmiddel vir gewasproduksie en voortplanting kan beskou word as' n goedkoper alternatief tot effektiewe mikro-organisme (EM) tegnologie wat uit plaaslike en natuurlike hulpbronne saamgestel is. Soos waargeneem in die studie, kan dit die beste gebruik word in kombinasie met 'n betroubare bron van plantvoedingstowwe. Dus, moet die gebruik daarvan vir die produksie van gewasse en voortplanting aangemoedig word.

As micorrizas arbusculares (MAs) são associações simbióticas mutualísticas entre fungos da ordem Glomales e as raízes da maioria das plantas terrestres. Os mecanismos moleculares que controlam o desenvolvimento e a eficiência da simbiose são pouco conhecidos, dificultando sua aplicação agrícola em larga escala. No entanto, a análise sistemática e em larga escala de genes expressos em raízes colonizadas por fungos micorrízicos arbusculares (FMAs) em diferentes condições ambientais poderia contribuir de forma significativa para o conhecimento da biologia dessa simbiose. O objetivo deste trabalho foi identificar genes com expressão induzida ou suprimida em raízes de cana-de-açúcar colonizadas por Glomus clarum em condições de baixo e alto fósforo (P) no substrato de cultivo, durante o desenvolvimento da simbiose. Usando hibridização subtrativa supressiva (\"Suppresive Subtractive Hybridization, SSH) foram encontrados 74 genes com expressão diferencial significativa em raízes micorrizadas de 12 semanas após inoculação (SAI). Por outra parte, um total de 386 genes foram arranjados em membranas de nylon e sua expressão diferencial avaliada por sondas sintetizadas a partir de cDNA extraído em 4 SAI e 12 SAI. A hibridização em macroarranjos mostrou na presença da micorriza acúmulo diferencial de transcritos de genes codificando proteínas putativas envolvidas no metabolismo de jasmonato (COI1) e de etileno (ET) em raízes com 4 SAI, e ácido salicílico (AS) em raízes com 12 SAI. Dos genes que apresentaram acúmulo de transcritos regulados pela micorrização foram escolhidos 12 genes: que codificam cisteina protease (meabolismo de proteínas); remorina (dinâmica celular); dehidroascorbato redutase, e proteína rica em hidroxiprolina (resposta ao estresse); duas proteína associadas a senecência; a horcolina (função não definida); COI1 (metabolismo de jasmonato); catalase, germina, glutationa (estresse oxidativo); quitinase (resposta de defesa), para analise de acúmulo de transcritos por qRT-PCR em 4, 6, 8, 10 e 12 SAI. Os resultados indicam que em etapas precoces do desenvolvimento das MAs são ativados genes que estão relacionados com a resposta ao estresse oxidativo e com metabolismo de fitohormônios, em etapas tardias esses genes mostraram diminução no acúmulo de transcritos. A análise dos padrões de expressão dos genes envolvidos no estabelecimento da simbiose poderia contribuir para elucidar os mecanismos genéticos que controlam a simbiose e poderiam facilitar a aplicação agrícola em larga escala dos FMAs / Arbuscular mycorrhizae (AM) are mutualistic symbiotic associations between fungi of the order Glomales and most of the terrestrial plants. The molecular mechanisms regulating AM development and efficiency are not well understood so that agricultural large scale application is so difficult. Although so many approaches are being used to the identification of differentially expressed genes in AMs, large scale analysis it must contributed to the knowledge of symbiosis molecular biology. The aim of this work was identify genes induced or suppressed in sugarcane roots colonized for G. clarum in low and high phosphorus (P) conditions, during symbiosis development. Using suppressive subtractive hybridization (SSH) was possible to detect 74 genes with differential significant expression in 12 weeks mycorrhized roots. In other hand a total of 386 genes were spotted into nylon membranes and differential expression was evaluated with probes of cDNA of 4 and 12 weeks. Macroarray hybridization shows increase of genes transcripts involved in jasmonate metabolism (COI1) and ethylene metabolism (ET) 4 weeks after inoculation, and in salycilic acid 12 weeks after inoculation. Twelve genes differentially expressed in mycorrhizal presences were choosen to analyse the expression with qRT-PCR in 4, 6, 8, 10 and 12 weeks after inoculation. A cistein protease (protein metabolism); a remorin (cellular dynamic), dehydroascorbate reductase, hidroxiproline rich protein (estress response); two proteins associated to senescence; a horcolin (unknown function); COI1 (jasmonate metabolism); catalase, germin, glutathione (oxidative estress); chitinase (defence response) were tested. The results showed that genes relationated with oxidative stress are activated in early stages of MAs development, and, in late stages these phytohormones metabolism related genes are suppressed. Patterns expression of genes involved in MAs development it must contributed to elucidate the genetics mechanisms controlling the symbiosis and it must facilitate large scale use of FMAs.

As micorrizas arbusculares (MAs) são associações simbióticas mutualísticas entre fungos da ordem Glomales e as raízes da maioria das plantas terrestres. Os mecanismos moleculares que controlam o desenvolvimento e a eficiência da simbiose são pouco conhecidos, dificultando sua aplicação agrícola em larga escala. No entanto, a análise sistemática e em larga escala de genes expressos em raízes colonizadas por fungos micorrízicos arbusculares (FMAs) em diferentes condições ambientais poderia contribuir de forma significativa para o conhecimento da biologia dessa simbiose. O objetivo deste trabalho foi identificar genes com expressão induzida ou suprimida em raízes de cana-de-açúcar colonizadas por Glomus clarum em condições de baixo e alto fósforo (P) no substrato de cultivo, durante o desenvolvimento da simbiose. Usando hibridização subtrativa supressiva (\"Suppresive Subtractive Hybridization, SSH) foram encontrados 74 genes com expressão diferencial significativa em raízes micorrizadas de 12 semanas após inoculação (SAI). Por outra parte, um total de 386 genes foram arranjados em membranas de nylon e sua expressão diferencial avaliada por sondas sintetizadas a partir de cDNA extraído em 4 SAI e 12 SAI. A hibridização em macroarranjos mostrou na presença da micorriza acúmulo diferencial de transcritos de genes codificando proteínas putativas envolvidas no metabolismo de jasmonato (COI1) e de etileno (ET) em raízes com 4 SAI, e ácido salicílico (AS) em raízes com 12 SAI. Dos genes que apresentaram acúmulo de transcritos regulados pela micorrização foram escolhidos 12 genes: que codificam cisteina protease (meabolismo de proteínas); remorina (dinâmica celular); dehidroascorbato redutase, e proteína rica em hidroxiprolina (resposta ao estresse); duas proteína associadas a senecência; a horcolina (função não definida); COI1 (metabolismo de jasmonato); catalase, germina, glutationa (estresse oxidativo); quitinase (resposta de defesa), para analise de acúmulo de transcritos por qRT-PCR em 4, 6, 8, 10 e 12 SAI. Os resultados indicam que em etapas precoces do desenvolvimento das MAs são ativados genes que estão relacionados com a resposta ao estresse oxidativo e com metabolismo de fitohormônios, em etapas tardias esses genes mostraram diminução no acúmulo de transcritos. A análise dos padrões de expressão dos genes envolvidos no estabelecimento da simbiose poderia contribuir para elucidar os mecanismos genéticos que controlam a simbiose e poderiam facilitar a aplicação agrícola em larga escala dos FMAs / Arbuscular mycorrhizae (AM) are mutualistic symbiotic associations between fungi of the order Glomales and most of the terrestrial plants. The molecular mechanisms regulating AM development and efficiency are not well understood so that agricultural large scale application is so difficult. Although so many approaches are being used to the identification of differentially expressed genes in AMs, large scale analysis it must contributed to the knowledge of symbiosis molecular biology. The aim of this work was identify genes induced or suppressed in sugarcane roots colonized for G. clarum in low and high phosphorus (P) conditions, during symbiosis development. Using suppressive subtractive hybridization (SSH) was possible to detect 74 genes with differential significant expression in 12 weeks mycorrhized roots. In other hand a total of 386 genes were spotted into nylon membranes and differential expression was evaluated with probes of cDNA of 4 and 12 weeks. Macroarray hybridization shows increase of genes transcripts involved in jasmonate metabolism (COI1) and ethylene metabolism (ET) 4 weeks after inoculation, and in salycilic acid 12 weeks after inoculation. Twelve genes differentially expressed in mycorrhizal presences were choosen to analyse the expression with qRT-PCR in 4, 6, 8, 10 and 12 weeks after inoculation. A cistein protease (protein metabolism); a remorin (cellular dynamic), dehydroascorbate reductase, hidroxiproline rich protein (estress response); two proteins associated to senescence; a horcolin (unknown function); COI1 (jasmonate metabolism); catalase, germin, glutathione (oxidative estress); chitinase (defence response) were tested. The results showed that genes relationated with oxidative stress are activated in early stages of MAs development, and, in late stages these phytohormones metabolism related genes are suppressed. Patterns expression of genes involved in MAs development it must contributed to elucidate the genetics mechanisms controlling the symbiosis and it must facilitate large scale use of FMAs.

Este é o primeiro relato da presença de bactérias diazotróficas dos gêneros Achromobacter e Zoogloea como endófitas de plantas de trigo, que foram identificadas pela análise das seqüências do DNA ribossomal 16S. Com o objetivo de estudar a localização dos isolados: IAC-AT-8- Azospirillum brasiliense, IAC-HT-11- Achromobacter insolitus, IAC-HT-12- Zoogloea ramigera nas plantas de trigo, foi realizado um experimento em condições axênicas. Após 15 dias da inoculação, as plantas foram observadas em microscópio eletrônico e visualizou-se a presença das bactérias externamente nas raízes e internamente no córtex, na região de alongamento. Para estudar possíveis benefícios propiciados por estes isolados à cultura do trigo em condições de campo, foram instalados dois experimentos no ano de 2002, com dois genótipos de trigo (ITD-19 e IAC-370) e três doses de N. As análises foram realizadas no estádio de quatro folhas e de perfilhamento. A inoculação promoveu maior massa de matéria seca e N acumulado e aumentou a produtividade de grãos, principalmente na presença de adubo nitrogenado adicional, porém, revertendo em lucro para o agricultor. As respostas variaram em relação ao local de cultivo. Em 2003, foi realizado outro experimento muito semelhante aos anteriores, entretanto, as análises foram realizadas no estádio perfilhamento e de maturidade fisiológica. A inoculação promoveu benefícios à cultura do trigo independentemente do genótipo utilizado, ou seja, aumento do N acumulado no grão, com a inoculação do isolado IAC-AT-8, e da produtividade de grãos na maior dose de N, com o isolado IACHT- 11. Os benefícios propiciados às plantas pela inoculação de bactérias diazotróficas podem estar relacionados à capacidade de síntese de fitormônios. Por isso instalou-se um experimento no ano de 2005 com o objetivo de relacionar a capacidade de síntese de indóis com os possíveis benefícios à cultura do trigo em condições de campo. Os isolados diferiram quanto a quantidade de indóis que sintetizaram, entretanto, houve benefícios para a cultura, principalmente com a adição de adubo nitrogenado, demonstrando que outros mecanismos podem ser responsáveis pelas respostas positivas. Assim, foi realizado um experimento em casa de vegetação para estudar a influência da inoculação no metabolismo do nitrogênio das plantas e avaliar a colonização em substrato não esterilizado. Os isolados foram marcados com resistência a antibióticos. Após 40 dias da inoculação todos os isolados estavam presentes no interior das raízes, entretanto, não foram detectados no solo. O isolado IAC-HT-11, influenciou o metabolismo do nitrogênio das plantas, propiciando maior atividade das enzimas redutase do nitrato e sintetase da glutamina, e maior teor de clorofila, resultando no aumento da eficiência de utilização do N. Apesar das respostas terem sido variáveis, em todos os experimentos realizados foram obtidas respostas positivas à inoculação do isolado IAC-HT-11 de Achromobacter insolitus, na presença de adubo nitrogenado adicionado. A inoculação é uma prática não onerosa, portanto, viável economicamente, aumentando a produtividade de grãos e gerando lucro para o agricultor. / This study reports for the first time the endophytic nature of Achromobacter and Zoogloea in wheat plants, that were identified by 16S rDNA gene sequence analyses. To find out the localization of the bacterial cells (IAC-AT-8- Azospirillum brasiliense, IACHT- 11- Achromobacter insolitus, IAC-HT-12- Zoogloea ramigera) in/on wheat plants, an experiment was set up under axenic conditions. Fifteen days after inoculation, the plants were observed under electron microscope. All the strains could be visualized on the external part of the root and in the inner part at the elongation zone, in the cortex cells. Two field experiments were carried out in year of 2002, with the aim of evaluating the benefits from inoculation of these strains in wheat plants, with two wheat genotypes (ITD-19 and IAC-370) and three levels of nitrogen fertilizer. The plants were evaluated at four leaves and at tillering stage. Inoculated plants presented increases in dry matter, N accumulation and grain yield, especially in combination with N fertilizer, generating financial profits for the farmer. The responses to inoculation varied according to the location. A field experiment was carried out, in year of 2003, with the same experimental set up used at the previous year. However, the plants were evaluated at tillering stage and at maturity stage. Grain yield and the main yield components were evaluated at harvest. Independent of the cultivar or the nitrogen dose, plants inoculated with the strain IAC-AT-8 presented increases in grain N accumulation and, at the highest nitrogen dose, the strain IAC-HT-11 increased grain yield. The plants? benefits due to the inoculation of endophytic diazotrophic bacteria may also result from their indole synthesis, a field experiment was carried out to find out which had been the main mechanisms for improvement of plant growth and yield, in 2005. The employed strains produced variable amounts of indoles, however, the inoculation brought benefits to wheat plants, mainly in the presence of added N, demonstrated that others mecanisms could be acting for the positive responses to inoculation. In this way, a greenhouse experiment was carried out with the aim of evaluating the influence of inoculation on nitrogen metabolism in wheat plants, and the colonization in unsterilized substratum. The strains were selectively market with antibiotic resistence to. Forty days after inoculation, all three strains had been established in the interior of the roots, however, they could not be detected in the soil. The strain IAC-HT-11 influenced the N metabolism of the plants, increased the nitrate reductase and glutamine synthetase activities, and the chlorophyll content, causing higher efficiency in N utilization. In spite of the great variability of the inoculation response, IAC-HT-11, Achromobacter insolitus, generated the greatest yield increase when combined with nitrogen fertilizer. Inoculation of diazotrophic bacteria is an inexpensive technique, that is economically sound, promoting the increase in grain yield and generating financial profits for the farmer.

Orientador: Fábio Silveira Nogueira / Resumo: O sistema radicular (SR) é importante pela ancoragem e obtenção de água e nutrientes. Em eudicotiledôneas, como Arabidopsis, o crescimento da raiz primária (RP) é afetado por fitormônios, especialmente pelo balanço entre auxina que controla a divisão celular, e citocinina que modula a diferenciação celular; também, os microRNAs (miRNAs), um sub-conjunto de pequenos RNAs que regulam pós-transcricionalmente seus alvos, regulam o crescimento da RP. O microRNA156 (miR156) e seus alvos, membros da família SQUAMOSA Promoter-Binding Protein-Like (SPL), constituem uma via genética que regula vários processos do desenvolvimento, incluindo desenvolvimento da raíz; porém, durante o crescimento da RP, não foi observado o efeito da via miR156/SPL, e da interação com auxina e citocinina; assim, foi avaliada essa interação durante o crescimento da PR regulado pelo tamanho do meristema da raiz (TMR) em Arabidopsis. Usando ferramentas genéticas e moleculares foi analizada a expressão de genes MIR156 e SPLs, o comprimento da RP, o TMR, as taxas de divisão celular, e as respostas de auxina e citocinina durante o crescimento da RP. Os genes MIR156 e SPLs possuem padrões de expressão opostos. Níveis altos do miR156 (nas plântulas p35S :: MIR156A), leva a menor comprimento da RP, TMR reduzido, menores taxas de divisão celular, respostas mais baixas e altas à auxina e citocinina respectivamente; em contraste, níveis severamente reduzidos do miR156 maduro disponível (nas plantas MIM156) conducem a e... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Light and phytohormones, such as cytokinins, have been known to play a pivotal role in numerous physiological processes in plant cells. Previous work in our laboratory has revealed the light- and cytokinin- modulated changes both in the levels of specific tRNA species and their modified nucleotide contents, in addition to the characterization of specific tRNAs and tRNA genes from higher plants. The plant hormone cytokinin, which is of particular interest to us has been implicated to be involved in processes such as induction of cell division, plastid biogenesis and delay of senescence. Ongoing work in our laboratory also points towards the role of Ca2+ as a second messenger in cytokinin mediated gene expression. With the objective of isolation of specific tRNA genes which could then be used as probes to study the light- and phytohormone- induced changes in the levels of respective functional mature tRNAs, a previously isolated clone containing a 6.6kb insert that hybridized with 3 end labeled cucumber total cellular tRNA was sequenced by the dideoxy chain termination method. Sequence analysis of the 6.6 kb DNA fragment has revealed a chloroplast genome DNA fragment containing the trnNGUU and trnRACG genes in addition to the genes coding for the ribosomal RNAs 4.5S, 5S and 23S as well as the protein coding genes ccsA (cytochrome c-synthesis) and ndhD(NADH plastoquinone oxidoreductase).These genes were found to be arranged in the order-23S-4.5S-5S-trnRACG-trnNGUU-ccsA-ndhD. This shows a divergence from the gene organization in the completely sequenced chloroplast genomes of other higher plant species such as tobacco, maize, rice and Arabidopsis, especially with regard to the absence of a highly conserved trnLUAG gene that has been shown to be present in the trnNGUU-ndhD intergenic region. The cucumber chloroplast trnNGUU and trnRACG genes have shown very high homology (>90%) whereas ccsA and ndhD show 50-61% similarity to corresponding genes from chloroplast genomes of other plant species. The relative levels of tRNAArg and tRNAAsn were determined by Northern analysis using the tRNA gene probes, in etiolated excised cucumber cotyledons treated with light or phytohormones, such as cytokinin (BA) and auxin (2,4-D). Light and phytohormones were found to significantly increase the levels of tRNAArg unlike in the case tRNAAsn where no significant changes in the levels were observed. This result points towards the regulation of relative levels of specific tRNA species by light and cytokinin so as to match the codon usage of the mRNA population during light- and cytokinin- induced plant development in cucumber. Northern analyses were also performed to monitor the relative transcript levels of the plastid encoded ccsA and ndhD in etiolated excised cucumber cotyledons treated with light or phytohormones. ccsA transcript levels were found to be significantly reduced in auxin treated cucumber cotyledons where as exogenous application of cytokinin to either dark-grown or light exposed cotyledons did not seem to have any pronounced effect. ndhD transcripts were found to be up-regulated by cytokinin treatment or light exposure in comparison to un-treated controls probably indicating a point of overlap in the light/ cytokinin mediated signal transduction pathways. Auxin treatment on the other hand was found to down-regulate ndhD transcript levels also. Recent studies from our laboratory have demonstrated the involvement of a calcium-dependent protein kinase(CDPK) in the cytokinin-signal transduction pathway associated with the induction of pathogenesis-related proteins (chitinase and β 1-3 modulation of nuclear-encoded CDPK transcripts in response to light and exogenously added phytohormones such as cytokinins and auxin. Towards this end, partial CDPK cDNAs were generated from Cucumis Sativus by RT-PCR using degenerate primers designed based on the conserved regions of the known CDPK proteins available in the database, cloned in pGEM-T and sequenced. Sequence analysis of twenty partial cDNA clones revealed the presence of at least four CDPK isoforms in Cucumis sativus (CuCDPK 1-4). Of the four partial CDPK cDNAs, the tissue-specific expression level of CuCDPK3 was studied using the highly sensitive Taqman Analysis (Quantitative RT-PCR). The results obtained indicate that, in excised dark-grown cucumber cotyledons light and cytokinin were found to up-regulate the levels of CuCDPK3 unlike auxin, which was found to have no significant effect. In cucumber hypocotyls, which had the highest levels of CuCDPK3, light was found to have a down-regulatory effect whereas cytokinin and auxin did not bring about any significant changes in the levels of CuCDPK3. In cucumber root tissue, both light and cytokinin were found to have a down-regulatory effect on the levels of CuCDPK3, unlike auxin. The southern analysis of cucumber genomic DNA revealed a CDPK multi-gene family in cucumber. Since cytokinins have been known to play a role in both etioplast and chloroplast biogenesis and since various groups have recently reported the presence of higher plant homologues of bacterial cell-division protein FtsZ and the requirement of plant nuclear-encoded FtsZs for plastid division, efforts were also made to isolate and to study the expression of cucumber FtsZ in dark-grown cucumber cotyledon tissue treated exogenously with light/phytohormones. Towards this end, a partial FtsZ cDNA was generated from cucumber by RT-PCR using degenerate primers designed based on conserved regions of known plant FtsZ proteins. Results of the Taqman Analysis indicate that cytokinin, unlike auxin, mimics the action of light by increasing the levels of CuFtsZ transcripts in dark-grown cotyledon tissue suggesting the involvement of FtsZ in cytokinin-induced plastid-biogenesis.

La diatomée d'eau douce A. formosa peut produire des lipides neutres en plus ou moins grandes quantités en fonction des conditions de culture. Ainsi, nous avons montré par exemple qu'une carence en silice stimule la production de triacylglycérols (TAGs) mais génère une diminution de la biomasse. En revanche, nous avons montré que l'addition de bicarbonate et de phytohormones augmente à la fois la biomasse et la production de TAGs. L'ajout de phytohormones dans les milieux de culture de cette diatomée résulte en une augmentation de l'activité d'enzymes dans les extraits et notamment celles du cycle de Benson-Calvin. Parmi ces enzymes, la GAPDH est une enzyme dont l'activité augmente significativement. Nous avons montré que chez A. formosa, cette enzyme forme un complexe ternaire avec la CP12 et la Férrédoxine NADP Réductase (FNR) et non pas avec la CP12 et la phosphoribulokinase comme chez la plupart des organismes photosynthétiques. La régulation de cette enzyme en est de fait modifiée. La phytohormone, 24-épibrassinolide conduit à une augmentation d'activité de la GAPDH qui résulte de la dissociation du complexe GAPDH-CP12 et la GAPDH n'est plus redox régulée. La GAPDH chez les diatomées est donc régulée par des interactions protéineprotéine. / A. formosa, a freshwater diatom, can produce different amounts of neutral lipids such as triacylglycerols (TAGs) under different growth conditions. We showed that as it is well-known for diatoms, starvation for silica increased the production of TAGs but decreased biomass. However, the addition of bicarbonate or phytohormones into the growth medium increased both biomass and TAGs. Addition of phytohormones increased the activities of enzymes in particular those of the Benson-Calvin cycle. Among the target enzymes of the Benson-Calvin cycle, GAPDH was strongly affected. We purified this enzyme and demonstrated that, in the diatom A. formosa, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the Calvin cycle, forms a complex with the small chloroplast protein CP12 and Ferredoxin NADP Reductase (FNR), which is involved in the photochemical phase of photosynthesis. In cells treated with the phytohormone, 24-epibrassinolide, GAPDH was "free", not redox-regulated and not associated anymore with CP12. Therefore GAPDH from this diatom is regulated by protein-protein interaction but the GAPDH/CP12/FNR complex replaces the one formed between GAPDH, CP12 and phosphoribulokinase found in most photoautotrophs.

Este é o primeiro relato da presença de bactérias diazotróficas dos gêneros Achromobacter e Zoogloea como endófitas de plantas de trigo, que foram identificadas pela análise das seqüências do DNA ribossomal 16S. Com o objetivo de estudar a localização dos isolados: IAC-AT-8- Azospirillum brasiliense, IAC-HT-11- Achromobacter insolitus, IAC-HT-12- Zoogloea ramigera nas plantas de trigo, foi realizado um experimento em condições axênicas. Após 15 dias da inoculação, as plantas foram observadas em microscópio eletrônico e visualizou-se a presença das bactérias externamente nas raízes e internamente no córtex, na região de alongamento. Para estudar possíveis benefícios propiciados por estes isolados à cultura do trigo em condições de campo, foram instalados dois experimentos no ano de 2002, com dois genótipos de trigo (ITD-19 e IAC-370) e três doses de N. As análises foram realizadas no estádio de quatro folhas e de perfilhamento. A inoculação promoveu maior massa de matéria seca e N acumulado e aumentou a produtividade de grãos, principalmente na presença de adubo nitrogenado adicional, porém, revertendo em lucro para o agricultor. As respostas variaram em relação ao local de cultivo. Em 2003, foi realizado outro experimento muito semelhante aos anteriores, entretanto, as análises foram realizadas no estádio perfilhamento e de maturidade fisiológica. A inoculação promoveu benefícios à cultura do trigo independentemente do genótipo utilizado, ou seja, aumento do N acumulado no grão, com a inoculação do isolado IAC-AT-8, e da produtividade de grãos na maior dose de N, com o isolado IACHT- 11. Os benefícios propiciados às plantas pela inoculação de bactérias diazotróficas podem estar relacionados à capacidade de síntese de fitormônios. Por isso instalou-se um experimento no ano de 2005 com o objetivo de relacionar a capacidade de síntese de indóis com os possíveis benefícios à cultura do trigo em condições de campo. Os isolados diferiram quanto a quantidade de indóis que sintetizaram, entretanto, houve benefícios para a cultura, principalmente com a adição de adubo nitrogenado, demonstrando que outros mecanismos podem ser responsáveis pelas respostas positivas. Assim, foi realizado um experimento em casa de vegetação para estudar a influência da inoculação no metabolismo do nitrogênio das plantas e avaliar a colonização em substrato não esterilizado. Os isolados foram marcados com resistência a antibióticos. Após 40 dias da inoculação todos os isolados estavam presentes no interior das raízes, entretanto, não foram detectados no solo. O isolado IAC-HT-11, influenciou o metabolismo do nitrogênio das plantas, propiciando maior atividade das enzimas redutase do nitrato e sintetase da glutamina, e maior teor de clorofila, resultando no aumento da eficiência de utilização do N. Apesar das respostas terem sido variáveis, em todos os experimentos realizados foram obtidas respostas positivas à inoculação do isolado IAC-HT-11 de Achromobacter insolitus, na presença de adubo nitrogenado adicionado. A inoculação é uma prática não onerosa, portanto, viável economicamente, aumentando a produtividade de grãos e gerando lucro para o agricultor. / This study reports for the first time the endophytic nature of Achromobacter and Zoogloea in wheat plants, that were identified by 16S rDNA gene sequence analyses. To find out the localization of the bacterial cells (IAC-AT-8- Azospirillum brasiliense, IACHT- 11- Achromobacter insolitus, IAC-HT-12- Zoogloea ramigera) in/on wheat plants, an experiment was set up under axenic conditions. Fifteen days after inoculation, the plants were observed under electron microscope. All the strains could be visualized on the external part of the root and in the inner part at the elongation zone, in the cortex cells. Two field experiments were carried out in year of 2002, with the aim of evaluating the benefits from inoculation of these strains in wheat plants, with two wheat genotypes (ITD-19 and IAC-370) and three levels of nitrogen fertilizer. The plants were evaluated at four leaves and at tillering stage. Inoculated plants presented increases in dry matter, N accumulation and grain yield, especially in combination with N fertilizer, generating financial profits for the farmer. The responses to inoculation varied according to the location. A field experiment was carried out, in year of 2003, with the same experimental set up used at the previous year. However, the plants were evaluated at tillering stage and at maturity stage. Grain yield and the main yield components were evaluated at harvest. Independent of the cultivar or the nitrogen dose, plants inoculated with the strain IAC-AT-8 presented increases in grain N accumulation and, at the highest nitrogen dose, the strain IAC-HT-11 increased grain yield. The plants? benefits due to the inoculation of endophytic diazotrophic bacteria may also result from their indole synthesis, a field experiment was carried out to find out which had been the main mechanisms for improvement of plant growth and yield, in 2005. The employed strains produced variable amounts of indoles, however, the inoculation brought benefits to wheat plants, mainly in the presence of added N, demonstrated that others mecanisms could be acting for the positive responses to inoculation. In this way, a greenhouse experiment was carried out with the aim of evaluating the influence of inoculation on nitrogen metabolism in wheat plants, and the colonization in unsterilized substratum. The strains were selectively market with antibiotic resistence to. Forty days after inoculation, all three strains had been established in the interior of the roots, however, they could not be detected in the soil. The strain IAC-HT-11 influenced the N metabolism of the plants, increased the nitrate reductase and glutamine synthetase activities, and the chlorophyll content, causing higher efficiency in N utilization. In spite of the great variability of the inoculation response, IAC-HT-11, Achromobacter insolitus, generated the greatest yield increase when combined with nitrogen fertilizer. Inoculation of diazotrophic bacteria is an inexpensive technique, that is economically sound, promoting the increase in grain yield and generating financial profits for the farmer.