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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

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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.

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The glucosinolate-derived diffusible HCN response is not general to the Brassicaceae. A. thaliana and B. juncea were investigated for their ability to provide a diffusible HCN response from endogenous glucosinolates or from exogenously added KSCN. The response in the König reaction-based diffusion assay was quantified relative to the mean of unspiked A. petiolata samples and a one-tailed unpaired t-test was performed on the log10-transformed ratios. Bars represent back-transformed mean ± SD. (**P < 0.01; n = 4).
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Figure 8: The glucosinolate-derived diffusible HCN response is not general to the Brassicaceae. A. thaliana and B. juncea were investigated for their ability to provide a diffusible HCN response from endogenous glucosinolates or from exogenously added KSCN. The response in the König reaction-based diffusion assay was quantified relative to the mean of unspiked A. petiolata samples and a one-tailed unpaired t-test was performed on the log10-transformed ratios. Bars represent back-transformed mean ± SD. (**P < 0.01; n = 4).

Mentions: Proposed biosynthetic origin of alliarinoside and HCN in A. petiolata. All illustrated metabolites were detected in either homogenate or microsomes prepared from A. petiolata leaves. Bold arrows signify biosynthesis demonstrated in planta, plain solid arrows signify reactions demonstrated in vitro, and dotted arrows indicate proposed reactions based on these reactions and other results obtained in the current study. The alliarinoside aglucon (12) is presumed to be produced by only one of the proposed routes via either 3,4-epithiobutanenitrile (18) or 3-butenenitrile (19). For further details on tested reactions, occurrence of compounds and abbreviations refer to Figures 2–5 regarding alliarinoside and Figures 7, 8 regarding HCN.


Diversified glucosinolate metabolism: biosynthesis of hydrogen cyanide and of the hydroxynitrile glucoside alliarinoside in relation to sinigrin metabolism in Alliaria petiolata
The glucosinolate-derived diffusible HCN response is not general to the Brassicaceae. A. thaliana and B. juncea were investigated for their ability to provide a diffusible HCN response from endogenous glucosinolates or from exogenously added KSCN. The response in the König reaction-based diffusion assay was quantified relative to the mean of unspiked A. petiolata samples and a one-tailed unpaired t-test was performed on the log10-transformed ratios. Bars represent back-transformed mean ± SD. (**P < 0.01; n = 4).
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Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4628127&req=5

Figure 8: The glucosinolate-derived diffusible HCN response is not general to the Brassicaceae. A. thaliana and B. juncea were investigated for their ability to provide a diffusible HCN response from endogenous glucosinolates or from exogenously added KSCN. The response in the König reaction-based diffusion assay was quantified relative to the mean of unspiked A. petiolata samples and a one-tailed unpaired t-test was performed on the log10-transformed ratios. Bars represent back-transformed mean ± SD. (**P < 0.01; n = 4).
Mentions: Proposed biosynthetic origin of alliarinoside and HCN in A. petiolata. All illustrated metabolites were detected in either homogenate or microsomes prepared from A. petiolata leaves. Bold arrows signify biosynthesis demonstrated in planta, plain solid arrows signify reactions demonstrated in vitro, and dotted arrows indicate proposed reactions based on these reactions and other results obtained in the current study. The alliarinoside aglucon (12) is presumed to be produced by only one of the proposed routes via either 3,4-epithiobutanenitrile (18) or 3-butenenitrile (19). For further details on tested reactions, occurrence of compounds and abbreviations refer to Figures 2–5 regarding alliarinoside and Figures 7, 8 regarding HCN.

View Article: PubMed Central

ABSTRACT

Alliaria petiolata (garlic mustard, Brassicaceae) contains the glucosinolate sinigrin as well as alliarinoside, a &gamma;-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