**2.3. Proteinase inhibitors**

against mustard aphid (*Lipaphis erysimi*) in comparison to GNA and ASAL (*Allium sativum* leaf agglutinin) [34]. Transgenic rice expressing ASAL exhibited protection against tungro disease also, after infestation with the *N. virescens* [35]. Vajhala et al. [36] recently demonstrated significant protection in ASAL expressing transgenic cotton against jassid and whitefly. ASAL is also reported to be toxic to chewing insects like *Helicoverpa armigera* and *Spodoptera litura* [27] and several other sucking insects like *Nephotettix virescens* and *Nilaparvata lugens* [37]. Studies related to the mechanism of toxicity showed that ASAL shares the similar receptors with Bt-Cry toxin [28], but both the proteins interact at different positions without steric hindrance and increased the toxicity of each other [29]. Therefore, they can be pyramided together for broad-

342 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

Legume lectins are purified from seeds and bind to carbohydrate structures like Thomsennouveau (Tn) antigen or complex N-glycan with terminal galactose and sialic acid residues. Pea lectin (*Pisum sativum* agglutinin, PSA) expressed in transgenic oilseed rape (*Brassica napus*) shows growth retardation of the pollen beetle larvae (*Meligethes aeneus*) [38] and no effect on the adult beetles [39]. A legume lectin known as Gleheda purified from ground ivy (*Glechoma hederacea*) exhibits high insecticidal activity against the Colorado potato beetle larvae (*Leptinotarsa decem-lineata*) [40]. GS-II lectin isolated from the seed of *Griffonia simplicifolia* shows toxicity to Cowpea weevil (*Callosobruchus maculates*) [41]. A mannose-binding legume lectin concanavalin A (ConA) from jackbean has shown toxicity to the hemipteran pea aphid

Hevein-related plant lectins exhibit specificity for chitin (chitin forms endo- and exo-arthro‐ pods, nematodes and fungi). These are also studied for insecticidal properties [45]. Due to the absence of chitin in mammals, hevein-related lectins are considered safe for the usage in genetically modified crops. Wheat germ agglutinin (WGA) has shown a negative effect on the development of the cowpea weevil (*Callosobruchus maculatus*) larvae and southern corn root worm (*Diabrotica undecimpunctata*) [46, 47]. WGA is active against lepidopteran insect larvae

Several other plant lectins have shown insecticidal property. Transgenic tobacco plants expressing tobacco leaf lectin (NICTABA) is detrimental to the cotton leafworm (*S. littoralis*) and the tobacco hornworm (*M. sexta*) [49]. Another protein, *phloem protein 2* (PP2) belonging to the NICTABA family, also possesses insecticidal activity [50, 51]. The amaranthins and the jacalin-related lectins have also shown the potential for insect control, especially against sapsucking insect pests. Transgenic cotton expressing *Amaranthus caudatus* agglutinin (ACA) under the control of a phloem-specific promoter shows a strong resistance against nymphs of the cotton aphid (*Aphis gossypii*) [52]. Transgenic tobacco expressing Heltuba, a jacalin-related lectin from the *Helianthus tuberosus*, showed reduced development and fecundity of the peach–

(*Acyrthosiphon pisum*) [42, 43] and tara plant hopper (*Tarophagous proserpina*) [44].

range insect resistance.

*2.2.3. Hevein-related lectins*

*2.2.4. Other insecticidal lectins*

also [47, 48].

*2.2.2. Legume lectins*

Proteinase inhibitors (PIs) are small molecular weight proteins which affect several metabolic pathways. They are the major components in seeds and storage organs of crops. Mickel and Standish [55] demonstrated the role of PIs in plant defense for the first time and noticed the abnormality in the development of larvae of certain insects fed on soybean products. The feature was attributed to trypsin inhibitors, and it was found to be toxic to the larvae of flour beetle (*Tribolium confusum*) [56].

PIs inhibit the digestion of proteins in midgut and cause mortality of insects due to nutritional imbalance [57, 58]. PIs also interfere with several metabolic processes (like moulting) by blocking the proteolytic activation of enzymes [59]. They affect growth and development, multiplication rate and insect life span [60–62]. PIs have been expressed in several transgenic plants for resistance against insect pests of several classes [63–65]. Pea and soybean trypsin– chymotrypsin inhibitors (PsTI-2, SbBBI) belonging to the Bowman–Birk family [66] and mustard-type trypsin–chymotrypsin variant Chy8 [67] cause significant mortality of pea aphid *A. pisum*. Plant-derived PIs have been used for the development of insect-resistant transgenic plants and projected as an alternative to Bt-Cry proteins [68, 69].

The majority of plant PIs originate from three main families, namely Solanaceae, Leguminosae and Gramineae [70]. Plant PIs can be grouped into four classes: serine, thiol, metallo and aspartyl. Most plant PIs are inhibitors of microbial and animal serine proteases, such as chymotrypsin, trypsin, elastase and subtilisin [71]. Specificity of protease inhibitor families is mainly based on the amino acid residues present in the active site [72].

### *2.3.1. Serine (Serpin) protease inhibitors*

It is found in almost all kingdoms of organisms [73–76]. Several serine PIs have been purified from plants and characterized [77, 78]. Plant serine PIs show inconsistent and varied specific‐ ities towards plant proteases [79]. *Hordeum vulgare* serine PI inhibits trypsin, chymotrypsin [80], thrombin, plasma kallikrein, Factor VIIa and Factor Xa [81]. *Triticum aestivum* serine PI inhibits chymotrypsin and cathepsin G [82]. Serine protease inhibitors have been used most commonly for the development of transgenic plants for the control of insect pests [83–85].

### *2.3.2. Cysteine protease inhibitors*

An inhibitor of cysteine proteinases was first described in egg white by Sen and Whitaker [86] and was later named cystatin [87]. Cysteine proteinases inhibitors are widely distributed in plants, animals and microorganisms [88]. Their role in defense has been explored by *in vitro* analysis on inhibition of digestive proteinases from insect pests and nematodes [89–91]. First plant cystatin was isolated from rice seeds and as of now, more than 80 members of different plant species have been characterized [92, 93]. Barley cystatin in artificial diets hampered the life cycle of two aphid species and also in transgenic *Arabidopsis* [94]. Expression of such inhibitors in maize enhanced the resistance against phytophagous mites [95]. Inhibition of these proteases provides a promising control on insects and therefore PIs can be employed as a potential source of defense in plants against insect pests.

## *2.3.3. Aspartyl protease inhibitors*

It is relatively less studied class, due to the rare occurrence [91]. Potato tubers possess cathepsin D, an aspartic proteinase inhibitor which showed substantial amino acid sequence similarity with the soybean trypsin inhibitor [96]. Aspartic proteases have been found in coleoptera species, such as *Callosobruchus maculatus* [97] and *H. hampei* [98], in which the acidic pH in midgut provides a favourable condition for these proteases [58].

### *2.3.4. Metallo-proteases inhibitors*

The metallo carboxypeptidase inhibitors (MCPIs) have been identified in solanaceaous plants tomato and potato [99]. The MCPIs are 38–39 amino acid residues long polypep‐ tide [100, 101]. Plants have evolved at least two families of metalloproteinase inhibitors, the metallo-carboxypeptidase inhibitor family in potato and tomato [102] and a cathepsin D inhibitor family in potato [103]. The inhibitor is produced in potato tubers and accumu‐ lates with potato inhibitor I and II families (serine proteinase inhibitors) during tuber development. The inhibitor also accumulates in potato leaf with inhibitor I and II in response to wounding and have the potential to inhibit all the major digestive enzymes (like trypsin, chymotrypsin, elastase, carboxypeptidase A and carboxypeptidase B) of higher animals and many insects [104].

### **2.4. α-Amylase inhibitors**

α-Amylases (α-1,4-glucan-4-glucanohydrolases) are hydrolytic enzymes, which catalyze the hydrolysis of α-1,4-glycosydic bonds in polysaccharides. They are present in microorganisms, animals and plants [105–107]. They are the most important digestive enzymes of many insects which feed exclusively on seed products. Inhibition of α-amylase impairs the digestion in an organism and causes shortage of free sugar for energy. α-Amylase inhibitors (α-AI) are found in many plants as a part of the defense system and abundant in cereals and legumes [108–111].

α-AI of *Phaseolus vulgaris* is the most studied amylase inhibitor and have shown toxic effects to several insect pests [110, 111]. Like lectins, they possess carbohydrate-binding property. There are at least four types of *Phaseolus* amylase inhibitors on the basis of α-AIs: AI-1, AI-2, AI-3 and the null type [112]. AI-1 is present in the most cultivated common bean varieties and inhibits mammalian α-amylases. It also inhibits α-amylases in insects like *C. chinensis, C. maculatus* and *B. pisorum* [106]. AI-2 is 78% homologous to AI-1 and found in few wild accessions. It inhibits the *Z. subfasciatus* larval α-amylase and pea bruchid α-amylase [106, 111, 113]. This inhibitor is a good example of co-evolution of insect digestive enzymes and plant defense proteins.

They are potential molecules for the development of insect-resistant transgenic plants [114, 115]. Seeds of transgenic pea and azuki, expressing α-AI-1 inhibitor of *P. vulgaris*, shows resistance against pea weevil (*Bruchus pisorum*), cowpea weevil (*C. maculatus*) and azuki bean weevil (*Callosobruchus chinensis*) [110, 113, 116].
