Phytochemistry 108 (2014) 35–46

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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

Profiling of secondary metabolites in root exudates of Arabidopsis

thaliana
Nadine Strehmel a,1, Christoph Böttcher a,b,⇑,1, Stephan Schmidt a, Dierk Scheel a
a
Leibniz Institute of Plant Biochemistry, Department of Stress and Developmental Biology, Weinberg 3, 06120 Halle (Saale), Germany
b
Julius Kühn-Institute, Federal Research Centre for Cultivated Plants, Institute for Ecological Chemistry, Plant Analysis and Stored Product Protection,
Königin-Luise-Strasse 19, 14195 Berlin, Germany

a r t i c l e i n f o a b s t r a c t

Article history: To explore the chemical composition of root exudates of the model plant Arabidopsis thaliana a workflow
Received 1 August 2014 for nontargeted metabolite profiling of the semipolar fraction of root exudates was developed. It com-
Received in revised form 1 October 2014 prises hydroponic plant cultivation and sampling of root exudates under sterile conditions, sample prep-
Available online 29 October 2014
aration by solid-phase extraction and analysis by reversed-phase UPLC/ESI-QTOFMS. Following the
established workflow, root exudates of six-week-old plants were profiled and a set of reproducibly occur-
Keywords: ring molecular features was compiled. To structurally elucidate the corresponding metabolites, accurate
Arabidopsis thaliana
mass tandem mass spectrometry and on-line hydrogen/deuterium exchange were applied. Currently, a
Brassicaceae
Root exudates
total of 103 compounds were detected and annotated by elemental composition of which more than
Secondary metabolism 90 were structurally characterized or classified. Among them, 42 compounds were rigorously identified
Metabolite profiling using an authenticated standard. The compounds identified so far include nucleosides, deoxynucleosides,
LC/MS aromatic amino acids, anabolites and catabolites of glucosinolates, dipeptides, indolics, salicylic and jas-
Structure elucidation monic acid catabolites, coumarins, mono-, di- and trilignols, hydroxycinnamic acid derivatives and oxy-
lipins and exemplify the high chemical diversity of plant root exudates.
Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Approximately 10% of the net photosynthetically fixed carbon is

To influence the biology and chemistry at the soil–root inter-
face, plant roots release a variety of compounds into the rhizo-
sphere (Badri and Vivanco, 2009; Baetz and Martinoia, 2014; Bais
et al., 2006). These so-called rhizodeposits include lysates of
sloughed-off root cap and border cells as well as exudates released
from intact root tissue. Root exudation is believed to occur either
passively by diffusion due to osmotic differences between the soil
solution and root cells, or actively by secretion. In the latter pro-
cess, multidrug and toxic compound extrusion (MATE) and ATP-
binding cassette (ABC) transporters are involved (Badri et al.,
2012; Fourcroy et al., 2014). Root exudates comprise low-molecu-
lar-weight primary and secondary metabolites (Dakora and
Phillips, 2002; Faure et al., 2009) as well as high-molecular-weight
compounds including mucilage and proteins (Wen et al., 2007).
⇑ Corresponding author at: Julius Kühn-Institute, Institute for Ecological Chem-
istry, Plant Analysis and Stored Product Protection, Königin-Luise-Strasse 19, 14195
Berlin, Germany. Tel.: +49 30 8304 2200; fax: +49 30 8304 2503.
E-mail addresses: Nadine.Strehmel@ipb-halle.de (N. Strehmel), Christoph.
Boettcher@jki.bund.de (C. Böttcher), Stephan.Schmidt@ipb-halle.de (S. Schmidt),
Dierk.Scheel@ipb-halle.de (D. Scheel).
1
These authors contributed equally to this work.
supposed to be released by soil-grown plants as root exudates into
the rhizosphere (Jones et al., 2009).
Root exudates were shown to be involved in various processes
within the rhizosphere. They play a pivotal role in the mobilization
and acquisition of nutrients (Ma et al., 2001; Schmid et al., 2014),
attract and repel certain microbe species (Rudrappa et al., 2008),
inhibit the growth of competing plant species (Biedrzycki et al.,
2010) or stimulate the germination of parasite seeds (Auger
et al., 2012). Certainly, their qualitative and quantitative composi-
tion depends on the plant species, the developmental stage
(Aulakh et al., 2001; Chaparro et al., 2013) as well as abiotic and
biotic factors (Bouwmeester et al., 2007).
Several techniques for the sampling of root exudates were pro-
posed in the past (for a survey see (Oburger et al., 2013)). However,
the collection of root exudates from soil-grown plants under con-
trolled abiotic and biotic conditions remains challenging. There-
fore, hydroponic cultivation systems are frequently used to study
root exudation. These systems have the advantage, that the exuded
compounds cannot be adsorbed onto soil particles and, when
applying sterile culture conditions, the metabolization of root exu-
dates by microorganisms can be excluded. For Arabidopsis thaliana,
several hydroponic culture systems were developed (Arteca and

http://dx.doi.org/10.1016/j.phytochem.2014.10.003
0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
36 N. Strehmel et al. / Phytochemistry 108 (2014) 35–46

Arteca, 2000; Conn et al., 2013; Gibeaut et al., 1997; Noren et al.,
2004; Schlesier et al., 2003; Tocquin et al., 2003; Toda et al.,
1999). However, most of them were not optimized for the
sampling of root exudates and carried out under nonsterile
conditions.
In previous studies root exudate profiles of A. thaliana were
comprehensively analyzed by GC/MS targeting polar primary
metabolites, such as amino acids, sugars, sugar alcohols and
organic acids (Badri et al., 2013; Chaparro et al., 2013). In contrast,
the profile of non-volatile secondary metabolites is by far less
explored. Data published so far exclusively focus on targeted anal-
yses of selected compounds (e.g. the phytoalexin camalexin (Millet
et al., 2010)) or compound classes (e.g. coumarins (Fourcroy et al.,
2014) and strigolactones (Kohlen et al., 2011)).
In the present manuscript a workflow is presented which allows
for the nontargeted LC/MS-based metabolite profiling of the semi-
polar fraction of root exudates of A. thaliana collected from a sterile
hydroponic plant cultivation system. Based on this workflow, more
than 100 compounds, mainly secondary metabolites, were
detected and structurally characterized by MS techniques.
2. Results and discussion
individual pre-cultivated plants were transferred to amber 50 mL
glass bottles filled with a modified Murashige and Skoog medium
(Supplementary Fig. 2). Using this setup for the main cultivation
period, the volume of the nutrient solution per plant was reduced
to a manageable amount in order to simplify the subsequent
enrichment of exuded components. For the sake of sterility, glass
bottles were kept in sealed plastic boxes and aeration of the nutri-
ent solution was omitted. Instead, it was exchanged once a week
and checked for microbial contamination.
To characterize the vegetative growth during the main cultiva-
tion period root morphology, root length and biomass as well as
shoot biomass were analyzed over a period of three weeks.
Short-day conditions were applied to avoid early flowering. After
the first week of the main cultivation period, roots only exhibited
a root cap (Fig. 1A). After additional two weeks, root hairs became
clearly visible (Fig. 1B). At this time point, a mean root biomass of
30 mg fresh weight per plant at a root/shoot biomass ratio of 0.75
was reached (Fig. 1C). Beyond the third week of the main cultiva-
tion period, several plants started to produce inflorescences. Since
the transition between vegetative growth and flowering is
reflected in the root exudate pattern (Chaparro et al., 2013), sam-
pling was restricted up to the third week of the main cultivation
period.

2.1. Hydroponic plant cultivation 2.2. Sample preparation

To sample root exudates from A. thaliana under sterile condi-
tions a two-step hydroponic cultivation system was adapted from
literature and optimized (Noren et al., 2004; Tocquin et al., 2003)
(Supplementary Fig. 1). In short, seeds were sown on bottom-cut
agar-filled PCR tubes and pre-cultivated in closed plastic boxes. Ini-
tial attempts with Murashige and Skoog medium as nutrient solu-
tion resulted in low germination rates of 625%. However, addition
of 1% (w/v) sucrose to the nutrient solution restored a reliable ger-
mination (germination rate P95%). After three weeks of pre-culti-
vation under short-day conditions, plantlets reached the six-leaf
stage and roots penetrated the agar-based solid support (root
length 2.9 ± 0.7 cm, n = 10). In contrast to previous hydroponic cul-
ture systems (Gibeaut et al., 1997; Noren et al., 2004; Tocquin
et al., 2003), in which voluminous aerated plastic containers are
utilized to grow multiple plants under non-sterile conditions,
The nutrient solution from the hydroponic cultivation system
contains inorganic macronutrients in the low millimolar concen-
tration range, whereas root exudates are expected to occur at a
3–6 orders of magnitude lower concentration (Jaitz et al., 2011).
Hence, nontargeted LC/MS-based profiling of root exudates
requires an extensive sample preparation which allows for an at
least 1000-fold enrichment of exuded components from the nutri-
ent solution and an efficient depletion of bulk components such as
inorganic salts at once.
In order to establish a solid-phase extraction procedure target-
ing the semipolar fraction of root exudates, two silica-based
reversed-phase sorbents (Strata C18-E, Bond Elute C18) and a poly-
meric mixed-mode sorbent (Strata-X) were evaluated using nutri-
ent solutions spiked with a set of 15 natural products with
highly diverse physico-chemical properties (Supplementary

A B

18 50
C 16 30
shoot biomass [mg]
root biomass [mg]

40
root length [cm]

14
20 30
12
20
10
10
8 10

6 0 0
1 2 3 1 2 3 1 2 3
time (main cultivation period) [weeks]

Fig. 1. Developmental stages of Arabidopsis thaliana (accession Col-0) during the main cultivation period. Plants were pre-cultivated for three weeks, transferred to glass
bottles and incubated under short-day conditions while exchanging the nutrient solution on a weekly basis. Rosette and root phenotypes after a main cultivation period of
one week (A) and three weeks (B) are shown. The scale bar equals 100 lm. Root and shoot growth kinetics during the main cultivation period are presented in (C). Error bars
indicate standard deviations (n = 48).
 N. Strehmel et al. / Phytochemistry 108 (2014) 35–46 37

Table 1). In our hands, the Bond Elute C18 sorbent showed the best
overall performance with a median process efficiency of 53%,
whereas only 36% were reached by the remaining two (Supple-
mentary Fig. 3). Consequently, this sorbent was selected for the
enrichment of root exudates. To test the repeatability of the sample
preparation protocol, aliquots of spiked nutrient solution were pro-
cessed. For 9 out of 15 test compounds which exhibited overall
process efficiencies of >40% the sample preparation procedure
resulted in good repeatabilities (mean relative standard deviation
(RSD) 8.5%, Supplementary Fig. 4). For the remaining test com-
pounds including amino acids (Phe and Trp), polar organic acids
(pantothenic and citric acid) and phenolic acids (salicylic and cin-
namic acid) low repeatabilities (mean RSD 40%) were observed in
the majority of cases. Hence, these compound classes elude a reli-
able analysis using the established sample preparation protocol.
2.3. Metabolite profiling of root exdudates
To determine an appropriate time point for root exudate analy-
sis, nutrient solutions were collected weekly in a pilot experiment
during the main cultivation period, worked up and analyzed by
UPLC/ESI-QTOFMS. Comparative analyses of appropriate blank
and exudate samples using the XCMS algorithm (Smith et al.,
2006) revealed that the number of features (molecular entities
with unique mass-to-charge ratio and retention time) significantly
increases with increasing plant age. In the nutrient solution col-
lected after the first week of the main cultivation period 242 exu-
date-related features (signal-to-noise ratio (S/N) >4) were detected
in positive ion mode. In contrast, analysis of nutrient solutions
obtained after the third week of the main cultivation period
resulted in the detection of 1137 exudate-related features. In the
negative ion mode, a 2.1-fold increase in the number of intense
features was registered when performing analogous comparisons.
Consequently, the study was focused on the analysis of nutrient
solutions collected after the third week of the main cultivation
period.
To obtain a set of reproducibly occurring exudate-related fea-
tures with intensities that facilitate reliable accurate mass mea-
surements and the acquisition of tandem mass spectra a total of
48 plants were cultivated in two independent experiments. Nutri-
ent solutions from six-week-old plants and corresponding blank
samples were collected, worked up and analyzed by UPLC/ESI-
QTOFMS in positive and negative ion mode. Representative base
peak chromatograms are shown in Fig. 2. With the help of the
XCMS algorithm, features which were consistently detectable in
at least 75% of the replicate exudate samples with a median inten-
sity of more than 5000 counts and an at least 8-fold higher inten-
sity in comparison to the blank (P 6 0.05, t-test) were screened for.
Applying these filter criteria, a total of 341 (389) features were
identified in positive (negative) ion mode. These were used to
reconstruct mass spectra of exuded compounds and to identify
corresponding quasi-molecular ion species. Subsequently, colli-
sion-induced dissociation (CID) mass spectra were acquired and
used in combination with the accurate mass and isotope pattern
of the fragmented quasi-molecular ion to determine the elemental
composition and to derive structural hypotheses. In addition, on-
line hydrogen/deuterium exchange experiments were performed
to validate the postulated molecular structures (Liu et al., 2001,
2007). Following this strategy, a total of 103 compounds were
annotated by elemental composition of which more than 90 were
structurally characterized or classified (Table 1, Fig. 3). With the
help of authenticated standards, 42 compounds were rigorously

A B

C D

Fig. 2. Representative base peak chromatograms (m/z 100–800) obtained from root exudates (A, C) and blank samples (B, D) by UPLC/ESI-QTOFMS in positive (A, B) and
negative ion mode (C, D). Root exudates were collected over a one-week period from six-week-old plants. Abundant peaks are numbered according to Table 1 is internal
standard (2,4-dichlorophenoxyacetic acid); 5 homologous series of polyethylene glycols; ; homologous series of polyethylene glycol monobutyl ethers; s polyethylene
glycol derivative; " dodecanediol sulfates; d dodecyl sulfate.
38 N. Strehmel et al. / Phytochemistry 108 (2014) 35–46
identified. Chromatographic and mass spectral data are detailed in
Supplementary Tables 2–4.
It should be noted, that despite the use of high purity solvents
and chemicals, LC/MS profiles obtained from blank nutrient solu-
tions contain numerous contaminants (Fig. 2). These were found
to preferably ionize in positive ion mode and comprise polyethyl-
ene glycols (PEGs), PEG monobutyl ethers and other PEG derived
compounds. In negative ion mode, dodecanediol sulfates and dode-
cyl sulfate were identified as contaminants.
2.4. Compounds identified in root exudates of A. thaliana
2.4.1. Nucleosides, amino acids and dipeptides
Although the established method was not intended to target
polar metabolites, several polar aromatic compounds including
three nucleosides (1–3), two deoxynucleosides (4, 5), a nucleoside
derivative (6) and the aromatic amino acids Phe (7), Tyr (8) and Trp
(9) were detected in root exudates of A. thaliana. In addition, a sim-
ple b-carboline alkaloid (10) previously reported to accumulate in
tomato fruits (Yahara et al., 2004) was identified using a synthetic
reference compound prepared from Trp and formaldehyde (Tilstra
et al., 1990). While other proteinogenic amino acids were not
detected using the established analytical method, the more hydro-
phobic side chain-elongated Met homologs (11, 12) and their S-
oxides (13, 14) were putatively identified based on the elemental
composition, the presence of three exchangeable protons and the
occurrence of characteristic neutral losses of 46.01 Da (HCOOH)
and 48.00 Da (CH3SH)/64.00 Da (CH3SOH) in the CID mass spectra
of the [M+H]+ ions. Compounds 11–14 represent biosynthetic
intermediates of corresponding x-methylsulfinylalkyl/x-meth-
ylthioalkyl glucosinolates of chain length 7 and 8 which are known
constituents in roots of the accession Col-0 (Brown et al., 2003;
Petersen et al., 2002).
Beside nucleosides and amino acids, an array of 20 dipeptides
(15–34) composed of the amino acids Gly, Val, Leu, Ile, Pro, Phe
and Tyr was identified in root exudates. Structural assignments
are in agreement with the elemental composition, the number of
exchangeable protons and the occurrence of characteristic y-type
fragment ions upon CID of the [M+H]+ ions. To discriminate Leu-
from Ile-containing peptides, a total of 16 dipeptides were synthe-
sized and used for authentication. The spectrum of dipeptides
detected in root exudates matches the set of 14 dipeptides
reported to accumulate in roots of A. thaliana in a cell-type specific
manner (Moussaieff et al., 2013).
2.4.2. Degradation products of methionine-derived glucosinolates
Met-derived glucosinolates are the predominating phytoantici-
pins in most organs of A. thaliana. Despite their significant accumu-
lation level in root tissue (Brown et al., 2003; Petersen et al., 2002)
glucosinolates were not consistently detected in root exudates
using the established experimental setup, which is in contrast to
a previous study on root exudates of Brassica rapa (Schreiner
et al., 2011). Instead of glucosinolates, a set of 8 putative glucosin-
olate degradation products (35–42) derived from 8-methylthiooc-
tyl, 8-methylsulfinyloctyl and 7-methylsulfinylheptyl gluco
sinolate were identified based on the occurrence of characteristic
neutral losses of 48.00 Da and 64.00 Da upon CID of the [M+H]+
ions which are indicative for methylthio and methylsulfinyl moie-
ties, respectively. Among them, classical glucosinolate breakdown
products known to be produced upon tissue damage were found
including two nitriles (35, 36) and an isothiocyanate (37). Related
to these compounds, a carboxylic acid (38) and its amide (39) as
well as three amines (40–42) were identified. It was suggested,
that these compounds represent glucosinolate breakdown prod-
ucts, which are formed in intact tissue by either nitrilase-mediated
hydrolysis of nitriles or by degradation of isothiocyanate–glutathi-
one conjugates (Bednarek et al., 2009; Wittstock and Burow, 2010).
2.4.3. Degradation products of tryptophan-derived glucosinolates and
other indolic compounds
In addition to aliphatic glucosinolates indolic glucosinolates
accumulate in A. thaliana and, in particular in roots, relatively large
amounts of these compounds are found (Brown et al., 2003). As
observed for Met-derived glucosinolates, the major Trp-derived
glucosinolates of roots (indol-3-yl methyl, 4-methoxy and 1-meth-
oxyindol-3-yl methyl glucosinolate) were absent in root exudates.
Again, corresponding degradation products including indol-3-
ylmethyl amine (43), its 4-methoxy (44) and 1-methoxy derivative
(45) were detected. Here, isomers 44 and 45 can be discriminated
based on the number of exchangeable protons determined for the
dominating [M+H-NH3]+-type in-source fragment ion. Amine 43
was recently identified as a product of the atypical myrosinase
PENETRATION2 (At2g44490) which is involved in an inducible
pre-invasion resistance mechanism conferring broad-spectrum
resistance to fungal pathogens (Bednarek et al., 2009; Lipka et al.,
2005). In addition, further indolic compounds were identified in
root exudates including indole-3-carbaldehyde (46), 4-hydroxyin-
dole-3-carbaldehyde (47), indole-3-carboxylic acid (48), a malo-
nyl-glucoside of 6-hydroxyindole-3-carboxylic acid (49) and an
unknown indole derivative (50). Compounds 46–49 represent
known metabolites which constitutively accumulate (in case of
47 and 48 as glucose conjugates) in leaf and root tissue of A. thali-
ana and exhibit a pathogen-inducible accumulation pattern
(Bednarek et al., 2005; Bottcher et al., 2014; Hagemeier et al.,
2001).
It should be noted that camalexin, the major indolic phytoalexin
of A. thaliana, was not detected as root exudate in sterile nutrient
solutions. In accordance to literature data describing the exudation
of this phytoalexin following treatment with a conserved 22-
amino-acid peptide from bacterial flagellin (flg22) (Millet et al.,
2010), camalexin became clearly detectable when analyzing
microbially contaminated nutrient solutions. This suggests that
the camalexin level could be used as an additional marker to judge
the sterility of the hydroponic culture system.
2.4.4. Salicylic and jasmonic acid degradation products
Based on their elemental composition and the occurrence of
consecutive neutral losses of 132.04 Da (C5H8O4) and 43.99 Da
(CO2) upon CID of the [MH] ions, two exuded compounds (51,
52) were annotated as dihydroxybenzoic acid (DHBA) pentosides.
Pentoside 51 was identified as 2,3-DHBA 3-O-b-D-xyloside using
an authenticated standard (Bartsch et al., 2010) whereas the other
was annotated as 2,5-DHBA conjugate based on acid-catalyzed
hydrolysis experiments. As recently shown, both DHBA pentosides
represent the major salicylic acid catabolites in young and senes-
cent A. thaliana rosette leaves (Zhang et al., 2013). In addition,
another plant hormone catabolite (53) was detected in root exu-
dates (Supplementary Fig. 5A). The CID mass spectrum obtained
from the [MH] ion of 53 exhibited a prominent neutral loss of
79.96 Da (SO3) and a corresponding fragment ion at m/z 96.96
(HSO 4 ) indicating the presence of a sulfate moiety (Supplementary
Fig. 5B). The elemental composition of 53 was determined to be
C12H20O7S suggesting that this compound might represent a sul-
fated dihydro-hydroxyjasmonic acid derivative. To prove this
hypothesis, A. thaliana seedlings were grown in liquid culture, trea-
ted for 24 h with [2-D2]-jasmonic acid, extracted and analyzed by
UPLC/ESI-QTOFMS. Following this treatment, 53 and its deuterated
isotopolgue became detectable (Supplementary Fig. 5C). On-line H/
D exchange experiments revealed the presence of two exchange-
able protons (Supplementary Fig. 5D). Thus, compound 53 pos-
sesses a saturated side chain and a ring keto group rather than
Table 1
Annotated compounds reproducibly detected by UPLC/ESI-QTOFMS in solid-phase extracted nutrient solution of hydroponically grown Arabidopsis thaliana Col-0. Nutrient solution was collected over a one-week period from six-week-
old plants.

No. Compound vl1 tr Elemental Quantifier ion # exchang. protons2 Observed fragment ions upon CID3
[s] composition m/z, precursor ion marked in bold
Type m/z
1 Adenosine S 44 C10H13N5O4 [M+H]+ 268.10 5 268, 136
2 Guanosine S 46 C10H13N5O5 [MH] 282.08 6 282, 150, 133
3 Uridine S 41 C9H12N2O6 [MH] 243.06 4 243, 200, 153, 152, 140, 122, 110
4 20 -Deoxyadenosine S 46 C10H13N5O3 [M+H]+ 252.11 4 252, 136, 117
5 Thymidine S 77 C10H14N2O5 [MH] 241.08 3 241, 198, 151, 125
6 20 -O-Methyladenosine S 64 C11H15N5O4 [M+H]+ 282.12 4 282, 136
7 Phe S 75 C9H11NO2 [M+H]+ 166.09 3 166, 149, 131, 120, 107, 103
8 Tyr S 42 C9H11NO3 [M+H]+ 182.08 4 182, 165, 147, 136, 123, 119, 91
9 Trp S 144 C11H12N2O2 [M+H]+ 205.10 4 205, 188, 159, 132
10 3-Carboxy-1,2,3,4-tetrahydro-b-carboline S 187 C12H12N2O2 [M+H]+ 217.10 3 217, 188, 173, 171, 144, 132
11 Pentahomo-Met I 276 C10H21NO2S [M+H]+ 220.14 a 220, 174, 126, 109
12 Hexahomo-Met I 327 C11H23NO2S [M+H]+ 234.15 3 234, 188, 140, 123
13 Pentahomo-Met S-Oxide I 86 C10H21NO3S [M+H]+ 236.13 3 236, 190, 172, 126
14 Hexahomo-Met S-Oxide I 164 C11H23NO3S [M+H]+ 250.15 3 250, 204, 186, 140
15 H-Phe-Gly-OH I 99 C11H14N2O3 [M+H]+ 223.11 4 223, 120, 103

N. Strehmel et al. / Phytochemistry 108 (2014) 35–46

16 H-Gly-Phe-OH I 138 C11H14N2O3 [M+H]+ 223.11 a 223, 177, 166, 120
17 H-Ile-Val-OH S,L 114 C11H22N2O3 [M+H]+ 231.17 4 231, 213, 185, 118, 86
18 H-Val-Ile-OH S,L 135 C11H22N2O3 [M+H]+ 231.17 4 231, 213, 185, 132, 86, 72
19 H-Leu-Val-OH S,L 139 C11H22N2O3 [M+H]+ 231.17 4 231, 213, 185, 118, 86
20 H-Val-Leu-OH S,L 159 C11H22N2O3 [M+H]+ 231.17 4 231, 213, 185, 132, 86, 72
21 H-Leu-Pro-OH S 154 C11H20N2O3 [M+H]+ 229.15 3 229, 116, 86
22 H-Leu-Tyr-OH S,L 166 C15H22N2O4 [M+H]+ 295.17 5 295, 277, 249, 182, 165, 136, 86
23 H-Tyr-Ile-OH S 174 C15H22N2O4 [M+H]+ 295.17 5 295, 278, 277, 250, 249, 136, 132, 130, 121, 119
24 H-Tyr-Leu-OH S 192 C15H22N2O4 [M+H]+ 295.17 5 295, 278, 277, 250, 249, 136, 132, 130, 121, 119
25 H-Phe-Val-OH I 179 C14H 20N2O3 [M+H]+ 265.15 4 265, 219, 120, 118
26 H-Val-Phe-OH I 196 C14H20N2O3 [M+H]+ 265.15 4 265, 219, 166, 120, 72
27 H-Ile-Ile-OH S,L 185 C12H24N2O3 [M+H]+ 245.19 4 245, 199, 132, 86
28 H-Leu-Ile-OH S,L 199 C12H24N2O3 [M+H]+ 245.19 4 245, 199, 132, 86
29 H-Ile-Leu-OH S,L 204 C12H24N2O3 [M+H]+ 245.19 4 245, 199, 132, 86
30 H-Leu-Leu-OH S,L 217 C12H24N2O3 [M+H]+ 245.19 4 245, 199, 132, 86
31 H-Phe-Ile-OH S,L 226 C15H22N2O3 [M+H]+ 279.17 4 279, 262, 261, 233, 132, 120, 86
32 H-Ile-Phe-OH S,L 235 C15H22N2O3 [M+H]+ 279.17 4 279, 261, 233, 166, 120, 86
33 H-Phe-Leu-OH S,L 247 C15H22N2O3 [M+H]+ 279.17 4 279, 262, 261, 233, 132, 120, 86
34 H-Leu-Phe -OH S,L 249 C15H22N2O3 [M+H]+ 279.17 4 279, 261, 233, 166, 120, 86
35 7-MeSO-Heptyl-CN I,L 266 C9H17NOS [M+H]+ 188.11 a 188, 124, 107, 105, 96, 91, 82, 81, 79, 77, 65
36 8-MeSO-Octyl-CN I,L 323 C10H19NOS [M+H]+ 202.13 0 202, 138, 121, 110, 105, 96, 95, 93, 91, 82, 81, 79, 77, 65
37 8-MeSO-Octyl-NCS S,L 483 C10H19NOS2 [M+H]+ 234.10 0 234, 175, 170, 161, 137, 136, 128, 122, 114,108, 69, 65, 55
38 8-MeSO-Octyl-CO2H S,L 296 C10H20O3S [M+H]+ 221.12 1 221, 203, 175, 139, 137, 121, 119, 111, 109, 95, 93
39 8-MeSO-Octyl-CONH2 I 233 C10H21NO2S [M+H]+ 220.14 2 220, 203, 175, 139, 137, 121
40 7-MeSO-Heptyl-NH2 S 61 C8H19NOS [M+H]+ 178.13 2 178, 161, 160, 114, 112, 97, 95
41 8-MeSO-Octyl-NH2 S,L 133 C9H21NOS [M+H]+ 192.14 2 192, 175, 174, 128, 111, 109, 97
42 8-MeS-Octyl-NH2 I 311 C9H21NS [M+H]+ 176.15 2 176, 159, 128, 111, 103
43 Indol-3-ylmethylamine (I3CH2NH2) S,L 111 C9H10N2 [M+H-NH3]+ 130.07 1b 130, 128, 103, 77
44 4-MeO-I3CH2NH2 S 192 C10H12N2O [M+H-NH3]+ 160.08 1b 160, 148, 145, 132, 130, 117
45 1-MeO-I3CH2NH2 I,L 223 C10H12N2O [M+H-NH3]+ 160.08 0b 160, 145, 132, 128, 117
46 Indole-3-carbaldehyde (I3CHO) S 289 C9H7NO [MH] 144.04 1 144, 142, 126, 116, 115
47 4-HO-I3CHO S 307 C9H7NO2 [MH] 160.04 2 160, 158, 132, 131, 130, 114, 104, 102
48 Indole-3-carboxylic acid (I3CO2H) S,L 284 C9H7NO2 [M+H]+ 162.06 2 162, 144, 118
49 6-(Malonyl-GlcO)-I3CO2H I,L 194 C18H19NO11 [M-H-CO2] 380.10 a 424, 380, 338, 320, 218, 176, 175, 132
50 Unknown indole derivative I 391 C10H9NO3 [M+H]+ 192.07 a 192, 177, 174, 161, 159, 148, 133, 132, 117,116, 105, 104
51 2,3-DHBA 3-O-Xyl S,L 176 C12H14O8 [MH] 285.06 5 285, 153, 109
52 2,5-DHBA Pent I,L 153 C12H14O8 [MH] 285.06 5 285, 153, 152, 108

(continued on next page)

39
 40
Table 1 (continued)

No. Compound vl1 tr Elemental Quantifier ion # exchang. protons2 Observed fragment ions upon CID3
[s] composition m/z, precursor ion marked in bold
Type m/z
53 9,10-Dihydrohydroxy-JA Sulfate I 222 C12H20O7S [MH] 307.09 2 307, 227, 97
54 Coniferin I,L 179 C16H22O8 [M+Na]+ 365.12 5 387 ([M-H+FA]), 341, 207, 179
55 Syringin I,L 193 C17H24O9 [M+Na]+ 395.13 5 417 ([M-H+FA]), 209, 207,194
56 G(8-O-4)G I 275/ C20H24O7 [MH] 375.15 4 375, 327, 195, 179, 165
281
57 G(8-O-4)SGlycerol I 188/ C21H28O10 [MH] 439.16 6 439, 391, 243, 195
192

58 G(8-5)G I 344 C20H22O6 [M-H-H2O] 339.12 3 357 ([MH]), 339, 327, 324
59 S(8-5)G I 340 C21H24O7 [M-H-H2O]- 369.13 3 387 ([MH]), 369, 357, 354
60 S(8-8)S I 377 C22H26O8 [MH] 417.16 2 417, 402, 387, 181, 166
61 Lariciresinol S,L 337 C20H24O6 [M-H- 329.14 3 359 ([MH]), 344, 329, 192, 178, 175, 164, 160, 159
CH2O]
62 Lariciresinol Hex I,L 272 C26H34O11 [MH] 521.20 6 521, 359, 329, 192, 178, 175, 160
63 G(8-O-4)S(8-5)G I 366/ C31H36O11 [MH] 583.22 5 583, 535, 505, 369, 357, 195, 165
379
64 G(8-O-4)S(8-8)S I 401/ C32H38O12 [MH] 613.23 a 613, 565, 535, 417, 402, 387, 195, 181, 165, 150
415

N. Strehmel et al. / Phytochemistry 108 (2014) 35–46

65 G(8-O-4)G(8-8)S/G(8-O-4)S(8-8)G I 407/ C31H36O11 [MH] 583.22 a 583, 535, 505, 387, 357, 195, 181, 180, 165, 150
421
66 Esculetin S,L 201 C9H6O4 [MH] 177.02 2 177, 149, 133, 121, 105, 93, 89, 81, 77
67 Scopoletin S,L 268 C10H8O4 [M+H]+ 193.05 1 191 ([MH]), 176, 148, 120, 104
68 Esculin S,L 166 C15H16O9 [MH] 339.07 5 339, 177, 133
69 Scopolin I,L 196 C16H18O9 [M-H+FA] 399.09 4 399, 353, 207, 191, 176
70 Scopoletin Hex-Pent I 197 C21H26O13 [MH] 485.13 6 485, 191, 176, 148
71 Scopoletin Benzoyl-Hex-Pent I 329 C28H30O14 [MH] 589.16 5 589, 467, 191, 148, 121
72 G(8-O-4)Scopoletin I 280 C20H20O8 [MH] 387.11 3 387, 339, 195, 191, 176, 165, 150
73 G(8-O-4)Esculetin I 377 C19H16O7 [MH] 355.08 2 355, 337, 325, 179, 177, 176
74 S(8-O-4)Esculetin I 370 C20H18O8 [MH] 385.09 2 385, 367, 355, 209, 177, 176
75 Scopoletin dehydrodimer I 315 C20H14O8 [MH] 381.06 2 381, 366, 337, 325, 322, 321, 307, 305
76 Unknown oligolignol I 300 C41H46O20 [MH] 857.25 8 857, 661, 485, 467, 389, 371, 341, 195, 193, 191, 176, 175, 165
77 G(8-O-4)FA Sulfate I 237/ C20H22O11S [MH] 469.08 4 469, 389, 341, 275, 245, 203, 195, 193
256
78 G(8-5)FA S 358 C20H20O7 [M-H-H2O] 353.10 3 371, 353, 341, 338, 326, 322, 309, 297, 294, 282, 235, 191
79 G(8-5)FA Sulfate I 323 C20H20O10S [MH] 451.07 3 451, 371, 353, 341, 338, 326, 309, 297, 294, 282, 267, 245, 97
80 FA dimer I 242/ C20H20O8 [MH] 387.11 4 387, 339, 324, 309, 235, 220, 217, 203, 202, 176, 151, 136
249
81 Didehydro-di(coumaroyl)spermidine Sulfate I 219 C25H29N3O7S [MH] 514.17 4 516 ([M+H]+), 436, 419, 362, 348, 347, 291, 290, 265, 203, 202, 145
82 Kaempferol 3-O-Rha(1?2)Glc 7-O-Rha I,L 228 C33H40O19 [MH] 739.21 11 739, 593, 430, 284
83 9,12,13-Trihydroxy-10(E),15(Z)- octadecadienoic S,L 420 C18H32O5 [MH] 327.22 4 327, 309, 291, 239, 229, 221, 211, 185, 183, 177, 171
acid
84 9,12,13-Trihydroxyoctadec-10-enoic acid I,L 445 C18H34O5 [MH] 329.23 4 329, 311, 293, 229, 211, 197, 193, 183, 171, 139
85 Fatty acid derivative I 634 C18H30O4 [MH] 309.21 2 309, 291, 273, 265, 247
86 Azelaoyl lysoPC I 232/ C17H34NO9P [MH] 426.19 2 428 ([M+H]+), 410, 258, 245, 184, 104
244
87 C4H10O Hex I 162 C10H20O6 [M+Na]+ 259.12 4 237 ([M+H]+), 163, 145, 127, 85
88 C4H10O Malonyl-Hex I 232 C13H22O9 [M+Na]+ 345.11 4 345, 301, 259
89 C4H10O Hex-DeoxyHex I 200 C16H30O10 [MH] 381.18 6 383 ([M+H]+), 309, 291, 273, 255, 237, 221, 147, 129
90 C4H10O Malonyl-Hex-DeoxyHex I 255 C19H32O13 [M-H-CO2] 423.19 6 467 ([MH]), 423, 381, 277, 235, 217, 161, 159, 143, 113, 101
91 C7H9N5O Hex I 63 C13H19N5O6 [MH] 340.13 5 342 ([M+H]+), 180, 135, 110
92 C9H10O3 Hex I 198 C15H20O8 [MH] 327.11 5 327, 165, 147, 101
93 C12H16O5 Hex I 202 C18H26O10 [MH] 401.14 a 447 ([M-H+FA]), 401, 255, 205, 161, 113
94 C7H14O4 Malonyl-Hex I 209 C16H26O12 [M-H-CO2] 365.15 a 409 ([MH]), 365, 323, 203, 161, 149, 143, 101
95 C12H16O6 Hex I 269/ C18H26O11 [M+Na]+ 441.14 4 463 ([M-H+FA]), 385, 241, 225, 179, 161, 153, 149, 143, 131, 119
279
 N. Strehmel et al. / Phytochemistry 108 (2014) 35–46 41

an unsaturated side chain and a ring hydroxy group. Beside 12-

number of exchangeable protons as determined by on-line H/D exchange: a number of exchangeable protons could not be unambigously determined due to low intensity, b compound does not form any quasi-molecular ion,
verification level: S, chromatographic and mass spectral behavior as observed for an authenticated standard; I, putative structure deduced from interpretation of elemental composition, collision-induced dissociation mass
445, 427, 409, 391, 353, 339, 325, 313, 311, 285, 283, 271, 269, 215
hydroxyjasmonic acid O-sulfate (Gidda et al., 2003), compound
53 represents a novel sulfated jasmonic acid catabolite produced
in A. thaliana.
471, 291, 179, 163, 145, 143, 125, 123, 119, 113, 107, 101

2.4.5. Phenylpropanoids
2.4.5.1. Lignols. Coniferin (54) and syringin (55) represent the pre-
dominant phenylpropanoids in dark-grown roots of A. thaliana
Pent, pentoside; Hex, hexoside; DeoxyHex, deoxyhexoside; DHBA, dihydroxybenzoic acid; JA, jasmonic acid; G, coniferyl alcohol; S, sinapyl alcohol; FA, ferulic acid; lysoPC, lysophosphatidylcholine.

(Bednarek et al., 2005) and were also detected in root exudates.

Both monolignol glucosides were accompanied by related di- and
239 ([M+H-H2O]+), 207, 179, 147, 119

496, 374, 345, 294, 223, 150, 121, 97

trilignols and derivatives thereof. The identity and the linkage type
443, 291, 151, 145, 125, 107, 101

of these compounds was derived from the analysis of CID mass

spectra obtained from corresponding [MH] ions (Morreel
237, 222, 193, 178, 149, 134

et al., 2010a,b). The CID mass spectra of the b-aryl ether-type

dimers 56 and 57 carrying an 8-O-4 linkage exhibited a character-
100 ([M+H]+), 83, 55
479, 437, 291, 161

istic neutral loss of 48.02 Da according to a combined loss of water

and formaldehyde from the [MH] ion. In addition, formation of
A and B fragment ions by cleavage of the aryl ether bond facili-
tated the identification of the aromatic units of these dimers
(Morreel et al., 2010b). For the phenylcoumaran-type dimers 58
and 59 with 8–5 linkage, fragmentation of the [MH] ions
resulted in dominating neutral losses of 18.01 Da (H2O) and
30.01 Da (CH2O) (Morreel et al., 2010b). Three resinol-type dilig-
nols with 8–8 linkage including syringaresinol (60), lariciresinol
spectra and online H/D exchange experiments; L, compound reported for A. thaliana in the literature, for references see Supplementary Table 2.

(61) and a derived hexoside (62) were found in root exudates. In

accordance with the literature, the CID mass spectrum obtained
from deprotonated 60 showed the product ions [MH-CH3] and
a
1

6
1
6
6
3
3

[MH-CH2O] as well as a 2,5X fragment ion at m/z 181.05

479.25

443.15
471.15

445.33
279.08

496.08
122.09

237.04

(Morreel et al., 2010b). The accumulation of glycosylated syring-

aresinol and lariciresinol in roots of A. thaliana was described
number of exchangeable protons determined for the [M+H-NH3]+ ion. For detailed information see Supplementary Table 3.

(Matsuda et al., 2010; Nakatsubo et al., 2008). Based on the frag-

[M-H-CO2]

mentation characteristics observed for dilignols, three trilignols

[M+Na]+

[M+Na]+

(63–65) with coniferyl and syringyl alcohol as monomeric untis

[MH]

[MH]
[MH]
[MH]
[M+H]+

were tentatively identified based on the fragmentation characteris-

tics observed for corresponding dilignols.

2.4.5.2. Coumarins. The coumarin glucoside scopolin represents

another abundant root phenylpropanoid (Bednarek et al., 2005;
C18H19N5O10S

Kai et al., 2006). It was recently shown, that the biosynthesis of

C21H28O12
C23H40O13

C20H28O11

coumarins in roots and their exudation can be stimulated inter alia

C12H16O6

C28H44O4
C11H10O6
C6H13N

upon iron deprivation indicating that coumarins are involved in

the acquisition of iron in A. thaliana (Fourcroy et al., 2014;
Schmid et al., 2014; Schmidt et al., 2014).
Esculetin (66), scopoletin (67) and its glucosides esculin (68)
326/
339

234

234
182

573
404

260

and scopolin (69) were detected in root exudates and identified

63

by authenticated standards. Beside these coumarins which are

already known to be exuded from roots (Fourcroy et al., 2014;
–

–
–
–
–
I
I
I

Schmid et al., 2014), further esculetin and scopoletin conjugates

were tentatively identified in root exudates on the basis of their
mass spectral fragmentation pattern. Scopoletin was found to be
conjugated to a hexose–pentose and to a benzoylated hexose–pen-
tose as indicated by a neutral loss of 294.10 Da (C11H18O9) from the
[MH] ion of 70 and consecutive neutral losses of 122.04 Da
(C7H6O2) and 294.10 Da from the [MH] ion of 71, respectively.
for details see Supplementary Table 4.

In addition, an 8-O-4-linked coniferylalcohol–scopoletin conjugate

Unknown secondary amine

(72) was annotated based on the occurrence of a characteristic

neutral loss of 48.02 Da from its [MH] ion and characteristic
C14H28O5 Malonyl-Hex

A and B fragment ions at m/z 195.07 and m/z 191.03. Compounds

C18H19N5O7 Sulfate

73 and 74 were tentatively identified as 8-O-4-linked cross-cou-

pling products of coniferyl alcohol with esculetin and syringyl alco-
Unknown

Unknown
Unknown
Unknown
Unknown

hol with esculetin, respectively. The presence of an unsubstitued 3-

OH group in the esculetin moiety thereby results in the formation
of a benzodioxane-type linkage motif. In accordance with the liter-
ature (Morreel et al., 2010b), CID of deprotonated 73 and 74
101
102
103
100
96

97
98
99

resulted in neutral losses of water and formaldehyde from the

1

[MH] ion and formation of A and B fragment ions at m/z

42 N. Strehmel et al. / Phytochemistry 108 (2014) 35–46

CO 2H R1 O
NH 2 R2 R1
CO2 H R R2
NH S R1
S
H2 N n
n N
R3 N
N R2 H
H 11 n=5, R=- 35 n=7, R1 =CN, R 2=O
12 n=6, R=- 36 n=8, R1 =CN, R 2=O 43 R1 =H, R 2 =H
10 13 n=5, R=O 46 R 1=H, R 2=H, R3 =H
37 n=8, R1 =NCS, R2 =O 44 R1 =OMe, R2 =H
14 n=6, R=O 47 R 1=H, R 2=OH, R 3=H
38 n=8, R1 =COOH, R2 =O 45 R1 =H, R 2 =OMe
39 n=8, 48 R 1=OH, R 2=H, R 3=H
R1 =CONH 2, R 2 =O
40 n=7, R1 =NH 2, R2 =O 49 R 1=OH, R 2=H, R 3=(6-O-Malonyl-β-Glc)O
41 n=8, R1 =NH 2, R2 =O OH OH
42 n=8, R1 =NH 2, R2 =-
HO
OH
R1 R2
CO2 H
O
R1 9
R
10 OH 4 4
OMe MeO OMe
β-Glc
R3 R2 O O O
1
HO 8
HO 8
COOH OMe
51 R1 =OH, R 2=β-XylO, R3 =OH OMe OMe
54 R=H HO HO
52 R1,3={OH, PentO}, R 2=OH 53 R 1,2
={H,OSO 3H}
55 R=OMe
OH OH
56 57
OH OH
OH R3 R2
OH O
MeO MeO OMe
OMe
8
HO 5
HO 5 8 O O
OMe
OMe O O R2 O O 8 8
8 O
O 8

MeO R1 4
R 4
MeO OMe
MeO O OMe O
HO OMe OMe OMe
HO O 8
OH
R1
8
58 R=H HO OH
HO OH
59 R=OMe 60 61 R 1=R 2=R 3=H OH
62 R 1,2,3 ={H,H,Hex} 63 MeO 64 R 1 =R 2=OMe
65 R1,2={H,OMe}
O O COOH COOH

R2 O O
O
4
OMe R1 O
R1 4 4 5
O O O O O OMe OMe
HO 8
8
HO O R1 O O
66 R =H, R =H
1 2
OMe 8 O 8
HO
67 R1 =H, R 2 =Me OMe
O
68 R =H, R =β-Glc
1 2
OH
R2 R3
69 R1 =β-Glc, R 2 =Me 72 R OMe O O OMe
70 R1 =(Hex-Pent), R 2=Me OH R2
77 R1,2,3={H,H,SO3 H}
71 R1 =(Benzoyl-Hex-Pent), R2 =Me 73 R=H 78 R1 =R2 =H
74 R=OMe 79 R1,2={H,SO 3H}
OH OH
OH
COOH R2
9 O
2 O O
R OH O O O
7
83 R1 P N+
3
R1 O O-
O
OH OH
OH O 86 R1,2={H,C(=O)(CH 2) 7COOH}
COOH
82 R =Rha(1->2)Glc, R =Rha
1 2 9

OH
84

Fig. 3. Molecular structures of identified and putatively annotated compounds detected in root exudates of Arabidopsis thaliana. For annotation levels see Table 1. Hex
Hexosyl; Pent Pentosyl.

179.07 and m/z 177.02 for 73 and m/z 209.08 and m/z 177.02 for 2.4.5.3. Hydroxycinnamic acid conjugates and flavonoids. Although
74. In addition, a scopoletin dehydrodimer (75) with unknown phenolic acids represent well-known constituents of root exudates
linkage motif and an unknown oligolignol (76) with four phenyl- (Lanoue et al., 2010), free hydroxycinnamic and hydroxybenzoic
propanoid units of which one was identified as scopoletin were acids could not be detected in root exudates of A. thaliana using
detected in root exudates. the established experimental system. Due to the low process effi-
 N. Strehmel et al. / Phytochemistry 108 (2014) 35–46
ciencies found for cinnamic and salicylic acid (vide supra), it
remains unclear, whether A. thalina roots do not exude these com-
pounds under sterile conditions or their detection is hampered by
the sample preparation protocol. However, three monolignol-feru-
lic acid conjugates (77, 78, 79) and a ferulic acid dimer (80) were
identified. Interestingly, two of them (77, 79) were detected as sul-
fate conjugates as indicated by a neutral loss of 79.96 Da from the
[MH] ions. For the 8-O-4 cross-coupled coniferyl alcohol–ferulic
acid conjugate 77 a characteristic neutral loss of 48.02 Da from the
[MH-SO3] ion was registered together with A and B fragments
at m/z 195.06 and m/z 193.05. As observed for phenylcoumaran-
type dilignols, the CID mass spectra of the 8–5 cross-coupled
coniferyl alcohol–ferulic acid conjugates 78 and 79 are character-
ized by neutral losses of water and formaldehyde from the [MH]
or [MH-SO3] ion, respectively.
Another sulfate conjugate was compound 81. Calculation of the
elemental composition of 81 suggested the presence of three nitro-
gen atoms. The CID mass spectrum obtained from [M+H]+ of 81
revealed neutral losses of 17.03 Da (NH3), 74.08 Da (C3H10N2),
88.10 Da (C4H12N2) and 145.16 Da (C7H19N3) from the [M+H-
SO3]+ ion, the latter resulting in a fragment ion at m/z 291.07 with
the elemental composition of C18H11O+4. This suggests that 81 rep-
resents a sulfated cyclic didehydro-di(coumaroyl)spermidine con-
jugate. The Arabidopsis genome contains a gene (At2g25150)
encoding a functional coumaroyl-CoA:spermidine coumaroyltrans-
ferase (Luo et al., 2009). This gene was shown to exhibit a root spe-
cific expression pattern and its overexpression resulted in the
accumulation of di(coumaroyl)spermidine and a related hexoside
in roots (Luo et al., 2009). Compound 81 could represent a metab-
olite which might be synthesized from di(coumaroyl)spermidine
by intramolecular oxidative phenol coupling and subsequent
sulfation.
Although flavonol glycosides are absent in dark-grown roots in
A. thaliana (Bednarek et al., 2005; Hemm et al., 2004), a kaempferol
triglycoside (82), which was found to be accumulating in rosette
leaves (Yonekura-Sakakibara et al., 2008) was identified in root
exudates.
2.4.6. Fatty acid derivatives
Among the more hydrophobic compounds of the semipolar root
exudate fraction three oxygenated C18 fatty acids (83–85) were
detected. For compounds 83 and 84 on-line H/D exchange experi-
ments revealed the presence of four exchangeable protons indicat-
ing that both fatty acid derivatives are functionalized by three
hydroxyl groups. The CID mass spectra obtained from the [MH]
ion of 83 and 84 showed fragment ions at m/z 229.14 (C12H21O 4)
and m/z 171.10 (C9H15O 3 ) suggesting that both fatty acid deriva-
tives are hydroxylated in position 12 and/or 13 and 9 and/or 10.
In a recent study, 9,12,13-trihydroxy-10,15-octadecadienoic acid
and 9,12,13-trihydroxy-10-dodecenoic acid were identified as
infection markers in the leaf apoplast washing fluid of A. thaliana
following infection by Verticillium longisporum (Floerl et al.,
2012). Authenticated standards of both compounds exhibited iden-
tical chromatographic and mass spectral properties as observed for
83 and 84.
The elemental composition of the fatty acid derivative 85 indi-
cated four oxygen atoms and four double bond equivalents, while
on-line H/D exchange experiments revealed the presence of only
two exchangeable protons. Unfortunately, the CID mass spectrum
obtained from deprotonated 85 showed only unspecific neutral
losses of water and carbon dioxide for which reason 85 could not
be characterized further.
In contrast, the CID mass spectrum obtained from [M+H]+ of 86
exhibited characteristic fragments at m/z 184.07 (C5H15NO4P+) and
m/z 104.11 (C5H15NO+4) indicating the presence of a phosphoryl-
choline moiety. The occurrence of fragments ions at m/z 187.10
43
(C9H15O 
4 ), m/z 169.09 (C9H13O3 ) and m/z 125.10 (C8H13O ) in

the CID mass spectrum obtained from deprotonated 86 was in
accordance with a lysophosphatidylcholine of an oxygenated C9
fatty acid. Since on-line H/D-exchange experiments revealed the
presence of two exchangeable protons 86 is postulated to be an
azelaoyl lysophosphatidylcholine. Azelaic acid itself was shown
to be produced by oxidative fragmentation of esterified 18:2 and
18:3 fatty acid species and proposed to be a general marker for
lipid peroxidation (Zoeller et al., 2012). In addition, this dicarbox-
ylic acid was identified as pathogen inducible metabolite from
the vascular sap of A. thaliana and shown to confer local and sys-
temic resistance against the pathogen Pseudomonas syringae
(Jung et al., 2009).
2.4.7. Unknown components
Among the compounds which were reproducibly detected in
root exudates of A. thaliana, 17 could not or only partially be struc-
turally characterized. Thereof, 11 compounds were annotated as
glycoconjugates with unknown aglycone based on characteristic
neutral losses and fragment ions. Among them, five hexose conju-
gates (87, 91, 92, 93, 95), three malonyl–hexose conjugates (88, 94,
97), a hexose–deoxyhexose conjugate (89) and a malonyl–hexose–
deoxyhexose conjugate (90) were detected. Beside the three sul-
fated phenylpropanoids and the sulfated jasmonic acid derivative,
another sulfate conjugate (98) was found.
3. Concluding remarks
In the present work a workflow for the LC/MS-based analysis of
the semipolar fraction of A. thaliana root exudates obtained from a
sterile hydroponic cultivation system was established. Using this
experimental procedure, more than 100 compounds were consis-
tently detected across two independent experiments and charac-
terized by elemental composition and fragmentation pattern
using UPLC/ESI-QTOFMS as analytical platform. In addition, on-line
H/D exchange experiments provided in numerous cases helpful
information to derive or validate structural hypotheses. More than
80% of the detected compounds were structurally characterized
and assigned to compound classes. The chromatographic and mass
spectral data assembled during this initial screening experiment
thereby provide a valuable basis for the development of targeted
analytical methods (e.g. by using triple quadrupole MS) to sensi-
tively quantify root exudates in subsequent studies. The develop-
ment of such methods should facilitate a significant reduction of
the sampling volume of the nutrient solution.
The spectrum of detected compounds impressively documents
a high metabolic diversity in the semi-polar fraction of root exu-
dates of A. thaliana. In general, the major secondary metabolite
classes known to be accumulated in root tissue were also present
in root exudates collected from six-week-old hydroponically
grown plants with the exception of glucosinolates. Currently, it
cannot be ruled out that glucosinolates are released at a later time
point. Detailed analytical studies are required in the future to com-
pare, to correlate and to precisely quantify the metabolic profiles of
root tissue and exudates in order to gain a qualitative and quanti-
tative understanding of root metabolism in relation to rhizosphere
biochemistry. For that purpose, hydroponic cultivation systems
will not be sufficient to study all aspects of rhizodeposition. How-
ever, they can be applied as manageable model systems to investi-
gate root metabolism and exudation e.g. under different
availability of inorganic micro- and macronutrients (Schmidt
et al., 2014).
Beside the multitude of glucosinolate degradation products, the
detection of an array of dipeptides in root exudates during this
study was most surprising, since dipeptides were considered as
44 N. Strehmel et al. / Phytochemistry 108 (2014) 35–46
source and transport form of nitrogen in plants (Komarova et al.,
2008). It remains to be elucidated whether the exudation of these
dipeptides is linked to a biological function.
The artificial hydroponic cultivation system results in a modi-
fied root architecture due to the lack of mechanical impedance,
and the different nutrient availability and O2/CO2 status in compar-
ison to soil-grown plants. Moreover, it is currently unclear,
whether the observed root exudate pattern from hydroponically
grown plants principally reflects that of plants from natural culti-
vation systems. Thus, different plant cultivation system and exu-
date sampling techniques have to be applied to study their
influence on the resulting exudation pattern.
4. Experimental
4.1. Chemicals
7-Methylsufinylheptyl isothiocyanate and 8-methylsulfinyloc-
tyl isothiocyanate (37) were purchased from LKT Laboratories
(www.lktlabs.com), 4-hydroxyindole-3-carbaldehyde (47) from
Apollo Scientific (www.apolloscientific.co.uk), 4-methoxyindole-
3-carbaldehyde from Chem-Impex International (www.chemim-
pex.com), 15-hydroxypentadecanoic acid from ABCR (www.ab
cr.de), 2-(2,4-dichlorophenoxy)acetic acid from Duchefa
(www.duchefa-biochemie.com), 20 -O-methyladenosine (6) from
TCI (http://www.tcichemicals.com/de/de/) and 3-[2-(4-hydroxy-
3-methoxyphenyl)-3-hydroxymethyl-7-methoxy-2,3-dihydroben-
zofuran-5-yl]acrylic acid (78) from Herbstandard (www.herbstan-
dard.com). 2,3-Dihydroxybenzoic acid 3-O-b-D-xyloside (51) was
kindly provided by Pawel Bednarek (Max Planck Institute for Plant
Breeding Research, Cologne) (Bartsch et al., 2010), 9,12,13-Trihy-
droxy-10(E),15(Z)-octadecadienoic acid (83) and 9,12,13-trihy-
droxy-10-octadecadienoic acid (84) by Ivo Feussner (University
of Göttingen) and [2-D2]-jasmonic acid by Claus Wasternack (Leib-
niz Institute of Plant Biochemistry, Halle).
3-Carboxy-1,2,3,4-tetrahydro-b-carboline (10) was obtained
from reaction of Trp with aqueous formaldehyde solution as
described (Tilstra et al., 1990).
Dipeptides were prepared from corresponding N-tert-butyloxy-
carbonyl amino acids and amino acid tert-butyl ester hydrochlo-
rides using O-(benzotriazol-1-yl)-N,N,N0 ,N0 -tetramethyluronium
tetrafluoroborate as coupling reagent. Deprotection was achieved
by treatment with trifluoroacetic acid/water, 95/5 (v/v).
7-Methylsufinylheptyl amine (40) and 8-methylsulfinyloctyl
amine (41) were prepared by acidic hydrolysis of 7-methylsufi-
nylheptylisothiocyanate and 8-methylsulfinyloctylisothiocyanate
in 6 M HCl for 16 h at 85 °C. 9-Methylsulfinylnonanoic acid (38)
was obtained by acidic hydrolysis of 8-methylsulfinyloctyl gluco-
sinolate (Olsen and Sorensen, 1980) which was isolated from A.
thaliana leaves. 4-Methoxyindol-3-ylmethylamine (44) was pre-
pared from 4-methoxyindole-3-carbaldehyde, which was first con-
verted into its oxime and afterwards reduced by NaBH4 in the
presence of NiCl26H2O (Bednarek et al., 2009). All other chemicals
were purchased from Sigma–Aldrich. All solvents used were of LC/
MS-grade quality (CHROMASOLV).
4.2. Hydroponic plant growth and collection of root exudates
Ultra-pure water (resistivity >18 MO cm) obtained from a Gen-
Pure system (TKA, www.tka.de) was used for the preparation of
nutrient solutions. Plant growth equipment (Supplementary
Fig. 2) and nutrient solutions were sterilized prior to use by auto-
claving. Plant transfer and nutrient solution exchange was per-
formed in a laminar flow hood.
Pre-cultivation: Seeds of A. thaliana (accession Col-0) were sur-
face sterilized for 1 h in an atmosphere of chlorine. Sterilized seeds
were put onto 0.2 mL PCR tubes which had been filled with agar
(8% (w/v) GELRITE, 1% (w/v) sucrose) and cut at the bottom. A total
of 48 tubes were placed into a yellow pipette tip box filled with
180 mL nutrient solution (one-half strength Murashige and Skoog
basal medium supplemented with Gamborg’s B-5 vitamins (Duch-
efa) and 1% (w/v) sucrose, pH 5.8). Afterwards, the box was closed,
sealed with leukoplast (Duchefa) and incubated in a growth cham-
ber under short day conditions (22 °C, 8 h light, 130 lmol/m2/s) for
three weeks.
Main cultivation: PCR tubes were transferred to amber 50 mL
DURAN bottles (cut-off wavelength approx. 500 nm) with perfo-
rated screw cap, which had been completely filled with approxi-
mately 70 mL modified Murashige and Skoog medium (1.0 mM
KH2PO4, 1 mM MgSO4, 0.25 mM K2SO4, 0.25 mM CaCl2, 2 mM NH4-
NO3, 0.1 mM Na-Fe-EDTA, 50 lM KCl, 30 lM H3BO3, 5 lM MnSO4,
1 lM ZnSO4, 1 lM CuSO4, 0.7 lM NaMoO4, pH 5.8) (von Wiren
et al., 1995). A total of 12 bottles were arrayed in a closed plastic
box (Araponics, www.araponics.com), sealed with leukoplast and
incubated in a growth chamber under short day conditions
(22 °C, 8 h light, 120–130 lmol/m2/s) for three weeks. The light
intensity reached about 100–110 lmol/m2/s in a sealed plastic
box while relative humidity was 100%. During this period, the
nutrient solution was exchanged on a weekly basis and checked
for bacterial contamination using LB agar plates. For the analysis
of root exudates, the content of four bottles (approximately
280 mL) was collected after the sixth week, pooled and stored at
4 °C in the dark until sample preparation. To obtain appropriate
blank nutrient solutions agar-filled PCR tubes without plants were
transferred to 50 mL DURAN bottles and processed in parallel as
described above.
Two independent experiments each comprising 24 bottles with
plants and 24 blanks were performed resulting in a total of 6 exu-
date and 6 blank samples per experiment.
4.3. Sample preparation
The pooled nutrient solution (280 mL) was spiked with 4 lg 2-
(2,4-dichlorophenoxy)acetic acid and successively evaporated
under reduced pressure at 40 °C to dryness using a 250 mL
round-bottom flask and a vacuum rotary evaporator. The remain-
ing residue was thoroughly reconstituted in 9 mL water/methanol,
95/5 (v/v), sonicated for 10 min at 20 °C, transferred into a 15 mL
polypropylene centrifuge tube and centrifuged at 6000g for
10 min. An aliquot of the supernatant was subjected to solid phase
extraction using a Bond Elut C18 cartridge (500 mg, 3 mL, Agilent,
www.agilent.com). For that purpose, the cartridge was conditioned
with 1 mL methanol, equilibrated with 1 mL water/formic acid, 98/
2 (v/v) and loaded with 4 mL concentrated nutrient solution. The
cartridge was washed with 1 mL water and retained compounds
eluted using 1 mL methanol/formic acid, 98/2 (v/v). The eluate
was evaporated to dryness at 40 °C using a vacuum centrifuge.
The remaining residue was reconstituted in 120 lL water/metha-
nol, 70/30 (v/v), sonicated for 5 min at 20 °C and centrifuged at
16,000g for 5 min. The supernatant was transferred into a glass
vial and subjected to UPLC/ESI-QTOFMS analysis.
4.4. UPLC/ESI-QTOFMS
Samples (2.6 lL, full loop injection) were separated at 40 °C on
an Acquity UPLC platform (Waters, www.waters.com) equipped
with a HSS T3 column (100  1.0 mm, particle size 1.8 lm, Waters)
applying the following gradient at a flow rate of 150 lL/min: 0–
1 min, isocratic 95% A (water/formic acid, 99.9/0.1 (v/v)), 5% B (ace-
 N. Strehmel et al. / Phytochemistry 108 (2014) 35–46
tonitrile/formic acid, 99.9/0.1 (v/v)); 1–16 min, linear from 5 to
95% B; 16–18 min, isocratic 95% B; 18–20 min, isocratic 5% B.
Eluting compounds were detected from m/z 90 to 1000 using a
MicrOTOF–Q I hybrid quadrupole time-of-flight mass spectrometer
equipped with an Apollo II electrospray ion source (Bruker Dalton-
ics, www.bruker.com) in positive and negative ion mode using the
following instrument settings: nebulizer gas, nitrogen, 1.6 bar; dry
gas, nitrogen, 6 L/min, 190 °C; capillary,5000 V (+4000 V); end
plate offset, 500 V; funnel 1 RF, 200 V; funnel 2 RF, 200 V; in-
source CID energy, 0 V; hexapole RF, 100 V; quadrupole ion energy,
3 eV (5 eV); collision gas, argon; collision energy, 3 eV (7 eV); col-
lision RF 100/200 V (timing 50/50); transfer time, 70 ls; pre pulse
storage, 5 ls; spectra rate, 3 Hz.
For the acquisition of CID mass spectra appropriate precursor
ions were isolated using an isolation width of ±3 m/z and frag-
mented inside the collision cell applying collision energies in the
range of 5–50 eV. Argon was used as collision gas. Product ions
were detected using the same parameter settings as described
above.
All mass spectra were acquired in centroid mode. Recalibration
of m/z scale was performed for individual raw data files on lithium
formate cluster ions obtained by automatic infusion of 20 lL
10 mM lithium hydroxide in isopropanol/water/formic acid, 49.9/
49.9/0.2 (v/v/v) at a gradient time of 18 min using a diverter valve.
For on-line H/D exchange experiments UPLC/ESI-QTOFMS was
performed under identical conditions, except eluent A for which
deuterium oxide/formic acid, 99.9/0.1 (v/v) was used.
4.5. Data analysis
For nontargeted data analysis, raw data files were converted
into mzData format using CompassXPort (Bruker Daltonics). Raw
data files were arrange in two sample classes (exudate, blank)
and processed using the R package XCMS (Smith et al., 2006). Fea-
ture detection was performed applying the centWave algorithm
(sntresh = 3, prefilter = (3, 100), ppm = 25, peak width = (5, 12))
(Tautenhahn et al., 2008). Alignment was accomplished by the
function group.density (minfrac = 0.75, bw = 2, mzwid = 0.05).
Missing feature intensities were filled in using the function fillPe-
aks. The resulting matrix was exported into Microsoft Excel and
feature intensities were log2-transformed. For the pairwise com-
parison of the exudate and blank sample class, medians of the fea-
ture intensity within a sample class as well as fold changes and P
values (Student’s t-test, two-sided, unequal variances) were deter-
mined and used for the identification of reproducibly occurring
exudate-related features.
DataAnalysis 4.0 (Bruker Daltonics) was used for generation of
extracted ion chromatograms, deconvolution of compound mass
spectra and calculation of elemental compositions. For the relative
quantification of compounds extracted ion chromatograms of cor-
responding quantifier ions were integrated applying QuantAnalysis
2.0 (Bruker Daltonics).
4.6. Determination of process efficiency and repeatability
A spiking mixture of 15 test compounds was prepared in water/
methanol, 70/30 (v/v) from individual 10 mM stock solutions. For
its qualitative and quantitative composition see Supplementary
Table 1. Afterwards, nine 280 mL aliquots of the modified MS
nutrient solution were fortified with 54 lL of the spiking mixture,
processed as described above and analyzed together with appro-
priately solvent diluted aliquots of the spiking mixture by UPLC/
ESI-QTOFMS in positive and negative ion mode. Test compounds
were relatively quantified using retention times and quantifier ions
from Supplementary Table 1. Process efficiency was expressed as
ratio of the medians calculated from the peak areas of a test
45
compound in processed samples to solvent diluted spiking mix-
tures. Repeatability was expressed as relative standard deviation
obtained from the peak areas of a test compound in processed
samples.
Acknowledgements
This work was supported by the Joint Initiative for Research and
Innovation of the Leibniz Association (SAW-2011-IPB-3 97, ‘‘Chem-
ical Communication in the Rhizosphere’’). Expert technical assis-
tance by Sylvia Krüger and Jessica Thomas is gratefully
acknowledged.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2014.
10.003.
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