Download Leaves of Six Small, Wound-lnducible, Proteinase lsoinhibitor

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Ryan, unpublished data).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
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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-
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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
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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.
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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-
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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.
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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
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