Abstract
M.Sc.
Proteinases are involved in many cellular reactions involving protein degradation, such as degradation of storage proteins and protein degradation during senescence processes. Their action can be inhibited by proteinase inhibitors. Information is still limited about the regulation of these inhibitors in plants and their possible interaction with proteinases under stress conditions. To obtain a better understanding of the physiological role of a proteinase inhibitor in plants under stress, the expression of a papaya cysteine proteinase inhibitor (cystatin) and its relation to proteinase expression was investigated in more detail. For this purpose, expression of the inhibitor was studied in papaya plants exposed to different physiological stress conditions, such as high/low temperature, and
treatment with selected chemicals, such as glutathione, OTC (L-2-
Oxothiazolidine-4-carboxylate), bestatin ([(2S, 3R)-3-amino-2-hydroxy-4-phenyl butanoylj-L-leu) and 2.4-D (2,4-dichiorophenoxyacetic acid). Using detection tools like activity gel electrophoresis, immunoblotting and enzymatic assays, the production of the cystatin under stress was monitored in different papaya explants, such as roots, leaves and embryos. Inhibitor production increased under different stress conditions when compared to untreated controls. However, this increase was not dramatic in any of the stresses applied. Exact quantification of the increase by using immunoblotting as the only specific tool to determine cystatin expression, was difficult. Neither activity gel electrophoresis
nor enzymatic assays were successful to further quantify the exact cystatin levels. Higher cystatin expression was accompanied with a decrease in proteinase activity. Transgenic tobacco plants carrying the gene for a rice cystatin had a significantly lower cysteine proteinase activity when compared to non-transgenic tobacco plants after prolonged cold stress. Furthermore, protein degradation and leaf yellowing as a consequence of cold treatment were prevented in transgenic plants. An attempt to obtain a transformed papaya plant to study silencing of cystatin expression under stress was unsuccessful. In this study, the protective role of a cystatin in cold stress was described for the first time.