DEV02554 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Amino acid sequence alignment of HVE and other CAND1 (TIP120) proteins. The HVE protein (At; NP_178360) of Arabidopsis thaliana appears aligned to homologous gene products from Drosophila melanogaster (Dm; NP_609389), Homo sapiens (Hs; NP_060918), Schizosaccharomyces pombe (Sp; NP_593286) and Caenorhabditis elegans (Ce; CAB04744). Residues conserved across three or more sequences are shaded black, and similar residues conserved across three or more sequences are shaded gray. Numbers correspond to amino acid positions. Mutations carried by the hve alleles are indicated. Dotted lines indicate the putative CUL1 binding domains of the CAND1 human protein, one of them encompassing 520 residues of the N-terminus, and the other 298 amino acids in the C-terminus, as described in Liu et al. (2002). The alignment was obtained using ClustalX v1.5b and shaded with Boxshade3.21 (http://www.ch.embnet.org/software/BOX_form.html).

• Supplemental Figure 2 -

  Fig. S2. (A) GST pull-down assay demonstrating direct interaction between CUL1 and HVE (CAND1) of Arabidopsis thaliana. GST-HVE, but not GST, was able to interact with CUL1, suggesting that, as its mammalian counterpart, CAND1 (HVE) regulates the activity of the SCF complexes by binding CUL1 in Arabidopsis thaliana. The “input” lane shows the signal from 10% of the amount of in vitro translated CUL1 protein present in each of the remaining samples. For the pull-down assay, the Arabidopsis thaliana At4g02570 gene, which encodes CUL1, was in vitro transcribed and translated using the Quick TNT7 rabbit reticulocyte lysate system (Promega) in the presence of 35S-Met, as described by del Pozo and Estelle (1999). To generate recombinant GST-HVE, the full-length cDNA of the At2g02560 gene was amplified by RT-PCR and cloned into the pGEM-TA vector (Promega). Afterwards, the At2g02560 cDNA was cloned into the pGEX-2TK vector (Pharmacia) and transferred to the BL21 expression strain. Expression of recombinant GST-HVE was induced for 2 hours with 1 mM IPTG at 30°C. Recombinant GST-HVE was then purified using Glutathione beads (Pharmacia) following the manufacturer’s instructions. For the pull-down, 1 ?g of GST or GST-HVE bound to beads was incubated with 25 ?l of 35S-CUL1 in 500 ?l of TBS (100 mM Tris-HCl, 150 mM NaCl, pH 7) plus 0.5% of NP40. After 2 hours of incubation at 30°C, beads were washed 4 times for 15 min each time and the pulled-down proteins were separated by SDS-PAGE and detected by autoradiography. (B) Visualization by semiquantitative RT-PCR of the transcription of the HVE gene in different tissues of wild-type Col-0 plants. gDNA: Genomic DNA. R: Root. Ro: Rosette. V: Vegetative leaf. C: Cauline leaf. S: Stem. B: Floral bud. F: Flower. M: Molecular weight marker. N: Negative control. OTC: ORNITHINE TRANSCARBAMYLASE gene, which was used as an internal control.

• Supplemental Figure 3 -

  Fig. S3. Transverse sections of the central region (A-D) and paradermal sections of the basal region (E-H) of Col-0 (A, B, E, F) and hve-3/hve-3 (C, D, G, H) first leaves, showing primary (pv; A, C, E, G) and secondary (sv; B, D, F, H) veins. In the mutant, the primary vein was narrower but the secondary veins looked normal. Cell shape and size were normal in the epidermis (ep), palisade mesophyll (pm) and spongy mesophyll (sm) of hve-3. However, the spongy mesophyll had larger air spaces and the palisade mesophyll was partially disorganized in the mutant (C, D). A midrib (mr) was clearly distinguishable abaxial to the midvein in the wild type (A), but seemed to be absent in the mutant (C). The structure of bundle sheath cells (arrowheads in F and H), sieve tubes and tracheary elements was normal in the mutant. Plant material was collected 21 days after sowing.

• Supplemental Figure 4 -

  Fig. S4. Visualization of ATHB-8-GUS transgene expression in (A-C) Ws-2 and (D-F) hve-1/hve-1 genetic backgrounds. First (A, D), third (B, E) and fifth (C, F) node leaves are shown. The hve-1 mutant shown here had been outcrossed twice to Ws-2. Pictures were taken 20 days after sowing.

• Supplemental Figure 5 -

  Fig. S5. Visualization of DR5-GUS transgene expression in the Col-0 and hve-3/hve-3 genetic backgrounds. (A-F) Col-0 first (A), third (B), seventh (C), ninth (D), tenth (E), and eleventh (F) leaves. (G-L) hve-3/hve-3 first (G), third (H), seventh (I), eleventh (J), thirteenth (K) and fourteenth (L) leaves. Plant material was collected 21 days after sowing. Scale bars represent 1 cm (A-C, G-I), 250 μm (D) and 100 μm (E, F, J-L).

• Supplemental Figure 6 -

  Fig. S6. Root growth inhibition by 2,4-D in the hve mutants. Error bars indicate standard deviations (n=10). The reduced leaf size, bushy inflorescence, simple venation pattern and suppressed root waving suggested that the hve mutants are deficient in auxin perception or biosynthesis. Hence, we studied in the hve mutants some responses that are known to be altered in auxin-related mutants. The figure shows that moderate 2,4-D resistance was displayed by hve-1/hve-1 and hve-3/hve-3 seedlings sown on non-supplemented medium and transferred 4 days later to 10, 50, 100, 200 and 500 nM 2,4-D medium. These results provide evidence supporting the involvement of HVE in auxin responsiveness. On the other hand, we found no differences between hve-1/hve-1, Ws-2 and En-2 plants in the gravitropic response of their roots, since all of them displayed a normal gravitropic response and were able to reorientate their growth in response to the rotation of the Petri dishes where the seeds had been sown. In addition, hve-1 and hve-3 did not cause a constitutive photomorphogenic response under our working conditions, as shown in homozygous plants grown for 8 days in the dark, which displayed elongated hypocotyls and unexpanded cotyledons, just as the wild-type ones did. Vascular patterning and leaf shape abnormalities similar to those already described for the wild type (Mattsson et al., 1999; Sieburth et al., 1999) were displayed by hve-1/hve-1 and Ws-2 plants grown in the presence of different concentrations of the auxin polar transport inhibitor TIBA (3,5-triiodobenzoic acid).

• Supplemental Figure 7 -

  Fig. S7. Rosettes and leaf venation patterns of single and double mutants showing altered vascular development. Rosettes and first leaf venation pattern photographs are shown for the Col-0 wild type and several single and double mutants. Pictures also include cotyledon vein patterns of cvp1-3, cvp2-1, hve-3 and their double mutants. All plants shown were homozygous for the indicated mutations. Plant material was collected 21 days after sowing. Venation images were obtained from cleared leaves using bright field optics. Scale bars represent 2 mm (rosettes) and 1 mm (venation patterns).