Abstract
Alliaria petiolata (garlic mustard, Brassicaceae) contains the glucosinolate sinigrin as well as alliarinoside, a γ-hydroxynitrile glucoside structurally related to cyanogenic glucosides. Sinigrin may defend this plant against a broad range of enemies, while alliarinoside confers resistance to specialized (glucosinolate-adapted) herbivores. Hydroxynitrile glucosides and glucosinolates are two classes of specialized metabolites, which generally do not occur in the same plant species. Administration of [UL-14C]-methionine to excised leaves of A. petiolata showed that both alliarinoside and sinigrin were biosynthesized from methionine. The biosynthesis of alliarinoside was shown not to bifurcate from sinigrin biosynthesis at the oxime level in contrast to the general scheme for hydroxynitrile glucoside biosynthesis. Instead, the aglucon of alliarinoside was formed from metabolism of sinigrin in experiments with crude extracts, suggesting a possible biosynthetic pathway in intact cells. Hence, the alliarinoside pathway may represent a route to hydroxynitrile glucoside biosynthesis resulting from convergent evolution. Metabolite profiling by LC-MS showed no evidence of the presence of cyanogenic glucosides in A. petiolata. However, we detected hydrogen cyanide (HCN) release from sinigrin and added thiocyanate ion and benzyl thiocyanate in A. petiolata indicating an enzymatic pathway from glucosinolates via allyl thiocyanate and indole glucosinolate derived thiocyanate ion to HCN. Alliarinoside biosynthesis and HCN release from glucosinolate-derived metabolites expand the range of glucosinolate-related defenses and can be viewed as a third line of defense, with glucosinolates and thiocyanate forming protein being the first and second lines, respectively.
Highlights
Cyanogenesis, defined as hydrogen cyanide (HCN) release, occurs from plants following β-glucosidase-catalyzed hydrolysis of cyanogenic glucosides (Figure 1A) (Morant et al, 2008; Gleadow and Møller, 2014)
Based on the chemical structure of alliarinoside (14), we hypothesized that this γ-hydroxynitrile glucoside is biosynthesized from methionine (1) via a pathway sharing the initial reactions of aliphatic chain elongation and oxime formation with the biosynthesis of sinigrin (15), a homomethionine-derived glucosinolate (Frisch and Møller, 2012) (Figure 2)
Our results suggest that the biosynthesis of alliarinoside and sinigrin in A. petiolata does not bifurcate at the level of the homomethionine-derived oxime (3) (Figure 2), meaning that CYP83 and CYP71E1 type enzymes do not co-exist in this plant
Summary
Cyanogenesis, defined as HCN release, occurs from plants following β-glucosidase-catalyzed hydrolysis of cyanogenic glucosides (Figure 1A) (Morant et al, 2008; Gleadow and Møller, 2014). Disrupted compartmentalization and subsequent hydrolysis, which release toxic degradation products, is caused by tissue-damaging herbivores (Pentzold et al, 2013) or possibly by regulated physiological mechanisms in intact tissues Evidence of such turnover of glucosinolates and hydroxynitrile glucosides in intact tissues indicate roles in defense against microbial pathogens (Bednarek et al, 2009; Clay et al, 2009; Møller, 2010). The biosynthetic studies reported here demonstrate that HCN release is derived from metabolism of sinigrin while alliarinoside is derived from methionine and may be derived from sinigrin turnover in intact tissue This adds another dimension of complexity to the diverse roles of glucosinolate-derived products in plant defense
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