DEV035535 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Molecular characterization of transgenic 35S::Cyclin-GUS Arabidopsis lines. (A) Transgenic Arabidopsis lines segregating at a 3:1 ratio for the resistance marker towards kanamycin were further analyzed by Southern blot analysis in order to select transgenic plants with a single insertion of the transgene. 2.5 µg of genomic DNA were digested by HindIII, separated by electrophoresis on an agarose gel, transferred to a nylon membrane and hybridized with the GUS probe. The results are only shown for the representative lines that were further used for the histochemical GUS staining. The left panel presents data for CycB-GUS n°15 and CycBΔDbox-GUS n°8 and the right panel for CycA-GUS n°15 and CycAΔDbox-GUS n°23. MW: molecular weight marker in kb. (B) The transgene expression level was analyzed by gel blot analysis of total RNAs extracted from 3-week-old plantlets grown in vitro. 20 µg of total RNA were separated by electrophoresis on an agarose-formaldehyde gel, transferred to a nylon membrane and hybridized successively with the indicated probes. pBI refers to a 35S::GUS transgenic line used as control. (C) The quantification of GUS activity was performed with 3-week-old plantlets grown in vitro. Triplicate assays are shown for the selected representative transgenic lines. WS: wild-type Wassilevskija plants.

• Supplemental Figure S2 -

  Fig. S2. Molecular analysis of APC/C hypomorphic lines. (A) Scheme of the constructs used to create APC6 and APC10 hypomorphic mutants. In co-suppression RNAi lines, fragments of 323 bp and 456 bp were overexpressed under the control of the 35S promoter to knockdown APC10 and APC6 genes, respectively (lines APC10S and APC6S). In hairpin RNAi lines, the same fragments were cloned on both sides of the chalcone synthase intron into opposite orientation. This construct generates hairpin structured RNA that triggers RNAi silencing of APC10 and APC6, respectively (lines RNAi APC10 and RNAi APC6). (B) Expression of the endogenous APC10 and APC6 genes in several independent APC10S and APC6S hypomorphic lines using RT-PCR. (C) Molecular analysis of several independent RNAi APC6 and RNAi APC10 lines. The upper panel shows the accumulation of APC6/APC10 siRNAs in the RNAi lines by northern blot using an APC6/APC10 probe. The bottom panel shows the expression of the endogenous APC6/APC10 gene by RT-PCR.

• Supplemental Figure S3 -

  Fig. S3. Reduced APC/C activity increases the accumulation of the artificial cyclin-GUS substrate. GUS staining of rosette leaves from plants originating from a cross between the 35S:CycB1-GUS line (upper panel) or the 35S::ΔDboxCycB-GUS line (bottom panel) and the APC6S and APC10S hypomorphic lines.

• Supplemental Figure S4 -

  Fig. S4. APC/C hypomorphic mutants have no root phenotype but display vascular tissue disorganization. (A) Root growth. One-week-old seedlings were transferred on MS plate and grown vertically for 4 days to measure the root growth. The length, indicated in cm, represents the mean of 20 measurements from wild type (Col0), RNAi APC6 n°18 and 27 and the strong hobbit mutant (hbt¹⁹³⁵). Scale bars: 0.5 cm. (B) (a,b) Transverse sections from hypocotyls from 8-day-old wild-type (a) and RNAi APC6 plants (b) stained with Toluidine Blue and viewed under bright-field illumination. e, endodermis; c, cortex; ep, epidermis; x+p, xylem+phloem. (c-f) Transverse sections from rosette leaf from 5-week-old wild-type (e,g) and RNAi APC6 plants (f,h) stained with Toluidine Blue and viewed under bright-field illumination. p, phloem; x, xylem. Scale bars: 50 µm.

• Supplemental Figure S5 -

  Fig. S5. P_VND7::GUS expression in APC/C hypomorphic mutants. GUS staining of APC/C hypomorphic lines crossed with the P_VND7::GUS marker line of the vascular tissues. (A-F) Comparison between P_VND7::GUS (A,C,E) and P_VND7::GUSxRNAi APC6 (B,D,F) lines. (A-D) Leaves and roots from 8-day-old seedlings. (E,F) Rosette leaves from 3-week-old plants. Scale bars: 0.5 mm. (G,H) Transverse sections from histochemical GUS staining of the apical part of the stem from 5-week-old P_VND7::GUSxRNAi APC6 plants, viewed under bright-field illumination. pi, pith; xy, xylem; co, cortex; ep, epidermis. Scale bars: 40 µm.