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Iron sulfur [Fe-S] proteins are essential components of many major biological processes including electron transport, respiration, photosynthesis, hormone biosynthesis, and environmental sensing. The process of [Fe-S] cluster assembly in living cells is a controlled mechanism that is highly conserved across all kingdoms. Considerable progress has been made in deciphering this mechanism in bacteria, yeast, and mammals. The key players are the NifS/IscS/SufS proteins, which act as the sulfur donor, and the NifU/IscU/SufU proteins, which serve as a scaffold that binds Fe and upon which the cluster is assembled. Additional proteins are involved in the maturation and transport of the clusters. In eukaryotes there is redundancy in the proteins involved in this mechanism and the process is compartmentalized. Not much is known about the [Fe-S] cluster assembly mechanism in plants. In addition to the redundancy and compartmentalization seen in this machinery in eukaryotes, plants present a further challenge by offering chloroplasts as an additional site for [Fe-S] cluster assembly. The objective of this project has been to characterize Arabidopsis AtNFS1 and AtISU1-3, which show high homology to NifS/IscS and NifU/IscU, respectively, and are hypothesized to be key players in [Fe-S] cluster biogenesis in plants. Subcellular localization results of the AtNFS1 and AtISU1-3 proteins fused to GFP from this study are consistent with the presence of dual machinery in plants, with both mitochondria and chloroplasts as sites for [Fe-S] cluster assembly. Furthermore, observations also showed that AtISU2 mRNA may be unstable. The results of these experiments, together with promoter analysis described in this dissertation using GUS fusions suggested that the genes encoding the AtISU scaffold proteins are regulated at the transcriptional and probably also at the posttranscriptional level. Gene silencing experiments performed in this dissertation research using antisense and RNAi constructs indicated that these genes have the potential to impact respiration, photosynthesis, phytohormone biosynthesis, and environmental sensing, diverse processes that rely on [Fe-S] proteins. These observations, together with previous in vitro evidence that AtNFS1 and AtISU1 can participate in [Fe-S] cluster assembly, provide strong evidence that these proteins are part of two distinct cluster assembly systems that function in different subcellular locations and perhaps under different environmental conditions. Information gathered here has made it possible to begin developing a detailed model of [Fe-S] cluster biogenesis in plants. / Ph. D.

TCP4 as an integrator of key developmental events A striking aspect of plant life is their sedentary life-style. Though it relieves them of the obligation of forming a complex body organization, it exposes them to environmental challenges. Plants have evolved a flexible pattern of post-embryonic growth. The major phases in their life cycle are photomorphogenesis, vegetative growth with phase transitions, reproductive growth and senescence. The phase transitions are coordinated temporally to ensure proper maturation of organism. Flexibility is built in the re-iterated programs of organogenesis, which provides a plant with an option to adopt an architecture best suited to prevailing environmental conditions. Organogenesis occurs by processes of cell division and maturation (expansion). Cell division determines the growth potential by generating the requisite number of cells and cell maturation fulfils the potential by elaborating the organ form. Organ growth requires spatially- and temporally-controlled cellular maturation. The TCP class of plant-specific transcription factors, conserved from bryophytes to angiosperms, control diverse developmental and morphological traits, such as plant architecture, floral asymmetry, seed germination, male and female gametophyte development and photomorphogenesis (Martín-Trillo and Cubas, 2010). Class II TCPs, which are targets of miR319, are best known for their role in leaf morphogenesis. They are believed to function by redundantly regulating the onset of cellular maturation ( Efroni et al., 2008; Koyama et al., 2007; Nath et al., 2003; Ori et al., 2007; Palatnik et al., 2003; Schommer et al., 2008). To establish the link between level of TCP activity and organ growth, we undertook the approach of hyper-activating the function of TCP4, a representative class II TCP, by fusing it with a strong transactivation domain. Enhanced level of TCP4 activity reduced organ growth by causing precocious cellular maturation. It also accelerated the process of organ initiation, maturation and its progression into the final stage of senescence. Hyper-active TCP4-expressing plants underwent faster maturation of shoot apex into reproductive phase. In general, hyper-activation of TCP4 advanced cellular, organ and organism maturation programs in Arabidopsis life cycle (Fig. 1). Traits such as organ initiation rate, organ size, flowering time and seed yield contribute to the fitness of the plant. Faster rate of organ initiation, bigger organ size, early onset of flowering and higher seed yield are obvious desirable traits. However, they rarely occur simultaneously in a mutant or a natural variant, suggesting that there is a trade-off among different traits. Studies have shown that such traits are linked and are controlled by multiple loci that contribute quantitatively to the phenotype. A change that benefits one trait may adversely affect another (Colautti et al., 2011; Kozlowski, 1992; Mendez-Vigo et al., 2010) . Our study shows that TCP4 activity can potentially coordinate these inter-connected traits. Though hyper-active TCP4-expressing plants have faster rate of organ initiation, the final organ size is reduced and senescence is advanced. These plants reach reproductive phase faster, but produce fewer seeds, hence limiting their propagation and lowering their fitness in comparison to the wild type. Such a genetic constraint on the traits limits the phenotypic variation that can be produced in plants and, hence, their adaptation to the environment. Our study suggests that TCP4 can link organ growth with that of the whole organism. It acts as a heterochronic regulator which possibly affects timing of multiple maturation programs. Any perturbation in the TCP activity may have far-reaching effects on plant growth and thus, optimal level of TCP activity is crucial for plant homeostasis. One possible explanation for the developmental pleitropy in TCP4 hyper-activation line is an alteration in hormone biosynthesis or sensitivity. A combination of microarray and hormone application studies on hyper-active TCP4-expressing line has indicated a reduction in the levels of GA and auxin and an increase in cytokinin and MeJA levels. There may also be an inhibition of auxin signaling and upregulation of MeJA and ethylene signaling. In addition, TCP4 appeared to regulate both GA biosynthesis and response in opposing manner. The molecular mechanisms involved in TCP4-mediated integration of hormonal pathways are still unclear. Answering these questions would require identification of its direct downstream targets.

published_or_final_version / abstract / toc / Botany / Master / Master of Philosophy

Dans ce travail de thèse, nous avons étudié le transport du fer dans la plante, en nous focalisant sur la dernière étape de circulation : le remplissage de la graine. Il nous a paru important de comprendre 1- comment le fer arrive à la graine, 2- la manière dont il est délivré à l'embryon en développement, 3- sous quelles formes et 4- où et comment il est stocké dans l'embryon. Nous avons choisi de nous appuyer sur deux espèces modèles: Pisum sativum pour les approches de biochimie et Arabidopsis thaliana pour les approches de génétique. Le pois a été choisi car ses graines sont de grande taille et ont la particularité d'accumuler un albumen liquide qui sert de soutien nutritionnel pour l'embryon. Le point de départ de ce projet est l'étude de la spéciation du fer, c'est-à-dire l'identification des ligands du fer, dans cet albumen. Sur la base de la connaissance des complexes de fer circulant jusqu'à la graine, nous avons proposé de nouvelles fonctions physiologiques. La grande taille des cellules de pois a permis d'étudier la localisation du fer et sa spéciation in situ, à l'échelle subcellulaire. Chez Arabidopsis, nous avons étudié le rôle de deux gènes candidats, potentiellement impliqués dans le transport du fer vers la graine. / In this work, we studied iron transport in plants and we focused on the last circulation step: the seed filling. It seemt important to understand 1- how iron is arriving to the seeds, 2- the way it is delivered to developping embryos, 3- in which form and 4- where and how it is stored in embryos.To adress these questions, we used two model species: Pisum sativum for biochemical approaches and Arabidopsis thaliana for genetic approaches. Pea was choosen because of the large size of its seeds, and their characteristic to accumulate a liquid endosperm that feed embryos.The project started with the study of iron speciation, meaning the identification of iron ligands, in this endosperm. On the basis of the knowledge of the iron complexes arriving to seeds, we highlighted new physiological functions. The large size of pea cells also allowed us to study iron localization and in situ speciation, at subcellular level. In Arabidopis, we studied the role of two candidate genes putatively involved in iron transport to seeds.

Pour lutter contre les maladies virales chez les plantes cultivées, il est important de développer des résistances génétiques. Dans ce contexte, les facteurs d’initiation de la traduction eIF4E jouent un rôle majeur dans la mise en place de résistance aux potyvirus, un groupe de virus à ARN nuisible pour les cultures. Fréquemment, des allèles naturels de résistances ont été sélectionnés dans les espèces à intérêt agronomique. Cependant, nombreuses sont les espèces ne possédant pas de résistance naturelle. Pour remédier à ce problème, j’étudie lors de ma thèse le pathosystème Arabidopsis thaliana-potyvirus afin d’élaborer de nouvelles sources de résistances, en vue d’étendre leur application aux plantes cultivables. Pour ceci, je crée artificiellement par mutagénèse dirigée des allèles de résistances dont je teste dans la plante la fonctionnalité ainsi que l’efficacité vis-à-vis de la résistance. En combinant ces allèles de résistance synthétiques avec d’autres résistances liées aux facteurs eIF4E, je vise à élargir le spectre de résistance aux potyvirus ainsi qu’à augmenter la durabilité de ces résistances. Cette étude permettra de prouver la faisabilité de ce système pour obtenir des plantes à large spectre de résistance avant de pouvoir ainsi l’appliquer aux plantes à intérêt agronomique. / The development of genetic resistance is important to avoid viral infections in cultivated crops. In this context, translation initiation factors eIF4E have a major role in resistance to potyviruses, a family of viruses damageable to crops. Although natural resistance alleles are often used in crops breeding, there are still species devoided of such natural resistance, making it impossible to develop genetic resistance. Using the Arabidopsis thaliana-potyviruses pathosystem, I aim at developing new sources of resistances as a proof of concept before considering their application to crop species. For this, I am developing artificial resistance alleles created by directed mutagenesis before testing them for both their functionality and their resistance efficiency in plants. By combining these synthetic resistance alleles with others eIF4E factors-mediated resistance, my aim is to enlarge resistance spectrum to potyviruses as well as to increase the resistance durability. This study will make proof of the feasibility of this system to obtain large spectrum resistance plants with the perspective of extending it to cultivated plants.

Les nématodes phytoparasites constituent une menace réelle pour la production alimentaire mondiale. Ils sont responsables de 14% des pertes de rendement de la production alimentaire globale, ce pourcentage est l’équivalent de 100 milliards de dollar américain par an. La lutte contre ces phytoparasites est devenue un défit majeur, notamment après l'interdiction de l'utilisation du nématicide le plus efficace, en raison de ses effets nocifs sur l’environnement. Par conséquent, des nouvelles sources pour la gestion des nématodes phytoparasites sont nécessaires et urgentes. Ce travail vise à déterminer si la plante modèle Arabidopsis thaliana pourrait servir comme une source naturelle de gènes de la résistance aux nématodes phytoparasites. Parmi des accessions d’Arabidopsis, on a trouvé une variation génétique naturelle de la résistance contre les nématodes à galles Meloidogyne javanica, on a également identifié plusieurs QTL de résistance aux nématodes, et finalement, on a réalisé une cartographie fine d’un des QTL détectés. / Plant-parasitic nematodes are a serious threat for global food production. They are responsible for 14% of global yield loss, equivalent to an economic value of more than 100 billion US$ per year. Pest management is challenging, in particular since the most efficient nematicide has been banned due to its devastating effect on the environment. Hence, novel sources for nematode management are urgently required. This work investigates whether the model plant Arabidopsis thaliana could serve as a natural source for resistance genes against plant-parasitic nematodes. It finds natural genetic variation among Arabidopsis accessions for resistance against the root-knot nematode Meloidogyne javanica, identifies several QTL for nematode resistance, and fine-maps one of these resistance QTL.

Li, Man Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 109-119). / Abstracts in English and Chinese. / Thesis Committee --- p.I / Statement --- p.II / Abstract --- p.III / 摘要 --- p.V / Acknowledgements --- p.VI / Abbreviations --- p.VIII / Abbreviations of Chemicals --- p.X / List of Tables --- p.XI / List of Figures --- p.XII / Table of Contents --- p.XIII / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- General amino acid control in yeast --- p.1 / Chapter 1.2 --- Mammalian eIF2α kinases --- p.7 / Chapter 1.2.1 --- Heme-regulated inhibitor kinase (EIF2AK1/HRI) --- p.7 / Chapter 1.2.2 --- Protein kinase dsRNA-dependent (EIF2AK2/PKR) --- p.8 / Chapter 1.2.3 --- PKR-like ER kinase (EIF2AK3/PERK) --- p.9 / Chapter 1.2.4 --- General control non-repressible 2 (EIF2AK4/GCN2) --- p.10 / Chapter 1.2.5 --- Activating transcription factor 4 (ATF4) --- p.11 / Chapter 1.3 --- Plant General Amino Acid Control --- p.12 / Chapter 1.3.1 --- Studies of the homolog of GCN2 in Arabidopsis thaliana --- p.12 / Chapter 1.3.2 --- Studies of the homolog of other eIF2a kinase in plant --- p.14 / Chapter 1.3.3 --- Studies of the homolog of other GAAC components --- p.14 / Chapter 1.4 --- Previous works in our lab --- p.15 / Chapter 1.5 --- Hypothesis and Objectives --- p.17 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.18 / Chapter 2.1.1 --- "Bacterial cultures, plant materials and vectors" --- p.18 / Chapter 2.1.2 --- Primers --- p.21 / Chapter 2.1.3 --- Commercial kits --- p.25 / Chapter 2.1.4 --- "Buffer, solution, gel and medium" --- p.25 / Chapter 2.1.5 --- "Chemicals, reagents and consumables" --- p.25 / Chapter 2.1.6 --- Enzymes --- p.25 / Chapter 2.1.7 --- Antibodies --- p.25 / Chapter 2.1.8 --- Equipments and facilities --- p.25 / Chapter 2.2 --- Methods --- p.26 / Chapter 2.2.1 --- Growth conditions of Arabidopsis thaliana --- p.26 / Chapter 2.2.1.1 --- Surface sterilize of Arabidopsis thaliana seed --- p.26 / Chapter 2.2.1.2 --- Growing of Arabidopsis thaliana --- p.26 / Chapter 2.2.1.3 --- Treatment of Arabidopsis seedling --- p.26 / Chapter 2.2.2 --- Basic molecular techniques --- p.27 / Chapter 2.2.2.1 --- Liquid culture of Escherichia coli --- p.27 / Chapter 2.2.2.2 --- Preparation of plasmid DNA --- p.27 / Chapter 2.2.2.3 --- Restriction digestion --- p.27 / Chapter 2.2.2.4 --- DNA purification --- p.28 / Chapter 2.2.2.5 --- DNA gel electrophoresis --- p.28 / Chapter 2.2.2.6 --- DNA ligation --- p.29 / Chapter 2.2.2.7 --- CaCl2 mediated E. coli transformation --- p.29 / Chapter 2.2.2.8 --- Preparation of DNA fragment for cloning --- p.29 / Chapter 2.2.2.9 --- PCR reaction for screening positive E. coli transformants --- p.30 / Chapter 2.2.2.10 --- DNA sequencing --- p.30 / Chapter 2.2.2.11 --- RNA extraction from plant tissue with tRNA --- p.31 / Chapter 2.2.2.12 --- Extraction of RNA without tRNA --- p.31 / Chapter 2.2.2.13 --- cDNA synthesis --- p.32 / Chapter 2.2.2.14 --- SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.33 / Chapter 2.2.2.15 --- Western blotting --- p.33 / Chapter 2.2.3 --- Sub-cloning of AtGCN2 --- p.34 / Chapter 2.2.3.1 --- Sub-cloning full length AtGCN2 into pMAL-c2 --- p.36 / Chapter 2.2.3.2 --- Sub-cloning of the N-terminal sequence of AtGCN2 into pMAL-c2 --- p.38 / Chapter 2.2.3.3 --- Sub-cloning of the C-terminal sequence of AtGCN2 into pMAL-c2 --- p.38 / Chapter 2.2.4 --- Cloning of the eIF2α candidates for the in vitro assay --- p.41 / Chapter 2.2.4.1 --- Cloning of At2g40290 (putative eIF2α candidate) --- p.41 / Chapter 2.2.4.2 --- Cloning of At5g05470 (putative eIF2α candidate) into pBlueScript KS II + --- p.43 / Chapter 2.2.4.3 --- Sub-cloning of At5g05470 into pGEX-4T-1 --- p.43 / Chapter 2.2.4 --- Expression and purification of fusion proteins --- p.45 / Chapter 2.2.5 --- Expression of fusion proteins in E. coli --- p.45 / Chapter 2.2.5.2 --- Extraction of E. coli soluble proteins --- p.45 / Chapter 2.2.5.3 --- Purification of GST tagged fusion protein --- p.46 / Chapter 2.2.5.4 --- Purification of MBP tagged fusion protein --- p.46 / Chapter 2.2.5.5 --- Concentration of purified fusion proteins --- p.46 / Chapter 2.2.5.6 --- MS/MS verification of purified fusion proteins --- p.47 / Chapter 2.2.6 --- Gel mobility shift assay --- p.47 / Chapter 2.2.6.1 --- Synthesis of short biotinylated RNA --- p.47 / Chapter 2.2.6.2 --- Ligation of short biotinylated RNA with tRNA --- p.48 / Chapter 2.2.6.3 --- Gel mobility shift assay --- p.48 / Chapter 2.2.6.4 --- Blotting of the sample on to nitrocellulose membrane --- p.48 / Chapter 2.2.6.5 --- Detection of the tRNA on the membrane --- p.49 / Chapter 2.2.6.6 --- Detection of the MBP fusion proteins on the membrane --- p.49 / Chapter 2.2.7 --- In vitro kinase assay of AtGCN2 --- p.49 / Chapter 2.2.8 --- In vitro translation inhibition assay --- p.50 / Chapter 2.2.8.1 --- In vitro transcription of HA mRNA --- p.50 / Chapter 2.2.8.2 --- In vitro translation --- p.51 / Chapter 2.2.8.3 --- Detection of the protein dot blot --- p.51 / Chapter 2.2.9 --- Gene expression analysis by real time PCR --- p.52 / Chapter 2.2.10 --- Total seed nitrogen analysis --- p.53 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Blast search results suggested that AtGCN2 may be the sole eIF2α kinase in Arabidopsis thaliana --- p.54 / Chapter 3.2 --- Existence of two eIF2α candidates in Arabidopsis thaliana genome --- p.59 / Chapter 3.3 --- Fusion proteins were successfully expressed and purified --- p.63 / Chapter 3.4 --- C-terminal of AtGCN2 has a higher affinity toward tRNA than rRNA --- p.67 / Chapter 3.5 --- Both eIF2α candidates can be phosphorylated by full length AtGCN2 in vitro --- p.70 / Chapter 3.6 --- AtGCN2 can inhibit translation in vitro --- p.72 / Chapter 3.7 --- Overexpression of AtGCN2 did not affect expression of selected genes --- p.74 / Chapter 3.8 --- Overexpression of AtGCN2 did not affect seed nitrogen content and C:N ratio under normal growth conditions --- p.83 / Chapter Chapter 4 --- Discussion --- p.85 / Chapter 4.1 --- Existing evidence supported that AtGCN2 is the sole eIF2α kinase in Arabidopsis thaliana --- p.85 / Chapter 4.2 --- Kinase activities of AtGCN2 and its two substrates in Arabidopsis --- p.86 / Chapter 4.3 --- C-terminal binds tRNA in the gel mobility shift assay --- p.88 / Chapter 4.4 --- Overexpression of AtGCN2 did not affect gene expression of the transgenic lines under nitrogen starvation and azerserine treatment --- p.90 / Chapter 4.5 --- Overexpression of AtGCN2 did not alter the seed nitrogen content --- p.91 / Chapter 4.6 --- Existence of GCN4 and ATF4 in plant --- p.92 / Chapter 4.7 --- Alternative model without GCN4 and ATF4 homolog --- p.93 / Chapter 4.8 --- Possible application of the in vitro kinase assay --- p.94 / Chapter 4.9 --- Possible application of the in vitro translation inhibition analysis platform in future study --- p.95 / Chapter Chapter 5 --- Conclusion and Future Prospective --- p.97 / Appendices / Appendix I Commercial kits used in this project --- p.98 / "Appendix II Buffer, solution, gel and medium" --- p.99 / "Appendix III Chemicals, reagents and consumables" --- p.102 / Appendix IV Enzymes --- p.103 / Appendix V Antibodies --- p.104 / Appendix VI Equipments and facilities --- p.105 / Appendix VII Supplementary Data --- p.106 / Appendix VIII Amplification efficiency of real time primers --- p.108 / References --- p.109

<p>In this thesis, we have firstly cloned and expressed the domains that harbours the putative catalytic GC domain in these receptor molecules and demonstrate that these molecules can convert GTP to cGMP in vitro. Secondly, we show that exogenous application of both Phytosulfokine and Brassinosteroid increase changes of intracellular cGMP levels in Arabidopsis mesophyll protoplast demonstrating that these molecules have GC activity in vivo and therefore provide a link as second messenger between the hormones and down-stream responses. In order to elucidate a relationship between the kinase and GC domains of the PSK receptor, we have used the AtPSKR1 receptor as a model and show that it has Serine/Threonine kinase activity using the Ser/Thr peptide 1 as a substrate. In addition, we show that the receptor`s ability to phosphorylate a substrate is affected by the product (cGMP) of its co-domain (GC) and that the receptor autophosphorylates on serine residues and this step was also observed to be affected by cGMP. When Arabidopsis plants are treated with a cell permeable analogue of cGMP, we note that this can affect changes in the phosphoproteome in Arabidopsis and conclude therefore that the cGMP plays a role in kinase-dependent downstream signalling. The obtained results suggest that the receptor molecules investigated here belong to a novel class of GCs that contains both a cytosolic kinase and GC domains, and thus have a domain organisation that is not dissimilar to that of atrial natriuretic peptide receptors NPR1 and NPR2. The findings also strongly suggest that cGMP has a role as a second messenger in both Brassinosteroid and Phytosulfokine signalling. We speculate that other proteins with similar domain organisations may also have dual catalytic activities and that a significant number of GCs, both in plants and animals, remain to be discovered and characterised.</p>

Thesis (Ph. D.)--Swedish University of Agricultural Sciences, 2001. / Includes bibliographical references.