The experiments described in this thesis were designed towards reaching an understanding of the control of flux through the arginine pathway in Neurospora crassa. The previous algebraic analysis of steady state enzyme networks has shown that the flux is a systemic property and that, typically, the relation between flux and effector is non-linear. It was therefore necessary in the present experimental analysis to apply the method of modulation, i.e. to determine the flux response to a series of changes in the concentration of a putative effector. It was already known that arginine affects the rate of each step in the arginine pathway by controlling the amount (induction or repression) and/or activity (activation or inhibition) of the individual enzymes. The principal and largely successful aim of this study, therefore, was to develop and apply an experimental method whereby the mycelial argtnine concentration could be predictably modulated over a wide range, under steady state conditions; then to monitor and interpret the systemic response, as indicated by the changes in i) the levels of other pathway intermediates, ii) the specific activities of the connecting enzymes and iii) the measured fluxes. This was achieved by controlling the rate of citrulline uptake and, consequently, the steady state concentration of mycelial arginine in the auxotrophic mutant ?. Ornithine then becomes an "end product" with its accumulated steady state concentration indicating the pathway flux. From the experimentally generated relationships between metabolic pools and between pools and enzyme specific activities the mechanisms of importance in the control of the pathway fluxes were revealed. It is shown that there is no unitary solution to the problem of flux control; a number of different mechanisms effect the control of flux through the arginine pathway with the proportion of the control exerted by each depending absolutely upon the level that the flux has.

Analysis of phytochrome null mutants has revealed roles for these photoreceptors throughout plant development, synchronising developmental events to daily and seasonal environmental changes. The roles of the phytochromes in shoot photomorphogenesis is relatively well characterised, but little is known about their influence on root development. Over the course of my PhD I have shown that phytochromes are in fact important regulators of root phenotype. My results demonstrate that application of low R:FR ration light, which reduces the active phytochrome pool (Pfr), changes the distribution of expression of the synthetic auxin reporter gene <i>DR5::</i>GUS, which in turn correlates with a reduction in lateral root emergence. Thus, I have identified a long distance signalling role for shoot phytochromes, acting collectively to control the emergence of lateral roots, at least partly by manipulating the early shoot-root auxin pulse. I have shown that phytochromes are present and light regulated in roots, and propose that phytochromes are able to act locally to regulate root hair elongation. My analysis of the interaction between <i>phyB</i> and <i>shy2-2</i> implicates these genes in the regulation of microtubule stability, and consequently of cytoskeleton organisation. I have also provided evidence that the Pr form of phytochrome, previously thought to be physiologically inactive, does in fact have important roles in the regulation of root development. I have taken a novel approach to understanding phytochrome signalling, and have opened many new lines of enquiry into an exciting new area of photobiology.

In this study potato plant with lowered D-enzyme activity were investigated. It was found previously that lack of D-enzyme resulted in a reduction in the tuber yield of these plants and a delay in tuber sprouting. In this study the reduction in tuber yield was characterised further. It was found that tuber fresh weight per plant was reduced but percentage dry matter and starch content were unaltered. In addition, the extent of the reduction in fresh weight tuber yield was dependent on irradiance. Lack of D-enzyme also resulted in a delay in sugar accumulation in tuber during prolonged storage. No differences were detected in the rates of starch synthesis and starch turnover in tubers with lowered D-enzyme activity compared to controls. One reason for this could have been that the labelling experiments employed to investigate tuber metabolism were not sensitive enough to detect small differences. To address this, D-enzyme activity was lowered in transgenic potato plants which exhibit exaggerated rates of starch synthesis and turnover due to the expression of a heterologous ADPglucose pyrophosphorylase gene. It was hypothesised that differences in starch metabolism would be more likely to be resolved in tubers from these plants. Surprisingly, no appreciable differences between the starch metabolism of tubers with exaggerated rates of starch turnover and lowered D-enzyme activity and control tubers were detected. However, the level of D-enzyme activity in these tubers was 14% of wild type and this could have been high enough for the <i>in vivo</i> role(s) of the enzyme to be fulfilled. Rates of starch breakdown in leaf tissue lacking D-enzyme were slightly reduced compared to controls during darkness. Lack of D-enzyme did not affect rates of starch synthesis during the light. These results are consistent with those from the D-enzyme mutant of <i>A. thaliana</i> and suggest that D-enzyme may have a role in potato leaf starch degradation during darkness and that this could in turn influence tuber growth.

Endogenous ascorbate concentrations remained unaffected following exposure of excised radish hypocotyls to various forms of stress that included aerobic, anaerobic, light, dark, 254 and 366 nm ultraviolet and contact with 33 mM sodium nitroprusside. Exposure of intact radish plants to a high-oxygen atmosphere produced significant drops in the ascorbate concentrations of the hypocotyl tissue. Irrigation of the roots of radish plants with 50 mM sodium ascorbate did not affect hypocotyl acorbate concentrations- Exposing radish plants to (50 mM) sodium ascorbate by watering through laterally trimmed roots and vacuum infiltration failed to increase endogenous ascorbate concentrations. Excised radish leaves fed 0.1 M L-galactonolactone showed a clear increase in L-gascorbate concentrations. Hypocotyls bathed in 0.1 M L-galactonolactone showed no increase in L-ascorbate concentrations. Radish plants fed L-galactonolactone through an incision in a leaf petiole showed a clear increase in L-ascorbate concentrations in the fed leaf as well as neighbouring leaves; however, no increase in ascorbate concentrations was seen in the hypocotyl. Hypocotyl disks bathed in de-gassed water for 24 hours showed production of L-aseorbate after being fed L-galactonolactone as well as clear uptake of L-[6­³H]galactonolactone with a K_m of 0.24 nM. A hypocotyl disk fed 17 MBq of L-[6-³H]galactonolactone produced cellular radiolabelled ascorbate as well as several other unknown metabolic products ‘The hypocotyl bathing solution contained several unknown products released by the cells into the extracellulax fluid. Compounds were detected by electrophoretic fluoragraphy at pH 6.5 and by chromatographic fluorography BuOH:HOAc:H₂O (12:3:5). Unknown compounds were detected by this method while tartrate, threonate and oxalate, metabolites of ascorbate metabolism were not detected.

Hydroxycinnamic acids are present in land plants as soluble and as insoluble (cell wall bound) esters. The most common hydroxycinnamic acids found in cell walls are ferulic and <I>p-</I>coumaric acids. These compounds are present particularly in Gramineae, where they are linked to arabinoxylan. The roles of cell wall bound ferulic acid have not been completely elucidated. Recently it has been reported that feruloylated arabinoxylans of cultured <I>Festuca </I>cells are relatively resistant to the enzyme mixture Driselase whereas Driselase can act successfully on other graminaceous cell walls (e.g. maize and barley) to release feruloylated oligosaccharides (e.g. ˜ 79% was solubilised from maize) such as Fer-Ara-Xyl (FAX) and Fer-Ara-Xyl<SUB>2</SUB> (FAXX). The resistance of <I>Festuca </I>feruloyl-arabinoxylans to Driselase means that only a very low percentage of the feruloyl groups were released as FAX and FAXX. The feruloylated material was solubilised by Driselase (˜80% as well) but in the form of relatively high molecular weight conjugates. The question of why <I>Festuca </I>feruloyl-arabinoxylan cannot be digested by Driselase to fragments such as FAX and FAXX to any great extent has now been investigated. The major feruloylated oligosaccharide (compound B) released from <I>Festuca arundinacea </I>cell walls by mild acid hydrolysis showed the unusual structure <I>5-O-(E)</I>-feruloyl-[<I>O-β</I>-D-xylopyranosyl-(1→)2]-L-arabinose. The (1→2)- linkage was established by NaIO<SUB>4</SUB>-oxidation and by PE of the NaBH<SUB>4</SUB>-reduced compound in molybdate-buffer alongside marker sugars with (1→2)- and (1→3)-linkages; the (<I>E</I>)-feruloyl ester group by its characteristic fluorescence and UV absorption; the <I>5-0</I>-linkage by partial hydrolysis of compound B to the known compound, 5-<I>O</I>-feruloyl-L-arabinose (compound A); and the β-linkage by hydrolysis with β-xylosidase. The structure was confirmed by NMR spectroscopy and methylation analysis.

Self-incompatibility (SI), the recognition and rejection of selfed pollen, is a classic study system for population geneticists and cell and molecular biologists interested in it's spectacular polymorphism and it's evolution. This plant mating system is one of the few systems where the selection forces maintaining the extreme level of polymorphism is well-defined, yet how SI evolves remains poorly understood. In <i>Brassica</i>, SI is controlled maternally by haplotypes involving at least two related genes; the S-related kinase (SRK) and S-glycoprotein (SLG), and paternally by the recently discovered cystein rich pollen gene (SCR). Consistent with frequency-dependent selection these loci exhibit exceptional levels of high amino-acid variability with some regions being 'hypervariable' (HV), similar to the MHC locus. A major question is whether HV regions are targeted by balancing selection, or merely regions of relaxed selective constraint. A second question relates to the role that recombination may play, if any, in the evolution of SI. The genes controlling SI are thought to be tightly physically linked such that maternal and paternal alleles maintain haplotype configurations necessary for correct recognition. In <i>Brassica,</i> I observed that linkage disequilibrium and nucleotide diversity patterns indicate recombination of some form, perhaps gene conversion, plays an evolutionary role at the SLG gene. It also appears now that this locus may not be essential for the recognition response. To assess the true level of nucleotide diversity and recombination in natural populations, and to address whether HV regions are neutral versus under selection, I identified and characterized seven loci in <i>Arabidopsis lyrata</i> that are homologous to the SI-genes in <i>Brassica. </i>Segregation analyses shows that all but one set of sequences is unlinked to self-incompatibility in <i>A. lyrata</i> and exhibit relatively low levels of nucleotide diversity. SLG- and SRK-like variants have been identified that segregate with incompatibility groups in three independent families, and exhibit extremely high levels of polymorphisms, similar to MHC and <i>Brassica </i>SI, suggesting that this locus is targeted by balancing selection.

The glyoxylate cycle is a key step in the conversion of storage lipids to sugars in germinating oilseeds. This conversion is thought to allow seedling growth prior to the development of photosynthetic organs. The glyoxylate cycle is thought not only to operate during this stage of development but also during senescence, starvation, pollen development and embryogenesis. It is not known how important the cycle is for plant physiology and development. Two enzymes isocitrate lyase (ICL, EC 4.1. 3.1 ) and malate synthase (MS, EC 4. 1. 3. 2), are central to the cycle. In order to elucidate the function of the glyoxylate cycle in plant development, it was proposed that the synthesis of MS and ICL be inhibited in transgenic plants transformed with chimaeric antisense <I>Ms </I>or <I>Icl </I>genes. Two plant species each offering specific advantages, <I>Nicotiana plumbaginifolia </I>and <I>Solanum tuberosum, </I>were selected for these experiments. Partial <I>Ms </I>and <I>Icl</I> cDNA fragments from both species were isolated and cloned. These were used to construct plant transformation plasmids in which each cDNA fragment was in the antisense orientation with respect to the CaMV 35S promoter. Transgenic <I>N. plumbaginifolia </I>and <I>S. tuberosum </I>plants were regenerated from tissue transformations using strains of <I>Agrobacterium tumefaciens </I>carrying the respective plasmids. The presence of either transgene did not detectably interfere with fertility, germination or post-germination growth in <I>N. plumbaginifolia.</I> In addition to the <I>Ms</I> and <I>Icl </I> mRNAs, the majority of <I>N. plumbaginifolia </I>transformants synthesised a transcript not detected in non-transformants, which was shown in antisense <I>Ms</I> transformants to be antisense RNA complementary to the <I>Ms</I> cDNA fragment. Despite the abundance of antisense RNA in some transformants, neither the steady-state levels of target gene mRNA, nor MS enzyme activities, were significantly affected. The relevance of these results to the mechanism of antisense inhibition is discussed. Evidence was obtained that suggests that the expression of <I>Ms</I> and <I>Icl</I> is influenced by multiple factors.

Using a biotin switch method we show that proteins are S-nitrosylated in <i>Arabidopsis </i>following challenge with both virulent <i>Pseudomonas syringae</i> pv.<i> tomato </i>strain <i>Pst </i>DC3000 and avirulent <i>Pst </i>carrying <i>(avrB). </i>Purification and MALDI analysis indicates that one of these S-nitrosylated proteins is a carbonic anhydrase. A single copy <i>Arabidopsis GS-FDH </i>was identified which shows 75% homology in amino acid sequence to mouse <i>GS-FDH. </i>The induction of <i>GS-FDH </i>by GSNO <i>in Arabidopsis </i>was confirmed by northern analysis.  <i>GS-FDH </i>expression in <i>Arabidopsis </i>is constitutive at basal levels. During the HR, which was induced by <i>Pst </i>DC3000 <i>(avrB), </i>GS-FDH expression and activity were found to be rapidly suppressed. Whereas no GS-FDH suppression takes place following infection with virulent <i>Pst </i>DC3000. Therefore GS-FDH expression and activity are tightly regulated during the HR. Three <i>GS-FDH </i>T-DNA insertion lines were identified from the SAIL and SIGnAL databases. Two of these lines <i>fdh1-1 </i>and <i>fdh1-2 </i>are allelic and contain a T-DNA insert in the promoter of GS-FDH. Northern analysis and enzyme activity assays found that GS-FDH is over-expressed in these lines. Characterisation of <i>fdhl-1 </i>revealed that it constitutively expresses the defence gene <i>PDF1.2. fdh1-1 </i>exhibits resistance against the virulent biotroph <i>Pst </i>DC3000. Moreover, <i>fdh1-1 </i>has a delayed HR response following <i>Pst </i>DC3000 <i>(avrB) </i>infiltration. In addition, an activation tagged population was screened to isolate mutants resistant to nitrosative stress, from which ten NO resistant mutant candidates were identified.

This project was an investigation of the regulation of PAL induction, following elicitation of cell cultures of<i>Capsicum frutescens</i>, and its effects on phenylpropanoid metabolism. It was shown that the response of PAL specific activity to elicitation increased throughout the culture cycle and consequently, stationary-phase cultures were used throughout the investigation. PAL activity in <i>Capsicum</i> cultures increased transiently after elicitation. Detailed investigation of the initial stages of elicitation showed that PAL activity was induced 40 min after the onset of the elicitor treatment and reached a maximum after <i>ca</i>. 8 h. <i>Capsicum</i> PAL protein sub-units were identified and the molecular size was estimated to be 77 kD, the same size as PAL sub-units isolated from parsley leaves. <i>In vivo</i> labelling techniques showed that the transient induction of PAL specific activity following elicitation resulted from a combination of the general induction of protein synthesis and a specific induction of PAL protein synthesis. Additionally, the synthesis of a 32 kD protein, probably a chitinase precursor, was transiently induced with a lag phase of 4-8 h. A 70 kD polypeptide, possibly a degradation product of PAL sub-units, was recognised by anti-(parsley PAL) serum in samples from elicited and control cultures of<i>Capsicum frutescens</i> but not in <i>Petroselinum</i> samples. PAL mRNA was identified and its molecular size was estimated to be 2.6 kb. Increased PAL mRNA steady state levels were detected following dilution and elicitation. The transient increase in PAL mRNA levels during elicitation followed the same pattern as PAL protein accumulation, thus indicating that the induction of PAL is regulated at the transcriptional level. Elicitation of <i>Capsicum</i> cultures increased PAL mRNA levels transiently resulting in <i>de novo</i> synthesis of PAL protein and, thus, increased PAL activity.

No description available.