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.

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.

Herbivore response in passion fruit (Passiflora edulis Sims) plants: induction of lipoxygenase activity in leaf tissue in response to generalist and specialist insect attack.

Lipoxygenases (LOXs, EC 1.13.11.12) are a class of non-heme iron containing dioxygenases which catalyze the regiospecific and stereospecific hydroperoxidation of polyunsaturated fatty acids with 1,4-pentadiene system such as linoleic acid and linolenic acid in plants. In this work we studied the LOX activity in damaged as well as in distal leaves in response to specialist (Agraulis vanillae vanillae) or generalist (Spodoptera frugiperda) insect attack. Enzymatic assays showed that induction of LOX activity occurred locally and systemically in response to both insects' attacks. Northern blot analysis revealed that LOX expression is also insect-inducible in agreement with enzymatic assay results. In addition, northern analysis corroborated previous reports that LOX activity is wound- and methyl jasmonate-inducible. These results suggest that the herbivore-response in passion fruit is mediated by jasmonates, since a key enzyme of the biosynthetic pathway of jasmonic acid is induced upon lepidopteran insects' attacks.