Regulation of programmed cell death in tomato endosperm and the involvement of a ricinosomal cysteine proteinase
This thesis investigates endosperm programmed cell death (PCD) in 'Solanum lycopersicum' L. (tomato) seeds following germination. The expression pattern of a cysteine proteinase-encoding gene, ' SlCysEP', and accumulation of the enzyme in imbibed whole seeds prior to, and following, germination are consistent with that of an enzyme involved in mobilization of the cell corpse following PCD. Further, immuno-gold labelling revealed that the enzyme localizes to a PCD-associated, endoplasmic reticulum-derived organelle, the ricinosome. Overexpression of a fluorescent protein-tagged SlCysEP construct in cultured tobacco cells induces formation of ricinosome-like structures (RLs). Removal of the cysteine proteinase-domain from the construct prevents the formation of RLs suggesting that this domain may be necessary for formation of ricinosomes 'in vivo'. The hormonal control of endosperm PCD was also investigated. This study confirms a previous report that gibberellic acid (GA) promotes endosperm PCD, and demonstrates that abscisic acid antagonizes the action of GA thereby preventing PCD. Limited evidence indicates that the C14 family of cysteine proteinases, in particular the metacaspases, participate in the regulation of some instances of PCD in plants. The latter portion of this thesis reports the cloning and initial characterization of a type II tomato metacaspase gene, 'SlMCA2 '. Transcript analysis of 'SlMCA2' indicate that it is not involved in endosperm PCD, but that it may be involved in instances of PCD in the developing seedling, and perhaps in dying tissues of the anther.