CHOROPLAST PROTEINS ARE DEGRADED BY CYSTEINE-PROTEASES IN SENESCENCE-ASSOCIATED VACUOLES

CARRION CRISTIAN; MARTINEZ DANA ETHEL; COSTA LORENZA; GUIAMET JUAN JOSÉ

Tucuman, Argentina

Congreso; SAIB 2009; 2009

Sociedad Argentina de Investigaciones Bioquimicas

The most obvious change during senescence of leaves is the degradation of chloroplasts, the photosynthetic organelles of plants. However, the mechanism underlying the breakdown of chloroplast proteins is poorly understood. “Senescence-associated vacuoles” (SAVs) with high proteolytic activity develop in senescing leaves of Arabidopsis, soybean and tobacco. These vacuoles contain chloroplast proteins and high levels of cysteine protease activity. The main goal of this work was to identify the cys-proteases present in tobacco SAVs, and to determine their role in Rubisco degradation. Cys-proteases were tagged with a biotynilated irreversible inhibitor; western blots showed three main Cys-protease bands in SAVs at 64, 41 and 32 kDa. These are also the most important Cys-proteases in crude extracts from senescing leaves. None of these proteases could be detected in isolated chloroplasts. In in vitro autodigestion experiments with isolated SAVs, Rubisco degradation was completely abolished by incubation with E-64, a specific, irreversible inhibitor of Cys-proteases. Likewise, in vivo E-64 effectively reduced Rubisco degradation in leaf disks induced to senesce in darkness. These data strongly argue that Cys-proteases in SAVs are involved in the degradation of chloroplast proteins during senescence of leaves.