Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Mutations in conserved basic residues outside of domain II affect auxin responsiveness of an IAA17(1-111):LUC:NLS protein. IAA17(1-111:K31Q,R32Q):LUC:NLS (QQ) shows a greater reduction in luciferase activity than the wild type IAA17(1-111):LUC:NLS (WT) fusion protein following a 2 hr incubation with the synthetic auxin 5 μM 2,4-D. The samples have statistically distinguishable ratios at a p value of 0.05 using a Student’s t-test. Error bars as in 2B. Data for IAA17(1-111):LUC:NLS from 2 lines, 3 exp; IAA17(1-111:K31Q, R32Q):LUC:NLS from 2 lines, 3 exp.
• Supplemental Figure 2 - Full-length IAA20:LUC is long-lived and not responsive to auxin. (A) A 12 hr treatment with cycloheximide does not diminish the levels of the IAA20:LUC fusion protein or promote luciferase cleavage, compared to samples mock-treated for 3 hr. Immunoblot performed with anti-luciferase antibodies. Arrow points to IAA20:LUC protein observed in Figure 5C. Two additional IAA20:LUC bands migrate more quickly but appear to be equally stable to the most slowly-migrating band. An asterisk marks a non-specific cross-reacting band used as a loading control. The lane without IAA20:LUC contains extract from mock-treated seedlings expressing the IAA20:4xMyc protein, and the adjacent lane contains markers (M). The IAA20:LUC fusion protein runs larger than its predicted size of ~80 kDa . The predicted size of luciferase is ~61 kDa. There is no evidence of free luciferase in the IAA20:LUC samples. (B) Luciferase activity did not drop in Arabidopsis seedlings expressing IAA20:LUC following a 12 hr treatment with 25 μM 2,4-D. Graphed as in 2C. Data for IAA20:LUC from 3 lines in a total of 4 experiments.
• Supplemental Figure 3 - IAA20:4xMyc is long-lived and its levels do not drop in response to auxin. (A) A 12 hr treatment with cycloheximide does not diminish the levels of the IAA20:4xMyc fusion protein. Immunoblot performed with anti-Myc antibodies. Arrows point to two forms of the IAA20:4xMyc protein with different mobilities. Upper band migrates above the predicted molecular mass of approximately 27 kDa. Asterisks mark non-specific cross-reacting bands used as loading controls. Markers (M) are indicated. Lane without IAA20:4xMyc contains extract from mock-treated seedlings expressing IAA20:LUC. (B) IAA20:4xMyc levels do not drop following a 12 hr treatment with 5 μM 2,4-D. Immunoblot performed with anti-myc antibodies and labeled as in (A). Lane without 10xMyc:IAA20 contains extract from mock-treated domain II (D2):LUC:NLS seedlings treated in parallel to verify cycloheximide effectiveness.
• Supplemental Figure 4 - Subcellular localization of GFP:IAA20. Arabidopsis seedling roots constitutively expressing smGFP alone(A-D) or sGFP:IAA20 in two different transgenic lines (E-H and I,J) were stained with DAPI and visualized by fluorescence microscopy. B, D, F, </bH show enlarged portions ofA, C, E, and G respectively.
• Supplemental Figure 5 - Solvent mock control for auxin treatment does not affect Aux/IAA:LUC degradation. Standard half-life experiments were performed using seedlings expressing IAA28:LUC following no pre-treatment, or a 2 hr pre-treatment and continuing incubation with 0.1 M KOH diluted in GM. 0.1 M KOH serves as the solvent for 2,4-D and this mock treatment represents a control solution for a 5 μM 2,4-D treatment. Degradation curves are plotted for the untreated (solid) and solvent mock-treated (dashed) experiments. Data for untreated samples from 2 lines, 3 exp. Data for solvent mock-treated samples from 2 lines, 3 exp.