Co-expression of AtbHLH17 and AtWRKY28 confers resistance to abiotic stress in Arabidopsis
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Stress adaptation in plants involves altered expression of many genes through complex signaling pathways. To achieve the optimum expression of downstream functional genes, we expressed AtbHLH17 (AtAIB) and AtWRKY28 TFs which are known to be upregulated under drought and oxidative stress, respectively in Arabidopsis. Multigene expression cassette with these two TFs and reporter gene GUS was developed using modified gateway cloning strategy. The GUS assay and expression analysis of transgenes in transgenic plants confirmed the integration of multigene cassette. The transgenic lines exhibited enhanced tolerance to NaCl, Mannitol and oxidative stress. Under mannitol stress condition significantly higher root growth was observed in transgenics. Growth under stress and recovery growth was substantially superior in transgenics exposed to gradual long term desiccation stress conditions. We demonstrate the expression of several downstream target genes under various stress conditions. A few genes having either WRKY or bHLH cis elements in their promoter regions showed higher transcript levels than wild type. However, the genes which did not have either of the motifs did not differ in their expression levels in stress conditions compared to wild type plants. Hence co-expressing two or more TFs may result in upregulation of many downstream target genes and substantially improve the stress tolerance of the plants.
KeywordsDrought Methyl viologen Multigene-construct Salinity Transcription factors
Basic helix loop helix
Authors acknowledge the financial support from Department of Biotechnology, Centre of Excellence programme support (BT/01/COE/05/03), Indian Council of Agricultural Research—Niche area of Excellence programme (F.No. 10-(6)/2005 EP&D) and Department of Science and Technology—Fund for Improvement of Science and Technology (SR/FST/LSI-051/2002).
- Andreasson E, Jenkins T, Brodersen P, Thorgrimsen S, Petersen NH, Zhu S, Qiu JL, Micheelsen P, Rocher A, Petersen M, Newman MA, Bjorn Nielsen H, Hirt H, Somssich I, Mattsson O, Mundy J (2005) The MAP kinase substrate MKS1 is a regulator of plant defense responses. EMBO J 24:2579–2589PubMedCrossRefGoogle Scholar
- Archana K, Rama N, Mamrutha HM, Nataraja KN (2009) Down-regulation of an abiotic stress related Nicotiana benthamiana WRKY TF induces physiological abnormalities. Indian J Biotechnol 8:53–60Google Scholar
- Govind G, Harshavardhan VT, Patricia JK, Dhanalakshmi R, Senthil-Kumar M, Sreenivasulu N, Udayakumar M (2009) Identification and functional validation of a unique set of drought induced genes preferentially expressed in response to gradual water stress in peanut. Mol Genet Genomics 281:591–605PubMedCrossRefGoogle Scholar
- Kiribuchi K, Jikumaru Y, Kaku H, Minami E, Hasegawa M, Kodama O, Seto H, Okada K, Nojiri H, Yamane H (2005) Involvement of the basic helix-loop-helix transcription factor RERJ1 in wounding and drought stress responses in rice plants. Biosci Biotechnol Biochem 69:1042–1044PubMedCrossRefGoogle Scholar
- Nakashima K, Yamaguchi-Shinozaki K (2005) Molecular studies on stress-responsive Gene expression in Arabidopsis and improvement of stress tolerance in crop plants by regulon biotechnology. JARQ 39:221–229Google Scholar
- Nakashima K, Tran LSP, Nguyen DV, Fujita M, Maruyama K, Todaka D, Ito Y, Hayashi N, Shinozaki K, Yamaguchi-Shinozaki K (2007) Functional analysis of a NAC-type transcription factor OsNAC6 involved in abiotic and biotic stress-responsive gene expression in rice. Plant J 51:617–630PubMedCrossRefGoogle Scholar
- Senthil-Kumar M, Srikanthbabu V, Mohanraju B, Kumar G, Shivaprakash N, Udayakumar M (2003) Screening of inbred lines to develop thermotolerant sunflower hybrid through temperature induction response (TIR) technique: a novel approach by exploiting residual variability. J Exp Bot 54:2569–2578PubMedCrossRefGoogle Scholar
- Strizhov N, Abraham E, Okresz L, Blickling S, Zilberstein A, Schell J, Koncz C, Szabados L (1997) Differential expression of two P5CS genes controlling proline accumulation during salt stress requires ABA and is regulated by ABA1, ABI1 and AXR2 in Arabidopsis. Plant J 12:557–569PubMedCrossRefGoogle Scholar
- Tran LSP, Nakashima K, Sakuma Y, Osakabe Y, Qin F, Simpson SD, Maruyama K, Fujita Y, Shinozaki K, Yamaguchi-Shinozaki K (2007) Co-expression of the stress-inducible zinc finger homeodomain ZFHD1 and NAC TFs enhances expression of the ERD1 gene in Arabidopsis. Plant J 49:46–63PubMedCrossRefGoogle Scholar
- Vemanna RS, Chandrashekar BK, Hanumantha Rao HM, Sathyanarayanagupta SK, Sarangi SK, Nataraja KN, Udayakumar M, Sarangi SK, Karaba NK, Udayakumar M (2012) A modified multisite gateway cloning strategy for consolidation of genes in plants. Mol Biotechnol. doi 10.1007/s12033-012-9499-6