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A 2-Oxoglutarate-Dependent Dioxygenase Mediates theBiosynthesis
of Glucoraphasatin in Radish1[OPEN]
Tomohiro Kakizaki*, Hiroyasu Kitashiba, Zhongwei Zou2, Feng Li3,
Nobuko Fukino, Takayoshi Ohara,Takeshi Nishio, and Masahiko
Ishida4
Division of Vegetable Breeding, Institute of Vegetable and
Floriculture Science, NARO, Ano, Tsu, Mie514-2392, Japan (T.K.,
N.F., T.O., M.I.); and Graduate School of Agricultural Science,
Tohoku University,Aoba-ku, Sendai, Miyagi 980-0845, Japan (H.K.,
Z.Z., F.L., T.N.)
ORCID IDs: 0000-0002-3636-6616 (T.K.); 0000-0002-8955-1153
(Z.Z.).
Glucosinolates (GSLs) are secondary metabolites whose
degradation products confer intrinsic flavors and aromas
toBrassicaceae vegetables. Several structures of GSLs are known in
the Brassicaceae, and the biosynthetic pathway andregulatory
networks have been elucidated in Arabidopsis (Arabidopsis
thaliana). GSLs are precursors of chemical defensesubstances
against herbivorous pests. Specific GSLs can act as feeding
blockers or stimulants, depending on the pestspecies. Natural
selection has led to diversity in the GSL composition even within
individual species. However, in radish(Raphanus sativus),
glucoraphasatin (4-methylthio-3-butenyl glucosinolate) accounts for
more than 90% of the total GSLs, andlittle compositional variation
is observed. Because glucoraphasatin is not contained in other
members of the Brassicaceae, likeArabidopsis and cabbage (Brassica
oleracea), the biosynthetic pathways for glucoraphasatin remain
unclear. In this report, weidentified and characterized a gene
encoding GLUCORAPHASATIN SYNTHASE 1 (GRS1) by genetic mapping using
amutant that genetically lacks glucoraphasatin. Transgenic
Arabidopsis, which overexpressed GRS1 cDNA,
accumulatedglucoraphasatin in the leaves. GRS1 encodes a
2-oxoglutarate-dependent dioxygenase, and it is abundantly
expressed inthe leaf. To further investigate the biosynthesis and
transportation of GSLs in radish, we grafted a grs1 plant onto a
wild-typeplant. The grafting experiment revealed a leaf-to-root
long-distance glucoraphasatin transport system in radish and
showedthat the composition of GSLs differed among the organs. Based
on these observations, we propose a characteristicbiosynthesis
pathway for glucoraphasatin in radish. Our results should be useful
in metabolite engineering for breeding ofhigh-value vegetables.
Glucosinolates (GSLs), the sulfur-containing
sec-ondarymetabolites, are precursors of chemical defensecompounds
of the Brassicaceae. When plants areattacked by herbivores and/or
pathogens, GSLs arerapidly hydrolyzed by endogenous
thioglucosidases(also known as myrosinases) to yield
isothiocyanates
(Rask et al., 2000; Wittstock and Halkier, 2002; Halkierand
Gershenzon, 2006). Isothiocyanates, such as sul-foraphane
(4-methylsulfinylbutane isothiocyanate),are known as
anticarcinogenic compounds that inducephase 2 detoxification
enzymes (Zhang et al., 1994).Specific isothiocyanates can act as
repellents depend-ing on the feeding species. In response to
natural se-lection by pests, qualitative variation of GSLs mayhave
arisen among Arabidopsis (Arabidopsis thaliana)accessions
(Giamoustaris and Mithen, 1995).
Plants contain more than 200 types of GSLs, which,on the basis
of their precursors, are classified into thefollowing three groups:
aliphatic, aromatic, and indolicGSLs. The main precursors of
aliphatic, aromatic, andindolic GSLs are Met, Phe, and Trp,
respectively (Faheyet al., 2001; Halkier and Gershenzon, 2006). The
struc-ture and composition of GSLs depend on plant species,variety,
developmental stage, and tissue (Mithen et al.,2000; Fahey et al.,
2001). In vegetables of the Brassica-ceae, GSLs not only act as
antipest compounds but alsoconfer specific flavors and tastes.
Radish (Raphanus sativus; 2n = 2x = 18) is an impor-tant root
vegetable of the Brassicaceae family thatgenerally contains
glucoraphasatin (4-methylthio-3-butenyl glucosinolate), one of the
aliphatic GSLsderived from Met, in the root. Glucoraphasatin isthe
predominant GSL in the root and accounts for
1 This work was supported by the Ministry of Agriculture,
For-estry, and Fisheries of Japan (Genomics-Based Technology for
Agri-cultural Improvement, grant no. HOR-1006).
2 Present address: Department of Plant Science, University of
Man-itoba, Winnipeg, R3T 2N2, Canada.
3 Present address: Division of Radiation Breeding, Institute
ofCrop Sciences, NARO, Hitachiomiya, Ibaraki 319-2293, Japan.
4 Present address: Institute of Vegetable and Floriculture
Science,NARO, Tsukuba, 305-8666, Japan.
* Address correspondence to [email protected] author
responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
de-scribed in the Instructions for Authors (www.plantphysiol.org)
is:Tomohiro Kakizaki ([email protected]).
T.K. and M.I. designed the study and performed most of the
ex-periments; H.K., Z.Z., and F.L. constructed the BAC library and
per-formed the screening; N.F. and T.O. performed the HPLC; T.K.
andT.N. wrote the article.
[OPEN] Articles can be viewed without a
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Plant Physiology�, March 2017, Vol. 173, pp. 1583–1593,
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more than 90% of the total GSLs present in Japanesecultivars
(Ishida et al., 2012). Recently, the genomesequence and
transcriptome profiles of radish havefacilitated studies on the GSL
biosynthetic pathway byprofiling of GSL synthesis-associated genes
of Ara-bidopsis (Wang et al., 2013; Kitashiba et al., 2014;Mitsui
et al., 2015; Wu et al., 2015). However, becauseglucoraphasatin is
characteristic of radish and notfound in other Brassica spp., the
pathway for its bio-synthesis remains unclear.
In a comprehensive screening of radish landracesfrom Japan using
their GSL composition, a mutantlacking glucoraphasatin was
identified in a previousstudy (Ishida et al., 2015). The mutant
contains glu-coerucin (4-methylthiobutyl glucosinolate) in roots
andshoots instead of glucoraphasatin. Glucoerucin is the
major GSL in cabbage (Brassica oleracea) and some ac-cessions of
Arabidopsis but is almost absent in wild-type radish. The
structural difference between glucor-aphasatin and glucoerucin is
the presence of a doublebond between third and fourth carbon chain
(Visentinet al., 1992; Barillari et al., 2005; Montaut et al.,
2010).Genetic analysis revealed that the absence of
glucor-aphasatin is controlled by a single recessive gene lo-cated
at the end of the Raphanus linkage group R1 andsuggested that the
mutant has a lesion in a gene en-coding an enzyme catalyzing the
synthesis of glucor-aphasatin from glucoerucin (Ishida et al.,
2015). Wenamed the mutant glucoraphasatin synthase1 (grs1)
andattempted to elucidate the function of GRS1. Here, wereport the
map-based cloning of GRS1 and its functionin the GSL biosynthetic
pathway in radish.
Table I. GSL content in radish plant
Aliphatic and indolic GSLs were measured in leaves of 3-week-old
soil-grown wild-type (cv. Taibyosoubutori), grs1-1, grs1-2, and F1
plants.Numbers are averages 6 SD (n = 10). Values given are mmol
g21 dry weight. Data within a column followed by the same letter
are not significantlydifferent (P , 0.05). n.d., Not detected.
Plant Glucoraphanin Glucoraphenin Glucoerucin Glucoraphasatin
Glucobrassicin4OH-
Glucobrassicin
4OMe-
GlucobrassicinTotal
Wild type 1.4 6 0.1 a 2.9 6 0.5 0.3 6 0.3 a 42.7 6 10.3 0.9 6
0.4 a n.d. n.d. 48.1 6 11.2 a
grs1-1 3.9 6 0.9 b n.d. 6.6 6 1.3 b n.d. 1.3 6 0.6 a,b n.d. 0.1
6 0.1 11.9 6 2.0 b
grs1-2 9.7 6 1.9 c n.d. 21.7 6 3.2 c n.d. 1.8 6 0.9 b 0.1 6 0.1
0.1 6 0.1 33.4 6 2.7 c
grs1-2 3 grs1-1 4.4 6 1.9 b n.d. 9.1 6 2.7 b n.d. 1.0 6 0.5 a
n.d. n.d. 14.6 6 3.1 b
Figure 1. Map-based cloning of GRS1. Themutation in GRS1 was
mapped between in-sertion/deletion markers s8D07 and ssG02
onRaphanus linkage group R1. Black bars, Ho-mozygous region for the
grs1-1 allele; whitebars, heterozygous region. The GSL content
inthe recombinants was measured by HPLC, andthe plants were
classified into glucoerucin-rich(E) and glucoraphasatin-rich (R)
types. Thenumber of recombinants with the same geno-type is
indicated in parentheses. Gray bars in-dicate bacterial artificial
chromosome clonesisolated using the linked markers. The pre-dicted
ORFs in this region are shown by blackarrows. cM, Centimorgan.
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RESULTS
Map-Based Cloning of GRS1
To identify GRS1, we employed two mutant plantsdeficient in
glucoraphasatin synthesis, grs1-1 and grs1-2.Both the mutants are
completely lacking in the accu-mulation of glucoraphasatin in
leaves (Table I). Thesemutants contain a high concentration of
glucoerucin,which is hardly detected in the wild-type plants.
More-over, glucoraphenin (4-methylsufinyl-3-butenyl
gluco-sinolate), an S-oxygenated product of glucoraphasatin,also
was not detected in the mutants. To determine theallelism of the
two mutants, F1 progeny (grs1-23 grs1-1)were obtained by
cross-pollination. The GSL profile ofthe F1 plant was similar to
that of the respective parents,indicating that these mutants have
lesions in the samegene that is involved in glucoraphasatin
biosynthesis(Table I).In our previous genetic mapping experiment,
GRS1
was mapped onto a 4.2-centimorgan interval end ofRaphanus
linkage group R1 using genome-wide single-nucleotide polymorphism
markers (Ishida et al., 2015).To fine-mapGRS1, we first screened a
large segregatingpopulation (;5,000 F2 and F3 plants) derived from
across between a grs1-1 mutant and the wild-typeHAGHN. The GSL
analysis of the segregating popu-lation revealed that three group
had recombinationbetween two insertion/deletion markers, s8D07
andssG02, putting GRS1 in a 23-kb genomic region (Fig. 1).By
screening a BAC library, constructed fromwild-typecv. Miyashige,
BAC contigs were built using theneighboring genetic markers (Fig.
1). The nucleotidesequence between s8D07 and ssG02 DNAmarkers
wasdetermined by shotgun sequencing of the P64M08 BACclone.
Although seven open reading frames (ORFs)were identified in this
region by the AUGUSTUS geneprediction program (Stanke and
Morgenstern, 2005),only ORF3 showed differences in the gene
expressionlevels between the leaf and root of grs1-1 and the
wildtype. The expression level of ORF3 was reduced dras-tically in
the grs1-1 mutant (Supplemental Fig. S1).These findings strongly
suggest that ORF3 is a candi-date GRS1.
Prediction of the Amino Acid Sequence andPhylogenetic Tree of
2-Oxoglutarate andFe(II)-Dependent Dioxygenases
Wenext determined the nucleotide sequence ofORF3in the grs1-1
and grs1-2 mutants. The 8.6- and 1.2-kbinsertions were identified
in the first exon of grs1-1 andthe third exon of grs1-2,
respectively (Fig. 2A). Thenucleotide sequence of the 8.6-kb
insertion in the grs1-1allele contained high similarity to a
Ty1-copia long ter-minal repeat retrotransposon. The 1.2-kb
insertion ofgrs1-2 showed no similarity to long terminal
repeatretrotransposons. Both the insertions led to an in-framestop
codon just downstream of the insertion site (Fig.2B). Real-time PCR
analysis revealed that the expression
of ORF3 in grs1-1 and grs1-2 was approximately 1:1,000and 1:10
of that in the wild type (HAGHN and cv. Tai-byosoubutori),
respectively (Fig. 2C). Therefore, the lesionin the accumulation of
glucoraphasatin in the two mu-tants was caused by the production of
a truncated ORF3protein and/or the suppression of ORF3
expression.
A BLAST search against the GenBank ConservedDomain Database
version 3.14 (http://www.ncbi.nlm.nih.gov/cdd/) indicated that the
372-amino acid proteinencoded by ORF3 is a member of the
2-oxoglutarateand Fe(II)-dependent dioxygenase (2OGD)
superfamily.2OGDs consist of a nonheme dioxygenase in
morphinesynthesis N-terminal (DIOX_N) in the N-terminal regionand a
2OG-Fe(II) oxygenase superfamily (2OG-FeII_Oxy)motif in the
C-terminal region (De Carolis and DeLuca, 1994). In plants, 2OGD
superfamily members are
Figure 2. Gene structure and expression ofORF3. A, Polymorphisms
inORF3. The introns (horizontal lines), exons (black boxes), and 59
and 39untranslated regions (white boxes) of theORF3 are shown.
Arrowheadsshow the large insertions. Bar = 100 bp. B, The insertion
sequence in-dicated by lowercase letters. Asterisks show the
in-frame stop codon. C,Quantitative analysis ofORF3mRNA expression
in wild-type (HAGHNand cv. Taibyosoubutori [TIB]) and grs1 mutant
plants. Each bar repre-sents the mean 6 SD (n = 3). The asterisk
above the bar indicates sig-nificant differences (P , 0.01) between
HAGHN and the respectivemutant, as determined by Student’s t
test.
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involved in various oxygenation/hydroxylation reac-tions.
Arabidopsis and rice (Oryza sativa) contain 130 and114 2OGD
proteins, respectively, classified into threeclasses, namely DOXA,
DOXB, and DOXC, based onsimilarity of the deduced amino acid
sequences (Kawaiet al., 2014). The domain organization of the
predictedORF3 protein suggested that it belongs to theDOXC
class.2OGDs of the DOXC class are classified into 57 phyloge-netic
clades and are involved in the biosynthesis of variousmetabolites.
To assess the biological function of ORF3, aphylogenetic tree was
constructed with 11 functionallycharacterized 2OGDs in the DOXC
class (Fig. 3). Phylo-genetic analysis revealed that ORF3 belongs
to theDOXC31 clade, which includes AtGSL-OH of Arabi-dopsis, CrD4H
of Catharanthus roseus, and ZmBX6 of Zeamays. AtGSL-OH is involved
in GSL biosynthesis andcatalyzes the conversion of 3-butenyl
glucosinolate to2-hydroxy-3-butenyl glucosinolate (Hansen et al.,
2008).Homology between the deduced amino acid sequences ofORF3 and
AtGSL-OHwas 50.1%. In the GSL biosyntheticpathway, another clade of
DOXC class 2OGDs, includingAOP1, AOP2, and AOP3 (for Arabidopsis
2-oxoglutarate-dependent dioxygenases), is involved (Kliebenstein
et al.,2001). However, these AOPs were classified into theDOXC20
clade. These data clearly showed that ORF3 is amember of the 2OGDs
and is classified into the same cladeas the enzyme that modifies
the aliphatic GSL side chain.However, it is difficult to infer a
distinct function from thephylogenetic analysis.
Analysis of Transgenic Arabidopsis Overexpressing ORF3
Further functional characterization of ORF3 wasachieved by
transgenic experiments inArabidopsis. It isknown that the
efficiency of transgenesis in radish is
extremely low compared with other plants of theBrassicaceae and
that accessions that show a high re-generation rate are limited.
Therefore, an overexpressionconstruct of ORF3 was introduced into
Arabidopsis. Asthe phenotype of grs1 mutants indicated that the
sub-strate of GRS1 is glucoerucin, we surveyed and selecteda
glucoerucin-rich accession, Ts-1, from the RIKENArabidopsis core
collection and generated transgenicplants overexpressing ORF3 in
the Ts-1 background. T3plants of three independent overexpression
and emptyvector lines were obtained. In HPLC for desulfo-GSLs
inleaf, an additional peak (peak 4) that was not detectedin the
vector controls was obtained in all the over-expression lines (Fig.
4A). We confirmed that the reten-tion time of the additional peak
was identical to that ofdesulfo-glucoraphasatin by comparing the
GSL profileswith those of wild-type radish plants. In
liquidchromatography-mass spectroscopy (LC-MS) analysis,peak 4 had
mass-to-charge ratio (m/z) values of 178, 340,and 362, which
corresponded to the previously reportedm/z values of
desulfo-glucoraphasatin for [M+H2Glc],[M+H], and [M+Na],
respectively (Fig. 4B; Kusznierewiczet al., 2013). Similarly, the
mass spectra of peak 3 wereidentical to that of desulfo-glucoerucin
(data not shown).The concentration of glucoraphasatin was 2.3 to
5.8mmol g21 dryweight in the overexpression lines; however,it was
not detected in the empty vector controls(Supplemental Table S1).
Contrary to the accumulation ofdesulfo-glucoraphasatin, the
deslufo-glucoerucin (peak 3)concentrationwas decreased in the
overexpression lines to40% of that in the vector control
(Supplemental Table S1).In the overexpression lines,
desulfo-glucoraphenin wasdetected byHPLC (Supplemental Table S1);
the samewasnot detected in the vector control. Both
glucoraphasatinand glucoraphenin contain a double bound in their
sidechains between the third and fourth carbons. These results
Figure 3. Phylogenetic tree of 2OGDproteins in the DOXC class.
Thededuced amino acid sequenceswere aligned with the MUSCLEprogram.
The phylogenetic tree wasconstructed by the maximum like-lihood
method using the MEGA6program (Tamura et al., 2013). Thebranches
indicate bootstrap valuescalculated by the 1,000-permutationtest.
The classificationwas accordingto Kawai et al. (2014).
ArabidopsisALKBH2 (At2g22260), which wasclassified into the DOXA5
class, wasused as an outgroup. The accessionnumbers and Arabidopsis
GenomeInitiative codes are indicated in pa-rentheses.
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indicated that GRS1 is a gene of ORF3 and is responsiblefor the
desaturation of the side chain in aliphatic GSLs, inparticular
glucoerucin (Fig. 5).
In Vitro Assay of GRS1
To determine if GRS1 encodes a glucoraphasatinsynthase, we
investigated its ability to desaturate
the side chain of glucoerucin. To this end, the full-lengthcDNA
of GRS1 was cloned into a pColdIII vector andthe recombinant
protein was expressed in Escherichiacoli. The recombinant GRS1
protein was solubilizedby sonication, and the crude supernatant was
used forthe in vitro assay. After incubation of the mixture
contain-ing recombinant GRS1, glucoerucin, Fe2+,
2-oxogulutarate,and ascorbate were analyzed by HPLC. However,
noenzymatic activity was detected.
Figure 4. ORF3 mediates the biosyn-thesis of glucoraphasatin in
transgenicArabidopsis. A, Chromatograms of desulfo-GSLs recorded at
229nm for the transgenicTs-1 containing the p35S::ORF3
construct,empty vector, andwild-type radish. Peak 1,Sinigrin
(internal control); peak 2, glucor-aphanin; peak 3, glucoerucin;
peak 4,glucoraphasatin. The leaves of 39-d-old T3transformants were
subsequently used inthe HPLC analysis. B, LC-MS and tandemmass
spectrometry (MS/MS) spectra show-ing peak 4 obtained in transgenic
Arabi-dopsis overexpressingORF3.
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Change in GSL Composition in Grafted Radish Plants
An analysis of GRS1 expression in cv. Taibyosoubu-tori, which is
a four-way cross cultivar, revealed anabundance of transcripts in
the aerial tissues like leaves,but not in roots and flower buds
(Fig. 6). In contrast,higher accumulation of aliphatic GSLs was
detected inroots and flower buds (Table II). These results
suggestthe long-distance transport of GSLs from leaves to rootsand
flower buds. The observation of low levels of GRS1expression in the
root during the thickening stage andthe abundant accumulation of
GSLs in the rootprompted us to hypothesize that glucoraphasatin
issynthesized in the leaf and accumulated in the root. Totest this
hypothesis, we reciprocally grafted grs1-1 andwild-type radish
plants on each other. For the graftingexperiment, cv. Karami199,
which has a high concen-tration of glucoraphasatin in the root, was
used. Scionsof grs1-1 were grafted on the stocks of both grs1-1and
cv. Karami199. Likewise, scions of cv. Karami199were grafted onto
the stocks of both grs1-1 and cv.Karami199. At the thickening stage
of the graftedplants, there were no significant differences in
themorphological traits between the heterografted andhomografted
plant roots (Fig. 7A).
In the analysis of GSL levels in the homografts, thelevels in
the leaves and roots were not significantlydifferent between the
homografts and nongrafts (datanot shown). These results indicate
that the distributionand accumulation of GSLs were not affected by
graft-ing. In the leaf, glucoerucin was the dominant GSL inthe
grafted GR/KA plants. Likewise, KA/GR plantsshowed high
accumulation of glucoraphasatin in theleaf (Fig. 7B). These
observations indicated the presence
of genotype-dependent GSL profiles in the leaf. Incontrast to
the GSL profile of the leaf, the GSL profile ofthe root was
affected by the genotype of the leaf (Fig.7C). Glucoraphasatin,
which was not detected in root ofGR/GR plants, was accumulated to
157.7 mmol g21 dryweight in the root of KA/GR plants. The total
amountof GSL in the root was not significantly different be-tween
KA/KA and KA/GR plants (SupplementalTable S2). In the roots of
KA/GR plants, glucor-aphasatin accounted for more than 85% of the
totalGSLs. These results clearly showed that the majority ofthe
aliphatic GSLs in the root were transported from theleaf.
DISCUSSION
Plants, including those of the Brassicaceae family,generally
contain GSLs as the precursors of defensivesubstances against pests
and pathogens. Nearly200 types of GSLs with different substituents
areknown. Combinations of GSL-associated genes lead tothis variety
of GSLs. A few transcription factors, MYBsand MYCs, control the
gene expression of a large set ofGSL-associated genes responsive to
environmentalcues (Gigolashvili et al., 2007, 2008; Hirai et al.,
2007;Schweizer et al., 2013). In recent genome projects inBrassica
spp. vegetables, the number of GSL-relatedgenes has been estimated
and species-specific biosyn-thesis pathways have been elucidated
(Wang et al.,2013; Liu et al., 2014; Mitsui et al., 2015). Mitsui
et al.(2015) identified three METHYLTHIOALKYLMALATESYNTHASE1
(MAM1)-like genes in the radish genome,but MAM3-like genes were
found to be absent. TheMAMs determine the side chain length of the
aliphaticGSLs in Arabidopsis (Kroymann et al., 2001). Among
Figure 5. Proposed GSL biosynthesis pathway in radish. GRS1 is
re-sponsible for the desaturation of the side chain of glucoerucin.
FMOGS-OXs, Flavin monooxygenases, which catalyze the S-oxygenation
ofthe side chain.
Figure 6. GRS1 expression. Quantitative analysis of GRS1
mRNAabundance was performed in various tissues. mRNA was
extractedfrom the listed tissues of wild-type cv. Taibyosoubutori
plants. ThemRNA levelswere analyzed by real-time PCR, and the
expression levelswere normalized to that of ACTIN. Each data point
represents themean 6 SD (n = 4).
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the several aliphatic GSLs in Arabidopsis, only the four-carbon
aliphatic GSLs, such as glucoraphasatin andglucoraphenin, have been
detected in radish. TheAOPs, which function in the oxidation of
glucor-aphenin in Arabidopsis, have not been identified in
theradish genome (Kliebenstein et al., 2001; Mitsui et al.,2015).
We identified a single genomic region that con-trols the GSL
composition in radish. This region, whichwas further delimited in
this study, contained a 2OGDinvolved in the biosynthesis of the
radish-specific GSL,glucoraphasatin. In plants, 2OGDs have awide
range ofbiochemical functions (Loenarz and Schofield, 2011;Kawai et
al., 2014). Although genes with similarity toGRS1 have been
identified in the genomes of both
Arabidopsis and cabbage, these species contain onlyglucoerucin
and not glucoraphasatin in abundance. Thedivergence of the
Arabidopsis lineage and the Brassica-Raphanus ancestor from a
common ancestor has beenreported to have occurred 38.8 million
years ago (Mitsuiet al., 2015). After the divergence, whole-genome
triplica-tion in the Brassica-Raphanus ancestor may have
occurredbetween 15.6 and 28.3 million years ago (Mitsui et
al.,2015). Geneswith similarity toGRS1have been retained inBrassica
spp., but the specificity of glucoraphasatin toradish indicates
that GRS1 was generated only in theRaphanus genome after the
whole-genome triplication.
In plants, 2OGDs belong to the second largest en-zyme family.
Its members facilitate numerous oxidative
Table II. GSL content in different tissues
Numbers are averages 6 SD (n = 3). Values given are mmol g21 dry
weight. Data within a column followed by the same letter are not
significantlydifferent (P , 0.05). n.d., Not detected.
Tissue Glucoraphanin Glucoraphenin Glucoerucin Glucoraphasatin
Glucobrassicin4OH-
Glucobrassicin
4OMe-
GlucobrassicinTotal
Leaf n.d. 2.3 6 1.3 a 0.1 6 0.2 a 24.8 6 16.4 a,b 0.1 6 0.1 a
n.d. n.d. 27.3 6 17.8 a
Stem 0.3 6 0.5 a 5.2 6 1.5 a n.d. 41.7 6 9.6 a 0.6 6 0.3 b 0.1 6
0.1 a n.d. 47.9 6 11.8 a,b
Hypocotyl n.d. 1.3 6 0.7 a n.d. 14.0 6 4.3 b n.d. n.d. n.d. 15.2
6 4.6 a
Root n.d. 3.0 6 1.2 a 0.4 6 0.2 a 72.6 6 11.8 c n.d. 0.1 6 0.1 a
0.2 6 0.1 76.2 6 12.9 b
Flower bud 2.1 6 0.3 b 64.2 6 22.0 b 1.1 6 0.1 b 132.6 6 5.6 d
0.9 6 0.3 b 0.3 6 0.1 b n.d. 201.2 6 16.9 c
Figure 7. GSL concentrations inleaves and roots of grafted
radishplants. A, Typical roots of recipro-cally grafted radish
plants. Lettersindicate grafted plants of grs1-1 ongrs1-1 (GR/GR),
cv. Karami199 ongrs1-1 (KA/GR), cv. Karami199on cv. Karami199
(KA/KA), andgrs1-1 on cv. Karami199 (GR/KA).The grafted plants were
harvested60 d after grafting. Bars = 1 cm. Band C, GSL
concentrations in theleaves (B) and roots (C) of graftedradish
plants. DW, Dry weight; n.d.,not detected.
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reactions, including hydroxylations, desaturations,
di-merizations, and cyclizations (Loenarz and Schofield,2008;
Farrow and Facchini, 2014). The Arabidopsis ge-nome contains 130
2OGD genes, corresponding to 0.5%of the total gene number (Kawai et
al., 2014). Structuralanalysis of some 2OGDs revealed that the
canonicalstructure contains a double-stranded b-helix core foldthat
supports the residues coordinating iron (Cliftonet al., 2006).
grs1-1 encodes a truncated protein con-taining an in-frame stop
codon caused by the insertionof a retrotransposon, resulting in the
deletion of iron-binding residues (Supplemental Fig. S2). In
grs1-2, theiron-binding domain is present but the
2-oxoglutarate-binding domain is missing as a consequence of the
2-kbinsertion (Supplemental Fig. S2).
GRS1 belongs to the DOXC31 clade, which containsfunctionally
diverse 2OGDs involved in various meta-bolic pathways, such as
ZmBX6 from Z. mays involvedin the biosynthesis of benzoxiazinoid,
which functionsin defense and allelopathy in graminaceous
plants(Frey et al., 2003). This enzyme catalyzes the hydrox-ylation
of DIBOA glucoside at position C7 (Jonczyket al., 2008). CrD4H from
C. roseus is involved in thebiosynthesis of monoterpenoid indole
alkaloids, whichcatalyze the hydroxylation of desacetoxyvindoline
atposition C4 in a later step of vindoline
biosynthesis(Vazquez-Flota et al., 1997). AtGSL-OH also belongsto
DOXC31, which catalyzes the hydroxylation of3-butenyl glucosinolate
(Hansen et al., 2008). All thepreviously characterized 2OGDs belong
to DOXC31and catalyze the hydroxylation of the substrate.
Inter-estingly, despite being a member of DOXC31, GRS1has a
desaturation activity. To determine the in vitroactivity of GRS1,
we tried to develop an assay usingrecombinant GRS1. When different
expression condi-tions were considered (e.g. expression vectors,
coex-pression with molecular chaperons, and temperatureduring
induction), we could not detect the enzymaticactivity of GRS1 using
commercial glucoerucin as asubstrate. Because glucoraphasatin was
accumulated inthe transgenic Arabidopsis lines overexpressing
theGRS1 cDNA, an additional factor might be necessaryfor the
desaturation of glucoerucin or for the desatu-ration of the side
chain occurring at the earlier stages ofthe GSL synthesis
pathway.
Our grafting experiments showed that glucoraphasatinwas
distributed from leaves to roots by the long-distancetransport
machinery (Fig. 7, B and C). Two nitrate/peptide transporters,
namely, GLUCOSINOLATETRANSPORTER1 (GTR1) and GTR2, are essential
forGSL translocation in Arabidopsis (Nour-Eldin et al.,2012). In
particular, short-chain aliphatic GSLs, suchas glucoerucin, have
higher affinity for GTR than thelong-chain aliphatic or indolic
GSLs (Andersen et al.,2013). In radish, some GSL
synthesis-associated genes(e.g. RsBCAT4, RsUGT74B1, and RsGS-OX1)
wereshown to be expressed weakly in roots, and abundanttranscripts
were detected in leaves and stems (Wanget al., 2013). As shown in
Figure 6, GRS1was stronglyexpressed in leaves but not in roots and
flower buds.
These results suggest that similar GSL transport sys-tems are
present in radish and Arabidopsis. The con-centrations of
glucoerucin and glucoraphasatin in rootswere 3 times higher in cv.
Karami199 than in grs1-1 (Fig.7C). The grafted plants with cv.
Karami199 leaves andgrs1-1 roots showed 2 times higher
concentration than theGR/KA plants. These findings suggest that not
only thecomposition but also the concentration of GSLs in theroots
is determined by the genotype of the leaf.
It is of interest to alter the composition of metabo-lites in
vegetables to confer new flavors, pest protec-tion, or anticancer
activity. Tattersall et al. (2001)reported that introducing the
entire cyanogenic glu-coside pathway of sorghum (Sorghum bicolor)
intoArabidopsis resulted in increased resistance to
specificinsects. In many pharmacological studies, sulforaphane,an
isothiocyanate derived from glucoraphanin(4-methylsulfinylbutyl
glucosinolate), has been shownto have health-promoting activities
(Juge et al., 2007).To increase the glucoraphanin composition, a
MYB28allele derived from the wild species Brassica villosawas
introgressed into broccoli (B. oleracea var italica;Traka et al.,
2013). These metabolite-engineering ef-forts permit the breeding of
new vegetables beneficialto human health. In previous studies,
using a radishvariety whose GSL composition was known to bepoor, we
found a single mutation leading to a quali-tative change in radish,
resulting in the accumulationof glucoerucin instead of
glucoraphasatin in the radishroot (Ishida et al., 2015). This
drastic change providesan advantage for dishes containing radish.
It is knownthat an isothiocyanate derived from
glucoraphasatinproduces a yellow pigment, methanethiol, which
im-parts color and flavor to the dishes (Takahashi et al.,2015).
These phenomena are among the reasons citedfor avoiding the cooking
of radish. Our results maybe of use in metabolite engineering for
breeding ofhigh-value vegetables.
MATERIALS AND METHODS
Plant Materials
The radish (Raphanus sativus) mutant lacking glucoraphasatin,
grs1-1, was asibling of NMR154N, described in our previous report
(Ishida et al., 2015). Theallelic mutant, grs1-2, was an inbred
line derived from Tohoku Karami daikon(R. sativus) and was
identified by GSL profiling. The wild-type inbred lineHAGHN was
used for genetic mapping. The commercial lines cv. Taibyosou-butori
(Takii seed) and cv. Karami199 (Kaneko Seeds) were used for gene
ex-pression analysis and for the grafting experiments.
Extraction and HPLC Analysis of GSLs
Extraction of GSLs from root and leaf was performed after
lyophilizationand pulverization using aMulti-Beads Shocker
following themethod describedby Ishida et al. (2015). GSLs were
desulfurized with sulfatase (Sigma) accordingto themethod of Bjerg
and Sørensen (1987), and desulfo-GSLs were subjected toHPLC, as
reported in our previous study (Ishida et al., 2012). HPLC was
per-formed on an LC-20A chromatograph (Shimadzu) fitted with a 5C
18-MS-IIcolumn (150 mm3 4.6 mm i.d., 5 mm; Nacalai Tesque). The
HPLC analysis wasperformed with a flow rate of 1.5 mL min21 at a
column oven temperature of30°C, and the absorbance was measured at
a wavelength of 229 nm. The mobilephase consisted of ultrapure
water (A) and 20% (v/v) acetonitrile (B). The
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mobile phase program was as follows: 1% (v/v) solvent B for 1
min, followedby a linear elution gradient over the next 20 min to
99% (v/v) solvent B, then99% (v/v) solvent B for 3 min, which was
changed to 1% (v/v) solvent B at24.1 min, and then 1% (v/v) solvent
B for 10 min (total, 35 min). The individualGSL contents were
calculated by the ratios of the individual desulfo-GSL peakareas to
the peak areas of an internal standard, sinigrin (Sigma), and a
responsefactor (ISO9167-1).
Genetic Mapping and BAC Screening
A population of 5,198 self-pollinated F2 and F3 progeny of
grs1-1 andHAGHN was used for fine-mapping of grs1. The leaves of
plants showing re-combination between the interval markers were
used for GSL analysis. TheBAC libraries constructed using total DNA
of the radish ‘Miyashige’ doubledhaploid line after partial
digestion with HindIII were used for screening(Kitashiba et al.,
2014).
Quantitative Real-Time PCR
Total RNA was extracted from various tissues with the RNeasy
Plant MiniKit (Qiagen). The first-strand cDNAwas synthesized with
500 ng of RNA usingthe PrimeScript RT Reagent Kit with gDNA Eraser
(TaKaRa) with randomhexamer and oligo(dT) primers in a volume of 20
mL. Quantitative real-timePCR was performed in a total volume of 25
mL, 1 mL of the cDNA, 0.4 mM gene-specific primers, and 12.5 mL of
SYBR Premix ExTaq (TaKaRa) on a ThermalCycler Dice Real-Time System
(TaKaRa) according to the manufacturer’s in-structions. The radish
ACTIN gene was used as an internal control (Zouet al., 2013). All
the expression data were based on at least three
biologicalreplications.
Full-Length cDNA Cloning and Transformation
Total RNAwas extracted fromHAGHNwith the RNeasy PlantMini Kit,
andRACEwas performedwith the FirstChoice RLM-RACE Kit (Life
Technologies).The gene-specific primer sequences are listed in
Supplemental Table S3. Theamplified fragments were cloned into a
pCR2.1-TOPO vector (Invitrogen) andsequenced with the BigDye
Terminator Version 3.1 Cycle Sequencing Kit (LifeTechnologies). A
coding sequence of ORF3 was amplified from total RNA ofHAGHN
leaves. The cauliflowermosaic virus 35S promoter and a terminator
ofnopaline synthase were amplified from the pBI121 binary vector.
Three am-plified fragments were fused into the
HindIII/EcoRI-digested pZK3B binaryvector (Kuroda et al., 2010)
using the In-Fusion HD Cloning Kit (TaKaRa). Thisconstruct was
transformed intoArabidopsis (Arabidopsis thaliana) accession
Ts-1using Rhizobium radiobacter GV3101::pMP90 by the floral dip
method (Cloughand Bent, 1998). To synchronize the germination, all
the seeds were kept at 4°Cfor 3 d after sowing. Plants were grown
on soil (expanded vermiculite) or on0.8% agarmediumwith
0.53Murashige and Skoog salts under a 16-h/8-h cycleof white light
(60–80 mmol m22 s21) and dark at 22°C in a growth chamber
(FLI-2000; Eyela). The primer sequences used for vector
construction are listed inSupplemental Table S3.
LC-MS Analysis of GSLs in Transformed Arabidopsis
The conditions for the detection and identification of
desulfo-GSLs byLC-MSwere as follows. The samples (15 mL) were
injected into 1200 Series HPLCequipment (Agilent) and desulfo-GSLs
were separated on a TSKgel super-ODScolumn (2 3 100 mm, 3-mm
particle size, 40°C) using a 0% to 20% acetonitrilegradient in
water (46 min) with a flow rate of 0.4 mL min21. The detection
wasdone online, first with a photodiode array detector at 230 nm
(wavelength 190–950 nm) and subsequently with the LTQ Orbitrap XL
MS/MS system (ThermoFisher Scientific) operated in electrospray
ionization positive ion mode (m/z =100–800; spray voltage, 3.5 kV;
temperature of the heated capillary, 300°C). Theflow rates for
nitrogen sheath gas and auxiliary gas were set to 50 and 10
ar-bitrary units min21, respectively. Desulfo-glucoraphasatin (m/z
= 178, 340, and362) was monitored by specific MS/MS scans in
addition to the full scan.
Phylogenetic Analysis
The amino acid sequences were deduced from nucleotide sequences
of thepredicted 2OGD genes and then aligned using the MUSCLE
program (Edgar,2004). The number of amino acids substituted between
each pair of 2OGD
proteins was estimated by the LG + G model (Le and Gascuel,
2008). From thenumber of estimated amino acid substitutions, a
phylogenetic tree was recon-structed by the maximum likelihood
method using MEGA version 6.06(Tamura et al., 2013). The bootstrap
values were calculated with 1,000 repli-cations. Arabidopsis ALKBH2
(At2g22260), which was classified in the DOXA5class, was used as an
outgroup.
Protein Assay of GRS1
The coding sequence ofGRS1was amplified fromwild-type HAGHN
usingPCR with the primers mentioned in Supplemental Table S3. The
amplifiedfragments were cloned into XhoI and SalI sites of the
pColdI vector (TaKaRa)using the In-Fusion HD Cloning Kit (TaKaRa).
The recombinant plasmids wereintroduced into Escherichia coli BL21
(DE3) pLysS strain.
Toproduce the recombinantGRS1protein, the bacteriawere cultured
at 37°Cin 10 mL of Luria-Bertani medium supplemented with
ampicillin (50 mg mL21)and chloramphenicol (30 mg mL21) until the
optical density at 600 nm reached0.5. The protein synthesis was
then induced by the addition of 0.1 mM iso-propylthio-b-galactoside
and further culturing for 20 h at 15°C. The culture wasthen
centrifuged at 5,000g for 5 min at 4°C. The cell pellets were
resuspended in1 mL of 13 phosphate-buffered saline buffer, pH 7.4,
sonicated by VP-300N(Taitec), and centrifuged at 15,000g for 10 min
at 4°C. The reaction mixture con-tained 100 mM NaH2PO4, pH 6.8, 10
mM a-ketoglutaric acid, 10 mM ascorbate,0.25 mM ferrous sulfate,
0.25 mM glucoerucin, and 20 mL of crude protein solution.The assays
were performed at 30°C for 30 min. The reactions were initiated by
theaddition of the enzyme and terminated by extraction with
methanol.
Reciprocal Grafting
Ten-day-old seedlings of grs1-1 and cv. Karami199 were used for
grafting.Seedlings with cotyledon and the first true leaf were cut
at the hypocotyl with arazor in a horizontal direction. The scions
and stocks were joined with a holder(superwith14; Nasunics). The
scions of grs1-1were grafted on the stocks of bothgrs1-1 and cv.
Karami199, and the scions of cv. Karami199 were grafted on
thestocks of both grs1-1 and cv. Karami199. The grafted plants were
named GR/GR, GR/KA, KA/GR, and KA/KA. They were grown in plastic
containers for10 d and transplanted into 15-cm pots in the
greenhouse. For GSL analysis, theleaves and roots were harvested 60
d after grafting.
Chemical Compounds Studied
Chemical compounds studiedwere as follows: glucoraphasatin
(PubChemCID: 6442557), glucoraphenin (PubChem CID: 15559531),
glucoerucin(PubChem CID: 656539), and glucoraphanin (PubChem CID:
9548633).
Accession Numbers
The sequence reported in this article has been deposited in the
GenBankdatabase with GenBank/EMBL/DNA Data Bank of Japan accession
numberLC077856 (GRS1 full-length cDNA, HAGHN).
Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. Reverse transcription-PCR analysis in
grs1-1 andthe wild type for seven ORFs linked to the grs
mutation.
Supplemental Figure S2. Alignment of the deduced amino acid
sequencesof GRS1 alleles.
Supplemental Table S1. Aliphatic GSL profile of the transgenic
plants.
Supplemental Table S2. Aliphatic and indolic GSLs in grafted
plants.
Supplemental Table S3. Primer sequences used in this work.
ACKNOWLEDGMENTS
We thank Y. Araki, S. Morimoto, Y. Kawamoto, C. Okuyama, Y.
Niina, andS. Negoro for technical assistance; Dr. H. Fukuoka (Takii
seed) for helpfuldiscussions; Dr. M. Kuroda (NARO) for providing
the binary vector; The
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Arabidopsis accession Ts-1was provided by RIKENBioResource
Center, whichis participating in the National Bio-Resource Project
of the Ministry of Educa-tion, Culture, Sports, Science and
Technology, Japan.
Received November 30, 2016; accepted January 16, 2017; published
January 18,2017.
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