Orientador: Ione Salgado / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T06:41:46Z (GMT). No. of bitstreams: 1 SantosFilho_PlinioRodriguesdos_D.pdf: 1637587 bytes, checksum: 89bdaa896ffa24aac0e2f5b00b8ece3a (MD5) Previous issue date: 2012 / Resumo: A nitrato redutase (NR) corresponde ao primeiro passo na assimilação do nitrato em plantas. Recentemente, essa enzima tem sido também relacionada à síntese de óxido nítrico (NO). Entre várias ações sinalizadoras para as plantas, o NO promove o acúmulo de fenilpropanóides pela ativação da expressão de enzimas iniciais dessa via. Contudo, uma correlação entre metabolismo de nitrogênio, emissão de NO e acúmulo de fenilpropanóides não foi estabelecida. Por isso, neste trabalho foi analisado o efeito do suprimento de nitrato e da deficiência na NR sobre a síntese de aminoácidos, a emissão de NO e o metabolismo de fenilpropanóides em diferentes tecidos de Arabidopis thaliana selvagem e mutante duplo deficiente para a NR (nia1 nia2). Análises cromatográficas mostraram que a mutante é deficiente na síntese de sinapoil malato (SM), fenilpropanóide predominante nas folhas, resultando no acúmulo de seu precursor sinapoil glicose (SG) e derivados de kaempferol. Essa deficiência não foi causada pela baixa assimilação do nitrato, já que a recuperação do conteúdo de aminoácidos na mutante não alterou seu perfil metabólico. Porém, a maior disponibilidade de nitrato aumentou a atividade da NR, a emissão de NO e os níveis de SM e diminuiu os níveis de SG, nos dois genótipos. O cultivo in vitro da mutante na presença de malato afetou a produção de SM de maneira dose-dependente, enquanto substâncias doadoras de NO causaram apenas um pequeno aumento em SG. Porém, a combinação malato/doador de NO promoveu a recuperação de SM ao nível da selvagem. Esse efeito sinergístico do NO com o malato também ocorreu quando as folhas da mutante foram infiltradas com esses compostos. Além disso, a atividade da enzima sinapoil glicose:malato sinapoil transferase (SMT) foi menor na mutante e a adição de NO aumentou a síntese de SM. Ainda, as folhas da mutante foram incapazes de acumular antocianinas sinapoiladas ao nível da selvagem quando submetidas a um estresse luminoso. Nos botões florais apenas derivados de kaempeferol e quercetina foram identificados e não houve diferença entre selvagem e mutante. Nas raízes não foram identificados fenilpropanóides, provavelmente porque esses compostos só são acumulados nesse órgão na presença de luz. Em relação ao acúmulo de aminoácidos, as folhas do mutante apresentaram níveis reduzidos de todos os aminoácidos parecendo atuar como fonte desses compostos para os botões florais, que não apresentaram nenhuma diferença em relação à selvagem. A glutamina recuperou os níveis de aminoácidos nas folhas, mas não causou diferença nos botões florais. Nas raízes, não houve diferença no conteúdo de aminoácidos entre selvagem e mutante, quando cultivadas no solo, mas in vitro, a mutante foi deficiente, provavelmente pela limitação de nutrientes nessa condição. Esses resultados indicam que o metabolismo dos ésteres de ácido sinápico nas folhas, controlado por aciltransferases dependentes de sinapoil glicose, está comprometido no mutante nia1 nia2 e sugere um potencial papel sinalizador para o NO na ativação dessas aciltransferases. Ainda, o efeito da deficiência na NR nos níveis de aminoácidos parece alterar as relações de fonte e dreno na planta e a folha foi o órgão mais afetado / Abstract: The nitrate reductase (NR) is the first step in nitrate assimilation in plants. Recently, this enzyme has also been related to the synthesis of nitric oxide (NO). Among various signaling actions for plants, NO promotes the accumulation of phenylpropanoids by activating the expression of the initial enzymes of this pathway. However, a correlation between nitrogen metabolism, NO emission and accumulation of phenylpropanoids has not been established. Therefore, this work analyzed the effect of nitrate supply and NR deficiency on the synthesis of amino acids, emission of NO and phenylpropanoid metabolism in different tissues of wild type and NR double-deficient (nia1 nia2) Arabidopsis thaliana plants. Chromatographic analysis showed that the mutant is deficient in the synthesis of sinapoylmalate (SM), the major phenylpropanoid in the leaves, resulting in accumulation of its precursor sinapoylglucose (SG) and kaempferol derivatives. This deficiency was not caused by the low nitrate assimilation, since the recovery of the amino acid content in the mutant did not change its metabolic profile. In contrast, an increased supply of nitrate enhanced NR activity and NO production, and increased SM and decreased SG levels in both genotypes. The in vitro cultivation of mutant in the presence of malate affected the production of SM in a dose-dependent manner, whereas NO donors caused only a slight increase in SG. However, the combination of malate/NO donor promoted the recovery of SM at the level of wild type plants. The synergistic effect of NO with malate in the recovery of SM also occurred when the mutant leaves were infiltrated with these compounds. Furthermore, sinapoylglucose:malate sinapoyltransferase (SMT) activity was reduced in the mutant, and the addition of NO increased SM synthesis. Additionally, mutant leaves were unable to accumulate sinapoylated anthocyanins at the level of wild type when exposed to light stress. In the flower buds just kaempeferol and quercetin derivatives were identified and there was no difference between wild type and mutant. In the roots, phenylpropanoids were not identified, probably because these compounds are accumulated in this organ only in the presence of light. Regarding the accumulation of amino acids, the mutant leaves showed reduced levels of all amino acids and appeared to act as a source of these compounds to the flower buds that showed no difference from the wild plant. Glutamine recovered the amino acid levels in leaves, but caused no difference in flower buds. In the roots, there was no difference in the amino acid content between wild type and mutant, when grown in soil, but in vitro, the mutant was deficient, probably due to nutrient limitation in this condition. These results indicate that hydroxycinnamate ester metabolism in leaves, controlled by the sinapoylglucose-dependent sinapoyltransferases, is compromised in nia1 nia2 mutant and suggests a potential signaling role for NO in the activation of these acyltransferases. Additionally, the effect of NR deficiency in the levels of amino acids appears to alter the relationship of source and sink in the plant and the leaf is the most affected organ / Doutorado / Bioquimica / Mestre em Biologia Funcional e Molecular

Introduction Yearly, 90 million people are infected with C. trachomatis. Even though it is easily treated with antibiotics the often-asymptomatic infection often spreads prior to detection. A vaccine is therefore of great interest. A chimeric major outer membrane protein (MOMP) of C. trachomatis has in earlier studies proved to contain the epitopes necessary for immunization. In this thesis the chimeric MOMP gene was cloned and expressed in E. coli. Furthermore, the expression of the protein was analyzed in previously transformed A. thaliana. Materials and Methods The chimeric MOMP gene was cloned into E.coli. Following vector amplification, the gene was expressed and the protein purified by affinity chromatography.  Seeds from different lines of previously transformed A. thaliana were screened by PCR. Hits were then analyzed by western blot.  Results The results show successful cloning and expression of the chimeric MOMP gene in E. coli. The following protein purification did result in purified protein, however in low concentration. For the A.thaliana lines, the presence and correct orientation of the gene was verified in some of the lines screened. The B7 line was verified to express the protein. Discussion The low concentration of purified protein in E.coli was probably due to un-optimized imunnoprecipitation conditions. In expression analysis of A. thaliana, purification of plant samples by immunoprecipitation prior to running western blot gave results, whereas running un-purified samples in urea buffer did not, probably due to interfering proteins in wild type plants.

Le travail présenté dans cette thèse consiste en la création et l'étude des propriétés d'un nouveau modèle computationnel de l'horloge circadienne végétale et de certains processus physiologiques qui en dépendent.L'horloge circadienne est un rythme endogène d'une période d'environ 24 heures que possèdent la plupart des êtres vivants. Il est généré au niveau moléculaire par des boucles de rétroaction transcriptionnelles, traductionnelles et/ou post-traductionnelles. L'horloge permet aux organismes de s'a- dapter à leur environnement. L'horloge des plantes se distingue par un nombre important de composants (gènes et protéines) dont la majorité sont régulés par la lumière.Dans un premier temps, un nouveau modèle computationnel qui combine une structure complexe et hautement interconnectée avec un nombre réduit d'équations et de paramètres est construit. Ce modèle reproduit correctement les profils d'expression des gènes de l'horloge du type sauvage ainsi que les altérations provoquées par une perte de fonction de chacun de ces gènes. Plusieurs extensions modélisant des processus physiologiques dépendant de l'horloge, à savoir la croissance de l'hypocotyle et la régulation de la floraison, sont également testées.Ensuite, la réponse particulièrement complexe de l'horloge végétale à la lumière est explorée en détail afin de déterminer l'utilité de multiples récepteurs lumineux. Pour ce faire, l'entraînement de l'oscillateur par des cycles jour-nuit de durée différente de 24 heures est mesuré et les différents comportements observés (entraînement périodique, quasipériodicité, chaos) sont caractérisés. Les simulations suggèrent que les multiples senseurs lumineux permettent d'allier une grande flexibilité et une résistance aux effets des fluctuations rapides de luminosité, améliorant ainsi l'adaptation des plantes à l'environnement.Enfin, plusieurs hypothèses permettant de rendre compte des différences observées entre l'horloge des racines et celle des feuilles sont explorées, et différents mécanismes de synchronisation entre ces deux oscillateurs sont testés. / The circadian clock is an endogenous timekeeper with a period of around 24 hours, found in most living beings, which helps organisms adapt to their environment by anticipating daily and seasonal variations. It originates at the molecular level, from transcriptional-translational feedback loops between a small number of genes.In this thesis, a computational model of the plant circadian oscillator is built based on current knowledge of the underlying genetic network. This network is highly complex and interconnected, but the new model needs only a small number of equations and parameters to accurately predict the expression profiles of the main clock genes in various light conditions, as well as the defects associated with a loss of function in those genes. Clock-regulated processes such as hypocotyl growth and flowering are also reproduced with good accuracy. One of the particularities of the plant clock is a large number of light-sensitive components. A study of the role of those multiple light sensors on the entrainment properties of the clock is presented. It uses the newly built model to subject the clock to a very large range of conditions and generate theoretical light-insensitive mutants. The combination of an intricate oscillator and a multiplicity of light sensors makes the plant clock highly flexible, able to adapt to a wide range of conditions but resistant to the disrupting effects of random fluctuations.Preliminary steps towards a more realistic depiction of the plant clock as multiple interacting oscillators are taken. These include modeling a heterogeneous population by changing parameter values, modifying the model to account for known differences between the clocks of the roots and shoots, and testing possible synchronizing mechanisms between those two organs. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Les aquaporines sont des canaux hydriques qui contrôlent la perméabilité à l'eau des membranes cellulaires, au cours du développement ou en réponse à des stress. La dynamique de l'expression des aquaporines de plantes et leur rôle physiologique ont été examinés dans deux organes modèles, le pollen et la racine d'Arabidopsis. Le pollen mature contient une cellule végétative et deux cellules de sperme. Des analyses transcriptomiques ont récemment identifié AtTIP1;3 et AtTIP5;1 comme deux aquaporines spécifiques du pollen. Dans ce travail, des protéines reportrices fluorescentes ont permis d'établir que AtTIP1;3 et AtTIP5;1 s'expriment spécifiquement dans la membrane vacuolaire de, respectivement, la cellule végétative et les cellules de sperme. Ces études révèlent aussi la grande plasticité dynamique des vacuoles, de la maturation du pollen jusqu'à la fécondation. Des approches de génétique inverse suggèrent un rôle des deux aquaporines dans la reproduction de la plante. La seconde partie de ce travail concerne les effets concomitants des stress oxydants, inhibant la conductivité hydraulique des racines et provoquant une accumulation intracellulaire des aquaporines initialement sur les membranes plasmiques. Le dernier processus a été disséqué par des approches de biochimie, pharmacologie et microscopie. La co-expression avec des marqueurs des endomembranes a révélé que l'isoforme AtPIP2;1 subit une accumulation dans les endosomes tardifs en réponse à l'H2O2. Ce processus peut être bloqué par l'auxine synthétique 1-NAA, mais non par l'inhibiteur d'endocytose tyrphostine A23. La grande stabilité des aquaporines internalisées suggère que l'H2O2 déclenche un mécanisme de séquestration réversible de celles-ci. Au-delà de données originales sur la régulation cellulaire des aquaporines, ce travail apporte un éclairage nouveau sur la dynamique des membranes intracellulaires des plantes, au cours du développement ou en réponse à des stress / Aquaporins are membrane water channel proteins that mediate the fine-tuning of cell membrane water permeability during development or in response to environmental stresses. The dynamic expression of aquaporins in planta, as well as their role in plant water relations, were investigated in two representative model organs, the pollen and roots of Arabidopsis. Mature pollen consists of a vegetative cell and two sperm cells. Transcriptomics recently identified AtTIP1;3 and AtTIP5;1 as two pollen exclusive aquaporins. Here, we investigated their in vivo temporal and spatial expression pattern. Fluorescently-tagged chimeras revealed that AtTIP1;3 and AtTIP5;1 have a distinct and specific localisation in the vacuolar membrane of the vegetative and sperm cells, respectively. The two aquaporins also revealed the dynamic plasticity of vacuoles from pollen maturation to embryo fecundation. Loss of function approaches suggest an implication of both proteins in plant reproduction. The second part of this work focused on the oxidative stress-induced internalisation of root plasma membrane aquaporins and its concomitant drop in root hydraulic conductivity. The former process was described in great detail by combined biochemical, pharmacological and microscopic approaches. Co-expression analyses of the AtPIP2;1 isoform with endomembrane markers revealed that H2O2 triggers AtPIP2;1 accumulation in late endosomal compartments. This process could be antagonized by the auxin analog 1-NAA, but not by the endocytosis blocker tyrphostin A23. Life-time analyses established the high stability of the internalised protein suggesting that H2O2 triggers a mechanism for intracellular and reversible sequestration of plasma membrane aquaporins. Besides information on cell regulation of aquaporins, the overall work gives novel and complementary insights into the dynamic remodelling of plant internal membranes during development and stress responses.

Understanding the strategies that plant populations implement to increase evolutionary responsiveness to better survive environmental changes induced by climate change is a critical challenge for ecology and evolutionary studies. This dissertation investigates the role of hybridization, local adaptation, and phenotypic plasticity in plant population responses to environmental change. Specifically, I utilized meta-analysis techniques to investigate the prevalence of local adaptation and phenotypic plasticity as the two main mechanisms used to adapt to heterogeneous environments, and experimentally explored the genetic pathway of plasticity in phenology traits such as bolting time in Arabidopsis thaliana under high temperatures. Furthermore, A. thaliana was used to create artificial hybrids to test if novel trait combinations allow hybrids to outperform their parental source in novel and stressful environments. In the second chapter, I included reciprocal transplant plant studies and found that local adaptation is more common than adaptive plasticity as an evolutionary response to environmental heterogeneity. Although local adaptation was more common, plastic responses have been reported as a mechanism to tolerate increases in global temperature; however, the underlying genetic and developmental mechanisms are only starting to be elucidated. To address this, the third chapter determined whether alternative splicing of the ambient temperature flowering pathway gene FLOWERING LOCUS-M (FLM), and expression of SHORT VEGETATIVE PHASE (SVP), can explain flowering time plasticity in ecotypes of A. thaliana under 18°C and 26°C. Although the expression of SVP and FLM-β tracks reaction norms, I failed to find evidence that alternative FLM splicing plays a role in phenotypic plasticity in intraspecific flowering time variation. Intraspecific hybridization (admixture) disrupts divergent genetic architectures between populations to generate phenotypic novelty and raw material for environmental selection to act upon. In order to understand the effect of this disruption to local adaptation of A. thaliana ecotypes separated along geographic and locally adaptive genetic distances, the fourth chapter used experimentally created F1-hybrids between geographically distant ecotypes, and used single nucleotide polymorphism (SNP) data to estimate (putatively neutral) background and adaptive genetic distances. My results suggest that disruption of locally adaptive genomic loci decreases the performance of offspring between distantly related parents, but that crosses between very closely related parents also reduce performance, suggesting that during admixture selection may have to balance the consequences of disrupting local adaption while also avoiding inbreeding depression. Lastly, I examined the effect of recombination events under limiting and novel growing conditions (i.e. drought, high temperatures, and freezing field over-wintering conditions) in A. thaliana F2-hybrids. I provide empirical data for the effect of limiting growing environment on phenology, growth, and fitness traits on the admixed and parental ecotypes. I found that recombination events generate novel phenotypes. Generally, offspring phenotypic variation increases and shifts from the parental ecotype phenotypes, and in some cases, offspring display transgressive segregation, heterosis, or outbreeding depression. This work provides a novel contribution towards understanding mechanisms that plant implement to deal with rapid environmental changes. Specifically, plastic responses and hybridization events may interplay to maintain and increase genotypic diversity.

Les sphingolipides et plus particulièrement les bases à longue chaîne (LCBs) jouent un rôle crucial dans l'induction de la mort cellulaire programmée. Chez les végétaux, la fumonisine B1 (FB1), une mycotoxine produite par le champignon nécrotrophe Fusarium moniliforme, perturbe la voie de biosynthèse des sphingolipides ce qui conduit à l'accumulation des deux LCBs majoritaires chez Arabidopsis thaliana, à savoir la phytosphingosine (PHS) et la dihydrosphingosine (DHS). Néanmoins, la voie de signalisation induite par la FB1 demeure largement inconnue à ce jour. En utilisant A. thaliana, l'équipe a récemment montré qu'en réponse aux LCBs ou à la FB1, les protéines 14-3-3 sont phosphorylées par la protéine kinase dépendante du calcium CPK3, à laquelle elles sont associées de manière constitutive. Cette phosphorylation conduit à la dissociation du complexe CPK3/14-3-3s et au clivage de la protéine kinase qui a été identifiée comme étant un régulateur majeur de cette voie de signalisation. L'objectif de mon travail de thèse s'est inscrit dans la continuité de ces travaux et a consisté, de manière générale, à une meilleure compréhension des processus impliquant la protéine kinase CPK3, notamment sa régulation par les 14-3-3s et son devenir après la dissociation du complexe en réponse aux LCBs. A travers mes travaux de thèse, j'ai pu montrer que CPK3 interagit préférentiellement avec les isoformes de 14-3-3s appartenant au groupe non-epsilon de manière phospho- et calcium-dépendante en condition contrôle. Suite à sa perte d'interaction avec les 14-3-3s, j'ai montré que le domaine variable N-terminal de CPK3 est clivé de manière LCB-dépendante. Ce clivage est à corréler avec l'activation, dépendante de la PHS et de la FB1, de la protéase à cystéine de type papaïne, RD21 (responsive-to-dessication 21). De manière intéressante, alors que la forme pleine longueur de CPK3 est principalement associée aux membranes en condition contrôle, la forme clivée de cette protéine kinase est retrouvée exclusivement au niveau de la fraction soluble. Une approche génétique associée à des analyses phénotypiques indique que RD21 agit en tant que régulateur négatif de la PCD induite par la FB1 chez A. thaliana. / The sphingolipids and more particularly the long chain bases (LCBs) play a crucial role in the induction of programmed cell death. In plants, the mycotoxin fumonisin B1 (FB1) produced by the necrotrophic fungus Fusarium moniliforme disrupts sphingolipid biosynthesis, leading to the accumulation of the two major LCBs in Arabidopsis thaliana, i.e. phytosphingosine (PHS) and dihydrosphingosine (DHS). However, the FB1-induced signaling pathway remains largely unknown. By using A. thaliana as a plant model, the team has recently shown that, upon LCB or FB1 treatment, 14-3-3 proteins are phosphorylated by the calcium-dependent protein kinase, CPK3, with which 14-3-3s are constitutively associated. This phosphorylation event leads to the dissociation of the CPK3/14-3-3 complex and to CPK3 cleavage, which was identified as a crucial regulator of this signaling pathway. In this context, the objectives of my thesis were to get a deep further in the knowledge of this signaling pathway involving the protein kinase CPK3, including its regulation by 14-3-3s and its becoming after complex dissociation in response to LCBs. Thus, I have shown that CPK3 preferentially binds to the non-epsilon 14-3-3 isoforms in a phospho- and calcium-dependent manner in control condition. After the CPK3/14-3-3 complex dissociation, I have demonstrated that the N-terminal variable domain of CPK3 is cleaved in a LCB-dependent manner. This cleavage can be correlated with the PHS/FB1-induced activation of the papain-like cysteine protease, RD21 (responsive-to-dessication 21). Interestingly, while full-length CPK3 is mainly associated to membranes in control condition, its FB1-induced cleaved form becomes soluble. A genetic approach associated to phenotype analyses indicates that RD21 acts as a negative regulator of FB1-induced cell death in A. thaliana.

M.Sc. / Plants produce volatile organic compounds in response to certain elicitors and environments. These compounds have a variety of functions, including the attraction of insects for pollination and seed dispersal, responses to both abiotic and biotic stresses and the priming or sensitizing of neighbouring plants for subsequent attack. The majority of the volatile blend is made up of terpenoid compounds and these compounds are formed through the action of an important class of enzymes termed Terpene Synthases. Lipopolysaccharides form part of the cell surface of Gram-negative bacteria and they are classed as “pathogen-associated molecular pattern molecules” and are thought to induce defence responses in plants by influencing different metabolic pathways that could ultimately result in the production of defence volatiles. LPS from Burkholderia cepacia that has been reported to induce the oxidative burst, the nitric oxide burst and changes in cytosolic calcium concentrations, was used in this study. In order to analyse the volatiles, Single-Drop Microextraction and Solid-Phase Microextraction were used as static headspace sampling techniques that allow the preconcentration of volatile analytes prior to analysis. Both these techniques are fast, simple and equilibrium based and both allow for minimal sample size and preparation. Luminometry was performed in order to test the efficacy of LPS and to determine if LPS is able to induce the oxidative burst in Arabidopsis thaliana. Histochemical staining of transgenic plants containing the PR1:GUS and PDF:GUS reporter gene constructs was performed in order to determine which signalling pathway LPS follows, either the jasmonic acid pathway or the salicylic acid pathway. SPME was then used to extract samples from both time and concentration studies. The time studies involved incubation times of 0 h, 2 h, 4 h and 6 h and 0 d, 1 d, 2 d and 3 d respectively, while the concentration studies involved using LPS concentrations of 0, 20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml and 100 μg/ml. SPME was also used for the comparision of two A. thaliana ecotypes (Columbia and C24) as well as two A. thaliana knock-out lines (At5g44630 – multi-product sesquiterpene synthase and At5g23960 – (E)-β-caryophyllene synthase), and finally it was used for the sampling of A. thaliana leaf tissue. SDME was used to compare two solvents, namely octane and toluene and these results were compared to the SPME results. GC-MS was used only for the identification of volatiles with both SPME and SDME. Finally, GC-MS was used with SPME to identify volatiles that are produced by leaf tissue after priming.

La caractérisation de la réponse au sel d'un mutant d'A.thaliana déficient dans la synthèse de la proline (p5cs1-4) a montré que la salinité affecte de la même manière les relations hydriques et l'homéostasie ionique chez les deux génotypes. La pulvérisation foliaire de proline sous contrainte saline améliore la capacité antioxydante chez le mutant p5cs1-4 et rétablit sa croissance ainsi que son activité photosynthétique. L'analyse des relations entre la production précoce de H2O2 par la NADPH oxydase et la défense antioxydante chez A. thaliana soumise à la salinité a montré que l'exposition des plantes sauvages à 200 mM NaCl pendant 24 h conduit à une accumulation transitoire de H2O2, suivie d'une augmentation des activités des enzymes Catalases, Ascorbate peroxydases et Glutathione reductases. En présence de sel, le prétraitement des plantes par le DMTU, un chélateur de H2O2, ou le DPI et l'imidazole, deux inhibiteurs des NADPH oxydases, conduit à de faibles activités des enzymes antioxydantes. Le double mutant affiche également des activités faibles de ses enzymes antioxydantes. La meilleure performance des plantes sauvages par rapport aux mutants se traduisant par une meilleure aptitude de protéger ces tissus contre le stress oxydatif. En outre, l'accumulation de la proline est précédée par des niveaux élevés de H2O2 et l'utilisation de DMTU supprime l'accumulation de la proline chez les plantes soumises aux contraintes osmotiques. Les résultats ont montré également que de DPI, ainsi que des mutants knock-out conduisent à une inhibition de l'activité de l'enzyme-clé de la synthèse de la proline. / Characterization of salt stress response in A. thaliana p5cs1-4 mutant defective in proline biosynthesis showed that no significant difference was observed in the leaf water status and Na+/K+ ratio between salt-treated WT and p5cs1-4 seedlings, suggesting that the salt hypersensitivity of the mutant was not due to the disruption of water uptake or Na+/K+ homeostasis. Foliar application of proline under salt stress increased the antioxidant activity in the p5cs1-4 mutant and restored its photosynthetic activity. The analysis of the relationship between the early production of H2O2 by the NADPH oxidase and the antioxidant defense in A. thaliana subjected to salinity showed that short-term salt exposure led to a transient and significant increase of H2O2 concentration, followed by a marked increase in Catalase, Ascorbate peroxidase and Glutathion reductase activities, pre-treatment with either dimethylthiourea, a chemical trap for H2O2, or two NADPH oxidase inhibitors such as imidazol and diphenylene iodonium, significantly decreased the above-mentioned enzyme activities under salinity. atrbohd/f double mutant plants failed to induce the antioxidant response under the culture conditions. The better performance of the WT was related to the plant ability to deal with the salt-induced oxidative stress as compared to atrbohd/f. In addition NaCl or mannitol stress resulted in a transient increase in H2O2 content followed by an accumulation of proline upon stress. In contrast DMTU and DPI were found to significantly inhibit proline accumulation. Expression level of the key enzyme involved in the biosynthesis of proline was observed to be diminished by DPI and in atrboh mutants.

Magister Scientiae - MSc / Stress responsive genes play a key role in shaping the manner in which plants process and respond to environmental stress. Their gene products are linked to DNA transcription and its consequent translation into a response product. However, whilst these genes play a significant role in manufacturing responses to stressful stimuli, transcription factors coordinate access to these genes, specifically by accessing a gene's promoter region which houses transcription factor binding sites. Here transcriptional elements play a key role in mediating responses to environmental stress where each transcription factor binding site may constitute a potential response to a stress signal. Arabidopsis thaliana, a model organism, can be used to identify the mechanism of how transcription factors shape a plant's survival in a stressful environment. Whilst there are numerous plant stress research groups, globally there is a shortage of publicly available stress responsive gene databases. In addition a number of previous databases such as the Generation Challenge Programme's comparative plant stressresponsive gene catalogue, Stresslink and DRASTIC have become defunct whilst others have stagnated. There is currently a single Arabidopsis thaliana stress response database called STIFDB which was launched in 2008 and only covers abiotic stresses as handled by major abiotic stress responsive transcription factor families. Its data was sourced from microarray expression databases, contains numerous omissions as well as numerous erroneous entries and has not been updated since its inception.The Dragon Arabidopsis Stress Transcription Factor database (DASTF) was developed in response to the current lack of stress response gene resources. A total of 2333 entries were downloaded from SWISSPROT, manually curated and imported into DASTF. The entries represent 424 transcription factor families. Each entry has a corresponding SWISSPROT, ENTREZ GENBANK and TAIR accession number. The 5' untranslated regions (UTR) of 417 families were scanned against TRANSFAC's binding site catalogue to identify binding sites. The relational database consists of two tables, namely a transcription factor table and a transcription factor family table called DASTF_TF and TF_Family respectively. Using a two-tier client-server architecture, a webserver was built with PHP, APACHE and MYSQL and the data was loaded into these tables with a PYTHON script. The DASTF database contains 60 entries which correspond to biotic stress and 167 correspond to abiotic stress while 2106 respond to biotic and/or abiotic stress. Users can search the database using text, family, chromosome and stress type search options. Online tools have been integrated into the DASTF, database, such as HMMER, CLUSTALW, BLAST and HYDROCALCULATOR. User's can upload sequences to identify which transcription factor family their sequences belong to by using HMMER. The website can be accessed at http://apps.sanbi.ac.za/dastf/ and two updates per year are envisaged. / South Africa

La paroi primaire végétale est une enveloppe dynamique impliquée dans le développement ainsi que la réponse aux contraintes environnementales. Elle est composée de réseaux de polysaccharides et de protéines dans lesquels interviennent des protéines multi-domaines de type LRX et des protéines à domaine PAC. Ce travail de thèse a consisté à rechercher la fonction de ces protéines dans les parois. Des analyses protéomiques réalisées sur des extraits de protéines pariétales de racines de plantes sauvages ou mutantes lrx1 d'A. thaliana ont permis d'identifier 424/434 protéines pariétales de plantes sauvages/lrx1 respectivement et 25 protéines candidates pouvant jouer un rôle dans la morphogenèse des poils absorbants. Par ailleurs, des protéines à domaine PAC ont été identifiées dans toutes les plantes terrestres étudiées. L'apparition des protéines à domaine PAC a pu être associée à la terrestrialisation. Une analyse phylogénique a permis de grouper les domaines PAC en 10 clades, chacun comportant un domaine PAC d'Amborella trichopoda. Outre les 6 résidus Cys caractérisant le domaine PAC, des motifs conservés ont été repérés dans les clades, ouvrant la voie pour des études fonctionnelles. Des tests in vitro ont montré que les domaines PAC interagissent avec différents types de polysaccharides pariétaux et permis de définir trois types de spécificité vis-à-vis de polysaccharides tels que les ß(1,4) galactanes/RGI, les mannanes, les xyloglucanes et/ou la cellulose. Un nouveau modèle d'interactions supramoléculaires dans les parois végétales faisant intervenir des protéines à domaine PAC et des polysaccharides pariétaux a été proposé / The plant primary cell wall is a dynamic envelope involved in development and in response to environmental constraints. It is composed of networks of polysaccharides and proteins to which multi-domain proteins like LRX (Leucine-Rich repeat Extensin) and PAC (Proline-rich Arabinogalactan Protein Cys-containing) domain proteins contribute. This work aimed at finding partners of such proteins in cell walls using different experimental approaches. Proteomics analyses have been performed on proteins extracted from cell walls of roots of wild type or lrx1 plants. They have allowed the identification of 424/434 cell wall proteins of wild type/lrx1 roots respectively as well as of 25 candidate proteins which could play a role in root hair morphogenesis. Besides, PAC domain proteins have been identified in all the studied terrestrial plants using a bioinformatic approach. The appearance of PAC domain proteins could be associated to terrestrialisation. A phylogenic analysis has allowed to group PAC domains in 10 clades, each of them containing a PAC domain of Amborella trichopoda, an ancestor of angiosperms. In addition to the 6 Cys residues which define the PAC domain, conserved motifs have been identified in each clade. This finding opens the way to functional studies. In vitro tests have shown that the PAC domains could interact with different kinds of cell wall polysaccharides. Three types of specificity could be defined towards ß(1,4) galactans/RGI, mannans, xyloglucans and/or cellulose. A new model of molecular interactions in plant cell walls including PAC domain proteins and polysaccharides has been proposed