The taxonomy of some species of the genus Arabis L. (including Cardaminopsis Hayek) has been investigated experimentally with particular reference to the British taxa of Arabis hirsuta s.l. and Cardaminopsis petraea (L.)Hiitonen. Cytological studies, hybridization experiments, ecological investigations, population analyses and observation of the behaviour of plants in cultivation, have contributed to an understanding of the origins of variation and the relationships of taxa. The A. hirsuta complex contains diploid and tetraploid taxa. The complete sterility of hybrids between the diploids and tetraploids effectively prevents gene-exchange between chromosome-levels. The wide-spread and variable A. hirsuta (L.)Scop. is suggested to have an allo-tetraploid origin from A. corymbiflora Vest. and A. sagittata DC. In Britain, A. hirsuta is differentiated into distinct, but interfertile, local biotypes as a consequence of facultative autogamy and the recent disjunction of its populations. The plants of the Atlantic calcareous- -dunes constitute an ecotype for which subspecific status is proposed. The A. alpina complex is a group of geographically isolated taxa which are little differentiated morphologically. Incompatibility barriers have developed between some taxa. The status of the components of the circum-boreal C. petraea complex is obscured by the extreme plasticity of the response of the plant to its environment. This plasticity is more obvious than the genetic variation of the species in Britain and is the origin of the varieties grandifolia (Druce) and faeroensis (Hornem.). The synthesis of a sterile hybrid between C. petraea (L.)Hiitonen and C. halleri (L.)Hayek and the possession of similar modes of vegetative reproduction indicates the close relationship of these species. C. arenosa (L.)Hayek, because of its morphological differences from these species and its ability to hybridize with species of Arabidopsis Heynhold, has greater affinities to the latter genus.

Two A. thaliana lines transgenic for the promoter-trap vector pgusBin19 were studied. Line AtEN101 lacked a phenotype but reporter gene (gus) expression exhibited temporal and spatial regulation, including in the developing embryo. This pattern was unaltered when AtEN101 was used as a functional marker in two embryo-defective mutant backgrounds tested. In roots of AtEN101 seedlings, gus expression was repressed by ABA and modified by auxins, in a sucrose-modulated manner. The tagged genomic region in the mutant and wildtype, and cDNAs mapping to it were isolated. The cDNAs derived from two overlapping genes (PKT1 and PKT2) almost identical except for their first exons and introns and encoding putative peroxisomal 3-ketoacyl-CoA thiolases. The T-DNA was inserted in the long intron1 (966 bp) of PKT1, and 0.3 kb upstream from exon1 of PKT2. gus copies were found at both T-NDA junctions. Fusion transcripts including exon1 of PKT1 and T-DNA sequences were identified, and a cryptic 3' splice site identified in the left border region. The genes were found to be independently expressed, at low levels, in all organs by Northern blot hybridization and RT-PCR. Both putative promoter regions were capable of driving the expression of gus in transient assays. That of PKT1 was also functional in reverse orientation. The thiolase genes are part of a family of at least four members, PKT1 being unique in lacking the characteristic N-terminal peroxisomal targeting signal, PTS2. A new T-DNA linked semi-dominant mutation causing dwarfism (bashful) was identified in line P26K4, gus-derived sequences were present in its genome but no GUS activity was observed. The mutation affected cell growth and was rescued by epibrassinolide but not gibberellic acid (GA3). Skotomorphogenic development is also affected in the mutant. The utility of pgusBin19 as a promoter-trap and insertional mutagen in Arabidopsis, and the possible functions of PKT1/PKT2 are discussed.

Here I describe how cycles of mathematical modelling and experimenting have advanced our quantitative understanding of two different processes: transcriptional regulation of the floral repressor FLOWERING LOCUS C (FLC ) in Arabidopsis thaliana and spatial positioning of low copy number plasmids in Escherichia coli. Despite the diversity in biological subjects, my spatiotemporal modelling approach provides a common ground. FLC regulation involves an antisense-mediated chromatin silencing mechanism, where alternative polyadenylation of antisense transcripts is linked to changed histone modifications at the locus and altered expression. Mathematical model predictions of FLC transcriptional dynamics are validated by measurements of total and chromatinbound FLC intronic RNA. This demonstrates that FLC regulation involves a quantitative coordination between transcription initiation and elongation, potentially a general feature of gene regulation in a chromatin context. A quantitative analysis of cellular RNA levels indicates that FLC processing and degradation are well described by Poisson processes. FLC transcription correlates with cell volume, which underlies the large cellular variation in transcript levels. Low copy number plasmids in bacteria require segregation for stable inheritance through cell division. This is often achieved by a parABC locus, comprising an ATPase ParA, DNA-binding protein ParB and a parC region, encoding ParB-binding sites. These components space plasmids equally over the nucleoid, yet the underlying mechanism has not been understood. Here I show mathematically that differences between competing ParA concentrations on either side of a plasmid can specify regular plasmid positioning. This can be achieved regardless of the exact mechanism of plasmid movement. Experimentally, parABC from E. coli plasmid pB171 increases plasmid mobility, inconsistent with models based on plasmid diffusion and immobilization. Instead this observation favours a directed motion model. These results unify previously contradictory models for plasmid segregation and provide a mechanistic basis for selforganized plasmid spacing.

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The regulation of programmed cell death (PCD) and autophagy are crucial for the fitness of plants when encountering extrinsic stress. Over two decades, research on plant metacaspases has mainly concentrated on the role of these cysteine proteases in PCD. Emerging data now indicate that metacaspases might also interfere with the autophagy regulatory network. In this thesis, I present the analysis of metacapase-5 (AtMC5) functions in regulating endoplasmic reticulum (ER) stress-induced PCD and autophagy in Arabidopsis thaliana. AtMC5 exhibited a positive regulation of PCD as both ion leakage and caspase-3-like activities are reduced in the atmc5 knockout seedlings. In response to ER stress, AtMC5 is required for the cleavage of Defender against Apoptotic Death-1 (AtDAD1) and potential cleavage site(s) have been identified. Furthermore, an AtDAD1 fusion with Green Fluorescent Protein (GFP) was loaded into autophagic bodies during prolonged ER stress. A selective autophagy of the ER, ER-phagy, was evident in ER-stressed root cells and TEM studies identified multiple-membrane vesicles containing ER whorls corresponding to ER-phagosomes. Interestingly, atmc5 loss of function caused an increase in autophagic bodies/ ER-phagosome counts per cell, suggesting that AtMC5 might apply a break on auto/ ER-phagy. Taken together, these data place firmly AtMC5 and the cleavage of AtDAD1 at the intersection of PCD and auto/ ER-phagy, opening new avenues to the study of the key interaction between these two cellular processes.

The plant cell wall is a complex and dynamic structure, involved in interaction with the environment, cell-cell interactions as well as plant growth and development. Although structure and architecture of the walls have been studied extensively, knowledge regarding the mechanisms required to generate the complexity and control dynamic changes of the walls is limited. In order to improve our understanding, this study employs a combination of genetic, molecular and biochemical methods to characterize the functions of three candidate genes, At1g65610, At3g44990 and At1g07260. They encode a putative membrane-bound endo-1,4-β-glucanase (KORRIGAN2), a putative xyloglucan endo-transferase/hydrolase (XTH31) and a putative UDP-glycosyltransferase (UGT71C3) from Arabidopsis thaliana. The expression of the candidate genes changes in response to treatment with the cellulose biosynthesis inhibitor isoxaben. Plants with loss of gene activity of the target genes show only subtle growth phenotypes under standard growth conditions. However, cell wall analysis of stems from mature plants found distinct differences between the mutant plants and their wild type controls. In addition, my research showed that during early stage phenotypes, germination, root growth and isoxaben hypersensitivity phenotypes were observed in korrigan2 and xth31 seedlings. Interestingly, ugt71c3 seedlings exhibited early germination phenotypes but no effects on root growth. In parallel, it was found that all mutant seedlings tested produce more ectopic lignin upon isoxaben treatment than wild type controls. These results show that the genes characterized are involved in cell wall metabolism and are required for the growth as well as the response to cell wall damage simulated by isoxaben treatment.

The plant cell wall is a dynamic structure that has an integral role during plant development and responses to biotic and abiotic stresses. A cell wall integrity (CWI) maintenance mechanism in plants has been inferred. The aim of this study was to investigate the signalling mechanism that regulates the response of Arabidopsis thaliana to CWI impairment. Here I show that JA and SA levels increase after 3 and 5 hours (respectively) of CWI impairment. In order to determine the downstream functions of the different signalling mechanisms and their cross-talk upon CWI impairment; lignin deposition, JA & SA induction and root growth inhibition (RGI) phenotypes were used as downstream readouts. My results show that ROS are required for lignin deposition whereas JA & SA-based mechanisms are required to inhibit lignin deposition. ROS and JA act antagonistically to regulate downstream lignin deposition. In addition, a bi-phasic ROS-based signal is induced after CWI impairment, with the initial signal required for JA induction and the later signal required for inhibition of JA induction. H2O2 increased at the plasma membrane in the elongation zone of the root during CWI impairment, as determined by the genetically encoded probe HyPer. The functions of several RLKs in CWI maintenance were investigated. These included BAK1 and BIK1. Most of the mutant RLKs investigated affected one or two of the downstream readouts, highlighting the complexity of CWI signalling and demonstrating the involvement of elements of the innate immunity signalling. However, THE1 and XII7, mutant seedlings display altered responsiveness to all CWI impairment downstream readouts. These results suggest that these two RLKs represent key regulators of CWI maintenance. To summarise, the research piresented here has provided novel insights into the signalling mechanisms mediating the response to CWI impairment; has clarified the involvement of several RLKs whose functions had not been defined before and has shown that THE1 and XII7 are key regulators required for mediating all responses to CWI impairment.

Two allelic mutants of Nicotiana plumbaginifolia have bee isolated, which display a hypocotyl which is long (hlg) when seedlings are grown in continuous white light. This can be accounted for by the decreased response of hypocotyl elongation rate in these mutants to red light. Both mutants are deficient in a phyB-like polypeptide that is immuno-detectable in the wild type; both have wild-type levels of a phyA-like polypeptide. These alleles are inherited in a partially dominant manner, and correspond to single base missense mutations in a gene highly homologous to N. tabacum PHYB, which codes for a phytochrome-B-type photoreceptor. When grown in white light, mature hlg mutants are not elongated with respect to the wild type; they also bolt and flower later. The shade-avoidance responses appear intact in these mutants.;The sensitivity of the Nicotiana plumbaginifolia wild type and hlg mutants to photoperiod were investigated. It was found that the hlg mutant shows later bolting than the wild type under long days, but not under short days. The endogenous rosette-leaf-movement rhythms of the mutants display a much greater amplitude than those of the wild type under long-day conditions, but the two behave indistinguishably under short-day conditions.;The Nicotiana plumbaginifolia pew mutants, which are deficient in phytochrome chromophore, were also investigated. They show diminished sensitivity to both red and far-red light, and flower at an earlier stage. The pew3 mutant may belong to a previously uncharacterised class of phytochrome chromophore mutants.;Finally, tobacco plants were created which overexpress the native PHYA gene under the control of the 35S and native PHYA promoters. Both sets of transgenic plants displayed enhanced far-red sensitivity, and were dwarfed as light-grown adults. However, the reversed shade-avoidance effect characteristic of Avena phyA overexpressers was not seen. The plants overexpressing native PHYA were all delayed in flowering.

In order to identify and isolate genomic sequences that direct gene expression in the vascular tissues a 'promoter trapping' approach was adopted. Populations of transgenic Arabidopsis thaliana have been generated that contain a promoter trap vector, comprising a promoterless gusA gene. The rationale is that the inserted gusA sequence is only expected to be activated when integrated downstream of a native gene promoter, creating a functional gene fusion. Five transgenic Arabidopsis lines that were previously demonstrated to exhibit GUS fusion activity in vascular tissues were selected for this study. The number of T-DNA inserts in these lines was determined, and three of the five lines were found to contain single T-DNA copies. One line containing a single T-DNA insert, designated AtVS-1, was selected for further study.;Promoterless gusA expression in AtVS-1 was analysed throughout development, and a temporal and spatial regulation of gusA gene expression was observed. A putative aberrant roof phenotype was also revealed. An inverse PCR strategy was used to amplify genomic sequences flanking the T-DNA left border in AtVS-1, to yield an IPCR product of 1.45kb. The IPCR product was used as a molecular probe to identify homologous clones in wild-type genomic and cDNA libraries, which have been partially sequenced and show no similarity with other known genes. AtVS-1 was also used as a marker line to analyse cellular organization in two embryonic morphogenesis mutants. These results demonstrate the potential of promoter trapping as a means of creating functional tags to analyse genomic sequences direction transgene expression in the vascular tissues, and to facilitate the isolation of those sequences.

Two phytochrome-like gene fragments have been isolated using a degenerate PCR strategy from the fern Pteridium aquilinum (bracken). Both share a high degree of similarity with each other and to previously isolated phytochromes. In an attempt to isolate larger phytochrome gene fragments and full length phytochrome genes a cDNA library was constructed from light-grown young bracken fronds. The library was screened with one of the bracken phytochrome fragments. The low level of abundance of phytochrome message, however, hindered the isolation of phytochrome genes from the cDNA library. The high degree of sequence homology between lower and high plant phytochromes has led to speculation on whether or not lower plant phytochromes can be functional in higher plants. This speculation can be addressed by expressing the lower plant phytochromes transgenically in higher plants and assaying for biological function. Arabidopsis thaliana ecotype Landsberg erecta has been transformed using an Agrobacterium mediated root explant transformation system with a phytochrome-like gene isolated from the fern Anemia phyllitidis. The phytochrome gene, PHY3 was placed under the control of a double 35S cauliflower mosaic virus (CaMV) promoter. A second construct has also been used to transform Arabidopsis containing the NH2-terminal half of Anemia PHY3 gene fused to the C-terminal half of oat PHYA. This construct was also attached to a CaMV promoter. Expression of these two constructs causes a reduction in sensitivity of the seedlings to continuous FR. This decreased sensitivity results in a long hypocotyl phenotype under FR, a reduction in anthocyanin production in FR and an inability of FR to block greening. The transgenic lines, however, showed a WT response to VLF light. It is apparent that the expression of the Anemia PHY3 gene is, in some way, interfering with the normal transduction of the phyA signalling pathway. The interference is restricted to the FR HIR.