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ORIGINAL PAPER
Hairy roots, callus, and mature plants of Arabidopsis thaliana
exhibit distinct glucosinolate and gene expression profiles
Anja Kastell •Iryna Smetanska •Monika Schreiner •
Inga Mewis
Received: 27 February 2013 / Accepted: 29 May 2013 / Published online: 8 June 2013
ÓSpringer Science+Business Media Dordrecht 2013
Abstract Hairy root cultures transformed with Agrobacte-
rium rhizogenes or undifferentiated callus cultures are used
for production of plant secondary metabolites. Glucosino-
lates (GS) are a group of secondary metabolites that produce
a variety of bioactive compounds upon hydrolysis. Several
studies report the successful production of high concentra-
tions of secondary metabolites in vitro. However, such
cultivation methods can significantly change metabolic
profiles, and the mechanism behind this to be rarely under-
stood. Therefore, we compared the GS and transcript pro-
files of Arabidopsis thaliana leaves and roots with hairy root
and callus in vitro cultures. Compared to the roots of intact
A. thaliana plants, overall, hairy roots contained lower GS
levels. In particular, lower quantities of short-chain aliphatic
GS were observed and a larger proportion of long-chain
aliphatic GS on total content. Corresponding, the transcript
levels of most aliphatic biosynthetic genes (MAM1,
CYP79F1, CYP83A1, UGT74C1,andSUR1)weresignifi-
cantly lower in hairy root cultures compared to roots of
intact plants. In callus culture, the lowest transcripts levels
were detected for overall GS biosynthetic genes with an
absence of aliphatic GS. From the indole group, 1-methoxy-
indol-3-ylmethyl GS was found to be a major component in
hairy root cultures and roots whereas indol-3-ylmethyl GS
dominated in leaves and 4-hydroxy-indol-3-ylmethyl GS in
callus cultures. Leaves of intact plants contained the highest
amounts of GS. Here, aliphatic short-chain GS dominated
which was in accordance with transcript levels of aliphatic
biosynthetic genes. The study reveals tissue-specific accu-
mulation of GS and transcript pattern in plants distinct from
in vitro culture systems.
Keywords In vitro culture Hairy roots Callus culture
Glucosinolates Gene expression Arabidopsis thaliana
Abbreviations
GS Glucosinolates
MS medium Murashige and Skoog medium
Trp Tryptophan
Introduction
Glucosinolates (GS) are typical plant secondary metabo-
lites in the order Brassicales consisting of a sulfur-linked
b-D-glucopyranose moiety and an amino acid-derived side
chain (Fahey et al. 2001). According to their side chain
structure, they are classified as aliphatic, aromatic, or
indole GS (Halkier and Gershenzon 2006). The precursor
amino acids of aliphatic and indole GS are methionine and
tryptophan, respectively. GS are synthesized in three sep-
arate phases: in the first chain elongation step in aliphatic
biosynthesis, methylene groups are inserted into side
Electronic supplementary material The online version of this
article (doi:10.1007/s11240-013-0338-7) contains supplementary
material, which is available to authorized users.
A. Kastell I. Smetanska
Department of Food Biotechnology, Berlin University of
Technology, Ko
¨nigin-Luise-Straße 22, 14195 Berlin, Germany
I. Smetanska
University of Applied Sciences Weihenstephan-Triesdorf,
Steingruberstr. 2, 91746 Weidenbach, Germany
M. Schreiner I. Mewis (&)
Department of Quality, Leibniz-Institute of Vegetable
and Ornamental Crops Großbeeren/Erfurt e.V.,
Theodor-Echtermeyer-Weg 1, 14979 Großbeeren, Germany
e-mail: inga@entomology.de
123
Plant Cell Tiss Organ Cult (2013) 115:45–54
DOI 10.1007/s11240-013-0338-7
chains of methionine (Supplementary Figure 1). Core GS
synthesis is catalyzed by cytochrome P450 (CYP) gene
products in the cytosol and the amino acid moiety is rec-
onfigured to an aldoxime. CYP79B2 and CYP79B3 cata-
lyze tryptophan-derived precursors (Hull et al. 2000),
CYP79F1 oxidates derivatives of methionine with one to
six methylene residues (preference for short-chain precur-
sors) and CYP79F2 metabolizes only precursors with five
to six methylene groups (Chen et al. 2003). In further steps,
aldoximes are oxidated to S-alkylthiohydroximates by
members of the CYP83 family of cytochrome P450 mon-
ooxygenases, with CYP83A1 showing a preference for
aliphatic and CYP83B1 for indolyl aldoximes (Bak et al.
2001). S-alkylthiohydroximates are cleaved by C–S-lyase
to form thiohydroximate (Mikkelsen et al. 2004). Follow-
ing desulfo GS are produced, catalyzed by glucos-
yltransferases of the UGT74 family. Here, UGT74C1
glucosylates methionine-derived thiohydroximates and
UGT74B1 metabolizes tryptophan-derived thiohydroxi-
mates (Grubb et al. 2004). The final step is sulfation by
sulfotransferases accomplished by SOT17 and SOT18 or
SOT16, which metabolize methionine-derived precursors
and tryptophan-derived precursors, respectively (Piotrow-
ski et al. 2004). Further secondary side chain modifications,
such as oxidation and hydroxylation of methionine-derived
GS and methoxylation and desaturation of indoles, lead to
the enormous structural variety of GS (Kliebenstein 2009;
Pfalz et al. 2011).
Glucosinolates produce different hydrolysis products
such as nitriles, isothiocyanates, and thiocyanates after
cleavage by the endogenous myrosinase enzyme (Fenwick
et al. 1983; Halkier and Gershenzon 2006). From these GS
breakdown products especially isothiocyanates are thought
to have several beneficial health effects, such as antimi-
crobial, antiflammatory, or anticarcinogeneic properties
(Jeffery and Araya 2009; Fahey et al. 2002). Especially the
isothiocyanate of 4-methylsulfinylbutyl GS, sulforaphane,
is thought to be a strong anticancer agent by inhibiting
phase-I enzymes and inducing phase-II enzymes as well as
apoptosis (Zhang et al. 1992; Fahey et al. 1997). However,
different groups of GS are associated with different effects
on human health (Verkerk et al. 2009). For these reasons, it
is of great interest to maintain systems with a defined GS
quantity or profile. For these studies, plant in vitro cultures
such as hairy root cultures or callus cultures are an optimal
system because they enable plant material to be grown
under defined conditions and the possibility for metabolic
engineering—altering gene expression and metabolic
pathways in cultures.
Hairy root cultures have been proven to be potent sys-
tems for producing valuable phytochemicals (Georgiev
et al. 2007; Srivastava and Srivastava 2007; Zhou et al.
2011). The formation of so-called hairy root phenotype is
caused by Agrobacterium rhizogenes mediated transfor-
mation with T-DNA carrying the genes rolA, rolB, rolC,
and rolD. In order to produce phytochemicals, hairy root
in vitro cultures offer advantages such as fast growth rates
in media without phytohormones, genetic and biochemical
stability, and controlled conditions. Root exudates can be
used for metabolite production from continuously culti-
vated hairy root cultures in order to obtain the desired
compounds (Schreiner et al. 2011; Cai et al. 2012). How-
ever, in vitro cultivation of plant cells can significantly
change metabolic profiles. This is partly due to stress
caused by cultivation methods. Moreover, rol-genes from
A. rhizogenes are potential activators of secondary
metabolism in transformed cells (Bulgakov 2008). Geor-
giev et al. (2010) reported a 20-fold higher total phenolic
concomitant compound in extract from hairy root cultures
in comparison to the parent plant Beta vulgaris. In our
previous study, the GS profile of hairy root cultures
obtained from two Brassica species differed from the
parent plant (Kastell et al. 2013). No aliphatic GS could be
found in hairy roots of Sinapis alba (white mustard) and
Brassica rapa subsp. rapa pygmeae teltowiensis (teltow
turnip); only indolic GS were detected. Gene expression
studies explaining the lack of aliphatic GS in hairy roots
are still missing. Alternative brassicaceous callus cultures
could be used as production systems for aliphatic and
indole GS. Plant callus cultures represent a potential source
of valuable secondary metabolites, which can be used as
food additives, nutraceuticals, and pharmaceuticals (Zhong
2001; Smetanska 2008). Several secondary metabolites,
such as betalains from Beta vulgaris (Trejo-Tapia et al.
2007) as well as anthocyanins from Vitis vinifera (Saw
et al. 2012) have been obtained from plant callus cultures.
The use of hairy root and callus cultures as production
systems of bioactive substances and model systems for
studies in biosynthesis pathways requires comparative
investigations into differences between plant and in vitro
GS biosynthesis in order to obtain systems with the desired
GS concentration and profile. Gene expression studies
provide further insights into how GS biosynthesis is regu-
lated by metabolic pathways in cultures compared to cor-
responding plants. Accordingly, the main objective of this
study was to compare the GS profile and expression profile
of GS biosynthetic genes in vitro cultures and plant organs
of Arabidopsis thaliana ecotype Columbia.
Materials and methods
Plant cultivation and generation of in vitro cultures
Arabidopsis thaliana (L.) ecotype Columbia (Col-0) was
selected for the GS comparison studies of in vitro cultures
46 Plant Cell Tiss Organ Cult (2013) 115:45–54
123
and plants. In order to obtain plants, seeds were sown in
pots filled with soil and stratified for 2 days at 4 °C in the
dark. The pots were transferred to a climate chamber with a
temperature of 21 ±1°C, a relative humidity of
60 ±5 %, and a light intensity of 200 lmol m
-2
s
-1
(10:14 day/night photoperiod). After germination, the
seedlings were transferred to single pots (7 cm diameter,
6 cm height) containing silica sand. The plants were
watered as needed with half-strength Hoagland (Hoagland
and Arnon 1950) solution.
For establishing hairy root cultures from A. thaliana,
seeds were surface-sterilized by immersion in 70 % etha-
nol (v/v), followed by treatment with 10 % sodium hypo-
chloride (v/v) solution each for 1 min. The seeds were
rinsed in sterile water and placed on petri dishes with a
hormone-free half-strength MS medium (Murashige and
Skoog 1962) for germination. Petri dishes were incubated
at 22 °C in 24 h light. Seedlings were used for infection
with the Agrobacterium rhizogenes strain A4. Prior to
transformation, A. rhizogenes strain was inoculated in 2 ml
liquid MYA medium (pH =6.6) on a shaker at 150 rpm at
37 °C for 24 h and the bacteria medium was transferred to
50 ml MYA medium and cultivated for another 24 h to
obtain an adequate bacteria density above OD
600
[1.6.
Roots from about 2-week-old seedlings were placed for
1 min in 2 ml tubes filled with the liquid bacterial solution.
The roots were then transferred to 250 ml flasks containing
60 ml hormone free half-strength MS medium supple-
mented with 3 % (w/v) sucrose. Cefotaxime (0.2 g l
-1
)
was added to eliminate the bacterium after 1 day. Only root
inoculates that developed the hairy root phenotype were
sub-cultivated. The hairy roots used for the biochemical
and molecular biological analysis were cultivated in a
hormone-free liquid half-strength MS nutrient medium,
supplemented with 3 % (w/v) sucrose in Erlenmeyer flasks
(250 ml) with 60 ml medium on a shaker (110 rpm) at
26 °C in the dark. The pH of the medium was adjusted to
5.8 by adding potassium hydroxide. The cultures were sub-
cultivated every 2 weeks and used for experiments
4 months after establishment.
Callus cultures were established by wounding 2-week-
old seedlings of A. thaliana and placing them on petri
dishes filled with half-strength MS medium, supplemented
with 0.4 mg/l 2,4-D, 3 % (w/v) sucrose, and 0.8 % (w/v)
agar. Callus cultures were sub-cultured every 3 weeks on
petri dishes with fresh half-strength MS medium supple-
mented with 0.4 mg/l 2,4-D, 3 % (w/v) sucrose, and 0.8 %
(w/v) agar and used in the experiments 4 months after
establishment.
Seven-week-old A. thaliana rosette leaves and roots
were harvested. Sand was removed from the roots by
rinsing them with water. Hairy root and callus cultures
were harvested for analysis 14 days after sub-cultivation.
After harvesting samples of A. thaliana, leaves, roots, hairy
root, and callus cultures were immediately frozen in liquid
nitrogen and stored at 80 °C until required for further use.
The tissues were compared with regarding to GS content
and relative gene expression of genes associated with GS
biosynthesis.
Glucosinolate extraction and HPLC analysis
GS extraction was performed using 20 mg of freeze-dried
ground sample each. The samples were incubated at 80 °C
with 750 ll of 70 % (v/v) methanol together with 60 llof
0.5 mM 4-hydroxybenzyl GS (sinalbin, purified from
Sinapis alba seeds) as internal standard. In order to pellet
the plant materials, samples were centrifuged at 4,500gfor
5 min. This step was repeated twice with 500 llof70%
boiling methanol, and the extracts were combined. Des-
ulfation was performed on DEAE Sephadex A-25 mini
columns washed and preconditioned according to the
method of Mewis et al. (2005). The extracts were desulf-
ated overnight with 75 ll purified aryl sulfatase solution
(H-1 from Helix pomatia, Sigma Aldrich). After elution
with 1 ml ultrapure water 40 ll of desulfo GS extracts
were analyzed with HPLC using UltiMate SR-3000 (Dio-
nex, Germany), equipped with LPG-3400SD pump, WPS-
3000SL automated sample injector, Acclaim 120 C18
Reversed-Phase LC Column (250 92.1 mm, 5 lm, Dio-
nex), DAD-3000 diode array detector (Dionex) and soft-
ware Chromeleon 6.8. The eluents used were ultrapure
water (A) and 40 % (v/v) acetonitrile in ultrapure water
(B) at a flow rate of 0.4 ml/min. The gradient run was as
follows: 0.5 % B (1 min), 0.5–20 % B (7 min), 20 % B
(2 min), 20–50 % B (9 min), 50 % B (3 min), 50–99 % B
(6 min), 99 % B (5 min), 99–0.5 % B (3 min), and a 7 min
equilibration at 0.5 % B. The eluent was monitored by
photodiode array detection between 190 and 360 nm.
Desulfo GS peaks were identified at 229 nm using reten-
tion time and UV spectra as compared to standards. Their
content was calculated using 4-hydroxybenzyl GS as an
internal standard and the response factor of each compound
relative to allyl GS.
Gene expression analysis by RT-PCR
Total RNA from shoots and callus cultures was isolated
using the Quiagen RNeasy Plant Mini Kit (Qiagen,
Valencia, CA, USA) and from roots and hairy root cultures
using TRIzol reagent (Invitrogen, Carlsbad, CA, USA)
following the standard protocols. It was necessary to use
different kits because the yield and quality of RNA were
not equally good for the different tissues. RNA extraction
procedures included a DNA digestion step. RNA was
quantified spectrophotometrically. 1 lg RNA was reverse
Plant Cell Tiss Organ Cult (2013) 115:45–54 47
123
transcribed using Moloney murine leukemia virus
(M-MLV) reverse transcriptase (Promega) and oligo dT
primer to obtain first-strand cDNA. The RT-PCR was
performed for 1 h at 42 °C and terminated heating to 70 °C
for 10 min.
PCR reaction was optimized to ensure that the PCR reaction
with all primers was in the linear range. 16 different genes were
analyzed: MAM1 (At5g23010), MAM3 (At5g23020), CYP79F1
(At1g16410), CYP79F2 (At1g16400), CYP79B2 (At4g39950),
CYP79B3 (At2g223300), CYP83A1 (At4g13770), CYP83B1
(At4g31500), UGT74B1 (At1g24100), UGT74C1 (At2g31
790), SUR1 (At2g20610), TGG1 (At5g26000), MYB28
(At5g61420), MYB76 (At5g07700), MYB34 (At5g60890), and
MYB51 (At1g18570). The sequences of primers were used as
shown in Supplementary Table 1. For PCR experiments, an
aliquot of 0.2 ll cDNA was used in 20 ll reaction volume. PCR
buffer contained 0.2 mM dNTP’s, 2.1 mM MgCl2, 0.5 mM of
the forward and reverse primer and 1 unit of Taq DNA poly-
merase (Promega). Actin8 (AC8, At1g49240) was used as a
reference gene and was designed to be intron spanning for the
possible detection of genomic DNA contamination. Actin8
forward primer was 50-ATGAAGATTAAGGTCGTGGCAC;
reverse had the sequence 50-GTTTTTATCCGAGTTTGAA-
GAGGC. PCR products were subjected to gel electrophoresis
using a 1.2 % agarose gel containing RedSafe DNA stain. For
quantification, they were visualized on a Biostep trans-illumi-
nator and band intensities were measured using TotalLab Quant
software. In order to calculate the quantity of the PCR product,
band intensities were computed to quantities of the low mass
ladder from Invitrogen in the software. Expression levels were
normalized relative to that of Actin8. RNA extraction and RT-
PCR were performed twice, and similar patterns of expression
were obtained.
Statistical analysis
Tukey’s honest significant difference (HSD) test after
analysis of variance was used to determine differences
among GS and transcript levels in different plant tissues, hairy
root, and callus culture by applying SYSTAT (SYSTAT 9,
SPSS Inc., Chicago, USA).
Results
Glucosinolate profiles of plant tissues and in vitro
cultures
The GS profiles of intact plants—roots and rosette leaves—
were compared with levels in hairy root and callus in vitro
cultures. In the leaves of A. thaliana (Col-0) plants, the
major group was aliphatic (Table 1). 4-methylsulfinylbutyl
GS (13.4 lmol g
-1
dry weight) was identified as the main
aliphatic GS in leaves, which was sixfold higher than levels
of the second highest aliphatic GS 3-methylsulfinylpropyl
GS (2.2 lmol g
-1
dry weight). 4-methylthiobutyl, 5-meth-
ylsulfinylpentyl, 6-methylsulfinylhexyl, and 7-methylsulfi-
nylheptyl GS were also detected as aliphatic GS in leaf
tissue. Four indole GS—4-hydroxyindol-3-ylmetyhl, indol-
3-ylmethyl, 4-methoxyindol-3-ylmethyl, and 1-methox-
yindol-3-ylmethyl-GS—were detected in all tissues. The
most abundant indole GS in leaves was indol-3-ylmethyl GS
(3.8 lmol g
-1
dry weight). The total content of indole GS in
leaves was about one-third of aliphatic GS levels. In A.
thaliana roots, the total GS content was approximately
8.0 lmol g
-1
dry weight, threefold lower compared to
24.2 lmol g
-1
dry weight present in leaves. Only five ali-
phatic GS (3-methylsulfinylpropyl, 4-methylthiobutyl,
4-methylsulfinylbutyl, 5-methylsulfinylpentyl, and 7-meth-
ylsulfinylheptyl GS) were detectable in roots. Indole GS
levels were also lower in roots compared to leaves. The
indole GS profile of leaves and roots was found to be dis-
tinct; 1-methoxy-indol-3-ylmethyl GS dominated in roots
while indol-3-ylmethyl GS and 4-methoxy-indol-3-ylmethyl
GS were abundant in leaves.
The aliphatic and indole GS profile of hairy root and
callus cultures was remarkably different from leaves and
roots, and total GS levels were generally lower (Table 1).
The main class of GS in hairy root and callus cultures was
indole with 4.1 and 2.4 lmol g
-1
dry weight, respectively,
and not aliphatic, as in leaves. While indol-3-ylmethyl GS
was the main indole GS in A. thaliana leaves, in roots of
intact plants and in hairy root cultures it was its derivate
1-methoxy-indol-3-ylmethyl GS. In callus culture the main
indole GS was detected to be the hydroxylated derivate of
indole-3-ylmethyl GS, 4-hydroxy-indol-3-ylmethyl GS. In
hairy root cultures, low levels of seven different aliphatic
GS (4-methylsulfinylbutyl, 4-methylthiobutyl, 5-meth-
ylsulfinylpentyl, 6-methylsulfinylhexyl, 7-methylsulfinyl-
heptyl, 8-methylsulfinyloctyl, and 8-methylthiooctyl GS)
were detected; long-chain aliphatic GS dominated with the
highest content of 8-methylsulfinyloctyl GS (0.5 lmol g
-1
dry weight). This is contrary to leaves and roots, in which
short-chain aliphatic GS are abundant. Two aliphatic GS
only—4-methylsulfinyl GS and 4-methylthiobutyl GS—
were found in small quantities in callus culture.
Transcription of glucosinolate genes
We determined the relative transcript levels of the main
genes involved in the biosynthesis of GS in 7-week-old
A. thaliana plants (leaves and root) and 2-week-old sub-
cultured in vitro hairy root and callus culture. The
expression levels in the four different tissues types varied,
underlying the molecular basis of their GS phenotype.
48 Plant Cell Tiss Organ Cult (2013) 115:45–54
123
Gene expression associated with aliphatic glucosinolate
biosynthesis
Expression levels of genes related to aliphatic GS biosyn-
thesis were generally highest in leaves, with one exception;
CYP79F2 encoding monooxygenase which metabolizes
long-chain elongated methionines—was significantly
higher expressed in roots and in vitro cultures (Fig. 1).
Transcript levels of CYP79F1—preference for short-chain
methionine precursors—were remarkable and significantly
higher in plant leaves compared to roots, hairy roots, and
callus cultures (Fig. 1). There were also differences in gene
expression between roots and hairy roots. Significantly
higher expression was determined in roots for MAM1,
CYP83A1, and SUR1 compared to hairy roots, whereas in
hairy roots higher expression was found for CYP79F1,
CYP79F2, and UGT74C1 (Fig. 1). Callus cultures exhib-
ited no expression, except for CYP79F2,UGT74C1, and
SUR1. It was not possible to detect MAM3 expression in
any analyzed tissue.
Gene expression associated with indole glucosinolate
biosynthesis
Of the two genes (CYP79B2 and CYP79B3) involved in
indole GS biosynthesis converting tryptophan to the cor-
responding aldoxime, CYP79B2 was highest expressed in
root tissue, followed by leaves and hairy roots (Fig. 2).
However, CYP79B2 was not expressed in callus culture
tissue (Fig. 2). Contrary to this, CYP79B3 was equally
expressed in callus culture and in leaves, with lower levels
in roots and significantly reduced levels in hairy roots
(Fig. 2). CYP83B1 encoding for an oxime-metabolizing
enzyme in the indole pathway was expressed in shoot, root,
and hairy root culture, but only marginally in callus culture.
Similarly for UGT74B1, the lowest expression was present
in callus culture. However, the expression in hairy root
cultures was also significantly lower when compared to
leaf and root tissues of the plant. Interestingly, the
expression levels of TGG1—encoding for the GS hydro-
lyzing enzyme myrosinase—was highly expressed only in
the leaf tissue (Fig. 2).
Transcription factor
The transcription factor associated with aliphatic GS bio-
synthesis—MYB28—was highly expressed in leaves, fol-
lowed by hairy root culture and roots; the lowest
expression was in callus culture (Fig. 3). Contrary to this,
another transcript factor of aliphatic biosynthesis—
MYB76—was equally expressed in leaves and the hairy
root culture, but transcripts were absent in roots and the
callus culture. From the transcript factors of indole GS
biosynthesis, MYB34 was expressed significantly higher in
leaves and hairy roots compared to roots and callus culture
(Fig. 2). In contrast, the transcription of another transcript
factor of indole GS biosynthesis—MYB51—was highest
expressed in rosettes, followed by roots and hairy roots,
with no detectable transcription in the callus culture.
Table 1 Aliphatic and indole
glucosinolate contents in
Arabidopsis thaliana leaves,
roots, hairy roots, and callus
cultures
Mean values (n =3) within
rows with different letters are
significantly different using
Tukey’s HSD test pB0.05.
‘‘n.d.’’ refers to non-detectable
levels
Glucosinolate (GS) Glucosinolate contents (lmol g
-1
dry weight)
Leaves Roots Hairy roots Callus
Aliphatic GS
3-Methylsulfinylpropyl 2.28 ±0.22
a
0.26 ±0.07
b
n. d. n. d.
4-Methylthiobutyl 0.84 ±0.07
a
0.28 ±0.10
b
0.05 ±0.04
c
0.01 ±0.01
c
4-Methylsulfinylbutyl 13.46 ±0.73
a
2.50 ±0.56
b
0.02 ±0.02
d
0.25 ±0.08
c
5-Methylsulfinylpentyl 0.44 ±0.05
b
1.04 ±0.64
a
0.01 ±0.01
c
n. d.
6-Methylsulfinylhexyl 0.37 ±0.05
a
n. d. 0.15 ±0.04
b
n. d.
7-Methylsulfinylheptyl 1.30 ±0.81
a
0.62 ±0.24
b
0.05 ±0.02
c
n. d.
8-Methylthiooctyl n. d. n. d. 0.47 ±0.14 n. d.
8-Methylsulfinyloctyl n. d. n. d. 0.52 ±0.23 n. d.
Total aliphatic GS 18.69 ±1.60
a
4.70 ±0.73
b
1.27 ±0.41
c
0.26 ±0.19
d
Indole GS
4-Hydroxy-indol-3-ylmethyl 0.14 ±0.03
b
0.16 ±0.05
b
0.13 ±0.04
b
1.35 ±0.34
a
indol-3-ylmethyl 3.82 ±0.76
a
0.84 ±0.29
b
0.16 ±0.07
c
0.84 ±0.22
b
4-Methoxy-indol-3-ylmethyl 1.18 ±0.24
a
0.25 ±0.08
b
1.09 ±0.39
a
0.13 ±0.06
b
1-Methoxy-indol-3-ylmethyl 0.40 ±0.19
b
2.07 ±0.49
a
2.70 ±0.77
a
0.08 ±0.01
c
Total indole GS 5.54 ±1.12
a
3.32 ±0.53
ab
4.08 ±1.47
ab
2.40 ±0.43
b
Total GS 24.23 ±2.69
a
8.02 ±1.83
b
5.35 ±1.34
bc
2.59 ±0.45
c
Plant Cell Tiss Organ Cult (2013) 115:45–54 49
123
Discussion
Hairy root or callus cultures have been the focus of studies
due to their utility as either production for plant secondary
metabolites or as a model system to study biochemical
processes. To evaluate the potential of in vitro cultures as
model system for glucosinolate biosynthesis or as pro-
duction systems for certain groups of glucosinolates it is
important to know the differences in biosynthetic pathways
that occur in the different systems—in vitro culture and
plants. In order to determine the differences between GS
biosynthetic pathways in different tissues of in vitro culture
and the plant, we compared the GS phenotype with asso-
ciated gene expression levels. For this purpose, tissue from
2-week-old sub-cultured hairy root and callus in vitro
cultures were compared to leaves and root tissue of the
corresponding 7-week-old parent A. thaliana plant (eco-
type Col-0).
Our findings reveal that the GS profile as well as gene
expression of corresponding biosynthetic pathways in hairy
roots and callus cultures of A. thaliana is considerable
distinct to the parent plant. The total GS content in leaves
of A. thaliana is considerably higher than in roots, hairy
roots, and callus cultures which is in line with the generally
highest transcript levels of genes related to GS biosynthesis
in this tissue. However, there are several contrasting
studies which report a higher productivity of different
secondary metabolites in hairy root cultures compared to
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1,8
2,0
MAM1 CYP79F1 CYP79F2 CYP83A1 UGT74C1 SUR1 TGG1
Relative Expression
Genes associated with aliphatic glucosinolate metabolism
Leaves Roots Hairy Roots Callus Cultures
a
a
a
a
a
a
a
aa
a
ab
b
bb
b
bc
bbb
bbb
b
c
cc
c
d
Fig. 1 Relative expression
levels of aliphatic GS
biosynthesis genes in leaves,
root, hairy root, and callus
culture of Arabidopsis thaliana
in relation to the reference gene
Actin8. For each gene, means
with different letters are
significantly different using
Tukey’s HSD test, pB0.05,
n=3
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1,8
2,0
CYP79B2 CYP79B3 CYP83B1 UGT74B1
Relative Expression
Genes associated with indole glucosinolate metabolism
Leaves Roots Hairy Roots Callus Cultures
a
a
a
ab
a
a
a
a
a
b
b
b
b
b
c
c
c
Fig. 2 Relative expression
levels of indole GS biosynthesis
genes in leaves, root, hairy root,
and callus culture of
Arabidopsis thaliana in relation
to the reference gene Actin8.
For each gene, means with
different letters are significantly
different using Tukey’s HSD
test, pB0.05, n =3
50 Plant Cell Tiss Organ Cult (2013) 115:45–54
123
the parent plant (Maldonado-Mendoza et al. 1993; Geor-
giev et al. 2007; Zhou et al. 2011). However, non-brassi-
caceous plants were used for these analyses. It should be
noted that hairy root cultures can release GS to the culture
medium (Cai et al. 2012). In the present study, we found
exclusively indole GS in the culture medium with about
5.3 lmol l
-1
medium (data not shown), indicating that the
productivity of indole GS in hairy root cultures may be
higher than in the parent plant.
Arabidopsis thaliana Col-0 exhibits an organ-specific
GS profile (Brown et al. 2003). The proportion of indole
GS in roots is higher than in leaves and siliques. Corre-
spondingly, in our study the aliphatic/indole ratio in leaves
of Col-0 was higher (3:1 ratio) than in roots (1.5:1 ratio). In
addition to genetic control, the accumulation of secondary
metabolites also depends on the cultivation conditions and
age of the plant: Indole GS are the dominating group in
younger Col-0 roots while aliphatic GS are the major group
older plants (Petersen et al. 2002). In contrast, in Col-0
leaves the aliphatic/indolic GS ratio declines with age, but
aliphatic GS are dominating all the time. For hairy root and
callus cultures, we found indole GS to be the main group
corresponding to our recent results with B. rapa and S. alba
and hairy root cultures (Kastell et al. 2013).
Arabidopsis thaliana Col-0 hairy roots and roots
exhibited a similar indole GS profile, whereby 1-meth-
oxyindol-3-ylmethyl GS dominated. However, 4-meth-
oxyindol-3-ylmethyl GS was the second most abundant
indole GS in hairy roots, while it was the precursor indol-3-
ylmethyl-GS in roots. Also the aliphatic GS profile of hairy
root culture was different compared to mature plants.
Short-chain aliphatic GS were the major group in roots,
whereas long-chain aliphatic GS dominated in hairy root
cultures. An explanation for this finding may be an altered
auxin biosynthesis in hairy root cultures: Auxin (indole-3-
aceticacid; IAA) has been thought to be synthesized from
tryptophan (Trp) by a two-step reaction, because Trp can
be converted by the aux1 gene product tryptophan mono-
oxygenase to indole-3-acetamide (IAM), which is then
converted by indole acetamide hydrolase, the aux2 gene
product, to IAA (Offringa et al. 1986; Thomashow
et al.1984). When applying auxin to A. thaliana plants,
Mikkelsen et al. (2003) found a reduced content of short-
chain aliphatic GS in leaves, with the most dramatic
decrease in 4-methylsulfinylbutyl GS. In contrast, con-
centrations of long-chain aliphatic and indole GS
increased, accompanied by an induction in expression of
CYP79B2 and CYP79B3 and a reduction of CYP79F1.
Knockout mutants in the GS biosynthetic gene CYP79F1
show increased levels of long-chain aliphatic GS and of
indole GS in A. thaliana leaves (Reintanz et al. 2001). The
altered GS profile of hairy root cultures compared to root
tissue of parent plant may be due to increased auxin levels
in hairy root cultures.
The GS profiles of roots and hairy roots do not exactly
reflect the different expression of CYP79 genes in these
tissues. The expression of CYP79B2 and CYP79B3
involved in indole GS pathway is higher in roots, which is
contrary to the slightly higher amount of indole GS
determined for hairy root cultures. This observation could
be explained by differences in gene expression for other
genes involved in GS biosynthesis and transcript factors
that regulate GS accumulation: No expression from tran-
script factor of indole GS biosynthesis ATR1/MYB34 was
found in roots, but expression of MYB51 was determined.
In hairy root culture, both transcript factors were equally
0,0
0,2
0,4
0,6
0,8
1,0
1,2
MYB28 MYB76 MYB34 MYB51
Relative Expression
Transcription factors of glucosinolate metabolism
Leaves Roots Hairy Roots Callus Cultures
ab
b
b
bbbb
b
b
c
c
a
aa
a
a
Fig. 3 Relative expression
levels of transcription factors of
GS biosynthesis in leaves, root,
hairy root, and callus culture of
Arabidopsis thaliana in relation
to the reference gene Actin8.
For each gene, means with
different letters are significantly
different using Tukey’s HSD
test, pB0.05, n =3
Plant Cell Tiss Organ Cult (2013) 115:45–54 51
123
transcribed. ATR1/MYB34 had previously been described
as a regulator of indole GS and indole-3-acetic acid
homeostasis (Bender and Fink 1998). Overexpression of
MYB51 resulted in the accumulation of only indole GS,
without affecting auxin metabolism (Gigolashvili et al.
2007). The increase of MYB34 together with changes in
gene expression of CYP79B2,CYP79B3, and UGT74B1
involved in indole GS biosynthesis and linked to auxin
metabolism in hairy root cultures indicates a change in
auxin homeostasis compared to parent plant on genomic
level. Another possible explanation for divergence in GS
quantity and gene expression in roots compared to in vitro
culture could be the transport mechanism through the
phloem which was shown between plant organs in A. tha-
liana through the application of radiolabeled p-hydroxy-
benzyl GS to the leaves and subsequent detection in the
seeds (Chen et al. 2001).
As with roots and hairy root cultures, CYP79F2 is also
expressed in callus culture of Col-0. However, no long-chain
GS but only small amounts of short-chain aliphatic GS were
detected in the callus culture. This is probably attributed to
the fact that genes encoding for aliphatic GS biosynthesis
were only marginally expressed or, in the case of MAM1 and
CYP79F1, transcription was absent. While several valuable
secondary metabolites are produced in unorganized callus or
suspension cultures, in other cases plant secondary metab-
olism has been linked to plant differentiation (Davioud et al.
1989). The low expression of genes involved in GS bio-
synthesis found in our study underlines the fact that the
accumulation of these secondary metabolites may also
require cell differentiation. The cellular localization of GS
has been the subject of considerable debate in which sub-
cellular and cellular location was discussed (Kelly et al.
1998). Koroleva et al. (2000) reported an existence of spe-
cific cells—so-called S-cells (sulfur-rich cells)—next to the
phloem bundles of A. thaliana flower stalk cells which were
rich in GS. Two contradicting studies with A. thaliana sus-
pension cells were performed recently: only transformed
callus cultures that overexpress the aliphatic transcription
factor MYB28 about twofold resulted in an increase in
expression of aliphatic biosynthetic genes and the produc-
tion of aliphatic GS in T87 cells (Hirai et al. 2007). Inter-
estingly, the transcription of MYB28 was comparably low in
callus cultures compared to the other tissues analyzed in our
study. The highest expression of MYB28 in leaves correlated
with the highest aliphatic GS levels. This also underlines the
key regulator function of MYB28 in aliphatic GS compared
to CYP76, which has been found equally expressed in leaves
and callus cultures. In another study by Alvarez et al. (2008),
suspension cultures of Col-0 showed a similar profile as that
found in our study, with a slightly higher total quantity of
GS. No expression for CYP79F1 was determined in sus-
pension culture and hypocotyls, which is consistent with our
findings. Interestingly, 4-hydroxy-indole-3-ylmethyl GS
was the major indole GS in callus cultures. This GS is the
precursor of the methoxylated derivative 4-methoxy-indole-
3-ylmethyl GS, suggesting that the indole glucosinolate
methyltransferases IGMT1 and IGMT2 that conduct the
methylation reaction (Pfalz et al. 2011) were only weakly
expressed in these undifferentiated cells.
The expression of TGG1 encoding for myrosinase 1,
which facilitates the hydrolysis of GS, was found to be very
low in root, hairy root and callus culture compared to leaves.
Jaquinod et al. (2007) detected no myrosinases in their
proteomic study of vacuoles from A. thaliana suspension
cells and Alvarez et al. (2008) detected only very low levels
in such suspension cells. A. thaliana suspension cells indeed
seem to contain only very low levels of myrosinase proteins
and activity compared to plant tissues. This is supported by
earlier results by Bones (1990), who found less myrosinase
activity in callus and tissue cultures compared to regenerate
plants of Brassica napus and S. alba.
This is the first study to compare GS and corresponding
gene transcription profiles of different plant organs with
those of hairy root and callus cultures. Such a comparison
may be of general utility for elucidating biosynthetic path-
ways in different culture systems or for targeted genetic
engineering. The results of this study clearly show that GS
production depends on the plant tissue and the type of culture
used. Hairy roots of A. thaliana produced higher levels of GS
and generally exhibited a higher gene expression compared
to unorganized callus tissue, suggesting that tissue differ-
entiation directly influences product accumulation. The
altered GS profile of hairy root cultures may be due to
increased auxin levels in hairy root cultures. Furthermore,
our study revealed the central regulative function of the
MYB28 transcript factor in aliphatic and MYB51 in indole GS
biosynthesis. Further studies should focus on genetic trans-
formation using genes that encode enzymes involved in GS
biosynthetic pathways to enhance the group of aliphatic GS
in plant in vitro cultures in particular.
Acknowledgments This work was funded by the German Federal
Ministry of Education and Research via Project Management Ju
¨lich,
Grant No. FKZ: 0315370B. We wish to thank Professor Dietrich
Knorr from Berlin University of Technology for supporting this work.
We also thank Irene Hemmerich from Berlin University of Tech-
nology and Andrea Maikath from Leibniz Institute of Vegetable and
Ornamental Crops Großbeeren for the technical assistance. We would
like to thank Professor David Tepfer INRA, France for providing the
A4 strain.
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