Isolation and characterization of proteinase inhibitor I from etiolated tobacco leaves

Kuo, Tsung Min; Pearce, Gregory; Ryan, Clarence A.

doi:10.1016/0003-9861(84)90430-2

Cited by 19 publications

(16 citation statements)

References 24 publications

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“…The type II inhibitors have a monomer Mr of 12,300 (Plunkett et al, 1982). PI I accumulates in etiolated tobacco (Nicofiana fabacum) leaves (Kuo et al, 1984), and elicitors from Phytophfhora parasifica var nicofianae were found to induce PI I accumulation in tobacco cell suspension cultures (Rickauer et al, 1989).…”

Section: Introductionmentioning

confidence: 99%

Proteinase inhibitors in Nicotiana alata stigmas are derived from a precursor protein which is processed into five homologous inhibitors.

et al. 1993

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Section: Introductionmentioning

confidence: 99%

Proteinase inhibitors in Nicotiana alata stigmas are derived from a precursor protein which is processed into five homologous inhibitors.

et al. 1993

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“…Inhibitory activity against trypsin and chymotrypsin was determined by titrating the enzymes with increasing amounts oftobacco leafextract (21). One microgram of trypsin or 3 ,g of chymotrypsin was used in each assay.…”

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confidence: 99%

Expression of proteinase inhibitors I and II in transgenic tobacco plants: effects on natural defense against Manduca sexta larvae.

Johnson

Narvaez

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

478

230

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Genes containing the cauliflower mosaic virus 35S promoter fused to open reading frames coding for tomato proteinase inhibitor I, tomato inhibitor II, and potato inhibitor II were expressed in transgenic tobacco plants. Inhibitor I and II proteins were identified by immunoblotting and quantified by immunoradial diffusion. Both inhibitors exhibited the molecular weights found for the native proteins in their natural environments. Extracts of leaves from transformed plants contained inhibitory activities against trypsin and chymotrypsin that reflected the levels of inhibitor I or H protein present. The results demonstrate that in tobacco leaves the introns of both inhibitor I and inhibitor II genes were excised correctly and that pre and prepro inhibitor I and II proteins were correctly processed. Growth of Manduca sexta larvae (tobacco hornworms) feeding on leaves of transgenic plants containing inhibitor II, a powerful inhibitor of both trypsin and chymotrypsin, was significantly retarded, compared to growth of larvae fed untransformed leaves. Levels of inhibitor H protein as low as 50 jg/g of tissue moderately affected larval growth, whereas levels above 100 ,ug/g severely reduced growth. The presence of tomato inhibitor I protein, a potent inhibitor of chymotrypsin but a weak inhibitor of trypsin, in transgenic tobacco leaves had little effect on the growth of the larvae. These experiments indicated that trypsin inhibitory activity, but not chymotrypsin inhibitory activity, was mainly responsible for the inhibition of larval growth.Potato and tomato plants contain two small multigene families that code for two powerful inhibitors of serine proteinases, called inhibitor I (monomer Mr 8100) and inhibitor II (monomer Mr 12,300) (1). Inhibitor I is an inhibitor of chymotrypsin that only weakly inhibits trypsin at its single reactive site (1), whereas inhibitor II contains two reactive sites, one of which inhibits trypsin and the other of which inhibits chymotrypsin (1). Members of both gene families are expressed in leaves in response to chewing insects or other severe mechanical damage (2). Both inhibitors are synthesized as precursors and undergo posttranslational modification (3)(4)(5) to form the mature proteins, which are sequestered in the vacuole (6). These inhibitors are thought to help defend the plant, by reducing the digestibility and nutritional quality of the leaves, against insect predators (7). Both cDNAs (4, 5) and genes (8, 9) that encode inhibitors I and II have been isolated and characterized. These are now being employed to further investigate the role of proteinase inhibitors in plant defense.It was previously shown (10) that transformation of tobacco plants with a gene encoding a cowpea trypsin inhibitor was able to confer increased resistance against predation by Heliothis virescens larvae. In order to assess the potential of inhibitor I and inhibitor II for increasing the natural defenses of crop plants through transformation, genes encoding these inhibitors were stably introduced ...

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“…Induction of PI by Phytophthora elicitor was not affected by transferring the tobacco cells from light to darkness. Tobacco plants, callus and cell suspension cultures were previously reported to accumulate PI I under the effect of environmental factors such as light and aging (10,24,26). In our work we have characterized a PI which is induced by a different stimulus (treatment with elicitors) and seems to differ from the PI I in that it has a lower mol wt and is not oligomeric.…”

Section: Discussionmentioning

confidence: 87%

Induction of Proteinase Inhibitors in Tobacco Cell Suspension Culture by Elicitors of Phytophthora parasitica var. nicotianae

Rickauer

Fournier²,

Esquerré-Tugayé³

1989

Plant Physiol.

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An elicitor preparation obtained from Phytophthora parasitica var. nicotianae, a pathogen of tobacco, induced an accumulation of proteinase inhibitors and a stimulation of ethylene synthesis in a tobacco (Nicotiana tabacum) cell suspension culture. About 30 micrograms per milliliter of elicitor were necessary for maximal induction of proteinase inhibitor accumulation, and the response was detectable after 12 hours of incubation with elicitor. Accumulation of proteinase inhibitors required de novo protein synthesis, since cycloheximide completely inhibited its elicitation, and actinomycin D inhibited it partially. One of the inhibitors was purified by a procedure that included heating, (NH4)2SO4 precipitation, ion-exchange chromatography, and affinity chromatography. The purified inhibitor was shown to be a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of about 10,500. It inhibited trypsin but not chymotrypsin.endopolygalacturonase and chitosan, which are potent elicitors of phytoalexin synthesis (25); on the other hand, PIIF could also induce phytoalexin synthesis. Accumulation of PIs has also been found in tomato plants after infection with Phytophthora infestans, showing differences in compatible and incompatible race-cultivar interactions (18). Thus, induction of PI seems to be an integral part of the plant defense system against pathogens.In the present work, we investigated the induction of PI by elicitors isolated from Phytophthora parasitica var. nicotianae, the causal agent of the black shank disease of tobacco plants. Helgeson and Haberlach (7) demonstrated that the tobacco-

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Isolation and characterization of proteinase inhibitor I from etiolated tobacco leaves

Cited by 19 publications

References 24 publications

Proteinase inhibitors in Nicotiana alata stigmas are derived from a precursor protein which is processed into five homologous inhibitors.

Proteinase inhibitors in Nicotiana alata stigmas are derived from a precursor protein which is processed into five homologous inhibitors.

Expression of proteinase inhibitors I and II in transgenic tobacco plants: effects on natural defense against Manduca sexta larvae.

Induction of Proteinase Inhibitors in Tobacco Cell Suspension Culture by Elicitors of Phytophthora parasitica var. nicotianae

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