Altered DREB1A Gene Expression in Arabidopsis thaliana Leads to Change in Root Growth, Antioxidant Enzymes Activity, and Response to Salinity but Not to Cold

Document Type: Research Article


1 Department of Biology, Hakim Sabzevari University, Sabzevar, Iran

2 Department of Biology, Golestan University, Gorgan, Iran

3 Department of Plant Biology, Southern Illinois University, IL, USA


DREB1A (Dehydration Responsive Element Binding 1A) transcription factor is involved in plant responses to abiotic stresses. An A. thaliana DREB1A T-DNA insertional mutant (dreb1a) alongside previously reported DREB1A over-expressing plants (OX28) were detailed in molecular and phenotypic characterizations. The T-DNA of the dreb1a line was inserted at position -253, and segregation ratio confirmed a single T-DNA locus in its T0 plant population. The RT-PCR analysis on dreb1a seedlings also revealed a null mutant in DREB1A gene. The phenotypes of the dreb1a seedlings subjected to cold stress were not different from those of the wild type (WT-Col0), but under salinity dreb1a plants showed about 11% less seed germination and the four times less survival rate, compared to WT-Col0 plants. Under normal growth conditions and in comparison to their wild type counterparts, there was direct correlation between DREB1A expression levels and the root length as the dreb1a, in contrast to the OX28 line, showing 29% longer roots than that in the WT-Col0 plants. Interestingly, this root phenotype had association with accumulation of reactive oxygen species (ROS) in dreb1a by 31% less, and in OX28 by 97% more than that in the control seedlings. In addition, the dreb1a plant possessed significantly higher activities in superoxide dismutase, peroxidase, polyphenol oxidase and significantly lower activity in catalase than WT-Col0, but no differences in extracellular peroxidase activity. On the other hand, the OX28 plant possessed a higher extracellular peroxidase activity. Overall, these results suggest that a precise expression level of DREB1A is required for proper growth and development in A. thaliana.


Afzal Z, Howton T, Sun Y, Mukhtar MS. 2016. The roles of aquaporins in plant stress responses. J Dev Biol 4: 9.

Agarwal PK, Agarwal P, Reddy M, Sopory SK. 2006. Role of DREB transcription factors in abiotic and biotic stress tolerance in plants. Plant Cell Rep. 25: 1263-1274.

Bartoli CG, Casalongué CA, Simontacchi M, Marquez-Garcia B, Foyer CH. 2013. Interactions between hormone and redox signalling pathways in the control of growth and cross-tolerance to stress. Environ Exp Bot 94: 73-88.

Beauchamp C, Fridovich I. 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44: 276-287.

Bhatnagar-Mathur P, Devi MJ, Reddy DS, Lavanya M, Vadez V, Serraj R, Yamaguchi-Shinozaki K, Sharma KK. 2007. Stress-inducible expression of AtDREB1A in transgenic peanut (Arachis hypogaea L.) increases transpiration efficiency under water-limiting conditions. Plant Cell Rep 26: 2071-2082.

Bilska-Kos A, Szczepanik J, Sowiński P. 2016. Cold induced changes in the water balance affect immunocytolocalization pattern of one of the aquaporins in the vascular system in the leaves of maize (Zea mays L.). J Plant Physiol 205: 75-79.

Boyes DC, Zayed AM, Ascenzi R, McCaskill AJ, Hoffman NE, Davis KR, and Gorlach J. 2001. Growth stage–based phenotypic analysis of Arabidopsis a model for high throughput functional genomics in plants. Plant Cell 13:1499-1510.

Chinnusamy V, Ohta M, Kanrar S, Lee B-h, Hong X, Agarwal M, Zhu J-K. 2003. ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. Gene Dev 17: 1043-1054.

Cong L, Zheng H-C, Zhang Y-X, Chai T-Y. 2008. Arabidopsis DREB1A confers high salinity tolerance and regulates the expression of GA dioxygenases in Tobacco. Plant Sci 174: 156-164.

Cook D, Fowler S, Fiehn O, Thomashow MF. 2004. A prominent role for the CBF cold response pathway in configuring the low-temperature metabolome of Arabidopsis. Proc Nat Acad Sci 101: 15243-15248.

Daudi A, Cheng Z, O’Brien JA, Mammarella N, Khan S, Ausubel FM, Bolwell GP. 2012. The apoplastic oxidative burst peroxidase in Arabidopsis is a major component of pattern-triggered immunity. The Plant Cell 24: 275-287.

Dunand C, Crèvecoeur M, Penel C. 2007. Distribution of superoxide and hydrogen peroxide in Arabidopsis root and their influence on root development: possible interaction with peroxidases. New Phytol 174: 332-341.

Fowler S, Thomashow MF. 2002. Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. The Plant Cell 14: 1675-1690.

Ghorbannejad H, Amooaghaie R. 2017. Differential changes of proline content and activities of antioxidant enzymes results in varied salt-tolerance in canola genotypes. J Genet Resour 3: 36-46.

Gill SS, Tuteja N. 2010. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem 48: 909-930.

Gilmour SJ, Sebolt AM, Salazar MP, Everard JD, Thomashow MF. 2000. Overexpression of the Arabidopsis CBF3Transcriptional Activator Mimics Multiple Biochemical Changes Associated with Cold Acclimation. Plant Physiol 124: 1854-1865.

Hoagland DR, Arnon DI. 1950. The water-culture method for growing plants without soil. Circular California Agri Experi Stat 347.

Huang B, Liu JY. 2006. A cotton dehydration responsive element binding protein functions as a transcriptional repressor of DRE-mediated gene expression. Biochem Biophys Res Commun 343: 1023-1031.

Jaglo-Ottosen KR, Gilmour SJ, Zarka DG, Schabenberger O, Thomashow MF. 1998. Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. Science 280: 104-106.

Jaspers P, Kangasjärvi J. 2010. Reactive oxygen species in abiotic stress signaling. Physiol Plant 138: 405-413.

Jia X, Li Y, Qi E, Ma S, Hu X, Wen G, Wang Y, Li J, Zhang X, Wang H. 2016. Overexpression of the Arabidopsis DREB1A. Russ J Plant Physiol 63: 523-531.

Kar M, Mishra D. 1976. Catalase, peroxidase, and polyphenoloxidase activities during rice leaf senescence. Plant Physiol 57: 315-319.

Kasajima I, Ide Y, Ohkama-Ohtsu N, Hayashi H, Yoneyama T, Fujiwara T. 2004. A protocol for rapid DNA extraction from Arabidopsis thaliana for PCR analysis. Plant Mol Bio Rep 22: 49-52.

Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K. 1999. Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nature Biotech 17: 287-291.

Kazan K. 2015. Diverse roles of jasmonates and ethylene in abiotic stress tolerance. Trends Plant Sci 20: 219-229.

Kelij S, Majd A, Nematzade G, Jonoubi P. 2015. Activation of lignin biosynthetic enzymes during internodal development of Aeluropus littoralis exposed to NaCl. J Genet Resour 1: 19-24.

Kim YH, Yang KS, Ryu SH, Kim KY, Song WK, Kwon SY, Lee HS, Bang JW, Kwak SS. 2008. Molecular characterization of a cDNA encoding DRE-binding transcription factor from dehydration-treated fibrous roots of sweet potato. Plant Physiol Biochem 46: 196-204.

Kohan-Baghkheirati E, Bagherieh-Najjar MB. 2011. DRE-binding Transcription factor (DREB1A) as a master regulator induced a broad range of abiotic stress tolerance in plant. Afr J Biotechnol 10: 15100-15108.

Kumar V, Parvatam G, Ravishankar GA. 2009. AgNO3: a potential regulator of ethylene activity and plant growth modulator. Electron J Biotechn 12: 8-9.

Li. X., Cheng X, Liu. J., Zeng H, Han. L., Tang W. 2011. Heterologous expression of the Arabidopsis DREB1A/CBF3 gene enhances drought and freezing tolerance in transgenic Lolium perenne plants. Plant Biotechnol Rep 5: 61-69.

Liszkay A, van der Zalm E, Schopfer P. 2004. Production of reactive oxygen intermediates (O2−, H2O2, and OH) by maize roots and their role in wall loosening and elongation growth. Plant Physiol 136: 3114-3123.

Liu JX, Srivastava R, Che P, Howell SH. 2007. Salt stress responses in Arabidopsis utilize a signal transduction pathway related to endoplasmic reticulum stress signaling. Plant J 51: 897-909.

Liu Q, Kasuga M, Sakuma Y, Abe H, Miura H, Yamaguchi-Shinozaki K, Shinozaki K. 1998. 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. Plant Cell 10: 1391–1406.

Malamy J. 2005. Intrinsic and environmental response pathways that regulate root system architecture. Plant Cell Environ 28: 67-77.

Maruyama K, Takeda M, Kidokoro S, Yamada K, Sakuma Y, Urano K, Fujita M, Yoshiwara K, Matsukura S, Morishita Y. 2009. Metabolic pathways involved in cold acclimation identified by integrated analysis of metabolites and transcripts regulated by DREB1A and DREB2A. Plant Physiol 150: 1972-1980.

Mayer AM. 2006. Polyphenol oxidases in plants and fungi: going places? A review. Phytochemistry 67: 2318-2331.

Mellersh DG, Foulds IV, Higgins VJ, Heath MC. 2002. H2O2 plays different roles in determining penetration failure in three diverse plant–fungal interactions. Plant J 29: 257-268.

Mika A, Minibayeva F, Beckett R, Lüthje S. 2004. Possible functions of extracellular peroxidases in stress-induced generation and detoxification of active oxygen species. Phytochem Rev 3: 173-193.

Mittler R. 2002. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7: 1360-1385.

Murashige T, Skoog F. 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15: 473-497.

Novillo F, Alonso JM, Ecker JR, Salinas J. 2004. CBF2/DREB1C is a negative regulator of CBF1/DREB1B and CBF3/DREB1A expression and plays a central role in stress tolerance in Arabidopsis. Proc Natl Acad Sci 101: 3985-3990.

Novillo F, Medina J, Salinas J. 2007. Arabidopsis CBF1 and CBF3 have a different function than CBF2 in cold acclimation and define different gene classes in the CBF regulon. Proc Natl Acad Sci 104: 21002-21007.

Park S, Lee CM, Doherty CJ, Gilmour SJ, Kim Y, Thomashow MF. 2015. Regulation of the Arabidopsis CBF regulon by a complex low-temperature regulatory network. Plant J 82: 193-207.

Rai GK, Rai NP, Rathaur S, Kumar S, Singh M. 2013. Expression of rd29A:: AtDREB1A/CBF3 in tomato alleviates drought-induced oxidative stress by regulating key enzymatic and non-enzymatic antioxidants. Plant Physiol Biochem 69: 90-100.

Rivas-San Vicente M, Plasencia J. 2011. Salicylic acid beyond defence: its role in plant growth and development. J Exp Bot 62: 3321-3338.

Rueden CT, Schindelin J, Hiner MC, DeZonia BE, Walter AE, Arena ET, Eliceiri KW. 2016. ImageJ: Image Analysis Interoperability for the Next Generation of Biological Image Data. BMC Bioinformatics 22: 2066-2067.

Saffar A, Bagherieh-Najjar MB, Mianabadi M. 2009. Activity of antioxidant enzymes in response to cadmium in Arabidopsis thaliana. J Biol Sci 9: 44-50.

Sarkar T, Thankappan R, Kumar A, Mishra GP, Dobaria JR. 2016. Stress Inducible Expression of AtDREB1A Transcription Factor in Transgenic Peanut (Arachis hypogaea L.) Conferred Tolerance to Soil-Moisture Deficit Stress. Front Plant Sci 7: 935.

Schneider CA, Rasband WS, Eliceiri KW. 2012. NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9: 671-675.

Seeley KA, Cheng JC, and Sung ZR. 2000. The two-stage theory of root development in Arabidopsis.

Seki M, Narusaka M, Ishida J, Nanjo T, Fujita M, Oono Y, Kamiya A, Nakajima M, Enju A, Sakurai T et al. 2002. Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. Plant J 31: 279-292.

Shi Y, Ding Y, Yang S. 2015. Cold signal transduction and its interplay with phytohormones during cold acclimation. Plant Cell Physiol 56: 7-15.

Shi Y, Tian S, Hou L, Huang X, Zhang X, Guo H, Yang S. 2012. Ethylene signaling negatively regulates freezing tolerance by repressing expression of CBF and type-A ARR genes in Arabidopsis. Plant Cell 24: 2578-2595.

Suo H, Ma Q, Ye K, Yang C, Tang Y, Hao J, Zhang ZJ, Chen M, Feng Y, Nian H. 2012. Overexpression of AtDREB1A causes a severe dwarf phenotype by decreasing endogenous gibberellin levels in soybean [Glycine max (L.) Merr.]. PloS One 7: e45568.

Tonkinson C, Lyndon R, Arnold G, Lenton J. 1997. The effects of temperature and the Rht3 dwarfing gene on growth, cell extension, and gibberellin content and responsiveness in the wheat leaf. J Exp Bot 48: 963-970.

Vadez V, Rao JS, Bhatnagar‐Mathur P, Sharma K. 2012. DREB1A promotes root development in deep soil layers and increases water extraction under water stress in groundnut. Plant Biol 15: 45-52.

Vaughn KC, Duke SO. 1984. Function of polyphenol oxidase in higher plants. Physiol Plant 60: 106-112.

Wan F, Pan Y, Li J, Chen X, Pan Y, Wang Y, Tian S, Zhang X. 2014. Heterologous expression of Arabidopsis C-repeat binding factor 3 (AtCBF3) and cold-regulated 15A (AtCOR15A) enhanced chilling tolerance in transgenic eggplant (Solanum melongena L.). Plant Cell Rep 33: 1951-1961.

Yamaguchi-shinozaki K, Shinozaki k. 1994. A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. Plant Cell. 6: 251-264.

Zhao C, Zhang Z, Xie S, Si T, Li Y, Zhu J-K. 2016. Mutational Evidence for the Critical Role of CBF Genes in Cold Acclimation in Arabidopsis. Plant Physiol 171: 2744-2759.


Volume 4, Issue 2
Summer and Autumn 2018
Pages 90-104