A review of the literature pertaining to the sites of synthesis, distribution and metabolism of cytokinins, established that there was a need to investigate the quantitative and qualitative changes in cytokinin levels during the growth cycle of a whole plant. An annual legume, Lupinus albus L. (the white lupin), was selected as the experimental plant for such a study. The levels of cytokinin activity in the root exudate, leaves and terminal (primary) apices of mature plants were examined between 5 and 17 weeks after planting. The results indicated that the total level of activity in the root exudate of a single plant is much greater than that which accumulates in the leaves and apices up to the time of flowering. This was considered to be indicative of rapid utilization of cytokinins in the actively growing shoot. It was also established that, after flowering, the level of activity in the terminal apices increases markedly, while that in the root exudate decreases. The latter finding was examined more closely in the next experiment, during the course of which cytokinin levels in the root exudate, leaves, fruits and fruit sap were determined at 2 weekly intervals after flowering. As in the previous experiment, the results showed that, although there may be a slight increase in the amount of activity in the root exudate, this activity was insufficiently high to account for the levels of cytokinin which accumulate in the fruiting apices. Cytokinin activity was however detected in the sap passing into the fruits (largely phloem sap), thereby indicating that at least some of the cytokinins detected in both pod wall and seed extracts could have originated in other parts of the plant. Embryonic development was followed at the gross morphological, anatomical and ultrastructural levels, and estimates were made of the cytokinin levels in the embryos and adjacent tissues. The results indicated that the cytokinin levels in the flowers, and young fruits, were low, but gradually increased with time. At 2 weeks after anthesis the level of activity in the pod wall was higher than that in the individual seeds. By 6 weeks, however, the activity in each seed was in excess of that recorded in the pod wall. Decreasing levels of activity in the seeds, and increasing levels in the pod wall, reversed this trend at later developmental stages. The level of activity recorded in the maturing embryo was low, while in the adjacent tissues it was high. This was especially noticeable at 6 weeks after anthesis when the cytokinin activity in the endosperm reached a high level. It would appear that these cytokinins facilitate nutrient mobilization to the tissues surrounding the embryo, and that they are possibly utilized in order to promote certain growth processes taking place in the embryonic cells during development. The cytokinin activity in the mature embryo was low, but increased slightly in the cotyledons, radicles and young shoots during germination and seedling establishment. The cytokinins present in all white lupin extracts had chromatographic properties, on paper and/or Sephadex LH-20, similar to the naturally occurring cytokinins zeatin and zeatin riboside and their glucosylated or phosphorylated derivatives. Mass spectrometric-analysis of the compound co-eluting with zeatin showed that this cytokinin was present in white lupin extracts, while analysis of the peak co-eluting with zeatin riboside indicated that the cytokinin at this elution volume was an adenine derivative. The possible presence of dihydroderivatives of these cytokinins was also indicated. The results did not exclude the possible presence of other cytokinin-like compounds. The significance of qualitative as well as quantitative changes in cytokinin activity were assessed. It is concluded thatcytokinins do not accumulate at excessively high levels in actively growing tissues, unless these tissues function as a transitory store for nutrients, as for example the endosperm, testa and pod wall. Cytokinins are probably involved in the control of a number of growth processes of which nutrient mobilization may be of considerable importance to the plant. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1978.

We studied how the endogenous and exogenous cytokinin and gibberellin functions in floral initiation and development in tuberose. In the aspect of cytokinins. The contents of endogenous cytokinin in tuberose corms (Polianthes tuberosa L. cv. Double) at vegetative, early floral initiation, and flower development stages were investigated. We also determined the influence of exogenous cytokinin treatment on the corm apex at three different growth stages in relation to floral initiation and development in tuberose. The exogenous cytokinin effectively induced floral initiation and development, especially at the early floral initiation and flower development stages. Endogenous cytokinins were higher in early floral initiation and development stages in comparison to the vegetative stage. During floral initiation stage, the zeatin and dihydrozeatin increased significantly, while the cytokinins, zeatin riboside, dihydrozeatin riboside, 6N-(£_2-isopentenyl) adenine, and 6N-(£_2-isope -ntenyl) adenine riboside at consistently low levels. The increase of cytokinin levels in tuberose corms during floral induction suggests a role for cytokinins in tuberose apex evocation. Moreover, these results indicate that cytokinins seem to promote the development of flower buds rather than inducing flowering in tuberose. Endogenous gibberellins (GAs) in tuberose corms were isolated using high performance liquid chromatography, bioassay and identified by combined capillary gas chromatography-mass spectrumetry. Gibber -ellins A19, A20 and A53 were quantified at the vegetative, early floral initiation and flower differentiation stages. The identification of these 13-hydroxylated GAs indicates the presence of the early-13-hydroxy -lation in tuberose corms. An increase in GA20 and decrease in GA19 coincided with the transition from the vegetative phase to the stages of early floral initiation and flower differentiation. GA53 maintained at constant levels at three different growth stages. When GA3, GA4, GA20 and GA32 were applied to corms at vegetative stage (plants about 5 cm in height ), floral initiation was induced and/or promoted by several, most notably by GA3, GA20 and GA32. It is suggest that hydroxylated C-19 GAs play an important role in flower induction in tuberose.

Rhodococcus fascians, a plant pathogenic actinomycete with a very broad host range, causes leafy galls and other malformations. The plant hormone, group, the cytokinins has been implicated in the alteration of host morphology. The aim of this project was to gain insight into the interaction of the cytokinin biosynthetic, isopentenyltransferase (IPT), cytokinin activating ( LOG (The Lonely Guy)) and the cytokinin metabolic, cytokinin oxidase/dehydrogenase (CKX) gene families of both Pisum sativum and R. fascians during infection of the plant. R. fascians colonisation and infection of pea were examined using scanning electron microscopy (SEM) and light microscopy. The expression of genes related to cytokinin biosynthesis, activation and metabolism were isolated and assessed with polymerase chain reaction (PCR) and real time-quantitative PCR (RT-qPCR) analysis. Primers were designed to discriminate between pea genes and R. fascians genes. In addition, the response of the pea cotyledons to R. fascians was measured through chlorophyll estimation and the expression of the transporter genes, sucrose transporter (SUT) and amino acid permease (AAP) which were assayed through RT-qPCR. The pea response regulators were monitored as an indirect measure of the level of endogenous cytokinins in pea. Two R. fascians strains, the avirulent strain 589 and the virulent strain 602, were selected for this project based on their virulence and similar growth rate under identical conditions. The virulence of R. fascians virulent strain 602 was also confirmed through Koch's postulates. The phenotypic alterations in the pea infected with the virulent strain 602 included stunted growth, multiple shoots, small leaves, thickened primary roots and reduced secondary root growth. Delayed senescence of shoots and dark green, intact cotyledons were also observed. Microscopic analyses revealed epiphytic colonisation by both the avirulent strain 589 and the virulent strain 602 in pea cotyledons, roots, shoots and leaves and endophytic colonisation in the seed coat from the time of seed inoculation to 45 days post inoculation (dpi). The expression of R. fascians genes was relatively high at 5 and 9 dpi in pea cotyledons and at 15 and 25 dpi in roots and shoots of pea infected with the virulent strain 602. The expression of RfIPT, RfLOG and RfCKX was not detected both in the control pea and the pea infected with the avirulent strain 589. The cytokinin biosynthesis, metabolism and response regulator (RR) multi-gene families of PsIPTs, PsLOGs, PsCKXs and PsRRs revealed differential and tissue-specific expression patterns. The expression of PsIPTs and PsLOGs was induced immediately after inoculation with the R. fascians virulent strain 602 in the cotyledon but not in roots and shoots, and the expression level reduced at later growth stages. The PsCKXs and PsRRs expression level increased with the growth of the host infected with the virulent strain 602. In pea infected with the avirulent strain 589 the expression of PsIPTs, PsLOGs and PsCKXs gene family members generally increased after 25 dpi in cotyledons, roots and shoots, whereas PsRRs expression was low at all time points. The up-regulation of PsIPTs and PsLOGs immediately after inoculation in cotyledons and at 15 dpi in roots and shoots by R. fascians virulent strain leads to elevated cytokinins which is reflected by the up-regulation of PsRRs. The plant responds to elevated cytokinin by producing phenotypic changes including shoot malformations. The plant activates its cytokinin homeostasis mechanism due to change in cytokinin level which is indicated by up-regulation of PsCKXs. Generally, the expression of the PsRRs was also up-regulated over time following infection by the R. fascians virulent strain. This indicates the presence of biologically active cytokinins in the host which maintain the symptoms. The outcome due to the avirulent strains indicates that, even though PsIPTs and PsLOGs are up-regulated at later growth stages (25 to 35 dpi), expression of PsCKX gene families were varied (either up-regulated or down regulated after 25 dpi). However, PsRRs expression was down-regulated suggesting low cytokinins levels in tissues which may be due to the activation of homeostatic mechanisms of the plant to reduce the level of biologically active cytokinins. The chlorophyll content increased in cotyledons infected with the virulent strain 602 and PsSUTs and PsAAPs expression pattern in pea cotyledon and shoot infected with the virulent strain 602 indicates that R. fascians converts the infected tissue into a sink for their establishment and growth.

No description available.

No description available.

Pak-chung Leung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 151-158). / Abstract --- p.i / Acknowledgments --- p.iii / Table of contents --- p.iv / List of Figures --- p.viii / List of Tables --- p.ix / List of Abbreviations --- p.xiv / Chapter / Chapter 1. --- General Introduction --- p.1 / Chapter 2. --- Literature Review: Greening process in higher plants --- p.4 / Chapter 3. --- Greening Bioassay in Excised Cucumber Cotyledons / Introduction --- p.13 / Materials and Methods / Plant material --- p.14 / Measurement of Ch1 synthesizing activity in excised cotyledons of different ages --- p.14 / Measurement of Ch1 accumulation in excised cotyledons after various pretreatment periods --- p.15 / Measurement of Ch1 accumulation in excised cotyledons under different light intensities and temperatures --- p.15 / Greening curve --- p.16 / Results and Discussion / Effect of seedling age on Ch1 accumulation --- p.16 / Effect of pretreatment period on Ch1 accumulation --- p.18 / Effects of light intensity and temperature on Ch1 accumulation --- p.18 / Effect of shaking (for oxygen replenishment) on Ch1 accumulation --- p.19 / Greening curve --- p.20 / Greening bioassay in excised cucumber cotyledons --- p.20 / Chapter 4. --- Regulation of Chlorophyll Accumulation in Excised Cucumber Cotyledons / Introduction --- p.27 / Materials and Methods / Materials --- p.28 / Seed germination --- p.28 / Harvesting and pretreatment --- p.28 / Illumination and Ch1 determination --- p.29 / Results / Effect of BA on Ch1 accumulation --- p.29 / Effect of Na2EGTA on Ch1 accumulation --- p.30 / "Effect of Na+, Ca2+ and K+ on Ch1 accumulation" --- p.32 / Effect of duration of pretreatment with Na+ ， Ca2+ and K+ on Ch1 accumulation --- p.34 / Effect of verapamil and Ca ionophore A23187 on Chl accumulation --- p.36 / Effect of TFP on Chl accumulation --- p.37 / Discussion / Effect of BA on Chl accumulation --- p.38 / "Effect of Na+, Ca2+ and K+ on Chl accumulation" --- p.39 / Demonstration of the involvement of Ca2+ in Chl accumulation --- p.43 / Chapter 5. --- The Calcium Effect on Benzyladenine-induced Stimulation of Chlorophyll Accumulation in Excised Cucumber Cotyledons / Introduction --- p.63 / Materials and Methods / Materials --- p.64 / Plant growth and treatment --- p.64 / Pretreatment experiment --- p.64 / Sequence experiment --- p.65 / Results / Pretreatment experiment --- p.65 / Sequence experiment --- p.66 / Discussion --- p.68 / Chapter 6. --- Regulation of 5-Aminolevulinic Acid and Chlorophyll Accumulation in Levulinic Acid-treated Cotyledons / Introduction --- p.80 / Materials and Methods / Materials --- p.81 / Plant growth and treatment --- p.81 / Chl determination and ALA assay --- p.82 / Measurement of ALA and Chl accumulation in LA-treated cotyledons --- p.83 / ALA accumulation curve --- p.83 / Measurement of Chl accumulation in 50 mM NaCl-treated cotyledons --- p.84 / "Measurement of ALA and Chl accumulation in BA-, cations-, Na2EGTA-, verapamil- and TFP-pretreated cotyledons" --- p.85 / Results / Concentrations effect of LA on ALA and Chl accumulation --- p.85 / ALA accumulation curve --- p.86 / Effect of 50 mM NaCl on Chl accumulation --- p.87 / Effect of BA on ALA and Chl accumulation in LA- treated cotyledons --- p.90 / "Effects of Na+, Ca2+ and K+ on ALA and Chl accumulation in LA-treated cotyledons" --- p.91 / "Effects of Na2EGTA, verapamil and TFP on ALA and Chl accumulation in LA-treated cotyledons" --- p.92 / Discussion / Measurement of ALA accumulation in LA-treated cotyledons --- p.93 / Effect of BA on ALA and Chl accumulation in LA-treated cotyledons --- p.96 / "Effects of Na+, Ca2+ and K+ on ALA and Chl accumulation in LA-treated cotyledons" --- p.98 / Demonstration of the involvement of Ca2+ in ALA accumulation --- p.99 / Chapter 7. --- Regulation of 5-Aminolevulinic Acid Accumulation in Isolated Developing Chloroplasts / Introduction --- p.123 / Materials and Methods / Materials --- p.124 / Plant materials --- p.124 / Chloroplast isolation --- p.125 / Incubation conditions --- p.126 / ALA assay --- p.127 / Measurement of ALA accumulation in LA-treated isolated developing chloroplasts --- p.128 / "Measurement of ALA accumulation in BA-, cations-, Na2EGTA-, verapamil- and TFP-treated isolated developing chloroplasts" --- p.128 / Results / Time course study of Chl accumulation in intact greening cotyledons of different ages --- p.129 / Measurement of ALA synthesizing activity in isolated developing chloroplasts incubated in the dark and light --- p.130 / Concentrations effect of LA on ALA accumulation in isolated developing chloroplasts --- p.130 / Effects of BA and the cations on ALA accumulation in isolated developing chloroplasts --- p.131 / "Effect of Na2EGTA, verapamil and TFP on ALA accumulation in isolated developing chloroplasts" --- p.132 / Discussion / Light regulation of ALA synthesizing activity in isolated developing chloroplasts --- p.132 / Regulation of ALA accumulation in isolated developing chloroplasts --- p.134 / Chapter 8. --- Conclusion --- p.147 / References --- p.151

Graduation date: 2003

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 119-129). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 119-129). Also available on the Internet.

Tagetes minuta L. is a weedy herb that has been a rich source of fragrant oils, used as in the perfume and flavour industry. T. minuta achenes germinate erratically under field conditions. However, at the optimal germination temperature of 25 °C, 100 % germination is attained within 48 h of imbibition. The achenes are thermoinhibited at 35 °C. The aims of this project were to assess the role of cytokinins (CKs) in normal germination at 25 °C, and to investigate the factors that regulate thermoinhibition at 35 °C. CKs were extracted from achenes germinating at 25 °C at 0, 24; 48; 96 and 144 h after imbibition. Two different purification techniques were used, namely Dowex cation exchange resin followed by paper chromatography, or high performance liquid chromatography (HPLC). CK-like activity was tested with the soybean callus bioassay. With both techniques, a peak in CK-like activity appeared 24 h after imbibition, which coincides with the period during which most of the achenes germinated. For quantitative analysis, HPLC\mass spectrometry (MS) techniques were used. The isoprenoid CKs were far more abundant in T. minuta achenes than the aromatic CKs. cis-Zeatin (cZ) and its derivatives were the most abundant CKs. In total, 19 CK compounds were detected, including 4 free bases and a number of corresponding conjugates. Benzyladenine (BA) was the only aromatic CK detected. There was no common time at which active free base maximal concentrations were detected, suggesting that different CKs may have specific roles in the germination process, and thus peak at different times. This in turn suggests that germination is not a single process, but rather a correlative process involving a number of events, with specific CKs having specific roles relating to these correlative events. There is sufficient evidence obtained from both the soybean callus bioassay and HPLC/MS analysis to suggest that CKs have an active role in T. minuta germination. A decline in free BA during germination without corresponding conjugation, suggests that BA is actively used in early germination processes, possibly in the stimulation of DNA synthesis. Secondly, there was a distinct dihydrozeatin (DHZ) peak obtained at 24 h. Roughly 75 % of the achenes germinate between 16 and 26 h, thus it is likely that DHZ has an active role during the germination of T. minuta. Although CKs are probably not involved in the breaking of dormancy per se, the distinct peak in CK-like activity obtained in the bioassays, 24 h after imbibition, suggests that CKs have an active role in the germination of T. minuta. With respect to the regulation of thermoinhibition, a number of exogenous treatments were applied, including hormones [gibberellins (GA₄₊₇), abscisic acid (ABA), ethylene and a number of CKs], adenosine triphosphate (ATP) and incubation in 100 % oxygen. ABA was extracted from thermoinhibited and germinating achenes to assess the role of ABA in thermoinhibition and germination. While exogenous 0.1 mg L¯¹ GA₄₊₇ application slightly improved normal germination at 25°C, no treatments were effective in alleviating thermoinhbibition in T. minuta achenes. Thermoinhibition in T. minuta achenes may be under hormonal regulation, as there is strong evidence for the role of ABA in the maintenance of dormancy and thermoinhbition. High ABA levels were found in dry control samples. Additionally, exogenous ABA application inhibited normal germination, and the commencement of germination was accompanied by a decrease in endogenous ABA levels. A number of experiments relating to the imposition of thermoinhibition were carried out. Thermoinhibition appears to be very rapidly imposed. Germination is rapidly inhibited following shifting to higher thermoinhibitory temperatures, even after prolonged exposure to optimal germination temperatures. Results suggest active de novo biosynthesis of ABA in thermoinhibited achenes. Active biosynthesis of ABA during thermoinhibition suggests that this phytohormone is essential in the maintenance of thermoinhibition of T. minuta achenes. It thus appears that ABA is synthesized in the achenes in response to elevated temperatures that are unfavourable for germination to proceed. Unfavourable environmental conditions result in an achene-mediated inhibition of germination, which appears to be initiated and maintained by elevated levels of endogenous ABA. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.