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Plant Physiol. (1 993) 102: 639-644 Purification and Characterization from Tobacco (Nicotiana tabacum) Leaves of Six Small, Wound-lnducible, Proteinase lsoinhibitors of the Potato lnhibitor II Family' Gregory Pearce, Scott Johnson, and Clarence A. Ryan* lnstitute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340 inducible trypsin inhibitor activity in tobacco leaves (WalkerSimmons and Ryan, 1977)but could not identify the inhibitor as a potato inhibitor I or I1 family member with the use of antibodies specific for these proteins (G. Pearce and C.A. Ryan, unpublished data). Recently, we found that the newly discovered polypeptide signal from tomato leaves, called systemin (Pearce et al., 1991), which activates inhibitor I and I1 genes in both tomato and potato leaves, did not activate the synthesis of the trypsin inhibitor activity in tobacco leaves. Therefore, we have isolated and characterized the wound-inducible trypsin inhibitor from tobacco leaves to further understand its structure, to obtain antibodies to quantify its wound inducibility in tobacco leaves, and to investigate the systemic signals that regulate its synthesis. We report the isolation and characterization of six isoinhibitor forms of TTI. The complete amino acid sequence of one isoinhibitor and partia1 sequence of another reveal that the systemically wound-inducible TTI isoinhibitors are members of the potato inhibitor I1 family. Six small molecular mass, wound-inducible trypsin and chymotrypsin inhibitor proteins from tobacco (Nicotiana tabacum) leaves were isolated to homogeneity. l h e isoinhibitors, cumulatively called tobacco trypsin inhibitor (TTI), have molecular masses of approximately 5500 to 5800 D, calculated from gel filtration analysis and amino acid content. l h e amino acid sequence of the entire 53 residues of one isoinhibitor, TTI-1, and the sequence of 36 amino acid residues from the N terminus of a second isoinhibitor, 111-5, were determined. The two isoinhibitors differ only at residue 11, which is threonine in TTI-1 and lysine in TTI-5. l h e isoinhibitors are members of the potato inhibitor II family and show considerable identity with the small molecular mass members of this family, which include the eggplant inhibitor, two small molecular m a s trypsin and chymotrypsin inhibitorsfrom potatoes, and an inhibitor from pistils of the ornamental plant Nicotiana alata. Antibodies produced against the isoinhibitors in rabbits were used in radial immunoassays to quantify both the systemic wound inducibility of TTI in tobacco leaves and its constitutive levels in flowers. Proteinase inhibitor proteins are among the defensive chemicals of plants directed against insects and pathogens (Ryan, 1990). At least eight families of Ser proteinase inhibitors in plants have been identified either in storage organs or in vegetative cells of virtually a11 plant families in which they have been sought (Garcia-Olmedo et al., 1987; Ryan, 1990). Members of four of the Ser proteinase inhibitor families are known to be systemically induced in various plants. These families include potato and tomato inhibitors I and I1 in solanaceous plants (Melville and Ryan, 1972; Bryant et al., 1976; Plunkett et al., 1982), Bowman-Birk inhibitors in alfalfa (Brown and Ryan, 1984), and a Kunitz inhibitor in poplar trees (Bradshaw et al., 1989). As part of our program to understand the signal transduction pathways that regulate the localized and systemic wound-inducible expression of proteinase inhibitor proteins in different plant genera in response to pest attacks (Ryan, 1992),we have isolated and characterized a wound-inducible proteinase inhibitor from tobacco (Nicotiana tabacum) leaves. In a previous study we had measured an oligogalacturonide- MATERIALS AND METHODS G-75 Sephadex was purchased from Pharmacia (Piscataway, NJ), and dialysis membranes were purchased from Spectrum Medica1 Industries, Inc. (Los Angeles, CA). SepPac Cls cartridges were purchased from Waters (Milford, MA). Tributyl phosphine and 4-vinylpyridine were purchased from Aldrich Chemical Co. (Milwaukee, WI), and BCA and TFA were purchased from Pierce Chemical Co. (Rockford, IL). Bovine trypsin (2X crystallized), bovine chymotrypsin (3X crystallized), carboxypeptidase P, p-tosyl-L-Arg methyl ester, N-benzoyl-L-Tyr ethyl ester, RSA, BSA, and Cyt c were purchased from Sigma Chemical Co. Kunitz trypsin inhibitor was purchased from Calbiochem (San Diego, CA), and V8 protease was from Boehringer Mannheim (Indianapolis, IN). Carboxypeptidase inhibitor and inhibitor I1 were purified from potato tubers as previously described (Bryant et al., 1976; Pearce and Ryan, 1983). HPLC was perfonned on a Beckman system consisting of two modelll2 pumps with a model420 gradient controller. Eluted peaks were detected with a modell65 variable wave- This research was supported in part by Washington State UNversity College of Agriculture and Home Economics, project 1791, and by National Science Foundation grants IBN-9244361 and IBN9117795. * Corresponding author; fax 1-509-335-7643. Abbreviations: BCA, bicinchoninic acid; PCI, polypeptide chymotrypsin inhibitor; PTI, polypeptide hypsin inhibitor; RP-HPLC, reverse-phase HPLC; RSA, rabbit serum albumin; SCX-HPLC, strong cation-exchange HPLC; TTI, tobacco trypsin inhibitor. 639 Downloaded from on August 1, 2017 - Published by www.plantphysiol.org Copyright © 1993 American Society of Plant Biologists. All rights reserved. 640 Pearce et al. length detector and displayed on a Hewlett-Packard model 3390A integrator. RP-HPLC was performed on a Vydac 218TP510 semipreparative column (10 X 250 mm, 5 pm, 300 A) purchased from The Nest Group (Southborough, MA). SCX-HPLC was performed on a polysulfoethyl aspartamide column (4.6 X 200 mm, 5 pm) purchased from The Nest Group. Affinity chromatography using immobilized trypsin was performed by the method of Cuatrecasas and Anfinson (1971). SDS-PAGE was performed in 7 M urea by the method of Swank and Munkres (1971). Inhibitor activities against trypsin and chymotrypsin were assayed spectrophotometrically with p-tosyl-L-Arg methyl ester and N-benzoyl-L-Tyr ethyl ester, respectively, as substrates by the method of Hummel (1959). Protein concentrations were determined using the BCA reagent with purified potato inhibitor I1 as the standard. TTI-1 was prepared for sequence analysis by reduction and alkylation using the method of Ruegg and Rudinger (1977). TTI-1 (200 pg) was dissolved in 20 pL of distilled water. To this was added 20 pL of 1 M triethylammonium acetate (pH lO), 20 pL of tributylphosphine (1%in 2-propanol), 2 pL of 4-vinylpyridine (neat), and 58 pL of 2-propanol. A blank containing no peptide was also prepared, and both vials were incubated at 37OC for 24 h. Reactants were separated on RPHPLC utilizing a linear gradient from 0.1% TFA in water at O min to 60% acetonitrile in 0.1% TFA at 60 min. Native TTI1 eluted at 33.47 min, and alkylated TTI-1 eluted at 35.00 min. The fraction containing the modified peptide was reduced in volume by vacuum centrifugation to 200 FL, and 1 M ammonium acetate was added dropwise to pH 4. V8 protease (20 pg) was added, and the sample was incubated at 22OC for 4 h. The digested peptide fragments were separated on RP-HPLC. Two fragments were identified, with retention times of 28.99 and 33.05 (Fig. I). The amino acid sequence of the intact alkylated TTI-1 through residue 35 w o z U 600 1-26 RETENTION TIME (min) Figure 1. lsolation of peptides from the V8 proteinase digestion of TTI-1 by RP-HPLC. The isoinhibitor protein was reduced and alkyl- ated as described in “Materialsand Methods.”The digested peptide (approximately 200 Ng) was applied to the column as described in Figure 3 and eluted with a gradient of O to 60% acetonitrile at 2 mL min-‘ for 60 min. Aliquots (50 pL) of each fraction were assayed for protein using BCA reagent. Plant Physiol. Vol. 102, 1993 and the two proteolytic fragments were determined using Edman chemistry on an Applied Biosystems model 475 sequencer with pulse-liquid update using the manufacturer’s protocol. To identify the carboxy-terminal residue of TTI-1, the native protein (100 pg in 150 pL of water) was incubated with carboxypeptidaseP (7.5 pg) in 15 pL of 100 m sodium citrate buffer (pH 4.3) at 37OC. Aliquots (55 pL) were removed at 10,30, and 120 min and diluted with 250 pL of 0.1% TFA in water and applied to a Sep-Pac cartridge equilibrated with 10% acetonitrile in 0.1% TFA/water. Free amino acids released by carboxypeptidase P were eluted from the cartridge using 1 mL of 10% acetonitrile in 0.1% TFA/water. The volume was reduced to 0.1 mL for amino acid analysis. Antibodies were produced by cross-linking the major peptide fraction from RP-HPLC to RSA (Doolittle, 1977). Briefly, 1 mg of RSA was dissolved in 40 pL of 0.4 M sodium phosphate (pH 7.5). TTI (5 mg) in 100 pL of water was added to the RSA solution. Glutaraldehyde (20 r m , 55 pL) was added witk stimng for 5 min, and the sample was allowed to stir for 30 min at room temperature. The remaining crosslinking sites were blocked by the addition of 14 r L of 1 M Gly, followed by stining for 30 min. The cross-linked proteins were recovered in the void peak from a G-75 gel filtration column equilibrated in 50 m ammonium bicarbonate. The void peak contents were analyzed by SDS-PAGE to ensure that cross-linking had occurred. The void fraction contents were injected into rabbits to elicit antibody production. The antibodies were used to quantify TTI by immunoradial diffusion analyses as described previously (Ryan, 1967; Trautman et al., 1971) using RP-HPLC-purified TTI as a standard. RESULTS AND DlSCUSSlON Oligouronide elicitors were previously shown to induce trypsin inhibitor activity in leaves of young, excised tobacco plants (Walker-Simmons and Ryan, 1977). Members of severa1 families of proteinase inhibitors are systemically induced in plant leaves in response to wounding. This includes the potato inhibitor I and I1 families in tomato and potato leaves (Green and Ryan, 1972), a member of the Bowman-Birk inhibitor family in alfalfa (Brown and Ryan, 1984), and a Kunitz inhibitor family member in poplar trees (Bradshaw et al., 1989). The identity of the tobacco trypsin inhibitor that was induced by oligouronides (Walker-Simmons and Ryan, 1977)was of interest, because antibodies raised against potato inhibitor I and I1 proteins, which strongly cross-reacted with the homologous inhibitors from tomato plants, did not crossreact with the extracts containing the oligouronide-inducible inhibitors from tobacco (C. Ryan, unpublished data), indicating that it might be from a different family of inhibitors. The oligouronide-inducibleinhibitory activity was subsequently found to be systemically wound inducible (G. Pearce and C.A. Ryan, unpublished data; cf. Fig. 7). To isolate the wound-inducible tobacco inhibitor, mature tobacco plants were grown to the flowering stage, each leaf was severely wounded along the edges using a hemostat, and the wounded leaves were harvested 48 h later for the isolation of TTI. The leaves (630 g) were homogenized for 3 min in 500 mL of cold buffer (0.01 M sodium citrate, 1 M NaCl [PH 4.31 with 3.5 g of sodium hydrosulfite) and squeezed through cheese- Downloaded from on August 1, 2017 - Published by www.plantphysiol.org Copyright © 1993 American Society of Plant Biologists. All rights reserved. Wound-lnducible Trypsin lnhibitor from Tobacco Leaves cloth. The extracted juice was centrifuged at 10,0008 for 20 min at 4OC. Ammonium sulfate was added to the supematant to 80% saturation and stirred at 4OC for 2 h, and the precipitate was pelleted at 10,OOOg for 10 min. The resulting pellets were taken up in 200 mL of distilled water, stirred for 30 min, and then heated to 8OoC on a steam bath. This solution was cooled to room temperature in an ice bath and then centrifuged at 10,OOOg for 15 min. The supematants were pooled and dialyzed against four changes of 0.01 M Tris, 0.1 M KC1 buffer (pH 8.1), using dialysis tubing with a molecular mass exclusion limit of 3500 D. The retained solution was applied to a trypsin-Sepharose CL-4B affinity column equilibrated with the same buffer used for dialysis. The adsorbed inhibitor protein was eluted with 8 M urea adjusted to pH 3 with HCl, dialyzed against 50 r m ammonium bicarbonate, and lyophilized. The lyophilized powder (18 mg) was applied to a G-75 gel filtration column (1.3 X 106 cm) equilibrated with 50 m~ ammonium bicarbonate, and the eluate were collected in 2mL fractions. The AzBOof fractions was determined, and inhibitory activities against trypsin were assayed. Tubes 53 to 68 (Fig. 2) were pooled and lyophilized. The elution profile of the TTI from Sephadex was compared with those of proteins of known mass (data not shown), and its molecular mass was estimated to be approximately5500 D. This molecular mass is comparable to those of small proteinase inhibitors from potatoes (Pearce et al., 1982) and eggplant (Richardson, 1979), suggesting that it may be a member of the potato inhibitor I1 family. The yield of TTI was 7.2 mg. The trypsin inhibitor fraction (200 pg) was dissolved in 0.1% TFA and applied to a semipreparative Cls RP-HPLC column. A linear gradient to 70% acetonitrile in 0.1% TFA was applied for 70 min, and 2-mL fractions were collected. One major and severa1 minor peaks of protein eluted from the column (Fig. 3), and a11 exhibited inhibitory activity against trypsin. The major fraction was pooled and lyophilized. This fraction was further purified using SCX-HPLC. O ‘ - 3 0 40 50 60 70 80 90 FRACTION NUMBER Figure 2. Gel filtration of tobacco trypsin inhibitory activity on Sephadex C-75. Affinity-purified tobacco trypsin inhibitor (18 mg in 1 mL of water) was applied to a 1.3- X 106-cm column of Sephadex G-75 equilibrated with 50 mM ammonium bicarbonate. Fractions (2 mL) were collected. Fractions containing trypsin inhibitory activity are indicated by the bar. 64 1 2.0 h E C v) cv hl Y 8 1.0 2 U m U O L v) m 4 O 5 3 . RETENTION TIME Figure 3. Separation of TTI on ClS RP-HPLC. An aliquot from the combined inhibitor fractions from Figure 2 containing 200 pg of protein was applied to the column and eluted with a gradient of O to 70% acetonitrile in 0.1% TFA for 70 min. The major absorbance peak (at 225 nm) containing the trypsin inhibitory activity was collected as marked above the elution profile. Six major peaks exhibited strong inhibitory activity against trypsin and were assigned numbers of 1 through 6 (Fig. 4A) Peaks 4 through 6 were desalted and lyophiliied. Peaks 1 through 3 were further purified using RP-HPLC with a shallow gradient (Fig. 4, B and C). The protein content of the six pure peptides was quantified using the BCA reagent with purified potato inhibitor 11 as a standard. Each inhibitor (5 Pg) was analyzed by SDS-PAGE and stained with Coomassie blue. Each inhibitor migrated as a single component (Fig. 5) with slight differences apparent in their migrations, indicating that small differences in molecular mass may exist among them. The apparent mass of the inhibitors, estimated from those of standard proteins, was less than 8 kD, but in this size range we have found that estimates of proteinase inhibitor molecular masses by SDS-PAGE analyses are often not accurate. The six purified peptides were assayed for their abilities to inhibit trypsin and chymotrypsin (Fig. 6). All of the polypeptides except number 3 were found to be strong inhibitors of trypsin. A11 six peptides were found to be .strong inhibitors of chymotrypsin. Rabbit antibodies were raised against the RP-HPLC fraction (Fig. 3) that contained the six isoinhibitors of TTI by cross-linking the proteins to RSA using glutaraldehyde. The antibodies were found to cross-react strongly with a11 six of the isolated inhibitors (data not shown). In double-diffusion assays in agar gels, no spurs were found between any of the precipitin lines among the six proteins. This indicated that a high degree of structural similarity was present among the Downloaded from on August 1, 2017 - Published by www.plantphysiol.org Copyright © 1993 American Society of Plant Biologists. All rights reserved. 642 Plant Physiol. Vol. 102, 1993 Pearce et al. Figure 4. A, SCX-HPLC separation of the trypsin inhibitory components of the major fraction from Figure 2. Protein from the Figure 3 fraction (120 Mg) was applied to an SCX-HPLC column, equilibrated with 5 mvi potassium phosphate (pH 3), containing 25% acetonitrile. The proteins were eluted for 60 min with a gradient of 500 mM KCI in the same buffer as above. Fractions (1 ml) were collected at a flow rate of 1 ml min"'. B, Elution profile of the RP-HPLC of peptide number 1 from A. The conditions were as in Figure 3 except a shallower gradient of 0 to 40% acetonitrile was used for an 80-min period. Peak number 1 was recovered. C, RPHPLC of combined peaks numbers 2 and 3 from A. Conditions were as described in B. 2.0 A. 123 C. B. 2.0 CM in CM UJ UJ CM o 1.0 CO tL O V) m 1 '° I m a: O J 5 CO m J min min. RETENTION TIME antigenic determinants of the proteins and confirmed the identity of all six proteins as isoinhibitor species. To demonstrate that TTI was the wound-inducible inhibitor, the antibodies were used to quantify the induction of TTI in leaves of wounded tobacco plants. In Figure 7 is shown the induction of TTI in leaves of young tobacco plants that had been wounded on their two lower leaves; the levels of TTI were quantified in all four leaves. The induction of TTI in the young, upper, unwounded leaves by wounding demonstrates the strong, systemic response of tobacco plants to wounding. The upper leaves were induced to accumulate about 40 /ig g~' of leaf tissue, whereas the inhibitor could not be detected in leaves of unwounded control plants. Lower wounded leaves were induced to accumulate about half of these levels of TTI. Mature tobacco plants in which all leaves were severely wounded accumulated about 80 ng of TTI g"1 of tissue in the youngest leaves. As in young plants, the lower leaves did not respond as well, but leaves from the middle of the plants accumulated about 25 /ng g~] of tissue. Leaves of unwounded plants did not exhibit the presence of TTI except for low levels in the uppermost leaves. The pattern of systemic induction in tobacco is similar to that found in tomato and potato plants, i.e. the younger, upper leaves respond more strongly to wounding than lower leaves. The amino acid sequence of the isoinhibitor TTI-1 was determined using the reduced and alkylated protein (Ruegg and Rudinger, 1977), repurified using RP-HPLC (see 'Materials and Methods'). The peptide was digested with V8 protease in ammonium acetate at pH 4, which specifically cleaves proteins with an internal Glu residue at the PI position. Two 1 2 3 4 5 6 0.5 1.0 5.0 0.5 1.0 2.0 0.5 1.0 INHIBITOR PER ASSAY (pg) Figure 6. Titration of trypsin and chymotrypsin by tobacco trypsin isoinhibitors 1 through 6. Details of the assays are in "Materials and Methods." Increasing quantities of isoinhibitor were added to a Figure 5. SDS-PACE of the six isoinhibitors purified in Figure 4. constant quantity of enzyme, and the remaining activities were Each peptide (5 jig) was loaded on the gel and analyzed by the assayed spectrophotometrically. Data are shown as percentages of method of Swank and Munkres (1971). The gel was stained with enzyme activity remaining. •, Trypsin activity. *, Chymotrypsin Coomassie blue dye. activity. Each is the average of three assays. Downloaded from on August 1, 2017 - Published by point www.plantphysiol.org Copyright © 1993 American Society of Plant Biologists. All rights reserved. Wound-lnducible Trypsin lnhibitor from Tobacco Leaves + 643 Table 1. Quantification of 7 7 1 and trypsin and chymotrypsin inhibition in various flower tissues Enzyme Inhibition' / P+2 Flower Part I 30 Ovary Sepal 20 Stamen Peta1 Mature fruit 10 O o 4 ã i2 16 20 a 24 HOURS AFTER WOUNDING Figure 7. Wound induction of TTI in 21-d-old tobacco plants. The two lower leaves of each plants were wounded around the perimeter, and the plants were incubated under light for 24 h. The juice was t h e n expressed from each leaf and assayed for the presence of TTI by radial immunodiffusion. Each point is the average from six plants. fragments of TTI were recovered (cf. Fig. 1) and subjected to sequence analysis by automated Edman degradation. The carboxy-terminal residue was determined to be a Ser by analyzing the release of amino acids by carboxypeptidase P. Only Ser was released, confirming that the C-terminal residue was Ser, as found by Edman degradation. The complete amino acid sequence of TTI-I is presented in Figure 8. TTI-I consists of 53 amino acid residues with a calculated molecular mass of 5868 D. The N-terminal sequence of 36 residues of isoinhibitor TTI-5 was obtained by Edman degradation. TTI-1 and TTI-5 (sequence not shown) exhibit nearly complete sequence identity with each other through the 36 residues available for comparison. The two isoinhibitors differ only at residue 11, where TTI-5 exhibits a Lys residue and TTI-1 a Thr residue. This is consistent with the longer retention time of TTI-5 on SCX-HPLC (Fig. 4A). The sequence data revealed that TTI isoinhibitors are members of the potato inhibitor I1 family. When the two isoinhibitors with known inhibitor I1 family members were compared (Fig. 8), the high percentage of sequence identity between this new member of the potato inhibitor I1 family and other members is clearly evident. TTI-1 appears to be most closely related to the eggplant inhibitor rather than to the small potato PCI. TTI-1 exhibits 83% identity with the eggplant 10 20 30 40 10 53 T?1-81 DRIC~CCAGTKGCKYI'SDDG?~VC~G~SDPRHPKACPRCDPRIAYGICPLS Comparisons of the amino acid sequence of TTI-1 (TTIsequence of the eggplant inhibitor (EP), potato PCI, and potato PTI. The arrow indicates the reactive site of the inhibitors. <E, Pyroglutamic acid. Figure 8. # 1 ) with t h e Protein" lnhibitorb mg mL-' mg-' protein 12.8 6.6 7.2 6.3 35.0 16.2 28.6 10.8 8.2 O Trypsin Chymotrypsin pg of enzyme inhibitedlmg oi protein 65.6 79.5 29.2 14.8 78.6 122.0 20.3 20.5 O O Protein was assayed using potato inhibitor I I as a stand- lnhibitor was assayed by immunoradial diffusion using ard. Determined by spectrophoRP-HPLC-pure TTI as a standard. tometer analysis (see "Materials and Methods"). inhibitor compared to 69% with PCI and PTI from potato tubers, 68% for potato inhibitor 11, and 60% homology with tomato inhibitor I1 (data not shown, Graham et al., 1985). The reactive site of TTI responsible for the proteinase inhibitor activity is assumed to be at the same residues as in the other members of the inhibitor I1 family. The P1-P1' residues at the reactive site would, therefore, be Arg-Asn. These are the identical residues found at the reactive sites of both the eggplant inhibitor and the PTI. In many plant species, the presence of defensive proteins in flowers has been reported. Recently, a small molecular mass proteinase inhibitor of the inhibitor I1 family was reported to be constitutively present in Nicotiana alata flowers in very high concentrations (Atkinson et al., 1993). Using anti-TTI serum to assay for the presence of TTI in the various flower parts, we have found that the flowers of N. tabacum also possess considerable quantities of TTI. Flowers were collected and dissected into various parts, and the tissues were crushed with a mortar and pestle to recover the expressed juice. The juice was centrifuged and assayed for soluble protein, for TTI protein levels, and for the presence of trypsin and chymotrypsin inhibitors. A11 flower parts exhibited high levels of TTI (Table I), with the sepals containing the highest levels. The mature fruit did not exhibit the presence of inhibitor. Modem tomato fruits do not contain proteinase inhibitor I or 11, but some wild species accumulate the two inhibitors to extraordinary levels of 1 to 2 mg g-' of tissue (Pearce et al., 1988). Stigmas from N. alata are reported to contain about 20 to 30% of their soluble proteins as TTI (Atkinson et al., 1993). The presence of trypsin inhibitor activity in leaves of tobacco plants was first reported (Walker-Simmons and Ryan, 1977) in a survey of oligogalacturonic acid-inducible trypsin inhibitors in plants. The wound-inducible trypsin inhibitors did not cross-react with rabbit antibodies raised against tomato or potato inhibitor I or I1 proteins, and it was assumed that the trypsin inhibitory protein(s) in tobacco was a member of a different inhibitor family. Rickauer et al. (1989) later reported the presence of an elicitor-inducible trypsin inhibitory activity in suspension cultures of N.taba- Downloaded from on August 1, 2017 - Published by www.plantphysiol.org Copyright © 1993 American Society of Plant Biologists. All rights reserved. Pearce et ai. 644 cum that appeared to be the same proteins a s the woundinducible proteins in leaves. Atkinson e t al. (1993) reported that the N. alata stigma trypsin inhibitor is a member of the potato inhibitor I1 family. The gene for this protein was m u c h larger t h a n that for t h e small inhibitor found in the stigma, indicating that t h e ínhibitor w a s proteolytically processed from the large precursor. Potato inhibitor I1 w a s t h e first member of this family to be isolated (Bryant e t al., 1976) a n d is t h e modem product of a n ancestral inhibitor gene that was duplicated a n d elongated to produce a n inhibitor of 12,300 kD with two similar domains, each possessing a reactive site (Garcia-Olmedo e t al., 1987). The sequences of two small, related inhibitors from potato tubers, called PCI and PTI (Hass e t al., 1982), are nearly identical with intemal sequences of potato a n d tomato inhibitor 11, suggesting that posttranslational t r i " i n g of t h e inhibitor takes place a t both t h e C terminus and the N terminus. PCI and PTI are composed of t h e last half of the N-terminal domain and the first half of t h e C-terminal domain (Hass et al., 1982), and their three-dimensional structure has been deduced by x-ray crystallography (Greenblatt et al., 1989). TTI has a sequence identical to the deduced sequence of t h e N. alata trypsin inhibitor (Atkinson et al., 1993) and is, therefore, likely to have been produced by a larger gene, as was found in N. alata. Our interest in TTI is to characterize t h e signal transduction in tobacco that regulates t h e systemic induction of this inhibitor in leaves in response t o wounding. This induction of TTI in tobacco leaves is similar in many ways t o t h e wound induction of inhibitor I and I1 proteins previously found in leaves of tomato a n d potato plants. In these latter species, a polypeptide signal, called systemin, appears t o be a n integral part of t h e signal transduction system (Pearce et al., 1991). The possibility exists that a polypeptide similar to tomato systemin may regulate t h e wound inducibility of tobacco TTI. The isolation of the wound-inducible TTI should facilitate our investigations of t h e signal transduction pathway that regulates t h e TTI gene in response t o pest attacks. ACKNOWLEDGMENTS The authors thank Greg Wichelns for growing the tobacco plants, Gerhardt Munske for the amino acid sequence analyses and helpful discussions, and S.E. Gurusaddaiah for amino acid analyses. Received January 21, 1993; accepted March 3, 1993. Copyright Clearance Center: 0032-0889/93/102/0639/06. LITERATURE CITED Atkinson AH, Heath RL, Simpson RJ, Clarke AE, Anderson MA (1993) Proteinase inhibitors in Nicotiana alata stigmas are derived from a precursor protein which is processed into five homologous inhibitors. Plant C e l l 5 203-213 Bradshaw HD, Hollick JB, Parsons TJ, Clarke HRG, Gordon M P (1989) Systemically wound-responsive genes in poplar trees encode proteins similar to sweet potato sporramins and legume Kunitz trypsin inhibitors. Plant Mo1 Bioll4 51-59 Brown WE, Ryan CA (1984) lsolation and characterization of a wound-induced trypsin inhibitor from alfalfa leaves. Biochemistry 2 3 3418-3422 Plant Physiol. Vol. 102, 1 9 9 3 Bryant J, Green TR, Gurusaddaiah S, Ryan CA (1976) Proteinase inhibitor I1 from potatoes: isolation and characterization of its promoter components. Biochemistry 1 5 3418-3424 Cuatrecasas P, Anfinson CB (1971) Affinity chromatography. Methods Enzymol22: 345-378 Doolittle RF (1977) Of Urfs and Orfs, A Primer on How to Analyze Derived Amino Acid Sequences. University Science Books, Mil1 Valley, CA, p 85 Garcia-Olmedo F, Salcedo G, Sanchez-Monge R, Gomez L, Royo J, Carbonero P (1987) Plant proteinaceous inhibitors of proteinases and a amylases. Oxf Surv Plant Mo1 Cell Biol4 275-334 Graham JS, Pearce G, Merryweather J, Titani K, Ericsson LH, Ryan CA (1985) Wound-induced proteinase inhibitors from tomato leaves. 11. The cDNA-deduced primary structure of preinhibitor 11. J Biol Chem 260 6561-6564 Green TR, Ryan CA (1972) Wound-induced proteinase inhibitor in plant leaves: a possible defense mechanism against insects. Sdence 1 7 5 776-777 Greenblatt HM, Ryan CA, James MNG (1989) Structure of the complex of Streptomyces griseus proteinase with polypeptide chymotrypsin inhibitor I from Russet Burbank potato tubers at 2.1 A resolution. J Mo1 Biol205 201-228 Hass GM, Hermodson MA, Ryan CA, Gentry L (1982) Primary structure of two low molecular weight proteinase inhibitors from potatoes. Biochemistry 21: 752-756 Hummel B (1959) A modified spectrophotometric determination of chymotrypsin, trypsin, and thrombin. Can J Biochem Physiol 37: 1393-1399 Melville CJ, Ryan CA (1972) Chymotrypsin inhibitor I from potatoes: large scale preparation and characterization of its subunit components. J Biol Chem 247: 3445-3453 Pearce G, Ryan CA (1983) A rapid, large-scale method for purification of the metallo-carboxypeptidaseinhibitor from potato tubers. Anal Biochem 1 3 0 223-225 Pearce G, Ryan CA, Liljegren D (1988) Proteinase inhibitors I and I1 in fruit of wild tomato species: transient components of a mechanism for defense and seed dispersal. Planta 1 7 5 527-531 Pearce G, Strydom D, Johnson S, Ryan CA (1991) A polypeptide from tomato leaves induces wound-indudble proteinase inhibitor proteins. Science 253 895-898 Pearce G, Sy L, Russell C, Ryan CA, Hass GM (1982) Isolation and characterization from potato tubers of two polypeptide inhibitors of serine proteinases. Arch Biochem Biophys 213 456-462 Plunkett G, Senear DF, Zuroske G, Ryan CA (1982) Proteinase inhibitors I and I1 from leaves of wounded tomato plants: purification and properties. Arch Biochem Biophys 213 463-472 Richardson M (1979) The complete amino acid sequence and the trypsin reactive (inhibitory) site of the major proteinase inhibitor from the fruits of the aubergine (Solanum melongena L.). FEBS Lett 104 322-326 Rickauer M, Fournier J, Esquerre-Tugaye MT (1989) Induction of proteinase inhibitors in tobacco cell suspension culture by elidtors of Phytophthora parasitica var nicotianae. Plant Physiol 9 0 1065-1070 Ruegg UT, Rudinger J (1977) Reductive cleavage of cystine disulfides with tibutylphosphine. Methods Enzymol47: 11 1-116 Ryan CA (1967) Quantitative determination of soluble cellular proteins by radial diffusion in agar gels containing antibodies. Anal Biochem 1 9 434-440 Ryan CA (1990) Protease inhibitors in plants: genes for improving defenses against insects and pathogens. Annu Rev Phytopathol 28: 425-449 Ryan CA (1992) The search for the proteinase inhibitor inducing factor, PIIF. Plant Mo1 Bioll9 123-133 Swank RT, Munkres KD (1971) Molecular weight analysis of oligopeptides by electrophoresis in polyaaylamide gels. Anal Biochem 39: 462-477 Trautman R, Cowan KM, Wagner GG (1971) Data for processing radial immunodiffusion. Immunochemistry 8: 901-916 Walker-Simmons M, Ryan CA (1977) Wound-induced accumulation of trypsin inhibitor activity in plant leaves. Survey of severa1 plant genera. Plant Physiol 5 9 437-439 Downloaded from on August 1, 2017 - Published by www.plantphysiol.org Copyright © 1993 American Society of Plant Biologists. All rights reserved.