1. Introduction
Low temperatures are a major ecological and environmental factor that strongly affects plant development and geographic distribution. Low temperature stress is categorized into chilling stress (0 –15 °C) and freezing stress (< 0 °C) [
1,
2]. Chilling and freezing stress are two kinds of different stresses, and require different solutions [
2]. Chilling damage is a directly temperature effect, which mainly restricts growth and development of plant, including wilting, chlorosis, sterility and even death. Nevertheless, freezing damage result from cellular dehydration and membrane injury triggered by extracellular ice crystallization. Thus, sudden freezing damage is more damaging to plants than chilling stress [
2]. However, plant have evolved a series of sophisticated regulatory mechanisms to adapt to low temperature stress. Among them, the most important regulatory mechanism termed cold acclimation (CA) (or cold hardening), which prior exposure plants to low but non-freezing temperature can enhance the freezing tolerance of plant [
2]. In this process, a series of physiological and molecular changes take place in cell, including synthesizing protective osmotic substances (soluble sugars, proline), and cold-resistance proteins (LEA, AFPs) [
3]. These substances and cold-resistance proteins participate in osmoregulation, reactive oxygen species (ROS) scavenging and ice crystal formation [
4]. In fact, cold stress triggered physiological and molecular changes rely in part on gene induction by transcriptional and post-transcriptional regulation. To date, the best characterized of cold signaling pathway is the ICE1-CBF-COR regulatory cascade.
In ICE1-CBF-COR cascade, the induction of cold-regulated (
COR) genes is an important biological event [
5].
COR genes encode key enzymes and cryoprotective proteins, such as soluble sugars, soluble proteins, and proline, which protect plant cells against cold-induced damage [
2]. Under low-temperature stress, the C-Repeat-Binding Factor (CBF) is rapidly induced and regulates the expression of downstream
COR genes by binding to their promoter regions [
6,
7,
8]. CBFs are key upstream transcription regulators of
COR genes, and their expression can be accurately controlled by upstream transcription factors. Among these factors, inducer of CBF expression 1 (ICE1), a MYC-like basic helix-loop-helix (bHLH) transcription factor, is the best-characterized positive regulator of
CBF genes identified to date [
6,
7,
8].
Under cold stress, ICE1 directly binds to the MYC recognition motif of the CBF3 promoter, leading to the activation of
CBF3 expression [
6]. In Arabidopsis, loss-of-function mutations in ICE1 lead to reduced resistance to cold stress, whereas ICE1 overexpression enhances the cold-induced upregulation of
CBFs [
6]. ICE2, a paralog of ICE1 with 61% identity, shares similar functions. Overexpression of either
ICE1 or
ICE2 enhances freezing resistance in transgenic Arabidopsis [
6,
9,
10]. Notably, ICE1 primarily regulates
CBF3 expression in cold signaling [
6], while ICE2 mainly targets
CBF1 [
10]. This suggests functional redundancy with distinct downstream targets. However, other studies using loss-of-function mutants suggest ICE1 may play a more dominant role compared with ICE2 [
11].
Although ICE1 is the key regulator in the ICE1–CBF–COR cold signaling pathway,
ICE1 expression itself is not responsive to cold at the transcriptional level [
6]. Its activity is controlled at the protein level by the 26S-proteasome pathway [
12,
13], highlighting the importance of post-translational modifications (PTM). Emerging evidence indicates that multiple PTMs control ICE1 cell turnover and duration at low temperatures. High expression of osmotically responsive gene 1 (HOS1), a ubiquitin E3 ligase with a RING finger, directly interacts with ICE1, promoting its degradation and negatively regulating cold resistance [
13]. Conversely, SAP and Miz1 domain-containing ligase 1 (SIZ1), a SUMO E3 ligase, enhances cold tolerance by stabilizing ICE1 through SUMOylation, which reduces HOS1-mediated ubiquitination [
14]. Open stomata 1 (OST1), a Ser/Thr protein kinase involved in abscisic acid (ABA) signaling, can be activated by cold stress. Cold-activated OST1 phosphorylates ICE1 and enhances its stability by interfering with the interaction between HOS1 and ICE1, thereby enhancing freezing tolerance [
12]. Beyond their role in cold signaling, ICE1/SCREAM (SCRM) and ICE2/SCRM2 are also involved in diverse processes, including stomatal development [
15,
16], flowering [
17], primary seed dormancy [
18], male fertility, and ABA signaling [
19]. These findings suggest that ICE1 is not only a central component in the ICE1–CBF–COR cold signaling pathway but also serves as a convergence point, integrating multiple signals to regulate both cold tolerance and plant growth.
Given its crucial role, ICE1 homologs have been identified in numerous plants, including wheat (
Triticum aestivum) [
20], rice (
Oryza sativa) [
21],
Saussurea involucrata [
22], maize (
Zea mays) [
23], and tomato (
Solanum lycopersicum) [
24]. Although these ICE1 homologous genes are involved in cold stress, different species may display diverse ICE1-dependent cold regulation mechanisms [
25,
26,
27]. For example, in Arabidopsis, cold-activated mitogen-activated protein kinase 3 (MPK3) and MPK6 kinases phosphorylate ICE1, targeting it for degradation and negatively regulating cold responses [
26,
27]. However, in rice, cold-activated OsMAPK3 phosphorylates and stabilizes OsbHLH002 (a rice ICE1 homolog), leading to trehalose-6-phosphate phosphatase (OsTPP1) activation and increased OsTPP1-regulated trehalose content [
25]. These contrasting findings indicate that ICE1 is evolutionarily conserved but contains functional divergence in cold signals in different species, particularly between Arabidopsis and rice. This functional differentiation of ICE1 is supported by a recent study in which
PsnICE1, a poplar ICE1 homolog (
Populus tomentosa Carr), was not only involved in the CBF-dependent pathway but also in reactive oxygen species (ROS) scavenging in response to cold stress by binding to different cis-acting elements [
28].
In addition to the functional diversity of different species, conflicting results exist regarding Arabidopsis ICE1's role in cold signaling. For example,
ice1 (a single substitution mutant of Arg-236 to His), a well-known dominant-negative mutant, exhibits reduced chilling and freezing tolerance, regardless of cold acclimation [
6]. However, another study found that
ice1-2 and
ice2-2, two T-DNA insertional mutants, did not exhibit any sensitive freezing tolerance phenotype in the absence of cold acclimation, suggesting that ICE1 and ICE2 may not be involved in the basal cold resistance of plants [
11].
Over the past three decades, the biological function of ICE1 has been well-understood in Arabidopsis and rice, however, as neither are winter plants, they cannot survive safely in winter. In contrast, some winter crops, such as winter rapeseed (
Brassica rapa), an important oilseed and economic crop worldwide, can survive at extremely low temperatures (-20 ℃ to -32 ℃) [
29,
30]. Theoretically, these winter
Brassica rapa crops may have evolved more effective cold acclimation mechanisms to respond to chilling and freezing stresses, however, the fundamental molecular mechanism remains elusive in
Brassica rapa. Until recently, using multi-omics technology, some cold-responsive differentially expressed genes (DEGs) [
29,
31,
32,
33], microRNA [
34,
35], differentially accumulated proteins (DAPs) [
36,
37] have been identified in
Brassica rapa. Nevertheless, these omics studies still cannot reveal the detail molecular mechanism, which
Brassica rapa how to response to the cold stress in molecular level. Unlike in Arabidopsis and rice, to data, only a few cold-regulated genes have been identified from
Brassica napus (
BN115, BnCBF17, BnHOS1) [
38,
39,
40], but not from
Brassica rapa. Recently, several studies have started to try to explore the molecular mechanism of
Brassica rapa to response to cold stress. For instance, Dong et al. found that overexpression
Brassica rapa antifreeze protein 1(BrAFP1) increased the cold tolerance of transgenic Arabidopsis [
41]. Our previous study using transcriptome analysis and immunoblotting assays revealed that MAPK kinase and Ca
2+-related protein kinase are important signaling molecule under low temperature stress in
Brassica rapa [
31]. Subsequently
, our study also found that
Brassica rapa EIN3-binding f-box 1 (BrEBF1) positively regulating cold tolerance, and BrEBF1 regulated cold tolerance is associated with ROS scavenging and MAPK kinase activity [
42].
Considering ICE1’s role in cold signaling, some researchers have investigated its physiological functions in Brassica rapa. However, unlike in Arabidopsis, the phylogenetic, evolutionary, and physiological functional divergence of ICE1 paralogs in cold signals remains unknown. In this study, the ICE1 homologs genes of six widely cultivated Brassica species were identified, and the role of BrICE1 and BrICE2 of Brassica rapa in cold signal were investigated. Our studies discovered that ICE1 homologous genes exhibit complex evolutionary relationships in Brassica species; two novel BrICE1 and BrICE2 paralogs of Brassica rapa positively regulate the cold tolerance via CBFs pathway and ROS scavenging mechanism, and balancing development and cold defense in transgenic Arabidopsis.
Figure 1.
Phylogenetic analysis of ICE1 homologous genes in Brassica species. The phylogenetic tree was constructed by neighbor-joining distance using MEGA 6.0. A total of 42 ICE1 homologous genes were identified from Brassica species. Well-known ICE1 and ICE2 homologous genes of dicotyledon Arabidopsis thaliana, tomato (Solanum lycopersicum), soybean (Glycine max), monocotyledon maize (Zea mays), foxtail millet (Setaria italica) and rice (Oryza sativa) were used as the outgroup. BrICE, BraICE, BoICE, BniICE, BnICE, BjuICE and BcaICE stand for the ICE1 homologous genes of Z1 (B. rapa, yellow sarson, as an oilseed crop), Chiifu-401-42 (B. rapa, Chinese cabbage, as a vegetable), B. oleracea, B. nigra, B. napus, B. juncea and B. carinata, respectively. Red filled triangle, red filled square and red filled circle represents ICE1 homologous of Arabidopsis, B. rapa (Chiifu-401-42, as a vegetable) and B. rapa (Z1, as an oilseed crop).
Figure 1.
Phylogenetic analysis of ICE1 homologous genes in Brassica species. The phylogenetic tree was constructed by neighbor-joining distance using MEGA 6.0. A total of 42 ICE1 homologous genes were identified from Brassica species. Well-known ICE1 and ICE2 homologous genes of dicotyledon Arabidopsis thaliana, tomato (Solanum lycopersicum), soybean (Glycine max), monocotyledon maize (Zea mays), foxtail millet (Setaria italica) and rice (Oryza sativa) were used as the outgroup. BrICE, BraICE, BoICE, BniICE, BnICE, BjuICE and BcaICE stand for the ICE1 homologous genes of Z1 (B. rapa, yellow sarson, as an oilseed crop), Chiifu-401-42 (B. rapa, Chinese cabbage, as a vegetable), B. oleracea, B. nigra, B. napus, B. juncea and B. carinata, respectively. Red filled triangle, red filled square and red filled circle represents ICE1 homologous of Arabidopsis, B. rapa (Chiifu-401-42, as a vegetable) and B. rapa (Z1, as an oilseed crop).
Figure 2.
Low temperature induces the expression of ICE1 homologous genes in Brassica Species. The 14-day-old seedlings were low temperature treated at 4 ℃ for 6 h, 12h, and 24 h, while the expression levels of ICE1 homologous genes were determined by qRT–PCR. BrACTIN2 was used as the control. (A) The expression profiles of six BnICE1 homologous genes in Westar. (B–D) The expression profiles of four BrICE1 homologous genes in Tianyou 2, Longyou 6, and Longyou 8. Values are shown as mean ± SD (n=3) of three independent experiments. Statistically significant differences are indicated by asterisks (Student’s t-test, *, p < 0.05).
Figure 2.
Low temperature induces the expression of ICE1 homologous genes in Brassica Species. The 14-day-old seedlings were low temperature treated at 4 ℃ for 6 h, 12h, and 24 h, while the expression levels of ICE1 homologous genes were determined by qRT–PCR. BrACTIN2 was used as the control. (A) The expression profiles of six BnICE1 homologous genes in Westar. (B–D) The expression profiles of four BrICE1 homologous genes in Tianyou 2, Longyou 6, and Longyou 8. Values are shown as mean ± SD (n=3) of three independent experiments. Statistically significant differences are indicated by asterisks (Student’s t-test, *, p < 0.05).
Figure 3.
Multiple sequence alignment and domain structure analysis of BrICE1 and BrICE2 of B. rapa. The DNAMAN v9.0 software was used to align the amino acid sequences of BrICE1 and BrICE2. Residues in red indicate the conserved serine-rich (S-rich) region sites, ZIP region domain, ICE-specific domain, potential SUMOylation site, and ACT-like domains. Residues in blue and green indicate the conserved MYC-like bHLH domain. Residues in purple indicate the specific glutamine-rich (Gin-rich) and leucine-rich (Leu-rich) domains of BrICE2.
Figure 3.
Multiple sequence alignment and domain structure analysis of BrICE1 and BrICE2 of B. rapa. The DNAMAN v9.0 software was used to align the amino acid sequences of BrICE1 and BrICE2. Residues in red indicate the conserved serine-rich (S-rich) region sites, ZIP region domain, ICE-specific domain, potential SUMOylation site, and ACT-like domains. Residues in blue and green indicate the conserved MYC-like bHLH domain. Residues in purple indicate the specific glutamine-rich (Gin-rich) and leucine-rich (Leu-rich) domains of BrICE2.
Figure 4.
BrICE1 and BrICE2 are nuclear-localized proteins. The 35S:BrICE1-GFP, 35S: BrICE2-GFP, 35S:AtICE1-GFP, and 35S:AtICE2-GFP plasmids were constructed and transiently expressed in tobacco leaves. The GFP signal was visualized under confocal microscope. Nuclei were indicated by DAPI staining, plasma membranes were indicated by FM4-64 staining, and autofluorescence of chloroplasts was indicated by chlorophyll b staining. From left to right, DAPI signal, green fluorescence GFP signal, chlorophyll autofluorescence signal, and merged image signal. Scale bar, 10 μm.
Figure 4.
BrICE1 and BrICE2 are nuclear-localized proteins. The 35S:BrICE1-GFP, 35S: BrICE2-GFP, 35S:AtICE1-GFP, and 35S:AtICE2-GFP plasmids were constructed and transiently expressed in tobacco leaves. The GFP signal was visualized under confocal microscope. Nuclei were indicated by DAPI staining, plasma membranes were indicated by FM4-64 staining, and autofluorescence of chloroplasts was indicated by chlorophyll b staining. From left to right, DAPI signal, green fluorescence GFP signal, chlorophyll autofluorescence signal, and merged image signal. Scale bar, 10 μm.
Figure 5.
Cold induces the degradation of BrICE1 and BrICE2. The three-day-old seedlings were grown on agar plates treated (4°C, 12 h), the roots were incubated in 0.02 mg/mL PI for 12 min, and the GFP signals in roots were visualized and photographed using confocal microscopy. (A) Visualization of AtICE1-GFP and BrICE1-GFP transgenic plants. (B) Visualization of AtICE2-GFP and BrICE2-GFP transgenic plants. Scale bar, 100 μm.
Figure 5.
Cold induces the degradation of BrICE1 and BrICE2. The three-day-old seedlings were grown on agar plates treated (4°C, 12 h), the roots were incubated in 0.02 mg/mL PI for 12 min, and the GFP signals in roots were visualized and photographed using confocal microscopy. (A) Visualization of AtICE1-GFP and BrICE1-GFP transgenic plants. (B) Visualization of AtICE2-GFP and BrICE2-GFP transgenic plants. Scale bar, 100 μm.
Figure 6.
Overexpression of BrICE1 and BrICE2 enhances the cold tolerance of transgenic Arabidopsis through the CBF-dependent pathway. The 14-day-old seedlings were subjected freezing at – 6 ℃ for 1 h with (CA, at 4 ℃ for 3 days) or without cold accumulation (NA). After 3 days recovery at 22 ℃, the survival rates, and ion leakage rates were determined. To test the expression levels of CBFs and their target genes, 14-day-old seedlings were low-temperature treated (at 4 ℃) for either 12 or 24 h and subjected to qRT–PCR analysis. ACTIN2 was used as the reference gene. (A) Freezing phenotypes. (B) Survival rates (n=120). (C) Ion leakage rates (n=30). (D–F) Expression levels of AtCBF1, AtCBF2 and AtCBF3 (n=3). (G–I) The expression levels of AtCOR15A, AtCOR47A, and AtKIN7 (n=3). Values are shown as mean ± SD of three independent experiments. Statistically significant differences are indicated by asterisks (Student’s t-test, *, p < 0.05).
Figure 6.
Overexpression of BrICE1 and BrICE2 enhances the cold tolerance of transgenic Arabidopsis through the CBF-dependent pathway. The 14-day-old seedlings were subjected freezing at – 6 ℃ for 1 h with (CA, at 4 ℃ for 3 days) or without cold accumulation (NA). After 3 days recovery at 22 ℃, the survival rates, and ion leakage rates were determined. To test the expression levels of CBFs and their target genes, 14-day-old seedlings were low-temperature treated (at 4 ℃) for either 12 or 24 h and subjected to qRT–PCR analysis. ACTIN2 was used as the reference gene. (A) Freezing phenotypes. (B) Survival rates (n=120). (C) Ion leakage rates (n=30). (D–F) Expression levels of AtCBF1, AtCBF2 and AtCBF3 (n=3). (G–I) The expression levels of AtCOR15A, AtCOR47A, and AtKIN7 (n=3). Values are shown as mean ± SD of three independent experiments. Statistically significant differences are indicated by asterisks (Student’s t-test, *, p < 0.05).
Figure 7.
Overexpression of BrICE1 and BrICE2 inhibits root growth in Arabidopsis. Seedlings were grown on half-strength MS at 22 ℃ for 7 days; roots length was measured using the ImageJ software and designated as L1. After 3 days at 22 ℃, seedlings were cold treated for 42 days at 22 ℃, and roots length was measured and designated as L2. The relative reduction rate of roots length was calculated as (L1-L2)/L1 × 100%. (A) Roots length phenotype. (B) Statistical analysis of roots length (n=90). (C) Relative reduction rate of roots length under low treatment. Values are shown as the mean ± SD of three independent experiments, each with three technical replicates. Statistically significant differences are indicated by asterisks (Student’s t-test, *, p < 0.05). Scale bar, 1 cm.
Figure 7.
Overexpression of BrICE1 and BrICE2 inhibits root growth in Arabidopsis. Seedlings were grown on half-strength MS at 22 ℃ for 7 days; roots length was measured using the ImageJ software and designated as L1. After 3 days at 22 ℃, seedlings were cold treated for 42 days at 22 ℃, and roots length was measured and designated as L2. The relative reduction rate of roots length was calculated as (L1-L2)/L1 × 100%. (A) Roots length phenotype. (B) Statistical analysis of roots length (n=90). (C) Relative reduction rate of roots length under low treatment. Values are shown as the mean ± SD of three independent experiments, each with three technical replicates. Statistically significant differences are indicated by asterisks (Student’s t-test, *, p < 0.05). Scale bar, 1 cm.
Figure 8.
Overexpression of BrICE1 and BrICE2 enhances ROS scavenging by elevating enzymatic antioxidants in Arabidopsis. The 10-day-old seedlings were chill and freeze treated for either 3 or 6 h, and the leaves were stained using an NBT solution. The phenotype was photographed, and the activities of SOD, CAT, POD, and O2-·, as well as the MDA, soluble sugar, and proline contents were detected. (A) The phenotype of ROS accumulation. (B) The changes of O2-· content. (C–E) The activity of SOD, CAT, and POD. (F–H) The MDA, soluble sugars, and proline contents. Values are shown as mean ± SD (n = 30) of three independent experiments, each with three technical replicates. Statistically significant differences are indicated by asterisks (Student’s t-test, *, p < 0.05). Scale bar, 2 mm.
Figure 8.
Overexpression of BrICE1 and BrICE2 enhances ROS scavenging by elevating enzymatic antioxidants in Arabidopsis. The 10-day-old seedlings were chill and freeze treated for either 3 or 6 h, and the leaves were stained using an NBT solution. The phenotype was photographed, and the activities of SOD, CAT, POD, and O2-·, as well as the MDA, soluble sugar, and proline contents were detected. (A) The phenotype of ROS accumulation. (B) The changes of O2-· content. (C–E) The activity of SOD, CAT, and POD. (F–H) The MDA, soluble sugars, and proline contents. Values are shown as mean ± SD (n = 30) of three independent experiments, each with three technical replicates. Statistically significant differences are indicated by asterisks (Student’s t-test, *, p < 0.05). Scale bar, 2 mm.
Figure 9.
Cold-induced degradation of BrICE1 and BrICE2 depends on the 26S-proteasome pathway. The 14-day-old wild-type and transgenic seedlings were treated at 4 °C for 1 to 24h with or without 100 mM CHX and 50 mM MG132, total protein was extracted and immunoblotting were performed using specific anti-ICE1 and anti-GFP antibodies. Coomassie brilliant blue (CBB) was used as the control for protein loading. The integrated optical density (IOD) values of ICE1bands were quantified. (A, B) Immunoblotting assays to assess the protein level in wild-type and transgenic seedlings without CHX and MG132 treatment using specific anti-ICE1 (A) and anti-GFP (B) antibodies. (C, D) Immunoblotting assays to assess the protein level in wild-type and transgenic seedlings with CHX and MG132 treatment using specific anti-ICE1 (A) and anti-GFP (B) antibodies.
Figure 9.
Cold-induced degradation of BrICE1 and BrICE2 depends on the 26S-proteasome pathway. The 14-day-old wild-type and transgenic seedlings were treated at 4 °C for 1 to 24h with or without 100 mM CHX and 50 mM MG132, total protein was extracted and immunoblotting were performed using specific anti-ICE1 and anti-GFP antibodies. Coomassie brilliant blue (CBB) was used as the control for protein loading. The integrated optical density (IOD) values of ICE1bands were quantified. (A, B) Immunoblotting assays to assess the protein level in wild-type and transgenic seedlings without CHX and MG132 treatment using specific anti-ICE1 (A) and anti-GFP (B) antibodies. (C, D) Immunoblotting assays to assess the protein level in wild-type and transgenic seedlings with CHX and MG132 treatment using specific anti-ICE1 (A) and anti-GFP (B) antibodies.