published_or_final_version / Botany / Doctoral / Doctor of Philosophy

published_or_final_version / Biological Sciences / Master / Master of Philosophy

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

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

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

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

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

In Arabidopsis thaliana, a gene family encodes acyl-CoA-binding proteins (ACBPs) conserved at the acyl-CoA-binding domain which facilitates the binding to acyl-CoA esters. These ACBPs, designated ACBP1 to ACBP6, range in size from 10.4 to 73.1 kD. Previous studies have shown that the the overexpression of ACBP1 or ACBP2 in Arabidopsis likely promotes repair of lipid membranes and result in enhanced tolerance to lead and cadmium, respectively. Microarray data (http://bar.utoronto.ca/) revealed that the expression of ACBP1 and ACBP2 is also regulated by other abiotic stresses, such as cold and drought, suggestive of their association with these environmental pressures. The aim of this study is to investigate and better understand the roles of ACBP1 and ACBP2 in different stress responses. It has been previously observed that the expression of both ACBP1 and ACBP4 is lead [Pb(II)]-inducible and recombinant ACBP1 and ACBP4 bind Pb(II) in vitro. In this study, ACBP1 and ACBP4 were overexpressed in Brassica juncea to test if these ACBPs could be extended for application in Pb(II) phytoremediation in transgenic B. juncea. On freezing (-12 to -8 °C) treatment, ACBP1-overexpressing Arabidopsis was freezing sensitive and accumulated more phosphatidic acid (PA), but less phosphatidylcholine (PC), in contrast to acbp1 mutant plants which were freezing tolerant and had reduced PA and elevated PC levels. Such changes in PC and PA were consistent with the expression of the mRNA encoding phospholipase D1 (PLD1), a major enzyme that promotes the hydrolysis of PC to PA. In contrast, the expression of phospholipase D (PLD), which plays a positive role in freezing tolerance, was up-regulated in acbp1 mutant plants and down-regulated in ACBP1-overexpressing plants. Reduced PLD1 expression and decreased hydrolysis of PC to PA may enhance membrane stability in the acbp1 mutant plants. Given that recombinant ACBP1 binds PA and acyl-CoA esters in vitro, the expression of PLD1 and PLD could be regulated by PA or acyl-CoAs maintained by ACBP1, if ACBP1 were to resemble the yeast 10-kD ACBP by its capability to modulate gene expression during stress responses. Interestingly, another membrane-associated ACBP, ACBP2, which shows high (76.9%) conservation in amino acid homology to ACBP1, did not appear to be affected by freezing treatment. Besides freezing stress, ACBP1, as well as ACBP2, have been observed to participate in abscisic acid (ABA) signaling. They both promote ABA signaling in seed germination and seedling development, while only ACBP2 is involved in the drought response. The overexpression of ACBP2 in Arabidopsis up-regulated reactive oxygen species (ROS) production culminating in reduction in stomatal aperture and water loss in guard cells, thereby enhancing drought tolerance. For tests in phytoremediation, B. juncea was selected for overexpression of ACBP1 and ACBP4 because it is fast-growing, has a higher biomass than Arabidopsis, and is known to be a good accumulator of Pb(II). However, results of Pb(II) treatment for two days showed that the overexpression of ACBP1 or ACBP4 in B. juncea did not significantly improve Pb(II) tolerance. Nevertheless, B. juncea overexpressing ACBP1 did accumulate Pb(II) in roots whereas ACBP4-overexpressing B. juncea lines accumulated Pb(II) in both shoots and roots. Given that B. juncea has a larger biomass than Arabidopsis, it is likely that the duration of Pb(II)-incubation tested in this study was not drastic enough for comparison, and the incubation time should be further extended for Pb(II) translocation. In addition, future studies on Arabidopsis should be conducted to better understand the mechanism of ACBP4-mediated Pb(II) accumulation using Arabidopsis acbp4 mutant and ACBP4-overexpressing plants. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Botany / Master / Master of Philosophy