TRAF1a and TRAF1b Are Required for Autophagosome Formation. TRAF1a and TRAF1b Are Required for Autophagosome Formation. (A) MDC staining of wild-type (WT) and traf1a/b root cells after nitrogen and carbon starvation. One-week-old seedlings were starvation treated (–C and –N) for 16 h with 1 μM CA, followed by staining with MDC. The labeled autophagosomes were visualized by epifluorescence microscopy. Bars = 50 µm. (B) Numbers of puncta per root section in the root tips (left graph) and mature root cells (right graph) of the wild type and traf1a/b in (A). Data are average values ± sd (n = 3) calculated from three independent experiments. For each experiment, 15 sections were used for the calculation for each genotype. Asterisks indicate significant differences from the wild type (**P < 0.01 by Student’s t test). (C) Confocal analysis of eGFP-ATG8e/WT and eGFP-ATG8e/traf1a/b-1 lines. One-week-old eGFP-ATG8e/WT and eGFP-ATG8e/traf1a/b-1 lines were exposed to N- and C-sufficient (MS) or N- and C-deficient (–C and –N) conditions with 1 μM CA for 16 h and visualized by fluorescence confocal microscopy. Bars = 50 μm. (D) Numbers of puncta per root section in the root cells of the wild type and traf1a/b in (C). Data are average values ± sd (n = 3) calculated from three independent experiments. For each experiment, 15 sections were used for the calculation for each genotype. Asterisks indicate significant differences from the wild type (**P < 0.01 by Student’s t test). (E) ATG (ATG8, ATG7, ATg1a, and ATG13a) protein levels in the wild type and traf1a/b-1 double mutant after carbon starvation (left images) and nitrogen starvation (right images) treatments for the indicated times. Ponceau S-stained membranes are shown below the blots to indicate the amount of protein loaded per lane. The numbers on the left indicate the molecular mass (kD) of the size markers. hpt, hours post-treatment. Hua Qi et al. Plant Cell 2017;29:890-911 ©2017 by American Society of Plant Biologists