Dehydration-responsive element/C-repeat Research Articles

Functional Characterization of Tea Plant (Camellia sinensis L.) CsCBF2 Gene Involved in Multiple Abiotic Stress Response in Tobacco (Nicotiana tabacum L.)

C-repeat binding factors/dehydration responsive element binding factors 1 (CBFs/DREB1s) are a small family of transcription factors that play important roles in plant resistance to various external stresses. However, functional characterization of tea plant (Camellia sinensis L.) CBF gene (CsCBF) was still seldom reported. Here, functional study of the cold-responsive CBF gene (CsCBF2) was done. Results showed that CsCBF2 had conserved AP2 DNA-binding domain and the typical PKK/RPAGRxKFxETRHP and DSAWR signature sequences of CBF/DREB1. Yeast one-hybrid and transcription activation assays revealed that the activation domain of CsCBF2 could activate the reporter gene expression, and the N terminal of CsCBF2 displayed an inhibitory effect. Although CsCBF2 was conserved to bind the C-repeat/dehydration-responsive element (CRT/DRE), intact CsCBF2 protein preferred the CRT cis element. Under normal growth conditions, CsCBF2-overexpressing tobacco plants (CsCBF2-OX) exhibited lighter green leaf color, growth retardation, and dwarfism. Smaller leaf of CsCBF2-OX was only seen in eight weeks after been sown in soil. Under cold, salinity, or drought stress, CsCBF2-OX displayed better growing with longer roots, heavier fresh weight, higher germination rate, and accumulated more proline and sugar contents, but lower electrolyte leakage. The results demonstrated that CsCBF2 enhanced plant tolerance to multiple abiotic stresses.

PpCBF6 Is Involved in Phytosulfokine α-Retarded Chilling Injury by Suppressing the Expression of PpLOX5 in Peach Fruit.

The involvement of PpCBF6 in phytosulfokine α (PSKα)-ameliorated chilling injury (CI) by suppressing the expression of lipoxygenase 5 (LOX5) in peach fruit was revealed. The peaches were immersed in distilled water and PSKα solution. PSKα application inhibited the progression of CI index and weight loss, and the reduction of firmness and total soluble solids content in peaches. The endogenous PSKα accumulation and gene expression of PSK receptor 1 (PSKR1) and PSKR2 were up regulated by PSKα application. The superoxide anion (O2–) production rate, hydrogen peroxide (H2O2) production and reactive oxygen species (ROS) content decreased by PSKα application. Furthermore, PSKα application reduced the gene expression of 12 PpLOXs and LOX activity. The gene expression of 6 PpCBFs was enhanced by PSKα application. Importantly, after PSKα application, among 12 PpLOXs, the decrease in gene expression of PpLOX5 was the lowest, and among 6 PpCBFs, the increase in gene expression of PpCBF6 was the highest. Further results suggested that PpCBF6 bound to the C-repeat/dehydration responsive element (CRT/DRE) motif in PpLOX5 promoter, and repressed its transcription. Thus, PpCBF6 was involved in the PSKα-retarded CI by inhibiting the expression of PpLOX5 in peaches.

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.

Changes of polyamines and CBFs expressions of two Hami melon (Cucumis melo L.) cultivars during low temperature storage

Hami melon (Cucumis melo L. var. inodorus Jacq) is susceptible to chilling injury (CI) during low temperature storage, which causes changes in physiological mechanisms and gene expression. In this study, cold tolerance of two Hami melon cultivars, namely Xinmi No. 3 and Xinmi No. 11 was evaluated. We assessed changes of membrane permeability, the content of polyamines (PAs), and the expression of C-repeat/dehydration-responsive element (CRT/DRE)-binding factors (CBFs) in different section of fruit of each cultivar stored at 3°C under 75–80% relative humidity. The results showed that Xinmi No. 11 was more cold tolerant than Xinmi No. 3, the content of PAs and the expression of CmCBFs in the cold-tolerant cultivar were significantly higher than in the cold-sensitive cultivar during storage at 3°C. CI was observed in the peel tissue, but not in the pulp tissue. The content of PAs and the expression of CmCBFs in the peel tissue were significantly higher than in the pulp tissue. The membrane permeability, putrescine (Put), spermine(Spm), and the expression of CmCBF1 and CmCBF3 in the peel tissue of Hami melon fruit increased with the degree of CI. Our results provide novel evidence about the positive correlation between the content of PAs, the expression of CmCBF1 and CmCBF3 and cold tolerance. The CI had major impact on Put, Spm and membrane permeability. The assessed parameters could provide sensitive indicators reflecting cultivar cold tolerance of Hami melon fruit under chilling stress.

Effect of nitric oxide treatment on chilling injury, antioxidant enzymes and expression of the CmCBF1 and CmCBF3 genes in cold-stored Hami melon (Cucumis melo L.) fruit

Short-term exposure to a low concentration of nitric oxide (NO) treatment can prevent or alleviate chilling injury (CI), and extend the postharvest life of various fresh fruits and vegetables. However no research on this topic has been reported for Hami melon fruit. In this study, Hami melon fruit were pre-treated with 60μL/L NO for 3h at 25°C, and then stored at 1±0.5°C with a relative humidity (RH) of 75–80% for 49d. The results showed that NO treatment effectively decreased the CI index and CI incidence, reduced the increases in membrane permeability and malondialdehyde (MDA) content, inhibited superoxide anion (O2−) production rates, reduced H2O2 content, and sustained higher activity of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) in the rind of Hami melon fruit during storage at 1±0.5°C. C-repeat/dehydration responsive element (CRT/DRE)-binding factors (CBFs) are the best-characterized transcription factors controlling cold-responsive gene expression and cold acclimation. To investigate the molecular regulation of cold resistance by NO treatment in Hami melon fruit, a 639-bp CmCBF1 gene and a 615-bp CmCBF3 gene were identified, and the effect NO on the expression of CmCBF1 and CmCBF3 was measured by RT-qPCR. The results showed that the expression of CmCBF1 and CmCBF3 were induced, the expression of the CmCBF1 and CmCBF3 in NO treated fruit was significantly higher than in control fruit during storage at 1±0.5°C. These results indicate that NO treatment can alleviate and delay CI in cold-stored Hami melon fruit via enhancing the activities of antioxidant enzymes and up-regulating the expression of CmCBF1 and CmCBF3.

Constitutive expression of DaCBF7, an Antarctic vascular plant Deschampsia antarctica CBF homolog, resulted in improved cold tolerance in transgenic rice plants

Deschampsia antarctica is an Antarctic hairgrass that grows on the west coast of the Antarctic peninsula. In this report, we have identified and characterized a transcription factor, D. antarctica C-repeat binding factor 7 (DaCBF7), that is a member of the monocot group V CBF homologs. The protein contains a single AP2 domain, a putative nuclear localization signal, and the typical CBF signature. DaCBF7, like other monocot group V homologs, contains a distinct polypeptide stretch composed of 43 amino acids in front of the AP2 motif. DaCBF7 was predominantly localized to nuclei and interacted with the C-repeat/dehydration responsive element (CRT/DRE) core sequence (ACCGAC) in vitro. DaCBF7 was induced by abiotic stresses, including drought, cold, and salinity. To investigate its possible cellular role in cold tolerance, a transgenic rice system was employed. DaCBF7-overexpressing transgenic rice plants (Ubi:DaCBF7) exhibited markedly increased tolerance to cold stress compared to wild-type plants without growth defects; however, overexpression of DaCBF7 exerted little effect on tolerance to drought or salt stress. Transcriptome analysis of a Ubi:DaCBF7 transgenic line revealed 13 genes that were up-regulated in DaCBF7-overexpressing plants compared to wild-type plants in the absence of cold stress and in short- or long-term cold stress. Five of these genes, dehydrin, remorin, Os03g63870, Os11g34790, and Os10g22630, contained putative CRT/DRE or low-temperature responsive elements in their promoter regions. These results suggest that overexpression of DaCBF7 directly and indirectly induces diverse genes in transgenic rice plants and confers enhanced tolerance to cold stress.

Construction of efficient, tuber-specific, and cold-inducible promoters in potato

Promoter activity is crucial for precise gene expression. Previously, a synthetic tuber-specific and cold-inducible promoter, pCL, containing a C-repeat/dehydration-responsive element (CRT/DRE) cassette and a tuber-specific fragment, was constructed in order to regulate cold-induced sweetening (CIS) in potatoes. However, the utility of pCL is limited due to its low activity. To improve its inducibility in response to low temperatures, we modified the CRT/DRE and flanking sequences. In particular, promoter activity was significantly improved by site-specific mutation of flanking sequences next to the core element (CCGAC) of CRT/DRE. We also inserted a modified CRT/DRE cassette into pCL; although this enhanced activity, it was not more effective than mutation of the flanking sequences. Indeed, up to 20-fold enhanced pCL activity could be achieved by replacing the CRT/DRE cassette in pCL with tandem repeats of two mutated CRT/DRE cassettes. This improvement was due to an enhanced affinity between the CRT/DRE cassette(s) and the StCBF1 transcription factor. Together, these data suggest that altering the structure of CRT/DRE can enhance CBF-related transcription complex formation and thus improve the activity of this cold-inducible promoter.

Isolation and Characterization of a OsRap2.4A Transcription Factor and its Expression in Arabidopsis for Enhancing High Salt and Drought Tolerance

Dehydration responsive element/C-repeat (DRE/CRT) is a cis-acting element involved in the regulation of abiotic stress-responsive gene expression in higher plants. Using a 50-nucleotide bait containing DRE cis-acting element localized on the downstream mini TATA box of glutamate dehydrogenase-like protein ( JRC2606 ) promoter in yeast one-hybrid screening, we have identified two transcription factors belonging to A6 subgroup of DREB subfamily - OsRap2.4A and OsRap2.4B. Expression of OsRap2.4A was induced by drought, high salt and temperature stress conditions, and also by abscisic acid treatment. Binding assay showed that OsRap2.4A bound specifically to DRE sequence in both in vivo and in vitro experiments. Transient expression of OsRap2.4A in protoplasts revealed that OsRap2.4A functioned as a transcriptional activator and upregulated expression of the GUS re-porter gene. Transgenic Arabidopsis plants expressing OsRap2.4A at low level showed no significant growth re-tardation under normal condition, whereas at high level expression of OsRap2.4A caused an obvious retardation. Expression of OsRap2.4A showed the tolerance against drought and salt stresses in comparison with control. These results suggested that OsRap2.4A gene may be involved in a new regulatory pathway in plant responses to abiotic stresses and it is potentially useful for the transformation into crop plants to improve tolerance against drought and high salt stresses.

DREB1A from Oryza sativa var. IR6: homology modelling and molecular docking

The family of AP2/ERF plant specific transcription factors, including dehydration-responsive element binding (DREB) proteins, has been reported to activate and bind with the dehydration responsive element/C repeat (DRE/CRT) of stress-inducible gene promoters. We amplified DREB1A from Oryza sativa var. IR6 (807 bp) and subjected it to protein homology modelling and phylogenetic analysis. BLAST sequence analysis indicated high similarity (99%) with the O. sativa japonica group. Phylogenetic analysis was carried out with other 12 DREB sequences from cereals/grasses using Clustal Omega. Protein secondary structure and 3D models were determined using SOPMA and MODELLER v9.10, respectively. PROSA was used to determine the excellence and consistency of models. The results indicated that the Z-score value was -2.94 and 79.5% of the residues were found in the favoured region. A psi/phi Ramachandran plot was determined using PROCHECK. The molecular docking of the GCC-box binding domain (GBD) of the protein under investigation was done using atomic coordinates of Arabidopsis thaliana GBD- and GCC-box containing DNA, respectively. The tertiary structure of the modelled protein and template were found to be very similar as predicted by a root mean square deviation of 0.968 A. It seemed that both the proteins interacted with the major groove of DNA using Beta-sheets. We attempted to expose the molecular basis of O. sativa var. IR6 DREB1A protein interaction with the target promoter sequence. The results highlighted that this gene could be a good candidate for production of abiotic stress-tolerant crop(s) in the future.

Strength comparison between cold-inducible promoters of Arabidopsis cor15a and cor15b genes in potato and tobacco

The cold-inducible promoter is ideal for regulating ectopic gene expression in plants to cope with the cold stress. The promoters of two cold-regulated genes, cor15a and cor15b, were cloned from Arabidopsis thaliana and their strengths were assayed in potato and tobacco. Although the cis-element composition and cold-inducible property were similar between the two promoters, the cor15b promoter showed significantly higher activity than the cor15a promoter in both potato and tobacco. In order to elucidate the factors determining this discrepancy, cor15a and cor15b promoters were separately truncated from 5'-end to construct short promoters with similar size containing a single C-repeat/dehydration-responsive element (CRT/DRE). Subsequently, two synthetic promoters were constructed by swapping the flanking sequences of CRT/DRE in the truncated promoters. The promoter strength comparison demonstrated that the flanking sequence could affect the promoter strength. These findings provide a potential regulatory mechanism to control the promoter strength without impact on other properties.

Expression of signalling and defence‐related genes mediated by over‐expression of JERF1, and increased resistance to sheath blight in rice

Sheath blight (SB) is one of the most destructive rice diseases that affect the yield and quality of rice. Jasmonate and Ethylene Response Factor 1, designated JERF1, can be induced by ethylene, methyl jasmonate (MeJA), abscisic acid (ABA) and salt stress. Previous research demonstrated that over‐expression of JERF1 enhanced drought tolerance by regulating the expression of stress‐related genes in rice. The present study reports that JERF1 could improve SB resistance of transgenic rice. At the molecular and physiological levels, JERF1 could up‐regulate the expression of signalling and defence‐related genes and increase the activities of lipoxygenase (LOX) and phenylalanine ammonia lyase (PAL). Analysis of cis‐elements showed that there were several DRE/CRT (dehydration‐responsive element/C‐repeat) and/or GCC boxes in the promoters of these candidate genes, indicating that JERF1 might regulate the expression of defence‐related genes via interaction with these cis‐elements. These results suggest that JERF1 might improve SB resistance by modulating the expression of defence‐related genes. This may prove to be a useful strategy for genetic engineering to improve SB resistance using transcription factor genes in rice.

Tomato SlDREB gene restricts leaf expansion and internode elongation by downregulating key genes for gibberellin biosynthesis.

Plants have evolved and adapted to different environments. Dwarfism is an adaptive trait of plants that helps them avoid high-energy costs under unfavourable conditions. The role of gibberellin (GA) in plant development has been well established. Several plant dehydration-responsive element-binding proteins (DREBs) have been identified and reported to be induced under abiotic and biotic stress conditions. A tomato DREB gene named SlDREB, which is a transcription factor and was cloned from cultivated tomato M82, was found to play a negative role in tomato plant architecture and enhances drought tolerance. Tissue expression profiles indicated that SlDREB was expressed mainly in the stem and leaf and could be induced by abscisic acid (ABA) but suppressed by GA and ethylene. SlDREB altered plant morphology by restricting leaf expansion and internode elongation when overexpressed, and the resulting dwarfism of tomato plants could be recovered by application of exogenous gibberellic acid (GA3). Transcriptional analysis of transgenic plants revealed that overexpression of SlDREB caused the dwarf phenotype by downregulating key genes involved in GA biosynthesis such as ent-copalyl diphosphate synthase (SlCPS) and GA 20-oxidases (SlGA20ox1, -2, and -4), thereby decreasing endogenous GA levels in transgenic plants. A yeast activity assay demonstrated that SlDREB specifically bound to dehydration-responsive element/C-repeat (DRE/CRT) elements of the SlCPS promoter region. Taken together, these data demonstrated that SlDREB can downregulate the expression of key genes required for GA biosynthesis and that it acts as a positive regulator in drought stress responses by restricting leaf expansion and internode elongation.

CsICE1 and CsCBF1: two transcription factors involved in cold responses in Camellia sinensis

C-repeat/dehydration-responsive element binding factors (CBFs) can induce the expression of a suite of cold-responsive genes to increase plant cold tolerance, and inducer of CBF expression 1 (ICE1) is a major activator for CBF. In the present study, we isolated the full-length cDNAs of ICE1 and CBF from Camellia sinensis, designated as CsICE1 and CsCBF1, respectively. The deduced protein CsICE1 contains a highly conserved basic helix-loop-helix (bHLH) domain and C-terminal region of ICE1-like proteins. CsCBF1 contains all conserved domains of CBFs in other plant species and can specifically bind to the C-repeat/dehydration-responsive element (CRT/DRE) as confirmed by electrophoretic mobility shift assay. The transcription of CsICE1 had no apparent alteration after chilling treatment (4°C). CsCBF1 expression was not detected in normal temperature (20°C) but was induced immediately and significantly by low temperature (4°C). Our results suggest that ICE1-CBF cold-response pathway is conserved in tea plants. CsICE1 and CsCBF1, two components of this pathway, play roles in cold responses in tea plants.

Identification of potential DREB2C targets in Arabidopsis thaliana plants overexpressing DREB2C using proteomic analysis.

The dehydration responsive element binding protein 2C (DREB2C) is a dehydration responsive element/C-repeat (DRE/CRT)-motif binding transcription factor that induced by mild heat stress. Previous experiments established that overexpression of DREB2C cDNA driven by the cauliflower mosaic virus 35S promoter (35S:DREB2C) resulted in increased heat tolerance in Arabidopsis. We first analyzed the proteomic profiles in wild-type and 35S:DREB2C plants at a normal temperature (22 degrees C), but could not detect any differences between the proteomes of wild-type and 35S:DREB2C plants. The transcript level of DREB2C in 35S:DREB2C plants after treatment with mild heat stress was increased more than two times compared with expression in 35S:DREB2C plants under unstressed condition. A proteomic approach was used to decipher the molecular mechanisms underlying thermotolerance in 35S:DREB2C Arabidopsis plants. Eleven protein spots were identified as being differentially regulated in 35S:DREB2C plants. Moreover, in silico motif analysis showed that peptidyl-prolyl isomerase ROC4, glutathione transferase 8, pyridoxal biosynthesis protein PDX1, and elongation factor Tu contained one or more DRE/CRT motifs. To our knowledge, this study is the first to identify possible targets of DREB2C transcription factors at the protein level. The proteomic results were in agreement with transcriptional data.

DEAR1, a transcriptional repressor of DREB protein that mediates plant defense and freezing stress responses in Arabidopsis

Plants have evolved intricate mechanisms to respond and adapt to a wide variety of biotic and abiotic stresses in their environment. The Arabidopsis DEAR1 (DREB and EAR motif protein 1; At3g50260) gene encodes a protein containing significant homology to the DREB1/CBF (dehydration-responsive element binding protein 1/C-repeat binding factor) domain and the EAR (ethylene response factor-associated amphiphilic repression) motif. We show here that DEAR1 mRNA accumulates in response to both pathogen infection and cold treatment. Transgenic Arabidopsis overexpressing DEAR1 (DEAR1ox) showed a dwarf phenotype and lesion-like cell death, together with constitutive expression of PR genes and accumulation of salicylic acid. DEAR1ox also showed more limited P. syringae pathogen growth compared to wild-type, consistent with an activated defense phenotype. In addition, transient expression experiments revealed that the DEAR1 protein represses DRE/CRT (dehydration-responsive element/C-repeat)-dependent transcription, which is regulated by low temperature. Furthermore, the induction of DREB1/CBF family genes by cold treatment was suppressed in DEAR1ox, leading to a reduction in freezing tolerance. These results suggest that DEAR1 has an upstream regulatory role in mediating crosstalk between signaling pathways for biotic and abiotic stress responses.

Diversification of the barley and wheat blt101/wpi6 promoters by the Xumet element without affecting stress responsiveness

BLT101-family plasma membrane proteins are found in a wide range of organisms from bacteria to nematodes and are involved in the regulation of cellular cation concentration under stress conditions. A comparison of the promoter regions of barley blt101 and its wheat ortholog, wpi6, revealed highly conserved nucleotide sequences between both genes and a unique insertion of a Xumet element in the blt101 promoter. The Xumet insertion occurred between a putative abscisic acid-responsive element (ABRE) and the dehydration-responsive element/c-repeat (DRE/CRT) within the blt101 promoter. However, blt101 and wpi6 were induced similarly in response to ABA, drought and low temperature, suggesting that the insertion does not affect promoter functions. The Xumet insertion in the blt101/wpi6 promoter region was detected in five barley cultivars, but absent in two wheat cultivars tested, suggesting that the insertion is barley-specific. Genomic Southern blot analysis revealed a large number of Xumet sequences interspersed in the barley genome, whereas only one or very few copies are present in the wheat genome. The data suggested that an expansion in copy number of Xumet elements occurred in the barley genome through evolution.

Identification of candidate CBF genes for the frost tolerance locus Fr-A m 2 in Triticum monococcum

A cluster of eleven CBF genes was recently mapped to the Frost resistance-2 (Fr-Am2) locus on chromosome 5 of diploid wheat (Triticum monococcum) using a cross between frost tolerant accession G3116 and frost sensitive DV92. The Fr-Am2 locus was mapped at the peak of two overlapping quantitative trait loci (QTL), one for frost survival and the other for differential expression of the cold regulated gene COR14b. Seven lines with recombination events within the CBF cluster were used to identify CBF candidate genes for these QTL. The lines carrying the critical recombination events were tested for whole plant frost survival and for differential transcript levels of cold induced COR14b and DHN5 genes. The strongest effect for these traits was associated to the linked TmCBF12, TmCBF14 and TmCBF15 genes, with the G3116 allele conferring improved frost tolerance and higher levels of COR14b and DHN5 transcript at mild cold temperatures (12-15 degrees C) than the DV92 allele. Comparison of CBF protein sequences revealed that the DV92 TmCBF12 protein contains a deletion of five amino acids in the AP2 DNA binding domain. Electrophoretic Mobility Shift Assays (EMSA) confirmed that the protein encoded by this allele cannot bind to the CRT/DRE (C-repeat/ dehydration-responsive element) motif present in the promoters of several cold induced genes. A smaller effect on frost tolerance was mapped to the distal group of CBF genes including TmCBF16. Transcript levels of TmCBF16, as well as those of TmCBF12 and TmCBF15 were up-regulated at mild cold temperatures in G3116 but not in DV92. Higher threshold induction temperatures can result in earlier initiation of the cold acclimation process and better resistance to subsequent freezing temperatures. The non-functional TmCBF12 allele in DV92 can also contribute to its lower frost tolerance.

Comparative Genomic Sequence and Expression Analyses ofMedicago truncatulaand Alfalfa Subspeciesfalcata COLD-ACCLIMATION-SPECIFICGenes

In Arabidopsis (Arabidopsis thaliana) the low-temperature induction of genes encoding the C-REPEAT BINDING FACTOR (CBF) transcriptional activators is a key step in cold acclimation. CBFs in turn activate a battery of downstream genes known as the CBF regulon, which collectively act to increase tolerance to low temperatures. Fundamental questions are: What determines the size and scope of the CBF regulon, and is this is a major determinant of the low-temperature tolerance capacity of individual plant species? Here we have begun to address these questions through comparative analyses of Medicago truncatula and Medicago sativa subsp. falcata. M. truncatula survived to -4 degrees C but did not cold acclimate, whereas Medicago falcata cold acclimated and survived -14 degrees C. Both species possessed low-temperature-induced CBFs but differed in the expression of the COLD-ACCLIMATION-SPECIFIC (CAS) genes, which are candidate CBF targets. M. falcata CAS30 was robustly cold-responsive whereas the MtCAS31 homolog was not. M. falcata also possessed additional CAS30 homologs in comparison to the single CAS31 gene in M. truncatula. MfCAS30 possessed multiple pairs of closely spaced C-REPEAT/DEHYDRATION RESPONSIVE ELEMENT (CRT/DRE) motifs, the cognate CBF binding site in its upstream region whereas MtCAS31 lacked one CRT/DRE partner of the two proximal partner pairs. CAS genes also shared a promoter structure comprising modules proximal and distal to the coding sequence. CAS15, highly cold-responsive in both species, harbored numerous CRT/DRE motifs, but only in the distal module. However, fusion of the MtCAS15 promoter, including the distal module, to a reporter gene did not result in low-temperature responsiveness in stably transformed Arabidopsis. In contrast, both MtCAS31 and MfCAS30 promoter fusions were low-temperature responsive, although the MfCAS31 fusion was less robust than the MfCAS30 fusion. From these studies we conclude that CAS genes harbor CRT/DRE motifs, their proximity to one another is likely key to regulatory output in Medicago, and they may be located kilobases distal to the transcriptional start site. We hypothesize that these differences in CRT/DRE copy numbers in CAS30/CAS31 upstream regions combined with differences in gene copy numbers may be a factor in determining differences in low-temperature tolerance between M. truncatula and M. falcata.

Combinatorial interactions of multiple cis‐elements regulating the induction of the Arabidopsis XERO2 dehydrin gene by abscisic acid and cold

Multiple combinations of mutations in the promoter of the XERO2 dehydrin gene were used to identify elements involved in ABA and cold induction. Mutating one of the three ACGT elements (ACGT1) increases expression in the absence of cold or ABA. An AT-rich element is a novel partner (coupling element) of ACGT-containing ABA-responsive cis-elements. A 12-bp palindrome also acts as a coupling element for ABA induction and includes one of the three dehydration-responsive element/C-repeat (DRE/CRT) elements and two overlapping motifs (TGTCG and TCGGC) previously shown to be statistically enriched in ABA-dependent and 'VP1 or ABA'-dependent activated genes (Plant Physiol. 2005; 139:437). At least two of the DRE/CRT elements are required for significant cold induction. During cold induction the AT-rich element also functions as a coupling element and ACGT1 is involved in repressing this induction. Two of the ACGT and DRE/CRT elements overlap, and mutating a single base in the ACGT of either of the two GCCGACGT sequences while retaining a DRE element reduced both ABA and cold induction. Changing the spatial relationships between the elements by deletion, inversion or insertion of DNA sequences reduced both cold and ABA induction. Overexpression of CBF1, -2 or -3 induced XERO2 expression in untreated plants. The ABI5 transcription factor may have a role in ABA-induced XERO2 expression, whereas ABI3 and ABI4 do not. The GCA2 gene was essential for both cold and ABA induction. A combination of the same overlapping and shared elements is used in the regulation of transcription by ABA and cold.

Perception, transduction, and networks in cold signaling

Cold temperature is one of the most critical environmental conditions that limit the geographic distribution of plants and account for significant reductions in the yields of agriculturally important crops. As part of their stress-adaptation, plants respond to cold by changing various aspects of their physiological processes, including gene expression. Numerous genes are induced by cold at the transcriptional level, encoding proteins that protect against freezing injury. While the expression of stress-inducible genes is mainly mediated by ABA-dependent and ABA-independent pathways, the C-repeat/dehydration-responsive element (CRT/DRE), containing a core sequence of-CCGAC-in the ABA-independent pathway, is essential for transcriptional activation in response to abiotic stresses, including cold. The CRT/DRE-binding factors (CBFs) and DRE-binding proteins (DREBs) can confer freezing and drought tolerances in transgenic plants by up-regulating the genes involved in enhancing stress tolerance when overexpressed. However, microarray analysis and characterization of variousArabidopsis mutants with altered response to cold and freezing temperatures suggest the existence of other cold-signaling pathways that do not involve CBF/DREB transcription factors. This review describes potential mechanisms for cold-perception, the functions of genes induced by cold temperatures in freezing tolerance, and the regulation of cold-responsive gene expression. Genetic approaches, including classical methods and reporter-gene-based screening, have revealed many signaling components in this stress response, suggesting that complicated, multiple pathways and cross-talk are involved. Recent advances in our understanding of the roles for these components as well as calcium-signaling networks are also discussed here.