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.