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

A sorghum gene SbLRR2, which is predicted to encode a simple extracellular leucine-rich repeat (LRR) protein, was previously isolated among a collection of fungal pathogen-induced sorghum cDNA clones generated by suppression subtractive hybridization. Phylogenetic analysis revealed that they are distinct from the simple extracellular LRR proteins reported previously. Subcellular localization analysis demonstrated that the SbLRR2-EYFP fusion protein was targeted to the extracellular space in tobacco leaf cells. Peptide N-Glycosidase F treatment revealed that the SbLRR2 is N-glycosylated with non-fucosylated oligosaccharides when transiently expressed in Nicotiana benthamiana leaves. Functional analysis was performed in SbLRR2 over-expression (OE) Arabidopsis plants which showed enhanced resistance against the necrotrophic pathogens Botrytis cinerea and Alternaria brassicicola. In addition, the OE lines were found to have elevated expression of several jasmonate acid (JA)-associated genes and higher endogenous JA contents. Hence, the SbLRR2-mediated defense responses in transgenic Arabidopsis are likely to be dependent on JA-signaling through increased JA production. On the other hand, the OE lines remained susceptible to Pseudomonas syringae pv. tomato as the wild type plants. Consistently, there was no up-regulation of salicylic acid (SA) defense marker gene expression or SA levels in the OE lines. Using yeast two-hybrid analysis, SbLRR2 was further shown to interact with Arabidopsis hypersensitive-induced response protein 1. Such interaction may suppress hypersensitive response which is known to enhance necrotrophic pathogen invasion. These data suggested a positive regulatory role of SbLRR2 in plant defense. Further phenotypic analysis of transgenic SbLRR2 revealed its novel role in enhancing lead [Pb(II)] tolerance in Arabidopsis. OE-lines were showed to alleviate Pb(II)-induced root inhibition, reduce the accumulation of Pb(II), and enhance transcription of AtPDR12 which was previously shown to function as a potential Pb(II) efflux pump contributing to Pb(II) detoxification. However, all the Pb(II) tolerance responses were abolished when SbLRR2 was transformed into the atpdr12 mutant. Meanwhile, the extracellular localization of SbLRR2 was shown to be essential for the enhanced Pb(II) tolerance in transgenic Arabidopsis. Together, these results indicated that SbLRR2-mediated Pb(II) tolerance was dependent on AtPDR12 via Pb(II) extrusion. Further investigations revealed the Pb(II)-induced transcriptional activation of SbLRR2 and several highly conserved AtPDR12 homologs in sorghum seedlings, suggesting the possibilities of a common molecular mechanism for Pb(II) tolerance in diverse plant species. Finally, an iTRAQ-based LC-MS/MS quantitative proteomics approach was used to investigate of lead responses in Arabidopsis. A total of 114 proteins showed significant changes in protein abundance with 58 up-regulated and 56 down-regulated proteins. Analysis of changes in the protein profile revealed that the photosynthesis, photorespiration and protein biosynthesis in Arabidopsis were inhibited under lead toxicity. On the other hand, abundances of proteins involved in the antioxidant system, glucosinolate-myrosinase system and JA biosynthesis pathway were elevated upon Pb(II) treatment. Further investigation revealed that Pb(II) stress induced a rapid increase of JA contents in Arabidopsis whereas a JA biosynthesis deficient mutant (AOS) showed hypersensitivity to Pb(II) toxicity, strongly implicating a significant role of JA in Pb(II) response. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

A network of three classes of proteins consisting of bHLH and MYB transcription factors and a WD40 repeat protein - TRANSPARENT TESTA GLABRA1 (TTG1) act in concert to activate trichome initiation and patterning in Arabidopsis. These proteins also regulate the flavonoid-based pigment biosynthetic pathway in almost all higher plants including Arabidopsis. Using TTG1-YFP translational fusions, I show that TTG1 is expressed ubiquitously in Arabidopsis leaves and is preferentially localized in the nuclei of trichomes at all developmental stages. Using conditional transgenic alleles I demonstrate that TTG1 directly regulates the same genes as GL3. In vivo binding of GL3, GL1 and TTG1 to the promoters of GL2, TTG2, CPC and ETC1 establishes that these genes are major transcriptional targets for the TTG1-bHLH-MYB regulatory complex. By co-precipitation, I confirm that TTG1 interacts with the GL3 (bHLH) and GL1 (Myb) proteins in vivo, forming a complex. The loss of members of the TTG1 complex through mutation, affects the subcellular distribution of other complex members. Using particle bombardment, I show that TTG1, GL3, GL1 and GL2 do not move between adjacent epidermal cells while CPC does move to neighboring cells. These data support a model for the TTG1 complex directly regulating activators and repressors and the movement of repressors to affect trichome patterning on the Arabidopsis leaf. In addition, I also show that GL3 is recruited to its own promoter in a GL1-independent manner, which results in decreased GL3 expression, suggesting the presence of a GL3 negative auto-regulatory loop. Expression studies using GL3-GR (GL3-glucocorticoid receptor) and TTG1-GR fusions reveal direct regulation of the late anthocyanin biosynthetic genes, but not of early biosynthetic genes. Taken together, our results provide insights on the molecular mechanisms by which the combinatorial TTG1-bHLH-MYB regulatory complexes activate and repress both developmental and biosynthetic pathways in Arabidopsis.

published_or_final_version / abstract / toc / Botany / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Botany / Master / Master of Philosophy

published_or_final_version / Biological Sciences / Master / Master of Philosophy

The discovery of the Arabidopsis ttg1 mutant almost three decades ago provided a unique opportunity for the study of how several cell fates and organ identity pathways are co-regulated. Besides showing a lack of flavonoid based pigments, the pleiotropic ttg1 mutant is also deficient for the development of several epidermal characters including plant hair cells (trichomes), the non-hair cells of the root and the mucilage-secreting cells of the seed coat epidermis. Ectopic expression of the maize R bHLH transcriptional regulator of the flavonoid pigment pathway could completely suppress all the ttg1 mutant phenotypes, providing the first clue to the nature of the control mechanisms governing TTG1-dependent traits. Because it was established that a bHLH and a Myb protein are required for the regulation of anthocyanin pigment production in several plant species and an Arabidopsis Myb gene was necessary for trichome initiation, the existence of bHLH and Myb proteins that would regulate all the TTG1-dependent developmental pathways was hypothesized. This study works towards the elucidation of the transcriptional control mechanisms that regulate the TTG1-dependent developmental pathways. The identification and characterization of a key regulator, EGL3, uncovered the redundant nature of bHLH proteins operating under the TTG1 regulatory umbrella. As a result, bHLH regulators were assigned to all TTG1-dependent epidermal cell fate pathways and new roles for previously identified bHLH proteins were revealed. Roles suggested in the literature for Arabidopsis Myb factors suspected of regulating the flavonoid pigment pathway were at odds with findings from other plant models. Analysis of Myb loss-of-function RNAi lines and TTG1:GR and GL3:GR fusion lines presented here provides a clarified understanding of the regulation of anthocyanin biosynthesis by the Myb/bHLH/WDrepeat complex in Arabidopsis. Missing from the combinatorial complex model is the Myb component controlling the differentiation of the seed coat epidermis. Work presented here characterizes Myb5 as the primary Myb regulator of this differentiation pathway and defines a new role for TT2 as partially redundant with Myb5 for testa epidermis development. Myb5 also plays a minor role in trichome development and PA biosynthesis. Thus pleiotropy among the TTG1-dependent Myb regulators previously unobserved is first noted here. A more complete Myb/bHLH combinatorial transcription factor network model for the regulation of the TTG1-dependent pathways is proposed based on the results of work presented in this dissertation. / text

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Artemisia annua L. is a common type of wormwood that grows throughout the world. Artemisinin, a terpene compound in A. annua, has recently been recognized as the most promising antimalaria drug. Artemisinin and other types of terpenoids are synthesized and accumulated in glandualr trichomes that appear on the surface of leaf, stem and flower bud. To identify new genes involved in artemisinin biosynthesis and trichome function in A. annua, a normalized glandular trichome cDNA collection was sequenced by Roche GS FLX pyrosequencing system. Two sequencing runs generated totally 85M nucleotides which were further assembled into 190,377 unigenes (42,678 contigs and 147,699 sigletons). Putative functions were assigned to the unigenes based on Blast search against GeneBank database. Many terpene biosynthesis pathway genes were identified from the pyrosequencing ESTs. Together with other identified A. annua terpene pathway genes, a global view of terpene biosynthesis in glandular trichomes of A. annua were re-established. Meanwhile, a WD repeat protein, AaWD40, which show high amino acid sequence similarity with its Arabidopsis ortholog, AtTTG1 (AT5G24520) was identified. To investigate the functional relevance of AaWD40 to its Arabidopsis counterpart, genetic complementation test using Arabidopsis mutants was conducted. When AaWD40 was transformed into Arabidopsis transparent testa glabrous1 (ttg1-1) mutant, the anthocyanins and proanthocyanidin (PAs) production in seeds were restored, and the trichomeless phenotype of ttg1-1 mutant was rescued. In addition, over-expression of AaWD40 and AtTTG1 modulated the expression of WUS and CLVs genes which are required to maintain the stem-cell niche of Arabidopsis shoot apex. Transcriptomic profiling of transgenic Arabidopsis over-expressing AaWD40, TTG1, or ttg1-1 mutant revealed lists of genes modulated by these two WD40 genes homologue and gene ontology (GO) analysis suggested that the top-ranked categories are defense, stress response and developmental programme. We hypothesize that WD40 repeat protein act as a crucial regulatory factor in a wide variety of cellular functions in A. thaliana. / Wang, Wei. / Advisers: Guo Dianjing; Jiang Liwen. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 82-105). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Yu, Pui Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 68-78). / Abstracts in English and Chinese. / Acknowledgements --- p.iv / Abstract --- p.v / Table of Contents --- p.ix / List of Figures --- p.xii / List of Tables --- p.xiv / List of Abbreviations --- p.xv / Chapter Chapter 1. --- General Introduction --- p.1 / Chapter Chapter 2. --- Literature Review --- p.5 / Chapter 2.1 --- The importance of terpenoids --- p.5 / Chapter 2.2 --- The difficulties in synthesizing terpenoids --- p.8 / Chapter 2.3 --- Structure and classification of terpenoids --- p.9 / Chapter 2.4 --- MVA and MEP pathways of terpenoid biosynthesis in higher plants --- p.12 / Chapter 2.4.1 --- The MVA pathway --- p.16 / Chapter 2.4.2 --- The MEP pathway --- p.18 / Chapter 2.5 --- The crosstalk between MVA and MEP routes --- p.20 / Chapter 2.6 --- The farnesyl diphosphate is a key enzyme in terpenoid biosynthetic pathway --- p.20 / Chapter 2.7 --- The glutaredoxin system --- p.22 / Chapter Chapter 3. --- Materials and Methods --- p.25 / Chapter 3.1 --- Plant materials and growth condition --- p.25 / Chapter 3.2 --- DNA extraction and screening of fps mutants --- p.25 / Chapter 3.3 --- Validation of the fps mutant by semi-quantitative RT-PCR --- p.26 / Chapter 3.4 --- Semi-quantitative RT-PCR analysis of the fps mutants --- p.28 / Chapter 3.5 --- Genechip analysis of fps mutants --- p.29 / Chapter 3.6 --- Enzyme assays --- p.29 / Chapter 3.7 --- Triterpene and sterol analysis of fps mutants --- p.30 / Chapter 3.8 --- Preparation of carotenoid standards for carotenoid analysis --- p.31 / Chapter 3.9 --- Carotenoids analysis of fps mutants by HPLC --- p.31 / Chapter 3.10 --- Subcellular localization of FPS 1 and FPS2 by transient expression --- p.33 / Chapter Chapter 4. --- Results --- p.36 / Chapter 4.1 --- Screening of fpsl and fps2 homozygous mutants --- p.36 / Chapter 4.2 --- Validation of fps mutants by RT-PCR and enzyme activity assay --- p.36 / Chapter 4.3 --- Genechip analysis of two fps mutants --- p.40 / Chapter 4.3.1 --- Quality control and normalization of microarray sample --- p.40 / Chapter 4.3.2 --- Normalization and identification of differentially expressed genes --- p.42 / Chapter 4.3.3 --- GO annotation of differentially expressed genes in fps mutants --- p.43 / Chapter 4.3.4 --- Genes participate in stress and defense response were differentially expressed in both fpsl and fps2 mutants --- p.48 / Chapter 4.3.5 --- Genes in the plastidial pathway were down-regulated --- p.51 / Chapter 4.4 --- Effects of FPS mutations on pathway enzymes --- p.53 / Chapter 4.5 --- Effects of fps mutants on terpenoids and sterol metabolism --- p.55 / Chapter 4.6 --- Comparison on carotenoids and chlorophyll contents --- p.55 / Chapter 4.7 --- Subcellular localization of FPS 1 and FPS2 --- p.61 / Chapter Chapter 5. --- Discussion --- p.62 / Chapter Chapter 6. --- Conclusion --- p.67 / Reference --- p.68 / Appendices --- p.79 / Appendix A. Primers designed for homozygous mutant screening for fps mutants --- p.79 / "Appendix B. Primer pairs designed for fps ORF, common sequence and fpsl specific region" --- p.80 / Appendix C. Primer pairs designed for studying expression level of the downstream genes of FPS --- p.81 / Appendix D. Annotations of differentially expressed genes in fpsl mutant --- p.82 / Appendix E. Annotations of differentially expressed genes in fps2 mutant --- p.84 / Appendix F. Log fold changes of terpenoid pathway genes involved in FPS mutants --- p.94