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. 2012 Aug 21;109(34):13853-8.
doi: 10.1073/pnas.1211418109. Epub 2012 Aug 6.

SAMBA, a plant-specific anaphase-promoting complex/cyclosome regulator is involved in early development and A-type cyclin stabilization

Nubia B Eloy  1 , Nathalie Gonzalez, Jelle Van Leene, Katrien Maleux, Hannes Vanhaeren, Liesbeth De Milde, Stijn Dhondt, Leen Vercruysse, Erwin Witters, Raphaël Mercier, Laurence Cromer, Gerrit T S Beemster, Han Remaut, Marc C E Van Montagu, Geert De Jaeger, Paulo C G Ferreira, Dirk Inzé
Affiliations

SAMBA, a plant-specific anaphase-promoting complex/cyclosome regulator is involved in early development and A-type cyclin stabilization

Nubia B Eloy et al. Proc Natl Acad Sci U S A. .

Abstract

The anaphase-promoting complex/cyclosome (APC/C) is a large multiprotein E3 ubiquitin ligase involved in ubiquitin-dependent proteolysis of key cell cycle regulatory proteins, including the destruction of mitotic cyclins at the metaphase-to-anaphase transition. Despite its importance, the role of the APC/C in plant cells and the regulation of its activity during cell division remain poorly understood. Here, we describe the identification of a plant-specific negative regulator of the APC/C complex, designated SAMBA. In Arabidopsis thaliana, SAMBA is expressed during embryogenesis and early plant development and plays a key role in organ size control. Samba mutants produced larger seeds, leaves, and roots, which resulted from enlarged root and shoot apical meristems, and, additionally, they had a reduced fertility attributable to a hampered male gametogenesis. Inactivation of SAMBA stabilized A2-type cyclins during early development. Our data suggest that SAMBA regulates cell proliferation during early development by targeting CYCLIN A2 for APC/C-mediated proteolysis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
SAMBA orthologs and in vivo expression profile. (A) ClustalW2 multiple sequence alignment of putative SAMBA orthologs identified in selected plant genomes. A low-complexity region (LCR) that separates two regions of increased sequence similarity is marked as SHR1 and SHR2. (B) Expression of the pSAMBA-GUS-GFP reporter gene at different developmental stages. Embryo at heart stage visualized by confocal microscopy. (C) GUS activity in mature embryo. (D) Seedling at 3 DAS. (E) Seedling at 5 DAS. (F) Seedling at 8 DAS. (G) Mature pollen grains. [Scale bars: 20 μm (B); 500 μm (C and D); 1 mm (E and F); 100 μm (G).]
Fig. 2.
Fig. 2.
Representative pictures of the phenotypical analysis of samba. (A) Seed size of WT and samba. (B) Mature embryos of WT and samba. (C) Seedlings of WT and samba at 15 DAS. (D) Root phenotype of WT and samba at 10 DAS. (E) Propidium iodide–stained root meristems of WT and samba at 5 DAS. Arrowheads mark the QC position and the meristem end, defined by the position where cells start elongating. (F) Side view of a reconstructed samba and WT SAM at 12 DAS. (G) Adaxial view of reconstructed primordia of leaves 1 and 2 of WT and samba at 4 DAS. [Scale bars: 100 μm (A and B); 1 mm (C and D); 20 μm (E); 50 μm (F and G).] The number of samples analyzed and the statistical analysis are provided in Fig. S2.
Fig. 3.
Fig. 3.
Kinematic analysis of leaf growth in samba and WT plants. (A) Leaf blade area of the first leaf pair of samba (gray) and WT (black) plants grown in vitro from 4 to 24 DAS. (Inset) Measurement at 4 and 5 DAS. (B) Number of cells on the abaxial side of leaves. (Inset) Cell numbers at 4 and 5 DAS. (C) Cell area. (D) Cell division rate. (E) Ploidy distribution of the first leaf pair of samba and WT plants. (Inset) Ploidy distribution measured by flow cytometry of the first leaves from 20-d-old samba and WT plants. (F) Transcript analysis of selected cell cycle genes in samba mutants measured by nCounter analysis. All values were normalized against the expression level of the housekeeping genes and expression was compared with the expression data in the WT. Data are means ± SE (n = 3).
Fig. 4.
Fig. 4.
CYCA2;3 stabilization. (A and B) Y2H interactions between SAMBA and different cyclins and CYCA2;3∆D-box (D-box–mutated), respectively. (C) Detection of CYCA2;3-HA protein levels in etiolated seedlings of WT CYCA2;3-HA (control line) and samba CYCA2;3-HA at 5 DAS.
Fig. 5.
Fig. 5.
Pollen phenotype of samba mutants and WT. The viability of pollen grains was tested by coloration with Alexander’s stain in WT (A) and samba (B) plants. The red staining indicates that the pollen is viable. (C and D) WT and samba pollen stained with DAPI and observed under UV fluorescence, respectively. Two densely stained sperm nuclei and one large diffuse vegetative nucleus are visible in the WT but frequently only one single vegetative nucleus in samba (arrows). [Scale bars: 500 μm (A and B); 10 μm (C and D).]
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