Two novel tomato cysteine proteinases: Characterization and analysis of their potential roles in programmed cell death
A variety of cysteine proteinases are implicated in plant programmed cell death, although their function in this process is not completely understood. Here, the cDNAs of two novel cysteine proteinases termed 'SLCysEP' and 'SLCYSPRO' were cloned and found to be members of the C1A superfamily. SLCysEP is somewhat unique as it contains a C-terminal KDEL motif that mediates retrieval of peptides to the ER. Both 'SLCysEP' and 'SLCYSPRO' were found to be single copy genes containing three introns each. Recombinant proteins expressed in 'E. coli' showed 'in vitro' proteolytic activity characteristic of C1A proteinases. Recombinant SLCysEP underwent self-hydrolysis when subjected to an acidic pH; recombinant SLCYSPRO did not. During stamen development, sporophytic cells surrounding the locules and within the stomium undergo programmed cell death, as do endospermic cells of the seed following germination. The ' SLCysEP' transcript and protein were detected at high levels in stamens of pre-dehiscent flower buds but not in any other floral whorl, and the ' SLCYSPRO' transcript and protein were detected in the endosperm of germinating seeds. SLCysEP was localized strictly to the stomium and the middle layer and endothecial cells surrounding the locules of pre-dehiscent anthers, and immunogold labeling with anti-SLCysEP antibodies, coupled with electron microscopy, localized the protein to endoplasmic reticulum-derived precursor protease vesicles.