Deficiency of CV and Autophagy trigger susceptibility to extended darkness
Taking into account that autophagy has a recognized role in plant energetic homeostasis under carbon starvation conditions (Ishida et al., 2013; Barros et al., 2017,2021; Hirota et al., 2018; McLoughlin et al., 2020), we further evaluated the autophagic process in the amircvplants. For this purpose, we measured the transcript levels ofATG8h , which encodes an isoform of the ATG8 protein required for autophagosome elongation. These increased from 3d of darkness in both WT and amircv mutants (Fig. 4D), suggesting a general activation of autophagy under these conditions. It was previously reported that CV and autophagy are independent pathways, occurring concomitantly in the cell (Wang and Blumwald, 2014). The lack of CV does not trigger any differential autophagy activation under extended darkness. However, theCV gene is upregulated in plants lacking autophagy during darkness treatment (Barros et al., 2017). Thus, our results suggest that CV triggers minor effects on energetic metabolism when autophagy is functional.
To test the coordination of these two pathways, we next generated double mutants for both CV and autophagy: amircv1 xatg5 (Fig. 5A). Double mutant plants and their parental lines were grown side-by-side under short-day and further submitted to extended darkness conditions. Interestingly, after 10d of darkness, the double mutants exhibited an early senescence phenotype similar to the atg5 single mutant (Fig. 5B). The sensitivity phenotype observed in both atg5 andamircv1 xatg5 mutants was followed by a massive reduction of chlorophyll levels (Fig. 5C). Since the sensitive phenotype was not recovered in amircv1 xatg5 mutants, it seems reasonable to suggest that other chloroplast catabolic pathways are likely activated in both atg5 and amircv1xatg5 plants under darkness.