Thesis (M. Sc.)--York University, 2001. Graduate Programme in Geography. / Typescript. Includes bibliographical references (leaves 87-102). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pMQ71607.

Thesis (Ph.D.)--Washington State University. / Includes bibliographical references.

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In the natural environment plants are confronted to a multitude of biotic and abiotic stress factors that must be perceived, transduced, integrated and signaled in order to achieve a successful acclimation that will secure survival and reproduction. Plants have to deal with excess excitation energy (EEE) when the amount of absorbed light energy is exceeding that needed for photosynthetic CO2 assimilation. EEE results in ROS formation and can be enhanced in low light intensities by changes in other environmental factors. The lesions simulating disease resistance (lsd1) mutant of Arabidopsis spontaneously initiates spreading lesions paralleled by ROS production in long day photoperiod and after application of salicylic acid (SA) and SA-analogues that trigger systemic acquired resistance (SAR). Moreover, the mutant fails to limit the boundaries of hypersensitive cell death (HR) after avirulent pathogen infection giving rise to the runaway cell death (rcd) phenotype. This ROS-dependent phenotype pointed towards a putative involvement of the ROS produced during photosynthesis in the initiation and spreading of the lesions. We report here that the rcd has a ROS-concentration dependent phenotype and that the light-triggered rcd is depending on the redox-state of the PQ pool in the chloroplast. Moreover, the lower stomatal conductance and catalase activity in the mutant suggested LSD1 was required for optimal gas exchange and ROS scavenging during EEE. Through this regulation, LSD1 can influence the effectiveness of photorespiration in dissipating EEE. Moreover, low and high SA levels are strictly correlated to lower and higher foliar H2O2 content, respectively. This implies an essential role of SA in regulating the redox homeostasis of the cell and suggests that SA could trigger rcd in lsd1 by inducing H2O2 production. LSD1 has been postulated to be a negative regulator of cell death acting as a ROS rheostat. Above a certain threshold, the pro-death pathway would operate leading to PCD. Our data suggest that LSD1 may be subjected to a turnover, enhanced in an oxidizing milieu and slowed down in a reducing environment that could reflect this ROS rheostat property. Finally, the two protein disulphide isomerase boxes (CGHC) present in the protein and the down regulation of the NADPH thioredoxin reductase (NTR) in the mutant connect the rcd to a putative impairment in the reduction of the cytosolic thioredoxin system. We propose that LSD1 suppresses the cell death processes through its control of the oxidation-reduction state of the TRX pool. An integrated model considers the role of LSD1 in both light acclimatory processes and in restricting pathogen-induced cell death.

<p>In the natural environment plants are confronted to a multitude of biotic and abiotic stress factors that must be perceived, transduced, integrated and signaled in order to achieve a successful acclimation that will secure survival and reproduction. Plants have to deal with excess excitation energy (EEE) when the amount of absorbed light energy is exceeding that needed for photosynthetic CO2 assimilation. EEE results in ROS formation and can be enhanced in low light intensities by changes in other environmental factors.</p><p>The lesions simulating disease resistance (lsd1) mutant of Arabidopsis spontaneously initiates spreading lesions paralleled by ROS production in long day photoperiod and after application of salicylic acid (SA) and SA-analogues that trigger systemic acquired resistance (SAR). Moreover, the mutant fails to limit the boundaries of hypersensitive cell death (HR) after avirulent pathogen infection giving rise to the runaway cell death (rcd) phenotype. This ROS-dependent phenotype pointed towards a putative involvement of the ROS produced during photosynthesis in the initiation and spreading of the lesions.</p><p>We report here that the rcd has a ROS-concentration dependent phenotype and that the light-triggered rcd is depending on the redox-state of the PQ pool in the chloroplast. Moreover, the lower stomatal conductance and catalase activity in the mutant suggested LSD1 was required for optimal gas exchange and ROS scavenging during EEE. Through this regulation, LSD1 can influence the effectiveness of photorespiration in dissipating EEE. Moreover, low and high SA levels are strictly correlated to lower and higher foliar H2O2 content, respectively. This implies an essential role of SA in regulating the redox homeostasis of the cell and suggests that SA could trigger rcd in lsd1 by inducing H2O2 production.</p><p>LSD1 has been postulated to be a negative regulator of cell death acting as a ROS rheostat. Above a certain threshold, the pro-death pathway would operate leading to PCD. Our data suggest that LSD1 may be subjected to a turnover, enhanced in an oxidizing milieu and slowed down in a reducing environment that could reflect this ROS rheostat property. Finally, the two protein disulphide isomerase boxes (CGHC) present in the protein and the down regulation of the NADPH thioredoxin reductase (NTR) in the mutant connect the rcd to a putative impairment in the reduction of the cytosolic thioredoxin system. We propose that LSD1 suppresses the cell death processes through its control of the oxidation-reduction state of the TRX pool. An integrated model considers the role of LSD1 in both light acclimatory processes and in restricting pathogen-induced cell death.</p>

En conditions de stress biotiques et abiotiques la production de plusieurs espèces réactives de l’oxygène (ERO) dans différents compartiments spécialisés de la cellule végétale est inévitable. L’oxygène singulet (1O2) a été identifié comme la principale ERO produite dans le chloroplaste au cours d’un stress lumineux. Cette ERO est très réactive et a une durée de vie courte d’environ 3 s dans les tissus biologiques, ce qui amène à penser que l’oxygène singulet agit principalement par cytotoxicité. Cependant, il a été récemment établi que l’oxygène singulet fonctionne aussi comme une molécule signal impliquée dans la signalisation rétrograde chloroplaste-noyau conduisant soit à la mort cellulaire programmée, soit à l’acclimatation. En raison des propriétés particulières de l’oxygène singulet, il est peu probable que cette ERO voyage en dehors du chloroplaste pour induire des changements d’expression de gènes nucléaires. Une possibilité est que l’oxygène singulet agisse via des médiateurs. Nous avons identifié un produit d’oxydation du β-carotène, le dihydroactinidiolide (dhA), comme intermédiaire dans la voie de signalisation de l’oxygène singulet, qui agit d’une manière similaire à un autre produit d’oxydation du β-carotène, le β-cyclocitral, précédemment identifié comme intermédiaire dans la voie de signalisation de l’oxygène singulet. Nous avons aussi mis en évidence le rôle dans la voie de signalisation régulée par le β-cyclocitral de la protéine MBS1 (METHYLENE BLUE SENSITIVITY 1), et montré que la mort cellulaire programmée induite par l’oxygène singulet chez l’Arabidopsis est controllée par une serine-threonine kinase, OXI1 (OXIDATIVE SIGNAL INDUCIBLE 1). / During biotic and abiotic stress conditions, the production of several reactive oxygen species (ROS) at different specialized compartments of the cell is inevitable. Singlet oxygen (1O2) was identified to be the predominant ROS produced in the chloroplast during high light stress. This molecule is highly reactive, with a short life time of about 3 µs in biological tissues. Such properties make believe that the predominant effect of 1O2 in plants is cytotoxicity. However, 1O2 has been identified to function as a chloroplast-to-nucleus retrograde signaling molecule, leading to acclimation or programmed cell death (PCD). Cognizant of the properties of 1O2, it is most unlikely to travel directly from the chloroplast to the nucleus to signal changes in nuclear gene expression. One possibility is that 1O2 carries out this signaling function with the help of mediators. We identify a β-carotene oxidation product, dihydroactinidiolide (dhA) as a 1O2 signaling intermediate, which function similarly to the β-carotene oxidation product β-cyclocitral, previously identified to be a mediator of 1O2 plastid-nuclear retrograde signaling in Arabidopsis. We reveal a dependence of the β-cyclocitral-mediated signaling pathway on the MBS1 (METHYLENE BLUE SENSITIVITY 1) protein, and show that Programmed cell death induced by 1O2 is mediated by the serine-threonine kinase, OXI1(OXIDATIVE SIGNAL INDUCIBLE 1).

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O objetivo do presente trabalho foi caracterizar aspectos fisiolÃgicos e bioquÃmicos que mostrem se plantas de arroz (Oryza sativa) deficientes da enzima citosÃlica peroxidase do ascorbato (cAPX) sÃo mais suscetÃveis ao estresse oxidativo do que as plantas com cAPX. A APX Ã uma importante enzima do metabolismo oxidativo de plantas, atuando na regulaÃÃo dos nÃveis endÃgenos do perÃxido de hidrogÃnio (H2O2). Para isso, plantas deficientes em cAPX (Apx1/2s) e plantas nÃo transformadas (WT) foram utilizadas para a experimentaÃÃo. As plantas foram silenciadas pela tÃcnica do RNA de interferÃncia (iRNA) e cultivadas por 35 dias em vasos de 1.5 L contendo soluÃÃo nutritiva sob condiÃÃes de casa de vegetaÃÃo. O experimento I foi realizado com segmentos de folhas imersos em metil-viologÃnio (MV) 50 &#61549;M durante 24h, e o experimento II foi realizado pela aplicaÃÃo dos seguintes estresses abiÃticos: salinidade, alta luminosidade e MV. Os resultados do experimento I mostraram que as plantas Apx1/2s possuem um nÃvel basal de H2O2 maior do que os nÃveis encontrados nas plantas WT, sugerindo que as plantas Apx1/2s apresentam um nÃvel de H2O2 prÃximo de um nÃvel de sinalizaÃÃo celular. O fato das plantas WT terem acumulado H2O2 apÃs 1h de tratamento sugere a necessidade de um nÃvel sinalizador de H2O2 para ativaÃÃo dos sistemas de defesa. As plantas Apx1/2s ao contrario das WT apresentaram uma queda constante no conteÃdo de H2O2, indicando uma provÃvel remoÃÃo do excesso de H2O2. ApÃs 3h de tratamento as enzimas SOD, APX e PHGPx de cloroplasto apresentaram atividade superior nas plantas Apx1/2s, tanto no controle quanto no estresse, comparadas com as plantas WT. Esses resultados sugerem existÃncia de um sistema antioxidante bastante ativado nas plantas Apx1/2s. No experimento II as plantas Apx1/2s nÃo apresentaram diferenÃas nos parÃmetros fotoquÃmicos quando comparadas com as plantas WT, mesmo possuindo uma menor fotossÃntese em condiÃÃes controle. A dissipaÃÃo do excesso de energia (NPQ) nas plantas Apx1/2s tratadas com luz foi, em mÃdia, maior que das plantas WT, indicando uma possÃvel maior eficiÃncia na dissipaÃÃo de energia. Mesmo com eficiente dissipaÃÃo de energia ambas as plantas nÃo conseguiram evitar energia excessiva no fotossistema e acabaram sofrendo fotoinibiÃÃo e danos no aparato fotossintÃtico (Fv/Fm). Em relaÃÃo Ãs plantas WT, as Apx1/2s apresentaram maior atividade das enzimas antioxidativas SOD, CAT e PHGPx nas condiÃÃes controle, na provÃvel tentativa de compensar a ausÃncia da cAPX. No tratamento com MV a isoforma cloroplÃstica da PHGPx foi estimulada em mais de 100% nas plantas Apx1/2s, indicando que essas plantas podem reparar danos oxidativos com mais rapidez que as WT. Os dados sugerem que as plantas Apx1/2s, apesar da ausÃncia da cAPX, ativam sistemas adicionais de proteÃÃo antioxidativa para compensar essa ausÃncia e responder mais rÃpida e eficientemente a situaÃÃes de estresse. / The aim of this study was to characterize physiological and biochemical aspects that show if rice plants (Oryza sativa) knockdown on cytosolic ascorbate peroxidase enzyme (cAPX) are more susceptible to oxidative stress than the wild type plants. APX is an important enzyme from oxidative metabolism of plants, acting on regulation of the endogenous levels of hydrogen peroxide (H2O2). For this, rice plants knockdown on cAPX (Apx1/2s) and wild type (WT) were used for the experimentation. The plants were silenced by interference RNA technical (iRNA) and were grown for 35 days into 1.5 L pots containing nutritive solution under greenhouse conditions. The experiment I was performed with leaves segments immersed in methyl viologen (MV) 50 &#956;M for 24h and the experiment II was performed by application of the following treatments: salt stress, high light and MV. The results from experiment I shown Apx1/2s plants have a higher level of H2O2 high than the levels found on WT rice plants, suggesting that Apx1/2s plants present a level of H2O2 near a level of cell signaling. The fact of WT plants had accumulated H2O2 1h after the treatment suggest the necessity of a signaling H2O2 level for stimulate defense systems. Apx1/2s plants unlike of WT plants presented a constant decline on H2O2 content, indicating a likely H2O2 scavenging excess. After 3h of treatment the chloroplastic enzymes SOD, APX and PHGPx presented upper active in Apx1/2s plants, in control and stress, compared with WT plants. These results suggest the existence of an antioxidant system quite active in the Apx1/2s plants. In experiment II the Apx1/2s plants presented no differences in the photochemical parameters when compared with WT plants, even possessing a smaller photosynthesis under controlled conditions. The energy dissipation (NPQ) in the Apx1/2s plants under high light was, in average, higher than WT plants, suggesting better energy dissipation. Even with efficient energy dissipation, the plants could not avoid the excess of energy in the photosystem and they suffered photoinhibition and damage in photosynthetic apparatus (Fv/Fm). In relation the WT plants, Apx1/2s plants presented a higher activity of antioxidant enzymes SOD, CAT and PHGPx under controlled conditions, probably intending compensate the lack of cAPX. In the MV treatment the chloroplastic PHGPx was stimulated above 100% in the Apx1/2s plants, indicating that these plants can repair oxidative damage faster than the WT plants. The results suggest that Apx1/2s plants, despite the absence of cAPX, activated additional security systems to compensate the lack of cAPX and respond quickly and efficiently to stress situations.

L’oxygène singulet ($^1O_2$) est la principale espèce réactive de l’oxygène produite dans le chloroplaste lors d'un stress photo-oxydant. L’$^1O_2$ est hautement cytotoxique s'attaquant aux protéines et lipides membranaires. L’$^1O_2$ est une molécule signal impliquée dans une signalisation rétrograde entre les chloroplastes et le noyau via des médiateurs, menant à la mort cellulaire programmée (MCP), ou à l’acclimatation. Nous avons caractérisé l’implication de la phytohormone salicylate, agissant en aval de la signalisation de la MCP dépendante d’OXI1 (OXIDATIVE SIGNAL INDUCIBLE 1) et du jasmonate. Nous avons mis en évidence une voie de régulation négative faisant intervenir des protéines inhibitrices de la MCP : DAD1 et DAD2. La sur-expression de ces protéines permet à la plante d’éviter de la MCP en inhibant la signalisation contrôlée par OXI1. Dans le Réticulum Endoplasmique (RE) a lieu la conformation d’une grande partie des protéines. Une perturbation dans ce compartiment induit l’activation d’une réponse adaptative appelée l’UPR (Unfolded Protein Response). Nous avons monté que la production d’$^1O_2$ active l’UPR. L’acclimatation à la forte lumière (FL) fait intervenir la branche bZIP28/BiP3 de l’UPR. Un pré-traitement modéré d’un inducteur de stress RE (tunicamycine), induisant l'UPR, déclenche une réponse d’acclimatation à l’$^1O_2$ permettant l’évitement de la MCP lors de l’exposition à la FL. Nous avons réalisé un crible génétique pour rechercher des révertants du mutant \textit{ch1} (un surproducteur d’1$^1O_2$) où l’inhibition de la croissance par l’$^1O_2$ est partiellement levée. Les gènes candidats identifiés feront l’objet d’études complémentaires. / Singlet oxygen ($^1O_2$) is a major reactive oxygen species produced within the chloroplasts during high light (HL) stress. $^1O_2$ has a cytotoxic effect due to its high reactivity towards macromolecules including proteins and membrane lipids. $^1O_2$ also acts as a signal molecule that plays a role in chloroplast-to-nucleus retrograde signaling involving mediators and leading either to programmed cell death (PCD) or to stress acclimation.We have shown the involvement of the phytohormone salicylic acid in HL-induced cell death, acting downstream of the OXI1 kinase and jasmonate. We have also shown a negative regulation of this signaling pathway by PCD inhibitory proteins: DAD1 and DAD2 (DEFENDER AGAINST CELL DEATH 1 and 2). Overexpressing those proteins inhibits OXI1-mediated PCD. Protein folding of most secreted proteins takes place in the Endoplasmic Reticulum (ER). Perturbations in this compartment lead to the activation of an adaptive response called UPR (Unfolded Protein Response). When ER stress is too intense, NRPs-mediated ER stress-induced cell death is activated. We have shown that 1O2 production activates UPR. In particular, the bZIP28/BiP3 UPR branch is activated during acclimation to HL. The induction of UPR by a chemical inducer of ER stress (Tunicamycin) can induce acclimation to $^1O_2$ production and can avoid HL-induced PCD.We performed a genetic screen to search for revertants of the $^1O_2$ overproducing \textit{ch1} mutants in which growth inhibition by$^1O_2$2 is partially released. The candidate genes will have to be confirmed by further phenotypic studies.