Down-regulation of CV has minor effects on starvation
response
To elucidate the role of the CV pathway in plant energetic metabolism,
we further conducted a metabolic characterization of amircvplants. Thus, the levels of starch, protein, sugars, and organic acids
were determined (Fig. 3). As might be expected, sugars (sucrose,
fructose, and glucose) and starch declined rapidly from 3d of dark
treatment in all genotypes (Fig. 3A-D). Both fumarate and malate were
initially reduced in WT and the amircv lines at 3d. Fumarate
levels were further slightly increased throughout the darkness treatment
(Fig. 3E-F). This result is in accordance with the dual pattern of TCA
cycle intermediates commonly observed during extended darkness
conditions (Ishizaki et al., 2005; Araújo et al., 2010; Barros et al.,
2017; Kamranfar et al., 2018). During both developmental and induced
senescence, plant cells undergo massive changes in cellular metabolism
and activate progressive hydrolysis of macromolecules (Watanabe et al.,
2013; Sade et al., 2018). Besides sugars, catabolism of proteins,
lipids, and chlorophyll provides energetic substrates that allow the
continued operation of mitochondrial respiration (Araújo et al., 2011;
Hortensteiner & Krautler, 2011; Barros et al., 2020). Accordingly,
following the exhaustion of carbohydrate reserves, during extended
darkness, a drop in protein content was observed (Fig. 3G). It is worth
mentioning that the amircv mutants presented a slightly higher
reduction of protein content in the early stage of darkness. Protein
degradation during stress conditions and senescence leads to a generally
increased amino acid accumulation (Araujo et al., 2011; Watanabe et al.,
2013). Consistently, the enhanced protein degradation resulted in a
slight increase of total amino acids in the amircv mutants after
3d of darkness (Fig. 3H). Despite this, the amircv lines and WT
presented relatively similar metabolic responses throughout the darkness
treatment.
Amino acids are the main alternative substrates to provide energy to
alternative respiration pathways under carbon starvation (Ishizaki et
al., 2005,2006; Araújo et al.,2010,2011; Cavalcanti et al., 2017). More
specifically, branched-chain amino acids (BCAA) and lysine have been
extensively reported to participate in energy generation by the
alternative pathway mediated by the ETF-ETFQO system (Araújo et al.,
2010, 2011). We previously demonstrated that both CV and alternative
respiratory pathways are activated in autophagy mutants submitted to
extended darkness (Barros et al., 2017). Thus, to investigate the
interplay between these processes, the expression of selected genes
related to alternative respiration, BCAA, and lysine degradation was
investigated in the amircv mutants (Fig. 4). This analysis
revealed that the ETFQO gene is induced during the darkness
treatment (Fig. 4A). In a similar vein, the transcript levels ofIVDH , a key enzyme of BCAA degradation, was also up-regulated
under these conditions (Fig. 4B). The induction of ETFQO and BCAA
catabolism related genes during low energy and senescence conditions
have been extensively reported previously (Izumi et al., 2013, Chrobok
et al., 2016; Barros et al., 2017,2022; Hirota et al., 2018).
Nevertheless, similar induction of ETFQO and IVDHtranscripts was observed in WT and amircv mutant plants.
Furthermore, we analyzed the Lys-ketoglutarate reductase/saccharopine
dehydrogenase (LKR/SDH ) gene, which encodes a key enzyme of
lysine catabolism. A strong induction of LKR/SDH was observed
during darkness in all genotypes, with a trend of minor induction ofLKR/SDH in the amircv mutants after 10d of darkness (Fig.
4C). The enzyme LKR/SDH controls most of the lysine homeostasis,
presenting a complex regulation both at the transcriptional and
post-translational level (Arruda and Barreto, 2020). It was recently
reported that both CV and LKR/SDH are activated during
aging and abiotic stress by a common regulator, the RD26 transcription
factor (Kamranfar et al., 2018). Additional studies are required to
understand the possible connection between CV and lysine metabolism.
However, our results collectively suggest that CV downregulation
exerts a minor influence on the activation of energetic amino acid
catabolism and ETF/ETFQO pathways during extended darkness conditions.