Sugarcane holds great potential as a biofactory for the tailored production of novel products of commercial value. In many cases however, the accumulation of an alien product within the cytoplasm interferes with essential cell metabolism. To avoid potential interference, targeting the accumulation of biofactory products into vacuoles may be beneficial. Vacuoles represent one endpoint of the plant endomembrane system where proteins destined for inclusion must contain appropriate targeting peptide signals. However, targeting peptide signals used previously to direct heterologous proteins to the vacuole have not yet been shown to function efficiently in sugarcane. The emphasis of the work described in this thesis was first to characterise the diversity of vacuole types in selected sugarcane tissues, and second to identify and test the function of putative vacuolar targeting signals identified in vacuolar proteins of sugarcane. In order to investigate vacuole diversity in sugarcane cells, a series of membrane-permeable fluorescent probes were used to assess both the acidity and proteolytic properties of vacuolar compartments. It is clear that even from early in development, large vacuoles filled most of the volume of storage parenchyma cells within the developing sugarcane stem. These vacuoles were intensely acidic and contained active aminopeptidases. In leaf cells, vacuoles labelled by chromogenic indicators and enzyme substrates appear much more diverse in pH and proteolytic intensity owing to the multiple functions that leaf cells participate in. As the predominant sugarcane vacuole in vegetative tissues appears to be proteolytic, sugarcane sequences showing homology to proteases and protease inhibitors in other plant species were aligned and compared to identify potential vacuolar targeting signals. This analysis revealed the presence of several candidate vacuolar targeting motifs which displayed high conservation across plant homologues. One such motif, represented by the sequence IRLPS, was identified in the N-terminal region of a legumain protein from sugarcane, which was homologous to known vacuolar processing enzymes in other species. To test the efficacy of the legumain targeting signal and to compare with other motifs, a series of GFP reporter constructs was synthesised and expressed in sugarcane. The sugarcane legumain vacuole targeting signal was particularly efficient at directing an otherwise secreted GFP fusion protein into a large acidic and proteolytic vacuole in sugarcane callus cells as well as in diverse plant species. In mature sugarcane transgenic plants, the stability of GFP fusion proteins in the vacuole appeared to be dependent on cell type, suggesting that the vacuolar environment can vary in its ability to degrade introduced proteins. The legumain vacuole targeting signal was further tested for its ability to direct an avidin protein and a fructosyltransferase enzyme into the lytic vacuole of transgenic sugarcane plants. Avidin, derived initially from chicken egg white, is a glycoprotein that displays a high affinity to the vitamin biotin. For this reason it has been investigated for use in sugarcane as a biocontrol agent against cane grub species. For the production of avidin in planta careful targeting to an appropriate subcellular location is required to avoid a detrimental depletion of available plant cell biotin reserves. When the legumain targeting signal was fused to avidin and expressed as a stably integrated transgene, the avidin protein was detected by immunoblotting but appeared to be proteolytically cleaved within the lytic vacuole in all sugarcane tissues analysed. These plants were phenotypically indistinguishable from controls, indicating that avidin did not appreciably deplete cellular biotin reserves while in transit through the endomembrane system. In contrast, when avidin was designed for either retention in the endoplasmic reticulum or for transit to a different type of vacuole using a heterologous targeting signal, stably transformed plants exhibited a biotin deficient phenotype. This suggests that the legumain vacuole targeting signal was efficient at directing heterologous proteins to a lytic type vacuole where they can be degraded and inactivated if susceptible to proteolysis. When the fructosyltransferase (ftf) gene from Streptococcus salivarius was stably transformed into sugarcane and directed into the lytic vacuole using the legumain vacuole targeting signal, no fructan product could be detected. The low pH and proteolytic environment of this vacuole together with low expression of this bacterial transgene most likely resulted in minimal Ftf activity. Taken together, evidence is presented that the legumain vacuolar targeting signal functions efficiently in directing transgene products such as GFP, avidin and a fructosyltransferase enzyme into a lytic type vacuole. This vacuole has been demonstrated to be both acidic and proteolytic and therefore strategies to improve the stability of heterologous proteins targeted to this vacuolar environment are required and may be specific to the product in question.
| Identifer | oai:union.ndltd.org:ADTP/254038 |
| Creators | Mark Jackson |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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