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Integration of Biosynthesis and Long-Distance Transport
Establish Organ-Specific Glucosinolate Profiles in
Vegetative Arabidopsis
W
Tonni Grube Andersen,a,b,1 Hussam Hassan Nour-Eldin,a,b,1 Victoria Louise Fuller,b Carl Erik Olsen,b
Meike Burow,a,b and Barbara Ann Halkiera,b,2
a DynaMo
Centre of Excellence, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen,
1871 Frederiksberg C, Denmark
b Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, 1871 Frederiksberg C, Denmark
Although it is essential for plant survival to synthesize and transport defense compounds, little is known about the coordination
of these processes. Here, we investigate the above- and belowground source-sink relationship of the defense compounds
glucosinolates in vegetative Arabidopsis thaliana. In vivo feeding experiments demonstrate that the glucosinolate transporters1
and 2 (GTR1 and GTR2), which are essential for accumulation of glucosinolates in seeds, are likely to also be involved in
bidirectional distribution of glucosinolates between the roots and rosettes, indicating phloem and xylem as their transport
pathways. Grafting of wild-type, biosynthetic, and transport mutants show that both the rosette and roots are able to synthesize
aliphatic and indole glucosinolates. While rosettes constitute the major source and storage site for short-chained aliphatic
glucosinolates, long-chained aliphatic glucosinolates are synthesized both in roots and rosettes with roots as the major storage
site. Our grafting experiments thus indicate that in vegetative Arabidopsis, GTR1 and GTR2 are involved in bidirectional longdistance transport of aliphatic but not indole glucosinolates. Our data further suggest that the distinct rosette and root
glucosinolate profiles in Arabidopsis are shaped by long-distance transport and spatially separated biosynthesis, suggesting
that integration of these processes is critical for plant fitness in complex natural environments.
INTRODUCTION
Plants are highly skilled organic chemists capable of synthesizing
a vast number of defense compounds. Typically, these compounds accumulate to their highest levels in those tissues that are
most likely to be attacked, which is assumed vital for plant survival by enabling them to adapt to their surroundings (Mckey,
1974). Specific distribution patterns of these important compounds may be obtained by in situ synthesis and/or long-distance
transport (Matsuda et al., 2010). The alkaloid nicotine in tobacco
(Nicotiana tabacum) is an example of long-distance transport of
a defense compound to organs distant from sites of synthesis, as
it is synthesized in roots but stored in leaf tissues (Shoji et al.,
2000; Yazaki, 2005; Morita et al., 2009). Hitherto, research on
plant defense compounds has mostly focused on elucidation of
their biosynthetic pathways, with much less attention paid to the
contribution of transport to the defense strategies.
The long-distance transport system in plants consists of xylem and phloem. Translocation in xylem is mainly driven by an
upward pull generated by transpiration from the aboveground
organs and capillary forces. Hence, flow of water and solutes
1 These
authors contributed equally to this work.
correspondence to [email protected].
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Barbara Ann Halkier
([email protected]).
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.113.110890
2 Address
through the xylem is unidirectional. As xylem is part of the
apoplast, the sole requirement for entry of compounds to the
ascending xylem sap is export across the plasma membrane. By
contrast, flow through the symplasmic phloem is driven by an
osmosis-regulated hydrostatic pressure difference between source
and sink tissues, primarily generated by the bulk flow of sucrose.
Depending on this relationship, transport of compounds through
the phloem can be considered as bidirectional. (Thompson, 2006).
Compared with xylem, an additional prerequisite for transport via
phloem is the import of compounds from the apoplast into the
symplasmic continuum of companion cells and their adjacent sieve
elements. In apoplastic loaders, such as Arabidopsis thaliana, this
import is predominantly facilitated by active transport through
transport proteins (Rennie and Turgeon, 2009).
In Arabidopsis, major components of the chemical defense
system are Met-derived and Trp-derived glucosinolates (aliphatic
and indole GLS, respectively) (Kliebenstein et al., 2001a; Halkier
and Gershenzon, 2006). Among the aliphatic GLS, a diversification
based on chain elongation of Met has occurred, resulting in up to
eight methylene groups in the side chain (Grubb and Abel, 2006).
Throughout leaf development, the highest GLS concentrations are
found in young leaves with the levels decreasing with leaf age until
GLS are virtually absent upon senescence, when accumulation
of GLS occurs in seeds (Brown et al., 2003). In comparison, in
roots, GLS content has been shown to increase at least until the
bolting stage (Petersen et al., 2002). The GLS profile of leaves
is mostly dominated by aliphatic GLS, whereas roots contain
higher amount of indole GLS (Petersen et al., 2002; Brown et al.,
2003). These observations indicate that accumulation of GLS in
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the various organs of Arabidopsis is under spatiotemporal regulation, although little is known about how these distribution patterns
are obtained. Recently, we identified two proton-driven transport
proteins of the nitrate/peptide transporter family in Arabidopsis,
glucosinolate transporter1 and 2 (GTR1 and GTR2), as high-affinity
transporters of GLS (Nour-Eldin et al., 2012). Trace levels of GLS in
seeds of a gtr1 gtr2 mutant and a concomitant increase in rosettes
and silique walls showed that these transporters are essential for
long-distance GLS transport to seeds. Based on their plasma
membrane localization, we suggested a role of GTR1 and GTR2
in importing GLS into companion cells for subsequent long-distance
phloem transport. Noticeably, in the gtr1 gtr2 mutant, only aliphatic
GLS and not indole GLS levels were increased in wilted rosettes
(Nour-Eldin et al., 2012), suggesting that different processes underlie
aliphatic and indole GLS distribution.
In this work, we investigate organ-specific distribution as well
as long-distance transport processes of GLS in Arabidopsis at
the vegetative growth stage. We use in vivo feeding of GLS and
micrografting of biosynthetic and transport knockout mutants to
examine the vascular mobility and the source-sink relationship
of GLS between above- and belowground organs. Our results
indicate that aliphatic GLS of different chain lengths differ with
respect to site of synthesis and storage and that GTR1 and GTR2
are essential for shaping their rosette/root distribution through
long-distance transport.
RESULTS
GLS Distribution between the Rosette and Roots Is
Dependent on GTR1 and GTR2
We initiated our investigations by analyzing GLS in leaves of
3-week-old soil-grown gtr1 and gtr2 single mutants as well as the
gtr1 gtr2 double mutant. No significant changes were observed in
the GLS profiles of leaves from gtr1 and gtr2 mutants when
compared with the wild type (Table 1). However, leaves of the gtr1
gtr2 double mutant contained approximately fivefold higher concentrations of aliphatic GLS with no significant changes observed
for indole GLS (Table 1). Interestingly, this buildup could mainly be
ascribed to 8-methylsulphinyloctyl GLS (see Supplemental Figure
1 online). Based on the proposed role of GTR1 and GTR2 in
phloem loading of GLS (Nour-Eldin et al., 2012), this overaccumulation of aliphatic GLS in rosette leaves at the vegetative
stage suggested that GTR1 and GTR2 are involved in transport of
GLS between the rosette and roots. To test this, we measured the
aliphatic GLS concentrations in roots using a hydroponic growth
system. Similar to those of soil-grown plants (Table 1), the entire
rosette of 3-week-old hydroponically grown gtr1 gtr2 mutants
accumulated significantly higher concentrations of aliphatic GLS
when compared with the wild type (P # 0.01) (Figure 1A; see
Supplemental Figure 2 online). When analyzing the roots of gtr1
gtr2 mutants, these were found to contain strongly reduced
concentrations of aliphatic GLS compared with the wild type
(P # 0.01) (Figure 1B). This reduction was mainly attributed to
8-methylthiooctyl GLS (8MTO) (see Supplemental Figure 2 online).
No GLS were detected in the growth medium. Interestingly, total
concentrations of aliphatic and indole GLS in gtr1 gtr2 mutants
Table 1. GLS Content in 3-Week-Old Soil-Grown gtr Mutants
Amount GLS per Leaf (nmol/mg Fresh Weight)
Wild type
gtr1
gtr2
gtr1 gtr2
Aliphatic GLS
0.11 (60.03)
0.16 (60.031)
0.19 (60.04)
0.57 (60.06)*
Indole GLS
0.044 (60.005)
0.064 (60.005)
0.052 (60.004)
0.040 (60.008)
Aliphatic and indole GLS in leaves of 4-week-old soil-grown Col-0 wild
type, gtr1, gtr2, and gtr1 gtr2 mutants. Numbers are average 6 SD; *twotailed t test (P < 0.01) versus the wild type (n = 12).
were significantly higher than the wild type (P # 0.01) (Figure 1C;
see Supplemental Figure 2 online), while the weight of the individual organs did not differ between the wild type and gtr1 gtr2
(see Supplemental Figure 3 online).
Both the Rosette and Roots Are Capable of Synthesizing
Aliphatic and Indole GLS
As a prerequisite for understanding the apparent role of GTR1 and
GTR2 in regulating the distribution of aliphatic GLS, the ability
of the above- and belowground organs to synthesize GLS was
investigated. We utilized micrografting to reciprocally combine
rosette and roots from the wild type and a mutant impaired in GLS
biosynthesis. The latter was a quadruple mutant (hereafter qko)
carrying T-DNA insertions in genes encoding the transcription
factors MYB28 and MYB29, which regulate the production of
aliphatic GLS, as well as in genes encoding the P450 enzymes
CYP79B2 and CYP79B3, which are essential for synthesis of
indole GLS (Müller et al., 2010). Scions and rootstocks of 4-d-old
wild-type and qko seedlings were reciprocally grafted and analyzed for GLS in the rosette and roots when 3 weeks old (i.e.,
before the onset of bolting). With rosette/root homografts of wildtype plants (wt/wt) set to 100%, qko homografts (qko/qko) were
found to contain only 1.8% 6 1.3% total GLS (Table 2), demonstrating that the grafting procedure did not induce GLS production in the qko mutant. In heterografts with impaired GLS
synthesis in roots (wt/qko), the total content of aliphatic and
indole GLS was reduced to 28.8% 6 5.1% and 20.7% 6 3.8% of
wild-type homografts levels, respectively (P # 0.05), constituting
;25% of the total GLS content in wild-type homografts (Table 2).
In grafts with impaired GLS synthesis in the rosette (qko/wt), aliphatic and indole GLS were reduced to 50.7% 6 4.4% and
56.6% 6 23.3%, respectively (P # 0,05), constituting ;50% of
total GLS content in wild-type homografts (Table 2). These observations demonstrate that both the rosette and roots of vegetative Arabidopsis are able to de novo synthesize aliphatic as well
as indole GLS. The content of individual GLS in all grafts is shown
in Supplemental Table 1 online.
Bidirectional Transport of Exogenously Applied
GLS between the Rosette and Roots Is GTR1
and GTR2 Dependent
With the apparent role of both the rosette and roots as sources
for GLS, interorgan transport might occur in a bidirectional
Glucosinolate Long-Distance Transport
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manner. Accordingly, we tested the GTR1- and GTR2-dependent
vascular mobility of GLS between the rosette and roots. We
performed in vivo feeding of 2-propenyl GLS, which although
not present in the ecotype Columbia-0 (Col-0), represents an
endogenous aliphatic GLS found in other Arabidopsis accessions (Kliebenstein et al., 2001b). 2-Propenyl GLS was infiltrated into a leaf of 3-week-old hydroponically grown plants.
After 72 h, wild-type plants contained ;8% of the total detected
2-propenyl GLS in roots, whereas ;59 and 33% were found in
the remaining rosette and the fed leaf, respectively (Figure 2A).
In contrast with the wild type, leaf infiltration of 2-propenyl GLS
into gtr1 gtr2 mutants led to no detectable accumulation in
roots, while ;45 and 55% of the total detected 2-propenyl GLS
was found in the remaining rosette and the fed leaf, respectively
(Figure 2A). Noticeably, although the fed amount of 2-propenyl
GLS was ;10 nmol, we recovered only around 8 nmol in either
genotype (Figure 2A), which is likely due to degradation. These
data support previous observations of long-distance transport of
GLS from the rosette to roots (Brudenell et al., 1999; Chen et al.,
2001) and demonstrate that GTR1 and GTR2 are key facilitators of
this transport. Based on the ability of roots to synthesize GLS, we
further examined if GLS can be transported from roots to rosette
and whether this involves GTR1 and GTR2 by incubating roots of
hydroponically grown plants in medium containing 2-propenyl
GLS. After 72 h incubation, ;3% of the recovered 2-propenyl
GLS was found in roots of wild-type plants. Interestingly, in gtr1
gtr2 mutants, virtually no 2-propenyl GLS was detected in roots,
although the total amount recovered from the plants was similar
to that of the wild type (Figure 2B). A similar GTR1- and GTR2dependent distribution was observed when feeding the aromatic
p-hydroxybenzyl GLS (p-OHB) (see Supplemental Figure 4 online),
which although being exogenous to Arabidopsis, has been demonstrated to undergo long-distance transport (Chen et al., 2001)
and to be an in vitro substrate for GTR1 and GTR2 (Nour-Eldin
et al., 2012). These observations show that GLS has the ability to
move from roots to the rosette and suggest that GLS accumulation in roots is dependent on GTR1 and GTR2.
GTR1 and GTR2 Are Expressed in the Root Vasculature with
Highest Expression in Lateral Branching Points
The possibility that GTR1 and GTR2 are involved in regulating
the accumulation of GLS in roots primed us to investigate their
expression in this organ. According to publically available cell
type–specific expression data (Mustroph et al., 2009; http://efp.
ucr.edu/), both transporters are translated in the vasculature
and cortex (see Supplemental Figure 5 online). To validate their
expression in the root vasculature, we analyzed transgenic plants
expressing nuclear localization signal (NLS)–linked green fluorescent
protein (GFP)–b-glucuronidase (GUS) fusion proteins driven by 2-kb
promoter sequences upstream of either GTR1 or GTR2 (Nour-Eldin
et al., 2012). GUS staining of 3-week-old plants and confocal laser
Figure 1. GLS Content in 3-Week-Old Hydroponically Grown Wild-Type
and gtr1 gtr2 Mutants.
HPLC analysis of GLS content in the rosette (A), roots (B), and in the entire
plant (C) of 3-week-old hydroponically grown Col-0 wild-type and gtr1 gtr2
mutants (black bars are the wild type; gray bars are gtr1 gtr2). Bars indicate
SD. *Two-tailed t test (P < 0.01) versus the wild type (n = 12 to 16).
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Table 2. GLS Content of Micrografted Plants with Impaired Biosynthesis in the Rosette or Roots
GLS Biosynthesis
Genotype
(Rosette/Roots)
wt/wt
qko/qko
wt/qko
qko/wt
GLS Content of Entire Plant (% of the Wild Type)
Rosette
Roots
Aliphatic GLS
Indole GLS
Total GLS
+
2
+
2
+
2
2
+
100
3.5 (62.6)**
28.8 (65.1)**
50.7 (64.4)**
100
nd
20.7 (63.8)**
56.6 (623.3)*
100
1.8 (61.3)**
23.8 (63.5)**
53.6 (612.0)**
Plants with impaired GLS synthesis in roots or the rosette were constructed by reciprocal micrografting of the myb28 myb29 cyp79b2 cyp79b3
quadruple mutant (qko) and Col-0 wild type (wt). GLS content of the entire 3-week-old grafted plants was analyzed by LC-MS and related to levels
found in the entire wild type. Numbers are averages, and SD is shown in parentheses. Two-tailed t test, *P < 0.05 and **P < 0.01 versus the wild type (n =
5 to 7). nd, none detected.
scanning microscopy on the nuclear-targeted GFP-GUS fusions
confirmed expression of both GTR1 and GTR2 in root vasculature
with the highest transcriptional activity observed in the lateral root
branching point areas (Figure 3).
(gtr1 gtr2/wt), showed similar levels of SC aliphatic GLS to those
of wild-type homografts in the rosette and roots (P > 0.05) (Figure
4A). In summary, these observations demonstrate that the rosette
is the major site for synthesis and storage of SC aliphatic GLS.
Rosettes Are the Major Site for Short-Chain Aliphatic
GLS Synthesis and Storage
LC Aliphatic GLS Are Synthesized in Above- and
Belowground Organs and Retained in Roots by
GTR1 and GTR2
The finding that in vivo–fed 2-propenyl GLS could move bidirectionally and that this was influenced by GTR1 and GTR2,
supports that these transporters are involved in regulating aboveand belowground distribution of endogenous GLS. The expression levels of GTR1 and GTR2 have previously been shown to be
unaffected by the absence of MYB28 and MYB29 (Sønderby
et al., 2010). This enabled us to use qko organs to examine GTR1and GTR2-dependent transport of endogenous GLS by micrografting wild-type, qko, and/or gtr1 gtr2 organs. For simplicity, in
the following, aliphatic GLS containing three to five methylene
groups (C3 to C5) and six to eight methylene groups (C6 to C8) in
their side chain will be referred to as short-chain (SC) and longchain (LC) aliphatic GLS, respectively. When analyzing the levels
of SC aliphatic GLS in homografts, none were detected in any qko
organs, while gtr1 gtr2 contained significantly reduced levels only
in the roots compared with wild-type homografts (P # 0.05)
(Figure 4A). For heterografts consisting of a wild-type rosette
grafted onto qko roots (wt/qko), the rosette accumulated SC
aliphatic GLS levels similar to those of wild-type homografts,
whereas a trend toward reduction was observed in the roots (P >
0.05) (Figure 4A). In a similar manner, heterografts consisting of
a gtr1 gtr2 rosette grafted onto qko roots (gtr1 gtr2/qko) contained
SC aliphatic GLS levels in the rosette indistinguishable from those
found in wild-type homografts (P > 0.05). While the gtr1 gtr2/qko
grafts contained significantly reduced SC aliphatic GLS levels in
roots when compared with wild-type homografts, these were in
the same range as those detected in roots of the wt/qko grafts
(P > 0.05) (Figure 4A). Heterografts consisting of a qko rosette
grafted onto either wild-type or gtr1 gtr2 roots (qko/wt or qko/gtr1
gtr2, respectively) contained strongly reduced total amounts of
SC aliphatic GLS, which was mainly due to reduced levels in the
rosette (Figure 4A). Thus, bidirectional transport of SC aliphatic
GLS can occur but may not be dependent on GTR1 and GTR2.
Noticeably, heterografts consisting of a wild-type rosette grafted
onto gtr1 gtr2 roots (wt/gtr1 gtr2), or the reciprocal combination
When analyzing LC aliphatic GLS in homografts, qko rosettes
contained levels below the detection limit, while roots had strongly
Figure 2. Translocation of the Aliphatic 2-Propenyl GLS Administered to
Leaves and Roots of Arabidopsis in the Vegetative Growth Phase.
GTR1- and GTR2-dependent translocation of the aliphatic 2-propenyl
GLS in the vasculature was investigated by administering 2-propenyl
GLS to a leaf and roots of hydroponically grown 3-week-old wild-type or
gtr1 gtr2 plants.
(A) 2-Propenyl GLS distribution measured 72 h after infiltration to a leaf.
The maximum injected amount was ;10 nmol.
(B) 2-Propenyl GLS distribution after 72 h of incubation in medium
containing 2-propenyl GLS; 100% represents total amount of 2-propenyl
GLS detected. Numbers are average 6 SD; *two-tailed t test (P < 0.01)
versus corresponding wild-type organ (n = 13 to 17). nd, none detected.
Glucosinolate Long-Distance Transport
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Figure 3. Expression of GTR1 and GTR2 at Root Branching Points of 3-Week-Old Plants.
Transgenic Arabidopsis plants expressing a NLS and GFP-GUS fusion under control of 2-kb promoter regions upstream of either GTR1 or GTR2 were
analyzed by GUS staining and counterstained with propidium iodide ([A],[C], and [E]). In similar sections, confocal laser scanning microscopy was used
to confirm presence of GFP in the nuclei ([B], [D], and [F]). The wild type ([A] and [B]),GTR1pro:NLS-GFP-GUS ([C] and [D]), and GTR2pro:NLS-GFPGUS ([E] and [F]). All plants were 3 weeks old and in the vegetative growth phase. LR, lateral root. Bars = 50 mm.
reduced levels when compared with wild-type homografts (Figure
4B). gtr1 gtr2 homografts overaccumulated LC aliphatic GLS in the
rosette and showed a strong reduction in root levels when compared with the wild type (Figure 4B). This distribution was also
observed in identically treated but nongrafted gtr1 gtr2 mutants
(see Supplemental Figure 2 online). When analyzing heterografts,
those consisting of a qko rosette grafted onto wild-type roots (qko/
wt) or the reciprocal combination (wt/qko) contained reduced total
amounts of LC aliphatic GLS (P # 0.05) (Figure 4A). This shows
that, in contrast with SC aliphatic GLS, both the rosette and roots
can contribute significantly to the synthesis of LC aliphatic GLS.
Furthermore, when comparing LC aliphatic GLS levels in heterografts consisting of wild-type or gtr1 gtr2 rosettes grafted onto qko
roots (wt/qko and gtr1 gtr2/qko), LC aliphatic GLS accumulation
in the roots was significantly higher in the wt/qko graft, demonstrating that endogenous GLS can move downward from rosette
to roots in a GTR1- and GTR2-dependent manner (Figure 4B).
Interestingly, in both wt/qko and qko/wt heterografts, LC aliphatic
GLS accumulated to levels indistinguishable from wild-type homografts in the rosette, while a significant reduction was found in
roots of both combinations (P # 0.05). This emphasizes that both
the rosette and roots represent sinks for LC aliphatic GLS (Figure
4B). Strikingly, when qko or wild-type rosettes were grafted onto
gtr1 gtr2 roots (qko/gtr1 gtr2 or wt/gtr1 gtr2), LC aliphatic GLS
were found to overaccumulate in rosettes to a similar extent to that
found in gtr1 gtr2 homografts (P < 0.05) (Figure 4B). This shows
that LC aliphatic GLS can be transported upwards from roots to
rosette but are retained in roots by a process that is dependent on
the presence of GTR1 and GTR2 in the roots. Noticeably, in the
qko/gtr1 gtr2 grafts, the total LC aliphatic GLS content was similar
to that in wild-type homografts, in contrast with the significantly
reduced total contents in all other heterografts containing qko
tissues (Figure 4B).
LC Aliphatic GLS Are in Vitro Substrates of GTR1 and GTR2
To biochemically validate LC aliphatic GLS as substrates of
GTR1 and GTR2, we compared uptake of 8MTO with that of the
previously characterized in vitro substrate 4-methylthiobutyl
GLS (4MTB) into Xenopus laevis oocytes. When accumulation of
4MTB in oocytes expressing GTR1 or GTR2 was set to 100%,
both showed ;40% uptake of 8MTO, while no accumulation
was observed in uninjected oocytes (Figure 5). This ability of
GTR1- or GTR2-expressing oocytes to take up 8MTO supports
the in planta evidence for GTR1 and GTR2 as being involved in
transport of LC aliphatic GLS.
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The Plant Cell
Figure 4. Relative Content of GLS in Micrografted Arabidopsis Plants in the Vegetative Growth Phase.
Glucosinolate Long-Distance Transport
Indole GLS Can Be Synthesized in Both Rosette and Roots
and Can Be Transported Bidirectionally
When analyzing homografts for indole GLS, levels in both the rosettes and roots were below the detection limit in qko homografts,
whereas homografted gtr1 gtr2 was found to contain levels similar to
those found in the homografted wild type (P > 0.05) (Figure 4C).
Indole GLS accumulated in all grafted qko organs, suggesting that
these GLS can move bidirectionally between the rosette and roots
(Figure 4C). Intriguingly, qko rosettes grafted to gtr1 gtr2 roots or
the reciprocal combination (i.e., qko/gtr1 gtr2 or gtr1 gtr2/qko, respectively) showed no reduction in indole GLS accumulation when
compared with qko organs grafted to the corresponding wild type
(i.e., qko/wt or wt/qko, respectively) (P > 0.05) (Figure 4C). Furthermore, heterografts consisting of a wild-type rosette and qko roots
(wt/qko) or the reciprocal combination (qko/wt) accumulated indole
GLS in the respective qko organ (Figures 4A to 4C). In summary, this
indicates that, in contrast with LC aliphatic GLS, distribution of indole
GLS is not regulated by GTR1 and GTR2 at the vegetative stage. An
overview of the individual GLS in all grafted and nongrafted rosettes
and roots can be found in Supplemental Tables 1 to 4 online.
GTR1 and GTR2 Show Highest Expression in Cells
Surrounding the Lateral Root Branching Points
To further investigate how GTR1 and GTR2 could be involved in
retaining GLS in the roots, localization of GTR1-YFP (for yellow
fluorescent protein) and GTR2-mOrange fusion proteins was investigated by confocal laser scanning microscopy. Root cross
sections ;2 cm below the hypocotyl of 3-week-old plants
showed GTR1-YFP and GTR2-mOrange to be expressed inside
the stele (Figures 6F and 6I). As both GTR1 and GTR2 were
found to have highest transcriptional activity in lateral root
branching points (Figure 3), we further investigated the presence
of GTR1-YFP and GTR2-mOrange in this area by cross-sectioning
the main root in areas containing lateral root branching points. In
these sections, both GTR1- and GTR2-specific signals were
mainly detected in areas that appeared to be cortex cells surrounding the lateral root branching points (Figures 6E and 6H).
DISCUSSION
Arabidopsis Roots Contribute to GLS Production
in the Vegetative Growth Phase
Investigations of GLS in various plant species have to a large
extent focused on aboveground organs (Dam et al., 2008). Consequently, the ability of roots to synthesize GLS has hitherto
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received little attention. In Arabidopsis, an apparent low expression
level of key biosynthetic genes in roots compared with leaves
(Brady et al., 2007) has led to the general assumption that GLS are
primarily made aboveground. Initially, and based on the proposed
role of GTR1 and GTR2 in phloem loading of GLS in leaves (NourEldin et al., 2012), we hypothesized that an overaccumulation of
aliphatic GLS in the rosette of gtr1 gtr2 mutants at the vegetative
stage (Figure 1) was caused by a decreased ability of this mutant
to facilitate downward GLS transport. With the assumption that
roots of Arabidopsis are incapable of GLS biosynthesis, this
suggested that roots constitute a sink for aliphatic GLS transported through the phloem. This was strengthened by the observation that, when fed to a leaf, the aliphatic 2-propenyl GLS
accumulated in roots of the wild type, but not of gtr1 gtr2 (Figure 2).
However, the finding that Arabidopsis roots are capable of synthesizing both aliphatic and indole GLS (Table 2) contradicted the
initial hypothesis and suggested that the source-sink relationship
of the rosette and roots for GLS in vegetative Arabidopsis is more
complex than originally assumed.
Phloem and Xylem as Transport Pathways for GLS
GLS are organic anions with a very low permeability coefficient
(Brudenell et al., 1999). According to the Kleier model (Kleier,
1988), they have physiochemical properties consistent with mobility in both phloem and xylem (Brudenell et al., 1999). In previous
experiments, radiolabeled p-OHB fed to leaves of Arabidopsis
and rape (Brassica napus) was shown to follow the phloem
pathway and accumulate in seeds and roots (Brudenell et al.,
1999; Chen et al., 2001). We previously showed transport of GLS
to seeds to be facilitated by GTR1 and GTR2 (Nour-Eldin et al.,
2012). In this study, feeding of both an aliphatic (2-propenyl) and
an aromatic (p-OHB) GLS to leaves of the gtr1 gtr2 mutant (Figure
2A; see Supplemental Figure 4 online) demonstrated that GTR1
and GTR2 are also essential for transport of GLS from rosette to
roots in the phloem. Interestingly, feeding of GLS to roots of the
wild type and gtr1 gtr2 resulted in upward transport to the rosette
(Figure 2B; see Supplemental Figure 4 online). Given the directional flow of the vascular transport pathways, and the physiochemical properties of GLS, this transport is expected to occur
either in the apoplastic matrix or in the ascending xylem sap.
Regardless of the route, upwards translocation in the gtr1 gtr2
mutant suggests the presence of a GTR1- and GTR2-independent
ability to translocate GLS from roots to the rosette. In wild-type
plants, part of the root fed GLS accumulated in the roots, whereas
in the gtr1 gtr2 mutant, this was only observed to a very low
degree (Figure 2B; see Supplemental Figure 4 online). This might
Figure 4. (continued).
Rosettes and roots from the wild type, the GLS synthesis impaired myb28 myb29 cyp79b2 cyp79b3 quadruple mutant (qko), and the gtr1 gtr2 mutant
were reciprocally grafted using 4-d-old seedlings. The grafts are depicted as the genotype of rosette and roots, respectively. GLS content in the rosette
and roots was analyzed by LC-MS in 3-week-old plants and related to the total amount found in wild-type homografts. Numbers represent the relative
amount in the entire plant. Bars and numbers are averages; error bars and parentheses are SD. Groups in subfigures are determined by one-way ANOVA
(P < 0.05) versus the wild type (n = 3 to 7). nd, none detected.
(A) SC aliphatic GLS.
(B) LC aliphatic GLS.
(C) Indole GLS.
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The Plant Cell
Figure 5. Uptake of 8MTO in GTR1- or GTR2-Expressing X. laevis
Oocytes.
8MTO accumulation in X. laevis oocytes expressing either GTR1 or GTR2
after 1 h incubation in medium containing in 100 mM 8MTO. Uptake after
1 h of 100 mM of the previously characterized (Nour Eldin et al., 2012).
GTR1 and GTR2 substrate 4MTB was set to 100%. Bars are averages;
error bars indicate SD (n = 4).
reflect a GTR1- and GTR2-dependent redistribution of GLS from
the rosette to roots following the upward transport. However, in
light of expression of GTR1 and GTR2 in the root vasculature
(Figures 3 and 6), we propose that GTR1 and GTR2 are involved
in removal of GLS from the xylem in roots. In support of this,
a study aimed at investigating transcriptional profiles associated
with different parts of the vasculature in roots and hypocotyls
suggested that the expression of GTR1 and GTR2 is associated
with the xylem rather than phloem in these tissues (Zhao et al.,
2005). The observed localization of GTR1 and GTR2 in cells outside
the stele in the lateral root branching points of the main root
(Figures 6E and 6H) suggests that GTR1- and GTR2-mediated
retention may occur via import of GLS into storage cells in these
areas. In Arabidopsis, GLS accumulates to concentrations up to
100 mM in sulfur-rich cells (S-cells) localized adjacent to the
vasculature in aboveground tissues (Koroleva et al., 2000, 2010;
Koroleva and Cramer, 2011; Sarsby et al., 2012). While S-cells in
roots have previously been described in the inner periderm of
the main root in field-grown B. napus (McCully et al., 2008), it remains to be investigated whether cells outside the stele expressing
GTR1 and GTR2 represent S-cells in Arabidopsis roots.
Distinct Above- and Belowground Source-Sink
Relationships Exist for SC and LC Aliphatic GLS
in Vegetative Arabidopsis
SC aliphatic GLS constitute the majority of GLS in the vegetative
rosette of Arabidopsis (Col-0) (Brown et al., 2003). Our grafting
experiments indicate that SC aliphatic GLS are primarily made
and stored in the rosette while the roots do not appear to serve
as a major source or sink at this growth stage (Figure 4A). In
comparison to SC aliphatic GLS, both the rosette and roots
were observed to contribute considerably to LC aliphatic GLS
production (Figure 4B). In support of this, transcriptional analyses have shown that MAM3 and CYP79F2, which are involved
in LC aliphatic GLS biosynthesis, are indeed expressed in both
above- and belowground organs, with highest expression in the
roots (Chen et al., 2003; Tantikanjana et al., 2004; Textor et al.,
2007; Redovnikovic et al., 2012). In contrast with SC aliphatic GLS,
the major storage site for LC aliphatic GLS in vegetative Arabidopsis appears to be in the roots (Figure 4B; see Supplemental
Figure 2 online), indicating that LC aliphatic GLS produced in rosettes might be transported to roots. In support of this, a similar
GTR1 and GTR2 dependency as for downward 2-propenyl GLS
transport was observed for the LC aliphatic GLS (Figure 2A), which
indicates that the movement of endogenous GLS from rosette to
roots occurs through the phloem. In the presence of GTR1 and
GTR2, LC aliphatic GLS are retained in the roots, whereas in the
absence of functional GTR1 and GTR2 in the roots, the majority of
these GLS are translocated via the xylem to the rosette (Figure 4B).
This suggests that for upward movement of GLS from roots to the
rosette, GTR1 and GTR2 activity in roots may represent a mechanism by which Arabidopsis regulates their distribution.
Integration of Biosynthesis and Transport in
Plant Defenses?
It has previously been suggested that differences in above- and
belowground GLS profiles are due to in situ biosynthesis rather
than transport (van Dam, 2009). In this study, plants with impaired GLS synthesis in rosettes accumulated LC aliphatic GLS
to only 35% of wild-type levels (Figure 4B, qko/wt). However,
impaired GLS biosynthesis in the rosette combined with abolishment of GTR1 and GTR2 in roots (qko/gtr1 gtr2) led to LC
aliphatic GLS levels in the entire plant indistinguishable from
those of wild-type homografts. Thus, the GTR1- and GTR2dependent retention of LC aliphatic GLS appears to feedback inhibit their biosynthesis in roots. This is supported by the increased
total level of aliphatic GLS in the ungrafted gtr1 gtr2 mutant
(Figure 1C; see Supplemental Figure 1 online). These observations indicate that an unknown crosstalk between aliphatic
GLS synthesis, transport, and storage exists in Arabidopsis.
In the example of nicotine, which is synthesized in roots and
transported to leaves in the xylem (Shoji et al., 2000; Yazaki, 2005;
Morita et al., 2009), root nicotine synthesis has been shown to
increase in response to insect attack of the leaves. Several other
compounds, including tropane alkaloids in Solanaceae species
(Ziegler and Facchini, 2008) and pyrrolizidine alkaloids in Asteraceae (Hartmann and Ober, 2000), have been shown to follow
a similar pattern of synthesis in roots followed by translocation to
leaves. These examples have led to the appreciation that roots
play important roles in the aboveground defensive mechanisms
(Erb et al., 2009). Only LC and not SC aliphatic GLS seem to be
primarily made in roots and distributed to other parts of the plant.
Differences in chain length of aliphatic GLS have also been shown
to play a role in both resistance toward the diamondback moth
(Plutella xylostella) (Kroymann et al., 2003) and the relative abundance of two specialist aphid species (Züst et al., 2012). This
illustrates that GLS chain lengths can be critical for survival of
plants. In a study with the green peach aphid (Myzus persicae)
on Arabidopsis, the LC aliphatic GLS 8-methylsulphinyloctyl was
Glucosinolate Long-Distance Transport
9 of 13
Figure 6. Localization of Fluorescent GTR1 and GTR2 Protein Fusions in Roots.
Roots of 3-week-old transgenic lines expressing full-length genomic regions of GTR1 or GTR2 C-terminally fused to YFP or mOrange, respectively,
under control of the 2-kb endogenous promoters used in Figure 3. (A), (D), and (G) show longitudinal optical sections along the xy axis of intact roots,
while subfigures (B), (E), and (H) show cross sections of the main root in lateral root branching points. Subfigures (C), (F), and (I) show cross sections of
the main root alone. LR, lateral root. Bars = 50 mm.
(A) to (C) Wild-type overlay of YFP and mOrange channels.
(D) to (F) YFP signal from a representative plant expressing GTR1-YFP.
(G) to (I) GTR2-mOrange signal from a representative plant expressing GTR2-mOrange. Arrows point to areas with expression outside the stele.
observed to increase significantly in aphid-infested leaves without
any changes in the systemically connected leaves (Kim and
Jander, 2007). As the roots are a major site of LC aliphatic GLS
storage, this suggests that LC aliphatic GLS may be translocated
from roots to leaves upon aphid attack. In another study, however,
transcriptional analysis of leaves infected with the pathogenic
ascomycete S. sclerotiorum showed an increased expression of
LC aliphatic GLS-specific genes in comparison to mock-treated
leaves (Stotz et al., 2011). This suggests that Arabidopsis may also
respond with in situ synthesis of LC aliphatic GLS. In light of our
data, these studies give rise to the hypothesis that Arabidopsis
responds to different biotic stresses by integrating both longdistance transport and in situ synthesis of LC aliphatic GLS. Under
the assumption that SC and LC aliphatic GLS differ in their
biological functions, the differences in major storage site and
source-sink relationships for SC and LC aliphatic GLS highlight
the notion that several different strategies may underlie the general flexibility in tissue-specific defense compound accumulation
in response to interacting organisms.
A Model for Spatial Organization of GLS Synthesis and
Long-Distance Transport between the Rosette and Roots
Based on our data, we propose a model for transport of GLS
between the rosette and roots in the vegetative growth phase of
Arabidopsis (Figure 7). According to the model, SC aliphatic GLS
are primarily produced and stored in the rosette, while both the
rosette and roots contribute to the synthesis and accumulation
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The Plant Cell
Figure 7. Model of GLS Transport between the Rosette and Roots in Vegetative Arabidopsis.
(A) Indole, SC, and LC aliphatic (SC: C3-C5 and LC: C6-C8, respectively) GLS accumulate in the rosette, whereas in roots, the predominant GLS are indole
and LC aliphatic. In this model, organ-specific GLS profiles are achieved through integration of in situ synthesis and long-distance bidirectional transport
between the rosette and roots in phloem and xylem. GTR1 and GTR2 are essential for establishing these profiles as they are involved in retention and
storage of LC aliphatic GLS in roots. GTR1- and GTR2-dependent storage of GLS is likely to occur in areas surrounding the lateral root branching points.
(B) Phloem loading of GLS across the plasma membrane is facilitated through the proton-dependent GTR1 and GTR2, and root retention can be
facilitated through the same mechanism. The transport mechanism(s) responsible for indole GLS transport and export of GLS across the plasma
membrane to the apoplast is currently unknown. Apo, apoplast; CC, companion cell; Pd, plasmodesmata; SE, sieve element; Sym, symplast.
of LC aliphatic and indole GLS (Figure 7A). While the mechanism
underlying transport of indole GLS is currently unknown, the
model proposes a complex source-sink relationship for LC aliphatic GLS where GTR1 and GTR2 are involved in obtaining
organ-specific accumulation through long-distance transport in
phloem as well as xylem. In roots, a GTR1- and GTR2-dependent
import of aliphatic GLS from the apoplast is likely to occur into
cells adjacent to xylem (Figure 7B), possibly into phloem cells as
well as storage cells in the lateral root branching points, thereby
attenuating upward xylem transport. The proposed model for
GLS transport further suggests an unknown mechanism for export of GLS into the apoplast in roots. Interestingly, transporters
involved in root to rosette distribution (Lin et al., 2008) and export
of nitrate (Segonzac et al., 2007) have been found within the
nitrate/peptide transporters. This family may hence also include
transporters facilitating GLS export. Furthermore, we hypothesize that transport mechanisms specific for indole GLS are likely
to exist in Arabidopsis.
In summary, this study has explored the role of synthesis and
transport processes in regulating the distribution of the GLS defense
compounds in Arabidopsis. We show that not only are aliphatic GLS
profiles coordinated differently from indole GLS, but organ-specific
SC and LC aliphatic GLS accumulation is independently regulated.
Our data support the proposal that distinct spatiotemporal distribution of defense compounds, such as GLS, is obtained through
a complex system capable of integrating long-distance transport
and in situ synthesis. Although GLS are characteristic for plants in
the Brassicales order (Fahey et al., 2001), insights gained on longdistance transport processes of GLS at the molecular level may
provide valuable knowledge for tissue-specific manipulation of not
only GLS in Brassica crops such as rape, but also other specialized
as well as primary metabolites in other plants.
METHODS
GLS Analysis
GLS were analyzed as desulfo GLS as previously described (Kliebenstein
et al., 2001b). The 96-well filter plates were charged with 45 mg of DEAE
Sephadex A25 and 300 mL of water per well and equilibrated at room
temperature for 2 h. The water was removed using a vacuum manifold
(Millipore). Plant material was harvested in either 300 mL or 5 mL 85% methanol
containing 50 mM allyl GLS (sinigrin) or p-hydroxybenzyl GLS (p-OHB)
added to each sample as an internal standard before sample aliquots
(200 mL) were applied to the filter plates and absorbed on the ion exchanger by vacuum filtration for 2 to 4 s. Sephadex material was washed
with 23 100 mL 70% methanol (v/v) and 23 100 mL water and briefly
centrifuged before addition of 20 mL of sulfatase solution (1.25 mg mL21,
sulfatase type H1; Sigma-Aldrich) on each filter. For analysis of growth
medium, 50 mL medium was run through filters charged with 150 mg
DEAE Sephadex A25 and 2 mL water per filter and equilibrated at room
temperature for 2 h before addition of 500 mL sulfatase solution. After
Glucosinolate Long-Distance Transport
incubation at room temperature overnight, desulfo GLS were eluted with 80 mL
water for 96-well filter plates and 250 mL for media samples. Media sample
elute was lyophilized and resuspended in 50 mL water. All samples were
analyzed by HPLC on an Agilent HP1200 Series instrument equipped with
a C-18 reversed phase column (Zorbax SB-Aq, 25-3 (4.6 mm), 5-mm particle
size; Agilent) using a water (solvent A)–acetonitrile (solvent B) gradient at a flow
rate of 1 mL min21 at 25°C (injection volume 45 µL). The gradient applied was
as follows: 1.5 to 7% B (5 min), 7 to 25% (6 min), 25 to 80% (4 min), 80%
B (3 min), 80 to 35% B (2 min), 35 to 1.5% B (2 min), and 1.5% B (3 min). The
eluent was monitored by diode array detection between 200 and 400 nm (2-nm
interval). Desulfo GLS were identified based on comparison of retention times
and UV absorption spectra with those of known standards (Brown et al., 2003)
and quantified in relation to the internal standard. For grafted plants, the GLS
content was measured by liquid chromatography–mass spectrometry (LCMS) using an Agilent 1100 Series liquid chromatograph coupled to a Bruker
HCT-Ultra ion trap mass spectrometer (Bruker Daltonics). A Zorbax SB-C18
column (Agilent; 1.8 mm, 2.1 mm i.d. 3 50 mm) was used at a flow rate of 0.2
mL/min, and the mobile phases were as follows: A, water with 0.1% (v/v) formic
acid and 50 mM NaCl; B, acetonitrile with 0.1% (v/v) HCOOH. The gradient
program was as follows: 0 to 0.5 min, isocratic 2% B; 0.5 to 7.5 min, linear
gradient 2 to 40% B; 7.5 to 8.5 min, linear gradient 40 to 90% B; 8.5 to 11.5 min
isocratic 90% B; 11.6 to 15 min, isocratic 2% B. The flow rate was increased to
0.3 mL/min in the interval 11.2 to 13.5 min. The mass spectrometer was run in
positive electrospray mode. The relative GLS content was determined by
relating all extracted peaks to the average wild-type content.
Xenopus laevis Oocyte Uptake Assay
Oocytes were kept in a Kulori-based solution containing 90 mM NaGluconate, 1 mM KGluconate, 1 mM CaGluconate2, 1 mM MgGluconate2, 1 mM
LaCl3, and 10 mM MES, pH 5. For uptake assays, 8MTO or 4MTB to a total
concentration of 100 mM was added to the solution. Solutions were
adjusted to 220 mOsmol kg21 using D-sorbitol. After 1 h of incubation in
medium containing GLS, the oocytes were washed and homogenized
individually in 100 mL 10% SDS and analyzed for GLS content by LC-MS as
described above. The content of 8MTO was normalized to the content of
similar oocytes subjected to 4MTB uptake.
GLS Feeding
p-OHB GLS was extracted from seeds of white mustard (Sinapis alba)
(SeedCom) as previously described (Thies, 1979; Zrybko et al., 1997). The
seeds were extracted with 80% (v/v) methanol, and the extract was
loaded onto a DEAE Sephadex A25 column from which intact GLS were
eluted with 0.5 M K2SO4 containing 3% (v/v) isopropanol. The samples
were concentrated to dryness and washed with 100% (v/v) methanol and
then with 100% (v/v) ethanol before resuspension in water. 2-propenyl
GLS was obtained from Sigma-Aldrich (lot 121K7063). Feeding of
3-week-old Arabidopsis thaliana plants was done by infiltrating a maximum of 1 mL Murashige and Skoog (MS) medium adjusted to pH 5.6,
containing 10 mM 2-propenyl GLS or 2.5 mM p-OHB directly into a leaf
using a 1-mL syringe with a 10-mL pipette tip. To evaluate root uptake,
2-propenyl GLS or p-OHB was added to growth medium to a final
concentration of 3 mM or 200 mM, respectively. After 72 h of incubation,
plant organs were harvested, washed three times in water, and analyzed
for 2-propenyl GLS or p-OHB content as described above.
Micrografting
Micrografting of Arabidopsis seedlings was performed based on Turnbull
et al. (2002). Two pieces of filter paper were added to a sterile Petri dish
and wet with 2 mL of sterile water. Two cellulose nitrate filters (Whatman
NC 45 ST) were place side-by-side on top of these, with additional 1 mL of
sterile water added to wet the membranes. Six to eight straight seedlings
11 of 13
of the respective genotype (2 to 3 cm total length) were placed in two rows
on each membrane oriented so that the cotyledons were horizontally
aligned. The cotyledons were removed using a sapphire knife (WPI
500313), and incisions were made as high as possible on the hypocotyls
to avoid adventitious root formation. Rootstocks from the top row and
scions from the bottom row were discarded and the respective counterparts were joined using sterile forceps. The plates were sealed using
Micropore tape (3M) and incubated vertically under long-day conditions
(light: 16 h, 20°C; darkness: 8 h, 16°C). The entire procedure was done in
a laminar flow cabinet using a dissection microscope. After 3 d, 1 mL of
sterile water was added, and after an additional 2 d, successfully joined
seedlings were transferred to MS agar plates containing 1% Suc and
grown under long-day conditions until the age of 3 weeks.
Cellular Localization
Arabidopsis lines harboring expression of a NLS and GUS-GFP under
control of 2 kb of either GTR1 or GTR2 promoters including the 59 untranslated regions (Nour-Eldin et al., 2012) were submerged in a solution
containing 0.5 mg/mL X-Glc (5-bromo-4-chloro-3-indolyl-b-D-Glucuronide), 0.05% Triton X-100, 20% (v/v) methanol, 50 mM NaH2PO4, pH 7.5,
10 mM K3[Fe(CN)6], 10 mM K4[Fe(CN)6] $3H2O), and incubated at 37°C in
darkness until blue staining appeared. Chlorophyll was cleared by washing
overnight in 70% ethanol and counterstained by incubation for 2 h in
100 mM propidium iodide. Generation of GTR1-YFP– and GTR2-mOrange–
expressing plant lines is described elsewhere (Nour-Eldin et al., 2012).
Localization experiments were done on 3-week-old hydroponically grown
plants; the expression was investigated ;2 cm below the hypocotyl using
a Leica SP5-X confocal microscope equipped with a HCX lambda blue PL
APO 320 objective (0.7 numerical aperture). Fluorophores were excited at
the respective absorption maxima (488 nm for GFP, 514 nm for YFP, and
546 nm for mOrange). Emission was collected at 500 to 515 nm (GFP), 525
to 540 nm (YFP), and 560 to 580 nm (mOrange).
Plant Growth Conditions
Arabidopsis (Col-0) plants were either grown on soil or in a hydroponic
system under long-day conditions (light: 16 h, 20°C; darkness: 8 h, 16°C).
Seeds were stratified for 2 d at 4°C in total darkness. Following stratification,
the seeds were either germinated on soil or on 1% (w/v) agar/50% (v/v)
nutrient solution (Gibeaut et al., 1997) in 0.5-mL Eppendorf tubes for hydroponics. After 1 week of germination, the tube bottom was cut off to allow
the roots to grow into sterile pipette tip containers containing ;400 mL
100% nutrient solution, which was changed weekly. No antibiotics were
added. For micrografting and localization analysis, seeds were surface
sterilized by washing in 70% (v/v) ethanol containing 0.05% (v/v) Triton X100
for 5 min followed by washing in 96% (v/v) ethanol, left on sterile Whatman
paper, and sown on half-strength MS agar plates containing 1% (w/v) Suc.
The plates were cold stratified for 2 d followed by vertical growth for 3 to 5 d
under long-day conditions (light: 16 h, 20°C; darkness: 8 h, 16°C).
Statistical Data Analysis
To represent the change of GLS levels in grafted plants, levels in all grafted
combinations were calculated as a percentage of the levels found in wildtype homografts grown under identical conditions. Significance levels of
GLS differences were tested by performing a one-way analysis of variance
(ANOVA). To achieve normal distributed data, a log10 transformation was
performed. The obtained data contain plants from at least three independent grafting experiments. Two-tailed Student’s t tests were used
to identify the variation in the GLS content between the wild type and
mutants in all other analyses. One-way ANOVA analysis was done using
Sigmaplot for Windows version 11 (www.systat.com), and Student’s t tests
were performed using Microsoft Excel 2010.
12 of 13
The Plant Cell
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession
numbers: At1g62500, Cortex specific transcript (pCO2); At4g39950
(CYP79B2); At2g22330 (CYP79B3); At1g09750 Endopeptidase (pPEP);
At1g79840 GLABRA2 (pGL2); At3g47960 (GTR1); At5g62680 (GTR2);
At5g61420 (MYB28), At5g07690 (MYB29); At3g54220 SCARECROW
(pSCR); At4g37650 SHORTROOT (pSHR); At1g22710 Sucrose transporter2 (pSUC2); and At1g77990 Sulfate transporter (pSULTR2;2).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Glucosinolate Content in Soil-Grown 3-Week-Old
gtr Mutants.
Supplemental Figure 2. Glucosinolate Content in Hydroponically
Grown gtr1 gtr2 Mutants.
Supplemental Figure 3. Tissue Weights of Hydroponically Grown
3-Week-Old gtr1 gtr2 Mutants.
Supplemental Figure 4. In Silico Microarray-Based Expression Profile
of GTR1 and GTR2 in Roots.
Supplemental Figure 5. Translocation of p-Hydroxybenzyl Glucosinolate Administered to Leaves and Roots of Arabidopsis in the
Vegetative Growth Phase.
Supplemental Table 1. Glucosinolate Content in Rosettes of Micrografted Plants.
Supplemental Table 2. Glucosinolate Content in Roots of Micrografted
Plants.
Supplemental Table 3. Glucosinolate Content in Rosettes of Micrografted and Ungrafted Plants.
Supplemental Table 4. Glucosinolate Content in Roots of Micrografted and Ungrafted Plants.
ACKNOWLEDGMENTS
We thank Rosemary White (Commonwealth Scientific and Industrial
Research Organization, Canberra, Australia) for her advice on plant
micrografting and Svend Roesen Madsen for his advice on hydroponic
growth setups. We also thank Nanna MacAulay and technicians
Charlotte Goos Iversen and Catia Correa Goncalves Andersen (The
Panum Institute, Copenhagen University) for providing X. laevis
oocytes. We thank Alexander Schulz for critical reading of the article.
Imaging data were collected at the Center for Advanced Bioimaging
Denmark, University of Copenhagen. Financial support for the work is
provided by FTP Grant 09-065827/274-08-0354 (H.H.N.-E.), FTP Grant
10-082395 (B.A.H.), Danish National Research Foundation for funding
DynaMo Center of Excellence (B.A.H.), and IEE Marie Curie stipend
PIEF-GA-2008-221236 (M.B.).
AUTHOR CONTRIBUTIONS
T.G.A. performed and designed all experiments and statistical analyses.
H.H.N.-E. contributed to experimental design and data analysis. C.E.O.
performed the LC-MS analysis. M.B. provided GLS. V.L.F. contributed to
GLS analysis. T.G.A., H.H.N.-E., M.B., and B.A.H. planned the research.
T.G.A., H.H.N.-E., and B.A.H. wrote the article based on a draft written by
T.G.A. M.B. commented on the article.
Received February 21, 2013; revised July 26, 2013; accepted August 9,
2013; published August 30, 2013.
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Integration of Biosynthesis and Long-Distance Transport Establish Organ-Specific Glucosinolate
Profiles in Vegetative Arabidopsis
Tonni Grube Andersen, Hussam Hassan Nour-Eldin, Victoria Louise Fuller, Carl Erik Olsen, Meike
Burow and Barbara Ann Halkier
Plant Cell; originally published online August 30, 2013;
DOI 10.1105/tpc.113.110890
This information is current as of June 15, 2017
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