Molecular Biology Reports

, Volume 43, Issue 1, pp 17–30 | Cite as

A novel PR10 promoter from Erianthus arundinaceus directs high constitutive transgene expression and is enhanced upon wounding in heterologous plant systems

  • M. Chakravarthi
  • Divya P. Syamaladevi
  • P. Harunipriya
  • Sruthy Maria Augustine
  • N. Subramonian
Original Article


In genetic engineering, inducible promoters play an important role as the expression of genes driven by them can be turned on or off under situations like biotic or abiotic factors. There are few reports on inducible promoters that can be employed in the development of transgenic plants, particularly in sugarcane. In the present study, four wound inducible genes (Chitinase, PR1A, PR10 and HRGP) were selected and were amplified from Erianthus arundinaceus, a distant relative of sugarcane. In order to determine the gene that is highly induced upon wounding, RT-qPCR was performed, which showed that PR10 gene expression was instantaneous and higher upon wounding when compared to the other three genes. Using the random amplification of genomic ends technique, a 592 bp promoter sequence was obtained and in silico analysis of the upstream regulatory region revealed a 469 bp promoter and 123 bp of 5′ untranslated region (UTR). Functional analyses of the promoter sequence (with and without 5′ UTR) in tobacco, rice and sugarcane using β-glucuronidase (GUS) as the reporter gene revealed the constitutive and inducible nature of the PR10 promoter. Our studies have demonstrated that the PR10 promoter, though highly constitutive, was quickly induced upon wounding as well as on treatment with abscisic acid and methyl jasmonate hormones. This is the first report on the isolation and characterization of a PR10 promoter from a wild grass and is expected to have application for development of transgenic plants.


Inducible promoter PR10 Wounding Erianthus 5′ regulatory sequence 



The authors would like to thank the Indian Council of Agricultural Research, New Delhi and Sugarcane Breeding Institute, Coimbatore, India for the funding and infrastructure.


  1. 1.
    Potenza C, Aleman L, Sengupta-Gopalan C (2004) Targeting transgene expression in research, agricultural, and environmental applications: promoters used in plant transformation. In Vitro Cell Dev Biol Plant 40(1):1–22CrossRefGoogle Scholar
  2. 2.
    van Loon LC, Rep M, Pieterse CM (2006) Significance of inducible defense-related proteins in infected plants. Annu Rev Phytopathol 44:135–162PubMedCrossRefGoogle Scholar
  3. 3.
    Zeevaart JAD, Creelman RA (1988) Metabolism and physiology of abscisic acid. Annu Rev Plant Physiol Plant Mol Biol 39:439–473CrossRefGoogle Scholar
  4. 4.
    Reymond P, Farmer EE (1998) Jasmonate and salicylate as global signals for defense gene expression. Curr Opin Plant Biol 1:404–411PubMedCrossRefGoogle Scholar
  5. 5.
    Turner JG, Ellis C, Devoto A (2002) The jasmonate signal pathway. Plant Cell 14(Suppl):S153–S164PubMedPubMedCentralGoogle Scholar
  6. 6.
    Delessert C, Wilson I, Van Der Straeten D, Dennis E, Dolferus R (2004) Spatial and temporal analysis of the local response to wounding. Plant Mol Biol 55(2):165–181PubMedCrossRefGoogle Scholar
  7. 7.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods 25:402–408PubMedCrossRefGoogle Scholar
  8. 8.
    Kuriakose B, Ganesan V, Thomas G, Viswanathan A, Anand N (2009) Random amplification of genomic ends (RAGE) as an efficient method for isolation and cloning of promoters and uncloned genomic regions. Afr J Biotechnol 8(19):4765–4773Google Scholar
  9. 9.
    Philip A, Syamaladevi DP, Chakravarthi M, Gopinath K, Subramonian N (2013) 5′ Regulatory region of ubiquitin 2 gene from Porteresia coarctata makes efficient promoters for transgene expression in monocots and dicots. Plant Cell Rep 32(8):1199–1210PubMedCrossRefGoogle Scholar
  10. 10.
    Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucl Acids Res 27:297–300PubMedCrossRefPubMedCentralGoogle Scholar
  11. 11.
    Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13:2498–2504PubMedCrossRefPubMedCentralGoogle Scholar
  12. 12.
    Kumar KK, Maruthasalam S, Loganathan M, Sudhakar D, Balasubramanian P (2005) An improved Agrobacterium-mediated transformation protocol for recalcitrant elite indica rice cultivars. Plant Mol Biol Rep 23(1):67–73CrossRefGoogle Scholar
  13. 13.
    Horsch RB, Fry JE, Hoffman NL, Eicholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231CrossRefGoogle Scholar
  14. 14.
    Arvinth S, Arun S, Selvakesavan RK, Srikanth J, Mukunthan N, Kumar PA, Premachandran MN, Subramonian N (2010) Genetic transformation and pyramiding of aprotinin-expressing sugarcane with cry1Ab for shoot borer (Chilo infuscatellus) resistance. Plant Cell Rep 29(4):383–395PubMedCrossRefGoogle Scholar
  15. 15.
    Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907PubMedPubMedCentralGoogle Scholar
  16. 16.
    Curtis MD, Rae AL, Rusu AG, Harrison SJ, Manners JM (1997) A peroxidase gene promoter induced by phytopathogens and methyl jasmonate in transgenic plants. Mol Plant-Microbe Interact 10(3):326–338PubMedCrossRefGoogle Scholar
  17. 17.
    Eulgem T, Rushton PJ, Robatzek S, Somssich IE (2000) The WRKY superfamily of plant transcription factors. Trends Plant Sci 5(5):199–206PubMedCrossRefGoogle Scholar
  18. 18.
    Xu X, Chen C, Fan B, Chen Z (2006) Physical and functional interactions between pathogen-induced Arabidopsis WRKY18 WRKY40 and WRKY60 transcription factors. Plant Cell 18(5):1310–1326PubMedCrossRefPubMedCentralGoogle Scholar
  19. 19.
    Nishiuchi T, Shinshi H, Suzuki K (2004) Rapid and transient activation of transcription of the ERF3 gene by wounding in tobacco leaves: possible involvement of NtWRKYs and autorepression. J Biol Chem 279(53):55355–55361PubMedCrossRefGoogle Scholar
  20. 20.
    Brown RL, Kazan K, McGrath KC, Maclean DJ, Manners JM (2003) A role for the GCC-box in jasmonate-mediated activation of the PDF12 gene of Arabidopsis. Plant Physiol 132(2):1020–1032PubMedCrossRefPubMedCentralGoogle Scholar
  21. 21.
    Maeda K, Kimura S, Demura T, Takeda J, Ozeki Y (2005) DcMYB1 acts as a transcriptional activator of the carrot phenylalanine ammonia-lyase gene (DcPAL1) in response to elicitor treatment, UV-B irradiation and the dilution effect. Plant Mol Biol 59(5):739–752PubMedCrossRefGoogle Scholar
  22. 22.
    Benfey PN, Chua N-H (1990) The cauliflower mosaic virus 35S promoter: combinatorial regulation of transcription in plants. Science 250:959–966PubMedCrossRefGoogle Scholar
  23. 23.
    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(5):577–585PubMedCrossRefGoogle Scholar
  24. 24.
    Stalberg K, Ellerstom 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(4):515–519PubMedCrossRefGoogle Scholar
  25. 25.
    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(5):1077–1090PubMedCrossRefPubMedCentralGoogle Scholar
  26. 26.
    Sandal NN, Bojsen K, Marcker KA (1987) A small family of nodule specific genes from soybean. Nucleic Acids Res 15(4):1507–1519PubMedCrossRefPubMedCentralGoogle Scholar
  27. 27.
    Kim HJ, Kim YK, Park JY, Kim J (2002) Light signalling mediated by phytochrome plays an important role in cold-induced gene expression through the C-repeat/dehydration responsive element (C/DRE) in Arabidopsis thaliana. Plant J 29(6):693–704PubMedCrossRefGoogle Scholar
  28. 28.
    Hudson ME, Quail PH (2003) Identification of promoter motifs involved in the network of phytochrome A-regulated gene expression by combined analysis of genomic sequence and microarray data. Plant Physiol 133:1605–1616PubMedCrossRefPubMedCentralGoogle Scholar
  29. 29.
    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(3):543–553PubMedCrossRefGoogle Scholar
  30. 30.
    Sablowski RW, Moyano E, Culianez-Macia FA, Schuch W, Martin C, Bevan M (1994) A flower-specific Myb protein activates transcription of phenylpropanoid biosynthetic genes. EMBO J 13(1):128PubMedPubMedCentralGoogle Scholar
  31. 31.
    Abe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell 15(1):63–78PubMedCrossRefPubMedCentralGoogle Scholar
  32. 32.
    Simpson SD, Nakashima K, Narusaka Y, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Two different novel cis-acting elements of erd1 a clpA homologous Arabidopsis gene function in induction by dehydration stress and dark-induced senescence. Plant J 33:259–270PubMedCrossRefGoogle Scholar
  33. 33.
    Shirsat A, Wilford N, Croy R, Boulter D (1989) Sequences responsible for the tissue specific promoter activity of a pea legumin gene in tobacco. Mol Gen Genet 215:326–331PubMedCrossRefGoogle Scholar
  34. 34.
    Yanagisawa S (2000) Dof1 and Dof2 transcription factors are associated with expression of multiple genes involved in carbon metabolism in maize. Plant J 21(3):281–288PubMedCrossRefGoogle Scholar
  35. 35.
    Ohme-Takagi M, Shinshi H (1995) Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element. Plant Cell 7(2):173–182PubMedCrossRefPubMedCentralGoogle Scholar
  36. 36.
    Rouster J, Leah R, Mundy J, Cameron-Mills V (1997) Identification of a methyl jasmonate-responsive region in the promoter of a lipoxygenase 1 gene expressed in barley grain. Plant J 11:513–523PubMedCrossRefGoogle Scholar
  37. 37.
    Shinozaki K, Yamaguchi-Shinozaki K (1996) Molecular responses to drought and cold stress. Curr Opin Biotechnol 7:161–167PubMedCrossRefGoogle Scholar
  38. 38.
    Somssich IE, Schmelzer E, Kawalleck P, Hahlbrock K (1988) Gene structure and its in situ transcript localization of pathogenesis related protein 1 in parsley. Mol Gen Genet 213(1):93–98PubMedCrossRefGoogle Scholar
  39. 39.
    Schmelzer E, Kruger-Lebus S, Hahlbrock K (1989) Temporal and spatial patterns of gene expression around sites of attempted fungal infection in parsley leaves. Plant Cell 1:993–1001PubMedCrossRefPubMedCentralGoogle Scholar
  40. 40.
    Warner SA, Scott R, Draper J (1992) Characterization of a wound-induced transcript from the monocot asparagus that shares similarity with a class of intracellular pathogenesis related (PR) proteins. Plant Mol Biol 19:555–561PubMedCrossRefGoogle Scholar
  41. 41.
    Lo SCC, Hipskind JD, Nicholson RL (1999) cDNA cloning of a sorghum pathogenesis-related protein (PR-10) and differential expression of defense-related genes following inoculation with Cochliobolus heterostrophus or Colletotrichum sublineolum. Mol Plant-Microbe Interact 12:479–489PubMedCrossRefGoogle Scholar
  42. 42.
    Handschuh L, Femiak I, Kasperska A, Figlerowicz M, Sikorski MM (2007) Structural and functional characteristics of two novel members of pathogensis-related multigene family of class 10 from yellow lupine. Acta Biochim Pol 54(4):783–796PubMedGoogle Scholar
  43. 43.
    Lu J, Sivamani E, Azhakanandam K, Samadder P, Li X, Qu R (2008) Gene expression enhancement mediated by the 5′ UTR intron of the rice rubi3 gene varied remarkably among tissues in transgenic rice plants. Mol Genet Gen 279:563–572CrossRefGoogle Scholar
  44. 44.
    Samadder P, Sivamani E, Lu J, Li X, Qu R (2008) Transcriptional and post-transcriptional enhancement of gene expression by the 5′ UTR intron of rice rubi3 gene in transgenic rice cells. Mol Genet Genomics 279:429–439PubMedCrossRefGoogle Scholar
  45. 45.
    Rakwal R, Agrawal GK, Yonekura M (2001) Light-dependent induction of OsPR10 in rice (Oryza sativa L.) seedlings by the global stress signaling molecule jasmonic acid and protein phosphatase 2A inhibitors. Plant Sci 161:469–479CrossRefGoogle Scholar
  46. 46.
    Wang CS, Huang JC, Hu JH (1999) Characterization of two subclasses of PR-10 transcripts in lily anthers and induction of their genes through separate signal transduction pathways. Plant Mol Biol 40:807–814PubMedCrossRefGoogle Scholar
  47. 47.
    McGee JD, Hamer JE, Hodges TK (2001) Characterization of a PR-10 pathogenesis-related gene family induced in rice during infection with Magnaporthe grisea. Mol Plant-Microbe Interact 14:877–886PubMedCrossRefGoogle Scholar
  48. 48.
    Jwa NS, Kumar AG, Rakwal R, Park CH, Prasad AV (2001) Molecular cloning and characterization of a novel Jasmonate inducible pathogenesis-related class 10 protein gene, JIOsPR10, from rice (Oryza sativa L.) seedling leaves. Biochem Biophys Res Commun 286:973–983PubMedCrossRefGoogle Scholar
  49. 49.
    Zhou XJ, Lu S, Xu YH, Wang JW, Chen XY (2002) A cotton cDNA (GaPR-10) encoding a pathogenesis-related 10 protein with in vitro ribonuclease activity. Plant Sci 162:629–636CrossRefGoogle Scholar
  50. 50.
    Kim ST, Yu S, Kang YH, Kim SG, Kim JY, Kim SH, Kang KY (2008) The rice pathogen-related protein 10 (JIOsPR10) is induced by abiotic and biotic stresses and exhibits ribonuclease activity. Plant Cell Rep 27(3):593–603PubMedCrossRefGoogle Scholar
  51. 51.
    Hwang SH, Lee IA, Yie SW, Hwang DJ (2008) Identification of an OsPR10a promoter region responsive to salicylic acid. Planta 227(5):1141–1150PubMedCrossRefPubMedCentralGoogle Scholar
  52. 52.
    Bahramnejad B, Goodwin PH, Zhang J, Atnaseo C, Erickson LR (2010) A comparison of two class 10 pathogenesis-related genes from alfalfa and their activation by multiple stresses and stress-related signaling molecules. Plant Cell Rep 29(11):1235–1250PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • M. Chakravarthi
    • 1
  • Divya P. Syamaladevi
    • 1
    • 2
  • P. Harunipriya
    • 1
  • Sruthy Maria Augustine
    • 1
  • N. Subramonian
    • 1
  1. 1.ICAR-Sugarcane Breeding InstituteCoimbatoreIndia
  2. 2.Directorate of Rice Research (ICAR)HyderabadIndia

Personalised recommendations