Degradation of ribulose-bisphosphate carboxylase by vacuolar enzymes of senescing French bean leaves: immunocytochemical and ultrastructural observations

The possible involvement of vacuolar cysteine proteinases in degradation of ribulose-bisphosphate carboxylase (Rubisco) in senescing French bean leaves was studied by ultrastructural and immunocytochemical analyses with antibodies raised against the large subunit (LSU) of Rubisco and SH-EP, a cysteine proteinase from Vigna mungo that is immunologically identical to one of the major proteinases of French bean plants. Primary leaves of 10-day-old plants were detached and placed at 25 degreesC in darkness for 0, 4, and 8 days to allow their senescence to proceed. The leaves at each senescence stage were subjected to the conventional electron microscopic and immunocytochemical studies. The results indicated that the chloroplasts of senescing French bean leaves were separated from the cytoplasm of the cell periphery and taken into the central vacuole and that the Rubisco LSU in the chloroplasts was degraded by vacuolar enzymes such as an SH-EP-related cysteine proteinase that developed in senescing leaves. The present results together with the results of previous biochemical studies using vacuolar lysates support the view that Rubisco is degraded through the association of chloroplasts with the central vacuole during the senescence of leaves that were detached and placed in darkness.