The OsMYB30 Transcription Factor Suppresses Cold Tolerance by Interacting with a JAZ Protein and Suppressing β-Amylase Expression

Abstract

Cold stress is one of the major limiting factors for rice (Oryza sativa) productivity. Several MYB transcriptional factors have been reported as important regulators in the cold stress response, but the molecular mechanisms are largely unknown. In this study, we characterized a cold-responsive R2R3-type MYB gene, OsMYB30, for its regulatory function in cold tolerance in rice. Functional analysis revealed that overexpression of OsMYB30 in rice resulted in increased cold sensitivity, while the osmyb30 knockout mutant showed increased cold tolerance. Microarray and quantitative real-time polymerase chain reaction analyses revealed that a few β-amylase (BMY) genes were down-regulated by OsMYB30. The BMY activity and maltose content, which were decreased and increased in the OsMYB30 overexpression and osmyb30 knockout mutant, respectively, were correlated with the expression patterns of the BMY genes. OsMYB30 was shown to bind to the promoters of the BMY genes. These results suggested that OsMYB30 exhibited a regulatory effect on the breakdown of starch through the regulation of the BMY genes. In addition, application of maltose had a protective effect for cell membranes under cold stress conditions. Furthermore, we identified an OsMYB30-interacting protein, OsJAZ9, that had a significant effect in suppressing the transcriptional activation of OsMYB30 and in the repression of BMY genes mediated by OsMYB30. These results together suggested that OsMYB30 might be a novel regulator of cold tolerance through the negative regulation of the BMY genes by interacting with OsJAZ9 to fine-tune the starch breakdown and the content of maltose, which might contribute to the cold tolerance as a compatible solute.

Figure 1.

Expression profiles and subcellular localization of OsMYB30. A, Expression levels of OsMYB30 under hormone and abiotic stress treatments, including ABA (time course, 0, 1, 3, and 12 h), JA (0, 1, 3, and 6 h), heat (0 and 10 min, 1 h, and 3 h), flooding (0, 6, 12, and 24 h), drought (0, 1, 2, and 3 d), salt (0, 2, 6, and 12 h), and cold (0, 6, 12, and 24 h). Seedlings at the four-leaf stage were treated with ABA (spraying 100 μm L⁻¹ ABA on the leaves), JA (spraying 100 μm L⁻¹ JA on the leaves), heat (42°C growth chamber), flooding (submerged with water), drought (stopping the water supply), salt (irrigation with 200 mm NaCl solution), or cold (4°C growth chamber). Error bars indicate the se based on three biological replicates. B, OsMYB30 colocalized with the GHD7 transcription factor in the nucleus of rice protoplasts. 35S::GFP was transformed as a control. Bars = 10 μm.

Figure 2.

Phenotypes of OsMYB30-OE plants and the osmyb30 mutant under cold stress conditions (4°C). A, Seedlings from three overexpression lines (OE2, OE8, and OE28) were grown on one-half-strength MS medium with ZH11 plants as a control. B, Survival rates of OsMYB30-OE plants and ZH11 after cold stress treatment. Error bars indicate the se based on three replicates. C, Electrolyte leakage (percentage of total electrolyte leakage) of ZH11 and overexpression plants under cold stress treatment. Error bars indicate the se of three replicates. **, P < 0.01 by Student’s t test. D, Seedling phenotypes of the homozygous mutant line of osmyb30 grown in MS medium with the wild-type Huayang (WT) as a control. E, Survival rates of the homozygous mutant plants and the wild type during cold stress treatment. Error bars indicate the se of three replicates. **, P < 0.01 by Student’s t test. F, Electrolyte leakage of homozygous mutant plants of osmyb30 and the wild type under cold stress treatment. Error bars indicate the se of three technical replicates. **, P < 0.01 by Student’s t test.

Figure 3.

Microarray analysis of the OsMYB30-OE and mutant plants before and after cold stress treatment. A, Venn diagram for the number of genes with expression levels affected in the overexpression and mutant plants before and after cold stress treatment. OsMYB30-up indicates genes up-regulated in the overexpression plants or down-regulated in the mutant; OsMYB30-down indicates genes down-regulated in the overexpression plants or up-regulated in the mutant. B, Gene Ontology classification of genes up- or down-regulated by OsMYB30 with a threshold of expression change 1.8-fold or greater or 0.6-fold or less. C, Expression patterns of OsMYB30 and the two BMY genes (BMY6 and BMY10) in OsMYB30-OE and the osmyb30 mutant under cold stress conditions, with ZH11 and Huayang as controls, respectively. Error bars indicate the se of three replicates. **, P < 0.01 by Student’s t test. WT, Wild type.

Figure 4.

OsMYB30 decreases maltose content by repressing BMY activity. A and B, BMY activity in the OsMYB30-OE plants (A) and the osmyb30 mutant (B) at the seedling stage under cold stress treatment. C and D, Maltose contents in OsMYB30-OE (C) and osmyb30 mutant (D) seedlings using Suc as an internal reference. E and F, F_v/F_m ratio in the OsMYB30-OE plants (E) and the osmyb30 mutant (F) under cold stress conditions. G and H, Electrolyte leakage analyses in the maltose treatment. Electrolyte leakage was determined in the OsMYB30-OE plants (G) and the osmyb30 mutant (F) under cold stress conditions (N) and cold stress plus 2.78 mm maltose treatment (M). Electrolyte leakage is presented as the ratio of R1 to R2, where R1 is the initial conductivity and R2 is the conductivity of the boiled leaf segments. Error bars indicate the se of three biological replicates. **, P < 0.01 by Student’s t test. WT, Wild type.

Figure 5.

OsMYB30 interacts with BMY promoters in vitro and in vivo. A, OsMYB30 bound to the promoters of the BMY genes in yeast cells through a yeast one-hybrid assay by growing the plasmids (pHis2-P_BMY plus pGADT7-OsMYB30) on selective medium (SD-Trp-Leu-His) containing 50 mm 3-AT along with the negative control (pHis2 plus pGADT7-OsMYB30) and the positive control (pHis2-P53 plus pGADT7-53). B, EMSA was carried by using the OsMYB30::GST protein and the BMY promoters as probes labeled with 5′ FAM. Competition for the labeled sequences was tested by adding different concentrations of unlabeled probes. The positions of the probes are indicated with red lines in C. C, ChIP-qPCR to confirm OsMYB30 binding to the BMY gene promoter. The fragments for ChIP-qPCR are indicated by forward (F) and reverse (R) primers; vertical black and red arrows indicate the positions of forward primers and probes, respectively. N and Cold indicate normal growth and cold stress conditions, respectively. The results are represented as relative values of immunoprecipitation (IP) relative to input. The immunoprecipitation and input samples were the chromatin from the four samples (N-osmyb30, N-WT, Cold-osmyb30, and Cold-WT) precipitated with and without OsMYB30 antibody, respectively. The osmyb30 mutant was used as a negative control. Error bars indicate the se of three replicates. **, P < 0.01 by Student’s t test.

Figure 6.

OsMYB30 interacts with OsJAZ9. A, BiFC assay indicates that OsMYB30 interacted with OsJAZ9 in rice. YFP fluorescence was detected in rice leaves with combinations of cYFP-OsMYB30 and OsJAZ9-nYFP. The combination of cYFP-OsMYB30 plus pVYNE was used as a negative control. Bars = 10 μm. B, Co-IP assay to verify the interaction of OsMYB30 and OsJAZ9 in vivo. OsMYB30-GFP was cotransformed with OsJAZ9-MYC (pVNYE) or the vector (pVNYE as a negative control) in rice protoplasts. Total protein extracts from the transient assay expressing OsMYB30-GFP plus OsJAZ9-MYC or OsMYB30-GFP plus the MYC-tag were immunoprecipitated (IP) using GFP beads and blotted with anti-OsMYB30 and anti-MYC antibodies. C, Pull-down assay to verify the interaction between OsMYB30 and OsJAZ9 in vitro. OsMYB30-GST fusion protein or GST alone was incubated with OsJAZ9-His in His beads. OsMYB30-GST but not GST was pulled down by the beads containing OsJAZ9-His. Western blot was used with anti-GST antibody.

Figure 7.

OsJAZ9 participates in the cold stress response and contributes to the negative regulation of OsMYB30. A, Expression levels of OsJAZ9 in the OsMYB30-OE lines (OE2, OE8, and OE28) under cold stress conditions. ZH11 is the wild-type control of the overexpression lines. B, Expression levels of BMY6 and BMY10 in the OsJAZ9-OE (OE8 and OE13) plants under cold stress conditions. Error bars indicate the se of three replicates. C, Effect of OsMYB30 on promoters of the GAL4 and BMY genes using a dual-luciferase transient assay in rice protoplasts with or without OsJAZ9 cotransformed. The main components of the vectors are displayed on the left. On the right, the effect of OsMYB30 on the promoters is compared with the control CK1, which is indicated by the solid line box. The effect of OsJAZ9 on the promoters, given the presence of OsMYB30, was evaluated based on the comparison of OsMYB30 + OsJAZ9 and OsMYB30 + CK2, which is indicated by the dotted line box. GAL4, BMY2, BMY6, and BMY10 represent reporter constructs containing the corresponding promoters. Error bars indicate the se of three replicates. **, P < 0.01 by Student’s t test.

Figure 8.

Schematic model of the OsMYB30 regulation mechanism in the cold stress response. Both OsMYB30 and OsJAZ9 were induced by cold stress. OsMYB30 directly bound to the promoter of the rice BMY genes. The interaction with OsJAZ9 may help OsMYB30 achieve the negative regulation on BMY genes. The BMY genes were repressed by OsMYB30-OsJAZ9 at the transcriptional level and BMY activity was decreased, then starch degradation and maltose accumulation were inhibited, resulting in the cell membrane protection being weakened, finally enhancing cold sensitivity.