Euclea natalensis A.DC. belongs to the Ebenaceae family, and is extensively distributed along the eastern coast of southern Africa. Many Euclea species are widely gathered by indigenous people because of their medicinal properties. Roots of these plant species are frequently used to treat respiratory complications such as chest pains, bronchitis, pleurisy and asthma. Ground root powder is topically applied in cases of leprosy and is used by some ethnic groups to treat toothache and headache. The bioactivity encountered is attributable to naphthoquinones, which are common phenolic compounds in the Ebenaceae family. Naphthoquinones isolated from E. natalensis (shinanolone, 7-methyljuglone, diospyrin, isodiospyrin and neodiospyrin) have exhibited a broad spectrum of antimicrobial activity. The demand for these products will escalate due the amount of plant material required to further research. We need to explore techniques that can maximize their productivity. The present study was conducted on E. natalensis, in an attempt to establish if there exists any correlation between the accumulation of naphthoquinones and stages of seedling growth, seasonal fluctuations and application of fertilizers. A possible correlation between seedling growth stages and the accumulation of naphthoquinones (shinanolone, 7-methyljuglone and diospyrin) was investigated in seeds and seedlings of Euclea natalensis. In this study, the seeds represented the first stage, whereas the second seedling stage was defined as the stage when the radicles were about 6 cm long. The lengths of the seedlings at the third, fourth and fifth seedling stages were 9 cm, 12 cm and 16 cm respectively. Plant materials collected from the five seedling stages were separately extracted using chloroform and the naphthoquinones were then quantified by means of High Performance Liquid Chromatography (HPLC). Mobile phase of MeCN: H2O: AcOH (62.5: 32.5: 5) was used as an eluent in an isocratic mode and at a flow rate of 0.8 ml/min. Standard curves of each of the four compounds were obtained by making a series of dilutions in the concentration range of 22.5 µg/ml to 2.25 µg/ml. Ten microlitres of each dilution was injected three times into the HPLC, and the run time for each injection was 20 minutes. Calibration curves were then generated and used for the quantification of each compound. Shinanolone, which was the only naphthoquinone detectible in seeds, accumulated at variable rates (P<0.01) and no trend could be established between its synthesis and seedling growth. The content of shinanolone ranged from 87.5 mg/kg dry weight (dw) in seeds to a high mean value of 1047 mg/kg (dw) during the fourth seedling stage. A significant correlation (P<0.01) was found between the mean concentrations of 7-methyljuglone and seedling growth. 7-Methyljuglone was quantified at a high mean level of 5003 mg/kg during the third seedling stage and was not detected in the seed samples. A positive correlation (P<0.01) was established between the concentration of diospyrin and seedling stages. Diospyrin was detected at an elevated mean concentration of 6182 mg/kg during the fifth seedling stage, which was higher than the other quantified naphthoquinones. Seasonal variation of naphthoquinones (shinanolone, 7-methyljuglone, diospyrin, isodiospyrin and neodiospyrin) was investigated from eleven plants of E. natalensis subsp. natalensis growing in natural populations, over a period of four seasons. The roots were harvested, dried, extracted and analysed as in the previous study. The mean levels of shinanolone and 7-methyljuglone were found to be uniform in all the seasons and no statistically significant variation could be found between seasonal changes and their mean concentrations. Accumulation of isodiospyrin and neodiospyrin varied significantly with seasonal changes (P<0.05). These two bioactive naphthoquinones were detected only in summer and autumn respectively, and not in winter. A statistically significant variation (P<0.05) was established between the levels of diospyrin and seasonal fluctuations. Diospyrin was detected at a mean concentration of 3190 mg/kg (dw) during spring, which was higher than the other naphthoquinones quantified in all four seasons. The effect of NPK fertilizers on growth performance and accumulation of naphthoquinones (shinanolone, 7-methyljuglone, diospyrin, isodiospyrin and neodiospyrin) in seedlings of E. natalensis grown in shade and under field conditions was investigated. Each group was subdivided into four subgroups, which were then subjected to four respective treatments of water-soluble foliar feed (2:1:2 (44) NPK) at three different concentrations. Treatments tested were as follows: Treatment 1 at 40 g/l, Treatment 2 and Treatment 3 at 20 g/l and 10 g/l respectively. The control group received only supplemental water. The first harvest was conducted after 6 months of application of fertilizers and the second one was done after 12 months of treatment. Roots and shoots were harvested and analysed separately. The naphthoquinones were quantified as previously described. The bioactivity of root extracts from seedlings was tested against Mycobacterium smegmatis and extracts with lower MIC were further tested on M. tuberculosis. Growth parameters differed between the two groups, with seedlings from the shadehouse showing more plant vigour than the field grown plants. No significant interaction could be established between the measured growth factors and treatment. A significant interaction (P<0.001) was found between Treatment 2 and shadehouse seedlings. Treatment 2 enhanced vegetative performance with the mean values of fresh weight of shoots and roots being twice as much as their respective control mean values. A significantly positive correlation was established between the concentration of shinanolone (P<0.01), isodiospyrin (P<0.05) and neodiospyrin (P<0.05) with fertilization from field-grown seedlings. Application of NPK fertilizers significantly (P<0.05) increased the accumulation of neodiospyrin in seedlings subjected to shadehouse conditions. The most potent naphthoquinone, 7-methyljuglone, was found to be abundant in all the extracts and was quantified at a high mean concentration of 10200 mg/kg from shadehouse seedlings. Root extracts of E. natalensis seedlings grown under field conditions were generally more active against the bacterial strain of M. smegmatis as compared to extracts acquired from roots of seedlings maintained under a shadehouse setting. A lowest minimum inhibitory concentration (MIC) of 0.78mg/ml against M. smegmatis was observed from the second harvest of field-cultivated seedlings of the control and Treatment 1 subgroups. The MIC values for shadehouse seedlings ranged from 1.6 to 6.3 mg/ml. Minimum bactericidal concentration (MBC) values from all the extracts tested were relatively higher than their respective MIC’s. Root extracts of E. natalensis were more active against M. tuberculosis and their MIC values were lower than the tested concentrations. Extracts acquired from field-grown seedlings were more active against M. smegmatis with a lowest MIC value of 0.78 mg/ml. Extracts from the control group and Treatment 1, which had less application of fertilizers were more active against strains of M. tuberculosis with MIC value of 10 µg/ml. This shows the selectivity of E. natalensis against the mycobacterial strain of M. tuberculosis. Based on the findings, synthesis and accumulation of naphthoquinones in E. natalensis is highly variable within individuals of the species investigated. Naphthoquinones accumulate in relatively higher amounts in roots of E. natalensis than in the aboveground structures, which validate their harvest by indigenous people. The concentration of shinanolone varied slightly and its production increased with seedling growth. The synthesis of 7-methyljuglone is independent of fertilisation as its accumulation was enhanced in seedlings subjected to control treatment. Neodiospyrin and isodiospyrin were always present in every sample obtained from the seedlings but they were not detectible in every profile of samples from mature plants. Diospyrin is the only naphthoquinone that was detected in every sample analysed and also quantified in high concentrations from mature plants harvested in spring. The study showed that depending on the requirement of a particular naphthoquinone for research, one could target the seasons and seedling stages recommended from this study. This study also showed that field-cultivated seedlings produced more potent naphthoquinones than the ones subjected to controlled environments. / Dissertation (MSc (Plant Science))--University of Pretoria, 2008. / Plant Production and Soil Science / unrestricted

Made available in DSpace on 2014-08-20T13:44:34Z (GMT). No. of bitstreams: 1 tese_caroline_borges_bevilacqua.pdf: 1981019 bytes, checksum: 797e667e01edbc569a7b08d3268c701b (MD5) Previous issue date: 2013-10-30 / Cold stress adversely modifies their physiology, metabolism plant growth and development, as well as, it limits crop productivity. The responses of rice (Oryza sativa L.) subjected to low temperatures are still poorly understood. A better understanding of stress tolerance mechanism in rice plants will help to develop rice germplasm with improved field level tolerance under variable temperature and sowing depth conditions. To characterize rice genotypes with variation in sensitivity to cold, these are the following objectives: to evaluate the applicability of different Stress Indices using seedling lengthas parameter; classify accessions cultivated rice and red rice as Indica or Japonica; compare response to rice cultivars cold-tolerant and cold-sensitive to cold stress according to the dry matter accumulation and possible changes in chlorophyll content; categorize different genotypes with regard to sensitivity to cold and to sowing depth stresses and, analyze the expression of cold-responsive genes, and also genes submergence-responsive. The seeds after seven days at 25°C were exposed at 4°C for 24h and after that, photosynthesis was measured later, the plants were 72h at 25°C (recovery period) to assess the dry mass and chlorophyll. For the other experiments, the seedlings were collected 7 and/or 14 days maintained at 25°C or 18/13°C day/night and different sowing depths (1.5cm, 5cm, 10cm and 15cm), differential gene expression were performed with those seedlings using different genes induced by cold. To evaluated gene expression using different genes induced by cold and anoxia, samples were collected after exposure to 10 ° C for 6, 24 and 96 h at 1.5 cm and 10 cm deep sowing. The results showed that is possible to identify superior genotypes for tolerance to these abiotic stresses based on the Tolerance Index (STI) and Media Geometric (GM) to select genotypes tolerant to cold or sowing depth, using as a parameter the seedling shoot length measurement. Japonica and Indica subspecies respond differently to abiotic stresses, however for some of these stress-responsive genes, these subspecies responded similarly. Furthermore, the analysis at the molecular level of cold tolerance and sowing depth indicated the importance of ABA- dependent and ABA-independent signal transduction pathways in plants under abiotic stress. / O estresse causado pelo frio interfere negativamente na fisiologia, metabolismo, crescimento e desenvolvimento das plantas e, portanto, limita a produtividade em lavouras de arroz. As respostas em nível de crescimento em arroz (Oryza sativa L.) submetido a baixas temperaturas ainda são pouco compreendidas. Um melhor entendimento do mecanismo de tolerância ao estresse em plantas de arroz pode ajudar na identificação, no germoplasma de arroz, de plantas com tolerância submetidas à temperatura variável, além de ser útil para outros estresses abióticos, como diferentes profundidades de semeadura. Para caracterizar genótipos de arroz, com variação na sensibilidade ao frio, tiveram-se como objetivos:avaliar a aplicabilidade de diferentes índices de estresse utilizando-se como parâmetro o comprimento de plântula; classificar acessos de arroz cultivado e vermelho como Japonica ou Indica; comparar a resposta ao frio de cultivares de arroz tolerante e sensível a esse estresse, com relação ao acúmulo de massa seca e possíveis alterações no teor de clorofila;categorizá-los com relação à sensibilidade ao frio e à profundidade de semeadura; e analisar a expressão de genes que respondem a frio, assim como genes responsivos a submersão, sob condições de frio e/ou tratamento constituídos por diferentes profundidades de semeadura. Para avaliar o acúmulo de massa seca e o teor de clorofila, as sementes, após sete dias a 25°C, foram expostas a 4°C durante 24 h e logo após, foi medida a fotossíntese e,posteriormente, as plantas ficaram 72 h a 25°C para sua recuperação. Já para os demais experimentos,as plântulas foram coletadas 7 e/ou 14 dias mantidas a 25°C ou 18/13°C dia/noite e diferentes profundidades de semeadura (1.5cm, 5cm, 10cm e 15cm); as avaliações da expressão gênica diferencial foram realizadas com essas amostras coletadas, para 4 diferentes genes induzidos pelo frio e também em amostras coletadas após exposição a 10°C durante 6, 24 e 96 h a 1.5 cm e 10 cm de profundidade de semeadura.Os resultados indicaram que é possível a identificação de genótipos superiores para a tolerância a esses estresses abióticos com base em seus índices de estresse, utilizando como parâmetro o comprimento da parte aérea, devido a habilidade das plantas tolerar estresses abióticos afetar a morfologia assim como a fisiologia da planta de arroz. Assim como é possível a utilização do Índice de Tolerância (STI) e da Média Geométrica (GM) para selecionar genótipos tolerantes ao frio ou profundidade de semeadura, baseado no comprimento de parte aérea de plântula. As subespécies Japonica e Indica respondem diferentemente aos estresses abióticos, no entanto, para alguns genes responsivos a esses estresses, essas subespécies apresentam o mesmo respondem semelhantemente. Além disso, as análises a nível molecular da tolerância ao frio e a profundidade de semeadura indicaram a importância das vias ABA-dependente e ABA-independente como vias de transdução do sinal em plantas sob estresse abiótico.