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.