Proteases play a crucial role in plant defence mechanisms as well as acclimation to changing metabolic demands and environmental cues. Proteases regulate the development of a plant from germination through to senescence and plant death. In this thesis the role of proteases and their inhibitors in plant response to cold stress and CO2 enrichment were investigated. The activity and inhibition of cysteine proteases (CP), as well as degradation of their potential target proteins was investigated in transgenic tobacco plants expressing the rice cystatin, OC-I. Expression of OC-I caused a longer life span; delayed senescence; significant decrease in in vitro CP activity; a concurrent increase in protein content; and protection from chilling-induced decreases in photosynthesis. An initial proteomics study identified altered abundance of a cyclophilin, a histone, a peptidyl-prolyl cis-trans isomerase and two RuBisCO activase isoforms in OC-I expressing leaves. Immunogold labelling studies revealed that RuBisCO and OC-I is present in RuBisCO vesicular bodies (RVB) that appear to be important in RuBisCO degradation in leaves under optimal and stress conditions. Plants need to respond quickly to changes in the environment that cause changes in the demand for photosynthesis. In this study the effect of CO2 enrichment on photosynthesis-related genes and novel proteases and protease inhibitors regulated by CO2 enrichment and/or development, was investigated. Maize plants grown to maturity with CO2 enrichment showed significant changes in leaf chlorophyll and protein content, increased epidermal cell size, and decreased epidermal cell density. An increased stomatal index in leaves grown at high-CO2 indicates that leaves adjust their stomatal densities through changes in epidermal cell numbers rather than stomatal numbers. Photosynthesis and carbohydrate metabolism were not significantly affected. Developmental stage affected over 3000 transcripts between leaf ranks 3 and 12, while 142 and 90 transcripts were modified by high CO2 in the same leaf ranks respectively. Only 18 transcripts were affected by CO2 enrichment exclusively. Particularly, two novel CO2 -modulated serine protease inhibitors modulated by both sugars and pro-oxidants, were identified. Growth with high CO2 decreased oxidative damage to leaf proteins. / Thesis (PhD)--University of Pretoria, 2009. / Plant Science / unrestricted

Establishing plant communities is essential for the restoration of contaminated land. As potential cover materials, fly ash and sewage sludge can prevent formation of acid mine drainage from sulfidic mine waste. The aim of the thesis was to i) screen for plants that can be established in, and prevent leakage of metals and nutrients from sludge on top of ash and tailings, and ii) investigate root growth into sealing layers of ash and sludge. Analyses were performed under laboratory, greenhouse and field conditions using selected plant species to examine the release of Cd, Cu, Zn, N, and P from the materials. Plant physiological responses and interactions with fly ash were also investigated. The data show that plants can decrease metal and nutrient leakage from the materials, and lower the elemental levels in the leachate, but with varying efficiencies among plant species. Plants capable of taking up both nitrate and ammonium were more efficient in preventing N leakage compared with those taking up primarily ammonium. Fast growing plants could raise the pH in acidic sludge leachate, but the initial pH decrease and N leakage was not counteracted by plants. Germination in fresh sludge was problematic, but enhanced by aeration of the sludge. In general, the accumulation of metals in plant shoots was low, especially if ash was located below the sludge. Fresh ash was phytotoxic (e.g., high alkalinity, salinity and metal levels) and induced the activity of stress-related enzymes in shoots. In sealing layers of aged and cured ash, roots could grow if the penetration resistance was low, or into the surface of stronger layers if the surface had become pulverized. The roots caused dissolution of calcium-rich minerals, possibly by exudation of saccharides. Addition of sludge to an ash layer increased root growth, likely due to decreased bulk density and pH, and nutrient addition. In conclusion, with selected plant species and a properly constructed cover, metal and nutrient leaching from the materials and root growth into the sealing layer can be restricted.

The increasing market demand for fresh produce promotes a keen interest in developing a rapid, sensitive and reliable method for monitoring plant health and determining the shelf-life of postharvest produce. The objective of this study is to explore the capability of Surface-enhanced Raman spectroscopy (SERS) in these applications. SERS integrates Raman spectroscopy which measures molecular vibrations and nanotechnology which enhances the weak Raman signals. Herein, we developed two SERS methods based on a surface detection approach using nanoparticles solution and a headspace detection approach using gold nanoparticles (AuNPs) fibers, to detect biochemical changes during postharvest storage of arugula leaves. Compared with surface detection, the headspace detection revealed significant spectral changes during the storage, particularly in the shifts around 500, 950 and 1030 cm-1. These changes analyzed using principal component analysis (PCA) to establish a prediction model for shelf-life determination. Through analyzing reference standard compounds, we identified the dimethyl disulfide (DMDS), 1-propanethiol and methanethiol (MT) were most likely to account for the signature spectra of headspace arugula at the late storage period due to the activities of spoilage bacteria. The headspace detection method was also applied to monitor the stress responses of living basil to abiotic stresses (pesticide/salinity). However, the volatile analysis of the basil plants response to abiotic stresses (pesticide/salinity) showed indistinctive results. In conclusion, the headspace detection based on SERS provides a new strategy for quality monitoring of fresh produce in the food industry.