, Volume 219, Issue 2, pp 233–242 | Cite as

Phloem-specific expression of the wound-inducible ribonuclease LE from tomato (Lycopersicon esculentum cv. Lukullus)

  • Margret Köck
  • Nadine Groß
  • Irene Stenzel
  • Gerd Hause
Original Article


Ribonuclease LE (RNaseLE) from tomato (Lycopersicon esculentum Mill. cv. Lukullus) belongs to the widespread RNase T2 family of ribonucleases. With the exception of S-RNases of the solanaceous self-incompatibility system the functions of other members of the RNase T2 family are only barely understood. Using a 2.6-kbp putative promoter sequence of RNaseLE in front of the uidA reporter gene, expression of β-glucuronidase in developing phloem tissue and, especially, in the meristematic and elongation zones at root tips was detected. The tissue-specific expression accords with the range of cis-acting elements detected in the RNaseLE promoter. RNaseLE mRNA was localized in developing phloem cells but not in mature phloem tissue, suggesting association of RNaseLE expression with phloem development. Histochemical staining of β-glucuronidase activity as well as detailed inspection of RNaseLE at mRNA, protein and enzyme activity levels revealed that the wound-induced expression of RNaseLE was also restricted to vascular tissue. RNaseLE transcript accumulation detected by in situ hybridization occurred preferentially in phloem and cambial cells of stem sections upon wounding. The data provide evidence for a role of RNaseLE in a tissue-specific wound response and in wound healing of tomato.


β-Glucuronidase Lycopersicon (wound response) Phloem Promoter study Ribonuclease Tissue specificity 



β-Glucuronidase (uidA gene)


Tomato ribonuclease LE


Inorganic phosphate



We thank Dr. M. Ganal (IPK Gatersleben) for providing the genomic tomato library and cultivating primary tomato transformants, Dr. S. Hertel and K. Lehmann (Biocenter) for regeneration and selection of PromLE2::uidA tomato lines, K. Eichhorst and K. Klar for technical assistance, and Prof. C. Wasternack (IPB Halle) for critical reading of the manuscript. The support of the Deutsche Forschungsgemeinschaft to M.K. (SFB 363 B9/B16) is gratefully acknowledged.


  1. Abel S, Köck M (2001) Secretory acid ribonucleases from tomato (Lycopersicon esculentum Mill.). Methods Enzymol 341:351–368PubMedGoogle Scholar
  2. Abel S, Krauss G-J, Glund K (1989) Ribonuclease in tomato vacuoles: high-performance liquid chromatographic analysis of ribonucleolytic activities and base specificity. Biochim Biophys Acta 998:145–150CrossRefGoogle Scholar
  3. Bariola PA, Howard CJ, Taylor CB, Verburg MT, Jaglan VD, Green PJ (1994) The Arabidopsis ribonuclease gene RNS1 is tightly controlled in response to phosphate limitation. Plant J 6:673–685PubMedGoogle Scholar
  4. Bevan M, Shufflebottom D, Edwards K, Jefferson R, Schuch W (1989) Tissue- and cell-specific activity of a phenylalanine ammonia-lyase promoter in transgenic plants. EMBO J 8:1899–1906PubMedGoogle Scholar
  5. Bosse D, Köck M (1998) Influence of phosphate starvation on phosphohydrolases during development of tomato seedlings. Plant Cell Environ 21:325–332CrossRefGoogle Scholar
  6. Dodds PN, Clarke AE, Newbigin E (1996) Molecular characterisation of an S-like RNase of Nicotiana alata that is induced by phosphate starvation. Plant Mol Biol 31:227–238PubMedGoogle Scholar
  7. Eschrich W, Heyser W (1975) Biochemistry of phloem constituents. In: Zimmermann MH, Milburn JA (eds) Transport in plants. I. Phloem transport. Encyclopedia of plant physiology, vol 1. Springer, Berlin Heidelberg New York, pp 101–136Google Scholar
  8. Hatton D, Sablowski R, Yung M-H, Smith C, Schuch W, Bevan M (1995) Two classes of cis sequences contribute to tissue-specific expression of a PAL2 promoter in transgenic tobacco. Plant J 7:859–876PubMedGoogle Scholar
  9. Hause B, Hause G, Kutter C, Miersch O, Wasternack C (2003) Enzymes of jasmonate biosynthesis occur in tomato sieve elements. Plant Cell Physiol 44:643–648CrossRefPubMedGoogle Scholar
  10. Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res 27:297–300PubMedGoogle Scholar
  11. Höfgen R, Willmitzer L (1988) Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res 16:9877PubMedGoogle Scholar
  12. Ide H, Kimura M, Arai M, Funatsu G (1991) The complete amino acid sequence of ribonuclease from the seeds of bitter gourd (Momordica charantia). FEBS Lett 284:161–164CrossRefGoogle Scholar
  13. Irie M (1999) Structure–function relationship of acid ribonucleases: lysosomal, vacuolar, and periplasmic enzymes. Pharmacol Ther 81:77–89CrossRefPubMedGoogle Scholar
  14. Ito H, Hiraga S, Tsugawa H, Matsui H, Honma M, Otsuki Y, Murakami T, Ohashi Y (2000) Xylem-specific expression of wound-inducible rice peroxidase genes in transgenic plant. Plant Sci 155:85–100CrossRefPubMedGoogle Scholar
  15. Jacinto T, McGurl B, Francheschi V, Delano-Freier J, Ryan CA (1997) Tomato prosystemin promoter confers wound-inducible, vascular bundle-specific expression of the β-glucuronidase gene in transgenic tomato plants. Planta 203:406–412CrossRefGoogle Scholar
  16. Jost W, Bak H, Glund K, Terpstra P, Beintema JJ (1991) Amino acid sequence of an extracellular, phosphate-starvation-induced ribonuclease from cultured tomato (Lycopersicon esculentum) cells. Eur J Biochem 198:1–6PubMedGoogle Scholar
  17. Kaletta K, Kunze I, Kunze G, Köck M (1998) The peptide HDEF as a new retention signal is necessary and sufficient to direct proteins to the endoplasmic reticulum. FEBS Lett 434:377–381CrossRefPubMedGoogle Scholar
  18. Kariu T, Sano K, Shimokawa H, Itoh R, Yamasaki N, Kimura M (1998) Isolation and characterization of a wound-inducible ribonuclease from Nicotiana glutinosa leaves. Biosci Biotechnol Biochem 62:1144–1151Google Scholar
  19. Kawata Y, Sakiyama F, Tamaoki H (1988) Amino-acid sequence of ribonuclease T2 from Aspergillus oryzae. Eur J Biochem 176:683–697PubMedGoogle Scholar
  20. Keller B, Baumgärtner C (1991) Vascular-specific expression of the bean Grp-1.8 gene is negatively regulated. Plant Cell 3:1051–1061PubMedGoogle Scholar
  21. Köck M, Löffler A, Abel S, Glund K (1995) Structural and regulatory properties of a family of starvation induced ribonucleases from tomato. Plant Mol Biol 27:477–485PubMedGoogle Scholar
  22. Köck M, Theierl K, Stenzel I, Glund K (1998) Extracellular administration of phosphate sequestering metabolites induces ribonucleases in cultured tomato cells. Planta 204:404–407CrossRefGoogle Scholar
  23. LeBrasseur ND, MacIntosh GC, Pérez-Amador MA, Saitoh M, Green PJ (2002) Local and systemic wound-induction of RNase and nuclease activities in Arabidopsis: RNS1 as a marker for a JA-independent systemic signaling pathway. Plant J 29:393–403CrossRefGoogle Scholar
  24. Lehmann K, Hause B, Altmann D, Köck M (2001) Tomato ribonuclease LX with the functional ER retention motif HDEF is expressed during physiological cell death processes including xylem differentiation, germination and senescence. Plant Physiol 127:436–449CrossRefGoogle Scholar
  25. Lers A, Khalchitski A, Lomaniec E, Burd S, Green PJ (1998) Senescence-induced RNases in tomato. Plant Mol Biol 36:439–449CrossRefGoogle Scholar
  26. Leon J, Rojo E, Sánchez-Serrano JJ (2001) Wound signalling in plants. J Exp Bot 52:1-9CrossRefGoogle Scholar
  27. Ling H-Q, Krieseleit D, Ganal MW (1998) Effect of ticarcillin/potassium clavulanate on callus growth and shoot regeneration in Agrobacterium-mediated transformation of tomato (Lycopersicon esculentum Mill.). Plant Cell Rep 17:843–847Google Scholar
  28. Löffler A, Abel S, Jost W, Beintema JJ, Glund K (1992) Phosphate-regulated induction of intracellular ribonucleases in cultured tomato (Lycopersicon esculentum) cells. Plant Physiol 98:1472–1478Google Scholar
  29. Löffler A, Glund K, Irie M (1993) Amino acid sequence of an intracellular, phosphate-starvation-induced ribonuclease from cultured tomato (Lycopersicon esculentum) cells. Eur J Biochem 214:627–633Google Scholar
  30. Lowry OH, Rosebrough AL, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  31. McClure BA, Haring V, Ebert PR, Anderson MA, Simpson RJ, Sakiyama F, Clarke AE (1989) Style self-incompatibility gene products of Nicotiana alata are ribonucleases. Nature 342:955–957PubMedGoogle Scholar
  32. Nishitani C, Demura T, Fukuda H (2001) Primary phloem-specific expression of a Zinnia elegans homeobox gene. Plant Cell Physiol 42:1210–1218CrossRefPubMedGoogle Scholar
  33. Nishitani C, Demura T, Fukuda H (2002) Analysis of early processes in wound-induced vascular regeneration using TED3 and ZeHB3 as molecular markers. Plant Cell Physiol 43:79–90CrossRefPubMedGoogle Scholar
  34. Nürnberger T, Abel S, Jost W, Glund K (1990) Induction of an extracellular ribonuclease in cultured tomato cells upon phosphate starvation. Plant Physiol 92:970–976Google Scholar
  35. Ohgi K, Shiratori Y, Nakajima A, Iwama M, Kobayashi H, Inokuchi N, Koyama T, Köck M, Löffler A, Glund K, Irie M (1997) The base specificities of tomato ribonuclease (RNase LE) and its Asp44 mutant enzyme expressed from yeast cells. Biosci Biotech Biochem 61:432–438Google Scholar
  36. Oparka KJ, Turgeon R (1999) Sieve elements and companion cells—traffic control centers of the phloem. Plant Cell 11:739–750CrossRefPubMedGoogle Scholar
  37. Quandt K, Frech K, Karas H, Wingender E, Werner T (1995) MatInd and MatInspector—new fast and versatile tools for detection of consensus matches in nucleotide sequence data. Nucleic Acids Res 23:4878–4884PubMedGoogle Scholar
  38. Rogers SW, Rogers JC (1999) Cloning and characterization of a gibberellin-induced RNase expressed in barley aleurone cells. Plant Physiol 119:1457–1464PubMedGoogle Scholar
  39. Ryan CA (2000) The systemin signaling pathway: differential activation of plant defensive genes. Biochim Biophys Acta 1477:112–121PubMedGoogle Scholar
  40. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  41. Sasaki K, Hiraga S, Ito H, Seo S, Matsui H, Ohashi Y (2002) A wound-inducible tobacco peroxidase gene expresses preferentially in the vascular system. Plant Cell Physiol 43:108–117Google Scholar
  42. Schulz A (1990) Wound-sieve elements. In: Behnke H-D, Sjölund RD (eds) Sieve elements. Comparative structure, induction and development. Springer, Berlin Heidelberg New York, pp 199–217Google Scholar
  43. Stenzel I, Hause B, Maucher H, Pitzschke A, Miersch O, Ziegler J, Ryan CA, Wasternack C (2003) Allene oxide cyclase dependence of the wound response and vascular bundle specific generation of jasmonate—amplification in wound-signalling. Plant J 33:577–589CrossRefPubMedGoogle Scholar
  44. Taylor CB, Bariola PA, DelCardayré SB, Raines RT, Green PJ (1993) RNS2: a senescence-associated RNase of Arabidopsis that diverged from the S-RNases before speciation. Proc Natl Acad Sci USA 90:5118–5122Google Scholar
  45. Xu Z-R, Qi W-Q, Ouyang X-Z, Yeung E, Chye M-L (2001) A proteinase inhibitor II of Solanum americanum is expressed in phloem. Plant Mol Biol 47:727–738CrossRefPubMedGoogle Scholar
  46. Ye Z-H, Droste DL (1996) Isolation and characterization of cDNAs encoding xylogenesis-associated and wounding-induced ribonucleases in Zinnia elegans. Plant Mol Biol 30:697–709Google Scholar
  47. Yin Y, Chen L, Beachy R (1997) Promoter elements required for phloem-specific gene expression from the RTBV promoter in rice. Plant J 12:1179–1188CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Margret Köck
    • 1
  • Nadine Groß
    • 1
  • Irene Stenzel
    • 1
    • 2
  • Gerd Hause
    • 1
  1. 1.BiocenterMartin Luther University of Halle-WittenbergHalleGermany
  2. 2.Department of Natural Product BiotechnologyInstitute of Plant BiochemistryHalleGermany

Personalised recommendations