Indian Journal o f Experimental Biology Vol. 37, March 1999, pp. 2 15-222

Review Article

Protein antimetabolites in legume seed defense

S Ignacimuthu

Entomology Research Institute, Loyo la Coll ege, Chennai 600034, Indi a

The search fo r insect resistan t chemicals in legume seeds has prompted scientists to identi ty many an timetabolites that impart resistance agai nst many stored product pests. Of the various fac tors contributing to defence mechan isms, the ro le of protein an timetabolites seems to be vel)' important. True resistance may be caused by the combined acti vity of several proteins each of which is present at a suboptimal level to confer resistance. Here an attempt is made to provide an insight into di fferent resistant factors, their biochemical, physiological and molecular aspects in confe rri ng resistance. The importance of protease inhi bitors. a - amylase inhi bitors, lectins and other plan t specific proteins in impart ing defence aga inst insects with special rcference to bruchids is discussed in the light of various fin dings.

Pul ses are the primary source of protein in the diet of the people in many countries. Substantial losses occur in pulses during storage due to infestation by pulse beetles. In India, about 8. 5% loss has been recorded in post harve.st handling of pul ses t

• The natural res istance exhibited by pulse varieties is one of the most important components of integrated pest control. Painter2 was among the pioneers who advocated the concept of host-plant res istance. The res istance may be attributed to some intrinsic as well as extrinsic properties of the pulse va rieties interacting with a spec ific insect spec ies. The identificati on of various characters/ factors that eli cit such res istance will fac ilitate enhancement of breeding programmes to produce varieties res istant to insect pests.

A few wild bean access ions from Mexico were found to be res istant to Acanlhosoelidus oblecllls afte r extensive screening' but it has not been poss ible to transfer the res istance to culti vated varieties, because of the complexity of the inheritance pattern of the trait4

. Attempts to identi fy the resi stance factor have fail ed so far5

. This limitatiol' makes it necessary to identity the biochemica l factors invo lved in th e res istance to deve lop an assay method to fac ilitate breedin g. Most plants are res istant to one or the other insect and when res istance breaks down, there is attack. The identifi cation of the res istance factors could also lead to c loning of such genes, opening the way to genetic transformati on. This approach could shorten otherwise lengthy breeding programme6

Thus genetic engineering helps to bring in res istance factors from other materi als aga inst which the pest has not developed res istance.

An Overview oj Pests oj Legume Seeds Amongst the various pests of pulses, the pulse

beetles (bruchids) are of major importance since they infest the grain, both in the fi elds and storehouses where they multiply rapidly causing heavy losses. Pulses are attacked by numerous spec ies of bruchid beetles. Callosobruchus maculalus (F.), Callosobruchus chinensis (L. ), Callosobruchus analis (Fab), Callosobruchus phaseoli (Gyllehal ) are some of the important pests of stored pulses which li ve in side the seeds and feed primarily on endosperm and thus make the seeds unfit for food and seeds. Substantial losses occur in pulses during storage due to infestation of pulse beetles . Annual post harvest losses caused by insect damage, microbial deteri orati on and other factors are estimated to be in the order of 10-25% world-wide. Of a ll the stored grain pests, particularly C. maculatus ex hibits a high degree of specific ity towards important leguminous seeds 7. Two major new world bruch id species affecting stored beans Phaseolus vulgaris (L.) are the Mex ican bean weevil , Zabroles subjascia(us (Boheman) and the bean weev i I, Acanlhoscelides oblec(us (Say).

Chickpea Cicer arielinul17 (L. ) is severe ly damaged by C. chinel1.\· /~· during storage . The fl our of benga l gram is prone to TriboliulI7 caslaneulI1 (Herbst) infestati on. Black gram, Vigna lI1ungo (L. ). Hepper and lenti I, Lens culinaris L. fai led to respond favourably for the growth and deve lopment of C. chillensis Linn . and C. maculalw,8 respecti ve ly. Although the pulse beetl es viz. C. chinensis and C. maculalus did not show any sign i fi cant va rietal

216 INDIAN J EXP BIOL, MARCH 1999

preference for the oviposition, there was a definite inter-pulse response on the oviposition of these two p~ts . The order of preference for C. chinensis was Cowpea > Black gram > Red gram > Benga l gram > Green gram > Pea9

; where as the order of preference of C. maculatus was Bengal gram > Black gram > Green gram > Cowpea > Red gram > Pea > Lentil 9

.

Resistant Factors Certain physical and chemical factors of pulse

seeds are known to play an important role either to favour or inhibit the oviposition as well as the development of pulse beetles . A number of attempts have been made to study the physical characteristics . These physical factors seem to differ from one pulse seed to the other and even among different varieties of the pulse commodity.

Studies have been made to investigate the chemical facto rs in the pulse seeds which might be responsible for res istance to storage insect attack 10. The brlJchid larvae can deve lop only in limited host seeds. Thi s is due to the presence o f certa in chemical s viz. protease inh ibi tors, phytohaemagglutinins, hetero­polysaccharides in legume seeds which act as antimetabo li tes . It has been observed that a lmost every bruchid spec ies is abl e to detox ify only a small number of compounds.

Methods of Screening Protein Antimetabolites In order to he lp Indian scientists to pursue thi s

work in a re lati ve ly easy way, I wou ld like to prov ide the va rious experimental protoco ls used in the ana lysis of prote in antimetabo lites.

A. Quantification aftrypsin inhibitors Seeds are ground and extracted with distilled water

(I : w/v) us ing chilled mortar and pestle. It is incubated at 4'!C for 3 hr with occasiona(stirring and subsequently centrifuged at 10,000 g for 20 min at 4°C. The supernatant co llected is diluted with di stilled water (I :9) and used as the inhibitor source for further studies . Different volumes ranging from 0-1 ml of this extract are made up to I ml with Tri s­HC I, pH 8.2 containing 2 mMCaCI2. Trypsin solution (20 Ilg/ml) is added to each tube. Reference standards i.e., without the sample, are also maintained . A ll the tubes are incubated at 37°C in a water bath for 5 min. Benzyol-DL-arginine-paranitro anilide (BAPNA) is used as substrate (0.4%) and added to each tube. The

reaction is allowed to proceed for 10 min at 37°C

after which it is stopped by adding 0.5 ml of 30% glacial acetic acid. Absorbance is read at 410 nm . Determination of inhibitor act ivity is calculated by the amount of aliquot of the extract required to inhibit 50% of trypsin activity, which is considered as one unit of trypsin inhibitor. Trypsin inhibitor activity is expressed as trypsin inhibitor units (TIU) per gram sample ll

•12

.

B. Quantification of chymotrypsin inhibitors.

Chymotrypsin inhibitor assay is done following the method of Erlanger et al13 Series of dilutions (0-1 ml) of the seed extract (in Tris HCI-CaC I2, pH 7.6) is made upto I III I using the same buffer. One ml o f chymotrypsin solution (400 Il g) is added to each tube. Reference standards are al so mainta ined. All the tubes are incubated in water bath at 37°C. After 5 min,S ml of substrate (N-glutaryl-DL-pheny la lanine, p-n itroanilide-GPNA dissolved in Tris HC I-CaC I2,

pH 7.6) is added to each tube. The reacti on is a ll owed to proceed fo r 10 min at 37°C after which it is stopped by adding I ml of 30% glac ia l acetic ac id . Absorbance is read at 410 nm . The a liquot o f the extract requi red to inhibit 50% of chymotryps in act ivity is determined and considered to be one unit of chymotrypsin inhi bitor. Chymotryps in inh ibi tor activity is expressed as chymotrypsin inhibitor uni ts (C IU) per gram sampl e.

c. Quantification o.fa-amylase inhibitors.

The method of Piergiovanni l4 is fo llowed to assay a -amylase inh ibitors. One gram dehull ed, dry, powdered seed is extracted in 25 ml of 0.9% NaC I for 2 hr at 4°C. 0.5 to 1.5 ml of this suspension is adjusted to 2 ml with di still ed water and incubated with pancreat ic porcine a-amy lase (3 0 Ill l mi in 0.05 mol/ L Tris-HCI buffer, pH 6.9 contain ing 0.0 I moliL CaC I2) (50 : '1) for 20 min at 30°C. Subsequently, 2 ml of starch solution (10 mglml) and 2 ml o f Tri s buffer (PH 6.9), containing 0.0 I moliL CaC I2 are added. After 40 min reaction at 30°C, suitable aliquots are added to 4 ml iodine solution (50 mg iodine and 500 mg potass ium iodide in I L 0.05 M HC I) . The ~bsorbance is measured at 563 nm . The blank digests i.e ., without seed extract are run under the same conditions. Results on inhibitor levels are ex pressed as fll of a -amylase inhibited per mg of dry sample .

IGNAC IM UTH U : PROTEIN ANTI METABOLITES IN LEGUME SEED DEFENCE 2 17

D. Quantitative analysis of trypsin and chymotrypsin inhibitors

Dehulled seeds are ground to fine powder and 100 mg is used to extract protease-inhibitors by incubating overnight at 4°C in 4 ml of Tri s-HCI­buffer, pH 8.1, containing 20 mM CaCI2. [t is centrifuged at 10000 g at 4°C for 15 min . The supernatant is used for electrophoretic analys is (Native PAGE). After electrophores is, the stacking gel is removed and the separating ge l stained for trypsin inhibitor and chymotrypsin inhibitor acti vities based on the method of Uriel and Bergas ' S with modifications as described by Kollipara and Hymowitz l 6

. The gels are washed in 0.1 I'll sodium phosphate buffer (pH 7.5) to equilibrate the ge l and subsequently incubated in phosphate buffe r, p H 7.5 containing 15 mg/ml of bov ine trypsin/chymotrypsin for 20 min on a shaker at room temperature. Following incubation, the gels are rinsed in di still ed water and stained using a so lution containing 20 mg of N-acetyl-DL-phenylalanine B- 1 naphthyl ester (APN E) in 8 ml of N ', N-d imethyl fo rmam ide and 40 mg of o-diani sidine, tetrazo iti zed (zinc chl oride complex) in 80 ml of di stilled water. The ge ls are stained for 4-10 hr in the dark . The stai ned ge ls are rinsed using di stilled water and stored in 7% acetic acid .

E. Extraction, isolation and partial purification of a­amylase inhibilors

a-Amylase inhibitors from seeds are iso lated using methods rep.orted by others with suitable modifications 7· 19 Dehulled seeds are powdered and extraction is carried out in 100 mM NaC I, 25 mM Tris-HCI buffer, pH 7.2 (1:4) for 12 hr. It is subsequently centrifuged at 12,000 g at 4°C fo r 30 min . The supernatant is prec ipitated with ammonium sulphate (0~90%) and kept at 4°C on a shaker for 4 hr. [t is then centrifuged at 12000 g at 4°C for 30 min . The supernatant is dialysed using Tri s-HCI buffer contai ning NaC I (PH 7.2) overni ght. The dialysate is collected and the protein content is estimated using the Bradford method21

. Equal quantiti es of di alysate is loaded on to SDS-PAGE ( 10%) gel along with molecular weight markers. Samples are al so subj ected to non-denatured electrophoretic analys is (68% Native PAGE).

F. Extraction, isolation and partial purification of lectins

Lectins in the seeds can be extracted and iso lated using standard procedure as described by Singh

el a12D. The seeds are dehulled and finely ground and

the seed powder is extracted in Tri s-HCI (25 mM) buffer containing 100 mMNaC I (PH 7.2) (I gram/3.3 ml buffer) at 4°C for 12 hr. It is then filtered thro~gh glass wool and centrifuged at 10000 g fo r 30 min and the supernatant collected. To the supernatant, ammonium sulphate (0-80%) is added . The sample is kept for 4 hr with constant stirring and subsequently centrifuged at 15000 g for 30 min at 4°(, The supernatant is discarded and the pellet redi sso lved in 25 mM Tris-HCI buffer containing 150 mMNaC I (PH 7.5). It is dialysed thoroughly using the same buffe r for 24 hr. The di alysate is co llected and the protein content measured usin g Bradfo rd method21

. Equa l quantiti es of the sample are subj ected to SDS-PAGE ( 10%) and Nati ve PAGE (8%). The molecular weight markers are also run for compari son.

Biochemical, physiological, cellular and molecular biological properties of protein antimetabolites

Protease inhibitors Protease inhibitors occur commonly in plants

prov iding a fo rm of natural defence against herbi vorous insects. Protease inhibitors act as competitive inhibitors of enzymes by binding ti ght ly to the ac tive site of the enzyme. Inhibitors that form complexes with proteases and inhibit their proteo lytic act ivity are widespread in nature. The anti metabolic activity of the protease inhibi tor is due to direct in hibiti on of larva l proteo lysis and utili sation of prote in leading to the death of the larvae by slow starvati on. Members of serine and cystein protease inhibitor fa milies have been more releva nt to the area of plant defence than meta llo and aspartyl proteinase inhibitors, since only a few of these latter two families of inhibitors have been fo und in plants22

Trypsin in l ibitors from different sources show marked spec ies spec ifi city in the ir inhibitory activity agn inst trypsin like enzymes2l

·24

. Protein ase inhibi tors are usually present in storage ti ssues such as seeds and tubers25

.26 and their ex pression are both

deve lopmentally regulated and induced in response to in sect and pathogen attack27

. Trypsin inhibitor is concentrated in the seed just below the seed testa28

.

Trypsin inhibitor acts by a mechanism other than inhibition of the insect' s di gesti ve proteinases29

.1 1

.

The phys iologica l role of the enzyme inhibi ti ng substances though not fully understood can be described as the e limination of un wanted hydro lys is of the macromolecul es such as protein , starch

218 INDIAN J EXP BlOL, MARCH 1999

and lipids32-34

. Baker et al. 35 suggested that C. maculatus resistance is controlled by two recessive genes implying that the chemical and/or physical factors responsible for resi stance should be present in all resistant lines and absent in all susceptible lines. A partial characterisation of the inhibitors revealed to be a mixture of several chymotrypsin as well as trypsin. Leaves of tob~cco plants transformed with a cowpea trypsin inhibitor gene did not support the growth of the tobacco budworm, a lepidopterous pest36 which is perhaps not surprising since it is known that lepidopterous larvae rely on trypsin like enzymes for protein digestion 37

.

Cysteine proteinase inh ibitors inhibit the cysteine mechanistic class of enzymes and they are ca lled "Cystatins". Several cystein proteinase inh ibitors have been isolated from plant spec ies. It has been mentioned that the leve ls of express ion of the cystein proteinase inhibitor and genomic organ isation in resistant and susceptible cultivar CE-31 (Pitiuba) Vigna unguiculata through molecular cloning using southern and northern blot analyses do not account for the differences in insect res istance of the two lines38

. However, it has been shown that there is poss ibly no correlation whatsoever between serine prote inases (tryps in , chymotrypsin and subtili sin) and cyste in protein (papai n) inhibitors and the resi stance/ susceptibil ity of cowpea seeds to Callosobruchus maculalus39

.

Knowledge on the ro le of aspartic proteinases in insect di gestion is more li mited th an that of cysteine proteinases. No aspartic protei nases have been iso lated from co leoptera, but Wolfson and Murdock40 demonstrated that pepstatin, a powerfu I and speci fic inhibitor of aspartyl prote inases, strongly in hibited proteolysis by the midgut enzymes of the co lorado potato beetle Leptinolarsa decimilineala, indicati ng that an aspartic proteinase was present in the extracts.

Alpha amylase inhibitors

Proteins that inhibi t a -amylase are fo und throughout the plant kingdom . Many of the abundant proteins in cerea l seeds are inhibitors of e ither amylases or proteinases or inhibitors of both41 . Like proteinase inhibitors, amylase inhibitors are considered to be part of the protect ive chemi ca ls of plants against in sect pests. a -amylase in hibitor proteins are members of a large super fam ily of storage prote in s. Many of the a-amylase inhibitors are homologoLis with four of the proteinase inhibitor

families: Kun itz, barley, Bowman-Birk and the ragi/maize bifunctional inhibitor fam ilies42

.

a-Amylase inhibitor has been characterised in I . . f b 4:145 I ' I . severa varietIes 0 eans -. . t IS a g ycoprotelll

(about 15% carbohydrate) and its native molecular weight has been estimated to be 43 to 50 KD. The inhibitor is either a trimer or a tetramer of identica l polypeptides or different polypeptides45

,46 . Moreno et al. 47 have reported that a-amylase inhibitor is encoded by a gene that encodes a LLP described by Haffman et al. 48 or by a closely re lated gene. The protein product of this gene is a glycoprotein with four Asn-linked glycons and that it is synthes ised on the rough ER. a-Amylase inhibitor. inhibits the a­amylase in the digestive tract of mammals and coleoptera49

.

Lectins Lectins are unique proteins wi th highly spec ific

carbohydrate (glycoconjugates) binding act ivity. Lectins are phytohaemagglutinins, fou nd in the seeds of many plant species . Lectins have specificity for N­acety lga lactosamine as we ll as N-acetylglucosamine. The genes encoding for effective plant lect ins could, in principle serve as anti bios is fac tors to use in host plant resistant programmes designed to bri ng res istance in to legumes through recombinant DNA techno logies. A major part of th e adaptive significance of phytohaemagglutini ns in legumes is to protect from seed predators50 Lectins play a protective rol e aga inst in sects particularly bruchids. The exact mechanisms of toxicity of lectin s appears to be analogous to that kn own to occllr in the rats whereby the ingested lectin molec ul es are ab le to bind to the midgu t epitheli um caus ing di rupti on of the ce ll surfaces and allowing the absorption of potentially harmful substances of lectin51. Indirect immunofl ourescence investigat ions using 1110no­spec ific antisera for globulin lectin s showed th at th e leet ins, when in gested by the larvae, bound to the midgut epitheli al ce ll s.

A general property of 1110St proteins, which are directly in volved in defen ce mechan isl11 of pl ants. is th eir marked stability. Many proteins become active after proteo lys is and thi s is part of the defense strategy. Protease inhibitors52

, a-a mylase inhibi tors)' . ribosome inacti vating proteins54 inactivate normal ce ll ular protei ns. All these proteins are stable ove r a broad pH range anci are resistant to a wide range of proteo lytic enzymes. In add ition, most of them are

IGNAC IMUTHU : PROTEIN ANTI METABOLITES IN LEGU ME SEED DEFENCE 2 19

fairly heat stable and lectins in particular can withstand heating much better than intra cellular enzymes. The extreme stability of the lectins is re(.a~d to the fact that the 16 cystein res idues of its polypeptide chai n (of 89 aminoac id residues) form eight di sulphide bridges55 .

Seeds are known to conta in multiple proteins that are inhibitory to in sects. Protease and a-amylase inhibitors are important seed defense proteins present in leguminous seeds. A single genotype often contains multi ple isoforms of these inhibitors perhaps resulting from gene dupl ication; and simple mutations can lead to changes in inhibitory specific ity. This process woul d create a range of genetic va ri abili ty th at could be exploited fo r crop improvement. True res istance may be caused by the combined activity of several proteins each of which is present at a suboptimal leve l to confer resistance. Express ion at hi gh leve ls o f a single inh ib itory protein can confer

. . . I 5657 res istance In transgeni c egumes . .

Vicilin The reserve prote ins of cowpea are mainly of the

vicilin type58 . The vici li n molecule is a tri mer fo rmed by three different po lypeptide chains of approx imately 50KO each 59

. They are deposited in special ised structures in coty ledonary cell s known as the protein bodies.

Vicilin (7S storage prote in s of cowpea seeds) of the line IT8 1 0 - 1045 and IT81 0-1 032 was fou nd to be more refract ory to digestion than vicili n of the cultivars CE-31 (pit iuba) and CE- II. This ditTerence in the digestion of vicilins by midgut proteinases could be ascribed to the storage proteins which confer res istance to Callosobruchus maculatlls of cowpea seeds bred from the resistant TVu 2027 variet/C) . The resi sta nce and detrimental effects of cowpea variant vicili ns on C. maculafus deve lopment can be related by complex formation of vicili ns with the peri trophic mcm brane in the insect 's midgut.

Seed proteins of wi nged bean (Psophocarpus fe fragol/olobus) constitute 3 fractions known as psophocarpin A, Band C. Psophocarpin ';B" was found to be very potentially insecticidal to bruchid deve lopment compared to other psophocarpi n fraction s. The prote in fract ion hav ing the most adverse effect upon bruchid development had the I . I I ' 6 1 lI g lest ectll1 content .

Legu me seeds particu lar ly Phaseolus species contain a family of evol utionarily related plant

defense proteins, composed of phytohaemogglutinin (pHA), arcelin (Arc) and a-amylase in hib itor (a­AI)62. Thi s protein family is encoded by homologous genes found at one complex locus in the bean genome, and it is therefore I ikely that these genes arose by duplication of a single ancestral gene6

.l . The mode of action of arce l in is unknown but the hi gh dose indicates that it is probab ly not we ll d igested by the insectM . Six a lle li c variants of the gene are known 63-65.

Experimental Evidences A hi gh dietary intake of proteinase inh ib itor leads

to loss of proteolyti c activ ity in the gut, thereby interfering with the process of digestion in insects1<J·66 . Preliminary studi es were conducted on the effect of proteinase inhib itors on insects as early as in 1954b7

Trypsin inh ib itors at 10% of the die t were toxic to larvae of the C. maculafus68

. Joh nson e f U/. (,9

transformed tobacco plants with genes coding fo r tomato and potato inhib itor " prote ins and a tomato inhibitor I prote in (havi ng on ly chymotrypsin inhibitory acti vities) regulated by CaMV promoters. Variet ies of cowpea Vigna unguiculata found in Nigeria showed med iu m to strong resistance to several strains of C. maculaflls70

. The resistance of one of these varieties TVu 2027 was thought to be linked to its high leve ls of trypsin inhibitors71. Thi s view seemed to be strengthened by a report that leaves of tobacco plants transformed with a cowpca trypsin inhibitor gene did not support the growth of the tobacco budwonn ; lepidopterous larvae rely on trypsin like enzymes for protein digestion72. CpTI gene amplified by PCR was introduced into tobacco by Agrobacterium mediated transformationn Several observations show that the larvae of C. macula flls utilise main ly cystei n proteinases for protein digesti on 74 ,75 ra ther than serine proteinases I ike tryps in.

In add ition, Roy and Bhat76 have observed low but posit ive correlati on between trypsin inhibitor activity and susceptibi lity of Lathyrus sativlIs seeds to C chinensis and Gupta 77 has esti mated the rich conten t of tryps in inh ibitors in legume seeds whic h are antagoni stic to digestion in insects. The potential of the gene fo r developing tnlllsgenics resistant to certain types of insect pests has already been demonstrated78. A tryps in inh ibi tor, member or Bowman-B irk family, was isolated and puri fied from Vigna unguiculata. Po lyc lonal ant ibod ies were rai sed

220 INDIAN ] EXP BIOL, MARCH 1999

against cowpea trypsin inhibitor (CpT!) protein in rabbits. The gene for CpTI was amplified by polymerase chain reaction and cloned in a bacterial expression vector P.VCA TFR 18416. The expression of C PTI protein in BL 21 (DE 3) strain of E. coli was confirmed by western blot studies. The CPTI gene was also sequenced and found to exhibit 100% homology with earl ier sequence of C PTI gene79

.

Gatehouse et al. 80 suggested that the res istance of seeds of a cowpea cultivar TVu 2027 towards its bruchid pest C maculatus was due to the high levels of tryps in inhibitors fo und in these seeds. However, Filho and Cam pos81 have found some cowpea cu ltivars whose seeds have trypsin inhibitory activity as high as the seeds of the cultivar TVu 2027, which are susceptible to C maculatus. Poor correlat ion between the levels. of proteinase inhibitors were found in seeds of differen t cultivars of cowpea and the resistance/susceptibi I ity to preda ti on by C maculatus82

.

Conversely a -amy lase inhibitors started to receive a systematic investigation in legumes in recent pase2,34,82.83, Amy lase inhibitors have been shown to

inhibit insect and mamma lian a_amylase83,84. The

deleterious effect of a-amylase inhibitor on the bruch id larvae development has beeD demonstrated

84.

L d 42 P d WI ' I 43 Marshal l and a u a, owers an lItaKe r

reported that the a-amy lase inhibitors was present at a level of 0 .4-0.5% in kidneybean seeds. The leve l of

the a-amylase inhibi tors in the seeds seems to be enough to protect them aga inst the attack o f azuk i

bean weevi l. When the a-amylase inhibitor was i'ncorporated at 0.2-0.5% in to the artificial beans, the larvae of C chinensis coul d not deve lop by feedin g

I d · d85 I I . d K ' 86·88 them and t ley Ie . s l lmoto an Itamura

confirmed that common bean a -amylase inhibitor comp letely suppressed the larva l growth of the azuki bean weevil (C chinensis) and cowpea weevi l (C maculatus) and marked ly inhibited the mi dgut a­amylase activ'ity of the Mexican bean weevil (Zabrotes subjasciatus (Boheman). A novel a­

amylase inhibitor: wh ich inhib its only the larva l a­amylase activity of Z sub{ascialus, has been identified in seeds of several wi ld common bean accessions res istant to Z subjasciatwP . Since only some of the res istant accessions contain the in hibi tor, it is considered that the inhi bi tor is not the only factor re ponsible fo r the resi stance61 . [t appears that the

large diversity of seed a-amylase inhibitor types, and

the presence of arcelin variants, both reflect the co­evolutionary adaptation of the common bean to bruchid attack in this region90

. Difference in the

primary structures of aAI-2 and a-AI-I may be the cause fo r distinct inhibitory activity towards

bruchids91. The gene coding of a-A I-2 is closely linked to that of arcelin-488. A close linkage

relationship among the genes of PHA, aAI-1 and four arcelin variants has been demonstrated90

. Suzuki et al. 92

,93 isolated and determined the sequence of 852

nucleotide (DNA of a bruchid res istant wild common

bean and designated as aAI-2 and fou nd it to contain a 720 base open reading frame (ORr). This ORF

encodes a 240 amino acid aA I-2 polypeptide 75 .8% identical with a-amylase inhibitor (aA[-I) and 50.6-55.6% with arcelin-I , phytohaemaggl utinin (PHA) and PH A-E of common bean. T he h igh degree of sequence homology suggests that there IS an evo lut ionary re lat ionship among these genes .

Murdock et al94 showed that for every 0.1 % increase in dose of wheatgerm lect in , which is the most potent, there was 1.47 days de lay in developmental time. Additionally fo r every 0.1 % increase in wheatgerm lectin there was a 1.79% increase in mortality . Resu lts obta ined by Gatehouse el al.95 on th e toxicity of Phaseolus vulgaris lectin when fed to C maculalus larvae were agreeable with the above hypothes is. However, in this study of Gatehouse it was observed th at a highly purified preparation (>95%) of P. vulgaris lectin was cons iderab ly less toxic to the deve lop ing larvae th an the commerc ia l preparations of lectins. A lthough later studies c laimed to confirm th e resu lts of Boulter96

more recent ex periments demonstrated that high Iy purified PHA has no inhibitory effect on the cowpea weev il. An explanation for the contrad ictory results obtai ned with PHA was g iven by Huesing and his co.ll aborators97 who demonstrated unamb iguous ly that the previously observed toxic effects of PHA were

due to an a-amy lase inhibitor contaminant.

Interesti ngly the a-amy lase, is structura lly related to PHA, a lthough it ha s no carbohydrate binding

properti es43. Moreover, bes ides the a-amy lase inhibitor some wi ld accession s of beans contain arce lin another PHA-l ike protein, which was shown to be tox ic for the bean weevi l (Z sub{ascialus)98. It appears therefore that beans do not rely on ly on lectin s for their defense against insects but also on lecti n- re lated proteins. Evidently the above described

IGNACIMUTHU : PROTEIN ANTI METABOLITES IN LEGUME SEED DEFENCE 22 1

example of PHA demonstrates that only the results of experiments with highly purified lectins are valid. Therefore, the observed entomotoxic activity of a lectin enriched fraction from tepary bean (Phaseolus acutifolius )99 against larvae of the bean bruchid beetle (Acanthoscelides obtectus) and the high insecticidal activ ity of a partially purified preparation of basic seed lectin from winged bean (Psophocarpus tetragonalobus)'on C. maculatus larvae44 do not prove that the respecti ve lectin s confer insect resistance.

Conclusion In recent years, immense work has been done on

the role of protein aAtimetabolites in defence mechanism against insects or microbes . Many research projects . are a imed at discovering proteinacious antimetabo li tes to control insect pests. Proteinase inhibitors have rece ived ample attention because of the ir s ize, abundance and stability which make them easy to work38

. Most of these prote in antimetabolites are involved in defense mechanism. Considering that the midgut represents the organ in in sects where critical functions like digestion and absorption take place, interference with the midgut function by disrupting the gut ce ll s seem to be the strategy adopted by the most effect ive insect ic ida l

. 100· 102 I I . . I 'b' prote lll s . n genera , protelllase III 11 Itors, u-amy lase inhibi tors, lectin s, plant spec ific defensive protei ns, ribosome inactivating proteins and thionins act synergistically in establishing highly efficient defense systems again st pl ant predators.

References I A.garwal A, Lal S & Gupta K C, Bull Grain Technology, 26

( 1'988) 154 . 2 Painter R H, Insect resistance in plants (Macmi ll an New

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