The core autophagy machinery is not required for chloroplast singlet oxygen-mediated cell death in the Arabidopsis thaliana plastid ferrochelatase two mutant

Abstract

Background Chloroplasts 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 cellular degradation including the selective degradation of damaged chloroplasts. This chloroplast quality control pathway can be monitored in the Arabidopsisthaliana mutant plastid ferrochelatase two (fc2) that conditionally accumulates chloroplast 1O2 under 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 was 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 of 1O2-stressed chloroplasts and cells has remained unexplored. Results To further understand 1O2-induced cellular degradation and determine what role autophagy may play, the expression of autophagy-related genes was monitored in 1O2-stressed fc2 seedlings and found to be induced. Although autophagosomes were present in fc2 cells, they did not associate with chloroplasts during 1O2 stress. Mutations affecting the core autophagy machinery (atg5, atg7, and atg10) were unable to suppress 1O2-induced cell death or chloroplast protrusion into the central vacuole, suggesting autophagosome formation is dispensable for such 1O2–mediated cellular degradation. However, both atg5 and atg7 led to specific defects in chloroplast ultrastructure and photosynthetic efficiencies, suggesting core autophagy machinery is involved in protecting chloroplasts from photo-oxidative damage. Finally, genes predicted to be involved in microautophagy were shown to be induced in stressed fc2 seedlings, indicating a possible role for an alternate form of autophagy in the dismantling of 1O2-damaged chloroplasts. Conclusions Our results support the hypothesis that 1O2-dependent cell death is independent from autophagosome formation, canonical autophagy, and chlorophagy. Furthermore, autophagosome-independent microautophagy may be involved in degrading 1O2-damaged chloroplasts. At the same time, canonical autophagy may still play a role in protecting chloroplasts from 1O2-induced photo-oxidative stress. Together, this suggests chloroplast function and degradation is a complex process utilizing multiple autophagy and degradation machineries, possibly depending on the type of stress or damage incurred.

Perturbation of protein homeostasis brings plastids at the crossroad between repair and dismantling

“…S1 A), whereas the expression of genes involved in ATG-independent micro-autophagy was mildly stimulated in oePRPS1 (Fig. S1 B) (Lemke et al ., 2021). Overall, these observations indicate that the reduced accumulation PRPS1 severely jeopardizes chloroplast integrity during leaf development, leading to the dismantling of damaged and misshapen chloroplasts with the final aim to remove reactive oxygen species–producing chloroplasts and redistributing nutrients to other tissues (Woodson, 2022).…”

“…Strikingly, both ATI1 (Michaeli et al, 2014) and ATG8f (Liu et al, 2021) transcripts were highly enriched in plantlets with increased PRPS1 transcript accumulation, driven by either CaMV35S-or DEX-induced promoters (Fig. S1 A), whereas NPC1 and VPS15 (Lemke et al, 2021) were the only genes of the ATG-independent pathway significantly up-regulated in oePRPS1 plantlets (Fig. S1 B).…”

Targeted for destruction: degradation of singlet oxygen-damaged chloroplasts

“…Neither mutation suppressed cell death or chloroplast degradation during 1 O 2 production. 24 Importantly, chloroplast blebbing into the central vacuole was still observed in fc2 atg double mutants. These analyses make clear that such hallmarks of 1 O 2 -induced chloroplast damage in fc2 are not dependent on autophagosomes and, thus, is distinct from ATG5- and ATG7-dependent chlorophagy.…”

“…Instead, autophagosomes only associated with fc2 chloroplasts under carbon starvation (dark) conditions that should lack 1 O 2 accumulation. 24 The role of autophagosome assembly in 1 O 2 -induced CQC was then tested by introducing atg5 and atg7 null mutations into the fc2 background. Neither mutation suppressed cell death or chloroplast degradation during 1 O 2 production.…”

“… 21 In 1 O 2 -stressed fc2 seedlings, several putative microautophagy-related genes (inferred from yeast homology 25 ) were induced, suggesting this degradation pathway is being activated and could be responsible for 1 O 2 -induced CQC. 24 It will be compelling to investigate the necessity of this process in CQC to determine if similar ATG-independent microautophagy-related processes are conserved in plants. However, the possibility remains that 1 O 2 -induced CQC may depend on an as-of-yet uncharacterized vacuolar transport mechanism.…”