Plant Defensive β-Glucosidases Resist Digestion and Sustain Activity in the Gut of a Lepidopteran Herbivore

Abstract

Two-component activated chemical defenses are a major part of many plants’ strategies to disrupt herbivory. The activation step is often the β-glucosidase-catalyzed removal of a glucose moiety from a pro-toxin, leading to an unstable and toxic aglycone. While some β-glucosidases have been well studied, several aspects of their roles in vivo, such as their precise sites of enzymatic activity during and after ingestion, and the importance of particular isoforms in plant defense are still not fully understood. Here, plant defensive β-glucosidases from maize, white mustard and almonds were shown to resist digestion by larvae of the generalist lepidopteran Spodoptera littoralis, and the majority of the ingested activities toward both general and plant pro-toxic substrates was recovered in the frass. Among other proteins potentially involved in defense, we identified specific plant β-glucosidases and a maize β-glucosidase aggregating factor in frass from plant-fed insects using proteomic methods. We therefore found that, while S. littoralis larvae efficiently degraded bulk food protein during digestion, β-glucosidases were among a small number of plant defensive proteins that resist insect digestive proteolysis. These enzymes remain intact in the gut lumen and frass and can therefore further catalyze the activation of plant defenses after ingestion, especially in pH-neutral regions of the digestive system. As most of the ingested enzymatic activity persists in the frass, and only particular β-glucosidases were detected via proteomic analyses, our data support the involvement of specific isoforms (maize ZmGlu1 and S. alba MA1 myrosinase) in defense in vivo.

Aggregation and Molecular Properties of β-Glucosidase Isoform II in Chayote (Sechium edule)

“…This suggests that the protein forms compact structures in some regions, which is a phenomenon described for other β-glucosidases for which we now have evidence as being in β-glucosidase II [20]. We think this property does not allow complete enzymatic digestion by trypsin [19], suggesting that it only hydrolyzes superficial peptides of the β-glucosidase II.…”

“…Of the reported amino acids 39% were non-polar and 61% polar. According to the data obtained, it is implied that the protein contains at least one-third of non-polar amino acids, as these amino acids have a strong tendency to associate with each other inside the protein promoting hydrophobic interactions [18,19]. This suggests that the protein forms compact structures in some regions, which is a phenomenon described for other β-glucosidases for which we now have evidence as being in β-glucosidase II [20].…”

“…Recent studies suggest that the BGAF does not protect β-glucosidase in the case of maize. Further studies are needed [19].…”

“…This is corroborated in line 3 by their presence in the same sample under standard conditions of SDS-PAGE in view of the high molecular weight component, at the top of the separating gel. This property is common in members GH1 family of β-glucosidase, particularly, in isoforms such as β-glucosidases from Zea mays (ZmGlu1) and Sinapis alba (MA1 myrosinase), which are also involved in the defense of plants in vivo [19].…”

“…Vassão et al describes the digestion resistance of three β-glucosidases in Zea mays and BGAF associated with this enzyme, myrosinase from Sinapis alba, and the β-glucosidase from Prunus dulcis (almonds). These proteins showed high resistance to the proteolytic enzymes from predator intestine (Spodoptera littoralis), maintaining the ability to catalyze the hydrolysis of glucosides [19]. However, there is no consensus on the belief that protein aggregates confer protection on the β-glucosidase.…”