DRE-binding Proteins Research Articles

Functional Characterization of Lobularia maritima LmTrxh2 Gene Involved in Cold Tolerance in Tobacco through Alleviation of ROS Damage to the Plasma Membrane.

Cold stress is a key environmental factor affecting plant growth and development, crop productivity, and geographic distribution. Thioredoxins (Trxs) are small proteins that are ubiquitously expressed in all organisms and implicated in several cellular processes, including redox reactions. However, their role in the regulation of cold stress in the halophyte plant Lobularia maritima remains unknown. We recently showed that overexpression of LmTrxh2, which is the gene that encodes the h-type Trx protein previously isolated from L. maritima, led to an enhanced tolerance to salt and osmotic stress in transgenic tobacco. This study functionally characterized the LmTrxh2 gene via its overexpression in tobacco and explored its cold tolerance mechanisms. Results of the RT-qPCR and western blot analyses indicated differential temporal and spatial regulation of LmTrxh2 in L. maritima under cold stress at 4 °C. LmTrxh2 overexpression enhanced the cold tolerance of transgenic tobacco, as evidenced by increased germination rate, fresh weight and catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) activities; reduced malondialdehyde levels, membrane leakage, superoxide anion (O2-), and hydrogen peroxide (H2O2) levels; and higher retention of chlorophyll than in non-transgenic plants (NT). Furthermore, the transcript levels of reactive oxygen species (ROS)-related genes (NtSOD and NtCAT1), stress-responsive late embryogenis abundant protein 5 (NtLEA5), early response to dehydration 10C (NtERD10C), DRE-binding proteins 1A (NtDREB1A), and cold-responsive (COR) genes (NtCOR15A, NtCOR47, and NtKIN1) were upregulated in transgenic lines compared with those in NT plants under cold stress, indicating that LmTrxh2 conferred cold stress tolerance by enhancing the ROS scavenging ability of plants, thus enabling them to maintain membrane integrity. These results suggest that LmTrxh2 promotes cold tolerance in tobacco and provide new insight into the improvement of cold-stress resistance to cold stress in non-halophyte plants and crops.

Characterization of TaDREB1 in wheat genotypes with different seed germination under osmotic stress

BackgroundThe cis-acting element DRE/CRT plays an important role in activating gene expression responsive to osmotic stress, low temperature and high-salinity. DREB1/CBF genes encode DRE-binding proteins with the function as transcript activators. TaDREB1 was also found to be induced by osmotic stress.MethodsThe dates of osmotic stress was assessed by seed germination drought resistance index; the full-length cDNA sequences of TaDREB1 gene were downloaded from NCBI datebase; identification of allelic variation and transcript expression were assessed by PCR and semi-quantitive RT-PCR analysis, respectively.ResultsTotal 13 new allele variations of TaDREB1 were identified in the germplasms tested in the paper, including 5 TaDREB1-A on chromosome 3AL, 4 TaDREB1-B on chromosome 3BL and 4 TaDREB1-D on chromosome 3DL. In each variety, there existed two loci of TaDREB1-D genes, named TaDREB1-D1 and TaDREB1-D2, both of which had the similar nucleotide sequence except an 11 bp insertion in the former. In wheat seeds under osmotic stress, we did not detect the transcript expression level of TaDREB1-A and TaDREB1-B, but that of TaDREB1-D.ConclusionsThe capacity of osmotic stress tolerance was closely correlated with the expression level and tendency of TaDREB1-D.

Soybean DREB1/CBF-type transcription factors function in heat and drought as well as cold stress-responsive gene expression.

Soybean (Glycine max) is a globally important crop, and its growth and yield are severely reduced by abiotic stresses, such as drought, heat, and cold. The cis-acting element DRE (dehydration-responsive element)/CRT plays an important role in activating gene expression in response to these stresses. The Arabidopsis DREB1/CBF genes that encode DRE-binding proteins function as transcriptional activators in the cold stress responsive gene expression. In this study, we identified 14 DREB1-type transcription factors (GmDREB1s) from a soybean genome database. The expression of most GmDREB1 genes in soybean was strongly induced by a variety of abiotic stresses, such as cold, drought, high salt, and heat. The GmDREB1 proteins activated transcription via DREs (dehydration-responsive element) in Arabidopsis and soybean protoplasts. Transcriptome analyses using transgenic Arabidopsis plants overexpressing GmDREB1s indicated that many of the downstream genes are cold-inducible and overlap with those of Arabidopsis DREB1A. We then comprehensively analyzed the downstream genes of GmDREB1B;1, which is closely related to DREB1A, using a transient expression system in soybean protoplasts. The expression of numerous genes induced by various abiotic stresses were increased by overexpressing GmDREB1B;1 in soybean, and DREs were the most conserved element in the promoters of these genes. The downstream genes of GmDREB1B;1 included numerous soybean-specific stress-inducible genes that encode an ABA receptor family protein, GmPYL21, and translation-related genes, such as ribosomal proteins. We confirmed that GmDREB1B;1 directly activates GmPYL21 expression and enhances ABRE-mediated gene expression in an ABA-independent manner. These results suggest that GmDREB1 proteins activate the expression of numerous soybean-specific stress-responsive genes under diverse abiotic stress conditions.

Analysis and application of RD29 genes in abiotic stress response

Abiotic stresses adversely affect the growth and productivity of plants and give rise to a series of morphological, physiological, biochemical and molecular changes. Molecular studies have shown that a number of genes with various functions are induced by abiotic stress. The RD29 (Responsive to Desiccation) genes RD29A and RD29B are such genes induced by desiccation, cold and high salt stresses. The genes encode hydrophilic proteins and endow plants with tolerance to these stresses. Two cis-acting elements, ABRE (ABA-responsive element) and DRE (dehydration-responsive element), are present, albeit in different numbers, in the promoter regions of the RD29 genes. Transcription factors, such as AREBs (ABA-responsive element-binding proteins) and DREBs (DRE-binding proteins), regulate the expression of RD29 genes through binding to ABRE and DRE, respectively. Therefore, the expression of RD29 genes can be divided into ABA-independent and ABA-dependent signal transduction pathways. RD29 sequences are used as markers to monitor stress-response pathways in plants. Furthermore, the RD29A promoter has been used widely in genetic engineering to improve plant adaptability to adverse environments. In addition, the chimeric gene consisting of the firefly luciferase (LUC) marker driven by the RD29A promoter is a powerful tool used to study stress signaling pathways and for reverse genetic analyses. In this review, the structures, expression and regulation patterns, and application in genetic engineering of RD29A and RD29B are introduced in detail.

Isolation and characterization of a novel cDNA encoding methyl jasmonate-responsive transcription factor TcAP2 from Taxus cuspidata

A novel transcription factor, TcAP2, was isolated from Taxus cuspidata by yeast one-hybrid strategy. This factor interacts with jasmonate- and elicitor-responsive element. Analysis of the deduced TcAP2 amino acid sequence revealed that TcAP2 contained a conserved AP2/ethylene-responsive element binding protein domain that consisted of 268 amino acids in a potential nuclear localization sequence. The factor of TcAP2 had a high homology, in its AP2 domain, to other AP2 family members. Based on phylogenetic analysis, it was different from other five DRE-binding proteins in their evolutionary relationship. The transcription of TcAP2 gene in yew accumulated primarily in young organs, such as young stems. Quantitative real-time RT-PCR analysis indicated that TcAP2 gene was inducible to express by treatments with methyl jasmonate plus salicylic acid, high salinity, and cold. This gene showed no response to either abscisic acid or drought treatment.

Differential Repression by Freud-1/CC2D1A at a Polymorphic Site in the Dopamine-D2 Receptor Gene

Freud-1/CC2D1A is a transcriptional repressor of the serotonin-1A receptor gene and was recently genetically linked to non-syndromic mental retardation. To identify new Freud-1 gene targets, data base mining for Freud-1 recognition sequences was done. A highly homologous intronic element (D2-DRE) was identified in the human dopamine-D2 receptor (DRD2) gene, and the role of Freud-1 in regulating the gene at this site was assessed. Recombinant Freud-1 bound specifically to the D2-DRE, and a major protein-D2-DRE complex was identified in nuclear extracts that was supershifted using Freud-1-specific antibodies. Endogenous Freud-1 binding to the D2-DRE in cells was detected using chromatin immunoprecipitation. The D2-DRE conferred strong repressor activity in transcriptional reporter assays that was dependent on the Freud-1 recognition sequence. In three different human cell lines, the level of Freud-1 protein was inversely related to DRD2 expression. Knockdown of endogenous Freud-1 using small interfering RNA resulted in an up-regulation of DRD2 RNA and binding sites, demonstrating a crucial role for Freud-1 in DRD2 regulation. A previously uncharacterized single nucleotide A/G polymorphism (rs2734836) was located adjacent to the D2-DRE and conferred allele-specific Freud-1 binding and repression, with the major G-allele having reduced activity. These studies demonstrate a key role for Freud-1 to regulate DRD2 expression and provide the first mechanistic insights into its transcriptional regulation. Allele-specific regulation of DRD2 expression by Freud-1 may possibly associate with psychiatric disorders or mental retardation.

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.

Isolated and characterization of a cDNA encoding ethylene-responsive element binding protein (EREBP)/AP2-type protein, RCBF2, in Oryza sativa L.

A transcription factor RCBF2 which interacts with C-repeat/DRE was isolated from Oryza sativa L. by a yeast one-hybrid method. Analysis of the deduced RCBF2 amino acid sequence revealed that RCBF2 contained a conserved ethylene-responsive element binding protein (EREBP)/AP2 domain of 59 amino acids and a potential nuclear localization sequence. RCBF2 showed a high level of homology with other CBF family members only in AP2 domain. Phylogenetic analysis showed that RCBF2 might be different from other eight DRE-binding proteins on evolutionary relationship. The semi-quantitative RT-PCR (s-Q RT-PCR) analysis indicated the expression of RCBF2 gene was induced by cold, dehydration and high-salinity, but not by abscisic acid, and the transcription of RCBF2 gene accumulated primarily in rice immature seeds, growing point and shoots.

OsDREB1 Gene from Rice Enhances Cold Tolerance in Tobacco

The OsDREB1 gene from rice encodes a transcription factor belonging to the DREBP transcription factor subfamily. Many DREBP transcription factors regulate gene expression in response to drought, high-salt, and cold stresses by binding specifically to the dehydration-responsive element (DRE). DRE-binding proteins, such as CBF1, DREB1A, and DREB2A, have been cloned from Arabidopsis thaliana and have been proved to play an important role in stress response of Arabidopsis and several other plants. In this study, the OsDREB1 gene was transferred to tobacco plants by the Agrobacterium-mediated transfer method, and 16 transgenic plants were identified. PCR analysis demonstrates that the foreign genes have been integrated into the tobacco genome. Results of freezing stress experiments indicate that the transgenic plants have enhanced cold tolerance.

Interaction between two cis-acting elements, ABRE and DRE, in ABA-dependent expression of Arabidopsis rd29A gene in response to dehydration and high-salinity stresses.

Many abiotic stress-inducible genes contain two cis-acting elements, namely a dehydration-responsive element (DRE; TACCGACAT) and an ABA-responsive element (ABRE; ACGTGG/TC), in their promoter regions. We precisely analyzed the 120 bp promoter region (-174 to -55) of the Arabidopsis rd29A gene whose expression is induced by dehydration, high-salinity, low-temperature, and abscisic acid (ABA) treatments and whose 120 bp promoter region contains the DRE, DRE/CRT-core motif (A/GCCGAC), and ABRE sequences. Deletion and base substitution analyses of this region showed that the DRE-core motif functions as DRE and that the DRE/DRE-core motif could be a coupling element of ABRE. Gel mobility shift assays revealed that DRE-binding proteins (DREB1s/CBFs and DREB2s) bind to both DRE and the DRE-core motif and that ABRE-binding proteins (AREBs/ABFs) bind to ABRE in the 120 bp promoter region. In addition, transactivation experiments using Arabidopsis leaf protoplasts showed that DREBs and AREBs cumulatively transactivate the expression of a GUS reporter gene fused to the 120 bp promoter region of rd29A. These results indicate that DRE and ABRE are interdependent in the ABA-responsive expression of the rd29A gene in response to ABA in Arabidopsis.

An EREBP/AP2-type protein in Triticum aestivum was a DRE-binding transcription factor induced by cold, dehydration and ABA stress.

We characterize one transcription factor of DRE-binding proteins (TaDREB1) that was isolated from a drought-induced cDNA library of wheat (Triticum aestivum L.). TaDREB1 contains one conserved EREBP/AP2 domain, and shows similarity with Arabidopsis thaliana DREB family members in both overall amino-acid sequences and the secondary structure arrangement within the DNA-binding motifs. In yeast one-hybrid system, TaDREB1, can specially activate the genes fused with the promoter containing three tandemly repeated copies of the wild-type DRE sequence: TACCGACAT. In different wheat cultivars, the Ta DREB1 gene is induced by low temperature, salinity and drought; and the expression of Wcs120 that contains DRE motifs in its promoter is closely related to the expression of TaDREB1. These results suggest that TaDREB1 functions as a DRE-binding transcription factor in wheat. We also observed the dwarf phenotype in transgenic rice (T0) overexpressing TaDREB1.

Maize DRE-binding proteins DBF1 and DBF2 are involved in rab17 regulation through the drought-responsive element in an ABA-dependent pathway.

The abscisic acid-responsive gene rab17 of maize is expressed during late embryogenesis, and is induced by ABA and desiccation in embryo and vegetative tissues. ABRE and DRE cis-elements are involved in regulation of the gene by ABA and drought. Using yeast one-hybrid screening, we isolated two cDNAs encoding two new DRE-binding proteins, designated DBF1 and DBF2, that are members of the AP2/EREBP transcription factor family. Analysis of mRNA accumulation profiles showed that DBF1 is induced during maize embryogenesis and after desiccation, NaCl and ABA treatments in plant seedlings, whereas the DBF2 mRNA is not induced. DNA-binding preferences of DBFs were analysed by electrophoretic mobility shift assays, and showed that both DBF1 and DBF2 bound to the wild-type DRE2 element, but not to the DRE2 mutant or to the DRE1 element which differs only in a single nucleotide. Transactivation activity using particle bombardment showed that DBF1 functioned as activator of DRE2-dependent transcription of rab17 promoter by ABA, whereas DBF2 overexpression had a repression action downregulating not only the basal promoter activity, but also the ABA effect. These results show that ABA plays a role in the regulation of DBF activity, and suggests the existence of an ABA-dependent pathway for the regulation of genes through the C-repeat/DRE element.

Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration- and high-salinity-responsive gene expression.

In plants, a cis-acting element, DRE/CRT, is involved in ABA-independent gene expression in response to dehydration and low-temperature stress. To understand signal transduction pathways from perception of the dehydration stress signal to gene expression, we characterized a gene family for DRE/CRT-binding proteins DREB2A and DREB2B in Arabidopsis thaliana. Northern analysis showed that both genes are induced by dehydration and high-salt stress. Organ-specific northern analysis with gene-specific probes showed that these genes are strongly induced in roots by high-salt stress and in stems and roots by dehydration stress. The DREB2A gene is located on chromosome 5, and DREB2B on chromosome 3. We screened an Arabidopsis genomic DNA library with cDNA fragments of DREB2A and DREB2B as probes, and isolated DNA fragments that contained 5'-flanking regions of these genes. Sequence analysis showed that both genes are interrupted by a single intron at identical positions in their leader sequence. Several conserved sequences were found in the promoter regions of both genes. The beta-glucuronidase (GUS) reporter gene driven by the DREB2 promoters was induced by dehydration and high-salt stress in transgenic Arabidopsis plants.

Overlapping but unique DNA binding specificities of the Ah receptor and constitutive dioxin-responsive element binding proteins from human keratinocytes

To understand the relationships between the protein architecture assembled on dioxin-responsive elements (DRE) and transcriptional regulation by dioxin in human keratinocytes, the nuclear DRE-binding proteins from human keratinocytes were identified and characterized. In addition to the aryl hydrocarbon receptor (AHR) complex inducible by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), nuclear extracts from 3 human keratinocyte cell lines also contained one or more proteins that bound specifically to the DRE but whose levels were unaffected by TCDD or by anti-AHR antibody pretreatment. Alteration of a conserved T, within the core DRE sequence needed for transcriptional activation by the AHR complex, did not affect AHR binding but severely affected the ability of the constitutive proteins to bind. These data suggest that the nonidentical interplay of the AHR and constitutive DRE-binding proteins on the DRE is important in the regulation of genes whose expression is controlled by DRE.

Identification of the DNA damage-responsive elements of the rhp51+ gene, a recA and RAD51 homolog from the fission yeast Schizosaccharomyces pombe.

The Schizosaccharomyces pombe rhp51+ gene encodes a recombinational repair protein that shares significant sequence identities with the bacterial RecA and the Saccharomyces cerevisiae RAD51 protein. Levels of rhp51+ mRNA increase following several types of DNA damage or inhibition of DNA synthesis. An rhp51::ura4 fusion gene was used to identify the cis-acting promoter elements involved in regulating rhp51+ expression in response to DNA damage. Two elements, designated DRE1 and DRE2 (for damage-responsive element), match a decamer consensus URS (upstream repressing sequence) found in the promoters of many other DNA repair and metabolism genes from S. cerevisiae. However, our results show that DRE1 and DRE2 each function as a UAS (upstream activating sequence) rather than a URS and are also required for DNA-damage inducibility of the gene. A 20-bp fragment located downstream of both DRE1 and DRE2 is responsible for URS function. The DRE1 and DRE2 elements cross-competed for binding to two proteins of 45 and 59 kDa. DNase I footprint analysis suggests that DRE1 and DRE2 bind to the same DNA-binding proteins. These results suggest that the DRE-binding proteins may play an important role in the DNA-damage inducibility of rhp51+ expression.