Purification and Characterization from Tobacco (Nicotiana ... · in solanaceous plants (Melville and Ryan, 1972; Bryant et al., 1976; Plunkett et al., 1982), Bowman-Birk inhibitors

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 I I Family'

Gregory Pearce, Scott Johnson, and Clarence A. Ryan* lnstitute of Biological Chemistry, Washington State University, Pullman, Washington 991 64-6340

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 inhibitors from 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-

This research was supported in part by Washington State UN- versity College of Agriculture and Home Economics, project 1791, and by National Science Foundation grants IBN-9244361 and IBN- 9117795.

* Corresponding author; fax 1-509-335-7643. 639

inducible trypsin inhibitor activity in tobacco leaves (Walker- Simmons 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 investi- gate 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.

MATERIALS AND METHODS

G-75 Sephadex was purchased from Pharmacia (Piscata- way, 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-

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.

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640 Pearce et al. Plant Physiol. Vol. 102, 1993

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 RP- HPLC utilizing a linear gradient from 0.1% TFA in water at O min to 60% acetonitrile in 0.1 % TFA at 60 min. Native TTI- 1 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 600 o z U 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 alkylated as described in “Materials and 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.

and the two proteolytic fragments were determined using Edman chemistry on an Applied Biosystems model 475 se- quencer 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 carboxypeptidase P (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 cross- linking 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; Traut- man 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 sev- era1 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 cross- react 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-inducible inhibitory activity was subse- quently 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-



Wound-lnducible Trypsin lnhibitor from Tobacco Leaves 64 1

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 precip- itate 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 2- mL fractions. The AzBO of 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 approximately 5500 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 ‘ -30 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.

2.0

h

E C v) cv hl Y

8 1.0 2 U m U O v) m 4

O L

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 polypep- tides 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



642 Pearce et al. Plant Physiol. Vol. 102, 1993

Figure 4. A, SCX-HPLC separation of the tryp-sin inhibitory components of the major fractionfrom 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 pro-teins were eluted for 60 min with a gradient of500 mM KCI in the same buffer as above. Frac-tions (1 ml) were collected at a flow rate of 1ml min"'. B, Elution profile of the RP-HPLC ofpeptide number 1 from A. The conditions wereas in Figure 3 except a shallower gradient of 0to 40% acetonitrile was used for an 80-minperiod. Peak number 1 was recovered. C, RP-HPLC of combined peaks numbers 2 and 3from A. Conditions were as described in B.

2.0

CM

UJo

COtLOV)m

1.0

A. 123 B.

J

C.

J5 minmin.

RETENTION TIME

2.0

inCMCMUJ

1'° Ima:OCOm

antigenic determinants of the proteins and confirmed theidentity of all six proteins as isoinhibitor species.

To demonstrate that TTI was the wound-inducible inhibi-tor, the antibodies were used to quantify the induction of TTIin leaves of wounded tobacco plants. In Figure 7 is shownthe induction of TTI in leaves of young tobacco plants thathad been wounded on their two lower leaves; the levels ofTTI were quantified in all four leaves. The induction of TTIin the young, upper, unwounded leaves by wounding dem-onstrates the strong, systemic response of tobacco plants towounding. The upper leaves were induced to accumulateabout 40 /ig g~' of leaf tissue, whereas the inhibitor couldnot be detected in leaves of unwounded control plants. Lowerwounded leaves were induced to accumulate about half ofthese levels of TTI. Mature tobacco plants in which all leaveswere severely wounded accumulated about 80 ng of TTI g"1

of tissue in the youngest leaves. As in young plants, the lowerleaves did not respond as well, but leaves from the middleof the plants accumulated about 25 /ng g~] of tissue. Leavesof unwounded plants did not exhibit the presence of TTIexcept for low levels in the uppermost leaves. The pattern of

1 2 3 4 5 6

Figure 5. SDS-PACE of the six isoinhibitors purified in Figure 4.Each peptide (5 jig) was loaded on the gel and analyzed by themethod of Swank and Munkres (1971). The gel was stained withCoomassie blue dye.

systemic induction in tobacco is similar to that found intomato and potato plants, i.e. the younger, upper leavesrespond more strongly to wounding than lower leaves.

The amino acid sequence of the isoinhibitor TTI-1 wasdetermined using the reduced and alkylated protein (Rueggand Rudinger, 1977), repurified using RP-HPLC (see 'Mate-rials and Methods'). The peptide was digested with V8 pro-tease in ammonium acetate at pH 4, which specifically cleavesproteins with an internal Glu residue at the PI position. Two

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 trypsinisoinhibitors 1 through 6. Details of the assays are in "Materials andMethods." Increasing quantities of isoinhibitor were added to aconstant quantity of enzyme, and the remaining activities wereassayed spectrophotometrically. Data are shown as percentages ofenzyme activity remaining. •, Trypsin activity. *, Chymotrypsinactivity. Each point is the average of three assays. www.plantphysiol.orgon January 27, 2020 - Published by Downloaded from

Copyright © 1993 American Society of Plant Biologists. All rights reserved.


Wound-lnducible Trypsin lnhibitor from Tobacco Leaves 643

+

30

20

10

O

/ P + 2 I

o 4 ã i 2 16 20 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 perim- eter, and the plants were incubated under light for 24 h. The juice was then 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

1 0 2 0 3 0 40 1 0 53 T?1-81 DRIC~CCAGTKGCKYI'SDDG?~VC~G~SDPRHPKACPRCDPRIAYGICPLS

Figure 8. Comparisons of the amino acid sequence of TTI-1 (TTI- # 1 ) with the sequence of the eggplant inhibitor (EP), potato PCI, and potato PTI. The arrow indicates the reactive site of the inhibitors. <E, Pyroglutamic acid.

Table 1. Quantification of 7 7 1 and trypsin and chymotrypsin inhibition in various flower tissues

Enzyme Inhibition'

Flower Part Protein" lnhibitorb Chymo- trypsin

Trypsin

pg of enzyme

protein mg-' inhibitedlmg oi

protein mg mL-'

Ovary 12.8 16.2 65.6 78.6

Stamen 7.2 10.8 29.2 20.3 Peta1 6.3 8.2 14.8 20.5 Mature fruit 35.0 O O O

Sepal 6.6 28.6 79.5 122.0

a Protein was assayed using potato inhibitor I I as a stand- lnhibitor was assayed by immunoradial diffusion using

Determined by spectropho- ard. RP-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-



644 Pearce et ai. Plant Physiol. Vol. 102, 1993

cum that appeared to be the same proteins as the wound- inducible 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 much larger than that for the small inhibitor found in the stigma, indicating that the ínhibitor was proteolytically processed from the large precursor.

Potato inhibitor I1 was the first member of this family to be isolated (Bryant e t al., 1976) and is the modem product of a n ancestral inhibitor gene that was duplicated and elongated to produce a n inhibitor of 12,300 kD with two similar do- mains, each possessing a reactive site (Garcia-Olmedo et 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 and tomato inhibitor 11, suggesting that posttranslational t r i " i n g of the inhibitor takes place a t both the C terminus and the N terminus. PCI and PTI are composed of the last half of the N-terminal domain and the first half of the 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 the 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 the signal transduction in tobacco that regulates the systemic induction of this inhibitor in leaves in response to wounding. This induction of TTI in tobacco leaves is similar in many ways to the wound induction of inhibitor I and I1 proteins previously found in leaves of tomato and potato plants. In these latter species, a polypeptide signal, called systemin, appears to be a n integral part of the signal transduction system (Pearce et al., 1991). The possibility exists that a polypeptide similar to tomato systemin may regulate the wound inducibility of tobacco TTI. The isolation of the wound-inducible TTI should facilitate our investigations of the signal transduction pathway that regulates the TTI gene in response to 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.

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Brown WE, Ryan CA (1984) lsolation and characterization of a wound-induced trypsin inhibitor from alfalfa leaves. Biochemistry 2 3 3418-3422

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Documents

Purification and Characterization from Tobacco (Nicotiana ... · in solanaceous plants (Melville and Ryan, 1972; Bryant et al., 1976; Plunkett et al., 1982), Bowman-Birk inhibitors