Stromal protein degradation is incomplete in Arabidopsis thaliana autophagy mutants undergoing natural senescence

In this work, levels of three stromal proteins (Rubisco large subunit, chloroplast glutamine synthetase and Rubisco activase) and one thylakoid protein (the major light harvesting complex protein of photosystem II) were measured during natural senescence in WT and in two autophagy T-DNA insertion mutants (atg5 and atg7). Thylakoid-localized protein decreased similarly in all genotypes, but stromal protein degradation was incomplete in the two atg mutants. In addition, degradation of two stromal proteins was observed in chloroplasts isolated from mid-senescence leaves.These data suggest that autophagy does contribute to the complete proteolysis of stromal proteins, but does not play a major degenerative role. In addition, support for in organello degradation is provided.Stromal proteins in C3 mesophyll chloroplasts contain approximately 55% of total cellular nitrogen, mostly in the form of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), while approximately 20% of total nitrogen is allocated to thylakoid proteins [1]. During senescence most of the nitrogen from these two sources is exported from the aging leaf [2,3], but the proteolytic process is not well understood [4-6]. Genetic approaches towards understanding senescence have focused on the isolation of stay-green mutants, and these studies have shown that stromal and thylakoid proteolysis can be uncoupled. One class of stay-green mutants, nonfunctional type C, retain thylakoid-localized light harvesting complex proteins while stromal proteins are degraded [7,8].The high nitrogen content of stromal proteins has led to extensive investigation of their proteolysis during leaf senescence. No chloroplast proteases specifically involved in Rubisco or other stromal protein degradation have been identified to date [9]. A Zn-dependent EP1 protease activity was partially purified [10], but no corresponding gene or gene product has been reported. Chloroplast stromal Clp proteases are likely candidates for stromal