Browsing by Author "Mtshali, Ntombizamatshali Prudence."

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Molecular characterization of acid phosphatase in the lichen Cladonia portentosa.
(2011) Mtshali, Ntombizamatshali Prudence.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
Acid phosphatases (apase) are important hydrolytic enzymes that function in the acquisition, production; transport and recycling of inorganic phosphate (Pi), thus making a significant contribution towards nutrients dynamic of many ecological niches. The aim of this study was to characterize the apase enzyme found in the lichen Cladonia portentosa at the molecular level. The initial experiment entailed cloning the apase gene by PCR using degenerate primers designed from close relatives of C. portentosa from the Ascomycete family. The isolation of apase gene from Cladonia portentosa using PCR was not successful. Attempts were then made to purify the secreted apase and to determine its biochemical and molecular properties and to allow comparison with already characterized secreted phosphatases from other fungal sources existing in the NCBI database. It was anticipated that the partial sequence of the purified enzymes would provide a corresponding apase gene. The acid phosphatase enzyme was partial purified to 45 fold by a gel filtration with a yield of 18%. It gave a single, broad glycoprotein band on native PAGE and SDS-PAGE corresponding in size to 250 and 148 kDa, respectively. Under reducing conditions, the purified enzyme migrated as two bands of 116 and 32 kDa, indicating the heterodimer nature of this enzyme. Only one distinct band, (pI 6.4) was observed after electrofocusing. The optimum temperature for the enzyme was 65 °C where an optimal pH was detected at 2.5. The enzyme was inhibited by known acid phosphatase inhibitors (fluoride, molybdate, orthovanadate and tartrate) and the metals (Cu²⁺ and Zn²⁺). The purified enzyme demonstrated broad substrates selectivity and had a KM of 31.2±0.25 μM for phytic acid. Peptide analysis by Mass Spectrometry (MS) MALDI-TOF indicated the presence of two apase proteins. The amino sequences of purified apase/s from Cladonia portentosa were FLAETNPAPFGH, AVGLGYVEELLAR and AQGLGYVQEVLAR. Comparing the amino acids of the sequenced protein with that of already known proteins confirmed the enzyme to be a secreted histidine acid phosphatase, resembling other acid phosphatases and phytase from several filamentous fungi with respect to amino acid composition. To investigate the effect of phosphorus on C. portentosa apase, the mycelium was grown under different concentrations of Pi [0.05, 1.0, 3.0, 10 and 100 mM (KH₂PO₄)]. The aim was to localize the apase enzyme and to screen for the occurrence of the gene coding for the acid phosphatase enzyme. A treatment of 3.0 mM Pi induced high levels of apase compared to all other treatments. In addition, cultures of C. portentosa were grown in axenic cultures to study the effect of pH and Pi versus menadione on the production of acid phosphatase and mycelia growth. A culture media of pH 4.8 and 6.0 resulted in higher apase secretion than when compared with pH 2.5 medium. The presence of 2.0 μM menadione marginally increased levels of the apase compared to the control treatment. Apase was further localized cytochemically using fluorescent substrate-enzyme-labelled fluorescence (ELF-97) which forms a fluorescent crystalline precipitate at the site of phosphate activity. Fluorescent microscope revealed that the enzyme was present in all treatments, irrespective of Pi concentration, however, the fluorescence signals were intense in low Pi concentrations (0.05 and 1.0 and 3.0 mM Pi). Ultrastructure localization using live mycelium under confocal microscopy using Vector blue III substrate revealed that the enzyme was localized in the cytoplasm, cell membrane, vacuole and small organelles, presumed to be endosomes. Co-staining with FM4-64, confirmed the punctuate structure to be secretory vesicles or a vacuolar network. To investigate the effect of P starvation on C. portentosa at a molecular level, the effect of Pi on the gene expression profile was examined. The generation of a cDNA library from axenic grown mycelium treated with P provided a foundation for the identification and characterization of genes expressed in the P treated mycelium through expressed sequence tags (ESTs). Several genes were identified whose transcriptional profiles have been significantly changed by phosphorus treatment and menadione. They include genes required for signal transduction and vesicular transport, cell biosynthesis and protein metabolism and stress response. In conclusion, this study constitutes the first step towards understanding the molecular mechanism governing acid phosphatase in C. portentosa.
Role of light and temperature in the flowering of Watsonia species.
(2006) Mtshali, Ntombizamatshali Prudence.; Van Staden, Johannes.; Thompson, David Ian.; Erwin, J.
The role of light and temperature on flowering of South African Watsonia species were evaluated to assess the potential for this genus as a commercial flower crop. Species were selected that represent different climatic regions of South Africa, with the aim of understanding how ecologically distinct species perform under cultivation. The four selected species were W. borbonica and W. tabularis (winter-rainfall area), W. angusta (shared rainfall) and W. pillansii (summer-rainfall area). In order to establish the optimum temperature required for flowering, plants were exposed for 12 weeks to three temperature regimes (12/7 °C, 21/15 °C and 29/21 °C) after attaining their first and/or second leaves. A temperature shift of 12/7 °C was used to assess if the plants had a vemalisation requirement. Controls were maintained under 25 % shade under natural conditions, with an average temperature of 24/7 °C. An elevated temperature of 29/21 °C was detrimental to plant growth. Moderate temperatures of 21/15 °C significantly (P<0.001) increased the height and the number of leaves produced per plant relative to the 12/7 °C treatment. These temperatures significantly (P<0.001) increased the total number of flowers produced per plant compared to low temperatures. However, flowering percentage and quality of flowers were reduced. A low temperature regime of 12/7 °C was efficient in satisfying vernalisation requirements and inducing flowering in four selected species. However, the total number of leaves produced per plant was signifcantly reduced. The summer-rainfall species, W pillansii, displayed a qualitative response to vernalisation, as no flowering was observed in non-vernalised plants. Two winter-rainfall species, W borbonica and W. tabularis, demonstrated a quantitative response to vernalisation. These species flowered at non-vernalising temperatures. W angusta behaved like the winter-rainfall species in terms of flowering. Overall, a vernalisaton treatment marginally reduced days to flower while flowering percentage was increased compared to other temperature regimes. However, there was no increase in the total number of flowers produced per plant. Low temperatures were not only effective for flower induction, but also for releasing corm dormancy, thus synchronising growth. Storing corms at either 4 or 10 QC resulted in 100 % sprouting within 4-6 weeks. The role of daylength in flowering of Watsonia plants was established by subjecting plants to long days (LO) of 16 h light and 8 h dark and to short days (SO) of 8 h light and 16 h dark. The number of leaves and flowering were significantly (P<0.01) promoted under the LO regime. However, there was strong temperature and daylength interaction in terms of flowering potential, as at low temperatures flowering was induced irrespective of daylength. In W. pillansii, flowering was obtained under both regimes (LO and SO) applied at the second leaf stage. Flowering in W. borbonica and W. tabularis was only observed under the LO regime at the second leaf stage. In both species, flowering was also obtained in SD-treated plants, provided treatment occurred after the formation of the third leaf. However, the total number and quality of flowers were reduced. To examine the effect of light intensity on flowering, plants at different developmental stages (first and/or second or beyond the third leaf stage) were exposed to photosynthetically active radiation (PAR) of 150 jJmol m-2s-1 or 39.5 jJmol m-2s-1 for 7 weeks. Exposure to low light intensity at either developmental stage compromised leaf quality. No flowering was observed following low light intensity treatment during the first to third leaf stages, even though plants were exposed to low temperature and LO regimes, both of which promoted flowering. Observation of the shoot apical meristem revealed that the second leaf stage was critical as the anatomical transition to flowering occurred at this level. When beyond the third leaf stage, low light intensity did not prevent flowering. However, the number of flowers produced per plant was reduced compared to plants maintained at 150 jJmol m-2s-1. Thus, light intensity played a role in both plant morphogenesis and flowering. LDs were effective in promoting vegetative growth whereas high light intensity and low temperature regimes played pivotal roles in flower induction. This makes them useful horticulture tools to produce desirable Watsonia plants for commercialisation.