Metabolite profiling of Arabidopsis seedlings in response to exogenous sinalbin and sulfur deficiency

Article abstract of DOI:10.1016/j.phytochem.2011.06.002

In order to determine how plant uptake of a sulfur-rich secondary metabolite, sinalbin, affects the metabolic profile of sulfur-deficient plants, gas chromatography time-of-flight mass spectrometry (GC-TOF-MS), in combination with liquid chromatography-mass spectrometry (LC-MS), was used to survey the metabolome of Arabidopsis seedlings grown in nutrient media under different sulfur conditions. The growth media had either sufficient inorganic sulfur for normal plant growth or insufficient inorganic sulfur in the presence or absence of supplementation with organic sulfur in the form of sinalbin (p-hydroxybenzylglucosinolate). A total of 90 metabolites were identified by GC-TOF-MS and their levels were compared across the three treatments. Of the identified compounds, 21 showed similar responses in plants that were either sulfur deficient or sinalbin supplemented compared to sulfur-sufficient plants, while 12 metabolites differed in abundance only in sulfur-deficient plants. Twelve metabolites accumulated to higher levels in sinalbin-supplemented than in the sulfur-sufficient plants. Secondary metabolites such as flavonol conjugates, sinapinic acid esters and glucosinolates, were identified by LC-MS and their corresponding mass fragmentation patterns were determined. Under sinalbin-supplemented conditions, sinalbin was taken up by Arabidopsis and contributed to the endogenous formation of glucosinolates. Additionally, levels of flavonol glycosides and sinapinic acid esters increased while levels of flavonol diglycosides with glucose attached to the 3-position were reduced. The exogenously administered sinalbin resulted in inhibition of root and hypocotyl growth and markedly influenced metabolite profiles, compared to control and sulfur-deficient plants. These results indicate that, under sulfur deficient conditions, glucosinolates can be a sulfur source for plants. This investigation defines an opportunity to elucidate the mechanism of glucosinolate degradation in vivo.

Full text of DOI:10.1016/j.phytochem.2011.06.002

Phytochemistry 72 (2011) 1767–1778
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Metabolite profiling of Arabidopsis seedlings in response to exogenous sinalbin
and sulfur deficiency
a
a
a
a
a
a
a
Jixiu Zhang , Xiumei Sun , Zhiping Zhang , Yuwen Ni , Qing Zhang , Xinmiao Liang , Hongbin Xiao ,
a,
⇑
b
Jiping Chen , James G. Tokuhisa
a
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
Department of Horticulture, Virginia Tech, Blacksburg, VA 24061, USA
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received in revised form 28 May 2011
Available online 2 July 2011
In order to determine how plant uptake of a sulfur-rich secondary metabolite, sinalbin, affects the
metabolic profile of sulfur-deficient plants, gas chromatography time-of-flight mass spectrometry (GC–
TOF-MS), in combination with liquid chromatography–mass spectrometry (LC–MS), was used to survey
the metabolome of Arabidopsis seedlings grown in nutrient media under different sulfur conditions.
The growth media had either sufficient inorganic sulfur for normal plant growth or insufficient inorganic
sulfur in the presence or absence of supplementation with organic sulfur in the form of sinalbin (p-
hydroxybenzylglucosinolate). A total of 90 metabolites were identified by GC–TOF-MS and their levels
were compared across the three treatments. Of the identified compounds, 21 showed similar responses
in plants that were either sulfur deficient or sinalbin supplemented compared to sulfur-sufficient plants,
while 12 metabolites differed in abundance only in sulfur-deficient plants. Twelve metabolites accumu-
lated to higher levels in sinalbin-supplemented than in the sulfur-sufficient plants. Secondary metabo-
lites such as flavonol conjugates, sinapinic acid esters and glucosinolates, were identified by LC–MS
and their corresponding mass fragmentation patterns were determined. Under sinalbin-supplemented
conditions, sinalbin was taken up by Arabidopsis and contributed to the endogenous formation of gluc-
osinolates. Additionally, levels of flavonol glycosides and sinapinic acid esters increased while levels of
flavonol diglycosides with glucose attached to the 3-position were reduced. The exogenously adminis-
tered sinalbin resulted in inhibition of root and hypocotyl growth and markedly influenced metabolite
profiles, compared to control and sulfur-deficient plants. These results indicate that, under sulfur defi-
cient conditions, glucosinolates can be a sulfur source for plants. This investigation defines an opportu-
nity to elucidate the mechanism of glucosinolate degradation in vivo.
Keywords:
Arabidopsis thaliana
Cruciferae
GC–TOF-MS
LC–MS
Metabolite profiling
Sinalbin
Sulfur
Ó 2011 Elsevier Ltd. All rights reserved.
1
. Introduction
sulfur deficiencies that include changes in glucosinolate metabo-
lism (Hirai et al., 2004; Nikiforova et al., 2005).
Sulfur is one of the essential macroelements in plants and is
assimilated by the roots as sulfate from the soil (Leustek et al.,
000). In planta, sulfate is first reduced to sulfide and then con-
Glucosinolates are plant secondary metabolites whose basic
skeleton consists of a b-thioglucose, an N-hydroxyiminosulfate
moiety, and a variable side-chain (R) (Halkier and Gershenzon,
2006) (Fig. 1). Glucosinolates are biochemically stable and non-
toxic compounds that accumulate in crucifers, yet they function
as chemical defense compounds. Tissue damage associated with
plant pest activity releases glucosinolates, which are hydrolyzed
by myrosinase yielding glucose, sulfate, and a variety of other
potentially cytotoxic products such as isothiocyanates, nitriles
and thiocyanates (Halkier and Gershenzon, 2006). Sulfatase is the
only other enzyme known to use all glucosinolates as substrates
converting them to their desulfated derivatives (desulfoglucosino-
lates) (Fig. 1). The enzyme is found in the guts of Plutella xylostella
larvae and the snail Helix pomatia, and modifies the ingested
glucosinolates resulting in the inactivation of the glucosinolate–
myrosinase defense system (Ratzka et al., 2002). Sinalbin (1)
2
verted into cysteine, with the latter serving as the principal sub-
strate for the biosynthesis of various other sulfur-containing
organic compounds which, in crucifer plants, include glucosino-
lates. One approach to understand sulfur metabolism has been to
compare plant metabolite differences between sulfur-depleted
and normal plants (Nikiforova et al., 2005). Under sulfur-deficient
conditions, it was anticipated that plants employ adaptive mecha-
nisms that compensate for nutrient deficiency. Interestingly,
integrative metabolomic and transcriptomic approaches have
identified aspects of the global responses of Arabidopsis plants to
⇑
Corresponding author. Tel./fax: +86 411 84379562.
E-mail address: chenjp@dicp.ac.cn (J. Chen).
0
031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2011.06.002

1
768
J. Zhang et al. / Phytochemistry 72 (2011) 1767–1778
OH
OH
OH
OH
content of endogenous glucosinolate formation was studied in
detail.
O
O
R1
S
R2
Sulfatase
S
C
N
C
N
HO
OH
HO
OH
2
. Results and discussion
OSO3H
OH
Glucosinolate
Desulfoglucosinolate
2.1. Comparison of symptoms
H2
C
1
In contrast to the morphology of 13-day-old Arabidopsis plants
grown under control conditions (+S medium) (Fig. 2A), plants
Sinalbin (p-hydroxybenzylglucosinolate) (1): R = HO
1
4
-Methylthiobutylglucosinolate (2): R = CH S (CH )
grown on inorganic sulfur-deficient (ꢀS) medium supplemented
3
2 4
with 846 lM sinalbin (1) were characterized by root and hypocotyl
H2
C
1
Benzylglucosinolate (3): R =
growth inhibition (Fig. 2B). The culturing experiments were repli-
cated six times with consistent results. Arabidopsis seedlings
grown in the presence of either other intact or desulfated glucosin-
olates, such as 4-methylthiobutylglucosinolate (2), benzylglucosin-
olate (3) and p-hydroxybenzyldesulfoglucosinolate (4) gave the
same results as plants supplemented with sinalbin (1) (data not
shown). Up to now, however little was known about the influence
of exogenous glucosinolates on plant morphology. A report by
Gijzen et al. (1989) found that the addition of 2-propenylglucosin-
olate to a growth medium containing rapeseed embryos resulted in
a decline in embryo fr. wt. In the present study, Arabidopsis plants
grown on sulfur-deficient media showed typical symptoms of sul-
fur deficiency such as stunted growth, leaf chlorosis and enhanced
root growth (Fig. 2C). Enhanced root growth is a developmental re-
sponse of plants to nutrient deficiency (Forde and Lorenzo, 2001;
Kutz et al., 2002).
H2
2
Desulfosinalbin (p-hydroxybenzyldesulfoglucosinolate) (4): R = HO
C
Fig. 1. Structures of glucosinolates and desulfoglucosinolates.
(
(
p-hydroxybenzylglucosinolate) is found in seeds of white mustard
Thies, 1988). The variable side-chain of the sinalbin structure (1)
is a p-hydroxybenzyl moiety derived from the amino acid tyrosine
Fig. 1). Because glucosinolates contain at least two sulfur atoms in
(
their structure, they potentially have an important role in sulfur
storage. Maruyama-Nakashita et al. (2003) regard the glucosino-
lates accumulating in root tissues as an alternative source for sul-
fur assimilation under sulfur-deficient conditions. Glucosinolates,
such as sinalbin (1), that have been purified and administered to
plants, are taken up, transported, and have been observed in the
phloem exudates of Arabidopsis petioles (Chen et al., 2001). How-
ever, the effect of exogenous glucosinolate on the metabolite pro-
file of sulfur-deficient plants is still unknown, as in the case when
glucosinolates are broken down in intact plants through the activ-
ity of myrosinase or other unknown factors.
It is a great challenge to obtain the whole metabolome using a
single analytical technique. GC–MS gives possibilities of analyzing
different classes of compounds, including organic and amino acids,
sugars, sugar alcohols, sterols, phosphorylated and lipophilic com-
pounds (Wagner et al., 2003; Lisec et al., 2006). However, LC–MS is
also a powerful technology to separate and analyze semi-polar sec-
ondary metabolites that can comprise large and unique groups of
compounds in plants (Brown et al., 2003; von Roepenack-Lahaye
et al., 2004; Stobiecki et al., 2006; de Vos et al., 2007). Therefore,
the combination of GC–MS and LC–MS allows for a broad view of
metabolic differences when comparing plants grown on different
nutrient media. In the present study, GC–TOF-MS and LC–MS were
used as profiling methods to study whether exogenously adminis-
tered glucosinolate, such as sinalbin (1), could be metabolized in
sulfur-deficient plants as a sulfur source and how such sulfur-
containing organic compounds affect the metabolic profiles under
sulfur-deficient conditions. Also, the effect of sinalbin (1) on the
2.2. Comparison of metabolite profiles obtained with GC–TOF-MS
To investigate differences in the primary metabolite levels of
samples prepared from plants grown on different nutrient media,
whole seedlings (eight biological replicates) were harvested and ex-
tracted, and the extract samples derivatized and analyzed. The aver-
age relative concentration of a metabolite was compared by
ꢀS/+S
calculating the response ratio (R or R+sinalbin/+s) of the average rel-
ative concentration on ꢀS mediumor +sinalbin medium to thaton +S
medium as a control. When the relative ratio (R or R+sinalbin/+s)
ꢀS/+S
was more than 2.0 or less than 0.5 with P < 0.05, the relative concen-
tration of metabolites was considered to be significantly altered,
which was similar to the criterion designated by Nikiforova et al.
(2005). The differences in the metabolite levels obtained with
GC–TOF-MS are listed in Table 1. A total of 90 metabolites
(1, 5–92) were identified, but the levels of some sulfur-containing
compounds (e.g., cysteine and methionine) were below limits of
detection.
Twenty-one metabolites (5–25) had similar responses (increased
or decreased abundance) to both sulfur-deficient and sinalbin-
supplemented nutrient conditions relative to the sulfur-sufficient
Fig. 2. The morphological appearance of 13-day-old Arabidopsis grown on Murashige and Skoog media with the sole sulfur source as (A) standard 846
medium, control), (B) 846
M sinalbin (1) (+sinalbin medium) or (C) no sulfur (ꢀS medium).
lM sulfate (+S
l

J. Zhang et al. / Phytochemistry 72 (2011) 1767–1778
1769
Table 1
Differences in metabolite levels obtained by GC–TOF-MS of extracts from Arabidopsis seedlings grown on different sulfur-containing media.
-S Avga
R
+Sinalbin/+S P
value
R
-S/+S
b
-S/+S P
value
No.
Name
Substance class
+S
Avg
+Sinalbin
Avg
+Sinalbin/
b
a
a
+S
1
5
6
7
8
9
Sinalbin
Glucosinolate
Amino acid
Amino acid
Amine
-
303.97
27.19
77.43
.-
26.18
94.31
-
-
-
-
Ornithine
b-Alanine
Putrescine
Raffinose
Mannose
Trehalose
Xylose
myo-Inositol
L(+)-ascorbate
Threonate
Glycerate
104.51
32.21
10.36
115.01
269.34
61.37
4.89
0.26
2.40
2.39
3.41
2.80
3.52
2.15
2.83
8.93
4.84
3.92
3.58
4.04
3.18
2.83
0.0446
7.54E-06
8.01E-06
0.0005
0.0016
1.29E-06
0.0174
3.61E-09
0.0036
3.04E-05
0.0002
0.0229
0.0097
0.0229
0.0025
0.25
2.93
3.01
11.32
3.05
2.07
4.60
2.07
6.99
2.03
5.80
3.75
0.0392
8.42E-07
0.0042
0.0005
0.0010
0.0025
0.0129
0.0009
0.0044
0.0002
1.31E-05
0.0009
0.0006
0.0167
0.0002
24.72
31.15
Trisaccharide
Monosaccharide
Disaccharide
Monosaccharide
Sugar alcohol
Organic acid
Organic acid
Organic acid
Organic acid
Amino acid
Amino acid
Amino acid
392.00
754.26
216.19
10.54
156.33
27.68
192.36
97.06
129.49
50.86
1301.70
822.63
126.81
22.49
114.23
21.65
10
11
12
13
14
15
16
17
18
19
55.25
3.10
39.71
24.75
36.14
12.60
4.81
80.66
143.54
135.36
1407.47
185.41
500.41
a
-Ketoglutarate
O-Acetyl- -serine
N-Acetyl- -serine
L
111.67
38.53
6.41
L
15.29
221.05
L
L
L
-Serine
78.13
2
2
0
1
-Threonine
-Isoleucine
Amino acid
Amino acid
29.96
51.24
79.12
190.03
2.64
0.0174
0.0041
6.34
7.62E-05
0.0054
909.00
1804.86 17.74
35.22
2
2
2
2
2
2
3
4
5
6
5-Oxoproline
Glutamine
1-Ethylglucopyranoside
Glucopyranose
Organic acid
Amino acid
Monosaccharide
Monosaccharide
Amino acid
415.57
22.79
4.62
298.01 1007.69
53.31
874.14
152.37
31.25
1122.28
183.59
10.69
1142.04
394.10
2.10
6.69
6.76
3.38
2.72
1.91E-05
0.0133
0.0014
1.34E-05
0.0817
2.70
8.06
2.31
3.83
7.39
9.05E-05
0.0035
0.0150
0.0004
0.0010
L
-Proline
144.96
2
2
7
8
Glycine
Amino acid
Amino acid
67.04
9.42
128.83
12.35
222.43
87.13
1.92
1.31
2.04E-05
0.6676
3.32
9.25
2.26E-06
0.0009
L
L
L
L
-Tryptophan
-Tyrosine
-Valine
2
3
3
9
0
1
Amino acid
Amino acid
Amino acid
49.56
26.55
44.78
55.33
47.26
67.16
213.24
210.52
132.68
1.12
1.78
1.50
0.6962
0.1280
0.0432
4.30
7.93
2.96
0.0029
0.0016
0.0006
-Asparagine
3
3
3
2
3
4
4-Aminobutyrate
Citrate
Fumarate
cis-Sinapinate
trans-Sinapinate
Organic acid
Organic acid
Organic acid
Organic acid
Organic acid
172.31
13.23
210.42
8.75
173.49
14.10
276.18
17.10
452.07
34.38
490.91
19.76
1.01
1.07
1.31
1.96
2.47
0.9634
0.5432
0.0320
0.0012
0.0024
2.62
2.60
2.33
2.26
1.66
2.84E-06
7.47E-05
0.0051
0.0096
0.1282
cis -35.
trans -
214.59
530.37
355.90
3
5
3
3
3
3
6
7
8
9
Erythronate
Maleate
Fructose
2-(4-
Organic acid
Organic acid
Monosaccharide
Nitrile
3.46
30.48
142.38
-
4.93
43.90
121.94
122.79
23.97
76.95
70.84
-
1.43
1.44
0.86
-
0.2762
0.1376
0.1052
-
6.93
2.52
0.50
-
2.65E-05
0.0015
3.85E-06
-
Hydroxyphenyl)acetonitrile
40
41
42
L
L
L
-Homoserine
-Phenylalanine
-Alanine
Amino acid
Amino acid
Amino acid
14.48
2.65
44.69
10.47
66.77
10.90
4.18
3.09
3.94
2.06
0.0018
0.0108
0.0089
0.75
1.58
1.88
0.5123
0.1134
0.0037
32.49
61.00
4
4
4
4
4
3
4
5
6
7
Sucrose
Maltose
Galactinol
Sorbitol
Fructose-6-phosphate
Disaccharide
Disaccharide
Sugar alcohol
Sugar alcohol
Phosphorylated
compound
57.25
1.19
28.42
2.09
193.11
3.03
58.38
6.00
87.46
1.15
39.11
2.66
3.37
2.55
2.05
2.86
2.00
7.55E-09
0.0197
9.95E-07
4.19E-05
0.0003
1.53
0.97
1.38
1.27
1.19
0.0212
0.9351
0.0897
0.3838
0.2502
5.09
10.18
6.05
48
49
50
L
-Glycerol-3-phosphate
Phosphorylated
compound
Phosphorylated
compound
Phosphorylated
compound
16.56
16.50
4.38
57.57
24.17
4.21
26.84
12.02
5.00
3.48
1.47
0.96
0.0003
0.1815
0.7484
1.62
0.73
1.14
0.0262
0.3970
0.4323
Glucose-6-phosphate
myo-Inositol-1-phosphate
5
5
5
5
5
5
1
2
3
4
5
6
Gluconate
Glucose
Dehydroascorbate
Erythritol
Shikimate
Organic acid
Monosaccharide
Organic acid
Sugar alcohol
Organic acid
Amino acid
12.91
165.33
41.10
7.27
2.56
77.11
13.10
153.57
80.04
2.48
4.57
77.03
12.16
116.09
37.03
0.60
2.81
331.79
1.01
0.93
1.95
0.34
1.78
1.00
0.9113
0.7336
5.50E-05
0.4278
1.56E-07
0.9983
0.94
0.70
0.90
0.08
1.10
4.30
0.7287
0.1987
0.4859
0.2668
0.2236
0.1198
L
-Leucine
5
5
7
8
Malate
Organic acid
Amino acid
330.15
372.58
370.76
536.74
439.07
569.65
1.12
1.44
0.2446
0.2321
1.33
1.53
0.0418
0.1471
L
L
L
-Asparate
-Lysine
5
6
9
0
Amino acid
Amino acid
19.23
16.13
27.36
0.84
1.32
0.6710
0.4442
1.42
1.76
0.4005
0.1042
-Glutamate
413.94
544.91
726.63
6
6
6
6
6
6
1
2
3
4
5
6
Ornithine; arginine
Spermidine
Dotriacontanol
Phytol
Triacontanol
9,12-(Z,Z)-Octadecadienoate
Amino acid
Amine
Alcohol
Alcohol
Alcohol
5.25
5.84
17.61
141.25
19.56
133.01
4.90
5.96
24.16
105.06
22.64
142.36
3.72
2.12
17.01
97.20
23.10
124.26
0.93
1.02
1.37
0.74
1.16
1.07
0.8355
0.9803
0.0923
0.1080
0.3610
0.6700
0.71
0.36
0.97
0.69
1.18
0.93
0.1203
0.3290
0.8819
0.0665
0.4744
0.7557
Fatty acid
(
continued on next page)

1
770
J. Zhang et al. / Phytochemistry 72 (2011) 1767–1778
Table 1 (continued)
b
No.
Name
Substance class
Fatty acid
+S
Avg
+Sinalbin
Avg
-S Avga
45.49
R
+Sinalbin/
b
+Sinalbin/+S P
value
R
-S/+S
-S/+S P
value
a
a
+S
6
7
9,12,15-(Z,Z,Z)-
Octadecatrienoate
Hexadecanoate
Octadecanoate
Oleate
1-Monohexadecanoylglycerol
Phosphorate
1,6-Anhydroglucose
Ribose
4-Hydroxybenzoate
Glycolate
Lactate
Glutarate
Ribonate
42.85
50.96
1.19
0.2549
1.06
0.7928
6
6
7
7
7
7
7
7
7
7
7
7
8
8
9
0
1
2
3
4
5
6
7
8
9
0
Fatty acid
Fatty acid
Fatty acid
Glyceride
Inorganic acid
Monosaccharide
Monosaccharide
Organic acid
Organic acid
Organic acid
Organic acid
Organic acid
Organic acid
126.13
16.43
5.70
164.42
122.35
9.48
121.55
22.14
8.16
178.57
78.51
6.21
123.82
21.65
4.18
120.65
235.55
6.98
0.96
1.35
1.43
1.09
0.64
0.66
1.05
2.33
2.06
1.41
0.49
1.43
1.81
0.7507
0.0640
0.0147
0.7071
0.0071
0.0973
0.9485
0.0625
0.0539
0.1799
0.4522
0.0001
0.1852
0.98
1.32
0.73
0.73
1.93
0.74
0.67
1.31
0.93
1.50
0.37
1.27
1.59
0.9160
0.2404
0.0720
0.1834
0.0166
0.1823
0.6671
0.6933
0.8906
0.1419
0.3571
0.0375
0.2539
9.09
8.44
9.57
6.08
19.63
66.40
519.67
0.85
35.67
80.64
11.04
30.17
552.64
0.63
31.77
70.75
32.29
368.41
1.71
24.98
44.50
trans-Threonic acid-1,4-
lactone
8
1
Sorbitol-6-phosphate
Phosphorylated
compound
Sterol
Sterol
Sterol
Sterol
Sterol
Sterol
Sugar alcohol
Sugar alcohol
Sugar alcohol
Thiol
9.03
17.66
38.28
1.96
0.4852
4.24
0.3578
82
83
84
85
86
87
88
89
90
91
a
-Tocopherol
89.56
59.62
29.63
26.21
6.75
25.65
9.04
3.00
123.98
68.60
47.66
26.13
7.60
17.72
9.89
2.79
156.95
71.63
40.55
30.04
7.41
27.05
13.15
0.20
1.38
1.15
1.61
1.00
1.13
0.69
1.09
0.93
0.60
0.92
0.0754
0.1083
0.0015
0.9760
0.3440
0.0074
0.5167
0.9129
0.1758
0.6630
1.75
1.20
1.37
1.15
1.10
1.05
1.45
0.07
0.96
1.89
0.0363
0.3360
0.2244
0.4526
0.6009
0.7867
0.1015
0.1422
0.9017
0.1060
b-Sitosterol
b-Tocopherol
Campesterol
Cholesterol
Stigmasterol
Octacosanol
Threitol
Xylitol
7.00
38.21
4.19
35.29
6.72
72.12
2,4,6-Tri-tert-
butylbenzenethiol
Urea
9
2.
Urea
778.16
0.71
2.10
0.08
0.3363
0.01 261.6600
a
Raw GC–TOF-MS data were corrected for variations in sample loading by dividing by the peak area for ribitol, added as an internal control during sample extraction.
Values for each biological sample were normalized with respect to the mean values of technical replicates run in each batch of samples.
b
R, ratio of average relative concentration at depleted sulfur or supplemented sinalbin to average relative concentration at normal sulfur (n = 8).
media. Only ornithine (5) showed decreased accumulation levels
under both conditions. Twenty metabolites (6–25) exhibited in-
creased accumulation levels and included not only those with
important roles in plant stress resistance such as b-alanine (6),
putrescine (7), and sugars such as raffinose (8), mannose (9), treha-
lose (10) and xylose (11) but also the sugar alcohol inositol (12), plus
in Arabidopsis, and which appeared only in sinalbin-supplemented
Arabidopsis viz., sinalbin (1) and 2-(4-hydroxyphenyl)acetonitrile
(39). The metabolites showing unique responses to sinalbin (1)
supplementation included the amino acids homoserine (40), phen-
ylalanine (41), and alanine (42), the organic acid trans-sinapinic
acid (trans-35), the sugars sucrose (43) and maltose (44), the sugar
alcohols galactinol (45) and sorbitol (46), and the phosphorylated
intermediates fructose-6-phosphate (47) and glycerol-3-
phosphate (48). The two metabolites unique to Arabidopsis,
sinalbin (1) and 2-(4-hydroxyphenyl)acetonitrile (40), which is a
breakdown product of sinalbin (1) hydrolysis catalyzed by myrosi-
nase, were identified by accurate mass and mass fragment deter-
minations, and sinalbin (1) was further confirmed by its
retention time and mass spectra relative to an authentic standard.
To determine whether 2-(4-hydroxyphenyl)acetonitrile (39)
was produced either as an artifact of tissue extraction or under
normal metabolism in vivo, the sinalbin (1)-supplemented plants
were processed at room temperature to allow for either post-
harvest myrosinase-mediated hydrolysis of glucosinolates or by a
boiling water extraction which inhibits post-harvest hydrolysis.
The results showed that 2-(4-hydroxyphenyl)acetonitrile (39)
was not observed in boiling water extracts, whereas in the room
temperature extracts not only 2-(4-hydroxyphenyl)acetonitrile
(39) was observed but also other glucosinolate-derived nitriles,
including 4-(methylthio)butanenitrile, 5-(methylthio)pentanenit-
rile, 8-(methylthio)octanenitrile, 2-(1H-indol-3-yl)acetonitrile,
and 2-(4-methoxy-1H-indol-3-yl)acetonitrile (data not shown).
Alternate glucosinolate breakdown products such as isothiocya-
nates were not detected in extracts prepared at either room
temperature or with boiling water. Therefore, the 2-(4-hydroxy-
phenyl)acetonitrile (39) detected was likely a product of limited
post-harvest hydrolysis of sinalbin by myrosinase.
organic acids such as
and -ketoglutarate (16). In addition, O-acetyl-
acetyl- -serine (18) showed increases of 111- and 38-fold, respec-
tively, under sulfur-deficient conditions, while exhibiting only
L
-ascorbate (13), threonate (14), glycerate (15)
a
L
-serine (17) and N-
L
ꢁ
4.0- and ꢁ3.2-fold increases under sinalbin-supplemented condi-
tions. Other metabolites demonstrated an increasing trend under
both conditions, such as serine (19) (a metabolic precursor of cys-
teine), threonine (20) and isoleucine (21), which are metabolites de-
rived from homoserine (40),
a precursor in common with
methioninebiosynthesis, and 5-oxoproline(22), a metaboliteof pro-
line (26).
Although 21 metabolites showed similar trends under both sul-
fur-deficient and sinalbin-supplemented conditions, other metabo-
lites showed significant differences under only one or the other
sulfur-deficient condition relative to the sulfur-sufficient condition
(Table 1, Fig. 3). In response solely to inorganic sulfur deficiency, 12
metabolites (26–34, cis-35, 36–37) exhibited increased levels
whereas fructose (38) showed a slightly decreased level. These
profile differences are similar to those observed by Nikiforova
et al. (2005). The amino acids which exhibited increased levels only
in response to sulfur deficiency included proline (26), glycine (27),
tryptophan (28), tyrosine (29), valine (30), asparagine (31) and 4-
aminobutyrate (32). In addition, the TCA cycle intermediates cit-
rate (33) and fumarate (34) and the phenylpropanoid pathway
intermediate cis-sinapinic acid (cis-35) showed increased levels.
Twelve metabolites (1, trans-35, 39–48) exhibited increased
levels in response only to sinalbin (1) supplementation including
two metabolites normally found in white mustard seed but not
Many of the 20 metabolites that showed increased levels in re-
sponse to both sulfur deficiency and sinalbin supplementation

J. Zhang et al. / Phytochemistry 72 (2011) 1767–1778
1771
galactinol (45)
raffinose (8)
maltose (44)
gluconate (51)
trehalose (10)
sucrose (43)
threonate (14)
glucose (52) fructose (38)
G6P (49)
mannose (9)
L-ascorbate (13)
dehydroascorbate (53)
mannitol
xylose (11)
erythritol (54)
O-Ac-serine (17)
galactose
F6P (47)
sorbitol (46)
cysteine
inositol (12)
inositol-1-P (50)
glycine (27)
serine (19)
3PGA
glycerate (15)
glycerol
glycerol-3-P (48)
tryptophan (28)
phenylalanine (41)
tyrosine (29)
shikimate (55)
PEP
sinapinate (cis-35)
sinapinate (trans-35)
leucine (56)
valine (30)
alanine (42)
pyruvate
β-alanine (6) asparagine (31)
Acetyl-CoA
citrate (33)
isocitrate
aspartate (58)
lysine (59)
oxaloacetate
malate (57)
methionine
homoserine (40)
fumarate (34)
succinate
oxalosuccinate
α-ketoglutarate (16)
threonine (20)
isoleucine (21)
5
-oxoproline (22)
proline (26)
glutamate (60)
glutamine (23)
-aminobutyrate (32)
4
ornithine (5)
spermidine (62)
putrescine (7)
citrulline
arginine (61)
Fig. 3. Changes in the mean metabolite levels of Arabidopsis seedlings in response to sulfur deficiency and sinalbin (1) supplementation (data from Table 1) arrayed by
pathways of central metabolism. Consistent increases or decreases in metabolite concentrations (with R > 2.0, P < 0.05 or R < 0.5, P < 0.05) of both sulfur-deficient and sinalbin
(1)-supplemented plants relative to control plants are indicated with red or green letters, respectively. The metabolites showing increases only under sulfur deficiency or
sinalbin (1) supplementation are depicted in blue or pink letters, respectively, and decreases only under sulfur deficiency in orange letters. Black letters indicate no significant
change, and grey letters denote metabolites that were not measured. Abbreviations are as follows: 3PGA, 3-Phosphoglycerate; PEP, Phosphoenolpyruvate; O-Ac-serine, O-
acetyl-L-serine (17); F6P, Fructose-6-phosphate (47); glycerol-3-P, Glycerol-3-phosphate (48); G6P, Glucose-6-phosphate (49); inositol-1-P, Inositol-1-phosphate (50).
Adapted from Messerli et al. (2007).
have similar responses to other environmental stresses. Raffinose
8) and galactinol (45), which accumulate in drought-, high
salinity- and cold-treated Arabidopsis plants (Taji et al., 2002),
are involved in stress tolerance by plants exposed to abiotic stress.
Herein, however, it was demonstrated that the levels of galactinol
(Kutchan, 2001). In extracts of Arabidopsis roots and leaves, ca.
2000 different mass signals were detected by CapLC-QqTOF-MS,
and a majority of which originate from secondary metabolites
(Von Roepenack-Lahaye et al., 2004). However, many of these sec-
ondary metabolites of Arabidopsis are as yet unidentified. Some
metabolites were identified from a comprehensive analysis of mass
fragmentation patterns and by comparison with published litera-
ture (Graham, 1998; Von Roepenack-Lahaye et al., 2004; Tohge
et al., 2005; Kerhoas et al., 2006; Routaboul et al., 2006; Stobiecki
et al., 2006; Besseau et al., 2007; Stracke et al., 2007). Additionally,
compounds were identified by molecular weight determinations
(
(
45) were elevated in response to sinalbin (1) supplementation
only. O-Acetyl- -serine (17) is general regulator of gene
expression associated with sulfur nutrition (Hirai et al., 2003).
The contents of O-acetyl- -serine (17) increased significantly in
L
a
L
sulfur-deficient Arabidopsis seedlings, consistent with earlier re-
ports (Nikiforova et al., 2003, 2005). Under short-term sulfur defi-
+
ꢀ
ciency or with exogenous supplementation of O-acetyl-
17), the endogenous content of O-acetyl- -serine (17) increased
significantly in both leaves and roots of Arabidopsis (Hirai et al.,
003). Under sulfur deficiency, the decreased levels of cysteine
resulted in the accumulation of its upstream metabolites, namely
O-acetyl- -serine (17), serine (19) and glycine (27) (Nikiforova
et al., 2005). In Arabidopsis seedlings, sinalbin (1) treatment led
to increased O-acetyl- -serine (17) levels, but the content was far
lower than that found in sulfur-deficient plants. This intermediate
level of O-acetyl- -serine (17) indicates that the sulfur deficiency
L-serine
from protonated [M+H] and deprotonated [MꢀH] molecular ions
in the first order mass spectra and then MS/MS spectra where suf-
ficient collision energy was applied to obtain maximum structural
information. The most abundant secondary metabolites identified
were flavonoid glycosides (93–100) and sinapinic acid esters
(101–102) (Fig. 4, Table 2, and Supplementary Fig. I). For flavonol
conjugates, identification was facilitated by the analysis of frag-
(
L
2
L
ꢀ
L
mentation pathways of [MꢀH] ions in the negative ion mode
and the observation that glycosidic bonds (to rhamnosyl 146u
and glucosyl 162u) were cleaved sequentially and generated char-
acteristic aglycone fragments. The major fragment ions, fragmen-
tation pathways and retention times of secondary metabolites
identified in extracts of Arabidopsis seedlings are listed in Table 2
and Supplementary Fig. II. In total, eight flavonol conjugates were
identified including five diglycosides, namely, kaempferol 3-O-
glucoside 7-O-rhamnoside (93), kaempferol 3-O-rhamnoside 7-O-
rhamnoside (94), quercetin 3-O-glucoside 7-O-rhamnoside (95),
quercetin 3-O-rhamnoside 7-O-rhamnoside (96), and isorhametin
L
was milder under sinalbin (1)-supplemented conditions and that
the exogenously applied sinalbin (1) provided organic sulfur for
the plant thus partially alleviating sulfur deficiency.
2.3. Identification of secondary metabolites obtained with LC–ESI-MS-
MS
Secondary metabolites play important roles in plant develop-
ment and in plant defense against biotic and abiotic stress
3
-O-glucoside 7-O-rhamnoside (97), and three triglycosides,

1
772
J. Zhang et al. / Phytochemistry 72 (2011) 1767–1778
R1
niques could be combined to identify those metabolites in sinal-
bin-supplemented Arabidopsis seedlings (Supplementary Fig. I).
OH
O
O
O
Rha
2
.4. Comparison of metabolite profiles obtained with LC–MS
O
2
OH
R
In comparison with control plants, sinalbin (1)-supplemented
1
2
Kaempferol 3-O-glucoside 7-O-rhamnoside (93): R = H, R = Glc
plants showed significantly decreased levels of flavonol diglyco-
sides with a glucose residue substituted at C3 such as kaempferol
3-O-glucoside 7-O-rhamnoside (93), quercetin 3-O-glucoside 7-O-
rhamnoside (95) and isorhametin 3-O-glucoside 7-O-rhamnoside
(97), while flavonol diglycosides with a rhamnose residue attached
to the 3-position such as kaempferol 3-O-rhamnoside 7-O-
rhamnoside (94) and quercetin 3-O-rhamnoside 7-O-rhamnoside
1
2
Kaempferol 3-O-rhamnoside 7-O-rhamnoside (94): R = H, R = Rha
1
2
Quercetin 3-O-glucoside 7-O-rhamnoside (95): R = OH, R = Glc
1
2
Quercetin 3-O-rhamnoside 7-O-rhamnoside (96): R = OH, R = Rha
1
2
Isorhametin 3-O-glucoside 7-O-rhamnoside (97): R = OCH
3
, R = Glc
1
2
Kaempferol 3-O-rhamnosyl-glucoside 7-O-rhamnoside (98): R = H, R = Glc-Rha
1
2
Kaempferol 3-O-glucosyl-glucoside 7-O-rhamnoside (99): R = H, R = Glc-Glc
1
2
Quercetin 3-O-glucosyl-glucoside 7-O-rhamnoside (100): R = OH, R = Glc-Glc
(96) clearly increased. Furthermore, levels of all flavonol triglyco-
sides (98–100) increased while those of the sinapinic acid esters
(101–102) were only slightly elevated (Table 3).
OCH3
H3CO
HO
H3CO
O
O
OH
O
O
OH
OCH3
In sulfur-deficient plants, all flavonol glycosides and sinapinic
acid esters were found in greater amounts compared to control
plants, but the flavonol diglycosides with glucose substituted at
C3 including kaempferol 3-O-glucoside 7-O-rhamnoside (93),
quercetin 3-O-glucoside 7-O-rhamnoside (95) and isorhametin 3-
O-glucoside 7-O-rhamnoside (97) and sinapinic acid esters (101–
O
O
HO
HO
HO
OH
OH
O
Sinapoyl glucose (101)
Sinapoyl malate (102)
Fig. 4. Structures of flavonoid glycosides (93–100) and sinapinic acid esters (101–
02) identified in Arabidopsis seedlings.
1
02) were only slightly higher than those in control plants. The
1
patterns of accumulation of flavonol diglycosides with rhamnose
attached at the 3-position and the flavonol triglycosides in the sul-
fur-deficient plants were identical to those in the sinalbin (1)-sup-
plemented plants, but the flavonol diglycosides with glucose
attached at the 3-position had opposite patterns of accumulation
under the two conditions.
Flavonoids and sinapinic acid esters, compounds derived from
the phenylpropanoid pathway, fulfill important functions of the
plant in development and interactions with the environment
(Milkowski et al., 2004; Taylor and Grotewold, 2005). Flavonoids
have crucial roles in cellular protection against UV light irradiation,
as well as in maintaining cellular redox balance (Laundry et al.,
1995; Pietta, 2000). Flavonoids also serve as phytoalexins to pro-
tect against damage caused by pathogen attack (Van Etten et al.,
1994). Sinapoyl glucose (101) and sinapoyl malate (102) serve as
UV protectants and constitute the major sinapate esters in Arabid-
opsis thaliana leaves (Milkowski et al., 2004). Because each gluco-
namely, kaempferol 3-O-rhamnosyl-glucoside 7-O-rhamnoside
98), kaempferol 3-O-glucosyl-glucoside 7-O-rhamnoside (99),
and quercetin 3-O-glucosyl-glucoside 7-O-rhamnoside (100). Char-
acteristic fragment ions of sinapoyl glucose (101) and sinapoyl ma-
late (102) at 223 m/z corresponding to deprotonated sinapinic acid
were found in the product ion spectra of both compounds (101-
(
1
02). In analyses of the secondary metabolites, sinalbin (1) was
also identified by comparing its CID spectrum and retention time
with those of an authentic sinalbin (1) standard. The desulfated
form of sinalbin (1), p-hydroxybenzyldesulfoglucosinolate (4)
was not detected. From the above, it can be deduced that after
exogenous intact sinalbin (1) is assimilated, it exists in an intact
rather than a desulfated form in the plant. In sinalbin (1)-supple-
mented Arabidopsis, the presence of sinalbin (1), the absence of
desulfosinalbin (4), and unchanged tyrosine (29) levels were noted.
These metabolite levels indicate that the high levels of sinalbin (1)
did not arise by de novo biosynthesis from tyrosine (29), but rather
by uptake from the culture medium as described by Chen et al.
sinolate contains a b-D-glucopyranose residue, the uptake of
exogenous sinalbin (1) is expected to have a significant effect on
the levels of the secondary metabolites, especially flavonol glyco-
sides. To illustrate the changes in flavonoid glycoside abundance,
a simplified scheme of the biosynthetic pathway of flavonoid gly-
cosides and sinapinic acid esters in the A. thaliana seedling is
shown in Fig. 5. Tyrosine (29) and phenylalanine (41) serve as
(
2001). In addition, significant changes in the concentration of
many other metabolites were also found in sulfur-deficient or
sinalbin (1)-supplemented plants, but the metabolites remain
unidentified. In a future study, LC–ESI-MS-MS and other tech-
Table 2
The major fragmentation ions of secondary metabolites identified in extracts of Arabidopsis seedlings.
No.
Namea
RT (min)
Major fragmentation ions
+
ꢀ
]+ 344 [M-SO
+2H]+ 346 [M-G SO
+
+ 3H]+ 150
1
Sinalbin
7.58
[M+H] 426 [MꢀH] 424 [M-SO
3
3
3
+3H] 184 [M-S-Glc-SO
3
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
9
9
9
9
9
9
9
1
1
1
3
4
5
6
7
8
9
00
01
02
K 3-O-glc 7-O-rha
K 3-O-rha 7-O-rha
Q 3-O-glc 7-O-rha
Q 3-O-rha 7-O-rha
I 3-O-glc 7-O-rha
K 3-O-glc-rha 7-O-rha
K 3-O-glc-glc 7-O-rha
Q 3-O-glc-glc 7-O-rha
Sinapoyl glucose
Sinapoyl malate
39.87
45.87
35.37
40.78
41.25
31.10
35.83
31.36
26.35
44.14
[M+H] 595 [MꢀH] 593 [M-Rha-H] 447 [M-Glc-H] 431 [M-Glc-2H] 430 [M-Rha-Glc-H] 285 [M-Rha-Glc-3H] 283
+
ꢀ
ꢀ
ꢀ
ꢀ
[M + H] 579 [MꢀH] 577 [M-Rha-H] 431 [M-2Rha-H] 285 [M-2Rha-3H] 283
+ ꢀ ꢀ ꢀ
ꢀ
ꢀ
[M+H] 611 [MꢀH] 609 [M-Rha-H] 463 [M-Glc-H] 447 [M-Glc-2H] 446 [M-Rha-Glc-3H] 299
+ ꢀ ꢀ ꢀ ꢀ
ꢀ
[M+H] 595 [MꢀH] 593 [M-Rha-H] 447 [M-Rha-2H] 446 [M-2Rha-H] 301 [M-2Rha-3H] 299
+ ꢀ ꢀ ꢀ
ꢀ
[M+H] 625 [MꢀH] 623 [M-Rha-H] 477 [M-Glc-H] 461 [M-Glc-Rha-H] 315
+ ꢀ ꢀ
ꢀ
ꢀ
ꢀ
[M+H] 741 [MꢀH] 739 [M-Rha-H] 593 [M-Rha-Glc-2H] 430 [M-2Rha-Glc-2H] 284 [M-2Rha-Glc-3H] 283
+ ꢀ ꢀ ꢀ
ꢀ
[M+H] 757 [MꢀH] 755 [M-Rha-H] 609 [M-Rha-Glc-H] 447 [M-Rha-2Glc-H] 285
+ ꢀ ꢀ ꢀ
ꢀ
[M+H] 773 [MꢀH] 771 [M-Rha-H] 625 [M-2Glc-3H] 445 [M-Rha-2Glc-3H] 301
+ ꢀ ꢀ ꢀ
ꢀ
[M+H] 387 [MꢀH] 385 [M-Glc] 223 [M-O-Glc-H] 206 [M-O-Glc-2H] 205
+
ꢀ
+
ꢀ
[M+H] 341 [MꢀH] 339 [M-Mal+H] 225 [M-Mal] 223
a
Abbreviations are as follows: K 3-O-glc 7-O-rha, kaempferol 3-O-glucoside 7-O-rhamnoside (93); K 3-O-rha 7-O-rha, kaempferol 3-O-rhamnoside 7-O-rhamnoside (94); Q
-O-glc 7-O-rha, quercetin 3-O-glucoside 7-O-rhamnoside (95); Q 3-O-rha 7-O-rha, quercetin 3-O-rhamnoside 7-O-rhamnoside (96); I 3-O-glc 7-O-rha, isorhametin 3-O-
3
glucoside 7-O-rhamnoside (97); K 3-O-glc-rha 7-O-rha, kaempferol 3-O-rhamnosyl-glucoside 7-O-rhamnoside (98); K 3-O-glc-glc 7-O-rha, kaempferol 3-O-glucosyl-gluco-
side 7-O-rhamnoside (99); Q 3-O-glc-glc 7-O-rha, quercetin 3-O-glucosyl-glucoside 7-O-rhamnoside (100).

J. Zhang et al. / Phytochemistry 72 (2011) 1767–1778
1773
Table 3
Differences in secondary metabolite levels identified in extracts of Arabidopsis seedlings grown on different sulfur-containing media (n = 8).
No.
Name
+S
Avg
+Sinalbin
Avg
ꢀS
R
+sinalbin/+S
+sinalbin/+S
P value
R
ꢀS/+S
ꢀS/+S
Avg
P value
9
9
9
9
9
9
9
1
1
1
3
4
5
6
7
8
9
00
01
02
K 3-O-glc 7-O-rha
K 3-O-rha 7-O-rha
Q 3-O-glc 7-O-rha
Q 3-O-rha 7-O-rha
I 3-O-glc 7-O-rha
K 3-O-glc-rha 7-O-rha
K 3-O-glc-glc 7-O-rha
Q 3-O-glc-glc 7-O-rha
Sinapoyl glucose
29.09
14.89
67.32
2.35
14.37
10.21
3.91
1.75
12.26
33.50
19.39
52.89
12.82
5.18
56.90
53.40
94.10
9.25
18.80
27.30
11.10
4.06
0.67
3.55
0.19
2.20
0.18
9.97
2.98
4.15
1.68
1.95
0.0172
1.96
3.59
1.40
3.94
1.31
2.67
2.84
2.31
1.35
1.85
6.13E-05
0.0022
0.0027
0.0289
0.0067
0.0007
0.0039
0.0065
0.2794
0.0039
8.21E-05
1.49E-07
4.94E-05
5.51E-08
1.33E-09
1.84E-07
2.21E-06
0.0029
2.62
101.80
11.64
7.27
20.56
65.25
16.50
62.00
Sinapoyl malate
0.0003
SGT
SMT
tyrosine (29)
phenylalanine (41)
sinapinc acid (35)
sinapoyl glucose (101)
sinapoyl malate (102)
F3'H
dihydrokaempferol
FLS
dihydroquercetin
FLS
OMT
kaempferol (K)
F3GT
quercetin (Q)
F3GT
isorhamnetin (I)
GT
K 3-O-glc
K 3-O-rha
F7RT
Q 3-O-glc
F7RT
Q 3-O-rha
F7RT
I 3-O-glc
GT
F7RT
K 3-O-glc 7-O-rha
K 3-O-rha 7-O-rha
Q 3-O-glc 7-O-rha Q 3-O-rha 7-O-rha
I 3-O-glc 7-O-rha
(
93)
(94)
(95)
(96)
(97)
FGT
K 3-O-glc-rha 7-O-rha
Q 3-O-glc-glc 7-O-rha
K 3-O-glc-glc 7-O-rha
(100)
(98)
(99)
Fig. 5. Simplified schema of the biosynthesis of flavonoid glycosides (93–100) and sinapinic acid esters (101–102) in Arabidopsis seedlings. Enzymes are indicated by capital
0
letters. Abbreviations are as follows: F3 H, flavanone 3-hydroxylase; FLS, flavonol synthase; GT, glycosyltransferase; F3GT, flavonol 3-O-glycosyltransferase; F7RT, flavonol 7-
O-rhamnosyltransferase; SGT, sinapate UDP-glucose sinapoyltransferase; SMT, sinapoylglucose malate sinapoyltransferase. K, kaempferol; Q, quercetin; I, isorhamnetin; K 3-
O-glc, kaempferol 3-O-glucoside; K 3-O-rha, kaempferol 3-O-rhamnoside; Q 3-O-glc, quercetin 3-O-glucoside; Q 3-O-rha, quercetin 3-O-rhamnoside; I 3-O-glc, Isorhamnetin
3
-O-glucoside; K 3-O-glc 7-O-rha, kaempferol 3-O-glucoside 7-O-rhamnoside (93); K 3-O-rha 7-O-rha, kaempferol 3-O-rhamnoside 7-O-rhamnoside (94); Q 3-O-glc 7-O-rha,
quercetin 3-O-glucoside 7-O-rhamnoside (95); Q 3-O-rha 7-O-rha, quercetin 3-O-rhamnoside 7-O-rhamnoside (96); I 3-O-glc 7-O-rha, isorhametin 3-O-glucoside 7-O-
rhamnoside (97); K 3-O-glc-rha 7-O-rha, kaempferol 3-O-rhamnosyl-glucoside 7-O-rhamnoside (98); K 3-O-glc-glc 7-O-rha, kaempferol 3-O-glucosyl-glucoside 7-O-
rhamnoside (99); Q 3-O-glc-glc 7-O-rha, quercetin 3-O-glucosyl-glucoside 7-O-rhamnoside (100). Adapted from Jones et al. (2003), Besseau et al. (2007), Stracke et al. (2007).
precursors of flavonol glycosides and sinapinic acid esters. Tyrosine
29) levels were almost constant and phenylalanine (41) levels
family of transcriptional factors, which participate in flavonol
accumulation in the A. thaliana seedling (Nikiforova et al., 2003;
Stracke et al., 2007), are especially upregulated and could account
for the increases of these flavonol glycosides. However, flavonol
diglycosides (93, 95, 97) with a glucose attached at the 3-position
did not significantly increase in amount. For further explanation of
the levels of flavonol glycosides, it will be necessary to identify and
quantify the expression levels of genes encoding flavonoid
glycosyltransferases.
(
were elevated ꢁ3.9-fold in the sinalbin (1)-supplemented plants
in comparison with those levels in control plants. In sulfur-
deficient plants, tyrosine (29) levels were increased 4.3-fold and
phenylalanine (41) was almost unchanged. The accumulation pat-
terns of flavonol diglycosides (93–97) were similar to those de-
scribed for the ugt78d2 mutant, which lacks the ability to
glucosylate flavonols at the 3-position as UDP-glucose: flavonoid
3
-O-glucosyltransferase (Tohge et al., 2005). In the sinalbin (1)-
supplemented plants, the decreased content of flavonol diglyco-
sides with glucose attached at the 3-position (93, 95, 97) was
correlated with increased levels of their downstream products,
the flavonol triglycosides (98–100). The result indicates that
UGT78D2 is the rate-limiting enzyme in the formation of flavonol
triglycosides under sinalbin-supplementation conditions. Conse-
quently, when the activity of enzymes of the competing branch
pathway and downstream of UGT78D2 increased relative to
UGT78D2 activity, the levels of the flavonol diglycosides (93, 95,
2.5. Principal component analysis
In addition to investigating differences in levels of individual
metabolites, principal component analysis (PCA) was performed
to compare differences in metabolic profiles of Arabidopsis seed-
lings grown on different media (Fig. 6). For the primary metabolite
profiles analyzed by GC–TOF-MS, PCA was applied to all peaks. The
samples clearly separated into three groups according to the cul-
ture media (Fig. 6A), indicating that exogenous sinalbin (1) had a
great effect on metabolite profiles of Arabidopsis seedlings ob-
tained by GC–TOF-MS. Replicate analyses of samples of each indi-
vidual treatment gave very similar profiles, which validated the
high reproducibility of the experimental procedure from plant har-
vest to data analysis. When only metabolites identified by LC–MS
9
7) with a glucose attached at the 3-position were markedly re-
duced and the flavonol diglycosides (94, 96) with rhamnose at-
tached at the 3-position and flavonol triglycosides with glucose
attached at the 3-position were significantly increased in abun-
dance. Under sulfur-deficient conditions, genes of the R2R3-MYB

1
774
J. Zhang et al. / Phytochemistry 72 (2011) 1767–1778
Fig. 6. Principal component analysis of the metabolic profiles from Arabidopsis. Seedlings were grown on Murashige and Skoog media containing sulfur as 846
blue triangle, +S medium), 846
by (A) GC–TOF-MS and (B) HPLC and HPLC–MS.
lM sulfate
(
lM sinalbin (1) (green circle, +sinalbin medium), or lacking sulfur (pink diamond, ꢀS medium). The metabolites were extracted and analyzed
+
+
+
were considered, the samples formed three clusters that were
dependent on the culture media. However, the metabolite profiles
of plants grown in +S and ꢀS media showed slightly similar pat-
terns (Fig. 6B). Altogether, these results indicate that the culture
media markedly affects the metabolite profile of Arabidopsis
seedlings.
[RCN] or [RCN+H] . The [R] ion was also observed in each indole
desulfoglucosinolate. In addition, m/z 130 was used to distinguish
4-methoxy-indol-3-yl-methyldesulfoglucosinolate (103) from 1-
methoxy-indol-3-yl-methyldesulfoglucosinolate (104) as shown
in Table 4. When CH
3
Oꢀ was substituted at the 1-position, an
ion was formed corresponding to m/z 130. However, fragment
ion m/z 130 was not detected in the CID spectra of 4-methoxy-in-
dol-3-yl-methyldesulfoglucosinolate (103). For short chain ali-
phatic desulfoglucosinolates, additional fragment ions were
observed such as m/z 118 of 3-methylthiopropyldesulfoglucosino-
late (105) and 3-methylsulfinylpropyldesulfoglucosinolate (106),
m/z 132 of 4-methylthiobutyldesulfoglucosinolate (107) and 4-
methylsulfinylbutyldesulfoglucosinolate (108), which are consis-
2.6. Identification of desulfoglucosinolates
Desulfoglucosinolates (4, 103–115) were identified by diode-
array UV spectra and mass fragmentation based on published
reports (Hogge et al., 1988; Griffiths et al., 2000; Kiddle et al.,
2
001). Glucosinolates have maximal UV absorptions at 229 nm,
tent with the loss of CH
the parent ions.
3 3
S- or CH SO- and glucosyl residues from
these being the characteristic absorptions of S-C = N structures. In-
dole glucosinolates have an additional absorbance maximum near
2
65 nm due to the characteristic absorbance of the conjugated
structure of the indole ring. The major fragmentation ions and
structures of desulfoglucosinolates (4, 103–115) are listed in
Table 4 and Supplementary Fig. III. In the absence of collision-in-
duced dissociation (CID), the base peak in the MS of each desulfog-
2.7. Comparison of glucosinolate and sulfate levels
Glucosinolates (1–2, 116–127) were quantified with respect to
an added internal standard using relative response factors derived
from pure glucosinolate standards (Brown et al., 2003). The abun-
dance of individual glucosinolates (1, 2, 116–127) in Arabidopsis
seedlings cultured in different media are summarized in Table 5.
Seedlings of Arabidopsis grown in sulfur-depleted media showed
+
lucosinolate was the [M+H] ion. Each spectrum also contained a
+
+
diagnostic ion corresponding to [M+H-162] or [M+2H-162] ,
which was attributed to the loss of glucosyl moiety from the parent
ion (Matthäus and Luftmann, 2000). Desulfoglucosinolates (4, 103–
1
15) also contained prominent diagnostic ions derived from the
a marked reduction of total glucosinolates from 26.36
wt at the normal nutrient condition to 1.62 lmol/g dry wt at the
l
mol/g dry
+
+
side-chain R, which correspond to [RCNOH] or [RCNOH+H] ,
Table 4
The major fragmentation ions of desulfoglucosinolates extracted from Arabidopsis thaliana.
No.
Desulfoglucosinolate
Major fragmentation ions (m/z)a
+
+
4
p-Hydroxybenzyl
346[M+H] 184[M+2H-162] 150[RCNOH]+
+
+
+
+
+
1
1
1
1
1
1
1
1
1
1
1
1
1
03
04
05
06
07
08
09
10
11
12
13
14
15
4-Methoxy-indol-3-yl-methyl
1-Methoxy-indol-3-yl-methyl
3-Methylthiopropyl
3-Methylsulfinylpropyl
4-Methylthiobutyl
4-Methylsulfinylbutyl
5-Methylsulfinylpentyl
6-Methylsulfinylhexyl
7-Methylthioheptyl
7-Methylsulfinylheptyl
8-Methylthiooctyl
399[M+H] 237[M+2H-162] 204[RCNOH+H] 186[RCN] 160[R]
399[M+H] 237[M+2H-162] 203[RCNOH] 186[RCN] 160[R] 130[R-OCH
3
]+
+
+
+
+
+
+
+
+
328[M+H] 166[M+H-162] 118[M-162 –CH
3
3
3
3
S+H]
SO+H]
S+H]
SO+H]
+
+
+
344[M+H] 182[M+H-162] 118[M-162 –CH
+
+
+
342[M+H] 180[M+H-162] 132[M-162 –CH
+
+
+
358[M+H] 196[M+H-162] 132[M-162 –CH
+
+
+
372[M+H] 210[M+2H-162]
386[M+H] 224[M+2H-162] 174[RCN+H]+
+
384[M+H] 222[M+2H-162] 188[RCNOH] 173[RCN+H]+
+
+
+
+
+
+
400[M+H] 238[M+H-162] 188[RCN+H]
+
+
+
+
+
398[M+H] 236[M+2H-162] 202[RCNOH] 186[RCN+H]
+
+
+
+
+
8-Methylsulfinyloctyl
Indol-3-yl-methyl
414[M+H] 252[M+2H-162] 218[RCNOH] 202[RCN+H] 175[R]
+
+
+
+
369[M+H] 207[M+2H-162] 174[RCNOH+H] 156[RCN] 130[R]
a
R: side-chain.

J. Zhang et al. / Phytochemistry 72 (2011) 1767–1778
1775
Table 5
Glucosinolate contents (lmol/g dry wt) of Arabidopsis seedlings grown on Murashige and Skoog media with different sulfur content.
No.
Glucosinolate
Glucosinolate content (l
mol/g)a
+
S
ꢀS
n.d.
n.d.
n.d.
+Sinalbin
1
2
p-Hydroxybenzyl
4-Methylthiobutyl
n.d.
35.14 ± 6.25
0.10 ± 0.02
n.d.
0.09 ± 0.01
0.04 ± 0.03
0.01 ± 0.01
0.32 ± 0.10
1.37 ± 0.29
0.25 ± 0.06
0.08 ± 0.03
0.14 ± 0.09
0.57 ± 0.17
1.04 ± 0.29
7.98 ± 1.25
2.91 ± 0.47
1
1
1
1
1
1
1
1
1
1
1
1
16
17
18
19
20
21
22
23
24
25
26
27
5-Methylsulfinylpentyl
6-Methylsulfinylhexyl
3-Methylthiopropyl
3-Methylsulfinylpropyl
4-Methylsulfinylbutyl
7-Methylthioheptyl
7-Methylsulfinylheptyl
8-Methylthiooctyl
8-Methylsulfinyloctyl
Indol-3-yl-methyl
4-Methoxy-indol-3-yl-methyl
1-Methoxy-indol-3-yl-methyl
Total
n.d.
0.09 ± 0.05
0.05 ± 0.03
0.22 ± 0.16
0.08 ± 0.01
0.79 ± 0.16
0.19 ± 0.06
0.11 ± 0.01
0.15 ± 0.11
2.85 ± 0.52
1.64 ± 0.29
4.00 ± 0.25
45.43 ± 6.84
0.01 ± 0.01
0.01 ± 0.004
0.01 ± 0.005
0.01 ± 0.01
0.03 ± 0.01
0.01 ± 0.01
0.01 ± 0.002
0.16 ± 0.10
0.30 ± 0.10
1.06 ± 0.65
1.62 ± 0.85
11.56 ± 5.17
26.36 ± 5.26
a
Mean values ± SD (n = 3). n.d. Not detected.
depleted sulfur condition. Under these conditions, the content of
individual glucosinolates (1–2, 116–127) decreased to ꢁ6–19% of
control levels and several such as 4-methylthiobutylglucosinolate
tent in the plant (Kopriva, 2006; Hawkesford and de Kok, 2006).
Moreover, as there is no sulfate available from the media, the sinal-
bin (1)-supplemented plants cannot assimilate sulfate from the
culture media. In Arabidopsis seedlings, sinalbin (1) treatment
led to increased O-acetyl-serine (17) levels compared to control
plants, but the content was far lower than that found in sulfur-
deficient plants. This indicates that the state of sulfur deficiency
was alleviated, yet the sulfate content was decreased relative to
sulfur-depleted plants. These results suggest that plants can use
sinalbin (1) as a sulfur source and under sulfur-deficient conditions
glucosinolate can be catabolized to release sulfur to support pri-
mary metabolic processes, such as protein synthesis. Glucosinolate
catabolism is evident in the fate of radiolabelled sinalbin (1) taken
up by germinating Arabidopsis seeds (Petersen et al., 2002). In
seedlings at the cotyledon stage, approximately 30% of radiola-
belled glucosinolate was lost and in young plants with rosettes of
six to eight leaves, radiolabelled glucosinolate could not be de-
tected. If glucosinolate catabolism occurs, this could be accelerated
under an inorganic sulfur shortage. In sulfur-depleted seedlings,
glucosinolates decreased significantly, which further demon-
strated that glucosinolate catabolism occurs in Arabidopsis.
However, the mechanism of glucosinolate catabolism is still un-
known. Increased turnover of sinalbin (1) was found to coincide
with elevated levels in activity of the glucosinolate-degrading en-
zyme myrosinase, which indicated the possibility that there was
a continuous turnover of glucosinolates mediated by myrosinase
during development and not only upon tissue disruption (Petersen
et al., 2002). However, in extracts of sinalbin (1)-supplemented
plants, although the nitrile breakdown product of sinalbin was de-
tected and shown to be an artifact of extraction, other nitriles or
isothiocyanates generated by in vivo myrosinase activity were
(2), 5-methylsulfinylpentylglucosinolate (116), and 6-meth-
ylsulfinylhexylglucosinolate (117) decreased below the limits of
detection. In addition, substantial amounts of sinalbin
(
35.10 lmol/g dry wt) (1) accounting for 77% of the total glucosin-
olates, accumulated in Arabidopsis seedlings grown in the sulfur-
deficient media containing exogenous sinalbin (1). Compared to
control conditions, the levels of endogenous glucosinolates were
reduced in sinalbin-supplemented media, but still higher than
those measured under sulfur starvation conditions.
Sulfate contents of Arabidopsis seedlings grown on different
conditions were analyzed and the results are shown in Fig. 7. Com-
pared to control conditions, sulfate contents under sulfur-deficient
and sinalbin (1)-supplemented conditions decreased significantly.
Sulfate contents in sinalbin (1)-supplemented plants relative to
sulfur-depleted plants were also reduced.
Relative to sulfur starvation conditions, high levels of endoge-
nous glucosinolates in sulfur-deficient conditions indicated that
exogenous sinalbin (1) could be catabolized in vivo to provide a
sulfur source for glucosinolate biosynthesis. Although the applica-
tion of sinalbin (1) might lead to a stress-induced activation of the
sulfur assimilation pathway, the increase in assimilated sulfur gen-
erally accounts for only a minor proportion of the total sulfur con-
2.0
1.6
1.2
0.8
0.4
0.0
2ꢀ
not detected and the contents of SO relative to sulfur-deficient
4
plants decrease. Furthermore, Barth and Jander (2006) found that
decreased levels of glucosinolates during senescence and germina-
tion are independent of TGG1 and TGG2 myrosinases, which are
expressed in above-ground tissues. Therefore, sinalbin (1) break-
down via myrosinase activity does not provide sulfur for plants.
3
. Conclusions
In conclusion, using GC–TOF-MS and LC–MS as the methods of
+
S
+Sinalbin
-S
metabolic profiling, the metabolites in Arabidopsis thaliania grow-
ing on sulfur-deficient, sinalbin (1)-supplemented and control con-
ditions were identified and their levels compared. The comparison
of individual metabolite levels of Arabidopsis seedlings in different
ꢀ
1
Fig. 7. Sulfate content
Murashige and Skoog media with different sulfur content. Error bars represent ± SD
n = 4).
(
l
mol
g
fr. wt) of Arabidopsis seedlings grown on
(

1
776
J. Zhang et al. / Phytochemistry 72 (2011) 1767–1778
media demonstrates that the exogenously supplied glucosinolate
sinalbin (1) had large effects on metabolite profiles and Arabidop-
sis could utilize exogenous sinalbin (1) as a sulfur source. Under
sulfur-deficient conditions, glucosinolates were catabolized to re-
lease sulfur for essential functions. The preliminary findings here,
to some extent, may lead to new and important opportunities to
elucidate glucosinolate catabolism in vivo.
tra against the Golm Metabolome database (Kopka et al., 2005) and
NIST library. In addition, the retention time indices were calibrated
using the n-alkane standards, which were also helpful for metabo-
lite identification. Following identification of metabolites, the mass
spectrometry data for metabolite profiles were automatically com-
pared using XCMS software, which incorporates nonlinear reten-
tion time alignment, matched filtration, peak detection, and peak
matching (Smith et al., 2006). The parameters for XCMS were
default
settings
(http://www.metlin.scripps.edu/download/;
4
. Experimental
Nordström et al., 2006; Smith et al., 2006). The relative concentra-
tions of metabolites were determined by normalizing the area inte-
gration of a characteristic fragment ion trace to the fresh weight of
the plant material extracted and the area of the internal standard
ribitol (m/z 319). The average relative concentration was compared
by calculating the response ratios (RꢀS/+S or R+sinalbin/+s) of the aver-
age relative concentration on ꢀS medium or +sinalbin medium to
that on +S medium as a control. When the relative ratio (RꢀS/+S or
4
.1. Plant material and growth conditions
Arabidopsis thaliana ecotype Wassilewskja (seeds provided by
Prof. Qi Xie, Institute of Genetics and Developmental Biology Chi-
nese Academy of Sciences, Beijing, China) was grown in a growth
chamber on a solidified agarose medium (0.5 ꢂ Murashige-Skoog
salts for control sulfur-sufficient medium) containing 846 lM sul-
R+sinalbin/+s) was more than 2.0 or less than 0.5 with P < 0.05, the rel-
fate, where they were subjected to a photosynthetic photon flux of
1
h day at 22 °C/8-h night at 18 °C. For sulfur-deficient (0
iments, all sulfate in the sulfur-sufficient medium was replaced
with chloride as described previously (Nikiforova et al., 2003). To
investigate the use of the exogenously provided glucosinolate
sinalbin (1) as a sulfur source for plant nutrition under sulfur-
deficient conditions, the sulfate was replaced by an equimolar con-
centration of sinalbin (1). After 13 days of growth, plant material
ꢀ2
ꢀ1
ative concentration of metabolites was considered to be signifi-
cantly altered, a criterion similar to that applied by Nikiforova
et al. (2005).
00
lmol m
s
, 65% relative humidity, and a photoperiod of, 16-
M) exper-
l
4.4. Data analysis
Statistical significance of the differences in relative concentra-
tions of metabolites was estimated using statistical analyses
including principal component analysis (PCA) and t-test. Missing
was harvested and immediately frozen in liq. N
80 °C until extraction.
2
and stored at
ꢀ
data were replaced with 0 for PCA. R was the changes between rel-
i
ative concentrations in plants grown in normal medium as a con-
trol and plants grown in different media. Statistical significance
of the differences in relative concentrations was analyzed with
the t test (Microsoft Excel). Changes in relative concentrations with
R > 2.0, P < 0.05 or R < 0.5, P < 0.05 were considered to be signifi-
cantly different.
4
.2. Extraction and GC–TOF-MS analysis of metabolites
To investigate the change in primary metabolite levels, eight
plants were harvested from each of the three different media
growth conditions. Individual Arabidopsis seedlings (100–
1
2
50 mg) were homogenized under liq. N and extracted with
MeOH–CHCl –H O (2 mL, 2.5:1:1, v/v/v). Ribitol was added as an
internal standard. The extracts were shaken for 15 min and centri-
3
2
4.5. Extraction and LC–MS analysis of secondary metabolites
fuged for 5 min at 23,000g. The supernatant was then concentrated
For LC–MS analysis of metabolites, MeOH-H
2
O (1.4 mL, 80:20,
L) as
to an aqueous phase under N
were derivatized with methoxyamine hydrochloride in pyridine
50 L, 20 mg/mL), and subsequently with N-methyl-N-trimethyl-
silyltrifluoroacetamide (MSTFA) (80 L) (Fiehn et al., 2000; Wagner
et al., 2003; Weckwerth et al., 2004; Lisec et al., 2006). A mixture of
11 to C36 n-alkanes was used for the determination of retention
2
and lyophilized. The dried samples
v/v, precooled at ꢀ20 °C) containing 0.5 mM genistein (30
l
internal standard was added to freshly ground Arabidopsis seed-
lings (ca 300 mg). The extracts were sonicated at 4 °C for 15 min
and centrifuged at 4 °C and 45,000g for 5 min. The supernatant
(
l
l
was concentrated to the aqueous phase under N
2
, lyophilized,
C
and then redissolved in H O (1.4 mL) for either LC–ESI-MS or LC-
2
time indices. The GC–TOF-MS system consisted of a gas chromato-
graph (Agilent 6890) and a time of flight mass spectrometer
PDA analysis. The apparatus and operation conditions were the
same as mentioned above, except for the following parameters:
mass scan: m/z 100–1100; scan mode: positive and negative ESI
(
Waters Micromass GCT PremierTM). The GC was operated at a con-
stant flow of 1 mL/min helium and an injector temperature of
50 °C. The samples were injected with a split ratio of 10:1 onto
a 60 m DB5 column (0.25 mm i.d., 0.25 m film thickness) using
the following temperature program: 80 °C (held for 2 min), 80–
80 °C (5 °C/min), 180–250 °C (3 °C/min), 250–300 °C (5 °C/min,
mode; gradient conditions (solvent A: 0.1% v/v HCOOH/H
vent B: CH CN): 0–10 min, 5–10% B; 10–40 min, 10–20% B; 40–
0 min, 20–30% B; 50–65 min, 30–50% B; 65–80 min, 50–95% B;
UV detection wavelength: 254 nm; collision energy (CE):
2
O, sol-
2
3
l
5
1
1
2
5ꢁ35 eV. UV/VIS absorption spectra were taken in the range of
held for 20 min). The ion source temperature of the TOF mass spec-
00–600 nm. Known metabolites were identified by their mass
trometer was 200 °C and the transfer line temperature was set at
spectrometric fragmentation patterns and relevant data reported
in the literature (Graham, 1998; von Roepenack-Lahaye et al.,
+
2
50 °C. Data were acquired by EI mode in the mass range of m/z
3
0–800.
2
2
004; Tohge et al., 2005; Kerhoas et al., 2006; Routaboul et al.,
006; Besseau et al., 2007; Stracke et al., 2007). The secondary
4
.3. Automated identification and quantification of primary
metabolites identified by LC–MS were quantified by integrating
the peak areas monitored at 254 nm using the CSASS (Complex
Sample Analysis Software System) software and by normalizing
them to the fresh wt of the extracted plant material and the peak
areas of the internal standard genistein. The CSASS software is tai-
lored to accurately quantify complex samples especially overlap-
ping peaks obtained by GC or LC using an automatic curve-fitting
method (Xue, 2004).
metabolites obtained with GC–TOF-MS
The GC–TOF-MS raw data files were converted to NetCDF for-
mat with Databridge (Waters). The mass spectra were automati-
cally refined by AMDIS (Automated Mass Spectral Deconvolution
and Identification Software; http://www.amdis.net). Then the tar-
get compounds were identified by matching the refined mass spec-

J. Zhang et al. / Phytochemistry 72 (2011) 1767–1778
1777
4.6. Extraction and analysis of sinalbin
Acknowledgments
Sinalbin (1) was extracted and purified from white mustard
We are grateful to Prof. Xun Che and Dr. Xingya Xue for help
with data analysis. We also thank Dr. Longxing Wang and Nan
Zhao for assistance in measuring the metabolites by LC–MS. This
work was supported by the National Science & Technology Pillar
Program (Grant No. 2006BAK02A12) and the National Natural Sci-
ence Foundation of China (Grant No. 20607022).
seeds (Sinapis alba (L.), a generous gift from Dr. M. Reichelt, Max
Planck Institute for Chemical Ecology, Jena, Germany) using a mod-
ification of the method described by Thies (1988). White mustard
seed (ca. 50 g) was dried in an oven at 120 °C for 2 h to inactivate
myrosinase. Dried samples were then ground to a powder and ex-
tracted with MeOH (2 ꢂ 100 mL). After 30 min gentle shaking at
room temperature (25 °C), samples were centrifuged at 1500g for
Appendix A. Supplementary data
5
min and the supernatant (200 mL) was loaded onto a (10 mL of
1
0 mg/mL) DEAE Sephadex A25 column. The column was rinsed
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.phytochem.2011.06.002.
with HCO
O (4 ꢂ 5 mL), and the eluates discarded. Samples were then
eluted using EtOH–H O (25 mL, 96:4, v/v) and 20 mM K SO /15%
i-PrOH (25 mL). After the eluent was agitated and cooled for
0 min at 4 °C, samples were centrifuged at 1500g for 5 min and
2 2
H–i-PrOH–H O (2 ꢂ 5 mL, 3:2:5, v/v/v) and deionized
H
2
2
2
4
References
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the supernatant was concentrated. EtOH (30 mL) was added to
the concentrate, followed by concentration to near-dryness. The
residue was transferred using MeOH (10 mL) and stored at ꢀ15 °C
for 15 min. The solution was centrifuged at 1500g for 5 min and
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sinalbin (1), 0.8 mol, 800 L of 1 mM). The other protocols of
2
(
l
l
extraction were the same as that of Reichelt et al. (2002). In the
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m, 4.6 ꢂ 250 mm column (Agela tech-
ꢀ1
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–30% B (10 min), 30–36% B (5 min), 36–40% B (3 min). The eluent
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3
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2
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9
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4
2
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