Isolation and characterization of dehydration-responsive element-binding factor 2 (DREB2) from Indian wheat (Triticum aestivum L.) cultivars

Plants have developed a sophisticated stress response system to cope with and adapt to different types of abiotic stress imposed by the frequenlty adverse environmets. It is essential to understand plants stress response mechanisms in the attempt to improve crop yield under stressful condition. Dehydration responsive element binding (DREB) proteins are important transcription factors known to regulate diverse processes of plant development and stress responses. In this study, DREB2 gene was amplified using specific primer from a diverse set of bread wheat cultivars. PCR products were purified using a gel extraction kit and sequenced. Obtained sequences were aligned and submitted to the NCBI database with HQ804651-55, FC556845-53 accession numbers. Alignment of corresponding amino acid sequences showed conservation of AP2 domain in Indian wheat genotypes. Transcripts level of the genes were shown to be most abundant (17%) in leaves, followed by flower (11%) and the least abundant in stem and seed (8%). To understand the structure function relationships, tertiary structure of DREB2 protein from wheat were built by homology modelling based on the crystal structure of GCC-box binding domain of Arabidopsis thaliana. Predicted models were refined by energy minimization and further validated by Procheck and root mean square deviation (RSMD) value. This is the first documentation of the 3-D model of DREB2 in wheat which is extremely reliable and stable. More similarities were found between AP2/EREBP protein of A. thaliana and T. aestivum by conducting protein docking with the DNA containing GCC-box. A protein was found to interact through their β-sheet, with the major DNA groove by hydrogen and hydrophobic bond, which provides structural stability to the molecule. Data generated in this study will be useful for conducting genomic research to determine the precise role of the AP2/ERF gene under water stress responses with the ultimate goal of improving crops.

[1]  R Sánchez,et al.  Advances in comparative protein-structure modelling. , 1997, Current opinion in structural biology.

[2]  A. Xiong,et al.  Isolation and characterization of a novel AP2/EREBP-type transcription factor OsAP211 in Oryza sativa , 2009, Biologia Plantarum.

[3]  K. Shinozaki,et al.  Organization of cis-acting regulatory elements in osmotic- and cold-stress-responsive promoters. , 2005, Trends in plant science.

[4]  P. Agarwal,et al.  Stress-inducible DREB2A transcription factor from Pennisetum glaucum is a phosphoprotein and its phosphorylation negatively regulates its DNA-binding activity , 2007, Molecular Genetics and Genomics.

[5]  S. Aras,et al.  Isolation of DNA for RAPD analysis from dry leaf material of someHesperis L. specimens , 2003, Plant Molecular Biology Reporter.

[6]  Jian-Kang Zhu,et al.  Salt and drought stress signal transduction in plants. , 2002, Annual review of plant biology.

[7]  Jaime Prilusky,et al.  FoldIndex copyright: a simple tool to predict whether a given protein sequence is intrinsically unfolded , 2005, Bioinform..

[8]  J. Beckmann,et al.  FoldIndex©: a simple tool to predict whether a given protein sequence is intrinsically unfolded , 2005 .

[9]  M. Nei,et al.  MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. , 2007, Molecular biology and evolution.

[10]  D. Rhodes,et al.  The solution structure of the first zinc finger domain of SWI5: a novel structural extension to a common fold. , 1996, Structure.

[11]  J M Thornton,et al.  LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. , 1995, Protein engineering.

[12]  A. Sarai,et al.  Unique Mode of GCC Box Recognition by the DNA-binding Domain of Ethylene-responsive Element-binding Factor (ERF Domain) in Plant* , 1998, The Journal of Biological Chemistry.

[13]  S. Hoving,et al.  Isoelectric focusing and two-dimensional gel electrophoresis. , 2009, Methods in enzymology.

[14]  David S. Wishart,et al.  SuperPose: a simple server for sophisticated structural superposition , 2004, Nucleic Acids Res..

[15]  Fadi Chen,et al.  Identification and characterization of two chrysanthemum (Dendronthema × moriforlium) DREB genes, belonging to the AP2/EREBP family , 2007, Molecular Biology Reports.

[16]  K. Shinozaki,et al.  Regulation and functional analysis of ZmDREB2A in response to drought and heat stresses in Zea mays L. , 2007, The Plant journal : for cell and molecular biology.

[17]  A. Wajid,et al.  DNA photolyase of enterococci: possible explanation for its low sunlight inactivation rate , 2009, Biologia.

[18]  G. He,et al.  AP2/ERF Transcription Factor in Rice: Genome-Wide Canvas and Syntenic Relationships between Monocots and Eudicots , 2012, Evolutionary bioinformatics online.

[19]  G. Sethi,et al.  DROUGHT TOLERANCE AMONG 30 CULTIVARS OF WHEAT (TRITICUM AESTIVUM L.) FOR WATER RELATION PARAMETERS , 2008 .

[20]  J. Boyer.  Plant Productivity and Environment , 1982, Science.

[21]  R. Sunkar,et al.  Drought and Salt Tolerance in Plants , 2005 .

[22]  J.-S. Zhang,et al.  An EREBP/AP2-type protein in Triticum aestivum was a DRE-binding transcription factor induced by cold, dehydration and ABA stress , 2003, Theoretical and Applied Genetics.

[23]  Zhiyong Li,et al.  Isolation and functional characterization of HvDREB1—a gene encoding a dehydration-responsive element binding protein in Hordeum vulgare , 2008, Journal of Plant Research.

[24]  Anil Kumar,et al.  PCR Primer Design: DREB Genes , 2008 .

[25]  C. Lata,et al.  Role of DREBs in regulation of abiotic stress responses in plants. , 2011, Journal of experimental botany.

[26]  K. Shinozaki,et al.  Two Transcription Factors, DREB1 and DREB2, with an EREBP/AP2 DNA Binding Domain Separate Two Cellular Signal Transduction Pathways in Drought- and Low-Temperature-Responsive Gene Expression, Respectively, in Arabidopsis , 1998, Plant Cell.

[27]  Lin Yuan,et al.  What is the solution? , 1984, Nature.

[28]  W. Shao.  Dehydration Responsive Element Binding (DREB) Transcription Activator and Its Function in Plant Tolerance to Environmental Stresses , 2004 .

[29]  Chin-Shang Li,et al.  Regulatory genes involved in the determination of frost tolerance in temperate cereals , 2009 .

[30]  Robert C. Edgar,et al.  MUSCLE: multiple sequence alignment with high accuracy and high throughput. , 2004, Nucleic acids research.

[31]  R. Harrison,et al.  Expression of soluble heterologous proteins via fusion with NusA protein , 2022 .

[32]  Anna Tramontano,et al.  The PMDB Protein Model Database , 2005, Nucleic Acids Res..

[33]  W. Delano.  The PyMOL Molecular Graphics System , 2002 .

[34]  Mark Isalan,et al.  Zinc-finger protein-targeted gene regulation: Genomewide single-gene specificity , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[35]  M. Bagajewicz,et al.  Predicting the solubility of recombinant proteins in Escherichia coli. , 2015, Methods in molecular biology.

[36]  K. Shinozaki,et al.  DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression. , 2002, Biochemical and biophysical research communications.

[37]  Rongfeng Huang,et al.  A novel ERF transcription activator in wheat and its induction kinetics after pathogen and hormone treatments. , 2007, Journal of experimental botany.

[38]  Kazuo Shinozaki,et al.  Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. , 2006, Annual review of plant biology.

[39]  Carsten Kutzner,et al.  GROMACS 4:  Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation. , 2008, Journal of chemical theory and computation.

[40]  C. Nakamura,et al.  Differential regulation of transcript accumulation and alternative splicing of a DREB2 homolog under abiotic stress conditions in common wheat. , 2006, Genes & genetic systems.

[41]  L. Cong,et al.  Characterization of the novel gene BjDREB1B encoding a DRE-binding transcription factor from Brassica juncea L. , 2008, Biochemical and biophysical research communications.

[42]  C. Morris,et al.  Identification of differentially expressed UniGenes in developing wheat seed using digital differential display , 2009 .

[43]  T. Zhu,et al.  Networks of transcription factors with roles in environmental stress response. , 2004, Trends in plant science.

[44]  N. Singh,et al.  DREB1/CBF transcription factors: their structure, function and role in abiotic stress tolerance in plants , 2012, Journal of Genetics.

[45]  Masashi Suzuki,et al.  A novel mode of DNA recognition by a β‐sheet revealed by the solution structure of the GCC‐box binding domain in complex with DNA , 1998, The EMBO journal.

[46]  K. Shinozaki,et al.  Molecular responses to dehydration and low temperature: differences and cross-talk between two stress signaling pathways. , 2000, Current opinion in plant biology.

[47]  D. Higgins,et al.  T-Coffee: A novel method for fast and accurate multiple sequence alignment. , 2000, Journal of molecular biology.

[48]  G. Gheysen,et al.  Transcriptome analysis of rice mature root tissue and root tips in early development by massive parallel sequencing. , 2012, Journal of experimental botany.

[49]  T. N. Bhat,et al.  The Protein Data Bank , 2000, Nucleic Acids Res..

[50]  A. Xiong,et al.  Expression of a rice DREB1 gene, OsDREB1D, enhances cold and high-salt tolerance in transgenic Arabidopsis. , 2009, BMB reports.

[51]  J. Palta,et al.  Responses to abiotic stresses. , 1998 .

[52]  P. Agarwal,et al.  Role of DREB transcription factors in abiotic and biotic stress tolerance in plants , 2006, Plant Cell Reports.