CV contributes to the abnormal chloroplast structure ofatg5 mutants under extended darkness
Given that both autophagy and CV can mediate chloroplast degradation, we next used transmission electron microscopy to investigate the effects of a simultaneous lack of autophagy and CV on chloroplast structure. Before darkness treatment, chloroplasts of all the genotypes appeared undamaged at the ultrastructural level, containing large starch granules, grana, stroma thylakoids and occasionally occurring small plastoglobules (Fig. 8A-D). After 7d of darkness, WT and the single mutant amircv-1chloroplasts displayed similar structures, containing remodeled thylakoid systems and granal stacking (Fig. 8E-F). However, the membrane organization of the amircv1 xatg5 chloroplasts was notably disturbed (Fig. 8H). We previously showed that ATG5 mutation compromises chloroplast integrity under darkness (Barros et al., 2021). Interestingly, when comparing the atg5 and amircv1xatg5mutants, differential changes in chloroplast structure were observed (Fig. 8G and H). In addition to the marked supersized plastoglobuli, atg5 chloroplasts lost their normal structure exhibiting a longish and curved shape containing deformed stroma and several vesicles (Fig. 8G). These vesicle structures resemble senescence-associated vacuoles (SAVs), which also mediate the recycling of chloroplast proteins by an autophagy-independent mechanism (Otegui et al., 2005). Additionally, the atg5 chloroplasts lost the curved thylakoid membrane response observed in WT and the amircv-1genotypes. Despite the presence of granal stacking in atg5 , the granum was narrow with more layers (Fig 8I and L). On the other hand, it seems that the amircv1xatg5 chloroplasts partially recovered the curved thylakoid membrane response, yet the of granal structure was highly compromised compared to WT and single mutants (Fig 8H and M). It is generally accepted that chloroplast stacking is governed by physicochemical forces between membranes and lipid-protein interactions (Armbruster et al., 2013; Johnson and Wientjes, 2019; Mazur et al., 2019). Since CV and autophagy operate in the selective degradation of chloroplast proteins, it is tempting to suggest that the distinct chloroplast responses observed are likely associated with differential protein degradation in these genotypes.