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Functional characterization of tomato Sl-IAA3 and Sl-hls genes. Role ...

Functional characterization of tomato Sl-IAA3 and Sl-hls genes. Role ...

Chapitre II:

Chapitre II: Sl-IAA3, a Tomato Aux/IAA at the Crossroads of Auxin and Ethylene Signalling Supplemental data Supplemental Figure 1. Supplemental Figure 1. Evolutionary Relationship of Sl-IAA3 and the Most Closely Related Tomato and Arabidopsis Aux/IAA Proteins. The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The optimal tree with the sum of branch length = 1.02850951 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method (Zuckerkandl and Pauling, 1965) and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). There were a total of 156 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4 (Tamura et al, 2007). Supplemental Figure 2. Supplemental Figure 2. Subcellular Localization of Sl-IAA3 Protein. Sl-IAA3:GFP fusion protein was transiently expressed in BY-2 tobacco protoplasts and sub-cellular localization was analyzed by confocal laser scanning microscopy. The merged pictures of the green fluorescence channel (left panels) and the corresponding bright field (middle panels) are shown (right panels). A. Control cells expressing GFP alone. B. Cells expressing the Sl-IAA3:GFP fusion protein. The scale bar indicates 10 µm. 81

Chapitre II: Sl-IAA3, a Tomato Aux/IAA at the Crossroads of Auxin and Ethylene Signalling Supplemental Figure 3. Mean fluorescence (a.u.) 60 50 40 30 20 10 0 DR5:GFP DR5:GFP + 35S-IAA3 DR5:GFP + 35S - 2,4-D + 2,4-D Supplemental Figure 3. Sl-IAA3 Protein Represses the In Vivo Activity of DR5. Tobacco protoplasts were transformed either with the reporter construct (DR5:GFP) alone or with both the reporter and effector constructs (35S-IAA3) and incubated in the presence or absence of 50 µM 2,4-D. GFP fluorescence was measured 16 h after transfection. A mock effector construct lacking Sl-IAA3 was used as a control for the cotransfection experiments. Transformations were performed in triplicate. Mean fluorescence is indicated in arbitrary unit (a.u.) ± SE. Supplemental Figure 4 Hypocotyl curvature (degree) A B 80 60 40 20 0 WT AS-IAA3-1 1 2 3 4 5 Time (hours) Root curvature (degree) 40 30 20 10 0 16 hours Supplemental Figure 4. Gravitropic response of hypocotyls and roots in wild type and antisense plants. Seedlings grown for 4 days in darkness on vertically held plates were turned 90°, and the angle of the hypocotyls ( A) and roots (B) curvature was measured at the indicated time. Values shown represent the mean ± SE of two independent experiments, in which 20 seedlings was measured. WT AS-IAA3-1 82

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