VaMIEL1-mediated ubiquitination of VaMYB4a orchestrates cold tolerance through integrated transcriptional and oxidative stress pathways in grapevine

Abstract

Cold stress poses a significant threat to viticulture, particularly under the increasing pressures of climate change. In this study, we identified VaMIEL1, a RING-type E3 ubiquitin ligase from Vitis amurensis, as a negative regulator of cold tolerance. Under normal temperature conditions, VaMIEL1 facilitates the ubiquitination and subsequent proteasomal degradation of the cold-responsive transcription factor VaMYB4a, thereby attenuating its regulatory role in the CBF-COR signaling cascade. However, under cold stress, VaMIEL1 expression is downregulated, leading to the stabilization of VaMYB4a and the activation of CBF-COR signaling. Through a combination of biochemical assays and functional analysis in Arabidopsis thaliana and grapevine calli, we demonstrate that VaMIEL1 overexpression reduces cold tolerance, as evidenced by increased oxidative stress, excessive reactive oxygen species (ROS) accumulation, and downregulated expression of cold-responsive genes. Conversely, silencing of VaMIEL1 enhances cold tolerance by stabilizing VaMYB4a and boosting antioxidant defenses. These findings uncover a previously unrecognized regulatory mechanism by which VaMIEL1 modulates cold tolerance through transcriptional and oxidative stress pathways, offering potential targets for the development of climate-resilient grapevine cultivars and other crops.

Figure 1

Interaction between VaMIEL1 and VaMYB4a. (A) Schematic representation of VaMIEL1 and VaMYB4a protein structures, including deletion constructs. VaMIEL1 contains a zinc finger (zf-CHY) and RING finger domain, while VaMYB4a features R2 and R3 MYB domains. (B) Y2H assay was employed to assess the interactions between VaMIEL1 and VaMYB4a. Yeast strain growth was observed on double dropout (DDO:SD/−Trp/−Leu) and quadruple dropout (QDO/X/A/3-AT:SD/−Leu/−Trp/-His/−Ade/X-α-gal/AbA/3-AT) selective media. For controls, the interaction between mouse p53 and SV40 large T-antigen was used as a positive control (PO), and the lamin C and SV40 large T-antigen interaction as a negative control (Ne). (C) BiFC assay confirmed the in vivo interaction between VaMIEL1 and VaMYB4a. VaMIEL1 was fused to the N-terminal YFP fragment (nYFP), while VaMYB4a was linked to the C-terminal YFP fragment (cYFP). The constructs were coexpressed with and without the proteasome inhibitor MG132. Fluorescence, indicating YFP reconstitution, verified the interaction. Negative controls consisted of constructs with unfused nYFP and cYFP fragments. Scale bars = 10 μm. (D) Co-IP assays showing that VaMIEL1-GFP coprecipitates with VaMYB4a. Immunoprecipitations (IP) were performed with anti-VaMYB4a antibody, and immunoblotting (IB) was conducted using anti-GFP antibody. UT, untransformed calli. ‘No antibody’ denotes the absence of MYB4a antibody immunoprecipitation.

Figure 2

Sequence analysis of VaMIEL1. (A) Genomic structure of VaMIEL1, displaying the gene’s genomic DNA (gDNA, 3222 bp) and cDNA (870 bp). The corresponding protein structure, consisting of 289 amino acids, includes a zinc finger CHY domain (zf-CHY), a RING domain, and a zinc ribbon domain (zinc_ribbon_6). (B) Phylogenetic analysis of plant MIEL1 proteins. A phylogenetic tree was generated using the maximum likelihood approach with 1000 bootstrap replicates and subsequently visualized in the NCBI Tree Viewer. Tree topology reflects the minimum sum of branch lengths (S), with sequence homology exceeding 75% among the selected sequences. The analysis categorizes sequences into monocots, dicots, and outgroups. (C) Protein sequence alignment of VaMIEL1 (V. amurensis: PP471211), VvMIEL1 (V. vinifera: XP_002274709.1), MnRZFP34 (M. notabilis: XP_024022996.1), and AtMIEL1 (A. thaliana: NP_197366.1). The conserved Zf-CHY, RING, and zinc ribbon domains are highlighted to indicate structural similarities across species.

Figure 3

Expression and GUS enzyme activity analysis of VaMIEL1 in various plant tissues and in response to cold stress. (A) RT–qPCR analysis of VaMIEL1 expression in various tissues of V. amurensis (root, shoot, stem, leaf, and tendril). VvActin1 was used as an internal reference. Data are expressed as mean ± SD from three biological replicates. Statistical significance was determined by one-way ANOVA, followed by Tukey’s HSD test for comparisons among multiple groups. Pairwise comparisons were performed using an independent t-test (^*P < 0.05, ^**P < 0.01, ^***P < 0.001). (B) Temporal expression profile of VaMIEL1 in the leaves of V. amurensis ‘ZuoShan-1’ seedlings subjected to cold stress (−2°C) for varying durations (0–48 h). VvActin1 served as an internal control. Values represent the mean ± SD of three independent replicates, with significance relative to the 0 h time point assessed by one-way ANOVA and LSD test (^**P < 0.01). (C) Histochemical GUS staining of calli transformed with the VaMIEL1 promoter-driven GUS reporter construct (Pro_VaMIEL1::GUS), following exposure to cold stress at 4°C for 0, 1, 3, 6, and 9 h. CaMV35S::GUS and pCAMBIA2301::GUS served as positive and negative controls, respectively. Scale bar = 1 mm. The images were digitally extracted for visual comparison. (D) GUS activity assay in calli expressing Pro_VaMIEL1::GUS during cold stress exposure. GUS enzyme activity was quantified as 4-MU production (μmol 4-MU/mg protein/min) in Pro_VaMIEL1::GUS transgenic calli at various time points following cold stress (0, 1, 3, 6, and 9 h). Error bars denote the SD of three biological replicates. Statistical significance was determined by t-test, comparing each time point (1, 3, 6, 9 h) to the 0-h control (^*P < 0.05, ^**P < 0.01).

Figure 4

Overexpression of VaMIEL1 in Arabidopsis suppresses cold tolerance. (A) Cold stress phenotypes of VaMIEL1-OE (VaMIEL1 overexpression) Arabidopsis seedlings. Twelve-day-old seedlings were grown on one-half MS plates at 22°C before being exposed to CA (−7°C for 1.5 h) or NA (−5°C for 1.5 h) conditions. WT: wild type, #2 and #4: VaMIEL1-OE lines, miel1: AtMIEL1 knockout mutant. Scale bars = 0.5 cm. (B) Cold phenotypes of 3-week-old VaMIEL1-OE and miel1 plants grown in soil under similar CA and NA conditions. Scale bars = 0.5 cm. (C) Survival rates of WT, VaMIEL1-OE lines (#2 and #4), and miel1 plants following cold stress. (D) Relative electrolyte leakage in WT, VaMIEL1-OE, and miel1 lines after CA or NA treatments. (E) Histochemical staining with 3,3′-diaminobenzidine (DAB, for H₂O₂ detection) and Nitrotetrazolium blue chloride (NBT, for O₂^·− detection) in leaves of WT, VaMIEL1-OE (#2 and #4), and miel1 plants under control (25°C) or cold stress conditions (4°C for 3 days). Scale bars = 0.5 cm. (F) Quantification of Pro content, MDA content, SOD activity, and POD activity in WT, VaMIEL1-OE, and miel1 mutants following 3 days of cold treatment at 4°C. Data represent the mean ± SD of three independent biological replicates, with 15 plants per replicate. FW, fresh weigh. (G) Expression levels of cold-responsive genes in the CBF-COR pathway (AtCBF1, AtCBF2, AtCBF3, AtCOR47, AtRD29A, and AtRD29B) in WT, VaMIEL1-OE (#2 and #4), and miel1 mutants at 3 and 24 h under control (25°C) or cold stress (4°C) conditions. Gene expression was measured by RT-qPCR, normalized to AtActin1 as an internal control. Data represent the mean ± SD of three independent experiments. Statistical significance was determined using one-way ANOVA followed by post hoc Tukey test (^*P < 0.05, ^**P < 0.01).

Figure 5

VaMIEL1 negatively regulates cold tolerance in grapevine calli. (A) Cold stress phenotypes of UT, VaMIEL1-OE, and VaMIEL1-RNAi grapevine calli. Calli were precultured at 25°C for 10 days and then subjected to chilling stress at 10°C for 20 days. Bars = 1 cm. The images were digitally extracted for visual comparison. (B) Fresh weight of UT, VaMIEL1-OE, and VaMIEL1-RNAi grapevine calli after cold treatment. Fresh weight at 25°C was used as the control. Data represent the mean ± SD of three independent biological replicates (n = 6 per replicate). (C) O₂^·− (Superoxide radicals) and H₂O₂ were detected in UT, VaMIEL1-OE, and VaMIEL1-RNAi calli via NBT and DAB staining. Calli following 3 days of cold treatment at 4°C. Bars = 1 mm. The images were digitally extracted for visual comparison. (D) Pro content, MDA content, SOD activity, and POD activity were quantified in UT, VaMIEL1-OE, and VaMIEL1-RNAi calli maintained at control conditions (25°C) or subjected to 3 days of cold stress (4°C). (E) Expression levels of cold-responsive genes in the CBF-COR pathway in transgenic calli under control (25°C) or following cold stress (4°C) for 3 and 12 h. Gene expression was normalized to internal reference genes. Data represent the mean ± SD of three independent experiments. Statistical significance was determined using one-way ANOVA followed by post hoc Tukey test (^*P < 0.05, ^**P < 0.01).

Figure 6

VaMIEL1 ubiquitinates and promotes the degradation of VaMYB4a. (A) In vitro ubiquitination of VaMYB4a by VaMIEL1. Ubiquitination assays were performed in the presence of ubiquitin (Ub), E1, E2, and His-tagged VaMIEL1, with MBP-tagged VaMYB4a as the substrate. The ubiquitination of VaMYB4a was detected using an Anti-Ub antibody. His- and MBP-tagged proteins were detected as controls. (B) In vivo ubiquitination of VaMYB4a by VaMIEL1. Total protein extracts from UT calli, VaMIEL1-OE, and VaMIEL1-RNAi transgenic calli were immunoprecipitated using an anti-VaMYB4a antibody. The ubiquitinated proteins were detected by WB using an Anti-Ub antibody. Anti-GFP and anti-Actin antibodies were used as controls for protein loading. (C) Degradation of VaMYB4a in cell-free degradation assays. Protein extracts from N. benthamiana leaves transiently expressing MYC-VaMYB4a or coexpressing MYC-VaMYB4a and VaMIEL1-GFP were treated with the protein synthesis inhibitor CHX, either alone or in combination with the proteasome inhibitor MG132. VaMYB4a protein levels were detected using an anti-VaMYB4a antibody. Actin was used as the internal reference.

Figure 7

Enhanced cold tolerance in grapevine calli mediated by VaMIEL1 and VaMYB4a interactions through the CBF-COR signaling pathway. (A) Cold stress phenotypes of grapevine calli overexpressing VaMIEL1, VaMYB4a, or coexpressing both. Calli were cultured at 25°C for 10 days prior to exposure to chilling stress at 10°C for 20 days. Scale bars = 1 cm. (B) Fresh weight of UT, VaMIEL1-OE, VaMYB4a-OE, and coexpressed VaMIEL1-OE/VaMYB4a-OE grapevine calli after cold treatment at 10°C. (C) Detection of O₂^·- and H₂O₂ in UT, VaMIEL1-OE, VaMYB4a-OE, and VaMIEL1-OE/VaMYB4a-OE calli using NBT and DAB staining. Calli were cold treated at 4°C for 3 days. Bars = 1 mm. The images were digitally extracted for visual comparison. (D) Quantification of Pro content, MDA content, SOD activity, and POD activity in transgenic calli maintained under control conditions (25°C) or exposed to cold stress (4°C) for 3 days. (E) Expression levels of cold-regulated genes in the CBF-COR pathway in transgenic calli under control (25°C) or after exposure to cold stress (4°C) for 3 and 12 h. Data represent the mean ± SD of three independent experiments. Statistical significance was determined using one-way ANOVA followed by post hoc Tukey test (^*P < 0.05, ^**P < 0.01).

Figure 8

Working model of the VaMIEL1-VaMYB4a module in response to cold stress in grapevine. Under normal temperature conditions (left panel), VaMIEL1 is highly expressed and promotes the degradation of VaMYB4a via the 26S proteasome pathway, leading to a suppression of CBF transcription. Consequently, the antioxidant enzyme levels (SOD, POD) remain at normal levels, and ROS are maintained at a steady state. Cold-responsive (COR) genes are not activated, and cold tolerance is not induced. Under cold stress conditions (right panel), VaMIEL1 expression is downregulated, reducing VaMYB4a ubiquitination and allowing its accumulation. The stabilized VaMYB4a binds to the CBF promoter, activating its expression. Increased CBF levels lead to the induction of downstream COR genes, enhancing cold tolerance. Additionally, the upregulation of SOD and POD contributes to ROS scavenging, further improving stress adaptation. The black arrows represent inducible expression, while flat-ended lines indicate inhibition. Red and blue arrows denote changes in enzyme activity and expression levels, respectively. The red ‘X’ indicates inhibited regulation, and Ub denotes ubiquitination modifications.