In transgenic research the precise control of transgene expression is crucial in order to obtain transformed organisms with expected desirable traits. A broad range of transgenic plants use the constitutive cauliflower mosaic virus (CaMV) 35S promoter to drive expression of selectable marker genes. Due to its strong enhancer function, this promoter can disturb the specificity of nearby eukaryotic promoters. When inserted immediately downstream of the 35S promoter in transformation vectors, special DNA sequences called insulators can prevent the influence of the CaMV35S promoter/enhancer on adjacent tissue-specific promoters for the transgene. Insulators occur naturally in organisms such as yeasts and animals but few insulators have been found in plants. Therefore, the goal of this study is to identify DNA sequences with insulator activity in Arabidopsis thaliana. A random oligonucleotide library was designed as an initial step to obtain potential insulators capable of blocking enhancer-promoter interactions in transgenic plants. Fragments from this library with insulator activity were identified and re-cloned into pB31, in order to confirm their activity. To date, one insulator sequence (CLO I-3) has been identified as likely possessing enhancer-blocking activity. Also, two other oligonucleotide sequences (CLO II-10 and CLO III-78) may possess insulator activity but more sampling is needed to confirm their activity. Further studies are needed to validate the function of plant insulator(s) and characterize their associated proteins.

Les milieux métallifères d’origine anthropique sont des habitats particulièrement stressants pour les êtres vivants du fait des fortes concentrations en métaux lourds et des changements environnementaux profonds qui y sont présents. Certaines espèces, dites métallophytes, sont capables de se développer dans ces milieux extrêmes. Parmi ces espèces, les pseudométallophytes (qui peuvent se développer à la fois dans des sites pollués et dans des sites non pollués) constitue des modèles particulièrement intéressants pour étudier l’adaptation locale et l’influence des différences forces évolutives dans la différentiation phénotypique entre populations. Arabidopsis halleri est une espèce pseudométallophyte modèle pour l’étude de la tolérance et de l’hyperaccumulation des métaux du fait de sa proximité avec l’espèce prééminente A. thaliana. Des études préalables sur la tolérance et l’hyperaccumulation du zinc chez cette espèce ont suggéré une possible évolution des populations métallicoles sous l’effet des fortes concentrations en métaux. Notre objectif a donc été de mieux comprendre les mécanismes évolutifs prenant place dans ces populations. Pour cela nous avons proposé d’utiliser la génomique des populations ainsi que l’expérimentation en milieu contrôlé et ce à une échelle locale. Nous avons tout d’abord cherché des traces de sélection dans le génome de cette espèce par une approche de criblage génomique sur 820 marqueurs AFLP. Nous avons pu identifier des locus potentiellement sous sélection dans les populations métallicoles qui constituent de bons candidats pour l’adaptation aux sites métallifères. De manière intéressante, certains candidats sont spécifiques à des populations ce qui suggère une évolution convergente ou des pressions de sélection différentes entre les sites. Une partie de ces candidats a été cartographiée chez A. thaliana dans le but de définir des gènes candidats. Nous avons dans un deuxième temps cherché à comprendre l’influence des différentes forces évolutives dans la distribution de la tolérance au zinc chez A. halleri. La tolérance au zinc de populations a été caractérisée par des mesures morphologiques et physiologiques et l’influence de la sélection par une approche Qst/Fst. Nos résultats suggèrent que, pour les populations échantillonnées, la tolérance au zinc augmente dans les populations métallicoles sous l’effet de la sélection. Cette sélection s’exerçant potentiellement sur la variabilité présente dans les populations non métallicoles ancestrales. Enfin, la dernière partie de cette étude s’intéresse à l’association entre les génotypes et les phénotypes de la tolérance au zinc. / Due to the high concentration of heavy metals and the important environmental modifications, ecological conditions of metalliferous sites are very stressing for most organisms. A few plant species, coined “metallophyte”, have acquired the capacity to grow on heavy metal contaminated soils. Among this group, the pseudometallophyte species (able to grow on contaminated as well as on non contaminated soils) constitute highly relevant models to study local adaptation and relative influence of evolutionary forces in shaping phenotypic differentiation among populations The pseudometallophyte plant Arabidopsis halleri is a model species for tolerance and hyperaccumulation of heavy metal because it is a close relative of the pre-eminent species A. thaliana. Previous studies on heavy metal tolerance and hyperaccumulation of A. halleri suggested evolution of metallicolous populations in response to high level of metal exposure. In order to investigate the evolutionary processes in neighboring metallicolous and non-metallicous A. halleri populations, we adopted population genomics and phenotyping approaches. First we explored the A. halleri genome to detect signatures of directional selection. We performed a genome-wide scan analysis using 820 AFLP markers. We identified some loci potentially under selection in metallicolous populations that constitute high-quality candidates for general adaptation to metalliferous sites. Interestingly, some candidates were population specifics, suggesting the possibility of convergent evolution or existence of different selection pressure in the different sites. Some of these candidates were mapped on A. thaliana genome to identify candidate genes. Our second objective was to evaluate the contribution of selection versus other forces in shaping distribution of zinc tolerance in A. halleri. We measured morphological and physiological traits to estimate zinc tolerance and role of selection was inferred using Qst/Fst approach. Our results suggest that, in the sampled populations, zinc tolerance has been increased in metallicolous populations through selection on standing variation within local non-metallicolous ancestral populations. The last part of this study focuses on looking for a link between genotype and phenotype of zinc tolerance.

Programmed Cell Death (PCD) is an essential process utilised for the defence and development of all multicellular organisms. In plants however, relatively little is known about the genes involved with the regulation and execution of this process. In particular, even less is known about the molecular components which act high up in the PCD pathway. In this thesis, we carried out an investigation into the novel peptide-encoding gene KISS OF DEATH (KOD). In Arabidopsis, KOD was found to be involved with mediating the elimination of the suspensor, an organ which undergoes developmental PCD. Two mutant alleles of KOD showed reduced PCD in both the suspensor and during heat-shock induced PCD of root hairs. Over-expression of KOD in plant tissues was sufficient to cause death in leaves or whole seedlings and involved the activation of caspase-like proteolytic activity. KOD-induced PCD was found to require light in leaves and is also sensitive to the PCD suppressor genes AtBI-1 and P35. We suggest that KOD acts high up in the PCD cascade as its expression resulted in depolarisation of the mitochondrial membrane, which is an early step in plant PCD. KOD appears to be a plant-specific peptide that is sufficient to induce PCD in Arabidopsis in the absence of external triggers. Typical BLAST searches yielded no obvious homolog for KOD, therefore a bioinformatics screen of the Arabidopsis genome was carried out. This screen for small genes similar in size to KOD enabled us to detect 10 previously unidentified genes, one of which may represent a putative KOD homolog. In summary, KOD appears to be a novel pro-PCD component of the Arabidopsis cell death machinery and represents the first plant peptide to be involved with a form of developmental PCD.

Photoacclimation is a process by which photosynthetic capacity is regulated in response to environmental adjustments in terms of light regime. Photoacclimation is essential in determining the photosynthetic capacity to optimize light use and to avoid potentially damaging effects. Previous work in our laboratory has identified a gene, gpt2 (At1g61800) that is essential for plants to acclimate to an increase in growth irradiance. Furthermore, we observed that the accession Columbia-0 (Col-0) is unable to respond to increases in light. Therefore, a Quantitative Trait Locus (QTL) mapping analysis was performed in Landsberg erecta (Ler)/Columbia (Col) recombinant inbred line population to identify novel genes responsible for this variation to acclimation. In order to investigate the photoacclimation in Arabidopsis thaliana, photosynthetic capacity was measured in plants of the accession Wassileskija (WS) and in plants lacking expression of the gene At1g61800 (WS-gpt2) during acclimation from high to low light. Plants were grown for 6 weeks under high light (400 μmol.m-2.s-1) and half of them were transferred to low light (100 μmol.m-2.s-1) after six weeks. Gas exchange measurements were performed in order to measure the maximum capacity for photosynthesis. Acclimation to a decrease in light resulted in a decrease in the photosynthetic capacity in WS and WS-gpt2 plants. This shows that under lower or limiting light, photosynthesis was slowed down. Chlorophyll fluorescence analysis was carried out to measure changes in the quantum efficiency of PSII (ΦPSII) and non-photochemical quenching (NPQ) during acclimation. ΦPSII decreased in both WS and WS-gpt2 plants showing that under low light, PSII is more saturated. However, it was found that there was no significant changes in NPQ level for both WS and WS-gpt2. To estimate the total chlorophyll and chl a/b ratio, a chlorophyll composition analysis was performed. There was no significant changes in the total chlorophyll for both WS and WS-gpt2. However, the chlorophyll a/b ratio was seen to be decreased in low light plants representing an increase in light harvesting complexes relative to reaction centre core. Plants of WS and WS-gpt2 were also grown under natural variable light in an unheated greenhouse in Manchester, UK. This experiment was carried out to study the photosynthetic acclimation of plants under fluctuating light condition. A preliminary work on gene expression of gpt2 was conducted by doing reverse transcriptase PCR (RT-PCR). It shows that the gene expression of gpt2 decreased following transfer to low light plants in WS. Microarray analysis was also performed to investigate the role of GPT2 (if any) and to identify any potential gene that is important in high to low light acclimation.

N-Acylethanolamines (NAEs) are a class of bioactive lipids, and FAAH is one of the enzymes responsible for degrading NAEs in both plants and animals. in plants, FAAH appears to be closely associated with ABA, a phytohormone which has long been associated with plant stress responses, since the overexpression of FAAH in Arabidopsis results in ABA hypersensitivity. Therefore, it is reasonable to speculate that alterations in FAAH transcript levels will result in altered stress responses in plants. to investigate this hypothesis experiments were carried out in which wild type (WT), FAAH-overexpressing (OE), and T-DNA insertional FAAH knockouts of Arabidopsis (faah) were grown in MS media under stress conditions. the stress conditions tested included chilling stress, heavy metal stress induced by cadmium or copper, nutrient limitations induced by low phosphorus or low nitrogen, salt stress induced with NaCl, and osmotic stress induced with mannitol. the OE plants were consistently hypersensitive to all stress conditions in relation to wild type plants. Inactive FAAH overexpressors did not have the hypersensitivity to the salt and osmotic stress of the active OE plants and were instead tolerant to these stresses. FAAH2 (faah2) knockouts and FAAH 1 and 2 double knockouts (faah 1+2) were based on some root development parameters somewhat more tolerant than WT plants, but more sensitive in terms of shoot growth. Collectively the data suggests that FAAH activity may interact with stress-responsive pathways in plants, perhaps including pathways involving ABA.

Plants are sessile organisms and constantly encounter a myriad of pathogens; therefore, they rely on highly effective defense system for their survival. Our understanding of how plant immunity is triggered and regulated has seen tremendous progress over the last two decades, with many important players identified in the model systems, Arabidopsis thaliana. Mitogen activated protein kinases play a central role in signal transduction in biotic and abiotic stresses. MAPK pathways are regulated by three-interlinked protein kinases (MAPKKK, MAPKK, MAPK), which are sequentially activated by phosphorylation. The activation of the three MAPKs MPK3, MPK4 and MPK6 is one of the earliest cellular responses following pathogen attack leading to the phosphorylation of appropriate cytosolic or nuclear targets to regulate cellular processes. However, only few targets of MPK3, MPK4 and MPK6 have been identified and validated so far and many MAPK substrates remain to be discovered. We performed largescale phosphoproteomics on mock treated and flg22 treated WT and the three loss-of-function mutants mpk3, mpk4 and mpk6 to identify novel MAPKs substrates and their cellular functions in response to pathogen attack. We identify and validated some of the differentially phosphorylated cytosolic and chromatin targets of MPK3, MPK4 and MPK6. DEK2, a nuclear protein involved in multiple chromatin-related processes, was identified in the phosphoproteomics screen as an in vivo target of MPK6 and it interacts in planta and is phosphorylated in vitro by the three immune MAPKs. dek2 loss-of-function mutants were susceptible to bacterial as well as fungal pathogens. Additionally, transcriptome data of the dek2-1 mutant show that DEK2 is a transcriptional repressor inclusive of defense related genes and hormone synthesis and signaling genes. We determined that DEK2 is a reader of the histone mark, H3K9me1, by Microscale thermophoresis. From ChIP-Seq analysis, DEK2 was found to be enriched at class I TCP binding motif regions. We further need to determine whether DEK2 binds to TCP transcription factors directly or indirectly. Finally, based on our data we postulate a hypothetical working model for the function of DEK2 as a transcriptional repressor and a reader of H3K9me1 mark.

The introduction of mass spectrometry techniques to the field of biology has made possible the exploration of the proteome as a whole system as opposed to prior techniques, such as anti-body based assays or yeast two-hybrid studies, which were strictly limited to the study of a few proteins at a time. This practice has allowed for a systems biology approach of exploring the proteome, with the possibility of viewing entire pathways over increments of time. In this study, the effect of treating Arabidopsis thaliana suspension culture cells with 3’,5’-cyclic guanosine monophosphate (cGMP), which is a native second messenger, was examined. Samples were collected at four time points and proteins were extracted and enriched for both oxidation and phosphorylation before analysis via mass spectrometry. Preliminary results suggest a tendency towards an increased number of phosphorylated proteins as a result of cGMP treatment. The data also showed a sharp increase in methionine oxidation in response to the treatment, occurring within the first ten minutes. This finding suggests that cGMP may utilize methionine oxidation as a mechanism of signal transduction. As such, this study corroborates a growing body of evidence supporting the inclusion of methionine oxidation in intracellular signaling pathways.

Drought (soil water deficit) as a major adverse environmental condition can result in serious reduction in plant growth and crop production. Plants respond and adapt to drought stresses by triggering various signalling pathways leading to physiological, metabolic and developmental changes that may ultimately contribute to enhanced tolerance to the stress. Here, a novel non-coding RNA (ncRNA) involved in plant drought stress tolerance was identified. We showed that increasing the expression of this ncRNA led to enhanced sensitivity during seed germination and seedling growth to the phytohormone abscisic acid. The mutant seedlings are also more sensitive to osmotic stress inhibition of lateral root growth. Consistently, seedlings with enhanced expression of this ncRNA exhibited reduced transiprational water loss and were more drought-tolerant than the wild type. Future analyses of the mechanism for its role in drought tolerance may help us to understand how plant drought tolerance could be further regulated by this novel ncRNA.

Recently global warming and increasing environmental temperature are discussed very frequently. Ambient temperature is an important factor regulating plant growth and global changes in temperature could significantly affect the yield of the agriculture. Our previous work revealed the role of plant hormones cytokinins in response to temperature and showed cytokinins as negative regulators of the thermomorphogenesis. Since gibberellins are known as positive regulators of the thermomorphogenesis this work was focused on their interaction in response to temperature in Arabidopsis thaliana. Analysis of the morphology of plant seedlings showed that the effect of cytokinins dominates over gibberellins in response to high temperature and is detectable soon after high-temperature treatment. Analysis of gene expression showed that cytokinins induce expression of gibberellin biosynthesis enzyme GA20ox1 and decrease the expression of gibberellin degradation enzyme GA2ox6. On the other hand, cytokinins have an inhibitory effect on the expression of the master regulator of thermomorphogenesis PIF4. Analysis of transgenic line overexpressing PIF4 suggests that cytokinins could affect the thermomorphogenesis by regulation of the PIF4 expression but this process is not the key mechanism of the regulation of the response to high temperature.

Dehydroabietinal (DA), an abietane diterpenoid, was previously demonstrated to be a potent activator of systemic acquired resistance (SAR). DA also promotes flowering time in Arabidopsis thaliana by repressing expression of the flowering repressor FLOWERING LOCUS C (FLC) while simultaneously upregulating expression of FLOWERING LOCUS D (FLD), FLOWERING LOCUS VE (FVE) and RELATIVE OF EARLY FLOWERING 6 (REF6), a set of flowering time promoters. To further understand the mechanism underlying signaling by abietane diterpenoids, Arabidopsis mutants exhibiting reduced responsiveness to abietane diterpenoids were identified. One such mutant plant, ems2/7, exhibited SAR-deficiency and delayed flowering, which were found to be associated with two independent, but linked loci. The gene responsible for the SAR defect in ems2/7 was identified as DEFECTIVE IN SYSTEMIC DEFENSE INDUCED BY ABIETANE DITERPENOID 1 (DSA1). Similar to the missense mutant dsa1-1 identified in the mutant screen, the T-DNA insertion bearing null allele dsa1-2 exhibited SAR deficiency that could be complemented by a genomic copy of DSA1. The gene responsible for the delayed flowering phenotype of ems2/7 remains to be identified. DSA1 encodes a protein that is homologous to human protein O-fucosyltransferase 2. DSA1 is required for long-distance transport of the SAR signal. It is hypothesized that DSA1 is involved in the O-fucosylation-facilitated channeling through the ER/Golgi network of a protein involved in long distance SAR signaling. In a yeast two-hybrid screen, all the DSA1-interacting proteins identified are chloroplast-localized proteins, thus raising the interesting possibility of ER interaction with chloroplast and its potential role in SAR signaling.