Abstract
Main conclusion
Multiplicity of protease inhibitors induced by predators may increase the understanding of a plant’s intelligent behavior toward environmental challenges.
Information about defense mechanisms of non-genomic model plant passion fruit (Passiflora edulis Sims) in response to predator attack is still limited. Here, via biochemical approaches, we showed its flexibility to build-up a broad repertoire of potent Kunitz-type trypsin inhibitors (KTIs) in response to methyl jasmonate. Seven inhibitors (20–25 kDa) were purified from exposed leaves by chromatographic techniques. Interestingly, the KTIs possessed truncated Kunitz motif in their N-terminus and some of them also presented non-consensus residues. Gelatin-Native-PAGE established multiple isoforms for each inhibitor. Significant differences regarding inhibitors’ activity toward trypsin and chymotrypsin were observed, indicating functional polymorphism. Despite its rarity, two of them also inhibited papain, and such bifunctionality suggests a recruiting process onto another mechanistic class of target protease (cysteine-type). All inhibitors acted strongly on midgut proteases from sugarcane borer, Diatraea saccharalis (a lepidopteran insect) while in vivo assays supported their insecticide properties. Moreover, the bifunctional inhibitors displayed activity toward midgut proteases from cowpea weevil, Callosobruchus maculatus (a coleopteran insect). Unexpectedly, all inhibitors were highly effective against midgut proteases from Aedes aegypti a dipteran insect (vector of neglected tropical diseases) opening new avenues for plant-derived PIs for vector control-oriented research. Our results reflect the KTIs’ complexities in passion fruit which could be wisely exploited by influencing plant defense conditions. Therefore, the potential of passion fruit as source of bioactive compounds with diversified biotechnological application was strengthened.
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Abbreviations
- BANA:
-
N-Benzoyl-l-arginine-2-naphthylamide
- BAPNA:
-
N-Benzoyl-dl-arginyl-plnitroanilide
- BSA:
-
Bovine serum albumin
- BTEE:
-
N-Benzoyl tyrosine ethyl ester
- eggCys:
-
Egg cystatin
- EST:
-
Expressed sequence tags
- KPIs:
-
Kunitz-type protease inhibitors
- KTI:
-
Kunitz-type trypsin inhibitors
- MeJa:
-
Methyl jasmonate
- NCBI:
-
National Center for Biotechnology Information
- PIs:
-
Proteinase inhibitors
- SDS:
-
Sodium dodecyl sulfate
- SoyKTI:
-
Soybean Kunitz inhibitor
- TFA:
-
Trifluoroacetic acid
- TIs:
-
Trypsin inhibitors
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Acknowledgments
The research was supported by Brazilian agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant no. 471848/2012-3) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ, grant no. E-26/110.942/2013). S.B.J. was recipient of a PhD fellowship from Universidade Estadual do Norte Fluminense (UENF) and V.A.P. was recipient of a PhD fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). We thank Dr. Turán Peter Ürmenyi, Universidade Federal do Rio de Janeiro (UFRJ) for critical reading of this manuscript.
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Supplementary Fig. S1 a SDS-PAGE (10 %) analysis of crude leaf extracts of plants exposed to MeJa by 48 and 96 h. b Gelatin-SDS-PAGE analyses (under semi-denaturing condition) of crude leaf extracts from exposed plants to MeJa by 48 and 96 h. For each lane were loaded 5 μg of protein. Trypsin inhibitory activities are shown by protein bands in a clear background (b, see Materials and methods).
Supplementary Fig. S2 a Native-PAGE (10 %) analysis of crude leaf extracts of plants exposed to MeJa by 48 and 96 h. b Gelatin-Native -PAGE analyses of crude leaf extracts from exposed plants to MeJa by 48 and 96 h. For each lane were loaded 5 μg of protein. Trypsin inhibitory activities are shown by protein bands in a clear background (b, see Materials and methods)
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Botelho-Júnior, S., Machado, O.L.T., Fernandes, K.V.S. et al. Defense response in non-genomic model species: methyl jasmonate exposure reveals the passion fruit leaves’ ability to assemble a cocktail of functionally diversified Kunitz-type trypsin inhibitors and recruit two of them against papain. Planta 240, 345–356 (2014). https://doi.org/10.1007/s00425-014-2085-3
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DOI: https://doi.org/10.1007/s00425-014-2085-3