Plant Growth Regulation

, Volume 88, Issue 3, pp 241–251 | Cite as

Functional characterization of OsHAK1 promoter in response to osmotic/drought stress by deletion analysis in transgenic rice

  • Guang ChenEmail author
  • Jiang Hu
  • Juan Lian
  • Yu Zhang
  • Li Zhu
  • Dali Zeng
  • Longbiao Guo
  • Ling Yu
  • Guohua XuEmail author
  • Qian QianEmail author
Original paper


The rice gene HAK1 (OsHAK1) is activated when the plant experiences drought stress. Here, a deletion analysis of the 3037 nt sequence lying upstream of the HAK1 translation initiation codon was carried out to identify which promoter region(s) are functionally important for its responsiveness to moisture stress. Four 5′ truncated sequences of the promoter (Dp1820, Dp1524, Dp1069 and Dp556) along with the intact sequence (Dp3037) were fused to GUS, and the resulting constructs transformed separately into rice. In the absence of any imposed abiotic stress, GUS activity was both ubiquitous and of similar intensity in plants carrying the Dp3037 and Dp1820 constructs, but was undetectable in those carrying either Dp1524, Dp1069 or Dp556. When the plants were subjected to moisture stress by the addition of polyethylene glycol to the culture medium, carriers of Dp3037 strongly expressed GUS, but carriers of Dp1820 did not respond, implying that a sequence(s) lying between the nucleotides − 3037 and − 1821 were responsible for osmotic stress inducibility. An in silico analysis of this key sequence revealed the presence of a number of putative cis-acting elements, including ABREs (abscisic acid-responsive) and DREs (dehydration-responsive). The 296 nt segment between the nucleotides − 1820 and − 1525 harbored both a CAAT-box and a TATA-box sequence. The Dp3037 sequence represents a potentially suitable candidate for regulating the expression of drought-responsive transgenes.


Oryza sativa Dehydration OsHAK1 GUS analysis Inducible promoter Cis-acting element 



This work was funded by National Natural Science Foundation of China (Grant Nos. 31601811, 31671666 and 31871594); Zhejiang Province Outstanding Youth Fund (Grant No. LR19C130001).

Supplementary material

10725_2019_504_MOESM1_ESM.docx (1.2 mb)
Supplementary material 1 (DOCX 1234 kb)


  1. Basak P, Sangma S, Mukherjee A, Agarwal T, Sengupta S, Ray S, Majumder AL (2018) Functional characterization of two myo-inositol-1-phosphate synthase (MIPS) gene promoters from the halophytic wild rice (Porteresia coarctata). Planta 248:1121–1141CrossRefGoogle Scholar
  2. Bate N, Twell D (1998) Functional architecture of a late pollen promoter: pollen-specific transcription is developmentally regulated by multiple stage-specific and co-dependent activator elements. Plant Mol Biol 37:859–869CrossRefGoogle Scholar
  3. Bhuria M, Goel P, Kumar S, Singh AK (2016) The promoter of AtUSP is co-regulated by phytohormones and abiotic stresses in Arabidopsis thaliana. Front Plant Sci 7:1957CrossRefGoogle Scholar
  4. Chen L, Jiang B, Wu C, Sun S, Hou W, Han T (2014) GmPRP2 promoter drives root-preferential expression in transgenic Arabidopsis and soybean hairy roots. BMC Plant Biol 14:245CrossRefGoogle Scholar
  5. Chen G, Feng H, Hu Q, Qu H, Chen A, Yu L, Xu G (2015a) Improving rice tolerance to potassium deficiency by enhancing OsHAK16p:WOX11-controlled root development. Plant Biotechnol J 13:833–848CrossRefGoogle Scholar
  6. Chen G, Hu Q, Luo LE, Yang T, Zhang S, Hu Y, Yu L, Xu G (2015b) Rice potassium transporter OsHAK1 is essential for maintaining potassium-mediated growth and functions in salt tolerance over low and high potassium concentration ranges. Plant, Cell Environ 38:2747–2765CrossRefGoogle Scholar
  7. Chen G, Liu C, Gao Z, Zhang Y, Jiang H, Zhu L, Ren D, Yu L, Xu G, Qian Q (2017) OsHAK1, a high-affinity potassium transporter, positively regulates responses to drought stress in rice. Front Plant Sci 8:1885CrossRefGoogle Scholar
  8. Chen G, Liu C, Gao Z, Zhang Y, Zhang A, Zhu L, Hu J, Ren D, Yu L, Xu G, Qian Q (2018a) Variation in the abundance of OsHAK1 transcript underlies the differential salinity tolerance of an indica and a japonica rice cultivar. Front Plant Sci 8:2216CrossRefGoogle Scholar
  9. Chen G, Liu C, Gao Z, Zhang Y, Zhu L, Hu J, Ren D, Xu G, Qian Q (2018b) Driving the expression of RAA1 with a drought-responsive promoter enhances root growth in rice, its accumulation of potassium and its tolerance to moisture stress. Environ Exp Bot 147:147–156CrossRefGoogle Scholar
  10. Chen G, Wu C, He L, Qiu Z, Zhang S, Zhang Y, Guo L, Zeng D, Hu J, Ren D, Qian Q, Zhu L (2018c) Knocking out the gene RLS1 induces hypersensitivity to oxidative stress and premature leaf senescence in rice. Int J Mol Sci 19:2853CrossRefGoogle Scholar
  11. Chen G, Zhang Y, Ruan B, Guo L, Zeng D, Gao Z, Zhu L, Hu J, Ren D, Yu L, Xu G, Qian Q (2018d) OsHAK1 controls the vegetative growth and panicle fertility of rice by its effect on potassium-mediated sugar metabolism. Plant Sci 274:261–270CrossRefGoogle Scholar
  12. Cheon BY, Kim HJ, Oh KH, Bahn SC, Ahn JH, Choi JW, Ok SH, Bae JM, Shin JS (2004) Overexpression of human erythropoietin (EPO) affects plant morphologies: retarded vegetative growth in tobacco and male sterility in tobacco and Arabidopsis. Transgenic Res 13:541–549CrossRefGoogle Scholar
  13. Cornejo MJ, Luth D, Blankenship KM, Anderson OD, Blechl AE (1993) Activity of a maize ubiquitin promoter in transgenic rice. Plant Mol Biol 23:567–581CrossRefGoogle Scholar
  14. De Wilde C, Van Houdt H, De Buck S, Angenon G, De Jaeger G, Depicker A (2000) Plants as bioreactors for protein production: avoiding the problem of transgene silencing. Plant Mol Biol 43:347–359CrossRefGoogle Scholar
  15. Dong Q, Jiang H, Xu Q, Li X, Peng X, Yu H, Xiang Y, Cheng B (2015) Cloning and characterization of a multifunctional promoter from Maize (Zea mays L.). Appl Biochem Biotechnol 175:1344–1357CrossRefGoogle Scholar
  16. Dutt M, Dhekney SA, Soriano L, Kandel R, Grosser JW (2014) Temporal and spatial control of gene expression in horticultural crops. Horticult Res 1:14047CrossRefGoogle Scholar
  17. Elmayan T, Tepfer M (1995) Evaluation in tobacco of the organ specificity and strength of the rolD promoter, domain A of the 35S promoter and the 35S2 promoter. Transgenic Res 4:388–396CrossRefGoogle Scholar
  18. Ezcurra I, Ellerström M, Wycliffe P, Stålberg K, Rask L (1999) Interaction between composite elements in the napA promoter: both the B-box ABA-responsive complex and the RY/G complex are necessary for seed-specific expression. Plant Mol Biol 40:699–709CrossRefGoogle Scholar
  19. Fehlberg V, Vieweg MF, Dohmann EM, Hohnjec N, Pühler A, Perlick AM, Küster H (2005) The promoter of the leghaemoglobin gene VfLb29: functional analysis and identification of modules necessary for its activation in the infected cells of root nodules and in the arbuscule-containing cells of mycorrhizal roots. J Exp Bot 56:799–806CrossRefGoogle Scholar
  20. Golldack D, Li C, Mohan H, Probst N (2014) Tolerance to drought and salt stress in plants: unraveling the signaling networks. Front Plant Sci 5:151CrossRefGoogle Scholar
  21. Gowik U, Burscheidt J, Akyildiz M, Schlue U, Koczor M, Streubel M, Westhoff P (2004) cis-Regulatory elements for mesophyll-specific gene expression in the C4 plant Flaveria trinervia, the promoter of the C4 phosphoenolpyruvate carboxylase gene. Plant Cell 16:1077–1090CrossRefGoogle Scholar
  22. Grotewold E, Drummond BJ, Bowen B, Peterson T (1994) The myb-homologous P gene controls phlobaphene pigmentation in maize floral organs by directly activating a flavonoid biosynthetic gene subset. Cell 76:543–553CrossRefGoogle Scholar
  23. Hamilton DA, Schwarz YH, Mascarenhas JP (1998) A monocot pollen-specific promoter contains separable pollen-specific and quantitative elements. Plant Mol Biol 38:663–669CrossRefGoogle Scholar
  24. Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res 27:297–300CrossRefGoogle Scholar
  25. Husebye H, Chadchawan S, Winge P, Thangstad OP, Bones AM (2002) Guard cell- and phloem idioblast-specific expression of thioglucoside glucohydrolase 1 (myrosinase) in Arabidopsis. Plant Physiol 128:1180–1188CrossRefGoogle Scholar
  26. Kagaya Y, Ohmiya K, Hattori T (1999) RAV1, a novel DNA-binding protein, binds to bipartite recognition sequence through two distinct DNA-binding domains uniquely found in higher plants. Nucleic Acids Res 27:470–478CrossRefGoogle Scholar
  27. Kaur C, Kumar G, Kaur S, Ansari MW, Pareek A, Sopory SK, Singla-Pareek SL (2015) Molecular cloning and characterization of salt overly sensitive gene promoter from Brassica juncea (BjSOS2). Mol Biol Rep 42:1139–1148CrossRefGoogle Scholar
  28. Kim DW, Lee SH, Choi SB, Won SK, Heo YK, Cho M, Park YI, Cho HT (2006) Functional conservation of a root hair cell-specific cis-element in angiosperms with different root hair distribution patterns. Plant Cell 18:2958–2970CrossRefGoogle Scholar
  29. Kovalchuk N, Smith J, Bazanova N, Pyvovarenko T, Singh R, Shirley N, Ismagul A, Johnson A, Milligan AS, Hrmova M, Langridge P, Lopato S (2012) Characterization of the wheat gene encoding a grain-specific lipid transfer protein TdPR61, and promoter activity in wheat, barley and rice. J Exp Bot 63:2025–2040CrossRefGoogle Scholar
  30. Kumpatla SP, Chandrasekharan MB, Iyer LM, Guofu L, Hall TC (1998) Genome intruder scanning and modulation systems and transgene silencing. Trends Plant Sci 3:97–104CrossRefGoogle Scholar
  31. Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouzé P, Rombauts S (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res 30:325–327CrossRefGoogle Scholar
  32. Lessard PA, Allen RD, Bernier F, Crispino JD, Fujiwara T, Beachy RN (1991) Multiple nuclear factors interact with upstream sequences of differentially regulated β-conglycinin genes. Plant Mol Biol 16:397–413CrossRefGoogle Scholar
  33. Manimaran P, Reddy MR, Rao TB, Mangrauthia SK, Sundaram RM, Balachandran SM (2015) Identification of cis-elements and evaluation of upstream regulatory region of a rice anther-specific gene, OSIPP3, conferring pollen-specific expression in Oryza sativa (L.) ssp. indica. Plant Reprod 28:133–142CrossRefGoogle Scholar
  34. Matzke MA, Matzke AJM (1995) Homology-dependent gene silencing in transgenic plants: what does it really tell us? Trends Genet 11:1–3CrossRefGoogle Scholar
  35. McElroy D, Zhang W, Cao J, Wu R (1990) Isolation of an efficient actin promoter for use in rice transformation. Plant Cell 2:163–171CrossRefGoogle Scholar
  36. Morita A, Umemura TA, Kuroyanagi M, Futsuhara Y, Perata P, Yamaguchi J (1998) Functional dissection of a sugar-repressed α-amylase gene (RAmy1A) promoter in rice embryos. FEBS Lett 423:81–85CrossRefGoogle Scholar
  37. Odell JT, Nagy F, Chua NH (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313:810–812CrossRefGoogle Scholar
  38. Pear JR, Ridge N, Rasmusgen R, Rose RE, Houck CM (1989) Isolation and characterization of a fruit-specific cDNA and the corresponding genomic clone from tomato. Plant Mol Biol 13:639–651CrossRefGoogle Scholar
  39. Plesch G, Ehrhardt T, Mueller-Roeber B (2001) Involvement of TAAAG elements suggests a role for Dof transcription factors in guard cell-specific gene expression. Plant J 28:455–464CrossRefGoogle Scholar
  40. Potenza C, Aleman L, Sengupta-Gopalan C (2004) Targeting transgene expression in research, agricultural, and environmental applications: promoters used in plant transformation. Vitro Cell Dev Biol Plant 40:1–22CrossRefGoogle Scholar
  41. Rai M, He C, Wu R (2009) Comparative functional analysis of three abiotic stress-inducible promoters in transgenic rice. Transgenic Res 18:787–799CrossRefGoogle Scholar
  42. Reyes JC, Muro-Pastor MI, Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice. Plant Physiol 134:1718–1732CrossRefGoogle Scholar
  43. Rogers HJ, Bate N, Combe J, Sullivan J, Sweetman J, Swan C, Lonsdale DM, Twell D (2001) Functional analysis of cis-regulatory elements within the promoter of the tobacco late pollen gene g10. Plant Mol Biol 45:577–585CrossRefGoogle Scholar
  44. Stålberg K, Ellerstöm M, Ezcurra I, Ablov S, Rask L (1996) Disruption of an overlapping E-box/ABRE motif abolished high transcription of the napA storage-protein promoter in transgenic Brassica napus seeds. Planta 199:515–519CrossRefGoogle Scholar
  45. Suzuki H, Fowler TJ, Tierney ML (1993) Deletion analysis and localization of SbPRP1, a soybean cell wall protein gene, in roots of transgenic tobacco and cowpea. Plant Mol Biol 21:109–119CrossRefGoogle Scholar
  46. Tester M, Langridge P (2010) Breeding technologies to increase crop production in a changing world. Science 327:818–822CrossRefGoogle Scholar
  47. Van Tunen AJ, Koes RE, Spelt CE, Van der Krol AR, Stuitje AR, Mol JN (1988) Cloning of the two chalcone flavanone isomerase genes from Petunia hybrida: coordinate, light-regulated and differential expression of flavonoid genes. EMBO J 7:1257–1263CrossRefGoogle Scholar
  48. Wang H, Fan M, Wang G, Zhang C, Shi L, Wei Z, Ma W, Chang J, Huang S, Lin F (2017) Isolation and characterization of a novel pollen-specific promoter in maize (Zea mays L.). Genome 60:485–495CrossRefGoogle Scholar
  49. Wang J, Song Z, Jia H, Yang S, Zhang H (2018) Characterization of wheat TaSnRK2. 7 promoter in Arabidopsis. Planta 248:1393–1401CrossRefGoogle Scholar
  50. Wu L, El-Mezawy A, Shah S (2011) A seed coat outer integument-specific promoter for Brassica napus. Plant Cell Rep 30:75–80CrossRefGoogle Scholar
  51. Xu W, Liu W, Ye R, Mazarei M, Huang D, Zhang X, Stewart CN (2018) A profilin gene promoter from switchgrass (Panicum virgatum L.) directs strong and specific transgene expression to vascular bundles in rice. Plant Cell Rep 37:587–597CrossRefGoogle Scholar
  52. Xue M, Long Y, Zhao Z, Huang G, Huang K, Zhang T, Jiang Y, Yuan Q, Pei X (2018) Isolation and characterization of a green-tissue promoter from common wild rice (Oryza rufipogon Griff.). Int J Mol Sci 19:2009CrossRefGoogle Scholar
  53. Yamaguchi-Shinozaki K, Shinozaki K (1993) Characterization of the expression of a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants. Mol Gen Genet 236:331–340CrossRefGoogle Scholar
  54. Yanagisawa S (2000) Dof1 and Dof2 transcription factors are associated with expression of multiple genes involved in carbon metabolism in maize. Plant J 21:281–288CrossRefGoogle Scholar
  55. Yevtushenko DP, Misra S (2018) Spatiotemporal activities of Douglas-fir BiP Pro1 promoter in transgenic potato. Planta 248:1569–1579CrossRefGoogle Scholar
  56. Yoshida T, Mogami J, Yamaguchi-Shinozaki K (2014) ABA-dependent and ABA-independent signaling in response to osmotic stress in plants. Curr Opin Plant Biol 21:133–139CrossRefGoogle Scholar
  57. Zhang H, Hou J, Jiang P, Qi S, Xu C, He Q, Ding Z, Wang Z, Zhang K, Li K (2016) Identification of a 467 bp promoter of maize phosphatidylinositol synthase gene (ZmPIS) which confers high-level gene expression and salinity or osmotic stress inducibility in transgenic tobacco. Front Plant Sci 7:42Google Scholar
  58. Zhang H, Jing R, Mao X (2017) Functional characterization of TaSnRK2.8 promoter in response to abiotic stresses by deletion analysis in transgenic Arabidopsis. Front Plant Sci 8:1198CrossRefGoogle Scholar
  59. Zhou J, Yang Y, Wang X, Yu F, Yu C, Chen J, Cheng Y, Yan C, Chen J (2013) Enhanced transgene expression in rice following selection controlled by weak promoters. BMC Biotechnol 13:29CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Rice BiologyChina National Rice Research InstituteHangzhouPeople’s Republic of China
  2. 2.State Key Laboratory of Crop Genetics and Germplasm Enhancement, MOA Key Laboratory of Plant Nutrition and Fertilization in Lower-Middle Reaches of the Yangtze RiverNanjing Agricultural UniversityNanjingPeople’s Republic of China

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