No description available.

The main aim was to investigate the cell-wall cross-links in Chinese water chestnut (CWC), in particular ferulic-acid-containing phenolic-polysaccharide cross-links. The secondary ~ims were: to understand the gross composition of C''!C cell walls froin the parenchyma, epidermis and sub-epidermis tissues of the corm and the -role of cell-wall composition in the plant's physiology, and to determine whether CWC contained higher oligomers of ferulic acid. Cell-wall composition was investigated using a range of chemical analyses including alkali phenolic extraction and methylation analysis. Chemical and biochemical methods were evaluated for their ability to produce oligosaccharide fragments attached to ferulic acid species. Mild acid hydrolysis followed by column chromatography using Biogel P-2 was the method chosen. lC-MS was used to identify compounds of interest. The compositions of the epidermal tissues differed particularly in the proportions of lignin and cellulose present. The relative amounts and proportions of the phenolics varied considerably, possibly indicating their functions in the different tissues. A multitude of phenolics were detected, a number of which now have detailed UV information recorded about them. The LC-MS results indicate that trimers and tetramers of ferulic acid are present, and provide some degree of structural information for the trimers. A reasonable level of solubilisation was achieved with mild acid hydrolysis, releasing ....70% of the arabinose, xylose and galactose present into the supernatant. LC-MS indicated that multiple species containing ferulic acid or diferulic acids linked to one or more pentose sugars are present in the TFA hydrolysate and Biogel P-2 fractions, indicating that ferulic acid and diferulic acid are linked to cell-wall sugars in CWC as in many other monocots. Trimers and tetramers of ferulic acid were detected in a nonmaize substrate for the first time, implying the possibility of higher oligomers of ferulic acid being present naturally in a wide range of cell walls.

No description available.

Ethylene is an established plant growth regulator linked with programmed cell death (PCD). To investigate the relationship between the cell cycle and PCD, ethylene was used to see if it induced mortality in a cell cycle specific manner. Tobacco BY-2 cultures synchronized with aphidicolin were treated with ethylene. Cell cycle progression and mortality, measured at hourly intervals, showed distinct peaks of mortality at the G2/M boundary and S-phase. In conjunction with this, DNA fragmentation increased at G2/M. Furthermore, ethylene caused a significant reduction in cell size of the cycling population. Simultaneous addition of silver nitrate with ethylene ameliorated ethylene-induced G2/M mortality, although a toxic effect of silver alone was evident. Due to the toxicity of silver, 1-MCP, an alternative chemical for blocking ethylene receptors was used. 1-MCP neither affected the BY-2 cell cycle nor mortality levels. In addition, 1-MCP ameliorated ethylene-induced G2/M mortality. To balance the chemical approaches to blocking ethylene receptors, tobacco BY-2 cells were transformed with Atetrl that encodes a dominant insensitive form of the Arabidopsis ETR1 ethylene receptor. Atetrl expression caused a massive perturbation to the tobacco BY-2 cell cycle, especially in S-phase, and resulted in high levels of mortality throughout the cell cycle. Ethylene treatment caused a doubling of G2 duration but did not affect temporal distribution of mortality. However, ethylene treatment generated a peak of mortality in S-phase. These results suggest that ethylene induces PCD at G2/M through the known ethylene signaling pathway. Furthermore, it confirms that 1-MCP and Atetrl result in ethylene insensitivity. To examine the G2/M transition, Spcdc25, a positive regulator of G2/M in fission yeast was transformed into the tobacco BY-2 cell line. This resulted in premature entry into mitosis, a shortened cell cycle, and reduced cell size. This was similar to Spcdc25 over-expression in fission yeast and suggests the presence of a CDC25-like phosphatase in plants.

Senescence is the final event in the life of many plant tissues. It is concerned with remobilisation of metabolites from the senescing organ and is distinct from other forms of cell death. It is a highly regulated process involving structural, biochemical and molecular changes. Gene expression is tightly regulated, with many genes down regulated and others induced. Conversely, ageing is a passive degradation. Petals and leaves are good model organs for developmental studies, as petal development is irreversible and tightly controlled, and leaves are an extensively studied senescent system. Similarities between these organs allow direct comparison of late developmental events to establish whether petals senesce, as leaves do, or age however, important differences in function lead to the hypothesis that petal deterioration and leaf senescence are distinct. Wallflowers were chosen as they are closely related to Arabidopsis and Brassica spp., have large petals, and are commercially relevant ornamental plants. Changes in wallflower morphology and physiology were examined over development. A high degree of gene homology was shown between wallflowers and Arabidopsis by northern blotting. 1632 genes upregulated in old wallflower tissues were cloned using subtractive selective hybridisation and a microarray of these genes, along with 91 Arabidopsis senescence associated genes, was constructed. Probing the microarray with RNA from different leaf and petal developmental stages identified 298 common genes significantly upregulated during both leaf senescence and late petal development, and expression patterns were compared between the two tissues. The expression patterns of selected genes were confirmed by RT-PCR. Late development in petals was shown to be an active process, and was tentatively concluded to be senescence due to upregulation of genes involved with nutrient remobilisation and the strong upregulation of the SAG 12 gene---a molecular marker for leaf senescence that is not upregulated during other forms of death in leaves.

Reactive oxygen species (ROS) together with other signalling molecules including phytohormones regulate plant growth and senescence as well as responses to abiotic stress and pathogens. One aim of this thesis was to investigate how ROS and phytohormones regulate progression of senescence in wallflower petals. A second aim of this thesis was to investigate how SAG21, previously found to be elevated in wallflower petals, functions during development, senescence and stress in the model plant, Arabidopsis thaliana. In wallflowers (Chapter 3), onset of petal senescence was characterized by a rise in ethylene and auxin, followed by ROS accumulation. In parallel, transcript levels for markers of senescence, ethylene biosynthesis and auxin-response also increased with age. However, SAG21 peaked earlier, before visible senescence. Treatments that aimed to increase cytokinin levels or reduce ethylene delayed progression of senescence and time to petal abscission compared to controls. In contrast, ethylene hastened both processes. Marker genes analysed displayed differential expression patterns. Patterns of ROS-related enzyme activity (catalase, ascorbate peroxidase and superoxide dismutase) were also investigated in relation to wallflower petal and leaf senescence. SAG21 (previously named AtLEA5 in Arabidopsis) belongs to the late embryogenesis- associated (LEA) protein family, implicated in growth and redox responses. A SAG21-YFP fusion was localized to Arabidopsis mitochondria (Chapter 4). Over-expression (OEX) of SAG21 in Arabidopsis resulted in increased root and shoot biomass, whereas antisense (AS) lines exhibited reduced biomass. Root and shoot development, flowering and senescence were altered in transgenic lines. Transgenic lines exhibited marked root hair phenotypes (Chapter 5). Abiotic stress induced stress induced SAG21 expression in roots but not leaves. Growth of B. cinerea and of a virulent bacterial pathogen {Pseudomonas syringae pv. tomato) was affected by SAG21 expression, however growth of an avirulent P.syringae strain was unaffected (Chapter 6). These data show that SAG21 is regulated by a complex signalling network.

Many herbicides are applied in the form of carboxylic esters to increase their hydrophobicity and hence aid their passage through the waxy cuticle. Hydrolysis in planta of these pro-herbicide esters releases the active acid or alcohol and the rate of this cleavage can be a factor in determining herbicide selectivity. Protein extracts from 13 crop and weed species were assayed for carboxylesterase activity toward multiple xenobiotic and pesticide ester substrates, including 2,4-D-methyl, aryloxyphenoxypropionate esters and p-nitrophenyl esters. A diversity of activities was exhibited by the different species, with Arabidopsis thaliana extracts showing high hydrolytic activity toward several xenobiotic esters, particularly 2,4-D- methyl.The major 2,4-D-methyl hydrolysing enzyme in arabidopsis cell cultures was purified through three rounds of chromatography, then selectively labelled with a biotinylated fluorophosphonate probe (FP-biotin). Following streptavidin affinity purification, the labelled protein was identified by proteomics as the previously uncharacterised serine hydrolase AtCXE12. Recombinant AtCXE12 was subsequently confirmed to effectively hydrolyse 2,4-D-methyl.A T-DNA insertion knockout line that did not express AtCXE12 was identified and characterised. Protein from the knockout plants did not contain AtCXE12 and was found to have a reduced rate of 2,4-D-methyl hydrolysis compared to wild-type plant extracts. This translated into a higher tolerance of 2,4-D-methyl in young atcxel2 plants, due to a lower rate of bioactivation of the pro-herbicide.The fluorophosphonate-based chemical probe was subsequently used to identify other major serine hydrolases in arabidopsis. AtCXE12 and three previously uncharacterised hydrolases were identified, each belonging to a distinct enzyme family.

No description available.

In flowering plants, the male gametophyte plays a vital role in plant fertility through the generation and delivery of two sperm cells to the embryo sac. A strict male germline is only established after meiosis when haploid microspores divide asymmetrically to form a small germ cell and large vegetative cell. This germ cell goes on to differentiate and divide once again to produce the functional twin sperm cells required for double fertilisation. Despite its importance in plant fertility and crop production, the mechanism integrating germ cell proliferation and specification during male gametogenesis has remained elusive. DUO1 is an evolutionary conserved unique R2R3-type MYB transcription factor that is specifically expressed in the male germline. DUO1 is the major determinant of male germline fate, regulating the expression of key specification genes required for fertilisation whilst integrating this with germ cell cycle progression through the regulation of the G2/M regulator CYCB1;1. DUO1 constitutes the major focus of this thesis and as such it addresses several aspects of DUO1 regulatory network. The first chapter of the thesis explores upstream regulatory mechanisms controlling male germline restricted expression in Arabidopsis. The expression of several genes, including DUO1, does not depend on a previously proposed derepression mechanism whilst DUO1 expression involves only positive promoter elements. The second chapter involves functional characterisation of the DUO1 protein, which has helped to delimit the transactivation domain of DUO1. This analysis has also provided insights into the evolutionary conserved supernumerary lysine residue present in the DUO1 MYB domain, which is likely to play a role in attenuating the rate of target gene transcription. The third part describes the analysis and verification of novel target genes in the DUO1 regulatory network. A mechanism for direct transactivation of target genes is also described whereby DUO1 binds to MYB sequences in target gene promoters. The final chapter concerns the functional analysis of two redundant DUO1-activated zinc finger (DAZ) EAR repressor proteins. Their intermediate role in coordinating male germ cell cycle progression is demonstrated by their ability to complement the cell cycle defect in the duo1 mutant. Furthermore, DAZ1 and DAZ2 also influence sperm cell specification, indicating that the DAZ subregulon also integrates specification with cell cycle progression. Together this data has provided compelling insights into the scale and architecture of the DUO1 male germline regulatory network controlling the production of functional sperm cells in flowering plants.

The shade avoidance response is of major adaptive significance to plants. Angiosperms in particular have developed a wide range of strategies to avoid shade. In canopy shade conditions, the ratio of red to far-red light wavelengths decreases (low R:FR ratio) this acts as a signal to instruct plants that they are in the proximity of neighbouring flora. A decrease in R:FR ratio is perceived through the phytochrome system, which results in onset of several developmental responses; such as elongation growth; that help promote escape from shade. Although much is known of the role phytochromes play in light perception and the concomitant physiological responses triggered by a decrease in the R:FR ratio, little is known of the downstream molecular components that bring about these changes. This study focuses on a small number of genes (DIN2, ENDO1 and XTH15) that whilst known to be R:FR ratio regulated, have as yet unknown roles during shade avoidance. This work demonstrates that all three genes respond with extreme rapidity and reversibility to changes in light quality conditions. The role phytochromes play in regulation of these genes is investigated. In addition a number of light regulated transcription factors, downstream of the phytochromes, are identified as intermediate regulators of these genes. As it has recently come to light that a number of R:FR ratio regulated genes are also under the control of the circadian clock the ability of this signalling pathway to influence expression of these genes was also investigated. Molecular analysis revealed that the circadian clock modulates two of the three genes examined within this study. Finally, systematic physiological characterisation of mutant and over-expressing lines was performed with an aim to determine the biological function of these genes during shade avoidance. Additionally, this study reveals that one of the genes studied; XTH15; not only exhibits R:FR ratio regulation of expression, but also blue light mediated transcriptional responses. This raises the possibility that R:FR ratio and blue light signaling pathways converge. Preliminary data suggests that XTH15 could represent a point of cross over with the phytochromes and the cryptochromes. This work, which characterises the response and potential role of three R:FR regulated genes downstream of the phytochromes, provides important and novel contributions to our understanding of the environmental regulation of plant development.