Characterization of proteolytic activity during senescence in daylilies

From 12 to 24 h after the opening of daylily flowers (Hemerocallis hybrid c v. Stella d'Oro), the petals begin to degrade and the protein levels of sol uble, microsomal- and plastid-enriched fractions decrease by 50% on a per p etal basis. To help determine some of the components for the cell death pro gram in daylily petals, we studied the mechanisms that regulate this loss o f protein. Enzyme activities capable of digesting native daylily protein, g elatin, and azocasein markedly increase after flower opening, and their app earance is inhibited by the translation inhibitor, cycloheximide. Protein h ydrolysis in vitro is prevented by inhibitors of cysteine, serine and metal loproteinases. Immunoblots using antibodies to ubiquitin pathway enzymes in dicate that the ubiquitin system is not senescence specific. However, ion l eakage is delayed by two inhibitors of the 26S proteasome. We propose that programmed cell death in daylily petals map involve the increase in activit y of at least three classes of proteinases, and discuss the possibility tha t these proteinases may operate in concert with the ubiquitin pathway.