Chlorophagy targets chloroplasts irreversibly damaged by photoinhibition

Chlorophagy removes photodamaged chloroplasts in plants, while individual chloroplasts are also protected by mechanisms that limit damage to the photosynthetic apparatus caused by excess light energy, known as photoinhibition. How chlorophagy is functionally coupled with chloroplast-level protective mechanisms during acclimation to photoinhibition remains unclear. We investigated acclimatory responses to photoinhibition at the intracellular level in Arabidopsis thaliana through an integrative analysis combining Chl fluorescence measurements, microscopy imaging, and genetic analyses using mutants defective in nonphotochemical quenching (NPQ) or the chloroplast avoidance response. The functionality of individual chloroplasts was further assessed by Chl fluorescence lifetime imaging and probe-based visualization of singlet oxygen and starch. Severe photoinhibition of photosystem II precedes the appearance of abnormally swollen chloroplasts, which are subsequently recognized by the autophagy machinery for degradation. The swollen chloroplasts represent irreversibly damaged chloroplasts that accumulate reactive oxygen species and lose photosynthetic function. The formation of these nonfunctional chloroplasts was enhanced in mutants defective in NPQ or the chloroplast avoidance response, and hyperaccumulation of nonfunctional chloroplasts led to necrotic cell death. Together, our findings illustrate how multilayered responses coordinate acclimation to photoinhibition: Chloroplast protective mechanisms minimize the formation of nonfunctional chloroplasts while chlorophagy removes them when they arise.