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First published online 31 March 2009
doi: 10.1242/dev.035535

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The APC/C E3 ligase remains active in most post-mitotic Arabidopsis cells and is required for proper vasculature development and organization

Katia Marrocco, Alexis Thomann^*, Yves Parmentier, Pascal Genschik and Marie Claire Criqui

Institut de Biologie Moléculaire des Plantes du CNRS, 12 rue du Général Zimmer, 67084 Strasbourg Cedex, France.

View larger version (50K): [in this window] [in a new window]   Fig. 1. CDH1-related and APC/C genes remain expressed in postmitotic cells. (A) GUS staining of 1 mm, 2 mm, 3 mm and 5 mm long leaves from a ProCycB1;1::NterCycB1;1-GUS line. Only dividing cells are stained blue. (B) RT-PCR on RNA extracted from leaves of the ProCycB1;1::NterCycB1;1-GUS line at different developmental stages using specific primers for CDC20-1, CDC20-2, CCS52A1, CCS52B, APC3/CDC27A, APC4, APC6, APC10, UBC19 and UBC20. RT-PCR on AtCycB1;1 and EF1 are used as controls. 1, 2, 3, 5 correspond to the leaf stages and G to genomic DNA.

View larger version (43K): [in this window] [in a new window]   Fig. 2. CycA-GUS and CycB-GUS reporter proteins are degraded in a cell cycle-dependent manner. (A) Schematic structure of the constructs inserted into the binary vector pBI121 under the control of the CaMV 35S promoter. Green and orange boxes represent the N-terminal domain of the cyclin A3 and cyclin B1, respectively, and blue boxes indicate the GUS sequence. Functional Dbox is highlighted in red, in contrast to the mutated Dbox. (B-F) Oscillation of GUS activity (blue curves) during the cell cycle in transgenic BY2 cell lines. After aphidicolin removal, the progression through the cell cycle was monitored by 3H-thymidine incorporation (green curves) and mitotic index determination (red curves). Quantitative GUS assays presented in this figure were obtained with the following transgenic cell lines 35S::GUS (B), 35S::CycA-GUS (C,E), 35S::CycB-GUS (D) and 35S::CycADbox-GUS (F).

View larger version (121K): [in this window] [in a new window]   Fig. 3. CycB-GUS reporter activity indicates that APC/C is active in post-mitotic plant cells. (A,B) Histochemical GUS staining of 6-day-old 35S::CycB-GUS (A) and 35S::CycBDbox-GUS (B) seedlings grown in vitro. The CycB-GUS reporter protein stains only regions of cell division, whereas the CycBDbox-GUS reporter protein accumulates in dividing and non-dividing cells. (C) Histochemical GUS staining of 35S::CycB-GUS seedlings treated with 100 µM MG132 (+) or mock treated (-) for 14 hours. (D-L) Histochemical GUS staining of 11-day-old 35S::CycB-GUS (D,K) and 35S::CycBDbox-GUS (E,L) plantlets, and 21-day-old 35S::CycBGUS (F,G,H,J) and 35S::CycBDbox-GUS (I) plantlets grown in vitro. The pictures show representative distribution pattern of GUS staining in leaves (D,E,F,G), trichomes (H,I) and roots (J,K,L). Scale bars: 1 mm in A,B; 5 mm in C; 100 µm in D-I.

View larger version (133K): [in this window] [in a new window]   Fig. 4. CycA-GUS reporter protein shows broader GUS staining distribution. (A,B) Histochemical GUS staining of 6-day-old 35S::CycA-GUS (A) and 35S::CycADbox-GUS (B) seedlings grown in vitro. (C-I) Histochemical GUS staining of 11-day-old 35S::CycA-GUS (D) and 35S::CycADbox-GUS (E) plantlets and 21-day-old 35S::CycA-GUS (C,F,G,H) and 35S::CycADbox-GUS (I) plantlets grown in vitro. The pictures show representative distribution pattern of GUS staining in hypocotyl (C), leaves (D-F) and roots (G-I). Scale bars: 1 mm in A,B; 100 µm in C-I.

View larger version (74K): [in this window] [in a new window]   Fig. 5. APC hypomorphic mutants show various leaf developmental alterations, including shape, cell size and endoreduplication. (A) One-week-old seedlings of in vitro grown wild-type and APC/C hypomorphic lines (upper panel) and 3-week-old plants of wild-type and APC/C hypomorphic lines grown in soil (bottom panel). (B) Venation patterns of cleared cotyledons from wild-type and RNAi APC6 (18) hypomorphic line. Scale bars: 1 mm. (C) Adult rosette leaves from wild-type and RNAi APC6 lines. Scale bar: 1 cm. (D) Epidermal cell sizes were measured in rosette leaf from 3-week-old wild-type (Col0) and different RNAi APC6 hypomorphic lines (numbered 1 to 20) and from 3-week-old wild-type (Col0) and APC6S-9 and APC10S-8 hypomorphic lines. T-tests were performed for each value compared with wild type to determine significant differences. Asterisks indicate values for which P<0.05. Scale bars: 40 µm in Col0 and line RNAi APC6-1; 50 µm in Col0, APC6S and APC10S. (E) DNA contents were measured on cells from the fifth rosette leaf of 4-week-old wild-type (Col0) and APC6 and APC10 hypomorphic lines (RNAi APC6-1, RNAi APC10-38, APC6S-4 and APC10S-6) using flow cytometry.

View larger version (92K): [in this window] [in a new window]   Fig. 6. APC/C hypomorphic mutants display a broomhead-like phenotype and vascular tissue disorganization. (A) Five-week-old plants of APC10S-6 hypomorphic line in comparison with wild type (a). Closer view of the `broomhead' phenotype of APC10S-6 (b), APC6S-4 (c), RNAi APC6-20 (d) and RNAi APC10-38 (e) hypomorphic lines. (B) Transverse and longitudinal sections from the apical part of the stem from 5-week-old wild-type (a,c,g) and APC10S plants (b,d,h) stained with Toluidine Blue and viewed under bright-field illumination. pi, pith; xy, xylem; p, phloem; pc, procambium; co, cortex; ep, epidermis; if, interfascicular cells; vb, vascular bundle; sc, sclerenchyma. Scale bars: 40 µm. Transverse sections from the base of the stem from 5-week-old wild-type (e) and RNAi APC6 (f) plants stained with Toluidine Blue and viewed under bright-field illumination. vc, vascular cambium. Scale bars: 50 µm.

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