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.

Environmental signals such as photoperiod and temperature provide plants with seasonal information, allowing them to time flowering to occur in favourable conditions. Most ecotypes of the model plant Arabidopsis thaliana flower earlier in long photoperiods and after prolonged exposure to cold (vernalization). The vernalized state is stable through mitosis, but is not transmitted to progeny, suggesting that the vernalization signal may be transmitted via a modification of DNA such as cytosine methylation. The role of methylation in the vernalization response is investigated in this thesis. ¶ Arabidopsis plants transformed with an antisense construct to the cytosine methyltransferase METI (AMT) showed significant decreases in methylation. AMT plants flowered significantly earlier than unvernalized wildtype plants, and the promotion of flowering correlated with the extent of demethylation. The flowering time of mutants with decreased DNA methylation (ddm1) was promoted only in growth conditions in which wildtype plants showed a vernalization response, suggesting that the early flowering response to demethylation operated specifically through the vernalization pathway. ¶ The AMT construct was crossed into two late flowering mutants that differed in vernalization responsiveness. Demethylation promoted flowering of the vernalization responsive mutant fca, but not of the fe mutant, which has only a slight vernalization response. This supports the hypothesis that demethylation is a step in the vernalization pathway. ¶ The role of gibberellic acid (GA) in the early flowering response to demethylation was investigated by observing the effect of the gai mutation, which disrupts the GA signal transduction pathway, on flowering time in plants with demethylated DNA. The presence of a single gai allele delayed flowering, suggesting that the early flowering response to demethylation requires a functional GA signal transduction pathway, and that demethylation increases GA levels or responses, directly or indirectly. ¶ In most transgenic lines, AMT-mediated demethylation did not fully substitute for vernalization. This indicates that part of the response is not affected by METI-mediated methylation, and may involve a second methyltransferase or a factor other than methylation. A second Arabidopsis methyltransferase, METIIa, was characterized and compared to METI. The two genes are very similar throughout the coding region, and share the location of their eleven introns, indicating that they diverged relatively recently. Both are transcribed in all tissues and at all developmental stages assayed, but the level of expression of METI is significantly higher than that of METIIa. The possible functions of METI, METIIa, and other Arabidopsis cytosine methyltransferase genes recently identified are discussed.

This dissertation describes the cloning and characterization of the TOUSLEDlike kinases genes of maize (ZmTLKs). The TOUSLED-like kinases (TLKs) are a conserved family of nuclear Ser/Thr kinases in higher eukaryotes. The maize genome has three TOUSLED-like kinase genes (ZmTLK1, ZmTLK2, and ZmTLK3). Based upon sequence similarity, the ZmTLKs are divided into two classes, the ZmTLK1 and the ZmTLK2/3 class. The origins of these genes can be inferred from their map positions and relationships with TLKs in other Zea species. The ZmTLK1 and ZmTLK2 genes occupy syntenous positions on chromosome arms 1L and 5S in the maize genome. There are two equivalent classes of TLK genes in other Zea species, altogether indicating that the two ZmTLK classes are orthologous genes from the precursor species of maize, an ancient allotetraploid. Gene expression studies of ZmTLKs show that there is a higher level of expression in tissues undergoing DNA synthesis. This is consistent with studies of TLKs in animal systems that show involvement in chromatin assembly/remodeling activities during DNA replication and repair, as well as in transcription. The highest level of gene expression for the ZmTLK2/3 class was observed during development of the endosperm, in a period of massive nuclear endoreduplication. ZmTLK1 is not upregulated in endoreduplicating endosperm, suggesting functional divergence between the two classes of ZmTLK genes. The function of the ZmTLKs was examined by testing whether maize TLK genes could complement the tousled mutant of Arabidopsis. In Arabidopsis thaliana, recessive mutations in the single copy TOUSLED (TSL) gene cause moderate vegetative and severe floral defects, suggesting that TLKs may play a role in gene expression modulation through chromatin remodeling. The ZmTLK proteins are 84% identical to TSL in the catalytic region and 45 - 49% at the N-terminal regulatory domain. However, structural features of the N-terminal region domains of the ZmTLKs are similar to that of TSL. Arabidopsis tsl-1 mutant plants were transformed with ZmTLK2, under the control of the CaMV 35S promoter. These plants showed wild-type Arabidopsis phenotype, indicating that in spite of their sequence differences, ZmTLK2 and TSL interact with the same substrates and regulatory partners and are functionally equivalent. / Graduation date: 2005

Graduation date: 2001

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 153-163).

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.