Abstract
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.
Similar content being viewed by others
Abbreviations
- CaMV:
-
cauliflower mosaic virus
- cDNA:
-
complementary DNA
- NOS:
-
nopaline synthase
- PAGE:
-
polyacrylamide gel electrophoresis
- PI:
-
proteinase inhibitor
- SaPIN2a:
-
Solanum americanum proteinase inhibitor IIa
- SDS:
-
sodium dodecyl sulphate
- T-DNA:
-
transferred DNA
References
Applebaum SW, Konijn AM (1966) The presence of a Tribolium-protease inhibitor in wheat. J Insect Physiol 12:665–669
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 8:2195–2202
Birk Y, Gertler A, Khalef S (1963) Separation of a Tribolium-protease inhibitor from soybeans on a calcium phosphate column. Biochim Biophys Acta 67:326–328
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 72:248–254
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 13:420–467
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 6:141–150
Curtis IS, Power JB, Blackhall NW, de Laat AMM, Davey MR (1994) Genotype-independent transformation of lettuce using Agrobacterium tumefaciens. J Exp Bot 45:1441–1449
Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1:19–21
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 24:83–96
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 10.1.17–10.1.23
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 3:49–63
Hendriks T, Vreugdenhil D, Stiekema WJ (1991) Patatin and four serine proteinase inhibitor genes are differentially expressed during potato tuber development. Plant Mol Biol 17:385–394
Holsters M, de Waele D, Depicker A, Messens E, van Montagu M, Schell J (1978) Transfection and transformation of Agrobacterium. tumefaciens. Mol Gen Genet 163:181–187
Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907
Jones JDG, Dunsmuir P, Bedbrook J (1985) High level expression of introduced chimaeric genes in regenerated transformed plants. EMBO J 4:2411–2418
Kollipara KP, Hymowitz T (1992) Characterization of trypsin and chymotrypsin inhibitors in the wild perennial Glycine species. J Agric Food Chem 40:2356–2363
Laskowski M Jr, Kato I (1980) Protein inhibitors of proteinases. Annu Rev Biochem 49:593–626
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 2:477–486
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 85:8012–8016
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 5:329–344
Mikola J, Pietila K (1972) Hydrolysis of ester substrates of trypsin and chymotrypsin by barley carboxypeptidase. Phytochemistry 11:2977–2980
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
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 227–235
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 B4:1–29
Odell JT, Nagy F, Chua NH (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313:810–812
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 113:79–89
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 3:963–972
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 202:368–373
Ryan CA (1981) Proteinase inhibitors. In: Marcus A (ed) The biochemistry of plants, vol 6. Academic Press, New York, pp 351–370
Ryan CA (1989) Proteinase inhibitor gene families: strategies for transformation to improve plant defenses against herbivores. BioEssays 10:20–24
Ryder EJ (1999) Lettuce, endive and chicory. CABI Publishing, New York
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
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 203:15–20
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 23:1–9
Shain Y, Mayer AM (1965) Proteolytic enzymes and endogenous trypsin inhibitor in germinating lettuce seeds. Physiol Plant 18:853–859
Shain Y, Mayer AM (1968) Activation of enzymes during germination — trypsin-like enzyme in lettuce. Phytochemistry 7:1491–1498
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 11:431–443
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 124:173–182
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 50:463–474
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 211:62–71
Walker-Simmons M, Ryan CA (1977) Wound-induced accumulation of trypsin inhibitor activities in plant leaves. Plant Physiol 59:437–439
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 90:1570–1576
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 3:371–380
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 47:727–738
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 130:257–261
Yang NS, Christou P (1990) Cell type specific expression of a CaMV 35S-GUS gene in transgenic soybean plants. Dev Genet 11:289–293
Acknowledgments
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.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Xu, ZF., Teng, WL. & Chye, ML. Inhibition of endogenous trypsin- and chymotrypsin-like activities in transgenic lettuce expressing heterogeneous proteinase inhibitor SaPIN2a. Planta 218, 623–629 (2004). https://doi.org/10.1007/s00425-003-1138-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00425-003-1138-9