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

The ‘epigenome’ refers to a difference in the transcriptional behaviour of a given allele that cannot be explained by differences in the genetic code, or ‘mutation’ at that locus. The epigenome is associated with certain biochemical marks, and generally exerts a silencing effect upon the transcription of genes under its influence. In Angiosperms such as Arabidopsis thaliana, fertilisation of both the egg and central cell– referred to as ‘double fertilisation’ – giving rise to the embryo and endosperm respectively, the latter of which is one of the most important tissues in the human food chain. Upon double fertilisation the gametes of each parent are known to contribute differing epigenetic ‘imprints’, where one gamete contributes a copy of a given allele in a transcriptionally unavailable state, while the other parent’s copy is in an available state. When a gene resides in such a region, the result is that only one parent’s copy is hence transcribed; such a gene is said to be ‘imprinted.’ Imprinting is known to affect the development of the placenta as well as some of the adult tissues in mammalian models, and in plants is most extensively found in the endosperm, where without the imprint of both parents the resulting seed exhibits reduced viability and defective endosperm development. A relative dearth in the number of known imprinted loci in the model angiosperm, A. thaliana makes is difficult to make reliable assessments of its role and regulation. This thesis initially aimed to extend the count of known imprinted genes using a model that proved insufficient to identify novel imprinted genes, and presents a meta-analysis showing that the reliable attribution of imprinted status to a gene is difficult using high-throughput methods as well. In addition, the further characterisation of a novel imprinted gene identified previously by this lab, MPC, with a view to acquiring a more detailed understanding of its role using mutants carrying point mutations in the MPC protein showed only a subtle phenotype to discern them from wild-type plants. There has also been recent speculation of a role for repeat elements in imprinting. This thesis presents findings suggesting that the apparent association of repeat elements with imprinted genes is an artefact rather of an association of endosperm-expressed genes with transposable elements, rather than genes that are specifically imprinted.

Cassava (Manihot esculenta Crantz) is an important tropical crop which provides a large portion of daily calories intake to hundreds of millions of people in Africa, Latin America, and tropical Asia. Cassava is grown for its starchy storage roots as staple food, as animal feed, and as industrial raw material. The utilisation of cassava is hindered by its characteristic physiological response, the post-harvest physiological deterioration (PPD). The inevitable wounding caused during harvesting and handling will trigger a series of physiological responses within 24 to 48 hours, which causes a blue-black discoloration in the storage roots, rendering these roots unmarketable and unpalatable in a few days. During the PPD response large amount of phenylpropanoid compounds, especially scopoletin and its glycoside, accumulate in the roots. Scopoletin may play an important role in PPD development but little work has been done on the possible relationship. Here we aim to examine the effects of altering scopoletin synthesis in cassava roots on the PPD response. In Arabidopsis thaliana, gene F6’H1 (feruloul CoA 6’-hydroxylase 1) is indispensable in the biosynthesis of scopoletin. Cassava F6’H1 candidate gene family involved in scopoletin synthesis were identified by their ability to functionally complement F6’H1 T-DNA insertion mutation in Arabidopsis thaliana that prevented synthesis of scopoletin. RNAi constructs targeting the identified cassava F6’H1 candidate gene family were designed, under the control of either constitutive CaMV 35S or root-specific StPAT promoters. These were used to transform wild-type cassava to down-regulate the expression of these scopoletin synthetic genes in F6’H1 gene family. The inhibition of cassava F6’H1 candidate gene expression and thus the scopoletin synthesis in transgenic cassava roots were confirmed by qRT-PCR and LC-MS, respectively. The RNAi transgenic cassava lines show less scopoletin accumulation and inhibited F6’H1 candidate genes expression during the PPD response. A reduced PPD discoloration development compared to that of the wild-type was also observed in the RNAi transgenic cassava lines.

The G-protein coupled receptors (GPCRs) are seven-transmembrane receptors that transduce signals from the cell surface to intracellular effectors. There are more than 1000 GPCRs in metazoans, while no GPCR has been definitively identified in plants. The most promising plant GPCR candidate, Arabidopsis G-protein coupled receptor 1 (GCR1), physically couples to the G-protein < subunit GPA1 and is involved in cell cycle regulation, blue light and phytohormone responses, but its signalling network remains largely unknown. This project aimed to achieve a better understanding of GCR1 signalling by identifying its interactors using a novel yeast two hybrid system – the Ras Recruitment System (RRS). Screening of an Arabidopsis cDNA library using a bait comprising intracellular loop 1 (i1) and 2 (i2) of GCR1 resulted in the isolation of 20 potential interactors. Extensive reconfirmation screening demonstrated that three of these interactors: Thioredoxin h3 (TRX3), Thioredoxin h4 (TRX4) and a DHHC type zinc finger family protein (zf-DHHC1) interact specifically with both i1 and i2 of GCR1. This was supported by the reverse RRS (rRRS) and 6xHis-pull-down assays. It is speculated that TRX3 and TRX4, which can reduce disulfide bridges of target proteins and act as powerful antioxidants, may regulate GCR1-mediated signalling events in response to oxidative stress. Alternatively, they may modulate GCR1 targeting or signalling through their chaperone activities. zf-DHHC1 has a predicted membrane topography that is shared by most DHHC domain-containing palmitoyl acyl transferases. It may modify GCR1 activity through palmitoylation of the two cysteines located at the cytoplasmic end of the first transmembrane domain. Together, these findings contribute to the growing understanding of the GCR1 signalling network, and provide valuable starting points for further investigation.

Most studies of plant hybridisation are concerned with documenting its occurrence in different plant groups. Many flowering plants are polyploids and seeds developed from crosses between individuals of different ploidies usually show abnormal features and often abort. The success or failure of interploidy crosses is very important to understanding the evolution of plants as well as to agriculture, but much remains to be learned about the nature of hybridisation barriers. Several mechanisms have been proposed to explain postzygotic barriers, including negative interactions between diverged sequences, global genome rearrangements, and widespread epigenetic reprogramming. Some recent advances in our understanding of the process of hybridisation are derived from different experimental studies on a series of A. thaliana ecotypes. Crosses between diploid (2x) and tetraploid (4x) individuals of the same ecotype can result in F1 lethality, and this dosage-sensitive incompatibility plays a major role in polyploidy speciation research. We have performed interploidy crosses between different diploid maternal A. thaliana ecotypes and tetraploid paternal Col-0 ecotype and identified a genetic variation in F1 lethality. We also found that maternal parents of some ecotypes such as Tsu-1 suppressed the F1 lethality caused by paternal-excess interploidy cross of Col-0 ecotype. A preliminary mapping exercise produced advanced backcross populations that are suitable for mapping maternal modifiers and for the identification of modifier genes. Furthermore, we studied the killer effect caused by Col-0 and identified three additive QTL that affect the rate of postzygotic lethality in F1 during interploidy crosses. This information will facilitate the identification of paternal genes that cause F1 lethality and contribute to reproductive isolation. The moa-1 (mosaic aneuploidy 1) mutant of A. thaliana was obtained in a screen of chemically (EMS) mutagenised seeds of Landsberg erecta (Ler). moa-1 has various phenotypic differences to wild type; the preliminary karyotype analysis showed that the cells of individual moa-1 mutant plants have a variable number of chromosomes (usually between 11-18). In contrast, the cells of wild type Arabidopsis plants and conventional aneuploids have a fixed number of chromosomes in each somatic cell. This data showed that all moa-1 plants have an abnormal number of chromosomes and thus they were termed as mosaic aneuploids.

During meiosis, homologous chromosomes pair and undergo reciprocal genetic exchange, producing crossovers. This generates genetic diversity and is required for balanced homolog segregation. Despite the critical functions of crossovers, their frequency and distribution varies extensively within and between species. This crossover variation can be caused by trans-modifiers within populations, which encode diffusible molecules that influence crossover formation elsewhere in the genome. This project utilised natural accessions of Arabidopsis thaliana to identify trans-modifying loci underlying crossover variation within the species. I performed Quantitative Trait Loci (QTL) mapping using a fluorescence-based crossover reporter system to measure recombination frequency in a genomic interval on chromosome 3, termed 420. Mapping in a Col-420 × Bur-0 F2 population revealed four major recombination QTLs (rQTLs) that influence crossover frequency. A novel recessive rQTL on chromosome 1 that reduced crossovers within the interval was fine-mapped to a premature stop codon in TATA Binding Protein (TBP)-associated factor 4b (TAF4b) in Bur-0 (taf4b-1). TAF4b is a subunit of the TFIID complex, a multi-protein general transcription factor complex comprising TBP and numerous TAFs that forms a component of the pre-initiation complex that recruits RNA polymerase II to promoters. Transformation-based complementation experiments and the isolation of several independent taf4b alleles provided genetic proof that TAF4b is essential for wild-type levels of crossover within 420. Analysis of the prevalence of the taf4b-1 mutation in the global Arabidopsis accession collection demonstrated its specificity to three accessions in the British Isles. A combination of cytology, genetic analysis using additional fluorescent reporter lines, and sequencing in F2 recombinant populations demonstrated a genome-wide reduction in crossover frequency in taf4b-1. In addition, RNA sequencing identified numerous transcriptional changes in taf4b-1. Both up- and down-regulated gene sets displayed significant enrichment for genes that are predominantly expressed in meiocytes, and several gene ontology terms pertaining to protein modification and meiotic processes. These results further demonstrate the existence of genetic modifiers of crossover frequency in natural populations of A. thaliana, and the characterisation of a novel trans-modifier of recombination, TAF4b. This signifies a novel function for TAF4b in Arabidopsis, and further enhances our understanding of the molecular factors controlling the frequency and distribution of meiotic crossovers in plants.

La phosphorylation/déphosphorylation des protéines est un mécanisme de signalisation intracellulaire commun. Parmi les kinases végétales, les Mitogen-Activated Protein Kinases (MAPKs) sont impliquées dans de nombreux processus biologiques importants, comme la réponse aux stress biotiques et abiotiques, le développement et la dynamique du cytosquelette. Chez Arabidopsis thaliana et ce malgré de nombreux efforts, les fonctions des kinases impliquées dans les cascades MAPK restent peu inconnues. L'activation des kinases en utilisant des mutations mimant la phosphorylation des sites normalement phosphorylés est une approchequi a fait ses preuves dans le cas de MAP2Ks et a largement contribué à élucider leurs fonctions. Cette stratégie s’est révélée impossible dans le cas des MAPKs, puisque les résidus à muter restent encore à identifier. Pour contourner ce problème, nous avons adapté un crible basé sur la complémentation fonctionnelle d’un mutant MAPK de levure avec des formes aléatoirement mutées de MPK6d’Arabidopsis dans le but d'identifier des mutants présentant une activité constitutive. Nous en avons identifiés plusieurs et avons montré que ces formes constitutivement actives (CA) de MPK6 sont actives sans phosphorylation par les MAP2Ks. Par ailleurs, les mutations des résidus équivalents dans d'autres MAPKs les rendent également hyperactives, ce qui indique que cette stratégie peut être utilisée comme approche générale pour activer les MAPKs afin d’en comprendre les fonctions. L’étude des interactions protéine-protéine et l’analyse des profils dephosphorylation indiquent que les MAPKs CA conservent leur spécificité envers leurs substrats et interacteurs. Comme preuve de concept, nous avons généré des formes actives du MPK4. La MPK4 CA exprimée sous son propre promoteur a parfaitement complémenté le mutant mpk4. La caractérisation des lignées exprimant MPK4 CA confirme le rôle négatif de cette kinase dans les réponses de défense aux pathogènes des plantes que ce soit dans la PTI (PAMP Triggered Immunity) ou dans la ETI (Effector Triggered Immunity). Globalement, ce travail permettra de fournir des informations directes sur les cibles des MAPKs et devrait contribuer à la compréhension globale de la transduction du signal chez les plantes. / Protein phosphorylations and dephosphorylations are common events occurring duringintracellular signaling processes. Among plant kinases, Mitogen-Activated Protein Kinases (MAPKs) are involved in signaling of many important biological processes, including biotic and abiotic stresses, development and cytoskeleton organization. Despite an abundant literature on MAPKs, the exact roles and direct targets of many Arabidopsis thaliana MAPKs are not clear yet. The activation of kinases using phospho-mimicking mutations of the phosphorylated residues was a successful approach in the case of MAP2Ks, helping to elucidate their functions. This strategy failed in the case of MAPKs since the necessary residues to mutate remain unclear. To bypass this problem, we adapted a screen based on the functional complementation of a MAPK yeast mutant with randomly mutated Arabidopsis MPK6 in order to identify the ones mutants showing constitutive activity. We identified several clones and showed that these constitutively active (CA) of MPK6 candidates are indeed active without phosphorylation by MAP2Ks. Interestingly, mutations of the equivalent residues in other MAPKs triggered constitutive activity as well, indicating that this strategy may be used as a general approach to activate MAPKs and identify their functions. Interaction and phosphorylation assays indicatedthat CA MAPKs retain their substrate and interactor specificity. As proof-of-concept, we generated active versions of MPK4. CA MPK4 expressed under itsown promoter successfully complements mpk4 mutant plants. Characterization of CA MPK4 lines further confirmed the negative role of MPK4 in plant pathogen defense responses and its implication in both PTI (PAMP Triggered Immunity) and ETI (Effector Triggered Immunity). Overall, the work will help to provide direct information on all MAPK targets and should be an important contribution to the overall understanding of signal transduction in plants.

The circadian clock is an endogenous timekeeper that synchronises biological processes with daily external rhythms such as light and temperature cycles. It provides organisms with a competitive advantage by allowing anticipation of daily events. The circadian clock encompasses a network of transcription-translational feedback loops (TTFLs) that orchestrates rhythmic expression of a large part of the genome. This network is regulated at post-transcriptional and post-translational level. Post-translational regulation of clock proteins is essential to ensure stable rhythms and robust timekeeping. Unlike the genes in the TTFL network, modifiers of clock proteins at post-translational level are conserved across taxa. SUMO, a small ubiquitin-related post-translational modifier, regulates timekeeping in mammals through modification of the clock transcription factor BMAL. In this study, SUMO is shown to contribute to oscillator function in Arabidopsis plants. Methods have been developed to prove that mutant lines defective in SUMO machinery, including SUMO-ligase and -protease mutants, display long circadian rhythms. Additionally, sumoylation on the crucial plant clock transcription factor CCA1 is observed in vivo. A fraction of the protein is sumoylated across the expression window of CCA1, with the phase of peak sumoylation in advance of peak total CCA1. The effect of sumoylation of CCA1 was investigated with respect to localisation, stability and DNA binding affinity of the protein, as these are previously described possible effects of sumoylation. The subcellular location of CCA1-YFP fusions in protoplasts was not altered in mutant lines of the SUMO machinery. In vitro experiments show that sumoylation negatively affects the affinity of CCA1 to its cognate promotor element, suggesting that SUMO could act as a reversible attenuator of CCA1 activity. Furthermore, effects of SUMO machinery mutations appear to be differential across a range of physiologically relevant temperatures, implying that sumoylation could be involved in the response to or buffering against fluctuating ambient temperatures. There is an increasing amount of evidence to suggest that metabolic oscillations are not only driven by transcriptional outputs of the clock, but are to some extent self-sustained and can feed timing information back into the clock. Glutathione was investigated as a possible metabolic feedback signal. Expression of clock gene CCA1 was found to be abolished in a mutant of the rate-limiting enzyme for glutathione synthesis (pad2-1). Surprisingly however, the amount of glutathione was not found to oscillate. Combined, the results discussed in this thesis provide a substantial advance on our understanding of post-translational regulation and the integration of metabolic and environmental information into the plant circadian clock.

No description available.