Inducer Of CBF Expression Research Articles

Functional profiling of EcaICE1 transcription factor gene from Eucalyptus camaldulensis involved in cold response in tobacco plants

A full-length cDNA designated as the EcaICE1 gene was isolated from Eucalyptus camaldulensis. The EcaICE1 gene was predicted to encode a MYC-like protein of 523 amino acids presenting a considerable degree of homology to currently identified plant inducer of CBF expression 1 (ICE1) proteins. RT-PCR assays indicated that EcaICE1 gene was expressed in a constitutive pattern in intact E. camaldulensis plants or the cold-treated (4 °C) plantlets. Strikingly, over-expression of the EcaICE1 gene in transgenic tobacco plants could enhance the tolerance to cold stress. While over-expression of the EcaICE1 gene resulted in a profound change of transcript level of C-repeat Binding Factor 1 (CBF1), CBF3 and group 2 LEA protein NtERD10C genes in transgenic tobacco plants. Further analysis revealed that EcaICE1 could function as a transcription activator in planta. Briefly, our results strongly suggest that the EcaICE1 gene is involved in the plant defense responses to low temperature.

Cold-induced modulation of CbICE53 gene activates endogenous genes to enhance acclimation in transgenic tobacco

Inducers of C-repeat binding factor (CBF) expression [inducer of CBF expression 1 (ICE1)-like], being MYC-type transcription factors, play an important role in plant tolerance to low temperatures and other abiotic environmental stresses. These ICEs are the key activators binding to C-repeat/dehydration-responsive elements, triggering the response of CBF signaling and other cold-related genes under cold stress. Our previous report documented a novel ICE transcription factor from Capsella bursa-pastoris, named CbICE53 (GenBank accession no. AY506804), which is responsive to cold, salt stress, and exogenous IAA, GA3, ABA, MeJA, and SA. In the present study, subcellular localization analyses reveal that CbICE53 is specifically localized to the nucleus. Similarly to AtICE1, CbICE53 exhibited slightly increased transcript level under cold induction. Compared with wild-type plants, transformants overexpressing CbICE53 showed increased tolerance to both chilling and freezing temperature according to the index of electrolyte leakage, relative water content and glucose content, and survival rate assay. The expressions of endogenous cold-responsive genes in transgenic tobacco (NtDREB1, NtDREB3, NtERD10a, and NtERD10b) were obviously upregulated only in cold condition. These results suggest that CbICE53 increases plant tolerance to low temperature via activating downstream cold-responsive pathways, which can be considered as a potential candidate for transgenic engineering in plant breeding for cold-tolerant crops.

Overexpression ofMtCAS31enhances drought tolerance in transgenic Arabidopsis by reducing stomatal density

• Dehydrins are a type of late embryogenesis abundant protein. Some dehydrins are involved in the response to various abiotic stresses. Accumulation of dehydrins enhances the drought, cold and salt tolerances of transgenic plants, although the underlying mechanism is unclear. MtCAS31 (Medicago Truncatula cold-acclimation specific protein 31) is a Y(2)K(4)-type dehydrin that was isolated from Medicago truncatula. • We analyzed the subcellular and histochemical localization of MtCAS31, and the expression patterns of MtCAS31 under different stresses. Transgenic Arabidopsis that overexpressed MtCAS31 was used to determine the function of MtCAS31. A yeast two-hybrid assay was used to screen potential proteins that could interact with MtCAS31. The interaction was confirmed by bimolecular fluorescence complementation (BiFC) assay. • After a 3-h drought treatment, the expression of MtCAS31 significantly increased 600-fold. MtCAS31 overexpression dramatically reduced stomatal density and markedly enhanced the drought tolerance of transgenic Arabidopsis. MtCAS31 could interact with AtICE1 (inducer of CBF expression 1) and the AtICE1 homologous protein Mt7g083900.1, which was identified from Medicago truncatula both in vitro and in vivo. • Our findings demonstrate that a dehydrin induces decreased stomatal density. Most importantly, the interaction of MtCAS31 with AtICE1 plays a role in stomatal development. We hypothesize that the interaction of MtCAS31 and AtICE1 caused the decrease in stomatal density to enhance the drought resistance of transgenic Arabidopsis.

低温条件下におけるイネICE1 (Inducer of CBF Expression)ホモログの解析

低温条件下におけるイネICE1 (Inducer of CBF Expression)ホモログの解析

Rice homologs of inducer of CBF expression (OsICE) are involved in cold acclimation

Cold stress on crops results in severe yield losses through growth retardation and irreversible damage. Recently, Inducer of CBF Expression 1 (ICE1) was identified as the master regulator inducing dehydration responsive element binding protein/C-repeat binding factor (DREB/CBF)-type transcriptional factors involved in the signaling of cold and osmotic stress in Arabidopsis. To examine whether rice ICE homologs function in cold acclimation via regulation of rice DREB homologs in response to cold stress, we assessed a polypeptide epitope containing an ICE-specific motif. Chilling stress on rice seedlings induced two ICE-related proteins with molecular masses of approximately 55 and 40 kDa. These sizes are consistent with those predicted for OsICE1 and OsICE2, respectively. In contrast to the proteins, cold stress had little or no effect on the expression of OsICE1 and OsICE2. Semi-quantitative RT-PCR indicated that both were constantly expressed, but that cold stress sequentially upregulated OsDREB1B, rice heat shock factor A3 (OsHsfA3), and trehalose-6- phosphate phosphatase (OsTPP1). Trehalose treatment enhanced the cold tolerance of seedlings. These results suggest that OsICE homologs function in transcriptional regulation at upstream of a cold-stress-induced transcription factor cascade involving OsDREB1B and OsHsfA3, leading to cold acclimation, possibly involving trehalose synthesis.

Induction of BAP1 by a Moderate Decrease in Temperature Is Mediated by ICE1 in Arabidopsis

Temperature variations at the nonextreme range modulate various processes of plant growth, development, and physiology, but how plants perceive and transduce these temperature signals is not well understood. Moderate cooling from 28 °C to 22 °C induces transcription of a number of genes in salicylic acid-dependent and -independent manners. Here, we report the study of the transcriptional control of the BON1-associated protein1 (BAP1) gene that is responsive to a moderate decrease of temperature as well as to many environmental stimuli. Using reporter genes under the control of series of regions of the BAP1 promoter, we identified a 35-bp fragment that is necessary and sufficient for the BAP1 transcript induction by a moderate cooling. This fragment also confers an induction of BAP1 by cold and reactive oxygen species-generating paraquat. Furthermore, the inducer of CBF expression1 (ICE1) protein that is involved in transcriptional control of cold responses is found to bind to a MYC element in this promoter and is required for the cooling induction of BAP1. The ice1 mutant has a low induction of BAP1 and enhanced resistance to a bacterial pathogen. Thus, responses to a moderate decrease in temperature may utilize components in the cold response as well as a potentiating signaling involving salicylic acid.

Dwarf apple MbDREB1 enhances plant tolerance to low temperature, drought, and salt stress via both ABA-dependent and ABA-independent pathways

In higher plants, DREB1/CBF-type transcription factors play an important role in tolerance to low temperatures, drought, and high-salt stress. These transcription factors bind to CRT/DRE elements in promoter regions of target genes, regulating their expression. In this study, we cloned and characterized a novel gene encoding a DREB1 transcription factor from dwarf apple, Malus baccata (GenBank accession number: EF582842). Expression of MbDREB1 was induced by cold, drought, and salt stress, and also in response to exogenous ABA. Subcellular localization analyses revealed that MbDREB1 localizes in the nucleus. A yeast activity assay demonstrated that the MbDREB1 gene encodes a transcription activator, which specifically binds to DRE/CRT elements. Compared with wild-type plants, transgenic Arabidopsis overexpressing MbDREB1 showed increased tolerance to low temperature, drought, and salt stresses. Analysis of the MbDREB1 promoter revealed an ABA-responsive element (ABRE), an inducer of CBF expression1 (ICE1)-like binding site, two MYB recognition sites, and three stress-inducible GT-1 boxes. GUS activities driven by the MbDREB1 promoter in transgenic Arabidopsis increased in response to ABA, cold temperature, drought, and salt treatments. Interestingly, the expression of both ABA-independent and ABA-dependent stress-induced genes (COR15a and rd29B, respectively) was activated under normal growth conditions in Arabidopsis overexpressing MbDREB1. These results suggest that MbDREB1 functions as a transcription factor and increases plant tolerance to low temperature, drought, and salt stress via both ABA-dependent and ABA-independent pathways.

HHP1, a novel signalling component in the cross-talk between the cold and osmotic signalling pathways in Arabidopsis

Heptahelical protein 1 (HHP1) is a negative regulator in abscisic acid (ABA) and osmotic signalling in Arabidopsis. The physiological role of HHP1 was further investigated in this study using transgenic and knock-out plants. In HHP1::GUS transgenic mutants, GUS activity was found to be mainly expressed in the roots, vasculature, stomata, hydathodes, adhesion zones, and connection sites between septa and seeds, regions in which the regulation of turgor pressure is crucial. By measuring transpiration rate and stomatal closure, it was shown that the guard cells in the hhp1-1 mutant had a decreased sensitivity to drought and ABA stress compared with the WT or the c-hhp1-1 mutant, a complementation mutant of HHP1 expressing the HHP1 gene. The N-terminal fragment (amino acids 1–96) of HHP1 was found to interact with the transcription factor inducer of CBF expression-1 (ICE1) in yeast two-hybrid and bimolecular fluorescence complementation (BiFC) studies. The hhp1-1 mutant grown in soil showed hypersensitivity to cold stress with limited watering. The expression of two ICE1-regulated genes (CBF3 and MYB15) and several other cold stress-responsive genes (RD29A, KIN1, COR15A, and COR47) was less sensitive to cold stress in the hhp1-1 mutant than in the WT. These data suggest that HHP1 may function in the cross-talk between cold and osmotic signalling.

Cucumber ( Cucumis sativus L.) over-expressing cold-induced transcriptome regulator ICE1 exhibits changed morphological characters and enhances chilling tolerance

Cold is an environmental factor that limits the growing season of cucumber and adversely affects fruit quality and productivity. ICE1 is a positive regulator of CBF3 and has a critical role in cold stress ( Chinnusamy et al., 2003). To test the function and potential use of cold-induced transcriptome regulator ICE1 (inducer of CBF expression) in improving the chill tolerance of cucumbers, the Arabidopsis ICE1 gene driven by super promoter was introduced into cucumbers by Agrobacterium-mediated transformation. Transgenic plants were obtained, and were confirmed by PCR and southern blot, then the expression of ICE1 was tested by RT-PCR. Transgenic plants exhibited dwarf phenotypes, and shorter internodes, with higher nodes in the first female flowers and a lower female flower rate of every node than wild type plants. Under cold stress, the chilling index of four transgenic lines (IF3, IF6, IF7, and IF10) was decreased significantly, indicating their improved chilling tolerance. Over-expression of ICE1 in cucumbers could induce the expression of cold-responsive genes, further activating the accumulation of soluble sugars and free proline, and inhibiting malondialdehyde (MDA) accumulation. This leads to electrolyte leakage and regulation of osmotic potential to osmotic stress under cold stress.

Molecular cloning, characterization and expression analysis of PtrHOS1, a novel gene of cold responses from trifoliate orange [Poncirus trifoliata (L.) Raf.

High expression of osmotically responsive genes 1 (HOS1) encodes an ubiquitin E3 ligase that promotes the degradation of transcription factor Inducer of CBF Expression 1 (ICE1). Inactivation of ICE1 reduces CBF-induced activation of many cold-responsive genes, and thus, HOS1 act as a negative regulator of cold-responsive genes. In this paper, a novel HOS1 gene, designated PtrHOS1 (Genebank accession number FJ844367), was cloned by RT-PCR and RACE-PCR from trifoliate orange [Poncirus trifoliata (L.) Raf.]. The full length of PtrHOS1 is 3,434 bp with an open reading frame of 2,922 bp, encoding a protein of 974 amino acids with a molecular weight of 110.2 kDa and a theoretical isoelectric point of 5.55. Sequence alignment showed that PtrHOS1 protein had a conserved RING finger domain in its N-terminal region and shared high identity with other plant species HOS1-like proteins. Semi-quantitative RT-PCR analysis revealed that PtrHOS1 could be constitutively expressed at high levels in leaves, stems and roots. Interestingly, the PtrHOS1 expression had a declined period in leaves, stems and roots after cold and ABA treatments, which suggested that the PtrHOS1 expression was down regulated both by cold and ABA. Moreover, the decline was first occurred in leaves (30 min), followed with stems (2 h) and roots (4 h) after cold treatments. These results probably suggest that the leaves of trifoliate orange first sense the cold stress, followed with stems and roots. Oppositely, after ABA treatments, the significant decline of PtrHOS1 expression was first occurred in roots (15 min), followed with stems and leaves (30 min). Our results provide useful information for further studies about cold acclimation mechanism in citrus.

SCREAM/ICE1andSCREAM2Specify Three Cell-State Transitional Steps Leading toArabidopsisStomatal Differentiation

Differentiation of specialized cell types in multicellular organisms requires orchestrated actions of cell fate determinants. Stomata, valves on the plant epidermis, are formed through a series of differentiation events mediated by three closely related basic-helix-loop-helix proteins: SPEECHLESS (SPCH), MUTE, and FAMA. However, it is not known what mechanism coordinates their actions. Here, we identify two paralogous proteins, SCREAM (SCRM) and SCRM2, which directly interact with and specify the sequential actions of SPCH, MUTE, and FAMA. The gain-of-function mutation in SCRM exhibited constitutive stomatal differentiation in the epidermis. Conversely, successive loss of SCRM and SCRM2 recapitulated the phenotypes of fama, mute, and spch, indicating that SCRM and SCRM2 together determined successive initiation, proliferation, and terminal differentiation of stomatal cell lineages. Our findings identify the core regulatory units of stomatal differentiation and suggest a model strikingly similar to cell-type differentiation in animals. Surprisingly, map-based cloning revealed that SCRM is INDUCER OF CBF EXPRESSION1, a master regulator of freezing tolerance, thus implicating a potential link between the transcriptional regulation of environmental adaptation and development in plants.

Structure and Functional Analysis of Wheat ICE (Inducer of CBF Expression) Genes

Two different inducers of CBF expression (ICE1-like genes), TaICE41 and TaICE87, were isolated from a cDNA library prepared from cold-treated wheat aerial tissues. TaICE41 encodes a protein of 381 aa with a predicted MW of 39.5 kDa while TaICE87 encodes a protein of 443 aa with a predicted MW of 46.5 kDa. TaICE41 and TaICE87 share 46% identity while they share 50 and 47% identity with Arabidopsis AtICE1 respectively. Expression analysis revealed that mRNA accumulation was not altered by cold treatment suggesting that both genes are expressed constitutively. Gel mobility shift analysis showed that TaICE41 and TaICE87 bind to different MYC elements in the wheat TaCBFIVd-B9 promoter. Transient expression assays in Nicotiana benthamiana, showed that both TaICE proteins can activate TaCBFIVd-B9 transcription. The different affinities of TaICE41 and TaICE87 for MYC variants suggest that ICE binding specificity may be involved in the differential expression of wheat CBF genes. Furthermore, analysis of MYC elements demonstrates that a specific variant is present in the wheat CBF group IV that is associated with freezing tolerance. Overexpression of either TaICE41 or TaICE87 genes in Arabidopsis enhanced freezing tolerance only upon cold acclimation suggesting that other factors induced by low temperature are required for their activity. The increased freezing tolerance in transgenic Arabidopsis is associated with a higher expression of the cold responsive activators AtCBF2, AtCBF3, and of several cold-regulated genes.

Interplay between cold-responsive gene regulation, metabolism and RNA processing during plant cold acclimation

Temperate plants are capable of developing freezing tolerance when they are exposed to low nonfreezing temperatures. Acquired freezing tolerance involves extensive reprogramming of gene expression and metabolism. Recent full-genome transcript profiling studies, in combination with mutational and transgenic plant analyses, have provided a snapshot of the complex transcriptional network that operates under cold stress. Ubiquitination-mediated proteosomal protein degradation has a crucial role in regulating one of the upstream transcription factors, INDUCER OF CBF EXPRESSION 1 (ICE1), and thus in controlling the cold-responsive transcriptome. The changes in expression of hundreds of genes in response to cold temperatures are followed by increases in the levels of hundreds of metabolites, some of which are known to have protective effects against the damaging effects of cold stress. Genetic analysis has revealed important roles for cellular metabolic signals, and for RNA splicing, export and secondary structure unwinding, in regulating cold-responsive gene expression and chilling and freezing tolerance.

Nuclear localization of sweet cherry DREB1/CBF ortholog (CIG) and low temperature-inducible activity of CIG promoter

The fluorescent signal of GFP fused to CIG-B, which is one of the three DREB1/CBF (dehydration responsive element binding protein 1/C-repeat binding factor) homologs isolated from sweet cherry, was observed only in nucleus. The expression of GFP driven by CIG-B promoter was up-regulated by low-temperature. These results suggested that nuclear localization signal (NLS) motif in the N-terminal region of CIG-B protein and inducer of CBF expression (ICE)-like motifs in CIG-B promoter could be responsible for nuclear targeting and low temperature perception, respectively.

TheArabidopsisCold-Responsive Transcriptome and Its Regulation by ICE1

To understand the gene network controlling tolerance to cold stress, we performed an Arabidopsis thaliana genome transcript expression profile using Affymetrix GeneChips that contain approximately 24,000 genes. We statistically determined 939 cold-regulated genes with 655 upregulated and 284 downregulated. A large number of early cold-responsive genes encode transcription factors that likely control late-responsive genes, suggesting a multitude of transcriptional cascades. In addition, many genes involved in chromatin level and posttranscriptional regulation were also cold regulated, suggesting their involvement in cold-responsive gene regulation. A number of genes important for the biosynthesis or signaling of plant hormones, such as abscisic acid, gibberellic acid, and auxin, are regulated by cold stress, which is of potential importance in coordinating cold tolerance with growth and development. We compared the cold-responsive transcriptomes of the wild type and inducer of CBF expression 1 (ice1), a mutant defective in an upstream transcription factor required for chilling and freezing tolerance. The transcript levels of many cold-responsive genes were altered in the ice1 mutant not only during cold stress but also before cold treatments. Our study provides a global picture of the Arabidopsis cold-responsive transcriptome and its control by ICE1 and will be valuable for understanding gene regulation under cold stress and the molecular mechanisms of cold tolerance.

Molecular cloning and characterization of a novel ice gene from Capsella bursa-pastoris

A new ice gene (designated as Cbice53, an inducer of CBF expression) was cloned from Capsella bursa-pastoris by rapid amplification of cDNA ends (RACE). The full-length cDNA of Cbice53 was 1811 bp long, with a 1476-bp open reading frame (ORF) encoding a Myc-like protein of 492 amino acids. The predicted CbICE53 protein contained a potential basic helix-loop-helix domain, a nuclear localization signal (NLS), an RNA-binding region (RGG box), and N-glycosylation and kinase phosphorylation sites. Bioinformatic analysis showed that CbICE53 was highly homologous to ICE1 from Arabidopsis thaliana. Transcription of Cbice53 gene was induced transiently during salt and cold treatments, suggesting that it was somehow involved in cold acclimation. The results of our study indicate that the Cbice53 gene is a new member of the ice gene family and may have a role in cold and salt responsiveness in C. bursa-pastoris.

Molecular genetic perspectives on cross-talk and specificity in abiotic stress signalling in plants.

The perception of abiotic stresses and signal transduction to switch on adaptive responses are critical steps in determining the survival and reproduction of plants exposed to adverse environments. Plants have stress-specific adaptive responses as well as responses which protect the plants from more than one environmental stress. There are multiple stress perception and signalling pathways, some of which are specific, but others may cross-talk at various steps. Recently, progress has been made in identifying components of signalling pathways involved in salt, drought and cold stresses. Genetic analysis has defined the Salt-Overly-Sensitive (SOS) pathway, in which a salt stress-induced calcium signal is probably sensed by the calcium-binding protein SOS3 which then activates the protein kinase SOS2. The SOS3-SOS2 kinase complex regulates the expression and activity of ion transporters such as SOS1 to re-establish cellular ionic homeostasis under salinity. The ICE1 (Inducer of CBF Expression 1)-CBF (C-Repeat Binding Protein) pathway is critical for the regulation of the cold-responsive transcriptome and acquired freezing tolerance, although at present the signalling events that activate the ICE1 transcription factor during cold stress are not known. Both ABA-dependent and -independent signalling pathways appear to be involved in osmotic stress tolerance. Components of mitogen-activated protein kinase (MAPK) cascades may act as converging points of multiple abiotic as well as biotic stress signalling pathways. Forward and reverse genetic analysis in combination with expression profiling will continue to uncover many signalling components, and biochemical characterization of the signalling complexes will be required to determine specificity and cross-talk in abiotic stress signalling pathways.