CONICET | Buscador de Institutos y Recursos Humanos

Chloroplast disassembly and the breakdown of photosynthetic proteins characterize leaf senescence. The mechanism involved in the massive degradation of plastid proteins is poorly understood. We previously reported that a novel class of "senescence-associated vacuoles "(SAVs) with high proteolytic activity accumulates in chloroplast-containing cells of senescing leaves of Arabidopsis and soybean (Otegui et al., 2005 Plant J. 41: 831-844). Using tobacco as a model plant, we found that the number of SAVs increases when chloroplast degradation rate is accelerated by treatment with ethephon. SAVs might be involved in chloroplast protein degradation. We used a transgenic line of tobacco expressing GFP fused to a chloroplast transit peptide (TP-GFP) to examine, by confocal microscopy, if chloroplast proteins are delivered to SAVs for degradation. While TP-GFP is correctly targeted to the plastid in non-senescent leaves, with no GFP signal outside chloroplasts, TP-GFP signal is readily detected in SAVs of senescing leaves. Spectral analysis of fluorescence emission confirms that the signal coming from SAVs corresponds to GFP. A SAV-enriched fraction was prepared by sucrose density centrifugation of a post-chloroplast supernatant. Isolated SAVs lacked the D1 protein of PSII, indicating that this fraction was free of chloroplasts or chloroplast fragments. On a protein basis, SAVs contained substantial amounts of the chloroplast-encoded, large subunit of Rubisco (RbcL). Levels of RbcL decreased in a time-dependent manner when SAVs were incubated at 30ºC for periods up to 4 hs, and this degradation was abolished by addition of protease inhibitors. Collectively, these data support the idea that SAVs are involved in the degradation of some chloroplast proteins during leaf senescence