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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.


Presence, production and consumption of 3-butenenitrile (19) and 2-butenenitrile (20) following incubation with homogenate or microsomes from A. petiolata. Homogenate (A) or microsomes (B) were incubated with or without addition of exogenous 3-butenenitrile or 2-butenenitrile (mixture of (Z)- and (E)-isomers), homogenate for 2 h and microsomes for 30 min. The content of 3-butenenitrile (green bars, m/z 67, 4.2 min) and 2-butenenitrile (blue striped bars, m/z 67, sum of isomers: 3.8+4.5 min) at the end of the incubation period was determined as the EIC peak area as obtained by SPME-GC-MS. The mean ± range of two technical replicates is depicted. For homogenate the incubated unspiked control is shown to evaluate whether endogenous sinigrin in this fraction led to an increase in the amounts of 3- or 2-butenenitrile. ND, not detected; t = 0, time point zero.
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Figure 5: Presence, production and consumption of 3-butenenitrile (19) and 2-butenenitrile (20) following incubation with homogenate or microsomes from A. petiolata. Homogenate (A) or microsomes (B) were incubated with or without addition of exogenous 3-butenenitrile or 2-butenenitrile (mixture of (Z)- and (E)-isomers), homogenate for 2 h and microsomes for 30 min. The content of 3-butenenitrile (green bars, m/z 67, 4.2 min) and 2-butenenitrile (blue striped bars, m/z 67, sum of isomers: 3.8+4.5 min) at the end of the incubation period was determined as the EIC peak area as obtained by SPME-GC-MS. The mean ± range of two technical replicates is depicted. For homogenate the incubated unspiked control is shown to evaluate whether endogenous sinigrin in this fraction led to an increase in the amounts of 3- or 2-butenenitrile. ND, not detected; t = 0, time point zero.

Mentions: GC-MS-based investigations were performed to identify potential intermediates in the conversion of sinigrin to alliarinoside. Allyl isothiocyanate (16), allyl thiocyanate (17), and 3,4-epithiobutanenitrile (18) were present in leaf homogenates and the amounts increased significantly following sinigrin addition, but also in this case with substantial biological variation in the amounts formed of the individual species (Figure S7). The simple nitrile (3-butenenitrile, 19) was not detectable at the conditions used, but using solid phase microextraction (SPME)-GC-MS we detected the presence of 3-butenenitrile in the leaf homogenate and 2-butenenitrile (20) in the microsomal fraction (Figure 5). As absorption onto the SPME fiber is not quantitative, no absolute measures could be calculated, and thus these samples were not spiked with sinigrin. When unspiked homogenate was incubated there was no evident increase in the concentration of 3-butenenitrile, the simple nitrile expected to be formed from the sinigrin present in this fraction (Figure 5A). This could be either because the increase was not sufficient to be detected by SPME-GC-MS, or because the nitrile was further metabolized. Due to the evident structural resemblance between the detected 2- and 3-butenenitrile and the alliarinoside aglucon (12) we tested these as potential intermediates in alliarinoside biosynthesis. In the microsomal fraction exogenously added 3-butenenitrile appeared to decrease substantially when NADPH was present, while 2-butenenitrile appeared to not be metabolized (Figure 5B). However, no formation of the alliarinoside aglucon or the (E)-isomer (13) was detected following addition of 3- or 2-butenenitrile to homogenate or microsomes supplemented with NADPH (Figure 2). Neither was 2-butenenitrile detected as a product from 3-butenenitrile metabolism (Figure 5). In contrast, 3-butenenitrile was found to be converted into 3-butenoic acid in homogenate and the microsomal fraction, which indicated the presence of nitrilase activity (Figure S8). Formation of the alternative nitrilase product, 3-butenamide, was not observed, but exogenously added 3-butenamide was metabolized into 3-butenoic acid by leaf homogenate indicating the presence of amidase activity. However, these conversions appear as unlikely events in sinigrin-derived alliarinoside formation.


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)

Presence, production and consumption of 3-butenenitrile (19) and 2-butenenitrile (20) following incubation with homogenate or microsomes from A. petiolata. Homogenate (A) or microsomes (B) were incubated with or without addition of exogenous 3-butenenitrile or 2-butenenitrile (mixture of (Z)- and (E)-isomers), homogenate for 2 h and microsomes for 30 min. The content of 3-butenenitrile (green bars, m/z 67, 4.2 min) and 2-butenenitrile (blue striped bars, m/z 67, sum of isomers: 3.8+4.5 min) at the end of the incubation period was determined as the EIC peak area as obtained by SPME-GC-MS. The mean ± range of two technical replicates is depicted. For homogenate the incubated unspiked control is shown to evaluate whether endogenous sinigrin in this fraction led to an increase in the amounts of 3- or 2-butenenitrile. ND, not detected; t = 0, time point zero.
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Related In: Results  -  Collection

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Figure 5: Presence, production and consumption of 3-butenenitrile (19) and 2-butenenitrile (20) following incubation with homogenate or microsomes from A. petiolata. Homogenate (A) or microsomes (B) were incubated with or without addition of exogenous 3-butenenitrile or 2-butenenitrile (mixture of (Z)- and (E)-isomers), homogenate for 2 h and microsomes for 30 min. The content of 3-butenenitrile (green bars, m/z 67, 4.2 min) and 2-butenenitrile (blue striped bars, m/z 67, sum of isomers: 3.8+4.5 min) at the end of the incubation period was determined as the EIC peak area as obtained by SPME-GC-MS. The mean ± range of two technical replicates is depicted. For homogenate the incubated unspiked control is shown to evaluate whether endogenous sinigrin in this fraction led to an increase in the amounts of 3- or 2-butenenitrile. ND, not detected; t = 0, time point zero.
Mentions: GC-MS-based investigations were performed to identify potential intermediates in the conversion of sinigrin to alliarinoside. Allyl isothiocyanate (16), allyl thiocyanate (17), and 3,4-epithiobutanenitrile (18) were present in leaf homogenates and the amounts increased significantly following sinigrin addition, but also in this case with substantial biological variation in the amounts formed of the individual species (Figure S7). The simple nitrile (3-butenenitrile, 19) was not detectable at the conditions used, but using solid phase microextraction (SPME)-GC-MS we detected the presence of 3-butenenitrile in the leaf homogenate and 2-butenenitrile (20) in the microsomal fraction (Figure 5). As absorption onto the SPME fiber is not quantitative, no absolute measures could be calculated, and thus these samples were not spiked with sinigrin. When unspiked homogenate was incubated there was no evident increase in the concentration of 3-butenenitrile, the simple nitrile expected to be formed from the sinigrin present in this fraction (Figure 5A). This could be either because the increase was not sufficient to be detected by SPME-GC-MS, or because the nitrile was further metabolized. Due to the evident structural resemblance between the detected 2- and 3-butenenitrile and the alliarinoside aglucon (12) we tested these as potential intermediates in alliarinoside biosynthesis. In the microsomal fraction exogenously added 3-butenenitrile appeared to decrease substantially when NADPH was present, while 2-butenenitrile appeared to not be metabolized (Figure 5B). However, no formation of the alliarinoside aglucon or the (E)-isomer (13) was detected following addition of 3- or 2-butenenitrile to homogenate or microsomes supplemented with NADPH (Figure 2). Neither was 2-butenenitrile detected as a product from 3-butenenitrile metabolism (Figure 5). In contrast, 3-butenenitrile was found to be converted into 3-butenoic acid in homogenate and the microsomal fraction, which indicated the presence of nitrilase activity (Figure S8). Formation of the alternative nitrilase product, 3-butenamide, was not observed, but exogenously added 3-butenamide was metabolized into 3-butenoic acid by leaf homogenate indicating the presence of amidase activity. However, these conversions appear as unlikely events in sinigrin-derived alliarinoside formation.

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.