INTRODUCTION
Under environmental and developmental conditions that lead to carbohydrate limitation, plants require alternative substrates to sustain metabolic reactions (Araujo et al., 2011). Such energetic demands may require the disassembly of organellar components and the redirection of alternative substrates for respiration. In plants, the major protein reserve is in the chloroplast, as approximately 80% of the total leaf nitrogen corresponds to photosynthetic proteins in C3 plants (Ishida et al., 2008). The degradation of chloroplasts is a hallmark of both natural and stress-induced plant senescence, and their catabolic products are used for energy production during carbon starvation conditions (Wada et al., 2009; Izumi et al., 2013, 2017, 2018). Accordingly, autophagy plays a key role in this process by targeting chloroplast proteins for degradation (Ishida et al., 2008; Xie et al.,2015; Izumi et al.,2017, 2018; Hirota et al., 2018).
Overall, autophagy is a well-characterized pathway by which cytoplasmic components are engulfed and delivered by a specialized double-membrane structure (autophagosome) to the vacuole for recycling (Michaeli et al., 2016; Magen et al., 2022). Interestingly, several autophagy-mediated chloroplast degradation pathways are differentially activated under distinct conditions (Izumi et al., 2018). The encapsulation of entire chloroplasts into ATG8-decorated autophagic vesicles and their subsequent delivery to the vacuole is termed “chlorophagy”. This process is dependent on ATG8 lipidation and is induced upon UV-B or high-light treatments (Izumi et al. , 2017; 2018). Rubisco-containing bodies (RCBs) are part of another type of chloroplast autophagy that provides piecemeal transport of stromal proteins, and is activated upon carbohydrate starvation (Ishida et al. 2008; Izumi et al., 2018; Hirota et al., 2018). By contrast, ATI (ATG8-Interacting 1) bodies, a type of chloroplast autophagy relying on a specific ATG8-binding protein, are initiated inside the chloroplast being associated with thylakoid, envelope, and stroma proteins (Michaeli et al., 2014; 2016). The appearance of plastid-associated ATI bodies has been observed in both senescent leaves and energy-starved seedlings (Michaeli et al., 2014; 2016).
Recent studies have also highlighted the relevance of autophagy-independent chloroplast degradation mechanisms, namely the Senescence Associated Vacuoles (SAVs) and Chloroplast Vesiculation (CV). SAVs are small proteolytic vacuolar compartments that degrade a subset of chloroplast components and accumulate in senescing leaves (Otegui et al., 2005; Martínez et al., 2008; Gomez et al., 2019). SAVs have been shown to contain stromal proteins and exhibit strong cysteine protease activity, as evidenced by the presence of the senescence-associated protease SAG12 (Otegui et al., 2005). In addition, CV was characterized as a chloroplast degradation pathway independent of either SAVs or autophagy (Wang and Blumwald, 2014). It was first described in rice as being strongly upregulated under abiotic stress and downregulated by cytokinin (Peleg et al., 2011). Later on, using Arabidopsis mutants, CV-containing vesicles (CCVs) were characterized as mobilizing thylakoid and stromal proteins to the vacuole for degradation (Wang and Blumwald, 2014). Furthermore, the disruption of CV has been associated with increased chloroplast stability, a delay in dark induced-senescence, and an enhanced tolerance to abiotic stress, whereas by contrast its overexpression results in premature leaf senescence (Wang and Blumwald, 2014; Sade et al., 2018; Ahouvi et al., 2022; Yu et al., 2022). In addition, a role for CV in mediating peroxisomal turnover and thereby contributing to the regulation of photorespiration and N assimilation in rice under elevated CO2, was also reported (Umnajkitikorn et al., 2020).
Although our knowledge of chloroplast degradation pathways has increased greatly during the last decade, the molecular hierarchy of these diverse pathways remains to be elucidated. Autophagy plays a key role in chloroplast degradation events, yet atg mutants have been shown to undergo early leaf senescence and accelerated degradation of chloroplast components upon diverse stress conditions (Thompson et al., 2005; Lee et al., 2013; Izumi et al., 2013; Barros et al., 2017, 2021; Hirota et al., 2018). These observations argue against a major role of autophagy in chloroplast degradation during senescence and highlight a possible connection between the different chloroplast degradation processes in response to stresses. In this context, we previously observed that the CV gene is highly induced in the absence of autophagy, contributing to the early-senescence phenotype observed in atg5 and atg7 mutants (Barros et al., 2017). Nevertheless, it remains unclear to which extent these pathways interact to control chloroplast stress responses.
Here, we investigated the significance of the CV pathway during carbon starvation. To this end, two previously described mutant lines with low expression of the CV pathway (amircv-1 and amircv-2 ) were characterized under dark-induced senescence conditions. Our results demonstrate that deficiency of CV alone only has minor effects on plant responses to extended darkness. We further assessed the relationship between CV and autophagy by analyzing amircv1xatg5 double-mutant plants that are characterized by a deficiency in both pathways. Although the amircv1xatg5 double mutants displayed a hypersensitive phenotype, similar to that observed in atg5 mutants under late stages of darkness, both CV and autophagy are likely required for chloroplast remodeling during these conditions. Our results further support the notion that autophagy is the preferred mechanism of chloroplast turnover under carbon-limiting conditions, while CV likely operates as a compensatory mechanism when autophagy is disrupted.