Fig. 5

ATHB8 Expression Domains and Auxin Levels. First leaves 2, 3, and 4 DAG. Columns 1–3: confocal laser scanning microscopy. Column 1: ATHB8::nQFP expression (red) associated with formation of midvein (2 DAG), first loop (3 DAG), or second loop (4 DAG) [16, 22, 44]. Column 2: Ratio of RPS5A::mDII:nRFP expression to RPS5A::DII:nYFP expression. Look-up table visualizes expression ratio levels: high RPS5A::mDII:nRFP/RPS5A::DII:nYFP ratio (green) indicates high auxin levels; low RPS5A::mDII:nRFP/RPS5A::DII:nYFP ratio (blue) indicates low auxin levels. Column 3: overlays of images in columns 1 and 2; blue: low RPS5A::mDII:nRFP/RPS5A::DII:nYFP ratio, i.e. low auxin levels; yellow: co-expression of ATHB8::nQFP (red) and high RPS5A::mDII:nRFP/RPS5A::DII:nYFP ratio (green), i.e. high auxin levels. Column 4: Ratio of RPS5A::mDII:nRFP expression levels to RPS5A::DII:nYFP expression levels (mean ± SE) in nuclei flanking ATHB8::nQFP-expressing nuclei (positions “-2”, “-1”, “+1”, and “+2”) relative to ratio of RPS5A::mDII:nRFP expression levels to RPS5A::DII:nYFP expression levels in nuclei co-expressing ATHB8::nQFP (position “0”) during formation of midvein (top), first loop (middle), or second loop (bottom). Difference between ratio of RPS5A::mDII:nRFP expression levels to RPS5A::DII:nYFP expression levels in nuclei at position -2, -1, +1, or +2 and ratio of RPS5A::mDII:nRFP expression levels to RPS5A::DII:nYFP expression levels in nuclei at position 0 was significant at P < 0.01 (**) or P < 0.001 (***) by One-Way ANOVA and Tukey’s Pairwise test. Sample sizes: 26 (2 DAG), 27 (3 DAG), or 29 (4 DAG) leaves; position -2: 56 (2 DAG), 42 (3 DAG), or 60 (4 DAG) nuclei; position -1: 52 (2 DAG), 37 (3 DAG), or 58 (4 DAG) nuclei; position 0: 74 (2 DAG), 85 (3 DAG), or 102 (4 DAG) nuclei; position +1: 44 (2 DAG), 44 (3 DAG), or 62 (4 DAG) nuclei; position +2: 42 (2 DAG), 25 (3 DAG), or 44 (4 DAG) nuclei. Scale bars (shown, for simplicity, only in column 2): 5 μm