Fig 1: Overexpression of FYVE1ΔC mutant impedes glutamine-responsive TORC1 activation(A and B) 35Spro:HA-FYVE1 and 35Spro:HA-FYVE1ΔC transgenic seedlings were treated with 1 mM L-glutamine for 20 min, and the total extracts were subjected to immunoblotting. UGPase was used as a loading control. The ratio of phosphorylated/total Rps6, normalized to the values for each line to which glutamine was not added, is shown for representative lines in B). Error bars represent the standard deviation (n = 3 independent experiments), and significance was determined by a two-tailed Student’s t test. Statistically significant differences are indicated with asterisks, (∗∗) representing p < 0.01 and (∗∗∗) representing p < 0.001.(C and D) Root-cultured cell derived protoplasts transformed with plasmids harboring both 35Spro:S6K and indicated 35Spro:FYVE1 mutants were treated with 1 mM L-glutamine for 10 min, and the total extracts were subjected to immunoblotting. The ratio of phosphorylated/total S6K normalized to the values for each transformant to which glutamine was not added in D). Error bars represent the standard deviation (n = 3 independent experiments). Significance was determined by a two-tailed Student’s t test. Statistically significant differences are indicated with (∗∗) representing p < 0.01 and (∗∗∗) representing p < 0.001.(E) Photographs of wild-type, 35Spro:HA-FYVE1, and 35Spro:HA-FYVE1ΔC seedlings grown for 7-day on 1/2 MS (+N−G), 1/4 MS with glucose (+N+G), and grown for 9 days on 1/4 MS without nitrogen but with glucose (−N+G). Scale bars: 1 cm.(F) Boxplots of primary root length of 7-day-old seedlings grown on 1/2 MS (+N−G), 1/4 MS with glucose (+N+G), and of 9-day-old seedlings grown on 1/4 MS without nitrogen but with glucose (−N+G) tested for wild-type (n = 55, 57, and 57 seedlings, respectively), 35Spro:HA-FYVE1 (n = 55, 55, and 54 seedlings, respectively), and 35Spro:HA-FYVE1ΔC (n = 53, 52, and 52 seedlings, respectively). Error bars represent standard deviation among the independent samples and the cross and the horizontal lines in each box represent mean and median values, respectively. Statistically significant differences between the lines are indicated with asterisk (∗) representing p < 0.05, (∗∗) representing p < 0.01, and (∗∗∗) representing p < 0.001, based on a two-tail t test assuming unequal variance between the lines.(G) PIN2 levels in seedlings of wild-type (Col-0) and indicated transgenic lines. Total extracts were subjected to immunoblotting with anti-PIN2 antibody. Actin was used as a loading control. See also Figures S2, S3, and S4.