Erwinia species cause soft rot in a range of economically important plants. The secretion of cellulases and pectinases, via the Out apparatus is considered a major pathogenic determinant. The Out proteins are encoded by a cluster of at least 13 genes, outC to outO, and show homology to the pullulanase secretion proteins of Klebsiella oxytoca, and secretion proteins of other plant and animal pathogens. This study concerns the nature of the interaction between the Out apparatus and the secreted enzymes, with a view to identification of the recognition motifs; on the secreted proteins that allow specific secretion. Secretion deficient mutants were isolated using hydroxylamine and PCR mutagenesis. These were characterised with respect to activity and stability of the Ce1V derivatives; ability to secrete other proteins; and DNA sequence. Two classes of mutant were identified. The first class exhibited accumulation of a cata1ytica11y active but conformationally defective product which did not affect the secretion of other proteins. These were therefore predicted to be defective at the level of recognition. The second class exhibited accumulation of a stable, full size product which interfered with the secretion of pectate lyase and were therefore predicted to be blocked within the translocation apparatus. The isolation of intragenic suppressors of some of these mutants is described. Several deleted derivatives of Ce1V were constructed, all of which were severely affected in their ability to be secreted, even when only-12 carboxy terminal amino acids were affected. In conclusion, recognition for secretion is highly dependent on conformational integrity of both of the functional domains of which the enzyme is composed. The pathways followed by cellulase and pectinases appear to be initially separate, converging to a common translocation apparatus. In addition, cellulase, although having limited independent macerating ability, was identified as playing a vital role in the collaborative action of exoenzymes during attack of potato tubers.

No description available.

No description available.

Rice (Oryza sativa) is well known as a biosilicifier though the mechanisms which underlie the silica deposition in them are still relatively unknown. Silica deposits in rice were imaged through various complementary techniques including PDMPO staining and fluorescence microscopy, scanning electron microscopy, micro-particle induced X-ray emission and low-energy X-ray fluorescence. These techniques showed silica deposition throughout all expected sites of silica deposition, as well as revealing novel areas of silica deposition in the xylem, a region not usually considered to be a zone of silicification despite its known role in transporting silicic acid. Silicon was found to be co-localised with aluminium at the silica cells through the use of low-energy X-ray fluorescence. This is, to the best of our knowledge, the first evidence of silicon/aluminium colocalisation in rice leaves. The link between the hemicellulose callose and silicification was investigated, with aniline blue and immunofluorescence staining revealing numerous sites of callose deposition in rice leaf tissue, all of which matched with known areas of silica deposition. It was demonstrated that silicic acid uptake in rice does occur at a rate which would require active transport through the use of a simple experiment where the silicic acid content of solutions with rice plants growing in them were measured over time. Silicification was studied in developing rice seedlings using PDMPO staining and fluorescence microscopy to image structures from different developmental stages of seedlings. Silicification was found to occur at all stages of development, with evidence of silicification of precursor leaf tissue inside ungerminated seeds. The implications of these findings were discussed and the possibility that silicon may be maintained in a form other than solid silica in certain areas of rice plants was put forward.

Nuclear-encoded thylakoid proteins are targeted into and across the thylakoid membrane by four distinct mechanisms. Precursors of lumenal proteins are translocated by either a Sec- or ΔpH-dependent mechanism. In both cases, thylakoidal processing peptidase (TPP) removes this signal peptide to release the mature protein. A structurally similar signal peptide is present in the fourth pathway used by a subset of integral membrane proteins including PSII-W, PSII-X and PsbY. In this case the integration process does not involve an identifiable energy source or known protein transport machinery. This Sec-independent direct insertion mechanism is unlike any other known, with the exception of a single protein, M13 procoat, which similarly inserts in the Escherichia coli plasma membrane by means of a signal peptide. This novel insertion mechanism has been probed by mutagenesis of TPP cleavage sites within pre-PSII-W, -PSII-X and -PsbY, in order to generate intermediates on the insertion pathway. TPP cleaves preferentially after Ala-X-Ala, and the terminal alanine was mutated to threonine by site-specific mutagenesis of the cDNA. Chapter 3 - In the case of pre-PSII-W and pre-PSII-X, TPP is the only proteinaceous component known to be involved at any stage of the insertion process. The PSII-W cleavage site mutant is imported and inserted into the thylakoid membrane, but cleavage by TPP is inhibited. Import into chloroplasts results in the accumulation of a mature size protein and an intermediate form that accumulates when TPP fails to complete the maturation (the precursor protein is processed to an intermediate in the stroma). Importantly, the intermediate is located exclusively in the thylakoid membrane, confirming that the action of TPP is not required for correct localisation. Protease-topology mapping presented, shows that this intermediate is in the form of a loop intermediate in which both the N- and C-termini are exposed on the stromal face of the membrane with the intervening region on the lumenal face. Preliminary data is also presented for a loop intermediate with the insertion of pre-PSII-X. Chapter 4 - Precursor PsbY has a more complex insertion mechanism and 6 mutants are presented that show various intermediates on the pre-PsbY insertion pathway. All the intermediates are stable in the thylakoid membrane. Furthermore, using this approach, it can be shown that pre-PsbY is in fact a polyprotein containing two similar single-span proteins, both of which are preceded by cleavable signal peptides. Pre-PsbY thus contains a single chloroplast targeting peptide, an initial thylakoid signal peptide and mature protein followed by a second thylakoid signal peptide and mature protein. This is therefore the first nuclear-encoded polyprotein targeted to the thylakoid membrane in higher plants, and the data suggest that this protein inserts as a double-loop form.

High soil salinity is a major environmental stress that adversely affects crop production throughout the world. It is now estimated that half of the world’s cropland is affected by salt stress. To cope with various environmental stresses, plants are able to spatially and temporally regulate gene expression through changes in DNA methylation and chromatin conformation, known as epigenetic modifications. Recent studies indicated that epigenetic modifications induced by environmental stress can be inherited over several generations, despite a genomewide epigenetic resetting of epigenetic imprints that takes place during plants reproduction. In this thesis, I evaluated in Arabidopsis thaliana the effect of multigeneration salt stress treatments on the genome-wide dynamics of DNA methylation and tolerance to high salinity. My results show that the immediate progenies of stressed plants displayed better germination and survival rate under high salinity, but contrary to current theories this effect is lost in the following non-stressed generation. Genome-wide DNA methylation analysis revealed that stress induced discrete de novo methylation and demethylation changes on epigenetically labile regions of the plant genome. These acquired tolerance and methylation marks are likely under parent-of-origin control as a result of a robust epigenetic reprogramming that takes place in the male germline. Stress-induced methylation marks identified are associated with transcriptional changes of stress responsive genes and correlated with antisense long-non coding RNA expression. Overall this work establish for the first time a link between differential DNA methylation, gene expression and shortterm adaptation to stress in plants.

Whilst juvenile during vegetative growth, plants are incapable of initiating flowering when grown under floral inductive conditions. Understanding juvenility is important since it influences flowering time, which in turn impacts on scheduling of crop production. Daily light integral (DLI or LI) is the total photosynthetically active radiation received in one day and is calculated from light intensity and light period (Korczynski et al., 2002). It is one of the factors that could regulate juvenility. The project aims are to determine the effect of light integral on (i) phase development, (ii) photosynthetic assimilation and partitioning in relation to juvenility, (iii) the expression of key flowering time genes, including FLOWERING LOCUS T (FT) and genes involved in carbohydrate metabolism and (iv) the expression of microRNA156 in relation to juvenility. Transfer experiments have been carried out to generate material differing in juvenility length for molecular and biochemical analyses. These utilised antirrhinum plants grown under different light integrals and Arabidopsis mutants defective in specific carbohydrate related genes. cDNAs representing genes involved in starch synthesis (PHOSPHOGLUCOMUTASE(PGM), PHOSPHOGLUCOISOMERASE(PGI), SUCROSE PHOSPHATE SYNTHASE(SPS) ) and degradation (STARCH EXCESS1(SEX-1), BETA AMYLASE3(BAM-3), ALPHA AMYLASE(AMY)) have been isolated from antirrhinum plants. Antirrhinum plants grown under reduced light integrals had extended juvenile and adult vegetative phases and reduced levels of AmFT, AmPGM, AmPGI, AmSEX-1, and AmBAM-3 during juvenility. Juvenility was shown to be extended in mutants defective in both starch synthesis and starch degradation. This indicates that light integral influences the time plants need to accumulate enough level of carbohydrate to support phase change and levels of oligosaccharide, released during starch degradation, may influence the length of juvenility. Consistently, in both Arabidopsis and antirrhinum, the timing of FT induction was shown to correlate with the end of juvenility and interestingly, light integral has a greater effect on AmFT induction and phase transition than photoperiod. Moreover, the current study shows that LI also affects the length of JP when there are highly expression of miR156 which means that miR156 possibly acts downstream of LI in the regulation of flowering.

In spite of constant technological advancements, world hunger remains a major challenge due to exponential population growth, and the loss of e effectiveness of crop treatments such as pesticides. As such, comprehending the plant response to stress is of great importance in breeding more resilient crops. Whilst different stresses elicit distinct responses from the plant, a core set of regulatory interactions are conserved across multiple responses and operate as networks. In this thesis, computational approaches were used to elucidate such regulatory interactions from time course expression datasets, predominantly through identification of genes co-expressed across multiple stimuli responses as a footprint of shared network co-regulation. The identification of such network footprints was tackled through Wigwams, a data mining algorithm capable of detecting groups of genes co-regulated across multiple datasets. In contrast to other algorithms, Wigwams assesses whether the co-expression it detects is likely to reflect co-regulation. The modules it found were significantly enriched in functionality and cis-regulatory elements, indicating actual co-regulation. Wigwams and other computational approaches were applied to time course expression data capturing Arabidopsis thaliana response to Pseudomonas syringae pv. tomato DC3000. The presence of a virulent and avirulent strain in the experiment allowed for the temporal deconstruction of the regulatory events underlying the virulent strain's attempts to overcome plant defence through effector action. This analysis led to the detection of a number of effector-specific transcription changes stifling the defence response and manipulating the host's gene and protein expression. A transcription factor-only regulatory network model was proposed to explain the detected network footprints. The inference of causal regulatory networks from expression data is a daunting task, and transcription factor-only models are a good computational compromise by capturing the key regulatory events taking place. However, they are lacking in target genes that carry out the functionality induced by the signalling, making functional assessment di cult. Wigwams was used to introduce the network footprint components into the corresponding transcription factor-only models, resulting in enhanced network models carrying information about downstream regulated genes. This allows for functional assessment to be used to identify nodes of interest within the network, and propose concise follow-up experiments.

A key question in biology is how the transfer of nutrients from mother to offspring is regulated. In mammals, the translocation of nutrients to the embryo is mediated by the placenta, while in flowering plants is governed by the seed endosperm. Current evidence shows that in plants, nutrient transfer from maternal tissues to developing embryos is regulated at two levels: Initially, from maternal integuments to endosperm through a layer of specialised transfer cells and secondly from endosperm to embryo through the embryo-surrounding cells (ESR). However, a remaining question is: how is the translocation of nutrients between these two layers coordinated? The aim of this work was to investigate small cysteine-rich proteins (CRP) and their involvement in the reproductive development of cereal crops. This was carried out by characterisation of ZmMlp1, a novel plant CRP gene, using promoter reporters, spatiotemporal gene expression profiling, biochemical protein characterisation and localisation. The function of ZmMlp1 was then investigated by knocking down its expression in maize through RNA interference and subsequent morphological and molecular phenotypic characterisation. The 3D structure of ZmMLP1 was resolved by solution state NMR, allowing investigation into key structural components and how they contribute to the functionality of ZmMLP1. The characterisation of ZmMLP1 demonstrated a novel expression pattern in maize, since ZmMLP1 was shown to be a secreted protein expressed in both the central cell of the ovule and the ESR of the developing endosperm between 0 and 10 DAP. Functionally, ZmMLP1 was shown to play a role in embryonic development, demonstrated by a defective embryo patterning phenotype displayed by the RNAi transgenics. Furthermore, seed weight and size was shown to be significantly reduced in ZmMlp1 –RNAi lines, linking ZmMlp1 to crop yield. The structure of ZmMLP1 was shown to contain a knottin-like core, stabilised by 4 disulphide bonds which support disordered sequence loops that form key sections of the molecular surface. Sequence chimeras formed from ZmMLP1 and AtESF1.3 induced a known suspensor phenotype in Arabidopsis, demonstrating that specific structural loops are critical to CRP functionality. Collectively these studies have furthered the understanding of nutrient translocation between maternal tissue to offspring and how CRPs are involved in these plant processes.

As a result of damage caused during the First Gulf War in 1990-1991, the Kuwaiti environment suffered from drastic pollution caused by massive petroleum hydrocarbon contamination resulting from the destruction and burning of 700 oil wells across Kuwait. A range of types of polluted soils, including fresh oil lakes, dry oil lakes, and tarcretes, damaged desert wildlife. The idea of phytoremediaton using native plants was introduced and concluded that the native species Haloxylon salicornicum (Amaranthaceae) had potential as a phytoremediator. In the initial phase of this study a follow-up survey of clean and polluted sites in 7 areas in Kuwait was undertaken: Bahra, Sabah Alahmad protected area, Burgan oil field, Um Alaish oil field, Um Alrros Military Base, Sabriya oil field, and Um Ghadaier oil field where 41 plant species were found to be present. TWINSPAN classification of the dataset identified four assemblages of plant species, occurring in four ecologically-distinguishable habitat types (represented by 7 sample-groups produced by the classification procedure); one of them is mostly north of Kuwait where Sabah Alahmad protected area is and Bahra and Um Alaish oil fields and tends to be more in the oil damaged areas and characterized by the presence of the Haloxylon salicornicum; the other one is in both the north (Um Alaish oil field) and south of Kuwait (Burgan oil fields) and is characterized by the presence of both Cyperus conglomeratus and Rhanterium epapposum while the third and fourth assemblages can be mostly considered variant and characterized by the presence of Pennisitum divisum. These native species (former 3) were hence selected as the focus for subsequent investigation. The survival of Haloxylon salicornicum plants in weathered oil-polluted soils was experimentally investigated under greenhouse conditions, using a random block design with 4 replicates and 5 treatments: pots containing 100%, 75%, 50%, and 25% polluted soil, mixed with clean soil, and clean soil only as a control. The results indicated that the plants could grow successfully even in 100% oil-polluted soil. The experimental results also provided evidence that water applied to the surface (simulating rainfall) could reach the root system in all of the treatments (even for the 100% oil contamination treatment). Following on from the greenhouse study, a field trial was undertaken to examine the survival and growth of Haloxylon plants introduced into clean and oil-polluted soils (in and adjacent to a weathered dry oil lake) under field conditions. Three replicate two-year old iii (nursery-grown) Haloxylon plants were planted at each randomly-chosen location in the dry oil lake soil, and the design was repeated at 10 random locations in clean soil close to the lake boundary. The experiment was set up in two different locations (Bahra area in the north; and in Burgan oil field in the south of Kuwait). there was no significant difference in growth rates between plants in clean and polluted soil, in either area. The biomass data showed a significant difference in fresh weight between plants from clean and polluted soils, with those growing in clean soil having higher moisture content (possibly less woody than those from the polluted sites). However there was no significant difference in either fresh or dry biomass between the two experimental areas in the north and south. Data produced by analysis of amount of TPH in the polluted soils in both experimental areas showed some variability, but overall there was no significant difference between the two polluted areas, in terms of their weathered petroleum hydrocarbon content. Successful phytoremediation usually, if not always, is a function not only of phytoremediator plant physiology, but also the activity of the phytoremediator species associated rhizosphere microflora. In order to gain some insight into the hitherto unknown rhizosphere microflora of Haloxylon salicornicum plants, bacterial and fungal isolation procedures were carried out on samples taken from the roots of wild Haloxylon salicornicum plants, and from cultured plants growing in oil-contaminated soils in the greenhouse experiment, using media enriched with petroleum hydrocarbons to encourage the culture to survive in such conditions. Bacteria organisms found to be associated with the rhizosphere of wild Haloxylon are Streptomyces spp. and Inquilinus sp., while in 100% oil contaminated soil Rhodococcus manshanensis, Agrobacterium tumefaciens, Nocardia cyriacigeorgica, Gordonia lacunae / Gordonia terrae and Lysobacter spp., occurred. In the 50% oil contamination treatment soil, around the roots of Haloxylon plants contained Gordonia lacunae / Gordonia terrae and Agrobacterium tumefaciens and finally the clean soil Sphingopyxis spp. was present. Fungi found in the Haloxylon rhizosphere included organisms known to be associated with petroleum hydrocarbon degradation, including Penicillium spp. in wild Haloxylon (and also 50%, 100% and clean soil from the greenhouse trial), as well as Trichoderma asperellum in the clean soil. The conclusion is that Haloxylon salicornicum, together with its rhizosphere microflora it contain, offers high potential for use in phytoremediation operations designed to assist in the clean-up of oil polluted desert soils in Kuwait.