Abstract
The open reading frame and terminator region of a wound-inducible tomato Inhibitor I gene, regulated by the CaMV 35S promoter, was stably integrated into the genomes of nightshade (Solanum nigrum), tobacco (Nicotiana tabacum), and alfalfa (Medicago sativa), using an Agrobacterium-mediated transformation system. The expression of the foreign Inhibitor I gene in leaves of each species was studied at the mRNA and protein levels. The levels of Inhibitor I protein present in leaves of each species correlated with the levels of mRNA. The average levels of both mRNA and Inhibitor I protein were highest in leaves of transgenic nightshade plants (over 125 μg of Inhibitor I per g tissue), less in tobacco plants (about 75 μg/g tissue), and lowest in leaves of transgenic alfalfa plants (below 20 μg/g tissue). Inhibitor I protein was observed in all tissues throughout transgenic plant species, but inhibitor concentration per gram of tissue was 2–3 times higher in young developing leaf tissues and floral organs. The differences in the expression of the CaMV-tomato Inhibitor I gene among the different plant genera suggests that either the rate of transcription of the foreign gene or the rate of degradation of the nascent Inhibitor I mRNA varies among genera. Using electron microscopy techniques, the newly synthesized pre-pro-Inhibitor I protein was shown to be correctly processed and stored as a mature Inhibitor I protein within the central vacuoles of leaves of transgenic nightshade and alfalfa. The results of these experiments suggest that maximal expression of foreign proteinase inhibitor genes, and perhaps other foreign defense genes, may require gene constructs that are fashioned with promoters and terminators that allow maximum expression in the selected plant species.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
An G, Mitra A, Choi HK, Costa MA, An K, Thornburg RW, Ryan CA: Functional analysis of the 3′ control region of the potato wound-inducible proteinase inhibitor II gene. Plant Cell 1: 115–122 (1989).
Applebaum SW: Biochemistry of digestion. In: Kerkut GA, Gilbert LI (eds.) Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 4, pp. 279–311. Pergamon, New York (1985).
Benfey PN, Chua NH: Regulated genes in transgenic plants. Science 244: 174–181 (1989).
Benfey PN, Chua NH: The cauliflower mosaic virus 35S promoter: combinatorial regulation of transcription in plants. Science 250: 959–966 (1990).
Bradford M: 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 (1976).
Broadway RM, Duffey SS: Plant proteinase inhibitors: Mechanism of action and effect on the growth and digestive physiology of larval Heliotis zea and Spodoptera exigua. J Insect Physiol 32: 827–833 (1986).
Broadway RM, Duffey SS, Pearce G, Ryan CA: Plant proteinase inhibitors: A defense against herbivorous insects? Entomol Exp Appl 41: 33–38 (1986).
Gamborg OL, Miller RA, Ojima K: Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50: 151–158 (1968).
Gatehouse AMR, Boulter D: Assessment of the antimetabolic effects of trypsin inhibitors from cowpea (Vigna unguiculata) and other legumes on development of the Bruchid beetle Callosobruchus maculatus. J Sci Food Agric 34: 345–350 (1983).
Graham JS, Hall G, Pearce G, Ryan CA: Regulation of synthesis of proteinase inhibitors I and II mRNAs in leaves of wounded tomato plants. Planta 169: 399–405 (1986).
Graham JS, Pearce G, Merryweather J, Titany K, Ericsson L, Ryan CA: Wound-induced proteinase inhibitors from tomato leaves. I. The cDNA deduced primary structure of pre-inhibitor I and its post-translational processing. J Biol Chem 260: 6550–6555 (1985).
Gustafson G, Ryan CA: Specificity of protein turnover in tomato leaves: Accumulation of proteinase inhibitors, induced with the wound hormone, PIIF. J Biol Chem 251, 7004–7010 (1976).
Hilder VA, Gatehouse AMR, Sheerman SE, Barker RF, Boulter D: A novel mechanism of insect resistance engineered into tobacco. Nature 330: 160–163 (1987).
Hummel BCW: A modified spectrophotometric determination of chymotrypsin, trypsin and thrombin. Can J Biochem Physiol 37: 1393 (1959).
Johnson R, Narvaez J, An G, Ryan CA: Expression of proteinase inhibitor I and II in transgenic tobacco plants: Effects on natural defense against Manduca sexta larvae. Proc Natl Acad Sci USA 86: 9871–9875 (1989).
Lawton MA, Dixon RA, Hahlbrock K, Lamb CJ: Elicitor induction of mRNA activity: Rapid effects of elicitor on phenylalanine ammonia-lyase and chalcone synthase mRNA activities in bean cells. Eur J Biochem 130: 131–139 (1983).
Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).
Melville JC, Ryan CA: Chymotrypsin inhibitor I from potatoes. J Biol Chem 247: 3445–3453 (1972).
Murashige T, Skoog F: A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol Plant 15: 473–495 (1962).
Narváez-Vásquez J: Expression of proteinase inhibitor genes in transgenic plants: Effects on insect resistance; levels of expression in four plant species; and cellular compartmentalization. Ph.D. thesis, Washington State University, Pullman, WA, USA (1991).
Odell JT, Nagy F, Chua N-H: Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313: 810–812 (1985).
Pearce G, Ryan CA, Lilegren D: Proteinase inhibitor I and II in fruit of wild tomato species: Transient components of a mechanism for defense and seed dispersal. Planta 175: 527–531 (1988).
Ryan CA: Quantitative determination of soluble cellular proteins by radial diffusion in agar gels containing antibodies. Anal Biochem 19: 434–440 (1967).
Ryan CA: Proteinase inhibitors. In: The Biochemistry of Plants, vol. 6, pp. 351–370. Academic Press, New York (1981).
Ryan CA: Protease inhibitors in plants: genes for improving defenses against insects and pathogens. Annu Rev Phytopath 28: 425–449 (1990).
Shenk RU, Hildebrant AC: Medium and techniques for induction and growth of monocotyledonous and dicotyledonous cell cultures. Can J Bot 50: 199–204 (1972).
Stuart DA, Nelsen J, Strickland SG, Nichol JW: Factors affecting developmental processes in alfalfa cell cultures. In: Henke R, Huges KW, Konstantin MP, Hollaender A (eds) Tissue Culture in Forestry and Agriculture, pp. 59–73. Plenum, New York (1985).
Swank RT, Munkres KD: Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. Anal Biochem 39: 462–477 (1971).
Trautman R, Cowan KM, Wagner GG: Data processing for radial immunodiffusion. Immunochemistry 8: 901–916 (1971).
Walker KA, Sato SJ: Morphogenesis in callus tissue of Medicago sativa: The role of ammonium ion in somatic embryogenesis. Plant Cell Tissue Organ Culture 1: 109–122 (1981).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Narváez-Vásquez, J., Orozco-Cárdenas, M.L. & Ryan, C.A. Differential expression of a chimeric CaMV-tomato proteinase Inhibitor I gene in leaves of transformed nightshade, tobacco and alfalfa plants. Plant Mol Biol 20, 1149–1157 (1992). https://doi.org/10.1007/BF00028901
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00028901