Interaction of cruciferous phytoanticipins with plant fungal pathogens: Indole glucosinolates are not metabolized but the corresponding desulfo-derivatives and nitriles are

Article abstract of DOI:10.1016/j.phytochem.2011.08.018

Glucosinolates represent a large group of plant natural products long known for diverse and fascinating physiological functions and activities. Despite the relevance and huge interest on the roles of indole glucosinolates in plant defense, little is known about their direct interaction with microbial plant pathogens. Toward this end, the metabolism of indolyl glucosinolates, their corresponding desulfo-derivatives, and derived metabolites, by three fungal species pathogenic on crucifers was investigated. While glucobrassicin, 1-methoxyglucobrassicin, 4-methoxyglucobrassicin were not metabolized by the pathogenic fungi Alternaria brassicicola, Rhizoctonia solani and Sclerotinia sclerotiorum, the corresponding desulfo-derivatives were metabolized to indolyl-3-acetonitrile, caulilexin C (1-methoxyindolyl-3-acetonitrile) and arvelexin (4-methoxyindolyl-3-acetonitrile) by R. solani and S. sclerotiorum, but not by A. brassicicola. That is, desulfo-glucosinolates were metabolized by two non-host-selective pathogens, but not by a host-selective. Indolyl-3-acetonitrile, caulilexin C and arvelexin were metabolized to the corresponding indole-3-carboxylic acids. Indolyl-3-acetonitriles displayed higher inhibitory activity than indole desulfo-glucosinolates. Indolyl-3-methanol displayed antifungal activity and was metabolized by A. brassicicola and R. solani to the less antifungal compounds indole-3- carboxaldehyde and indole-3-carboxylic acid. Diindolyl-3-methane was strongly antifungal and stable in fungal cultures, but ascorbigen was not stable in solution and displayed low antifungal activity; neither compound appeared to be metabolized by any of the three fungal species. The cell-free extracts of mycelia of A. brassicicola displayed low myrosinase activity using glucobrassicin as substrate, but myrosinase activity was not detectable in mycelia of either R. solani or S. sclerotiorum.

Full text of DOI:10.1016/j.phytochem.2011.08.018

Phytochemistry 72 (2011) 2308–2316
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Interaction of cruciferous phytoanticipins with plant fungal pathogens: Indole
glucosinolates are not metabolized but the corresponding desulfo-derivatives
and nitriles are
⇑
M. Soledade C. Pedras , Sajjad Hossain
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, SK, Canada S7N 5C9
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 April 2011
Received in revised form 21 July 2011
Available online 13 September 2011
Glucosinolates represent a large group of plant natural products long known for diverse and fascinating
physiological functions and activities. Despite the relevance and huge interest on the roles of indole gluc-
osinolates in plant defense, little is known about their direct interaction with microbial plant pathogens.
Toward this end, the metabolism of indolyl glucosinolates, their corresponding desulfo-derivatives, and
derived metabolites, by three fungal species pathogenic on crucifers was investigated. While glucobrass-
icin, 1-methoxyglucobrassicin, 4-methoxyglucobrassicin were not metabolized by the pathogenic fungi
Alternaria brassicicola, Rhizoctonia solani and Sclerotinia sclerotiorum, the corresponding desulfo-deriva-
tives were metabolized to indolyl-3-acetonitrile, caulilexin C (1-methoxyindolyl-3-acetonitrile) and arve-
lexin (4-methoxyindolyl-3-acetonitrile) by R. solani and S. sclerotiorum, but not by A. brassicicola. That is,
desulfo-glucosinolates were metabolized by two non-host-selective pathogens, but not by a host-selec-
tive. Indolyl-3-acetonitrile, caulilexin C and arvelexin were metabolized to the corresponding indole-3-
carboxylic acids. Indolyl-3-acetonitriles displayed higher inhibitory activity than indole desulfo-glucosin-
olates. Indolyl-3-methanol displayed antifungal activity and was metabolized by A. brassicicola and R.
solani to the less antifungal compounds indole-3-carboxaldehyde and indole-3-carboxylic acid. Diindol-
yl-3-methane was strongly antifungal and stable in fungal cultures, but ascorbigen was not stable in solu-
tion and displayed low antifungal activity; neither compound appeared to be metabolized by any of the
three fungal species. The cell-free extracts of mycelia of A. brassicicola displayed low myrosinase activity
using glucobrassicin as substrate, but myrosinase activity was not detectable in mycelia of either R. solani
or S. sclerotiorum.
Keywords:
Alternaria brassicicola
Brassicaceae
Crucifer
Indole glucosinolate
Secondary metabolism
Myrosinase activity
Rhizoctonia solani
Sclerotinia sclerotiorum
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
sinolate producing plant species have enormous economic impor-
tance (family Brassicaceae, common name crucifer) as sources of
Glucosinolates (GLs) represent a large group of plant natural
products long known for diverse and fascinating physiological func-
tions and activities (Fenwick and Heaney, 1983; Fahey et al., 2001;
Traka and Mithen, 2009; Vig et al., 2009; Hopkins et al., 2009). The
functional group glucosinolate contains an O-sulfated thiohydroxi-
mate connected with a b-glucopyranosyl residue through the sulfur
atom (Fig. 1). The R moiety of the molecule is derived from protein
amino acids and their homologues (Sønderby et al., 2010; Halkier
and Gershenzon, 2006). Many glucosinolate producing plant species
have been credited with both beneficial and detrimental health ef-
fects. For example, metabolic products of GLs have been shown to in-
duce detoxifying enzymes in humans and suggested to protect
against cancer (Traka and Mithen, 2009; Vig et al., 2009). On the
other hand, harmful goitrogenic effects have also been attributed
to GLs (Fenwick and Heaney, 1983; Traka and Mithen, 2009). Gluco-
healthy oils (canola and rapeseed), vegetables (cabbage, cauliflower,
broccoli, Brussels sprouts, turnip, rutabaga, and other Brassica spp.)
and condiments (mustard an wasabi). Central to the huge interest
generated by GLs, and their metabolic products, is their involvement
in plant defense pathways against very destructive organisms,
including a broad range of insects, nematodes, and microbes (Fen-
wick and Heaney, 1983; Traka and Mithen, 2009; Vig et al., 2009;
Hopkins et al., 2009; Ahuja et al., 2010).
In general, the defensive layer of plant metabolites is composed
of both phytoanticipins (constitutive) and phytoalexins (inducible)
(Smith, 1996). GLs are phytoanticipins of crucifers as metabolic
products of their enzymatic degradation are effective in protecting
against pests and microbes. A correlation between defense signal-
ling pathways and GL profiles observed in Arabidopsis thaliana L.
indicated that indole GLs (IGLs) were involved in defense, although
the function of each particular compound was not examined (Mik-
kelsen et al., 2003). Furthermore, IGLs (R = indolyl-3-methyl, 1–3)
are potential intermediates in the biosynthesis of cruciferous
⇑
Corresponding author. Tel.: +1 306 966 4772; fax: +1 306 966 4730.
E-mail address: s.pedras@usask.ca (M.S.C. Pedras).
0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2011.08.018

M.S.C. Pedras, S. Hossain / Phytochemistry 72 (2011) 2308–2316
2309
to indolyl-3-methylene carbocations 13–15. In the plant, these
carbocations react with nucleophiles such as water, ascorbate, glu-
tathione, etc., to yield indolyl-3-methanols 22–24 and ascorbigens
16–18 (Fig. 2) (Agerbirk et al., 2009). In addition, from the thiohy-
droximate intermediates 4–6, indolyl-3-acetonitriles 10–12 are
formed by enzymatic catalyses that require particular proteins
(e.g. ESP = epithiospecifier protein plus Fe2+; NSP = nitrile specifier
protein).
Fig. 1. General chemical structure of glucosinolates.
While altering GL profiles has been shown to affect plant disease
resistance to microbialpathogens (Brader et al., 2006; Agerbirket al.,
2009), despite speculation, very little is known about the direct
interaction of plant fungal pathogens with GLs. By contrast, there
is substantial evidence suggesting that the degradation products of
GLs, namely isothiocyanates and nitriles, are strongly antifungal
and antibacterial (Manici et al., 1997; Góralska et al., 2009; Aires
et al., 2009). Interestingly, transformed A. thaliana (expressing
CYP79D2) accumulating isopropyl and methylpropyl GLs showed
enhanced resistance against the bacterial soft-rot pathogen Erwinia
carotovora, whereas transformed A. thaliana (expressing CYP79A1 or
over-expressing the endogenous CYP79A2) accumulating p-
hydroxybenzyl or benzyl GLs, respectively, showed enhanced sus-
ceptibility to the plant fungal pathogen Alternaria brassicicola and
enhanced resistance to the bacterial pathogen Pseudomonas syringae
phytoalexins, indicating that their production is crucial to plant fit-
ness (Pedras et al., 2010, 2011). Therefore, IGLs are among the most
important components of the cruciferous defense arsenal.
The metabolic products of GLs result from hydrolyses catalyzed
by enzymes known as myrosinases (thioglucoside glucohydrolases,
EC 3.2.1.147), which are brought together with their substrates
only upon tissue damage. Enzyme catalyzed hydrolysis is followed
by additional enzymatic and chemical transformations of the thio-
hydroximate intermediates to yield volatile isothiocyanates as ma-
jor products (Bones and Rossiter, 2006); however, the chemical
structures of these products depend on various factors, including
the structures of the R groups. For example, in the case of IGLs
1–3 (e.g. R = indolyl-3-methyl, known as glucobrassicin (1)), the
isothiocyanates 7–9 are neither volatile nor stable, decomposing
Fig. 2. Metabolism of indole glucosinolates 1–3 in plants: (i) hydrolyses catalyzed by myrosinases; (ii) chemical rearrangement; (iii) enzymatic reactions catalyzed by Fe2+
/
ESP (epithiospecifier protein)/NSP (nitrile specifier protein); (iv) reaction with ascorbic acid; (v) hydrolyses catalyzed by NIT (nitrilases); (vi) reaction with water; (vii)
chemical rearrangement.

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M.S.C. Pedras, S. Hossain / Phytochemistry 72 (2011) 2308–2316
(Brader et al., 2006). Because fungal pathogens of Brassica spp., and
crucifers in general, overcome the protection provided by phytoal-
exins and related metabolites through detoxification reactions (Ped-
ras and Ahiahonu, 2005; Pedras, 2008; Pedras and Yaya, 2010;
Pedras et al., 2011), it is of great importance to establish if such reac-
tions occur with IGLs and their metabolites. The paucity of studies
dealing with metabolism of IGLs may be due to the commercial
unavailability of these compounds as their chemical syntheses are
not trivial (Rollin and Tatibouet, 2011) and the isolation and purifi-
cation of intact products require extensive chromatography, afford-
ing insufficient amounts for such studies (e.g. ca. 5 mg from 1.3 kg of
leaf tissue, Pedras et al., 2008).
To better understand the roles of IGLs in the interaction of cru-
cifers with their fungal pathogens, their potential metabolism, and
that of the corresponding desulfo-derivatives, by the phytopatho-
genic fungi A. brassicicola, Rhizoctonia solani and Sclerotinia sclero-
tiorum was investigated. In addition, the products of enzymatic
and chemical reactions of IGLs by the three fungal species was
determined. It was established that antifungal plant metabolites
derived from IGLs such as indolyl-3-acetonitriles and indolyl-3-
methanol are metabolized to products with lower antifungal activ-
ity. In addition, indole glucosinolates and desulfo-derivatives
showed no inhibitory activity against any of the three plant patho-
gens. Interestingly, myrosinase activity was detected in mycelia of
Fig. 3. Metabolism of indole desulfo-glucosinolates 28 and 29 in cultures of
Rhizoctonia solani (i) and Sclerotinia sclerotiorum (ii) and nitriles 10–12 in cultures of
R. solani (i), S. sclerotiorum (ii) and Alternaria brassicicola (iii). Dashed arrows
represent hypothetical steps.
Fig. 4. Kinetics of transformation of indole desulfo-glucosinolates in cultures of: (A) Alternaria brassicicola incubated with 28; (B) Rhizoctonia solani incubated with 28 and
formation of nitrile 10; (C) Sclerotinia sclerotiorum incubated with 28; (D) A. brassicicola incubated with 29; (E) R. solani incubated with 29 and formation of nitrile 11; (F) S.
sclerotiorum incubated with 29 and formation of nitrile 11; control curves indicate the stability of compounds 28 and 29 incubated in water under similar conditions but
without the fungus.

M.S.C. Pedras, S. Hossain / Phytochemistry 72 (2011) 2308–2316
2311
A. brassicicola, but no detectable activity was found in mycelia of R.
solani or S. sclerotiorum.
were further metabolized to the corresponding carboxylic acids, as
described below in Section 2.3. Because no potential intermediates
of the transformations of 28 and 29 to 10 and 11 were detected in
any of the cultures, we hypothesize that a glucosyl hydrolase(s)
catalyzes these transformations to yield thiohydroxamic acids 34
and 35, which are further transformed on standing (enzymatically
or spontaneously) to the corresponding nitriles 10 and 11. Similar
transformation of isotopically labeled 34–10 were observed in
planta (Pedras and Okinyo, 2008).
2. Results and discussion
Three fungal species pathogenic to crucifers were chosen to carry
out metabolic studies of IGLs and derivatives, based on their host-
range and amenabilityto cultureunder aseptic conditions. A. brassic-
icola (Schwein.) Wiltshire, together with A. brassicae, causes one of
the most economically important diseases of Brassica species, the
so-called Alternaria black spot (also called dark leaf spot) (Cooke
et al., 1997). A. brassicicola is quite selective in its host-range, infect-
ing mainly cultivars of B. juncea L. or other Brassica spp. (Rimmer and
Buchwaldt, 1995; Vishwanathet al., 1999). By contrast, both R. solani
Kuhn, and S. sclerotiorum (Lib.) de Bary infect crucifers and a wider
range of plant families, but while S. sclerotiorum infectsmainly above
ground plant organs (Bolton et al., 2006), R. solani infects mainly
underground organs (Luebeck, 2004).
To the best of our knowledge, this is the first report of the trans-
formation of desulfo-IGLs by fungi. Previously, a soil isolate of the
fungus Aspergillus flavus was shown to convert enzymatically 2-
propenyl and 2-phenylethyl glucosinolates and their desulfo-
derivatives to nitriles, that is sulfatase and myrosinase activities
were reported in A. flavus (Galletti et al., 2008). The metabolism
of desulfo-IGLs 28 and 29 suggests that both R. solani and
S. sclerotiorum produce glucosyl hydrolases that do not have myro-
sinase activity, and do not produce sulfatases, as intact IGLs are not
metabolized. However, these transformations were not detoxifica-
tion processes, since the nitriles were more inhibitory to each fun-
gal species than the corresponding desulfo-IGLs (Tables 1 and 2).
Although some Aspergillus spp. have been reported to produce
myrosinases (Ohtsuru et al., 1973; Smits and Knol, 1993; Rakariya-
tham et al., 2006), this enzymatic activity does not appear to be a
known trait of plant fungal pathogens. Hence, the myrosinase activ-
ity of cell-free extracts of mycelia of A. brassicicola, R. solani and S.
sclerotiorum grown under various conditions was determined using
glucobrassicin (1) as substrate. Under the conditions tested (Chevol-
leau et al., 1997), low but consistent myrosinase activity was de-
tected in cell-free extracts of mycelia of A. brassicicola (0.4 nmoles/
mg/min of indolyl-3-methanol (22) and dindolylmethane (25)
formed), but not in mycelia of R. solani or S. sclerotiorum. However,
since glucobrassicin (1) was not transformed in cultures of A. brassic-
icola, the myrosinase activity detected in cell-free extracts is likely
notsecretedintotheculturemedia, aspreviouslyobservedforAsper-
gillus spp. (Ohtsuru et al., 1973). Although ascorbate is a known sta-
bilizer of myrosinase activity (Shikita et al., 1999), we found that
when ascorbate was used in enzyme assays using glucobrassicin
(1)as substrate, itreactedwiththereactionproductindolyl-3-meth-
anol (22), thus ascorbate was not used in subsequent assays. Myro-
sinase activity was not induced by 1-methoxyglucobrassicin (2).
Considering that myrosinase activity has not been reported for any
other plant fungal pathogens, it is difficult to draw conclusions be-
fore further work with other fungal species under various conditions
is carried out.
2.1. Chemical synthesis and antifungal activity of compounds
The chemical synthesis of compounds was carried out as de-
scribed in the experimental and those commercially available were
used as such, after confirming their purity by HPLC analysis. IGLs
1–3, desulfo-IGLs 28 and 29 and known derivatives of indole gluc-
osinolates (compounds 10–12, 16, 22 and 25, Fig. 2) were assayed
for antifungal activity. A. brassicicola, R. solani and S. sclerotiorum
were used in standard mycelial growth assays described in the
experimental. As summarized in Table 1, neither IGLs 1–3 nor their
desulfo-derivatives 28 and 29 displayed detectable antifungal
activity. While diindolyl-3-methane (25) was the most active of
all compounds, inhibiting growth of the three fungal species com-
pletely at 0.50 mM, ascorbigen (16) displayed the lowest activity.
Among the nitriles, caulilexin C (1-methoxyindolyl-3-acetonitrile)
(11) was the most active, inhibiting completely the growth of R.
solani at 0.50 mM.
2.2. Metabolism of indole glucosinolates 1–3, desulfo-glucosinolates
28 and 29 and myrosinase activity
Liquid cultures of A. brassicicola, R. solani and S. sclerotiorum
grown for 72 h were incubated with IGLs 1–3 for various time peri-
ods, samples were withdrawn from cultures immediately after
addition of each compound and up to 5 days and immediately fro-
zen and then lyophilized (Pedras and Suchy, 2005). Media incu-
bated with each compound (control solutions) were treated
similarly to determine the chemical stability of compounds during
the incubation experiments. HPLC analysis of the concentrated cul-
ture samples indicated no transformation of any of IGLs 1–3.
Although a slight decrease in the concentration of 2 was detected,
this decrease was similar to that observed in control solutions and
attributed to its slow decomposition. The final results indicated
that none of the IGLs were metabolized by any of the fungal
species.
Similar experiments conducted with desulfo-IGLs 28 and 29
indicated that these compounds were transformed by R. solani
and S. sclerotiorum to the corresponding nitriles (Fig. 3) at slower
rate in S. sclerotiorum (t1/2 120 h for both 28 and 29) than in R.
solani (t1/2 24 h for 28, t1/2 90 h for 29) (Fig. 4B, C, E and F). By con-
trast, A. brassicicola did not metabolize any of the desulfo-IGLs 28
and 29, which remained in culture for the duration of the experi-
ments (5 and 6 days, respectively, Fig. 4A and D). Nitriles 10 and
11 accumulated in cultures of R. solani and could be quantified
after 12-h incubations (Fig. 4B and E), but in cultures of S. sclerotio-
rum 10 was detected only in trace amounts (Fig. 4C) and nitrile 11
was quantifiable only after 72-h incubations (Fig. 4F). These nitriles
2.3. Metabolism of indolyl-3-acetonitriles 10–12
Cultures of A. brassicicola were incubated with nitriles 10–12
and cultures of R. solani and S. sclerotiorum were incubated with ni-
triles 11 and 12 (because studies with 10 were previously reported,
Pedras and Montaut, 2003) and their transformations were moni-
tored by HPLC (photodiode array and ESI detection). Samples were
withdrawn from cultures immediately after addition of each com-
pound, were lyophilized, and analyzed by HPLC, as reported in the
experimental. Media samples incubated with nitriles 10–12 (con-
trol solutions) were analyzed similarly, to determine the chemical
stability of each compound during the incubation experiments. Ni-
triles 10–12 were metabolized by each species to the correspond-
ing indole-3-carboxylic acids 31–33 (Fig. 3). As shown in Fig. 5 and
summarized in Table 3, the rates of transformation of nitriles 10
and 11 were faster in A. brassicicola than in R. solani or S. sclerotio-
rum, whereas transformations of 4-methoxy nitrile 12 were similar
in the three species. The identity of each acid was confirmed by di-
rect comparison with authentic samples synthesized as reported in
the experimental. Interestingly, the inhibitory activity of nitriles 10

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M.S.C. Pedras, S. Hossain / Phytochemistry 72 (2011) 2308–2316
Table 1
Antifungal activity of compounds 1–3, 10–12, 16, 22, 25 and 28 and 29 against the plant fungal pathogens Alternaria brassicicola, Rhizoctonia solani and Sclerotinia sclerotiorum (as
described in Section 4.3) under continuous light.
Compound name (#)
Concentration (mM)
% Inhibitiona (standard error)
A. brassicicola
R. solani
S. sclerotiorum
Glucobrassicin (1)
0.50
0.50
0.50
0.50
0.20
0.10
0.50
0.20
0.10
0.50
0.20
0.10
0.50
0.20
0.10
0.50
0.20
0.10
0.50
0.20
0.10
0.50
0.50
NI
NI
NI
NI
NI
NI
NI
NI
NI
1-Methoxyglucobrassicin (2)
4-Methoxyglucobrassicin (3)
Indolyl-3-acetonitrile (10)
27 (2) a, x
13(1)
41 (2) a, y
15 (2)
3 (2)
27 (0) a, x
16 (0.3)
4 (0.3)
54 (0) b, z
13 (1)
0 (0)
77 (0) c, y
46 (0)
24 (1)
10 (0) d, y
7 (0)
3 (0.3)
43 (1) b, x
29 (0)
18 (0)
59 (1) c, x
15 (1)
0 (0)
36 (1) d, x
12 (0)
0 (0)
43 (1) b, x
29 (0)
18 (0)
100 (0) e, x
77 (1)
60 (1)
NI
1-Methoxyindolyl-3-acetonitrile (11)
4-Methoxyindolyl-3-acetonitrile (12)
Ascorbigen (16)
100 (0) b, y
45 (1)
29 (1)
70 (1) c, y
46 (0)
26 (1)
12 (0.3) d, y
6 (0)
0 (0)
3 (0)
Indolyl-3-methanol (22)
44 (0) a, x
27 (0.3)
11 (0)
100 (0) b, x
61 (1)
32 (1)
NI
73 (0.3) c, y
60 (0)
41 (0.3)
100 (0) e, x
81 (2)
72 (2)
NI
Diindolyl-3-methane (25)
Desulfoglucobrassicin (28)
1-Methoxydesulfoglucobrassicin (29)
NI
NI
NI
a
Percentage of growth inhibition calculated using the formula:% inhibition = 100 À [(growth on amended medium/growth in control medium) Â 100]. Data are the
mean SE; for statistical analysis, one-way ANOVA tests were performed followed by Tukey’s test with adjusted set at 0.05; n = 3; different letters in the same column (a–e)
a
indicate significant differences (P < 0.05); different letters in the same row (x–z) indicate significant differences (P < 0.05).
Table 2
Antifungal activity of compounds 19, 31–33 and 36 against the plant fungal pathogens Alternaria brassicicola, Rhizoctonia solani and Sclerotinia sclerotiorum (as described in
Section 4.3) under continuous light.
Compound name (#)
Concentration (mM)
% Inhibitiona (standard error)
A. brassicicola
R. solani
S. sclerotiorum
Indolyl-3-acetic acid (19)
0.50
0.20
0.10
0.50
0.20
0.10
0.50
0.20
0.10
0.50
0.20
0.10
0.50
0.20
0.10
28 (2) a, x
13 (2)
NI
22 (0) b, x
11 (0)
6 (0)
48 (1) c, x
24 (0)
12 (0)
18 (0) b, x
11 (1)
7 (1)
19 (1) a, y
12 (0.3)
7 (0)
9 (0.3) b, y
5 (0.3)
NI
14 (0) c, y
7 (0)
3 (0.3)
14 (0) c, y
6 (0.3)
NI
18 (1) a, y
6 (0)
NI
34 (2) b, z
9 (0)
4 (0)
67 (0) c, z
43 (0.3)
27 (0)
11 (0.3) d, z
3 (0)
Indole-3-carboxylic acid (31)
1-Methoxyindole-3-carboxylic acid (32)
4-Methoxyindole-3- carboxylic acid (33)
Indole-3-carboxaldehyde (36)
NI
29 (1) a, x
21 (1)
6 (1)
32 (1) d, x
17 (1)
7 (1)
16 (0.3) a, y
6 (0)
NI
a
Percentage of growth inhibition calculated using the formula: % inhibition = 100 À [(growth on amended medium/growth in control medium) Â 100]. Data are the
mean SE; for statistical analysis, one-way ANOVA tests were performed followed by Tukey’s test with adjusted set at 0.05; n = 3; different letters in the same column (a–d)
indicate significant differences (P < 0.05); different letters in the same row (x–z) indicate significant differences (P < 0.05).
a
and 11 was stronger against R. solani, than that of acids 31 (41% vs.
9% at 0.50 mM) and 32 (100% vs. 14% at 0.50 mM), respectively,
whereas acid 32 was more inhibitory to S. sclerotiorum than the
corresponding nitrile 11 (54% vs. 67% at 0.50 mM) (Tables 1 and
2). By contrast, the 4-methoxy nitrile 12 was more inhibitory to
the three species than the corresponding acid (59% vs. 18%, 70%
vs. 14%, 77% vs. 11% at 0.50 mM) (Tables 1 and 2).
analyses of lyophilized samples. The compounds were considered
sufficiently stable to incubate with cultures of A. brassicicola, R.
solani and S. sclerotiorum, although ascorbigen (16) showed ca.
40% decomposition in 4 days and indolyl-3-methanol (22) showed
<10% decomposition in 2 days. The potential transformations of
each compound were monitored by HPLC (photodiode array and
ESI detection) analyses of concentrated cultures. Ascorbigen (16)
was not metabolized by any of the pathogens and was found to
decompose slowly in cultures or in water to indolyl-3-methanol
(22). Indolyl-3-methanol (22) was metabolized in ca. 12 h to in-
dole-3-carboxaldehyde (36) and then to indole-3-carboxylic acid
(31) by both A. brassicicola and R. solani (Figs. 6 and 7). We suspect
that S. sclerotiorum metabolizes 22 similarly; however, because 22
2.4. Metabolism of ascorbigen (16), indolyl-3-methanol (22) and
diindolyl-3-methane (25)
Initially, the stability of ascorbigen (16), indolyl-3-methanol
(22) and diindolyl-3-methane (25) in water was evaluated by HPLC

M.S.C. Pedras, S. Hossain / Phytochemistry 72 (2011) 2308–2316
2313
Fig. 8. Non-enzymatic transformation of indolyl-3-methanol (22) in cultures of
Sclerotinia sclerotiorum.
decomposed quickly (ca. 90% in 2 h) in cultures of S. sclerotiorum,
no evidence could be obtained. Isolation of the products of decom-
position of 22 and NMR spectroscopic data showed that the main
products resulted from dimerization and trimerization of 22 to
yield 25 and 37, respectively, as shown in Fig. 8; transformations
are likely to occur via the indolyl-3-methylene cation (13) (Grose
and Bjeldanes, 1993). This decomposition was eventually found
to be due to the acidic pH of the culture solution; it was deter-
mined that in water, mycelia of S. sclerotiorum excreted sufficient
amounts of acids to decrease the pH of the culture solution to
3.8. In the case of A. brassicicola and R. solani the pH of the culture
solution remained around 6.5. Production of oxalic acid by S. scle-
rotiorum has been documented under various conditions (Bolton
et al., 2006). In control solutions, indolyl-3-methanol (22) decom-
posed mainly to 25 and 37, albeit at much slower rates (Fig. 7, <10%
in 48 h) than in acidic medium. Indolyl-3-methanol (22) displayed
stronger antifungal activity against each of the three fungal species
than indole-3-carboxylic acid (31). Oxidation of indolyl-3-metha-
nol (22) to 36 and 31 has been reported in mammalian systems
and in plants, but to the best of our knowledge, has not been re-
ported in fungi (Agerbirk et al., 2009).
Fig. 5. Kinetics of transformation of nitriles 10–12 in cultures of: A, Alternaria
brassicicola incubated with 10 and formation of 19; B, A. brassicicola, Rhizoctonia
solani and Sclerotinia sclerotiorum incubated with 11; C, A. brassicicola, R. solani and
S. sclerotiorum incubated with 12; control curves indicate the stability of nitriles
10–12 incubated in water under similar conditions but without the fungus.
In similar time-course experiments, incubation of diindolyl-3-
methane (25) with each pathogen and analyses of concentrated
cultures by HPLC indicated no additional compounds present in
the culture. The recovery of diindolyl-3-methane (25) from cultures
was ca. 90%, 50% was recovered from the broth and 40% from the
fungal mycelia (mycelial adsorption likely due to its lipophilicity).
3. Conclusion
The first investigation of the fate of three IGLs (1–3) in cultures
of three plant fungal pathogens, differing on their host-range and
preferred plant tissues (A. brassicicola, R. solani and S. sclerotiorum),
Fig. 6. Metabolism of indolyl-3-methanol (22) by Alternaria brassicicola and
Rhizoctonia solani.
Fig. 7. Kinetics of transformation of indolyl-3-methanol (22) and product formation in cultures of: A, Alternaria brassicicola; B, Rhizoctonia solani; control curves indicate the
stability of 22 incubated in water under similar conditions but without the fungus.

2314
M.S.C. Pedras, S. Hossain / Phytochemistry 72 (2011) 2308–2316
Table 3
Metabolism of compounds 10–12, 22, 28 and 29 by the plant fungal pathogens Alternaria brassicicola, Sclerotinia sclerotiorum, and Rhizoctonia solani.
Compound name
Metabolic products
A. brassicicola
R. solani
S. sclerotiorum
Indolyl-3-acetonitrile (10)
Indole-3-carboxylic acid (31); 100%
transformation in 12 h
Indole-3-carboxylic acid (31); 75%
Indole-3-carboxylic acid (31); 100%
transformation in 5 daysa
transformation in 24 ha
1-Methoxyindolyl-3-acetonitrile
(11)
4-Methoxyindolyl-3-acetonitrile
(12)
Indolyl-3-methanol (22)
1-Methoxyindole-3-carboxylic acid (32); 1-Methoxyindole-3-carboxylic acid (32); 1-Methoxyindole-3-carboxylic acid (32);
50% transformation in 20 h 50% transformation in 42 h 50% transformation in 54 h
4-Methoxyindole-3-carboxylic acid (33); 4-Methoxyindole-3-carboxylic acid (33); 4-Methoxyindole-3-carboxylic acid (33);
50% transformation in 48 h
Indolyl-3-carboxylic acid (31); 100%
transformation in 12 h
50% transformation in 14 h
Indolyl-3-carboxylic acid (31); 100%
transformation in 12 h
50% transformation in 36 h
Unstable in acidic medium, not
determined
Desulfoglucobrassicin (28)
No transformation
Indolyl-3-acetonitrile (10); 50%
transformation in 24 h
Indolyl-3-acetonitrile (10); 50%
transformation in 108 h
1-Methoxydesulfoglucobrassicin
(29)
No transformation
1-Methoxyindolyl-3-acetonitrile (11);
50% transformation in 84 h
1-Methoxyindolyl-3-acetonitrile (11);
50% transformation in 72 h
a
Results from previous work Pedras and Montaut (2003).
indicated that none of the three species metabolized these com-
pounds. By contrast, the corresponding indole desulfo-glucosino-
lates 28 and 29 were metabolized by the non-host-selective
pathogens R. solani and S. sclerotiorum to indolyl-3-acetonitriles
10 and 11, but not by the host-selective A. brassicicola (Table 3).
These results suggest that both R. solani and S. sclerotiorum produce
glucosyl hydrolases that do not have myrosinase activity, since in-
tact IGLs are not metabolized. This conclusion is consistent with
the lack of myrosinase activity in mycelia of these pathogens. To
date, there is no evidence of metabolism of IGLs 1–3 by plant fun-
gal pathogens; however, considering that IGLs do not appear to
have detrimental effects on the fungal species investigated, the
lack of metabolizing enzymes should not be surprising. Consistent
with previous work, our overall results indicate that indolyl-3-
acetonitriles 10–12 and indolyl-3-methanol (22) are antifungal
and thus potentially able to provide additional protection against
some fungal species. In general, the methoxy substituted nitriles
11 and 12 were more inhibitory than nitrile 10; however while
the 4-methoxy nitrile 12 was more inhibitory to both A. brassicicola
and S. sclerotiorum, the 1-methoxy nitrile 11 was more inhibitory
to R. solani. That is, cruciferous species that contain larger amounts
of 11 in the roots are likely to show lower susceptibility to R. solani,
a root pathogen. Nonetheless, since these pathogens are able to
detoxify 10–12 and 22, such protection can only be temporary.
Of all metabolites derived from IGLs, diindolyl-3-methane (25) ap-
pears the most useful to the plant, since it is not metabolized and is
a good growth inhibitor of fungal growth. Nonetheless, because 25
arises from non-enzymatic transformation of 22, it is difficult to
predict which metabolic modifications (e.g. increase the levels of
IGL 1, decrease formation of nitrile 10 by removing any nitrile
specifier activity, or both) are necessary to increase its concentra-
tion. Hence, to improve the natural resistance traits of Brassica
spp. to microbial pathogens, pathways regulating both IGL and
phytoalexin biosynthesis ought to be carefully examined.
diode array detector (DAD, wavelength range 190–600 nm), degas-
ser, and a column, having an in-line filter. Elution method A: column
Eclipse XDB-C18 (5
l
m particle size silica, 4.6 i.d. Â 150 mm), mo-
bile phase 50% H2O – 50% CH3OH to 100% CH3OH, for 25.0 min, linear
gradient, and at a flow rate 0.75 ml/min; elution method B (indolyl
glucosinolates and other polar metabolites): column Zorbax SB-
C18 (3.5
l
m particle size silica, 100 Â 3.0 mm i.d.), equipped with
an in-line filter, with the mobile phase H2O (with 0.1% TFA) – CH3OH
(with 0.1% TFA) from 85:15 to 70:30 in 25 min, to 50:50 in 5 min, to
40:60 in 5 min and a flow rate of 0.40 ml/min. HPLC–DAD–ESI-MS
analysis was carried out with an Agilent 1100 series HPLC system
equipped with an autosampler, binary pump, degasser, and a diode
array detector connected directly to a mass detector (Agilent
G2440A MSD-Trap-XCT ion trap mass spectrometer) with an elec-
trospray ionization (ESI) source. Chromatographic separation was
carried out at room temperature using an Eclipse XDB C-18 column
(5
l
m particle size silica, 4.6 i.d. Â 150 mm). The mobile phase con-
sistedof a lineargradientof 0.2%formicacidinwaterand 0.2%formic
acid in CH3CN (75:25 to 25:75 in 35 min, to 0:100 in 5 min) and a
flow rate of 1 ml/min. Data acquisition was carried out in positive
and negative polarity modes in a single LC run. All other experimen-
tal conditions as reported previously (Pedras et al., 2010).
4.2. Synthesis and characterization of new compounds
All compounds gave satisfactory spectroscopic data. The com-
mercially available compounds 10, 19, 22, 31 and 36 were used
as such; compounds 1–3 (Rollin and Tatibouet, 2011), 11 and 12
(Pedras et al., 2007), 16 (Piironen and Virtanen, 1962), 25 (Nagara-
jan and Perumal, 2004), 28 and 29 (Robertson and Botting, 1999),
and 32 and 33 (Somei et al., 2001) were synthesized according to
literature procedures and their purity was established by 1H
NMR spectroscopy and HPLC analysis. The first synthesis and spec-
troscopic characterization of compound 33 is reported below and
NMR spectra are provided in Supplementary data.
4. Experimental
4.2.1. 4-Methoxyindole-3-carboxylic acid (33)
4.1. Chemicals and instrumentation
Indole-3-carboxaldehyde (100.2 mg; 0.69 mmol, 36) was added
to a solution of thallium trifluoroacetate (562.1 mg, 1.03 mmol) in
TFA (1.2 ml), and the mixture was stirred at 30 °C for 3 h. After
evaporation of the solvent under reduced pressure, I2 (525.8 mg,
2.07 mmol), CuI (525.8 mg, 2.76 mmol), and DMF (5.0 ml) were
added to the residue. After stirring the reaction mixture at r.t. for
1 h, NaOCH3 (15% w/v, 5 ml) was added and the stirring continued
at 100–110 °C for 3 h. The reaction was cooled to room tempera-
ture, diluted with CH2Cl2–MeOH (95:5) and filtered through Celite.
The filtrate was washed with water and brine, dried and concen-
trated under reduced pressure. Purification of the product by FCC
All solvents were HPLC grade and used as such, except for any sol-
vents used in synthetic procedures (dried as necessary for each pro-
cedure). Unless otherwise noted, materials were obtained from
commercial suppliers and used without further purification. Flash
columnchromatography (FCC):silicagel, grade60, 230–400 lm. Or-
ganic extracts were dried over Na2SO4 and the solvents were re-
moved under vacuum using a rotary evaporator. HPLC analysis
was carried out with Agilent high performance liquid chromato-
graphs equipped with quaternary pump, automatic injector, and

M.S.C. Pedras, S. Hossain / Phytochemistry 72 (2011) 2308–2316
2315
over silica using EtOAc:hexane (3:1) as the eluent yielded pure 4-
methoxyindole-3-carboxaldehyde (62.1 mg, 75%).
activity assays. Glucobrassicin (1) in buffer (500
added to the cell-free extract (4.5 ml) and samples (1 ml) were col-
l
l, 2.1 mM) was
A solution of NaHClO2 (870.5 mg, 8.0 mmol) and NaH2PO4Á2H2O
(940.2 mg, 6.0 mmol) in H2O was added to a solution of 4-meth-
oxyindole-3-carboxaldehyde (70.2 mg, 0.40 mmol) in t-BuOH
(4.2 ml) and 2-methoxy-2-butene (4.2 ml) at 0 °C. After stirring
at r.t. for 48 h, the reaction mixture was extracted with Et2O
(30 ml  2), dried and the product purified by FCC over silica using
CH2Cl2–CH3OH (99:1), to yield 69.5 mg (91%) of the desired com-
pound 33 (Somei et al., 2001).
lected after 1, 3 and 6 h, extracted with Et2O (2 Â 2 ml), dried, dis-
solved in CH3OH (200 ll) and analyzed by HPLC. The specific
activity was calculated as the number of nmoles of product (indo-
lyl-3-methanol (22) and diindolyl-3-methane (25)) formed per mg
of protein per min. The amounts of product formed were quantified
using calibration curves built with authentic synthetic samples.
The Bradford assay method was used to calculate the protein
content of cell-free extracts using a standard BSA calibration
curve (wavelength 595 nm). Similar assays were carried out using
mycelia grown in the absence of 1-methoxyglucobrassicin (2).
HPLC tR = 5.2 min (method A). 1H NMR d (500 MHz, CD3CN):
11.6 (1H, s), 10.2 (1H, s, br), 7.99 (1H, d, J = 3.1 Hz), 7.24–7.20
(2H, m), 6.84–6.80 (1H, m), 4.09 (3H, s). 13C NMR (125.8 MHz,
CD3CN) 165.0, 152.0, 139.7, 135.1, 125.3, 114.9, 109.1, 108.4,
103.8, 57.7. HREI-MS m/z: calc. for C10H9NO3: 191.0582, found
191.0583; m/z (%): 191.1 [M]+ (100), 172.0 (12), 162.1 (8), 144.0
(33), 132.0 (17), 118.1 (15), 104.1 (64). UV (HPLC, MeOH–H2O) kmax
Acknowledgements
We thank P.B. Chumala for HPLC–MS data, K. Thoms for HRMS-
ESI data and K. Brown for NMR data. Financial support for the
authors’ work was obtained from the Natural Sciences and Engi-
neering Research Council of Canada (Discovery Grant to M.S.C.P.),
the Canada Research Chairs program, Canada Foundation for Inno-
vation, the Saskatchewan Government, and the University of Sas-
katchewan (teaching assistantship to S.H.).
(nm): 210, 230, 290. FTIR (KBr, cmÀ1
1245, 1089, 740, 735.
) mmax: 3183, 1696, 1526, 1395,
4.3. Mycelial radial growth antifungal bioassays
Three-day-old cultures of S. sclerotiorum, 4-day-old cultures of
R. solani and 7-day-old cultures of A. brassicicola grown on PDA un-
der constant light at 23 1 °C were used for mycelial radial growth
assays. Plugs (4 mm) were cut from the edges of mycelia and
placed inverted onto six-well agar plates (for S. sclerotiorum and
R. solani) or twelve-well agar plates (A. brassicicola) amended with
test compounds (dissolved in DMSO or H2O). The final concentra-
tions of each compound in agar varied from 0.10 to 0.50 mM, with
a DMSO or H2O concentration of 1%. The plates were allowed to
grow under constant light at 23 1 °C; the diameter of the mycelial
mat was measured after 24 h (S. sclerotiorum), 48 h (R. solani) and
96 h (A. brassicicola) and compared to control mycelia grown on
plates containing DMSO or H2O only.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.phytochem.2011.08.018.
References
Agerbirk, N., De Vos, M., Kim, J.H., Jander, G., 2009. Indole glucosinolate breakdown
and its biological effects. Phytochem. Rev. 8, 101–120.
Ahuja, I., Rohloff, J., Bones, A.M., 2010. Defence mechanisms of Brassicaceae:
implications for plant–insect interactions and potential for integrated pest
management. Agron. Sustain. Dev. 30, 311–348.
Aires, A., Mota, V.R., Saavedra, M.J., Monteiro, A.A., Simoes, M., Rosa, E.A.S., Bennett,
R.N., 2009. Initial in vitro evaluations of the antibacterial activities of
glucosinolate enzymatic hydrolysis products against plant pathogenic
bacteria. J. Appl. Microb. 106, 2096–2105.
4.4. Liquid fungal cultures and metabolism studies
Bolton, M.D., Thomma, B.P.H.J., Nelson, B.D., 2006. Sclerotinia sclerotiorum (lib.) de
Bary: biology and molecular traits of a cosmopolitan pathogen. Mol. Plant
Pathol. 7, 1–16.
Bones, A.M., Rossiter, J.T., 2006. The enzymic and chemically induced
decomposition of glucosinolates. Phytochemistry 67, 1053–1067.
Brader, G., Mikkelsen, M.D., Halkier, B.A., 2006. Altering glucosinolate profiles
modulates disease resistance in plants. Plant J. 46, 758–767.
Chevolleau, S., Gasc, N., Rollin, P., Tulliez, J., 1997. Enzymatic, chemical, and thermal
breakdown of 3H-labeled glucobrassicin, the parent indole glucosinolate. J.
Agric. Food Chem. 1997 (45), 4290–4296.
Cooke, D.L., Jenkins, P.D., Lewis, D.M., 1997. Production of phytotoxic spore
germination liquids by Alternaria brassicae and A. brassicicola and their effect
on species of the family Brassicaceae. Ann. Appl. Biol. 131, 413–426.
Fahey, J.W., Zalcmann, A.T., Talalay, P., 2001. The chemical diversity and distribution
of glucosinolates and isothiocyanates among plants. Phytochemistry 56, 5–51.
Fenwick, G.R., Heaney, R.K., 1983. Glucosinolates and their breakdown products in
cruciferous crops, foods and feeding stuffs. Food Chem. 11, 249–271.
Galletti, S.S., Leoni, E.O., Cinti, S., Cerato, C., 2008. Aspergillus flavus transformation of
glucosinolates to nitriles by an arylsulfatase and a b-thio-glucosidase. Soil Biol.
Biochem. 40, 2170–2173.
Góralska, K., Dynowska, M., Ciska, E., 2009. Fungistatic properties of glucosinolates
– a reconnaissance study. Polish J. Environ. Stud. 18, 377–382.
Grose, K.R., Bjeldanes, L.F., 1993. Oligomerization of indole-3-carbinol in aqueous
acid. Chem. Res. Toxicol. 5, 188–193.
Halkier, B.A., Gershenzon, J., 2006. Biology and biochemistry of glucosinolates.
Annu. Rev. Plant Biol. 57, 303–333.
Liquid cultures of each pathogen were grown in 250 ml Erlen-
meyer flasks containing 100 ml of minimal medium inoculated
fungal spores for a final concentration of 1 Â 106 per 100 ml for
A. brassicicola, or six mycelial plugs (6 mm diameter) per 100 ml
for R. solani and S. sclerotiorum, and cultures were incubated on a
shaker at 120 rpm. After 48–72 h at 23 1 °C, under constant light
the cultures were filtered aseptically and the mycelia washed with
sterile water. Then the mycelia from each culture were transferred
to 250 ml flasks containing sterile water (100 ml), and a solution of
each compound (1–3, 10–12, 16, 22, 25, 28, and 29) in sterile water
or CH3CN (100–250 ll depending on solubility) was added to the
cultures, for a final concentration of 0.10 mM. Similar control solu-
tions containing compounds only or mycelia were prepared. The
flasks were returned to the shaker, and samples (5–10 ml) were
withdrawn at various times and immediately frozen and lyophi-
lized. The residue was dissolved in MeOH, CH3CN, or MeOH–H2O
and analyzed by HPLC, using method A for compounds 10–12,
16, 22, and 25, and method B for compounds 1–3, 28, and 29, as de-
scribed previously (Pedras et al., 2010).
Hopkins, R.J., van Dam, N.M., van Loon, J.J.A., 2009. Role of glucosinolates in insect–
plant relationships and multitrophic interactions. Annu. Rev. Entomol. 54, 57–
83.
4.5. Myrosinase activity
Luebeck, M., 2004. Molecular characterization of Rhizoctonia solani. Appl. Mycol.
Biotechnol. 4, 205–224.
Manici, L.M., Lazzeri, L., Palmieri, S., 1997. In vitro fungitoxic activity of some
glucosinolates and their enzyme derived products toward plant pathogenic
fungi. J. Agric. Food Chem. 45, 2768–2773.
Mikkelsen, M.D., Petersen, B.L., Glawischnig, E., Jensen, A.B., Andreasson, E., Halkier,
B.A., 2003. Modulation of CYP79 genes and glucosinolate profiles in Arabidopsis
by defense signaling pathways. Plant Physiol. 131, 298–308.
For induction, 5-day-old fungal cultures were incubated with 1-
methoxyglucobrassicin (2) to a final concentration of 0.1 mM for
24 h. The mycelia were filtered, washed and dried under vacuum,
weighed and frozen immediately. The frozen mycelia (6.5 g) were
ground with NaH2PO4 buffer (pH 7.0, 13 ml) at 4 °C, centrifuged
and the supernatant (cell-free extract) was used for myrosinase

2316
M.S.C. Pedras, S. Hossain / Phytochemistry 72 (2011) 2308–2316
Nagarajan, R., Perumal, P.T., 2004. Potassium hydrogen sulfate-catalyzed reactions
of indoles: a mild, expedient synthesis of bis-indolylmethanes. Chem. Lett. 33,
288–289.
Ohtsuru, M., Tsuruo, I., Hata, T., 1973. The production and stability of intracellular
myrosinase from Aspergillus niger. Agric. Biol. Chem. 37, 967–971.
Pedras, M.S.C., 2008. The chemical ecology of crucifers and their fungal pathogens:
boosting plant defenses and inhibiting pathogen invasion. Chem. Rec. 8, 109–
115.
Piironen, E., Virtanen, A.I., 1962. The synthesis of ascorbigen from ascorbic acid and
3-hydroxymethylindole. Acta Chem. Scand. 16, 1286–1287.
Rakariyatham, N., Butr-Indr, B., Niamsup, H., Shank, L., 2006. Improvement of
myrosinase activity of Aspergillus sp. NR4617 by chemical mutagenesis.
Electron. J. Biotechnol. 9, 379–385.
Rimmer, S.R., Buchwaldt, L., 1995. Diseases. In: Kimber, D.S., McGregor, D.I. (Eds.),
Brassica Oilseeds: Production and Utilization. CAB International, Wallingford,
Oxon, UK, pp. 111–140.
Pedras, M.S.C., Ahiahonu, P.W.K., 2005. Metabolism and detoxification of phyto-
alexins and analogs by phytopathogenic fungi. Phytochemistry 66, 391–411.
Pedras, M.S.C., Montaut, S., 2003. Probing metabolic pathways in fungal pathogens
Robertson, A.A.B., Botting, N.P., 1999. Synthesis of deuterium labeled
desulfoglucosinolates as internal standards of LC–MS analysis. Tetrahedron
55, 13269–13284.
of
crucifers:
biotransformation
of
indole-3-acetaldoxime,
4-
Rollin, P., Tatibouet, A., 2011. Glucosinolates: the synthetic approach. C. R. Chim. 14,
194–210.
Shikita, M., Fahey, J.W., Golden, T.R., Holtzclaw, W.D., Talalay, P., 1999. An unusual
case of ‘uncompetitive activation ’ by ascorbic acid: purification and kinetic
properties of a myrosinase from Raphanus sativus seedlings. Biochem. J. 341,
725–732.
hydroxyphenylacetaldoxime, and their metabolites. Bioorg. Med. Chem. 11,
3115–3120.
Pedras, M.S.C., Okinyo, D.P.O., 2008. Remarkable incorporation of the first sulfur
containing indole derivative: another piece in the biosynthetic puzzle of
crucifer phytoalexins. Org. Biomol. Chem. 6, 51–54.
Pedras, M.S.C., Suchy, M., 2005. Detoxification pathways of the phytoalexins
brassilexin and sinalexin in Leptosphaeria maculans: isolation and synthesis of
the elusive intermediate 3-formylindolyl-2-sulfonic acid. Org. Biomol. Chem. 3,
2002–2007.
Pedras, M.S.C., Yaya, E.E., 2010. Phytoalexins from Brassicaceae: news from the
front. Phytochemistry 71, 1191–1197.
Pedras, M.S.C., Yaya, E., Glawischnig, E., 2011. The phytoalexins from cultivated and
wild crucifers: Chemistry and biology. Nat. Prod. Rep., 28, 1381–1405.
Pedras, M.S.C., Yaya, E., Hossain, S., 2010. Unveiling the phytoalexin biosynthetic
puzzle in salt cress: unprecedented incorporation of glucobrassicin into
wasalexins A and B. Org. Biomol. Chem. 8, 5150–5158.
Pedras, M.S.C., Zheng, Q.A., Gadagi, R.S., Rimmer, S.R., 2008. Phytoalexins and polar
metabolites from the oilseeds canola and rapeseed: differential metabolic
responses to the biotroph Albugo candida and to abiotic stress. Phytochemistry
69, 894–910.
Smith, C.J., 1996. Accumulation of phytoalexins: defence mechanism and stimulus
response system. New Phytol. 132, 1–45.
Smits, J.P., Knol, W.B.J., 1993. Glucosinolate degradation by Aspergillus clavatus and
Fusarium oxysporum in liquid and solid-state fermentation. Appl. Microbiol.
Biotechnol. 38, 696–701.
Somei, M., Tanimoto, A., Orita, H., Yamada, F., Toshiharu, O., 2001. Synthesis of
wasabi phytoalexin (methyl-1-methoxyindole-3-carboxylate) and its 5-iodo
derivative, and their nucleophilic substitution reactions. Heterocycles 54, 425–
432.
Sønderby, I.E., Geu-Flores, F., Halkier, B.A., 2010. Biosynthesis of glucosinolates –
gene discovery and beyond. Trends Plant Sci. 15, 283–290.
Traka, M., Mithen, R., 2009. Glucosinolates, isothiocyanates and human health.
Phytochem. Rev. 8, 269–282.
Vig, A.P., Rampal, G., Thind, T.S., Arora, S., 2009. Bio-protective effects of
glucosinolates – a review. LWT – Food Sci. Technol. 42, 1561–1572.
Vishwanath, S.J.K., Singh, M.P., Awasthi, R.P., 1999. Induction of resistance in
mustard (Brassica juncea) against Alternaria black spot with an avirulent
Alternaria brassicae isolate-D. Eur. J. Plant Pathol. 105, 217–220.
Pedras, M.S.C., Zheng, Q.A., Sarma-Mamillapalle, V.K., 2007. The phytoalexins from
Brassicaceae: structure, biological activity, synthesis and biosynthesis. Nat.
Prod. Commun. 2, 319–330.