Figure 3. PP2-A13 works in parallel to autophagy.

(A) GUS staining of the PP2-A13_promoter::GUS transgenic line. Right: zoom-in view of the white box area in the left image. Scale bar = 1mm. (B) PP2-A13-GFP was co-expressed with a nuclear marker mCherry-VirD2NLS in Arabidopsis protoplasts. Scale bar = 10 μm. (C) qRT-PCR of ATG8a from 6-week-old wild-type (Col, black) and pp2-a13–1 mutant (orange) grown in 8L:16D. IPP2 was used as an internal control. Error bars indicate SD (n = 3). **, p⩽0.01 (Welch’s t-test) (D) Immunoblot analysis of the pp2-a13–1 mutant. Crude protein extracts of 11-week-old wide-type (Col) and pp2-a13–1 mutant were subjected to SDS-PAGE and immunoblot analysis with anti-ATG8a antibody. Equal protein loads were confirmed by immunoblot analysis with anti-Actin antibody. (E) Phenotypes of Col (WT), pp2-a13–1, atg5–1, and atg7–2 mutants in response to nitrogen starvation and continuous darkness. One-week-old Col (WT), pp2-a13–1, atg5–1, and atg7–2 seedlings germinated on 1/2 MS media plates were transferred to nitrogen rich (+N) or nitrogen deficient (−N) media and grown in 8L:16D or 24D growth conditions. The seedlings were photographed at 7 days after treatment. (F) Representative images of wild-type (Col), pp2-a13–1, atg5–1, atg7–2, atg5–1 pp2-a13–1, and atg7–2 pp2-a13–1 mutant plants grown in 16L:8D for 28 days or 8L:16D for 87 days. Scale bar = 2 cm in 16L:8D and 3 cm in 8L:16D. (G) Aerial fresh weight of wild-type (Col), pp2-a13–1, atg5–1, atg7–2, atg5–1 pp2-a13–1, and atg7–2 pp2-a13–1 mutant plants grown in 16L:8D and 8L:16D. Different letters indicate significant differences as determined by one-way ANOVA followed by Dunnett’s T3 multiple comparison test; p⩽0.05. Error bars indicate SD (n = 3–5). (H) Immunoblot analysis of ATG5 in wild-type (Col), pp2-a13–1, atg5–1, and atg7–2 mutant plants. Asterisk indicates protein cross-reacting with ATG5 antibody.