CDKB1;1 forms a functional complex with CYCA2;3 to suppress endocycle onset

Abstract

The mitosis-to-endocycle transition requires the controlled inactivation of M phase-associated cyclin-dependent kinase (CDK) activity. Previously, the B-type CDKB1;1 was identified as an important negative regulator of endocycle onset. Here, we demonstrate that CDKB1;1 copurifies and associates with the A2-type cyclin CYCA2;3. Coexpression of CYCA2;3 with CDKB1;1 triggered ectopic cell divisions and inhibited endoreduplication. Moreover, the enhanced endoreduplication phenotype observed after overexpression of a dominant-negative allele of CDKB1;1 could be partially complemented by CYCA2;3 co-overexpression, illustrating that both subunits unite in vivo to form a functional complex. CYCA2;3 protein stability was found to be controlled by CCS52A1, an activator of the anaphase-promoting complex. We conclude that CCS52A1 participates in endocycle onset by down-regulating CDKB1;1 activity through the destruction of CYCA2;3.

Figure 1.

In vivo interaction between CDKB1;1 and CYCA2;3. A, Subcellular localization of CDKB1;1 (CDKB-eGFP), CYCA2;3 (CYCA2;3-eGFP), and the CYCA2;3-CDKB1;1 (CYCA2;3-nGFP + CDKB1;1-cGFP) complex. Tobacco epidermal cells were transfected with constructs encoding the indicated fusion proteins. DIC, Differential interference contrast. B, Confocal images of a root of an Arabidopsis plant coexpressing CDKB1;1-cGFP and CYCA2;3-nGFP.

Figure 2.

Effect of coexpression of CYCA2;3-nGFP and CDKB1;1-cGFP. Col-0 seedlings (A and C) and plants coexpressing CYCA2;3-nGFP and CDKB1;1-cGFP (B and D) were grown for 12 DAG on agar medium, and cotyledons were harvested. The cotyledons were stained with PI and observed with a confocal laser-scanning microscope (A and B) or subjected to flow cytometry (C and D).

Figure 3.

Effect of CYCA2;3-GFP induction on the EI in transgenic plants ectopically expressing CDKA;1, CDKB1;1, or CDKB1;1.N161. Cotyledons were harvested at 7 DAG. Seedlings were grown on agar medium without (− inducer) or with (+ inducer) 10 μm β-estradiol and subjected to flow cytometry. Col-0 was used as a control. Values are means ± sd (n = 3). Each biological repeat was carried out with seedlings obtained from independent F1 crosses.

Figure 4.

Phenotypes of cotyledon epidermal cells in Col-0 × CYCA2;3-GFP plants (control) and plants coexpressing CYCA2;3-GFP and CDKA;1 (CDKA;1 × CYCA2;3-GFP), CDKB1;1 (CDKB1;1 × CYCA2;3-GFP), or CDKB1;1.N161 (CDKB1;1.N161 × CYCA2;3-GFP). Seedlings were grown for 14 DAG on agar medium containing 10 μm β-estradiol. At 7 DAG, seedlings were transferred to fresh 10 μm β-estradiol-containing medium in order to sustain the induction of CYCA2;3-GFP. At left are images obtained with differential interference contrast microscopy; at right are drawings of the outlines of the epidermal cells obtained by tracing the microscopy images with Adobe Photoshop 6. The arrow marks aberrant stomatal morphology.

Figure 5.

High kinase activity in CDKB1;1 × CYCA2;3-GFP plants. Kinase activity assays of purified CYCA2;3-GFP complexes in Col-0 × CYCA2;3-GFP, CDKA;1 × CYCA2;3-GFP, and CDKB1;1 × CYCA2;3-GFP plants (F1 generation of approximately five independent crosses). Relative CYCA2;3-GFP-associated CDK activity was measured with histone H1 as substrate. For quantification, the control was arbitrarily set at 100%.

Figure 6.

Stabilization of CYCA2;3 in the absence of CCS52A1. Fluorescence in inducer-treated roots expressing CYCA2;3-GFP or mDB-CYCA2;3-GFP in Col-0 or ccs52a1-1 (CYCA2;3-GFP × ccs52a1-1 and mDB-CYCA2;3-GFP × ccs52a1-1) background and CYCA2;3-GFP in ccs52a2-1 (CYCA2;3-GFP × ccs52a2-1) background. Seedlings were grown for 5 d on agar without β-estradiol and for 2 d on agar medium with 10 μm β-estradiol. Cell walls were stained with PI. The fluorescence from the GFP fusion proteins was observed using confocal laser-scanning microscopy.

Figure 7.

CYCA2;3 stabilization in ccs52a1-1 roots. Fluorescence in inducer-treated roots expressing CYCA2;3-GFP or in Col-0 or ccs52a1-1 (CYCA2;3-GFP × ccs52a1-1) background. Seedlings were grown for 5 d on agar medium without β-estradiol and for 2 d on agar medium containing 10 μm β-estradiol. Cell walls were stained with PI. The fluorescence from the GFP fusion proteins was observed with confocal laser-scanning microscopy.

Figure 8.

Effect on endoreduplication of CYCA2;3-GFP stabilization in ccs52a1-1. A and B, Cotyledons (A) and roots (B) from seedlings harvested at 7 DAG. Seedlings were grown on agar medium without (− inducer) or with (+ inducer) 10 μm β-estradiol; the respective tissues were subjected to flow cytometry. Values are means ± sd (n = 3).

Figure 9.

Phenotype of cotyledon adaxial epidermal cells in Col-0 (control), CYCA2;3-GFP, ccs52a1-1, and CYCA2;3-GFP × ccs52a1-1 plants. Seedlings were grown for 14 d on agar medium containing 10 μm β-estradiol. At 7 DAG, seedlings were transferred to fresh 10 μm β-estradiol-containing medium to sustain the induction of CYCA2;3-GFP. At left are images obtained with differential interference contrast microscopy; at right are drawings of the outlines of the epidermal cells obtained by tracing the microscopy images using Adobe Photoshop 6.