| This study investigates the involvement of a cysteine proteinase in Vicia faba L. leaf senescence. The enzyme, VFCYSPRO, was detected in protein extracts from both aging and senescing leaves, but not in young leaves. This correlates the presence of VFCYSPRO with leaf senescence temporally. The sub-cellular localization of the enzyme was studied by transient expression of chimeric fluorescent protein constructs in a novel Vicia faba mesophyll protoplast system and in onion epidermal peels. The results demonstrate that the VFCYSPRO pro-peptide is a necessary and sufficient vacuolar sorting determinant (VSD), and that it routes to a large, acidic, central vacuole via the endoplasmic reticulum and, likely, the Golgi. VFCYSPRO is therefore a bona fide vacuolar enzyme, a localization that is consistent for an enzyme participating in programmed cell death. |
