The Indeterminate Domain Protein ROC1 Regulates Chilling Tolerance via Activation of DREB1B/CBF1 in Rice

Abstract

Abiotic stress, including salinity, drought and cold, severely affect diverse aspects of plant development and production. Rice is an important crop that does not acclimate to cold; therefore, it is relatively sensitive to low temperature stress. Dehydration-responsive element-binding protein 1s (DREB1s)/C-repeat binding factors (CBFs) are well known for their function in cold tolerance, but the transcriptional regulation of CBFs remains elusive, especially in rice. Here, we performed a yeast one-hybrid assay using the promoter of CBF1, a cold-induced gene, to isolate transcriptional regulators of CBF1. Among the seven candidates identified, an indeterminate domain (IDD) protein named ROC1 (a regulator of CBF1) was further analyzed. The ROC1 transcript was induced by exogenously-treated auxin, while it was not altered by cold or ABA stimuli. ROC1-GFP was localized at the nucleus, and ROC1 showed trans-activation activity in yeast. The electrophoretic mobility shift assay (EMSA) and ChIP analyses revealed that ROC1 directly bound to the promoter of CBF1. Furthermore, ROC1 mutants exhibited chilling-sensitive symptoms and inhibited cold-mediated induction of CBF1 and CBF3, indicating that ROC1 is a positive regulator of cold stress responses. Taken together, this study identified the CBF1 regulator, and the results are important for rice plant adaptation to chilling stress.

Keywords: CBF1; ROC1; cold stress; indeterminate domain; rice.

Figure 1

Identification of a regulator of the CBF1 gene. (a) A rice complementary DNA (cDNA) library was generated using a pGAD424 vector in which the coding sequences of the protein of interest were C-terminally fused to the activation domain (AD). A 2.0-kb section of the CBF1 gene promoter was cloned into the pHISi vector in which His was a reporter gene; (b) A yeast one-hybrid assay was performed to analyze the regulator of the CBF1 gene (ROC1) activation of the CBF1 promoter. Yeast cells harboring either AD-ROC1 and pCBF1-His or AD and pCBF1-His were grown on SD media lacking Leu or His and containing the indicated concentrations of 3-amino-1,2,4-triazole (3AT), a competitive inhibitor of HIS3.

Figure 2

ROC1 directly binds to the promoter of CBF1. (a) The schematic diagram shows the locations of the putative IDD binding motifs (red oval) in the CBF1 promoter and regions (blue line) tested in the electrophoretic mobility shift assay (EMSA) and ChIP assays; (b) EMSA was performed to evaluate the ROC1 affinities to each of the putative IDD binding motifs located in the CBF1 promoter; (c) ROC1-Myc expression in Ubiquitin:ROC1-Myc transgenic rice plants was analyzed by immunoblotting using an anti-cMyc antibody; (d) A CHIP assay was performed by amplifying immunoprecipitated DNA to detect the P1 and P2 regions in the CBF1promoter; immunoprecipitated DNA was normalized to input DNA after ChIP-PCR. Data represent the means ± SE (n = 3); non-transgenic plants (wild-type) were used as controls. Ab: IgG; cMyc: Myc antibody. ** p < 0.01; the p-value of the ROC1-Myc sample was calculated with respect to that of the controls.

Figure 3

Trans-activation and sub-cellular localization of ROC1. (a) ROC1 consists of 495 amino acids and encodes an indeterminate domain. Gray and black boxes indicate the C2H2 and C2HC zinc finger motifs, respectively; (b) Trans-activation activity of ROC1. DNA encoding full-length ROC1 was C-terminally fused to the GAL4 DNA-binding domain (BD) and expressed in yeast cells. Rice ID1 and an empty vector were used as the positive and negative control, respectively. Yeast cells expressing the indicated constructs were grown on SD media lacking Trp or His; (c) Localization of free GFP (a) and ROC1-GFP (c) in onion epidermal cells. GFP indicates the green fluorescence of proteins; (b,d) DIC indicates the differential interference contrast phase. Bars = 100 µm.

Figure 4

Genomic structure and phenotypic expression of ROC1 mutants. (a) The diagram shows the genomic structure of the ROC1 T-DNA insertional mutant (roc1). Black and white boxes indicate the exons and UTR regions, respectively. The triangle in the second intron indicates the T-DNA insertion sites; (b) The ROC1 expression levels in wild-type (WT) and ROC1 mutants were analyzed by qRT-PCR. The expression levels were normalized against that of Ubiquitin mRNA. A significant difference between the wild-type and mutant was shown (*** p < 0.001); (c) Fifteen-day-old wild-type and ROC1 mutant plants grown in a rice growth chamber (28 °C, upper side) were further grown in a low temperature growth chamber (4 °C) for four days. The plants were then moved to a 28 °C growth chamber and photographed after 10 days of recovery (lower side); (d) The ROC1 expression levels were monitored in four independent wild-type ROC1 RNAi transgenic plants (Ri1, Ri3, Ri4 and Ri5). Significant differences between the wild-type and RNAi lines are shown (* p < 0.05, ** p < 0.01); (e) Wild-type and two RNAi lines (Ri3 and Ri5) grown in a rice growth chamber (28 °C) were further grown in a low temperature growth chamber (4 °C) for four days. The plants were then moved to a 28 °C growth chamber and photographed after 10 days of recovery; (f) The survival rates of each line were calculated. Black columns indicate roc1 and its corresponding wild-type plants while grey columns indicate RNAi lines and their corresponding wild-type plants. Experiments were repeated at least three times, and data represent the mean ± SE (n > 10 plants). Significant differences between the wild-type and mutants or RNAi lines are shown (* p < 0.05, ** p < 0.01).

Figure 5

Cold stress and hormone-dependent expression of ROC1. (a) Fifteen-day-old wild-type seedlings were grown in a normal rice growth chamber (28 °C) and were moved into a low temperature growth chamber (4 °C), and then, the seedlings were sampled after 0, 1, 3, 6, 12 and 24 h. ROC1 expression levels were monitored with qRT-PCR; (b) Hormonal-regulation of ROC1 transcription. Fifteen-day-old seedlings were treated with 1 μM ABA, 1 μM NAA, 0.1 μM 2,4-epiBL, 1 μM GA and 1 μM ACC for 3 h. qRT-PCR was performed to analyze the expression patterns of ROC1 upon hormone treatment. The expression levels were normalized against that of Ubiquitin mRNA. Significant differences between NAA-treated and -untreated samples (control, CK) are shown (* p < 0.05).

Figure 6

Cold stress-mediated expressions of CBF1 and CBF3 in ROC1 mutants. (a) Fifteen-day-old wild-type and roc1 seedlings were grown in a normal rice growth chamber (28 °C) and were moved into a low temperature growth chamber (4 °C). The seedlings were sampled after 0, 1, 3, 6, 12, 24 and 48 h. The expression levels of CBF1 and CBF3 were detected by Northern blot analysis; (b) The levels of CBF1 and CBF3 were analyzed in wild-type and two ROC1 RNAi lines (Ri3 and Ri5) after 0, 1 and 3 h of cold treatment. Ethidium bromide (EtBr) staining of rRNA is shown as the loading control.