C-repeat Binding Factor Pathway Research Articles

The NAC056 transcription factor confers freezing tolerance by positively regulating expression of CBFs and NIA1 in Arabidopsis

Freezing stress can seriously affect plant growth and development, but the mechanisms of these effects and plant responses to freezing stress require further exploration. Here, we identified a NAM, ATAF1/2, and CUC2 (NAC)-family transcription factor (TF), NAC056, that can promote freezing tolerance in Arabidopsis. NAC056 mRNA levels are strongly induced by freezing stress in roots, and the nac056 mutant exhibits compromised freezing tolerance. NAC056 acts positively in response to freezing by directly promoting key C-repeat-binding factor (CBF) pathway genes. Interestingly, we found that CBF1 regulates nitrate assimilation by regulating the nitrate reductase gene NIA1 in plants; therefore, NAC056–CBF1–NIA1 form a regulatory module for the assimilation of nitrate and the growth of roots under freezing stress. In addition, 35S::NAC056 transgenic plants show enhanced freezing tolerance, which is partially reversed in the cbfs triple mutant. Thus, NAC056 confers freezing tolerance through the CBF pathway, mediating plant responses to balance growth and freezing stress tolerance.

Identification of Brassica rapa BrEBF1 homologs and their characterization in cold signaling

EIN3-binding F-box 1 (EBF1) is involved in cold tolerance in Arabidopsis; however, its exact roles in cold signaling in Brassica rapa remain uncertain. Herein, we demonstrated that EBF1 homologs are highly conserved in Brassica species, but their copy numbers are diverse, with some motifs being species specific. Cold treatment activated the expression of EBF1 homologs BrEBF1 and BrEBF2 in B. rapa; however, their expression schemas were diverse in different cold-resistant varieties of the plant. Subcellular localization analysis revealed that BrEBF1 is a nuclear-localized F-box protein, and cold treatment did not alter its localization but induced its degradation. BrEBF1 overexpression enhanced cold tolerance, reduced cold-induced ROS accumulation, and enhanced MPK3 and MPK6 kinase activity in Arabidopsis. Our study revealed that BrEBF1 positively regulates cold tolerance in B. rapa and that BrEBF1-regulated cold tolerance is associated with ROS scavenging and MPK3 and MPK6 kinase activity through the C-repeat binding factor pathway.

SlHSP17.7 Ameliorates Chilling Stress-Induced Damage by Regulating Phosphatidylglycerol Metabolism and Calcium Signal in Tomato Plants

Tomatoes (Solanum lycopersicum L.) are sensitive to chilling temperatures between 0 °C and 12 °C owing to their tropical origin. SlHSP17.7, a cytoplasmic heat shock protein, interacts with cation/calcium exchanger 1-like (SlCCX1-like) protein and promotes chilling tolerance in tomato fruits (Zhang, et al., Plant Sci., 2020, 298, 1–12). The overexpression of SlHSP17.7 can also promote cold tolerance in tomato plants, but its specific mechanism remains unclear. In this study, we show that the overexpression of SlHSP17.7 in tomato plants enhances chilling tolerance with better activity of photosystem II (PSII). Metabolic analyses revealed that SlHSP17.7 improved membrane fluidity by raising the levels of polyunsaturated fatty acids. Transcriptome analyses showed that SlHSP17.7 activated Ca2+ signaling and induced the expression of C-repeat binding factor (CBF) genes, which in turn inhibited the production of reactive oxygen species (ROS). The gene coexpression network analysis showed that SlHSP17.7 is coexpressed with SlMED26b. SlMED26b silencing significantly lowered OE-HSP17.7 plants’ chilling tolerance. Thus, SlHSP17.7 modulates tolerance to chilling via both membrane fluidity and Ca2+-mediated CBF pathway in tomato plants.

Identification and function analysis of bHLH genes in response to cold stress in sweetpotato

Basic/helix–loop–helix (bHLH) transcription factors are involved in various metabolic and physiological processes in plants. Sweetpotato (Ipomoea batatas (L.) Lam.) is an important crop in China but is highly susceptible to cold stress. However, little information on the bHLH gene family is available, and the function of this family in response to cold stress has not been revealed in sweetpotato. Here, 110 IbbHLHs were identified and classified into 17 categories based on phylogenetic relationships, conserved motifs and gene structure analyses. Except for 5 IbbHLHs, 90 IbbHLHs were putative E-box-binding proteins including 70 IbbHLHs belonging to G-box, whereas 15 IbbHLHs were putative non-E box-binding proteins based on DNA-binding analysis. In total, 37 pairs of segmental duplicated genes and 5 pairs of tandem duplication genes were identified within the IbbHLH gene family. The transcript level of 20 IbbHLHs was regulated by cold stress based on RNA-seq data, and 8 genes were selected for further quantitative real-time PCR (qRT-PCR) analysis. IbHLH8 and IbHLH92 are involved in network interaction with several genes related to abiotic and biotic stresses under cold treatment. IbbHLH79, an ICE1-like gene, was isolated and overexpressed in sweetpotato. The IbbHLH79 protein can activate the CBF (C-repeat Binding Factor) pathway, and IbbHLH79-overexpressing transgenic plants display enhanced cold tolerance. Taken together, these results provide valuable information on the IbbHLH gene family; in addition, several IbbHLHs may regulate cold stress, and the results suggest IbbHLH79 will be useful for molecular breeding of enhanced cold tolerance in sweetpotato.

Positive Interaction between H2O2 and Ca2+ Mediates Melatonin-Induced CBF Pathway and Cold Tolerance in Watermelon (Citrullus lanatus L.).

Cold stress is a major environmental factor that detrimentally affects plant growth and development. Melatonin has been shown to confer plant tolerance to cold stress through activating the C-REPEAT BINDING FACTOR (CBF) pathway; however, the underlying modes that enable this function remain obscure. In this study, we investigated the role of H2O2 and Ca2+ signaling in the melatonin-induced CBF pathway and cold tolerance in watermelon (Citrullus lanatus L.) through pharmacological, physiological, and genetic approaches. According to the results, melatonin induced H2O2 accumulation, which was associated with the upregulation of respiratory burst oxidase homolog D (ClRBOHD) during the early response to cold stress in watermelon. Besides, melatonin and H2O2 induced the accumulation of cytoplasmic free Ca2+ ([Ca2+]cyt) in response to cold. This was associated with the upregulation of cyclic nucleotide-gated ion channel 2 (ClCNGC2) in watermelon. However, blocking of Ca2+ influx channels abolished melatonin- or H2O2-induced CBF pathway and cold tolerance. Ca2+ also induced ClRBOHD expression and H2O2 accumulation in early response to cold stress in watermelon. Inhibition of H2O2 production in watermelon by RBOH inhibitor or in Arabidopsis by AtRBOHD knockout compromised melatonin-induced [Ca2+]cyt accumulation and melatonin- or Ca2+-induced CBF pathway and cold tolerance. Overall, these findings indicate that melatonin induces RBOHD-dependent H2O2 generation in early response to cold stress. Increased H2O2 promotes [Ca2+]cyt accumulation, which in turn induces H2O2 accumulation via RBOHD, forming a reciprocal positive-regulatory loop that mediates melatonin-induced CBF pathway and subsequent cold tolerance.

Variation in ICE1 Methylation Primarily Determines Phenotypic Variation in Freezing Tolerance in Arabidopsis thaliana.

Cold stress is a major abiotic factor plants face during their life cycle. Although plants often exhibit phenotypic variation in cold tolerance, the underlying mechanism remains poorly understood. In the present study, the 50% lethal temperature (LT50) values of 37 Arabidopsis thaliana accessions at latitudes from 15° to 58° ranged from -13.2°C to -4.9°C and were closely correlated with the cold climates of the collection sites. According to a methylation analysis of all C-repeat (CRT)-binding factor (CBF) pathway genes, the coding and promoter regions of AtICE1, a regulator of CBF genes, exhibited the greatest variability in methylation levels among the accessions and included 5-122 methylated cytosine residues. In contrast, unmethylated or only slightly methylated genes in the CBF pathway showed little variation among the accessions. According to a gene expression analysis of four selected A. thaliana populations with distinct methylation patterns, except for the down-regulated gene AtCBF2, the expression levels of all members of the CBF pathway were negatively correlated with AtICE1 gene methylation levels. Treatment of the four A. thaliana populations with the DNA methylation inhibitory reagent 5-azacytidine resulted in a 30.0-78.3% enhancement of freezing tolerance and decreases in LT50 values of approximately 1.9-3.6°C. Similar effects were observed in drm2 mutants, including 30.0-48.3% increases in freezing tolerance and decreases in LT50 values of approximately 0.7-3.4°C. Thus, the AtICE1 methylation-regulated transcription of CBF pathway genes is responsible for the phenotypic variation in the freezing tolerance observed in A. thaliana.

PIF3 is a negative regulator of the CBF pathway and freezing tolerance in Arabidopsis

Light and temperature are major environmental factors that coordinately control plant growth and survival. However, how plants integrate light and temperature signals to better adapt to environmental stresses is poorly understood. PHYTOCHROME-INTERACTING FACTOR 3 (PIF3), a key transcription factor repressing photomorphogenesis, has been shown to play a pivotal role in mediating plants' responses to various environmental signals. In this study, we found that PIF3 functions as a negative regulator of Arabidopsis freezing tolerance by directly binding to the promoters of C-REPEAT BINDING FACTOR (CBF) genes to down-regulate their expression. In addition, two F-box proteins, EIN3-BINDING F-BOX 1 (EBF1) and EBF2, directly target PIF3 for 26S proteasome-mediated degradation. Consistently, ebf1 and ebf2 mutants were more sensitive to freezing than were the wild type, and the pif3 mutation suppressed the freezing-sensitive phenotype of ebf1 Furthermore, cold treatment promoted the degradation of EBF1 and EBF2, leading to increased stability of the PIF3 protein and reduced expression of the CBF genes. Together, our study uncovers an important role of PIF3 in Arabidopsis freezing tolerance by negatively regulating the expression of genes in the CBF pathway.

Soybean GmNFYB1 transcription factor confers abiotic stress tolerance in transgenic Arabidopsis

In the present study, the nuclear factor-Y subunit B (GmNFYB1) gene, induced by abscisic acid (ABA), 8% polyethylene-glycol (PEG) 6000, and NaCl, was cloned from soybean. GmNFYB1 is located in the cell nucleus. Two independent transgenic Arabidopsis lines overexpressing GmNFYB1 and GmNFYB1-GR (GmNFYB1 fused with the glucocorticoid receptor) were generated to investigate the function of GmNFYB1 in abiotic stress tolerance. The GmNFYB1 lines exhibited a higher seed germination rate and longer root lengths, with increased proline accumulation in leaves and decreased water loss under drought and salt stress conditions. In contrast, the GmNFYB1-GR transgenic lines displayed almost the same growth pattern as the wild-type plants under ABA, salt, and drought treatments in the absence of dexamethasone (DEX). However, after DEX application as an inducer, the GmNFYB1-GR lines showed positive responses to drought and salt resistance, similar to the GmNFYB1 lines. The inhibition of seed germination under ABA stress indicates that GmNFYB1 is involved in the ABA signalling pathway. Furthermore, the quantitative reverse transcription real-time polymerase chain reaction (qRT-PCR) analysis suggested that GmNFYB1 boosts the stress tolerance of plants through the C-repeat binding factor pathway. Taken together, these findings demonstrate the characteristics of GmNFYB1 as a transcription factor that lead to improved drought and salt tolerance in plants.

Jasmonates: Emerging Players in Controlling Temperature Stress Tolerance.

The sedentary life of plants has forced them to live in an environment that is characterized by the presence of numerous challenges in terms of biotic and abiotic stresses. Phytohormones play essential roles in mediating plant physiology and alleviating various environmental perturbations. Jasmonates are a group of oxylipin compounds occurring ubiquitously in the plant kingdom that play pivotal roles in response to developmental and environmental cues. Jasmonates (JAs) have been shown to participate in unison with key factors of other signal transduction pathway, including those involved in response to abiotic stress. Recent findings have furnished large body of information suggesting the role of jasmonates in cold and heat stress. JAs have been shown to regulate C-repeat binding factor (CBF) pathway during cold stress. The interaction between the integrants of JA signaling and components of CBF pathway demonstrates a complex relationship between the two. JAs have also been shown to counteract chilling stress by inducing ROS avoidance enzymes. In addition, several lines of evidence suggest the positive regulation of thermotolerance by JA. The present review provides insights into biosynthesis, signal transduction pathway of jasmonic acid and their role in response to temperature stress.

Phytochrome A and B Function Antagonistically to Regulate Cold Tolerance via Abscisic Acid-Dependent Jasmonate Signaling.

Light signaling and phytohormones both influence plant growth, development, and stress responses; however, cross talk between these two signaling pathways in response to cold remains underexplored. Here, we report that far-red light (FR) and red light (R) perceived by phytochrome A (phyA) and phyB positively and negatively regulated cold tolerance, respectively, in tomato (Solanum lycopersicum), which were associated with the regulation of levels of phytohormones such as abscisic acid (ABA) and jasmonic acid (JA) and transcript levels of ABA- and JA-related genes and the C-REPEAT BINDING FACTOR (CBF) stress signaling pathway genes. A reduction in the R/FR ratio did not alter cold tolerance, ABA and JA accumulation, and transcript levels of ABA- and JA-related genes and the CBF pathway genes in phyA mutant plants; however, those were significantly increased in wild-type and phyB plants with the reduction in the R/FR ratio. Even though low R/FR treatments did not confer cold tolerance in ABA-deficient (notabilis [not]) and JA-deficient (prosystemin-mediated responses2 [spr2]) mutants, it up-regulated ABA accumulation and signaling in the spr2 mutant, with no effect on JA levels and signaling in the not mutant. Foliar application of ABA and JA further confirmed that JA functioned downstream of ABA to activate the CBF pathway in light quality-mediated cold tolerance. It is concluded that phyA and phyB function antagonistically to regulate cold tolerance that essentially involves FR light-induced activation of phyA to induce ABA signaling and, subsequently, JA signaling, leading to an activation of the CBF pathway and a cold response in tomato plants.

RDM4 modulates cold stress resistance in Arabidopsis partially through the CBF-mediated pathway.

The C-REPEAT-BINDING FACTOR (CBF) pathway has important roles in plant responses to cold stress. How the CBF genes themselves are activated after cold acclimation remains poorly understood. In this study, we characterized cold tolerance of null mutant of RNA-DIRECTED DNA METHYLATION 4 (RDM4), which encodes a protein that associates with RNA polymerases Pol V and Pol II, and is required for RNA-directed DNA methylation (RdDM) in Arabidopsis. The results showed that dysfunction of RDM4 reduced cold tolerance, as evidenced by decreased survival and increased electrolyte leakage. Mutation of RDM4 resulted in extensive transcriptomic reprogramming. CBFs and CBF regulon genes were down-regulated in rdm4 but not nrpe1 (the largest subunit of PolV) mutants, suggesting that the role of RDM4 in cold stress responses is independent of the RdDM pathway. Overexpression of RDM4 constitutively increased the expression of CBFs and regulon genes and decreased cold-induced membrane injury. A great proportion of genes affected by rdm4 overlapped with those affected by CBFs. Chromatin immunoprecipitation results suggested that RDM4 is important for Pol II occupancy at the promoters of CBF2 and CBF3. We present evidence of a considerable role for RDM4 in regulating gene expression at low temperature, including the CBF pathway in Arabidopsis.

Natural variation in the C-repeat binding factor cold response pathway correlates with local adaptation of Arabidopsis ecotypes.

The natural range of Arabidopsis thaliana (Arabidopsis) encompasses geographical regions that have greatly differing local climates, including harshness of winter temperatures. A question thus raised is whether differences in freezing tolerance might contribute to local adaptation in Arabidopsis. Consistent with this possibility is that Arabidopsis accessions differ in freezing tolerance and that those collected from colder northern latitudes are generally more tolerant to freezing than those collected from warmer southern latitudes. Moreover, recent studies with Arabidopsis genotypes collected from sites in Sweden (SW) and Italy (IT) have established that the two accessions are locally adapted, that the SW ecotype is more tolerant of freezing than the IT ecotype, and that genetic differences between the two ecotypes that condition local adaptation and freezing tolerance map to a region that includes the C-repeat binding factor (CBF) locus. The CBF locus includes three genes - CBF1, CBF2 and CBF3 - that are induced by low temperature and encode transcription factors that regulate a group of more than 100 genes, the CBF regulon, which impart freezing tolerance. Here we show that cold induction of most CBF regulon genes is lower in IT plants compared with SW plants, and that this is due to the IT CBF2 gene encoding a non-functional CBF2 protein. The non-functional IT CBF2 protein also contributes to the lower freezing tolerance of the IT plants compared with the SW plants. Taken together, studies on the SW and IT ecotypes provide evidence that natural variation in the CBF pathway has contributed to adaptive evolution in these Arabidopsis populations.

The Arabidopsis RCC1 Family Protein TCF1 Regulates Freezing Tolerance and Cold Acclimation through Modulating Lignin Biosynthesis.

Cell water permeability and cell wall properties are critical to survival of plant cells during freezing, however the underlying molecular mechanisms remain elusive. Here, we report that a specifically cold-induced nuclear protein, Tolerant to Chilling and Freezing 1 (TCF1), interacts with histones H3 and H4 and associates with chromatin containing a target gene, BLUE-COPPER-BINDING PROTEIN (BCB), encoding a glycosylphosphatidylinositol-anchored protein that regulates lignin biosynthesis. Loss of TCF1 function leads to reduced BCB transcription through affecting H3K4me2 and H3K27me3 levels within the BCB gene, resulting in reduced lignin content and enhanced freezing tolerance. Furthermore, plants with knocked-down BCB expression (amiRNA-BCB) under cold acclimation had reduced lignin accumulation and increased freezing tolerance. The pal1pal2 double mutant (lignin content reduced by 30% compared with WT) also showed the freezing tolerant phenotype, and TCF1 and BCB act upstream of PALs to regulate lignin content. In addition, TCF1 acts independently of the CBF (C-repeat binding factor) pathway. Our findings delineate a novel molecular pathway linking the TCF1-mediated cold-specific transcriptional program to lignin biosynthesis, thus achieving cell wall remodeling with increased freezing tolerance.

The MYB96-HHP module integrates cold and abscisic acid signaling to activate the CBF-COR pathway in Arabidopsis.

Various environmental stresses limit plant growth, development, and reproductive success. Plants have therefore evolved sophisticated adaptive responses to deal with environmental challenges. The responses of plants to environmental stresses are mainly mediated by abscisic acid (ABA)-dependent and ABA-independent signaling pathways. While these two pathways have been implicated to play discrete roles in abiotic stress responses, accumulating evidence suggests that they are also intertwined. Here, we report that an R2R3-type MYB transcription factor, MYB96, integrates the ABA and cold signaling pathways. In addition to its role in ABA-mediated drought responses, MYB96 is also induced by cold stress in an ABA-independent manner and subsequently activates freezing tolerance. Notably, MYB96 regulates HEPTAHELICAL PROTEIN (HHP) genes by binding to their promoters. The HHP proteins, in turn, interact with C-REPEAT BINDING FACTOR (CBF) upstream regulators, such as INDUCER OF CBF EXPRESSION 1 (ICE1), ICE2, and CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 3 (CAMTA3). The specific interactive networks of HHPs with the CBF upstream regulators are necessary to facilitate transcriptional activation of the CBF regulon under stressful conditions. Together, the MYB96-HHP module integrates ABA-dependent and ABA-independent signals and activates the CBF pathway, ensuring plant adaptation to a wide range of adverse environmental fluctuations.

Regulation of the Arabidopsis CBF regulon by a complex low-temperature regulatory network.

Exposure of Arabidopsis thaliana plants to low non-freezing temperatures results in an increase in freezing tolerance that involves action of the C-repeat binding factor (CBF) regulatory pathway. CBF1, CBF2 and CBF3, which are rapidly induced in response to low temperature, encode closely related AP2/ERF DNA-binding proteins that recognize the C-repeat (CRT)/dehydration-responsive element (DRE) DNA regulatory element present in the promoters of CBF-regulated genes. The CBF transcription factors alter the expression of more than 100 genes, known as the CBF regulon, which contribute to an increase in freezing tolerance. In this study, we investigated the extent to which cold induction of the CBF regulon is regulated by transcription factors other than CBF1, CBF2 and CBF3, and whether freezing tolerance is dependent on a functional CBF-CRT/DRE regulatory module. To address these issues we generated transgenic lines that constitutively overexpressed a truncated version of CBF2 that had dominant negative effects on the function of the CBF-CRT/DRE regulatory module, and 11 transcription factors encoded by genes that were rapidly cold-induced in parallel with the 'first-wave' CBF genes, and determined the effects that overexpressing these proteins had on global gene expression and freezing tolerance. Our results indicate that cold regulation of the CBF regulon involves extensive co-regulation by other first-wave transcription factors; that the low-temperature regulatory network beyond the CBF pathway is complex and highly interconnected; and that the increase in freezing tolerance that occurs with cold acclimation is only partially dependent on the CBF-CRT/DRE regulatory module.

OST1 Kinase Modulates Freezing Tolerance by Enhancing ICE1 Stability in Arabidopsis

Cold stress is a major environmental factor that limits plant growth and development. The C-repeat-binding factor (CBF)-dependent cold signaling pathway is extensively studied in Arabidopsis; however, the specific protein kinases involved in this pathway remain elusive. Here we report that OST1 (open stomata 1), a well-known Ser/Thr protein kinase in ABA signaling, acts upstream of CBFs to positively regulate freezing tolerance. The ost1 mutants show freezing hypersensitivity, whereas transgenic plants overexpressing OST1 exhibit enhanced freezing tolerance. The OST1 kinase is activated by cold stress. Moreover, OST1 interacts with both the transcription factor ICE1 and the E3 ligase HOS1 in the CBF pathway. Cold-activated OST1 phosphorylates ICE1 and enhances its stability and transcriptional activity. Meanwhile, OST1 interferes with the interaction between HOS1 and ICE1, thus suppressing HOS1-mediated ICE1 degradation under cold stress. Our results thus uncover the unexpected roles of OST1 in modulating CBF-dependent cold signaling in Arabidopsis.

C-repeat binding factor gene family identified in non-heading Chinese cabbage is functional in abiotic and biotic stress response but different from that in Arabidopsis

C-repeat binding factor (CBF) signaling pathway is involved in cold acclimation responsive to low temperature and some other stresses. CBF transcription factor family is the key component of this pathway. In this study, eight CBF-like genes, BrCBF1, BrCBF2, BrCBF3, BrCBF4, BrCBF5, and BrCBF6A/B/C were isolated from non-heading Chinese cabbage (Brassicacampestris ssp. chinensis L. Makino, NHCC). The deduced CBF proteins shared high similarity with their Arabidopsis orthologs and localized to the nucleus. Furthermore, quantitative real-time PCR (qPCR) analysis showed that BrCBF1~3 were induced by cold (4 °C) but not drought or abscisic acid (ABA), indicating that they are involved in an ABA-independent pathway; however, BrCBF4~6 were regulated by both drought and ABA, suggesting that they were involved in an ABA-dependent pathway. Nevertheless, unlike Arabidopsis, BrCBF4~6 showed response to both cold and ABA, indicates ABA-independent and ABA-dependent parts of CBF pathway in NHCC might not be completely separate, and these genes may act as the connection points in the network. BrCBFs were also accumulated in response to salicylic acid (SA), methyljasmonate (MeJA), and ethylene (ET), indicating that BrCBF genes might participate in the response to biotic stresses. Taken together, eight CBF genes were isolated from NHCC which compose a functional CBF signaling pathway by participating in response to multiple stresses and performing roles from Arabidopsis to some extent.

The Influence of Light Quality, Circadian Rhythm, and Photoperiod on the CBF-Mediated Freezing Tolerance

Low temperature adversely affects crop yields by restraining plant growth and productivity. Most temperate plants have the potential to increase their freezing tolerance upon exposure to low but nonfreezing temperatures, a process known as cold acclimation. Various physiological, molecular, and metabolic changes occur during cold acclimation, which suggests that the plant cold stress response is a complex, vital phenomenon that involves more than one pathway. The C-Repeat Binding Factor (CBF) pathway is the most important and well-studied cold regulatory pathway that imparts freezing tolerance to plants. The regulation of freezing tolerance involves the action of phytochromes, which play an important role in light-mediated signalling to activate cold-induced gene expression through the CBF pathway. Under normal temperature conditions, CBF expression is regulated by the circadian clock through the action of a central oscillator and also day length (photoperiod). The phytochrome and phytochrome interacting factor are involved in the repression of the CBF expression under long day (LD) conditions. Apart from the CBF regulon, a novel pathway involving the Z-box element also mediates the cold acclimation response in a light-dependent manner. This review provides insights into the progress of cold acclimation in relation to light quality, circadian regulation, and photoperiodic regulation and also explains the underlying molecular mechanisms of cold acclimation for introducing the engineering of economically important, cold-tolerant plants.

Photoperiodic regulation of the C-repeat binding factor (CBF) cold acclimation pathway and freezing tolerance inArabidopsis thaliana

The CBF (C-repeat binding factor) pathway has a major role in plant cold acclimation, the process whereby certain plants increase in freezing tolerance in response to low nonfreezing temperatures. In Arabidopsis thaliana, the pathway is characterized by rapid cold induction of CBF1, CBF2, and CBF3, which encode transcriptional activators, followed by induction of CBF-targeted genes that impart freezing tolerance. At warm temperatures, CBF transcript levels are low, but oscillate due to circadian regulation with peak expression occurring at 8 h after dawn (zeitgeber time 8; ZT8). Here, we establish that the CBF pathway is also regulated by photoperiod at warm temperatures. At ZT8, CBF transcript levels in short-day (SD; 8-h photoperiod) plants were three- to fivefold higher than in long-day plants (LD; 16-h photoperiod). Moreover, the freezing tolerance of SD plants was greater than that of LD plants. Genetic analysis indicated that phytochrome B (PHYB) and two phytochrome-interacting factors, PIF4 and PIF7, act to down-regulate the CBF pathway and freezing tolerance under LD conditions. Down-regulation of the CBF pathway in LD plants correlated with higher PIF4 and PIF7 transcript levels and greater stability of the PIF4 and PIF7 proteins under LD conditions. Our results indicate that during the warm LD growing season, the CBF pathway is actively repressed by PHYB, PIF4, and PIF7, thus mitigating allocation of energy and nutrient resources toward unneeded frost protection. This repression is relieved by shortening day length resulting in up-regulation of the CBF pathway and increased freezing tolerance in preparation for coming cold temperatures.

The cold-induced basic helix-loop-helix transcription factor gene MdCIbHLH1encodes an ICE-like protein in apple

BackgroundPlant growth is greatly affected by low temperatures, and the expression of a number of genes is induced by cold stress. Although many genes in the cold signaling pathway have been identified in Arabidopsis, little is known about the transcription factors involved in the cold stress response in apple.ResultsHere, we show that the apple bHLH (basic helix-loop-helix) gene MdCIbHLH1 (Cold-Induced bHLH1), which encodes an ICE-like protein, was noticeably induced in response to cold stress. The MdCIbHLH1 protein specifically bound to the MYC recognition sequences in the AtCBF3 promoter, and MdCIbHLH1 overexpression enhanced cold tolerance in transgenic Arabidopsis. In addition, the MdCIbHLH1 protein bound to the promoters of MdCBF2 and favorably contributed to cold tolerance in transgenic apple plants by upregulating the expression of MdCBF2 through the CBF (C-repeat-binding factor) pathway. Our findings indicate that MdCIbHLH1 functions in stress tolerance in different species. For example, ectopic MdCIbHLH1 expression conferred enhanced chilling tolerance in transgenic tobacco. Finally, we observed that cold induces the degradation of the MdCIbHLH1 protein in apple and that this degradation was potentially mediated by ubiquitination and sumoylation.ConclusionsBased on these findings, MdCIbHLH1 encodes a transcription factor that is important for the cold tolerance response in apple.