3 Biotech

, 8:380 | Cite as

Enhanced vascular activity of a new chimeric promoter containing the full CaMV 35S promoter and the plant XYLOGEN PROTEIN 1 promoter

  • Yu-mei Chen
  • Yi-hu Dong
  • Zhi-bin Liang
  • Lian-hui ZhangEmail author
  • Yi-zhen DengEmail author
Original Article


To develop a new strategy that controls vascular pathogen infections in economic crops, we examined a possible enhancer of the vascular activity of XYLOGEN PROTEIN 1 promoter (Px). This protein is specifically expressed in the vascular tissues of Arabidopsis thaliana and plays an important role in xylem development. Although Px is predicted as vascular-specific, its activity is hard to detect and highly susceptible to plant and environmental conditions. The cauliflower mosaic virus 35S promoter (35S) is highly active in directing transgene expression. To test if 35S could enhance Px activity, while vascular specificity of the promoter is retained, we examined the expression of the uidA reporter gene, which encodes β-glucuronidase (GUS), under the control of a chimeric promoter (35S-Px) or Px by generating 35S-Px-GUS and Px-GUS constructs, which were transformed into tobacco seedlings. Both 35S-Px and Px regulated gene expression in vascular tissues. However, GUS expression driven by 35S-Px was not detected in 30- and 60-day-old plants. Quantitative real-time PCR analysis showed that GUS gene expression regulated by 35S-Px was 6.2–14.9-fold higher in vascular tissues than in leaves. Histochemical GUS staining demonstrated that 35S-Px was strongly active in the xylem and phloem. Thus, fusion of 35S and Px might considerably enhance the strength of Px and increase its vascular specificity. In addition to confirming that 35S enhances the activity of a low-level tissue-specific promoter, these findings provide information for further improving the activity of such promoters, which might be useful for engineering new types of resistant genes against vascular infections.


Plant vascular pathogens XYLOGEN PROTEIN 1 CaMV 35S promoter Chimeric promoters Promoter engineering 



The study was financed by the National Basic Research Program of China 973 (Grant No. 2015CB150600).

Compliance with ethical standards

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Supplementary material

13205_2018_1379_MOESM1_ESM.tif (4.8 mb)
Supplementary material 1: Figure S1 DNA sequence of the AtXYP1 promoter (Px). The promoter sequence contains part of AtXYP1 coding region and its 5′region. Symbol: +1, the first codon of AtXYP1 coding region. Important cis-elements were identified and labeled: (1) CAAT box, core promoter element; (2) TATA box, core promoter element; (3) AC elements, AC-rich region responsible for vascular-specific activity; (4) TCA element, element involved in salicylic acid response; (5) Ocs-element, element involved in auxin and salicylic acid response. (TIF 4874 KB)
13205_2018_1379_MOESM2_ESM.eps (4.6 mb)
Supplementary material 2: Figure S2 PCR analysis of the reporter sequences with T1 generation of transgenic tobacco. a, Detection of GUS gene (upper panel, 1.7 kb) and Px sequence (lower panel, 2.4 kb) by PCR amplification in Z4 (Px-GUS) transgenic lines (lanes 1–14). b, Detection of GUS gene (upper panel) and Px sequence (lower panel) by PCR amplification in Z5 (35S-Px-GUS) transgenic lines (lanes 1–13). c, Detection of GUS gene by PCR amplification in 35S (35S-GUS) transgenic lines (lanes 1–15). Symbols: M, DNA size markers; W, wild type tobacco as a negative control; P, Z5 plasmid as a positive control. (EPS 4748 KB)


  1. Benfey PN, Ren L, Chua NH (1989) The CaMV 35S enhancer contains at least two domains which can confer different developmental and tissue-specific expression patterns. EMBO J 8(8):2195–2202CrossRefPubMedPubMedCentralGoogle Scholar
  2. Benfey PN, Ren L, Chua NH (1990) Combinatorial and synergistic properties of CaMV 35S enhancer subdomains. EMBO J 9(6):1685–1696CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bevan M, Shuffle bottom D, Edwards K et al (1989) Tissue- and cell- specific activity of a phenylalanine ammonia-lyase promoter in transgenic plants. EMBO J 8(7):1899–1906CrossRefPubMedPubMedCentralGoogle Scholar
  4. Boatwright JL, Pajerowska-Mukhtar K (2013) Salicylic acid: an old hormone up to new tricks. Mol Plant Pathol 14(6):623–634CrossRefPubMedGoogle Scholar
  5. Bustin SA, Benes V, Garson JA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55(4):611–622CrossRefPubMedGoogle Scholar
  6. Campos L, Granell P, Tárraga S et al (2014) Salicylic acid and gentisic acid induce RNA silencing-related genes and plant resistance to RNA pathogens. Plant Physiol Biochem 77:35–43CrossRefPubMedGoogle Scholar
  7. Comai L, Moran P, Maslyar D (1990) Novel and useful properties of a chimeric plant promoter combining CaMV 35S and MAS elements. Plant Mol Biol 15(3):373–381CrossRefPubMedGoogle Scholar
  8. Dong Y, Wang L, Xu J (2002) Quenching quorum-sensing dependent bacterial infection by an N-acyl homoserine lactonase. Nature 411:813–816CrossRefGoogle Scholar
  9. During K (1993) Can lysozymes mediate antibacterial resistance in plants? Plant Mol Biol 23(1):209–214CrossRefPubMedGoogle Scholar
  10. Florack D, Allefs S, Bollen R et al (1995) Expression of giant silk moth cecropin B genes in tobacco. Transgenic Res 4(2):132–141CrossRefPubMedGoogle Scholar
  11. Fukuda H (2004) Plant cell biology: Signals that control plant vascular cell differentiation. Nature Rev Mol Cell Biol 5(5):379–391CrossRefGoogle Scholar
  12. Gómez-Ros LV, Gabaldón C, López Núñez-Flores MJ et al (2012) The promoter region of the Zinnia elegans basic peroxidase isoenzyme gene contains cis-elements responsive to nitric oxide and hydrogen peroxide. Planta 236(2):327–342CrossRefPubMedGoogle Scholar
  13. Gray-Mitsumune M, Molitor EK, Cukovic D et al (1999) Developmentally regulated patterns of expression directed by poplar PAL promoters in transgenic tobacco and poplar. Plant Mol Biol 39(4):657–669CrossRefPubMedGoogle Scholar
  14. Hauffe KD, Paszkowski U, Schulze-Lefert P et al (1991) A parsley 4CL-1 promoter fragment specifies complex expression patterns in transgenic tobacco. Plant Cell 3(5):435–443CrossRefPubMedPubMedCentralGoogle Scholar
  15. Hauffe K, Lee SP, Subramaniam R (1993) Combinatorial interactions between positive and negative cis-acting elements control spatial patterns of 4CL-1 expression in transgenic tobacco. Plant J 4(2):235–253CrossRefPubMedGoogle Scholar
  16. Horsch R (1985) A simple and general method for transferring genes into plants. Science 227(4691):1229–1231CrossRefGoogle Scholar
  17. Janes KA (2015) An analysis of critical factors for quantitative immunoblotting. Sci Signal 8(371):rs2CrossRefPubMedPubMedCentralGoogle Scholar
  18. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6(13):3901–3907CrossRefPubMedPubMedCentralGoogle Scholar
  19. Keller B, Heierli D (1994) Vascular expression of the grp1.8 promoter is controlled by three specific regulatory elements and one unspecific activating sequence. Plant MolBiol 26:747–756Google Scholar
  20. Keller B, Schmid J, Lamb CJ (1989) Vascular expression of a bean cell wall glycine-rich protein–β-glucuronidase gene fusion in transgenic tobacco. EMBO J 8(5):1309–1314CrossRefPubMedPubMedCentralGoogle Scholar
  21. Kobayashi Y, Motose H, Iwamoto K et al (2011) Expression and genome-wide analysis of the xylogen-type gene family. Plant Cell Physiol 52(6):1095–1106CrossRefPubMedGoogle Scholar
  22. Lescot M, Déhais P, Thijs G et al (2002) Plant CARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res 30(1):325–327CrossRefPubMedPubMedCentralGoogle Scholar
  23. Liu ZZ, Wang JL, Huang X et al (2003) The promoter of a rice glycine-rich protein gene, Osgrp-2, confers vascular-specific expression in transgenic plants. Planta 216(5):824–833PubMedGoogle Scholar
  24. Liu W, Yuan JS, Stewart CJ (2013) Advanced genetic tools for plant biotechnology. Nat Rev Genet 14(11):781–793CrossRefPubMedGoogle Scholar
  25. Lorang JM, Shen H, Kobayashi D et al (1994) avrA and avrE in Pseudomonas syringaepv. tomato PT23 play a role in virulence on tomato plants. Mol Plant-Microbe Interact 7(4):508–515CrossRefGoogle Scholar
  26. Mansfield J, Genin S, Magori S et al (2012) Top 10 plant pathogenic bacteria in molecular plant pathology. Mol Plant Pathol 13(6):614–629CrossRefPubMedGoogle Scholar
  27. Motose H, Sugiyama M, Fukuda H (2004) A proteoglycan mediates inductive interaction during plant vascular development. Nature 429(6994):873–878CrossRefPubMedGoogle Scholar
  28. Odell JT, Nagy F, Chua NH (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313(6005):810–812CrossRefPubMedGoogle Scholar
  29. Osakabe Y, Chiang K (2009) Isolation of 4-coumarate co-A ligase gene promoter from Loblolly pine (Pinus taeda) and characterization of tissue-specific activity in transgenic tobacco. Plant PhysiolBiochem 11(47):1031–1036Google Scholar
  30. Rivero M, Furman N, Mencacci N (2012) Stacking of antimicrobial genes in potato transgenic plants confers increased resistance to bacterial and fungal pathogens. J Biotechnol 157(2):334–343CrossRefPubMedGoogle Scholar
  31. Robert CA, Erb M, Hitpold I et al (2013) Genetically engineered maize plants reveal distinct costs and benefits of constitutive volatile emissions in the field. Plant Biotechnol J 11(5):628–639CrossRefPubMedGoogle Scholar
  32. Rombauts S, Déhais P, Van Montagu M, Rouzé P (1999) Plant CARE, a plant cis-acting regulatory element database. Nucleic Acids Res 27(1):295–296CrossRefPubMedPubMedCentralGoogle Scholar
  33. Sandelin A, Alkema W, Engstrom P, Wasserman WW, Lenhard B (2004) JASPAR: an open-access database for eukaryotic transcription factor binding profiles. Nucleic Acids Res 32(Database issue):91–94CrossRefGoogle Scholar
  34. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3(6):1101–1108CrossRefPubMedGoogle Scholar
  35. Tyagi W, Rajagopal D, Singla-Pareek SL et al (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(8):1411–1422CrossRefPubMedGoogle Scholar
  36. Wang GL, Song WY, Ruan DL et al (1996) The cloned gene, Xa21, confers resistance to multiple Xanthomonas oryzae pv. oryzae isolates in transgenic plants. Mol Plant Microbe Interact 9(9):850–855CrossRefPubMedGoogle Scholar
  37. Xu W, Liu W, Ye R et al (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(4):587–597CrossRefPubMedGoogle Scholar
  38. Zhang B, Singh KB (1994) ocs element promoter sequences are activated by auxin and salicylic acid in Arabidopsis. Proc Natl Acad Sci USA 91(7):2507–2511CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.State Key Laboratory for Conservation and Utilization of Subtropical Agro-BioresourcesSouth China Agricultural UniversityGuangzhouChina
  2. 2.Guangdong Province Key Laboratory of Microbial Signals and Disease Control, College of AgricultureSouth China Agricultural UniversityGuangzhouChina
  3. 3.Integrative Microbiology Research CenterSouth China Agricultural UniversityGuangzhouChina
  4. 4.Institute of Molecular and Cell BiologySingapore CitySingapore

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