Protoplasts were isolated from 20‐d‐old leaves and transformed as described previously (Wu et al., 2009 (link)). Protoplasts were observed by epifluorescence microscopy (Carl Zeiss Axio Imager.A2; Carl Zeiss) using a fluorescein isothiocyanate (FITC) filter, and protoplasts with more than three visible autophagosomes were counted as active for autophagy as previously described (Yang et al., 2016 (link); Pu et al., 2017 (link)). One hundred protoplasts were analyzed per treatment per genotype and the experiment was repeated three times. For sucrose starvation, transformed protoplasts were incubated in W5 solution without sucrose or with 0.5% (w/v) sucrose as control at room temperature for 36 h in dark before assessing autophagy.
Significance of basal autophagy levels was assessed by two‐sample t‐test whereas a generalized linear model with treatment and genotype as factors and controlling for random effects of replicates was used for autophagy levels under sucrose starvation. A P‐value < 0.05 was used as a cut‐off on both cases.
Significance of basal autophagy levels was assessed by two‐sample t‐test whereas a generalized linear model with treatment and genotype as factors and controlling for random effects of replicates was used for autophagy levels under sucrose starvation. A P‐value < 0.05 was used as a cut‐off on both cases.