Autophagy Is Accelerated by Nitrogen or Fixed-Carbon Starvation in Maize.(A) atg12 mutants block the release of free YFP from YFP-ATG8a. Autophagy Is Accelerated by Nitrogen or Fixed-Carbon Starvation in Maize.(A)atg12 mutants block the release of free YFP from YFP-ATG8a. Wild-type W22, atg12-1, and atg12-2 seedlings expressing YFP-ATG8a were grown on soil for 2 weeks and the second leaf blade (L2) was either kept in the light (+) or subjected to fixed-carbon (C) starvation by covering a section with aluminum foil for 2 d (−). Total protein extracted from a mix of three to six leaves was subjected to SDS-PAGE followed by immunoblot analysis with anti-GFP antibodies. YFP-ATG8a and free YFP are indicated by closed and open arrowheads, respectively. Immunodetection of histone H3 was used to confirm near equal protein loading. Two independent experiments gave similar results.(B) Vacuolar release of free YFP from YFP-ATG8a is increased as leaves age or upon nitrogen (N) or fixed-C starvation. Tissue analyzed included the L1, L2, and L3 leaves (numbers correspond to the order of emergence) of plants grown in N-rich soil (left panel), the L2 leaf blade subjected to fixed-C starvation as in (A), and roots from plants grown hydroponically in high-N liquid medium for 10 d and then either kept on high-N (+) or transferred to low-N (−) liquid medium for 2 d (right panel). Total protein extracts were subjected to SDS-PAGE followed by immunoblot analysis as in (A). YFP-ATG8a and free YFP are indicated by closed and open arrowheads, respectively.(C) N starvation induces the accumulation of autophagic vesicles. YFP-ATG8a plants were grown hydroponically in high-N liquid medium for 10 d and then either kept on high-N (+N) or transferred to low-N (−N) liquid medium for 2 d. Leaf epidermal cells were examined for autophagosomes and autophagic bodies by confocal fluorescence microscopy. Bar = 10 μm. Faqiang Li et al. Plant Cell 2015;27:1389-1408 ©2015 by American Society of Plant Biologists