Bacillus thuringiensis (Bt) is a gram-positive common soil bacterium that produces crystals (Cry) containing proteins that are toxic to certain insects, in particular larvae of Lepidoptera and Diptera. The Bt toxin in the past has been widely used as a bioactive compound for the biological control of mainly lepidopteran pests. Most recently a variety of crops, including cotton and maize, have been genetically modified to express a Bt toxin to confer resistance to lepidopteran pests. However, the effect of abiotic environmental stress, such as drought and heat, which are typical for Africa, on Bt toxin expression in a genetically modified crop has so far not been fully evaluated. This study focuses on the expression and stability of the Cry1Ac insecticidal protein from Bacillus thuringiensis in genetically modified cotton plants under drought and heat stress. These include the physiological and biochemical characterization of the expressed Bt toxin gene under drought stress as well as the biological activity against first-instar larvae of the African cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae). Non-genetically modified cotton (Gossypium hirsutum cv. Opal), as well as genetically modified cotton (cv. Nuopal) expressing the Bt toxin Cry1Ac, were exposed to drought and heat stress. Drought stress was induced by withholding watering plants until the soil moisture content reached 25- 30 % of field capacity. Non-stressed control plants were watered and soil moisture content to 80-100 % of field capacity was maintained. For heat stress, plants were grown at 38 to 32 DC during the day and night, respectively, whereas control plants were grown in a growth cabinet at a 28/25 DC day/night cycle. For growth analysis plants were harvested every second week after planting. At each harvest, different parts of the plant were collected and their fresh and dry weight determined. For biochemical analysis and determining biological activity against first-instar larvae of H. armigera, two types of experiments were carried out, the first experiment four weeks after treatment induction and the second experiment eight weeks after treatment induction. Different plant material (leaves, flowers and immature green bolls) were used for Bt detection as well as for determining biological activity against first-instar larvae of H. armigera. Under drought stress conditions a reduction in leaf area and leaf dry weight were found in both Bt toxin expressing and non-expressing cotton plants, but no significant difference in physiological performance between Bt-expressing and non-expressing cotton plants was found. This study shows that the Bt toxin (Cry1Ac) level decreases in senescent plants and that drought stress did not affect the growth and development of genetically modified Bt plants when compared to non-Bt plants. Although the expression of Bt toxin (Cry1Ac) in Bt cotton plants decreased under drought stress no effect on the efficacy of the toxin against H. armigera was observed. In addition, no significant decrease of Bt toxin content was found in Bt cotton leaves after exposure to heat stress when compared to leaves from nonheat stressed plants. / Dissertation (MSc)--University of Pretoria, 2008. / Plant Science / unrestricted

Plant cysteine proteinase inhibitors or also called phytocystatins inhibit the action of cysteine proteinases in plants. These proteinases are involved in many developmental processes by degrading proteins. In this study possible effects of an exogenous oryzacystatin-I (OC-I) expressed in transformed tobacco has been investigated. By challenging OC-I expressing and non-expressing tobacco with drought and heat stress, OC-I transcription and translation were not affected in OC-I expressing plants and plant extracts from stressed plants containing the inhibitor inhibited papain activity in vitro. Further, plant growth and photosynthesis was not greatly different under the selected growth conditions in both plant types under stress and non-stress conditions. However, OC-I expressing plants showed slightly lower photosynthetic rate, were shorter and had a higher lower dry mass production under non-stress condition. By applying cDNA Representational Difference Analysis (cDNA-RDA) to detect differentially expressed genes in the two types of plants, a gene coding for the light harvesting chlorophyll a/b binding protein gene (lhcb1) of photosystem II (LHC II) was isolated from non-OCI expressing plants. Northern blot analysis showed lower transcript accumulation of the lhcb gene in OCI-expressing plants both under non-stress and stress conditions, which was accompanied by lower chlorophyll content in OC-I expressing plants. Furthermore, plants benefited from OC-I expression by protection of a variety of expressed proteins against degradation. Identification of possible target cysteine proteinases for OC-I in tobacco resulted in the isolation, cloning and characterization of two new papain-like cysteine proteinases from tobacco designated NtCP1 and NtCP2. NtCP1 was expressed only in senescent leaves and it was not induced in mature green leaves upon exposure to drought or heat stress. NtCP1 has therefore a possible potential as a developmental senescence marker in tobacco. In contrast, NtCP2, which was expressed in mature green leaves, has a high similarity to KDEL-tailed cysteine proteinases that are involved in programmed cell death. Both drought and heat decreased NtCP2 transcript abundance in mature green leaves. Overall, this study has provided evidence that expression of exogenous OC-I does not significantly improve plant performance in tobacco in terms of physiological traits under drought and heat stress but provides protection in terms of stability of protein expression by possibly interacting with endogenous tobacco cysteine proteinases. Further detailed studies are suggested on the interaction of endogenous cysteine proteinases and exogenous phytocystatins to elucidate in more detail the type of interaction. Copyright 2006, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Beyene, G 2006, Gene expression and plant performance in oryzacystatin-I expressing transformed tobacco (Nicotiana tabacum L. cv Samsun) plants under abiotic stress, PhD thesis, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-12052006-144409 / > / Thesis (PhD (Botany))--University of Pretoria, 2006. / Plant Science / unrestricted

Considerando (1) a importância da bananicultura no Vale do Ribeira, (2) o destaque da Mata Atlântica no contexto da conservação da fauna e flora mundial, (3) a necessidade da adoção de práticas agrícolas alternativas mais eficientes do ponto de vista ambiental e econômico, (4) o papel dos hormônios etileno, acido indol-3-acético (AIA) e ácido abscísico (ABA) no contexto das respostas dos vegetais a diferentes condições ambientais e nos atributos de qualidade da banana, (5) a falta de metodologias otimizadas para a extração de compostos fenólicos solúveis totais (CFST) em bananeiras e (6) a importância do estudo da relação entre os CFST e fatores de estresse, o presente trabalho teve como objetivo avaliar a influência da proximidade do fragmento florestal de Mata Atlântica com a área de cultivo da banana (Musa acuminata AAA cv. Nanicão) sobre o amadurecimento da fruta e os CFST em folhas de bananeiras, além de otimizar uma técnica para a extração destes últimos. Foi observado que bananas colhidas próxima ao fragmento florestal apresentam vida-verde (período compreendido entre a colheita do fruto e o início do seu amadurecimento) maior quando comparados a frutos com a mesma idade fisiológica, porém colhidos em áreas sem a influência da floresta nativa. Este fato pode ser explicado, pelo menos em parte, pela diferença nos perfis de etileno, ABA e AIA ao longo do amadurecimento das bananas provenientes das diferentes áreas, que também influenciam no metabolismo amido-sacarose. Quanto aos CFST nas folhas, foi observado que a utilização de acetona 80% em água (v/v) e posterior emprego de hexano para a remoção do excesso de clorofilas é capaz de obter um bom rendimento de extração de CFST, sem extrair compostos que interferem significativamente no método de Folin-Ciocalteu. Além disso, a utilização da metodologia otimizada mostrou que bananeiras podem apresentar diferenças na sua composição de fenólicos quando influenciadas ou não pela presença de biodiversidade. Assim, a avaliação dos CFST em folhas pode fornecer informações importantes sobre as condições ambientais da planta. / Considering (1) the importance of banana production in Ribeira Valley, (2) the Atlantic Rainforest in the context of fauna and flora conservation, (3) the need for the adoption of more sustainable agricultural practices, (4) the ethylene, indole 3-acetic acid and abscisic acid responses in acclimation mechanisms of plants and in the quality attributes of the banana, (5) the lack of methodologies optimized for the extraction of total soluble phenolics compounds (TSPC) in banana leaves and (6) the importance of the relationship between the TSPC content and stress factors, the present work aimed to evaluate the influence of the Atlantic Forest fragments proximity in the banana (Musa acuminata AAA cv. Nanicão) crop area on fruit ripening and leaves TSPC levels, using a optimized methodology. It was observed that bananas harvested near to the forest fragment presented a longest greenlife (period between the harvest and the climacteric) when compared with the fruits with the same phisiologycal age, but without the influence of the native forest. This fact can be explained, at least partly, by the difference on ethylene, ABA and IAA profiles in the ripening of bananas from the different areas, which also influence the starch-sucrose metabolism. Moreover, it was observed that the extraction with acetone (80% v/v in water) and posterior hexane cycle to remove chlorophylls excess was able to obtain a good TSPC extraction yield in leaves, without extracting compounds that interfere significantly with Folin-Ciocalteu method. In additional, the use of optimized methodology showed that bananas leaves can present different TSPC amount when influenced by the presence of native forest. Thus, the evaluation of leaves TSPC profile can provide important information about the environmental conditions of the plant.

Cette étude vise à explorer les mécanismes de tolérance des plantes aux xénobiotiques organiques de la famille des HAPs (phénanthrène), à travers l’analyse de l’impact des évènements de spéciation par hybridation et duplication génomique (allopolyploïdie). Nous avons pour cela mené une approche comparative sur un modèle de spéciation allopolyploïde récente, constitué des espèces parentales hexaploïdes S. alterniflora et S. maritima, et de l’allopolyploïde S. anglica qui résulte de la duplication du génome de leur hybride F1 S. x townsendii. Une approche intégrative basée sur des analyses physiologiques et moléculaires nous a permis de montrer que chez Spartina l’hybridation et le doublement du génome augmentent la tolérance aux xénobiotiques. Le parent paternel S. maritima se montre particulièrement sensible au phénanthrène par rapport au parent maternel S. alterniflora. Différentes analyses transcriptomiques ont permis l’identification de novo de transcrits spécifiquement exprimés en condition de stress, et l’annotation des petits ARNs (miARNs, leurs gènes cibles, et siARNs) agissant en tant que régulateurs de l’expression des gènes et la régulation des éléments transposables. Les analyses d’expression différentielle en réponse au stress ont permis de générer un modèle de régulation (miARN/gènes cibles) en réponse aux HAPs, testé par validation fonctionnelle en système hétérologue chez Arabidopsis. Un travail exploratoire de profilage du microbiome de la rhizosphère des spartines exposées au phénanthrène a été réalisé pour préciser les mécanismes de dégradation des xénobiotiques dans l’environnement en vue d’une application dans les stratégies de remédiation verte. / We explored mechanisms involved in tolerance to organic xenobiotics belonging to PAHs (phenanthrene), in the context of allopolyploid speciation (hybrid genome duplication). We developed a comparative approach, using a recent allopolyploidization model including the hexaploid parental species S. alterniflora and S. maritima, and the allopolyploid S. anglica, which resulted from genome doubling of the F1 hybrid S. x townsendii. Integrative approach based on physiological and molecular analyses highlights that hybridization and genome doubling enhance tolerance to xenobiotics in Spartina. The paternal parent S. maritima exhibits higher sensitivity compared to the maternal parent S. alterniflora. Various transcriptomic analyses were performed, to identify de novo stress responsive transcripts, and to annotate small RNAs (miRNAs, their target genes, and siRNAs) involved in gene expression and transposable element regulations. Differential expression analyses in response to stress allowed us to develop a putative miRNA regulatory network (miRNA/target genes) in response to PAH, functionally validated in Arabidopsis as heterologous system. An exploratory profiling of Spartina rhizosphere microbiome exposed to phenanthrene was also performed to characterize environmental degradation abilities, in the perspective of optimizing green remediation strategies.

Constitutive and alternative splicing of pre-mRNAs from multiexonic genes controls the diversity of the proteome; these precisely regulated processes also fine-tune responses to cues related to growth, development, and biotic and abiotic stresses. Recent work showed that AS is pervasive across plant species, with more than 60% of intron-containing genes producing different isoforms. Mammalian cell-based assays have discovered various AS small-molecule inhibitors that perturb splicing and thereby provide invaluable tools for use as chemical probes to uncover the molecular underpinnings of splicing regulation and as potential anticancer compounds. Here, I show that the macrolide Pladienolide B (PB) and herboxidiene (GEX1A) inhibits both constitutive and alternative splicing, mimics an abiotic stress signal, and activates the abscisic acid (ABA) pathway in plants. Moreover, PB and GEX1A activate genome-wide transcriptional patterns involved in abiotic stress responses in plants. PB and GEX1A treatment triggered the ABA signaling pathway, activated ABA-inducible promoters, and led to stomatal closure. Interestingly, PB and GEX1A elicited similar cellular changes, including alterations in the patterns of transcription and splicing, suggesting that these compounds might target the same spliceosome complex in plant cells. This work establishes PB and GEX1A as potent splicing inhibitors in plants that can be used to probe the assembly, dynamics, and molecular functions of the spliceosome and to study the interplay between splicing stress and abiotic stresses, as well as having potential biotechnological applications.

Bacterial volatile organic compounds (VOCs) are signal molecules that may have beneficial roles in the soil-plant-microbiome ecosystem. In this Ph.D. thesis, I aimed to assess and characterize the role of bacterial VOCs in plant tolerance to drought and in the biocontrol of fungal pathogens. I started by studying two root endophytic bacteria isolated from pepper plants cultivated under desert farming conditions. They showed an enhancement of pepper tolerance to drought stress and an amelioration of its physiological status. Moreover, they induced the expression of a vacuolar pyrophosphatase proton pump (V-PPase), implicated in the regulation of the vacuolar osmotic pressure, facilitating water uptake. Besides, the exposure of Arabidopsis thaliana plants, grown under salinity stress, to the volatile 2,3-butanediol, described for its plant growth promotion (PGP) potential, enhanced the plants tolerance to salinity, proving the potential involvement of this volatile in the osmotic stress resistance mechanism. Then, I studied VOCs released by three bacteria associated to healthy rice plants. Their released VOCs mixtures modified the color pattern of Magnaporthe oryzae, the agent of the rice blast disease, and protected rice from the pathogen infection. A significant reduction of melanin production, sporulation and appressoria formation was measured in presence of the bacterial VOCs, without major effects on mycelial proliferation. 1-butanol-3-methyl, one of the nine VOCs co-produced by the studied bacteria, proved its potential of reducing M. oryzae melanin in vitro. In vivo tests confirmed the infection inhibition effects mediated by the rice-bacterial VOCs, with a reduction of 94% of the disease incidence. Lastly, I compared the genomes of the five bacteria considered in the previous experimental studies. The PGP traits and the VOCs pathways identified from the genome analyses confirmed the effects observed with the in vitro and in vivo assays, revealing a complex mode of promotion and protection offered by the studied plant-associated bacteria. In conclusion, plant-associated bacterial VOCs can play potentially important roles in modulating plant drought tolerance and reducing fungal virulence. Such biological resources represent novel tools to counteract the deleterious effects of abiotic and biotic stresses and have the potential to be exploited for sustainable approaches in agriculture.

One of the major challenges for agricultural research in the 21st century is to increase crop productivity to meet the growing demand for food and feed. Biotic (e.g. plant pathogens) and abiotic stresses (e.g. soil salinity) have detrimental effects on agricultural productivity, with yield losses being as high as 60% for major crops such as barley, corn, potatoes, sorghum, soybean and wheat, especially in semi-arid regions such as Saudi Arabia. Plant growth promoting bacteria isolated from pioneer desert plants could serve as an eco-friendly, sustainable solution for improving plant growth, stress tolerance and health. In this dissertation, culture-independent amplicon sequencing of bacterial communities revealed how native desert plants influence their surrounding bacterial communities in a phylogeny-dependent manner. By culture-dependent isolation of the plant endosphere compartments and a number of bioassays, more than a hundred bacterial isolates with various biochemical properties, such as nutrient acquisition, hormone production and growth under stress conditions were obtained. From this collection, five phylogenetically diverse bacterial strains were able to promote the growth of the model plant Arabidopsis thaliana under salinity stress conditions in a common mechanism of inducing transcriptional changes of tissue-specific ion transporters and lowering Na+/K+ ratios in the shoots. By combining a number of in vitro bioassays, plant phenotyping and volatile-mediated inhibition assays with next-generation sequencing technology, gas chromatography–mass spectrometry and bioinformatics tools, a candidate strain was presented as a multi-stress tolerance promoting bacterium with potential use in agriculture. Since recent research showed the importance of microbial partners for enhancing the growth and health of plants, a review of the different factors influencing plant-associated microbial communities is presented and a framework for the successful application of microbial inoculants in agriculture is proposed. The presented work demonstrates a holistic approach for tackling agricultural challenges using microbial inoculants from desert plants by combining culturomics, phenomics, genomics and transcriptomics. Microbial inoculants are promising tools for studying abiotic stress tolerance mechanisms in plants, and they provide an eco-friendly solution for increasing crop yield in arid and semi-arid regions, especially in light of a dramatically growing human population and detrimental effects of global warming and climate change.

L'oxydase terminale plastidiale (PTOX) est présente uniquement chez les organismesphotosynthétiques. PTOX oxyde le plastoquinol (PQH2) et réduit l'oxygène en eau.PTOX est impliquée dans la synthèse des caroténoïdes, dans le transportphotosynthétique d'électrons et dans la chlororespiration. De plus, son activité estconsidérée comme pouvant jouer un rôle en tant que soupape de sécurité, permettant de maintenir oxydé le pool de plastoquinones (PQ) et d'éviter la surréduction duchloroplaste et ainsi la photoinhibition. Chez la majorité des plantes testées, les niveaux de PTOX sont plus élevés dans des conditions de stress (une exposition à forte intensité lumineuse, par exemple). D'autre part, la surexpression de PTOX chez Arabidopsis thaliana n'a pas rendu les plantes moins sensibles à la photoinhibition. Par ailleurs, il semble que PTOX surexprimée chez Nicotiana tabacum a induit la génération des espèces réactives de l'oxygène (ERO) et une photoinhibition importante sous forte lumière.Le but de cette thèse était la caractérisation de l'activité enzymatique de PTOX enutilisant la protéine purifiée et de comprendre pourquoi PTOX protège du stressphotooxydant dans certaines conditions et pourquoi elle augmente ce stress quand elle est surexprimée in planta.L'analyse biochimique de PTOX recombinante purifiée a démontré que l'enzymeexiste principalement sous forme tétramérique. Cette forme se dissocie partiellement,principalement en dimères. Le turnover maximal de l'enzyme purifié correspond à 320électrons par seconde et par molécule de PTOX. Nous avons démontré que PTOXgénère des ERO dans une réaction secondaire dépendante de la concentration dusubstrat (PQH2) et du pH de la solution. À pH 8 (représentant le pH du stroma deschloroplastes actifs), PTOX a une activité antioxydante quand la concentration de PQH2 est basse et prooxydante quand cette concentration est élevée.En mesurant la fluorescence de la chlorophylle a, nous avons démontré quePTOX est active lorsqu'elle est ajoutée aux membranes enrichies en PSII.L'attachement aux membranes dépend du pH et de cations de la solution: lorsque le pHdiminue ou lorsque la solution est riche en cations monovalents, la quantité de PTOXattachée à la membrane diminue.L'activité de PTOX in planta et son effet sur le transport des électronsphotosynthétiques ont été analysés en utilisant Arabidopsis thaliana surexprimant laphytoène désaturase bactérienne (CRTI) et Nicotiana tabacum surexprimant PTOX1 deChlamydomonas reinhardtii. Arabidopsis thaliana surexprimant CRTI a un niveau plusimportant de PTOX et de production d'ERO et le transport cyclique des électrons estsupprimé chez les transformants. Cela implique que PTOX est en compétition avec letransfert cyclique pour les électrons du pool PQ et que PTOX joue un rôle importantdans le contrôle de l'état rédox de ce pool. En utilisant Nicotiana tabacum surexprimant PTOX1, nous avons démontré que PTOX fait concurrence au transfert linéaire d'électrons photosynthétique, mais que PTOX est inactivée quand le pH du stroma est neutre. Grâce aux résultats obtenus, nous proposons un modèle où l'association de PTOX avec la membrane est contrôlée par le pH du stroma. Quand le pH est neutre, PTOX est soluble et n'est pas active, ce qui évite l'interférence avec le transfert linéaire d'électrons. Quand le pH du stroma est alcalin et la chaîne des transporteurs photosynthétiques est surréduite (lors des conditions du stress), PTOX s'attache à la membrane, devient active et joue le rôle de soupape de sécurité. / The plastid terminal oxidase PTOX is encoded by higher plants, algae and some cyanobacteria. PTOX is a plastid-localized plastoquinol (PQH2) oxygen oxidoreductase. PTOX was shown to be implicated in plant carotenoid biosynthesis, photosynthetic electron transport and chlororespiration and may act as a safety valve protecting plants against photo-oxidative stress. PTOX protein levels increase during abiotic stress indicating a function in stress acclimation. But overexpression of PTOX in Arabidopsis did not attenuate the severity of photoinhibition or, when overexpressed in tobacco, even increased the production of reactive oxygen species (ROS) and exacerbated photoinhibition.Biochemical analysis of recombinant purified PTOX (PTOX from rice fused to the maltose-binding protein) showed that the enzyme exists mainly as a tetramer, which dissociated to a certain extent during electrophoresis, mainly into a dimeric form. The PTOX activity was 320 electrons s−1 PTOX−1. It was also shown that PTOX generates ROS in a side reaction in a substrate (decylPQH2) and pH-dependent manner when liposomes were used: at the basic stromal pH of photosynthetically active chloroplasts, PTOX was antioxidant at low decylPQH2 gaining prooxidant properties with increasing quinol concentrations. It is concluded that PTOX can act as a safety valve when the steady state [PQH2] is low while a certain amount of ROS is formed at high light intensities.It was shown by chlorophyll a fluorescence that recombinant purified PTOX is active when added to photosystem II (PSII)-enriched membrane fragments. PTOX attached tightly to the PSII-enriched membrane fragments. The amount of PTOX attaching to the membrane depended on pH and salts: an alkaline pH and monovalent compared to divalent cations increased PTOX attachment.PTOX activity in planta and its effect on photosynthetic electron transport were investigated using Arabidopsis expressing bacterial phytoene desaturase and tobacco expressing PTOX1 from Chlamydomonas. Arabidopsis expressing bacterial phytoene desaturase (CRTI lines) showed a higher PTOX content and increased PTOX related ROS generation. Furthermore, cyclic electron flow was suppressed in these lines. This implicates that PTOX competes efficiently with cyclic electron flow for PQH2 in the CRTI-expressing lines and that it plays a crucial role in the control of the reduction state of the plastoquinone pool. Using tobacco expressing PTOX1 from Chlamydomonas, it was shown that PTOX competes efficiently with photosynthetic electron flow, but gets inactive when the stromal pH is neutral. Based on the in vitro and in vivo results, a model is proposed, where the association of PTOX to the membrane is controlled by the stromal pH: When the stromal pH is neutral, PTOX exists as a soluble form and is enzymatically inactive avoiding the interference of PTOX with linear electron flow. When the stromal pH is alkaline and the photosynthetic electron chain is highly reduced under stress conditions as high light, PTOX binds to the membrane, gets enzymatically active and can serve as safety valve.

La photorespiration est un processus essential chez tous les organismes photosynthétiques. Elle est déclenchée par l’activité oxygénase de la Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RuBisCO) menant à la production d’une molécule de 3-phosphoglycerate and une molécule de 2-phosphoglycolate (2PG). Le 2PG est toxique et sera recyclé par la photorespiration qui implique huit principales enzymes et prend place dans les chloroplastes, les peroxysomes, les mitochondries et le cytosol. Bien que la photorespiration aboutisse à une efficacité réduite de l’assimilation du CO₂ photosynthétique et soit considérée comme un processus inutile, le phénotype de croissance des mutants d’enzymes photorespiratoires (croissance réduite, chlorose) reflète l’importance de ce processus dans la croissance et le développement normal car il interagit avec plusieurs voies métaboliques primaires. Les données actuelles montrent que sept des huit principales enzymes photorespiratoires pourraient être phosphorylées et qu’ainsi la phosphorylation pourrait être un élément régulateur essentiel du cycle photorespiratoire. Afin de mieux comprendre la régulation du cycle photorespiratoire, nous avons étudié l’effet d’une phosphorylation/ absence de phosphorylation sur la sérine hydroxyméthyltransférase 1 mitochondriale (SHMT1) et de l’hydroxypyruvate réductase peroxisomale en utilisant des versions de ces enzymes mimant une phosphorylation (sérine ou la thréonine mutée en acide aspartique) ou une absence de phosphoryaltion (sérine ou thréonine mutée en alanine).Deux sites sont phosphorylés chez HPR1: S229 et T335. La mutation de ces sites montre que seule la version mimant une phosphorylation sur le site T335 (HPR1 T335D) entraîne une activité réduite de la protéine recombinante HPR1. Ce résultat a été confirmé in vivo puisque le mutant Arabidopsis hpr1 exprimant HPR1 T33D était incapable de totalement complémenter le phénotype photorespiratoire du mutant hpr1.Par complémentation du mutant d’Arabidopsis shm1-1 par une forme sauvage de SHMT1, d’une version mimant (S31D) ou non (S31A) une phosphorylation, les résultats ont montré que toutes les formes de SHMT1 pouvaient presque totalement complémenter le phénotype de croissance de shm1-1. Cependant, chaque ligne transgénique n'avait que 50% de l'activité de SHMT normale. En réponse à un stress dû au sel ou à la sécheresse, les lignées Compl-S31D ont montré un déficit de croissance plus accentué que les autres lignées transgéniques. Cette sensibilité au sel semble refléter les quantités réduites de protéines SHMT1-S31D ainsi qu’une activité plus faible ayant un impact sur le métabolisme des feuilles, entraînant une sous-accumulation de proline et une suraccumulation de polyamines. La mutation S31D de la protéine SHMT1 a également entraîné une réduction de la fermeture stomatique induite par le sel et l'ABA. Ainsi, nos résultats soulignent l’importance du maintien de l’activité du SHMT1 photorespiratoire dans des conditions de stress dû au sel et à la sécheresse et indiquent que la phosphorylation de SHMT1 S31 pourrait être impliquée dans la modulation de la stabilité de la protéine SHMT1. / Photorespiration is an essential process in oxygenic photosynthetic organisms, and it is triggered by the oxygenase activity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RuBisCO) to produce one molecular 3-phosphoglycerate and one molecular 2-phosphoglycolate. The toxic 2-PG is recycled by the photorespiratory pathway which includes eight core enzymes and takes place in chloroplasts, peroxisomes and metochondria and cytosol. Although the photorespiration leads to a reduced efficiency of the photosynthetic CO₂ assimilation and thereby is considered as a wasteful process, the growth phenotype of the photorespiratory enzymes can reflect the importance of this process in normal growth and development of air-grown plants. Normally, for most photorespiratory enzyme mutants, they exhibit small, chlorotic plants sometimes non-viable in air which are not observed when the mutants are grown under high CO₂ condition that limit the photorespiration by reducing the RuBisCO oxygenase activity. Photorespiratory cycle interacts with several major primary metabolic pathways, thus is a highly regulated and extensive works. Current data show that seven of eight core photorespiratory enzymes could be phosphorylated and the protein phosphorylation seems to be a critical regulatory component of the photorespiratory cycle. In order to better understand the regulation of the photorespiratory cycle, we explored the effect of SHMT1 and HPR1 phosphorylation/non-phosphorylation events on plant physiology and metabolism by several methods: Site-directed mutagenesis assay, complementation assay, activity assay, stomatal aperture assays, plant salt/drought resistance assays, metabolites measurement, gas exchange measurement. The results show the phosphorylation mimicking version of HPR1 at T335 results to a less HPR1 activity and retarded growth at the ambient air condition. For the phosphorylation mimicking version of SHMT1 at S31 resulted in a less stability leading to a reduced resistance to drought and salt stress. The decline of resistance against abiotic stress was mainly due to impairment in the closure of stomata which were unable to respond properly to ABA probably because of a default in the PLC pathway. So there results indicate that the phosphorylation of SHTM1 leads to a negative effect for the plant growth especially under stress condition. Thus, we propose that the SHMT1 can be phosphorylated at a basic level under normal growth conditions, once the photorespiratory flux is increased such under salt stress condition, the SHMT1 should be dephosphorylated to stabilize SHMT1 and sustain a high photorespiration flux to cope with reduced CO₂ availability.

Fresh fruits and vegetables (FFVs) are the most frequently wasted foods because of their perishability and handling requirements. However, there is a lack of information on how much each step of the supply chain impacts FFVs quality, particularly on tomatoes, and what measures need to be taken for an immediate and effective impact on waste reduction. There is also no information on how the supply chain affects the proteome of the tomato and what proteins are differentially regulated by the most impactful steps of the supply chain. The objectives of the work presented on this thesis were to evaluate the decline in the overall quality and quantify tomato waste at each step of the supply chain, from the farm to consumer; and to determine what proteins are impacted by the decline in quality that is associated with temperature abuse. To determine overall quality and tomato waste, light-red tomatoes were exposed to an optimum temperature (13 °C) and eighteen different time-temperature scenarios, normally encountered during supply chain, and sensory and physicochemical attributes measured at each step. To determine the impact of chilling and non-chilling temperatures normally encountered during tomato supply chain, on the proteome, light-red tomatoes were exposed to an optimum temperature (13 °C) and to two time and temperature supply chain scenarios (2 °C and 25 °C) that showed the most negative impact on tomato overall quality, and physicochemical and proteomic attributes were measured at each step. For the first tomato harvest, the steps with the highest impact on quality and waste were shipping to distribution center (DC; 20°C), cooling at the grower (25°C) and storing at the consumer (4°C). For the second tomato harvest, shipping to the store (2°C), cooling at the farm (10°C) and displaying at the store (20°C) negatively impacted quality. High temperatures during cooling, shipping and store display impacted sensory quality and resulted in increased weight loss, and decreased sugar, carotenoids, and ascorbic acid contents. Although low temperatures during shipping, cooling and consumer did not impact tomato sensory quality, they contributed to a decline in sugar, carotenoids and ascorbic acid contents. Overall, the most impactful steps on tomato quality and waste, regardless of the temperature, were shipping to DC, cooling, shipping to stores, displaying at the store, and consumer storage. Analysis of the differentially expressed proteins in the tomato showed that metabolic proteins were greatly impacted by temperature abuses such as phosphomannomutase, heme oxygenase 1, and MAP kinase; and that proteins regulating cellular membrane integrity such as vacuolar protein sorting-associated protein were also impacted.