Purification of trypsin inhibitor from seeds of Cicer arietinum (L.) and its insecticidal potential against Helicoverpa armigera (Hübner)

Nair, Meera; Sandhu, Sardul Singh; Babbar, Anita

Abstract

Trypsin inhibitors (TI) have crucial functions in plant defense against pests, and recent studies reveal their diversity in stress response. Disease and herbivore infestations are harmful to plant growth, but the response of TI in Cicer arietinum (L.) in Central India has been less studied. In the present study, we partially characterized the TI from Cicer arietinum (L.) seeds and evaluated its insecticidal potential against Helicoverpa armigera (Hübner). A 20 kDa trypsin inhibitor was purified to homogeneity by ammonium sulfate precipitation and chromatographies with Sephadex G-100 and diethylaminoethyl cellulose (DEAE-cellulose-52) ion-exchange column. TI assay categorized 17 cultivars in three groups: with high (more than 70%), intermediate (16-70%) and very low (0-15%) TI activity. Moreover, results from the TI activity coincided with herbivore attack studies carried out in J.N.K.V.V. fields earlier. Together, our results suggest that the presence of TI triggers a signal that leads the dynamics of stress response and, in turn, regulates pest resistance in plants. Feeding experiments conducted with 5th instar larvae suggested dose-dependent decrease for both the larval weight and the survival of the larva.

bioassay; Helicoverpa armigera gut proteinase (HGP); resistant; susceptible; protein

Arumuganathan K, Earle ED (1991) Nuclear DNA content of some important plant species. Plant Molecular Biology Reporter 9:208-218.
Belew M, Eaker D (1976) The trypsin and chymotrypsin inhibitors in chickpeas. Identification of trypsin-reactive site, partial amino acid sequence and further physico chemical properties of the major inhibitor. European Journal of Biochemistry 62:499-508.
Bhattacharyya A, Babu CR (2009) Purification and biochemical characterization of a serine proteinase inhibitor from Derris trifoliata Lour. seeds: insight into structural and antimalarial features. Phytochemistry 70:703-12.
Bown DP, Wilkinson HS, Gatehouse JA (1997) Differentially regulated inhibitor sensitive and insensitive protease genes from the phytophagous insect pest, Helicoverpa armigera, are members of complex multigene families. Insect Biochemistry and Molecular Biology 27:625-638.
Chaturvedi I (2007) Economic threshold level and monitoring Systems: reality and practicality in Helicoverpa armigera (Lepidoptera: Noctuidae). Electronic Journal of Environmental, Agricultural and Food Chemistry 6:2199-2206.
De Leo F, Bonadé-Botino M, Ceci RL, Galerani R, Jouanin L (2001) Effects of mustard trypsin inhibitors expressed in different plants on three different lepidopteran pests. Insect Biochemistry and Molecular Biology 31:593-602.
Dunse K, Anderson M (2011) Towards the Next Generation of Pest Resistant Plants. ISB News Report, Virginia Polytechnic Institute and State University June.
Ellis J, Jones D (1998) Structure and function of proteins controlling strain-specific pathogen resistance in plants. Current Opinion in Plant Biology 1:288-293.
Erlanger BF, Kokowesky N, Cohen W (1964) The preparation and properties of two new chromogenic substrates of trypsin. Archives of Biochemistry and Biophysics 95:271-278.
Gatehouse AMR, Norton E, Davison GM, Babbe SM, Newell CA, Gatehouse JA (1999) Digestive proteolytic activity in larvae of tomato moth, Lacanobia oleracea: Effects of plant proteinase inhibitor in vitro and in vivo Journal of Insect Physiology 45:545-558.
Girard C, Le Metayer M, Bonade-Bottino M, Pham-Delegue MH, Jouanin L (1998) High level of resistance to proteinase inhibitors may be conferred by proteolytic cleavage in beetle larvae. Insect Biochemistry and Molecular Biology 28:229-237.
Giri AP, Harsulkar AM, Deshpande VV, Sainani MN, Gupta VS, Ranjekar PK (1998) Chickpea defensive proteinase inhibitors can be inactivated by pod borer gut proteinases. Plant Physiology 116:393-401.
Giri AP, Kachole MS (1998) Amylase inhibitors of pigeonpea (Cajanus cajan L.) seeds. Phytochemistry 47:197-202.
Gomes A de PG, Dias SC, Jr Bloch C, Melo FR, Jr Furtado JR, Monnerat RG, Grossi-de-Sá MF, Franco OL (2005) Toxicity to cotton boll weevil Anthonomus grandis of a trypsin inhibitor from chickpea seeds. Comparative Biochemistry and Physiology, Part B: Biochemistry and Molecular Biology 140:313-9.
Harsulkar AM, Giri AP, Gupta VS, Sainani MN, Deshpande VV, Patankar AG, Ranjekar PK (1998) Characterization of Helicoverpa armigera gut proteinases and the interaction with proteinase inhibitors using gel X-ray film contact print technique. Electrophoresis 19:1397-1402.
Harsulkar AM, Giri AP, Kothekar VS (1997) Protease inhibitors of chickpea (Cicer arietinum L.) during seed development. Journal of the Science of Food and Agriculture 74:509-512.
Harsulkar AM, Giri AP, Patankar AG, Gupta VS, Sainani MN, Ranjekar PK, Deshpande VV (1999) Successive use of non-host plant proteinase inhibitors required for effective inhibition of Helicoverpa armigera gut proteinases and larval growth. Plant Physiology 121:497-506.
Headey SJ, Macaskill UK, Wright MA, Claridge JK, Edwards PJ, Farley PC, Christeller JT, Laing WA, Pascal SM (2010) Solution structure of the squash aspartic acid proteinase inhibitor (SQAPI) and mutational analysis of pepsin inhibition. Journal of Biological Chemistry 285:27019-25.
Jibson MD, Birk Y, Bewley TA (1981) Circular Dichroism spectra of trypsin and chymotrypsin complexes with Bowman-Birk or chickpea trypsin inhibitor. International Journal of Protein Research 18:26-32.
Johnston KA, Gatehouse JA, Anstee JH (1993) Effects of soybean protease inhibitors on the growth and development of larval Helicoverpa armigera Journal of Insect Physiology 39:657-664.
Johnston KA, Lee M, Gatehouse JA, Anstee JH (1991) The partial purification and characterization of serine proteinase activity in midgut of larval Helicoverpa armigera Insect Biochemistry 21:389-397.
Kakade ML, Rackis JJ, McGhee JE, Puski G (1974) Determination of trypsin inhibitor activity of soy products: A collaborative analysis of an improved procedure. Cereal Chemistry 51:376-382.
Kansal R, Kumar M, Kuhar K, Gupta RN, Subrahmanyam B, Koundal KR, Gupta VK (2008) Purification and characterization of trypsin inhibitor from Cicer arietinum L. and its efficacy against Helicoverpa armigera. Brazilian Journal of Plant Physiology 20:313-322.
Koiwa H, Shade RE, Zhu-Salzman K, Subramanian L, Murdock LL, Nielsen SS, Bressan RA, Hasegawa PM (1998) Phage display selection can differentiate insecticidal activity of soybean cystatins. Plant Journal 14:371-379.
Lawrence PK, Koundal KR (2002) Plant protease inhibitors in control of phytophagous insects. Electronic Journal of Biotechnology 5:5-6.
Lowry OH, Rosenbrough NJ, Farr A, Randall RJ (1951) Protein measurement by folin phenol reagent. Journal of Biological Chemistry 193:265-267.
Maiti RK (2001) The Chickpea crop. In: Maiti R, Wesche-Ebeling P (eds), Advances in Chickpea Science, pp.1-32. Science Publishers Inc., Enfield, NH.
Mulimani VH, Kulkarni S, Giri AP (2002) Detection of Legume Protease Inhibitors by the Gel-X-ray Film Contact Print Technique. Biochemistry and Molecular Biology Education 30:40-44.
Nagashima Y, Takeda M, Ohta I, Shimakura K, Shiomi K (2004) Purification and properties of proteinaceous trypsin inhibitors in the skin mucus of pufferfish Takifugu pardalis. Comparative Biochemistry and Physiology, Part B: Biochemistry and Molecular Biology 138:103-10.
Parde VD, Sharma HC, Kachole MS (2010) In vivo inhibition of Helicoverpa armigera gut pro-proteinase activation by non-host plant protease inhibitors. Journal of Insect Physiology 56:1315-24.
Prasad ER, Dutta-Gupta A, Padmasree K (2010) Purification and characterization of a Bowman-Birk proteinase inhibitor from the seeds of black gram (Vigna mungo). Phytochemistry 71:363-72.
Rai S, Aggarwal KK, Babu CR (2008) Isolation of a serine Kunitz trypsin inhibitor from leaves of Terminalia arjuna Current Science 94:1509-1512.
Ryan CA (1990) Protease inhibitors in plants-genes for improving defenses against insects and pathogens. Annual Review of Phytopathology 28:425-449.
Saadati F, Bandani AR (2011) Effects of serine protease inhibitors on growth and development and digestive serine proteinases of the Sunn pest, Eurygaster integriceps. Journal of Insect Science 11:1-12.
Saini HS, Weder JKP, Knights EJ (1992) Inhibitor activities of chickpeas (Cicer arietinum L.) against bovine, porcine and human trypsin and chymotrypsin. Journal of the Science of Food and Agriculture 60:287-295.
Sastry MC, Murray DR (1987) The contribution of trypsin inhibitors to the nutritional value of chick pea seed protein. Journal of the Science of Food and Agriculture 40:253-261.
Satheesh LS, Murugan K (2012) Purification of protease inhibitor from leaves of Coccinia grandis (L.) Voigt. and its insecticidal potentiality against Spodoptera litura Fabricus. Asian Pacific Journal of Tropical Biomedicine 1:1-9.
Schuler TH, Poppy GM, Kerry BR, Denholm I (1998) Insect resistant transgenic plants. Trends in Biotechnology 16:168-175.
Sharma U, Suresh CG (2011) Purification, crystallization and X-ray characterization of a Kunitz-type trypsin inhibitor protein from the seeds of chickpea (Cicer arietinum). Acta Crystallographica -Structural Biology and Crystallization Communications 67:714-7.
Smirnoff P, Khalef S, Birk Y, Applebaum SW (1979) Trypsin and chymotrypsin inhibitor from chickpeas-selective chemical modification of the inhibitor and isolation of two isoinhibitors. International Journal of Protein Research 14:186-192.
Srinivasan A, Giri AP, Harsulkar AM, Gatehouse JA, Gupta VS (2005) A Kunitz trypsin inhibitor from chickpea (Cicer arietinum L.) that exerts anti-metabolic effect on podborer (Helicoverpa armigera) larvae. Plant Molecular Biology 57:359-74.
Thangwana NM, Ogola, JBO (2012) Yield and yield components of chickpea (Cicer arietinum): Response to genotype and planting density in summer and winter sowings. Journal of Food, Agriculture and Environment 10:710-715.
Vain P, Worland B, Clarke MC, Richard G, Beavis M, Liu H, Kohli A, Leech M, Snape J, Christou P (1998) Expression of an engineered cysteine proteinase inhibitor (Oryzacystatin-I delta D86) for nematode resistance in transgenic rice plants. Theoretical and Applied Genetics 96:266-271.
Valueva TA, Parfenov IA, Revina TA, Morozkina EV, Benevolensky SV (2012) Structure properties of the potato chymotrypsin inhibitor. Plant Physiology and Biochemistry 52:83-90.
Wang CZ, Qin JD (1996) Effect of soybean trypsin inhibitor, gossypol, and tannic acid on the midgut protease activities and growth of Helicoverpa armigera larvae. Acta Entomologica Sinica 39:337-341.
Wu Y, Llewellyn D, Mathews A, Dennis ES (1997) Adaptation of Helicoverpa armigera (Lepidoptera: Noctuidae) to a proteinase inhibitor expressed in transgenic tobacco. Molecular Breeding 3:371-380.

Publication Dates

Publication in this collection
15 Oct 2013
Date of issue
2013

History

Received
27 Feb 2013
Accepted
20 June 2013

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

[1] Arumuganathan K, Earle ED (1991) Nuclear DNA content of some important plant species. Plant Molecular Biology Reporter 9:208-218.

[2] Belew M, Eaker D (1976) The trypsin and chymotrypsin inhibitors in chickpeas. Identification of trypsin-reactive site, partial amino acid sequence and further physico chemical properties of the major inhibitor. European Journal of Biochemistry 62:499-508.

[3] Bhattacharyya A, Babu CR (2009) Purification and biochemical characterization of a serine proteinase inhibitor from Derris trifoliata Lour. seeds: insight into structural and antimalarial features. Phytochemistry 70:703-12.

[4] Bown DP, Wilkinson HS, Gatehouse JA (1997) Differentially regulated inhibitor sensitive and insensitive protease genes from the phytophagous insect pest, Helicoverpa armigera, are members of complex multigene families. Insect Biochemistry and Molecular Biology 27:625-638.

[5] Chaturvedi I (2007) Economic threshold level and monitoring Systems: reality and practicality in Helicoverpa armigera (Lepidoptera: Noctuidae). Electronic Journal of Environmental, Agricultural and Food Chemistry 6:2199-2206.

[6] De Leo F, Bonadé-Botino M, Ceci RL, Galerani R, Jouanin L (2001) Effects of mustard trypsin inhibitors expressed in different plants on three different lepidopteran pests. Insect Biochemistry and Molecular Biology 31:593-602.

[7] Dunse K, Anderson M (2011) Towards the Next Generation of Pest Resistant Plants. ISB News Report, Virginia Polytechnic Institute and State University June.

[8] Ellis J, Jones D (1998) Structure and function of proteins controlling strain-specific pathogen resistance in plants. Current Opinion in Plant Biology 1:288-293.

[9] Erlanger BF, Kokowesky N, Cohen W (1964) The preparation and properties of two new chromogenic substrates of trypsin. Archives of Biochemistry and Biophysics 95:271-278.

[10] Gatehouse AMR, Norton E, Davison GM, Babbe SM, Newell CA, Gatehouse JA (1999) Digestive proteolytic activity in larvae of tomato moth, Lacanobia oleracea: Effects of plant proteinase inhibitor in vitro and in vivo Journal of Insect Physiology 45:545-558.

[11] Girard C, Le Metayer M, Bonade-Bottino M, Pham-Delegue MH, Jouanin L (1998) High level of resistance to proteinase inhibitors may be conferred by proteolytic cleavage in beetle larvae. Insect Biochemistry and Molecular Biology 28:229-237.

[12] Giri AP, Harsulkar AM, Deshpande VV, Sainani MN, Gupta VS, Ranjekar PK (1998) Chickpea defensive proteinase inhibitors can be inactivated by pod borer gut proteinases. Plant Physiology 116:393-401.

[13] Giri AP, Kachole MS (1998) Amylase inhibitors of pigeonpea (Cajanus cajan L.) seeds. Phytochemistry 47:197-202.

[14] Gomes A de PG, Dias SC, Jr Bloch C, Melo FR, Jr Furtado JR, Monnerat RG, Grossi-de-Sá MF, Franco OL (2005) Toxicity to cotton boll weevil Anthonomus grandis of a trypsin inhibitor from chickpea seeds. Comparative Biochemistry and Physiology, Part B: Biochemistry and Molecular Biology 140:313-9.

[15] Harsulkar AM, Giri AP, Gupta VS, Sainani MN, Deshpande VV, Patankar AG, Ranjekar PK (1998) Characterization of Helicoverpa armigera gut proteinases and the interaction with proteinase inhibitors using gel X-ray film contact print technique. Electrophoresis 19:1397-1402.

[16] Harsulkar AM, Giri AP, Kothekar VS (1997) Protease inhibitors of chickpea (Cicer arietinum L.) during seed development. Journal of the Science of Food and Agriculture 74:509-512.

[17] Harsulkar AM, Giri AP, Patankar AG, Gupta VS, Sainani MN, Ranjekar PK, Deshpande VV (1999) Successive use of non-host plant proteinase inhibitors required for effective inhibition of Helicoverpa armigera gut proteinases and larval growth. Plant Physiology 121:497-506.

[18] Headey SJ, Macaskill UK, Wright MA, Claridge JK, Edwards PJ, Farley PC, Christeller JT, Laing WA, Pascal SM (2010) Solution structure of the squash aspartic acid proteinase inhibitor (SQAPI) and mutational analysis of pepsin inhibition. Journal of Biological Chemistry 285:27019-25.

[19] Jibson MD, Birk Y, Bewley TA (1981) Circular Dichroism spectra of trypsin and chymotrypsin complexes with Bowman-Birk or chickpea trypsin inhibitor. International Journal of Protein Research 18:26-32.

[20] Johnston KA, Gatehouse JA, Anstee JH (1993) Effects of soybean protease inhibitors on the growth and development of larval Helicoverpa armigera Journal of Insect Physiology 39:657-664.

[21] Johnston KA, Lee M, Gatehouse JA, Anstee JH (1991) The partial purification and characterization of serine proteinase activity in midgut of larval Helicoverpa armigera Insect Biochemistry 21:389-397.

[22] Kakade ML, Rackis JJ, McGhee JE, Puski G (1974) Determination of trypsin inhibitor activity of soy products: A collaborative analysis of an improved procedure. Cereal Chemistry 51:376-382.

[23] Kansal R, Kumar M, Kuhar K, Gupta RN, Subrahmanyam B, Koundal KR, Gupta VK (2008) Purification and characterization of trypsin inhibitor from Cicer arietinum L. and its efficacy against Helicoverpa armigera. Brazilian Journal of Plant Physiology 20:313-322.

[24] Koiwa H, Shade RE, Zhu-Salzman K, Subramanian L, Murdock LL, Nielsen SS, Bressan RA, Hasegawa PM (1998) Phage display selection can differentiate insecticidal activity of soybean cystatins. Plant Journal 14:371-379.

[25] Lawrence PK, Koundal KR (2002) Plant protease inhibitors in control of phytophagous insects. Electronic Journal of Biotechnology 5:5-6.

[27] Lowry OH, Rosenbrough NJ, Farr A, Randall RJ (1951) Protein measurement by folin phenol reagent. Journal of Biological Chemistry 193:265-267.

[28] Maiti RK (2001) The Chickpea crop. In: Maiti R, Wesche-Ebeling P (eds), Advances in Chickpea Science, pp.1-32. Science Publishers Inc., Enfield, NH.

[29] Mulimani VH, Kulkarni S, Giri AP (2002) Detection of Legume Protease Inhibitors by the Gel-X-ray Film Contact Print Technique. Biochemistry and Molecular Biology Education 30:40-44.

[30] Nagashima Y, Takeda M, Ohta I, Shimakura K, Shiomi K (2004) Purification and properties of proteinaceous trypsin inhibitors in the skin mucus of pufferfish Takifugu pardalis. Comparative Biochemistry and Physiology, Part B: Biochemistry and Molecular Biology 138:103-10.

[31] Parde VD, Sharma HC, Kachole MS (2010) In vivo inhibition of Helicoverpa armigera gut pro-proteinase activation by non-host plant protease inhibitors. Journal of Insect Physiology 56:1315-24.

[32] Prasad ER, Dutta-Gupta A, Padmasree K (2010) Purification and characterization of a Bowman-Birk proteinase inhibitor from the seeds of black gram (Vigna mungo). Phytochemistry 71:363-72.

[33] Rai S, Aggarwal KK, Babu CR (2008) Isolation of a serine Kunitz trypsin inhibitor from leaves of Terminalia arjuna Current Science 94:1509-1512.

[34] Ryan CA (1990) Protease inhibitors in plants-genes for improving defenses against insects and pathogens. Annual Review of Phytopathology 28:425-449.

[35] Saadati F, Bandani AR (2011) Effects of serine protease inhibitors on growth and development and digestive serine proteinases of the Sunn pest, Eurygaster integriceps. Journal of Insect Science 11:1-12.

[36] Saini HS, Weder JKP, Knights EJ (1992) Inhibitor activities of chickpeas (Cicer arietinum L.) against bovine, porcine and human trypsin and chymotrypsin. Journal of the Science of Food and Agriculture 60:287-295.

[37] Sastry MC, Murray DR (1987) The contribution of trypsin inhibitors to the nutritional value of chick pea seed protein. Journal of the Science of Food and Agriculture 40:253-261.

[38] Satheesh LS, Murugan K (2012) Purification of protease inhibitor from leaves of Coccinia grandis (L.) Voigt. and its insecticidal potentiality against Spodoptera litura Fabricus. Asian Pacific Journal of Tropical Biomedicine 1:1-9.

[39] Schuler TH, Poppy GM, Kerry BR, Denholm I (1998) Insect resistant transgenic plants. Trends in Biotechnology 16:168-175.

[40] Sharma U, Suresh CG (2011) Purification, crystallization and X-ray characterization of a Kunitz-type trypsin inhibitor protein from the seeds of chickpea (Cicer arietinum). Acta Crystallographica -Structural Biology and Crystallization Communications 67:714-7.

[41] Smirnoff P, Khalef S, Birk Y, Applebaum SW (1979) Trypsin and chymotrypsin inhibitor from chickpeas-selective chemical modification of the inhibitor and isolation of two isoinhibitors. International Journal of Protein Research 14:186-192.

[42] Srinivasan A, Giri AP, Harsulkar AM, Gatehouse JA, Gupta VS (2005) A Kunitz trypsin inhibitor from chickpea (Cicer arietinum L.) that exerts anti-metabolic effect on podborer (Helicoverpa armigera) larvae. Plant Molecular Biology 57:359-74.

[43] Thangwana NM, Ogola, JBO (2012) Yield and yield components of chickpea (Cicer arietinum): Response to genotype and planting density in summer and winter sowings. Journal of Food, Agriculture and Environment 10:710-715.

[44] Vain P, Worland B, Clarke MC, Richard G, Beavis M, Liu H, Kohli A, Leech M, Snape J, Christou P (1998) Expression of an engineered cysteine proteinase inhibitor (Oryzacystatin-I delta D86) for nematode resistance in transgenic rice plants. Theoretical and Applied Genetics 96:266-271.

[45] Valueva TA, Parfenov IA, Revina TA, Morozkina EV, Benevolensky SV (2012) Structure properties of the potato chymotrypsin inhibitor. Plant Physiology and Biochemistry 52:83-90.

[46] Wang CZ, Qin JD (1996) Effect of soybean trypsin inhibitor, gossypol, and tannic acid on the midgut protease activities and growth of Helicoverpa armigera larvae. Acta Entomologica Sinica 39:337-341.

[47] Wu Y, Llewellyn D, Mathews A, Dennis ES (1997) Adaptation of Helicoverpa armigera (Lepidoptera: Noctuidae) to a proteinase inhibitor expressed in transgenic tobacco. Molecular Breeding 3:371-380.

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Purification of trypsin inhibitor from seeds of Cicer arietinum (L.) and its insecticidal potential against Helicoverpa armigera (Hübner)

Abstract

Publication Dates

History