CDKB2 is involved in mitosis and DNA damage response in rice

Abstract

DNA damage checkpoints delay mitotic cell-cycle progression in response to DNA stress, stalling the cell cycle to allow time for repair. CDKB is a plant-specific cyclin-dependent kinase (CDK) that is required for the G₂/M transition of the cell cycle. In Arabidopsis, DNA damage leads the degradation of CDKB2, and the subsequent G₂ arrest gives cells time to repair damaged DNA. G₂ arrest also triggers transition from the mitotic cycle to endoreduplication, leading to the presence of polyploid cells in many tissues. In contrast, in rice (Oryza sativa), polyploid cells are found only in the endosperm. It was unclear whether endoreduplication contributes to alleviating DNA damage in rice (Oryza sativa). Here, we show that DNA damage neither down-regulates Orysa;CDKB2;1 nor induces endoreduplication in rice. Furthermore, we found increased levels of Orysa;CDKB2;1 protein upon DNA damage. These results suggest that CDKB2 functions differently in Arabidopsis and rice in response to DNA damage. Arabidopsis may adopt endoreduplication as a survival strategy under genotoxic stress conditions, but rice may enhance DNA repair capacity upon genotoxic stress. In addition, polyploid cells due to endomitosis were present in CDKB2;1 knockdown rice, suggesting an important role for Orysa;CDKB2;1 during mitosis.

Figure 1

DNA damage response in rice calli following X-ray irradiation. (a) Quantitative analysis of DSBs by the comet assay. Wild-type rice calli were irradiated with 5, 25 and 100 Gy doses of X-rays at 133 Gy h⁻¹, and tail moment values were measured just after irradiation. (b) Quantification of DSBs by the comet assay at various time points after irradiation of calli with a 5 Gy dose of X-rays. (c) Transcription level of OsRAD51A2 determined by real-time quantitative PCR. (d) Ploidy of X-ray-irradiated rice calli. The DNA content of nuclei prepared from calli was analyzed by flow cytometry at various times following irradiation. (e) Transcription levels of CDKB2;1 determined by real-time quantitative PCR. (f) Immunological detection of CDKB2;1. Upper panel: Western blot analysis of protein extracts from non-irradiated (Non-IR) or X-ray-irradiated rice calli using antibodies against Orysa;CDKB2;1. Middle panel: stained membrane showing equal loading of protein samples. Lower panel: quantification of CDKB2;1 protein.

Figure 2

DNA damage response in rice suspension-cultured cells after bleomycin treatment. (a) Immunoblot analysis of CDKB2;1 using rice suspension-cultured cells. Suspension-cultured cells were blocked in S phase for 24 h using aphidicolin (left panel). The DNA-damaging agent bleomycin (1 mg L⁻¹) was added to the culture medium 4 h after aphidicolin block removal (right panel). Crude proteins were prepared at 2 h intervals after release from the aphidicolin block. (b) PI staining of suspension-cultured cells with or without 2 h bleomycin treatment. (c) Transcription level of CDKB2;1 determined by real-time quantitative PCR.

Figure 3

Characterization of Orysa;CDKB2;1 knockdown calli. (a) Schematic representation of CDKB2;1 structure and RNAi silencing target region. (b) Northern blot analysis of CDKB2;1. Upper panel: OsCDKB2;1 mRNA. Lower panel: RNAs transferred to nylon membrane to visualize equal loading of total RNA in each lane. (c) Transcription level of CDKB2;1 determined by real-time quantitative PCR. The region amplified is indicated in (a). (d) Ploidy of CDKB2;1 knockdown rice calli (B2RNAi) grown on selection medium for 36 days. (e) Immunological detection of CDKB2;1. (f) Growth of wild-type (WT) and B2RNAi calli on N6D solid medium at 0 days (upper panel) and 12 days (lower panel) after transfer.

Figure 4

Chromosome observation of Orysa;CDKB2;1 knockdown calli. Chromosome spreads of nuclei from Orysa;CDKB2;1 knockdown line B2RNAi-5. Nuclei containing 24 (a), 48 (b) or 96 (c) chromosomes were found.

Figure 5

Phenotypic analysis of a regenerated plant from the Orysa;CDKB2;1 inducible knockdown line. (a)Tetraploid rice plant regenerated from CDKB2;1 inducible RNAi transformed callus (B2RNAiID-1). (b) Flow cytometry measurements of DNA content from leaf cells of wild-type and B2RNAiID-1. (c) Scanning electron micrographs of the epidermal leaf surface of wild-type (diploid) and B2RNAiID-1 (tetraploid) plants. Arrowheads indicate stomata.

Figure 6

X-ray sensitivity of Orysa;CDKB2;1 knockdown plants. (a) Western blot analysis of CDKB2;1. Crude protein was prepared from roots of non-irradiated wild-type (WT) and B2RNAi-7 plants. (b) WT and CDKB2;1 knockdown plants (B2RNAi-7-1 and B2RNAi-16-1) grown on MS culture medium were irradiated with 100 Gy X-rays. Non-irradiated and irradiated plants were grown on MS culture medium for 5 days. (c) Morphology of primary root tip response with or without 100 Gy X-ray irradiation.

Figure 7

Effect of constitutive DNA damage by bleomycin on growth and ploidy of the calli. (a) Proliferation of calli on medium supplemented with 0, 0.5 or 2mg L⁻¹ bleomycin. (b) Ploidy of rice calli grown on medium containing 0, 0.5 or 2 mg L⁻¹ bleomycin for 10 days.