Drought Response Element Binding Research Articles

Exploration of Oryza sativa drought-responsive element binding protein 2A (OsDREB2A) gene in several local Indonesian rice varieties

Abstract. Yuliza, Salamah A, Puspitaningrum H. 2024. Exploration of Oryza sativa drought-responsive element binding protein 2A (OsDREB2A) gene in several local Indonesian rice varieties. Biodiversitas 25: 574-582. Drought is one of the abiotic stress factors that reduces rice productivity in Indonesia. OsDREB2A is a member of the DREBP subfamily of AP2/ERF transcription factors and participates in drought stress by directly binding to DRE elements to regulate downstream gene expression. However, further research is still needed to observe each OsDREB2A gene in local Indonesian rice varieties. This research aims to explore the OsDREB2A gene in several local Indonesian rice varieties, namely from Java (Ciherang, Situbagendid, Way Apo, Inpara 32 and Inpari 42), Kalimantan (Black Rice), Aceh (Sigupai) and West Sumba (Pare Lambem and Pare Bakato Kaka). DNA was isolated from the leaves of each variety, amplified using PCR, and then electrophoresed and sequenced. Sequencing data were analyzed using DNA Baser, BioEdit and then visualized using the SWISS-MODEL server, Rice Genome Annotation Project Database, and chromosome map tools. The results showed that nine samples had 100% query cover with the Pokkali OsDREB2A (KU159743.1) cultivar sequence, 99.86% similar percent identity compared to cultivar R180 and WAB540-1-B-P-6-1-1, 99.75% similar percent identity compared to cultivar FL-478 and 99.62% similar percent identity compared to cultivar Nona Bokra. The amino acid structure of each cultivar refers to chromosome 1 at the LOC_Os01g07120 locus.

Introducing sorghum DREB2 gene in maize (Zea mays L.) to improve drought and salinity tolerance.

Salinity and drought are significant abiotic stresses causing a considerable loss of seed and biomass yield in most commercial crops. Some of the most critical players in the abscisic acid pathway are drought responsive element binding (DREB) proteins that are a part of AP2/ethylene response factor transcription factors that bind to promoters of some family genes needed to be expressed under abiotic stresses. In this study, salt- and drought-tolerant maize plants were produced from immature maize embryos bombarded by the sorghum (Sorghum bicolor L.) DREB2 gene that is linked to hygromycin resistance (hpt) genes. The putative transgenic calli were transferred to an N6 medium containing 1mg/L benzylaminopurine and 50mg/L hygromycin. Regeneration was completed after 4 weeks on selective media under a 16/8h light/dark condition at 25°C. Polymerase chain reaction (PCR) and reverse transcription-PCR approved the existence of upstream promoter (rd29a), hpt gene, and the expression of the DREB2 in transgenes up to the third generation (T2). It was found that the K+/Na+ ratio and the amount of proline as a screening indicator were higher in transgenic plants compared to their wild types. This result is a promising model to enhance maize tolerance to abiotic stressors.

Over-expression of DREB46 enhances drought tolerance in Populus trichocarpa

The drought responsive element binding (DREB) gene family has a significant role in plant abiotic stress responses. Here, we cloned a drought-inducible DREB gene, DREB46 (Potri.019G075500), and investigated its function in drought tolerance in Populus trichocarpa. Under treatment with exogenous abscisic acid and 6% PEG6000, DREB46 was rapidly and abundantly expressed. We successfully inserted P. trichocarpa DREB46 constructs into P. trichocarpa. After 11 d of drought stress and 3 d of rehydration treatment, the DREB46 over-expression (OE) lines exhibited significantly increased survival rates relative to the wild type (WT). Histochemical staining showed that the accumulation of reactive oxygen species (ROS) in transgenic plants under drought stress was lower than that in WT plants. Furthermore, OE plants displayed higher superoxide dismutase, peroxidase, and catalase activities and proline content, but lower malondialdehyde content than the WT plants under drought stress. In contrast, DREB46-RNA interference (RNAi) lines exhibited the opposite phenotype. Under PEG-6000 stress, OE plants produced significantly more adventitious roots (ARs) than WT plants. In contrast, RNAi-mediated DREB46-inhibited poplar exhibited fewer ARs. Quantitative real-time PCR indicated that WOX11/12a (Potri.013G066900), a gene related to root growth and development regulation, was significantly increased in OE plants. Additionally, yeast two-hybrid (Y2H) assays showed that DREB46 could interact with protein kinase MPK1 (Potri.002G032100) and protein phosphatase PP2C47 (Potri.007G058700), respectively, and this result was also verified by luciferase complementation assay. Transient co-expression results of leaves showed that PP2C47 and DREB46 Agrobacterium-transformed leaves had strong drought tolerance. These results show that DREB46 plays a key role in drought tolerance by inducing the ROS scavenging system and increasing the number of ARs.

Influence of Light Intensity on Tobacco Responses to Drought Stress

The influence of high irradiance, drought stress and their cross-talk were explored in tobacco plants (Nicotiana tobaccum) grown under low light (250 μmol m-2 s-1) irradiance (LL) and high (1600 μmol m-2 s-1) irradiance (HL) then exposed to water deficient condition for 7 or 14 days. The detached leaves of HL-treated plants showed less water loss compared to LL plants. The HL-treated and 7 days drought-stressed plants had higher fresh and dry weights, as well as water content than the LL and drought-stressed leaves. The survival rate in 21 days drought-stressed plants after 3 days of re-watering was 50% in HL-grown and 0% in LL-grown plants. A transcriptome profiling analysis of the tobacco responses to light intensity highlights the increased abundance of a large group of drought-related transcripts including DROUGHT-RESPONSIVE ELEMENT BINDING FACTORS (DREBs), C-REPEAT/DROUGHT-RESPONSIVE BINDING FACTOR 1 (CBF1), GLYCINE-RICH RNA BINDING PROTEINS (GRPs), WRKY33 and MYCs transcription factors, as well as zeaxanthin epoxidase, which play as a regulator of plant responses to water deficient condition. These findings identify light-dependent changes in the cell redox state that limit water loss and enhance plant responses to drought stress.

Influence of Light Intensity on Tobacco Responses to Drought Stress

The influence of high irradiance, drought stress and their cross-talk were explored in tobacco plants (Nicotiana tobaccum) grown under low light (250 ?mol m-2 s-1) irradiance (LL) and high (1600 ?mol m-2 s-1) irradiance (HL) then exposed to water deficient condition for 7 or 14 days. The detached leaves of HL-treated plants showed less water loss compared to LL plants. The HL-treated and 7 days drought-stressed plants had higher fresh and dry weights, as well as water content than the LL and drought-stressed leaves. The survival rate in 21 days drought-stressed plants after 3 days of re-watering was 50% in HL-grown and 0% in LL-grown plants. 
 A transcriptome profiling analysis of the tobacco responses to light intensity highlights the increased abundance of a large group of drought-related transcripts including DROUGHT-RESPONSIVE ELEMENT BINDING FACTORS (DREBs), C-REPEAT/DROUGHT-RESPONSIVE BINDING FACTOR 1 (CBF1), GLYCINE-RICH RNA BINDING PROTEINS (GRPs), WRKY33 and MYCs transcription factors, as well as zeaxanthin epoxidase, which play as a regulator of plant responses to water deficient condition.
 These findings identify light-dependent changes in the cell redox state that limit water loss and enhance plant responses to drought stress.

Two generation reproduction toxicity study of GmDREB3 gene modified wheat in Wistar rats

To study reproductive toxicity of gene modified wheat generated by introducing DREB3 (drought response element binding protein 3) gene, Wistar rats of were allocated into 3 groups and fed with DREB3 gene modified wheat mixture diet (GM group), non-gene modified wheat mixture diet (Non-GM group) and AIN-93 diet (Control group) from parental generation (F0) to the second offspring (F2). GM wheat and Non-GM wheat, Jimai22, were both formulated into diets at a ratio of 69.55% according to AIN93 diet for rodent animals. Compared with non-GM group, no biologically related differences were observed in GM group rats with respect to reproductive performance such as fertility rate, gestation rate, mean duration, hormone level, reproductive organ pathology and developmental parameters such as body weight, body length, food consumption, neuropathy, behavior, immunotoxicity, hematology and serum chemistry. In conclusion, no adverse effect were found relevant to GM wheat in the two generation reproduction toxicity study, indicating the GM wheat is a safe alternative for its counterpart wheat regarding to reproduction toxicity.

Overexpression of oil palm EgDREB1 in tomato decreased fruit sizeand produced parthenocarpic fruits

Drought-responsive element binding (DREB) is involved in the regulation of stress-responsive gene expressions in plants through abscisic acid (ABA)-independent pathway. In this study, constitutive expression of oil palm (Elaeis guineensis) EgDREB1 driven by double strength cauliflower mosaic virus 35S promoter in tomato (Solanum lycopersicum) reduced seed number, produced parthenocarpic fruits, changed morphology of leaves, and increased root biomass of transgenic plants. Early flowering and fruiting of the transgenic lines were observed in the culture vessels. EgDREB1 was specifically expressed in the fruits and its expression was not detected in vegetative tissues (leaves and roots). Altered expression of several endogenous tomato genes involved in the biosynthesis of phytohormones including jasmonic acid, ethylene, auxin, cytokinin, gibberellin (GA) and ABA were observed compared to wild type plants. The expression of AP2-like-ethylene transcription factor (LeAP2), allene oxide synthase (LeAOS), allene oxide cyclase (LeAOC), aminocyclopropane-1-carboxylic acid synthase (LeACS), 1-aminocyclopropane-1-carboxylate oxidase 1 (LeACO), auxin responsive factor 8 (LeARF8), auxin/indole-3-acetic acid (LeAux/IAA), cytokinin oxidase/dehydrogenase-like (LeSlCKX1), adenylate isopentenyltransferase (LeSlIPT1), gibberellin 2-oxidase 2 (LeGa2ox2), gibberellin 20-oxidase 4 (LeGa20ox4) and ABA-aldehyde oxidase (LeAAO) were different in fruits with reduced seed number compared to parthenocarphic fruits. These results suggest that their expression has significant effects on fruit development in transgenic tomato. EgDREB1 may mediate the expression of some of these genes as dehydration-responsive element binding (DRE) motif were found in their promoter sequences. These data indicate that the EgDREB1 controls fruit development in trsngenic plants by regulating the expression of hormone-associated genes.

The effect of cadmium on transgenic potato (Solanum tuberosum) plants overexpressing the StDREB transcription factors

Cadmium (Cd) is a toxic heavy metal naturally present in soils. It causes environmental pollution because of the increase of mining and industrial activities in the twentieth century. Although plants do not require Cd, they can accumulate high levels of this metal. The response of plants to Cd pollution involves several signaling pathways involving stress responsive transcription factors. Among these transcription factors, it has been reported that DREB (drought-responsive element binding) factors are involved in plant response to abiotic constraints. In this context, in previous studies, we isolated and characterized two DREB genes from potato that were designated StDREB1 and StDREB2. Their overexpression in transgenic potato lines led to enhanced drought and salt stress tolerance in comparison to untransformed plants. These DREB transcription factors play crucial regulatory roles in different stress responsive pathways in potato. The goal of this report is to study the response of these transgenic potato plants overexpressing StDREB1 and StDREB2 to Cd stress. Therefore, transgenic plants and control plants were cultivated in vitro in MS (Murashige and Skoog) medium supplemented with 50–150 µM CdCl2. After 7 and 14 days of treatment, plant tissues were used to evaluate a number of physiological parameters. Transgenic plants submitted to Cd stress, showed lower decrease in growth, chlorpophyll and carotenoid content than wild-type (WT) plants. Similarly, almost no loss of water content was measured in BF3, SP1 and SPM lines. All transgenic plants exhibited lower H2O2 content and higher antioxidant enzyme expression than WT. A higher proline content was also measured in these plants after Cd treatment in comparison to WT. All these data suggest that overexpression of the StDREB genes improves tolerance of potato plants to Cd by improving plant growth, proline and antioxidant production leading to low oxidative stress damage.

Melatonin-induced CBF/DREB1s are essential for diurnal change of disease resistance and CCA1 expression in Arabidopsis

Melatonin (N-acetyl-5-methoxytryptamine) is an important regulator of circadian rhythms and immunity in animals. However, the diurnal changes of endogenous melatonin and melatonin-mediated diurnal change of downstream responses remain unclear in Arabidopsis. Using the publicly available microarray data, we found that the transcript levels of two melatonin synthesis genes (serotonin N-acetyltransferase (SNAT) and caffeate O-methyltransferase (COMT)) and endogenous melatonin level were regulated by diurnal cycles, with different magnitudes of change. Moreover, the transcripts of C-repeat-binding factors (CBFs)/Drought response element Binding 1 factors (DREB1s) were co-regulated by exogenous melatonin and diurnal changes, indicating the possible correlation among clock, endogenous melatonin level and AtCBFs expressions. Interestingly, diurnal change of plant immunity against Pst DC3000 and CIRCADIANCLOCK ASSOCIATED 1 (CCA1) expression were largely lost in AtCBFs knockdown line-amiR-1. Taken together, this study identifies the molecular pathway underlying the diurnal changes of immunity in Arabidopsis. Notably, the diurnal changes of endogenous melatonin may regulate corresponding changes of AtCBF/DREB1s expression and their underlying diurnal cycle of plant immunity and AtCCA1.

Melatonin induces the transcripts of CBF/DREB1s and their involvement in both abiotic and biotic stresses in Arabidopsis.

Melatonin (N-acetyl-5-methoxytryptamine) is a naturally occurring small molecule that acts as an important secondary messenger in plant stress responses. However, the mechanism underlying the melatonin-mediated signaling pathway in plant stress responses has not been established. C-repeat-binding factors (CBFs)/Drought response element Binding 1 factors (DREB1s) encode transcription factors that play important roles in plant stress responses. This study has determined that endogenous melatonin and transcripts level of CBFs (AtCBF1, AtCBF2, and AtCBF3) in Arabidopsis leaves were significantly induced by salt, drought, and cold stresses and by pathogen Pseudomonas syringe pv. tomato (Pst) DC3000 infection. Moreover, both exogenous melatonin treatment and overexpression of CBFs conferred enhanced resistance to both abiotic and biotic stresses in Arabidopsis. Notably, AtCBFs and exogenous melatonin treatment positively regulated the mRNA expression of several stress-responsive genes (COR15A, RD22, and KIN1) and accumulation of soluble sugars content such as sucrose in Arabidopsis under control and stress conditions. Additionally, exogenous sucrose also conferred improved resistance to both abiotic and biotic stresses in Arabidopsis. Taken together, this study indicates that AtCBFs confer enhanced resistance to both abiotic and biotic stresses, and AtCBF-mediated signaling pathway and sugar accumulation may be involved in melatonin-mediated stress response in Arabidopsis, at least partially.

The cysteine2/histidine2-type transcription factor ZINC FINGER OF ARABIDOPSIS THALIANA 6-activated C-REPEAT-BINDING FACTOR pathway is essential for melatonin-mediated freezing stress resistance in Arabidopsis.

Melatonin (N-acetyl-5-methoxytryptamine) is not only a widely known animal hormone, but also an important regulator in plant development and multiple abiotic stress responses. Recently, it has been revealed that melatonin alleviated cold stress through mediating several cold-related genes, including C-REPEAT-BINDING FACTORs (CBFs)/Drought Response Element Binding factors (DREBs), COR15a, and three transcription factors (CAMTA1, ZINC FINGER OF ARABIDOPSIS THALIANA 10 (ZAT10), and ZAT12). In this study, we quantified the endogenous melatonin level in Arabidopsis plant leaves and found the endogenous melatonin levels were significantly induced by cold stress (4 °C) treatment. In addition, we found one cysteine2/histidine2-type zinc finger transcription factor, ZAT6, was involved in melatonin-mediated freezing stress response in Arabidopsis. Interestingly, exogenous melatonin enhanced freezing stress resistance was largely alleviated in AtZAT6 knockdown plants, but was enhanced in AtZAT6 overexpressing plants. Moreover, the expression levels of AtZAT6 and AtCBFs were commonly upregulated by cold stress (4 °C) and exogenous melatonin treatments, and modulation of AtZAT6 expression significantly affected the induction AtCBFs transcripts by cold stress (4 °C) and exogenous melatonin treatments. Taken together, AtZAT6-activated CBF pathway might be essential for melatonin-mediated freezing stress response in Arabidopsis.

Role of melatonin in alleviating cold stress in Arabidopsis thaliana

Melatonin (N-acetyl-5-methoxytryptamine) has been implicated in abiotic and biotic stress tolerance in plants. However, information on the effects of melatonin in cold-stress tolerance in vivo is limited. In this study, the effect of melatonin was investigated in the model plant Arabidopsis thaliana challenged with a cold stress at 4⁰C for 72 and 120 hr. Melatonin-treated plants (10 and 30 μm) had significantly higher fresh weight, primary root length, and shoot height compared with the nontreated plants. To aid in the understanding of the role of melatonin in alleviating cold stress, we investigated the effects of melatonin treatment on the expression of cold-related genes. Melatonin up-regulated the expression of C-repeat-binding factors (CBFs)/Drought Response Element Binding factors (DREBs), a cold-responsive gene, COR15a, a transcription factor involved in freezing and drought-stress tolerance CAMTA1 and transcription activators of reactive oxygen species (ROS)-related antioxidant genes, ZAT10 and ZAT12, following cold stress. The up-regulation of cold signaling genes by melatonin may stimulate the biosynthesis of cold-protecting compounds and contribute to the increased growth of plants treated with exogenous melatonin under cold stress.

Overexpression of StDREB1 Transcription Factor Increases Tolerance to Salt in Transgenic Potato Plants

It has been established that drought-responsive element binding (DREB) proteins correspond to transcription factors which play important regulatory roles in plant response to abiotic and biotic stresses. In this study, a novel cDNA encoding DREB transcription factor, designated StDREB1, was isolated from potato (Solanum tuberosum L.). This protein was classified in the A-4 group of DREB subfamily based on multiple sequence alignments and phylogenetic characterization. Semi-quantitative RT-PCR showed that StDREB1 is expressed in leaves, stems, and roots under stress conditions and it is greatly induced by NaCl, drought, low temperature, and abscisic acid (ABA) treatments. Overexpression of StDREB1 cDNA in transgenic potato plants exhibited an improved salt and drought stress tolerance in comparison to the non-transformed controls. The enhanced stress tolerance may be associated with the increase in P5CS-RNA expression (δ (1)-pyrroline-5-carboxylate synthetase) and the subsequent accumulation of proline osmoprotectant in addition to a better control of water loss. Overexpression of StDREB1 also activated stress-responsive genes, such as those encoding calcium-dependent protein kinases (CDPKs), in transgenic potatoes under standard and high salt conditions. These data suggest that the StDREB1 transcription factor is involved in the regulation of salt stress tolerance in potato by the activation of different downstream gene expression.

CgDREBa transgenic chrysanthemum confers drought and salinity tolerance

The drought and salinity tolerances of a creeping ground-cover chrysanthemum variety ‘Yuhuaxunzhang’ were compared to the performance of its two derived transgenic lines carrying a drought-responsive element binding (DREB) factor from chrysanthemum designated as CgDREBa. The over-expression of CgDREBa conferred a measure of tolerance to both stresses. The transgenic lines showed a higher survival rate and were better able to retain fresh weight in the presence of stress. Activities of superoxide dismutase and peroxidase and the proline content were all higher in the leaves of the transgenic plants after a prolonged period of stress, but they accumulated less malondialdehyde. CgDREBa appears to function as a transcription activator of genes within the oxidative and osmotic homeostasis transduction pathways, and represents a promising candidate for a biotechnological approach to improve the level of abiotic stress tolerance in plants.

The Arabidopsis thaliana Med25 mediator subunit integrates environmental cues to control plant development

Development in plants is controlled by abiotic environmental cues such as day length, light quality, temperature, drought, and salinity. These signals are sensed by a variety of systems and transmitted by different signal transduction pathways. Ultimately, these pathways are integrated to control expression of specific target genes, which encode proteins that regulate development and differentiation. The molecular mechanisms for such integration have remained elusive. We here show that a linear 130-amino-acids-long sequence in the Med25 subunit of the Arabidopsis thaliana Mediator is a common target for the drought response element binding protein 2A, zinc finger homeodomain 1, and Myb-like transcription factors which are involved in different stress response pathways. In addition, our results show that Med25 together with drought response element binding protein 2A also function in repression of PhyB-mediated light signaling and thus integrate signals from different regulatory pathways.

Supra‐optimal expression of the cold‐regulated OsMyb4 transcription factor in transgenic rice changes the complexity of transcriptional network with major effects on stress tolerance and panicle development

The R2R3-type OsMyb4 transcription factor of rice has been shown to play a role in the regulation of osmotic adjustment in heterologous overexpression studies. However, the exact composition and organization of its underlying transcriptional network has not been established to be a robust tool for stress tolerance enhancement by regulon engineering. OsMyb4 network was dissected based on commonalities between the global chilling stress transcriptome and the transcriptome configured by OsMyb4 overexpression. OsMyb4 controls a hierarchical network comprised of several regulatory sub-clusters associated with cellular defense and rescue, metabolism and development. It regulates target genes either directly or indirectly through intermediary MYB, ERF, bZIP, NAC, ARF and CCAAT-HAP transcription factors. Regulatory sub-clusters have different combinations of MYB-like, GCC-box-like, ERD1-box-like, ABRE-like, G-box-like, as1/ocs/TGA-like, AuxRE-like, gibberellic acid response element (GARE)-like and JAre-like cis-elements. Cold-dependent network activity enhanced cellular antioxidant capacity through radical scavenging mechanisms and increased activities of phenylpropanoid and isoprenoid metabolic processes involving various abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), ethylene and reactive oxygen species (ROS) responsive genes. OsMyb4 network is independent of drought response element binding protein/C-repeat binding factor (DREB/CBF) and its sub-regulons operate with possible co-regulators including nuclear factor-Y. Because of its upstream position in the network hierarchy, OsMyb4 functions quantitatively and pleiotrophically. Supra-optimal expression causes misexpression of alternative targets with costly trade-offs to panicle development.

Identification of the amino acids crucial for the activities of drought responsive element binding factors (DREBs) of Brassica napus

We have previously identified two homologous groups of BnDREBs in Brassica napus, the trans-active BnDREBI and the trans-inactive BnDREBII, which provided an ideal system to study the trans-activation of DREB1/CBF. Deletion analysis indicated that the two additional regions in BnDREBI contributed little to the transcriptional activity. Domain swapping analysis indicated that all the domains contributed to the activity of BnDREBI, including the ERF/AP2 DNA binding domain. Through site-directed mutagenesis, we identified nine residues that were involved in the activity of BnDREBI, among which six residues are specific to BnDREBI, and three are common to DREB1A.

Stress-inducible DREB2A transcription factor from Pennisetum glaucum is a phosphoprotein and its phosphorylation negatively regulates its DNA-binding activity

Abiotic stress-mediated gene expression is regulated via different transcription factors of which drought-responsive element-binding (DREB) proteins play an important role. There are two types of DREBs. Presently, the function of DREB1 type protein is well studied; however, much less information is available for DREB2. In this study, a cDNA with an open reading frame of 332 amino acids, encoding the transcription activation factor DREB2A, was cloned from Pennisetum glaucum, a stress tolerant food grain crop. Phylogenetic tree revealed that PgDREB2A is more close to DREBs isolated from monocots, though it forms an independent branch. The PgDREB2A transcript was up-regulated in response to drought within 1 h of the treatment, whereas the induction was delayed in response to cold and salinity stress. However, during cold stress, the transcript was induced more as compared to drought and salinity. The recombinant PgDREB2A protein having a molecular mass of 36.6 kDa was purified using Ni-NTA affinity chromatography. Gel mobility shift assays using the purified protein and two cis elements of rd29A (responsive to dehydration 29A) gene promoter of Arabidopsis revealed that PgDREB2A binds to drought-responsive element (DRE) ACCGAC and not to GCCGAC. PgDREB2A is a phosphoprotein, which has not been reported earlier. The phosphorylation of PgDREB2A in vitro by P. glaucum total cell extract occurred at threonine residue(s). The phosphorylated PgDREB2A did not bind to the DREs. The present data indicate that stress induction of genes could occur via post-translational modification by phosphorylation of DREB2A.

SSCP-SNP-based conserved ortholog set (COS) markers for comparative genomics in cassava (Manihot esculenta crantz)

The ever increasing body of information on genomics and functional genomics from model plants, and new tools of comparative genomics, provide an opportunity to accelerate the development of molecular markers for increasing the efficiency of breeding of lesser studied crops, so-called “orphan crops.” Conserved ortholog set (COS) markers represent orthologous genes in widely divergent plant species, and are currently the principal tool of choice for comparative genomics. EST sequences of 3 drought tolerance related genes—chalcone synthase (CHS), dihydroflavonol-4-reductase (DHRF) and drought responsive element binding factor 1 (DREB-1) fromMusa sp—were used to identify cassava EST homologs that were then scanned against the Arabidopsis genome database to identify them as COS markers. The CHS and DHRF ESTs were demonstrated to be COS markers, while the DREB EST was shown to belong to a gene family. The three genes were evaluated as single strand conformation polymorphism—single nucleotide polymorphism (SSCP-SNP) markers in the parents of an F1 mapping population and subsequently in the progenies. The DHRF COS marker mapped to linkage group R of the female-derived map while the DREB-1 EST mapped at an end of the male-derived linkage group K. The CHS COS marker could not be mapped because it was not polymorphic in the parents of the mapping population. These new marker tools should accelerate the development of markers associated with genes controlling traits of agronomic interest via the candidate gene loci (CGL) QTL-mapping approach.

Regulating the Drought-responsive Element (DRE)-mediated Signaling Pathway by Synergic Functions of Trans-active and Trans-inactive DRE Binding Factors in Brassica napus

DREB1/C-repeat binding factor (CBF) is a plant-specific family of transcription factors and plays a crucial role in freeze tolerance. In the present work, two groups of drought-responsive element binding factor (DREB)-like genes were isolated from Brassica napus, named Group I and Group II. The two groups of genes were both induced by low temperature, but the expression of Group I preceded that of Group II. The Group I DREBs could specifically bind with the DRE cis-acting element and activate the expression of downstream genes, but Group II factors were trans-inactive although they still had the ability to bind with DRE, which was confirmed by electrophoretic mobility shift assay. Fluorescence quenching assays indicated that the DRE binding ability of the two groups was similar. Co-expression of Group II could depress the trans-activation activity of Group I DREB in a concentration-dependent manner. These results strongly suggested that the trans-active Group I DREBs were expressed at the early stage of cold stress to open the DRE-mediated signaling pathway in cold stress, whereas the trans-inactive Group II DREBs were expressed at the later stage to close the signal pathway in a competitive manner. The results herein provide a new insight into the regulation mechanisms of the DRE-mediated signaling pathway in response to cold stress.