There is a growing need to engineer an increased yield in food crops coupled with greater resistance to environmental stresses, such as drought, to meet the requirement of a growing population. As plant responses to drought (and other abiotic stress) are complex, many studies have attempted to understand the drought response in a holistic manner, for example, by analysing transcriptomics data obtained from plants subjected to drought. Preliminary work of obtaining time-series microarray data from a slow-drying experiment of Arabidopsis was used to construct Gene Regulatory Networks using Variational Bayesian State Space Modelling. This led to the identification of a number of transcription factors RAP2.12, FD, BHLH038, ANL2, and two unknown genes, UKTF and POZ, as possibly being important regulatory genes in the drought response. Loss- and gain-of-function mutants of these genes, as well as those of AGL22 identified by Bechtold et al. (2016), were phenotyped under drought conditions. Only the flowering time AGL22 showed a drought phenotype. Network connections in the gene network of AGL22 were tested by qPCR. Drought responsive transcription factors, such as DREB1A and WRKY20 were found to be induced by AGL22.

Intraspecific variation within the genetic model plant Arabidopsis thaliana has been used to research numerous potentially adaptive and economically important traits. In this thesis I used this tool to investigate root morphology and adaptation of plants to edaphic conditions. Firstly I tested local adaptation of chosen A. thaliana wild genotypes collected from north eastern Scotland, to two soil types with contrasting textures. When local adaptation is defined as fitness advantage (reproductive output) in local soil, I did not find clear signs of local adaptation. Additionally, I observed significant phenotypic variation between collected accessions for both mineral nutrient uptake and growth in the two soil types. Together these results may suggest the mixing of adapted genotypes due to extensive human disturbance in north eastern Scotland. Secondly I focused on two linked aspects of root biology: endodermal development and root system architecture. The characteristic features of the endodermis include Casparian strips (CS) that form a barrier to apoplastic transport. Some mutants with an altered CS are sensitive to growth on media with elevated magnesium (Mg) and reduced calcium (Ca). In order to potentially identify novel alleles of genes involved in CS biosynthesis I took advantage of this growth phenotype and performed genome wide association (GWA) analysis on response of plants to low Ca/Mg ratio of the growth medium. As a result I compiled a list of genes for the future research by choosing candidate genes identified in the GWA study for both plant weight and elemental composition. This list was further refined using knowledge on gene expression in the endodermis. Accessions with the most extreme response to this low Ca/Mg treatment were analysed further. I identify a link between both lateral root number and total root length with performance on growth medium containing a low Ca/Mg ratio.

The global success of plants depends largely on their ability to perceive and respond to light, mainly in two regions of the electromagnetic spectrum. Phytochromes are light sensors for the red and far-red wavelengths of light while cryptochromes, phototropins and members of the ZTL/ADO family respond to blue and UV-A wavelengths of light. Phototropins are UV-A/blue-light receptor kinases found ubiquitously in plants from the unicellular green alga Chalmydomonas reinhardtii through bryophytes and pteridophytes up to angiosperms. The model plant Arabidopsis possesses two phototropins (phot1 and phot2) and is the subject of the work presented in this thesis. The general structure of the phototropin protein comprises a photosensory region at the N-terminal that contains two LOV (light, oxygen and voltage sensing) domains and a C-terminal kinase domain belonging to the large AGC family of protein kinases. The LOV domains form a covalent adduct with the chromophore flavin mononucleotide (FMN) in response to illumination with blue light which in turn leads to structural changes throughout the protein resulting in autophosphorylation of the N-terminal region by the kinase domain. Phototropins function redundantly to mediate a number of physiological responses in planta which serve to promote plant fitness and maximise photosynthetic potential. Phototropism, chloroplast accumulation, blue light-induced stomatal opening, leaf expansion and leaf movements can be induced through the activation of both phot1 and phot2 in response to different intensities of light, with phot1 being more light sensitive than phot2. In addition to the functionally redundant responses, phot1 alone is responsible for destabilisation of certain mRNA transcripts and the rapid inhibition of hypocotyl elongation when etiolated seedlings are transferred to blue light, while phot2 is solely responsible for the high light induced chloroplast avoidance response. While much is known about the mechanisms of light perception by the phototropins at the molecular level, and the responses mediated by them have been well described, little is known about their methods of signalling to induce these physiological responses upon photoactivation by blue light. Therefore, the aims of this study were to identify novel phot-interacting proteins and to investigate the modes of phot1 signalling by structure/function analyses in order to better understand the way phototropins elicit signal transduction to downstream components in order to bring about the responses described above. Initially, a yeast two-hybrid screen was carried out to try to identify immediate interacting partners for phot1. The results of the yeast two-hybrid screen are described in Chapter 3. One hundred and thirty yeast colonies containing putative phot1-interacting proteins were identified from the screen and preliminary characterisation of six of these proteins are described in this chapter. Two of the proteins investigated are members of the ADP-ribosylation family which is involved in the regulation of membrane trafficking. The ARF proteins identified show a blue-light-sensitive interaction with phot1 and also interact with phot2. These proteins are of interest given the subcellular movement of phototropins from the plasma membrane after exposure to blue light. The C-terminal kinase domain of phot1 was found to interact with p-glycoprotein 19 (PGP19), a protein involved in polar auxin transport. The interaction between these proteins is interesting because of the role auxin plays in phot1-mediated responses such as phototropism and leaf expansion, and preliminary characterisation of the interaction in vitro is shown in Chapter 3. The implications of a direct link between phototropins and the proteins involved in auxin transport are discussed. A further two proteins identified from the screen are members of the NPH3/RPT2-Like (NRL) family. RPT2 has already been identified as a phot1-interacting protein and identification of this protein increased confidence in the efficacy of the screen to identify genuine interacting proteins. A novel member of the NRL family, designated NPH3-L, was also identified from the screen. Chapter 4 describes the tissue specific and subcellular localisation of NPH3-L and contains results of preliminary investigations into the function of NPH3-L in planta. 14-3-3λ was identified from the screen using full-length phot1 as bait. A 14-3-3 protein has been shown previously to bind to autophosphorylated phototropin in Vicia faba (Kinoshita et al., 2003). Chapter 5 details the localisation of 14-3-3λ at tissue and subcellular levels and shows that 14-3-3λ binding to plant-derived phot1-GFP is both light dependent and induced by receptor autophosphorylation. Creation of GFP-14-3-3λ overexpressing lines in wild-type and phot1-5phot2-1 backgrounds allowed investigation into the roles that light and phototropins play in regulating the subcellular localisation of 14-3-3λ. It is shown that light-induced movement of 14-3-3λ at the plasma membrane is dependent on the presence of endogenous phototropins. Physiological implications of this interaction are discussed. Finally, in order to determine the modes of phototropin signalling, structure/function studies were carried out by expressing different regions of phot1 in a variety of Arabidopsis backgrounds. The results of the structure/function studies are described in Chapter 6. Known phot1-mediated responses were investigated in the transgenic plants to determine the effects of individual domains of phot1. Particular attention was paid to the role of receptor autophosphorylation in phot1-mediated responses to light. A transgenic line overexpressing the LOV2-kinase region of phot1 demonstrates that phot1 autophosphorylation is not the primary signalling event involved in phot1-mediated responses to light and shows that the truncated version of phot1 is sufficient to complement most phot1-mediated responses. This also shows that the LOV1 domain is dispensable and suggests phot1 may signal through phosphorylation of substrates. Comparisons are drawn between phot1 kinase overexpressing lines and inactive phot1 kinase overexpressing lines. Preliminary observations of a transgenic line overexpressing phot1 in a wild-type background indicate that overexpression of phot1 may alter polar auxin transport. Together these studies provide new insights into possible mechanisms of phot1 signalling and the function of major domains of phot1.

Global climate change is predicted to alter the weather patterns around the world, as climatic zones shift, forest carbon sequestration projects (e.g. the UK woodland carbon code) need to take into account the specific requirements of planted species. In the UK, oaks are an important charismatic group of trees favoured in recent planting programmes. The English oak (Quercus robur L.), has poor water conservation, but is a major component of natural forests in lowland UK. On the other hand, Holm oak (Quercus ilex L.) is a Mediterranean oak that has high water conservation and can also tolerate cold despite being restricted by minimum temperatures. At local scales, there may be advantages of planting either evergreen or deciduous oak species for forestry and climate mitigation. Alternatively, a comparative assessment of non native versus native productivity, may give clues to the invasiveness potential of Holm oak and its ability to out compete the deciduous oak along an urban to upland gradient. This thesis documents a series of field based experiments intended to analyse differences in carbon sequestration potential and photosynthetic efficiency between these two species and in relation to their environment within the Southwest UK. 520 one year saplings were planted, half in pots and half in nursery field beds situated on Dart- moor, the east Devon Dartmoor fringe, Totnes, and Plymouth city centre. Originally two sites were chosen for their relative ‘urban’ qualities, two at ‘rural’ localities, one upland and a control site with access to a polytunnel for comparisons with well-watered and non nutrient limited trees. However, data analyses showed that sapling characteristics were site specific with the five sites falling along an urban, rural to upland gradient. The field experiments included monthly height and diameters (ground level diameter or DAG), monthly assimilation rates and analy- sis of chlorophyll fluorescence to aid interpretation of photosystem II functioning and sapling ‘vitality’. Further laboratory experiments analysed specific leaf area (SLA), mass based leaf Nitrogen (Nleaf ) and carbon (Cleaf), with differences between sun and shade leaves included, to aid comparisons between species and sites. The final experiment was a destructive harvest and this was used to find total biomass estimates and carbon allocation to different root shoot fractions. In order to quantify differences between saplings and adult trees a smaller experiment was con- ducted in the canopy using experienced climbers and leaf level productivity analysed. Intrinsic water use (iWUE), stomatal conductance (Gs), means net assimilation rates (An) and chloro- phyll fluorescence parameters; Variable fluorescence over maximum fluorescence (Fv/Fm) and performance index (PI) were measured and relative carbon assimilation rates and productivity assessed and compared between species at one urban , rural and upland site. Results showed that Q. ilex allocated relatively more carbon to branches and leaves as a sapling which in turn increased growth rate and whole tree assimilation rates to larger values than the deciduous oak despite Q. robur being able to increase maximum assimilation rates in response to increasing temperatures. This gives Q. ilex the advantage and overall biomass was higher at all sites than Q. robur apart from the upland site where there were no differences in biomass accumulation between species. However, despite no significant difference in biomass at this site Q. robur had greater survival and photosystem II functioning. In mature trees Q. ilex was under stress and Nleaf and carbon sequestration potential were higher in the deciduous species at the urban site. In contrast, Q. robur was under stress at the upland site at Castle Drogo where thin and nutrient poor soils have made it more vulnerable to drought stress. Here, mature Q. ilex showed reduced photosynthetic efficiency in relation to cold and drought, but was able to recover when milder temperatures occurred. The results were site specific, with a reduction in both SLA and relative allocation to the leaf weight fraction (LWF) in Q.robur the only common urban related effect seen. The potential for Q. ilex to perform well at sapling stage is due to its morphological plasticity and drought tolerance. This species may become more prevalent within the Southwest as local climates continue to push it northwards from its natural Mediterranean range. In contrast, if Q. robur continues to suffer from defoliation and fungal attack and this may leave it more vulnerable to competition throughout less fertile and drier areas of its natural range.

The Arabidopsis thaliana MALE STERILITY1 (MS1) gene, encodes a plant homeodomain (PHD) transcription factor critical for viable pollen formation (Wilson et al., 2001). In the ms1 mutant, there are alterations in the production of pollen wall materials, as well as a failure of tapetal programmed cell death (PCD) (Vizcay-Barrena and Wilson, 2006). This ultimately results in the failure to produce viable pollen. Large numbers of genes are down-regulated in the ms1 mutant indicating that MS1 plays a key role in regulating late tapetal expression and pollen wall deposition (Ito et al., 2007; Yang et al., 2007). Two putative MS1 interacting proteins At1g58210/NET2A (termed as Y2H54) and AT2G46260/ LRB1 (termed as POB2) were identified from a previous Arabidopsis stamen specific yeast-2-hybrid screen, using a truncated version of the MS1 protein without the PHD motif. POB2 and MS1 were found co-localised in the nucleus, while Y2H54 was specifically located at the plasma membrane. Further confirmation of the interaction using Förster resonance energy transfer (FRET) assay methods showed that POB2 failed to interact with MS1 in planta, however, the association between the two proteins occurred in vitro, as confirmed by protein pull-down assays. Additionally, enhanced general plant growth and floral development were seen in the overexpression lines of Y2H54. However, no significant phenotypes were observed in the RNAi silencing lines. Chromatin Immunoprecipitation (ChIP) analysis uncovered that MS1 directly regulated the expression of MYB DOMAIN PROTEIN 99 (MYB99) by binding to its promoter. Other putative MS1 direct targets identified by ChIP include 3-KETOACYL-COA SYNTHASE 7 (KCS7), 3-KETOACYL-COA SYNTHASE 15 (KCS15), SPERMIDINE HYDROXYCINNAMOYL TRANSFERASE (SHT) and TAPETUM-SPECIFIC METHYLTRANSFERASE 1 (TSM1). Histone extraction and western blotting assays suggest a role for MS1 in facilitating detrimethylation of H3 marks. H3K36me3 deposition was enhanced at MYB99 in ms1 compared with the wild type, suggesting that MS1 may regulate MYB99 via H3K36me3. A new model for the MS1 regulatory network in pollen wall formation has therefore been proposed.

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.

Brassica crops are grown worldwide for food, oil, medicinal and crop rotation properties. They suffer from insect pests which cause large yield and economic losses. Application of insecticides is the preferred way of dealing with insect problems, but it is not only hazardous to the environment, it also affects humans as the chemicals easily get incorporated into the food chain. As a result, new more resistant varieties are urgently needed to meet the demand of growing populations. A set of 200 accessions were classified as resistant (non-preferred) or susceptible (preferred) in response to cabbage aphid feeding in the field. Fifteen accessions were further assessed to characterize and identify the level and location of resistance factors by investigating feeding behaviour of cabbage aphid using the Electrical Penetration Graph (EPG) technique. The feeding behaviour assessment revealed the presence of interspecific & intraspecific variation and presence of resistance factors at multiple levels. The transcriptional response of these accessions under presence and absence of aphid feeding for 24h showed that gene expression is highly regulated in response to aphid feeding. Gene ontology (GO) enrichment study helped identify strong candidate genes for aphid resistance. In addition to this, the gene expression differences between CWR and landraces indicated adaptations of landraces during the process of domestication. Lastly, Gene expression data was used to develop models to predict insect resistance status. In conclusion, the combination of EPG and transcriptomics provides an opportunity to assess brassica germplasm for further research into defence mechanisms of cabbage aphids.

An analysis of the photoperiodic induction of flowering in Arabidopsis thaliana (L.) Heyne, Landsberg erecta ecotype, was carried out. This revealed that 4 day old seedlings, at which time the cotyledons where expanding and greening, could differentiate between a LD and a SD. At this stage the critical daylength was between 8 and 10 hours. Plants grown in daylengths of 8 hours, a short day, flowered after 50-70 days and when grown in 16 hour daylengths, a long day, flowered after 24-27 days. At 7 days seedlings required five long days to fully induce flowering and as the seedlings aged in short days they became more sensitive to interposed long days, so that by day 20 one long day was fully inductive. It was found that there were two different photomorphogenic responses shown by plants grown in short days, firstly flowering was delayed and secondly further leaves were induced. The short days delay of flowering occurred in newly germinated seedlings older than 4 days and further leaves were induced in plants older than 10 days. From light quality experiments it was concluded that both a blue light photoreceptor and phytochrome promoted flowering. The induction of flowering by phytochrome was through a HIR mode. Three of the photoreceptor mutants, hy, isolated by Koornneef et al. (1980) were used. In daylength transfer experiments all the hy mutants studied showed delay in flowering by short days and all responded to long days by flowering earlier. Both hy2 and hy3 produced far fewer leaves than Ler when grown in short days. The hy4 mutants flowered later in both long days and short days than Ler and had an increased leaf number. A scheme is proposed in which photoperiodic induction depends on the ability of the plant to sense photoperiod, the stage of development and the photobiological input. It also proposes that phyA or C and the blue light photoreceptor promote flowering whereas phyB promotes vegetative development. Two screens were set up to isolate novel photoperiodic mutants. Six mutants were isolated, from ethtylmethane sulphonate mutated seed, which all flowered earlier than Ler in SD. They were called FUN 1-6, flowering pjcoupled. Genetic analysis showed that all were non allelic and that they were recessive except .tun4 which was semi-dominant. Physiological studies showed that there were two types of mutants: firstly funl and 2 whose flowering was not significantly delayed by SD in comparison to long day treatments and flowered early in the dark; and secondly fun3-6 which all showed a delay in flowering when grown in SD. The .funl, and 2 mutants had poorly developed leaves as did fun5. The other mutants did not show any other clearly defined phenotypes. These results suggest that funl and 2 mutants are constitutive flowering mutants and the remaining mutants are transduction chain mutants.

Begonia is one of the ten largest plant genera and is found throughout the tropics. I have used Begonia section Gireoudia to study the genetics underlying vegetative diversity in tropical herbaceous plants. Section Gireoudia is a large Central American group. The section is remarkably diverse in morphology and habitat preference. It ranges from wet rainforests to seasonally dry forests. I have investigated variation in morphological, anatomical and ecophysiological differences for 21 species in Begonia section Gireoudia. Based on the observed variation, species in Begonia section Gireoudia form a complex and unique group that stands out from currently analysed taxa in the global scale of variation on the basis of leaf function and resource use strategy traits as well as their peculiar leaf anatomy. Traits directly related to leaf function such as photosynthesis and stomatal conductance has very low values which overlap with those of CAM and aquatic plants. Values for traits indicative of resource use such as leaf mass area (LMA) and leaf dry matter content (LDMC) are also very low in Begonia when compared with the values observed globally. The trait- trait correlations across the species in section Gireoudia were also investigated and revealed patterns in micromorphology and ecophysiology. Some of the traits measured are correlated with each other in apparently straightforward, well charaterised biological relationships e.g., the variation among Begonia species in stomatal conductance and net assimilation rate are positively correlated. On the other hand, the linkage of high Amass with high Nmass which is in large part the result of a direct causal relationship, has been observed at the global scale but this relationship is not significant in Begonia section Gireoudia. I examined B. plebeja and B. conchifolia, two very closely related though ecologically divergent species from Meso-America, in more detail. I detected significant differences between the species for a number of phenotypic variables which may be related to their habitat preferences. This suggested that environmental conditions have driven divergent evolution of phenotypic traits for these two species. Using a mapping population generated from hybrids between these two species I was able to examine the genetic basis of these differences. This revealed that although some traits (such as anthocyanin accumulation) appear to be under simple genetic control, most of the variation between species has complex genetic inheritance patterns. I used QTL analysis to identify significant QTLs for 20 physiological, anatomical and morphological traits which varied between these two species. Leaf shape traits appear to be largely influenced by a few loci of large effect, making these good potential targets for further analysis. The study also identified clusters of coincident QTLs for different correlated traits identifying pleiotropic genes or suites of linked loci.

The Polycomb group (Pc-G) and trithorax group (trx-G) genes play crucial roles in development by regulating expression of homeotic and other genes that control cell fate. Both groups catalyse modifications in chromatin, including histone methylation, leading to epigenetic changes in gene activity. The trx-G antagonises the function of Pc-G genes by activating Pc-G target genes, and consequently trx-G mutants suppress Pc-G mutants. The trx-G genes are relatively poorly characterised in plants. We identified a novel trx-G candidate SUPRESSOR OF POLYCOMB 12 (SOP12) by a genetic screen for suppressors of mutants for the Arabidopsis Pc-G gene CURLY LEAF (CLF). Thus sop12 mutations have no discernible phenotype in wild type backgrounds but partially suppress the leaf curling and early flowering phenotypes of clf mutants. Molecular cloning shows that SOP12 encodes a Harbinger transposase nuclease-like protein which is conserved in green plants, although key residues required for the catalytic activity of the nuclease domain are not conserved. In sop12 clf double mutants, many CLF target genes are down-regulated relative to clf mutant, which suggests SOP12 is a general activator of Pc-G target genes instead of a target of CLF or a late flowering suppressor. The CLF gene encodes an H3K27me3 histone methyltransferase, however chromatin immunoprecipitation (ChIP) analysis indicates that SOP12 does not antagonise Pc-G by removing H3K27me3 methylation, which is consistent with the fact that sop12 suppresses mutants for another Pc-G gene, LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), which is not involved in H3K27me3 deposition. . Rather, genetic analysis shows that sop12 enhances the phenotype of mutants of EARLY FLOWERING IN SHORT DAYS (EFS), a trx-G gene involved in deposition of H3K36me3, and of ULTRAPETALA 1 (ULT1), a plant specific trx-G gene. The enhancement indicates SOP12 may act together with ULT or EFS proteins, or at least regulate the same targets in synergistic ways. For example, SOP12 activates AP3 expression, a role which overlaps with EFS. Yeast two hybrid screening and imunoprecipitation followed by Mass spectrometry were performed to identify numerous potential SOP12 interacting proteins but await further validation. One protein (SUP1) identified through yeast two hybrid screens was independently identified by another group as a Pc-G suppressor, suggesting that SOP12 and SUP1 may act in a common complex to regulate Pc-G targets. Collectively, my data suggests that SOP12 represents a domestic transposase that has acquired a role as a novel, plant specific trx-G members.