The plant Golgi apparatus plays a central role in the secretory pathway, being the site of protein glycosylation, sorting and the synthesis of cell wall polysaccharides and glycolipids. Golgi matrix proteins are involved in the formation and maintenance of the Golgi stack and putative plant homologues have been identified, but their general function and role in plant Golgi biogenesis still is unknown. To study Golgi biogenesis, the Golgi was disrupted using both the drug Brefeldin A (BFA) and a mutated GTP-Iocked form of the small GTPase Sarlp, blocking protein transport from the endoplasmic reticulum to the Golgi. As prolonged expression of Sarl-GTP is toxic to cells, the gene was inserted into a dexamethasone-inducible promoter system, allowing controlled expression and Golgi disruption. The system was characterised by live cell imaging of fluorescent Golgi and ER exit site markers and at the ultrastructural level by electron microscopy. A novel method to reverse the dexamethasone-inducib'e system in tobacco using the glucocorticoid antagonist RU486 was established. The subcellular location of the Golgi matrix proteins GFP-AtCASP, GFP-golgin-84, AtGRIP-GFP and GFP-TMF and other Golgi markers fused to fluorescent proteins was studied during Golgi disruption and reformation. After disruption of Golgi membranes, the trans-Golgi matrix proteins GFP-TMF and AtGRIP-GFP were redistributed into the cytoplasm. The cis-Golgi matrix proteins GFP-AtCASP and GFP-golgin-84 labelled not only the ER and cytoplasm respectively, but also remnant punctate structures. Those remnants were found to co-locate with the ER exit site marker Sarl-GTP-YFP, indicating a close relationship between the cis-Golgi matrix and ER exit sites. After BFA washout, cis-Golgi matrix proteins labelled the reforming Golgi before transGolgi matrix proteins. Interactions between Golgi matrix proteins and regulatory proteins were visualised in vivo in tobacco leaf epidermal cells using fluorescence lifetime Imagmg (FUM) combined with fluorescence resonance energy transfer (FRET).

No description available.

No description available.

Pyruvate, orthophosphate dikinase and phospho<i>enol</i>pyruvate carboxykinase both catalyse the synthesis of phospho<i>enol</i>pyruvate. Single and double <i>Arabidopsis PPDK </i>and <i>PCK1 </i>T-DNA insertional knock-out mutants were used to investigate whole plant enzyme pools. Vein specific <i>Arabidopsis</i> knock-down lines were created by using a combined approach of RNAi and enhancer trapping to reduce activity of the two enzymes in a cell-specific manner. Developmental analyses of the two mutant classes indicated an important role for both enzymes during mature aerial tissue development and early seedling growth. Metabolic profiling of mature leaves of vein specific knock-down lines suggested that veinal pools of the two enzymes may be involved in amino acid interconversions in these tissues. Microarray analysis of etiolated knock-out seedlings implied that PPDK may act to supplement the gluconeogenic role of PEPCK during seedling establishment under limiting conditions. PPDK is regulated by a bifunctional regulatory protein which catalyses inactivation of PPDK via phosphorylation, and its activation via dephosyphorylation. Both activities proceed via atypical mechanisms, but despite these unusual properties, little is known about the structure of RP. The location of the unidentified kinase and dephosphorylase domains of <i>Arabidopsis </i>RP, and the mechanism of interaction with PPDK, were investigated using mutagenised AtRP1 and chimaeric AtRP1-AtRP2 variants. Results did not support the proposal that separate sites are involved in RP kinase and dephosphorylase activities, or the current view that a putative P-loop is likely to be only critical for kinase activity.

The biological tissues used were cauliflower and soybean cells capable of synthesizing their own inositol. The existing assays for monitoring IMPase activity were investigated, and a major achievement of the project was the development of a dual-label radiochemical assay which measured both IMPase and 'other phosphatase' activities simultaneously. A number of methods for the purification of IMPase were investigated and developed. Adopting ammonium sulphate precipitation as the first step, soybean IMPase was found to precipitate in the 30% to 50% saturation fraction and a purification of 2.4 fold obtained. Ion exchange chromatography, using Q-Sepharose Fast Flow, resulted in a purification of 12 fold. Various types of immobilized ligand and salts were tested to improve separation by hydrophobic interaction chromatography. A purification of 13 fold was achieved. Purification using preparative iso-electric focusing by the Rotofor cell proved to be the most successful; a purification of 115 fold was obtained. Gel filtration chromatography was also examined as a possible step in the purification strategy as well as preparative native acrylamide gel electrophoresis. The <I>M</I><SUB>r</SUB> of soybean IMPase was found to be 60 kDa and the pI 5.3 Soybean IMPase was shown to be less thermostable than its mammalian counter-part and heat treatment proved not to be an effective technique to use in the purification of IMPase. Inhibitor studies on IMPase revealed that 6-deoxy-6-phosphonoglucose, an isosteric analogue of Ins3<I>P, </I>was a weak competitive inhibitor. Partially purified soybean IMPase was insensitive to Li<SUP>+</SUP>, with marked inhibition at 250mM Li<SUP>+</SUP>. Other inhibitors tested were: a methylene bisphosphonic acid derivative, NaF, HgC1<SUB>2</SUB> and β-mercaptoethanol. Overall, considerable progress has been made in achieving the aims of this project. Different techniques which exploit different characteristics of the protein have been successfully applied. As a result, purification of soybean IMPase may be expected when the techniques are assembled in the optimal order.

The work presented in this thesis involves the development of a proteomic method for protein localization that is not dependent on the preparation of pure organelles. This method has subsequently been named localization of organelle proteins by isotope tagging (LOPIT). Organelles are first partially separated using centrifugation through density gradients. Distributions of proteins within such gradients are then assessed by measuring the relative abundance of proteins between fractions along the length of the gradients. This is achieved using isotope-coded tags for protein quantitation by mass spectrometry. The subcellular localizations of proteins can then be determined by comparing their distributions to those of previously localized proteins, since proteins that belong to the same organelle will co-fractionate in the density gradients. Application of the LOPIT technique to the study of the Arabidopsis endomembrane system has resulted in the simultaneous assignment of 527 proteins to the endoplasmic reticulum, Golgi apparatus, vacuole, plasma membrane or to the mitochondria and plastids. These results demonstrate that proteomic analysis of the major endomembrane components can be performed in parallel, enabling protein steady state distribution between these organelles to be determined. Consequently, for the first time by proteomics, genuine residents of the endoplasmic reticulum, Golgi apparatus, vacuole and plasma membrane have been distinguished from contaminants and proteins that are in transit through the secretory pathway.

The aim of the work described in this thesis was to assess the role of invertase in carbohydrate metabolism in two red-fruited and hexose-accumulating species, <I>Lycopersicon esculentum</I> and <I>Lycopersicon pimpinellifolium</I>. Fruit from the latter had previously been reported to show a much greater soluble acid invertase activity and correspondingly greater sugar accumulation than that of the former. The pigments chlorophyll a and b, and the carotenoids were measured in developing fruit with the aim of finding a marker for development other than age of the fruit (in days after anthesis). Immuno-gold electron microscopy and immuno-blots were used to determine the intra- and inter-cellular localisation of invertase. Maximal extractable soluble invertase from <I>L. pimpinellifolium</I> PI 126436 fruit was correlated with sugar accumulation during tomato fruit development, whereas no correlation was found during the development of either <I>L. esculentum</I> FM 6203 or <I>L. pimpinellifolium</I> LA 722 fruit. There were three major isoforms of soluble invertase present in <I>L. pimpinellifolim</I> PI 126436 fruit compared with only two major isoforms in <I>L.esculentum</I> FM 6203 or <I>L. pimpinellifolim</I> LA 722 fruit. The activity of invertase appeared earlier in development (i.e. in green fruit) in <I>L. pimpinellifolim</I> PI 126436 fruit than in <I>L. esculentum </I>or <I>L. pimpinellifolium</I> LA 722, where it is virtually absent from green fruit. Tomato fruit produced from progeny of cross between <I>L. esculentum</I> FM 6203 and <I>L. pimpinellifolium </I>PI 126436 (which were selected for the vacuolar invertase gene present) were the used for further investigation. All fruit contained similar fruit sugars and invertase activities regardless of the origin of the gene. It was concluded that the higher soluble solids trait of <I>L. pimpinellifolim </I>PI 126436 fruit may be attributed, at least in part, to invertase activity, as fruit contained at least one major isoform of invertase that was not found in quantity in <I>L. esculentum</I> FM 6203.

Cyclin dependent kinases (CDKs) are threonine-serine kinases that, in complex with cyclins, regulate the progression of cell division in eukaryotic organisms. There are 14 CDKs and 49 cyclins in <i>Arabidopsis</i>, but the pairings between them are not well understood, and the substrates of these complexes remain largely unknown. To better understand the various CDK-cyclin interactions and to ascertain the substrates of these kinases, two different methods were used to isolate interacting proteins from <i>Arabidopsis</i>. Isolations of CDKA and CDKB complexes were carried out through immunoaffinity purifications using antisera specific to either type of CDK. A two-step tandem affinity purification (TAP) method was used to purify CYCD3;1 complexes. Subsequent tandem mass spectrometric analysis of purified proteins identified 51 putative interactors from duplicate purifications by CDKA and 63 from duplicate purifications by CDKB. Both CDKA and CDKBs interact with ribosomal proteins, though the overlap is very small, suggesting the two types of CDKs have distinct functions. Of the proteins pulled down with CDKA, 24% are involved in unknown biological processes, indicating that CDKA may be involved in cellular processes not yet elucidated. From CDKB purifications, 51% and 32% of the proteins identified are involved in protein metabolism and cell organisation, respectively, suggesting a role for CDKBs in protein degradation, regulation of the cytoskeleton, and organisation of microtubules. Surprisingly, CYCD4;1 was the only cyclin co-purified with CDKA. Moreover, two proteins were identified to be binding to CYCD3;1-TAP. CDKA was found to be the exclusive partner for CYCD3;1 and the two proteins are potentially in complex with heat shock protein 70. Both methods are successful in purifying CDK-cyclin complexes from <i>Arabidopsis </i>and therefore provide a tool to explore the composition of these complexes and to identify putative targets. Together with further validation, these purification strategies can provide insight into control of the cell cycle and other mechanisms by CDK-cyclin kinases.

A long term study of the variation in d¹²C of bulk leaf material was undertaken under 3 light regimes (3%, 12% and 100% sunlight) using 2 different species displaying contrasting shoot phenologies: <i>Betula pendula </i>Roth. an early successional species with a rapid leaf turnover (“successive” phenology) and <i>Fagus sylvatica</i> L. a late successional species with a single flush of long-lived leaves (“flush” phenology). It was hypothesised that there would be greater seasonal changes in the isotopic composition of the leaves of <i>B. pendula, </i>within a single leaf cohort (due to rapid leaf turnover of that species) and that the different light regimes and shade tolerances of the two species would further extend variation. Physiological and phenological parameters, alongside bulk leaf d¹³C, were measured over the first growth season (2002) on 4 leaf cohorts in the <i>B. pendula </i>and the single flush of the <i>F. sylvatica</i>. The d¹³ C of the initial leaf was consistently more enriched in the heavier isotope than bud material.  Further changes were observed both within (up to 2‰) and between (up to 4‰) leaf cohorts in both species as well as substantial shifts between light regime (up to 6‰). During the following growth season, measurements were focused at 2 time points: early season and mild season to attempt to explain the variation within leaf cohort and in the change in isotopic signal between the bud and initial leaf. Alongside the continuation of measurements from the previous season, the d¹³C of the soluble carbohydrates and starch fractions were measured, as well as the d¹⁸ O of leaf organic material to follow the mobile carbon fractions as the organic signal changed and to partition the d¹³C signal into stomatal and biochemical influences. Carbohydrate d¹³C values showed that the within cohort variation could be explained by fractionation processes during phloem unloading and, later in the season, the progressive incorporation of new carbohydrate with a more negative delta.

The present work shows that the maize Rb homologue, ZmRb, interacts with human E2F1, <I>Drosophila</I> dE2F and the <I>Drosophila</I> dE2F/dDP heterodimer, while this activity is absent from a ZmRb mutant. Genomic and cDNA sequences with the potential to encode <I>Arabidopsis </I>E2F homologues were identified in the <I>Arabidopsis thaliana</I> database. Amplification of <I>Arabidopsis</I> sequences by PCR with degenerated primers, based on the database sequence information, followed by DNA hybridisation library screening led to the isolation of cDNA sequences for three <I>Arabidopsis</I> E2F clones-AtE2F1, AtE2F2 and AtE2F3. Phylogenetic analysis of the AtE2F sequences indicated that the AtE2Fs are related to human E2Fs (30% - 44% identity), but form a distinct group. The structural domain organisation of the AtE2Fs is the same as for other E2Fs, with the DNA-binding domain, leucine zipper and marked box conserved. A putative Rb-binding domain was identified in the C-termini of the AtE2Fs. Preliminary characterisation of AtE2F1 has revealed that AtE2F1 activated transcription in yeast cells and that ZmRb repressed AtE2F1-mediated transactivation. Direct interaction of AtE2F1 with ZmRb, but not a ZmRb mutant, was shown. As a heterodimer with the human DP1 protein, AtE2F1 bound to the E2F-binding sites in the promoters of a putative <I>Arabidopsis</I> CDC6 homologue and the human dihydrofolate reductase gene. The expression of the three <I>AtE2Fs</I> was followed in a partially synchronised <I>Arabidopsis</I> cell suspension culture re-entering the cell cycle, and was found to be cell cycle-dependent, with peak expression during S phase. The isolation of three <I>Arabidopsis</I> E2F homologues which share characteristics with mammalian E2Fs, indicates that this aspect of cell cycle regulation has been conserved between plants and mammals.