Stress-inducible DREB2A transcription factor from Pennisetum glaucum is a phosphoprotein and its phosphorylation negatively regulates its DNA-binding activity
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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.
KeywordsDRE DREB2A Pennisetum glaucum Phosphorylation Transcription factors
The research was supported by NATP, ICAR, New Delhi, and Department of Biotechnology Govt. of India and internal grants from ICGEB.
- Dubouzet JG, Sakuma Y, Ito Y, Kasuga M, Dubouzet EG, Miura S, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, high-salt- and cold-responsive gene expression. Plant J 33:751–763PubMedCrossRefGoogle Scholar
- Jaglo KR, Kleff S, Amundsen KL, Zhang X, Haake V, Zhang JZ, Deits T, Thomashow MF (2001) Components of the Arabidopsis C-repeat/dehydration-responsive element binding factor cold-response pathway are conserved in Brassica napus and other plant species. Plant Physiol 127:910–917PubMedCrossRefGoogle Scholar
- Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, 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 10:1391–1406PubMedCrossRefGoogle Scholar
- Medina J, Bargues M, Terol J, Perez-Alonso M, Salinas J (1999) The Arabidopsis CBF gene family is composed of three genes encoding AP2 domain-containing proteins whose expression is regulated by low temperature but not by abscisic acid or dehydration. Plant Physiol 119:463–469PubMedCrossRefGoogle Scholar
- Mudgil Y, Singh BN, Upadhyaya KC, Sopory SK, Reddy MK (2002) Cloning and characterization of a cell cycle-regulated gene encoding topoisomerase I from Nicotiana tabacum that is inducible by light low temperature and abscissic acid. Mol Genet Genomics 267:382–390Google Scholar
- Riechmann JL, Heard J, Martin G, Reuber L, Jiang C, Keddie J, Adam L, Pineda O, Ratcliffe OJ, Samaha RR, Creelman R, Pilgrim M, Broun P, Zhang JZ, Ghandehari D, Sherman BK, Yu G (2000) Arabidopsis transcription factor: genome wide comparative analysis among eukaryotes. Science 290:2105–2110PubMedCrossRefGoogle Scholar
- Shen YG, Zhang WK, He SJ, Zhang JS, Liu Q, Chen SY (2003a) An EREBP/AP2-type protein in Triticum aestivum was a DRE-binding transcription factor induced by cold, dehydration and ABA stress. Theor Appl Genet 106:923–930Google Scholar
- Shen YG, Zhang WK, Yan DQ, Du BX, Zhang JS, Liu Q, Chen SY (2003b) Characterization of a DRE-binding transcription factor from a halophyte Atriplex hortensis. Theor Appl Genet 107:155–161Google Scholar
- Stockinger EJ, Gilmour SJ, Thomashow MF (1997) Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc Natl Acad Sci USA 94:1035–1040PubMedCrossRefGoogle Scholar
- Tyagi W, Rajagopal D, Singla-Pareek SL, Reddy MK, Sopory SK (2005) Cloning and regulation of a stress-regulated Pennisetum glaucum vacuolar ATPase c gene and characterization of its promoter that is expressed in shoot hairs and floral organs. Plant Cell Physiol 46:1411–1422PubMedCrossRefGoogle Scholar
- Yamaguchi-Shinozaki K, Kasuga M, Liu Q, Nakashima K, Sakuma Y, Abe H, Shinwari ZK, Seki M, Shinozaki K (2002) Biological mechanisms of drought stress response. JIRCAS Working Report 1–8Google Scholar