DEV030098 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Subcellular localization of VAN3 in veins. (A-C) VAN3-VENUS in veins. YFP fluorescence (A), DIC image (B) and their merged image (C). Hached square in A shows vein-forming cells. (D-F) VAN3-sGFP in veins of mutant plants. Control (D), cvp2cvl1-1 (E) and cvp2vab-1 (F). Scale bars: 50 µm in A-C; 30 µm in D-F.

• Supplemental Figure S2 -

  Fig. S2. GUS activity in van3-1 mutants complemented by the pVAN3::VAN3:GUS translational fusion construct. (A) Overview of a 14-day-old seedling. (B) A cotyledon. (C) A first leaf. (D) A third leaf. (E) Stomata. (F) Trichoms. (G) Flowers of a 37-day-old plant. (H) A torpedo stage embryo. (I) A mature embryo. (J) Lateral root primordium. (K) A primary root. (L) magnification of the image in K.

• Supplemental Figure S3 -

  Fig. S3. Vein patterns in Arabidopsis mutants defective in vascular continuity. (A-C) Vein patterns of cotyledon, the 1st leaves and the 2nd leaves in sfc9 mutants (A), sfc9cvp2cvl1-1 triple mutants (B), and sfc9vab-1 double mutants (C). (D-G) Vein patterns of cotyledon of vab-2 (D) and van3-2vab-2 (E) and the 1st leaves of vab-2 (F) and van3-2vab-2 (G). Scale bars: 1 mm.

• Supplemental Figure S4 -

  Fig. S4. (A-X) Continuous PIN1-expressing-cell fate specification in the leaf primodium in Arabidopsis mutants defective in vascular continuity. Wild type (A-C), cvp2cvl1-1 (D-H), vab-1 (I-L), sfc-9 (M-P), sfc-9cvp2cvl1-1 (Q-T) and sfc-9vab-1 mutants (U-X). Note that discontinuous files of cells expressing PIN1 were observed in mutants 5 days after germination. Fractions in the lower left corner showed proportions of continuous PIN1 expression veins in each genotype. Region of non-connective veins in G left (enlarged in D), G right (enlarged in H), K (enlarged in L), O (enlarged in P), S (enlarged in T) and W (enlarged in X) were outlined by hatched square. Scale bars: 10 µm in A,E,I,M,Q,U; 20 µm in B,F,J,N,R,V; 50 µm in C,G,K,O,S,W.

• Supplemental Figure S5 -

  Fig. S5. Yeast two-hybrid analysis of the interactions between VAN3 and VAB. VAN3 and VAB were used as either bait or pray. Interactions between two proteins were tested using the HIS3 reporter gene. AD, transcriptional activation domain; BD, DNA-binding domain.

• Supplemental Figure S6 -

  Fig. S6. Changes in autofluorescence of intrinsic tryptophan of AtARF1. (A,B) VAN3-dependent GTP hydrolysis in GTP-loaded AtArf1 was measured in the presence of 50 nM of GST-VAN3, 100 nM of different phospholipids and 0.5 µM myristoylated GTP-loaded AtARF1 (A) or GMPPNP-loaded AtARF1 (B). (C) Nucleotide exchange in the AtArf1 was monitored as the change in autofluorescence of intrinsic tryptophan in the presence of 50 nM of GST, 100 nM of different phospholipids and 0.5 µM myristoylated GTP-loaded AtARF1.