Enhanced tolerance to chilling stress in OsMYB3R-2 transgenic rice is mediated by alteration in cell cycle and ectopic expression of stress genes

Abstract

MYB transcription factors play central roles in plant responses to abiotic stresses. How stress affects development is poorly understood. Here, we show that OsMYB3R-2 functions in both stress and developmental processes in rice (Oryza sativa). Transgenic plants overexpressing OsMYB3R-2 exhibited enhanced cold tolerance. Cold treatment greatly induced the expression of OsMYB3R-2, which encodes an active transcription factor. We show that OsMYB3R-2 specifically bound to a mitosis-specific activator cis-element, (T/C)C(T/C)AACGG(T/C)(T/C)A, a conserved sequence that was found in promoters of cyclin genes such as OsCycB1;1 and OsKNOLLE2. In addition, overexpression of OsMYB3R-2 in rice led to higher transcript levels of several G2/M phase-specific genes, including OsCycB1;1, OsCycB2;1, OsCycB2;2, and OsCDC20.1, than those in OsMYB3R-2 antisense lines or wild-type plants in response to cold treatment. Flow cytometry analysis revealed an increased cell mitotic index in overexpressed transgenic lines of OsMYB3R-2 after cold treatment. Furthermore, resistance to cold stress in the transgenic plants overexpressing OsCycB1;1 was also enhanced. The level of cellular free proline was increased in the overexpressed rice lines of OsMYB3R-2 and OsCycB1;1 transgenic plants compared with wild-type plants under the cold treatment. These results suggest that OsMYB3R-2 targets OsCycB1;1 and regulates the progress of the cell cycle during chilling stress. OsCPT1, which may be involved in the dehydration-responsive element-binding factor 1A pathway, showed the same transcription pattern in response to cold as did OsCycB1;1 in transgenic rice. Therefore, a cold resistance mechanism in rice could be mediated by regulating the cell cycle, which is controlled by key genes including OsMYB3R-2.

Figure 1.

Identification of OsMYB3R-2 transgenic rice and its expression pattern. A, Northern-blot assay of rice transgenic plants. Total RNA isolated from wild-type (WT) or transformed plants underwent hybridization with a [α-³²P]dCTP-labeled probe of OsMYB3R-2 cDNA as described in “Materials and Methods.” B, Real-time RT-PCR of the expression of OsMYB3R-2 in antisense lines. C and D, Southern-blot assay of transformed rice plants. Genomic DNA isolated from wild-type or transformed plants was digested with EcoRI (E) or HindIII (H). The blot was hybridized with the open reading frame of the GUS gene labeled with [α-³²P]dCTP. OL3, OL5, OL7, and OL8 and AL1, AL2, AL4, and AL5 represent overexpression (O) and antisense (A) lines of OsMYB3R-2 transgenic rice. E, Expression pattern of OsMYB3R-2 in vivo. GUS staining shows expression pattern of OsMYB3R-2 in vivo in various tissues from the T1 generation of OsMYB3R-2 promoter∷GUS transgenic rice. a, Root; b, young internode; c, mature internode; d, node; e, mature leaf; f, lamina joint; g, leaf sheath; h, flower; i, immature seed.

Figure 2.

Phenotype analysis of the T2 generation of OsMYB3R-2 transgenic rice. A, The germination and growth of OsMYB3R-2 transgenic rice at 30°C. Shelled seeds of rice at 0 d; germinating seeds at 1, 2, and 3 d; young seedlings transferred to light (12 h of light/12 h of dark, 30°C/26°C) at 4 and 6 d. B, Two-week-old seedlings (T2 generation) of OsMYB3R-2 transgenic rice. C, Transgenic overexpressing seedlings of OsMYB3R-2 at the tillering stage 35 d after germination. AL1, OsMYB3R-2-antisense rice; OL5, OsMYB3R-2-overexpressing rice; WT, wild type. Bars = 10 cm.

Figure 3.

Tolerance response of the OsMYB3R-2 transgenic lines to cold stress. A, Wild-type and OsMYB3R-2 transgenic 2-week-old rice seedlings at the same stage before the 2°C treatment. B, Seedlings were grown in the greenhouse for 2 weeks after 2°C treatment for 72 h. C, Survival rate of seedlings grown for 2 weeks in the greenhouse after 2°C treatment for 72 h. D, Time course for cold treatment in survival rate of seedlings grown for 2 weeks in the greenhouse. In C and D, the error bars show se and are from three independent replications in the same experiment. The phenotype was confirmed by further experiments that were repeated more than four times. AL1 and AL4, OsMYB3R-2-antisense lines; OL3, OL5, and OL7, OsMYB3R-2-overexpressing lines; WT, wild type.

Figure 4.

Gene expression patterns and free Pro levels of transgenic rice plants in response to cold. A, Expression pattern of OsCPT1 in wild-type and OsMYB3R-2 transgenic rice at room temperature (25°C) or low temperature (4°C). Tubulin was used as an internal control. The expression pattern of OsCPT1 to cold stress was confirmed with two independent experiments. B, Cellular free Pro level. Data represent means and se of experiments performed in triplicate. Error bars show se and are from three independent replications of the same experiment. The Pro content determination was confirmed by experiments that were repeated twice. AL1, AL4, and AL5, OsMYB3R-2-antisense lines; FW, fresh weight of materials; OL3, OL5, and OL7, OsMYB3R-2-overexpressing lines; RT, room temperature (25°C); WT, wild type.

Figure 5.

Transcription activation analysis of OsMYB3R-2 protein. A, Different pGBKT7-OsMYB3R-2 vector constructs. The truncated cDNA fragments of OsMYB3R-2 were sequenced and inserted into the NdeI-PstI sites, with ATG added at the end of the NdeI site in every forward primer. For numbers at left, 1 represents full-length OsMYB3R-2 protein and 2 to 14 represent different truncated OsMYB3R-2 protein fragments; numbers at right represent the positions of different truncated OsMYB3R-2 protein fragments. The broken line represents the deleted fragment (amino acids 351 to 449) of OsMYB3R-2. The transcription activation of OsMYB3R-2 was confirmed twice. B, The corresponding positions of transformed yeast thalli daubed on the plates. CK, pGBKT7 vector used as a control. C, a, The transformed yeast thalli grew on the SD/-His/-Trp plates with solid SD medium. C, b, X-Gal activation detection of transformed yeast thalli on the SD/-His/-Trp plates with solid SD medium shown in C, a. C, c, The transformed yeast thalli grew on the SD/-Ade/-Trp plates with solid SD medium. C, d, X-Gal activation detection of transformed yeast thalli on the SD/-Ade/-Trp plates with solid SD medium shown in C, c. C, e, The transformed yeast thalli grew on the SD/-His/-Ade/-Trp plates with solid SD medium. C, f, X-Gal activation detection of transformed yeast thalli on the SD/-His/-Ade/-Trp plates with solid SD medium shown in C, e.

Figure 6.

DNA binding affinity of OsMYB3R-2 protein. A, The alignment of MSA-like sequences shown in the promoters of type B cyclin genes: rice OsCycB1;1 and OsKNOLLE2, Arabidopsis cyc1bAt (Day et al., 1996) and cyc2aAt (Ferreira et al., 1994), and tobacco NtCYM (Ito et al., 1997), NACK1, and NACK2 (Ito et al., 1998) encoding kinesin-like proteins. The boxed 11-bp sequences share high homology with each other. The nucleotide positions are numbered from the transcription start sites. The motifs of binding sites of c-Myb (Howe and Watson, 1991) and v-Myb (Grotewold et al., 1994) are also shown. B, Probes and competitors used in EMSA. BP, A 378-bp fragment of OsCycB1;1 promoter upstream of the transcription start site ATG; RT1, an MSA cis-acting element of BP; RT2, an MSA trans-acting element of BP; RT3, an MSA cis-acting element of OsKNOLLE2 promoter upstream of the transcription start site ATG; RT1mut, mutant of the RT1 motif; BPWT and RT1WT, competitors of biotin-labeled probes of BP and RT1, respectively. C to E, EMSA assays of OsMYB3R-2 protein. Abbreviations are the same as in B. Binding reaction mixtures were incubated with the probes and mock-translated product (mock = probe + no protein) or in vitro-synthesized OsMYB3R-2, with GST as a control, in the presence or absence of a 200-fold molar excess of unlabeled oligonucleotide competitors. DNA binding affinity of OsMYB3R-2 was confirmed experimentally twice. AL1 and AL4, OsMYB3R-2-antisense lines; OL5 and OL7, OsMYB3R-2-overexpressing lines.

Figure 7.

Expression patterns of cyclin genes in the OsMYB3R-2 transgenic rice, cold response and survival ratio, and cellular free Pro in the OsCycB1;1 transgenic rice lines. A, Expression patterns of cyclin-responsive genes in wild-type (WT) and OsMYB3R-2 transgenic rice under room temperature (25°C) or cold treatment (4°C) for 24 h. The method of obtaining seedlings at the same stage is described in “Materials and Methods.” Tubulin was used as an internal control. AL1 and AL4, OsMYB3R-2-antisense lines; OL5, OL7, and OL8, OsMYB3R-2-overexpressing lines. The experiments on the response of cyclin genes to cold were repeated at least twice. B, The response of the OsCycB1;1 transgenic rice lines to cold stress. AT, Grown for 1 week after the cold treatment (3°C) for 72 h; BT, before the cold treatment (3°C), which is a control without cold treatment; OE, OsCycB1;1-overexpressing lines; RNAi, OsCycB1;1-RNAi lines. C, The determination of cellular free Pro in OsCycB1;1 transgenic rice lines. AT, After the cold treatment (2°C) for 24 h; BT, before the cold treatment (2°C); FW, fresh weight of materials. Error bars in B and C show se and are from three independent replications. Data represent means and se of experiments performed in triplicate.

Figure 8.

Cell cycle progression response to cold in flow cytometry assay in the OsMYB3R-2 transgenic lines. A, The wild type at 28°C. B, The wild type at 4°C. C, Overexpressing line 7 (OL7) of OsMYB3R-2 transgenic rice at 28°C. D, Overexpressing line 7 at 4°C. Seedlings 5 d after germination were treated with low temperature (4°C) or room temperature (control, 28°C) for 24 h. Cell nuclei (10,000) taken from the root apical meristem were stained with DAPI (1 μg mL⁻¹) and analyzed by flow cytometry. 2C and 4C represent the DAPI signals that correspond to nuclei with different DNA contents. E, Cell mitotic index in root apical meristem (RAM) in rice. Numbers above the black histograms represent the percentage of decrease in the mitotic index at 4°C. The error bars show se and are from three independent replications of the same experiment. Flow cytometry determination was repeated twice. AL1 and AL4, OsMYB3R-2-antisense lines; OL5 and OL7, OsMYB3R-2-overexpressing lines; WT, wild type.