Dehydration-responsive Element-binding Factor Research Articles

A Proximal Promoter Region of Arabidopsis DREB2C Confers Tissue‐specific Expression under Heat Stress

The dehydration-responsive element-binding factor 2C (DREB2C) is a member of the CBF/DREB subfamily of proteins, which contains a single APETALA2/Ethylene responsive element-binding factor (AP2/ERF) domain. To identify the expression pattern of the DREB2C gene, which contains multiple transcription cis-regulatory elements in its promoter, an approximately 1.4 kb upstream DREB2C sequence was fused to the β-glucuronidase reporter gene (GUS) and the recombinant p1244 construct was transformed into Arabidopsis thaliana (L.) Heynh. The promoter of the gene directed prominent GUS activity in the vasculature in diverse young dividing tissues. Upon applying heat stress (HS), GUS staining was also enhanced in the vasculature of the growing tissues. Analysis of a series of 5'-deletions of the DREB2C promoter revealed that a proximal upstream sequence sufficient for the tissue-specific spatial and temporal induction of GUS expression by HS is localized in the promoter region between -204 and -34 bps relative to the transcriptional start site. Furthermore, electrophoretic mobility shift assay (EMSA) demonstrated that nuclear protein binding activities specific to a -120 to -32 bp promoter fragment increased after HS. These results indicate that the TATA-proximal region and some latent trans-acting factors may cooperate in HS-induced activation of the Arabidopsis DREB2C promoter.

Isolation and characterization of a dehydration responsive element binding factor MsDREBA5 in Malus sieversii Roem.

A dehydration responsive element binding (DREB) protein homologous gene, designated MsDREBA5 (GenBank ID: JQ655768), was isolated from Malus sieversii Roem. Based on similarity with AP2 domains, MsDREBA5 was cladeded into the A-5 subgroup in DREB subfamily in AP2/ERF family. Subcellular localization analyses revealed that MsDREBA5 was subcellularly localized into the nucleus when transiently expressed in onion epidermis cells. The MsDREBA5 protein might show no transcriptional activation ability. The 1624-bp-long promoter contained putative cis-acting elements and transcription factor binding motifs, including elements predicted to respond to abiotic stresses. MsDREBA5 was expressed in almost all the tissues and organs examined under normal conditions. Additionally, expression of MsDREBA5 was induced by cold, drought and exogenously applied abscisic acid, but down-regulated by salt stress. Transgenic Arabidopsis plants overexpressing MsDREBA5 appeared to down-regulate the expression levels of the stress-induced effecter genes under drought stress. Our findings may provide an important basis for further studies of the acclimation and tolerance of M. sieversii to abiotic stresses.

Constitutive expression of Arabidopsis DREB1B in transgenic potato enhances drought and freezing tolerance

DNA cassette consisting of an Arabidopsis dehydration-responsive element binding factor 1 (DREB1B) cDNA, driven by a cauliflower mosaic virus 35S promoter, was introduced into potato plants (Solanum tuberosum L.) through Agrobacterium tumefaciens-mediated gene transfer. The presence and expression of the gene in transgenic plants were confirmed by the PCR and RT-PCR techniques, respectively. Northern hybridization using a DREB1B cDNA probe revealed high levels of DREB1B expression among the most transgenic lines. Overexpression of DREB1B imparted a significant freezing and drought tolerance gain in the transgenic potato lines. In comparison with the wild-type plants, the transgenic potatoes contained higher proline content under drought and freezing conditions, and maintained their relative water content higher under water stress. The enhancement of tolerance in transgenic potato highlights the presence of genes responding to the transcription factor DREB1B in this plant.

Constitutive expression of Arabidopsis DREB1B in transgenic potato enhances drought and freezing tolerance

DNA cassette consisting of an Arabidopsis dehydration-responsive element binding factor 1 (DREB1B) cDNA, driven by a cauliflower mosaic virus 35S promoter, was introduced into potato plants (Solanum tuberosum L.) through Agrobacterium tumefaciens-mediated gene transfer. The presence and expression of the gene in transgenic plants were confirmed by the PCR and RT-PCR techniques, respectively. Northern hybridization using a DREB1B cDNA probe revealed high levels of DREB1B expression among the most transgenic lines. Overexpression of DREB1B imparted a significant freezing and drought tolerance gain in the transgenic potato lines. In comparison with the wild-type plants, the transgenic potatoes contained higher proline content under drought and freezing conditions, and maintained their relative water content higher under water stress. The enhancement of tolerance in transgenic potato highlights the presence of genes responding to the transcription factor DREB1B in this plant.

Characterization of a new dehydration responsive element binding factor in central arctic cowberry

The C-repeat binding factor/dehydration responsive element binding factor 1 (CBF/DREB1) is a critical regulator of the development of tolerance to abiotic stress in plants. Here we report the cloning of the homologous gene, VviDREB1, from a central arctic plant, the cowberry Vaccinium vitis-idaea L., using rapid amplification of cDNA ends and genome walking techniques. The cDNA and genomic DNA are 887 and 3790 bp long, respectively. Alignment and phylogenetic tree analysis suggested that VviDREB1 should be classified into the A-1 group of the DREB subfamily. The 1522-bp-long promoter contains putative cis-acting elements and transcription factor binding motifs, including elements predicted to respond to phytohormones, abiotic stress, and light. The deduced amino acid sequence of the VviDREB1 cDNA contains a predicted DNA-binding domain with a three-stranded anti-parallel β-sheet and an α-helix, similar to that of the Arabidopsis thaliana ethylene-responsive-element-binding factor AtERF1, suggesting that VviDREB1 might bind to the dehydration responsive and ethylene responsive element. The expression of VviDREB1 was mainly localized to the nuclei of onion epidermal cells. Importantly, VviDREB1 expression in the cowberry was up-regulated by cold, high-salt, and abscisic acid treatment. Our findings may provide a novel basis for further studies of the acclimation and tolerance of cowberry to abiotic stresses.

Analysis of DNA methylation of maize in response to osmotic and salt stress based on methylation-sensitive amplified polymorphism

Water stress is known to alter cytosine methylation, which generally represses transcription. However, little is known about the role of methylation alteration in maize under osmotic stress. Here, methylation-sensitive amplified polymorphism (MSAP) was used to screen PEG- or NaCl-induced methylation alteration in maize seedlings. The sequences of 25 differentially amplified fragments relevant to stress were successfully obtained. Two stress-specific fragments from leaves, LP166 and LPS911, shown to be homologous to retrotransposon Gag-Pol protein genes, suggested that osmotic stress-induced methylation of retrotransposons. Three MSAP fragments, representing drought-induced or salt-induced methylation in leaves, were homologous to a maize aluminum-induced transporter. Besides these, heat shock protein HSP82, Poly [ADP-ribose] polymerase 2, Lipoxygenase, casein kinase (CK2), and dehydration-responsive element-binding (DREB) factor were also homologs of MSAP sequences from salt-treated roots. One MSAP fragment amplified from salt-treated roots, designated RS39, was homologous to the first intron of maize protein phosphatase 2C (zmPP2C), whereas - LS103, absent from salt-treated leaves, was homologous to maize glutathione S-transferases (zmGST). Expression analysis showed that salt-induced intron methylation of root zmPP2C significantly downregulated its expression, while salt-induced demethylation of leaf zmGST weakly upregulated its expression. The results suggested that salinity-induced methylation downregulated zmPP2C expression, a negative regulator of the stress response, while salinity-induced demethylation upregulated zmGST expression, a positive effecter of the stress response. Altered methylation, in response to stress, might also be involved in stress acclimation.

Overexpression of two chrysanthemum DgDREB1 group genes causing delayed flowering or dwarfism in Arabidopsis

We isolated 13 DREB1 (dehydration responsive element binding factor 1) genes from chrysanthemum and further divided them into three groups, DgDREB1A, DgDREB1B and DgDREB1C, based on the phylogenetic analysis. Each group showed their unique expression patterns under cold, dehydration and salt stress conditions. Arabidopsis plants overexpressing DgDREB1A (1A plants) exhibited significantly stronger tolerance to freezing and drought than those overexpressing DgDREB1B (1B plants) and the control plants. In addition, 1A plants showed delayed flowering, but not dwarfism; while 1B plants showed dwarfism, but not delayed flowering. In 1A plants, the expression of three stress-related DREB1-downstream genes, COR47, COR15A, and RD29A, was strongly induced while the expression of CO and FT, two photoperiod responsive flowering-time genes, was inhibited. In 1B plants, the expression of GA2ox7, a GA-deactivation enzyme gene, was dramatically enhanced. The results above strongly suggest that members from different DgDREB1 groups may have distinct effects on plant development: DgDREB1A may be involved in photoperiod-related flowering-time determination and DgDREB1B in GA-mediated plant development.

Enhanced Tolerance to Chilling Stress in OsMYB3R-2 Transgenic Rice Is Mediated by Alteration in Cell Cycle and Ectopic Expression of Stress Genes

MYB transcription factors play central roles in plant responses to abiotic stresses. How stress affects development is poorly understood. Here, we show that OsMYB3R-2 functions in both stress and developmental processes in rice (Oryza sativa). Transgenic plants overexpressing OsMYB3R-2 exhibited enhanced cold tolerance. Cold treatment greatly induced the expression of OsMYB3R-2, which encodes an active transcription factor. We show that OsMYB3R-2 specifically bound to a mitosis-specific activator cis-element, (T/C)C(T/C)AACGG(T/C)(T/C)A, a conserved sequence that was found in promoters of cyclin genes such as OsCycB1;1 and OsKNOLLE2. In addition, overexpression of OsMYB3R-2 in rice led to higher transcript levels of several G2/M phase-specific genes, including OsCycB1;1, OsCycB2;1, OsCycB2;2, and OsCDC20.1, than those in OsMYB3R-2 antisense lines or wild-type plants in response to cold treatment. Flow cytometry analysis revealed an increased cell mitotic index in overexpressed transgenic lines of OsMYB3R-2 after cold treatment. Furthermore, resistance to cold stress in the transgenic plants overexpressing OsCycB1;1 was also enhanced. The level of cellular free proline was increased in the overexpressed rice lines of OsMYB3R-2 and OsCycB1;1 transgenic plants compared with wild-type plants under the cold treatment. These results suggest that OsMYB3R-2 targets OsCycB1;1 and regulates the progress of the cell cycle during chilling stress. OsCPT1, which may be involved in the dehydration-responsive element-binding factor 1A pathway, showed the same transcription pattern in response to cold as did OsCycB1;1 in transgenic rice. Therefore, a cold resistance mechanism in rice could be mediated by regulating the cell cycle, which is controlled by key genes including OsMYB3R-2.

Characterization of the TaAIDFa gene encoding a CRT/DRE-binding factor responsive to drought, high-salt, and cold stress in wheat

Dehydration responsive element-binding factors (DBFs) belong to the AP2/ERF superfamily and play vital regulatory roles in abiotic stress responses in plants. In this study, we isolated three novel homologs of the DBF gene family in wheat (Triticum aestivum L.) by screening a drought-induced cDNA library and designated them as TaAIDFs (T. aestivum abiotic stress-induced DBFs). Compared to TaAIDFb and TaAIDFc, TaAIDFa lacks a short Ser/Thr-rich region, a putative phosphorylation site, following the AP2/ERF domain. The TaAIDFa gene, located on chromosome 3BS, is interrupted by a single intron at the 17th Arg (R) in the N-terminal domain. The N-terminal region of the TaAIDFa protein modulates nuclear localization. The TaAIDFa protein is capable of binding to CRT/DRE elements in vitro and in vivo, and of trans-activating reporter gene expression in yeast cells. The TaAIDFa promoter, with various stress-related cis-acting elements, drives expression of the GUS reporter gene in wheat calli under stress conditions. This was further confirmed by responses of TaAIDFa transcripts to drought, salinity, low-temperature, and exogenous ABA. Furthermore, overexpression of TaAIDFa activated CRT/DRE-containing genes under normal growth conditions, and improved drought and osmotic stress tolerances in transgenic Arabidopsis plants. These results suggested that TaAIDFa encodes a CRT/DRE element-binding factor that might be involved in multiple abiotic stress signal transduction pathways.

Identification and characterization of CBF/DREB1-related genes in Populus hopeiensis

The dehydration-responsive element-binding factor (DREB) is a plant-specific family of transcription factors and plays an important role in plant response and adaptation to abiotic stress. In the present work, two highly similar CBF/DREB1-like genes, designated as PhCBF4a and PhCBF4b, were identified from Populus hopeiensis. These two genes contain all conserved domains known to exist in other CBF/DREB1 genes. In the AP2 domain, there is only one different amino acid residue between PhCBF4a and PhCBF4b, alanine or valine, a nonpolar amino acid, suggesting that PhCBF4a and PhCBF4b may have similar DNA binding ability. Their expression is induced by water-loss treatment and their expression patterns are similar. Moreover, with a genomic DNA as template, the presence of the same bands in PCR products as those in expression pattern analysis indicated that PhCBF4a and PhCBF4b exist in the genome of P. hopeiensis. Their detailed functions are discussed and will need further study.

Cis-regulatory element-based genome-wide identification of DREB1/CBF targets in Arabidopsis

Microarray analysis is used to identify transcriptional targets. However, the direct targets of transcription factors cannot be distinguished from indirect ones; further, genes with low-expression levels cannot be identified by this method. In the present study, we exploit the cis-element dehydration-responsive element (DRE) that is known to be responsible for the transcription of DRE binding factor 1 (DREB1) targets in the promoter region of all Arabidopsis genes. Putative targets whose promoters contain the elements were verified by both microarray and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Five new DREB1/CBF direct targets were identified. Compared with traditional microarray analysis, our method is convenient and cost-effective for identifying the downstream targets of transcription factors.

Arabidopsis CBF1 and CBF3 have a different function than CBF2 in cold acclimation and define different gene classes in the CBF regulon.

The C-repeat-binding factor (CBF)/dehydration-responsive element-binding factor (DREB1) proteins constitute a small family of Arabidopsis transcriptional activators (CBF1/DREB1B, CBF2/DREB1C, and CBF3/DREB1A) that play a prominent role in cold acclimation. A fundamental question about these factors that remains to be answered is whether they are functionally equivalent. Recently, we reported that CBF2 negatively regulates CBF1 and CBF3 expression, and that CBFs are subjected to different temporal regulation during cold acclimation, which suggested this might not be the case. In this study, we have analyzed the expression of CBF genes in different tissues of Arabidopsis, during development and in response to low temperature, and characterized RNA interference (RNAi) and antisense lines that fail to accumulate CBF1 or/and CBF3 mRNAs under cold conditions. We found that CBF1 and CBF3 are regulated in a different way than CBF2. Moreover, in contrast to CBF2, CBF1 and CBF3 are not involved in regulating other CBF genes and positively regulate cold acclimation by activating the same subset of CBF-target genes. All these results demonstrate that CBF1 and CBF3 have different functions than CBF2. We also found that the CBF regulon is composed of at least two different kind of genes, one of them requiring the simultaneous expression of both CBF1 and CBF3 to be properly induced. This indicates that CBF1 and CBF3 have a concerted additive effect to induce the whole CBF regulon and the complete development of cold acclimation.

Gene expression profiles during heat acclimation in Arabidopsis thaliana suspension-culture cells

Thermotolerance is induced by moderated heat acclimation. Suspension cultures of heat-acclimated Arabidopsis thaliana L. (Heynh.), ecotype Columbia, show thermotolerance against lethal heat shock (9 min, 50 degrees C), as evidenced by a chlorophyll assay and fluorescein diacetate staining. To monitor the genome-wide transcriptome changes induced by heat acclimation at 37 degrees C, we constructed an A. thaliana cDNA microarray containing 7,989 unique genes, and applied it to A. thaliana suspension-culture cells harvested at various times (0.5, 1, 2.5, 6, and 16 h) during heat acclimation. Data analysis revealed 165 differentially expressed genes that were grouped into ten clusters. We compared these genes with published and publicly available microarray heat-stress-related data sets in AtGenExpress. Heat-shock proteins were strongly expressed, as previously reported, and we found several of the up-regulated genes encoded detoxification and regulatory proteins. Moreover, the transcriptional induction of DREB2 (dehydration responsive element-binding factor 2) subfamily genes and COR47/rd17 under heat stress suggested cross-talk between the signaling pathways for heat and dehydration responses.

The DNA-binding activity of an AP2 transcriptional activator HvCBF2 involved in regulation of low-temperature responsive genes in barley is modulated by temperature.

An HvCBF2 cDNA was isolated from barley leaves. It encoded a protein containing an AP2 DNA-binding domain homologous to C-repeat (CRT)/dehydration-responsive element (DRE) binding factors (CBF/DREB1). In contrast to the previously reported cold-inducible CBF/DREB1 genes, HvCBF2 was expressed in barley leaves under non-stress conditions. Only a transient increase in the HvCBF2 transcript level was observed during cold treatment. Transactivation analysis showed that HvCBF2 was a transcriptional activator, capable of activating expression of a reporter gene driven by a low-temperature and drought-responsive HVA1s promoter in barley leaves. The activity of HvCBF2 as a transcriptional activator was upregulated by low temperature. DNA-binding analysis revealed that HvCBF2 did not bind to the CRT/DRE motif at 30 degrees C. A low, but detectable, binding activity was observed at 25 degrees C and the binding activity gradually increased as the temperature decreased. The binding activity at 0 degrees C was the highest and more than 10 times higher than that at 25 degrees C. The activation and inactivation of HvCBF2 activity were reversible and were achieved in a cell-free system simply by temperature change. Analysis of the binding sequence showed that HvCBF2 bound to a (G/a)(T/c)CGAC core motif, where the lower-case letters are less efficient bases. These data suggest that HvCBF2 is a transcription factor interacting with the core CRT/DRE motif containing a preferred sequence of GTCGAC and its DNA-binding activity is regulated by temperature. This represents a new type of activation mechanism for transcriptional activators.