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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.

Frisch T, Motawia MS, Olsen CE, Agerbirk N, Møller BL, Bjarnholt N - Front Plant Sci (2015)

Bottom Line: Hydroxynitrile glucosides and glucosinolates are two classes of specialized metabolites, which generally do not occur in the same plant species.Hence, the alliarinoside pathway may represent a route to hydroxynitrile glucoside biosynthesis resulting from convergent evolution.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.

View Article: PubMed Central - PubMed

Affiliation: Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen Copenhagen, Denmark ; VILLUM Research Center for Plant Plasticity, Department of Plant and Environmental Sciences, University of Copenhagen Copenhagen, Denmark.

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-(14)C]-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

The diffusible HCN response from sinigrin and organic/inorganic thiocyanate is dependent on the presence of A. petiolata enzymes. (A) Colorimetric HCN detection based on the König reaction was used to quantify the diffusible HCN response in homogenates of A. petiolata leaves. The effect of spiking the homogenate with alliarinoside (14), sinigrin (15) or available glucosinolate degradation products was quantified relative to the untreated samples (n = 4, except alliarinoside: n = 2). The relative diffusible response was significantly increased by addition of sinigrin, benzyl thiocyanate (**P < 0.01) or KSCN (*P < 0.05), whereas other compounds had no significant effect (one-tailed unpaired t-test of log10-transformed ratios). The back-transformed mean ± SD is depicted. (B) Feigl-Anger paper, a different colorimetric method for HCN detection, was used to confirm the effect of KSCN on the diffusible response from leaf homogenates of A. petiolata. Results of triplicates are shown. As positive control, highly cyanogenic Lotus japonicus leaf material was used (4 mg FW sample−1). Larger amounts of leaf material were applied to obtain a detectable response from A. petiolata (60 mg FW sample−1). There was a tendency to more intense color development in A. petiolata samples added KSCN.
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Figure 7: The diffusible HCN response from sinigrin and organic/inorganic thiocyanate is dependent on the presence of A. petiolata enzymes. (A) Colorimetric HCN detection based on the König reaction was used to quantify the diffusible HCN response in homogenates of A. petiolata leaves. The effect of spiking the homogenate with alliarinoside (14), sinigrin (15) or available glucosinolate degradation products was quantified relative to the untreated samples (n = 4, except alliarinoside: n = 2). The relative diffusible response was significantly increased by addition of sinigrin, benzyl thiocyanate (**P < 0.01) or KSCN (*P < 0.05), whereas other compounds had no significant effect (one-tailed unpaired t-test of log10-transformed ratios). The back-transformed mean ± SD is depicted. (B) Feigl-Anger paper, a different colorimetric method for HCN detection, was used to confirm the effect of KSCN on the diffusible response from leaf homogenates of A. petiolata. Results of triplicates are shown. As positive control, highly cyanogenic Lotus japonicus leaf material was used (4 mg FW sample−1). Larger amounts of leaf material were applied to obtain a detectable response from A. petiolata (60 mg FW sample−1). There was a tendency to more intense color development in A. petiolata samples added KSCN.

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.

Frisch T, Motawia MS, Olsen CE, Agerbirk N, Møller BL, Bjarnholt N - Front Plant Sci (2015)

The diffusible HCN response from sinigrin and organic/inorganic thiocyanate is dependent on the presence of A. petiolata enzymes. (A) Colorimetric HCN detection based on the König reaction was used to quantify the diffusible HCN response in homogenates of A. petiolata leaves. The effect of spiking the homogenate with alliarinoside (14), sinigrin (15) or available glucosinolate degradation products was quantified relative to the untreated samples (n = 4, except alliarinoside: n = 2). The relative diffusible response was significantly increased by addition of sinigrin, benzyl thiocyanate (**P < 0.01) or KSCN (*P < 0.05), whereas other compounds had no significant effect (one-tailed unpaired t-test of log10-transformed ratios). The back-transformed mean ± SD is depicted. (B) Feigl-Anger paper, a different colorimetric method for HCN detection, was used to confirm the effect of KSCN on the diffusible response from leaf homogenates of A. petiolata. Results of triplicates are shown. As positive control, highly cyanogenic Lotus japonicus leaf material was used (4 mg FW sample−1). Larger amounts of leaf material were applied to obtain a detectable response from A. petiolata (60 mg FW sample−1). There was a tendency to more intense color development in A. petiolata samples added KSCN.
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Figure 7: The diffusible HCN response from sinigrin and organic/inorganic thiocyanate is dependent on the presence of A. petiolata enzymes. (A) Colorimetric HCN detection based on the König reaction was used to quantify the diffusible HCN response in homogenates of A. petiolata leaves. The effect of spiking the homogenate with alliarinoside (14), sinigrin (15) or available glucosinolate degradation products was quantified relative to the untreated samples (n = 4, except alliarinoside: n = 2). The relative diffusible response was significantly increased by addition of sinigrin, benzyl thiocyanate (**P < 0.01) or KSCN (*P < 0.05), whereas other compounds had no significant effect (one-tailed unpaired t-test of log10-transformed ratios). The back-transformed mean ± SD is depicted. (B) Feigl-Anger paper, a different colorimetric method for HCN detection, was used to confirm the effect of KSCN on the diffusible response from leaf homogenates of A. petiolata. Results of triplicates are shown. As positive control, highly cyanogenic Lotus japonicus leaf material was used (4 mg FW sample−1). Larger amounts of leaf material were applied to obtain a detectable response from A. petiolata (60 mg FW sample−1). There was a tendency to more intense color development in A. petiolata samples added KSCN.
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.

Bottom Line: Hydroxynitrile glucosides and glucosinolates are two classes of specialized metabolites, which generally do not occur in the same plant species.Hence, the alliarinoside pathway may represent a route to hydroxynitrile glucoside biosynthesis resulting from convergent evolution.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.

View Article: PubMed Central - PubMed

Affiliation: Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen Copenhagen, Denmark ; VILLUM Research Center for Plant Plasticity, Department of Plant and Environmental Sciences, University of Copenhagen Copenhagen, Denmark.

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-(14)C]-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