MdbHLH4 negatively regulates apple cold tolerance by inhibiting MdCBF1/3 expression and promoting MdCAX3L-2 expression

Abstract

Low temperature affects the yield and quality of crops. Inducer of CBF expression 1 (ICE1) plays a positive role in plant cold tolerance by promoting the expression of CRT binding factor (CBF) and cold-responsive (COR) genes. Several ICE1-interacting transcription factors (TFs) that regulate plant cold tolerance have been identified. However, how these TFs affect the function of ICE1 and CBF expression under cold conditions remains unclear. Here, we identified the MYC-type TF MdbHLH4, a negative regulator of cold tolerance in Arabidopsis (Arabidopsis thaliana) and apple (Malus domestica) plants. Under cold conditions, MdbHLH4 inhibits the expression of MdCBF1 and MdCBF3 by directly binding to their promoters. It also interacts with MdICE1L, a homolog of AtICE1 in apple, and inhibits the binding of MdICE1L to the promoters of MdCBF1/3 and thus their expression. We showed that MdCAX3L-2, a Ca2+/H+ exchanger (CAX) family gene that negatively regulates plant cold tolerance, is also a direct target of MdbHLH4. MdbHLH4 reduced apple cold tolerance by promoting MdCAX3L-2 expression. Moreover, overexpression of either MdCAX3L-2 or MdbHLH4 promoted the cold-induced ubiquitination and degradation of MdICE1L. Overall, our results reveal that MdbHLH4 negatively regulates plant cold tolerance by inhibiting MdCBF1/3 expression and MdICE1L promoter-binding activity, as well as by promoting MdCAX3L-2 expression and cold-induced MdICE1L degradation. These findings provide insights into the mechanisms by which ICE1-interacting TFs regulate CBF expression and ICE1 function and thus plant cold tolerance.

Figure 1

Protein characteristics and expression analysis of MdbHLH4. A, Subcellular localization of MdbHLH4-GFP in N. benthamiana leaves. Bars = 30 μm. B, Relative expression levels of MdbHLH4 in different apple tissues. C, Expression analysis of MdbHLH4 in apple leaves under cold treatment. Different letters in B and C represent significant differences based on one-way ANOVA and Duncan’s test (P < 0.05). Error bars indicate the SD of three biological replicates. D, Sequence alignment of the bHLH domain of MdbHLH4 and bHLH proteins in Arabidopsis. Rectangular boxes represent the bHLH domain. Triangles indicate the HER motif. Dots and stars represent highly conserved amino acids (R10, R12, L23, L64, and P28) in the bHLH family. E and F, The 3D structure diagram of the dimer formed by the bHLH domain (E) or the C-terminal domain (F) of MdbHLH4.

Figure 2

Functional characterization of MdbHLH4 in regulating cold tolerance in transgenic Arabidopsis and apple plants. A, Growth phenotype and survival rate of Arabidopsis seedlings after cold treatment. NA, without cold acclimation; CA, with cold acclimation (4°C for 12 h). Scale bars, 1 cm. B, The growth phenotype and survival rate of Arabidopsis plants (3 weeks old) after cold treatment. Scale bar, 2 cm. C, Expression analysis of COR genes in Arabidopsis under cold treatment. D–F, Freezing phenotype (D), survival rate (E), and leaf REL (F) of WT and MdbHLH4 transgenic apple plants under cold treatment. Scale bars, 9 cm. G, Expression analysis of COR genes in apple plants. Error bars indicate the SD of three biological replicates in all graphs. Bars labeled with * in each panel are significantly different from the Col-0 in C and WT in G (P < 0.05, Student’s t test). Different letters in A, B, E, F, and G indicate significant differences at P < 0.05 based on one-way ANOVA and Duncan’s tests.

Figure 3

MdbHLH4 directly represses MdCBF1/3 expression under cold stress. A, Schematic diagram of the constructed reporter and effector vectors. B, Fluorescence observations and relative LUC/REN activity measurements in dual-LUC assays. The relative LUC/REN value is the average of three biological replicates, with each replicate containing three N. benthamiana plants. Error bars indicate the sd of three biological replicates. 1, empty reporter and effector; 2, empty reporter+35S::MdbHLH4; 3, proMdCBF1::LUC+empty effector; 4, proMdCBF1::LUC+35S::MdbHLH4; 5, proMdCBF3::LUC+empty effector; and 6, proMdCBF3::LUC+35S::MdbHLH4. C, Identification of the promoter-binding ability of MdbHLH4 on the promoters of MdCBF1/3 by Y1H assays. D, Diagram of the potential MdbHLH4-binding sites within the MdCBF1/3 promoters. E, EMSAs showing the binding of MdbHLH4 to the MdCBF1/3 promoters. Numbers indicate the potential binding sites in D. F, Verification of the binding specificity of MdbHLH4 by competing EMSAs. Competitor, probes without biotin labeling. Mut, mutant probes with core sequences within E-box altered. 5×, 10×, and 50× represent the rates of competitor probes. Sequences of different probes are provided in Supplemental Table S1.

Figure 4

MdbHLH4 interacts with MdICE1L/MdICE1. A, Identification of the self-activation activity of full-length and truncated MdbHLH4 (MdbHLH4-N55) based on Y2H assays. B and C, Protein interaction identification between MdbHLH4 and MdICE1L/MdICE1 by Y2H (B) and Co-IP (C) assays. D, Prediction of the effects of point mutations, functional region division, and self-activation activity identification of different regions of MdbHLH4. Detailed results of the effects of point mutations are provided in Supplemental Figure S7. E, Identification of crucial regions for the MdbHLH4–MdICE1L/MdICE1 interaction in MdbHLH4. AD, pGAD424 and BD, pGBT9. F, Identification of crucial regions for the MdbHLH4–MdICE1L/MdICE1 interaction in MdICE1L/MdICE1. AD, pGADT7; BD, pGBKT7; DDO, SD medium without leucine and tryptophan; and QDO, SD medium without leucine, tryptophan, histidine, and adenine.

Figure 5

MdbHLH4 represses the binding activity of MdICE1L on MdCBF1/3 promoters. A, EMSAs showing that MdICE1L directly binds to the promoters of MdCBF1/3. Numbers indicate the binding sites in Figure 3. B, Dual-LUC assays showing that MdbHLH4 inhibits the transcriptional activation of MdCBF1/3 by MdICE1L under cold stress. C, Identification of the MdICE1L-binding sites that are most significantly affected by MdbHLH4 through dual-LUC assays. pro2, pro4, and pro5 represent the effector vectors containing the corresponding binding sites in MdCBF1/3 promoters in A. Significant differences (*) were determined using Student’s t test: P < 0.05. D, EMSAs showing that MdbHLH4 inhibits the promoter-binding activity of MdICE1L. MdbHLH4-GST and MdICE1L-HIS proteins were added to the binding buffer at the indicated molar ratios. E and F, MdbHLH4 reduced the enhancement effect of MdICE1L on the cold tolerance of transgenic apple calli. Growth phenotype (E) and fresh weight (F) after cold treatment are shown. EV-HA indicates apple calli transformed with the empty vector pCambia35S-3HA. Bars represent 1 cm. G, ChIP-qPCR assays showing that MdbHLH4 inhibits the binding of MdICE1L to the promoters of MdCBF1/3 in apple. Apple calli treated with 4°C/25°C for 3 h were harvested for ChIP assays. Immunoprecipitation was performed with or without anti-HA antibody. The relative enrichment was calculated as the ratio of MdbHLH4/MdICE1L transgenic to control (EV-HA) samples. H, Expression analysis of MdCBF1/3 in transgenic calli. Apple calli treated with 4°C/25°C (control) for 3 h were harvested for RT-qPCR analysis. Error bars indicate the SD of three biological replicates in all graphs. Different letters in B, F, G, and H represent significant differences based on one-way ANOVA and Duncan’s tests (P < 0.05).

Figure 6

MdbHLH4 directly binds to the promoter of MdCAX3L-2 and activates its expression. A, Y1H assays showing the interaction between MdbHLH4 and the MdCAX3L-2 promoter. B, EMSAs showing that MdbHLH4 directly binds to the MdCAX3L-2 promoter. Putative binding sites in the MdCAX3L-2 promoter are shown in the schematic diagram. C, Identification of MdbHLH4-binding specificity to sites 1 and 3 in B by competing EMSAs. Sequences of different probes can be found in Supplemental Table S1. D and E, Dual-LUC assays showing the transcriptional activation activity of MdbHLH4 on the MdCAX3L-2 promoter in N. benthamiana. 1, empty reporter and effector; 2, empty reporter + 35S::MdbHLH4; 3, proMdCAX3L-2::LUC + empty effector; and 4, proMdCAX3L-2::LUC + 35S::MdbHLH4. Error bars indicate the sd of three biological replicates. Different letters represent significant differences based on one-way ANOVA and Duncan’s tests (P < 0.05).

Figure 7

MdCAX3L-2 enhances the negative effect of MdbHLH4 on cold tolerance. A and B, Localization of MdCAX3L-2 protein in N. benthamiana epidermal cells (A) and yeast cells (B). AtCBL2-mCherry was used as a tonoplast location marker. GFP protein was used as a control. Scale bars, 30 μm in A and 3 μm in B. C–E, OE of MdCAX3L-2 reduced Arabidopsis cold tolerance. Growth phenotype (C), survival rate (D), and REL (E) of WT and MdCAX3L-2 transgenic Arabidopsis seedlings (1-mouth-old) after cold treatment. Scale bar, 4 cm. F, MdbHLH4 increased the sensitivity of MdCAX3L-2-OE transgenic apple calli to cold stress. 1, WT; 2, MdCAX3L-2-RNAi; 3, MdCAX3L-2-Flag; 4, MdbHLH4-GFP; 5, proMdCAX3L-2::MdCAX3L-2-Flag + MdbHLH4-GFP; and 6, MdCAX3L-2-Flag+MdbHLH4-GFP. Bars represent 1 cm. G, Fresh weight of calli in F. H, Expression analysis of MdCAX3L-2 and MdCBF1/3 in WT and transgenic apple calli in F. Apple calli treated at 4°C for 3 h were harvested for RT-qPCR analysis. Error bars indicate the sd of three biological replicates in all graphs. Different letters in D, E, G, and H represent significant differences based on one-way ANOVA and Duncan’s tests (P < 0.05).

Figure 8

MdbHLH4 and MdCAX3L-2 promote the cold-induced ubiquitination and degradation of MdICE1L. A, Identification of the MdICE1L-MdMPK6a/b interaction using Y2H assays. B, Identification of the MdICE1L–MdMPK6a/b interaction through split-LUC assays. C, MdCAX3L-2 and MdbHLH4 promoted the cold-induced degradation of MdICE1L protein. The protein levels of MdICE1L-HA in transgenic calli (incubated with CHX) were detected at specified time points under 4°C treatment with the anti-HA antibody. Apple calli treated with CHX plus MG132 were sampled at 9 h of cold treatment. Numbers under the upper panel represent the band intensity, which was determined using ImageJ software. D and E, OE of either MdCAX3L-2 (D) or MdbHLH4 (E) promotes the ubiquitination of MdICE1L under cold stress. The ubiquitination level of MdICE1L-HA was detected using anti-ubiquitin (left) and anti-HA (right) antibodies.

Figure 9

A working model showing the roles of MdbHLH4 in cold stress signaling. When plants are exposed to cold stress, MdICE1L is activated rapidly, and the expression of MdCBF1/3 is initiated, a process that as least partially depends on cold-induced Ca²⁺ signaling. Subsequently, cold signaling activates the expression of MdbHLH4. The accumulated MdbHLH4 protein directly inhibits MdCBF1/3 expression by binding to their promoters. MdbHLH4 also inhibits their expression by inhibiting the promoter-binding activity of MdICE1L through the MdbHLH4–MdICE1L interaction. Besides, MdbHLH4 activates the expression of MdCAX3L-2 to promote the compartmentalization of cytosolic Ca²⁺ into vacuoles and thus attenuate the cold-induced Ca²⁺ signature. This reduces the cold-induced expression of CBF and COR genes and thus plant cold tolerance. This also leads to the enhanced ubiquitination and degradation of MdICE1L in response to cold, a process that may be related to MdMPK6a/b in apple.