DISCUSSION
To cope with energetically challenging conditions, plants activate
strategic mechanisms of organelle disassembly, which generally culminate
in a senescence-induced response. During both developmental and
stress-induced senescence, chloroplast proteins are massively degraded,
and the amino acids released can be either remobilized for other tissues
or used as respiratory substrates (Araujo et al., 2011, Watanabe et al.,
2013, Hildebrandt et al., 2015; Chrobok et al., 2016). It is well known
that chloroplasts contain their own proteolytic machinery, comprising
several types of proteases that operate in their protein quality control
(Buet et al., 2019). This fact aside, recent studies have reported the
existence of extra-plastidial pathways for chloroplast turnover (Otegui
2017; Izumi and Nakamura, 2018; Woodson 2022). Three main
extra-plastidic processes involved in the degradation of chloroplasts
were identified: autophagy (Ishida et al., 2008; Wada et al., 2009),
SAVs (Otegui et al., 2005; Martínez et al., 2008), and CV (Wang and
Blumwald, 2014). It remains unknown, however, how these distinct
pathways are regulated and under which molecular hierarchy they ensure
proper chloroplast turnover. We previously suggested that CV might
function as a compensatory mechanism contributing to the sensitive
phenotype of atg mutants under carbon starvation (Barros et al.,
2017). Here, we provide additional experimental evidence of the
importance of CV and autophagy pathways for both metabolic responses and
chloroplast remodeling under extended darkness.
We first investigated the effects of the disruption of CV alone, on
plant tolerance to extended darkness. Thus, we focused on phenotypic and
metabolic characterization of previously described mutant lines
exhibiting deficient expression of the CV gene (Wang and
Blumwald, 2014). Accordingly, we verified that WT and amircv-1and amircv-2 plants displayed similar phenotypes under conditions
of extended darkness (Fig. 2). However, the reduced protein content
coupled with an elevated total free amino acid level after 3d of
darkness indicates that mechanisms of protein turnover are more rapidly
induced in the amircv mutants (Fig. 3). Furthermore, minor
metabolic differences were observed between the amircv mutants
and WT plants throughout the darkness treatment (Fig. 3 and 6).
Additionally, the accumulation of amino acids was not correlated with
differential activation of alternative respiration pathways in theamircv lines (Fig. 4). Collectively, these findings suggest that
the altered protein response observed in the amircv mutants does
not trigger significant effects on energetic metabolism.
It was previously demonstrated that silencing CV resulted in increased
chloroplast stability, whereas CV overexpression led to chloroplast
degradation by destabilizing the photosynthetic apparatus during abiotic
stress (Wang and Blumwald, 2014; Sade et al., 2018; Ahouvi et al., 2022;
Yu et al., 2022). By contrast, plants with enhanced autophagy were
fitter and displayed enhanced tolerance to oxidative stress (Minina et
al., 2018). Autophagy is a versatile mechanism of chloroplast
degradation since it mediates the turnover of piecemeal stroma
components and the degradation of entire chloroplasts by the chlorophagy
mechanism (Izumi and Nakamura, 2018). Experimental evidence has
suggested that the selective degradation of stromal proteins mediated by
autophagy, the RCB pathway, is preferentially activated to provide amino
acids in energy-starved plants (Hirota et al., 2018; Izumi et al.,
2019). Taken together, the data described above, coupled with our
results, suggest that autophagy is more likely to operate as a
pro-survival mechanism by the turnover of stromal components under
starvation conditions, while CV leads to the widespread degradation of
chloroplasts under specific abiotic stress conditions. Additionally, our
previous study indicated a higher activation of the CV pathway inatg mutants submitted to extended darkness (Barros et al., 2017).
Therefore, it is tempting to suggest that autophagy operates as a
primary chloroplast degradation pathway, while CV is most likely a
complementary mechanism activated in the absence of autophagy. By
contrast, a recent study revealed that CV‐silenced tomato plants display
stable chloroplast structure, ROS control, and delayed senescence in
response to extended darkness (Yu et al., 2022). Therefore, these
results open new questions regarding possibly contrasting roles of CV in
metabolic reprogramming in different plant species under starvation
conditions.
To unravel how these pathways modulate chloroplast turnover and
stress-induced senescence response, we further characterized double
mutants for both CV and autophagy pathways. Surprisingly, under extended
darkness, these amircv1xatg5 mutants displayed an early
senescence response accompanied by a reduction of chlorophyll levels,
resembling the atg5 single mutant phenotype (Fig. 5). The
metabolic analysis also demonstrated that tCV deficiency alone only
resulted in minor effects on metabolic reprogramming, whereas the
disruption of both autophagy and CV triggered similar effects as
deficiency inatg alone(Fig. 6 and 7).
Nevertheless, levels of specific
amino acids differed between amircv1xatg5 and atg5mutants. The amircv1xatg5 double mutants displayed more reduced
levels of arginine, aspartate, glutamate, isoleucine, serine and valine
after 10d of darkness compared to the atg5 mutants (Fig. 6).
Glutamine, glutamate, asparagine, aspartate, and their derivatives have
been long documented to be key compounds of nitrogen metabolism
(Masclaux-Daubresse et al., 2006; Gaufichon et al., 2016). The fact that
the amircv1xatg5 mutant displayed reduced levels of arginine,
aspartate and glutamate suggests a possible impairment of N
remobilization pathways in the amircv1xatg5 mutants.
Interestingly, the link between CV and N assimilation was previously
observed in rice CV mutants under water stress (Sade et al., 2018).
Under this condition, RNAiOsCV plants were characterized by altered
expression of N metabolism genes and differential activity of nitrate
reductase (NR) and glutamate dehydrogenase (GDH). It was additionally
demonstrated that CV interacts with glutamine synthase 2 (GS2) mediating
its degradation. Altogether these results were associated with the
activation of N assimilation in RNAiOs CV plants ensuring a
superior performance under water stress (Sade et al., 2018).
In the context of darkness-induced carbon starvation , the shift in
amino acid metabolism by CV deficiency is likely capable of compromising
plant survival, once amino acids are extensively used as energetic
substrates under these conditions. Indeed, the impairment of amino acid
release in atg mutants leads to energetic failure and
differential respiratory response under extended darkness conditions
(Barros et al., 2017). Interestingly, the amircv1xatg5 mutants
displayed ed lower accumulation of TCA cycle intermediates compared to
the atg5 single mutant (Fig. 7). Our previous study associated
the higher levels of organic acids to a higher flux through the
tricarboxylic acid cycle as a consequence of the respiratory activity in
Arabidopsis atg mutants (Barros et al., 2017). The accumulation
of TCA cycle intermediates during dark and developmental senescence has
previously been suggested as a symptom of higher respiration (Araujo et
al., 2010, 2011). In this context, the minor increases of organic acids
observed in the amircv1xatg5 mutant suggest that the respiratory
activation of the atg5 mutant is somehow restrained in the
absence of CV. In addition to chloroplasts, it has been shown that CV is
also important in peroxisome and possibly mitochondrial maintenance. In
this context, the OsCV rice mutant was characterized by a reduced number
of peroxisomes and mitochondria under elevated CO2levels (Umnajkitikorn et al., 2020). Accordingly, the activation of OsCV
mediates the removal of the OsPEX11 protein to the vacuole, triggering a
reduced number of peroxisomes and decreased catalase activity under
elevated CO2 conditions (Umnajkitikorn et al., 2020).
The role of CV in peroxisomal protein turnover was also demonstrated in
tomato plants submitted to extended darkness wherein SlCV interacts with
SlCAT3 leading to excessive accumulation of ROS (Yu et al., 2022).
Considering that energy-related pathways occur in different cell
compartments, it may be expected that CV participates in the
maintenance/degradation of other organelles in addition to chloroplasts.
Therefore, the reduced levels of TCA cycle intermediates in the double
mutants may also be triggered by a possible differential degradation of
peroxisome and mitochondria in the amircv1xatg5 mutants. The
accumulation of peroxisome and mitochondrial proteins in atgmutants has been extensively investigated (Shibata et al., 2013;
Yoshimoto et al., 2014; Li et al., 2014; Ma et al, 2021; Kacprzak and
van Aken, 2022). Despite that, the exact connection between CV and
autophagy in maintaining energy-related organelles still needs to be
addressed.
To decipher the importance of autophagy and CV pathways in chloroplast
maintenance, we paid particular attention to the analysis of chloroplast
ultrastructure. It was previously reported that atg5 mutants
presented marked changes in chloroplast structure following extended
darkness conditions (Barros et al., 2021). Here, we further demonstrated
that the disruption of both CV and autophagy leads to a differential
chloroplast ultrastructure under extended darkness. Accordingly, theatg5 mutant displayed deformed chloroplasts with a more
compromised stromal structure (Fig. 8). Although we observed certain
conservation of grana in the chloroplasts of the atg5 mutants,
the thylakoid membrane system was static and missed the typical
reorganization of darkened chloroplast observed in the WT andamircv-1 lines, characterized by curvature of thylakoid
membranes. On the other hand, theamircv1xatg5 chloroplasts partially recovered thylakoid membrane
organization, despite showing compromised granal stacking (Fig. 8). It
was previously observed that CV overexpression induced
chloroplast structural changes, characterized by the unstacking and
swelling of the thylakoid membranes which compromised chloroplast
stability (Wang and Blumwald, 2014; Yu et al., 2022). Interestingly, our
results show that downregulation of CV combined with the absence of
autophagy results in more compromised thylakoid and grana structures
compared to WT and to the correspondent single mutants under extended
darkness. It is worth mentioning that CV was previously proposed to
mediate the target of PsbO protein, altering the structure of the
photosystem II, and facilitating the access of the thylakoid-associated
proteases, such as DEGP1 and FTSH, to chloroplast core proteins (Wang
and Blumwald, 2014). The lack of chloroplast repair mediated by
proteases results in the accumulation of damaged chloroplast proteins,
generating ROS (Kato and Sakamoto et al., 2009), and possibly thereby
leading to the deregulation of chloroplast machinery. Therefore, it is
tempting to suggest that the selective turnover of envelope proteins by
the CV pathway contributes to chloroplast remodeling in the absence of
autophagy. It should also be highlighted that lipid composition directly
influences the biophysical properties of thylakoid membranes (Mazur et
al., 2019). Indeed, recent studies have reported massive changes in
galactolipids in plants lacking autophagy (Havé et al., 2019; McLoughlin
et al., 2020; Barros et al., 2021). However, the potential roles of CV
in chloroplast lipid composition remain to be addressed.
Collectively, the findings described here refine our understanding of
chloroplast degradation events during dark-induced senescence. By
contrast to the situation observed when autophagy is disrupted, the
impairment of CV has a minor impact on plant response to energy
deprivation. This highlights the preferential activation of
autophagy-mediated pathways to ensure chloroplast maintenance and
starvation response under extended darkness. Our data further suggest
that CV deficiency triggers minor energetic consequences when autophagy
is still present and activated. The further characterization ofamircv1xatg5 double mutants revealed a potential role of CV in
the metabolic response and chloroplast remodeling of the atg5mutant during extended darkness. Noteworthy, the early senescence
phenotype of the amircv1xatg5 mutants under extended darkness
highlights the possible operation of other catabolic pathways. The
presence of SAVs in atg5 chloroplast indicates the relevance of
this pathway in chloroplast turnover during these conditions. Dissecting
the intertwined mechanisms regulating chloroplast turnover are still
required to fully understand the exact relation between autophagy, CV
and SAVs on chloroplast maintenance and plant stress tolerance.
METHODS
Plant material and dark
treatment
Two Arabidopsis RNAi mutant lines for CV gene,amircv-1 and amircv-2 (Wang and Blumwald, 2014), the T-DNA
line atg5-1 (SAIL_129B079) and its correspondent WT (Columbia 0
ecotype) were used in this study. Seeds were surface-sterilized and
imbibed for 4 days at 4°C in the dark and subsequently germinated.
Seedlings grown at 22°C under short-day conditions (8 h light/16 h
dark), 60% relative humidity with 150 μmol photons
m-2 s-1. amircv mutants were
selected by application of Glufosinate-ammonium (120 mg/L) in ten-old
day seedlings. For dark treatments, selected seedlings were grown at
22°C under short-day for 4 weeks. Afterwards, plants were maintained in
dark in the same growth cabinet. The rosettes were harvested at
intervals of 0, 3, 7 and 10 days after transition to darkness and
immediately frozen in liquid nitrogen and stored at -80°C until further
analysis.