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 leave s was studied by ultrastructural and immunocytochemical analyses with antib odies raised against the large subunit (LSU) of Rubisco and SH-EP, a cystei ne proteinase from Vigna mungo that is immunologically identical to one of the major proteinases of French bean plants. Primary leaves of 10-day-old p lants were detached and placed at 25 degreesC in darkness for 0, 4, and 8 d ays to allow their senescence to proceed. The leaves at each senescence sta ge were subjected to the conventional electron microscopic and immunocytoch emical studies. The results indicated that the chloroplasts of senescing Fr ench bean leaves were separated from the cytoplasm of the cell periphery an d taken into the central vacuole and that the Rubisco LSU in the chloroplas ts was degraded by vacuolar enzymes such as an SH-EP-related cysteine prote inase 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.