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Diversified glucosinolate metabolism: biosynthesis of hydrogen cyanide and of the hydroxynitrile glucoside alliarinoside in relation to sinigrin metabolism in Alliaria petiolata

View Article: PubMed Central

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.

No MeSH data available.


Related in: MedlinePlus

Biosynthetic experiments documenting that alliarinoside (14) is methionine (1)-derived. [UL-14C]-methionine was administered to A. petiolata leaves and formation of radiolabelled products was monitored by TLC using two different solvent systems to separate hydroxynitrile glucosides (A) and glucosinolates (B), respectively. Representative results obtained with a single leaf are shown. The negatively charged sulfate group of sinigrin is prone to interact with the matrix and consequently cause different migration of sinigrin in the plant extract compared to an external standard. To more accurately determine the migration of alliarinoside and sinigrin in the plant extract, unlabeled internal standards were added to aliquots of leaf extract before samples were applied to the TLC plates (left panels). The spot showing increased staining following charring of the TLC plate indicated the position of the internal standard. O, origin; F, MeOH focus line.
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Figure 3: Biosynthetic experiments documenting that alliarinoside (14) is methionine (1)-derived. [UL-14C]-methionine was administered to A. petiolata leaves and formation of radiolabelled products was monitored by TLC using two different solvent systems to separate hydroxynitrile glucosides (A) and glucosinolates (B), respectively. Representative results obtained with a single leaf are shown. The negatively charged sulfate group of sinigrin is prone to interact with the matrix and consequently cause different migration of sinigrin in the plant extract compared to an external standard. To more accurately determine the migration of alliarinoside and sinigrin in the plant extract, unlabeled internal standards were added to aliquots of leaf extract before samples were applied to the TLC plates (left panels). The spot showing increased staining following charring of the TLC plate indicated the position of the internal standard. O, origin; F, MeOH focus line.

Mentions: Fortunately, a small amount of [UL-14C]-methionine was later obtained enabling us to test directly whether methionine is the amino acid precursor of alliarinoside. [UL-14C]-Methionine was administered to excised A. petiolata leaves through the petiole. After 24 h incubation in the light, methanol extracts of each leaf were analyzed by TLC (Figure 3). As observed in the experiments with [UL-14C]-aspartate, administration of [UL-14C]-methionine resulted in strong labeling of alliarinoside. Simultaneous strong labeling of sinigrin, a known methionine-derived glucosinolate, demonstrates that the [UL-14C]-methionine administered was taken up and efficiently incorporated in the sinigrin biosynthesis of the leaves.


Diversified glucosinolate metabolism: biosynthesis of hydrogen cyanide and of the hydroxynitrile glucoside alliarinoside in relation to sinigrin metabolism in Alliaria petiolata
Biosynthetic experiments documenting that alliarinoside (14) is methionine (1)-derived. [UL-14C]-methionine was administered to A. petiolata leaves and formation of radiolabelled products was monitored by TLC using two different solvent systems to separate hydroxynitrile glucosides (A) and glucosinolates (B), respectively. Representative results obtained with a single leaf are shown. The negatively charged sulfate group of sinigrin is prone to interact with the matrix and consequently cause different migration of sinigrin in the plant extract compared to an external standard. To more accurately determine the migration of alliarinoside and sinigrin in the plant extract, unlabeled internal standards were added to aliquots of leaf extract before samples were applied to the TLC plates (left panels). The spot showing increased staining following charring of the TLC plate indicated the position of the internal standard. O, origin; F, MeOH focus line.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4628127&req=5

Figure 3: Biosynthetic experiments documenting that alliarinoside (14) is methionine (1)-derived. [UL-14C]-methionine was administered to A. petiolata leaves and formation of radiolabelled products was monitored by TLC using two different solvent systems to separate hydroxynitrile glucosides (A) and glucosinolates (B), respectively. Representative results obtained with a single leaf are shown. The negatively charged sulfate group of sinigrin is prone to interact with the matrix and consequently cause different migration of sinigrin in the plant extract compared to an external standard. To more accurately determine the migration of alliarinoside and sinigrin in the plant extract, unlabeled internal standards were added to aliquots of leaf extract before samples were applied to the TLC plates (left panels). The spot showing increased staining following charring of the TLC plate indicated the position of the internal standard. O, origin; F, MeOH focus line.
Mentions: Fortunately, a small amount of [UL-14C]-methionine was later obtained enabling us to test directly whether methionine is the amino acid precursor of alliarinoside. [UL-14C]-Methionine was administered to excised A. petiolata leaves through the petiole. After 24 h incubation in the light, methanol extracts of each leaf were analyzed by TLC (Figure 3). As observed in the experiments with [UL-14C]-aspartate, administration of [UL-14C]-methionine resulted in strong labeling of alliarinoside. Simultaneous strong labeling of sinigrin, a known methionine-derived glucosinolate, demonstrates that the [UL-14C]-methionine administered was taken up and efficiently incorporated in the sinigrin biosynthesis of the leaves.

View Article: PubMed Central

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.

No MeSH data available.


Related in: MedlinePlus