"Senescence-associated vacuoles" are involved in the degradation of chloroplast proteins in tobacco leaves

MARTINEZ, DE; COSTA, ML; GOMEZ, F; OTEGUI, M; GUIAMET, JJ

PLANT JOURNAL

Blackwell-Synergy

Año: 2008 vol. 56 p. 196 - 206

0960-7412

Massive degradation of photosynthetic proteins is the hallmark of leaf senescence, however the mechanism involved in chloroplast protein breakdown is not completely understood. Since small “senescence-associated vacuoles” (SAVs) with intense proteolytic activity accumulate in senescing leaves of soybean and Arabidopsis, the main goal of this work was to test if SAVs are involved in the degradation of chloroplastic components. SAVs with protease activity were readily detected through confocal microscopy of naturally senescing leaves of tobacco (Nicotiana tabacum L.). In detached leaves incubated in darkness, acceleration of chloroplast degradation rate by ethylene treatment correlated with a two-fold increase of the number of SAVs per cell, compared to untreated leaves. In a tobacco line expressing GFP targeted to plastids, we detected GFP re-located to SAVs in senescing leaves. SAVs were isolated by sucrose density gradient centrifugation. Isolated SAVs contained chloroplast-targeted GFP, and the chloroplast stromal proteins Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) and  glutamine synthetase, but they lacked the thylakoid proteins D1 and LHCII of the PSII reaction center and PSII antenna, respectively. In SAVs incubated at 30ºC there was a steady decrease in Rubisco levels, which was completely abolished by addition of protease inhibitors. Thus, SAVs are likely involved in the degradation of the soluble photosynthetic proteins of the chloroplast stroma during senescence of leaves.Nicotiana tabacum L.). In detached leaves incubated in darkness, acceleration of chloroplast degradation rate by ethylene treatment correlated with a two-fold increase of the number of SAVs per cell, compared to untreated leaves. In a tobacco line expressing GFP targeted to plastids, we detected GFP re-located to SAVs in senescing leaves. SAVs were isolated by sucrose density gradient centrifugation. Isolated SAVs contained chloroplast-targeted GFP, and the chloroplast stromal proteins Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) and  glutamine synthetase, but they lacked the thylakoid proteins D1 and LHCII of the PSII reaction center and PSII antenna, respectively. In SAVs incubated at 30ºC there was a steady decrease in Rubisco levels, which was completely abolished by addition of protease inhibitors. Thus, SAVs are likely involved in the degradation of the soluble photosynthetic proteins of the chloroplast stroma during senescence of leaves.