Additional file 1 of Control of vein-forming, striped gene expression by auxin signaling
Creators: name: Krishna, Anmol
name: Gardiner, Jason
name: Donner, Tyler J.
name: Scarpella, Enrico
Publisher: name: figshare
Year: 2021
Subjects: subject: Genetics
subject: FOS: Biological sciences
subject: Plant Biology

description: Additional File 1: Figure S1. ATHB8-, SHR-, and MP-Promoter-Driven Expression. (A–F) First leaves 4 DAG. Confocal laser scanning microscopy. Dashed white line: leaf outline. Top right: genotype. Bottom left: reproducibility index (number of samples with the displayed features / number of analyzed samples). (C) Co-expression of ATHB8::nCFP and SHR::nYFP during second loop formation. (E,F) Look-up table — ramp in G — visualizes YFP expression levels; blue: autofluorescence. Scale bars: (A,B,D–F) 25 μm; (C) 10 μm. Figure S2. MP::MP:YFP and MP::MP Functionalities in Vein Network Formation. Dark-field illumination of cleared first leaves 14 DAG. Top right: genotype. Scale bars: 0.5 mm. Figure S3. ATHB8 Expression Domains and MP and RIBO Expression Levels. First leaves 4 DAG. Confocal laser scanning microscopy. Top right: reporter. Dashed green outline: second loop nuclei expressing ATHB8::nCFP (A,B) or ATHB8::nYFP (D,E). (B,E) Look-up table — ramp in C — visualizes expression levels. Scale bars (shown, for simplicity, only in A and D): 5 μm. Figure S4. ATHB8 Expression Domains and RIBO Expression Levels. (A–E) First leaves 4 DAG. (A) Schematic of 4-DAG leaf — imaged in B–E — illustrating onset of ATHB8 expression (red) — imaged in B — associated with second loop formation [16, 22, 44]. Increasingly darker gray: progressively older ATHB8 expression domains. (B–E) Confocal laser scanning microscopy. (B) ATHB8::nYFP expression. (C) RIBO::nCFP expression. (D) Autofluorescence. (E) Overlay of images in B–D; red: ATHB8::nYFP expression; green: RIBO::nCFP expression; blue: autofluorescence. (F) RIBO::nCFP expression levels (mean ± SE) in nuclei at positions -2, -1, +1, and +2 — as defined in legend to Fig. 2 — relative to RIBO::nCFP expression levels in nuclei at position 0 — as defined in legend to Fig. 2 — during second loop formation. Difference between RIBO::nCFP expression levels in nuclei at position -2 or -1 and RIBO::nCFP expression levels in nuclei at position 0 was significant at P &lt; 0.001 (***) by One-Way ANOVA and Tukey’s Pairwise test. Sample population sizes: 27 leaves; position -2, 42 nuclei; position -1, 64 nuclei; position 0, 69 nuclei; position +1, 50 nuclei; position +2, 28 nuclei. Scale bars (shown, for simplicity, only in column 2): 5 μm. Figure S5. mp-11 and MP::MP Effects on MP Expression. MP transcript levels in mp-11 and MP::MP seedlings relative to MP transcript levels in WT (mean ± SE of three technical replicates for each of three biological replicates). Seedlings 4 DAG. RT-qPCR. Difference between mp-11 and WT, and between MP::MP and WT was significant at P &lt; 0.001 (***) by F-test and t-test with Bonferroni correction. Figure S6. ATHB8 Expression Domains and Levels in iaa12-1 and MP::VP16:bdlΔI;iaa12-1. (A,B) First leaves 4 DAG. Confocal laser scanning microscopy. Dashed white line: leaf outline. ATHB8::nYFP expression (look-up table — ramp in C — visualizes expression levels). Top right: genotype. Bottom left: reproducibility index (number of samples with the displayed features / number of analyzed samples). Scale bars: (A,B) 25 μm. Figure S7. Summary and Interpretation. A three-gene incoherent type-I feedforward loop [77] activates ATHB8 expression in narrow preprocambial stripes and leads to vein formation. MP receives the auxin input and activates expression of intermediate-loop AUX/IAA genes, which in turn inhibit MP expression [60, 64]. MP and AUX/IAA genes jointly regulate expression of the stripe gene ATHB8, which converts the auxin input into vein formation output. Arrows indicate positive effects. Blunt-ended lines indicate negative effects.