Journal of Chemical Ecology, Vol. 9, No. 2, 1983

INVESTIGATIONS OF TRYPSIN INHIBITORS IN LEAVES OF FOUR NORTH AMERICAN PRAIRIE GRASSES

C.W. ROSS 1 and J.K. DETLING 2 ~Department o f Botany and Plant Pathology and 2Natural Resource Ecology Laboratory Colorado State University Fort Collins, Colorado 80523 (Received March 29, 1982; revised June 10, 1982)

Abstract--Leaves of four important North American prairie grasses ( Agropyron smithff, Andropogon gerardii, A. scoparius, and Bouteloua gracilis) were examined for the presence of trypsin inhibitors which are thought to protect some plant species from herbivory. Leaves of all four species had significant inhibitor activity at levels comparable to those in tomato leaves. Our evidence suggests that part of the inhibitor activity was proteinaceous and part may have been polyphenolic. Young leaves of A. smithii had more inhibitory activity than old leaves, but no leaf age differences were observed in B. gracilis. Mechanical wounding of leaves of A. smithii caused no consistent increase in inhibitor activity, in contrast to reports for some plant species. Plants of A. smithii collected from areas heavily grazed by prairie dogs had trypsin inhibitor levels comparable to those in plants collected from a grazing exclosure. Thus, the ecological role of proteinase inhibitors in these Great Plains dominants remains to be demonstrated. Key Words--Agropyron smithii, Bouteloua gracilis, Andropogon gerardii, Andropogon scoparius, Lycopersicon esculentum, herbivory, trypsin inhibitors, polyphenols.

INTRODUCTION

Plants have developed chemical defense systems against certain herbivores (e.g., Rosenthal and Janzen, 1979; Schoonhoven, 1972). Among many potential defensive chemicals, inhibitors of proteinases such as trypsin have gained recent credence (Ryan, 1978). Numerous investigations, largely with 247 0098-0331/83/0200-0247503.00/09 1983PlenumPublishingCorporation

248

Ross AND DETLING

seeds, showed that such inhibitors are commonly polypeptides or lowmolecular-weight proteins relatively stable to heat and rich in disulfide bonds (Richardson, 1977; Ryan, 1981). Green and Ryan (1972, 1973) found that chewing by Colorado potato beetles or mechanical wounding of tomato and potato leaves significantly raised the levels of an inhibitor of trypsin and chymotrypsin within 24 hr. Furthermore, either treatment caused inhibitor levels to rise in adjacent noninjured leaves, indicating transport of some factor that induces proteinase inhibitor formation. Evidence for such a factor in leaves from 37 of 39 species representing 20 plant families, including three cereal grains, was subsequently presented (McFarland and Ryan, 1974). These results suggest that vegetative damage caused by numerous herbivores might enhance production of certain proteinase inhibitors in leaves of many species. If so, such inhibitors could influence evolution of both plants and herbivores. However, Walker-Simmons and Ryan (1977) showed that among 23 plant species, only a few responded either to endogenous proteinase inhibitor inducing factor preparations or preparations from tomato. None of the three cereals they tested responded, a result consistent with those of Kirsi and Midola (1977) and Weiel and Hapner (1976) for barley leaves. Apparently, no native grass species have been tested for the presence of proteinase inhibitors in vegetative tissues. METHODS AND MATERIALS

Plant Materials. Agropyron srnithii Rydb. (western wheatgrass), Andropogon gerardii Vitman (big bluestem), and Andropogon scoparius Michx. (little bluestem) were obtained from sods of plants collected in Wind Cave National Park in southwestern South Dakota (Detling and Painter, 1983). Bouteloua gracilis (H.B.K.) Griffiths (blue grama) was obtained from sods collected at the USDA Central Plains Experimental Range near Nunn, Colorado. Tomato plants (Lycopersieon esculentum L.) were grown from seeds. All plants were grown in pots in a greenhouse. Because environmental conditions varied between summer and winter, months in which analyses were performed are given in tables. For the grasses, only green portions of leaves from plants without visible florets were used. For tomato, young leaves of plants just forming visible flowers were used. Extraction of Trypsin Inhibitors. Leaves were excised, weighed, cut into sections about 1 cm long, and homogenized in distilled H20 (1.0 g fresh wt/12.5 ml) at 5-10 ~C with a Vir-Tis or Polytron homogenizer. Homogenates were centrifuged at 5 ~ either once or twice for 20 min at 20,000 g. The green-colored (A. smithii and B. gracilis), greenish-tan (L. esculentum), or tan (A. gerardii and A. scoparius) final supernatant solutions were used directly

249

T R Y P S I N I N H I B I T O R S I N GRASSES

for spectrophotometric protein analysis (595 nm) by the Biorad Coomassie blue dye-binding method (Bradford, 1976; Robinson, 1979) and for trypsin inhibitor analysis. Trypsin Inhibitor Analyses. A sensitive and specific method (Hummel, 1959) that measures the increase in absorbance at 247 nm as bovine pancreatic trypsin (Sigma Chemical Co.) hydrolyzes the ester bond ofp-toluenesulfonylL-arginine methyl ester (TAME) was used. Assays were performed at 37~ with 0.1 ml of leaf extract (0.04-0.2 mg protein) present in 3.0 ml of a solution also containing 1.65 mM TAME, 8 mM CaCI2, 40 mM Tris HC1, pH 8.1, and 0.18 t~g/ml trypsin. Reactions were started by addition of trypsin (prepared and stored at 5~ 1 mM HC1) and stopped after 20 min with 0.2 ml of cold 5 N acetic acid. Absorption (247 rim) was then measured immediately against corresponding blanks in which H20 or leaf extract replaced trypsin. Analyses from each extract were performed in duplicate or triplicate. Inhibitory activity is reported on a tissue weight basis (micrograms trypsin inhibited per gram fresh weight) and on a protein basis (specific activity = micrograms trypsin inhibited per milligram protein). Separation of Inhibitors by Gel-Filtration Chromatography. Sephadex G-25 from Pharmacia was hydrated by boiling for 3 hr in H20, then a 1.6 • 15.5-cm column was equilibrated overnight at 5~ with 50 mM Tris HC1 (pH 8.1) containing 10 mM CaCI2. The column's void volume (10 ml, determined with blue dextran) and elution volumes for marker proteins of known molecular weights were measured at 5~ according to Pharmacia's instructions. All protein markers and plant extracts were made 10% (w/v) with sucrose before they were added to the column. For A. smithii, !.0 ml of an extract made from 2 g of leaf tissue and 10 ml of H20 was used. Inhibitor analyses were made with duplicate 0.3-ml aliquots from eluted fractions of 2.0 ml. For A. scoparius, 2.0 ml of a similar extract (2 g tissue/10 ml H20) were added to the column, and duplicate 0.4-ml aliquots from eluted 2.0 ml fractions were analyzed for trypsin inhibitors. Aliquots (0.3 ml) remaining in each fraction collected were used for protein analysis.

RESULTS

Preliminary results (data not shown) to evaluate the overall inhibitor assay method indicated that leaves homogenized in distilled H20 had more apparent inhibitor activity than leaves homogenized in 5 m M dithiothreitol. Although dithiothreitol provided greener (less brown) supernatant solutions, such solutions were 20-50% less inhibitory on a leaf weight basis, depending upon the species. Distilled HzO also yielded slightly more trypsin inhibitor activity than did 50 mM Tris HCI as a homogenizing medium, at least for A.

250

Ross AND DETLING

smithii. I n h i b i t i o n by H 2 0 extracts was p r o p o r t i o n a l to the v o l u m e of e x t r a c t with a l i q u o t sizes v a r y i n g f r o m 0.025 to 0.3 ml.

Comparisons o f Inhibitor Levels A m o n g Species. Significant i n h i b i t o r activity was f o u n d in leaves of all f o u r grass species at levels c o m p a r a b l e to those in y o u n g L. esculentum leaves (Table 1). Highest activities on a fresh weight basis o c c u r r e d in A. smithii, while activities in the o t h e r three grasses were n o t significantly different f r o m each o t h e r or f r o m L. esculentum. On a specific a c t i v i t y (protein) basis, the two Andropogon species h a d the highest activities, b e c a u s e less p r o t e i n was detected in extracts f r o m t h e m t h a n f r o m all o t h e r species. These low p r o t e i n values likely resulted f r o m p r e c i p i t a t i o n of p r o t e i n s by phenolics in the t a n c o l o r e d Andropogon extracts ( L o o m i s a n d Battaile, 1966). Chemical Nature o f Grass Trypsin Inhibitors. P a r t of the i n h i b i t o r activity in each grass species is a p p a r e n t l y p r o t e i n a c e o u s . Evidence t h a t this is true for A. smithii a n d B. gracilis was o b t a i n e d f r o m heat d e n a t u r a t i o n e x p e r i m e n t s s u m m a r i z e d in T a b l e 2. H e a t i n g e x t r a c t s to 7 0 ~ for 10 min (followed by c e n t r i f u g a t i o n but p r i o r to i n h i b i t o r assay) caused loss of a b o u t t w o t h i r d s of the i n h i b i t o r activity f r o m A. smithii a n d all activity f r o m B. gracilis. C o n c o m i t a n t l y , a b o u t 90% of the p r o t e i n was m a d e i n s o l u b l e in each case. F o r the Andropogon species, however, h e a t i n g m a d e insoluble only a b o u t one f o u r t h of the t o t a l p r o t e i n a n d actually increased a p p a r e n t i n h i b i t o r levels on either a leaf weight basis or a specific activity basis.

TABLE 1. COMPARISON OF TRYPSIN INHIBITOR ACTIVITIES IN FIVE SPECIES a

Species

A. smithii

Inhibition (~g trypsin/g fresh wt)

Protein extracted (mg/ml)

Specific activity (tzg trypsin/rag protein)

1.6 -+ 0.6

3.0 -+ 1.3 a

43 +_12 b

2.0 -+0.2

1.7 +-0.5 b

41 -+ 11 b

0.4 -+ 0.1

8.0 • 2.5 c

44 +_4.2 b

0.4 -+ 0.1

10.0 -+ 2.2 c

44 _+8.7 b

1.7 -+ 0.3

2.1 +-0.3 b

59 _+11

ab

(N = 19)

B. gracilis (N =

14)

A. gerardii (N=7)

A. ~coparius (N= 7)

L. esculentum (N= 10)

aExperiments with Andropogon spp. were performed in late summer; others were performed in various seasons. bMeans followed by different letters are significantly different at the 95% confidence level (Student's t test).

1.90 0.19 2.1 0.0

2.7 9.8

50 0

1.71 0.18

63 22

Boutelouagracilis

aAndropogon experiments were performed in July; others were performed in various seasons.

Inhibition (tzg/g fresh wt) Nonheated Heated Protein concentration (/~g/ml) Nonheated Heated Specific activity (/~g trypsin/mg protein) Nonheated Heated

Agropyron smithii

6.4 10.5

0.40 0.29

32 38

Andropogongerardii

8.0 12.0

0.38 0.32

38 48

Andropogonscoparius

TABLE 2. TYPICAL EFFECTS OF HEATING ON TRYPSIN INHIBITOR ACTIVITIES IN LEAF EXTRACTS a

1,3

N

>

252

Ross AND DETLING '

I

|

I

Inhibition

A. smith#

40

.8

~---Protein (A595) 30

.6

--: 20

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0

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= 'o-

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~" ~ cyt.

c

0

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.o_ ~: -

Inhibition

,~

i,

5

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..~

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20

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30

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