Effects of autophagy and CV deficiency on metabolic adjustment under extended darkness
To gain more insight into the differential impacts of CV and autophagy deficiency under extended darkness, we further analyzed the metabolic profile of amircv1xatg5 along with its parental lines,amircv-1 and atg5 (Fig. 6 and 7). In agreement with the phenotype observed, amircv-1 presented similar metabolic changes to WT plants, while atg5 and amircv1xatg5 exhibited more comparable responses. While the majority of amino acids were highly accumulated in WT and amircv-1 plants, only minor accumulations of arginine, glutamate, glycine, isoleucine, lysine, serine, threonine and valine were observed in atg5 and amircvxatg5 after 10d of extended darkness (Fig. 6). We also found that the TCA cycle intermediates α-ketoglutarate, citrate, fumarate, malate, and succinate were highly accumulated in both atg5 and amircvxatg5mutants after 10d of darkness (Fig. 7). These results are consistent with previous findings demonstrating the impairment of amino acid provision coupled with an altered respiratory response in atgmutants under extended darkness (Barros et al., 2017; Hirota et al., 2018).
Despite the similarities with the atg5 mutant,amircv1xatg5 also showed specific metabolic signatures. The general accumulation of organic acids was lower in theamircv1xatg5 mutant compared to the atg5 mutant after 10d of darkness (Fig. 7). Additionally, the amircv1xatg5 mutant displayed lower accumulation of arginine, aspartate, glutamate, isoleucine, serine and valine levels in the later stages of darkness (Fig. 6). Glutamate, glutamine, asparagine, aspartate are important components of nitrogen assimilation, recycling, transport and storage in plants (Gaufichon et al., 2010). The coordination between CV and nitrogen assimilation pathways was previously proposed during water stress and elevated CO2 conditions wherein rice CV mutants presented altered levels of amino acids (Sade et al., 2018; Umnajkitikorn et al., 2020). On the other hand, the disruption of autophagy impacts the supply of amino acids and respiratory rates under carbon depletion (Izumi et al., 2013; Avin-Wittenberg et al., 2015; Barros et al., 2017; Hirota et al., 2018). Collectively, our results indicate that a deficiency solely of CV has only minor effects on plant responses to extended darkness, and suggests that the metabolic reprogramming of atg5 mutants under extended darkness is, at least partially, dependent on the operation of a functional CV pathway.