AbstractBackgroundChloroplasts respond to stress and changes in the environment by producing reactive oxygen species (ROS) that have specific signaling abilities. The ROS singlet oxygen (1O2) is unique in that it can signal to initiate selective degradation of damaged chloroplasts and then cell death. This chloroplast quality control pathway can be monitored in theArabidopsismutantplastid ferrochelatase two(fc2) that conditionally accumulates chloroplast1O2under diurnal light cycling conditions leading to rapid chloroplast degradation and eventual cell death. The cellular machinery involved in such degradation, however, remains unknown. Recently it has been demonstrated that whole damaged chloroplasts can be transported to the central vacuole via a process requiring autophagosomes and core components of the autophagy machinery. The relationship between this process, referred to as chlorophagy, and the degradation of1O2-stressed chloroplasts and cells has remained unexplored.ResultsTo further understand1O2-induced cellular degradation and determine what role autophagy may play, the expression of autophagy-related genes were monitored in1O2-stressedfc2seedlings and found to be induced. Although autophagosomes were present infc2cells, they did not associate with chloroplasts during1O2stress. Mutations blocking the core autophagy machinery (atg5, atg7, andatg10) were unable to suppress1O2-induced chloroplast degradation or cell death in thefc2mutant, suggesting autophagosome formation and macroautophagy are dispensable for1O2–mediated cellular degradation. However, bothatg5andatg7led to specific defects in chloroplast ultrastructure and photosynthetic efficiencies, suggesting macroautophagy may be involved in protecting chloroplasts from photo-oxidative damage. Finally, genes predicted to be involved in microautophagy were shown to be induced in stressedfc2seedlings, indicating a possible role for an alternate form of autophagy in the dismantling of1O2-damaged chloroplasts.ConclusionsOur results support the hypothesis that1O2-dependent chloroplast degradation is independent from autophagosome formation, canonical macroautophagy, and chlorophagy. Instead, ATG-independent microautophagy may be involved in such degradation. However, canonical macroautophagy may still play a role in protecting chloroplasts from1O2-induced photo-oxidative stress. Together, this suggests chloroplast function and degradation is a complex process that utilizes multiple autophagy and degradation machineries, possibly depending on the type of stress or damage incurred.