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.