Article Figures & Data
Figures
- Fig. 1.
PLPIs separation on C-18 RP-HPLC. One-fourth of the inhibitors eluted from the chymotrypsin affinity column was loaded into the C-18 column and eluted with a gradient of 0% to 40% (v/v) acetonitrile in 0.1% (v/v) TFA for 50 min. The seven major peaks were eluted at approximately 34, 35, 40, 41, 43, 45, and 46 min and were purified to homogeneity using a combination of C-18 RP-HPLC and SCX-HPLC.
- Fig. 2.
SDS/urea-PAGE of the seven purified PLPIs. Fifteen micrograms of each PLPI was loaded on the gel. The gel was silver stained and part of the gel containing the lanes with PLPIs 43, 45, and 46 was developed longer.
- Fig. 3.
Titration of chymotrypsin and trypsin by each purified PLPI. Trypsin and chymotrypsin activities were measured spectrophotometrically in the presence of increasing amounts of the inhibitors. ●, Chymotrypsin activity. ▵, Trypsin activity.
- Fig. 4.
Sequence alignment of the PLPIs. A, Alignment of PLPIs 34, 35, 40, 45, and 46 with the GenBank accession no. AF221097. B, Alignment of PLPIs 34, 35, 40, 41, and 43 with the GenBank accession number AF039398. C, Alignment of deduced proteins from tobacco stigma inhibitors II precursor (GenBank accession no. JQ2153), tomato inhibitor II (GenBank accession no. P05119), and pepper (GenBank accession nos. AF221097 and AF039398) signal sequences were omitted for clarity. Reactive sites are indicated by amino acids in bold and conserved cysteines are indicated by bars between sequences.
- Fig. 5.
Wound-inducibility of PLPIs in pepper leaves. Pepper plants were wounded with a hemostat across the midvein and in three different locations along the borders of the lower leaf. Tissues were collected at times indicated and leaf juice was extracted for PLPI evaluation. PLPIs levels were evaluated using immunodiffusion assay. Six plants were used for each time point evaluated. ♦, Wounded plants. ▪, Control plants.
- Fig. 6.
Analyses of PLPI protein levels in eight different varieties of pepper. Each variety was wounded with a hemostat across the midvein and in three different locations along the lower leaf. PLPI protein levels of the wounded plants (black bars) and untreated plants (white bars) were evaluated by radial immunodiffusion. The results are the average of 12 plants.
- Fig. 7.
Gel-blot analyses of PLPI mRNA induction in control plants and upon wounding. Cotyledons and leaves were harvested at the times indicated and were immediately frozen in liquid nitrogen for RNA extraction. Fifteen micrograms of total RNA was loaded and separated on a 1.5% (w/v) agarose gel. The RNA was salt transferred to nylon membranes that were used for hybridization with DNA probes. 18S rRNA was used as loading control.
- Fig. 8.
Gel-blot analyses of PLPI mRNA induction in systemin (A), water (B), and MeJ-treated plants (C). Cotyledons and leaves were harvested at the times indicated and were immediately frozen in liquid nitrogen for RNA extraction. Fifteen micrograms of total RNA was loaded and separated on a 1.5% (w/v) agarose gel. The RNA was salt transferred to nylon membranes that were used for hybridization with DNA probes. 18S rRNA was used as loading control.
Tables
Purification Steps Volume Protein Inhibitors Activity Specific Activity mL mg CUI1-a CUI1-a/mg protein Crude extract 3,300 2,425 54.7 440,000 182 Ammonium sulfate precipitation. 1,620 2,284 53.3 438,000 192 Heat treatment + dialysis 2,110 527 38.7 298,000 566 Chymotrypsin affinity column 90 36 36.0 259,000 7,194 HPLC of most abundant inhibitors 3.3 9.94 9.94 -
↵F1-a CUI, Chymotrypsin units inhibited.
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- Table II.
Quantities obtained for each PLPI after using C-18 RP-HPLC and SCX-HPLC purification
PLPI Retention Time on C-18 RP-HPLC Inhibitor Protein Percent min mg 34 2.27 23 35 1.87 19 40 2.87 30 41 0.96 9 43 0.64 6 45 0.48 5 46 0.85 8 Inhibitor Retention Time N-Terminal Sequence3-a Mass Inhibitor of Chymotrypsin Inhibitor of Trypsin KiChymotrypsin Ki Trypsin min D m 34 RICTNCCAGRKGCNYYSADG 5,646 Yes Yes 8 × 10−11 5 × 10−9 35 .................... 5,671 Yes Yes 3 × 10−10 4 × 10−9 40 .................... 5,681 Yes No 1 × 10−10 – 41 AKEP................ 5,952 Yes No 6 × 10−10 – 43 EP.................. 5,755 Yes No 7 × 10−10 – 45 .L.................. 5,716 Yes Yes 1 × 10−9 1 × 10−8 46 .L.................. 5,586 Yes Yes 3 × 10−10 6 × 10−9 -
↵F3-a A dot represents an amino acid identical to PLPI 34.
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Treatments4-a Inhibitor Level4-b μg/mL MeJ 84 ± 8 Systemin 60 ± 8 Water/Buffer 49 ± 16 Untreated 34 ± 5 SA 33 ± 4 SA +Wounding 30 ± 4 -
↵F4-a Control plants for systemin and SA treatment were supplied with water or buffer, control plants for MeJ were left undisturbed, untreated. Pepper leaves were harvested 24 h after the experiments started and PLPI levels were evaluated by radial immunodiffusion assay.
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↵F4-b Average of six plants for each treatment. Treatments with the same letter do not differ statistically at the significance level of 0.05 (Duncan, 1955).
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