Molecular and physiological investigation of trace metal stress in seagrass, Zostera muelleri

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Despite the vast research on the negative effects of anthropogenic pollution on marine organisms, little is known about the toxicity responses of seagrasses to such perturbations. Understanding seagrass responses at the molecular level will ensure adequate conservation strategies to mitigate the increasing decline rate of seagrasses as a result of climate change and anthropogenic driven disturbances. The meadows of the Southern hemisphere seagrass species, ๐˜ก๐˜ฐ๐˜ด๐˜ต๐˜ฆ๐˜ณ๐˜ข ๐˜ฎ๐˜ถ๐˜ฆ๐˜ญ๐˜ญ๐˜ฆ๐˜ณ๐˜ช, encounter similar threats, which led to a significant loss along the Australia and New Zealand coasts. Trace metal pollution and most specifically copper (Cu), have been previously reported in industrial, agricultural and domestic run-off waste which often finds their way to the ocean and jeopardise the health of the seagrass meadows. Although we have a firm undersetting of the deleterious effect of Cu stress at the physiological and ecological level, no current knowledge exists on how ๐˜ก. ๐˜ฎ๐˜ถ๐˜ฆ๐˜ญ๐˜ญ๐˜ฆ๐˜ณ๐˜ช responds to elevated levels of Cu at the molecular level. Upon our investigation of the physiological responses of ๐˜ก. ๐˜ฎ๐˜ถ๐˜ฆ๐˜ญ๐˜ญ๐˜ฆ๐˜ณ๐˜ช to 250 ฮผg Cu Lโปยน and 500 ฮผg Cu Lโปยน over a 7 day period of exposure, the Cu accumulation in the leaves, the continual production of ROS and the decline of photosynthetic efficiency were observed in ๐˜ก. ๐˜ฎ๐˜ถ๐˜ฆ๐˜ญ๐˜ญ๐˜ฆ๐˜ณ๐˜ช at both above mentioned Cu concentrations. However, the responses were concentration-dependent illustrating 250 ฮผg Cu Lโปยน and 500 ฮผg Cu Lโปยน as a tolerable and a toxic level for ๐˜ก. ๐˜ฎ๐˜ถ๐˜ฆ๐˜ญ๐˜ญ๐˜ฆ๐˜ณ๐˜ช, respectively. The results of our molecular investigations indicated regulation shifts in the expression of genes and the abundance of proteins mainly at 500 ฮผg Cu Lโปยน were associated with energy metabolism, carbon fixation, photosynthesis and defence mechanism. While the expression of genes (and the abundance of proteins) involved in energy metabolism (mainly glycolysis) and defence mechanism have been shown to be mainly increased, the opposite was observed in the photosynthetic process and carbon fixation. As a result, whilst these results offers a new level of understanding into the seagrass toxicity responses at transcriptomic and proteomic levels, it also provides candidate molecular markers for future toxicology studies and seagrass monitoring. This PhD thesis also evaluates a protein-centric and four peptide-centric proteomic methods and proposed an optimised peptide desalting protocol. Additionally, major alterations in photosynthesis process as a result of Cu stress has led us to report on an optimised intact chloroplast isolation method that can be used for future proteomic-based studies.
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