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Comparative functional analysis of six drought-responsive promoters in transgenic rice

Abstract

Rice production is greatly affected by environmental stresses such as drought and high salinity. Transgenic rice plants tolerant to such stresses are expected to be produced. Stress-responsive promoters with low expression under normal growth conditions are needed to minimize the adverse effects of stress-tolerance genes on rice growth. We performed expression analyses of drought-responsive genes in rice plants using a microarray, and selected LIP9, OsNAC6, OsLEA14a, OsRAB16D, OsLEA3-1, and Oshox24 for promoter analysis. Transient assays using the promoters indicated that AREB/ABF (abscisic acid (ABA)-responsive element-binding protein/ABA-binding factor) transcription factors enhanced expressions of these genes. We generated transgenic rice plants containing each promoter and the β-glucuronidase (GUS) reporter gene. GUS assays revealed that the LIP9 and OsNAC6 promoters were induced by drought, high salinity, and ABA treatment, and both promoters showed strong activity under normal growth conditions in the root. The other promoters were strongly induced by stresses and ABA, but showed low activity under normal growth conditions. In seeds, GUS staining showed that Oshox24 expression was low and expressions of the other genes were high. Transgenic rice plants overexpressing OsNAC6 under the control of the Oshox24 promoter showed increased tolerance to drought and high salinity, and no growth defects. These data suggest that the Oshox24 promoter is useful to overexpress stress-tolerance genes without adversely affecting growth.

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Abbreviations

ABA:

Abscisic acid

ABRE:

ABA-responsive element

AREB:

ABRE-binding protein

CaMV:

Cauliflower mosaic virus

CRT:

C-repeat

CBF:

CRT-binding factor

DRE:

Dehydration-responsive element

DREB:

DRE-binding protein

GUS:

β-Glucuronidase

LEA:

Late embryogenesis abundant

TF:

Transcription factor

UBI:

Ubiquitin

References

  1. Agalou A, Purwantomo S, Overnas E, Johannesson H, Zhu X, Estiati A, de Kam RJ, Engstrom P, Slamet-Loedin IH, Zhu Z et al (2008) A genome-wide survey of HD-Zip genes in rice and analysis of drought-responsive family members. Plant Mol Biol 66:87–103

  2. Bang SW, Park SH, Jeong JS, Kim YS, Jung H, Ha SH, Kim JK (2013) Characterization of the stress-inducible OsNCED3 promoter in different transgenic rice organs and over three homozygous generations. Planta 237:211–224

  3. Becker D (1990) Binary vectors which allow the exchange of plant selectable markers and reporter genes. Nucl Acids Res 18:203

  4. Behnam B, Kikuchi A, Celebi-Toprak F, Kasuga M, Yamaguchi-Shinozaki K, Watanabe KN (2007) Arabidopsis rd29A:DREB1A enhances freezing tolerance in transgenic potato. Plant Cell Rep 26:1275–1282

  5. Bhatnagar-Mathur P, Devi MJ, Reddy DS, Lavanya M, Vadez V, Serraj R, Yamaguchi-Shinozaki K, Sharma KK (2007) Stress-inducible expression of At DREB1A in transgenic peanut (Arachis hypogaea L.) increases transpiration efficiency under water-limiting conditions. Plant Cell Rep 26:2071–2082

  6. Bray EA (2004) Genes commonly regulated by water-deficit stress in Arabidopsis thaliana. J Exp Bot 55:2331–2341

  7. Bray EA, Bailey-Serres J, Weretilnyk E (2000) Responses to abiotic stresses. In: Buchanan BB, Gruissem W, Jones RL (eds) Biochemistry and molecular biology of plants. American Society of Plant Physiologists, Rockville, pp 1158–1203

  8. Buchanan CD, Klein PE, Mullet JE (2004) Phylogenetic analysis of 5′-noncoding regions from the ABA-responsive rab16/17 gene family of sorghum, maize and rice provides insight into the composition, organization and function of cis-regulatory modules. Genetics 168:1639–1654

  9. Chakrabortee S, Boschetti C, Walton LJ, Sarkar S, Rubinsztein DC, Tunnacliffe A (2007) Hydrophilic protein associated with desiccation tolerance exhibits broad protein stabilization function. Proc Natl Acad Sci USA 104:18073–18078

  10. Cornejo MJ, Luth D, Blankenship KM, Anderson OD, Blechl AE (1993) Activity of a maize ubiquitin promoter in transgenic rice. Plant Mol Biol 23:567–581

  11. Datta K, Baisakh N, Ganguly M, Krishnan S, Yamaguchi-Shinozaki K, Datta SK (2012) Overexpression of Arabidopsis and rice stress genes’ inducible transcription factor confers drought and salinity tolerance to rice. Plant Biotech J 10:579–586

  12. 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. Plant Cell 14:1675–1690

  13. Fujita Y, Fujita M, Satoh R, Maruyama K, Parvez MM, Seki M, Hiratsu K, Ohme-Takagi M, Shinozaki K, Yamaguchi-Shinozaki K (2005) AREB1 is a transcription activator of novel ABRE-dependent ABA signaling that enhances drought stress tolerance in Arabidopsis. Plant Cell 17:3470–3488

  14. Furihata T, Maruyama K, Fujita Y, Umezawa T, Yoshida R, Shinozaki K, Yamaguchi-Shinozaki K (2006) ABA-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1. Proc Natl Acad Sci USA 103:1988–1993

  15. Hadiarto T, Tran LS (2011) Progress studies of drought-responsive genes in rice. Plant Cell Rep 30:297–310

  16. Hong B, Tong Z, Ma N, Li J, Kasuga M, Yamaguchi-Shinozaki K, Gao J (2006) Heterologous expression of the AtDREB1A gene in chrysanthemum increases drought and salt stress tolerance. Sci China Series C, Life Sci/Chinese Acad Sci 49:436–445

  17. Hsieh TH, Lee JT, Charng YY, Chan MT (2002a) Tomato plants ectopically expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress. Plant Physiol 130:618–626

  18. Hsieh TH, Lee JT, Yang PT, Chiu LH, Charng YY, Wang YC, Chan MT (2002b) Heterology expression of the Arabidopsis C-repeat/dehydration response element binding factor 1 gene confers elevated tolerance to chilling and oxidative stresses in transgenic tomato. Plant Physiol 129:1086–1094

  19. Hu H, Dai M, Yao J, Xiao B, Li X, Zhang Q, Xiong L (2006) Overexpressing a NAM, ATAF, and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice. Proc Natl Acad Sci USA 103:12987–12992

  20. Hundertmark M, Hincha DK (2008) LEA (late embryogenesis abundant) proteins and their encoding genes in Arabidopsis thaliana. BMC Genomics 9:118

  21. Ito Y, Katsura K, Maruyama K, Taji T, Kobayashi M, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2006) Functional analysis of rice DREB1/CBF-type transcription factors involved in cold-responsive gene expression in transgenic rice. Plant Cell Physiol 47:141–153

  22. Jeong JS, Kim YS, Baek KH, Jung H, Ha SH, Do Choi Y, Kim M, Reuzeau C, Kim JK (2010) Root-specific expression of OsNAC10 improves drought tolerance and grain yield in rice under field drought conditions. Plant Physiol 153:185–197

  23. Jeong JS, Kim YS, Redillas MC, Jang G, Jung H, Bang SW, Choi YD, Ha SH, Reuzeau C, Kim JK (2013) OsNAC5 overexpression enlarges root diameter in rice plants leading to enhanced drought tolerance and increased grain yield in the field. Plant Biotech J 11:101–114

  24. 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. Nat Biotech 17:287–291

  25. Kasuga M, Miura S, Shinozaki K, Yamaguchi-Shinozaki K (2004) A combination of the Arabidopsis DREB1A gene and stress-inducible rd29A promoter improved drought- and low-temperature stress tolerance in tobacco by gene transfer. Plant Cell Physiol 45:346–350

  26. 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 10:1391–1406

  27. Maggio A, Zhu JK, Hasegawa PM, Bressan RA (2006) Osmogenetics: Aristotle to Arabidopsis. Plant Cell 18:1542–1557

  28. Maruyama K, Todaka D, Mizoi J, Yoshida T, Kidokoro S, Matsukura S, Takasaki H, Sakurai T, Yamamoto YY, Yoshiwara K et al (2012) Identification of cis-acting promoter elements in cold- and dehydration-induced transcriptional pathways in Arabidopsis, rice, and soybean. DNA Res 19:37–49

  29. Nakashima K, Yamaguchi-Shinozaki K (2002) Use of β-glucuronidase to show dehydration and high-salt gene expression. In: Jackson JF, Linsken HF, Inman RB (eds) Molecular methods of plant analysis: Testing for genetic manipulation in plants, vol 22. Springer-Verlag, Berlin, pp 37–61

  30. Nakashima K, Yamaguchi-Shinozaki K (2013) ABA signaling in stress-response and seed development. Plant Cell Rep 32:959–970

  31. Nakashima K, Tran LS, 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 51:617–630

  32. Nakashima K, Ito Y, Yamaguchi-Shinozaki K (2009) Transcriptional regulatory networks in response to abiotic stresses in Arabidopsis and grasses. Plant Physiol 149:88–95

  33. Nakashima K, Takasaki H, Mizoi J, Shinozaki K, Yamaguchi-Shinozaki K (2012) NAC transcription factors in plant abiotic stress responses. Biochim Biophys Acta 1819:97–103

  34. Nuruzzaman M, Manimekalai R, Sharoni AM, Satoh K, Kondoh H, Ooka H, Kikuchi S (2010) Genome-wide analysis of NAC transcription factor family in rice. Gene 465:30–44

  35. Odell JT, Nagy F, Chua NH (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313:810–812

  36. Olsson AS, Engstrom P, Soderman E (2004) The homeobox genes ATHB12 and ATHB7 encode potential regulators of growth in response to water deficit in Arabidopsis. Plant Mol Biol 55:663–677

  37. Pellegrineschi A, Reynolds M, Pacheco M, Brito RM, Almeraya R, Yamaguchi-Shinozaki K, Hoisington D (2004) Stress-induced expression in wheat of the Arabidopsis thaliana DREB1A gene delays water stress symptoms under greenhouse conditions. Genome 47:493–500

  38. Polizel AM, Medri ME, Nakashima K, Yamanaka N, Farias JR, de Oliveira MC, Marin SR, Abdelnoor RV, Marcelino-Guimaraes FC, Fuganti R et al (2011) Molecular, anatomical and physiological properties of a genetically modified soybean line transformed with rd29A:AtDREB1A for the improvement of drought tolerance. Genet Mol Res 10:3641–3656

  39. Rabbani MA, Maruyama K, Abe H, Khan MA, Katsura K, Ito Y, Yoshiwara K, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Monitoring expression profiles of rice genes under cold, drought, and high-salinity stresses and abscisic acid application using cDNA microarray and RNA gel-blot analyses. Plant Physiol 133:1755–1767

  40. Rai M, He C, Wu R (2009) Comparative functional analysis of three abiotic stress-inducible promoters in transgenic rice. Transgenic Res 18:787–799

  41. Redillas MC, Jeong JS, Kim YS, Jung H, Bang SW, Choi YD, Ha SH, Reuzeau C, Kim JK (2012) The overexpression of OsNAC9 alters the root architecture of rice plants enhancing drought resistance and grain yield under field conditions. Plant Biotech J 10:792–805

  42. Saint Pierre C, Crossa JL, Bonnett D, Yamaguchi-Shinozaki K, Reynolds MP (2012) Phenotyping transgenic wheat for drought resistance. J Exp Bot 63:1799–1808

  43. Sakuma Y, Maruyama K, Osakabe Y, Qin F, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2006) Functional analysis of an Arabidopsis transcription factor, DREB2A, involved in drought-responsive gene expression. Plant Cell 18:1292–1309

  44. Su J, Shen Q, David Ho TH, Wu R (1998) Dehydration-stress-regulated transgene expression in stably transformed rice plants. Plant Physiol 117:913–922

  45. Takano M, Egawa H, Ikeda JE, Wakasa K (1997) The structures of integration sites in transgenic rice. Plant J 11:353–361

  46. Takasaki H, Maruyama K, Kidokoro S, Ito Y, Fujita Y, Shinozaki K, Yamaguchi-Shinozaki K, Nakashima K (2010) The abiotic stress-responsive NAC-type transcription factor OsNAC5 regulates stress-inducible genes and stress tolerance in rice. Mol Genet Genomics 284:173–183

  47. Todaka D, Nakashima K, Shinozaki K, Yamaguchi-Shinozaki K (2012) Toward understanding transcriptional regulatory networks in abiotic stress responses and tolerance in rice. Rice 5:6

  48. Tran LS, Nakashima K, Sakuma Y, Simpson SD, Fujita Y, Maruyama K, Fujita M, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2004) Isolation and functional analysis of Arabidopsis stress-inducible NAC transcription factors that bind to a drought-responsive cis-element in the early responsive to dehydration stress 1 promoter. Plant Cell 16:2481–2498

  49. Valdés AE, Overnas E, Johansson H, Rada-Iglesias A, Engstrom P (2012) The homeodomain-leucine zipper (HD-Zip) class I transcription factors ATHB7 and ATHB12 modulate abscisic acid signalling by regulating protein phosphatase 2C and abscisic acid receptor gene activities. Plant Mol Biol 80:405–418

  50. Wang X-S, Zhu H-B, Jin G-L, Liu H-L, Wu W-R, Zhu J (2007) Genome-scale identification and analysis of LEA genes in rice (Oryza sativa L.). Plant Sci 172:414–420

  51. Xiang Y, Tang N, Du H, Ye H, Xiong L (2008) Characterization of OsbZIP23 as a key player of the basic leucine zipper transcription factor family for conferring abscisic acid sensitivity and salinity and drought tolerance in rice. Plant Physiol 148:1938–1952

  52. Xiao F-H, Xue G-P (2001) Analysis of the promoter activity of late embryogenesis abundant protein genes in barley seedlings under conditions of water deficit. Plant Cell Rep 20:667–673

  53. Xiao B, Huang Y, Tang N, Xiong L (2007) Over-expression of a LEA gene in rice improves drought resistance under the field conditions. Theor Applied Genet 115:35–46

  54. Xiao BZ, Chen X, Xiang CB, Tang N, Zhang QF, Xiong LZ (2009) Evaluation of seven function-known candidate genes for their effects on improving drought resistance of transgenic rice under field conditions. Mol Plant 2:73–83

  55. Xu DQ, Huang J, Guo SQ, Yang X, Bao YM, Tang HJ, Zhang HS (2008) Overexpression of a TFIIIA-type zinc finger protein gene ZFP252 enhances drought and salt tolerance in rice (Oryza sativa L.). FEBS Lett 582:1037–1043

  56. Yang M, Xiong L (2011) Isolation and characterization of a drought-inducible promoter Oshox24P in rice. J Huazhong Agr Univ 30:525–531

  57. Yi N, Kim YS, Jeong MH, Oh SJ, Jeong JS, Park SH, Jung H, Choi YD, Kim JK (2010) Functional analysis of six drought-inducible promoters in transgenic rice plants throughout all stages of plant growth. Planta 232:743–754

  58. Yi N, Oh SJ, Kim YS, Jang HJ, Park SH, Jeong JS, Song SI, Choi YD, Kim JK (2011) Analysis of the Wsi18, a stress-inducible promoter that is active in the whole grain of transgenic rice. Transgenic Res 20:153–163

  59. Yokotani N, Ichikawa T, Kondou Y, Matsui M, Hirochika H, Iwabuchi M, Oda K (2009) Tolerance to various environmental stresses conferred by the salt-responsive rice gene ONAC063 in transgenic Arabidopsis. Planta 229:1065–1075

  60. Yoshida T, Fujita Y, Sayama H, Kidokoro S, Maruyama K, Mizoi J, Shinozaki K, Yamaguchi-Shinozaki K (2010) AREB1, AREB2, and ABF3 are master transcription factors that cooperatively regulate ABRE-dependent ABA signaling involved in drought stress tolerance and require ABA for full activation. Plant J 61:672–685

  61. Yu SM, Ko SS, Hong CY, Sun HJ, Hsing YI, Tong CG, Ho TH (2007) Global functional analyses of rice promoters by genomics approaches. Plant Mol Biol 65:417–425

  62. Zheng X, Chen B, Lu G, Han B (2009) Overexpression of a NAC transcription factor enhances rice drought and salt tolerance. Biochem Biophys Res Commun 379:985–989

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Acknowledgments

We are grateful for technical support provided by Kaori Amano, Emiko Kishi, Kyoko Murai, and Kyouko Yoshiwara of JIRCAS. We thank Masami Toyoshima for her excellent editorial support. This work was supported in part by the grants from the Ministry of Agriculture, Forestry and Fisheries (MAFF; in part by Genomics for Agricultural Innovation, Development of Abiotic Stress Tolerant Crops by DREB Genes), and the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN) of Japan.

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Correspondence to Kazuo Nakashima or Kazuko Yamaguchi-Shinozaki.

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Nakashima, K., Jan, A., Todaka, D. et al. Comparative functional analysis of six drought-responsive promoters in transgenic rice. Planta 239, 47–60 (2014). https://doi.org/10.1007/s00425-013-1960-7

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Keywords

  • Drought
  • High salinity
  • Transgenic rice
  • Stress-responsive promoter
  • Stress tolerance