Lotus japonicus flowers are defended by a cyanogenic β-glucosidase with highly restricted expression to essential reproductive organs

Abstract

Flowers and leaves of Lotus japonicus contain α-, β-, and γ-hydroxynitrile glucoside (HNG) defense compounds, which are bioactivated by β-glucosidase enzymes (BGDs). The α-HNGs are referred to as cyanogenic glucosides because their hydrolysis upon tissue disruption leads to release of toxic hydrogen cyanide gas, which can deter herbivore feeding. BGD2 and BGD4 are HNG metabolizing BGD enzymes expressed in leaves. Only BGD2 is able to hydrolyse the α-HNGs. Loss of function mutants of BGD2 are acyanogenic in leaves but fully retain cyanogenesis in flowers pointing to the existence of an alternative cyanogenic BGD in flowers. This enzyme, named BGD3, is identified and characterized in this study. Whereas all floral tissues contain α-HNGs, only those tissues in which BGD3 is expressed, the keel and the enclosed reproductive organs, are cyanogenic. Biochemical analysis, active site architecture molecular modelling, and the observation that L. japonicus accessions lacking cyanogenic flowers contain a non-functional BGD3 gene, all support the key role of BGD3 in floral cyanogenesis. The nectar of L. japonicus flowers was also found to contain HNGs and additionally their diglycosides. The observed specialisation in HNG based defence in L. japonicus flowers is discussed in the context of balancing the attraction of pollinators with the protection of reproductive structures against herbivores.

Cyanogenic Glucosides and Derivatives in Almond and Sweet Cherry Flower Buds from Dormancy to Flowering

“…In the case of Lotus japonicus , the two aliphatic CNglcs linamarin and lotaustralin are present throughout in the flower tissue (Lai et al, 2015). As mentioned previously ( Figure 1B ), bioactivation of the CNglc takes place only when specific β-glucosidases come into contact with their corresponding substrate.…”

“…As mentioned previously ( Figure 1B ), bioactivation of the CNglc takes place only when specific β-glucosidases come into contact with their corresponding substrate. In L. japonicus the reproductive organs are only cyanogenic when a specific β-glucosidase BGD3 is expressed (Lai et al, 2015). Hydrogen cyanide release was derived specifically from the keel and enclosed reproductive organs of the flower.…”

“…Hydrogen cyanide release was derived specifically from the keel and enclosed reproductive organs of the flower. Sepals, wings, buds, and pods also contained the cyanogenic glucosides linamarin and lotaustralin, but no release of any hydrogen cyanide from these tissues was observed because the β-glucosidases were not present in these tissues (Lai et al, 2015). It needs to be investigated whether or not a particular β-glucosidase might also be expressed in almond and cherry flower buds.…”

Sequestration and biosynthesis of cyanogenic glucosides in passion vine butterflies and consequences for the diversification of their host plants

“…Samples collected in the field were homogenized in the solution where they were soaked: 4 ml 80% (v/v) methanol. A boiling extraction method (Lai et al, ) was used for the plant samples, where leaf pieces of each species were added to microtubes containing 0.5 ml 85% (v/v) methanol and boiled for 5 min in a water bath. All samples were subsequently centrifuged at 10,000 g for 5 min and the supernatant filtered (Anapore 0.45 µm, Whatman) to remove insoluble components.…”

“…Samples collected in the field were homogenized in the solution where they were soaked: 4 ml 80% (v/v) methanol. A boiling extraction method (Lai et al, 2015) was used for the plant samples,…”