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Many of the anabolic effects of growth hormone (GH) are indirect, occurring through GH-stimulated production of insulin-like growth factor-I (IGF-I) by the liver. As well as being GH regulated, plasma IGF-I concentrations have been demonstrated to be dependent upon protein nutrition, with low protein diets being associated with reduced plasma IGF-I concentrations. This effect cannot be reversed by GH, suggesting that liver sensitivity to GH is impaired. To investigate the mechanisms through which protein supply affects GH sensitivity, primary cultures of ovine hepatocytes were grown in defined media. In a first experiment the media contained various fractions (0.2, 1.0, 5.0) of portal vein amino acid concentrations in fed sheep. In the second 24h incubation period, unstimulated IGF-I secretion was highly sensitive the concentration of amino acids in the media, with significantly greater release of basal IGF-I in 5x compared to either 1x (P<0.05) or 0.2x amino acid containing media. In a second series of experiments the effects of specific amino acid depletions was examined. Methionine depletion of 0.2x portal amino acid concentrations ablated the GH response second 24h of culture without affecting basal IGF-I release. By comparison 3H-leucine incorporation into secreted protein, following 20 hours of culture in defined media was significantly reduced in 0.2x aa (P<0.01) and 1.0x aa (P<0.05) media compared with 5.0x aa media, however secretory protein synthesis was unaffected by methionine depletion to 0.2x portal concentrations. The results suggest that amino acid availability regulates both basal and GH stimulated IGF-I release in ovine hepatocytes. Furthermore reducing methionine concentrations in the culture media to 0.2x portal concentrations diminishes GH response without compromising protein secretion.

Different aspects of the mode of action of cytokinin in the moss Physcomitrella patens were studied. In the search for a cytokinin receptor, a particulate fraction was isolated which had cytokinin-binding activity . Cytokinin-binding a c t ivi ty is described as the amount of binding of a radioactive cytokinin that is prevented from binding by excess unlabel led cytokinin. The cytokinin-binding activity was only detected with a tritiated cytokinin of high specific activity. Two assays were used, an equil ib r ium d ia ly s i s and a c ent r i fugat ion assay. These assays were used to show that the cytokinin-binding a c t iv i ty was heatlab i le and can be solubilised by the detergent Triton X100, but not by acetone. Cytokinin-binding a c t iv i ty was higher in phosphate-starved t is sue which consists mainly of caulonemata, the target cells for cytokinin action. A 13 ,0 0 0-8 0 ,000g pel let appeared to contain a component which has some of the characteristic s expected for a cytokinin receptor that was a membrane bound protein. Mutagenised spores were screened for cytokinin nonresponding mutants. No such mutants were obtained from 25,000 plants. A temperature-sensitive mutant, ove 409, was isolated which produced normal leafy shoots at I7°C, but many abnormal buds at 2 4 ° C . T h i s mutant was found to over-produce isopentenyladenine at the higher temperature. The level of cytokinin production in wild type and ove 78 was also found to be temperature-dependent. The change in cytokinin production by ove 409 was over twice that with wild type and ov.e 78, and dropped to wi ld type levels at 17°C. By the use of protoplast fusion, ove 409 was found to be recessive to wild type and in the same complementation group as ove A78. The role of light was studied in the induction of the gametophore. In the presence of supplied cytokinin, bud induction was caused by red light around 613 to 687nm. When the cytokinin-induced bud induction occurs in the dark after exposure to red light , bud induction is reduced by a short exposure to far-red light or a dark period before the addit ion of the cytokinin- This indicates that phytochrome is involved in bud induction. However, another factor appears to be involved in bud induction as this was greater when the cytokinin was applied to the cultures 2 hours after the exposure to red light. The possible role of calcium ions in bud induction was investigated. Calcium ions have been reported to induce the first stages of buds in the moss Funaria hygrometrica. Using the calcium ionophore A23187, calcium ions were shown to induce chloronemal branching in Physcomitre 11a patens. Therefore calcium ions appear to have a role in branching, but not in the differentiation of a bud.

Carotenoid biosynthesis and accumulation in plants are tightly coordinated to maintain cellular homeostasis by regulating several processes, including plastid development, production of phytohormones and signalling molecules associated with metabolic regulation and environmental stress response. The EPSILON LYCOPENE CYCLASE (εLCY) and BETA LYCOPENE CYCLASE (βLCY) enzymes control metabolic flux through the α- and β-branches of the carotenoid pathway and regulate the production and accumulation of downstream carotenoids. Previous research showed that the changes in εLCY gene expression might be associated with an altered abundance of downstream carotenoids such as β-carotene or controlled with an apocarotenoid retrograde signal originated from cis-carotenes. Here we characterise the substrate of the apocarotenoid signal (ACS) and describe a molecular mechanism regulating εLCY expression. Analysis using cis-carotene mutants, ccr2, ziso and ccr2 ziso showed that the εLCY regulatory ACS could not be derived from a cis-carotene intermediate downstream of ZISO metabolic step. The exogenic expression of a bacterial carotenoid isomerase (CrtI) bypassed the metabolic steps between PDS and CRTISO and decreased the cis-carotene content and enhanced the accumulation of β-branch carotenoids as well as εLCY expression in dark-grown ccr2 seedlings. The de-etiolation of seedlings under constant light caused higher accumulation of β-branch carotenoids and enhanced the εLCY expression. Metabolic and gene expression analysis with green leaves at different stages of development demonstrated that carotenoid accumulation, as well as εLCY expression, was higher in leaves at an early stage of development. These findings suggest that feedback control of εLCY expression was required throughout the plant development. Subsequent analysis with norflurazon (NFZ) treated dark-grown wild-type, ccr2 and CrtI expressing transgenic seedlings revealed that cis-carotenes, including phytoene and phytofluene, could not be involved in the regulation of εLCY expression. The εLCY expression was upregulated in β-carotene-rich cotyledon tissues of dark-grown Arabidopsis ccd4 mutants with wild-type and ccr2 background. These pieces of evidence showed that an unknown β-apocarotenoid retrograde signal might control the εLCY expression in order to maintain the balance between α- and β-carotene accumulation. Treatment with D15, an inhibitor of CCD catalytic activity, revealed that CCDs were not involved in generating an εLCY regulatory ACS; however, they might have enhanced the β-carotene substrate accumulation in embryonic tissues to generate β-apocarotenoids. Therefore, we conclude that the β-apocarotenoid signal which regulates εLCY was generated by the oxidative cleavage of β-carotene rather than by enzymatic catalysis. Previous research shows that the disruption of εLCY 5’end by mutations or transposon insertion enhances the β-carotene abundance in crop species. We hypothesized that the εLCY 5’end may harbour a regulatory unit that might interact with the β- apocarotenoid signal to control εLCY expression. In order to test this, we generated Arabidopsis transgenic lines harbouring an εLCY promoter-reporter gene fusion and confirmed that luciferase transcript levels were reduced in etiolated tissues of ccr2 and NFZ treated seedlings, and up-regulated by 30-fold following light exposure. The regulation of reporter gene expression was parallel to the changes in β-carotene abundance in plant tissues. In-silico and in-vitro analysis of the εLCY promoter region revealed three alternative transcription start sites (TSS) separated by a highly conserved 60-70bp AT-rich segments and a putative RNA regulatory switch in the 5’ untranslated leader region (UTR) having two alternative secondary structures resembling an RNA riboswitch. A mutated version of the εLCY 5’UTR having an altered secondary structure reduced CaMV35S promoter-driven luciferase activity in light-grown tissues. In-silico analysis of RNA secondary structures in εLCY 5’UTR showed that a conserved hairpin motif, which is eliminated in the mutated version of the 5’UTR, might be involved in shifting the RNA structure between transcriptionally active and inhibitor form of RNA structures. We propose that a β- apocarotenoid signal (ligand) can move out of the chloroplast and regulate εLCY gene expression through an RNA structural switch (ligand-binding domain) in the 5’UTR in order to fine-tune carotenoid flux through the pathway branch during development and in response to environmental changes.

Gibberellins (GA) are phytohormones involved in regulating plant growth and development. DELLAs negatively regulate GA signaling and are degraded in the presence of bioactive GA. Loss of DELLA function result in increased GA response and mutants are insensitive to GA application. Gain of DELLA function result in decreased GA response. DELLAs are considered master regulators of GA signaling but evidence in tomato suggests a DELLA-independent GA signaling pathway could exists. Tomato has one DELLA protein called PROCERA (PRO) and the putative loss-of-function mutant is responsive to GA treatment. In this thesis, I used the of the genome editing tool called TALEN to induce site-specific insertion/deletion events in PRO. TALENs were successfully employed and seven heritable alleles (proTALEN) with premature stop codons were generated. All homozygous mutants exhibited phenotypes consistent with increased GA response. A new pro loss-of-function mutant (proΔGRAS) was also identified and characterized in addition to proTALEN. Complementation assays demonstrated proTALEN and proΔGRAS are on the same gene. Both mutants are tall and insensitive GA treatment. However, gene expression analysis identified GA2oxidase4 to be up-regulated along with five other genes. These results demonstrate that DELLAs are master regulators of GA signaling with respect to plant growth but some genes may have DELLA-independent regulation. I also describe how DELLA activity may be regulated by the protein SECRET AGENT (SEC). SEC is an O-linked N-acetylglucosamine transferase proposed to positively regulate GA signaling. Early studies of sec-1 and sec-2 found only subtle growth defects. A new sec-3 dwarf mutant was identified but the dwarf plant height was only partially rescued with GA treatment. This suggests SEC may be involved in regulating GA signaling and not necessarily GA levels. Thus, I investigated SEC function in tomato and identified two SEC genes (SlSEC1 and SlSEC2). Preliminary studies suggest SlSEC1 and SlSEC2 have redundant function since homozygous CRISPR/Cas9-induced mutant (slsec1_CRISPR1) exhibit wild-type phenotypes. SlSEC1 and SlSEC2 expression were also repressed using microRNAs and the suppression lines were dwarf, consistent with reduced GA signaling. Taken together, these investigations demonstrate that DELLAs are master regulators of GA signaling and DELLA repressive activity may be fine-tuned by SEC.

Plant hormones are small molecules that regulate various aspects of plant growth and development. Plant hormones are often chemically modified in the cell to affect their storage, degradation, or transport. We had previously discovered that two genes belonging to the Arabidopsis thaliana SABATH methyltransferase (MT) family encode enzymes that catalyze the methylation of the plant hormones salicylic acid (SA) and jasmonic acid. The Arabidopsis genome contains 22 other genes belonging to the SABATH family, whose catalytic functions at the time were unknown. Using a biochemical genomic approach, I, together with co-workers, have identified a SABATH MT that methylates the plant hormone indole-3-acetic acid (IAA), and two other SABATH MTs that methylate another class of plant hormones, the gibberellins (GAs). Biochemical characterizations of these MTs have shown that they are specific for the respective hormone substrate. In addition, mutant plants with ectopic expression of IAA or GA methyltransferases show a disruption in hormonal homeostasis. The discoveries that IAA and GAs could be methylated are novel. However, the in vivo functions of methylated plant hormones such as SA methylester (MeSA) and IAA methylester (MeIAA) had not yet been determined. I have therefore characterized several plant esterases that hydrolyze these methylated plant hormones. The tobacco protein SABP2 had been originally identified as a SA-binding protein involved in plant defense responses, and I demonstrated that it is an esterase that hydrolyzes McSA to SA. Our results suggested that McSA may function as the long-sought mobile signal for the activation of the SA-dependent pathogen-induced defense responses in plants. The genome of Arabidopsis contains 20 genes encoding enzymes homologous to SABP2. I have shown that several of these enzymes can hydrolyze McIAA in vitro, and that at least one such esterase is capable of hydrolyzing McIAA in vivo to the active hormone IAA. My results suggested that McIAA itself does not possess hormonal functions. Overall, my work contributed to the discoveries and the elucidation of the role of methylated plant hormones, and helped to understand the mechanism by which hormone homeostasis is maintained.

The following experiments were done with the aim of developing a technique of finding out whether or not auxin stimulates the uptake of radioactivelly labelled gibberellin into plant cells. Experiments in this area in the past have used whole plants and portions of plant tissue. In this experiment we have attempted to use a relatively homogenous population of cells by the isolation of cells from a leaf. Protoplasts were used instead of cells because this eliminated a possible problem of the radioactivelly labelled hormone being adsorbed onto the cell wall.

It has been suggested that vegetative organs, i.e. the shoot apex, buds, stem tissue, and roots have some measure of control on apical dominance in Phaseolus vulgaris L. by contributing to the balance of hormones available for bud growth. The inhibitory influence, auxin, was produced by the shoot apex, leaves, to a lesser extent small lateral buds, and possibly also by the stem tissue. The shoot produced auxin interacted with cytokinin and gibberellin from the root system in the control mechanism. The root produced hormones were possibly acting to promote lateral growth. In the inhibited stage of lateral growth, the supply of root cytokinin, as studied by the use of ¹⁴C-kinetin was directed primarily to the apex and only small amounts of ¹⁴C -kinetin activity accumulated in the lateral buds. When the apex was removed significantly greater amounts of ¹⁴C -kinetin activity accumulated in the lateral buds. Only when the promotory factor became available to the buds, did significant outgrowth of lateral buds commence. The supply of hormones from their site of production in vegetative organs probably modifies the local hormone balance at the buds and regulates bud outgrowth.

Flowering and the factors controlling flowering have intrigued scientists and laymen alike for centuries. Over the years, developments in scientific research have revealed that flowering is controlled or at least affected by a wide range of environmental and physiological factors. This should have suggested that flower induction and evocation results from a complicated interaction of physiological processes. However many plant physiologists hoped to find a substance common to all plants which caused flowering. Some were less ambitious and thought that each species or genus may have a specific floral hormone. Such ideas have never been supported by more than a few grafting experiments and many photoperiod studies using the presence or absence of flowers as the criterion of success. These methods gave no information of the biochemical processes operating in intact plants. As techniques were refined the search for specific flowering hormones became intensified only to reveal more and more information which did not support the specific hormone theory. However this intense research has yielded very little information on biochemical and physiological processes. “Quite often as compensation.........there is intensive speculation involving a host of unproved assumptions. Speculation and hypothesis are, of course, an essential part of experimental work. But this does not mean that each hypothesis needs to be published” (Lang, 1970). A decade before writing this Lang (1959) discussed the state of knowledge about flowering physiology and today. Little of significance can be added. When reporting their studies of strawberry plant responses to varying environmental conditions Porlingis and Boynton (1961b) emphasised the need for sequential analyses of endogenous GA-like substances in plants under different photoperiod regimes. The following year, Moore and Hough (1962) applied this concept to endogenous auxin actively in strawberries. But since that time there have been few studies which have attempted to relate the activity of endogenous hormones to growth processes. The present study is an attempt to partially fill this gap. The GA-like activity in strawberry plant tissues is determined and related to the process of floral induction in response to SD conditions. As the study progresses the techniques being used were found to be inadequately accurate and sensitive for a study of this nature. This suggested a reason for the slow advance in the state of knowledge about flowering physiology. A review of the literature revealed a lack of standardisation of techniques making it virtually impossible to realistically compare results from different research laboratories. New techniques now coming into use and the standardisation of the old methods should help to provide the important biochemical and physiological facts now so scarce in the field of flowering physiology.