Phenotypes of amircv mutant lines
To examine the metabolic consequences of CV impairment, we used two previously described micro-RNA mutants lines, namely amircv-1 andamircv-2 (Wang and Blumwald 2014). The low expression of theCV gene was initially confirmed by RT-qPCR (Supplemental Fig. S1). We next investigated the physiological consequences of CV impairment under optimal conditions. Photosynthetic parameters were determined in mature plants of amircv mutants and WT grown under short-day conditions. No differences were observed in net assimilation rate (Fig. 1A), stomatal conductance (Fig. 1B), and internal CO2 concentration (Fig. 1C) of amircv-1 andamircv-2 lines compared to WT. This result is in line with previous data, demonstrating that the CV pathway functions in chloroplast degradation mainly under abiotic stress (Wang and Blumwald 2014; Sade et al., 2018; Ahouvi et al., 2022). Accordingly, rice and tomato CV knockdown mutants exhibited higher photosynthesis rates only when submitted to water and salt stress, while similar photosynthetic rates were observed under optimal conditions (Sade et al., 2018; Ahouvi et al., 2022).
Given that the induction of the CV gene during energy depletion conditions has been previously demonstrated (Barros et al., 2017, 2022; Yu et al., 2022), we next transferred 4-week-old plants to extended darkness, a system previously used to prove the metabolic importance of autophagy and alternative pathways of respiration in plants (Ishizaki et al., 2005; Araújo et al., 2010; Barros et al., 2017). After 10d of continuous darkness, both WT and amircv plants exhibited only limited signs of leaf senescence (Fig. 2A). To obtain a detailed analysis of leaf senescence parameters, we measured the total chlorophyll content and the maximum variable fluorescence/maximum yield of fluorescence (F v/F m). Chlorophyll loss coupled with reductions of F v/F m were observed throughout the experiment (Fig. 2B and C). In agreement with the visual phenotype reported, no significant differences in chlorophyll levels andF v/F m were verified in amircv lines under extended darkness conditions.