Petunia hybrida belongs to the family Solanaceae and is an important horticultural crop grown worldwide. Petunia hybrida and one of its parental species, Petunia inflata, are also established model organisms for the study of gametophytic self-incompatibility (SI). All members of the Solanaceae, including Petunia, share the S ribonuclease mechanism of self-incompatibility. The pistil phenotype is determined by an extracellular stylar ribonuclease or “S-RNase”. The pollen phenotype is determined by several S-linked F-box genes (SLFs). All these genes are tightly linked at the S-locus to form an “S-haplotype”. Most cultivated Solanaceae exhibit self-compatibility (SC) as this greatly facilitates the production of seed for annual crops. SC can arise from the breakdown of a functional SI system during domestication and breeding. In this PhD project, we have attempted to develop approaches to control SI in Petunia. Several stocks of Petunia that exhibit SI are available at the University of Nottingham. These carry five distinct S-haplotypes with corresponding S-RNase sequence data, three in P. inflata (PiS3, PiSk1, PiSd) and two in P. hybrida (PhS3L, PhSv). Our approach to engineer SC into lines with SI involved the introduction of a heterologous SLF gene from Antirrhinum hispanicum. It has previously been shown that transformation of P. hybrida with the AhSLF-S2 gene under the control of a pollen-specific promoter (LAT52) causes SI to breakdown (Qiao et al, 2004b). This is explained as a “competitive interaction”. We have obtained the LAT52: AhSLF S2 construct used by the group of Prof. Yongbiao Xue (Chinese Academy of Sciences, Beijing). In addition, we have obtained three constructs containing other SLF family members (AhSLF-S2C, AhSLF-S4D and AhSLF-S1E) all expressed specifically in pollen (Zhou et al, 2003). The aim of this project is to extend the analysis of these heterologous SLF genes to test whether they offer a general approach to overcome SI in P. hybrida. This involved using the wider range of S haplotype stocks available (5) and the full range of constructs available (4). Initial experiments confirmed the genotype and phenotype of the P. inflata stocks. Crosses were made between P.hybrida and P.inflata and the resulting F1 progeny were tested for SI. It was noticed that some progeny inheriting the PiSd allele of P. inflata have a tendency towards SC, but others have a stable SI phenotype. This observation was further analysed in the F2 generation and based on reciprocal crosses it was determined that the pollen part of the PiSd allele had lost its function resulting in compatibility. Prior to transformation the constructs were checked by sequencing plasmid DNA and colony PCR products using specific primers. The expected fusions of SLF gene and LAT52 promoter were confirmed. An established protocol was used to transform the S3/Sv genotype of P. hybrida. Different numbers of transgenic plants have been identified for each construct (6, 17, 3 and 11 for AhSLF-S2, AhSLF-S2C, AhSLF-S1E and AhSLF-S4D respectively). However, in the T0 generation competitive interaction was not observed. Transgenic plants were crossed with Petunia inflata but F1 hybrid plants remained SI. Transgenic plants obtained for AhSLF-S2 and AhSLF-S1E were analysed in the T1 generation. In spite of the fact that all plants derived from PhSLF-S2 remained SI, one plant derived from AhSLF-S1E became SC. It was predicted that the compatibilty in this particular plant arises as a result of homozygosity. Based on this observation a hypothesis was proposed for a relationship between compatibility and homozygosity and several techniques were used to test this hypothesis. It has been concluded that there is a relationship between homozygosity and transgene behaviour in specific epigenetic situations.

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.

UV-B is a natural component of the sunlight spectrum. As a result of the potentially harmful effects of this radiation, plants have evolved a highly effective suit of protective and repair mechanisms. However, the signalling pathways that control such responses are not yet well known. For example while the photoreceptors responsible for red and blue light responses are well characterised, no such UV-B photoreceptor has yet been identified. Despite this particularly large gap in our knowledge, previous work identified the first UV- B specific signalling component which, unlike the more general stress-associated pathways often seen at high doses, specifically regulates expression of genes in response to even very low fluence rates of UV-B. This protein, UV-RESISTANCE LOCUS 8 (UVR8) regulates the induction of a number of photoprotective genes mostly via the transcription factors ELONGATED HYPOCOTYL 5 (HY5) and HY5 HOMOLOGUE (HYH). The end result of this pathway is the production of photoprotective compounds such as the flavonoids which enhance a plants ability to withstand UV-B stress. Thus UVR8 promotes plant fitness under these conditions. While we know that UVR8 binds to chromatin in the promoter region of HY5 and that it accumulates in the nucleus under UV-B, many other questions about this particular protein remain unanswered. For example, we do not yet know if UVR8-mediated UV-B signalling involves other factors which interact with UVR8 nor do we understand the mechanism by which UVR8 localisation is mediated. In addition, although we are aware of the importance of UVR8 in UV-B acclimation, it is unclear what roles might be played by other genes and proteins acting independently of this pathway. Therefore, the aims of this study were to investigate low fluence UV-B pathways that may act independently of UVR8 and to further examine the UVR8 protein itself both in terms of its interactions with other proteins and also in the role of the N-terminal region in regulation of its localisation. To achieve the first of these aims, RNA samples derived from plants treated with low fluence UV-B were submitted for microarray analysis. It was initially determined that the total number of genes induced was roughly equal in both low fluence treated samples and also to that found in the previous microarray performed by Brown et al. (2005) at a comparatively higher fluence. Thus, as only 72 genes have currently been linked to UVR8, there do appear to be many low-fluence UV-B induced pathways besides that regulated by UVR8. Several genes were analysed further using RT-PCR and qPCR methods in order to confirm their independence from the UVR8 signalling pathway components as well as assess their dependence on other hypothesised UV-B sensory mechanisms. It was found that while some genes did seem to be expressed independently of known photoreceptors, DNA damage signals as well as UVR8, HY5, HYH and COP1; one gene was expressed in a COP1-dependent but UVR8 independent manner. It therefore appears that at least four classes of genes are induced by UV-B; low fluence UVR8/HY5/HYH independent COP1 dependent, low fluence UVR8/HY5/HYH/COP1 dependent, low fluence UVR8/HY5/HYH/COP1 independent and finally high fluence non- specific signalling. The second portion of this thesis examined the structure and function of UVR8 in greater detail. To assist in this analysis, the BLAST sequence homology tool was used to probe both the Arabidopsis genome and available green plant sequences. It was found that 23 UVR8-like sequences exist in Arabidopsis but none of these appear to have similar N or C-terminal sequences to UVR8. As these two regions have previously been shown to be of vital importance in UVR8 function (Kaiserli and Jenkins, 2008; Kaiserli unpublished data) it is unlikely that any are acting in a redundant fashion to UVR8. A number of similar proteins to UVR8 can be found in other plant species. These potential homologues however fall into two categories based on their closer similarity with either UVR8 or its close homologue in humans REGULATOR OF CHROMATIN CONDENSATION 1 (RCC1). The wide variety of plant species that did show UVR8-like proteins suggests that this particular means of UV-B acclimation may have arisen relatively early with the colonisation of land plants. Interestingly, many of these likely homologues had a conserved N terminal. The N-terminal of UVR8 has previously been show to have a role in UV-B dependent nuclear accumulation (Kaiserli and Jenkins, 2008). This was examined further in Chapter 4 through the generation of a number of deletion and addition constructs in both a stable Arabidopsis uvr8-1 background as well as transiently in tobacco. From analysis of localisation of these constructs via confocal microscopy it was determined that the first 12 amino acids are sufficient but not necessary for nuclear accumulation, while the first 20 appear to be both necessary and sufficient. Indeed, it was shown that the initial 32 amino acids also confer constitutive localisation of a GFP tag in the nucleus regardless of light condition and despite the presence of a nuclear exclusion signal (NES). It therefore appears that this region, which shows strong conservation with UVR8-like proteins in other plant species, is of vital importance to the nuclear accumulation seen under UV-B. Finally, in Chapter 5, the possibility that UVR8 may be acting as part of a complex was explored. This involved use of size exclusion chromatography to provide approximate sizes of the UVR8 protein complex. It was found that native UVR8 appears to exist in a complex of about 70-90 kDa in size. This suggests that at least one other protein interacts stably with UVR8. Other fusion constructs were also analysed in this way, however the results were more difficult to interpret due the apparent artificial dimerisation of the GFP tag. In summary, the work presented here has shown that although UVR8 dependent pathways are predominant, a variety of low fluence UV-B induced genes and pathways may exist. Homology searches and mutational analyses suggest that the N-terminal region of UVR8 plays a critical role in its function and localisation. Finally, size exclusion chromatography suggests that UVR8 forms a complex in vivo with as yet uncharacterised partner proteins. In total these results provide further insight into the mechanisms UVR8 action and highlight new avenues for both UVR8 dependent and independent UV-B signalling.

Rhododendrons are economically important plants in horticulture, and many species are threatened in the wild by habitat degradation. It is therefore doubly important that their nutritional needs should be understood.

Garcinia section Garcinia is one of 14 sections of the species-rich pantropical genus Garcinia (Guttiferae/Clusiaceae). In its most recent circumscription the section comprised 43 species, mostly of rain forest understorey trees, distributed from eastern India to Fiji, and in Madagascar (Jones, 1980: unpublished Phd. Thesis, University of Leicester). Its most famous member is the fruit tree, mangosteen (G. mangostana). Taxonomic revision of section Garcinia reduces the number of species from 43 to 16 with five varieties, with distribution from eastern India to Malesia. A total of nine species are excluded, 19 species are reduced to synonyms and five species are insufficiently known to be classified. Within sect. Garcinia, G. acuticosta, G. discoidea, G. exiguous, G. ochraceus and G. sangudsangud are newly described, G. diospyrifolia var. arborea, G. diospyrifolia var. minor and G. malaccensis var. pseudomangostana are new varieties, and G. cataractalis, previously unassigned to any section by Jones (1980) is newly included. Species limits are defined on the basis of combinations of characters such as shape of stamens and presence of pistillode, fruit type, and leaf characters such as shape, size, venation pattern and type of glandular lines. A total of 83 accessions representing 42 Garcinia species were sequenced for the nuclear ribosomal internal transcribed spacer (ITS) region, while 50 accessions (30 spp.) and 24 accessions (16 spp.) were sequenced for the chloroplast non-coding regions trnS-G and trnD-T respectively. Of these, 30 accessions or ten spp. (ITS), 23 accessions or nine spp. (trnS-G) and 17 accessions or nine spp. (trnD-T) belong to sect. Garcinia. Parsimony and Bayesian analyses show that the ITS data are more phylogenetically informative and provide better resolution for sectional and species relationships compared to trnS-G and trnD-T. Most of the sections delimited by Jones (1980) proved to be monophyletic but sect. Garcinia is not. Garcinia maingayi, G. trianii and G. costata, assigned to sect. Garcinia by Jones, are in a separate clade that is strongly supported as sister to sect. Brindonia. These species have significantly different morphological characters from sect. Garcinia, and should be excluded from it. Characters that are important for sectional delimitation are inflorescences that are simple cymes, stamen bundles that are 4-angled or 4-lobed, and fruits with a smooth surface. Four major clades in sect. Garcinia are supported by the shape of the stamen bundles, the shape of the fruit and the stigma, and fruit wall characters. ITS trees are significantly incongruent with plastid trees because of the placement of G. rigida, which could reflect a hybrid origin. Another hybrid species, the cultivated mangosteen, could be the product of hybridisation events between varieties of G. malaccensis if mangosteen is proven to be an obligate agamosperm. However, if mangosteen is a facultative agamosperm, G. malaccensis is likely to be the female parent, but any other Garcinia species could be the putative paternal species. Phylogenetic trees of ITS show that most Garcinia species from east of Wallace’s Line are nested within species from the west. This might reflect dispersal of species across this biogeographic division from west to east when the Sahul and Sunda shelves converged (c. 20 MY).This hypothesis is supported by the estimated divergence of accessions of G. rigida from the east of Wallace’s Line, from their most recent common ancestor to the west of the line, not later than 21.58+/-2.90 MY.

No description available.

A study has been made, by light and electron microscopy, of the structure of the phloem tissue and sieve tubes of Sallx oaprsea. and the relevance of the findings to the electrokinetic theory of transport has been discussed. The anatomical features of the phloem at the cellular level do not seem particularly favourable to the theory; on the other hand, the ultra structure of the sieve tubes and in particular the prevalence of "slime" in this species and many other species being composed of banded fibrils, seems to favour the electro kinetic theory at least as much, as any other. A supporting calculation is given.

The distribution of 45Ca, 42K and 32P was studied in the hypocotyls of Helianthus annuus and also in the coleoptiles of Zea mays in relation to geotropic stimulation and response. The concentration of calcium was higher in the upper side and the concentration of potassium and phosphorus was higher in the lower side of the hypocotyls placed in horizontal position. This differential distribution was correlated with the curvature. The redistribution of ions is not connected to geotropic perception since 0.5 hour horizontal orientation at 25 C and subsequent exposure at 4 C did not result in any redistribution. No differential distribution was observed in the hypocotyls placed in horizontal position when curvature was stopped by pre-treatment with NPA. After unilateral IAA application (0.1% W/W in lanolin) the concentration of calcium was higher in the concave side whereas the concentration of potassium and phosphorus was higher in the convex side. IAA probably affects the ion pumps via differential growth which results in redistribution of ions. In unilaterally illuminated coleoptiles illuminated (concave) side showed higher concentration of calcium, whereas shaded (convex) side showed higher concentration of potassium and phosphorus. NPA inhibited phototropic curvature as a result no differential distribution of ion was observed. Unilateral application of mersalyl (10-4M in hydrated lanolin) resulted some movement of calcium away from the donor side and movement of potassium into the donor side. Mersalyl affects the distribution of ions by altering ion pumps and its mechanism of ion pump operation must be different from that of IAA as mersalyl does not produce a differential growth response and the IAA effect is dependent on differential growth. Hence, the results show a correlation between differential distribution of ions and curvature. It is concluded, therefore, that differential distribution is probably caused by curvature.

Recent trials of Antirrhinum majus cultivars have revealed a wide range of susceptibility to rust (Puccinia antirrhini). The best plants were used as parents in a breeding programme to produce a useful level of durable field resistance. The F1 generation was self-pollinated but the F2 to F4 were selectively cross-pollinated within lines. The segregating generations were grown outdoors at two sites in Surrey, where they were subject to natural rust epidemics encouraged by the use of spreader rows. Individual plants were selected for rust-resistance and horticultural quality. By the F4 generation, lines showed greater resistance to rust than existing varieties and were becoming uniform in flower colour, growth habit and horticultural quality. Many floral abnormalities arose, especially in the F3. Their expression was usually highly variable and was enhanced by environmental stress. Tests of rust isolates against individual antirrhinum plants showed that there is considerable genetic variation within the rust population. Some monitoring of resistant varieties is desirable to identify future changes in the rust population. The spread of spores between widely spaced patches of susceptible host was simulated using a computer, which indicated that small, isolated patches may escape infection. The effect of rate-reducing resistance is increased when plants are widely separated. This is in agreement with practical experience. The urediniospores of Puccinia antirrhini cannot survive the winter in Britain and, though teliospores are regularly produced, no alternate host is known. However, overwintered antirrhinum plants produce viable urediniospores which start epidemics in early summer: there is no need to postulate an alternate host in the British Isles. Treating antirrhinum as a summer annual might be an important hygienic measure, reducing the local build up of inoculum early in the season. Acceptable control of rust should be possible, provided only the more resistant varieties are grown.

Thirty species from the family Caryophyllaceae were found to contain inhibitors of local lesion production by TNV. The crude extracts of 20 species totally eliminated lesion production. Dilution experiments revealed the presence of at least two materials in the extracts of some species, an inhibitor which decreased and an augmenter which increases the number of lesions. Heat treatment decreases inhibitory activity in some species and not in other. Two species were studied in detail, Gypsophila paniculata which showed powerful inhibitory activity was partially thermostable and Minuratia capillacea which showed weak thermolabile inhibition. The constituents of Gp extracts acted as virus inhibitors rather than inactivators, survived at room temperature for 60 days, and were stable to a wide range of pH. Dialysis experiments confirmed that the inhibitory activity resided in both high and low molecular weight compounds. Ethanol or ammonium sulphate precipitation, and disc electrophoresis suggested that the inhibitors are composed in part, of protein and glycoprotein. Furthermore, inhibitory activity could not be attributed to RNA, but remained unchanged after incubation with each of three proteolytic enzymes, Sephadex G-100 gel filtration showed that Gp extract inhibitors to have a molecular weights of about 12,600 - 27,500 and 2,000 for the materials which eluted slowly. Ion exchange column chromatography on CM-52 cellulose showed that extracts contained at least three virus inhibitor fractions, one neutral and two basic in nature. The low molecular weight fraction, dialysable part, contained 17 free amino acids most of which had inhibitory activity against TNV, and 3 free sugars all of which stimulated local lesion production. Aqueous extracts prepared from G.paniculata and M.capillacea each inhibit local lesion production by TNV, TMV and PVX viruses. The G.paniculata inhibitors seem to have direct effects on TNV and some was precipitated on centrifugation. Minuratia inhibitors are not precipitated by centrifugation. Inhibition in both extracts influences host plants and acts either by altering the susceptibility of the cells to virus attachment, or perhaps allow penetration and prevent early events of virus replication. The crude extracts from Gp induced local and systemic resistance in untreated parts of the plant. Systemic resistance was induced on French bean leaves when Gp extracts were applied to their roots. The sample taken from resistant leaves contained an inhibitor of virus infection and showed 3 additional protein bands with molecular weights of 105, 94 and 40 x10-3daltons on SDS polyacrylamide gel electrophoresis. Gp = Gypsonhila paniculata TMV = tobacco mosaic virusTNV = tobacco necrosis virus PVX ss potato virus X.