C-repeat Binding Factor Expression Research Articles

Genome-Wide Identification of the CBF Gene Family and ICE Transcription Factors in Walnuts and Expression Profiles under Cold Conditions.

Cold stress impacts woody tree growth and perennial production, especially when the temperature rapidly changes in late spring. To address this issue, we conducted the genome-wide identification of two important transcription factors (TFs), CBF (C-repeat binding factors) and ICE (inducers of CBF expression), in three walnut (Juglans) genomes. Although the CBF and ICE gene families have been identified in many crops, very little systematic analysis of these genes has been carried out in J. regia and J. sigillata. In this study, we identified a total of 16 CBF and 12 ICE genes in three Juglans genomes using bioinformatics analysis. Both CBF and ICE had conserved domains, motifs, and gene structures, which suggests that these two TFs were evolutionarily conserved. Most ICE genes are located at both ends of the chromosomes. The promoter cis-regulatory elements of CBF and ICE genes are largely involved in light and phytohormone responses. Based on 36 RNA sequencing of leaves from four walnut cultivars ('Zijing', 'Lvling', 'Hongren', and 'Liao1') under three temperature conditions (8 °C, 22 °C, and 5 °C) conditions in late spring, we found that the ICE genes were expressed more highly than CBFs. Both CBF and ICE proteins interacted with cold-related proteins, and many putative miRNAs had interactions with these two TFs. These results determined that CBF1 and ICE1 play important roles in the tolerance of walnut leaves to rapid temperature changes. Our results provide a useful resource on the function of the CBF and ICE genes related to cold tolerance in walnuts.

PUB25 and PUB26 dynamically modulate ICE1 stability via differential ubiquitination during cold stress in Arabidopsis.

Ubiquitination modulates protein turnover or activity depending on the number and location of attached ubiquitin (Ub) moieties. Proteins marked by a lysine 48 (K48)-linked polyubiquitin chain are usually targeted to the 26S proteasome for degradation; however, other polyubiquitin chains, such as those attached to K63, usually regulate other protein properties. Here, we show that 2 PLANT U-BOX E3 ligases, PUB25 and PUB26, facilitate both K48- and K63-linked ubiquitination of the transcriptional regulator INDUCER OF C-REPEAT BINDING FACTOR (CBF) EXPRESSION1 (ICE1) during different periods of cold stress in Arabidopsis (Arabidopsis thaliana), thus dynamically modulating ICE1 stability. Moreover, PUB25 and PUB26 attach both K48- and K63-linked Ub chains to MYB15 in response to cold stress. However, the ubiquitination patterns of ICE1 and MYB15 mediated by PUB25 and PUB26 differ, thus modulating their protein stability and abundance during different stages of cold stress. Furthermore, ICE1 interacts with and inhibits the DNA-binding activity of MYB15, resulting in an upregulation of CBF expression. This study unravels a mechanism by which PUB25 and PUB26 add different polyubiquitin chains to ICE1 and MYB15 to modulate their stability, thereby regulating the timing and degree of cold stress responses in plants.

High-throughput miRNA sequencing and identification of a novel ICE1-targeting miRNA in response to low temperature stress in Eucalyptus camaldulensis.

MicroRNAs (miRNAs) play a crucial role in the growth, development, morphogenesis, signal transduction, and stress response in plants. The ICE (Inducer of CBF expression)-CBF (C-repeat binding factor)-COR (Cold-regulated gene) regulatory cascade is an important signalling pathway in plant response to low temperature stress, and it remains unknown whether this pathway is regulated by miRNAs. In this study, high-throughput sequencing was employed for predicting and identifying the miRNAs that were likely to target the ICE-CBF-COR pathway in Eucalyptus camaldulensis. A novel ICE1-targeting miRNA, eca-novel-miR-259-5p (nov-miR259), was further analysed. A total of 392 conserved miRNAs and 97 novel miRNAs were predicted, including 80 differentially expressed miRNAs. Of these, 30 miRNAs were predicted to be associated with the ICE-CBF-COR pathway. The full-length of mature nov-miR259 was 22 bp and its precursor gene was 60 bp in length, with a typical hairpin structure. The RNA ligase-mediated 5' amplification of cDNA ends (5'-RLM-RACE) and Agrobacterium-mediated tobacco transient expression assays demonstrated that nov-miR259 could cleave EcaICE1 in vivo. Moreover, qRT-PCR and Pearson's correlation analysis further revealed that the expression levels of nov-miR259 were almost significantly negativelycorrelated with those of its target gene, EcaICE1, and the other genes in the ICE-CBF-COR pathway. We first identified the nov-miR259 as a novel ICE1-targeting miRNA, and the nov-miR259-ICE1 module may be involved in regulating the cold stress response in E.camaldulensis.

Integration of chromatin accessibility and gene expression reveals new regulators of cold hardening to enhance freezing tolerance in Prunus mume.

Low temperature is one of the most important abiotic factors limiting the growth, development and geographical distribution of plants. Prunus mume is an attractive woody ornamental plant that blooms in early spring in Beijing. However, the molecular mechanisms underlying cold hardening to enhance freezing tolerance in Prunus genus remains elusive. This study examined the dynamic physiological responses induced by cold hardening, and identified freezing-tolerance genes by RNA-seq and ATAC-seq analyses. Cold hardening elevated the content of soluble substances and enhanced freezing resistance in P. mume. Transcriptome analysis indicated that the candidate differentially expressed genes (DEGs) were those enriched in Ca2+ signalling, mitogen-activated protein kinase (MAPK) cascade, abscisic acid signalling, and inducer of CBF expression 1 (ICE)-C-repeat binding factor (CBF) signalling pathways. The openness of gene chromatin positively correlated with the expression level of these genes. Thirteen motifs were identified in the open chromatin regions in the treatment group subjected to freezing after cold hardening. The chromatin opening of transcription start site at the proximal -177 region of cold-shock protein CS120-like (PmCSL) was markedly increased, while the expression level of PmCSL was significantly up-regulated. Overexpression of PmCSL in Arabidopsis significantly improved the freezing tolerance of transgenic plants. These findings provide new insights into the regulatory mechanism of freezing tolerance to improve breeding of cold-hardy P. mume plants.

Transcriptome and Metabolome Analyses Reveal Molecular Responses of Two Pepper (Capsicum annuum L.) Cultivars to Cold Stress.

Low temperature is a significant factor affecting field-grown pepper. The molecular mechanisms behind peppers’ response to cold stress remain unknown. Transcriptomic and metabolomic analyses were used to investigate the responses of two pepper cultivars, XS (cold-sensitive) and GZ (cold-resistant), to cold stress; these were screened from 45 pepper materials. In this study, compared with the control group (0 h), we identified 10,931 differentially expressed genes (DEGs) in XS and GZ, 657 differentially expressed metabolites (DEMs) in the positive ion mode, and 390 DEMs in the negative ion mode. Most DEGs were involved in amino acid biosynthesis, plant hormone signal transduction, and the mitogen-activated protein kinase (MAPK) signaling pathway. Furthermore, metabolomic analysis revealed that the content of free polyamines (PAs), plant hormones, and osmolytes, mainly contained increased putrescine, spermine, spermidine, abscisic acid (ABA), jasmonic acid (JA), raffinose, and proline, in response to cold stress. Importantly, the regulation of the ICE (inducer of CBF expression)-CBF (C repeat binding factors)-COR (cold regulated) pathway by Ca2+ signaling, MAPK signaling, and reactive oxygen species (ROS) signaling plays a key role in regulating responses of peppers to cold stress. Above all, the results of the present study provide important insights into the response of peppers to cold stress, which will reveal the potential molecular mechanisms and contribute to pepper screening and breeding in the future.

The network centered on ICEs play roles in plant cold tolerance, growth and development.

ICEs are key transcription factors in response to cold in plant, they also balance plant growth and stress tolerance. Thus, we systematize the information about ICEs published to date. Low temperature is an important factor affecting plant growth and development. Exposing to cold condition results in a suit of effects on plants including reduction of plant growth and reproduction, and decrease in crop yield and quality. Plants have evolved a series of strategies to deal with cold stress such as reprogramming of the expression of genes and transcription factors. ICEs (Inducer of CBF Expression), as transcription factors regulating CBFs (C-repeat binding factor), play key roles in balancing plant growth and stress tolerance. Studies on ICEs focused on the function of ICEs on cold tolerance, growth and development; post-translational modifications of ICEs and crosstalk between the ICEs and phytohormones. In this review, we focus on systematizing the information published to date. We summarized the main advances of the functions of ICEs on the cold tolerance, growth and development. And we also elaborated the regulation of ICEs protein stability including phosphorylation, ubiquitination and SUMOylation of ICE. Finally, we described the function of ICEs in the crosstalk among different phytohormone signaling pathway and cold stress. This review provides perspectives for ongoing research about cold tolerance, growth and development in plant.

ICE-CBF-COR Signaling Cascade and Its Regulation in Plants Responding to Cold Stress.

Cold stress limits plant geographical distribution and influences plant growth, development, and yields. Plants as sessile organisms have evolved complex biochemical and physiological mechanisms to adapt to cold stress. These mechanisms are regulated by a series of transcription factors and proteins for efficient cold stress acclimation. It has been established that the ICE-CBF-COR signaling pathway in plants regulates how plants acclimatize to cold stress. Cold stress is perceived by receptor proteins, triggering signal transduction, and Inducer of CBF Expression (ICE) genes are activated and regulated, consequently upregulating the transcription and expression of the C-repeat Binding Factor (CBF) genes. The CBF protein binds to the C-repeat/Dehydration Responsive Element (CRT/DRE), a homeopathic element of the Cold Regulated genes (COR gene) promoter, activating their transcription. Transcriptional regulations and post-translational modifications regulate and modify these entities at different response levels by altering their expression or activities in the signaling cascade. These activities then lead to efficient cold stress tolerance. This paper contains a concise summary of the ICE-CBF-COR pathway elucidating on the cross interconnections with other repressors, inhibitors, and activators to induce cold stress acclimation in plants.

Nitric Oxide Regulates Mitochondrial Fatty Acids and Promotes CBF Expression of Peach Fruit during Cold Storage

Low-temperature storage is convenient for postharvest preservation of peach fruit, but peach fruit is sensitive to cold damage, which lowers its quality. Nitric oxide (NO) has the potential to improve the bitter resistance of peach fruit. In this work, peach fruit was treated with 15 μmol L−1 NO and 5 μmol L−1 c-PTIO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxo-3-oxide], to study changes in mitochondrial fatty acids and expression of the C-repeat binding factor (CBF). The results showed that 15 μmol L−1 exogenous NO significantly maintained fruit quality, reduced peroxidation of mitochondrial fatty acids, increased the activities of the antioxidants superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbic acid peroxidase (APX), and reduced the content of hydrogen peroxide (H2O2). Meanwhile, NO treatment suppressed the increase in browning index and ion leakage rate, increased the activity of S-nitrosoglutathione reductase (GSNOR), the contents of S-nitrosothiols (SNOs), and the ratios of mitochondrial NAD+/NADH and NADP+/NADPH, increased the expression levels of PpCBF1/5/6. However, the expression levels of PpCBF2/3/4 were not significantly regulated by exogenous NO. Peaches treated with c-PTIO showed opposite effects to those treated with exogenous NO. These results suggest that exogenous NO can improve antioxidant capacity, preserve mitochondrial fatty acids, and upregulate the expression of PpCBF1/5/6 to alleviate cold tolerance and maintain the peach quality during storage.

An R2R3-MYB Transcription Factor RmMYB108 Responds to Chilling Stress of Rosa multiflora and Conferred Cold Tolerance of Arabidopsis

A sudden cooling in the early spring or late autumn negatively impacts the plant growth and development. Although a number of studies have characterized the role of the transcription factors (TFs) of plant R2R3-myeloblastosis (R2R3-MYB) in response to biotic and abiotic stress, plant growth, and primary and specific metabolisms, much less is known about their role in Rosa multiflora under chilling stress. In the present study, RmMYB108, which encodes a nuclear-localized R2R3-MYB TF with a self-activation activity, was identified based on the earlier published RNA-seq data of R. multiflora plants exposed to short-term low-temperature stress and also on the results of prediction of the gene function referring Arabidopsis. The RmMYB108 gene was induced by stress due to chilling, salt, and drought and was expressed in higher levels in the roots than in the leaves. The heterologous expression of RmMYB108 in Arabidopsis thaliana significantly enhanced the tolerance of transgenic plants to freezing, water deficit, and high salinity, enabling higher survival and growth rates, earlier flowering and silique formation, and better seed quantity and quality compared with the wild-type (WT) plants. When exposed to a continuous low-temperature stress at 4°C, transgenic Arabidopsis lines–overexpressing RmMYB108 showed higher activities of superoxide dismutase and peroxidase, lower relative conductivity, and lower malondialdehyde content than the WT. Moreover, the initial fluorescence (Fo) and maximum photosynthetic efficiency of photosystem II (Fv/Fm) changed more dramatically in the WT than in transgenic plants. Furthermore, the expression levels of cold-related genes involved in the ICE1 (Inducer of CBF expression 1)-CBFs (C-repeat binding factors)-CORs (Cold regulated genes) cascade were higher in the overexpression lines than in the WT. These results suggest that RmMYB108 was positively involved in the tolerance responses when R. multiflora was exposed to challenges against cold, freeze, salt, or drought and improved the cold tolerance of transgenic Arabidopsis by reducing plant damage and promoting plant growth.

Crosstalk between Brassinosteroid and Redox Signaling Contributes to the Activation of CBF Expression during Cold Responses in Tomato.

Brassinosteroids (BRs) play a critical role in plant responses to stress. However, the interplay of BRs and reactive oxygen species signaling in cold stress responses remains unclear. Here, we demonstrate that a partial loss of function in the BR biosynthesis gene DWARF resulted in lower whilst overexpression of DWARF led to increased levels of C-REPEAT BINDING FACTOR (CBF) transcripts. Exposure to cold stress increased BR synthesis and led to an accumulation of brassinazole-resistant 1 (BZR1), a central component of BR signaling. Mutation of BZR1 compromised the cold- and BR-dependent increases in CBFs and RESPIRATORY BURST OXIDASE HOMOLOG 1 (RBOH1) transcripts, as well as preventing hydrogen peroxide (H2O2) accumulation in the apoplast. Cold- and BR-induced BZR1 bound to the promoters of CBF1, CBF3 and RBOH1 and promoted their expression. Significantly, suppression of RBOH1 expression compromised cold- and BR-induced accumulation of BZR1 and related increases in CBF transcripts. Moreover, RBOH1-dependent H2O2 production regulated BZR1 accumulation and the levels of CBF transcripts by influencing glutathione homeostasis. Taken together, these results demonstrate that crosstalk between BZR1 and reactive oxygen species mediates cold- and BR-activated CBF expression, leading to cold tolerance in tomato (Solanum lycopersicum).

Apple B-box protein BBX37 regulates jasmonic acid mediated cold tolerance through the JAZ-BBX37-ICE1-CBF pathway and undergoes MIEL1-mediated ubiquitination and degradation.

The plant hormone jasmonic acid (JA) is involved in the cold stress response, and the inducer of CBF expression 1 (ICE1)- C-repeat binding factor (CBF) regulatory cascade plays a key role in the regulation of cold stress tolerance. In this study, we showed that a novel B-box (BBX) protein MdBBX37 positively regulates JA-mediated cold-stress resistance in apple. We found that MdBBX37 bound to the MdCBF1 and MdCBF4 promoters to activate their transcription, and also interacted with MdICE1 to enhance the transcriptional activity of MdICE1 on MdCBF1, thus promoting its cold tolerance. Two JA signaling repressors, MdJAZ1 and MdJAZ2 (JAZ, JAZMONATE ZIM-DOMAIN), interacted with MdBBX37 to repress the transcriptional activity of MdBBX37 on MdCBF1 and MdCBF4, and also interfered with the interaction between MdBBX37 and MdICE1, thus negatively regulating JA-mediated cold tolerance. E3 ligase MdMIEL1 (MIEL1, MYB30-Interacting E3 Ligase1) reduced MdBBX37-improved cold resistance by mediating ubiquitination and degradation of the MdBBX37 protein. The data reveal that MIEL1 and JAZ proteins co-regulate JA-mediated cold stress tolerance through the BBX37-ICE1-CBF module in apple. These results will aid further examination of the post-translational modification of BBX proteins and the regulatory mechanism of JA-mediated cold stress tolerance.

Varying Atmospheric CO2 Mediates the Cold-Induced CBF-Dependent Signaling Pathway and Freezing Tolerance in Arabidopsis.

Changes in the stomatal aperture in response to CO2 levels allow plants to manage water usage, optimize CO2 uptake and adjust to environmental stimuli. The current study reports that sub-ambient CO2 up-regulated the low temperature induction of the C-repeat Binding Factor (CBF)-dependent cold signaling pathway in Arabidopsis (Arabidopsis thaliana) and the opposite occurred in response to supra-ambient CO2. Accordingly, cold induction of various downstream cold-responsive genes was modified by CO2 treatments and expression changes were either partially or fully CBF-dependent. Changes in electrolyte leakage during freezing tests were correlated with CO2′s effects on CBF expression. Cold treatments were also performed on Arabidopsis mutants with altered stomatal responses to CO2, i.e., high leaf temperature 1-2 (ht1-2, CO2 hypersensitive) and β-carbonic anhydrase 1 and 4 (ca1ca4, CO2 insensitive). The cold-induced expression of CBF and downstream CBF target genes plus freezing tolerance of ht1-2 was consistently less than that for Col-0, suggesting that HT1 is a positive modulator of cold signaling. The ca1ca4 mutant had diminished CBF expression during cold treatment but the downstream expression of cold-responsive genes was either similar to or greater than that of Col-0. This finding suggested that βCA1/4 modulates the expression of certain cold-responsive genes in a CBF-independent manner. Stomatal conductance measurements demonstrated that low temperatures overrode low CO2-induced stomatal opening and this process was delayed in the cold tolerant mutant, ca1ca4, compared to the cold sensitive mutant, ht1-2. The similar stomatal responses were evident from freezing tolerant line, Ox-CBF, overexpression of CBF3, compared to wild-type ecotype Ws-2. Together, these results indicate that CO2 signaling in stomata and CBF-mediated cold signaling work coordinately in Arabidopsis to manage abiotic stress.

The HY5 and MYB15 transcription factors positively regulate cold tolerance in tomato via the CBF pathway.

The induction of C-repeat binding factors (CBFs) is crucial for plant survival at low temperatures. Therefore, understanding the mechanisms that regulate CBF transcription is vital for the future development of crops with increased cold tolerance. Here, we provide evidence for the existence of a LONG HYPOCOTYL 5 (HY5)-MYB15-CBFs transcriptional cascade that plays a crucial role in the cold response in tomato. The exposure of tomato plants to cold (4°C) increased the levels of HY5, MYB15 and CBFs transcripts. Moreover, mutations in HY5 or MYB15 decreased the levels of CBF transcripts. In contrast, overexpression of HY5 or MYB15 increased CBF transcript abundance. Crucially, the HY5 transcription factor activated the expression of MYB15 by directly binding to the promoter region, while both HY5 and MYB15 activated the expression of CBF1, CBF2 and CBF3. Taken together, these data show that HY5 can directly regulate CBF transcript levels, and also influence CBF expression indirectly via MYB15. The coordinated action of HY5 and MYB15 allows precise regulation of CBF expression and subsequent cold tolerance. These findings provide an improved understanding of the molecular mechanisms affording transcriptional regulation of CBFs, which can be exploited in the future to enhance cold tolerance in crops.

CsICE1 Functions in Cold Tolerance by Regulating Polyamine levels May through Interacting with Arginine Decarboxylase in the Tea Tree

Background: The identification of C-repeat binding factor (CBF), and the characterization as an inducer of CBF Expression 1 (ICE1), and a major activator for C-repeat binding factor, were important breakthroughs in the cold signaling network. Methods: In the present study, the full length cDNA of ICE1 was isolated from the tea tree (Camellia sinensis). CsICE1 protein was located in the cell nucleus as revealed by subcellular localization analysis. To investigate the biological functions of CsICE1, a transgenic line fused with the CsICE1 gene in Arabidopsis thaliana (arabidopsis) was generated by the floral dip method. Results: The CsICE1 was expressed differentially in various tea tree tissues, mostly in buds and leaves, and the transcript level of CsICE1 was increased after 1 h and peaked at 2 h under cold treatment. Transcription activity assay indicated that the spermine synthase (SPMS) and arginine decarboxylase (ADC) genes were possible targets of CsICE1. In addition, the values of net photosynthetic rate, transpiration rate, stomatal conductance in transgenic lines declined by less extent than wild-type plants under low temperatures. Furthermore, transcript levels of ADC genes in the transgenic lines had no apparent alteration under normal growth conditions but substantially increased under cold conditions, consistent of changes in free polyamine levels. Taken together, these results demonstrated that CsICE1 plays a positive role in cold tolerance, which may be due to the modulation of polyamine levels through interacting with CsADC.

Corrigendum: ATBS1-INTERACTING FACTOR 2 negatively regulates dark- and brassinosteroid-induced leaf senescence through interactions with INDUCER OF CBF EXPRESSION 1.

ATBS1-INTERACTING FACTOR 2 (AIF2) is a non-DNA-binding basic helix-loop-helix (bHLH) transcription factor. We demonstrated that AIF2 retards dark-triggered and brassinosteroid (BR)-induced leaf senescence in Arabidopsis thaliana. Dark-triggered BR synthesis and the subsequent activation of BRASSINAZOLE RESISTANT 1 (BZR1), a BR signaling positive regulator, result in BZR1 binding to the AIF2 promoter in a dark-dependent manner, reducing AIF2 transcript levels and accelerating senescence. BR-induced down-regulation of AIF2 protein stability partly contributes to the progression of dark-induced leaf senescence. Furthermore, AIF2 interacts with INDUCER OF CBF EXPRESSION 1 (ICE1) via their C-termini. Formation of the AIF2-ICE1 complex and subsequent up-regulation of C-REPEAT BINDING FACTORs (CBFs) negatively regulates dark-triggered, BR-induced leaf senescence. This involves antagonistic down-regulation of PHYTOCHROME INTERACTING FACTOR 4 (PIF4), modulated through AIF2-dependent inhibition of ICE1's binding to the promoter. PIF4-dependent activities respond to dark-induced early senescence and may promote BR synthesis and BZR1 activation to suppress AIF2 and accelerate dark-induced senescence. Taken together, these findings suggest a coordination of AIF2 and ICE1 functions in maintaining stay-green traits.

Genes underlying cold acclimation in the tea plant (<i>Camellia sinensis</i> (L.) Kuntze)

The article reviews the latest studies showing the diversity of genetic mechanisms and gene families underlying the increased cold and frost tolerance of tea and other plant species. It has been shown that cell responses to chilling (0…+15°C) and freezing (< 0°C) are not the same and gene expression under cold stress is genotype-specific. In recent decades, progress has been made in understanding the genetic mechanisms underlying the cold response of plants – ICE1 (inducer of CBF expression 1), CBF (C-repeat-binding factor), COR (cold-regulated genes) pathways and signaling have been discovered. The ICE, CBF and DHN gene groups play a key role in the cold acclimation of the tea plant. The accumulation of CBF transcripts occurs after 15 min of chilling induction, and longer cold stress leads to accumulation of CBF transcripts. It is shown that the transcripts of the CsDHN1, CsDHN2 and CsDHN3 genes accumulate at a higher level in resistant genotypes of tea in comparison with susceptible cultivars during freezing. CBF-independent pathways include genes involved in metabolism and transcription factors such as HSFC1, ZAT12, CZF1, PLD (phospholipase D), WRKY, HD-Zip, CsLEA, LOX, NAC, HSP, which are widely distributed in plants and are involved in the basic mechanisms of tea resistance to cold and frost. The most recent studies show an important role of miRNA in the mechanisms of response to chilling and freezing in tea. The data obtained on different plant species may correlate with the mechanisms of frost tolerance of tea and are the basis for future studies of the signaling pathways of response to cold in the tea plant. The results of the research emphasize the need to further explore the ways in which various genes regulate the tolerance of tea to cold stress to find the molecular markers of frost tolerance.

ATBS1-INTERACTING FACTOR 2 negatively regulates dark- and brassinosteroid-induced leaf senescence through interactions with INDUCER OF CBF EXPRESSION 1.

ATBS1-INTERACTING FACTOR 2 (AIF2) is a non-DNA-binding basic helix–loop–helix (bHLH) transcription factor. We demonstrated that AIF2 retards dark-triggered and brassinosteroid (BR)-induced leaf senescence in Arabidopsis thaliana. Dark-triggered BR synthesis and the subsequent activation of BRASSINAZOLE RESISTANT 1 (BZR1), a BR signaling positive regulator, result in BZR1 binding to the AIF2 promoter in a dark-dependent manner, reducing AIF2 transcript levels and accelerating senescence. BR-induced down-regulation of AIF2 protein stability partly contributes to the progression of dark-induced leaf senescence. Furthermore, AIF2 interacts with INDUCER OF CBF EXPRESSION 1 (ICE1) via their C-termini. Formation of the AIF2–ICE1 complex and subsequent up-regulation of C-REPEAT BINDING FACTORs (CBFs) negatively regulates dark-triggered, BR-induced leaf senescence. This involves antagonistic down-regulation of PHYTOCHROME INTERACTING FACTOR 4 (PIF4), modulated through AIF2-dependent inhibition of ICE1’s binding to the promoter. PIF4-dependent activities respond to dark-induced early senescence and may promote BR synthesis and BZR1 activation to suppress AIF2 and accelerate dark-induced senescence. Taken together, these findings suggest a coordination of AIF2 and ICE1 functions in maintaining stay-green traits.

Characterization and expression profiling of the ICE-CBF-COR genes in wheat

Cold stress is one of the major abiotic stresses that limit crop production. The ICE-CBF-COR pathway is associated with cold stress response in a wide variety of crop species. However, the ICE-CBF-COR genes has not been well characterized in wheat (Triticum aestivum). This study identified, characterized and examined the expression profiles of the ICE, CBF and COR genes for cold defense in wheat. Five ICE (inducer of CBF expression) genes, 37 CBF (C-repeat binding factor) genes and 11 COR (cold-responsive or cold-regulated) genes were discovered in the wheat genome database. Phylogenetic trees based on all 53 genes revealed that CBF genes were more diverse than ICE and COR genes. Twenty-two of the 53 genes appeared to include 11 duplicated pairs. Twenty rice (Oryza sativa) genes and 21 sorghum (Sorghum bicolor) and maize (Zea mays) genes showed collinearity with the wheat ICE, CBF and COR genes. Transcriptome data and qRT-PCR analyses revealed tissue-specific expression patterns of the ICE, CBF and COR genes, and identified similarities in the expression pattern of genes from the same family when subjected to drought, heat, drought plus heat, and cold stress. These results provide information for better understanding the biological roles of ICE, CBF, COR genes in wheat.

The Methylation Patterns and Transcriptional Responses to Chilling Stress at the Seedling Stage in Rice.

Chilling stress is considered the major abiotic stress affecting the growth, development, and yield of rice. To understand the transcriptomic responses and methylation regulation of rice in response to chilling stress, we analyzed a cold-tolerant variety of rice (Oryza sativa L. cv. P427). The physiological properties, transcriptome, and methylation of cold-tolerant P427 seedlings under low-temperature stress (2–3 °C) were investigated. We found that P427 exhibited enhanced tolerance to low temperature, likely via increasing antioxidant enzyme activity and promoting the accumulation of abscisic acid (ABA). The Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq) data showed that the number of methylation-altered genes was highest in P427 (5496) and slightly lower in Nipponbare (Nip) and 9311 (4528 and 3341, respectively), and only 2.7% (292) of methylation genes were detected as common differentially methylated genes (DMGs) related to cold tolerance in the three varieties. Transcriptome analyses revealed that 1654 genes had specifically altered expression in P427 under cold stress. These genes mainly belonged to transcription factor families, such as Myeloblastosis (MYB), APETALA2/ethylene-responsive element binding proteins (AP2-EREBP), NAM-ATAF-CUC (NAC) and WRKY. Fifty-one genes showed simultaneous methylation and expression level changes. Quantitative RT-PCR (qRT-PCR) results showed that genes involved in the ICE (inducer of CBF expression)-CBF (C-repeat binding factor)—COR (cold-regulated) pathway were highly expressed under cold stress, including the WRKY genes. The homologous gene Os03g0610900 of the open stomatal 1 (OST1) in rice was obtained by evolutionary tree analysis. Methylation in Os03g0610900 gene promoter region decreased, and the expression level of Os03g0610900 increased, suggesting that cold stress may lead to demethylation and increased gene expression of Os03g0610900. The ICE-CBF-COR pathway plays a vital role in the cold tolerance of the rice cultivar P427. Overall, this study demonstrates the differences in methylation and gene expression levels of P427 in response to low-temperature stress, providing a foundation for further investigations of the relationship between environmental stress, DNA methylation, and gene expression in rice.

BRASSINOSTEROID-INSENSITIVE2 Negatively Regulates the Stability of Transcription Factor ICE1 in Response to Cold Stress in Arabidopsis.

Cold acclimation is a crucial strategy for plant survival at freezing temperatures. C-REPEAT BINDING FACTOR (CBF) genes are rapidly and transiently induced by low temperature and play important roles in cold acclimation. However, the mechanism underlying the attenuation of CBF expression during the later stages of the cold stress response is obscure. Here, we show that the protein kinase BRASSINOSTEROID-INSENSITIVE2 (BIN2) interacts with and phosphorylates INDUCER OF CBF EXPRESSION1 (ICE1) in Arabidopsis (Arabidopsis thaliana) under prolonged cold stress, facilitating the interaction between ICE1 and the E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE1 and thereby promoting ICE1 degradation. The kinase activity of BIN2 is inhibited during the early stages of the cold stress response and is subsequently restored, suggesting that BIN2 mainly downregulates ICE1 abundance when CBF expression is attenuated. A loss-of-function mutation of ICE1 partially suppresses the cold-induced expression of CBFs and compromises the enhanced freezing tolerance of bin2-3 bil1 bil2 These findings reveal an important role for BIN2 in fine-tuning CBF expression, and thus in balancing plant growth and the cold stress response.