Plant Science, 72 (1990) 37--44 37 Elsevier Scientific Publishers Ireland Ltd.

a-Amylase activities of agricultural insect pests are specifically affected by different inhibitor preparations from wheat and

barley endosperms

C a r m e n Gut i e r r ez a, R o s a S a n c h e z - M o n g e b, Luis i G o m e z b, M a n u e l R u i z - T a p i a d o r a, P e d r o Cas ta f i e ra c and Gabr i e l Sa lcedo b,*

*Departamento de Proteccion Vegetal, CIT-Instituto Nacional de Investigaciones Agrarias, Carretera de la Coruna s/n, 28040 Mad- rid, bDepartamento de Bioquiraica, E.T.S. Ingenieros Agronomos, Ciudad Universitaria, 28040 Madrid and cUnidad de Fitopatolo-

gia, Centro Investigaciones Biologicas-CSIC, Velazquez 115, 28006 Madrid (Spain)

(Received March 9th, 1990; revision received July 2nd, 1990; accepted July 2nd, 1990)

In vitro a-amylase (EC 3.2.1.1) activity from 23 agricultural insect pests, including moths, cereal aphids, pentatomids, cereal thrips, stored cereal and legume grain pests and colorado potato beetle, have been determined. Enzyme activity per unit of body weight was higher in extracts from stored cereal pests as compared with the other insect groups. Crude inhibitor preparations from the endosperms of Triticum aestivum, Hordeum vulgate and Triticum monococcum were decreasingly active in that order. The three classes of a-amylase inhibitors from T. aestivum - - monomeric, dimeric and tetrameric - - were differentially active against crude enzyme preparations from different insect species. This was further confirmed by testing the purified dimeric (0.19 and 0.53) and monomeric (0.28) inhibitors. The monomeric inhibitor was considerably more active against the a-amylases from Tenebrio molitor and Sitophilus oryzae than the dimeric ones, whereas the opposite situation occurs for the a-amylase from Leptinotarsa decemlineata and Oryzaephilus surinamensis. Both types of purified inhibitors were about equally active against other insect enzyme preparations tested.

Key words: agricultural insect pests; a-amylase inhibitors; Hordeum (endosperm); specificity (enzyme inhibition); Triticum (endosperm)

Introduction

Proteinaceous inhibitors of digestive enzymes from phytophagous organisms are widespread in plants and might play an important role in their protection [1]. Certain proteinase inhibitors are induced in response to wounding by insects or fungi [2]. In the case of a proteinase inhibitor from the cowpea, a correlation was found between inhibitor level and pest resistance [3] and transformation of tobacco with the corresponding gene conferred resistance to bud worm [4].

Inhibitors of heterologous a-amylases are particularly abundant in cereal endosperm,

*To whom correspondence should be sent. Abbreviations: IEF, isoelectrofocusing; electrophoresis.

SGE, starch-gel

0168-9452/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Printed and Published in Ireland

specially in that of wheat [1,5]. These inhibitors are encoded by a disperse multi-gene family which includes not only monomeric, dimeric and tetrameric a-amylase inhibitors but also proteinase inhibitors. Previous evidence suggests that inhibitors of a-amylases are implicated in plant defense mechanisms. Increased mortality of larvae from Tribolium castaneum has been observed when crude inhibitor preparations from wheat flour were added to synthetic diets [6,7], and while high inhibitor concentrations were required in this case, quite low concentrations were effective against the legume storage pest Cal- losobruchus maculatus [8]. A correlation between resistance to the larvae of Sitophilus oryzae among wheat cultivars and 'in vitro' inhibition of the insect a-amylase has been observed [9]. Cereal storage pests generally have a markedly higher

Ltd.

38

a-amylase activity than those that are not able to feed on cereal flours [10]. Although the specificity of different inhibitors towards a-amylases from various sources has not been investigated in detail, it has been recently shown that partially purified wheat inhibitors had differential effects towards different isoamylases from Sitophilus oryzae I11]. We report here the 'in vitro' effects of crude inhib- itor preparations from barley and from two wheat species on the a-amylases of 23 insect pests of agri- cultural importance and the specificity of inhibitor classes and pure inhibitors towards the a-amylases of representative insect species.

Material and Methods

Pest insects All species used are listed in Table I. Moths

(larvae), cereal aphids (nymphs) and stored cereal and legume pests (adults and larvae) were from laboratory cultures. Colorado potato beetle (larvae), pentatomids (adults) and cereal thrips (larvae) came from field populations. In all cases, late instar larvae and nymphs were used.

Plant material Flours from Triticum aestivum (cv. Chinese

Spring), Hordeum vuigare (cv. Bomi), and Triticum monococcum (accession UP-l) were used in this study.

Insect extracts Whole insects were homogenized with 20 mM

sodium acetate, 100 mM NaCl, 0.1 mM CaCI2, pH 5.4 buffer (1:10 w/v) in a small Potter homogenizer at 4°C. After 20 min at the same temperature, with shaking, the suspensions were centrifuged at 11 000 × g for 10 min and the supernatants were used for amylase activity and inhibition assays.

Inhibition tests Inhibitory activity against insect a-amylases

was tested essentially as in [12], using a 20 mM sodium acetate, 100 mM NaC1, 0.1 mM CaCI, pH 5.4 buffer. Two hundred thirty microliters of this buffer, containing the amylase and appropriate

amounts of inhibitor preparation, were incubated at 25 °C for 30 min and then 230/A of 1% starch solution was added. The mixture was incubated at 25oc for 30 min. After adding 460 /A of 3,5- dinitrosalicylic acid reagent, heating in a boiling water-bath for 5 min, cooling on ice, and diluting with 2 ml of water, the absorbance of the final mixture was measured at 550 nm. All tests were carried out using approximately 1 unit of a- amylase, defined as the activity of enzyme required to produce the reducing equivalents of 1 /~mol of maltose in our experimental conditions.

Extraction and fractionation of inhibitors Crude inhibitor preparations from wheats and

barley endosperms were obtained as previously described [13]. The preparation from T. aestivum was fractionated by gel filtration as in Gomez et al. [13]. Purification of inhibitors 0.19, 0.28 and 0.53 from the appropriate gel filtration fractions was according to Sanchez-Monge et al. [14,15]. Protein concentration was quantitated by the methods of Lowry et al. [16] and Smith et al. [17].

Electrophoretic procedures Two-dimensional electrophoresis by combined

electrofocusing and starch-gel electrophoresis was carried out as described by Gomez et al. [18].

Results and Discussion

The a-amylase activity of crude homogenates of 23 insect species was determined and its inhibition by crude inhibitor preparations from hexaploid wheat, Triticum aestivum, diploid wheat, Triticum monococcum, and barley, Hordeum vuigare, was investigated (Table I). Higher activity was found in the stored cereal pests tested than in the other insects investigated, except in Carpocoris pudicus, which suggests a relationship between enzyme activity per unit of fresh weight and feeding habits. No inhibition of the a-amylases from the moths and aphids studied was observed for any of the crude inhibitor preparations tested (50 /~g/unit of a-amylase). Both groups of insects are among those with the lowest activity per unit of body weight, whereas high inhibitory effects were observed against the

39

Table I. Inhibition of insects a-amylase activities by crude inhibitors preparations from wheats and barley endosperms. All tests

were carried out using approximately 1 unit of a-amylase (see Materials and Methods).

Species Stage" U.A. /mg b Inhibition c

Ta Hv Tm

Moths Agrotis ipsilon Hufn. L 0.027 _+ 0.001 Agrotissegetum Schiff. L 0.046 .+ 0.003 Heliothis armigera Hb. L 0.034 -+ 0.001 Mamestra oleracea L. L 0.028 _ 0.004

Colorado potato beetle Leptinotarsa decemlineata L.

m

m

m

L 0.13 .+ 0.01 + + + +

Cereal aphids DiuraphisnoxiaMordw. N 0.12 _+ 0.01 Metopolophium dirhodum Walk. N 0.12 .+ 0.01

Rhopalosiphumpadi L. N 0.14 .+ 0.02 Schizaphisgraminum Rond. N 0.13 .+ 0.01 SitobionavenaeF. N 0.14 _+ 0.02

u

m

m

Pentatomids Aelia rostrata Boh. A 0.90 .+ 0.26 + + + CarpocorispudicusPodr. A 3.3 .+ 0.3 + + + + + + + Eurygasteraustriacus Schr. A 0.79 .+ 0.04 + - -

Cereal thrips Haplothrips triticiKurdj. L 0.16 .+ 0.01 + + + +

L 4.7 .+ 0.4 + + + - - A 10.0 .+ 0.7 + + + - - A 0.39 .+ 0.02 + + + - A 27.6 _+ 0.4 + + + + + + -

A 4.4 _ 0.4 + + + + + + - L 3.7 ___0.2 + + + + + + + L 10.2 --. 1.1 + + + + + + + A 3.7 .+ 0.4 + + + + + + +

L 0.47 _ 0.07 + + + + + + + A 0.10 _+ 0.01 - - -

Stored cereal pest Oryzaephilus surinamensis L.

A 0.090 _+ 0.013 + + + + + A 0.12 -¢- 0.01 + + + + + + +

Rhizopertha dominica F. Sitophilus granarius L. Sitophilus oryzae L. Tenebrio molitor L. Tribolium castaneum Hbst.

Trogoderma glabrum Hbst.

Legumes grain pest Acanthoscelides obtectus Say. Callosobruchus maculatus F.

• N = Nymphs; L = Larvae; A = Adults. In all cases, both late instar nymphs and larvae were used. b Units of a-amylase per mg of fresh weight. Means _+ S.E. (n = 4).

¢ Each inhibition assay was carried out using 50/ag of crude inhibitors preparations from Triticum aestivum (Ta), Hordeum vulgare (Hv) or Triticum monococcum (Tm). Extract from all insect species were tested in, at least, two different experiments (n = 4). Inhi- bition values < 5o/0 were recorded as no inhibition ( - ). + = 5--330?0 inhibition; + + = 33--660?0 inhibition; + + + = 66-- 100070 inhibition.

a - a m y l a s e s o f c e r e a l s t o r a g e p e s t s , a n d , t o a

l e s s e r e x t e n t , a g a i n s t t h o s e f r o m p e n t a t o m i d s ,

c e r ea l t h r i p s , l e g u m e g r a i n p e s t s , a n d C o l o r a d o

p o t a t o be t t l e , m o s t o f w h i c h h a v e a h i g h e r a c t i v i t y

p e r u n i t o f f r e s h w e i g h t . W h i l e t h e s e r e s u l t s a r e in

a g r e e m e n t w i t h t h e g e n e r a l c o n c l u s i o n o f S i l a n o et

al . [10] c o n c e r n i n g t h e h i g h e r s e n s i t i v i t y t o w h e a t

i n h i b i t o r s o f a - a m y l a s e s f r o m s t o r e d w h e a t p e s t s ,

40

they also suggest a complex specificity pattern. The crude inhibitor preparations from the three cereal species show differential effects not only among groups of insects (i.e. storage cereal pests vs, others) but also among species within a group (C. pudicus vs. other pentatomids) and among developmental stages (adults vs. larvae in T. glabrum). This last result could be related to the lack of feeding activity in adults of this species [19], and probably to changes in the major isoamylases. The crude inhibitor preparations from T. aestivum, H. vulgare, and T. monococcum were decreasingly active in that order.

All the a-amylase extracts that were susceptible to inhibition in the first screening were tested again using different amounts of the three inhibitor preparations (Table II). While some

enzyme preparations were almost completely inhibited or showed increasing inhibition with increasing amounts of inhibitors, other preparations such as those from L. decemlineata, A. rostrata, H. tritici, T. glabrum and C. maculatus, were only partially inhibited and the inhibition did not increase with increasing amounts of inhibitors. The most likely explanation would be that part of the a-amylase activity (possibly one or more isozymes) is insensitive to the inhibitors, as suggested by the data of Campos et al. [20]. All species in the first group are stored cereal pests, while only one of the second (T. glabrum) belongs to this group. The relative activity of the three inhibitor preparations against the a-amylases of the different insects is not constant (compare, for example, the effects on extracts from C. pudicus, H. tritici or O.

Table I I . Inh ib i t ion (%) o f insects a -amylase act ivi t ies by d i f fe ren t p ro te in levels o f c rude inhib i tor p repa ra t ions f rom T. aestivum, H. vulgare and T. monococcum endosperms . Al l tests were car r ied out us ing a p p r o x i m a t e l y 1 uni t o f a -amylase (see Mater ia l s and

Methods) . Means ± S.E. (n = 4).

Species • Inh ib i t i on (%)

T. aestivum H. vulgate T. monococcum

Pro te in ~ g ) of c rude inh ib i to r p repa ra t ion

1 5 50 1 5 50 1 5 50

Leptinotarsa decemlineata

A elia rostrata Carpocoris pudicus Eurygaster austriacus

4 0 ± 9 5 4 ± 4 5 3 ± 5 - - 3 8 ± 5

1 5 ± 1 2 0 ± 5 1 9 ± 5 1 6 ± 4 ~ ± 5 1 7 ± 3

9 1 ± 3 9 3 ± 1 9 5 ± 1 1 7 ± 3 4 6 ± 9 7 7 ± 1

- - 1 0 ± 4

Haplothrips tritici 48 ± 6 55 ± 8

OryzaephilussurinamensisL 39 -+ 1 66 ± 2 A 55 ± 1 68 ± 4

Rhizoperta dominica 28 ± 6 44 ± 3 Sitophilusgranarius 54 ± 7 81 ± 2

Sitophilus oryzae 83 - 6 92 ± 2 Tenebrio molitor 62 ± 6 94 ± 2

Tribolium castaneum L 69 ± 6 84 ± 6 A 8 1 ± 1 9 0 ± 1

TrogodermaglabrumL 72 ± 2 71 ± 5 A

5 4 ± 8 - - 1 3 ± 1

8 3 ± 1 8 9 ± 2

5 4 ± 3 - - 1 1 ± 2 9 5 ± 1 1 6 ± 3 8 4 ± 5 9 6 ± 2 ~ ± 1 2 1 ± 4 7 7 ± 2 9 6 ± 1

9 3 ± 1 1 7 ± 4 7 8 ± 5 9 0 ± 1

9 2 ± 1 - - 5 0 ± 9 9 4 ± 1 - - 2 7 ± 6 5 5 ± 1 7 3 ± 3 1 6 ± 1 2 2 ± 5 5 9 ± 7

Acanthoscelidesobtectus 57 ± 5 55 ± 8 50 Callosobruchus maculatus 70 ± 8 69 ± 4 67

± 6 1 3 ± 4 2 8 ± 5 4 1 ± 4 ± 6 4 8 ± 9 5 9 ± 6 5 9 ± 9

m

m

m

1 5 ± 1

1 4 ± 5 1 7 ± 2

1 6 ± 3

1 9 ± 5

1 2 ± 5

D

p

2 1 ± 3

41__ .5 4 4 ± 4

3 6 ± 1

2 9 ± 9 5 5 ± 4

a L = Larvae ; A = Adul t s . b Inh ib i t ion values < 5°70 were recorded as no inh ib i t ion ( - ).

surinamensis) , w h i c h s u g g e s t t h a t d i f f e r e n t

c o m p o n e n t s o f t h e i n h i b i t o r p r e p a r a t i o n s a r e

m a i n l y r e s p o n s i b l e f o r t h e i n h i b i t i o n in t h e

d i f f e r e n t cases . I n g e n e r a l , t h e i n h i b i t o r

p r e p a r a t i o n f r o m T. m o n o c o c c u m is 5 0 - - 1 0 0

t i m e s less a c t i v e t h a n t h a t o f T. aest ivum, w h i c h

e x p l a i n s w h y n o i n h i b i t o r y a c t i v i t y w a s f o u n d in

T. m o n o c o c c u m a t c o n c e n t r a t i o n s t h a t w e r e

c l ea r ly i n h i b i t o r y f o r t h e T. aes t i vum p r e p a r a t i o n

[13,21] .

T h e i n h i b i t i o n o f t h e s a m e e n z y m e e x t r a c t s

l i s t ed in T a b l e I I b y m o n o m e r i c , d i m e r i c , a n d

t e t r a m e r i c i n h i b i t o r s f r o m T. aes t ivum w as a l so

i n v e s t i g a t e d ( T a b l e I I I ) . T h e c r u d e i n h i b i t o r

p r e p a r a t i o n w a s f r a c t i o n a t e d as p r e v i o u s l y

d e s c r i b e d (Fig . 1A) . T h e t h r e e i n h i b i t o r c lasses

we re n o t c r o s s - c o n t a m i n a t e d w i t h e a c h o t h e r , as

41

j u d g e d b y t w o - d i m e n s i o n a l e l e c t r o p h o r e s i s (F ig .

1 B - - D ) , a l t h o u g h e a c h c o n t a i n e d d i f f e r e n t

p r o p o r t i o n s o f n o n - i n h i b i t o r y p r o t e i n s [13]. A l l

t h r e e i n h i b i t o r c lasses w e r e a c t i v e a g a i n s t m o s t o f

t h e e n z y m e e x t r a c t s t e s t e d , b u t e a c h e x t r a c t was

p r e f e r e n t i a l l y i n h i b i t e d b y o n e o r m o r e i n h i b i t o r

c lasses ( u n d e r l i n e d v a l u e s in T a b l e I I I ) . T h i s is in

c o n t r a s t w i t h t h e p r e v i o u s c o n s i d e r a t i o n o f t h e

m o n o m e r i c i n h i b i t o r s as t h e m o s t a c t i v e a g a i n s t

in sec t a - a m y l a s e s [1 ,5 ,10 ,22 ] . T o e l u c i d a t e

w h e t h e r i n d i v i d u a l m e m b e r s o f t h e i n h i b i t o r

c lasses s h o w e d t h e s a m e t y p e o f spec i f i c i ty , t h e

m a j o r h o m o d i m e r i c (0 .19 a n d 0 .53) a n d

m o n o m e r i c (0 .28) i n h i b i t o r spec ies w e r e p u r i f i e d

t o h o m o g e n e i t y b y p r e v i o u s l y d e s c r i b e d m e t h o d s

[14,15] a n d t e s t e d a g a i n s t s e l ec t ed e n z y m e e x t r a c t s

(Fig . 2). W h i l e , as p r e v i o u s l y o b s e r v e d , t h e p u r e

Table IIl. Inhibition (o70) of insects a-amylase activities by gel-filtration fractions corresponding to tetrameric, dimeric and mon- omeric inhibitors from T. aestivum endosperm (see Fig. 1). All tests were carried out using approximately l unit of a-amylase (see Materials and Methods). Means ± S.E. (n = 4).

Species i Inhibition (o7o)

Tetrameric Dimeric Monomeric

Protein (pg) of gel filtration fraction

1 5 1 5 l 5

Leptinotarsa decemlineata 26 ± 9 44 ± 1 44 _ 5 52 _+ I 25 ± 1 29 ± 4 Aeliarostrata 19 ± 3 23 ± 5 10 ± 4 24 _+ 1 14 ± 2 20 ± 2 Carpocorispudicus 51 ± 1 78 _ 1 92 ± 1 94 ± 2 91 ± 3 99 ± 1 Eurygaster austriacus - - - - 10 ± 2 10 _+ 4 - - - -

Haplothripstritici 32 ± 13 35 _ 16 46 ± 2 50 ± 3 15 ± 1 26 __ 8

OryzaephilussurinamensisL 22 ± 5 43 ± 2 53 ± 2 57 _+ 4 - - 13 ± 5 A 41 ± 1 4 9 2 5 5 2 2 8 6 9 2 7 11 ± 6 19_+ 7

Rhizopertadominica 10 ± 4 23 ± 1 27 ± 5 47 ± l 48 ± 2 61 _+ l Sitophilusgranarius 51 ± 3 69 ± l0 25 ± 3 73 ± 4 60 ± 3 96 ± 1 Sitophilusoryzae 68 ± 2 85 ± 1 72 ± 5 88 ± 1 95 ± 1 97 ± l Tenebriomolitor 48 ± 9 87 ± 7 58 ± 7 96 ± 1 77 ± 4 95 ± 3 TriboliumcastaneumL 51 ± 1 60 ± 4 76 ± 2 88 ± 3 71 ± 5 75 _+ 2

A 66 ± 11 79 ± 7 87 ± 1 93 ± 1 65 ± 3 76 ± 1 TrogodermaglabrumL 67_+ 4 72 ± 2 71 ± 2 7 0 _ 1 56 ± 1 60 ± 2

A . . . . . .

Acanthoscelides obtectus 46 ± 10 59 ± 1 59 ± 4 60 ± 7 58 ± 3 60 ± 1 Callosobruchusmaculatus 68 ± 9 65 ± 2 71 ± 4 71 ± 3 72 ± 3 72 ± 3

L = Larvae; A = Adults. b Inhibition values < 5% were recorded as no inhibition ( - ). Inhibition values corresponding to the inhibitor class(es) with highest

activity against a given enzyme preparation are underlined.

42

A • •

20- / ~ T O

I,-

i l / A

2OO 30O ELUTION

67 25 12.3 kDa

M

VOLUME (ml)

¢4 I z G. i ' " ' • A

pH6 IEF pH8

Fig. 1. (A) Gel filtration on Sephadex G-100 of a crude inhibitor preparation from T. aestivum endosperm. The fractions indicated include tetrameric (T), dimeric (D) and monomeric (M) a-amylase inhibitors. Bovine serum albumin (67 kDa), chymotrypsinogen (25 kDa) and cytochrome c (12.3 kDa) were the protein standards used to calibrate the column. (B--E) Two-dimensional electrophoretic maps (IEF pH 6--8 x SGE pH 3.2) of a crude inhibitor preparation from T. aestivum (B) and gel filtration fractions that include tetrameric (C), dimeric (D) and monomeric (E) inhibitors as indicated in Fig. IA. Only appropriate zones of the protein map are shown. Arrows point to previously described subunits of tetrameric inhibitors (~), and to homodimeric (V) and monomeric (A) inhibitors. The position of purified inhibitors used in this study, 0.19, 0.53 and 0.28, are also indicated.

monomeric inhibitor was 7- -10 times more active than the dimeric inhibitors against T. molitor a- amylase, as well as against that f rom S. granarius, the dimeric inhibitors were more active than the monomeric one against the a-amylase f rom L.

decemlineata and, to a lesser extent, against those from O. surinamensis. The two classes of purified inhibitors were about equally effective against the a-amylases of the other insects tested (Fig. 2). Considering that all three inhibitors are

100

110.19 ~0 .53

80 t-I 0.28

60

r- , o

20

0 Ld Cp OsL OsA Sg TcL TcA Tm

Fig. 2. Inhibition (o/0) of insect a-amylase activities by purified dimeric, 0.19 and 0.53, and monomeric, 0.28, inhibitors from T. aestivum. All test were carried out using approximately 1 unit of a-amylase (see Material and Methods) and 0.25/ag of purified inhibitor. Means and S.E. (n = 4) are represented. Extracts used correspond to the following insect species: Ld = Leptinotarsa decemlineata: Cp = Carpocoris pudicus; Os = Oryzaephilus surinamensis; Sg = Sitophilus granarius; Tc = Tribolium castaneum; Tm = Tenebrio molitor. L = Larvae; A = Adults.

homologous, with at least 55°7o of sequence similarity [1,23], the present results indicate that the sequence divergence has led to the evolution (co-evolution?) of inhibitor specificity towards enzymes from different phytophagous insects and suggest that possession of distinctive types of homologous inhibitors represents a protective arsenal that should confer a considerable adaptative advantage to hexaploid wheat. The data presented here indicate that this protein family from the Triticeae offers a good opportunity to investigate structure/activity relationships in an inhibitor/enzyme system. The plant protection implications of these results are susceptible of further exploration by the trans- specific expression of the corresponding genes.

10

43

Acknowledgements

We would like to thank Dr. F. Garcia-Olmedo for helpful comments and his advice in writing the manuscript, Dr. F. Garcia-Arenal for critical reading of the manuscript and J. Garcia-Guijarro and D. Lamoneda for technical assistance. We also acknowledge the population of moths and stored product pests supplied by the Departamento de Entomologia, E.T.S. Ingenieros Agr6nomos of Cordoba and Madrid (Spain), respectively. This work was supported by the Comision Interministerial de Ciencia y Tecnologia (grant No. AL189-121).

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