Comprendre l’évolution des espèces, et notamment l’adaptation locale, est un enjeu majeur dans le contexte des changements globaux actuels. Certaines espèces végétales se développent à la fois dans des environnements présentant de fortes concentrations en zinc, plomb et cadmium (populations métallicoles, M), et sur des sols non pollués (populations non-métallicoles, NM). Ces espèces, appelées pseudo-métallophytes, montrent des caractéristiques adaptives très étudiées depuis quelques années : la tolérance et l’hyperaccumulation de métaux lourds. Afin de mieux comprendre l’adaptation d’Arabidopsis halleri aux milieux métallifères, nous avons conduit une étude sur un ensemble de populations M et NM. Parce que l’adaptation nécessite notamment des variations phénotypiques et qu’elle résulte de l’action de pressions de sélection caractéristiques d’un milieu, nous avons utilisé une approche où écologie, phénotypage et génétique se rejoignent. Nous mettons en évidence que (1) d’autres facteurs environnementaux que les métaux distinguent les populations et pourraient agir comme des pressions de sélection sur l’accumulation et la tolérance au zinc, (2) séparer systématiquement les populations en M/NM ne semble pas judicieux pour étudier l’adaptation locale des populations, (3) les réponses des plantes sont traits et environnement-dépendantes, (4) l’architecture génétique de la tolérance au zinc est un ensemble d’effets additifs et épistatiques et enfin plus globalement (5) nous suggérons que l’adaptation aux milieux métallifères a évolué en réponse à de multiples pressions de sélection qui ont conduit à la mise en en place de tolérance à de multiples facteurs écologiques. / In the context of global changes, understanding the evolution of species, and especially local adaptation, is a major challenge. Some species are present in polluted areas where zinc, cadmium and leab are present in high and toxic concentrations (metallicolous populations, M) but also in non-polluted areas (non-metallicolous populations, NM). They are called pseudo-metallophytes and are metal tolerant, and sometimes metal hyperaccumulating. In order to understand better the adaptation of Arabidopsis halleri to polluted areas we studied M and NM populations at a local scale. Because the adaptation implies some phenotypic variations and results from the action of selective pressures we used an ecological, phenotypical and genetical approach. We showed that (1) populations differed not only by metal concentrations but also by other environmental parameters which could act as selective pressures on zinc tolerance and accumulation, (2) separating populations in M or NM groups is not adequate to study local adaptation, (3) plant responses are trait and environmental dependant, (4) the genetic architecture of zinc tolerance is a network of epistatic and additive effects and more globally (5) we suggest that the adaptation to metalliferous environments has evolved in response to a lot of selective pressures which have led to multi-tolerance to numerous ecological factors.

The effect of mechanical stimulation in plants has been studied in depth for more than a century. This type of stress has been shown to trigger alterations in development such as stunting, thickened stems and differential cell wall deposition. These responses are very likely to be initiated at a subcellular level, but the molecular mechanisms transducing mechanical signals into intracellular responses still remain unknown in plants. In this thesis I test the hypothesis that the membrane anchored protein Defective Kernel 1 (DEK1) could act as a plant-specific mechanosensor in plants. Constitutive overexpression of the cytoplasmic CALPAIN domain DEK1 causes a phenotype in Arabidopsis, that that resembles that of mechanically stressed plants. The CALPAIN domain of DEK1 shows a very high homology with animal calpains; a class of calcium-dependent Cysteine proteases which undergo a calciumstimulated CALPAIN domain-releasing autolytic cleavage event during activation. A similar autolytic cleavage event has been observed in DEK1 which, together with the fact that the CALPAIN domain alone can rescue the embryo-lethality associated with loss of DEK1 function, has led to the suggestion that this domain represents an activated form of the protein. I show that like mechanically stressed plants, CALPAIN overexpressing plants show a modified call wall composition. Consistent with this, transcriptional analysis of these plants shows a deregulation of genes encoding cell wall modifying enzymes, amongst others. Other characteristics of mechanically stimulated plants which I have characterized in CALPAIN overexpressing lines include late flowering and thickened stems. Therefore, I proposed a model in which the CALPAIN domain of DEK1 acts as an effector which is normally activated by mechanical stimulation. In this model, the transmembrane domains of DEK1 would regulate activation (cleavage) of the CALPAIN domain, potentially in response to mechanical stress. In order to test this model further, CALPAIN overexpressing lines were generated in a dek1 mutant background. If the model is correct, these plants should not only behave as if responding constitutively to mechanical stimulation, but should also lack appropriate responses to applied mechanical stimuli due to lack of the mechanosensory integral membrane domain of DEK1. My results confirm that the absence of the transmembrane domains of DEK1 is indeed translated into a lack of some, but not all responses to mechanical stimulation compared to wild-type plants. Furthermore, the lack of the transmembrane domains of DEK1 correlates with the absence of a mechanically-triggered calcium flux in the plant. Thus my work suggests that the transmembrane domains of DEK1 are involved in sensing mechanical stimulation, via the regulation activity of a mechano-sensitive calcium flux at the plasma membrane. In summary, my proposal is that Defective Kernel 1 (DEK1) acts both as a key mechanosensory cellular component, and as the first effector of the signalling cascade in response to mechanical stimulation, via an autolytic activation in response to mechanical stress.

RNAs endógenos competitivos (ceRNAs) são transcritos naturais que atuam como esponjas endógenas de miRNAs, modulando a ação de miRNAs sobre mRNAs-alvo. RNA circular (circRNA) é uma dentre as várias classes de ceRNA. circRNAs são produzidos a partir de um processo chamado backsplicing. CircRNAs já foram identificados em diversos eucariotos; no entanto, nas plantas, ainda não foi demonstrado se estes são capazes de atuar como esponjas eficazes de miRNAs. Dados públicos de RNAseq de bibliotecas de imunoprecipitação de Argonauta (AGO-IP) a partir de flores de Arabidopsis thaliana foram utilizados em um método multi-comparativo de análises de bioinformática para identificar potenciais RNAs circulares. Utilizando cinco diferentes métodos, foram preditos de 15 a 27812 circRNAs com pelo menos dois reads na junção do backsplicing. Posteriormente, a plataforma psRNAtarget foi utilizada para discriminar os circRNAs com sítios de ligação de miRNAs, que potencialmente possam estar atuando como esponjas. Como a AGO forma um complexo ternário com miRNA e mRNA-alvo, também foram comparadas as contagens de alvos em bibliotecas AGO-IP e controle, demonstrando que os mRNAs-alvo desses miRNAs também estão enriquecidos nas bibliotecas AGO-IP. Neste trabalho, foram encontrados dois prováveis circRNAs que podem potencialmente funcionar como esponjas de miRNA. Esse achado contribui para um melhor entendimento desta nova forma de regulação transcricional e pós-transcricional em plantas, ampliando o conhecimento e aplicações do tema. / Competing endogenous RNAs (ceRNAs) are natural transcripts that act as endogenous sponges of miRNAs, modulating the action of miRNAs on mRNAs targets. Circular RNA (circRNA) is one among the various classes of ceRNA. They are produced from a process called backsplicing. CircRNAs have been identified in several eukaryotes; however, in plants, it has not yet been demonstrated whether they are able to act as effective sponges for miRNAs. Public RNAseq data from Argonaute immunoprecipitation libraries (AGO-IP) from Arabidopsis thaliana flowers were used with a multi-comparative method of bioinformatics analyzes to identify putative circular RNAs. Using five different methods, 15 to 27812 circRNAs with at least two reads at the backsplicing junction were predicted. Subsequently, the psRNAtarget platform was used to discriminate circRNAs harboring miRNA binding sites, which could potentially be acting as sponges. Identities and amounts of mRNAs present in AGO-IP and control libraries were quantified, as AGO can also form a ternary complex among miRNA and their target mRNAs. It showed that target mRNAs from circRNA:miRNA pairs were also enriched in the AGO-IP libraries. Two circRNAs were positively identified, corroborating their action as potential miRNA sponges. This finding leads to a better understanding of a new form of transcription and post-transcription regulation in plants, increasing the knowledge and applications of the topic.

Les habitats naturels sont de plus en plus détruits et fragmentés par l'expansion urbaine et les activités humaines. La fragmentation des espaces naturels et agricoles par les bâtiments et les nouvelles infrastructures affecte la taille, la connectivité et la qualité des habitats. Les populations d’organismes vivants sur ces territoires anthropisés sont alors plus isolées. Or, la différenciation entre populations d’un même organisme dépend de processus démographiques et génétiques tels que la dérive génétique, le flux génétique, la mutation et la sélection naturelle. La persistance et le développement des populations dans des conditions environnementales modifiées dépendent de mécanismes de tolérance. Dans ce contexte, l'introduction de contaminants tels que des métaux dans l'environnement peut influencer l'évolution des plantes et des animaux en modifiant les forces évolutives et en créant des différences entre populations. Dans ce travail, l’attention a été portée sur les conséquences génétiques de la pollution métallique sur deux espèces, le ver de terre Lumbricus terrestris et une plante modèle Arabidopsis halleri. Deux approches différentes ont été utilisées pour étudier la réponse génétique à la contamination métallique : une approche de génétique des populations chez L. terrestris et une approche de génétique quantitative chez A. halleri. Tout d’abord, il s’est agi d’identifier et de valider de nouveaux marqueurs microsatellites chez L. terrestris. Ensuite, ces marqueurs ont été utilisés afin de caractériser la diversité génétique neutre chez des vers collectés sur des sites agricoles et urbanisés. Parallèlement, l'architecture génétique de la tolérance et de l'hyperaccumulation de Zn chez A. halleri a été explorée à l’aide d’un croisement intraspécifique entre une population métallicole et une population non métallicole. Une densité élevée de marqueurs SNP a été utilisée pour procéder à l'étape de cartographie QTL. / Natural habitats are more and more destructed and fragmented by urban expansion and human activities. The fragmentation of natural and agricultural areas by buildings and new infrastructures affects the size, connectivity and the quality of habitats. The populations of organisms inhabiting these anthropized territories are then more isolated. However, differentiation between populations of the same organism depends on demographic and genetic processes such as genetic drift, gene flow, mutation and natural selection. Only species that have developed special tolerance mechanisms can persist under changed environmental conditions. The introduction of contaminants such as metals in the environment may influence plants and animals evolution by modifying the evolutionary forces and thus generating differences between populations. In this work, attention was focused on the genetic consequences of metallic pollution on two species, the earthworm Lumbricus terrestris and the plant model Arabidopsis halleri. Two different approaches have been used to study the genetic response to metallic contamination: a population genetic approach was performed in L. terrestris and a quantitative genetic approach was carried on in A. halleri. First, it was a question of identifying and validating new microsatellite markers in L. terrestris. These markers were then used to characterize the neutral genetic diversity in worms collected from agricultural and urban sites. Secondly, genetic architecture of Zn tolerance and Zn hyperaccumulation was conducted investigated for the first time using an intraspecific crossing between metallicolous and non-metallicolous individuals of A. halleri. High density of SNP markers was used to proceed to the QTL mapping step.

Angiosperm sexual reproduction is unique and remarkable. Unlike for other living organisms, fertilisation involves the fusion of two sperm cells to two cell types of female gametophyte, the egg and central cell. This fertilization process is called double fertilisation. Fusion of the egg and the sperm yields a zygote whereas the interaction of the central cell and the sperm gives the nutritive endosperm. This fertilization process has been studied extensively for many years. However, despite these studies, relatively little is known at the molecular level about either of the plant gametes. This is primarily due to the difficulties in plant gamete isolation. Both plant gametes reside within enclosed tissues, pollen grains and embryo sacs. In this study, sperm isolation techniques were successfully developed for Brassica oleracea and Arabidopsis thaliana which ultimately utilised fluorescence-activated cell sorting (FACS) to obtain pure cell samples. Proteomic studies utilising two-dimensional protein electrophoresis and mass spectrometry (MS) were carried out on both semi-purified and FACS-purified sperm cells. In parallel with the proteomic studies a bioinformatics approach was taken which used sperm transcriptome data of maize, Plumbago zeylanica, rice and tobacco to identify homologues in Arabidopsis. Transcriptional analyses, RT-PCR and GFP translational fusion experiments were used to investigate these Arabidopsis sperm cell-expressed gene candidates. As a result, two sperm cell-expressed genes (At1g10090 and At5g39650) were identified and these are being analysed to confirm their functions in the reproductive process. Moreover, the sperm cell-expressed gene candidates derived from the bioinformatics were also screened for roles in plant reproduction by a reverse genetics approach (Arabidopsis T-DNA insertion mutagenesis plant screening) and eight genes were identified. In addition, for the first time, sperm cell size dimorphism was identified for Arabidopsis in this study utilising a GFP-labelled sperm line and confocal microscopy. Overall the techniques for sperm cell-expressed gene candidate selection were proven to be effective and will certainly facilitate further sperm cell-specific gene identification studies. Further the Arabidopsis sperm purification technique successfully developed in this project will surely be useful for any plant sperm cell studies in the future.

葉綠體是一重要的植物細胞器。除在光合作用上扮演重要角色外，葉綠體同時也與其他生理過程有關，例如油脂，氨基酸及植物賀爾蒙的生成等等。葉綠體的發育及作用由一系列的調控因子所調控。如有任何一個調控因子無法正常運作，都可能導致葉綠體什至整棵植物無法正常生長及發育。本人在碩士論文研究期間利用T-DNA激活標籤法進行了一項的bzr1-1D抑制子的突變體篩選實驗。在篩選過程中，意外發現一個幼苗階段致死的隱性白化突變體，命名為bt20。bzr1-1D是一個對油菜素内酯（brassinosteroids, BRs）高度敏感的單點突變體，而該單點突變發生於BR信號傳導中的轉錄因子BZR1基因上。我在該碩士論文研究中取得的主要結果如下：白化突變體的T-DNA被確定插入於擬南芥的第二染色體上，而該插入點與白化的表現形相連鎖。本人曾嘗試用多種方法克隆有關基因，並已發現若干候選基因。生理學研究顯示該白化突變體的葉綠體無法正常發育，而其光合作用也無法正常進行。該些實驗數據顯示BT20極有可能在葉綠體的生物发生及發育當中扮演重要角色。綜上所述，本人在該碩士論文的研究中發現一全新的、可能受BR調節的葉綠體發育調控因子，對於將來研究BR與葉綠體發育的關係具重要科學價值。 / Chloroplasts are a type of organelles in plants that not only capture light for photosynthesis but are also involved in other important biological processes such as the synthesis of lipids, amino acids and phytohormones. The development and functioning of a chloroplast are coordinated by multiple regulators. Loss of function of any of the regulators may result in abnormal development of chloroplasts and even the whole plant. In my thesis project, I characterized a recessive seedling-lethal albino mutant, named bt20, which was isolated from a genetic mutant screen for bzr1-1D suppressor mutants screen using a T-DNA activation tagging approach in Arabidopsis thaliana. bzr1-1D is a brassinosteroid (BR) hypersensitive mutant that was caused by a dominant mutation in the transcription factor BZR1 in the BR signaling pathway. I confirmed that the T-DNA insertion in the bt20 mutant is located in the chromosome 2 of Arabidopsis and is linked with the albino phenotype. Attempts for the mutant gene cloning have identified several candidate genes but the exact responsible gene(s) remain to be determined. Physiological studies indicated impairment of chloroplasts and photosynthesis within the mutant, suggesting that BT20 gene plays an important role in regulating chloroplast development. We anticipate that our study may lead to the identification of a novel regulator involved in the biogenesis and development of chloroplasts and may establish a molecular link between BR and chloroplast development. / Detailed summary in vernacular field only. / Wong, King Shing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 87-95). / Abstracts also in Chinese. / Thesis/Assessment Committee --- p.i / Statement --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Acknowledgements --- p.vi / Table of Contents --- p.viii / List of Figures --- p.xi / List of Tables --- p.xiii / Chapter Part 1 --- Introduction --- p.1 / Chapter 1.1 --- Brassinosteroids --- p.1 / Chapter 1.1.1 --- Brassinosteroids and its discovery --- p.1 / Chapter 1.1.2 --- Brassinosteroid signal transduction --- p.2 / Chapter 1.1.3 --- The transcription factor BZR1 --- p.5 / Chapter 1.1.4 --- bzr1-1D suppressor mutant screen and the identification of an albino mutant --- p.6 / Chapter 1.2 --- Chloroplast --- p.7 / Chapter 1.2.1 --- Chloroplasts in plants and their origin --- p.7 / Chapter 1.2.2 --- Structure of chloroplasts in plants --- p.8 / Chapter 1.2.3 --- Chloroplast development in cotyledons --- p.11 / Chapter 1.2.4 --- Nuclear gene transcription in chloroplast development --- p.14 / Chapter 1.2.5 --- Protein import for chloroplast development --- p.15 / Chapter 1.2.6 --- Chloroplast gene transcription --- p.16 / Chapter 1.2.7 --- Chloroplast RNA processing and protein translation --- p.18 / Chapter 1.2.8 --- Chloroplast protein processing --- p.19 / Chapter 1.2.9 --- Defects in regulators involving chloroplast development and biogenesis --- p.20 / Chapter 1.2.10 --- Potential relationship between BR and chloroplast development --- p.21 / Chapter Part 2 --- Materials and Methods --- p.22 / Chapter 2.1 --- Plant materials and growing conditions --- p.22 / Chapter 2.2 --- bzr1-1D suppressor mutant screen --- p.23 / Chapter 2.3 --- Genotypic PCR for bzr1-1D background and T-DNA insertion --- p.24 / Chapter 2.4 --- Fresh weight measurement of Arabidopsis plants --- p.26 / Chapter 2.5 --- Genetic crossing of Arabidopsis plants --- p.27 / Chapter 2.6 --- T-DNA insertion site identification --- p.28 / Chapter 2.7 --- Gene Cloning --- p.33 / Chapter 2.8 --- Reverse Transcription PCR (RT-PCR) --- p.36 / Chapter 2.9 --- Gene Cloning and vector constructions --- p.39 / Chapter 2.10 --- Gene transformation into Agrobacteria and Arabidopsis --- p.42 / Chapter 2.11 --- Transgenic plant screen --- p.44 / Chapter 2.12 --- Chlorophyll extraction and measurement --- p.45 / Chapter 2.13 --- Chlorophyll fluorescence assay --- p.46 / Chapter 2.14 --- Electronic microscopy (EM) --- p.48 / Chapter 2.15 --- Brassinolide (BL) and brassinozole (brz) treatments --- p.49 / Chapter Part 3 --- Results --- p.50 / Chapter 3.1 --- Summary of the bzr1-1D suppressor mutant screen --- p.50 / Chapter 3.2 --- Isolation of the albino bt20 mutant from the suppressor mutant screen --- p.52 / Chapter 3.3 --- T-DNA insertion site of bt20 is located in the chromosome 2 of Arabidopsis and is linked to the phenotype --- p.57 / Chapter 3.4 --- Genes flanking the insertion site are over-expressed --- p.59 / Chapter 3.5 --- T-DNA insertion does not cause base pair change in genes flanking the insertion site --- p.60 / Chapter 3.6 --- Over-expression of the selected candidate genes do not reproduce the albino phenotype --- p.62 / Chapter 3.7 --- bt20 seedlings have very low chlorophyll content --- p.64 / Chapter 3.8 --- bt20 has defect in chloroplast development --- p.65 / Chapter 3.9 --- bt20 has very low photosynthesis efficiency --- p.67 / Chapter 3.10 --- The expression of genes encoding plastid proteins in the bt20 mutant --- p.71 / Chapter 3.11 --- bt20 can respond to BL and brz treatment --- p.73 / Chapter 3.12 --- bt20 mutation does not affect the expression of BR biosynthetic gene --- p.75 / Chapter Part 4 --- Discussion --- p.77 / Chapter 4.1 --- bzr1-1D suppressor mutant screen and identification of the bt20 albino mutant --- p.77 / Chapter 4.2 --- Characterization of the bt20 albino mutant and cloning of the BT20 gene --- p.79 / Chapter 4.3 --- The possible role of BT20 role in plant growth and chloroplast development --- p.82 / Chapter 4.4 --- BR can improve the growth of the albino bt20 mutant --- p.85 / Chapter Part 5 --- Conclusion --- p.86 / Chapter Part 6 --- Reference --- p.87

H₂O₂作为信号分子参与基因表达调控和植物生理与发育过程。在本论文中，我在拟南芥中发现并详细研究了H₂O₂的一个新功能--诱导黑暗中的形态建成。光呼吸是植物内源H₂O₂的主要来源，因此我构建了GOX基因过量表达转基因植株，并在黑暗中模拟了光呼吸H₂O₂产生机制，显著诱导了幼苗的黑暗形态建成。细胞周期报告株系的研究表明，在黑暗中细胞周期停滞在G2/M过渡点，而H₂O₂的处理能帮助细胞通过G2/M时期，从而在黑暗中生长出顶端分生组织。这些结果提示H₂O₂可能参与细胞周期调控。 / 然后，我研究了拟南芥中H₂O₂的产生和分布模式。H₂O₂主要集中分布于维管组织、根冠、叶毛和子叶末端。GOX1和GOX2基因恰恰在H₂O₂分布区域较高表达。在gox1-4和gox2-1突变体中，H₂O₂水平显著下降，表明GOX1和GOX2是拟南芥中主要的H₂O₂产生基因。GOX1和GOX2在发芽早期就较高表达，并伴随有H₂O₂的积累。时序基因表达数据表明在发芽过程中GOX酶的底物并非产生于光呼吸，而是脂肪代谢。因此，我提出了两阶段H₂O₂产生机制模型，即：发芽过程中脂肪代谢提供底物，而生长过程中光呼吸提供底物。 / 最后，我探索了H₂O₂诱导黑暗形态建成过程中的信号通路。利用基因芯片分析和生物信息学方法从H₂O₂和光诱导基因的启动子序列中预测了GGGCC（R-box）调控元件。该元件集中分布于-50到-250bp区域，并且在小鼠和人的基因组的相同区域中也大量分布。EMSA实验验证了该调控元件是H₂O₂可诱导的。随后从其结合的核因子中分离鉴定了2个snoRNP蛋白：fibrillarin 2和NOP56，同时还克隆了数个C/D家族snoRNA序列。这些结果表明，在拟南芥中存在着一个参与H₂O₂信号通路的snoRNP/ R-box调控机制。 / For years H₂O₂ has been known as signal molecule involved in gene expression regulation. which affect particular physiological and developmental processes. In this study, I identified and characterized a new function of H₂O₂ in Arabidopsis development. Specifically, I found that H₂O₂ can induce seedling establishment in the dark. Nitrogen and sucrose have substantial influence on the effect of H₂O₂ induced seedling establishment. Because photorespiration is a major source of H₂O₂ production, when photorespiratory H₂O₂ producing pathway was mimicked in glycolate oxidase (GOX) over-expression lines, and significantly increased seedling establishment in the dark was observed. Using reporter lines, I found that the cell cycle is largely blocked at G2/M transition checkpoint in the dark, and H₂O₂ treatment helps the cells pass through the G2/M transition and finally establish shoot apical meristem. These results provide clues on the role of H₂O₂ roles in cell cycle regulation. / Then I characterized the H₂O₂ production and distribution pattern in Arabidopsis. H₂O₂ is accumulated at high level in root vasculature, root cap, veins and distal end in cotyledons, as well as leaf trichomes. The expression sites of GOX1 and GOX2 are in accordance with the places where H₂O₂ is distributed. Both peroxisomal and cytosol H₂O₂ share the same distribution pattern, indicating that the peroxisomal H₂O₂ might be the major source of H₂O₂ in Arabidopsis. In Arabidopsis gox1-4 and gox2-1 mutants, the H₂O₂ levels are both significantly reduced in roots and shoots, the sites where GOX1 and GOX2 are highly expressed, suggesting that GOX1 and GOX2 are likely the major H₂O₂ producing enzymes in Arabidopsis plants. Both GOX1 and GOX2 genes show early expression during germination, along with the H₂O₂ accumulation. Temporal expression data suggest the GOX substrates are not derived from photorespiration, rather from glyoxylate cycle that consumes lipid metabolites to provide carbon sources during germination. A working model is proposed to explain the possible H₂O₂ production mechanism. / Finally, I explored the signaling pathway involving H₂O₂ induced seedling establishment. A GGGCC motif (R-box) was identified from the upstream sequences of H₂O₂ and light induced genes in Arabidopsis. This motif is highly enriched within the -50 to -250 bp region of the induced genes, and it is also specifically distributed in the same region in mouse and human genome. EMSA experiments revealed that several nuclear factors (NF) are bound to this motif, and the binding activities alter under the H₂O₂ treatment. Two C/D family snoRNP proteins, fibrillarin 2 and NOP56 were identified from the R-box binding NFs. Several C/D family snoRNA, including R63, U24a and Z15, were also cloned from R-box binding NFs. These data suggest a snoRNP/ R-box regulation pathway may associate with H₂O₂ signaling in Arabidopsis. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Cheng, Han. / "December 2012." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 101-114). / Abstract also in Chinese. / Thesis/Assessment Committee --- p.ii / Statement: --- p.iii / Abstract --- p.iv / 摘要 --- p.vi / Acknowledgements --- p.vii / Table of Contents --- p.viii / List of Tables --- p.x / Lists of Figures --- p.xi / List of Abbreviations --- p.xii / Chapter 1 --- p.1 / General Introduction --- p.1 / Chapter 1.1 --- H₂O₂ metabolism and homeostasis in plants --- p.2 / Chapter 1.2 --- H₂O₂ Signaling pathway --- p.4 / Chapter 1.3 --- H₂O₂ roles on Plant Growth and Development --- p.6 / Chapter 1.4 --- Project Objectives --- p.7 / Chapter 2 --- p.9 / H₂O₂ Promote Post-germinative Establishment by Accelerating G2/M Transition --- p.9 / Chapter 2.1 --- Introduction --- p.10 / Chapter 2.2 --- Materials and Methods --- p.10 / Chapter 2.2.1 --- Establishment experiment in dark conditions --- p.10 / Chapter 2.2.2 --- GUS staining --- p.10 / Chapter 3.2.4 --- Constructs and generation of transgenic plants --- p.11 / Chapter 2.2.5 --- Ammonia content measurement --- p.12 / Chapter 2.2.6 --- Glycolate oxidase activity measurement --- p.12 / Chapter 2.3 --- Results --- p.12 / Chapter 2.3.1 --- H₂O₂ facilitated Post-germinative establishment in Arabidopsis --- p.12 / Chapter 2.2.2 --- Sucrose and nitrogen source are involved in the establishment --- p.13 / Chapter 2.3.3 --- Overexpression of glycolate oxidase in Arabidopsis --- p.14 / Chapter 2.3.4 --- The H₂O₂ facilitate the establishment by promoting the G2/M transition in the shoot meristem --- p.15 / Chapter 2.4 --- Discussion --- p.16 / Chapter 3 --- p.24 / Glycolate Oxidases mediate H₂O₂ distribution in Arabidopsis --- p.24 / Chapter 3.1 --- Introduction --- p.25 / Chapter 3.2 --- Materials and Methods --- p.26 / Chapter 3.2.1 --- Plant Material and growth conditions --- p.26 / Chapter 3.2.2 --- Phenotype analyses --- p.27 / Chapter 3.2.3 --- Constructs and generation of transgenic plants --- p.27 / Chapter 3.2.4 --- Transient expression --- p.29 / Chapter 3.2.5 --- Confocal microscopy, GUS staining, and stereomicroscopy --- p.29 / Chapter 3.2.6 --- PI, HPF and H2DCFDA staining --- p.30 / Chapter 3.2.7 --- RNA extraction, RT-PCR and quantitative RT-PCR --- p.30 / Chapter 3.2.8 --- Mutants Genotyping --- p.31 / Chapter 3.2.9 --- Expression Data Analysis --- p.32 / Chapter 3.3 --- Results --- p.32 / Chapter 3.3.1 --- Characterization of glycolate oxidase family genes --- p.32 / Chapter 3.3.2 --- Tissue-specific expression of GOX family genes --- p.34 / Chapter 3.3.3 --- H₂O₂ distribution pattern is in accord with GOX1 and GOX2 expression in root and leaf --- p.36 / Chapter 3.3.4 --- The H₂O₂ distribution pattern in the cytosol is similar to that in the peroxisome --- p.37 / Chapter 3.3.5 --- Gox1 and gox2 mutants show decreased H₂O₂ distribution in root and leaf --- p.38 / Chapter 3.3.7 --- A working model for H₂O₂ patterning in Arabidopsis --- p.39 / Chapter 3.3.6 --- Gox1 and gox2 mutants show delayed vegetative-to-reproductive transition --- p.41 / Chapter 3.4 --- Discussions --- p.42 / Chapter 3.4.1 --- H₂O₂ production in plant --- p.42 / Chapter 3.4.2 --- Glycolate can be derived from lipid breakdown during germination --- p.43 / Chapter 3.4.3 --- GOX functions in developmental phases transition regulation --- p.44 / Chapter 4 --- p.63 / The Identification of a New H₂O₂ signaling pathway in Arabidopsis --- p.63 / Chapter 4.1 --- Introduction --- p.64 / Chapter 4.2 --- Materials and Methods --- p.65 / Chapter 4.2.1 --- Plant materials and culture conditions --- p.65 / Chapter 4.2.2 --- Microarray Experiment and Data Analysis --- p.65 / Chapter 4.2.3 --- Crude Nuclear Protein Extraction --- p.66 / Chapter 4.2.4 --- Gel Mobility Shift Assay --- p.67 / Chapter 4.2.5 --- Purification and Identification of R-box Binding NF proteins --- p.68 / Chapter 4.2.6 --- Antibodies --- p.69 / Chapter 4.2.7 --- Western blotting --- p.69 / Chapter 4.2.8 --- Depletion tests --- p.69 / Chapter 4.2.9 --- Shift-Western assay --- p.70 / Chapter 4.2.10 --- Cloning and Detection of snoRNAs --- p.70 / Chapter 4.2.11 --- Bioinformatic Analysis --- p.71 / Chapter 4.3 --- RESULTS --- p.72 / Chapter 4.3.1 --- Transcriptome profiling revealed that H₂O₂ responsive genes also respond to light --- p.72 / Chapter 4.3.2 --- Identification of a putative novel motif from the promoters of co-upregulated genes --- p.73 / Chapter 4.3.3 --- Verification of the GGGCC Motif by Gel Mobility Shift Assay --- p.74 / Chapter 4.3.4 --- R-box is widely found In eukaryotic gene promoters --- p.75 / Chapter 4.3.5 --- R-box is ROS responsive in higher eukaryotic species --- p.76 / Chapter 4.3.6 --- NOP56 and fibrillarin 2 proteins were identified from NF+ samples --- p.77 / Chapter 4.3.7 --- Molecular cloning and detection of box C/D family snoRNA from NF+ samples --- p.79 / Chapter 4.4 --- Discussion --- p.80 / Chapter 5 --- p.99 / Conclusions and Perspectives --- p.99 / Chapter 5.1 --- Conclusions --- p.100 / Chapter 5.2 --- Perspectives --- p.101 / References --- p.103

Um dos grandes desafios da pesquisa agrícola atual é desenvolver estratégias para obtenção de plantas mais tolerantes, portanto, continuar ampliando o conhecimento sobre os mecanismos pelos quais as plantas respondem à seca, é essencial para a identificação de rotas metabólicas envolvidas no processo de defesa e o desenvolvimento de genótipos cada vez mais adaptados. Neste trabalho, para a obtenção de plantas de soja tolerantes à seca, a construção RD29A:AtDREB1A a qual confere tolerância ao déficit hídrico foi inserida em cultivares de soja e a biossegurança alimentar da proteína DREB1A/CBF3 de A. thaliana foi avaliada. Também, os extratos protéicos das plantas submetidas ao estresse hídrico foram comparados para a identificação de proteínas de soja diferencialmente expressas que podem estar envolvidas nas respostas à seca. A avaliação de biossegurança alimentar da proteína AtDREB1A in silico mostrou que a proteína não possui características de toxicidade, alergenicidade, antinutricionais, ou sítios de N-glicosilação, assim como, de atividade hemolítica sob eritrócitos de humanos atestado com a proteína AtDREB1A produzida in vitro, indicando ausência de efeitos adversos. A inserção da construção RD29A:AtDREB1A e RD29A:GUS em soja, demonstrou que o promotor RD29A e o fator de transcrição AtDREB1A de A. thaliana são ativados aumentando a tolerância ao déficit hídrico em soja. Vinte linhagens transgênicas foram obtidas por biobalística e apresentaram estabilidade do transgene. Análises histoquímicas confirmaram a indução do promotor RD29A em condições de desidratação e o aumento de expressão de genes de soja GmPip1 GmGols regulados pela proteína DREB1A também foi confirmado por RT-qPCR. Diferenças anatômicas significativas não foram observadas. Em média, os parâmetros fisiológicos foram superiores nas linhagens transgênicas, quando comparadas à sua isolinha BR16. Embora características agronômicas mais adaptativas relacionadas à produção não foram evidentes em casa de vegetação, há uma boa indicação de que a estratégia pode melhorar a tolerância à seca em plantas de soja. Para isso, experimentos a campo já estão sendo conduzidos para uma melhor caracterização agronômica e fisiológica. Na busca por ampliar os conhecimentos sobre os eventos envolvidos nas respostas à seca, a comparação dos extratos protéicos de soja BR16 não transgênica e BR16(P58) geneticamente modificada (GM) com a construção RD29A:AtDREB1A em condições controle e sob déficit hídrico, possibilitou a identificação de “spots” comuns aos dois tratamentos assim como de “spots” diferencialmente expressos, através da metodologia de 2-DE. Nos tratamentos de déficit hídrico, 25 “spots” foram identificados em BR16 não GM e 34 “spots” em P58 GM, entretanto, análises de espectrometria de massa ainda estão em andamento e pesquisas em bancos dados serão fundamentais para a identificação das proteínas diferencialmente expressas em resposta ao déficit hídrico. / One of the challenges in agriculture is to develop strategies to obtain plants with higher drought tolerance. Therefore, the identification of metabolic pathways and understanding of the mechanisms by which plants respond to drought is an essential tool for the development of more adapted genotypes. In this work, the RD29A:AtDREB1A genetic construct which was reported to confer water deficit tolerance was inserted into soybean cultivars, and the food safety of Arabidopsis thaliana AtDREB1A/CBF3 protein was evaluated. Also, protein extracts from soybean plants submitted to water stress conditions were compared to identify differentially expressed soybean proteins involved in drought responses. The in silico evaluation of AtDREB1A protein showed that the protein has no evidence of toxicity, allergenicity, antinutritional features or N-glycosylation sites. No hemolytic activity on human erythrocytes assayed in vitro with AtDREB1A protein was detected, indicating no adverse effects. The insertion of RD29A:AtDREB1A and RD29A:GUS constructs in soybean showed that the RD29A stress-inducible promoter and the AtDREB1A transcription factor were activated and improved drought tolerance in soybean. Using bioballistic transformation, we obtained twenty stably transformed soybean lines. Histochemical analysis confirmed the induction of the RD29A promoter under dehydration conditions and increased expression of two soybean genes activated by AtDREB1A GmPip1 and GmGols were confirmed by RTqPCR. No anatomical differences were observed. On average, physiological parameters were superior in the transgenic line BR16(P58) when compared to the isoline BR16. Although agronomic traits related to higher adaptive production were not evident in greenhouse, there is a good indication that the strategy can improve drought tolerance in soybean. To establish trait efficacy, field experiments are already being conducted to obtain relevant agronomic and physiological data. To better understand the molecular events involved in drought responses, a comparison using protein extracts from genetically modified (GM) BR16(P58) and non-GM soybean BR16 in watered control conditions and under water deficit treatment allowed the identification of common proteins to both treatments as well as of proteins that are differentially expressed between treatments using 2-dimension gel electrophoresis (2-DE). In the water deficit treatment 25 differentially expressed protein spots were identified in non-GM isoline BR16 and 34 spots in BR16(P58). Subsequent analysis of these spots using mass spectrometry is still been conducted and peptide information in available public databases will be essential for identification of differentially expressed proteins.

Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 123-132). Also available on the World Wide Web.

Telomeres are important protein-DNA structures at the ends of linear eukaryotic chromosomes that are necessary to prevent chromosome fusions and exonuclease attack. We found that telomere tracts in Arabidopsis are fairly uniformly distributed throughout a size range of 2-9kb. Unexpectedly, telomeres in WS plants displayed a bimodal size distribution with some individuals exhibiting 4-8 kb telomeres and others 2-5 kb telomeres. We also examined the dynamics of telomere tracts on individual chromosome ends. Following the fate of telomeres in plants through successive generations, we found that the shortest telomeres were typically elongated in the subsequent generation, while the longest telomeres were usually shortened. Thus, telomere length homoeostasis is achieved through intermittent telomerase action on shorter telomeres to attain an optimal size.Single-strand telomere binding proteins were also analyzed. Four major telomere binding protein complexes from cauliflower were identified and their DNA-binding properties characterized. The DNA-binding component of one of the complexes was purified and analyzed by mass-spectrometry. Peptide mass data was used to search for putative protein candidates from the Arabidopsis thaliana database. Additionally, two Arabidopsis genes, AtPot1 and AtPot2, were identified and characterized. The genes encode two single-strand telomeric DNA binding proteins. AtPot1 and AtPot2 proteins can homo- and heterodimerize in vitro. Pot1 protein predominantly localizes to the nucleolus, whereas Pot2 is exclusively nuclear. Plants over-expressing full-length Pot1 and Pot2 proteins had no obvious phenotype, while over-expression of P2DBD and P1∆DBD caused moderate telomere shortening. Plants over-expressing P2DBD had severe morphological and reproductive defects, multiple chromosome abnormalities and aneuploidy. Over-expression of a chimeric protein DBD-P1∆DBD led to rapid telomere shortening, confirming the involvement of Arabidopsis Pot proteins in telomere length maintenance. Intriguingly, telomerase in DBD-P1∆DBD-EYFP plants is inactivated, suggesting that Pot proteins are also involved in regulation of telomerase activity. The analysis of Arabidopsis telomeres and telomere binding proteins will provide additional information towards understanding the role of the telomeric nucleoprotein complex in eukaryotic chromosome biology.