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. 2017 Jan 18;173(3):1583–1593. doi: 10.1104/pp.16.01814

A 2-Oxoglutarate-Dependent Dioxygenase Mediates the Biosynthesis of Glucoraphasatin in Radish1,[OPEN]

Tomohiro Kakizaki 1,2,*, Hiroyasu Kitashiba 1,2, Zhongwei Zou 1,2,2, Feng Li 1,2,3, Nobuko Fukino 1,2, Takayoshi Ohara 1,2, Takeshi Nishio 1,2, Masahiko Ishida 1,2,4
PMCID: PMC5338677  PMID: 28100450

Biosynthesis of glucoraphasatin, a major glucosinolate in radish, is mediated by 2-oxoglutarate-dependent dioxygenase.

Abstract

Glucosinolates (GSLs) are secondary metabolites whose degradation products confer intrinsic flavors and aromas to Brassicaceae vegetables. Several structures of GSLs are known in the Brassicaceae, and the biosynthetic pathway and regulatory networks have been elucidated in Arabidopsis (Arabidopsis thaliana). GSLs are precursors of chemical defense substances against herbivorous pests. Specific GSLs can act as feeding blockers or stimulants, depending on the pest species. 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, and little compositional variation is observed. Because glucoraphasatin is not contained in other members of the Brassicaceae, like Arabidopsis and cabbage (Brassica oleracea), the biosynthetic pathways for glucoraphasatin remain unclear. In this report, we identified and characterized a gene encoding GLUCORAPHASATIN SYNTHASE 1 (GRS1) by genetic mapping using a mutant that genetically lacks glucoraphasatin. Transgenic Arabidopsis, which overexpressed GRS1 cDNA, accumulated glucoraphasatin in the leaves. GRS1 encodes a 2-oxoglutarate-dependent dioxygenase, and it is abundantly expressed in the leaf. To further investigate the biosynthesis and transportation of GSLs in radish, we grafted a grs1 plant onto a wild-type plant. The grafting experiment revealed a leaf-to-root long-distance glucoraphasatin transport system in radish and showed that the composition of GSLs differed among the organs. Based on these observations, we propose a characteristic biosynthesis pathway for glucoraphasatin in radish. Our results should be useful in metabolite engineering for breeding of high-value vegetables.

Glucosinolates (GSLs), the sulfur-containing secondary metabolites, are precursors of chemical defense compounds of the Brassicaceae. When plants are attacked by herbivores and/or pathogens, GSLs are rapidly hydrolyzed by endogenous thioglucosidases (also known as myrosinases) to yield isothiocyanates (Rask et al., 2000; Wittstock and Halkier, 2002; Halkier and Gershenzon, 2006). Isothiocyanates, such as sulforaphane (4-methylsulfinylbutane isothiocyanate), are known as anticarcinogenic compounds that induce phase 2 detoxification enzymes (Zhang et al., 1994). Specific isothiocyanates can act as repellents depending on the feeding species. In response to natural selection by pests, qualitative variation of GSLs may have 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 the following three groups: aliphatic, aromatic, and indolic GSLs. The main precursors of aliphatic, aromatic, and indolic GSLs are Met, Phe, and Trp, respectively (Fahey et al., 2001; Halkier and Gershenzon, 2006). The structure 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 Brassicaceae, GSLs not only act as antipest compounds but also confer specific flavors and tastes.

Radish (Raphanus sativus; 2n = 2x = 18) is an important root vegetable of the Brassicaceae family that generally contains glucoraphasatin (4-methylthio-3-butenyl glucosinolate), one of the aliphatic GSLs derived from Met, in the root. Glucoraphasatin is the predominant GSL in the root and accounts for more than 90% of the total GSLs present in Japanese cultivars (Ishida et al., 2012). Recently, the genome sequence and transcriptome profiles of radish have facilitated studies on the GSL biosynthetic pathway by profiling of GSL synthesis-associated genes of Arabidopsis (Wang et al., 2013; Kitashiba et al., 2014; Mitsui et al., 2015; Wu et al., 2015). However, because glucoraphasatin is characteristic of radish and not found in other Brassica spp., the pathway for its biosynthesis remains unclear.

In a comprehensive screening of radish landraces from Japan using their GSL composition, a mutant lacking glucoraphasatin was identified in a previous study (Ishida et al., 2015). The mutant contains glucoerucin (4-methylthiobutyl glucosinolate) in roots and shoots instead of glucoraphasatin. Glucoerucin is the major GSL in cabbage (Brassica oleracea) and some accessions of Arabidopsis but is almost absent in wild-type radish. The structural difference between glucoraphasatin and glucoerucin is the presence of a double bond between third and fourth carbon chain (Visentin et al., 1992; Barillari et al., 2005; Montaut et al., 2010). Genetic analysis revealed that the absence of glucoraphasatin is controlled by a single recessive gene located at the end of the Raphanus linkage group R1 and suggested that the mutant has a lesion in a gene encoding an enzyme catalyzing the synthesis of glucoraphasatin from glucoerucin (Ishida et al., 2015). We named the mutant glucoraphasatin synthase1 (grs1) and attempted to elucidate the function of GRS1. Here, we report the map-based cloning of GRS1 and its function in the GSL biosynthetic pathway in radish.

RESULTS

Map-Based Cloning of GRS1

To identify GRS1, we employed two mutant plants deficient in glucoraphasatin synthesis, grs1-1 and grs1-2. Both the mutants are completely lacking in the accumulation of glucoraphasatin in leaves (Table I). These mutants contain a high concentration of glucoerucin, which is hardly detected in the wild-type plants. Moreover, glucoraphenin (4-methylsufinyl-3-butenyl glucosinolate), an S-oxygenated product of glucoraphasatin, also was not detected in the mutants. To determine the allelism of the two mutants, F1 progeny (grs1-2 × grs1-1) were obtained by cross-pollination. The GSL profile of the F1 plant was similar to that of the respective parents, indicating that these mutants have lesions in the same gene that is involved in glucoraphasatin biosynthesis (Table I).

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 ± sd (n = 10). Values given are µmol g−1 dry weight. Data within a column followed by the same letter are not significantly different (P < 0.05). n.d., Not detected.

Plant Glucoraphanin Glucoraphenin Glucoerucin Glucoraphasatin Glucobrassicin 4OH-Glucobrassicin 4OMe-Glucobrassicin Total
Wild type 1.4 ± 0.1 a 2.9 ± 0.5 0.3 ± 0.3 a 42.7 ± 10.3 0.9 ± 0.4 a n.d. n.d. 48.1 ± 11.2 a
grs1-1 3.9 ± 0.9 b n.d. 6.6 ± 1.3 b n.d. 1.3 ± 0.6 a,b n.d. 0.1 ± 0.1 11.9 ± 2.0 b
grs1-2 9.7 ± 1.9 c n.d. 21.7 ± 3.2 c n.d. 1.8 ± 0.9 b 0.1 ± 0.1 0.1 ± 0.1 33.4 ± 2.7 c
grs1-2 × grs1-1 4.4 ± 1.9 b n.d. 9.1 ± 2.7 b n.d. 1.0 ± 0.5 a n.d. n.d. 14.6 ± 3.1 b
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In our previous genetic mapping experiment, GRS1 was mapped onto a 4.2-centimorgan interval end of Raphanus linkage group R1 using genome-wide single-nucleotide polymorphism markers (Ishida et al., 2015). To fine-map GRS1, we first screened a large segregating population (∼5,000 F2 and F3 plants) derived from a cross between a grs1-1 mutant and the wild-type HAGHN. The GSL analysis of the segregating population revealed that three group had recombination between two insertion/deletion markers, s8D07 and ssG02, putting GRS1 in a 23-kb genomic region (Fig. 1). By screening a BAC library, constructed from wild-type cv. Miyashige, BAC contigs were built using the neighboring genetic markers (Fig. 1). The nucleotide sequence between s8D07 and ssG02 DNA markers was determined by shotgun sequencing of the P64M08 BAC clone. Although seven open reading frames (ORFs) were identified in this region by the AUGUSTUS gene prediction program (Stanke and Morgenstern, 2005), only ORF3 showed differences in the gene expression levels between the leaf and root of grs1-1 and the wild type. The expression level of ORF3 was reduced drastically in the grs1-1 mutant (Supplemental Fig. S1). These findings strongly suggest that ORF3 is a candidate GRS1.

Figure 1.

Figure 1.

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Map-based cloning of GRS1. The mutation in GRS1 was mapped between insertion/deletion markers s8D07 and ssG02 on Raphanus linkage group R1. Black bars, Homozygous region for the grs1-1 allele; white bars, heterozygous region. The GSL content in the recombinants was measured by HPLC, and the plants were classified into glucoerucin-rich (E) and glucoraphasatin-rich (R) types. The number of recombinants with the same genotype is indicated in parentheses. Gray bars indicate bacterial artificial chromosome clones isolated using the linked markers. The predicted ORFs in this region are shown by black arrows. cM, Centimorgan.

Prediction of the Amino Acid Sequence and Phylogenetic Tree of 2-Oxoglutarate and Fe(II)-Dependent Dioxygenases

We next determined the nucleotide sequence of ORF3 in the grs1-1 and grs1-2 mutants. The 8.6- and 1.2-kb insertions were identified in the first exon of grs1-1 and the third exon of grs1-2, respectively (Fig. 2A). The nucleotide sequence of the 8.6-kb insertion in the grs1-1 allele contained high similarity to a Ty1-copia long terminal repeat retrotransposon. The 1.2-kb insertion of grs1-2 showed no similarity to long terminal repeat retrotransposons. Both the insertions led to an in-frame stop 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 1:1,000 and 1:10 of that in the wild type (HAGHN and cv. Taibyosoubutori), respectively (Fig. 2C). Therefore, the lesion in the accumulation of glucoraphasatin in the two mutants was caused by the production of a truncated ORF3 protein and/or the suppression of ORF3 expression.

Figure 2.

Figure 2.

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Gene structure and expression of ORF3. A, Polymorphisms in ORF3. The introns (horizontal lines), exons (black boxes), and 5′ and 3′ untranslated regions (white boxes) of the ORF3 are shown. Arrowheads show the large insertions. Bar = 100 bp. B, The insertion sequence indicated by lowercase letters. Asterisks show the in-frame stop codon. C, Quantitative analysis of ORF3 mRNA expression in wild-type (HAGHN and cv. Taibyosoubutori [TIB]) and grs1 mutant plants. Each bar represents the mean ± sd (n = 3). The asterisk above the bar indicates significant differences (P < 0.01) between HAGHN and the respective mutant, as determined by Student’s t test.

A BLAST search against the GenBank Conserved Domain Database version 3.14 (http://www.ncbi.nlm.nih.gov/cdd/) indicated that the 372-amino acid protein encoded by ORF3 is a member of the 2-oxoglutarate and Fe(II)-dependent dioxygenase (2OGD) superfamily. 2OGDs consist of a nonheme dioxygenase in morphine synthesis N-terminal (DIOX_N) in the N-terminal region and a 2OG-Fe(II) oxygenase superfamily (2OG-FeII_Oxy) motif in the C-terminal region (De Carolis and De Luca, 1994). In plants, 2OGD superfamily members are involved in various oxygenation/hydroxylation reactions. Arabidopsis and rice (Oryza sativa) contain 130 and 114 2OGD proteins, respectively, classified into three classes, namely DOXA, DOXB, and DOXC, based on similarity of the deduced amino acid sequences (Kawai et al., 2014). The domain organization of the predicted ORF3 protein suggested that it belongs to the DOXC class. 2OGDs of the DOXC class are classified into 57 phylogenetic clades and are involved in the biosynthesis of various metabolites. To assess the biological function of ORF3, a phylogenetic tree was constructed with 11 functionally characterized 2OGDs in the DOXC class (Fig. 3). Phylogenetic analysis revealed that ORF3 belongs to the DOXC31 clade, which includes AtGSL-OH of Arabidopsis, CrD4H of Catharanthus roseus, and ZmBX6 of Zea mays. AtGSL-OH is involved in GSL biosynthesis and catalyzes the conversion of 3-butenyl glucosinolate to 2-hydroxy-3-butenyl glucosinolate (Hansen et al., 2008). Homology between the deduced amino acid sequences of ORF3 and AtGSL-OH was 50.1%. In the GSL biosynthetic pathway, another clade of DOXC class 2OGDs, including AOP1, AOP2, and AOP3 (for Arabidopsis 2-oxoglutarate-dependent dioxygenases), is involved (Kliebenstein et al., 2001). However, these AOPs were classified into the DOXC20 clade. These data clearly showed that ORF3 is a member of the 2OGDs and is classified into the same clade as the enzyme that modifies the aliphatic GSL side chain. However, it is difficult to infer a distinct function from the phylogenetic analysis.

Figure 3.

Figure 3.

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Phylogenetic tree of 2OGD proteins in the DOXC class. The deduced amino acid sequences were aligned with the MUSCLE program. The phylogenetic tree was constructed by the maximum likelihood method using the MEGA6 program (Tamura et al., 2013). The branches indicate bootstrap values calculated by the 1,000-permutation test. The classification was according to Kawai et al. (2014). Arabidopsis ALKBH2 (At2g22260), which was classified into the DOXA5 class, was used as an outgroup. The accession numbers and Arabidopsis Genome Initiative codes are indicated in parentheses.

Analysis of Transgenic Arabidopsis Overexpressing ORF3

Further functional characterization of ORF3 was achieved by transgenic experiments in Arabidopsis. It is known that the efficiency of transgenesis in radish is extremely low compared with other plants of the Brassicaceae and that accessions that show a high regeneration rate are limited. Therefore, an overexpression construct of ORF3 was introduced into Arabidopsis. As the phenotype of grs1 mutants indicated that the substrate of GRS1 is glucoerucin, we surveyed and selected a glucoerucin-rich accession, Ts-1, from the RIKEN Arabidopsis core collection and generated transgenic plants overexpressing ORF3 in the Ts-1 background. T3 plants of three independent overexpression and empty vector lines were obtained. In HPLC for desulfo-GSLs in leaf, an additional peak (peak 4) that was not detected in the vector controls was obtained in all the overexpression lines (Fig. 4A). We confirmed that the retention time of the additional peak was identical to that of desulfo-glucoraphasatin by comparing the GSL profiles with those of wild-type radish plants. In liquid chromatography-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 reported m/z values of desulfo-glucoraphasatin for [M+H−Glc], [M+H], and [M+Na], respectively (Fig. 4B; Kusznierewicz et al., 2013). Similarly, the mass spectra of peak 3 were identical to that of desulfo-glucoerucin (data not shown). The concentration of glucoraphasatin was 2.3 to 5.8 µmol g−1 dry weight in the overexpression lines; however, it was not detected in the empty vector controls (Supplemental Table S1). Contrary to the accumulation of desulfo-glucoraphasatin, the deslufo-glucoerucin (peak 3) concentration was decreased in the overexpression lines to 40% of that in the vector control (Supplemental Table S1). In the overexpression lines, desulfo-glucoraphenin was detected by HPLC (Supplemental Table S1); the same was not detected in the vector control. Both glucoraphasatin and glucoraphenin contain a double bound in their side chains between the third and fourth carbons. These results indicated that GRS1 is a gene of ORF3 and is responsible for the desaturation of the side chain in aliphatic GSLs, in particular glucoerucin (Fig. 5).

Figure 4.

Figure 4.

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ORF3 mediates the biosynthesis of glucoraphasatin in transgenic Arabidopsis. A, Chromatograms of desulfo-GSLs recorded at 229 nm for the transgenic Ts-1 containing the p35S::ORF3 construct, empty vector, and wild-type radish. Peak 1, Sinigrin (internal control); peak 2, glucoraphanin; peak 3, glucoerucin; peak 4, glucoraphasatin. The leaves of 39-d-old T3 transformants were subsequently used in the HPLC analysis. B, LC-MS and tandem mass spectrometry (MS/MS) spectra showing peak 4 obtained in transgenic Arabidopsis overexpressing ORF3.

Figure 5.

Figure 5.

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Proposed GSL biosynthesis pathway in radish. GRS1 is responsible for the desaturation of the side chain of glucoerucin. FMO GS-OXs, Flavin monooxygenases, which catalyze the S-oxygenation of the side chain.

In Vitro Assay of GRS1

To determine if GRS1 encodes a glucoraphasatin synthase, we investigated its ability to desaturate the side chain of glucoerucin. To this end, the full-length cDNA of GRS1 was cloned into a pColdIII vector and the recombinant protein was expressed in Escherichia coli. The recombinant GRS1 protein was solubilized by sonication, and the crude supernatant was used for the in vitro assay. After incubation of the mixture containing recombinant GRS1, glucoerucin, Fe2+, 2-oxogulutarate, and ascorbate were analyzed by HPLC. However, no enzymatic activity was detected.

Change in GSL Composition in Grafted Radish Plants

An analysis of GRS1 expression in cv. Taibyosoubutori, which is a four-way cross cultivar, revealed an abundance 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 in roots and flower buds (Table II). These results suggest the long-distance transport of GSLs from leaves to roots and flower buds. The observation of low levels of GRS1 expression in the root during the thickening stage and the abundant accumulation of GSLs in the root prompted us to hypothesize that glucoraphasatin is synthesized in the leaf and accumulated in the root. To test this hypothesis, we reciprocally grafted grs1-1 and wild-type radish plants on each other. For the grafting experiment, cv. Karami199, which has a high concentration of glucoraphasatin in the root, was used. Scions of grs1-1 were grafted on the stocks of both grs1-1 and cv. Karami199. Likewise, scions of cv. Karami199 were grafted onto the stocks of both grs1-1 and cv. Karami199. At the thickening stage of the grafted plants, there were no significant differences in the morphological traits between the heterografted and homografted plant roots (Fig. 7A).

Figure 6.

Figure 6.

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GRS1 expression. Quantitative analysis of GRS1 mRNA abundance was performed in various tissues. mRNA was extracted from the listed tissues of wild-type cv. Taibyosoubutori plants. The mRNA levels were analyzed by real-time PCR, and the expression levels were normalized to that of ACTIN. Each data point represents the mean ± sd (n = 4).

Table II. GSL content in different tissues.

Numbers are averages ± sd (n = 3). Values given are µmol g−1 dry weight. Data within a column followed by the same letter are not significantly different (P < 0.05). n.d., Not detected.

Tissue Glucoraphanin Glucoraphenin Glucoerucin Glucoraphasatin Glucobrassicin 4OH-Glucobrassicin 4OMe-Glucobrassicin Total
Leaf n.d. 2.3 ± 1.3 a 0.1 ± 0.2 a 24.8 ± 16.4 a,b 0.1 ± 0.1 a n.d. n.d. 27.3 ± 17.8 a
Stem 0.3 ± 0.5 a 5.2 ± 1.5 a n.d. 41.7 ± 9.6 a 0.6 ± 0.3 b 0.1 ± 0.1 a n.d. 47.9 ± 11.8 a,b
Hypocotyl n.d. 1.3 ± 0.7 a n.d. 14.0 ± 4.3 b n.d. n.d. n.d. 15.2 ± 4.6 a
Root n.d. 3.0 ± 1.2 a 0.4 ± 0.2 a 72.6 ± 11.8 c n.d. 0.1 ± 0.1 a 0.2 ± 0.1 76.2 ± 12.9 b
Flower bud 2.1 ± 0.3 b 64.2 ± 22.0 b 1.1 ± 0.1 b 132.6 ± 5.6 d 0.9 ± 0.3 b 0.3 ± 0.1 b n.d. 201.2 ± 16.9 c
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Figure 7.

Figure 7.

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GSL concentrations in leaves and roots of grafted radish plants. A, Typical roots of reciprocally grafted radish plants. Letters indicate grafted plants of grs1-1 on grs1-1 (GR/GR), cv. Karami199 on grs1-1 (KA/GR), cv. Karami199 on cv. Karami199 (KA/KA), and grs1-1 on cv. Karami199 (GR/KA). The grafted plants were harvested 60 d after grafting. Bars = 1 cm. B and C, GSL concentrations in the leaves (B) and roots (C) of grafted radish plants. DW, Dry weight; n.d., not detected.

In the analysis of GSL levels in the homografts, the levels in the leaves and roots were not significantly different between the homografts and nongrafts (data not shown). These results indicate that the distribution and accumulation of GSLs were not affected by grafting. In the leaf, glucoerucin was the dominant GSL in the grafted GR/KA plants. Likewise, KA/GR plants showed high accumulation of glucoraphasatin in the leaf (Fig. 7B). These observations indicated the presence of genotype-dependent GSL profiles in the leaf. In contrast to the GSL profile of the leaf, the GSL profile of the root was affected by the genotype of the leaf (Fig. 7C). Glucoraphasatin, which was not detected in root of GR/GR plants, was accumulated to 157.7 µmol g−1 dry weight in the root of KA/GR plants. The total amount of GSL in the root was not significantly different between KA/KA and KA/GR plants (Supplemental Table S2). In the roots of KA/GR plants, glucoraphasatin accounted for more than 85% of the total GSLs. These results clearly showed that the majority of the aliphatic GSLs in the root were transported from the leaf.

DISCUSSION

Plants, including those of the Brassicaceae family, generally contain GSLs as the precursors of defensive substances against pests and pathogens. Nearly 200 types of GSLs with different substituents are known. Combinations of GSL-associated genes lead to this variety of GSLs. A few transcription factors, MYBs and MYCs, control the gene expression of a large set of GSL-associated genes responsive to environmental cues (Gigolashvili et al., 2007, 2008; Hirai et al., 2007; Schweizer et al., 2013). In recent genome projects in Brassica spp. vegetables, the number of GSL-related genes has been estimated and species-specific biosynthesis pathways have been elucidated (Wang et al., 2013; Liu et al., 2014; Mitsui et al., 2015). Mitsui et al. (2015) identified three METHYLTHIOALKYLMALATE SYNTHASE1 (MAM1)-like genes in the radish genome, but MAM3-like genes were found to be absent. The MAMs determine the side chain length of the aliphatic GSLs in Arabidopsis (Kroymann et al., 2001). Among the several aliphatic GSLs in Arabidopsis, only the four-carbon aliphatic GSLs, such as glucoraphasatin and glucoraphenin, have been detected in radish. The AOPs, which function in the oxidation of glucoraphenin in Arabidopsis, have not been identified in the radish genome (Kliebenstein et al., 2001; Mitsui et al., 2015). We identified a single genomic region that controls the GSL composition in radish. This region, which was further delimited in this study, contained a 2OGD involved in the biosynthesis of the radish-specific GSL, glucoraphasatin. In plants, 2OGDs have a wide range of biochemical functions (Loenarz and Schofield, 2011; Kawai et al., 2014). Although genes with similarity to GRS1 have been identified in the genomes of both Arabidopsis and cabbage, these species contain only glucoerucin and not glucoraphasatin in abundance. The divergence of the Arabidopsis lineage and the Brassica-Raphanus ancestor from a common ancestor has been reported to have occurred 38.8 million years ago (Mitsui et al., 2015). After the divergence, whole-genome triplication in the Brassica-Raphanus ancestor may have occurred between 15.6 and 28.3 million years ago (Mitsui et al., 2015). Genes with similarity to GRS1 have been retained in Brassica spp., but the specificity of glucoraphasatin to radish indicates that GRS1 was generated only in the Raphanus genome after the whole-genome triplication.

In plants, 2OGDs belong to the second largest enzyme family. Its members facilitate numerous oxidative reactions, including hydroxylations, desaturations, dimerizations, and cyclizations (Loenarz and Schofield, 2008; Farrow and Facchini, 2014). The Arabidopsis genome contains 130 2OGD genes, corresponding to 0.5% of the total gene number (Kawai et al., 2014). Structural analysis of some 2OGDs revealed that the canonical structure contains a double-stranded β-helix core fold that supports the residues coordinating iron (Clifton et al., 2006). grs1-1 encodes a truncated protein containing an in-frame stop codon caused by the insertion of a retrotransposon, resulting in the deletion of iron-binding residues (Supplemental Fig. S2). In grs1-2, the iron-binding domain is present but the 2-oxoglutarate-binding domain is missing as a consequence of the 2-kb insertion (Supplemental Fig. S2).

GRS1 belongs to the DOXC31 clade, which contains functionally diverse 2OGDs involved in various metabolic pathways, such as ZmBX6 from Z. mays involved in the biosynthesis of benzoxiazinoid, which functions in defense and allelopathy in graminaceous plants (Frey et al., 2003). This enzyme catalyzes the hydroxylation of DIBOA glucoside at position C7 (Jonczyk et al., 2008). CrD4H from C. roseus is involved in the biosynthesis of monoterpenoid indole alkaloids, which catalyze the hydroxylation of desacetoxyvindoline at position C4 in a later step of vindoline biosynthesis (Vazquez-Flota et al., 1997). AtGSL-OH also belongs to DOXC31, which catalyzes the hydroxylation of 3-butenyl glucosinolate (Hansen et al., 2008). All the previously characterized 2OGDs belong to DOXC31 and catalyze the hydroxylation of the substrate. Interestingly, despite being a member of DOXC31, GRS1 has a desaturation activity. To determine the in vitro activity of GRS1, we tried to develop an assay using recombinant GRS1. When different expression conditions were considered (e.g. expression vectors, coexpression with molecular chaperons, and temperature during induction), we could not detect the enzymatic activity of GRS1 using commercial glucoerucin as a substrate. Because glucoraphasatin was accumulated in the transgenic Arabidopsis lines overexpressing the GRS1 cDNA, an additional factor might be necessary for the desaturation of glucoerucin or for the desaturation of the side chain occurring at the earlier stages of the GSL synthesis pathway.

Our grafting experiments showed that glucoraphasatin was distributed from leaves to roots by the long-distance transport machinery (Fig. 7, B and C). Two nitrate/peptide transporters, namely, GLUCOSINOLATE TRANSPORTER1 (GTR1) and GTR2, are essential for GSL translocation in Arabidopsis (Nour-Eldin et al., 2012). In particular, short-chain aliphatic GSLs, such as glucoerucin, have higher affinity for GTR than the long-chain aliphatic or indolic GSLs (Andersen et al., 2013). In radish, some GSL synthesis-associated genes (e.g. RsBCAT4, RsUGT74B1, and RsGS-OX1) were shown to be expressed weakly in roots, and abundant transcripts were detected in leaves and stems (Wang et al., 2013). As shown in Figure 6, GRS1 was strongly expressed in leaves but not in roots and flower buds. These results suggest that similar GSL transport systems are present in radish and Arabidopsis. The concentrations of glucoerucin and glucoraphasatin in roots were 3 times higher in cv. Karami199 than in grs1-1 (Fig. 7C). The grafted plants with cv. Karami199 leaves and grs1-1 roots showed 2 times higher concentration than the GR/KA plants. These findings suggest that not only the composition but also the concentration of GSLs in the roots is determined by the genotype of the leaf.

It is of interest to alter the composition of metabolites in vegetables to confer new flavors, pest protection, or anticancer activity. Tattersall et al. (2001) reported that introducing the entire cyanogenic glucoside pathway of sorghum (Sorghum bicolor) into Arabidopsis resulted in increased resistance to specific insects. In many pharmacological studies, sulforaphane, an isothiocyanate derived from glucoraphanin (4-methylsulfinylbutyl glucosinolate), has been shown to have health-promoting activities (Juge et al., 2007). To increase the glucoraphanin composition, a MYB28 allele derived from the wild species Brassica villosa was introgressed into broccoli (B. oleracea var italica; Traka et al., 2013). These metabolite-engineering efforts permit the breeding of new vegetables beneficial to human health. In previous studies, using a radish variety whose GSL composition was known to be poor, we found a single mutation leading to a qualitative change in radish, resulting in the accumulation of glucoerucin instead of glucoraphasatin in the radish root (Ishida et al., 2015). This drastic change provides an advantage for dishes containing radish. It is known that an isothiocyanate derived from glucoraphasatin produces a yellow pigment, methanethiol, which imparts color and flavor to the dishes (Takahashi et al., 2015). These phenomena are among the reasons cited for avoiding the cooking of radish. Our results may be of use in metabolite engineering for breeding of high-value vegetables.

MATERIALS AND METHODS

Plant Materials

The radish (Raphanus sativus) mutant lacking glucoraphasatin, grs1-1, was a sibling of NMR154N, described in our previous report (Ishida et al., 2015). The allelic mutant, grs1-2, was an inbred line derived from Tohoku Karami daikon (R. sativus) and was identified by GSL profiling. The wild-type inbred line HAGHN was used for genetic mapping. The commercial lines cv. Taibyosoubutori (Takii seed) and cv. Karami199 (Kaneko Seeds) were used for gene expression analysis and for the grafting experiments.

Extraction and HPLC Analysis of GSLs

Extraction of GSLs from root and leaf was performed after lyophilization and pulverization using a Multi-Beads Shocker following the method described by Ishida et al. (2015). GSLs were desulfurized with sulfatase (Sigma) according to the method of Bjerg and Sørensen (1987), and desulfo-GSLs were subjected to HPLC, as reported in our previous study (Ishida et al., 2012). HPLC was performed on an LC-20A chromatograph (Shimadzu) fitted with a 5C 18-MS-II column (150 mm × 4.6 mm i.d., 5 µm; Nacalai Tesque). The HPLC analysis was performed with a flow rate of 1.5 mL min−1 at a column oven temperature of 30°C, and the absorbance was measured at a wavelength of 229 nm. The mobile phase consisted of ultrapure water (A) and 20% (v/v) acetonitrile (B). The mobile phase program was as follows: 1% (v/v) solvent B for 1 min, followed by a linear elution gradient over the next 20 min to 99% (v/v) solvent B, then 99% (v/v) solvent B for 3 min, which was changed to 1% (v/v) solvent B at 24.1 min, and then 1% (v/v) solvent B for 10 min (total, 35 min). The individual GSL contents were calculated by the ratios of the individual desulfo-GSL peak areas to the peak areas of an internal standard, sinigrin (Sigma), and a response factor (ISO9167-1).

Genetic Mapping and BAC Screening

A population of 5,198 self-pollinated F2 and F3 progeny of grs1-1 and HAGHN was used for fine-mapping of grs1. The leaves of plants showing recombination between the interval markers were used for GSL analysis. The BAC libraries constructed using total DNA of the radish ‘Miyashige’ doubled haploid 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 Mini Kit (Qiagen). The first-strand cDNA was synthesized with 500 ng of RNA using the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa) with random hexamer and oligo(dT) primers in a volume of 20 µL. Quantitative real-time PCR was performed in a total volume of 25 µL, 1 µL of the cDNA, 0.4 µm gene-specific primers, and 12.5 µL of SYBR Premix ExTaq (TaKaRa) on a Thermal Cycler Dice Real-Time System (TaKaRa) according to the manufacturer’s instructions. The radish ACTIN gene was used as an internal control (Zou et al., 2013). All the expression data were based on at least three biological replications.

Full-Length cDNA Cloning and Transformation

Total RNA was extracted from HAGHN with the RNeasy Plant Mini Kit, and RACE was performed with the FirstChoice RLM-RACE Kit (Life Technologies). The gene-specific primer sequences are listed in Supplemental Table S3. The amplified fragments were cloned into a pCR2.1-TOPO vector (Invitrogen) and sequenced with the BigDye Terminator Version 3.1 Cycle Sequencing Kit (Life Technologies). A coding sequence of ORF3 was amplified from total RNA of HAGHN leaves. The cauliflower mosaic virus 35S promoter and a terminator of nopaline synthase were amplified from the pBI121 binary vector. Three amplified fragments were fused into the HindIII/EcoRI-digested pZK3B binary vector (Kuroda et al., 2010) using the In-Fusion HD Cloning Kit (TaKaRa). This construct was transformed into Arabidopsis (Arabidopsis thaliana) accession Ts-1 using Rhizobium radiobacter GV3101::pMP90 by the floral dip method (Clough and Bent, 1998). To synchronize the germination, all the seeds were kept at 4°C for 3 d after sowing. Plants were grown on soil (expanded vermiculite) or on 0.8% agar medium with 0.5× Murashige and Skoog salts under a 16-h/8-h cycle of white light (60–80 µmol m−2 s−1) and dark at 22°C in a growth chamber (FLI-2000; Eyela). The primer sequences used for vector construction are listed in Supplemental Table S3.

LC-MS Analysis of GSLs in Transformed Arabidopsis

The conditions for the detection and identification of desulfo-GSLs by LC-MS were as follows. The samples (15 µL) were injected into 1200 Series HPLC equipment (Agilent) and desulfo-GSLs were separated on a TSKgel super-ODS column (2 × 100 mm, 3-µm particle size, 40°C) using a 0% to 20% acetonitrile gradient in water (46 min) with a flow rate of 0.4 mL min−1. The detection was done online, first with a photodiode array detector at 230 nm (wavelength 190–950 nm) and subsequently with the LTQ Orbitrap XL MS/MS system (Thermo Fisher Scientific) operated in electrospray ionization positive ion mode (m/z = 100–800; spray voltage, 3.5 kV; temperature of the heated capillary, 300°C). The flow rates for nitrogen sheath gas and auxiliary gas were set to 50 and 10 arbitrary units min−1, respectively. Desulfo-glucoraphasatin (m/z = 178, 340, and 362) 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 the predicted 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 the number of estimated amino acid substitutions, a phylogenetic tree was reconstructed by the maximum likelihood method using MEGA version 6.06 (Tamura et al., 2013). The bootstrap values were calculated with 1,000 replications. Arabidopsis ALKBH2 (At2g22260), which was classified in the DOXA5 class, was used as an outgroup.

Protein Assay of GRS1

The coding sequence of GRS1 was amplified from wild-type HAGHN using PCR with the primers mentioned in Supplemental Table S3. The amplified fragments were cloned into XhoI and SalI sites of the pColdI vector (TaKaRa) using the In-Fusion HD Cloning Kit (TaKaRa). The recombinant plasmids were introduced into Escherichia coli BL21 (DE3) pLysS strain.

To produce the recombinant GRS1 protein, the bacteria were cultured at 37°C in 10 mL of Luria-Bertani medium supplemented with ampicillin (50 µg mL−1) and chloramphenicol (30 µg mL−1) until the optical density at 600 nm reached 0.5. The protein synthesis was then induced by the addition of 0.1 mm isopropylthio-β-galactoside and further culturing for 20 h at 15°C. The culture was then centrifuged at 5,000g for 5 min at 4°C. The cell pellets were resuspended in 1 mL of 1× 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 contained 100 mm NaH2PO4, pH 6.8, 10 mm α-ketoglutaric acid, 10 mm ascorbate, 0.25 mm ferrous sulfate, 0.25 mm glucoerucin, and 20 µL of crude protein solution. The assays were performed at 30°C for 30 min. The reactions were initiated by the addition 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 a razor in a horizontal direction. The scions and stocks were joined with a holder (superwith14; Nasunics). The scions of grs1-1 were grafted on the stocks of both grs1-1 and cv. Karami199, and the scions of cv. Karami199 were grafted on the stocks 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 for 10 d and transplanted into 15-cm pots in the greenhouse. For GSL analysis, the leaves and roots were harvested 60 d after grafting.

Chemical Compounds Studied

Chemical compounds studied were as follows: glucoraphasatin (PubChem CID: 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 GenBank database with GenBank/EMBL/DNA Data Bank of Japan accession number LC077856 (GRS1 full-length cDNA, HAGHN).

Supplemental Data

The following supplemental materials are available.

Supplementary Material

Supplemental Data
supp_173_3_1583__index.html (1.2KB, html)

Acknowledgments

We thank Y. Araki, S. Morimoto, Y. Kawamoto, C. Okuyama, Y. Niina, and S. Negoro for technical assistance; Dr. H. Fukuoka (Takii seed) for helpful discussions; Dr. M. Kuroda (NARO) for providing the binary vector; The Arabidopsis accession Ts-1 was provided by RIKEN BioResource Center, which is participating in the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Glossary

GSL

glucosinolate

BAC

bacterial artificial chromosome

LC-MS

liquid chromatography-mass spectroscopy

MS/MS

tandem mass spectrometry

ORFs

open reading frames

Footnotes

1

This work was supported by the Ministry of Agriculture, Forestry, and Fisheries of Japan (Genomics-Based Technology for Agricultural Improvement, grant no. HOR-1006).

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Supplemental Data
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