Environmental and Developmental Regulation of Trypsin Inhibitor ...

Journal of Chemical Ecology, Vol. 26, No. 6, 2000

ENVIRONMENTAL AND DEVELOPMENTAL REGULATION OF TRYPSIN INHIBITOR ACTIVITY IN Brassica napus

DONALD F. CIPOLLINI* and JOY BERGELSON Department of Ecology and Evolution, University of Chicago 1101 East 57th Street, Chicago, Illinois 60637 (Received August 16, 1999; accepted February 1, 2000) Abstract—We examined several environmental and developmental influences on trypsin inhibitor (TI) activity in leaves of young Brassica napus seedlings in a series of greenhouse experiments. In seedlings of B. napus cv. Westar, TI activity is constitutively present and exhibits a rise then fall through time in the first true leaves of young plants. TI activity is induced by wounding in the first true leaves, but the degree of induction is relatively insensitive to the degree of wounding over a gradient of 5–15% of leaf area damage. TI activity is enhanced in first true leaves of plants in which the cotyledons have been wounded relative to plants in which the cotyledons have not been wounded. TI activity is also enhanced in the second true leaves on plants in which the first true leaves have been wounded. The degree of systemic induction in second true leaves declines additively with plant age, but local induction in the first true leaves is not affected by age. In B. napus cv. Gido, TI activity is constitutively present but is not locally wound-inducible in first true leaves of young plants exposed to the same wounding gradient as cv. Westar. In unwounded plants at the six-leaf stage, TI activity is higher in second true leaves than in fifth true leaves, indicating that TI activity is developmentally regulated in this cultivar. Key Words—Brassica napus, defense-related proteins, environmental effects, induced defenses, proteinase inhibitors, regulation, resistance, trypsin inhibitors, variation, wounding.

*To whom correspondence should be addressed at current address: Department of Biological Sciences, Wright State University, 3640 Colonel Glenn Highway, Dayton, Ohio 45435-0001.

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INTRODUCTION

It is now widely recognized that constitutive and inducible chemical defenses are major determinants of plant resistance to natural enemies (e.g., Rosenthal and Berenbaum, 1992; Karban and Baldwin, 1997). As such, variation in the expression of chemical defenses among plants and plant parts may partly explain the observed variation in the amount of damage inflicted by natural enemies in the field within and among plant tissues and plant populations. To fully understand the mechanistic basis of variation in plant resistance, it is important to understand how environmental, developmental, and genetic factors may regulate the production of specific defense traits in plants. Serine proteinase inhibitors are a class of defense-related proteins found widely throughout the plant kingdom (Koiwa et al., 1997). They function by binding specifically with the protein binding site of proteinases of both plant and nonplant origin (e.g., trypsin and chymotrypsin), thereby competitively inhibiting their ability to bind and cleave proteins (see Koiwa et al., 1997, for review). Although their effectiveness as a defense against natural enemies can vary by plant and herbivore species (Broadway, 1995), proteinase inhibitors reduce the growth and survival of many insect herbivores when present in artificial diets (Broadway and Duffey, 1986) and reduce both insect feeding rate and performance when expressed in transgenic plants (Johnson et al., 1990; McManus et al., 1994). Moreover, the induction of serine proteinase inhibitors by wounding or natural enemy attack, along with the induction of other classes of defense proteins, is commonly associated with induced resistance to herbivores (Green and Ryan, 1972; Broadway et al., 1986; Thaler et al., 1996). Regulation of proteinase inhibitors has been well studied in crop plants in the Solanaceae (e.g., tomato) and Fabaceae (e.g., soybean), partly because of the ability of proteinase inhibitors to produce physiological disorders when in the diet of humans and livestock, and partly because of their role in defense against natural enemies (Koiwa et al., 1997). Although proteinase inhibitors are known to occur in many wild and cultivated members of the Cruciferae (Broadway, 1989), environmental and developmental regulation in crucifers has not been well characterized (but see Broadway and Missurelli, 1990). This is probably because much of the research on the ecology of chemical defenses in crucifers has focused on the glucosinolates. Such work has surprisingly shown only infrequently that glucosinolates directly mediate plant resistance to insects (Chew, 1988; Bodnaryk, 1992, 1997; Hopkins et al., 1998). In a series of greenhouse experiments, we examined some of the potential factors regulating the expression of proteinase inhibitor activity in leaves of the crucifer Brassica napus L. We first characterized the time course of the expression of constitutive and wound-induced trypsin inhibitor (TI) activity in first true leaves of 10-day-old Brassica napus seedlings. We then examined the effects of

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wound location, plant and leaf age, soil volume, and genotype on the expression of constitutive and wound-induced TI activity in seedlings.

METHODS AND MATERIALS

B. napus L. (Brassicacae) is a self-compatible annual plant that is grown commercially as an oilseed and forage crop and that occasionally naturalizes in some environments (Duke, 1983). We studied an agronomic cultivar of B. napus (cv. Westar), commonly used in agroecological and physiological studies (Bodnaryk, 1992), and another less well studied cultivar (cv. Gido). Plants were grown from seed in a greenhouse, in 300-ml square pots containing ProMix BX potting soil, unless stated otherwise. To reduce genetic variation among replicate plants, we used seeds gathered from one self-pollinated maternal plant that had been grown under the same greenhouse conditions as those used in this experiment. Greenhouse photoperiod during experiments was controlled at 16L : 8D. Natural daylengths were extended as necessary with sodium vapor lamps, which were programmed to turn on whenever natural light levels dipped below 400 mmol photons/ m2 / sec (photosynthetically active radiation) during the light period. Mean daytime irradiance during these experiments was 1100 mmol photons/ m2 / sec PAR. Temperatures averaged 26 ± 38 C during the light period and 21.5 ± 18 C during the dark period. Plants were watered daily with tap water and fertilized as indicated in each experiment. In all experiments, plants were grown in a completely randomized fashion on the greenhouse benches and moved randomly within the benches at least three times over the course of each experiment. Experiment 1: Time Course of TI Activity in Wounded and Unwounded Plants. In this experiment, 10-day-old plants (cv. Westar) were wounded by crushing the distal 5, 10, or 15% of the total area of the first true leaf with sterilized needle-nosed pliers. Control plants were left unwounded. When treatments were applied (day 0 of the time course), the first true leaves were ∼4–5 days old. The induction of TI activity in Brassica oleraceae (Broadway and Missurelli, 1990), and Brassica rapa (D. Cipollini, J. Busch, K. Stowe, E. Simms, and J. Bergelson, unpublished data) by this type of mechanical wounding closely simulates that produced in response to feeding by larval Trichoplusia ni. We expect that TI induction by mechanical wounding also closely simulates induction by natural herbivory in B. napus, but the degree to which this is true is not known. Samples of the first true leaves were harvested on days 0, 1, 2, 3, and 6 following wounding from separate sets of plants receiving each treatment. At harvest, leaf samples from wounded and unwounded plants were placed in 1.7-ml microfuge tubes, flash frozen in liquid nitrogen, and stored at − 208 C until analysis. Treatments were replicated 6–10 times. Effects of day, wounding, and their

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interaction on TI activity were analyzed with a two-way fixed effects ANOVA. SAS (Release 6.12, SAS Institute, Cary, North Carolina) was used to analyze the data from this and all other experiments. Experiment 2: Systemic Induction from Cotyledon to First True Leaf (cv. Westar). In this experiment, 10-day-old plants were either wounded by crushing 10% of the area of both cotyledons with needle-nosed pliers or left unwounded. Three days following wounding, the first true leaves were harvested from all plants, stored, and analyzed for TI activity as below. Treatments were replicated seven to eight times. The effect of wounding on TI activity was analyzed with a one-way fixed effect ANOVA. Experiment 3: Effect of Plant Age on Local and Systemic Induction (cv. Westar). In this experiment, a staggered planting was used to produce plants that were either 10 or 15 days old at the start of the experiment. Plants of each age were either wounded by crushing 10% of the area of the first true leaf with needle-nosed pliers or left unwounded. Three days following wounding, both the first and the second true leaves were harvested from all plants, stored, and analyzed for TI activity as below. Treatments were replicated 8–10 times. Effects of plant age, wounding, and their interaction on TI activity were analyzed by using separate two-way fixed effects ANOVAs for data from the first and second true leaves. Experiment 4: Effect of Pot Size and Wounding on Trypsin Inhibitor Activity (cv. Gido). In this experiment, plants (cv. Gido) were grown from seed in either 300-ml or 600-ml pots. First true leaves on 10-day-old plants were exposed to the same wounding gradient as in experiment 1 and harvested three days following wounding. Treatments were replicated 9–10 times. Leaves were stored and analyzed for TI activity as below. Effects of pot size, wounding, and their interaction on TI activity were analyzed with a two-way fixed effects ANOVA. Experiment 5: Effect of Leaf Age on TI Activity (cv. Gido). In this experiment, the second and fifth true leaves on 25-day-old plants (cv. Gido) were harvested from unperturbed plants, stored, and analyzed for TI activity as below. Leaves were harvested from 17 plants. The effect of leaf age on TI activity was analyzed with a one-way fixed effect ANOVA. Protein Extraction and Analysis of TI Activity. To extract soluble proteins, we first ground individual leaf samples directly in the microfuge tubes without buffer with a Teflon minipestle. A 0.150-ml aliquot of ice-cold 1 mM HCl (Broadway and Missurelli, 1990) was placed in each tube, and the tubes were vortexed for 10 sec. Samples were centrifuged at 12,000g for 12 min in a microfuge cooled to 48 C. After centrifugation, the cleared supernatants were transferred into new tubes and used as the soluble protein extracts. Extracts were kept on ice throughout the procedure, and used immediately in chemical analyses. We analyzed TI activity of individual extracts by using a radial diffusion


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assay (Jongsma et al., 1993, 1994). First, 100 ml of a melted agar (Bacto-Agar, Difco, Detroit, Michigan) solution (1.8% w/ v in 100 mM Tris Cl buffer, pH 7.6) was cooled to 558 C and mixed with a solution of bovine trypsin (Sigma Chemical Co., St. Louis, Missouri) to a final concentration of 0.001 mg/ ml in the agar. Immediately after adding the enzyme, the melted solution was poured into a 24 × 24 cm square plastic bioassay dish (Nunc, Denmark) and allowed to solidify at 48 C for 4 hr. We punched wells 4 mm in diameter in the agar to accommodate each sample. Sample extracts (28 ml) were introduced into the wells randomly throughout the gel and were allowed to diffuse at 48 C for 18 hr. After incubation, we rinsed the gel for 2 min in 100 mM Tris Cl, pH 7.6, containing 10 mM CaCl2 . A solution consisting of 48 mg Fast Blue B Salt (O-dianisidine) in 90 ml of 100 mM Tris Cl, pH 7.6, at 378 C was mixed with 24-mg N-acetyl-DL-phenylalanine-naphthyl ester in 10 ml of N,N-dimethylformamide, and immediately poured onto the gel. The gel was incubated with the staining solution at 378 C for 30 min and then rinsed four times with tap water. With this technique, zones with TI activity around each well remain clear, while the rest of the gel stains a bright pink–purple. We quantified TI activity by measuring the diameter of the clear zones around each well with digital vernier calipers, while the gel was placed on a transilluminator. Samples were compared to a standard curve made with purified soybean TI in 1 mM HCl that was run in the same gel with the sample extracts. Soluble protein contents of each extract were quantified by the method of Bradford (1976) by using the Bio-Rad protein dye reagent. We expressed TI content of each extract as micrograms TI per milligram extract protein. All chemicals were purchased from Sigma Chemical Co. (St. Louis, Missouri).

RESULTS

Experiment 1. The expression of TI activity in first true leaves of wounded and unwounded B. napus (cv. Westar) seedlings was followed for six days (Figure 1). TI activity was constitutively present, but also enhanced by wounding in first true leaves of this cultivar (wound: F 3, 123 c 2.68, P c 0.049). Induction of TI activity was evident one day following wounding and became significant two days following wounding. TI activity exhibited a rise and then fall through time in both wounded and unwounded plants (time: F 3, 123 c 72.23, P c 0.0001), but wound-induced levels remained higher throughout the time course. The degree of induction of TI activity by wounding did not vary with the degree of wounding over the gradient that we imposed. Experiment 2. The systemic induction of TI activity was examined in first true leaves of B. napus (cv. Westar) seedlings on which the cotyledons had been wounded. TI activity was higher in plants on which the cotyledons had been

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FIG. 1. Time-course of trypsin inhibitor activity in first true leaves of wounded and unwounded Brassica napus (cv. Westar) seedlings. On the day plants were wounded (day 0), plants were 10 days old and first true leaves were 4–5 days old. Each point is the mean (±SE) of 6–10 replicate plants.

wounded than in unwounded plants (wounded: 20.12 ± 1.09 mg TI/ mg prot, unwounded: 17.27 (± 0.84) mg TI/ mg prot; F 1, 13 c 3.87, P c 0.05). Experiment 3. The local and systemic wound-induction of TI activity was examined in 10- and 15-day-old B. napus (cv. Westar) seedlings (Figures 2 and 3). Wounding enhanced TI activity in the first true leaves of both 10- and 15day-old seedlings (wound: F 1, 30 c 19.57, P c 0.0001). TI activity in first true leaves was unaffected by both plant age (age: F 1, 30 c 0.53, P c 0.474), and the interaction between wounding and plant age (wound × age: F 1, 30 c 0.04, P c 0.835). Wounding of the first true leaves also enhanced TI activity in the second true leaves of both 10- and 15-day-old plants (wound: F 1, 30 c 9.52, P c 0.0043). Plant age marginally affected TI activity in second true leaves (age: F 1, 30 c 3.31, P c 0.070). No interaction was observed between wounding and plant age on TI activity in second true leaves (age × wound: F 1, 30 c 2.06, P c 0.162). The degree of systemic induction tended to decline with plant age, owing to the increase in constitutive levels of TI activity in second true leaves of 15-day-old plants. Experiment 4. The presence and wound-inducibility of TI activity was examined in first true leaves of B. napus (cv. Gido) grown at two pot sizes (Figure 4). TI activity was constitutively present in all plants, but wounding (F 3, 68 c 0.23, P c 0.634), pot size (F 1, 68 c 0.83, P c 0.4795), and their interaction (F 3, 68 c 1.94, P c 0.132) had no significant effect on TI activity in this cultivar.


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FIG. 2. Effect of plant age on local induction of trypsin inhibitor activity in first true leaves of Brassica napus (cv. Westar) seedlings. Bars are the mean (±SE) of 8–10 replicate plants.

FIG. 3. Effect of plant age on systemic induction of trypsin inhibitor activity in second true leaves of Brassica napus (cv. Westar) seedlings on which the first true leaves had been wounded or unwounded. Bars are the mean (±SE) of 8–10 replicate plants.

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FIG. 4. Trypsin inhibitor activity in first true leaves of wounded and unwounded Brassica napus (cv. Gido) seedlings grown at two pot volumes. Bars are the mean (±SE) of 9–10 replicate plants.

Experiment 5. TI activity was compared between the second and fifth true leaves on unperturbed 25-day-old seedlings of B. napus (cv. Gido). TI activity was higher in the older, second true leaves, than in the younger, fifth true leaves (second: 20.93 (± 0.60) mg TI/ mg prot, fifth: 18.46 (± 0.55) mg TI/ mg prot; F 1, 33 c 9.22, P c 0.0047). DISCUSSION

In this study, TI activity in seedlings of B. napus was shown to be under complex control by several environmental, developmental, and genetic factors, all of which can contribute to variation in the chemical phenotype. First, TI activity, on a per milligram protein basis, exhibits significant changes through time in the first true leaves. TI activity rises with leaf age to an apparent peak when the leaf is about 1 week old and then declines over the next several days (Figure 1). Proteinase inhibitors are known to be stored after synthesis in vacuoles and to have slow turnover rates (Gustafson and Ryan, 1976). Thus, most of the constitutive synthesis of TI in B. napus leaves probably occurs early in the life of the leaf, after which its synthesis declines relative to the synthesis of other soluble leaf proteins, leading to a reduction in TI activity per milligram protein with time (a dilution effect). Although this experiment focused on the first true leaves, there is likely a similar time course to the expression of TI


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activity in each successive leaf as it appears and ages, although absolute levels of TI activity may vary by leaf position. While TI activity was constitutively present in all plants in our study, mechanical wounding significantly enhanced TI activity not only locally, but also systemically in leaves of young B. napus (cv. Westar). Enhanced TI activity in wounded plants also remained for at least six days (and probably longer) following a single wounding event. In contrast, TI activity in B. oleraceae is not systemically inducible by wounding, although it is constitutively expressed and is locally wound-inducible at certain developmental stages (Broadway and Missurelli, 1990). TI activity is also constitutively present, as well as locally and systemically wound-inducible in Arabidopsis thaliana (D. Cipollini and J. Bergelson, unpublished data), but the dearth of studies on the induction of TI activity in other crucifers prevents further comparisons. Unlike cruciferous plants, TI activity in the foliage of solanaceous plants like tomato is typically present at negligible levels constitutively, but is induced greatly both locally and systemically by wounding (Koiwa et al., 1997). In accordance with other studies, we found that systemic induction of TI activity also declined with plant age, which is thought to result from a general decline in the ability of plants to systemically signal defense as they age (Alarcon and Malone, 1995). These findings illustrate that patterns of the deployment of TI activity in plants can share similarities, but also exhibit important differences, among and within plant families. For example, while TI activity in cruciferous plants is moderately inducible, it appears to be primarily a constitutive defense (Broadway and Missurelli, 1990). In contrast, TI activity in solanaceous plants is generally absent constitutively and appears to be primarily an inducible defense (Koiwa et al., 1997). Although the induction of TI activity and other chemical defenses in plants typically shows some sort of dose–response to abiotic or biotic elicitors of defense (e.g., Bodnaryk, 1992; Thaler et al., 1996), induction of TI activity was relatively insensitive to the amount of wounding that we imposed. This indicates that either similar amounts of wound signal were produced by each of our wound treatments or that an upper limit on TI production exists, regardless of the amount of wound signal generated (DeWitt et al., 1998). It is not known how TI activity might respond to higher levels of wounding B. napus, but resource depletion caused by leaf area removal could begin to constrain induction at higher amounts of damage (Baldwin and Schmelz, 1994). Our study illustrates that the effects of even a single wounding event on TI activity can be significant and long lasting, but it is possible that chronic gradual wounding (such as that produced by insect feeding) could more greatly affect TI activity (Jongsma et al., 1994). While our results and discussion are based primarily on a cultivar of B. napus (cv. Westar) that is clearly wound-inducible, our experiments with B. napus cv. Gido illustrate that genotypic variation in wound-inducibility exists

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in this species. In cv. Gido, TI activity was constitutively present at similar levels as cv. Westar but was not wound-inducible when exposed to the same wound gradient and examined at a similar plant age and leaf stage. Soil volume also had no effect on the constitutive and wound-induced expression of TI activity in this cultivar, which was not surprising given that plants exhibited no differences in growth at either of the two pot sizes (data not shown). Genetic variation in inducibility of chemical defenses has been little studied in any plant (Cipollini, 1998) but is known to exist for furanocoumarins in wild parsnip (Zangerl and Berenbaum, 1990) and pyrrolizidine alkaloids in Cynoglossum officinale (van Dam and Vrieling, 1994). While genetic variation in constitutive TI activity has been identified in the seeds of some legumes (Domoney et al., 1994; Kollipara et al., 1994), the extent of genetic variation in inducibility of TI activity in B. napus or any other plant remains to be determined and would require the inclusion of many more genotypes that the two used in our comparison. Overall, our results demonstrate that the expression of a putative defense, TI activity, can be affected by many environmental, developmental, and genotypic factors. In a companion paper, we show that other environmental factors, such as plant density and nutrient availability, can also profoundly affect constitutive and wound-inducible TI activity in B. napus (cv. Westar) (D. Cipollini and J. Bergelson, in review). As with other defenses, the effectiveness of TIs as an antiherbivore defense can depend on the ability of an herbivore species to acclimate to TIs in the diet by adjusting midgut physiology (Broadway, 1995; Jongsma and Bolter, 1997). The presence of certain plant components in the diet can also affect the ability of TIs to act as antiherbivore defenses (Felton et al., 1989). However, although the relative importance of TIs in defense is still unclear, variation in TI expression could contribute to the variation in the amount of herbivore damage seen within and among Brassica populations in the field. Although wound inducibility is thought to be a cost-saving form of adaptive phenotypic plasticity in plants (Cipollini, 1998; Baldwin, 1999), the tremendous variation in chemical phenotypes that can exist within and among plants in a population, regardless of their inducibility, is thought to be adaptive in itself. Variation in food quality may decrease herbivore performance and/ or the ability of an herbivore population to adjust its physiology to track a plant population in ecological time or to adapt to a plant population through natural selection (Karban et al., 1997). While our study illustrates that even agronomic Brassica cultivars that have undergone artificial selection for certain performance traits can exhibit significant phenotypic variation in TI activity, variation in TI activity within and among wild Brassica species and populations may be even greater than that found in our experiment, and is the subject of current investigation. Acknowledgments—We thank Jeremiah Busch and Rebecca Steinberg for technical assistance, Roxanne Broadway and Maarten Jongsma for advice on extraction and quantification of TI activity,


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and Kendra Cipollini for editorial assistance. Comments by two anonymous reviewers substantially improved this manuscript. This work was supported by grants from the National Science Foundation and a Packard Fellowship to J.B. REFERENCES ALARCON, J.-J., and MALONE, M. 1995. The influence of plant age on wound induction of proteinase inhibitors in tomato. Physiol. Plant. 95:423–427. BALDWIN, I. T. 1999. Inducible nicotine production in native Nicotiana as an example of adaptive phenotypic plasticity. J. Chem. Ecol. 25:3–30. BALDWIN, I. T., and SCHMELZ, E. A. 1994. Constraints on an induced defense: The role of leaf area. Oecologia 97:424–430. BODNARYK, R. P. 1992. Effects of wounding on glucosinolates in the cotyledons of oilseed rape and mustard. Phytochemistry 31:2671–2677. BODNARYK, R. P. 1997. Will low glucosinolate cultivars of the mustards Brassica juncea and Sinapis alba be vulnerable to insect pests? Can. J. Plant. Sci. 77:283–287. BRADFORD, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254. BROADWAY, R. M. 1989. Tryptic inhibitory activity in wild and cultivated crucifers. Phytochemistry 28:755–758. BROADWAY, R. M. 1995. Are insects resistant to plant proteinase inhibitors? J. Insect Physiol. 41:107–116. BROADWAY, R. M., and DUFFEY, S. S. 1986. Plant proteinase inhibitors: Mechanism of action and effect on the growth and digestive physiology of larval Heliothis zea and Spodoptera exigua. J. Insect Physiol. 32:827–833. BROADWAY, R. M., and MISSURELLI, E. L. 1990. Regulatory mechanisms of tryptic inhibitory activity in cabbage plants. Phytochemistry 29:3721–3725. BROADWAY, R. M., DUFFEY, S. S., PEARCE, G., and RYAN, C. A. 1986. Plant proteinase inhibitors: A defense against insects? Entomol. Exp. App. 41:33–38. CHEW, F. S. 1988. Searching for defensive chemistry in the Cruciferae, or, do glucosinolates always control interactions of Cruciferae with their potential herbivores and symbionts? No! pp. 81–112, in K. C. Spencer (ed.). Chemical Mediation of Coevolution. Academic Press, San Diego. CIPOLLINI, D. F. 1998. Induced defenses and phenotypic plasticity. Trends Ecol. Evol. 13:200. DEWITT, T. J., SIH, A., and WILSON, D. S. 1998. Costs and limits of phenotypic plasticity. Trends Ecol. Evol. 13:77–81. DOMONEY, C., WELHAM, T., ELLIS, N., and HELLENS, R. 1994. Inheritance of qualitative and quantitative trypsin inhibitor variants in Pisum. Theor. Appl. Genet. 89:387–391. DUKE, J. 1983. Handbook of Energy Crops. Unpublished. Purdue University Library. FELTON, G. W., BROADWAY, R. M., and DUFFEY, S. S. 1989. Inactivation of proteinase inhibitor activity by plant-derived quinones: Complications for host plant resistance against noctuid herbivores. J. Insect Physiol. 35:981–990. GREEN, T. R., and RYAN, C. A. 1972. Wound-induced proteinase inhibitors in plant leaves: A possible defense mechanism against insects. Science 175:776–777. GUSTAFSON, G., and RYAN, C. A. 1976. The specificity of protein turnover in tomato leaves: The accumulation of proteinase inhibitors, induced with the wound hormone PIIF. J. Biol. Chem. 251:7004–7010. HOPKINS, R. J., EKBOM, B., and HENKOW, L. 1998. Glucosinolate content and susceptibility for insect attack of three populations of Sinapis alba. J. Chem. Ecol. 24:1203–1216.

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