Proteome analysis of glandular trichome from Artemisia annua L.

Wu, Ting.Thesis (M.Phil.)--Chinese University of Hong Kong, 2011.Includes bibliographical references (leaves 56-70).Abstracts in English and Chinese.ACKNOWLEDGEMENTS --- p.IllABSTRACT --- p.VTABLE OF CONTENTS --- p.IXLIST OF ABBREVIATIONS --- p.XIIChapter CHAPTER 1. --- LITERATURE REVIEW --- p.1Chapter 1.1 --- THE DISEASE OF MALARIA --- p.1Chapter 1.1.1 --- Pathogenesis --- p.2Chapter 1.1.2 --- The treatment of malaria --- p.4Chapter 1.2 --- THE PLANT OF ARTEMISIA ANNUA L --- p.5Chapter 1.2.1. --- Horticulture --- p.5Chapter 1.2.2. --- Historical Importance --- p.6Chapter 1.3 --- ARTEMISININ --- p.7Chapter 1.3.1 --- The content and distribution of artemisinin --- p.7Chapter 1.3.2 --- The biosynthesis of artemisnin --- p.8Chapter 1.4 --- TRICHOMES --- p.13Chapter 1.4.1 --- Structure and function of trichomes --- p.13Chapter 1.4.2 --- Trichome investigation in A. annua --- p.14Chapter 1.5 --- PROTEOMICS --- p.17Chapter 1.5.1 --- The basic principle of proteomics --- p.17Chapter 1.5.2 --- Two-dimensional gel electrophoresis --- p.18Chapter 1.5.3 --- Mass Spectrometry --- p.19Chapter 1.5.4 --- Gel-free proteomics --- p.20Chapter 1.6 --- OBJECTIVES --- p.21Chapter CHAPTER 2. --- MATERIALS AND METHODS --- p.23Chapter 2.1 --- CHEMICALS --- p.23Chapter 2.2 --- PLANT MATERIALS --- p.23Chapter 2.3 --- ISOLATION OF GLANDULAR TRICHOMES --- p.23Chapter 2.4 --- PROTEIN EXTRACTION . --- p.25Chapter 2.5 --- Two DIMENSIONAL GEL ELECTROPHORESIS --- p.25Chapter 2.6 --- IMAGINE ANALYSIS --- p.26Chapter 2.7 --- IN GEL DIGESTION AND PROTEIN IDENTIFICAIOTN BY MASS SPECTROMETRY --- p.27Chapter CHAPTER 3. --- RESULTS AND DISCUSSION --- p.29Chapter 3.1 --- THE ISOLATION OF GLANDULAR TRICHOMES --- p.29Chapter 3.2 --- 2DE PATTERNS OF A. ANNUA LEAVE TRICHOMES AND LEAF TISSUE --- p.32Chapter 3.3 --- IDENTIFICATION OF PROTEINS IN GLANDULAR TRICHOMES --- p.34Chapter 3.3.1 --- Protein involved in electron transport chain --- p.47Chapter 3.3.2 --- Protiens invovled in metabolism --- p.48Chapter 3.3.2.1 --- artemisinin biosynthesis --- p.48Chapter 3.3.2.2 --- glycolysis --- p.49Chapter 3.3.2.3 --- other metabolic enzymes --- p.50Chapter 3.3.3 --- Proteins involved in transcription and translation --- p.51Chapter 3.3.4 --- Protein involved in proteolysis --- p.51Chapter 3.3.5 --- "Detoxificaiton, stress related protein" --- p.52Chapter 3.4 --- PERSPECTIVE --- p.53Chapter 3.5 --- CONCLUSION --- p.53REFERENCES --- p.5

Identification of transcription factors controlling the expression of paclitaxel biosynthesis genes in cambial meristematic cells of Taxus cuspidata

Paclitaxel is an antitumor diterpene from Taxus spp. that binds tubulin, stabilizes microtubules and induces apoptosis in dividing human cells. It was originally isolated from the bark of Taxus brevifolia and approved for clinic uses by the FDA in 1992. Because of its excellent activity in treatment of various cancers, a significant supply shortage has been created by the enormous demand for this natural product. Thus, researchers have been focusing on the development of effective ways to increase the production of paclitaxel and related bioactive molecules. This shortage was initially solved by over-harvesting of T. brevifolia bark; however, it is not an environment-friendly, effective and sustainable way to supply paclitaxel. A semisynthetic route was then developed to convert the more readily available and renewable 10-deacetylbacatin III into paclitaxel. As an alternative, plant cell cultures have been employed to commercially produce paclitaxel and it is a more environment-friendly and sustainable route to end the supply crisis. However, problems associated with plant cell culturing at an industrial scale, such as cell aggregation and variability in yield, significantly affect paclitaxel production. Therefore, a discovery of a better-performing Taxus cell line might be a solution to overcome these culturing-associated problems. A cambial meristematic cell (CMC) line of Taxus cuspidata has been isolated, cultured and demonstrated to be a cost-effective and environmentally friendly platform for the sustainable production of paclitaxel (Lee et al. 2010). Compared to dedifferentiated cell (DDC) lines, CMC lines are undifferentiated cells and proved to have stem cell-like properties. When cultured at an industrial scale, this cell line contains much smaller cell aggregates with many cells appearing as singletons, the biomass of which is still increasing after 22-month culturing, and has much greater paclitaxel production after elicitation (Lee et al. 2010). In my project, we aimed to identify the transcription factors (TFs) that regulate the expression of paclitaxel biosynthesis genes. We performed Illumina Solexa sequencing on cDNA libraries derived from methyl jasmonate (MeJA)-elicitated CMCs to digitally profile gene expression. Analysis of differentially expressed gene (DEG) abundance led to the discovery of 19 putative TFs and bioinformatic analysis further showed that these 19 TFs belong to 5 different TF families. Further, the DNA binding motifs associated with these TFs can be found in the promoters of the two early, taxadiene synthase (TASY) and taxadiene 5α hydroxylase (T5αH), and three late, 10-deacetylbaccatin III-10-O-acetyltransferase (DBAT), phenylpropanoyltransferase (PAM) and 3’-N-debenzoyl-2-deoxytaxol-Nbenzoyltransferase (DBTNBT), paclitaxel biosynthesis pathway genes. Then, yeast one-hybrid analysis, gel shifting assays and plant transient expression assays (TEA) were employed to assay TFs that interact with these promoters. Although Y1H screening did not show any convincing TF-promoter interactions, the attempted plant transient expression assay in the leaves of Nicotiana benthamiana might be a more suitable system to screen the positive regulators. Finally, the elucidation of a TF regulatory network that controls paclitaxel biosynthesis will guide the rational engineering of CMCs to ultimately increase yields of this important pharmaceutical

Functional Characterization of an Organ Specific Effector See1 of Ustilago Maydis

Ustilago maydis is the causative agent of the corn smut. This basidiomycetous fungus is a biotophic plant pathogen that succeeds by colonizing living tissue and establishes a biotrophic interaction which results in the formation of enormous tumors. This tumor formation is a result of efficient host immune suppression and nutrient efflux during disease progression. The fungus secretes several hundreds of effector proteins which are expressed at various stages of colonization to modulate the host. Previous studies have revealed that the effector proteins of U. maydis are acting in an organ specific manner and deletion of one organ specific effector does not hamper the symptom formation in non-target organ (Skibbe et al., 2010; Schilling et al., 2014). The previous study of Schilling et al., 2014 identified leaf specific effectors, which are induced in juvenile leaves. An interesting candidate among these that showed a perfect organ specificity was see1 (Seedling efficient effector 1, um02239), which is required in the colonized leaves. Deletion mutants for see1 are able to penetrate and colonize the seedling but fail to induce expansion of tumors. The deletion mutant is seen to be actively blocked in mesophyll and vascular cell layers of the leaf, which may indicate that the effector function may be confined to a specific cell or tissue type. In contrast, see1 deletion does not affect tumor formation in the floral parts of the host. Aim of this thesis was the functional characterization of See1. Monitoring of the DNA synthesis in host, showed that See1 is specifically required to induce DNA synthesis in colonized host cells and re-direct them to form tumors. Yeast-two-hybrid analysis showed that See1 interacts with a nucleo-cytoplasmic host protein SGT1, which is a cell cycle and immune response modulator and which also shows a leaf specific transcriptional regulation. Constitutive overexpression of see1 caused tassel base abnormality specifically showing tumors in the vegetative base of the tassel pointing towards an active role of see1 in inducing tumor in vegetative maize tissues. Electron microscopy showed that See1 is translocated to the plant cell and is localized in the cytoplasm and nucleus of the host cell. Furthermore, it was demonstrated that See1 blocks the phosphorylation of maize SGT1 at a monocot specific site which is necessary to activate the signaling cascade upon pathogen perception. Experiments indicate that see1 specifically activates the host cell cycle release thereby activating the colonized cells to undergo a tumor pathway. Hence organ specific effectors like see1, not only manipulate the defense responses, but also the metabolic state of the host cell leading to tumor development