Mitochondria are ubiquitous eukaryotic organelles which carry out a range of essential functions, most notably the production of ATP through the process of oxidative phosphorylation. While the main biochemical function of mitochondria was established over 50 years ago, the processes which control mitochondrial morphology are, at present, poorly understood. The thesis aims to add to our knowledge of the factors that control mitochondrial morphology and dynamics in the model plant species, Arabidopsis thaliana. The phenotypic characteristics of two novel mitochondrial morphology mutants, motley mitochondria I (mmtl) and network mitochondria (nmt), were examined and quantified. mmtl has an increased heterogeneity of mitochondrial plan area relative to wild-type, which is matched by a similar chloroplast phenotype. nmt exhibits a reticular mitochondrial morphology, similar to the mitochondria found in yeast and animals. Genetic mapping of the two mutant loci has established that mmtl resides on a short region of chromosome 11w, hile nmt was mapped to a small area of chromosome V. This thesis describes the identification and functional analysis of two novel orthologs of yeast and animal mitochondrial division genes. Using TDNA reverse genetics, it is shown that disruption of the dynamin-like DRP3A or BIGYIN (an Arabidopsis orthologue of yeast FISI) led to an increase in mitochondrial plan area, which is coupled with a decrease in the number of physically discrete mitochondria per cell. Finally, the morphology and behaviour of Arabiclopsis mitochondria is investigated upon the induction of cell death. Abiotic stress treatments that induce cell death led to fast and irreversible changes in mitochondrial morphology. The role of these changes, as possible early indicators of cell death, are discussed.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Arabidopsis thaliana Acyl-CoA-Binding Protein 3, one of six acyl-CoA-binding proteins, is unique by the C-terminal location of its acyl-CoA-binding (ACB) domain. It promotes autophagy (ATG)-mediated leaf senescence and confers resistance to Pseudomonas syringae pv. tomato DC3000. To understand the regulation of ACBP3, a 1.7 kb 5’-flanking region of ACBP3 and its deletion derivatives were characterized using β-glucuronidase (GUS) reporter gene fusions. A 374 bp minimal fragment (-151/+223) could drive GUS expression while a 1698 bp fragment (-1475/+223) conferred maximal activity. Further, histochemical GUS staining analysis on transgenic Arabidopsis harboring the largest (1698 bp) ACBP3pro::GUS fusion displayed ubiquitous expression in floral organs and vascular bundles of leaves and stems, consistent with previous results that extracellularly localized ACBP3 functions in plant defense. A 160 bp region (-434/-274) induced GUS expression in extended darkness and conferred down-regulation in extended light. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting assay showed that the DNA binding with one finger box (Dof-box, -341/-338) interacted specifically with leaf nuclear proteins from dark-treated Arabidopsis while GT-1 (-406/-401) binds both dark- and light-treated Arabidopsis, suggesting that Dof and GT-1 motifs are required to mediate circadian regulation of ACBP3. Moreover, GUS staining and fluorometric measurements revealed that a 109 bp region (-543/-434) was responsive to phytohormones and pathogens. Within this 109 bp region, an S-box of AT-rich sequence (-516/-512) was identified to bind nuclear proteins from pathogen-infected Arabidopsis leaves, providing the basis for pathogen-inducible regulation of ACBP3 expression. Hence, three cis-responsive elements (Dof, GT-1 and S-box) in the 5’-flanking region of ACBP3 were demonstrated to participate in the regulation of ACBP3. The regulation of ACBP3 by circadian control is not surprising given that defense genes are now known to be circadian-regulated; infection being anticipated at dawn coinciding with pathogen activity in spore dispersal during the light period. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

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.

Currently research has focused on insulators from non-plant species such as the fruit fly, Drosophila melanogaster. The accumulated data suggests that many different insulator sequences exist in D. melanogaster, each one containing its own different primary binding protein, while sharing similar secondary binding proteins. Together, they produce chromatin loops separating enhancers and promoters into distinct domains preventing cross-talk between them. Is this the case in plants? To approach this question, we have investigated enhancer-blocking insulators in the model plant Arabidopsis thaliana using two unrelated approaches. Firstly, we have developed an assay for the direct selection of insulators in Arabidopsis thaliana using a random oligonucleotide library. This assay helped us to define four novel insulator sequences named InI-3, InII-12, InIII-50, and InIII-78. Secondly, we have used genetic analyses to characterize potential insulator sequences originally from three non-plant species: UASrpg from the fungus Ashbya gossypii, BEAD1c from human T-cell receptors, and gypsy from D. melanogaster, that have been reported to function in A. thaliana. Our findings suggest that non-plant insulators and their protein binding sites function in plants and support the model of multiple, functional, different insulator sequences as was found in D. melanogaster. They also argue for the conservation of insulator mechanisms across species.

在酵母和动物细胞中，内膜蛋白（EMP）隶属于进化上保守的九跨膜结构域（TMD）蛋白家族，此类蛋白的共同结构特征是有一个很长的N 末端，紧接着九个跨膜结构域后面连着暴露于胞质的C 末端短肽。在黏菌以及酵母中，EMP 蛋白被发现参与蛋白分泌功能以及细胞的贴壁生长。拟南芥基因组中有12 个EMP 编码基因，关于它们所编码蛋白质的定位以及功能甚少有研究报道。在此项研究中，借助于不同的生化以及细胞生物学手段，包括瞬时表达、共聚焦成像、电子显微镜分析、pull down 相互作用蛋白捕获以及质谱分析，我将主要研究拟南芥中EMP12 蛋白的亚细胞定位以及分选信号和蛋白靶定机理。通过研究我发现：1）在拟南芥植物中，内源性的EMP12 蛋白（通过EMP12 特异性抗体标记）和绿色荧光蛋白标记的GFP-EMP12 蛋白都定位于高尔基体；2）C 末端连接的GFP 导致 EMP12-GFP 融合蛋白错误地定位到后高尔基体细胞器，并最终被运送到液泡而降解；3）EMP12 蛋白的C 末端有两个分选信号：内质网输出信号（FV/Y）和一个新发现的高尔基体滞留信号（KXD/E），这两个分选信号分别和COPII 和COPI 囊泡相互作用从而实现其功能；4）把EMP12 的高尔基体滞留信号连接到其他后高尔基体定位的膜蛋白时可以滞留它们在高尔基体。EMP12 中发掘的内质网输出信号和高尔基体滞留信号在所有的植物EMP 蛋白家族中都是非常保守的，这也预测了植物EMP 蛋白家族都通过类似的分选途径而定位于高尔基体并且预示这些保守的分选信号对于植物EMP 蛋白家族的靶定是非常重要的。 / Endomembrane Proteins (EMPs), belonging to the evolutionarily conserved transmembrane nine superfamily in yeast and mammalian cells, are characterized by the presence of a large lumenal N-terminus, nine transmembrane domains (TMD) and a short cytoplasmic tail (CT). In the slime mold and yeast, it has been reported that EMP family proteins are involved in protein secretion function and cell adhesion growth. The Arabidopsis genome contains 12 EMP members (EMP1 to EMP12) with little information about their protein subcellular localization and function. Here I studied the subcellular localization and targeting mechanisms of EMP12 in Arabidopsis through a combination of biochemical and cell biological approaches including transient expression, confocal observation, electron microscopy, pull down and mass spectrometry. I found that 1) both endogenous EMP12 (detected by EMP12 antibodies) and green fluorescent protein (GFP)-EMP12 fusion localized to the Golgi apparatus in transgenic Arabidopsis plants; 2) GFP fusion at the C-terminus of EMP12 caused mis-localization of EMP12-GFP to reach post-Golgi compartments and vacuoles for degradation in Arabidopsis cells; 3) EMP12 CT contained dual sorting signals: an ER export motif (FV/Y) and a novel Golgi retention signal (KXD/E) that interacted with COPII and COPI subunits respectively to achieve their ER export or Golgi retention functions; 4) the Golgi-retention motif of EMP12 retained several post-Golgi membrane proteins within the Golgi apparatus in gain-of-function analysis. These sorting signals are highly conserved in all the plant EMP isoforms, thus likely representing a general mechanism for EMP targeting in plant cells. / Detailed summary in vernacular field only. / Gao, Caiji. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 86-94). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract 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 Tables --- p.XI / List of Figures --- p.XII / List of Abbreviations --- p.XIV / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- The Plant Secretory Pathway --- p.2 / Chapter 1.2 --- Sorting Signals for Membrane Protein Trafficking between ER and Golgi --- p.5 / Chapter 1.3 --- Endomembrane Proteins (EMPs) in Non-plant Species --- p.7 / Chapter 1.4 --- Endomembrane Proteins (EMPs) in Plants --- p.8 / Chapter 1.5 --- Objectives of this Research --- p.11 / Chapter Chapter 2 --- Materials and Experimental Procedures --- p.12 / Chapter 2.1 --- Plasmid Construction --- p.13 / Chapter 2.2 --- Plant Materials and Transient Expression in Protoplasts --- p.17 / Chapter 2.3 --- Immunofluorescence Study and Confocal Microscopy --- p.17 / Chapter 2.4 --- Electron Microscopy (EM) Study --- p.18 / Chapter 2.5 --- Antibodies, Protein Preparation and Western Blot Analysis --- p.21 / Chapter 2.6 --- In Vitro Binding Assay of COPI and COPII Coat Proteins to Sorting Motifs --- p.22 / Chapter 2.7 --- Mass Spectrometry (MS) Identification of Binding Proteins --- p.23 / Chapter 2.8 --- Topology Analysis and Protease Protection Assay --- p.23 / Chapter Chapter 3 --- Results --- p.25 / Chapter 3.1 --- At EMP12 is a Golgi-localized Multiple TMDs Protein --- p.26 / Chapter 3.1.1 --- Trypsin Digestion and Topology Analysis of EMP12 --- p.26 / Chapter 3.1.2 --- Golgi Localization of Endogenous EMP12 and GFP-EMP12 Fusion in Arabidopsis Plant --- p.29 / Chapter 3.1.3 --- Golgi Localization of GFP-EMP12 Fusion in Arabidopsis Protoplasts --- p.36 / Chapter 3.2 --- The Cytosolically Exposed C-terminal Region Contains Essential ER Export Signals for the Trafficking of EMP12 from the ER to the Golgi --- p.38 / Chapter 3.2.1 --- C-terminus is Essential for ER export of EMP12 --- p.38 / Chapter 3.2.2 --- Identification of FV/Y Residues as the ER Export Signals for EMP12 --- p.40 / Chapter 3.2.3 --- The ER Export Signals, FV/Y, Can Interact with COPII Subunit Sec24 --- p.46 / Chapter 3.3 --- The C-terminal Fused GFP-tag Causes Mislocalization of EMP12-GFP Fusion to TGN, PVC and Vacuole --- p.48 / Chapter 3.4 --- The EMP12 CT Contains a Novel KXD/E Motif for Golgi Retention --- p.51 / Chapter 3.4.1 --- Identification of the KXD/E Motif for Retaining the Mislocalized EMP12-GFP in the Golgi Apparatus --- p.51 / Chapter 3.4.2 --- The Mislocalized EMP12-GFP Fusions Go to Vacuole via PVC for Degradation --- p.58 / Chapter 3.4.3 --- Western Blot Analysis of Protoplasts Expressing various EMP12-GFP fusions --- p.61 / Chapter 3.5 --- The KXD/E Motif Shows Similar Golgi Retention Function for Other Plant EMP Homologues --- p.63 / Chapter 3.6 --- The KXD/E Motif Interacts with COPI Vesicle to Achieve its Golgi Retention Function --- p.65 / Chapter 3.7 --- The RNIKCD Functions as a Golgi Retention Motif for Post-Golgi Membrane Proteins --- p.69 / Chapter 3.7.1 --- The RNIKCD Can Retain the SCAMP1-GFP in the Golgi Apparatus --- p.69 / Chapter 3.7.2 --- The RNIKCD Motif Causes Partial Golgi Retention of TPK1-GFP --- p.71 / Chapter 3.8 --- Localization Patterns of Singly-expressed Various EMP12 Fusions and Their Mutants in Arabidopsis Protoplasts --- p.74 / Chapter Chapter 4 --- Discussions, Conclusions and Perspectives --- p.76 / Chapter 4.1 --- Discussions --- p.77 / Chapter 4.1.1 --- The Position of GFP Tag Affects the Proper Golgi Localization of EMP12 --- p.77 / Chapter 4.1.2 --- Multiple Sorting Signals and Proper COPII Vesicle Function are Involved in ER Export of EMP12 --- p.79 / Chapter 4.1.3 --- KXD/E Motif and COPI Vesicle Mediate Golgi Localization of EMP12 --- p.80 / Chapter 4.2 --- Conclusions and Working Model of EMP12 Trafficking --- p.83 / Chapter 4.3 --- Future Perspectives --- p.85 / References --- p.86 / List of Publications --- p.95

No description available.

WRKY transcription factors (TFs) belong to a large family of regulatory proteins in plants that modulate many plant processes. Extensive studies have been conducted on WRKY-mediated defense response in Arabidopsis thaliana and many crop species. This study aims to investigate the potential roles and contributions of WRKY TFs regulation in improving defense response in the resynthesized Arabidopsis allotetraploids (Arabidopsis suecica) from two related autotetraploid progenitors, Arabidopsis thaliana (At4) and Arabidopsis arenosa (Aa). Upon infection by Pseudomonas syringae (Pst), the allotetraploids has showed enhanced resistance against the pathogen when compared to the parents. Rapid induction of WRKY18, WRKY40, WRKY38, WRKY53, WRKY6; MAP kinase pathway related genes, WRKY33, PAD3; SA-pathway related genes, ICS1, EDS1, PBS3, MYB31; was evident in response to Pst and salicylic acid treatment in the allotetraploids. Cleaved amplified polymorphic sequences analysis further revealed that the AtWRKY18, AaWRKY40, AtWRKY33, and AtWRKY60 alleles expressed at higher levels when compared to their respective homoeologs in the allotetraploids, suggesting potential altered protein-protein interaction networks in the hybrids. Therefore, a split-luciferase complementation assay was used to characterize and quantify protein-protein interaction among these homoeologous WRKYs in the allotetraploids. Results showed that preferential protein-protein interactions exist for the cis-interacting AtWRKY18/AtWRKY18 homodimer or trans-interacting AtWRKY18/AaWRKY40 heterodimer when compared to the respective interacting complexes. In addition, differential affinities of WRKY18 and WRKY40 homo- and hetero- dimers toward the W-boxes at the WRKY60 promoter were observed. In the allotetraploids, PR1 expression was repressed under basal state when compared to the progenitors. Although PR1 is expressed at a higher level in A. thaliana, its expression fold change was higher and faster in the all otetraploids upon salicylic acid treatment. Transient expression of WRKY18 or WRKY40 homodimer in various combinations induced differential expression of PR1 gene in their respective wrky18 and wrky40 Arabidopsis thaliana mutants. In contrast, similar PR1 induction by homodimer in various combinations was observed when they were transiently expressed in the allotetraploids. In addition, transgenic AtWRKY18 overexpression plant displayed enhanced disease resistance against Pst when compared to AaWRKY18 overexpression lines. Such enhanced disease resistance was found to associate with the higher expression of PR1 and PR2 in AtWRKY18 transgenic lines. Moreover, differential Pst-induced expression of the direct targets (ICS1, EDS1 and PBS3) of WRKY18 in the Arabidopsis AtWRKY18 and AaWRKY18 overexpressors supported a biological difference between the At and Aa homodimers in mediating the targets regulation, thus contributing to the difference in disease responses. Overall, our findings suggested that the rapid differential alleles expression and altered protein-protein or protein-DNA interactions of WRKY transcription factors could contribute to the improved defense in the allotetraploids, providing a molecular basis of for heterotic phenotype development in hybrids.

Cui, Yong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 73-84). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vi / Table of Contents --- p.vii / List of Figures --- p.xi / List of Supplemental Tables --- p.xiii / List of Abbreviations --- p.xiii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- The plant secretory and endocytosis pathways --- p.2 / Chapter 1.2 --- Rab proteins --- p.4 / Chapter 1.2.1 --- Overview of the small GTPases --- p.4 / Chapter 1.2.2 --- Function of Rab proteins in Arabidopsis --- p.6 / Chapter 1.3 --- Prevacuolar compartments --- p.9 / Chapter 1.3.1 --- PVCs in mammalian and yeast cells --- p.9 / Chapter 1.3.2 --- PVCs in plant cells --- p.9 / Chapter 1.4 --- Vacuolar Sorting Receptors --- p.10 / Chapter 1.5 --- Project objectives --- p.10 / Chapter CHAPTER 2 --- Early and Late Prevacuolar Compartments in Arabidopsis thaliana Cells --- p.12 / Chapter 2.1 --- Introduction --- p.13 / Chapter 2.2 --- MATERIALS AND METHODS --- p.19 / Chapter 2.2.1 --- Plasmid Construction --- p.19 / Chapter 2.2.2 --- Plants materials and growth conditions --- p.19 / Chapter 2.2.3 --- Transient Expression of Arabidopsis suspension cultured cells --- p.20 / Chapter 2.2.4 --- Confocal imaging studies --- p.21 / Chapter 2.3 --- RESULTS --- p.23 / Chapter 2.3.1 --- Organelle markers serve as a tool to study biogenesis and turnover of PVCs --- p.23 / Chapter 2.3.2 --- AtRab5 and AtRab7 proteins show distinct but closely associated patterns in the PVC-to-Vacuole pathway --- p.26 / Chapter 2.3.3 --- AtRab5 and AtRab7 proteins localize on the distinct organellein Arabidopsis thaliana protoplasts --- p.32 / Chapter 2.3.4 --- AtRab5 proteins are closely associated with AtRab7 proteins --- p.35 / Chapter 2.3.5 --- ARA7-Q69L proteins recruit a SNARE complex onto the enlarged PVCs --- p.37 / Chapter 2.4 --- Discussion --- p.40 / Chapter 2.4.1 --- PVC dynamics in Arabidopsis cells --- p.40 / Chapter 2.4.2 --- AtVSR and its point mutation form defined different stages of PVCs in Arabidopsis thaliana protoplasts --- p.41 / Chapter 2.4.3 --- AtRab7 proteins localized on the tonoplast and newly defined late PVCs --- p.41 / Chapter CHAPTER 3 --- AtRab7 proteins play a critical role in mediating vacuolar trafficking in Arabidopsis thaliana Cells --- p.43 / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- MATERIALS AND METHODS --- p.45 / Chapter 3.2.1 --- Plasmid Construction --- p.45 / Chapter 3.2.2 --- Plants materials and growth conditions --- p.45 / Chapter 3.2.3 --- Transient Expression of Arabidopsis suspension cultured cells --- p.45 / Chapter 3.2.4 --- Confocal imaging studies --- p.45 / Chapter 3.2.5 --- Drug treatment --- p.46 / Chapter 3.3 --- RESULTS --- p.48 / Chapter 3.3.1 --- Mutations at GTP-binding motifs and the effector domain affect the subcellular localization of AtRabG3e --- p.48 / Chapter 3.3.2 --- "AtRabG3e-T22N induced vacuolation of YFP-ARA7 marked PVCs, which remains separated from ER, Golgi and TGN but colocalizes with early PVC markers" --- p.51 / Chapter 3.3.3 --- AtRab7-T22N inhibits vacuolar trafficking of cargo proteins --- p.54 / Chapter 3.3.4 --- Wortmannin-induced vacuolation of late PVCs in transgenic plants --- p.57 / Chapter 3.4 --- Discussion --- p.59 / Chapter 3.4.1 --- The proper targeting of AtRab7 proteins --- p.59 / Chapter 3.4.2 --- AtRab5 and AtRab7 proteins are essential for vacuolar protein trafficking --- p.59 / Chapter CHAPTER 4 --- Summary and Future Perspectives --- p.61 / Chapter 4.1 --- Summary --- p.62 / Chapter 4.1.1 --- Localization of AtRab5 and AtRab7 proteins on different populations of PVCs --- p.62 / Chapter 4.1.2 --- Functions of AtRab7 proteins in Arabidopsis cells --- p.63 / Chapter 4.1.3 --- The Rab conversion maturation model --- p.63 / Chapter 4.2 --- Future perspectives --- p.64 / References --- p.73

Arabidopsis has emerged as an important model system for molecular plant biology. The extensive resources available for Arabidopsis make it an attractive system to study the molecular mechanisms involved in early seed development. During the early stages of seed development Arabidopsis endosperm is syncytial and proliferates rapidly through repeated rounds of mitosis without cytokinesis. This stage of endosperm development is both important in determining final seed size and is a model for studying various aspects of cellular and molecular biology, such as the cell cycle and genomic imprinting. However, the small size of Arabidopsis seed, the syncytial nature of the proliferative endosperm, and the surrounding maternal tissues make high throughput molecular analysis of the early endosperm technically difficult. To get around this we used laser capture microdissection to enable transcript analysis of the early proliferative endosperm of Arabidopsis at 4 days after pollination (DAP). Microarray results identified several thousand genes with endosperm expression, including many that were endosperm preferred. A number of genes were validated by relative quantification PCR and were consistent with the findings of the microarray. Meta analysis of the endosperm transcriptome revealed a developmental program dominated by mitosis and under the influence of several phytohormones, predominated by cytokinin signaling. The list of endosperm-preferred genes included all characterised imprinted genes in Arabidopsis. Imprinting is an epigenetic phenomenon by which genes are expressed predominantly from either their paternal or their maternal allele and very few imprinted genes have been identified in plants. The mono-allelic expression of the characterised imprinted genes appears to be limited to the endosperm where they provide important regulatory controls for seed development via direct effects on endosperm development. Genes from the endosperm-preferred list were screened for mono-allelic expression using sequence polymorphisms between the Colombia and Landsberg erecta ecotypes. We generated PCR products that spanned the polymorphisms of 67 genes from template obtained by laser capture of endosperm tissue from hybrid seed. Sequence analysis revealed three genes which gave strong allelic bias toward the maternal allele (At2g32460, At1g55550 and At2g21420) and one biased for the paternal allele (At1g47840). In summary, laser capture microdissection has enabled high-resolution transcript analysis of the proliferative stage of Arabidopsis endosperm development. The data generated provides a useful resource providing novel insight into early seed development, facilitating both identification of endosperm expressed and novel imprinted genes.