Dehydration Responsive Element Binding Genes Research Articles

Characteristics of Rapeseed (Brassica rapa L.) genome DREB family demonstrate their roles in stress

Dehydration-responsive element-binding (DREB) proteins are crucial transcription factors in plant regulatory mechanisms, playing a key role in responding to environmental stresses. Dehydration-responsive element-binding (DREB) proteins are crucial transcription factors in plant regulatory mechanisms, playing a key role in responding to environmental stresses. This study employs bioinformatics research methods to identify and characterize the DREB gene family in rapeseed (Brassica rapa L.) under abiotic stress conditions. The approach facilitates a detailed exploration of the genomic architecture and potential regulatory mechanisms that these genes may exhibit in response to environmental stresses. A whole-genome survey identified 75 DREB genes in rapeseed, which were classified into six subgroups (Ⅰ-Ⅵ) based on phylogenetic comparison with Arabidopsis homologs. The amino acid lengths of these genes ranged from 144 to 530, with predicted molecular weights between 15.63kDa and 60.36kDa, and isoelectric points from 4.48 to 9.32. Chromosomal localization revealed that these genes are distributed across 10 chromosomes, predominantly near telomeric regions, with more than 10 DREB genes located on Chr3 and Chr9 respectively. Structural domain analysis indicated that 57 DREB genes possess a single exon. Promoter cis-element analyses suggested that BrDREB genes could respond to diverse stressors, including drought, heat, hormones, and light. Transcriptomic analysis under cold stress revealed significant upregulation of BrDREB17, 24, 49, 50, and 51 in response to low temperature, while five BrDREB genes were not expressed. Specifically, BrDREB17, 24, 49, 50, and 51 showed high expression levels at 3 hours of cold stress, whereas BrDREB30, 42, and 67 were highly expressed at 24 hours of cold stress. Gene Ontology (GO) analysis demonstrated that the majority of biological processes mediated by BrDREB are associated with plant stress resistance. Protein interaction network analysis revealed that BrDREB9 and BrDREB26 interact with 15 proteins each, underscoring their significant biological roles within the DREB family in rapeseed. These findings provide a theoretical framework for further examination of the physiological and biochemical properties of the DREB gene family components in rapeseed. Our results may help to screen candidate genes for cold resistance in rapeseed and select rapeseed varieties better suited to the Tibetan locality to improve quality and yield in agricultural production.

The positive impact of the NtTAS14-like1 gene on osmotic stress response in Nicotiana tabacum.

NtTAS14-like1 enhances osmotic tolerance through coordinately activating the expression of osmotic- and ABA-related genes. Osmotic stress is one of the most important limiting factors for tobacco (Nicotiana tabacum) growth and development. Dehydrin proteins are widely involved in plant adaptation to osmotic stress, but few of these proteins have been functionally characterized in tobacco. Here, to identify genes required for osmotic stress response in tobacco, an encoding dehydrin protein gene NtTAS14-like1 was isolated based on RNA sequence data. The expression of NtTAS14-like1 was obviously induced by mannitol and abscisic acid (ABA) treatments. Knock down of NtTAS14-like1 expression reduced osmotic tolerance, while overexpression of NtTAS14-like1 conferred tolerance to osmotic stress in transgenic tobacco plants, as determined by physiological analysis of the relative electrolyte leakage and malonaldehyde accumulation. Further expression analysis by quantitative real-time PCR indicated that NtTAS14-like1 participates in osmotic stress response possibly through coordinately activating osmotic- and ABA-related genes expression, such as late embryogenesis abundant (NtLEA5), early responsive to dehydration 10C (NtERD10C), calcium-dependent protein kinase 2 (NtCDPK2), ABA-responsive element-binding protein (NtAREB), ABA-responsive element-binding factor 1 (NtABF1), dehydration-responsive element-binding genes (NtDREB2A), xanthoxin dehydrogenase/reductase (NtABA2), ABA-aldehyde oxidase 3 (NtAAO3), 9-cis-epoxycarotenoid dioxygenase (NtNCED3). Together, this study will facilitate to improve our understandings of molecular and functional properties of plant TAS14 proteins and to improve genetic evidence on the involvement of the NtTAS14-like1 in osmotic stress response of tobacco.

Genome-wide identification, comprehensive characterization of transcription factors, cis-regulatory elements, protein homology, and protein interaction network of DREB gene family in Solanum lycopersicum.

Tomato is a drought-sensitive crop which has high susceptibility to adverse climatic changes. Dehydration-responsive element-binding (DREB) are significant plant transcription factors that have a vital role in regulating plant abiotic stress tolerance by networking with DRE/CRT cis-regulatory elements in response to stresses. In this study, bioinformatics analysis was performed to conduct the genome-wide identification and characterization of DREB genes and promoter elements in Solanum lycopersicum. In genome-wide coverage, 58 SlDREB genes were discovered on 12 chromosomes that justified the criteria of the presence of AP2 domain as conserved motifs. Intron-exon organization and motif analysis showed consistency with phylogenetic analysis and confirmed the absence of the A3 class, thus dividing the SlDREB genes into five categories. Gene expansion was observed through tandem duplication and segmental duplication gene events in SlDREB genes. Ka/Ks values were calculated in ortholog pairs that indicated divergence time and occurrence of purification selection during the evolutionary period. Synteny analysis demonstrated that 32 out of 58 and 47 out of 58 SlDREB genes were orthologs to Arabidopsis and Solanum tuberosum, respectively. Subcellular localization predicted that SlDREB genes were present in the nucleus and performed primary functions in DNA binding to regulate the transcriptional processes according to gene ontology. Cis-acting regulatory element analysis revealed the presence of 103 motifs in 2.5-kbp upstream promoter sequences of 58 SlDREB genes. Five representative SlDREB proteins were selected from the resultant DREB subgroups for 3D protein modeling through the Phyre2 server. All models confirmed about 90% residues in the favorable region through Ramachandran plot analysis. Moreover, active catalytic sites and occurrence in disorder regions indicated the structural and functional flexibility of SlDREB proteins. Protein association networks through STRING software suggested the potential interactors that belong to different gene families and are involved in regulating similar functional and biological processes. Transcriptome data analysis has revealed that the SlDREB gene family is engaged in defense response against drought and heat stress conditions in tomato. Overall, this comprehensive research reveals the identification and characterization of SlDREB genes that provide potential knowledge for improving abiotic stress tolerance in tomato.

Pan-Genome-Wide Identification and Transcriptome-Wide Analysis of DREB Genes That Respond to Biotic and Abiotic Stresses in Cucumber

The production of cucumber (Cucumis sativus L.) is often harmed by biotic and abiotic stresses. Although the dehydration-responsive element-binding (DREB) transcription factors, playing vital roles in stress responses, have been characterized in several plant species, little is known about the pan-genome characteristics of DREB genes and their expression patterns under different stresses in cucumber. In this study, we identified 55 CsDREBs from the cucumber pan-genomes of 13 accessions, but only four accessions had all the genes. Most of the CsDREB proteins had sequence length and/or amino acid variations, and only four of them had no variation among different accessions. Using the 55 CsDREBs from ‘9930’, we analyzed their gene structures, conserved domains, phylogenetic relationships, gene promoter’s cis-elements and syntenic relationships, and classified them into six groups. Expression pattern analysis revealed that eight CsDREBs showed constitutive expression (FPKM > 1 in all samples), and different CsDREBs showed specifically high expression in root, stem, leaf, tendril, male-flower, female flower, and ovary, respectively, suggesting that these genes might be important for morphogenesis and development in cucumber. Additionally, a total of 31, 22, 30 and nine CsDREBs were differentially expressed in responding to the treatments of heat, NaCl and/or silicon, power mildew and downy mildew, respectively. Interestingly, CsDREB33 could respond to all the tested stresses. Our results provide a reference and basis for further investigation of the function and mechanism of the DREB genes for resistance breeding in cucumber.

Osa-miR1320 targets the ERF transcription factor OsERF096 to regulate cold tolerance via JA-mediated signaling.

MicroRNAs play key roles in abiotic stress response. Rice (Oryza sativa L.) miR1320 is a species-specific miRNA that contributes to miR168-regulated immunity. However, it is still unknown whether miR1320 is involved in rice response to abiotic stress. In this study, we illustrated that the miR1320 precursor generated two mature miR1320s, miR1320-3p, and miR1320-5p, and they both displayed decreased expression under cold stress. Genetic evidence showed that miR1320 overexpression resulted in increased cold tolerance, while miR1320 knock down (KD) reduced cold tolerance. Furthermore, an APETALA2/ethylene-responsive factor (ERF) transcription factor OsERF096 was identified as a target of miR1320 via 5'-RACE and dual luciferase assays. OsERF096 expression was altered by miR1320 overexpression and KD and exhibited an opposite pattern to that of miR1320 in different tissues and under cold stress. Consistently, OsERF096 negatively regulated cold stress tolerance. Furthermore, we suggested that OsERF096 could bind to the GCC and DRE cis-elements and act as a transcriptional activator in the nucleus. Based on RNA-sequencing and targeted metabolomics assays, we found that OsERF096 modified hormone content and signaling pathways. Finally, phenotypic and reverse transcription-quantitative PCR assays showed that jasmonic acid (JA) methyl ester application recovered the cold-sensitive phenotype and JA-activated expression of three Dehydration Responsive Element Binding genes in the OsERF096-OE line. Taken together, our results strongly suggest that the miR1320-OsERF096 module regulates cold tolerance by repressing the JA-mediated cold signaling pathway.

Genome-Wide Analysis of DREB Genes Identifies a Novel Salt Tolerance Gene in Wild Soybean (Glycine soja).

Salt stress is a major factor limiting the growth and yield of soybean (Glycine max). Wild soybeans (Glycine soja) contain high allelic diversity and beneficial alleles that can be re-introduced into domesticated soybeans to improve adaption to the environment. However, very few beneficial alleles have been identified from wild soybean. Here, we demonstrate that wild soybean is more salt tolerant than cultivated soybean and examine dehydration responsive element-binding (DREB) family transcription factor genes to look for advantageous alleles that might improve drought tolerance in cultivated soybean. Our genome-wide analysis identified 103 DREB genes from the Glycine max genome. By combined RNA-sequencing and population genetics of wild, landrace, and cultivated soybean accessions, we show that the natural variation in DREB3a and DREB3b is related to differences in salt tolerance in soybean accessions. Interestingly, DREB3b, but not DREB3a, appears to have undergone artificial selection. Soybean plants carrying the wild soybean DREB3b allele (DREB3b39Del) are more salt tolerant than those containing the reference genome allele (DREB3bRef). Together, our results suggest that the loss of the DREB3b39Del allele through domestication of cultivated soybean may be associated with a reduction in salt tolerance. Our findings provide crucial information for improving salt tolerance in soybean through molecular breeding.

Genome-wide promoter analysis, homology modeling and protein interaction network of Dehydration Responsive Element Binding (DREB) gene family in Solanum tuberosum.

Dehydration Responsive Element Binding (DREB) regulates the expression of numerous stress-responsive genes, and hence plays a pivotal role in abiotic stress responses and tolerance in plants. The study aimed to develop a complete overview of the cis-acting regulatory elements (CAREs) present in S. tuberosum DREB gene promoters. A total of one hundred and four (104) cis-regulatory elements (CREs) were identified from 2.5kbp upstream of the start codon (ATG). The in-silico promoter analysis revealed variable sets of cis-elements and functional diversity with the predominance of light-responsive (30%), development-related (20%), abiotic stress-responsive (14%), and hormone-responsive (12%) elements in StDREBs. Among them, two light-responsive elements (Box-4 and G-box) were predicted in 64 and 61 StDREB genes, respectively. Two development-related motifs (AAGAA-motif and as-1) were abundant in StDREB gene promoters. Most of the DREB genes contained one or more Myeloblastosis (MYB) and Myelocytometosis (MYC) elements associated with abiotic stress responses. Hormone-responsive element i.e. ABRE was found in 59 out of 66 StDREB genes, which implied their role in dehydration and salinity stress. Moreover, six proteins were chosen corresponding to A1-A6 StDREB subgroups for secondary structure analysis and three-dimensional protein modeling followed by model validation through PROCHECK server by Ramachandran Plot. The predicted models demonstrated >90% of the residues in the favorable region, which further ensured their reliability. The present study also anticipated pocket binding sites and disordered regions (DRs) to gain insights into the structural flexibility and functional annotation of StDREB proteins. The protein association network determined the interaction of six selected StDREB proteins with potato proteins encoded by other gene families such as MYB and NAC, suggesting their similar functional roles in biological and molecular pathways. Overall, our results provide fundamental information for future functional analysis to understand the precise molecular mechanisms of the DREB gene family in S. tuberosum.

Genome-wide analysis, identification, evolution and genomic organization of dehydration responsive element-binding (DREB) gene family in Solanum tuberosum.

BackgroundThe dehydration responsive element-binding (DREB) gene family plays a crucial role as transcription regulators and enhances plant tolerance to abiotic stresses. Although the DREB gene family has been identified and characterized in many plants, knowledge about it in Solanum tuberosum (Potato) is limited.ResultsIn the present study, StDREB gene family was comprehensively analyzed using bioinformatics approaches. We identified 66 StDREB genes through genome wide screening of the Potato genome based on the AP2 domain architecture and amino acid conservation analysis (Valine at position 14th). Phylogenetic analysis divided them into six distinct subgroups (A1–A6). The categorization of StDREB genes into six subgroups was further supported by gene structure and conserved motif analysis. Potato DREB genes were found to be distributed unevenly across 12 chromosomes. Gene duplication proved that StDREB genes experienced tandem and segmental duplication events which led to the expansion of the gene family. The Ka/Ks ratios of the orthologous pairs also demonstrated the StDREB genes were under strong purification selection in the course of evolution. Interspecies synteny analysis revealed 45 and 36 StDREB genes were orthologous to Arabidopsis and Solanum lycopersicum, respectively. Moreover, subcellular localization indicated that StDREB genes were predominantly located within the nucleus and the StDREB family’s major function was DNA binding according to gene ontology (GO) annotation.ConclusionsThis study provides a comprehensive and systematic understanding of precise molecular mechanism and functional characterization of StDREB genes in abiotic stress responses and will lead to improvement in Solanum tuberosum.

Impact of the overexpression of the StDREB1 transcription factor on growth parameters, yields, and chemical composition of tubers from greenhouse and field grown potato plants.

Potato plants are often exposed to biotic and abiotic stresses that negatively impact their growth, development, and yield. Plants respond to different stresses by inducing large numbers of stress-responsive genes, which can be either functional or regulatory genes. Among regulatory genes, Dehydration Responsive Element Binding (DREB) genes are considered as one of the main groups of transcriptional regulators. The overexpression of these factors in several transgenic plants leads to enhancement of abiotic stress tolerance. However, a number of reports showed that the overexpression of DREB factors under control of constitutive promoter, affects their morphology and production. Therefore, it becomes interesting to evaluate the effect of the overexpression of this StDREB1 transcription factor on plant growth, morphology, yield and tuber composition under both greenhouse and field culture conditions. To our knowledge, there is no available data on the effect of DREBA-4 overexpression on potato plants morphology and yield. Indeed, most studies focused on DREB genes from A-1 and A-2 groups for other plant species. Our results showed that StDREB1, a A-4 group of DREB gene from potato (Solanum tuberosum L.), overexpressing plants did not show any growth retardation. On the contrary, they seem to be more vigorous, and produced higher tuber weight in greenhouse and field culture than the wild type (WT) plants. Moreover, the overexpression of StDREB1 transcription factor seemed to have an effect on tuber quality in terms of dry matter, starch contents and reducing sugars in comparison to the WT tubers. These data suggest that the StDREB1 gene from A-4 group of DREB subfamily can be a good candidate in potato breeding for stress tolerance.

Characterization of the AP2/ERF Transcription Factor Family and Expression Profiling of DREB Subfamily under Cold and Osmotic Stresses in Ammopiptanthus nanus.

APETALA2/ethylene-responsive factor (AP2/ERF) is one of the largest transcription factor (TF) families in plants, which play important roles in regulating plant growth, development, and response to environmental stresses. Ammopiptanthus nanus, an unusual evergreen broad-leaved shrub in the arid region in the northern temperate zone, demonstrates a strong tolerance to low temperature and drought stresses, and AP2/ERF transcription factors may contribute to the stress tolerance of A. nanus. In the current study, 174 AP2/ERF family members were identified from the A. nanus genome, and they were divided into five subfamilies, including 92 ERF members, 55 dehydration-responsive element binding (DREB) members, 24 AP2 members, 2 RAV members, and 1 Soloist member. Compared with the other leguminous plants, A. nanus has more members of the DREB subfamily and the B1 group of the ERF subfamily, and gene expansion in the AP2/ERF family is primarily driven by tandem and segmental duplications. Promoter analysis showed that many stress-related cis-acting elements existed in promoter regions of the DREB genes, implying that MYB, ICE1, and WRKY transcription factors regulate the expression of DREB genes in A. nanus. Expression profiling revealed that the majority of DREB members were responsive to osmotic and cold stresses, and several DREB genes such as EVM0023336.1 and EVM0013392.1 were highly induced by cold stress, which may play important roles in cold response in A. nanus. This study provided important data for understanding the evolution and functions of AP2/ERF and DREB transcription factors in A. nanus.

Two promoter regions confer heat-induced activation of SlDREBA4 in Solanum lycopersicum

Dehydration-responsive element binding (DREB) transcription factors activate the expression of downstream functional genes in combination with a dehydration-responsive element (DRE), and thereby improve the resistance of plants to abiotic stresses such as heat. However, the upstream regulatory mechanism of DREB genes under heat is unclear. A DREBA4 subfamily transcription factor (SlDREBA4), which is heat-responsive and improves heat resistance, was isolated from Solanum lycopersicum ‘Microtom’. In this study, promoter truncation experiments were performed to verify changes in β-glucuronidase (GUS) enzyme activity and GUS gene expression levels in transgenic plants with different lengths of promoter fragments under heat and to identify specific regions in the promoter that respond to heat. Our results showed that the GUS reporter gene was constitutively expressed in tissues of the full-length promoter transgenic ‘Microtom’ plants, with higher expression in conducting tissues of root, stem, and leaf, as well as sepals of flowers and fruits. Under heat treatment, GUS enzyme activity and GUS gene expression levels in tissues of the full-length promoter transgenic plants increased. Promoter deletion analysis identified two positive regulatory regions (−1095 to −730 bp and −162 to −38 bp) responsible for the promoter’s response to heat. These results indicated that the heat shock element (HSE) and MYC recognition sequences may cooperate in heat-induced activation of SlDREBA4 promoter.

Independent and combined abiotic stresses affect the physiology and expression patterns of DREB genes differently in stress-susceptible and resistant genotypes of banana.

In tropics, combined stresses of drought and heat often reduce crop productivity in plants like Musa acuminata L. We compared responses of two contrasting banana genotypes, namely the drought-sensitive Grand Nain (GN; AAA genome) and drought tolerant Hill banana (HB; AAB genome) to individual drought, heat and their combination under controlled and field conditions. Drought and combined drought and heat treatments caused greater reduction in leaf relative water content and greater increase in ion leakage and H2 O2 content in GN plants, especially in early stages, while the responses were more pronounced in HB at later stages. A combination of drought and heat increased the severity of responses. Real-time expression patterns of the A-1 and A-2 group DEHYDRATION-RESPONSIVE ELEMENT BINDING (DREB) genes revealed greater changes in expression in leaves of HB plants for both the individual stresses under controlled conditions compared to GN plants. A combination of heat and drought, however, activated most DREB genes in GN but surprisingly suppressed their expression in HB in controlled and field conditions. Its response seems correlated to a better stomatal control over transpiration in HB and a DREB-independent pathway for the more severe combined stresses unlike in GN. Most of the DREB genes had abscisic acid (ABA)-responsive elements in their promoters and were also activated by ABA suggesting at least partial dependence on ABA. This study provides valuable information on physiological and molecular responses of the two genotypes to individual and combined drought and heat stresses.

Dehydration responsive element binding transcription factors and their applications for the engineering of stress tolerance.

Dehydration responsive element binding (DREB) factors or CRT element binding factors (CBFs) are members of the AP2/ERF family, which comprises a large number of stress-responsive regulatory genes. This review traverses almost two decades of research, from the discovery of DREB/CBF factors to their optimization for application in plant biotechnology. In this review, we describe (i) the discovery, classification, structure, and evolution of DREB genes and proteins; (ii) induction of DREB genes by abiotic stresses and involvement of their products in stress responses; (iii) protein structure and DNA binding selectivity of different groups of DREB proteins; (iv) post-transcriptional and post-translational mechanisms of DREB transcription factor (TF) regulation; and (v) physical and/or functional interaction of DREB TFs with other proteins during plant stress responses. We also discuss existing issues in applications of DREB TFs for engineering of enhanced stress tolerance and improved performance under stress of transgenic crop plants.

Mulberry Transcription Factor MnDREB4A Confers Tolerance to Multiple Abiotic Stresses in Transgenic Tobacco.

The dehydration responsive element binding (DREB) transcription factors have been reported to be involved in stress responses. Most studies have focused on DREB genes in subgroups A-1 and A-2 in herbaceous plants, but there have been few reports on the functions of DREBs from the A-3–A-6 subgroups and in woody plants. Moreover, mulberry trees are ecologically and economically important perennial woody plants, but there has been little research on its stress physiology, biochemistry and molecular biology. In this study, a DREB gene from the mulberry tree, designated as MnDREB4A, classified into the A-4 subgroup by our previous study, was selected for further characterization. Our results showed that the MnDREB4A protein was localized to the nucleus where it activated transcription. The promoter of MnDREB4A can direct prominent expression downstream of the β-glucuronidase (GUS) gene under heat, cold, drought and salt stress, and GUS staining was deepest after 12 h of stress treatment. The MnDREB4A-overexpression transgenic tobacco showed the improved growth phenotype under untreated conditions, such as greener leaves, longer roots, and lower water loss and senescence rates. Overexpression of MnDREB4A in tobacco can significantly enhance tolerance to heat, cold, drought, and salt stresses in transgenic plants. The leaf discs and seedlings of transgenic plants reduced leaf wilting and senescence rates compared to the wild type plants under the different stress conditions. Further investigation showed that transgenic plants also had higher water contents and proline contents, and lower malondialdehyde contents under untreated condition and stress conditions. Our results indicate that the MnDREB4A protein plays an important role in plant stress tolerance.

Cloning of four DREB genes from Tibetan Sophora moorcroftiana and analysis of their expression during abiotic stress

Sophora moorcroftiana is an endemic, drought-resistant shrub that grows in Tibet and has some degree of resistance to salt, cold, heat, and drought. In the present study, four dehydration responsive element-binding (DREB) genes (SmDREB1, SmDREB2, SmDREB and SmDREB1) were isolated from S. moorcroftiana for the first time and their expression and proline content under abiotic stress were analyzed. Proline accumulated in seedlings under drought, salt, cold, and heat stress treatments. The four genes were variously expressed in response to the four abiotic stresses. SmDREB1 was induced by drought, cold, and heat stresses; SmDREB2 and SmDREB4 were both induced by salt, cold, and heat stresses, whereas SmDREB3 was induced by drought and heat stresses. Thus, these four genes may participate in conferring tolerance to these four abiotic stresses and are candidate genes for genetic engineering in the future.

Cloning and stress response analysis of the PeDREB2A and PeDREB1A genes in moso bamboo (Phyllostachys edulis).

Moso bamboo is a large woody bamboo with the highest ecological, economic, and cultural value among all bamboos in Asia. However, environmental stress influences its growth and development and limits its geographic distribution. Therefore, improving its resistance to environmental stress is extremely important. Dehydration responsive element binding (DREB) transcription factors perform an important role in the regulation of stress-related genes, enhancing the resistance of plants to abiotic stress. In the current study, two novel DREB genes, PeDREB2A and PeDREB1A (Gene ID No. PH01000046G1730 and PH01000668G0350), were isolated from moso bamboo and the sequences were identified and characterized (coding sequence lengths were 795 and 825 bp, respectively). The PeDREB2A and PeDREB1A proteins were estimated to have typical AP2/ERF domains, molecular weights of 28.96 and 28.84 kDa, and isoelectric points of 9.47 and 5.34, respectively. RT-PCR analysis revealed that PeDREB2A and PeDREB1A were tissue-specific genes, expressed in leaves, young stems, and roots, with similar expression levels in leaves and young stems. qRT-PCR analysis of leaves demonstrated that PeDREB2A transcription levels rapidly accumulate following exposure to drought and salt stress, peaking at 12 and 0.5 h, respectively, but only low expression levels were observed under cold stress. PeDREB1A exhibited a strong response to cold stress, reaching a peak in expression 3 h after exposure, but demonstrated only a slight response to drought and salt stress. In roots, PeDREB2A was down-regulated, and PeDREB1A was initially upregulated but then declined, under stress conditions. Two plant expression vectors, pCAMBIA2300- CaMV35S-PeDREB2A and pCAMBIA2300-CaMV35S-PeDREB1A were also successfully constructed.

Molecular cloning and characterization of two novel DREB genes encoding dehydration-responsive element binding proteins in halophyte Suaeda salsa

The dehydration-responsive element-binding (DREB) proteins play an important role in regulating expression of stress-inducible genes under abiotic stresses. In this study, two genes encoding putative DREB proteins, named SsDREBa and SsDREBb, were cloned from halophyte Suaeda salsa L. using RACE method. The deduced SsDREBa and SsDREBb proteins contain a typical AP2/ERF domain. Multiple sequence alignments and phylogenetic analysis revealed that the two SsDREB genes of S. salsa were highly similar in AP2/ERF domains at the nucleotide and amino acid levels and belong to the A-6 subgroup of the DREB transcription factor subfamily. A subcellular localization assay showed that both SsDREBs localized to the nucleus. Yeast one-hybrid experiments testified that both proteins were able to specifically bind to the DRE sequence and activate the expression of the down-stream HIS reporter gene in yeast. Quantitative real-time PCR analysis demonstrated that under normal conditions, the expression level of SsDREBa was the most high in the roots and no SsDREBa mRNAs were detected in the stems; SsDREBb expressed at relatively higher levels in the leaves than in the roots and stems. The expression of SsDREa and SsDREBb genes in S. salsa roots and leaves was remarkably induced by high-salt and dehydration treatments, but not by cold and ABA, and exhibited stronger induction in roots and leaves, respectively. These results indicate that the SsDREBa and SsDREBb are novel stress-responsive transcription factors, which are involved in the drought and high-salt stress responses through ABA-independent pathways and could be used for production of stress-tolerant transgenic crops.

In silico analysis of DREB transcription factor genes and proteins in grasses.

Plants are exposed to various environmental stresses, including drought, salinity, low temperature, etc. Dehydration responsive element binding (DREB) genes, the members of AP2/ERF transcription factor family, regulate the biological processes against cold and dehydration stresses. In this study, we analyzed a total of 19 DREB transcription factor genes and proteins from 14 grass species by using bioinformatic approaches, including their physiochemical properties, conserved motif structures, homology models, and phylogenetic relationships. The domain analysis showed that all grass species contained an AP2 domain whereas some residual substitutions and/or insertions were observed in the AP2 domains of some grasses. The physiochemical analysis revealed that many DREB proteins (89.5%) were of acidic character while the number of amino acids ranged from 213 (Aegilops speltoides subsp. speltoides) to 394 (Triticum aestivum). Based on the subcellular prediction, 16 of 19 DREB proteins were predicted to be localized in the nuclear region. According to the sequence analysis of grass DREBs, the average value of pairwise distance was found to be 0.588, while nucleotide diversity (π) was found to be 0.435. Thus, among all DREB proteins, two most divergent ones (Oryza sativa and Avena sativa) were selected for 3D structure and protein cavity comparison. In addition, 19 DREB proteins were analyzed according to their phylogenetic relationships, and as a consequence, two main groups were observed. In this study, our analyses could be a scientific base to understand DREB genes and proteins to further wet lab studies in plants, particularly in grass species.

Association of an allele-specific marker with dehydration stress tolerance in foxtail millet suggests SiDREB2 to be an important QTL

Dehydration-responsive element binding (DREB) genes assist in improving stress tolerance of plants by activating the expression of several stress-responsive genes. Therefore, development of functional markers for useful alleles utilizing DREB genes is crucial for crop improvement strategies. Earlier we reported a synonymous single nucleotide polymorphism (SNP) associated with dehydration tolerance at 558th bp (an A/G transition) in the SiDREB2 gene of foxtail millet (Setaria italica L.) and developed an allele-specific marker (ASM) for SiDREB2. In the present study, we validated this ASM using a set of 122 foxtail accessions, of which 45 were investigated in an earlier study. The QTL associated with SiDREB2 contributed to ~20 % of the total phenotypic variation (PV) for relative water content (RWC) and this signified the importance of this QTL for dehydration tolerance in foxtail millet.

Genome-wide analysis and expression profiling of the DREB transcription factor gene family in Malus under abiotic stress

The dehydration responsive element binding (DREB) transcription factor family is one of the most promising regulons for genetic engineering of plant responses to abiotic stresses. However, knowledge about apple DREB genes is limited. In the present study, we found, for the first time, 68 MdDREB genes that could be further classified into six subgroups against the entire genome of apple. All putative proteins from those genes contained a typical APETALA 2 domain and shared similar motifs. The predicted MdDREBs were distributed with different densities over 12 chromosomes, with five tandem duplication sites occurring simultaneously. Both Genevestigator and expressed sequence tags were used for preliminary investigations of expression patterns. Results from quantitative real-time PCR showed that transcript levels of some putative MdDREB genes were up-regulated significantly under various abiotic-stress treatments, which indicated their vital roles during stress adaptation. Identifying these genes and profiling their expression provides useful information and constitutes a foundation for their further, practical utilization in apple through gene-transfer techniques.