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1
McGouran, Joanna. "Probing sugar-plant-soil signalling." Thesis, University of Oxford, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.526091.
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Truman, William Matthew Donald. "Signalling pathways underylying plant innate immunity." Thesis, Imperial College London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.429264.
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de, Vos Sarah. "Phosphatidylinositol 3-phosphates in plant cell signalling." Thesis, University of East Anglia, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.251889.
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Sukiran, Nur Afiqah Binti. "Exploring plant hormonal signalling through chemical perturbation." Thesis, Durham University, 2018. http://etheses.dur.ac.uk/12625/.
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Plant growth and development is tightly regulated by a set of plant hormones that includes abscisic acid (ABA) and gibberellic acid (GA). Understanding how this is achieved is challenging due to a complex interplay between the various signalling pathways involved. A chemical genetics approach was used in this study to explore this hormonal crosstalk with a focus on plant growth. A small library of analogues, modified from the calmodulin inhibitor W7, was generated and the effect of these compounds was tested in root growth assays. One particular compound, eW5, was identified as providing enhanced and prolonged root growth even when plants were subsequently removed from the compound. Further analysis suggested that the phenotype induced upon eW5 application was due to modification of DNA methylation. Therefore it was hypothesized that eW5 might affect gene expression, which was tested using a RNA-seq experiment. Results from this suggested that eW5 regulates hormone signalling pathways, with a particular positive correlation to the GA signalling pathway observed. Interestingly, eW5 binds to the ABA receptor PYR1, thus potentially functioning as an ABA antagonist and also promotes DELLA (a negative regulator of plant growth) degradation, in a similar fashion to that observed with GA. Further work was performed to investigate the effect of eW5 on ABA and GA independent pathways. In ABA signalling, eW5 showed inhibition of some ABA responses such as stomatal opening, however no recovery in PP2C phosphatase activity suggests that it does not promote growth by inhibiting ABA perception. In addition, due to the specific GA-mediated response in hypocotyl growth, the eW5 effect was further explored in this particular process. With regard to eW5’s potential role in GA signalling, it was found to enhance sensitivity to GA that leads to DELLA protein degradation and growth promotion. Moreover, it was suggested that the promotion of eW5 in stomatal opening occurs through GA signalling. Having established the positive effect of eW5 on root and hypocotyl growth and stomatal opening, a further small library of analogues of eW5 was generated to further explore its mode-of-action. The position of sulfonamide was identified as a potential site that is responsible for hypocotyl growth promotion.
5
Walker, J. Ross. "Presenilin complexes in Arabidopsis : novel plant cell-signalling components?" Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/4090.
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Intercellular signalling is essential for multicellular organisms to coordinate growth and development, and is mediated by a huge variety of proteins. Some signalling pathways rely on the proteolytic cleavage of membrane proteins by a relatively newly discovered process of regulated intramembrane proteolysis (RIP), the cleavage of proteins within a transmembrane domain. There are four classes of intramembrane cleaving proteases (ICliPs) – Rhomboids, Site-2-proteases, Signal peptide peptidases and γ-secretase. Of all the ICliPs studied to date, γ-secretase is unique, as it is comprised of a four-protein complex, and is only found in multicellular organisms. A vast amount of research is carried out on the γ-secretase complex, not just because of its role in developmentally important pathways, such as NOTCH signalling, but also due to its role in Alzheimer’s disease. The β-amyloid precursor protein (APP) is cleaved by γ-secretase, and defects in this process result in the release of abnormal peptides that form the senile plaques in the brains of Alzheimer’s disease patients. Homologues of the four components of γ-secretase (PRESENILIN (PS), NICASTRIN (NCT), ANTERIOR PHARYNX DEFECTIVE-1 (APH-1) and PRESENILIN ENHANCER-2 (PEN-2)) are found in plants. The aim of this thesis was to characterise the potential γ-secretase components in Arabidopsis thaliana, to determine whether they form a complex, and to analyse what role, if any, they play in plant signalling. The members of the putative Arabidopsis γ-secretase complex (AtPS1 and 2, AtNCT, AtAPH1 and AtPEN2) were identified through BLAST searches, and found to be uniformly expressed. Analysis of T-DNA insertion mutants in each of these genes, and combinations there of, revealed no gross morphological differences to wild type under normal growth conditions and when subjected to a range of stresses. Protein fusions to GFP under the control of the 35S promoter were constructed and stably transformed into plants. AtPEN2:GFP is expressed throughout the plant, and accumulates in BFA sensitive Golgi bodies in roots. AtPS1:GFP, only accumulates strongly in developing seeds. Native blue PAGE was used to look for high molecular weight complexes (HMW) containing AtPEN2:GFP and AtPS1:GFP. Both fusion proteins were found in similar sized HMW complexes. A variety of methods were used to look for substrates of the iv putative γ-secretase complex in Arabidopsis, and although no specific substrates were identified, a potential role in seed development has been established.
6
Kneeshaw, Sophie. "Molecular mechanisms of redoxin-mediated signalling in plant immunity." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/18754.
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Posttranslational modification (PTM) of proteins is essential to creating a diverse proteome with the complex functions necessary to regulate key cellular processes. Redox-based PTMs exhibit many desirable characteristics to finely modulate transcriptional regulators; they occur rapidly and can alter protein conformation, localisation and activity. The plant immune system offers an excellent model in which to study redox-based modifications due to the rapid accumulation of oxidising agents that occurs during immune invasion. This so-called “oxidative burst” causes spontaneous oxidation of cysteine residues that are present in many regulatory proteins. These modifications fine-tune the activities of proteins that harbour them, enabling them to act in a concerted effort to reprogram the transcriptome, prioritising the expression of immune-related genes over housekeeping genes. Disulphide bonds (S-S) and S-nitrosothiols (SNO, i.e. the addition of an NO group to a cysteine moiety) have been shown to play particularly important roles in plant immunity. However, what still remains unclear is how these redox-based PTMs are rendered reversible, enabling them to act as molecular signalling switches. The work presented in this thesis explores a class of enzymes that are responsible for controlling the cellular levels of protein oxidation: the Thioredoxins. In addition to their well-established role in reducing disulphide bonds, I demonstrate in Chapter 3 that Thioredoxins are able to reverse protein S-nitrosylation during plant immune signalling. Immune-inducible Thioredoxin-h5 (TRXh5) was shown to be unable to restore immunity in gsnor1 mutants that display excessive accumulation of the NO donor S-nitrosoglutathione, but rescued impaired immunity and defence gene expression in nox1-mutants that exhibit elevated levels of free NO. This data indicates that TRXh5 discriminates between protein-SNO substrates to provide previously unrecognized specificity and reversibility to protein-SNO signalling in plant immunity. Furthermore, data is presented to show that TRXh5 reversed the effects of S.nitrosylation on many immune-related transcriptional regulators in vitro, forming the initial stages of an investigation into which proteins and pathways might be controlled by reversible S-nitrosylation in plant immunity (Chapters 3 & 4). Although the majority of transcriptional regulators are likely modified at their site of action, the nucleus, very little is currently known about nuclear redox signalling in plants. Therefore, in Chapter 5 a subclass of theThioredoxin superfamily was studied, the Nucleoredoxins, which have previously been shown to display disulphide reduction activity and localise in part to the nucleus. Here it is revealed that the activity and nuclear accumulation of Nucleoredoxin 1 (NRX1) is induced by the plant leaf pathogen Pseudomonas syringae, suggesting a key role for this protein in immune signalling. Target-capture experiments and subsequent mass spectrometry analysis identified the first in vitro targets of NRX1 and revealed many proteins with roles in oxidative stress, including the hydrogen peroxide scavenger Catalase 2 (CAT2). Moreover, overexpression of NRX1 was shown to be able to rescue the enhanced cell death phenotype of cat2 knockout mutants in response to the oxidative stressor, methyl viologen. Accordingly, nrx1 knockout mutants also exhibited an enhanced cell death phenotype in response to methyl viologen treatment. Together, these data indicate that NRX1 plays a key role in the control of oxidative stress-mediated cell death, potentially through direct regulation of Catalase proteins. Taken together, the work in this thesis implicates members of the Thioredoxin family as key regulators of transcriptional reprogramming during plant immunity and uncovers a novel role for Thioredoxin superfamily member, NRX1, in the control of oxidative stress.
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Vincent, Thomas. "The role of calcium signalling in plant-aphid interactions." Thesis, University of East Anglia, 2016. https://ueaeprints.uea.ac.uk/62931/.
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Myzus persicae is one of the most successful insects on the planet. It is the world’s most pesticide-resistant insect, feeds on hundreds of plant species and acts as a vector for over 100 viruses. Upon perception of M. persicae feeding, plants activate pattern-triggered immunity (PTI), a pivotal part of which is believed to be calcium signalling. The aim of this thesis is to uncover the role that calcium signalling might be playing in the interaction between M. persicae and one of its hosts: the model plant Arabidopsis. Using a fluorescent calcium sensor (GCAMP3), in vivo imaging of calcium dynamics was performed on leaves infested with M. persicae. There is a rapid and highly localised calcium burst around the feeding site in the epidermal and mesophyll cells, making it as one of the first plant responses to aphid attack. This calcium burst is triggered after perception of the aphid by the defence co-receptor BRASSINOSTEROID INSENSITIVE-ASSOCIATED KINASE 1 (BAK1), establishing it as part of PTI. Calcium is released from the extracellular space into the cell by GLUTAMATE-LIKE RECEPTORS 3.3 and 3.6 (GLR3.3 and GLR3.6), in combination with the release of intracellular calcium from the vacuole by TWO-PORE CHANNEL 1 (TPC1). Loss of BAK1, GLR3.3/GLR3.6 or TPC1 significantly attenuates the aphid-induced calcium burst and has an effect on the induction of anti-aphid defence responses. Downstream of the burst, CBL-INTERACTING PROTEIN KINASES 3, 9, 23 and 26 are activated by calcium and together mediate plant resistance to aphid attack. Furthermore, the M. persicae effector Mp10 partially suppresses the feeding site calcium burst, suggesting that the aphid is manipulating this pathway as part of its successful colonisation of the plant. Together, the data presented in this thesis provide evidence for the significant involvement of calcium signalling in the plant response to aphid attack.
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Li, Xiaoqing. "Plant root development and hormone signalling during drought stress." Thesis, Lancaster University, 2016. http://eprints.lancs.ac.uk/79357/.
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The plant root system is crucial for plant survival, growth and development, and it plays an important role in plant resistance to drought stress. Drought is one of the primary factors that restrict plant growth and yield, and its threat to crop yields will increase along with the growing food demand by the population of a world experiencing a changing climate. In response to drought in plants, various hormones are vital regulators, because they are able to manipulate plant development and in some cases minimise the adverse impact of drought. Therefore, understanding how the plant root system will adapt to a soil drying challenge is crucial. Of particular importance is the plant response to a non-lethal drought stress, which is often encountered in the field. Elucidation of the mechanisms underlying such responses, including hormonal regulations, may help crop scientists improve the plant performance under drought. A six-day progressive soil drying pot experiment was designed to examine the synchronisation of physiological responses in maize (Zea mays L.) roots and leaves during soil drying. It was found that maize roots showed earlier responses to soil drying than leaves in changing growth rates, water potentials and hormone levels. Root growth was stimulated at soil water content of 25−32% (ca. 41% in well-watered pots), while both root growth and leaf elongation were inhibited when soil water content was below 20%. Root abscisic acid (ABA) level gradually increased when soil water content was lower than 32% during soil drying. The stimulation and inhibition of root growth during soil drying may be regulated by root ABA, depending on the degree of the concentration increase. The ethylene release rates from leaves and roots were inhibited during soil drying, which occurred later than the increase in ABA levels. In a subsequent root phenotyping study on 14 maize genotypes, significant genetic variation was observed in root angle and size (root length, surface area and dry weight), and in the plasticity of these traits under mild and severe drought stress. Genotypes with a steeper root angle under well-watered conditions tended to display more promotion or less inhibition in root size under drought. Further analysis showed that combined traits of maize root angle, its plasticity and the root size plasticity under drought may be a better predictor for maize drought resistance than a single one of these traits. Moreover, root angle was found positively related to the leaf and root ABA levels and negatively related to the root tZ (a cytokinin) level under well-watered conditions. In another study on the crosstalk of drought-related hormones using the model plant Arabidopsis thaliana L., the biphasic responses of root elongation to ABA were confirmed, i.e. low external ABA concentrations stimulated root growth while high ABA concentrations inhibited it. Furthermore, ethylene and auxin were found to be involved in these responses. The inhibitory effect of high ABA levels on root growth was reduced or even eliminated when Arabidopsis was chemically treated to inhibit the ethylene biosynthesis or signalling, or to block auxin influx carriers. This was confirmed using mutants with blocked ethylene or auxin signalling, or a defect in the auxin influx carrier AUX1. On the other hand, the stimulatory effect of low ABA levels on root growth was lost when Arabidopsis seedlings were chemically treated to inhibit the auxin efflux carriers, and in mutants with blocked auxin signalling or with a defect in the PIN2/EIR1 auxin efflux carrier. These results indicate that ABA regulates root growth through two distinct pathways. The inhibitory effect that operates at high ABA concentrations is via an ethylene-dependent pathway and requires auxin signalling and auxin influx through AUX1. The stimulatory effect that operates at low ABA concentrations is via an ethylene-independent pathway and also requires auxin signalling and auxin efflux through PIN2/EIR1. This research contributes to our understanding of the responses of plant root system to different degrees of non-lethal drought stress, and it highlights the importance of root traits that may be important to plant drought resistance. The potential involvement of hormones (ABA, ethylene, auxin and cytokinin) in these processes is clarified. The knowledge gained may be integrated in novel crop management strategies to plan irrigation and help in the development of drought resistant crop varieties.
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Dove, Stephen K. D. "Inositol lipids and signalling in higher plants." Thesis, University of East Anglia, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338295.
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Barker, Richard. "Gibberellin biosynthesis and signalling in Arabidopsis root growth." Thesis, University of Nottingham, 2011. http://eprints.nottingham.ac.uk/12202/.
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Using targeted expression of a constitutively active repressor of GA signalling Susana Ubeda-Tomas et al., (2007) demonstrated that GA action in endodermal cells is necessary for correct root growth. However, GUS studies have shown the final and penultimate GA-biosynthetic genes are not expressed in the endodermis, indicating movement of GAs may be required. This study used the targeted mis-expression of GA degrading enzymes in Col-0 and the attempted targeted rescue of GA biosynthetic and signalling mutants, using the corresponding GA metabolic or signalling component, to gain an insight into the localisation of important GA biosynthesis and signalling sites. This study has demonstrated that GA12 can be made by epidermal, cortical and endodermal cells. However, the ground tissue of the elongation zone does not contain GA12 due to the early GA biosynthetic enzymes only being expressed within cells with a close proximity to the QC. Subsequently the 20-oxidation converts GA12 to GA15, to GA24 to GA9. These reactions mobilise GA allowing it to move from the meristematic region to the elongation zone. GA20ox and GA3ox activity is required in both the meristematic region and the elongation zone for correct root growth to occur. In addition, GA metabolic components are subject to tissue specific GA feedback regulation as a result of post-transcriptional processing and/or post-translational modifications to their protein stability. GA perception in any tissue of the elongation zone can promote complete cell elongation, suggesting that any one tissue can elongate it neighbours, or that each cell is capable of releasing a signal to ensure they all elongate proportionally. The transcriptional network within the endodermis has a disproportionately important role in GAs regulation of cell division within the root proximal meristem but GA action in other cell types is also required. The cambian and bundle sheath cells in aerial tissue like the endodermis in the root contain an important transcriptional network that promotes GA dependant growth.
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Fleck, Barbara. "Functional analysis of GAI, an Arabidopsis gibberellin signalling protein." Thesis, University of East Anglia, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.247096.
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Mellor, Nathan L. "Multiscale modelling of plant hormone signalling : auxin regulated lateral root emergence." Thesis, University of Nottingham, 2013. http://eprints.nottingham.ac.uk/30420/.
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The formation of lateral roots is an important post-embryonic developmental process that allows plants to adapt to their environment via exploitation of soil mineral resources. New lateral roots initiate as lateral root primordia (LRP) in the pericycle cell layer adjacent to the central vascular tissue in the primary root, and must pass through the outer cell layers of endodermis, cortex and epidermis to emerge as mature roots. A key regulator of emergence is the plant hormone auxin and it has been shown previously that in Arabidopsis the auxin induced expression of the auxin influx carrier LAX3 in specific cortical cells over LRP is required for emergence to occur, as this leads to the expression of cell wall remodelling enzymes such as polygalacturonase (PG). By developing mathematical models of auxin transport and LAX3 expression the work in the thesis aims to test the existing conceptual models for lateral root emergence, and provide testable hypotheses for the existence of additional gene regulatory components. An initial single cell model demonstrates that hysteresis and bistability may explain the experimentally observed 'all-or-nothing' LAX3 spatial expression pattern in cortical cells containing a gradient of auxin concentrations. By fitting model parameters against experimental data, the model is then used to show that some auxin homeostasis mechanism is present, with both endogenous and exogenous sources of homeostasis investigated. The single cell model also investigates the validity of several alternative gene regulatory networks for LAX3, and its apparent repression by a key mediator of the auxin response, ARF19. Finally, the model is extended to a multicellular context, in which the auxin distribution from a simulated LRP source cell is used as a basis for the expression of LAX3, leading to the expression of PG in specific cells between which the LRP must pass.
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Allison, Andrew. "Systemic signalling in the wound response of the tomato plant." Thesis, University of East Anglia, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.361728.
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Johansson, Åke Henrik. "Investigation into temperature effects on the plant light signalling pathways." Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/12231.
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The ability to withstand environmental temperature variation is essential for plant survival. Formative studies in Arabidopsis have revealed that light signalling pathways has a potentially unique role in shielding plant growth and development from seasonal and daily fluctuations in temperature. In this thesis we further investigate the integration of the light signalling networks and temperature signalling on the molecular level in Arabidopsis. First, we identified the transcript of the bHLH transcription factor LONG HYPOCOTYL IN FAR-RED 1 (HFR1) to be highly dependent on the ambient temperature and under strong control of the red light photoreceptor PHYTOCHROME B (phyB). We found that the long hypocotyl phenotype of the hfr1 mutant was exaggerated in warm conditions, specifically in blue light, downstream of cryptochrome 1. We further show that HFR1 acts in the warm by suppressing the function of PHYTOCHROME INTERACTING FACTOR 4 and 5 (PIF4, PIF5). PIF4 appears to act as a master regulator of several temperature responses and is directly regulated by the phytochromes. Thus, we define a molecular network where red light and blue light signals together with temperature merge on the regulation of PIF4. In the second part of this thesis, we investigate the relationship between temperature and the fluence rate of light on the inhibition of hypocotyl elongation in Arabidopsis. We find that the response to increasing fluence rates of light is highly dependent on the ambient temperature and that PIF4 and PIF5 acting downstream of the major red light photoreceptor, phyB, are essential for this response. In addition, we provide evidence that in cool conditions, PIF activity is under strong suppression by the gibberellin and HY5 pathways specifically at high fluence rates of red light. The collected work of this thesis highlights the importance of the PIF proteins as integrators of temperature and light signals and furthermore, demonstrates that the response to temperature is highly dependent on both the quality and quantity of light.
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Movahedi, Mahsa. "Identifying stomatal signalling genes to improve plant water use efficiency." Thesis, University of Sheffield, 2013. http://etheses.whiterose.ac.uk/4539/.
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Water is lost from higher plants via transpiration through stomatal pores the aperture of which is regulated by pairs of guard cells. Genetic engineering of the guard cell abscisic acid (ABA) signalling network that induces stomatal closure under drought stress is a key target for improving crop water use efficiency. In this study experiments were designed to investigate whether the biochemical mechanisms associated with the N-end rule pathway of targeted proteolysis could be involved in the regulation of stomatal apertures. The results indicate that the gene encoding the plant N-recognin, PRT6 (PROTEOLYSIS6), and the N-end rule pathway, are important in regulating stomatal ABA-responses in addition to their previously described roles in germination and hypoxia. Direct measurements of stomatal apertures showed that plants lacking PRT6 exhibit hypersensitive stomatal closure in response to ABA, and IR thermal imaging revealed reduced evapotranspiration under drought-stress. Together with a reduction in stomatal density, these properties result in drought tolerant plants. Plants lacking PRT6 are able to synthesis NO but their stomata do not close in response to NO suggesting that PRT6 is required for stomatal aperture responses to NO. Double mutant studies suggested that PRT6 (and by implication the N-end rule pathway) genetically interacts with known guard cell ABA signalling components OST1 and ABI1, and that it may act either downstream in the same signalling pathway or in an independent pathway. Several other enzymatic components of the plant N-end rule pathway were also shown to be involved in controlling stomatal ABA sensitivity including arginyl transferase and methionine amino peptidase activities. These results indicate that at least one of the N-end rule protein substrates which mediates ABA sensitivity has a methionine-cysteine motif at its N-terminus. A separate set of experiments were designed to investigate whether stomatal ABA-signalling pathways could have been conserved throughout land plant evolution. Cross-genetic complementation experiments were carried out to determine whether Physcomitrella stomatal apertures are able to respond to ABA and CO2 using a similar signalling pathway to that of flowering plants. The results demonstrated involvement of OST1 and ABI1 orthologues indicating that the stomata of the moss respond to ABA and CO2 using a signalling pathway that appears to be directly comparable to that of the model flowering plant Arabidopsis thaliana.
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Vaughan-Hirsch, John. "The role of cytokinin signalling in rice root vascular patterning." Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/48433/.
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The vascular anatomy of rice roots is an important factor in drought tolerance, but little is known about the mechanisms governing its patterning. The Arabidopsis thaliana root vascular tissues are patterned by a mutually inhibitory feedback loop between auxin and cytokinin signalling (Mähönen et al. 2006). Cytokinin signalling in the procambium promotes accumulation of auxin in neighbouring cells which consequently differentiate as xylem. In these cells, high auxin levels promote expression of the pseudo-histidine phosphotransfer protein (PHP) AHP6, an inhibitor of cytokinin signalling (Bishopp et al. 2011). Whereas the root vasculature of Arabidopsis consists of a single xylem axis with phloem poles on either side, that of rice roots show a central metaxylem vessel surrounded by multiple xylem poles, between which are phloem. I hypothesised that the basic interaction between auxin and cytokinin in vascular patterning is conserved between Arabidopsis and rice, but is adapted to enable development of these different tissue patterns. Chemical treatments revealed that auxin and cytokinin are central regulators of root vascular patterning in rice, as is the case in Arabidopsis. In an effort to test whether this conservation extended to individual components of the molecular circuitry, I examined the role of the three OsPHPs, homologues of AHP6. These genes are not auxin inducible and are not able to rescue the Arabidopsis AHP6 mutant, suggesting they do not function in rice root vascular patterning. However, screening known inhibitors of cytokinin signalling for in auxin inducibility in the root tip revealed a subset of the type-A RRs (OsRR1, OsRR6 and OsRR7) are auxin inducible, and were able to rescue the Arabidopsis ahp6 mutant, suggesting these genes may have been recruited to function in the hormonal crosstalk regulating root vascular patterning. Phylogenetic analyses revealed a single amino acid substitution in a conserved region of the PHPs which differs between the dicots and the monocots. To directly determine activity of the OsPHPs, and the relevance of these substitutions, in-vitro phosphotransfer assays were performed. Preliminary results suggest these substitutions do not affect protein function. To characterise candidate genes, transcriptional reporters and mutant plants were generated. Reporters show expression of most candidate genes in the root, with some localised to root protoxylem. Mutants were generated using CRISPR and homozygous and heterozygous mutants were identified. Together, results described here give insight into how regulatory networks may be adapted to bring about differing responses.
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Jackson, Robert. "Chemical dissection of hormone signalling in Arabidopsis." Thesis, University of Nottingham, 2014. http://eprints.nottingham.ac.uk/14409/.
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The plant hormone gibberellin (GA) regulates many developmental processes during a plant’s life cycle, including root and hypocotyl growth. Bioactive GAs promote GA-responsive growth and development by targetting DELLA proteins for degradation. Whilst the early steps of GA signalling are well understood it is not yet clear how the DELLA proteins alter the expression of GA-responsive genes. As other steps of the signalling pathway are encoded by multi-gene families it is possible that genetic redundancy is masking the transcription factors that act downstream of DELLAs. Using a chemical screen based on DELLA protein’s control of GA biosynthesis, 28 chemicals which blocked the GA-mediated downregulation of GA20ox1::GUS activity were identified. Using GA-mediated RGA degradation as a marker, 11 chemicals were identified as acting downstream of DELLAs in the GA signalling pathway. One of the chemicals (N23) identified in the screen was found to induce agravitropic root growth, a response more often associated with perturbation of auxin signalling. However, N23 had no effect on auxin signalling based on the characterisation of its effect on auxin-inducible genes and AUX/IAA degradation. The mode of action of N23 requires further investigation. However, N23 represents a potential for studying the role of GA in modulating gravitropism. The compound N16 potentially perturbs GA signalling by altering GA transport. It was found to block the uptake of both radiolabelled and fluorescent labelled GA into the root. Five days of exposure to N16 was required before any inhibition was observed on Col-0 roots but root elongation in ga1-3 seedlings was inhibited after only 24 hours suggesting that roots of wild type plants are saturated for GA. The site of action of N16 was not identified, but a putative oligopeptide transporter OPT6 was which is rapidly downregulated in the roots in response to GA application was investigated as a potential novel GA transporter. However, GA uptake assays in yeast strains overexpressing OPT6 proved inconclusive.
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Zhong, Silin. "Protein localization and interactions in the tomato ethylene signalling pathway." Thesis, University of Nottingham, 2007. http://eprints.nottingham.ac.uk/10405/.
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Early studies of the tomato ethylene signalling network using yeast two-hybrid screen previously identified three novel proteins (IntCR22, 242 and 266) that could interact with a putative ethylene kinase LeCTR2 (Lin et al., 2003). In this study, it has been demonstrated that IntCR22 is a cytoplasmic UDP-glycosyltransferase and IntCR266 is a chloroplast metallo-proteinase homologue to the Arabidopsis FtSH5/VAR1, whereas IntCR242 encodes a novel chloroplast protein with a C-terminal histidine-rich domain. In order to gain more insight into the tomato ethylene signalling mechanism, the sub-cellular localization and protein-protein interactions of the tomato ethylene signalling components have been investigated by fluorescent protein labelling and yeast two-hybrid experiments. Three tomato ethylene receptors (ETR1, NR and ETR4) and a downstream regulator EIN2 have been found in the endoplasmic reticulum (ER). Three putative downstream MAPKK kinases (CTRs) could interact with the C-terminus of the ethylene receptor possibly on the cytoplasmic side of the ER, whereas a novel ethylene signalling component GREEN-RIPE was located in the Golgi. It was therefore concluded from the localization study that IntCR242 and IntCR266 were false positives from the yeast two-hybrid screen and could not interact in vivo with the ethylene signalling components. The results presented in this study, in line with previous ethylene research suggest a possible involvement of the plant endomembrane system in the ethylene signalling network. However, the question as to how the ethylene signal moves from the ER localized receptors to promote activation of genes for the transcription factors within the nucleus remains unsolved.
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Walia, Ankit. "Involvement of mitogen-activated protein kinase signalling in plant microtubule function." Thesis, University of British Columbia, 2009. http://hdl.handle.net/2429/21739.
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Plants have developed sophisticated signalling networks that are involved in mediating
developmental transitions and environmental signals. Mitogen-activated protein kinases (MPKs)
are a class of signalling proteins that are involved in cellular processes that help plants to detect and initiate appropriate responses to numerous development and environmental inputs. The
microtubule cytoskeleton plays a pivotal role in plant development and morphogenesis, although
the mechanisms that regulate the microtubule-associated proteins and microtubule functions in
plant cells are not well understood. I investigated whether perturbations in the microtubule organization triggered by the MICROTUBULE ORGANIZATION] temperature-sensitive mutant(morl-l) could lead to altered transcriptional activity, with a particular interest in the genes
encoding signal transduction components. I showed that perturbations in the microtubule
cytoskeleton, achieved through the microtubule disruption phenotype of mor1-1, led to changes
in the expression of gene transcripts associated with diverse cellular processes, including
changes in the expression of the PROPYZAMIDE HYPERSENSITIVE 1(PHS1) gene, a member
of MPK-specific signal transduction pathway, which has been previously implicated in
mediating cortical microtubule functions in plant cells. Through biochemical, cell biological and
genetic tools, I identified MPK18 as one of the MPKs that interacts with the PHS1 phosphatase
and demonstrated through reverse genetics analysis that manipulation of MPK18 results in
conditional and subtle defects in the microtubule functionality. In contrast, analysis of MPK12,
which was shown to also interact with PHS1, identified no microtubule-specific function. My
live cell imaging studies revealed that the absence of MPK18 protein appears to have no effect
on microtubule plus end growth and shrinkage rates, indicating that MPK18 indirectly influences
microtubule functions. Based on the genetic analysis, MOR1 itself does not appear to be a target of the putative MPK18 signalling module. Preliminary attempts to obtain evidence for direct
impacts of PHS activity on MOR1 failed to demonstrate that manipulation of PHS1 altered
either subcellular localization or phosphorylation status of the MOR1 protein. These results
provide a platform that should facilitate future investigations aimed at understanding the role of
MPK signalling in the regulation of plant microtubule functions.
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Lee, Jennifer C. "Role of phosphoinositide signalling pathways in plant interactions with their environment." Thesis, University of Sheffield, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.419599.
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Elphick, Carmen Heather. "Na'+ transport and Ca'2'+ signalling in higher plant salt tolerance." Thesis, University of York, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341845.
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Wu, Chih-Hang. "A complex NLR signalling network mediates immunity to diverse plant pathogens." Thesis, University of East Anglia, 2016. https://ueaeprints.uea.ac.uk/62253/.
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Both plants and animals rely on nucleotide-binding domain leucine-rich repeat-containing (NLR) proteins to respond to invading pathogens and activate immune responses. An emerging concept in NLR biology is that “sensor” NLR proteins are often paired with “helper” NLR proteins to mediate immune signalling. However, the degree to which NLRs form signalling networks beyond sensor and helper pairs is poorly understood. In this thesis, I discovered that a large NLR immune signalling network with a complex architecture mediates immunity to oomycetes, bacteria, viruses, nematodes, and insects. Helper NLRs in the NRC (NLR-required for cell death) family are functionally redundant but display distinct specificities towards diverse sensor NLRs. Several sensor NLRs, including Rx, Bs2 and Sw5b, signal via interchangeable NRC2, NRC3 or NRC4, whereas some other sensor NLRs have a more limited downstream spectrum. For example, Prf signals via interchangeable NRC2 or NRC3 but not NRC4, and Rpi-blb2 signals via only NRC4. These helper/sensor NLRs form a unique phylogenetic superclade, with the NRC clade sister to the sensor NLR clades. The network has emerged over 100 million years ago from an NLR pair that diversified into up to one half of the NLRs of asterids. I propose that this NLR network increases evolvability and robustness of immune signalling to counteract rapidly evolving plant pathogens.
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Yin, Minghui. "Genetic dissection of nitric oxide signalling network in plant defence response." Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/10462.
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Following pathogen recognition, nitric oxide (NO) is rapidly produced in plants, this small molecule has emerged as a key signal in plant defence responses. S-nitrosylation is the major route of NO signal transduction in plants, a redox-based modification by addition of an NO moiety on cysteine thiol to form an S-nitrosothiol (SNO). S-nitrosoglutathione reductase (GSNOR) regulates cellular levels of S-nitrosylation and displays a key role in regulating the plant defence response. In this context, NO is important to orchestrate both defence gene expression and the hypersensitive response (HR) during attempted microbial infection. However, how the plant immune system recognizes NO and how NO level could elicit plant defence responses are poorly understood. The Arabidopsis thaliana (Arabidopsis) mutant NO overproducing 1 (nox1) was employed to characterize how NO level elicits defence dynamics. In response to microbial infection, resistance (R) gene-mediated defence and basal resistance were found to be compromised in the nox1 mutant relative to wild type Col-0 plants. Interestingly, nox1 mutant exhibit similar levels of HR and pathogen susceptibility to the GSNOR loss-of-function mutant atgsnor1-3. This phenomenon suggests that NO might regulate defence responses via GSNOR-mediated S-nitrosylation. Therefore, the nox1 atgsnor1-3 double mutant was generated and characterized to clarify this hypothesis. Accelerated HR and increased pathogen susceptibility are shown in the double mutant, which implies that increased NO mediated by nox1 and elevated SNOs resulting from atgsnor1-3, are additive with respect to the plant defence response. To identify genes responsible for NO perception, forward genetic screens were developed to identify Arabidopsis mutants with abnormal NO recognition. NO marker genes for genetic screens were identified from both lab and open source microarray data. Two genes, At3g28740 and At1g76600 were selected and experimentally confirmed to be strongly induced by NO. Transgenic Arabidopsis plants were generated carrying a NO reporter cassette, which consist of a luciferase reporter gene (LUC) driven by the promoter of NO marker gene. This forward genetic approach might be a powerful tool to identify genes integral to NO signal transduction. Three C2H2 zinc finger transcription factors (ZnTFs) ZAT7, ZAT8 and ZAT12 were identified as being rapidly and strongly induced by NO donors, which could be modulators of redox/NO-dependent signalling pathway. T-DNA insertion mutants within these ZnTFs have been identified. Basal resistance against Pseudomonas syringae pv tomato (Pst) DC3000 is compromised in all single knockout lines. Therefore, the full characterisation of defence phenotype of these mutants would be necessary to explore the role of these TFs in the plant defence. Furthermore, zat8 mutant is more sensitive to nitrosative stress when compared to wild type Col-0. This suggests that ZAT8 may be involved in protecting plants against nitrosative stress. However, the molecular mechanisms that underpin this function remain to be determined. In conclusion, NO and SNOs might regulate plant disease resistance via distinct pathways. Our work has also established NO-reporter lines to identify genes responsible for NO perception. In addition, three NO-induced ZnTFs have been identified that participate in regulation of basal resistance, which might unveil aspects of NO signalling related to the regulation of transcription.
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Larkindale, Jane. "Cell signalling in response to heat shock in Arabidopsis thaliana." Thesis, University of Oxford, 2001. http://ora.ox.ac.uk/objects/uuid:dc24f743-a69b-4aa9-92a4-fbdcbcff2bef.
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Increases in temperature damage plant cells, and plants react to heat stress by inducing of a number of protective mechanisms. In this study, it has been shown that heat damages cells both directly while the plants are being heated, and indirectly through heat induced oxidative stress during recovery from heating. Different stress response pathways are induced in each case. Evidence implicates salicylic acid, abscisic acid, ACC (a precursor of ethylene), calcium ions and active oxygen species in pathways resulting in thermotolerance (i.e. increased survival at high temperature). Addition of these potential second messengers results in increased survival and decreased oxidative damage after heating (as measured using the TBARS assay), while silencing the pathways through use of mutants, inhibitors or transgenes results in decreased thermotolerance. In vivo calcium measurements show cytosolic calcium transients only at initiation of cooling after heat stress. Calcium chloride also induces thermotolerance when added after heating, as does ACC. These substances can induce early increases in ascorbate peroxidase activity after heating, and induce expression of antioxidant genes. Thus they may play a role in heat induced oxidative stress response pathways in recovery. Two different pathways induced during heating appear to result in the expression of genes for heat shock proteins. The predominant pathway induced at 30oC involves ABA, while that at 35-40oC involves an oxidative burst generated through the NADPH oxidase, atrbohB. Addition of SA can induce HSP (heat shock protein) expression, but there is little evidence that this occurs endogenously in plant cells, although nahG plants unable to signal via SA are thermosensitive. Results in this study indicate that there are several signalling pathways associated with heat shock. The pathway induced during recovery from heating involves calcium ions and ethylene, and results in increased antioxidant capacity. During heating two pathways induce expression of HSPs: one involving abscisic acid and possibly salicylic acid and one involving an oxidative burst. At least one further pathway is believed to exist, which involves protein kinases and phosphatases as heat shock causes up-regulation of expression of certain genes for these signalling components.
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Murphy, Evan. "Characterising signalling components mediating root architecture in Arabidopsis thaliana." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/31976/.
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Our planet is growing rapidly in population and with that comes a demand for resources. To address issues in food security, scientists are looking to the underground parts of plants for novel mechanisms that will eventually lead to enhanced crop traits. Scientists are examining the underlying genetic frameworks to identify which genes play key roles in specific developmental processes. In this study we examined the model plant Arabidopsis thaliana as the roots are easily visualised, the genome has been sequenced, and there are many tools broadly available to work with. This thesis has used a multidisciplinary approach to uncover the signalling cascades revolving around the small signalling peptide RALF34, which is significantly involved in primary and lateral root growth. We have demonstrated, in the following chapters that RALF peptides are inherent to normative lateral root initiation, potentially regulated through shoot derived auxin. Furthermore, RALF4 and 34 peptides play a strong role in restricting primary root growth, and that together these peptides have an additive effect on cell elongation. Lastly, we identify several leucine-rich repeat receptor-like proteins, kinase proteins, and cell wall remodelling enzymes, which putatively play unique and diverse roles during primary and lateral root development. Taken together, this thesis provides novel and unique insights into new signalling pathways during root growth, which may in future aid in agronomic enterprises.
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Peñalosa-Barbero, Andrés. "New signalling network in plant abiotic stress discovered through a genetic approach." [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=97211131X.
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Kaliff, Maria. "Genes, hormones and signalling pathways implicated in plant defence to Leptosphaeria maculans /." Uppsala : Dept. of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, 2007. http://epsilon.slu.se/2007119.pdf.
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Panagopulos, Michael. "The role of long-chain sphingoid base kinases in plant cell signalling." Thesis, University of Bristol, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.520228.
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Johnston, M. J. G. "The neuropeptide signalling system as a drug target in plant parasitic nematodes." Thesis, Queen's University Belfast, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.431591.
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McKinney, S. L. "The neuropeptidergic signalling system as a drug target in plant parasitic nematodes." Thesis, Queen's University Belfast, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.411746.
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Audley, Matthew David. "Understanding the role of gibberellin signalling in wheat anther development during heat stress." Thesis, University of Nottingham, 2017. http://eprints.nottingham.ac.uk/39335/.
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High temperature (HT) stress during wheat male reproductive development causes irreversible damage to the anther tapetum layer and the developing microspores it supports, resulting in reduced yield. With the frequency of pre-flowing temperature stress events likely to increase, a better understanding of the effects of high temperature stress on anther developmental regulation is required. Gibberellin (GA) signalling has been shown to regulate tapetum programmed cell death (PCD) and pollen coat formation via the transcription factor (TF) GAMYB. This project aimed to investigate the function of two putative GA-signalling components in wheat anther development and characterise the global hormonal and transcriptional anther responses to HT. RNAi and TILLInG mutants for TaGAMYB and a putative orthologue of a rice tapetum PCD component, TabHLH141, revealed that both are required for male fertility. Tagamyb mutants displayed stunted anther development with irregular tapetum vacuolisation and reduced pollen viability. An interaction between RHT-D1 and TabHLH141 suggests that GA may mediate anther development through regulation of DELLA-TF interactions. Having characterised and developed a non-destructive staging method for wheat anther development, RNA-Seq and global hormone analysis was used to investigate the response to HT stress around pollen mother cell meiosis. Significant changes in expression of tapetum metabolism and PCD annotated transcripts and anther GA, auxin and jasmonate concentrations indicates that hormonal regulation of HT-responsive transcription may contribute to defective anther development. The work in this project demonstrates that advanced functional genomics techniques can be now be applied to the dissection of complex signalling pathways in hexaploid wheat.
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Smith, Stephanie. "Functional characterisation of small signalling peptides in the regulation of Arabidopsis thaliana development." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/33853/.
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Root architecture governs the uptake of water and nutrients from the soil and thus is essential for plant growth and survival. The control of lateral root branching is a crucial aspect of determining root architecture, and is a process largely controlled by the phytohormone auxin, which promotes lateral root organogenesis from founder cells within the vasculature and also subsequent lateral root elongation. A small posttranslationally modified peptide, CTERMINALLY ENCODED PEPTIDE1 (CEP1) is a bioactive 15-amino acid peptide cleaved from a larger precursor protein previously shown to negatively affect primary root elongation and lateral root branching in Arabidopsis thaliana. Within this thesis, genetic, biochemical and pharmacological approaches are used to demonstrate that another member of the CEP family, CEP5, reduces primary root length and negatively regulates lateral root branching in Arabidopsis thaliana with minor effects on aboveground architecture. CEP5 is also demonstrated to have an antagonistic relationship with auxin, with increased abundance of CEP5 leading to downstream stabilisation of the auxin signalling repressor AUXIN/INDOLE ACETIC ACID (AUX/IAA) proteins. Correspondingly, reduced abundance of CEP5 increases auxin responses, including enhanced lateral root progression. These studies suggest CEP5 regulates lateral root branching through attenuation of auxin responses.
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Byers, J. "Degradation of acyl-homoserine lactone signalling molecules in the plant pathogen Erwinia carotovora." Thesis, University of Cambridge, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597203.
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Erwinia carotovora is a Gram-negative plant pathogen affecting several commercially important crops such as potatoes and tobacco. Virulence in many strains of E. carotovora is controlled by the quorum sensing signalling molecule N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) which accumulates as the bacteria multiply and activates exoenzyme and secondary metabolite production. Mutant strains lacking OHHL are avirulent, suggesting a preventative treatment for infection. It had previously been observed in E. carotovora that OHHL accumulates in a cell density dependent fashion until stationary phase when the concentration falls in an equally rapid manner. This work shows that the stationary phase decline in OHHL is due to alkalisation of the growth media and that growing in buffered media can prevent this decline. Functional genomics was used to explore the possibility that E. carotovora subsp. atroseptica might possess an OHHL degrading enzyme similar to AttM from Agrobacterium tumefaciens. Four AttM orthologues were identified in the Eca strain 1043 genome, with one of these proteins (209-2), showing some degrading activity against synthetic HHL. Mutants were created in each of the orthologues but they had no effect on OHHL accumulation or exoenzyme production. The mutation in the 209-1 gene reduced motility, while mutation in the 91 gene reduced virulence in potato tubers. Further investigation using proteomic analysis found three of the mutants had reduced levels of Re1A, which is involved in regulation of the attM operon in A. tumefaciens. This study shows that E. carotovora would exhibit reduced virulence in basic environments due to increased AHL lability. Only one of the AttM orthologues identified possessed AHL degrading ability under the conditions tested but three did effect the expression of AttM, suggesting potential involvement in AHL regulation.
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Bacon, Sandra. "Investigation of plant derived compounds mediating cell signalling effects on FOXO1a and AMPK." Thesis, University of Dundee, 2012. https://discovery.dundee.ac.uk/en/studentTheses/b6abc6a4-de82-430a-ad30-015e8f69c8e8.
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In recent years there has been a considerable increase in human obesity levels with an associated increase in the incidence of type 2 diabetes mellitus (T2DM), a disorder of glucose metabolism characterised by insulin resistance. It is unclear why obesity and insulin resistance should frequently exist comorbidly but the current prevalence of polypharmacy suggests the underlying mechanisms may be multifactorial in origin. This project has highlighted plant compounds and extracts with: Insulin-like properties in cell culture experiments in that they induce phosphorylation and therefore inactivation of the transcription factor FOXO1a, which is a major downstream effector insulin and Properties similar to the T2D drugs metformin and pioglitazone, which both activate AMOK signalling and reduce phosphorylation of the ribosomal protein S6. The project began with an analysis of plant extracts which can mediate intracellular cell signalling effects on FOXO1a and AMPK. The pilot data established that one extract (grape seed, GSE) induces regulation of AMPK and FOXO1a much more readily than another (pine bark, PBE). GSE and PBE were subjected to multiple fractionation methods and mass spectrometry to learn more about the active agent(s) in the extracts. Another chapter adopted a candidate-approach, investigating effects of the plant compound gallic acid (GA) on AMPK. Although GA cannot explain the effects of GSE on AMPK, the availability of a variety of analogues of GA allowed investigation of structural requirements for cell responses. Two more extracts, cranberry and lingonberry, were then investigated using the techniques established earlier with GSE and PBE. These studies discovered that fractions containing B-type linkages were more effective at phosphorylating of FOXO1a than those containing A-type linkages, suggesting B-type linkages may be required for these effects. Metformin itself is a synthetic analogue of a plant compound and metformin analogues known as diguanides were synthesised and structure/activity relationships were assessed. Diguanides and biguanides were found ti induce similar responses but diguanides were much more toxic than biguanides, suggesting they may be less specific in their mechanism of action, or alternatively that they have different intramitochondrial targets. The aims of this project were 1. To extract polyphenolic compounds from grape seed, pine bark and berries using chromatography techniques and to characterise these compound using liquid chromatography/mass spectrometry 2. To exclude or confirm insulin-like or metformin-like properties by application to cell culture models to assess effects on regulation of glucose and energy homeostasis by measuring phosphorylation of the transcription factor FOXO1a, AMPK and the ribosomal protein S6.
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Foster, Rosie. "Plants signalling to herbivores : is there a link between chemical defence and visual cues?" Thesis, University of Sussex, 2013. http://sro.sussex.ac.uk/id/eprint/45168/.
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The use of visual cues by insect herbivores is likely to be an important component of plant-herbivore interactions in the wild, yet has until recently received little attention from researchers. In the last decade, however, interest in this topic has intensified following Hamilton & Brown's (2001) autumn colouration hypothesis, which proposes that the intensity of colouration of trees at autumn time is a signal of their defensive commitment to potential herbivores. This idea remains controversial and to date robust empirical data linking colouration with chemical defence and herbivory have been lacking. This thesis begins with a meta-analysis, in which I synthesize and analyse previously published data to determine the evidence for the use of host plant colouration by herbivores. I then move to explore the relationship between chemical defences and colouration in a classic plant-herbivore system: the wild cabbage (Brassica oleracea) and its herbivores the cabbage white butterfly (Pieris rapae) and the cabbage aphid (Brevicoryne brassicae). Both species have colour vision, and I use spectral sensitivity data to model the colour of the host ‘through the eyes' of the herbivores. First, I present data from a field study of wild cabbage populations showing significant relationships between herbivory, plant colouration and levels of glucosinolates defensive compounds. These results suggest that plant colouration could be used by herbivores to gain information about plant chemical defence. I then show colouration has a fixed genetic component in a common garden experiment; a necessary requirement for evolution of a colour signal. I explore the use of colouration in host choice by herbivores in more detail in a series of behavioural experiments. I show that cabbage aphids do not use leaf brightness as a cue when selecting among plants, but they do respond to different leaf colours. I also show that cabbage white butterflies do not choose hosts based on particular colour cues, even though this colouration potentially provides important information about host defence levels, which are shown to impact upon offspring fitness. Together, these results provide a clear demonstration of a link between plant chemistry and colouration in the wild cabbage system. However, the data presented in this thesis indicate that the use of colouration as a guide to host defence is limited, and I conclude by discussing possible reasons why this might be the case.
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Hey, Sandra Janet. "Guard cell gene expression in Pisum sativum L." Thesis, University of the West of England, Bristol, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.319258.
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Ghneim, Thaura. "Interaction of cytokinin, nitrogen and carbon metabolism in the control of growth and leaf senescence in Arabidopsis thaliana." Thesis, University of Exeter, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.370013.
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Matthus, Elsa. "Phosphate starvation alters calcium signalling in roots of Arabidopsis thaliana." Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/290260.
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Low bioavailability of phosphate (P) due to low concentration and high immobility in soils is a key limiting factor in crop production. Application of excess amounts of P fertilizer is costly and by no means sustainable, as world-wide P resources are finite and running out. To facilitate the breeding of crops adapted to low-input soils, it is essential to understand the consequences of P deficiency. The second messenger calcium (Ca2+) is known to signal in plant development and stress perception, and most recently its direct role in signalling nutrient availability and deficiency has been partially elucidated. The use of Ca2+ as a signal has to be tightly controlled, as Ca2+ easily complexes with P groups and therefore is highly toxic to cellular P metabolism. It is unknown whether Ca2+ signals P availability or whether signalling is altered under P starvation conditions. The aim of this PhD project was to characterise the use of Ca2+ ions, particularly cytosolic free Ca2+ ([Ca2+]cyt), in stress signalling by P-starved roots of the model plant Arabidopsis thaliana. The hypothesis was that under P starvation and a resulting decreased cellular P pool, the use of [Ca2+]cyt may have to be restricted to avoid cytotoxic complexation of Ca2+ with limited P groups. Employing a range of genetically encoded Ca2+ reporters in Arabidopsis, P starvation but not nitrogen starvation was found to strongly dampen the root [Ca2+]cyt increases evoked by mechanical, salt, osmotic, and oxidative stress as well as by extracellular nucleotides. The strongly altered root [Ca2+]cyt response to extracellular nucleotides was shown to manifest itself during seedling development under chronic P deprivation, but could be reversed by P resupply. Fluorescent imaging elucidated that P-starved roots showed a normal [Ca2+]cyt response to extracellular nucleotides at the apex, but a strongly dampened [Ca2+]cyt response in distal parts of the root tip, correlating with high reactive oxygen species (ROS) levels induced by P starvation. Excluding iron, as well as P, rescued the altered [Ca2+]cyt response, and restored ROS levels to those seen under nutrient-replete conditions. P availability was not signalled through [Ca2+]cyt. In another part of this PhD project, a library of 77 putative Ca2+ channel mutants was compiled and screened for aberrant root hair growth under P starvation conditions. No mutant line showed aberrant root hair growth. These results indicate that P starvation strongly affects stress-induced [Ca2+]cyt modulations. The data generated in this thesis further understanding of how plants can integrate nutritional and environmental cues, adding another layer of complexity to the use of Ca2+ as a signal transducer.
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Ayyar, Priya Vijay. "Uncovering the role of S-nitrosylation in jasmonic acid signalling during the plant immune response." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25783.
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Plants have evolved a plethora of effective mechanisms to protect themselves from biotic stresses. Jasmonates (JAs) are employed as vital defence signals against both insect and pathogen attack. Jasmonic acid (JA) signalling plays a central role in plant defence and development. S-nitrosylation, a redox-based post-translational modification plays an important role in plant disease resistance. S-nitrosoglutathione (GSNO) is formed by the reaction of antioxidant glutathione (GSH) and nitric oxide (NO) and acts as a mobile reservoir of NO bioactivity. The Arabidopsis thaliana S-NITROSOGLUTATHIONE REDUCTASE (AtGSNOR1) controls multiple modes of disease resistance via S-nitrosylation. In this context, the Arabidopsis lossof- function mutant atgsnor1-3 exhibits higher susceptibility to Botrytis cinerea a necrotrophic pathogens and Pieris rapae insect attack. Accumulation of JA was reduced in atgsnor1-3 after mechanical wounding. JA marker genes were also downregulated in atgsnor1-3 compared to Col-0 after Methyl Jasmonate (Me-JA) treatment. The relative gene expression of Vegetative Storage Protein (VSP) was reduced in atgsnor1-3 compared to wild type. Further, protein-protein interaction experiments in yeast two hybrid assays revealed an inhibition of Coronatine-insensitive 1 (COI1) and Jasmonate ZIM domain (JAZ1) interactions upon NO donor application. Interestingly it was also shown that Nitric oxide donor may inhibited the degradation of JAZ1-β-glucoronidase (GUS) fusion protein driven by a CaMV35s:: JAZ1-GUS transgene in GUS histochemical analysis but not in flurometric assay. A biotin switch assay of recombinant JAZ1-Maltose-binding protein (MBP) has shown that JAZ1-MBP was S-nitrosylated and mass spectrometry suggested Cysteine229 (Cys229) was the site of this modification. Further, CaMV35S::JAZ1-Flag transgene expressed in either a wild-type or atgsnor1-3 genetic background, suggested that JAZ1 was S-nitrosylated in vivo. Collectively, our data imply that JA-signalling engaged in response to either insect predation or attempted B. cinerea infection is under redox control as high SNO in atgsnor1-3 has disrupted the JA signalling pathway. Furthermore, our data suggest that S-nitrosylation of Cys-229 of JAZ1 may control JA-mediated signalling by blocking the interaction of this protein with COI1, thus reducing the turnover of JAZ1 by the 26S proteasome and consequently enabling continued JAZ1-mediated repression of JA-dependent gene expression in the presence of Me-JA. Thus our findings highlight the importance of NO and associated S-nitrosylation in JA signalling during plant immune response.
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Schnaubelt, Daniel. "The roles of glutathione in the control of plant growth, development and signalling in Arabidopsis thaliana." Thesis, University of Leeds, 2013. http://etheses.whiterose.ac.uk/6864/.
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Reduced glutathione (GSH) is an abundant low molecular weight thiol that fulfils multiple functions in plants, many of which remain poorly characterised. The following studies were undertaken in order to characterise the roles of GSH in growth, development and signalling in Arabidopsis thaliana. In the first experiments, a phenomics approach was used to investigate the effects of GSH deficiency on growth and stress tolerance using mutants that are either defective either in GSH synthesis (cad2-1, pad2-1 and rax1-1) or the export of -glutamylcysteine and GSH from the chloroplast (clt1clt2clt3). Whereas the clt1clt2clt3 mutant had a greater rosette area than the wild-type under low light growth conditions, the GSH deficient mutants were significantly smaller. Moreover, lateral root densities were significantly decreased in GSH deficient and clt1clt2clt3 mutants. The redox potentials of the nucleus and cytosol in the root cells of the wild-type seedlings measured using roGFP were over -300mV. However, in roots grown in the presence of the GSH synthesis inhibitor buthionine sulfoximine (BSO), the redox potentials of the nucleus and cytosol increased to approximately -260mV. Low GSH-responsive genes were identified by transcript profiling analysis of the GSH-deficient root meristemless 1-1 (rml1-1) mutant. These included a large number of transcription factors, proteins involved in cell division, redox regulation and auxin signalling. Many transcripts modified by low GSH influence plant growth and development, and explain the altered root development observed the low GSH mutants. These results demonstrate that low GSH leads to significant increases in the redox states of the nucleus and cytosol and results in specific responses in gene expression that are distinct from those observed under oxidative stress. Moreover, the findings suggest that the cytosolic/nuclear GSH pool is important in the control of root development and that low GSH per se does not enhance overall sensitivity to abiotic stresses.
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Brewer, Helen Caroline. "Functional evaluation of plant defence signalling against Fusarium graminearum and F. culmorum in Arabidopsis floral tissue." Thesis, University of Exeter, 2014. http://hdl.handle.net/10871/16552.
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Fusarium Ear Blight (FEB) is a globally important floral disease of cereal crops such as wheat, maize and barley. The predominant causal agents of FEB disease of wheat in the UK are Fusarium culmorum and F. graminearum. Wheat infecting isolates of both of these fungal species infect the floral and silique tissues of the model plant Arabidopsis thaliana, providing a tractable model for analysis of factors determining plant susceptibility or resistance to Fusarium infection. The effect of F. culmorum infection on the metabolic composition (metabolome) of Arabidopsis pedicel tissue following silique inoculation was investigated in a collection of mutants with altered defence responses to F. culmorum and/or other plant pathogens, using a 1¬H-NMR/ESI-MS (+/-) triple fingerprinting approach. These mutants showed differing metabolomic fingerprints in the absence of F. culmorum infection, as well as differences in accumulation or depletion of metabolites in response to F. culmorum colonisation. A number of metabolites were also identified which were induced by F. culmorum infection irrespective of plant genotype. Quantitative differences in compound accumulation were also observed between genotypes in the Columbia and Landsberg erecta accessions following F. culmorum infection. One of the genotypes investigated was eds11, which has enhanced susceptibility to F. culmorum floral infection. Mapping of the mutation responsible for the eds11 phenotype was initiated using an isogenic mapping by sequencing approach. This resulted in a list of potential candidates for the EDS11 gene. Additional Arabidopsis mutants were investigated for altered defence responses to F. culmorum floral infection. Multiple mutant alleles of the Arabidopsis homoserine kinase gene DMR1 were found to have enhanced resistance to F. culmorum silique infection and rosette leaf colonisation, associated with accumulation of homoserine in siliques and delayed leaf senescence. Exogenous homoserine application enhanced resistance in wild type and dmr1 plants. Collectively, these findings form a novel contribution to current knowledge of the Fusarium-Arabidopsis interaction. This may have applications for improvement of FEB resistance in cereals.
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Czyzewicz, Nathan. "Functional characterisation of small signalling peptides and a receptor kinase involved in root architecture development in Arabidopsis and crop species." Thesis, University of Nottingham, 2017. http://eprints.nottingham.ac.uk/41776/.
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Post-embryonic root development is a plastic process by which plants are able to interface with the rhizosphere in order to provide anchorage, and increase surface area available for acquisition of nutrients and water. While root growth is governed primarily by auxin/cytokinin interactions, roots are able to sense the presence of surrounding nutrient deposits and changes to environmental conditions – directing growth accordingly – due to the action of signalling cascades. This thesis presents data pertaining to the characterisation of two signalling elements involved in governing root architecture; the small signalling peptide CLAVATA/EMBRYO SURROUNDING REGION 26 (CLE26), and the receptor kinase ARABIDOPSIS CRINKLY 4 (ACR4). Initially, the origins and evolutionary history of root architecture are explored, and an overview of signalling elements involved in root architectural development is provided, before discussing the potential benefits that manipulation of signalling events may allow in targeted crop improvement. To provide background on peptide signalling, the physiological and biochemical effects of small signalling peptides are discussed in view of the current literature, demonstrating the diverse range of developmental processes which are known to be regulated by these ligands and their known receptors. Following this, functional characterisation analyses indicate CLE26 as a novel, potent inhibitor of primary root growth and protophloem development in Arabidopsis, and is also shown to induce a similar effect upon exogenous application to several crop species. Furthermore, data is presented demonstrating the clear requirement for functional analysis during the development phase of creating antagonistic peptides, as a previously described antagonistic peptide technology was not applicable in all cases. Concluding the exploration of CLE26 signalling, a phosphoproteomics screen was conducted to probe further into CLE26 function, determining 23 putative effectors of CLE26 signalling, which are discussed in view of their potential to mediate CLE26 signalling, according to current literature. Next, the known roles of ACR4 and its orthologues are reviewed, demonstrating the importance of ACR4 signalling in many developmental processes, including regulation of asymmetric cell division during postembryonic root development. Although ACR4 activity is known to regulate asymmetric cell division in both columella stem cells and lateral root primordia, little is known about the downstream mediators of ACR4 signalling. In an attempt to fill this gap in knowledge, yeast 2-hybrid and co-immunoprecipitation approaches were employed. These two parallel proteomics screens together resulted in identification of 19 putative interactors of ACR4 signalling (PAIPs), which are discussed as potential mediators of ACR4 signalling in view of current literature. Of the identified PAIPs, three were further characterised by loss of function analysis, demonstrating that loss of PHOSPHOLIPASE Iγ2 and a PROTEIN OF UNKNOWN FUNCTION (At1g49840/UNK) was able to affect root architecture. Further in-silico characterisation of UNK reveals its similarity to soluble phospholipase receptors, which, alongside PLA-Iγ2, may potentially implicate ACR4 as a key player in a novel mechanism involved in regulation of bioactive lipid production.
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Tomaz, Juarez Pires. "Envolvimento dos quatro genes bZIPs do Grupo C de Arabidopsis thaliana na sinalização por glicose, manose e ABA." [s.n.], 2008. http://repositorio.unicamp.br/jspui/handle/REPOSIP/317162.
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Orientador: Michel Georges Albert VincentzTese (outorado) - Universidade Estaulal de Campinas, Instituto de Biologia
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Resumo: Na planta modelo eudicotiledónea A. thaliana quatro genes para fatores de transcrição do tipo bZIP que são homólogos a Opaco-2 (O2) do milho, uma monocotiledônea, foram identificados. O2 é um regulador chave do metabolismo coordenado de carbono e nitrogênio e da síntese de prolaminas de reserva durante o desenvolvimento da semente. Estes quatro genes, AtbZIP9, o par de parálogos AtbZIP10 e AtbZIP25, e AtbZIP63, o provável ortólogo de O2, formam o Grupo C de genes bZIP de Arabidopsis. Sabe-se que AtbZIP9 provavelmente desempenhe um papel no processo de desenvolvimento do floema, AtbZIP10 está associado com e resposta à estresses, além de, junto com AtbZIP25, participar na regulação de genes de proteínas de reserva na semente e que AtbZIP63 pode estar envolvido com o balanço energético da planta. Para acrescentar novas informações relevantes sobre a função dos bZIPs do Grupo C e, a longo prazo, entender como a função de O2 evoluiu em angiospermas, iniciou-se neste trabalho uma caracterização detalhada da regulação dos membros do Grupo C em resposta a diversos sinais hormônais e a açúcares. Mostramos que apenas as hexoses glicose e manose e o ácido abscísico (ABA) regulam de maneira transiente a expressão dos genes bZIP do Grupo C, sugerindo que eles representam intermediários mediando as respostas a estes sinais. A glicose reprime a expressão de AtbZIP9 e de AtbZIP63 e induz a expressão de AtbZIP25, ABA reprime a expressão de AtbZIP63 e manose reprime a expressão de AtbZIP25 e de AtbZIP63. Em Arabidopsis, a hexoquinase1 (HXK1) é um sensor da glicose que ativa a síntese e sensibilidade ao ABA para inibir o desenvolvimento da plântula em resposta a glicose. Reportamos aqui que as repressões em curto prazo de AtbZIP9 e AtbZIP63 por glicose e de AtbZIP25 e AtbZIP63 por manose estão mediadas por vias de sinalização independentes de HXK1 e envolvem elementos relacionados a ABA. AtbZIP25 apresenta uma indução por glicose dependente de ABI5 e repressão por manose dependente de ABA2 e ABI4. A repressão de AtbZIP63 por glicose envolve uma via dependente de ABA2 e de ABI5 que é reprimida por ABI4. Já a repressão de AtbZIP63 por manose e de AtbZIP9 por glicose estão inseridas em vias independentes de ABA2, ABI4 e ABI5. A dependência diferencial de ABI5 e de ABI4 na regulação por glicose e manose de AtbZIP25 e de AtbZIP63, permite inferir que ambas hexoses atuam através de vias de transdução distintas e enfatiza a importância de manose como sinal metabólico de regulação. Observou-se ainda que ação conjunta de ABA e glicose apresenta um efeito sinérgico na repressão de AtbZIP63, provavelmente refletindo regulações pós-transcricionais da expressão deste gene. Os dados sugerem que AtbZIP63 representa um importante nó da comunicação entre a sinalização por ABA (estresse abiótico) e por glicose (nível energético) permitindo adequar eficientemente a resposta a estresse abiótico que seja compatível com o estado energético da organismo.
Abstract: In the model eudicot organism A. thaliana (Arabidopsis), four genes encoding bZIP transcription regulatory factors that are homologous to the maize Opaque-2 (O2) locus were identified. O2 is a key regulator of the carbon to nitrogen balance and of the prolamine type storage proteins synthesis during seed development. The Arabidopsis genes, AtbZIP9, the two paralogues AtbZIP10 and AtbZIP25 and AtbZIP63, the most probable O2-ortholgue, together form group C bZIP genes. AtbZIP9 is likely to be involved in phloem development while AtbZIP10 is related to stress responses but is also required for the regulation of seed storage protein genes very much like AtbZIP25. Finally, AtbZIP63 seems to be involved in the control of the energetic balance. In order to get new and relevant information about the role of the group C bZIP genes and consequently obtain new insight into the evolution of the O2-related functions in angiosperms, we initiated a detailed characterization of the regulation of group C members in response to hormonal signals and sugars. We show here that two hexoses, glucose and mannose as well as abscisic acid (ABA) are the only signals that transiently modulated the expression of group C bZIP genes, suggesting they are players in the response induced by these signals. While glucose is shown to repress the expression of AtbZIP9 and AtbZIP63 and to induce AtbZIP25 expression ABA is able to repress the expression of AtbZIP63 and mannose represses the expression of AtbZIP25 and AtbZIP63. In Arabidopsis, hexokinase1 (HXK1) is a glucose sensor that may trigger abscisic acid (ABA) synthesis and sensitivity to mediate glucose-induced inhibition of seedling development. We report that the short term regulation of the expression of AtbZIP9, AtbZIP63 by glucose and the repression of AtbZIP25 and AtbZIP63 by mannose are HXK1-independent and for AtbZIP25 and AtbZIP63, these regulations partly rely on ABA synthesis. It also shown that the activation of AtbZIP25 expression by glucose relies on ABI5 while its repression by mannose appears to be ABA2- and ABI4-dependent. Glucose repression of AtbZIP63 expression seems to involve an ABA2- and ABI5-dependent pathway which is repressed by ABI4. We also reveal that the regulations of AtbZIP63 by mannose and of AtbZIP9 by glucose do not require ABA, ABI4 or ABI5. The differential dependence of glucose and manose-induced regulation of AtbZIP63 and AtbZIP25 expression for ABI5 and ABI4 indicates that both hexoses act through distinct transduction pathways and highlights the importance of mannose as a regulatory metabolite. A synergetic repression of AtbZIP63 by ABA and glucose, which possibly reflects a post-transciptional regulatory scheme of AtbZIP63 expression, was uncovered. Together, the data suggests that AtbZIP63 is a key nod of the ABA (abiotic stress) and glucose (energetic balance) crosstalk network allowing to efficiently adjust the response to abiotic stresses according to the energetic status of the organism.
Tese (outorado) - Universidade
Genetica Vegetal e Melhoramento
Doutor em Genetica e Biologia Molecular
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Babíková, Zděnka. "How do interactions between herbivores and mycorrhizal fungi regulate production of plant signalling compounds and parasitoid behaviour?" Thesis, University of Aberdeen, 2013. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=203957.
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The aim of this PhD was to investigate major gaps in our understanding of how mycorrhizal fungi and aphids interact via their effects on plants, and how these interactions regulate emission of plant volatiles and consequently aphid and parasitoid host location. A series of experiments was designed using broad bean (Vicia faba L.), pea aphids (Acyrthosiphon pisum) and their parasitoid wasp, Aphidius ervi and mixed or single spore cultures of AM fungi as a model system. This PhD has determined that arbuscular mycorrhizal fungi are more important drivers of above-ground ecological interactions than ever considered before. They have key roles in specialist aphid host location and in influencing their development. The antagonistic effect of aphids on functioning of mycorrhizal association suggests that the interactions operate in both directions. However, if plants were supplied with phosphorus the aphids did not affect mycorrhizal colonisation suggesting that at sufficient phosphorus availability plants can tolerate the effect of aphids on mycorrhizal colonisation. This demonstrates how dynamic the multi-trophic systems are and that their outcomes are also influenced by soil nutrient availability, with implications for agricultural practices. This PhD has discovered that underground signals carried through common mycelial networks warn neighbouring plants of aphid attack. This signalling allows plants that receive the signal to initiate their defence system by changing their profiles of volatiles emissions and repel aphids and attract their parasitoids so that they may prevent the attack. Because the signal transfer is rapid it incurs the greatest fitness benefit for the receiving plant and potentially also for the donor of the signal and for the fungi. We now need to determine the wider ecosystem implications of this phenomenon, how the signalling is regulated in nature and in agroecosystems and what the fitness consequences are for each component organism.
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Mühlenbock, Per. "Genetic and Molecular Mechanisms Controlling Reactive Oxygen Species and Hormonal Signalling of Cell Death in Response to Environmental Stresses in Arabidopsis thaliana." Doctoral thesis, Stockholms universitet, Botaniska institutionen, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-1358.
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In the present work the regulation of environmentally induced cell death and signaling of systemic acquired acclimation (SAA) in Arabidopsis thaliana is characterized. We used the lesion simulating disease1 (lsd1) mutant as a model system that is deregulated in light acclimation and programmed cell death (PCD). In this system we identify that redox status controlling SAA and cell death is controlled by the genes LSD1, EDS1, EIN2 and PAD4 which regulate cellular homeostasis of salicylic acid (SA), ethylene (ET), auxin (IAA) and reactive oxygen species (ROS). Furthermore we propose that the roles of LSD1 in light acclimation and in biotic stress are functionally linked. The influence of SA on plant growth, short-term acclimation to high light (HL), and on the redox homeostasis of Arabidopsis leaves was also assessed. SA impaired acclimation of wild-type plants to prolonged conditions of excess excitation energy (EEE). This indicates an essential role of SA in acclimation and regulation of cellular redox homeostasis. We also show that cell death in response to EEE is controlled by specific redox changes of photosynthetic electron transport carriers that normally regulate EEE acclimation. These redox changes cause production of ET that signals through the EIN2 gene and regulon. In the lsd1 mutant, we found that propagation of cell death depends on the plant defence regulators EDS1 and PAD4 operating upstream of ET production. We conclude that the balanced activities of LSD1, EDS1, PAD4 and EIN2 regulate chloroplast dependent acclimatory and defence responses. Furthermore, we show that Arabidopsis hypocotyls form lysigenous aerenchyma in response to hypoxia and that this process involves H2O2 and ET signalling. We found that formation of lysigenous aerenchyma depends on LSD1, EDS1 and PAD4. Conclusively we show that LSD1, EDS1 and PAD4, in their functions as major plant redox and hormone regulators provide a basis for fundamental plant survival in natural contitions.
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Ordonez, Herrera Natalia Maria [Verfasser], and Ute [Gutachter] Hoecker. "Analysis of COP1/SPA signalling events in plant developmental responses / Natalia Maria Ordonez Herrera ; Gutachter: Ute Hoecker." Köln : Universitäts- und Stadtbibliothek Köln, 2017. http://d-nb.info/1151298174/34.
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Vatsa, Parul. "Involvement of a putative glutamate receptor mediated calcium signalling in tobacco : a new link in plant defence." Thesis, Dijon, 2010. http://www.theses.fr/2010DIJOS009.
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Chez les mammifères, le glutamate est un neuromédiateur bien connu au niveau du système nerveux central et plus récemment un rôle immunomodulateur lui a été reconnu. Le glutamate est le ligand de récepteurs ionotopiques (iGluRs) qui sont des récepteurs-canaux perméables à divers cations dont le calcium (non-selective cation channels, NSCC). Chez Arabidopsis thaliana, une famille de 20 gènes de iGluRs homologues des iGluRs de mammifères a été identifiée et leur implication dans divers processus biologiques est suggérée. Dans ce travail où nous utilisons des suspensions de cellules de tabac (Nicotiana tabacum var Xanthi), divers arguments suggèrent que ces iGluR sont fonctionnels dans le tabac : influx de calcium et élévation rapide et transitoire de la concentration en calcium cytosolic libre en réponse à l’addition de glutamate, inhibition de ces effets par 4 antagonistes de iGluRs animaux (compétitifs ou non compétitifs), désensibilisation, et pH dépendance des effets. Pour la première fois chez les plantes nous montrons que le glutamate induit la production de NO très vraisemblablement via l’activation de iGluRs. De plus, nous démontrons que ce(s) iGluRs sont impliqués dans le mode d’action, via les flux de calcium, de la cryptogéine une protéine de 10 kDa de Phytophthora cryptogea, éliciteur des réactions de défense chez le tabac. Néanmoins, à ce niveau, les iGluRs ne sont pas impliqués dans la plupart des événements calcium-dépendants induits par la cryptogéine dont l’activation des MAPKs et de canaux anioniques, la production de H2O2 (activation de la NADPH-oxydase) et la réponse hypersensible. En revanche, ils sont tout ou partiellement responsables de la production de NO décrite pour la première fois par le passé en réponse à la cryptogéine. Ces résultats suggèrent que différents types de canaux calciques activés par divers médiateurs, génèrent, via le calcium, des messages spécifiques décodés par des protéines associées à chacun de ces types de canaux et impliquées dans des réponses biologiques différentes. Dans le mode d’action de la cryptogéine, nous démontrons que l’activation des iGluRs est possible grâce à l’exocytose de glutamate dans l’apoplaste, induite par la cryptogéine. Ainsi, ce travail est la première démonstration du rôle de iGluRs potentiels dans la défense chez les plantes et de leur implication dans la production de NO. Nos résultats sont un argument supplémentaire à la conservation des mécanismes de la défense dans le monde vivant et posent le problème du rôle du glutamate dans la signalisation chez les plantesGlutamate is recognized as the primary excitatory neurotransmitter in the mammalian central nervous system (CNS) but recent studies have shown that glutamate has an important additional immunomodulator role. Glutamate is the ligand of ionotropic glutamate receptors (iGluRs), which are non-selective cation channels (NSCC), permeable to calcium. In plants, animal iGluR homologs were found that were involved in many developmental processes. Here we demonstrate the involvement of putative iGluRs in calcium signalling in response to cryptogein which is a 10 kDa protein secreted by the oomycete Phytophthora cryptogea and is an elicitor of defence in tobacco. Using transformed tobacco cell suspensions expressing aequorin in the cytosol or in the nucleus, our results have shown that glutamate induces a strong and transient [Ca2+]cyt elevation without [Ca2+]nuc changes. Glutamate-induced [Ca2+]cyt elevation was a result of calcium influx from the extracellular medium and was inhibited by different GluR inhibitors. This data suggest the presence of functional calcium channels of GluRs-type in tobacco. Nevertheless, glutamate does not induce some of the calcium-dependent characteristic events of the defence pathways, which are H2O2 production, MAPK activation and hypersensitive response, but promoted NO production. Further, Ca2+ influx,[Ca2+]cyt elevation and NO production induced by cryptogein were shown to be partially inhibited by the glutamate receptor inhibitors, suggesting that cryptogein treatment could activate a calcium channel of the GluR-type leading to plant defense signalling through NO production. We have also demonstrated that cryptogein induces an efflux of glutamate in the apoplast by the process of exocytosis thus activating the GluRs in tobacco. This is the first demonstration for a potential GluR(s) involvement in plant defense signalling, furthermore by mechanisms that showed homology with glutamate effect on neuronal cells
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Parfitt, D. "A study of glucose signalling and gene expression in early flower development in the short day plant Pharbitis nil." Thesis, Cardiff University, 2003. http://eprints.worc.ac.uk/746/.
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In this thesis I present a study on floral determination in the short day plant Pharbitis nil. Shoot apical meristems are determined if, following induction, they form floral organs even if isolated in non-inductive conditions. Five day old P. nil is fully induced by a 48 h dark period. P. nil apices are determined with respect to carpels 24 h after the 48 h inductive treatment if cultured in glucose medium but not until 5 d after induction if cultured in sucrose. I found that similar differential effects existed in the determination times of the other floral organs by excising shoot apices periodically through a 48 h dark treatment and the following 24 h of continuous light, culturing them in glucose or sucrose medium and measuring the degree of floral development. I cultured apices with glucose analogues instead of glucose. The analogue 3 oxymethylglucose, which is transported into the cell but not phosphorylated, mimicked the effect of glucose so that glucose entry into the cell probably acts as a signal for floral development. Glucose was as prevalent as sucrose in the sap extracted from seedlings, regardless of induction. Structural homologues of the Arabidopsis thaliana genes LEAFY, AGAMOUS and CRABS CLAW were cloned in P. nil. The homologues PnAG1, PnAG2, PnCRC2 and PnLFY1 were found to be expressed more strongly in induced than in non-induced apices in vivo and more strongly in induced apices cultured in glucose medium than in sucrose medium using semi- quantitative RT-PCR. I conclude that PnAG1, PnAG2, PnCRC2 and PnLFY1 are floral homeotic genes and that glucose is involved in signalling for floral development and signals for the increased expression of these genes. Finally a model of floral determination, based on these conclusions, is proposed.
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Tunn, Ruth Elizabeth. "Expression of two-pore channels in mammalian primary cells and tissues, and their role in adipose tissue formation and function." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:6c0b970d-6133-4752-987a-e21f6e2dc69c.
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Two-pore channels (TPCs, gene name Tpcn) have recently been identified as endolysosomal cation channels modulated by the potent calcium (Ca2+) releasing messenger nicotinic acid adenine dinucleotide phosphate (NAADP). Gene knockout (KO) and RNA knockdown studies have implicated TPCs in fundamental cellular processes, including secretion, of insulin in pancreatic islets, and differentiation, of skeletal myoblasts and osteoclasts. Investigations of Tpcn1 and Tpcn2 mRNA expression have indicated widespread tissue distribution, but a lack of suitable antibodies has impeded study of the endogenous proteins. In this study, an anti-TPC1 antibody was purified from immune sera and used in immunoblotting investigations to demonstrate TPC1 protein expression in a wide range of mouse tissues, with highest expression levels observed in kidney, liver and adipose tissue. Endogenous mouse TPC1 was demonstrated to be glycosylated, with apparent differences in the extent of glycosylation in different tissues based on the indicated molecular weight before and after treatment with a deglycosylating enzyme, which may have implications for the functional regulation of channel activity. Given the increasing prevalence of type 2 diabetes and obesity, an understanding of the molecular basis of glucose homeostasis and adipose tissue formation and function is an important scientific goal. Tpcn KO mice have been developed; in both Tpcn1 KOs and Tpcn2 KOs, impaired pancreatic β-cell Ca2+ signalling and reduced insulin secretion from the whole pancreas were demonstrated. However, the whole-animal phenotype has not been extensively researched. In this study, intraperitoneal glucose tolerance tests were conducted in Tpcn KO mice. These indicated that glucose homeostasis was not significantly affected in Tpcn2 KOs or Tpcn1/2 double KOs (DKOs), and only mildly impaired in Tpcn1 KOs, despite the defects previously observed at the cellular and tissue level. In addition, body composition was investigated in Tpcn1 KO, Tpcn2 KO and Tpcn1/2 DKO animals using magnetic resonance spectroscopy and time domain-nuclear magnetic resonance. Single Tpcn KOs were found to have lower adipose tissue levels as a percentage of body composition, while Tpcn1/2 DKOs were shown to have increased bodyweight but normal body composition. To investigate potential roles for TPCs in adipose tissue formation, Tpcn expression during adipogenesis was investigated using an in vitro multipotent mesenchymal stem cell line model of adipogenic differentiation. Tpcn2 mRNA levels were demonstrated by quantitative PCR to be transiently increased during the early stages of adipogenic differentiation, and cyclic AMP (cAMP) was identified as the factor that induced this upregulation. Lentiviruses were developed to express fluorescently-tagged TPCs, and overexpression of TPC2 was demonstrated to partially overcome the requirement for the cAMP-inducing agent in the medium used for the induction of adipogenesis. Collectively, these data suggest that TPCs may play a role in the formation and/or function of adipose tissue.
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Unsworth, Amanda J. "The role of protein kinase C in platelet activation." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:114582b8-185a-41f5-958c-77038fb185df.
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The Protein kinase C (PKC) superfamily is a key regulator in platelet activation with individual isoforms playing distinct roles. This thesis focuses on the role of the novel PKC isoforms downstream of several agonists using both pharmacological and genetic approaches and human and mouse platelets. Quantification of the protein levels of PKC isoforms identified different levels of the five major PKC isoforms expressed in human platelets and also differences between levels of the same isoform in human and mouse platelets. Use of a selection of broad spectrum and isoform-specific inhibitors, identified both positive and negative novel roles for PKC in the regulation of human and mouse platelets. A net positive role for PKC was found in GPVI, Clec-2, and PAR receptor signalling, with classical isoforms of PKC playing a major role in aggregation and dense granule secretion. A novel negative regulatory role was also identified in the regulation of ADP-induced platelet activation for PKC~, and both PKCE and PKC~ in human and mouse platelets respectively. Gene knock-out mouse models confirmed a positive regulatory role for PKCe in allb~3 outside-in signalling but identified no other regulatory role for PKCe in agonist induced platelet activation. Despite this relatively minor role, functional redundancy was identified between PKCe and PKCE isoforms in haemostasis, as tail bleeding was significantly increased in mice deficient in both novel isoforms. The work presented here identifies key roles for the PKC superfamily in the complex regulation of platelet activation, with different isoforms supporting and limiting the process of thrombus formation and haemostasis.