, Volume 218, Issue 4, pp 623–629 | Cite as

Inhibition of endogenous trypsin- and chymotrypsin-like activities in transgenic lettuce expressing heterogeneous proteinase inhibitor SaPIN2a

  • Zeng-Fu Xu
  • Whei-Lan Teng
  • Mee-Len Chye
Original Article


SaPIN2a, a proteinase inhibitor II from American black nightshade (Solanum americanum Mill.) is highly expressed in the phloem and could be involved in regulating proteolysis in the sieve elements. To further investigate the physiological role of SaPIN2a, we have produced transgenic lettuce (Lactuca sativa L.) expressing SaPIN2a from the CaMV35S promoter by Agrobacterium-mediated transformation. Stable integration of the SaPIN2a cDNA and its inheritance in transgenic lines were confirmed by Southern blot analysis and segregation analysis of the R1 progeny. SaPIN2a mRNA was detected in both the R0 and R1 transformants on northern blot analysis but the SaPIN2a protein was not detected on western blot analysis using anti-peptide antibodies against SaPIN2a. Despite an absence of significant inhibitory activity against bovine trypsin and chymotrypsin in extracts of transgenic lettuce, the endogenous trypsin-like activity in each transgenic line was almost completely inhibited, and the endogenous chymotrypsin-like activity moderately inhibited. Our finding that heterogeneously expressed SaPIN2a in transgenic lettuce inhibits plant endogenous protease activity further indicates that SaPIN2a regulates proteolysis, and could be potentially exploited for the protection of foreign protein production in transgenic plants.


Chymotrypsin Lactuca Protease Proteolysis Solanum Trypsin 



cauliflower mosaic virus


complementary DNA


nopaline synthase


polyacrylamide gel electrophoresis


proteinase inhibitor


Solanum americanum proteinase inhibitor IIa


sodium dodecyl sulphate


transferred DNA



This work was supported by funds from The University of Hong Kong (to M.-L.C.). Z.-F.X. received a postgraduate studentship from The University of Hong Kong.


  1. Applebaum SW, Konijn AM (1966) The presence of a Tribolium-protease inhibitor in wheat. J Insect Physiol 12:665–669Google Scholar
  2. 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 8:2195–2202Google Scholar
  3. Birk Y, Gertler A, Khalef S (1963) Separation of a Tribolium-protease inhibitor from soybeans on a calcium phosphate column. Biochim Biophys Acta 67:326–328CrossRefPubMedGoogle Scholar
  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  5. Brzin J, Kidric M (1995) Proteinases and their inhibitors in plants: role in normal growth and in response to various stress conditions. Biotechnol Genet Eng Rev 13:420–467Google Scholar
  6. Cordero MJ, Raventos D, Segundo BS (1994) Expression of a maize proteinase inhibitor gene is induced in response to wounding and fungal infection: systemic wound-response of a monocot gene. Plant J 6:141–150CrossRefPubMedGoogle Scholar
  7. Curtis IS, Power JB, Blackhall NW, de Laat AMM, Davey MR (1994) Genotype-independent transformation of lettuce using Agrobacterium tumefaciens. J Exp Bot 45:1441–1449Google Scholar
  8. Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1:19–21Google Scholar
  9. Florack DEA, Dirkse WG, Visser B, Heidekamp F, Stiekema WJ (1994) Expression of biologically active hordothionins in tobacco. Effects of pre- and pro-sequences at the amino and carboxyl termini of the hordothionin precursor on mature protein expression and sorting. Plant Mol Biol 24:83–96PubMedGoogle Scholar
  10. Gallagher SR (1995) Separation of proteins on gradient gels. In: Coligan JE, Dunn BM, Ploegh HL, Speicher DW, Wingfield PT (eds) Current protocols in protein science, vol 1. Wiley, New York, pp 10.1.17–10.1.23Google Scholar
  11. Gatehouse AMR, Davison GM, Newell CA, Merryweather A, Hamilton WDO, Burgess EPJ, Gilbert RJC, Gatehouse JA (1997) Transgenic potato plants with enhanced resistance to the tomato moth, Lacanobia oleracea: growth room trials. Mol Breed 3:49–63CrossRefGoogle Scholar
  12. Hendriks T, Vreugdenhil D, Stiekema WJ (1991) Patatin and four serine proteinase inhibitor genes are differentially expressed during potato tuber development. Plant Mol Biol 17:385–394PubMedGoogle Scholar
  13. Holsters M, de Waele D, Depicker A, Messens E, van Montagu M, Schell J (1978) Transfection and transformation of Agrobacterium. tumefaciens. Mol Gen Genet 163:181–187PubMedGoogle Scholar
  14. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907PubMedGoogle Scholar
  15. Jones JDG, Dunsmuir P, Bedbrook J (1985) High level expression of introduced chimaeric genes in regenerated transformed plants. EMBO J 4:2411–2418Google Scholar
  16. Kollipara KP, Hymowitz T (1992) Characterization of trypsin and chymotrypsin inhibitors in the wild perennial Glycine species. J Agric Food Chem 40:2356–2363Google Scholar
  17. Laskowski M Jr, Kato I (1980) Protein inhibitors of proteinases. Annu Rev Biochem 49:593–626CrossRefPubMedGoogle Scholar
  18. Lorberth R, Dammann C, Ebneth M, Amati S, Sanchez-Serrano JJ (1992) Promoter elements involved in environmental and developmental control of potato proteinase inhibitor II expression. Plant J 2:477–486CrossRefPubMedGoogle Scholar
  19. Margossian LJ, Federman AD, Giovannoni JJ, Fischer RL (1988) Ethylene-regulated expression of a tomato fruit ripening gene encoding a proteinase inhibitor I with a glutamic residue at the reactive site. Proc Natl Acad Sci USA 85:8012–8016PubMedGoogle Scholar
  20. McCabe MS, Mohapatra UB, Debnath SC, Power JB, Davey MR (1999) Integration, expression and inheritance of two linked T-DNA marker genes in transgenic lettuce. Mol Breed 5:329–344CrossRefGoogle Scholar
  21. Mikola J, Pietila K (1972) Hydrolysis of ester substrates of trypsin and chymotrypsin by barley carboxypeptidase. Phytochemistry 11:2977–2980CrossRefGoogle Scholar
  22. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497Google Scholar
  23. Nagy F, Odell JT, Morelli G, Chua NH (1985) Properties of expression of the 35S promoter from CaMV in transgenic tobacco plants. In: Zaitlin M, Day P, Hollaender A (eds) Biotechnology in plant science: relevance to agriculture in the eighties. Academic Press, New York, pp 227–235Google Scholar
  24. Nagy F, Kay SA, Chua NH (1988) Analysis of gene expression in transgenic plants. In: Gelvin SB, Schilperoort RA (eds) Plant molecular biology manual. Kluwer, Dordrecht, pp B4:1–29Google Scholar
  25. Odell JT, Nagy F, Chua NH (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313:810–812PubMedGoogle Scholar
  26. Passelegue E, Kerlan C (1996) Transformation of cauliflower (Brassica oleracea var. botrytis) by transfer of cauliflower mosaic virus genes through combined cocultivation with virulent and avirulent strains of Agrobacterium. Plant Sci 113:79–89CrossRefGoogle Scholar
  27. Pena-Cortes H, Willmitzer L, Sanchez-Serrano JJ (1991) Abscisic acid mediates wound induction but not developmental-specific expression of the proteinase inhibitor II gene family. Plant Cell 3:963–972CrossRefPubMedGoogle Scholar
  28. Rosahl S, Eckes P, Schell J, Willmitzer L (1986) Organ-specific gene expression in potato: isolation and characterization of tuber-specific cDNA sequences. Mol Gen Genet 202:368–373Google Scholar
  29. Ryan CA (1981) Proteinase inhibitors. In: Marcus A (ed) The biochemistry of plants, vol 6. Academic Press, New York, pp 351–370Google Scholar
  30. Ryan CA (1989) Proteinase inhibitor gene families: strategies for transformation to improve plant defenses against herbivores. BioEssays 10:20–24PubMedGoogle Scholar
  31. Ryder EJ (1999) Lettuce, endive and chicory. CABI Publishing, New YorkGoogle Scholar
  32. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  33. Sanchez-Serrano JJ, Schmidt R, Schell J, Willmitzer L (1986) Nucleotide sequence of proteinase inhibitor II encoding cDNA of potato (Solanum tuberosum) and its mode of expression. Mol Gen Genet 203:15–20Google Scholar
  34. Seymour GB, Fray RG, Hill P, Tucker GA (1993) Down-regulation of two non-homologous endogenous tomato genes with a single chimaeric sense gene construct. Plant Mol Biol 23:1–9PubMedGoogle Scholar
  35. Shain Y, Mayer AM (1965) Proteolytic enzymes and endogenous trypsin inhibitor in germinating lettuce seeds. Physiol Plant 18:853–859Google Scholar
  36. Shain Y, Mayer AM (1968) Activation of enzymes during germination — trypsin-like enzyme in lettuce. Phytochemistry 7:1491–1498CrossRefGoogle Scholar
  37. Solomon M, Belenghi B, Delledonne M, Menachem E, Levine A (1999) The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants. Plant Cell 11:431–443PubMedGoogle Scholar
  38. Stevens LH, Stoopen GM, Elbers IJW, Molthoff JW, Bakker HAC, Lommen A, Bosch D, Jordi W (2000) Effect of climate conditions and plant developmental stage on the stability of antibodies expressed in transgenic tobacco. Plant Physiol 124:173–182CrossRefPubMedGoogle Scholar
  39. Sunilkumar G, Mohr L, Lopata-Finch E, Emani C, Rathore KS (2002) Developmental and tissue-specific expression of CaMV 35S promoter in cotton as revealed by GFP. Plant Mol Biol 50:463–474CrossRefPubMedGoogle Scholar
  40. Tamayo MC, Rufat M, Bravo JM, Segundo BS (2000) Accumulation of a maize proteinase inhibitor in response to wounding and insect feeding, and characterization of its activity toward digestive proteinases of Spodoptera littoralis larvae. Planta 211:62–71CrossRefPubMedGoogle Scholar
  41. Walker-Simmons M, Ryan CA (1977) Wound-induced accumulation of trypsin inhibitor activities in plant leaves. Plant Physiol 59:437–439Google Scholar
  42. Williamson JD, Hirsch-Wyncott ME, Larkins BA, Gelvin SB (1989) Differential accumulation of a transcript driven by the CaMV 35S promoter in transgenic tobacco. Plant Physiol 90:1570–1576Google Scholar
  43. Wu Y, Llewellyn D, Mathews A, Dennis ES (1997) Adaptation of Helicoverpa armigera (Lepidoptera: Noctuidae) to a proteinase inhibitor expressed in transgenic tobacco. Mol Breed 3:371–380CrossRefGoogle Scholar
  44. Xu ZF, Qi WQ, Ouyang XZ, Yeung E, Chye ML (2001) A proteinase inhibitor II of Solanum americanum is expressed in phloem. Plant Mol Biol 47:727–738CrossRefPubMedGoogle Scholar
  45. Yamauchi Y, Ejiri Y, Sugimoto T, Sueyoshi K, Oji Y, Tanaka K (2001) A high molecular weight glutamyl endopeptidase and its endogenous inhibitors from cucumber leaves. J Biochem 130:257–261PubMedGoogle Scholar
  46. Yang NS, Christou P (1990) Cell type specific expression of a CaMV 35S-GUS gene in transgenic soybean plants. Dev Genet 11:289–293Google Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  1. 1.Department of BotanyThe University of Hong KongHong KongChina
  2. 2.Key Laboratory of Gene Engineering of the Ministry of EducationZhongshan (Sun Yat-sen) UniversityGuangzhou 510275China

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