Eukaryotic cells maintain a compartmental cellular organization of membrane-enclosed organelles that communicate with each other through the exchange of trafficking vesicles. Members of a superfamily of membrane proteins, the so-called SNAREs, are essential for the necessary fusion of vesicle membranes to the membrane of target organelles. SNAREs are needed to overcome the energy barrier that prevents spontaneous membrane fusion events. A number of studies from the past decade indicated that SNARE proteins might fulfill a function beyond merging membranes. The mammalian plasma membrane SNARE Syntaxin1A was shown to directly interact with and through this interaction modify the activity of, for example, a calcium ion channel and a potassium ion channel. In its classical function as SNARE protein, Syntaxin1A mediates specialized vesicle fusion events such as synaptic transmission in neurons or secretion of insulin from pancreatic cells. These specialized vesicle fusion events require precise timing that is controlled by intracellular signaling events. These intracellular signaling events involve the coordinated action of members from different families of ion channels. Current models suggest that the dual functions of a SNARE protein in ion channel regulation and membrane fusion serve to fine-tune highly regulated vesicle fusion events. This thesis provides evidence for the first direct interaction between a SNARE protein and an ion channel from plants and suggests a function for this interaction in Arabidopsis potassium nutrition. Three different protein-protein interaction assays for full-length membrane proteins that comprised a yeast mating based split-ubiquitin assay, co-immunoprecipitation after expression in insect cells and bi-molecular fluorescence complementation after transient Arabidopsis root transformation, confirm that the Arabidopsis plasma membrane SNARE SYP121 interacts in vitro and in vivo with the Shaker ion channel subunit KC1. Furthermore, the interaction between KC1 and SYP121 is specific over the closest homologue of Syp121, namely SYP122. Shaker channels are plasma membrane proteins with four subunits that transport the essential macronutrient potassium in response to changes in membrane voltage. The KC1 subunit is unique among the Shaker channels. It can only act as a regulatory subunit that modifies channel properties when forming heterotetramers with other Shaker subunits such as AKT1, not as functional homotetramer. AKT1 is expressed predominantly in the root epidermis, i.e. root hairs, where it overlaps with the more broadly expressed KC1 and SYP121. Previous publications showed that a low external potassium concentration combined with high levels of ammonium that is used to block all root potassium uptake systems apart from AKT1, causes akt1 null mutants to display strongly reduced main root length as well as whole plant potassium content compared to wild type plants. It is shown here that the phenotype of both syp121 and kc1 null mutants is identical to the akt1 mutant under these growth conditions. The design of new antibodies against native AKT1 and KC1 and an optimized protocol for root plasma membrane protein enrichment and solubilisation allowed for the first time visualization of native Arabidopsis AKT1 protein. This technical advance made it possible to confirm that both Shaker channel subunits are present in equal amounts in the plasma membrane of roots cells from syp121 mutant and wild type plants. It is concluded that the potassium uptake phenotype of the syp121 mutant is not caused by the absence of channel proteins from the plasma membrane due to a disruption of the vesicle trafficking function of the SNARE SYP121. An alternative function for SYP121 in potassium nutrition that involves direct interaction with AKT1-KC1 heterotetrameric channels is supported by electrophysiological measurements after heterologous expression in Xenopus leavis oocytes. SYP121 modifies the voltage-dependent potassium uptake characteristics of AKT1-KC1 heterotetramers in a way most easily understood in context of a conformational change within the voltage sensing protein parts of the Shaker channel that are caused by the direct interaction with the SNARE protein. It is concluded that the identical potassium uptake phenotype of the akt1, kc1 and syp121 mutants is caused by the inability to form a functional tripartite complexes. As KC1 is able to form heterotetrameric channels with several different Shaker channel subunits, for example KAT1 that is highly expressed in guard cells, it is likely that this novel interaction between KC1 and SYP121 might modulate channel activities in different tripartite complexes to affect various cellular functions.

The rhizosphere is defined as the area of soil surrounding plant roots, which is influenced by plant exudates. The rhizosphere hosts a diverse and dynamic microbiome, which is shaped by both plant and environmental factors. The plant-microbe and microbe-microbe functional interactions which occur in the rhizosphere can have significant impacts on plant growth. Developing understanding of the composition, functions and interactions of the rhizosphere microbiome and the factors which shape it, may prove valuable to improve agricultural sustainability. The rhizosphere and bulk soil microbiomes of contrasting Brassica napus genotypes growing in the field under high and low N inputs were characterised using amplicon sequencing. Taxonomic identification, functional prediction tools and network analysis were used to gauge how nutrient availability and plant genotype influenced the microbiome. N availability was seen to have a greater influence on composition, function and connectivity of the microbiome than crop genotype, with varying effects on microbes from different Kingdoms. Metatranscriptome analysis enables analysis of the functioning of the microbiome. The effectiveness of different methods for the separation of root and rhizosphere soil for metatranscriptome analysis was compared. Washing roots in water to separate roots and rhizosphere soil followed by freeze drying prior to RNA extraction was shown to be the best method to avoid distorting the metatranscriptome profile. Metatranscriptome analysis of field grown B. napus revealed increases in the rhizosphere relative to soil for protein metabolism functions, and the root compartment contained a high proportion of transcripts related to phage activity. Plant rhizosphere functions were investigated using transcriptomic analysis of a diverse range of cultivated and wild Brassica oleracea plants. Uptake of PO4 is a vital plant process but the identity of PO4 transporters is unknown in B. oleracea. A number of putative PHT1 PO4 transporter genes were identified. Significant differences in expression of the putative PHT1 genes were found between cultivated and wild lines, which may inform future plant breeding strategies.

Wheat yields need to rise to meet growing demands due to population growth and changing diets. Additionally, the resilience of crop yields to climate change and rising temperatures needs to be improved. Inhibition of photosynthesis under sub-optimal environmental conditions decreases carbon fixation, reducing crop yields. Heat stress inhibits photosynthesis, in part due to a decrease in the activation state of Rubisco. Rubisco activase (Rca) is required to restore and maintain the catalytic activity of Rubisco. Rca has a relatively low temperature optimum; improving its thermal tolerance would maintain Rubisco activity and enhance photosynthesis at higher temperatures, with predicted positive impacts on grain yields under moderate heat stress. Two approaches were taken to improve the thermal tolerance of Rca in wheat. Firstly, natural variation in the thermal tolerance of Rca in wheat was investigated. Cultivars exhibiting differences in their photosynthetic performance were identified, but the complexity in breeding for increased thermal tolerance was highlighted, with both advantageous and disadvantageous characteristics being identified. The second approach was to introduce the more thermally stable Rca from cotton into wheat in an attempt to broaden the range of temperatures at which photosynthesis operates. Transgenic plants were produced but the cotton Rca protein was undetectable in the wheat lines investigated. Two genes encoding Rca in wheat were identified; one gene is alternatively spliced to produce α and β isoforms. Virus-Induced Gene Silencing of the Rca isoforms in wheat indicated that the Rca genes in wheat may be co-regulated. A non-radioactive activity assay was developed for use in Rubisco and Rca research, allowing high-throughput of samples and avoiding the difficulties some labs may have in completing radioactive assays. The information gained in this study will guide future approaches to optimise the thermal stability of Rca and generate temperature-resilient crops.

Plants can vary in their mating systems from completely inbreeding to completely outcrossing, with intermediate forms referred to as mixed mating systems. Arabidopsis lyrata is a strongly outcrossing perennial due to a sporophytic self incompatibility (SI) system. The species occurs in temperate regions of the Northern hemisphere where in Europe its SI system is fully working but around the Great Lakes of North America some populations of A. lyrata show a breakdown in SI. Consequently these North American populations are inbreeding or have a mixed mating system next to outcrossing populations with a working SI system. In this thesis I used North American A. lyrata to investigate the evolutionary consequences involving variation in mating systems. First of all I was interested in the time that populations had been isolated from each other in the past that could explain differences in mating systems. In order to determine whether populations experienced a breakdown of SI independently or whether this originated from a single event I used chloroplast DNA (cpDNA) markers to reveal deep phylogeny and microsatellite markers to determine recent population genetic patterns. The results showed a loss of SI in populations from all three detected cpDNA haplotypes. Microsatellite data showed that predominantly inbreeding populations sharing one of these haplotypes showed high levels of homozygosity and that in all three haplotype lineages self-compatible individuals always had reduced heterozygosity compared to self-incompatible individuals. The data further showed that there had likely been at least two independent postglacial colonization routes to the north of the great lakes. This was consistent with phylogeographic studies of other organisms with limited dispersal such as reptiles and amphibians. The next question was the role of inbreeding depression in the loss of SI. Inbreeding depression is defined as the decline of fitness after an inbreeding event. Inbreeding causes an increase in homozygosity that exposes recessive deleterious mutations, which would normally be sheltered in a heterozygous state, and causes a fitness decline. Individuals experiencing a loss of SI will have higher inbreeding levels and can result in inbreeding depression, which is thought to maintain the SI system. To gain more insight into the role of inbreeding depression in the shift from self-incompatibility to self-compatibility, I conducted an experiment in which I created outcrossed and selfed offspring from self-compatible and self-incompatible mothers from populations with different outcrossing histories. I monitored the offspring for early- and late acting fitness traits like germination rate, growth and time to flowering. I found inbreeding depression in only one late acting fitness trait, the increase in leaves 5 weeks after germination, to be significantly higher for self-incompatible than self-compatible individuals. I also conducted a regression analysis where relative fitness (the ratio of the fitness trait values of selfed and outcrossed offspring) per mother was regressed against population heterozygosity and found a significantly negative regression. This result suggested that individuals from a population with a relatively high heterozygosity suffered more from inbreeding depression than individuals from populations with a relatively low heterozygosity. This indicated that the history of outcrossing of a population, or purging, played an important role in the shift from outcrossing to inbreeding. The detection of inbreeding depression could not be evident by only looking at life history traits under greenhouse conditions. But stressful environmental conditions like a pathogen infection could magnify inbreeding depression. I would expect that predominantly outcrossing populations would have a higher heterozygosity than predominantly inbreeding populations and therefore be able to show a higher fitness when exposed to a pathogen. To test this hypothesis I used four outcrossing and four inbreeding populations, which I infected with the crucifer pathogen Albugo candida and measured relative growth rates (RGR) and monitored resistance rates. The results showed that there were three infection phenotypes: resistant (no signs of infection), partially resistant (only the initially infected parts showed symptoms) and susceptible (symptoms present on the whole plant). The inbreeding populations showed a bimodal distribution of resistance as two populations showed a high rate of resistance and two showed a low rate of resistance. The outcrossing populations showed a much more uniform distribution of resistant individuals with a higher rate of partially infected individuals across populations than inbreeding populations. Resistant and partially resistant individuals did not differ significantly in their RGR from each other but both had a significantly lower RGR than the untreated control group and a significantly higher RGR than the susceptible individuals. This suggested a cost of resistance that was lower than a cost of being susceptible in the presence of a pathogen. There was no effect of mating system on RGR, which was primarily caused by the fact that two inbreeding populations contained a high amount of resistant individuals and an outcrossing population that showed a very low amount of partially resistant and resistant individuals. The difference in resistance to A. candida in A. lyrata differed much more between inbreeding than between outcrossing populations. This suggested that alleles responsible for resistance were concentrated in homozygous form in inbreeding populations and both homozygous and heterozygous form in outcrossing populations. This would mean that mating system plays a role in susceptibility, as resistance genes would be concentrated in certain individuals in inbreeding populations as opposed to a more modal distribution in outcrossing populations. A shift in mating system often has an effect on floral traits, as there is a lack of necessity to attract pollinators. I wanted to test whether these changes were apparent in A. lyrata by comparing pollinator attractants and sexual floral traits between strongly outcrossing and strongly inbreeding populations. I hypothesized that individuals depending on pollinators for outcrossing would show a higher emission of volatiles and floral traits that had evolved to optimize pollen transmission to conspecifics. Autonomously selfing individuals would be independent of pollinators so should show a reduced volatile emission pattern, a floral trait composition that evolved to transmit pollen to their own stigma, and a reduction in floral display compared to outcrossers. My results showed a somewhat contradicting pattern as self-compatible individuals showed higher volatile emission than self-incompatible individuals but self-incompatible individuals showed larger petal size than self-compatible individuals. Pistil height and stamen length were strongly correlated but petal size seemed to co-vary relatively independent from pistil and stamen length. I found no effect of mating system on the evolvement of floral traits to optimize pollen to the stigma and contradicting patterns for pollinator attractant traits. Due to low sample sizes this study turned out to be a pilot study for further research so the results in this study were not conclusive at this stage. Finally I conclude that SI has been lost independently several times and the low observed genetic load in the North American populations compared to the European populations could be responsible for that. There have probably been two independent colonization routes to the North of the Great Lakes following the last glaciation in which a Northern distributed cpDNA haplotype lineage seems to have a lower frequency of SC individuals than a southern cpDNA haplotype lineage.

The root system is the plant’s principal organ for water and mineral nutrient supply. Root growth follows an endogenous, developmental programme. Yet, this programme can be modulated by external cues which makes root system architecture (RSA), the spatial configuration of all root parts, a highly plastic trait. Presence or absence of nutrients such as nitrate (N), phosphate (P), potassium (K) and sulphate (S) serve as environmental signals to which a plant responds with targeted proliferation or restriction of main or lateral root growth. In turn, RSA serves as a quantitative reporter system of nutrient starvation responses and can therefore be used to study nutrient sensing and signalling mechanisms. In this study, I have analysed root architectural responses of various Arabidopsis thaliana genotypes (wildtype, mutants and natural accessions) to single and multiple nutrient deficiency treatments. A comprehensive analysis of combinatorial N, P, K an S supply allowed me to dissect the effect of individual nutrients on individual root parameters. It also highlighted the existence of interactive effects arising from simultaneous environmental stimuli. Quantification of appropriate RSA parameters allowed for targeted testing of known regulatory genes in specific nutritional settings. This revealed, for example, a novel role for CIPK23, AKT1 and NRT1.1 in integrating K and N effects on higher order lateral root branching and main root angle. A significant contribution to phenotypic variation also arose from P*K interactions. I could show that the iron (Fe) concentration in the external medium is an important driving force of RSA responses to low-P and low-K. In fact, P and K deprivation caused Fe accumulation in distinct parts of the root system, as demonstrated by Fe staining and synchrotron X-Ray fluorescence. Again, selected K, P and Fe transport and signalling mutants were tested for aberrant low-K and/or low-P phenotypes. Most notably, the two paralogous ER-localised multicopper oxidases LPR1 and LPR2 emerged as important signalling components of P and K deprivation, potentially integrating Fe homeostasis with meristematic activity under these conditions. In addition to the targeted characterisation of specific genotype-environment interactions, I investigated novel RSA responses to low-K via a non-targeted approach based on natural variation. A morphological gradient spanned the entire genotype set, linking two extreme strategies of low-K responses. Strategy I accessions responded to low-K with a moderate reduction of main root growth but a severe restriction of lateral root elongation. In contrast, strategy II genotypes ceded main root growth in favour of lateral root proliferation. The genetic basis of these low-K responses was then subsequently mapped onto the A. thaliana genome via quantitative trait loci (QTL) analysis using recombinant inbred lines derived from parental accessions that either adopt strategy I (Col-0) or II (Ct-1). In sum, this study addresses the question how plants incorporate environmental signals to modulate developmental programmes that underly RSA formation. I present evidence for novel phenotypic responses to nutrient deprivation and for novel genetic regulators involved in nutrient signalling and crosstalk.

Plant performance under reduced water availability has traditionally been assessed as drought resistance and more recently as water use efficiency (WUE). An extensive body of work has been established over the past 15 years where the natural variation of water use efficiency has been studied in the model species Arabidopsis thaliana (Arabidopsis). At the same time, a substantial degree of criticism has arisen with respect to the use of drought resistance and WUE as measures of plant performance, due to the lack of relatedness of these parameters to reproductive performance, i.e. yield. The work in this thesis is centered on understanding the physiological and genetic basis of water use and water productivity as alternative measures of plant performance under the context of reduced water availability. The first part of this study describes an extensive assessment of the natural variation of water use and water productivity in Arabidopsis in relation to numerous key physiological, phenological, and developmental parameters. Furthermore, this work concisely relates plasticity of key traits to historical climatic variation. A fundamental aspect of this work was the clarification that it is possible to estimate long term water use to a high degree of accuracy based on short term water use, i.e. soil drying rate, and flowering time. Flowering time was demonstrated to be the predominant driver of vegetative performance and water use, however it appeared to be genetically uncoupled from reproductive performance. This is in contrast to previous work that suggests WUE, measured as the ratio of C12 to C13 isotopes (δ13C), is positively associated with flowering time. Additionally, it was demonstrated that multiple commonly employed proxies of reproductive performance including total biomass, WUE, and flowering time, were not sufficient at predicting seed yield in Arabidopsis across multiple environments. The second part of this study involved the genetic dissection of water use and productivity related traits in Arabidopsis through a quantitative trait loci (QTL) mapping study and a genome wide association study (GWAS). QTL mapping using a recombinant inbred line (RIL) population developed from the ecotypes Col-0 and C24 revealed two key flowering time genes, FLOWERING LOCUS C (FLC) and FRIGIDA (FRI), as key regulators of water use. It was demonstrated that a combination of non-functional alleles of both FLC and FRI reduced long term water use via a shorted life cycle, which is again in contrast to previous work relating to the genetic dissection of WUE in Arabidopsis. Crucially, it was observed that reduced water use mediated in this fashion did not detrimentally impact upon reproductive performance. GWAS was employed subsequent to the QTL mapping in order to identify candidate genes underlying the variation for productivity as a unique trait and also as a factor of water use, i.e. water productivity. GWAS identified multiple promising candidate genes that potentially underlie the heritable genetic variation for flowering time, water use, and water productivity.

No description available.

Liriomyza bryoniae is an economically important pest of vegetable and ornamental crops in European glasshouse agriculture. Diglyphus isaea is a parasitoid of Liriomyza leaf miners and is commercially available as a biological control agent. Anecdotal reports made to commercial producers of the parasitoid suggest that the efficacy of D. isaea varies between crops. This study examines the tritrophic interactions between crop plant, L. bryoniae and D. isaea. Host plant was found to influence the abundance of L. bryoniae and D. isaea with larger populations establishing in the culturing host than in the novel host, tomato. Individual size of L. bryoniae also varies with host plant. These patterns are consistent in L. bryoniae across three generations of rearing on tomato. Habituation of L. bryoniae to tomato does not affect D. isaea efficacy nor does the natal plant host of D. isaea. Both L. bryoniae and D. isaea are affected by plant host ontogenetic stage, becoming most numerous on juvenile plants. The D. isaea natal insect-plant complex showed no effect on D. isaea olfactory preferences. Diglyphus isaea demonstrated greater thermal tolerance than its host. These results are discussed in relation to biological control and also in terms of their wider ecological implications.