ABSTRACT The original objective of this work was to compare the cellular processes in salt tolerant and salt sensitive plants cells to gain insight into the mechanisms that confer halotolerance. Halotolerant and salt sensitive cell lines were derived from the model glycophyte Arabidopsis thaliana; in addition cell suspension cultures from the dicot halophytes Beta vulgaris and Atriplex halimus were also generated. Unfortunately, severe disruptions were encountered following a serious fire; persistent power failures, and failures of new equipment hampered progress with this work. For this reason, only comparisons between the Arabidopsis cell lines were completed. The halotolerant (HHS) cell lines survival strategy is to prevent Na accumulation when grown in < 100 mM NaCl. Wild type (WT) cells grow faster than HHS cells in the absence of NaCl, but rapidly take up Na in 50 mM NaCl where their growth is severely affected, and fail to grow completely above 100 mM NaCl. No evidence was found to suggest this growth impairment arose from osmotic stress or nutrient ion deficiencies. Protein profiling of HHS cells identified a number of proteins whose abundance is regulated by salt stress. These included proteins involved in ion transport, central metabolism, and general stress responses. The implications of these findings are discussed. In a separate project, a whole plant approach was taken to establish the physiological mechanisms that account for the reported difference in halotolerance between two commercially grown barley lines originating from China. Measurements on growth and development, plant water status, tissue ion profiling, photosynthesis rates, and transpiration rates suggested the tolerant line (Zhou 1) enters the reproductive phase of its life cycle approximately one week earlier than the sensitive line (Zhou 85), and this critical period allows floral development resulting in improved yields. This early ii flowering is not associated with the well characterized PpD-H1 locus controlling early flowering in cereals. The main conclusion from this study is that for glycophytes that do not complete a full life cycle above 100 mM NaCl (which includes all of the world’s major crops), it is the ionic component of salinity stress that impairs growth and yield. Further research on salinity stress in crops should focus on understanding the processes that control ionic balance rather than osmoregulation.

The ability of plants to convert energy provided by the sun into a form accessible by heterotrophic organisms ensures that they form a basal part of most ecosystems. However, in addition to being a vital energy source light can also serve as an environmental indicator. In order to maximise light perception, plants have evolved a suite of photosensors with differing sensitivities, which in combination provide detailed information regarding light availability and quality. The major photoreceptor families identified in plants include the phytochromes, which are most sensitive to red and far-red light, and the cryptochromes and phototropins, which are UV-A and blue light receptors. Additional photosensors, including the recently identified ZEITLUPE/ADAGIO family, have roles in modulating the action of these main components. Plants can also respond to UV-B and green light, although the photoreceptors responsible for their detection remain elusive. Phototropins are blue light sensors that are responsible for a range of responses (including phototropism, chloroplast movement and stomatal opening) that combine to increase the photosynthetic efficiency of plants. Initially identified in the model plant Arabidopsis thaliana, phototropins have since been characterised in the unicellular alga Chalmydomonas reinhardtii, pteridophytes and angiosperms. The light sensitivity of phototropins is derived from the action of two highly conserved regions known as LOV domains which subsequently induce activity of an integral serine/threonine kinase domain via movement of a conserved alpha-helix (Jalpha-helix). Although little is known regarding phototropin signal transduction an obvious biochemical consequence of phototropin light stimulation is autophosphorylation. Phototropin autophosphorylation has previously been studied using a baculovirus/insect cell expression system, with the consequences of phototropin mutation on phototropin kinase activity within this heterologous system having comparable effects to those when identically-mutated phototropins were introduced into transgenic Arabidopsis. Whilst the ability of phototropins to act as light-regulated kinases is well established, the mechanism by which this occurs remains unclear. In this study a baculovirus/insect cell expression system is used to further characterise the mode of phototropin autophosphorylation and the functionality of a mutated phototropin 1 which demonstrates increased autophosphorylation activity in this system is assessed in planta using transgenic Arabidopsis. It was initially of interest to further evaluate the mode of phototropin autophosphorylation. For example, it is unclear whether phototropin autophosphorylation occurs via an intramolecular mechanism or whether this process involves cross-phosphorylation between phototropin molecules. In Chapter 3 the mode of phototropin autophosphorylation activity was further examined using the baculovirus/insect cell expression system to assess the effect of protein truncations and specific point mutations on phototropin kinase activity. Such mutational analysis reveals that phototropin 1 is capable of intermolecular phosphorylation in vitro and also suggests that further phototropin autophosphorylation sites exist in addition to those previously mapped. Additionally, the importance of LOV2 and the Jalpha-helix as components of the phototropin receptor activation are confirmed. The implications of such findings for our understanding of phototropin autophosphorylation are discussed. LOV domains bind flavin mononucleotide as a chromophore. The light-induced formation of a covalent adduct between the LOV domain and the associated chromophore is thought to induce conformational changes culminating in phototropin kinase activation. In Chapter 4 the two leading hypotheses proposed to permit signal transmission between the light-sensitive LOV domains and the integral kinase domain are examined. Previous work has shown that photosensitivity within one of the two LOV domains (LOV2) is sufficient for light-regulated kinase activity, suggesting that light-induced kinase activation is primarily induced via LOV2. This allowed the effect of point mutations within LOV2 on the light-regulated kinase activation of the full-length phototropin protein to be assessed using the baculovirus/insect cell expression system. Work presented here suggests that a single point mutation within LOV2 (Gln575 to Leu; Q575L) is sufficient to reduce signal transmission between LOV2 and the phototropin kinase domain whilst not altering the photosensitivity of the LOV2 domain. In contrast, mutation of a highly conserved salt bridge at the surface of LOV2 does not alter LOV2 domain signal transmission. Interestingly, the reduced light-induced kinase activity of the phot1 Q575L mutant could be counteracted by an additional mutation within the Jalpha-helix (Ile608 to Glu; I608E) which has previously been shown to increase the autophosphorylation of mock-treated phot1 in vitro in comparison with a wild-type control. Such findings suggest that conformational changes may occur in sequence to induce phototropin kinase activation. The final component of this work involved transgenic Arabidopsis to examine the in planta functionality of phot1 I608E, which has been shown to have increased autophosphorylation in vitro when expressed using the baculovirus/insect cell system. However, rather than demonstrating phenotypes consistent with a constitutively-active form of phototropin, transgenic Arabidopsis expressing phot1 I608E appeared to have only partial functionality in planta and indeed appeared to inhibit phot-mediated phenotypes when expressed in a wild-type background. Possible explanations of the observed phenotypes are discussed. The studies presented here further advance our knowledge of the light-induced mechanism which results in activation of the phototropin photoreceptor and also provide insight into the potential differing roles of phototropin autophosphorylation in planta.

Potamogeton rutilus Wolfg. is a rare threatened macrophyte which, within the British Isles, is confined to oligo-mesotrophic Scottish lochs. The species has been lost from some of its former recorded sites, thought due to eutrophication and other environmental habitat changes. The plants limited distribution may be partly related to its lack of seed production that may limit long distance bird seed dispersal. P. rutilus appears to be reliant on clonal growth for reproduction, so may have a reduced genetic diversity that can reduce fecundity. To assist the development of strategies for the future conservation of P. rutilus, a series of investigations was carried out to reveal the autecological factors that influence the plant’s distribution and survival. In summing up the main findings of the investigation, it was found that Scottish and Finnish P. rutilus have different trophic plant community types, with Scottish P. rutilus inhabiting less nutrient rich oligo-mesotrophic lochs than the more nutrient rich, eutrophic, Finnish P. rutilus habitats. Salinity may have caused P. rutilus to be lost from some of its former machair lochs. P. rutilus abundance and macrophyte diversity significantly declined with reduced light availability in lochs. The growth experiments revealed that P. rutilus turion size is a good measure of plant fitness and fecundity and eutrophic phosphate conditions, under a half-light regime, produced optimum P. rutilus turion growth. The genetic investigations suggest there is limited gene flow between P. rutilus populations and this could be due to the lack of seed production for inter-population seed dispersal. However, P. rutilus does not totally rely on clonal growth for reproduction, as genetic evidence suggested there was some degree of sexual reproduction in some populations.

No description available.

The methanolic crude extract of aerial parts of the plant Scrophularia nodosa was shown to have potent DPPH radical scavenging activity (IC50 = 48.75 ± 7.00 μg/ml). Activity-guided fractionation resulted in the isolation of three principal antioxidant compounds; acteoside, angoroside C and angoroside A. Acteoside (yield = 1.21%, IC50 = 15.2 μM) appeared to be the most abundant and most antioxidant-active. The potent antioxidant activity is in support of the traditional use of the plant for wound healing and anti-inflammatory conditions. The methanolic extract of aerial parts of Tanacetum vulgare has potent DPPH radical scavenging activity (IC50 = 37.00 ± 1.20 μg/ml). Activity-guided fractionation on the methanolic extract of T. vulgare resulted in the isolation of 3,5-di-caffeoylquinic acid (3,5-DCQA), axillarin and luteolin. 3,5-DCQA appeared to be the most abundant and most antioxidant-active compound (yield = 7.28%, IC50 = 9.7 μM). The potent antioxidant activity is in support of the traditional use of the herb for fever, rheumatic conditions and anti-inflammatory conditions. The methanolic crude extract of Cassia auriculata and its fractions were shown to have potent scavenging activity on DPPH, hydroxyl and hydroperoxide radicals, moderate superoxide radical scavenging activity and potent ion(III) reducing power. The activity-directed studies resulted in the isolation of kaempferol-3-0-β-D-rutinoside, kaempferol, luteolin, quercetin and unknown antioxidant inactive compound. The previously reported pharmacological aspects of the above flavonoids and flavonoid glycosides (anti-carcinogenic, anti-inflammatory, hyperglycaemic, antidiabetic) along with the shown antioxidant behaviour explain the traditional medicinal values of the plant. Cassia alata L crude extract and its fractions showed potent radical scavenging activity against formation of lipid peroxide radicals. The activity directed isolations resulted in the isolation of kaempferol along with p-hydroxybenzoic acid. These may contribute towards the traditional medicinal values of the plant as an antidiabetic, anti-microbial and for skin diseases.

The adhesion between neighbouring plant cells is established as cells are formed during cytokinesis through the middle lamella that is made principally of pectins and proteins. Pectins are secreted into the cell wall in a highly methylesterified form and subsequently de-esterified in muro by pectin methyl esterase (PME, E.C. 3.1.11). The present study reports on the biochemical characterization and immunochemical analyses of phosphate buffer/EDTA pectic extracts associated with cell-cell adhesion in suspension cultures of wild type (WT), salt tolerant (HHS) cell lines and synchronized Arabidopsis suspension cultures. Using the synchronized cultures, The PME-mediated configuration of pectins at the onset of adhesion during cytokinesis, was assessed through the analysis of the expression patterns of the PME isoforms annotated to be expressed throughout the cell cycle The wild type Arabidopsis seemed to maintain the intercellular adhesion through the gelling of the highly methylated JIM7 recognized homogalacturonans that were shown to be abundant in the primary cell walls, middle lamellae and cellular junctions, possibly due to the hydrophobic interactions between the methoxy groups. The rhamnogalacturonan-I fraction was rich in arabinan side chains reflecting the proliferative state of the cells. The increase in arabinan content was accompanied by a reduction in the galactan content 4 days after subculturing. The cell walls of salt tolerant Arabidopsis contained the JIM7 and LM7recognized epitopes along with a high degree of branching of rhamnogalacturonan-I carrying galactans and arabinans as side chains. The change in the detected epitopes is thought to play a role in the ability of the cells to withstand the high osmotic pressure and increase the in the level of adhesion between cells. The JIM5 low methylesterified HGs were less abundant in both cultures, and the absence of the 2F4 antibody recognizing the Ca2+ egg boxes could be attributed to the scarce amounts of Ca2+ present in the culturing medium The immunochemical studies of the pectin extracted from the synchronized Arabidopsis suspension cultures after washing out aphidicolin indicated that the recognition of both of JIM7 and JIM5 varied in parallel during the cell cycle, whereas, the recognition of arabinan increased during the cell division. The sequence and phylogenetic analysis of ten PME isoforms that were annotated to be expressed at one or more phases of the cell cycle of synchronized Arabidopsis thaliana suspension cultures (Menges and Murray, 2002 and 2003), revealed that only five of these genes could be PMEs. The genes At4g02330, At1g02810, At2g26440, and At2g47550 were thought to be of type II PMEs which have a pre-pro-catalytic domains and At5g47500 is a type I PME that lack the pro-region. The amino acid sequence of At4g12390 showed similarities with the N-terminal pro-peptides of plant PME and invertase inhibitors. The expression of several PME genes was studied in suspension cultures of Arabidopsis thaliana synchronised using aphidicolin. Semi-quantitative PCR experiments showed that the expression of At5g47500 transcript was always detected during M phase of the cell cycle. The rest of the genes failed to show consistent patterns of expression. Northern blots revealed that mRNA coding for At5g47500 decreases during S and G2 phases and accumulates during the M phase of the cell cycle. Our results suggest that this PME isoform is involved in the modulations of the cell walls as the cells are going through division and cytokinesis.

This thesis describes the characterisation of the PQ-loop repeat family, a novel family of membrane proteins from Arabidopsis thaliana. The family consists of six members (AtPQL1-6) all predicted to contain seven transmembrane segments and two copies of the PQ-loop domain. The repeated PQ-loop domain is a well conserved feature present in several eukaryotic proteins. However, the functional significance of the domain remains to be determined. Although most PQ-loop repeat proteins are of unknown function some have been characterised from non-plant species. Those studied include CTNS - a lysosomal cystine transporter from humans, STM1 - a putative G-protein coupled receptor from Schizosaccharomyces pombe, ERS1 - a Saccharomyces cerevisiae protein isolated as a suppressor of a trafficking mutant and LEC35/MPDU1 - an endoplasmic reticulum protein from mammals involved in the utilisation of dolichol monosaccharides during glycosylation. The AtPQL family can be divided into three groups based on similarity with their non-plant homologues. Detailed sequence analysis was carried out to investigate conserved motifs, topology, domain organisation and genome distribution of the six genes. The implications of these findings for putative functions are discussed. AtPQL gene expression was investigated using promoter::GUS fusions and quantitative PCR. Whereas AtPQL4 and AtPQL6 appear to be expressed ubiquitously, AtPQL1, AtPQL2 and AtPQL5 are all preferentially expressed in floral tissues and AtPQL3 is expressed primarily in roots. Several family members showed rhythmic expression over the course of a day and induction by both viral infection and wounding. Localisation at the sub-cellular level was investigated using green fluorescent protein fusions and confocal microscopy. At least four AtPQL proteins are targeted to intracellular compartments. AtPQL1 and AtPQL5-GFP fusions both highlight the tonoplast whereas AtPQL4 and AtPQL6-GFP appear to highlight the ER. A number of knockout and over-expresser mutants were isolated for each gene. Although no physiological phenotype could be found, mutants with altered expression of AtPQL4 and AtPQL6, the two closest A. thaliana homologues of MPDU1/LEC35, showed a clear molecular phenotype. These mutants displayed differential regulation of a number of genes encoding N-glycoproteins as well as defence, cell-surface, lipid biosynthesis and ER stress associated proteins. Finally, the functional importance of the AtPQL family is discussed and a role for AtPQL4 and AtPQL6 in glycosylation proposed.

The Begonia flora of Southeast Asia comprises more than 540 species. This exceptional species diversity and the wide distribution of the genus in tropical rainforests offers the opportunity to address biogeographical questions and to investigate the processes which underlie modern patterns of biodiversity, but also poses major taxonomic challenges. Only few apomorphies characterising infrageneric taxa in this large genus have been identified and delimitation of Asian Begonia sections is highly problematic. A robust phylogenetic framework of Asian Begonia informing taxonomic monographs and facilitating biogeographical and evolutionary studies is currently lacking. Maximum parsimony, maximum likelihood and Bayesian analyses of plastid (ndhA intron, ndhF-rpl32 spacer, rpl32-trnL spacer; 115 taxa) and nuclear ribosomal (ITS; 89 taxa) sequence data were used to reconstruct the phylogeny of Southeast Asian Begonia and to determine whether major Asian sections are monophyletic. Morphological characters which are crucial in current sectional circumscriptions were mapped on the phylogeny to determine their degree of homoplasy and to assess their suitability in infrageneric classifications. Relaxed molecular clock analyses of a Cucurbitales-Fagales datatset (cpDNA: matK gene, rbcL gene, trnL intron, trnL-F spacer; 92 taxa; five fossil calibrations) and a Begoniaceae datatset (cpDNA: ndhA intron, ndhF-rpl32 spacer, rpl32-trnL spacer; 110 taxa; two alternative secondary calibrations), as well as ancestral area reconstructions were employed to elucidate temporal and spatial diversification patterns in Asian Begonia. The results indicate that Asian and Socotran Begonia species form a well supported clade. Most major Asian sections are not supported as monophyletic and the strong systematic emphasis placed on single, homoplasious characters such as undivided placenta lamellae (section Reichenheimia) and fleshy pericarps (section Sphenanthera), and the recognition of sections primarily based on a plesiomorphic fruit syndrome and the absence of characteristic features of other taxa (section Diploclinium) has resulted in the circumscription of several highly polyphyletic sections. Ovary and fruit characters have traditionally played a major role in sectional delimitation, however the high level of homoplasy associated with these has obscured systematic relationships in Asian Begonia. Gene trees derived from separate analyses of the plastid and nuclear ribosomal data show congruent support for several major clades, but there is hard incongruence within the clades comprising species of the species-rich sections Platycentrum s.l. (including section Sphenanthera) and Petermannia s.l. (including section Symbegonia), indicating that hybridization might have had a significant impact on the evolution of the genus. The molecular divergence ages and the biogeographical analyses indicate an initial diversification of Asian Begonia on the Indian subcontinent and in continental Southeast Asia in the Middle Miocene, and subsequent colonization of Malesia by multiple lineages. The predominant directional trend of the reconstructed dispersals between continental Asia and Malesia and within Malesia is from west to east including four independent dispersal events from continental Southeast Asia and the Malesian Sunda Shelf region to Wallacea dating from the Late Miocene to the Pleistocene. Dispersal across the ancient deep water channels separating intervening islands of the Sunda Shelf and Wallacea and subsequent successful colonisation of Wallacean islands seem to have been infrequent events during this period. This suggests that the water bodies which have separated the Sunda Shelf region from Wallacea have been distinct, yet porous barriers to the predominantly anemochorous dispersal in Begonia. The inferred timing of dispersals from the Sunda Shelf region to Wallacea is generally concordant with hypotheses about the geological history of the region, which indicate that the period from the Late Miocene onwards offered opportunities for dispersal to Wallacea and across Wallacea to New Guinea as substantial land masses emerged in Sulawesi and New Guinea, and newly emergent volcanic islands along the Sunda Arc, the Banda Arc and the Halmahera Arc formed potential routes for dispersal by island hopping. The results further suggest that Begonia section Petermannia (>270 spp.) originated in the Malesian Sunda Shelf region, and subsequently dispersed to Wallacea, New Guinea and the Philippines. Lineages within this section diversified rapidly since the Pliocene with diversification peaking in the Pleistocene. The timing of diversifications coincides with orogenesis on Sulawesi and New Guinea, as well as pronounced glacioeustatic sea-level and climate fluctuations. It can be hypothesised that a complex interplay of extrinsic and intrinsic factors including the presence and formation of suitable microhabitats by orogenesis, cyclical vicariance by frequent habitat fragmentations and amalgamations caused by sea-level and climate fluctuations, as well as only weakly developed mechanisms to maintain species cohesion in fragmented habitats in Begonia could have driven speciation in allopatry and could have resulted in the remarkable Begonia species diversity found in Southeast Asia today.

Investigating the individual proteins involved in plant defence and the pathways these proteins are part of is important to gain an understanding of infection and disease resistance processes. It is hoped that this knowledge will help develop solutions to prevent crop infection and increase crop yield. More specifically, CMPG1 was first found to be important in pathogen responses by Kirsch et al. (2001) in parsley. It has further been found to have homology to NbACRE74 and AtPUB20 and AtPUB21 implying it is common to many plant species. CMPG1 is an U-Box E3 Ubiquitin ligase and involved in a number of different defence responses (INF1, Cf-9 & Pto/AvrPto). PiAvr3a is an RxLR cytoplasmic effector protein from potato late blight pathogen Phytophthora infestans. Its virulence function was shown by its ability to suppress INF1 cell death by Bos et al. (2006). This P. infestans protein and the plant protein CMPG1 were found to interact in Yeast-two-hybrid studies performed by Dr M Armstrong (Bos et al., 2010). The aims of this project were to determine the nature of this interaction, functionally and biochemically. Does the interaction found in the Yeast-two-hybrid take place in the plant? What are the biological implications of PiAvr3a and CMPG1 interaction? What is the biochemical nature of the interaction? This project used in planta studies to investigate the CMPG1-PiAvr3a interaction. The biological reason for why this interaction occurred was studied using virus induced gene silencing (VIGS) and hypersensitive response/cell death suppression assays. The biochemical nature of this interaction was investigated using ubiquitination assays, and purified proteins, in vitro. This thesis aimed to provided data to increase the overall understanding of the function of CMPG1 and PiAvr3a during infection. Firstly, this project found evidence for the stabilisation of StCMPG1 by PiAvr3a as well as possible direct interaction inside plant cells. The stronger interaction with, and stabilisation of StCMPG1 by PiAvr3aKI, is in accordance with the stronger suppression of INF1 cell death by this form of the effector (Bos et al., 2006; 2009). Secondly, evidence for the role of CMPG1 in disease resistance to multiple plant pathogens, including oomycetes, bacteria and fungi was found. PiAvr3a suppresses all of these cell death responses. It is likely that CMPG1 is a target for other effectors from other pathogens. Cell death suppression by PiAvr3a is caused by stabilisation, and thus altered function, of CMPG1. Thirdly, promising and surprising data revealed that PiAvr3a may act as an E2 conjugating enzyme. Moreover, it appeared to act via both lysines 48 and 63, perhaps suggesting that it forms a mixed chain on CMPG1 substrates.

Plant adaptation to stress is dependent upon the initialisation of molecular signalling networks that regulate the expression of stress-related genes. By examining high-resolution microarray datasets it has been possible to track gene expression changes over time during senescence and in response to infection by fungal pathogen Botrytis cineria in the model organism Arabidopsis thaliana. Dramatic variations in gene expression are observed at the onset of stress with different groups of genes showing different expression time-courses. This observation must, for a large part, be down to the action of different transcription factors (TFs) binding to the cis-regulatory DNA in the promoters of genes in each group and it is this regulatory code that underpins the gene regulatory networks that regulate stress responses. This thesis presents an interdisciplinary investigation of the regulatory codes that are responsible for controlling plant stress responses. Computational analysis of non-coding sequences provides a powerful approach to identify patterns within DNA that may function to regulate gene expression. This thesis covers the development of Analysis of Plant Promoter-Linked Elements (APPLES), an object-orientated software framework for the analysis of non-coding DNA. Within this environment, methods were developed to probe the regulatory codes that exist within these non-coding sequences and identify regulatory motifs that may function to regulate stress responses in Arabidopsis. APPLES methods were used to identify a novel motif that is likely to play a role in regulating drought responses in Arabidopsis, with experimental approaches providing support for this view. Using known motifs that describe previously characterised TF binding sites, it was possible to identify motifs that are associated with clusters of co-regulated genes identified from the senescence and Botrytis microarray time-course datasets. This analysis revealed cis-regulatory elements that may contribute to generating the observed expression patterns. In a contrasting approach to in silico identification of regulatory elements, the Yeast-1-Hybrid (Y1H) assay was used to experimentally identify interactions between TFs and non-coding DNA. The use of a TF library allowed the ability of approximately 1400 Arabidopsis TFs to interact with a given DNA sequence in a single assay. Using the stress-associated ANAC092 promoter as a test case, it was possible to use this highthroughput procedure to identify TFs that can bind to the promoter of this gene. This high-throughput Y1H system was then used to perform a detailed mapping of protein- DNA interactions that can occur across the core promoters of three highly related stress inducible TF-encoding genes, ANAC019, ANAC055 and ANAC072. Microarrays were used to assess the regulatory consequence of a subset of these interactions by perturbing the expression of interacting TFs and observing the effect on target gene expression during multiple stresses. This approach confirmed predicted regulatory relationships and therefore enhanced the current understanding of the transcriptional regulatory networks that operate during stress responses in Arabidopsis.