Cyanide in the Chemical Arsenal of Garlic Mustard, Alliaria petiolata

Article (PDF Available)inJournal of Chemical Ecology 33(1):85-94 · February 2007with191 Reads
DOI: 10.1007/s10886-006-9205-x · Source: PubMed
Abstract
Cyanide production has been reported from over 2500 plant species, including some members of the Brassicaceae. We report that the important invasive plant, Alliaria petiolata, produces levels of cyanide in its tissues that can reach 100 ppm fresh weight (FW), a level considered toxic to many vertebrates. In a comparative study, levels of cyanide in leaves of young first-year plants were 25 times higher than in leaves of young Arabidopsis thaliana plants and over 150 times higher than in leaves of young Brassica kaber, B. rapa, and B. napus. In first-year plants, cyanide levels were highest in young leaves of seedlings and declined with leaf age on individual plants. Leaves of young plants infested with green peach aphids (Myzus persicae) produced just over half as much cyanide as leaves of healthy plants, suggesting that aphid feeding led to loss of cyanide from intact tissues before analysis, or that aphid feeding inhibited cyanide precursor production. In a developmental study, levels of cyanide in the youngest and oldest leaf of young garlic mustard plants were four times lower than in the youngest and oldest leaf of young Sorghum sudanense (cv. Cadan 97) plants, but cyanide levels did not decline in these leaves with plant age as in S. sudanense. Different populations of garlic mustard varied moderately in the constitutive and inducible expression of cyanide in leaves, but no populations studied were acyanogenic. Although cyanide production could result from breakdown products of glucosinolates, no cyanide was detected in vitro from decomposition of sinigrin, the major glucosinolate of garlic mustard. These studies indicate that cyanide produced from an as yet unidentified cyanogenic compound is a part of the battery of chemical defenses expressed by garlic mustard.

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Cyanide in the Chemical Arsenal of Garlic Mustard,
Alliaria petiolata
Don Cipollini & Bill Gruner
Received: 3 July 2006 / Revised: 30 August 2006 / Accepted: 18 September 2006 /
Published online: 5 December 2006
#
Springer Science + Business Media, Inc. 2006
Abstract Cyanide production has been reported from over 2500 plant species, including
some members of the Brassicaceae. We report that the important invasive plant, Alliaria
petiolata, produces levels of cyanide in its tissues that can reach 100 ppm fresh weight
(FW), a level considered toxic to many vertebrates. In a comparative study, levels of
cyanide in leaves of young first-year plants were 25 times higher than in leaves of young
Arabidopsis thaliana plants and over 150 times higher than in leaves of young Brassica
kaber, B. rapa, and B. napus. In first-year plants, cyanide levels were highest in young
leaves of seedlings and declined with leaf age on individual plants. Leaves of young plants
infested with green peach aphids (Myzus persicae) produced just over half as much cyanide
as leaves of healthy plants, suggesting that aphid feeding led to loss of cyanide from intact
tissues before analysis, or that aphid feeding inhibited cyanide precursor production. In a
developmental study, levels of cyanide in the youngest and oldest leaf of young garlic
mustard plants were four times lower than in the youngest and oldest leaf of young
Sorghum sudanense (cv. Cadan 97) plants, but cyanide levels did not decline in these leaves
with plant age as in S. sudanense. Different populations of garlic mustard varied moderately
in the constitutive and inducible expression of cyanide in leaves, but no populations studied
were acyanogenic. Although cyanide production could result from breakdown products of
glucosinolates, no cyanide was detected in vitro from decomposition of sinigrin, the major
glucosinolate of garlic mustard. These studies indicate that cyanide produced from an as yet
unidentified cyanogenic compound is a part of the battery of chemical defenses expressed
by garlic mustard.
Keywords Allelopathy
.
Alliaria petiolata
.
Cyanide
.
Invasive plants
.
Plant defenses
.
Sorghum sudanense
Introduction
Cyanide is a well-known inhibitor of respiratory electron transport that is produced by over
2500 plant species including many food plants (Jones 1998). Although substantial debate
J Chem Ecol (2007) 33:8594
DOI 10.1007/s10886-006-9205-x
D. Cipollini (*)
:
B. Gruner
Department of Biological Sciences, Wright State University, 3640 Colonel Glenn Highway, Dayton,
Ohio 45435, USA
e-mail: don.cipollini@wright.edu
exists on the role of cyanogenic compounds and cyanide in plants (Jones 1998; Gleadow
and Woodrow, 2002), cyanide production has been invoked as an herbivore- and pathogen-
resistance mechanism in many plants including clover, black cherry, and sorghum (Lindroth
et al., 2000; Busk and Møller, 2002; Fitzgerald et al., 2002; Gleadow and Woodrow, 2002).
In addition, cyanogenic compounds in plants like cassava appear to have roles in storage
and mobilization of nitrogen (Gleadow and Woodrow, 2002; Jorgensen et al., 2005).
Cyanide production in each of the aforementioned species typically reaches levels that can
impact animal or human health (Jones 1998). Cyanide production has been reported in
some members of the Brassicaceae, including Brassica oleraceae, B. juncea, B. rapa, B.
napus, and Raphanus sativus (Sexton and Howlett, 2000; Chandra et al., 2004), but levels
found thus far in plants in this family have been minimal and generally well below those
considered to affect plant or animal health. In turn, the source of the detectable cyanide in
brassicaceous plants i s unknown, although the decomposition of glucosinolates, a
ubiquitous group of defensive secondary compounds in this family, is one candidate
(Sexton and Howlett, 2000).
Garlic mustard Alliaria petiolata [(M. Bieb) Cavara and Grande; Brassicaceae] is a
Eurasian biennial herb that is an important invasive plant in North America. This plant has
several known or presumed impacts on the flora and fauna in its invaded range that are
apparently chemically mediated. These include allelopathic interactions with plants (Prati
and Bossdorf, 2004), mycorrhizal fungi (Roberts and Anderson, 2001; Stinson et al., 2006),
and herbivorous insects (Porter 1994; Haribal and Renwick, 1998; Haribal et al., 2001).
Garlic mustard is also grazed little by mammalian or avian herbivores (D. Cipollini,
personal observation). Like all brassicaceous plants, garlic mustard produces glucosinolates
and defense proteins in both a constitutive and inducible fashion (Vaughan and Berhow,
1999; Cipollini et al., 2005 ). Garlic mustard is also rich in flavone gycosides, including
isovitexin 6-O-β-
D-glucopyranoside, and it produces a novel cyanoallyl glycoside,
alliarinoside; both have demonstrable effects on insect resistance (Haribal and Renwick,
1998; Haribal et al., 2001; Cipollini et al., 2005). It is likely that this suite of defenses
contributes to the invasive success of this plant in novel habitats.
In this paper, we report that garlic mustard produces substantial quantities of cyanide
from its tissues, an as yet undescribed component of the chemical arsenal of this plant. We
first examined cyanide production in leaves and roots of young plants from a single
population of garlic mustard, and then compared levels found in shoots to those of several
members of the Brassicaceae. Second, we compared temporal variation in cyanide production
in the youngest and oldest leaves of garlic mustard to that of a known cyanide producer,
Sorghum sudanense (Sudan grass). Third, we examined leaf age-specific cyanide
production in garlic mustard in the presence and absence of aphid herbivory. Fourth, we
examined variation among garlic mustard populations in constitutive and inducible cyanide
production. Finally, we examined whether the decomposition of sinigrin, the primary
glucosinolate in garlic mustard (Vaughan and Berhow, 1999), could be the source of
cyanide produced by this plant.
Methods and Materials
Experiment 1: Demonstration of Cyanide in Roots and Shoots of Garlic Mustard Seeds of
garlic mustard were collected from plants growing naturally in the Wright State University
forest preserve, cold stratified, and grown in the greenhouse in Pro Mix BX potting
86 J Chem Ecol (2007) 33:8594
medium, as described by Cipollini et al. (2005). Most experiments were conducted with the
Wright State population, although experiment 5 includes several other populations. In this
experiment, four plants were grown for 3 wk at which time all stems, leaves, and petioles
present on each plant were collected and pooled for cyanide analysis, as described below.
Roots of each plant were carefully extracted from the soil at the same time and rinsed free
of soil with distilled water before cyanide analysis. Cyanide production by above-ground
tissues was compared to that of roots with one-way analysis of variance (ANOVA) on SAS
(version 9.1). Means were compared using Tukeys test in this and all other experiments.
Experiment 2: Comparisons of Cyanide Production in Leaves of Garlic Mustard with Other
Brassicaceous Plants Seedlings of Arabidopsis thaliana (ecotype Columbia), Brassica
kaber (wild collected), B. rapa (wild collected), and B. napus (cv. Westar), were grown
from seed as described above, but without cold stratification. Garlic mustard was grown
from cold-stratified seed as described above. All leaf material present on five 3-wk-old
plants of each species was collected and pooled for analysis of cyanide. Cyanide production
was compared among species with one-way ANOVA.
Experiment 3: Temporal Variation in Cyanide Production in Leaves of Garlic Mustard in
Comparison to Sudan Grass This experiment was designed to compare variation in
cyanide production in garlic mustard to a known cyanide-producing plant. Garlic mustard
and Sudan grass [S. sudanense (cv. Cadan 97)] were grown from seed individually in pots
in Pro Mix BX, as described above. Starting 2 wk after germination, the youngest fully
expanded leaf and the oldest fully expanded leaf were collected from five plants of each
species for cyanide analysis. Harvests continued in this manner from a subset of previously
unsampled plants once per week for the next 8 wk, although the oldest leaves on each plant
could be sampled for only 4 wk until they were too senescent to sample. This collection
scheme isolates the effect of plant age on cyanide production in leaves of two distinct
developmental stages. Cyanide production through time was compared between garlic
mustard and Sudan grass with repeated-measures ANOVA, with time as the within-subject
effect and species as the between-subjects effect. As the youngest leaves on each plant were
sampled for 8 wk, and the oldest for only 4 wk, cyanide production was statistically
examined separately for the youngest and oldest leaves.
Experiment 4: Leaf-specific Cyanide Production in the Presence of Aphid Herbivory This
experiment examined within-plant variation in cyanide production among leaves in garlic
mustard, but the plants were impacted by an outbreak of green peach aphids (Myzus persicae)
in our greenhouse. When plants grown as described above were 40-d-old, we classified
subsets of them as noninfested or infested (2030 aphids per plant). Infested plants were
affected by aphids for approximately 2 wk at the time they were sampled. Fully expanded
third (old), fourth (intermediate), and fifth (young) true leaves of garlic mustard plants were
collected from 1013 plants in each aphid infestation group for analysis of cyanide.
Cyanide production was compared among aphid exposures and leaf ages by using two-way
ANOVA, with aphid exposure and leaf age nested within aphid exposures as effects.
Experiment 5: Population Variation in Constitutive and Salicylic Acid-induced Levels of
Cyanide Seeds of five populations of garlic mustard collected in the wild from Dalsland,
Sweden; Marmontweg and Elderveld, the Netherlands; Hanau, Germany; and Ligonier,
Pennsylvania, were cold-stratified and grown as described above. When plants were 8 wk
J Chem Ecol (2007) 33:8594 87
old, five plants from each population were sprayed with a 0.05-M solution of salicylic acid,
as described by Cipollini et al. (2004). Salicylic acid induces many defense-related genes in
plants (Mauch-Mani and Metraux, 1998), but its effect on cyanide production has never
been examined. Leaf material was collected from the fully expanded fourth true leaf of each
of these plants along with each of five control plants. Cyanide production was compared
among populations and salicylate treatments with two-way ANOVA.
Experiment 6: Assessment of Sinigrin as a Source of Cyanide In this experiment, we
evaluated the ability of sinigrin, the primary glucosinolate in garlic mustard (Vaughn and
Berhow, 1999), to liberate cyanide upon enzymatic decomposition with thioglucosidase.
We incubated 0.1 ml of sinigrin (Sigma Chemical, St. Louis, MO, USA) at concentrations
of 0.125, 0.25, 0.5, 1.0, and 2.0 mg/ml with 0.1 ml of 3 mg/ml thioglucosidase (E.C.
3.2.3.1 from Sinapis alba; Sigma Chemical) suspended in sodium phosphate buffer in
25 ml center-well incubation flasks, and followed the protocol for cyanide trapping and
analysis described below.
Cyanide Analysis We analyzed endogenous cyanide production in our tissues using a
method modified from Lambert et al. (1975). Briefly, a weighed amount of fresh plant
material (0.020.05 g depending on tissue and species) was ground in a cold mortar with
0.5 ml of ice-cold 0.1-M sodium phosphate buffer. The homogenate was quickly transferred
to a 25-ml center-well incubation flask, and the mortar was rinsed into the flask with an
additional 0.5 ml of phosphate buffer. To trap cyanide in the flask, 0.2 ml of 0.1 M NaOH
was placed in the center well, and the flasks were sealed and incubated at room temperature
for 18 hr. To quantify cyanide present in the trap solution, 0.1 ml of 1 M acetic acid were
added to the 0.2-ml trap solution (which was diluted as needed), followed by 1 ml of 0.25%
succinimide/0.025% N-chlorosuccinimide reagent and 0.2 ml of 3% barbituric acid
pyridine solution. After incubation at room temperature for 10 min, absorbance at 580 nm
was recorded for each sample in a spectrophotometer. Cyanide levels were quantified
relative to a standard curve made with potassium cyanide and are expressed as milligram
cyanide per kilogram tissue fresh weight (=ppm FW).
Results
Experiment 1 Three-week-old garlic mustard plants produced a mean level of 44.4
(±2.17) ppm cyanide from their aboveground tissues (including stems, leaves, and petioles)
and 5.4 (±2.13) ppm from their roots. These levels were significantly different from one
another (F
1, 6
=212.5, P<0.001).
Experiment 2 Leaf cyanide production varied among the five brassicaceous species
examined (F
4, 14
=526.0, P<0.001, Table 1). Levels of cyanide in leaves of 3-wk-old
garlic mustard plants were 25 times higher than in leaves of 3-wk-old A. thaliana plants and
over 150 times higher than in leaves of 3-wk-old B. kaber, B. rapa, and B. napus plants.
Experiment 3 Changes in cyanide production through time by the youngest fully expanded
leaf varied between garlic mustard and Sudan grass (time × species: F
7, 2
=54.02, P=0.018,
Fig. 1). Cyanide production in the youngest leaf of Sudan grass was five times higher than
88 J Chem Ecol (2007) 33:8594
in garlic mustard on the first sampling date, and it steadily declined to minimal levels
through the 8-wk-time course as plants aged. In contrast, cyanide production declined little
through time in garlic mustard, and by the fifth week of the time course through the end,
cyanide levels in the youngest leaves of garlic mustard plants were significantly higher than
in Sudan grass. A similar pattern was observed in cyanide production in the oldest fully
expanded leaf of garlic mustard and Sudan grass through the 4-wk-time course (time ×
species: F
3, 6
=150.54, P<0.001, Fig. 2). The decline in cyanide production in the oldest
leaves in Sudan grass through time was even more dramatic than in the youngest leaves.
Experiment 4 Cyanide production varied among aphid-infested and noninfested plants
(F
1, 64
=25.56, P<0.001) and among leaf ages within each aphid treatment (F
4, 64
=10.72, P<
0.001, Table 2). Cyanide production was higher in young leaves than in older leaves in
general and was lower in aphid-infested plants across all leaf ages.
Experiment 5 Significant variation in constitutive and salicylic acid (SA)-inducible cyanide
production in leaves of 8-wk-old garlic mustard plants was detected among populations
(pop: F
4, 40
=4.21, P=0.006; pop × SA: F
4, 40
=8.4, P<0.001, Table 3). Constitutive
cyanide production was higher in plants from Dalsland, Marmontweg, and Elderveld than
0
25
50
75
100
125
150
175
200
225
250
275
12345678
Week of samplin
g
Cyanide concentration (ppm)
Sorghum
Garlic mustard
Fig. 1 Mean (±1 SE) cyanide levels in the youngest fully expanded leaf of garlic mustard and Sudan grass
(Sorghum sudanense) through an 8-wk time course as plants aged. Plants were 2 wk old at the first sampling
Table 1 Mean (±1 SE) cyanide levels in leaves of 3-wk-old seedlings of five brassicaceous species
Species Cyanide (ppm)
Alliaria petiolata 108.9 (7.82)
a
Arabidopsis thaliana 4.19 (2.83)
b
Brassica kaber 0.733 (0.571)
c
Brassica rapa 0.617 (0.562)
c
Brassica napus 0.433 (0.096)
c
Means followed by different letters are significantly different at P=0.05.
J Chem Ecol (2007) 33:8594 89
in plants from Hanau and Ligonier. Although SA had little effect on cyanide production in
plants from Dalsland, Marmontweg, and Ligonier, it strongly induced cyanide production in
plants from Hanau and strongly reduced production in plants from Elderveld.
Experiment 6 Cyanide production was not detectable from the decomposition of sinigrin at
any concentration in our experimental setup.
Discussion
Garlic mustard, an important invasive plant of forest understories throughout North
America, produces a wealth of chemical defenses (e.g., Cipollini et al., 2005). We
demonstrate for the first time here that garlic mustard produces substantial amounts of
cyanide from its tissues.
0
25
50
75
100
125
150
175
200
225
250
275
1234
Week of sampling
Cyanide concentration (ppm)
Sorghum
Garlic mustard
Fig. 2 Mean (±1 SE) cyanide levels in the oldest fully expanded leaf of garlic mustard and Sudan grass
(Sorghum sudanense) through a 4-wk time course as plants aged. Plants were 2 wk old at the first sampling
Table 2 Mean (±1 SE) cyanide levels in young, intermediate, and old leaves of 5-wk-old seedlings of garlic
mustard in the presence and absence of green peach aphids
Treatment Leaf age Cyanide (ppm)
Aphids absent Young 66.2 (8.74)
a
Intermediate 38.7 (5.39)
b
Old 40.4 (5.00)
b
Aphids present Young 47.9 (6.68)
a
Intermediate 14.9 (2.13)
b
Old 16.9 (2.1)
b
Means within an aphid treatment with different letters are significantly different at P=0.05.
90 J Chem Ecol (2007) 33:8594
Cyanide contents reached by garlic mustard in some experiments were over 100 ppm
FW in leaves, a level considered to be acutely toxic to humans (and other vertebrates) for
plants like cassava (Jorgensen et al., 2005). In other experiments, levels in leaves ranged
from 20 to 85 ppm, levels that are still considered to be moderately toxic (Jorgensen et al.,
2005). As cyanide has known biological activity against animals, plants, and microbes
(Jones 1998; Blenis et al., 2004; Gonzalez and Sotomayor, 2005), cyanide may contribute
to some of the reported allelopathic effects of garlic mustard on other plants, insects, and
mycorrhizal fungi (e.g., Porter 1994; Prati and Bossdorf, 2004; Stinson et al., 2006), and to
its known deterrence of mammalian or avian her bivores (D. Cipo llini, personal
observation). It should be noted that we expressed cyanide content in garlic mustard per
unit fresh weight, although many authors express cyanide content per unit dry weight (e.g.,
Lindroth et al., 2000). Expressing cyanide content in garlic mustard per unit dry weight
would increase levels substantially, accounting for the average moisture content of leaves.
Within each plant, cyanide production was much higher in leaves than in roots and
generally declined with leaf age. These findings are consistent with patterns of cyanide
production in the foliage and roots of many other cyanogenic plants (e.g., Vickery et al.,
1987; Busk and Møller, 2002; Fitzgerald et al., 2002; Jorgensen et al., 2005). In plants
where the roots contain high levels of cyanide, like cassava, cyanide production by leaves is
thought to be the source of cyanogenic compounds that are subsequently transported to
roots (Jorgensen et al., 2005). Unlike S. sudanense studied here, cyanogenic potential in the
youngest leaf of garlic mustard did not decline as plants aged. This indicates that garlic
mustard consistently maintains the potential for cyanide toxicity in young leaves throughout
much of its first season of growth, a pattern also seen in some other cyanogenic plants (e.g.,
Gleadow and Woodrow, 2000).
Interestingly, cyanide production was lower in aphid-infested plants across all leaf ages
than in noninfested plants. Because we classified plants as infested or noninfested after the
aphids had time to choose plants, it is possible that the patterns we observed resulted from
aphid selection of the more weakly cyanogenic individuals in our experiment. However, as
can be seen in the mean cyanide contents of the noninfested plants, individuals do not vary
to a large extent in cyanide production in this population, which is known to be relatively
genetically homogenous for other growth and defense characteristics (Cipollini 2002).
Thus, it is more likely that aphid feeding somehow caused the differences in cyanide
contents that we observed. For example, aphid feeding may have led to the volatilization of
Table 3 Mean (±1 SE) cyanide levels in leaves of 8-wk-old plants of five garlic mustard populations treated
with 0.05 M salicylic acid or a control solution
Population Salicylate Treatment Cyanide (ppm)
Dalsland, Sweden 30.0 (4.2)
a
+ 25.4 (4.3)
Elderveld, Netherlands 32.0 (4.6)
a
*
+ 11.4 (4.0)
Marmontweg, Netherlands 27.0 (4.7)
a
+ 23.1 (4.1)
Hanau, Germany 14.4 (4.1)
b
*
+ 36.7 (4.3)
Ligonier, Pennsylvania 17.2 (4.0)
b
+ 23.4 (4.6)
Means followed by different letters differ significantly at P=0.05 in the absence of salicylic acid. Means
followed by asterisks indicate populations where salicylic acid significantly affected cyanide levels.
J Chem Ecol (2007) 33:8594 91
cyanide from intact tissues before their analysis by enabling cyanide precursors and
enzymes to mix in planta, thus lowering the cyanogenic potential of the tissue at the time of
analysis. Alternatively, aphid feeding may have decreased cyanide precursor production
either directly by downregulating gene expression or indirectly through nutrient limitation
of precursor production. As cyanide production is increased by nitrogen fertilization in
many plants (e.g., Busk and Møller, 2002; Jorgensen et al., 2005), aphid feeding may have
reduced cyanide content by lowering tissue nitrogen content.
Populations varied to some extent in the constitutive and inducible production of
cyanide, similar to other cyanogenic plant species (e.g., Lindroth et al., 2000; Schappert and
Shore, 2000), but none of the six total populations studied here was acyanogenic. We have
observed differences in defense production among populations of garlic mustard (Cipollini
et al., 2005), but not enough populations were sampled in the current study to place our
observations in an evolutionary or ecological context. Salicylic acid increased, decreased,
or had no effect on cyanide production, depending upon the population studied. Salicylic
acid is involved in the induction of numerous defenses in plants in response to biotic stimuli
(Mauch-Mani and Metraux, 1998) and is known to induce cyanide-resistant respiration in
plants (Kapulnik et al., 1992). Its effect on cyanide production has not been examined in
any plant to our knowledge, but it has been shown to suppress the expression of dhurrinase
mRNA in Sorghum bicolor (Salzman et al., 2005), which would presumably decrease
cyanide production from the cyanogenic glycoside, dhurrin, found in these plants.
Garlic mustard produces substantially more cyanide than any brassicaceous plant studied
to date (e.g., Sexton and Howlett, 2000; Chandra et al., 2004), including A. thaliana and the
three Brassica species examined in this study. This appears to make garlic mustard unique
among members of this family, which are characterized by their product ion of
glucosinolates; cyanide production, however, has not been widely surveyed in this family.
Only a few plant species are known as producers of both glucosinolates and cyanogenic
glycosides, including Carica papaya in the Caricaceae family (Bennett et al., 1997), which
is a member of the Capparales order along with members of the Brassicaceae family
(Rodman et al., 1998). It has been suggested that glucosinolate production arose in an
already cyanogenic species (Rodman et al., 1998; Halkier and Gershenzon, 2006), and it is
perhaps surprising that so few members of the Brassicaceae have been confirmed to be
cyanogenic.
Regardless of origin, cyanide production by garlic mustard may contribute to its
unpalatability toward mustard specialist herbivores that typically prefer glucosinolate-
containing plants (Porter 1994; Haribal and Renwick, 1998; Haribal et al., 2001) and some
of its other allelopathic effects. We have noted anecdotally that treatment of garlic mustard
leaves by freezing followed by subsequent thawing and incubation at room temperature
makes them substantially more palatable and fosters better growth of larvae of the cabbage
looper, Trichoplusia ni (D. Cipollini, unpublished data). This effect may be explained, in
part, by the freezing-induced loss of cyanide from tissues. Interestingly, garlic mustard is
also distinguished among members of its family by a characteristic garlic odor when its
leaves are crushed (thus its common name). To our knowledge, the chemical profile and
significance of this volatile blend has not been examined, but cyanide is certainly a part of it.
The source of cyanide in garlic mustard could not be determined in this study. The
decomposition of glucosinolates has been suggested as a possible source in brassicaceous
plants, but this does not appear to be the source in garlic mustard. Garlic mustard contains
sinigrin (2-propenyl glucosinolate) as its primary glucosinolate (Vaughan and Berhow,
1999), but neither we nor Sexton and Howlett (2000) found that the thioglucosidase-
92 J Chem Ecol (2007) 33:8594
catalyzed hydrolysis of this compound could liberate cyanide in vitro. Moreover, the
Brassica species and A. thaliana studied here are each well known as producers of
glucosinolates (e.g., Cipollini et al., 2003, 2004; Chandra et al., 2004), yet each gave rise to
negligible levels of cyanide in this study. The conversion of 1-amino-cyclopropane 1-
carboxylic acid (ACC) into ethylene results in some cyanide production in plants, but levels
seen here are much higher than those noted to be produced in this manner in other plant
tissues (Yip and Yang, 1998). Although not initially considered a cyanogenic compound,
garlic mustard contains a novel cyanoallyl glycoside, alliarinoside, with a CN group as
part of the molecule (Haribal et al., 2001). It is possible that either the enzymatic or
nonenzymatic decomposition of this compound could yield volatile cyanide. If this
compound is not the source of cyanide, then either other glucosinolates or an as yet
undescribed cyanogenic compound may be the source.
Acknowledgments We thank Stephanie Enright for technical assistance, the Ohio Board of Regents for
funding, and a simple curiosity that motivated this project. Martin Heil, Nicole van Dam, and Harriet
Hillstrom kindly provided seeds of European garlic mustard plants. Two anonymous reviewers substantially
improved the manuscript.
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    • "Garlic mustard, like other plants in the mustard family, has secondary chemicals, primarily glucosinolates, which are toxic to aerobic organisms, including potential herbivores and soil microbes. The cyanide-like moiety that results from glucosinolate degradation in damaged garlic mustard tissues (Cipollini and Gruner, 2007 ) is likely responsible for its anti-herbivore activity and potential impacts on soils. In its introduced range, the 0929-1393/$ – see front matter © 2012 Elsevier B.V. All rights reserved. "
    Dataset · Jan 2016 · Journal of Chemical Ecology
    • "These attempts did not provide any indication of the presence of cyanogenic glucosides in A. petiolata (Figure S9). To confirm the reported cyanide release from A. petiolata (Cipollini and Gruner, 2007), the HCN potential in Danish populations was determined. HCN can be detected by various colorimetric assays as described in this section, but a number of compounds can cross-react in these assays. "
    [Show abstract] [Hide abstract] 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.
    Full-text · Article · Oct 2015
    • "Both alliarinoside and sinigrin tend to be concentrated in new leaves, and alliarinoside peaks during mid­summer in rosette leaves (Frisch et al. 2014 ; Haribal and Renwick 2001 ). Finally, A. petiolata is unique in its production of other compounds that may be involved in insect resistance, such as cyanide (Cipollini and Gruner 2007 ). Davis and Cipollini ( 2014 ) "
    [Show abstract] [Hide abstract] ABSTRACT: As it pertains to insect herbivores, the preference-performance hypothesis posits that females will choose oviposition sites that maximize their offspring's fitness. However, both genetic and environmental cues contribute to oviposition preference, and occasionally "oviposition mistakes" occur, where insects oviposit on hosts unsuitable for larval development. Pieris virginiensis is a pierine butterfly native to North America that regularly oviposits on an invasive plant, Alliaria petiolata, but the caterpillars are unable to survive. Alliaria petiolata has high concentrations of the glucosinolate sinigrin in its tissues, as well as a hydroxynitrile glucoside, alliarinoside. We investigated sinigrin as a possible cause of mistake oviposition, and sinigrin and alliarinoside as possible causes of larval mortality. We found that sinigrin applied to leaves of Cardamine diphylla, a major host of P. virginiensis that does not produce sinigrin, had no effect on oviposition rates. We tested the effect of sinigrin on larval performance using two host plants, one lacking sinigrin (C. diphylla) and one with sinigrin naturally present (Brassica juncea). We found no effect of sinigrin application on survival of caterpillars fed C. diphylla, but sinigrin delayed pupation and decreased pupal weight. On B. juncea, sinigrin decreased survival, consumption, and caterpillar growth. We also tested the response of P. virginiensis caterpillars to alliarinoside, a compound unique to A. petiolata, which was applied to B. oleracea. We found a significant reduction in survival, leaf consumption, and caterpillar size when alliarinoside was consumed. The 'novel weapon' alliarinoside likely is largely responsible for larval failure on the novel host A. petiolata. Sinigrin most likely contributes to the larval mortality observed, however, we did not observe any effect of sinigrin on oviposition by P. virginiensis females. Further research needs to be done on non-glucosinolate contact cues, and volatile signals that may induce P. virginiensis oviposition.
    Full-text · Article · Sep 2015
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