7 8 9 10 11 Methionine γ-Lyase€¦ · 97 that in ordinary soybean cultivars. 98 Met is a biosynthetic member of the aspartate (Asp) family (Fig. 1) (Galili et 99 al., 2016, Amir

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Short title: Met γ-lyase suppression leads to high S-methyl Met 1 2 Corresponding Author: 3 Kenji Matsui 4 Graduate School of Sciences and Technology for Innovation, Yamaguchi University, 5 Yamaguchi 753-8515, Japan 6 Telephone: +81-83-933-5850 7 Fax: +81-83-933-5820 8 Email: [email protected] 9 10 Suppressed Methionine γ-Lyase Expression Causes Hyperaccumulation of 11 S-Methylmethionine in Soybean Seeds 12 13 Takuya Teshimaa, Naohiro Yamadab, Yuko Yokotac, Takashi Sayamac,f, Kenji Inagakid, 14 Takao Koedukaa, Masayoshi Uefunee, Masao Ishimotoc, Kenji Matsuia,1 15 16 aDivision of Agricultural Sciences, Graduate School of Sciences and Technology for 17 Innovation, Yamaguchi University, Yamaguchi 753-8515, Japan 18 bNagano Vegetable and Ornamental Crops Experiment Station, Shiojiri, Nagano 19 399-6461, Japan 20 cInstitute of Crop Science, National Agriculture and Food Research Organization 21 (NARO), 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8518, Japan 22 dDepartment of Biofunctional Chemistry, Graduate School of Environmental and Life 23 Science, Okayama University, Okayama700-8530, Japan 24 eDepartment of Agrobiological Resources, Faculty of Agriculture, Meijo University, 25 Nagoya, Aichi, 468-8502, Japan 26 27 One-sentence Summary: 28 Suppression of the methionine catabolism in soybean seeds causes hyperaccumulation 29 of S-methylmethionine. 30 31 Author Contributions: 32

Plant Physiology Preview. Published on April 28, 2020, as DOI:10.1104/pp.20.00254

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K.M. conceived this project, designed experiments, and wrote the article. T.T. analyzed 33 metabolites, characterized recombinant enzyme, N.Y. performed crossbreeding and 34 harvested seeds, M.I., T. S., and Y. Y. performed genotyping and positional cloning, K.I. 35 provided enzymes, T.K. mined database and constructed phylogenetic trees, M.U. 36 performed statistical analyses. 37 38 Funding: 39 This work was supported partly by JSPS KAKENHI Grant Number 16H03283 and 40 19H02887, Tojuro Iijima Foundation for Food Science and Technology, and Fuji 41 Foundation for Protein Research (to K. M.). The authors declare that they have no 42 conflict of interest with the contents of this article. 43 44 fPresent address: Western Region Agricultural Research Center, NARO, 6-12-1 45 Nishifukatsu, Fukuyama, Hiroshima 721-8514, Japan 46 47 1Address correspondence to [email protected] 48 The author responsible for the distribution of materials to the findings presented in this 49 article in accordance with the policy described in the Instructions for Authors 50 (www.plantphysiol.org) is: Kenji Matsui ([email protected]). 51 52



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53 Abstract 54 Several soybean (Glycine max L.) germplasms, such as Nishiyamahitashi 98-5 (NH), 55 have an intense seaweed-like flavor after cooking because of their high seed 56 S-methylmethionine (SMM) content. In this study, we compared the amounts of amino 57 acids in the phloem sap, leaves, pods, and seeds between NH and the common soybean 58 cultivar Fukuyutaka (FY). This revealed a comparably higher SMM content alongside a 59 higher free methionine (Met) content in NH seeds, suggesting that the 60 SMM-hyperaccumulation phenotype of NH soybean was related to Met metabolism in 61 seeds. To investigate the molecular mechanism behind SMM hyperaccumulation, we 62 examined the phenotype-associated gene locus in NH plants. Analyses of the 63 quantitative trait loci in segregated offspring of the cross between NH and the common 64 soybean cultivar Williams82 indicated that one locus on chromosome 10 explains 65 71.4% of SMM hyperaccumulation. Subsequent fine-mapping revealed that a 66 transposon insertion into the intron of a gene, Glyma.10g172700, is associated with the 67 SMM-hyperaccumulation phenotype. The Glyma.10g172700-encoded recombinant 68 protein showed Met-γ-lyase (MGL) activity in vitro, and the transposon-insertion 69 mutation in NH efficiently suppressed Glyma.10g172700 expression in developing 70 seeds. Exogenous administration of Met to sections of developing soybean seeds 71 resulted in transient increases in Met levels, followed by continuous increases in SMM 72 concentrations, which was likely caused by Met methyltransferase activity in the seeds. 73 Accordingly, we propose that the SMM-hyperaccumulation phenotype is caused by 74 suppressed MGL expression in developing soybean seeds resulting in transient 75 accumulation of Met, which is converted into SMM to avoid the harmful effects caused 76 by excess free Met. 77 78 Introduction 79 In the 2017/2018 market year, 346.2 million tons of soybean (Glycine max L. Merr.) 80 were produced (FAOSTAT; http://www.fao.org/faostat/en/#home). This large amount of 81 soybean production was driven by the high oil and protein contents of the plant. 82 Soybean meal is widely used as animal feed and has high protein contents and a 83 well-balanced amino acid profile. However, its nutritional value for monogastric 84 animals could be improved by increasing its cysteine (Cys) and methionine (Met) 85



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contents. Accordingly, the regulatory mechanisms relating to Met and Cys biosynthesis 86 in soybean have been investigated extensively (Hesse & Hoefgen, 2003, Galili et al., 87 2016, Krishnan & Jez, 2018, Amir et al., 2019). In the central region of Japan, specific 88 soybean seeds have been cultivated for their seaweed odor, which is strongly related to 89 dimethyl sulfide. Dimethyl sulfide is formed spontaneously from S-methylmethionine 90 (SMM) during heating of the seeds for cooking (Morisaki et al., 2014). In a 91 representative cultivar, Nishiyamahitashi 98-5 (NH), the SMM level in the seeds is 92 more than 100-fold higher than in common soybean cultivars, such as Fukuyutaka (FY) 93 (Morisaki et al., 2014). Because SMM is a direct product of Met metabolism by Met 94 methyltransferase (MMT), it is assumed that the cultivars with higher SMM content are 95 likely to metabolize Met and its derivatives via mechanisms that are distinctive from 96 that in ordinary soybean cultivars. 97 Met is a biosynthetic member of the aspartate (Asp) family (Fig. 1) (Galili et 98 al., 2016, Amir et al., 2019). Cystathionine γ-synthase (CGS) performs the crucial 99 regulatory step in Met biosynthesis and determines the rate of Met production from 100 O-phosphohomoserine (Galili et al., 2016). Free Met is used to form proteins, but a 101 portion of Met is converted to SAM by a SAM synthetase. Another portion of free Met 102 accepts a methyl group from SAM through MMT and is converted into SMM. 103 Homocysteine methyltransferase (HMT) converts SMM back to Met. MMT and HMT 104 constitute the SMM cycle, which seems to operate throughout plant tissues, including 105 reproductive tissues of various plant species (Ranocha et al., 2001). In several flowering 106 plants, SMM is produced in the leaves by MMT and is transported through the phloem 107 toward the reproductive organs, where it is reconverted to Met by HMT (Bourgis et al., 108 1999, Cohen et al., 2017a). With developing seeds of Medicago truncatula, SMM is 109 converted back to Met via HMT in seed coats, and Met released into the seed apoplast is 110 taken up by seeds (Gallardo et al., 2007). Both the in situ formation of Met through Asp 111 family enzymes and the biosynthesis of SMM in the leaves following phloem transport 112 likely regulate Met contents simultaneously in the seeds (Cohen et al., 2017a, Amir et 113 al., 2019). Met catabolism also controls Met levels in plant tissues. Met γ-lyase (MGL) 114 is a pyridoxal phosphate (PLP)-dependent enzyme that metabolizes Met into 115 2-ketobutyric acid, methanethiol, and ammonia (Sato & Nozaki, 2009). 2-Ketobutyric 116 acid can be metabolized to form isoleucine (Ile) even though this pathway is auxiliary to 117 the major Ile biosynthetic pathway through threonine (Thr) deaminase (Joshi & Jander, 118



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2009). 119 Based on accumulating studies of Met regulation in plant tissues, numerous 120 attempts to improve Met levels in crops have been made through genetic engineering 121 particularly of the Asp family pathway (Hacham et al., 2008, Hanafy et al., 2013, Song 122 et al., 2013, Cohen et al., 2014, Kumar & Jander, 2017, Amir et al., 2019). Acceleration 123 of SMM transport from non-seed tissues to seeds was also attempted to increase Met 124 levels in seeds (Lee et al., 2008, Cohen et al., 2017a). As such, attempts to increase seed 125 Met levels are becoming more successful but are sometimes disturbed by abnormal 126 phenotypes of the plants (Krishnan & Jez, 2018, Amir et al., 2019). Severe growth 127 retardation was observed in potato (Solanum tuberosum) plants overexpressing the 128 feedback-insensitive CGS to form more Met and β-zein to store Met (Dancs et al., 129 2008). Tobacco (Nicotiana tabacum) plants overexpressing CGS and with elevated free 130 Met levels also had increased sensitivity to oxidative stress (Hacham et al., 2017). In 131 addition, Arabidopsis seeds overexpressing a mutant CGS accumulated Met to 2.5-fold 132 higher levels, and these conditions were associated with increased expression of 133 stress-related transcripts (Cohen et al., 2014, Cohen & Amir, 2017). These studies 134 suggest that Met levels are tightly controlled in plant tissues and that excessive free Met 135 is deleterious to plant health. Yet, the mechanisms by which Met levels are regulated in 136 some tissues remain poorly understood. 137

The soybean SMM-hyperaccumulation phenotype, like that of NH, was 138 assumed to be attributable to a genotype related to Met metabolism in seeds. We 139 investigated the genotype of SMM-hyperaccumulating soybean plants and identified the 140 gene that is responsible for SMM accumulation in soybean. Through analyses of gene 141 function and NH phenotypes, we propose a mechanism underlying SMM 142 hyperaccumulation. 143 144 145



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RESULTS 146 147 S-Methylmethionine and Free Methionine Levels 148 149 The quantities of SMM and free Met were determined in the leaves, pods, and seeds of 150 two soybean cultivars, Fukuyutaka (FY) and Nishiyamahitashi 98-5 (NH) at the 151 flowering stage, immature green seed stage, mature green seed stage, and in dry seeds 152 (Fig. 2). The seed coats were removed from the other parts of seeds (i.e., cotyledons, 153 plumules, and radicles) before analyses to avoid mixing tissues of maternal genotype 154 (i.e., seed coats) and those of offspring genotypes (cotyledons, plumules, and radicles). 155 The contents of SMM and free Met were low in the leaves, and no significant 156 differences were identified between FY and NH. The SMM level in the pods seemed to 157 be a little higher than that found in the leaves, but a statistically significant difference in 158 its level between FY and NH was hardly detected. The free Met level in the NH pods at 159 the immature green stage was significantly lower than that of FY, but the level in the FY 160 pods lowered at the mature green stage to the level found in the NH pods. The levels of 161 SMM in the NH seeds were 8.0- and 15.6-fold higher at the immature and mature green 162 stage, respectively, than those found in the FY seeds. A substantial amount of SMM was 163 detected in dry NH seeds, whereas it was under the detection limit in the dry FY seeds. 164 The levels of free Met in the NH seeds were significantly higher at the mature green 165 stage and in dry seeds than those found in the FY seeds. 166 The SMM level in the phloem exudate collected through a cut petiole of NH 167 at the seed-developing stage was significantly higher than that found for FY, whereas 168 the level of free Met was lower than that found for FY (Table 1). The levels of SMM 169 and free Met in phloem exudate collected using the same procedure through 170 Arabidopsis petioles at the seed-filling stage were more than 93-fold higher than those 171 found for NH and FY. 172

The free amino acid contents of mature seeds were mostly similar between FY 173 and NH, yet the histidine (His), Met, phenylalanine (Phe), Thr, and homoserine contents 174 were significantly higher in NH than FY seeds (Fig. 3). Homocysteine was under the 175 detection level (4.2 µg g-1) in both NH and FY. The total protein contents, the protein 176 profiles examined using Coomassie Brilliant Blue staining after SDS-PAGE 177 (Supplemental Fig. S1), and total amino acid contents showed no significant differences 178



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between the FY and NH seeds (Supplemental Table S1). 179 180 Positional Cloning of the Gene Responsible for Hyperaccumulation of 181 S-Methylmethionine 182 183 The contents of SMM and free Met were determined with four F1 seeds after reciprocal 184 crossing of the FY and NH cultivars. The hyperaccumulation of SMM was only evident 185 in self-pollinated NH seeds, and the maternal and paternal genotypes did not play a 186 significant role in the accumulation of SMM in seeds (Fig. 4). Moreover, F2 seeds of the 187 FY × NH cross were segregated into high SMM/low SMM at a ratio of 3/17 with a 188 consistent segregation ratio of 1:3 (Chi-squared test, P = 0.30; Supplemental Fig. S2A). 189 These data indicate that hyperaccumulation of SMM is essentially regulated by a single 190 recessive allele. In order to identify the gene responsible for hyperaccumulation of 191 SMM, we crossbred NH to Williams 82 (WI) cultivar. WI was used because the SMM 192 contents in WI seeds were as low as those in FY seeds (see below) and also because the 193 reference genome sequence was produced with WI (Schmutz et al., 2010). A total of 194 156 F5 recombinant inbred lines (RILs) were generated from the cross between NH and 195 WI, and SMM levels in their mature seeds were determined (Supplemental Fig. S2B). 196 Quantitative trait loci (QTL) analyses with molecular markers indicated that an allele 197 near a simple sequence repeat marker (Satt477) on chromosome 10 explained 71.4% of 198 the phenotypic variation (Fig. 5A). No other QTL with a significant influence on the 199 hyperaccumulation of SMM was detected. Fine-mapping of the allele responsible for 200 the hyperaccumulation of SMM in F6 and F7 residual heterozygous lines narrowed 201 down this region to a 12-kb sequence on chromosome 10 (Fig. 5B). Furthermore, 202 examination of the Phytozome soybean genome sequence database 203 (https://phytozome.jgi.doe.gov/pz/portal.html) revealed only one open reading frame 204 (ORF) of Glyma.10g172700 in this region. Glyma.10g172700 comprises two exons 205 flanking a single intron. Finally, sequence analyses of Glyma.10g172700 in NH 206 (GenBank acc. no. MK887190) indicated that a copia-type retrotransposon (AB370254, 207 Liu et al. 2008) was inserted into the intron. 208 209 Hyperaccumulation of SMM Correlates Well with Transposon Insertion in the 210 Glyma.10g172700 Gene 211



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212 We collected local soybean cultivars in Nagano Prefecture, Japan (Supplemental Fig. 213 S3). These were previously shown to have differing SMM levels (Morisaki et al. 2014). 214 We extracted genomic DNA and then amplified the Glyma.10g172700 gene using 215 primers for 5′- and 3′-termini of its deduced ORF. In normal soybean cultivars, such as 216 FY and WI, the resulting DNA fragment was 2885 bp, as expected from genome 217 sequences in the Phytozome. The Glyma.10g172700 gene length was the same in five of 218 the ten local cultivars as that detected in normal cultivars, but was longer in the other 219 five cultivars and was similar in length to that amplified from the NH cultivar (ca. 9.0 220 kb; Fig. 6A). Therefore, it was expected that these latter five soybean cultivars carry an 221 inserted transposon in the Glyma.10g172700 gene of the same size as the inserted 222 transposon in NH. Determinations of SMM levels in seeds of these cultivars showed 223 higher SMM levels in cultivars harboring the Glyma.10g172700 gene with the 224 transposon insertion than in those without the transposon-insertion (Fig. 6B). Free Met 225 levels were higher in some seeds having the transposon insertion in Glyma.10g172700 226 gene than those without the insertion, but the correlation was not always evident. 227 Reverse transcription quantitative PCR (RT-qPCR) analyses on RNA extracted from 228 maturing seeds showed that the lower levels of transcript derived from 229 Glyma.10g172700 were detected with cultivars that had the transposon insertion in 230 Glyma.10g172700 (Fig. 6C). The product sizes obtained with RT-PCR with NH and FY 231 seeds with excess amplification cycles using primers for 5′- and 3′-termini of GmMGL1 232 ORF were same (Supplemental Fig. S4), and no sign of alternative splicing was 233 detected. 234 235 Glyma.10g172700 Encodes a Functional Methionine γ-Lyase 236 237 The ORF of Glyma.10g172700 encodes a protein of 48,069 Da, yet the deduced protein 238 sequence had no predictable targeting signal in TargetP analyses 239 (http://www.cbs.dtu.dk/services/TargetP/), suggesting a cytosolic location of the protein. 240 The Glyma.10g172700 gene was tentatively assigned as a gene encoding an MGL, and 241 the deduced protein sequence had 77.2% and 78.6% identities with Arabidopsis MGL 242 (Q9SGU9, AEE34271.1) and melon (Cucumis melo) MGL (M1NFB7, 243 NP_001315378.1), respectively (Rébeillé et al. 2006, Gonda et al. 2013) (Supplemental 244



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Fig. S5). Moreover, the protein sequence has a motif (Ser237-Xaa-Xaa-Lys240) that is 245 conserved in pyridoxal-5’-phosphate (PLP) enzymes of the γ subfamily and associates 246 with the cofactor PLP (Martel et al. 1987, Sato & Nozaki, 2009) (Supplemental Fig. S5). 247 Tyr142, Asp216, and Arg410 residues are also involved in substrate binding and 248 catalysis at appropriate positions (Goyer et al. 2007), and Gly144 is conserved as in 249 Arabidopsis and melon MGLs that retain restricted substrate specificity for L-Met 250 (Gonda et al. 2013) (Supplemental Fig. S5). 251 Glyma.10g172700 cDNA was cloned using RNA that was extracted from 252 developing WI seeds. We expressed the recombinant protein as an N-terminal 253 His-tagged protein and purified it using Ni2+-affinity chromatography (Fig. 7A). 254 Subsequently, L-Met reacted with the recombinant protein in the presence of PLP, and 255 the products were converted into their 3-methyl-2-benzothiazolinone hydrazone 256 derivatives. This derivatization resulted in increased absorption at 320 nm (Fig. 7B), 257 suggesting the formation of an aliphatic carbonyl compound (Esaki & Soda, 1987, 258 Inoue et al., 1995). To confirm its structure, the reaction product that was extracted 259 using ethyl acetate was reacted with N,O-bis(trimethylsilyl)trifluoroacetamide and was 260 then analyzed using GC-MS. A peak at the retention time of 9.5 min was assigned as 261 trimethylsilylated 2-ketobutyric acid by comparing its MS profile and retention time 262 with that prepared from a standard compound (Fig. 7C and 7D). Accordingly, we 263 concluded that Glyma.10g172700 encodes MGL that catalyzes γ-elimination of L-Met. 264 We denoted the gene GmMGL1. This reaction had optimal activity at pH 7.0 and 265 followed Michaelis–Menten kinetics, with Km and Vmax values of 7.72 mM and 0.55 266 µmol mg−1 min−1, respectively (Supplemental Fig. S6). 267 268 Comparison of Methionine Metabolism Genes in Fukuyutaka and 269 Nishiyamahitashi 98-5 270 271 BLAST searches for GmMGL1 indicated that the soybean genome encodes the 272 MGL-like genes Glyma.02g087900 and Glyma.13g001200 (hereafter referred to as 273 GmMGL2 and GmMGL3, respectively). These genes encode proteins with the amino 274 acid signatures that are conserved among the MGLs described above (Supplemental Fig. 275 S5). Among the three GmMGLs, GmMGL2 and -3 showed higher sequence similarity 276 than the other combinations. The phylogenetic analyses with MGL and MGL-like 277



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sequences found in several plant species indicated that GmMGL1 is located in a clade 278 different from the one GmMGL2 and GmMGL3 belong to (Supplemental Fig. S7). 279 The RT-qPCR analyses of the FY seeds showed that GmMGL1 mRNA 280 expression was enhanced at the early stage of seed maturation (from stages one to two) 281 and remained constant thereafter until the matured green stage (stage five; Fig. 8A). 282 However, GmMGL1 expression was considerably lower in NH seeds than in FY seeds 283 throughout seed development and differed little between developmental stages. 284 GmMGL2 and GmMGL3 expression levels were transiently induced during stage three, 285 but only in NH seeds, and they were not significantly different between FY and NH 286 cultivars at the other stages. The MGL activity in crude protein extracts prepared from 287 developing seeds (at stage four) of NH (5.86 ± 0.81 nmol h-1 g-1) was significantly 288 lower than that detected in FY seeds (12.1 ± 2.28 nmol h-1 g-1) (P < 0.05, Student’s t-test, 289 n=4). GmMGL1 expression was significantly lower in the leaves, stems, and roots of 290 NH plants than in the leaves, stems, and roots of FY plantlets at the leaf-expansion stage 291 before flowering (Fig. 8B). Among these, the transcript levels of GmMGL2 and 292 GmMGL3 were highest in the leaves and did not differ significantly between the 293 soybean cultivars. 294 Because the genes of Met metabolism are regulated coordinately (Liao et al. 295 2012), we examined the effects of GmMGL1 suppression on the expression of 296 cystathionine γ-synthase (CGS), which catalyzes a key regulatory step of the Met 297 biosynthetic pathway (Hesse & Hoefgen, 2003), and of Met methyltransferase (MMT) 298 and homocysteine methyltransferase (HMT), which are directly involved in the 299 formation and decomposition of SMM (Fig. 1; Cohen et al., 2017a). In SoyBase 300 BLAST searches using AtCGS (At3g01120), AtMMT (At5g49810), and AtHMT1 301 (At3g25900) as queries, two CGS homologs (Glyma.18g261600 and Glyma.09g235400; 302 referred to as GmCGS1 and GmCGS2, respectively), two MMT homologs 303 (Glyma.12g163700 and Glyma.16g000200; GmMMT1 and GmMMT2, respectively), 304 and three HMT homologs (Glyma.08g261200, Glyma.19g158800, and 305 Glyma.20g148900; GmHMT1, GmHMT2, and GmHMT3, respectively) were identified. 306 RT-qPCR analyses of mRNA expression from soybean seeds at different developing 307 stages revealed no significant differences between FY and NH, except for GmCGS1/2, 308 GmHMT2, and GmHMT3 at stage one (Supplemental Fig. S7). 309 310



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Administration of Methionine Causes Accumulation of S-Methylmethionine in 311 Developing Seeds 312 313 Suppression of GmMGL1 expression in developing soybean seeds might lead to the 314 accumulation of Met, which would otherwise be catabolized to ammonia, methanethiol, 315 and 2-ketobutyric acid. One of the alternative fates of free Met is the formation of SMM 316 via the activity of MMT (Fig. 1), which is likely to occur in developing soybean seeds 317 because of the substantial expression levels of GmMMT1 and -2 (Supplemental Fig. S8). 318 To examine whether MMTs are active in developing soybean seeds, we conducted a 319 Met-feeding experiment. We fed free Met solution onto slices of immature green 320 soybean seeds of the FY and NH cultivars and determined SMM and Met contents using 321 LC-MS/MS (Fig. 9). Inclusion of 1 or 5 mM Met in the solution covering the cut 322 surfaces of the FY seeds yielded incremental increases in the SMM contents, and after 323 24 h of treatment, the SMM levels increased up to 37.9 and 135 µg g-1 for the 1 and 5 324 mM Met solutions, respectively. The SMM levels of the NH seeds also showed similar 325 incremental increases, but in a more prominent manner, and after 24 h, the SMM levels 326 increased up to 214 and 316 µg g-1 for the 1 and 5 mM Met solutions, respectively. The 327 SMM level in the NH seeds treated only with water also significantly increased to 80.0 328 µg g-1 after 24 h. No significant difference in the Met levels was observed for FY and 329 NH seeds treated with 1 mM Met in comparison with the levels in seeds treated with 330 water except those after 24 h with NH seeds; however, following feeding with 5 mM 331 Met solution, the Met levels increased significantly in both FY and NH, with more 332 prominent increases in NH. The highest Met level for FY was 9.71 µg g-1 at 8 h and for 333 NH was 27.2 µg g-1 at 24 h. 334 To confirm substrate–product relationships, we performed feeding 335 experiments using 13C-Met (C5, 99 atom %) with NH seeds. Subsequent LC-MS/MS 336 analyses of SMM in the extract confirmed that it was predominantly formed from 337 13C-Met, as indicated by m/z 169.1 and 106.1 that were generated from 13C5-labeled 338 SMM (Fig. 9B). 339 340 341 342



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DISCUSSION 343 344 Using a molecular genetic approach to locate the allele responsible for 345 hyperaccumulation of SMM, we found that a transposon insertion into the intron of 346 GmMGL1 is strongly associated with SMM hyperaccumulation in soybean seeds. 347 Expression of the GmMGL1 gene and, accordingly, MGL activity in seeds were 348 suppressed due to the transposon insertion. Under these conditions, Met catabolism 349 would be low in seeds, leading to Met accumulation. Because excess Met levels have 350 been associated with various adverse effects in plant tissues, we hypothesized that 351 surplus Met in soybean seeds with MGL deficiencies was converted to the 352 better-tolerated compound SMM by MMT activity (Fig. 10). In line with this hypothesis, 353 Met-feeding experiments showed that surplus Met was efficiently converted into SMM 354 in green mature soybean seeds and that the conversion was more prominent with 355 MGL-deficiency. 356 357 The Transposon Insertion Suppresses Expression of GmMGL1 358 359

The insertion of a transposon into the intron of GmMGL1 strongly suppressed its 360 mRNA expression, whereas processing of the corresponding precursor mRNA through 361 splicing at the inherent positions was little affected by the intronic insertion. Intronic 362 insertion of transposons generally have minimal impacts on gene expression levels or 363 splicing events (Hirsch & Springer, 2017). For example, insertion of the retrotransposon 364 Ty1-copia, which is approximately 5000 bp in length, had little impact on transcription 365 with flax (Linum usitatissimum) (Galindo-Gonzákez et al. 2016). Hence, the present 366 marked repressive effects of transposon insertion into the intron of GmMGL1 are unique. 367 Alternatively, in a previous study of soybeans, transposons in or near a gene were 368 related to increased CHG/CHH methylation, and consequently, lower expression levels 369 (Kim et al., 2015). Hence, epigenetic regulatory mechanisms likely play roles in the 370 present repression of GmMGL1. As such, analyses of DNA methylation should be one 371 of the next priority research areas to reveal more details about the mechanisms of this 372 type of gene suppression. 373 374 Suppression of GmMGL1 Accounts for SMM Hyperaccumulation 375



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376 In plant tissues, Met levels are tightly regulated through biosynthesis and catabolism 377

(Fig. 1). Higher free Met contents (in addition to SMM contents) in NH seeds than in 378 FY seeds prompted us to assume that GmMGL1 participated in controlling free Met 379 levels in seeds. If this is the case, in the absence of substantial MGL activity as found 380 with NH, free Met levels should increase, and surplus Met could be converted into 381 SMM by the MMT activity in seeds. This scenario showed no fundamental 382 inconsistency in the results obtained in this study about the Met metabolism of NH. The 383 function of MGL to adjust free Met levels has been demonstrated with Arabidopsis, in 384 which knock-out of the AtMGL gene increased free Met contents in leaves, flowers, and 385 seeds (Goyer et al., 2007, Joshi & Jander, 2009). Notably, the Arabidopsis knock-out 386 mutant contained 4.5-fold higher SMM contents in leaves than its parental wild type; 387 therefore, it is presumed that conversion of surplus Met to SMM is common among 388 plants. In support of this hypothesis, SMM accumulation has been reported in multiple 389 transgenic plants with high free Met levels (Kim et al., 2002, Hacham et al., 2008, 390 Hacham et al., 2017). Taken together, it is suggested that GmMGL1 was involved in 391 controlling free Met levels in developing soybean seeds. SMM-hyperaccumulation is 392 likely to be a consequence of suppressed GmMGL1, and a subsequent “fail-safe” 393 system employing MMT activity to avoid the adverse effect of excess Met. 394

One of the MGL products, 2-ketobutyric acid, is partly converted to Ile in 395 Arabidopsis, especially under drought stress (Rébeillé et al., 2006, Joshi & Jander, 396 2009). However, we found no significant difference in either free or total Ile content 397 between NH and FY seeds, suggesting that GmMGL1 accounted little for Ile formation 398 in developing seeds. On the contrary, dry NH seeds had higher free Thr, Phe, His, and 399 homoserine levels in addition to increased free Met and SMM levels. Therefore, the 400 MGL deficiency is likely to cause pleiotropic effects on the metabolism of other amino 401 acids. Accumulation of free amino acids was often observed in several transgenic plants 402 generated to enhance Met levels (Hanafy et al., 2013, Cohen et al., 2014, Hacham et al., 403 2017, Huang et al., 2014). Accordingly, it is suggested that the adverse effect of surplus 404 Met in NH induced a stress response that led to higher Thr, Phe, His, and homoserine 405 levels. 406 407 Limited Significance of Phloem Transport of SMM for SMM Hyperaccumulation 408



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409 It has been reported for several plant species, including Arabidopsis and wheat 410

(Triticum aestivum), that SMM formed in vegetative tissues is transported to seeds 411 through the phloem (Bourgis et al., 1999, Lee et al., 2008, Frank et al., 2015, Cohen et 412 al., 2017b). Therefore, it was assumed that the phloem transportation of SMM formed in 413 vegetative tissues to seeds could also be accountable for hyperaccumulation of SMM in 414 NH seeds. The level of SMM in the phloem exudate collected from leaves of NH was 415 higher than that found for FY plants. Therefore, transportation of SMM through the 416 phloem toward the seeds is likely to be at least partly accountable for the 417 hyperaccumulation of SMM in NH seeds. The SMM levels in the pods of both NH and 418 FY showed a tendency to decrease during maturation; thus, the transportation of SMM 419 from the pods to the seeds should also be taken into consideration. However, it was 420 remarkable that the SMM levels found in the phloem exudate of NH plants were 421 291-fold lower than that in Arabidopsis phloem exudate. Concordant with the fact that 422 amino acid levels in soybean phloem exudate were 8-fold lower than those in 423 Arabidopsis and wheat (Bourgis et al., 1999), our observation of low levels of SMM in 424 soybean phloem exudate prompted us to consider that the contribution of phloem 425 transport of SMM toward seeds for SMM hyperaccumulation is not negligible, but is 426 limited. Furthermore, the results of the reciprocal crossing of NH and FY indicated that 427 maternal as well as paternal genotypes did not play a substantial role in determining the 428 seed phenotype of SMM hyperaccumulation, which was caused only when the genotype 429 of the seeds was homozygous for mgl1, and thus, the involvement of vegetative organs 430 in SMM hyperaccumulation in the seeds of NH is likely limited. 431

The extensively lower levels of SMM and Met in the soybean phloem, compared to 432 levels in the Arabidopsis phloem, are noteworthy because the levels of free and total 433 amino acids including Met are more than 10-fold higher in soybean seeds than in 434 Arabidopsis seeds (Cohen et al., 2017c). Source-sink transport of amino acids from 435 vegetative organs to seeds, and in situ synthesis of amino acids in seeds, might be 436 accountable in different ways for accumulation of amino acids in seeds in these two 437 plant species. 438 439 Exogenous Methionine is Converted into S-Methylmethionine 440 441



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The addition of Met at 1 mM onto developing seeds of FY had only a slight effect 442 on Met or SMM concentrations, probably because Met, supplied exogenously, was 443 appropriately catabolized in part by intrinsic GmMGL1 activity in FY seeds. This 444 Met-catabolizing system was, however, overwhelmed by treatments with 5 mM Met 445 solution, as indicated by transient increases in free Met in developing FY seeds, 446 followed by a significant increase in the amount of SMM. The accumulation of Met and 447 SMM seemed to be further emphasized for NH. This result indicated that both NH and 448 FY seeds exhibited enough MMT to convert Met supplied exogenously into SMM. The 449 exaggerated responses of accumulation of Met and SMM in NH seeds could be 450 explained by the lower activity of GmMGL1 in the seeds, and the surplus Met left 451 behind in the seed tissues being converted into SMM by MMT. SMM has been 452 considered to be a tentative storage form of Met to avoid excessive Met concentrations 453 (Mudd & Datko, 1990). In agreement, the SMM concentrations in soybean seeds treated 454 with 5 mM Met solution were much higher than the Met concentrations, suggesting that 455 SMM is a safer storage form of Met. In summary, SMM-hyperaccumulation was caused 456 exclusively by suppression of GmMGL1 that regulates free Met levels in developing 457 soybean seeds. MMT activity in developing soybean seeds should be sufficient to 458 convert surplus Met into SMM, irrespective of MGL activity (Fig. 10). 459

The present data indicate that the genetic suppression of MGL in soybean seeds 460 affects Met metabolism, favors hyperaccumulation of SMM, and provides further 461 insights into the regulatory mechanisms of Met metabolism. This knowledge should be 462 taken into consideration when attempting to modify Met metabolism in soybean seeds. 463 464 465 466 MATERIALS AND METHODS 467 468 Plant Materials. Seeds of the soybean cultivars NH, FY, and WI were grown and 469 harvested during 2016 in an experimental field at the Nagano Vegetable and Ornamental 470 Crops Experiment Station, Shiojiri City (E 137°57', N 36°06'; annual mean temperature, 471 11°C). For the quantification of SMM levels in leaves, pods, and seeds, NH and FY 472 plants were grown and harvested in 2019 in an experimental field at the Yoshida campus 473 of Yamaguchi University, Yamaguchi City (E 131°47', N 34°15'; annual mean 474



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temperature, 15°C). To prepare samples at a similar developmental stage, each organ 475 was collected at slightly different dates because FY showed a little early growth 476 phenotype when compared with NH. For collecting seeds for the RT-qPCR analyses and 477 Met-feeding experiments, plants were grown and harvested in 2017 and 2019 in an 478 experimental field at the Yoshida campus of Yamaguchi University. For extraction of 479 RNA from leaves, stems, and roots, plantlets (NH and FY) were germinated with 480 vermiculite and were transplanted to the hydroponic culture system (Kuroda & Ikenaga, 481 2015) with a 12-h light (at 27°C)/12-h dark (22°C) cycle. NH and FY plants were 482 cultured under these conditions for 40 and 34 days, respectively. Seeds of the cultivars 483 Shinano-Kurakake, Nishiyamahitashi 94-5, Sinanomachi Arasebara Kurakake, 484 Togakushi Morozawa Ganimame, Usuda Zairai, Shinano Midori, Nishiyamahitashi 94-1, 485 Tousanhitashi 94-1, Ogawa Zairai 5, and Chino Zairai 4 were harvested in 2012 and 486 2015 from the experimental field at the Nagano Vegetable and Ornamental Crops 487 Experiment Station, or in 2019 from the experimental field at the Yoshida campus of 488 Yamaguchi University. 489 490 Determination of SMM contents 491 Seed coats were carefully removed and soybean seeds containing hypocotyls were then 492 powdered using a multi-beads shocker (PM2000, Yasui Kikai, Osaka, Japan) equipped 493 with stainless metal cones (MC-0316S, Yasui Kikai) and operated at 2500 rpm for two 494 30-s periods with a 10-s interval. Powder samples of 20 mg were then mixed with 1 mL 495 of distilled water containing 50 µg mL−1 L-methionine-S-methyl d6 sulfonium chloride 496 (d6-SMM; Toronto Research Chemicals Inc. Toronto, Ontario, Canada) and were then 497 placed in a water bath sonicator (US-2, SND Co. Ltd., Suwa City, Nagano, Japan) for 498 10 min. The resulting suspensions were centrifuged at 15,000 rpm (20,000 × g) for 10 499 min at 4°C. Subsequently, 100-µL aliquots were added to Strata C18-E (100 mg mL−1; 500 Phenomenex Inc. Torrance, CA, USA), and SMM was eluted twice with 0.5-mL 501 aliquots of distilled water. Elutes were cleared using an Ekicrodisc 3 (0.45 µm, 3 mm, 502 Pall Co., Tokyo, Japan). 503 Liquid chromatography with tandem mass spectrometry (LC-MS/MS) 504 analyses were performed using an AB Sciex (Framingham, MA, USA) 3200 Q-TRAP 505 LC-MS/MS system equipped with a Prominence UFLC (Shimadzu, Kyoto, Japan) in 506 multiple reaction monitoring (MRM) mode with positive electrospray ionization (ESI). 507



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Chromatography was conducted using a Discovery HS F5 column (15 cm × 2.1 mm, 3 508 µm; Supelco, Bellefonte, PA, USA), and HPLC and MS analyses were performed using 509 previously described conditions (Morisaki et al., 2014). To quantify 13C-labeled SMM 510 formation from 13C-Met (Cambridge Isotope Laboratories, Tewksbury, MA, USA), MS 511 analyses were performed in the enhanced production mode with m/z 169.2 (for labeled 512 SMM) or 164.2 (for non-labeled SMM) using positive electron spray ionization (ESI) 513 with a capillary voltage of 4500 V, an arbitrary source temperature, a curtain gas of 10 514 (arbitrary units), ion source gases 1 and 2 of 16 and 0 (arbitrary units), respectively, a 515 declustering potential of 26 V, and an entrance potential of 2.5 V. The level of free Met 516 was also analyzed using the same LC-MS/MS condition but with different MRM 517 transitions. The detailed parameters for LC-MS/MS analysis are shown in Supplemental 518 Table S2. 519 To collect phloem exudate, fully expanded leaves of NH and FY plants at 520 their seed-filling stage were detached with the base of the petiole under a solution of 20 521 mM EDTA (pH 7.0) and immersed into 0.2 mL of the same solution in 0.5-mL 522 microtubes, and placed in humid chambers in the dark at 25°C (Urquhart & Joy, 1981). 523 After 5 h, the EDTA solution was collected and used for the LC-MS/MS analyses as 524 described above to estimate the concentration of SMM and free Met. As a comparison, 525 fully expanded rosette leaves of Arabidopsis (ecotype Ws-0) were used at its 526 seed-filling stage. 527 528 Determinations of amino acid and protein contents 529 Soluble amino acids were extracted from dry seed flour (20 mg) as described by Hanafy 530 et al. (2013). Flour samples were suspended in 240 µL of 3% (w/v) sulfosalicylic acid 531 and were suspended with vigorous shaking for 30 min. After centrifugation at 12,000 × 532 g for 10 min at 25°C, precipitates were extracted two more times as described above. 533 Combined supernatants were then filtered and analyzed using LC-MS/MS with an ESI 534 interface (Tomita et al., 2016) as detailed above. An Intrada amino acid column (100 × 3 535 mm i.d., 3 µm; Imtakt, Kyoto, Japan) was used with a column temperature of 40°C. 536 Mobile phases were applied at 0.4 mL min − 1 and comprised solvents A 537 (acetonitrile/formic acid at 100/0.3, v/v) and B (0.1 M acetonitrile/ammonium formate 538 at 20/80, v/v) at 15% B for 10 min, followed by a linear increase from 15% B to 60% B 539 over 15 min, then from 60% B to 100% B over 5 min, and then 100% B for 10 min. The 540



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injection volume was 4 µL. The MS system was operated in MRM mode using positive 541 ESI with a capillary voltage of 3000 V, a source temperature at 550°C, a curtain gas of 542 35 (arbitrary units), ion source gases 1 and 2 of 80 and 60 (arbitrary units), respectively, 543 a declustering potential of 16 V, and an entrance potential of 5 V. MRM transitions of 544 the precursor to product ions used for the quantification and collision energy are 545 summarized in Supplementary Table S2. Quantification was done using calibration 546 curves constructed using amino acid mixture standard solution (Wako Pure Chemicals, 547 Osaka, Japan) supplemented with homoserine and homocysteine (Wako Pure 548 Chemicals). 549

Proteins were extracted from 20-mg samples of soybean flour using 500-µL 550 aliquots of 10 mM Tris HCl (pH 8.0) containing 2.5% (w/v) sodium dodecyl sulfate and 551 10 mM 2-mercaptoethanol. Proteins were extracted for 10 min with vigorous vortexing 552 at the highest speed using a Micro Tube Mixer (MT-360, Tomy Seiko, Tokyo, Japan) 553 and subsequent treatment with a water bath sonicator (120 W, 38 kHz, US-2, SND Co. 554 Nagano, Japan) for 10 min at 25°C. A clear protein solution was obtained after 555 centrifugation at 20,000 × g for 20 min at 4°C. Protein contents were determined using 556 Protein Assay (Bio-Rad, Hercules, CA, USA). After the separation of proteins with 557 SDS-PAGE, each protein band was quantified using ImageJ 1.48v 558 (http://imagej.nih.gov/ij). 559

Total amino acids were determined according to the method employed by 560 Ishimoto et al. (2019). In brief, 10 mg of soybean seed flour was hydrolyzed with 1 mL 561 of 6N HCl at 100°C for 22 h under argon. The dried hydrolysate was dissolved in 0.02N 562 HCl and served to LC-MS/MS analysis, as described above. To analyze total Cys and 563 Met, the flour was oxidized with 1 mL of performic acid at 0°C for 16 h to give cysteic 564 acid and methionine sulfone prior to acid hydrolysis. 565 566 Analyses of quantitative trait loci for S-Methylmethionine contents 567 RILs, including 155 F5 lines were developed from a single seed descendant of the cross 568 between NH and WI. Total DNA extraction and linkage map construction by simple 569 sequence repeat markers (WSGP ver. 2) were performed as described previously (Fujii 570 et al., 2018). SMM contents in each RIL were quantified using bulked F6 seeds that 571 were derived from the F5 individual. Because SMM contents varied widely among RILs, 572 QTL analysis was conducted with the common logarithm (log) value for contents. 573



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QTL analyses were performed using composite interval mapping, as 574 implemented in QTL Cartographer 2.5 software (Wang et al., 2005). The genome was 575 scanned at 1-cM intervals. One thousand permutation tests were conducted to determine 576 the threshold value of the limit of detection score. 577 578 Mapping of responsible genes 579 F6 and F7 progenies of parent individuals with hetero genotypes in the chromosomal 580 region that corresponded with QTL were used for fine genetic mapping of responsible 581 genes. Initial QTL analyses indicated a region of around 486 kb ranging from Satt 477 582 and Satt592 on chromosome 10. A population of F7 progeny was then used to delimit 583 the locus using marker-genotyping and SMM quantification. Gene mapping was 584 performed using the BARCSOYSSR markers (BSSR) described by Song et al., 2010. 585 The tail sequence CACGACGTTGTAAAACGAC was added to the 5’ end of the 586 reverse primer and oligonucleotides that were complementary to tail sequences were 587 fluorescently labeled with 6-FAM, VIC, NED, and PET (Thermo Fisher Scientific, 588 Waltham, MA, USA) before addition to PCR reaction solutions. PCRs and PCR 589 fragment length analyses were conducted following Fujii et al. (2018). Only one ORF 590 (i.e., Glyma.10g172700) was identified in the region after the second round of 591 fine-mapping. 592 593 cDNA cloning and expression of recombinant proteins 594 Total RNA was extracted from developing seeds of WI using Qiagen RNeasy Plant 595 Mini Kits (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. 596 DNA was degraded using DNA-free Kits (Ambion, Thermo Fischer Scientific, Waltham, 597 MA, USA), and cDNA was synthesized using SuperScript VILO cDNA Synthesis Kits 598 (Invitrogen). Subsequently, Glyma.10g172700 (GmMGL1) cDNA was PCR amplified 599 using primers for 5′ and 3′ ends of the translation initiation site (Supplementary Table 600 S3). The resulting PCR products were cloned into pGEM T-easy vectors (Promega, 601 Madison, WI, USA) for sequencing. PCR products were then sub-cloned into the 602 EcoRI-XhoI site of the pET24a vector (Merck) and the resulting plasmid was 603 transfected into Escherichia coli Rosetta2 (DE3) pLysS cells (Merck). Cells were 604 subsequently grown in Luria broth supplemented with kanamycin (50 µg mL−1) and 605



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chloramphenicol (30 µg mL−1) at 37°C to an optical density of 0.6–0.8 at 600 nm. After 606 chilling the cultures on ice for 15 min, isopropyl β-D-1-thiogalactosylpyranoside was 607 added to a concentration of 1 mM, and cells were then cultured at 30°C for 16 h. 608

Cells from 50-mL cultures were recovered by centrifugation at 4,000 × g for 609 20 min at 4°C and were resuspended in 5 mL of 100 mM potassium phosphate buffer 610 (pH 7.5) containing 0.01% (w/v) dithiothreitol and 1.3 mM pyridoxal phosphate (PLP). 611 After the addition of 5-µL aliquots of 100 mM phenylmethane sulfonyl fluoride and 50 612 mg mL−1 lysozyme, suspensions were kept on ice for 15 min, and cells were then 613 disrupted using a tip-type ultrasonic disruptor (UD-211, Tomy Seiko, Tokyo, Japan). 614 After centrifugation at 12,000 × g for 10 min, supernatants were directly applied to a 615 column (2 mL) of Ni-NTA agarose (Nacalai Tesque, Kyoto, Japan) that had been 616 equilibrated with 100 mM potassium phosphate buffer (pH 7.5) containing 0.01% (w/v) 617 dithiothreitol and 10 µM pyridoxal phosphate (PLP). The column was then washed with 618 10 mL of the same buffer containing 10 mM imidazole, and His-tagged recombinant 619 proteins were eluted with 10 mL of the same buffer containing 250 mM imidazole. 620 Active fractions were finally combined and desalted using a PD-10 column (GE 621 Healthcare, Chicago, IL, USA). 622

623 Enzyme assays 624 Methionine-γ-lyase enzyme assays were performed according to previous reports (Esaki 625 and Soda, 1987, Takakura et al., 2004). Given volumes of purified enzyme solution 626 were mixed with 100 mM K-phosphate buffer (pH 7.5) containing 10 µM PLP and 40 627 mM L-Met and were incubated at 30°C with gentle shaking. Aliquots of 2 mL were 628 taken every two min and were added to 100 µL of 50% (w/v) trichloroacetic acid. After 629 centrifugation at 20,000 × g for 8 min at 25°C, 0.8-mL supernatants were mixed with 630 1.6-mL aliquots of 1 M sodium acetate buffer (pH 5.0) and 0.6-mL aliquots of 0.1 % 631 (w/v) 3-methyl-2-benzothiazolone hydrazine hydrochloride (MBTH). Reaction tubes 632 were then tightly closed and incubated at 50°C for 40 min. The MGL product 633 2-ketobutyric acid was quantified according to absorbance at 278 nm, which is derived 634 from the MBTH derivative of 2-ketobutyric acid. The absorbance at 0 min was 635 subtracted from later measurements, and a calibration curve was generated using 636 authentic 2-ketobutyric acid (Sigma-Aldrich). The structure of 2-ketobutyric acid was 637 confirmed using GC-MS after converting the acid into a trimethylsilylated product 638



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(Gonda et al. 2013). After incubating the recombinant enzyme with L-Met and PLP 639 overnight in a total volume of 3 mL, reactions were terminated by adding 40-µL 640 aliquots of 6N HCl. Products were then extracted in 2 mL of ethyl acetate and extracts 641 were washed once with 1 mL of water prior to removing the solvent under a stream of 642 nitrogen-gas. After confirming complete dryness, extracts were incubated with 100-µL 643 aliquots of anhydrous pyridine at 25°C for 90 min. Thereafter, 100-µL aliquots of 644 O-bis(trimethylsilyl)trifluoroacetamide (BSTFA, Tokyo Chemical Industry, Tokyo, 645 Japan) were added and incubated for 90 min. GC-MS was performed using a QP-5050 646 (Shimadzu, Kyoto, Japan) instrument equipped with a 0.25 mm × 30 m DB-5MS column 647 (film thickness; 0.25 µm, Restek, Bellefonte, PA, USA). The column temperature was 648 programmed as follows: 50°C for 1 min, increasing by 5°C min−1 to 120°C, then by 649 20°C min−1 to 280°C, and then maintenance at 280°C for 1 min. The carrier gas (He) 650 was delivered at a flow rate of 30 kPa. Injector and interface temperatures were 240 °C 651 and 300 °C, respectively. The mass detector was operated in electron impact mode with 652 an ionization energy of 70 eV. Compounds were assigned by comparing MS profiles and 653 retention times with those of TMS-derivatized 2-ketobutyric acid that was prepared 654 separately. 655 To determine MGL activity in developing soybean seeds, the seeds of NH and 656 FY at seed developmental stage four (cf. the photo in Fig. 8) were homogenized with 657 four volumes of 50 mM sodium phosphate buffer (pH 7.5) containing 5% (w/v) sorbitol, 658 10 mM dithiothreitol, 5 mM sodium metabisulfite, and 2.5 µM PLP. After centrifugation 659 at 20,000 × g for 20 min at 4°C, the cleared supernatant (0.5 mL) was mixed with 0.25 660 mL of 0.2 M Met in a buffer (50 mM sodium phosphate, pH 7.5 containing 2.5 µM PLP) 661 in a total volume of 4.5 mL, and incubated at 30°C with a shaking water bath for 17 h. 662 After the reaction, the reaction mixture was acidified by adding 66.6 µL of 6N HCl, and 663 then, the products were extracted with 4 mL of ethylacetate. After washing the 664 ethylacetate extract with 1 mL of saturated NaCl solution, the extract was used for 665 derivatization with BSTFA as described above. The amount of 2-ketobutyric acid was 666 determined using GC-MS analysis and a calibration curve constructed with authentic 667 2-ketobutyric acid. Molecular ion chromatograms with m/z 73 and 115 were used for 668 quantification. 669 670 Genomic PCR and Reverse Transcription Quantitative PCR Analysis 671



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Genomic DNA was isolated according to Hanafy et al. (2013). Total RNA was isolated 672 using the Qiagen RNeasy Plant Mini Kit according to the manufacturer’s instruction. 673 Total RNA (0.25 µg) was then reverse transcribed with 2.5-µM aliquots of oligo(dT)15 674 primer (Invitrogen) and ReverTra Ace (derived from moloney murine leukemia virus 675 reverse transcriptase; Toyobo, Osaka, Japan) according to the manufacturer’s 676 instructions. Reverse transcription quantitative PCR (RT-qPCR) was performed with an 677 Eco Real-Time PCR System (Illumina). Ct values for the genes of interest were 678 normalized to means of the reference gene for the 20S proteasome subunit beta 679 (Glyma.06g078500; Pereira Lima et al. 2017). Expression levels were calculated as 680 relative amounts using ΔΔCt values. The lowest ΔΔCt value in each experiment was set 681 at 1. 682

Homologs of MGL, CGS, MMT, and HMT were searched using BLASTP 683 analysis on SoyBase (https://soybase.org/) with GmMGL1 (Glyma.10g17200), AtCGS 684 (At3g01120), AtMMT (At5g49810), and AtHMT1 (At3g25900) as queries, respectively. 685 Primers for genomic PCR and RT-qPCR are shown in Supplemental Table S3. 686 687 Met-feeding 688 Pods harboring the seeds of developmental stage two (cf. the photo in Fig. 8) were 689 collected and were gently removed with their seed coats. Thin sections of 1-mm 690 thickness were excised at the short axis using a razor blade, and they were immediately 691 placed on a sheet of Parafilm (Bemis Flexible Packaging, Chicago, IL, USA) in a glass 692 Petri dish. The inner surface of the Petri dish was covered with a moistened paper towel. 693 Fifty-microliter aliquots of 0, 1, or 5 mM Met or 13C-Met in water were then placed on 694 the surfaces of seed sections at 11:00 am, and the Petri dish was immediately closed and 695 incubated at 25°C for 0, 4, 8, and 24 h under light/dark conditions of 14-h light (8:00 am–696 10:00 pm)/10-h dark (10:00 pm–8:00 am). To determine Met and SMM concentrations, 697 sections were carefully washed with water and were mixed with 1-mL aliquots of 698 distilled water containing 1-µg mL−1 d6-SMM. Sections were then homogenized in a 699 mortar, and homogenates were placed in a water bath sonicator (US-2, SND Co. Ltd., 700 Suwa City, Nagano, Japan) for 10 min to facilitate extraction of Met and SMM. 701 Suspensions were centrifuged at 15,000 rpm (20,000 × g) for 10 min at 4°C, and 702 100-µL aliquots were applied to a Strata C18-E (100 mg mL−1) cartridge (Phenomenex 703 Inc. Torrance, CA, USA). Met and SMM were eluted twice with 0.5 mL of distilled 704



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water and eluted solutions were cleared with Ekicrodisc 3 (0.45 µm, 3 mm, Pall Co., 705 Tokyo, Japan) prior to LC-MS/MS analyses as described above. 706 707 Accession numbers 708 Sequence data from this article can be found in the GenBank/EMBL data libraries under 709 accession number MK887190 (Glyma.10g172700 in NH). 710 711 SUPPLEMENTAL DATA 712 713 The following supplemental materials are available. 714 715 Supplemental Figure S1. Protein contents and profile of soybean cultivars used in this 716 study. 717 718 Supplemental Figure S2. The inheritance pattern of S-methylmethionine (SMM) 719 contents. 720 721 Supplemental Figure S3. Appearance of local soybean cultivars collected in Nagano 722 Prefecture, Japan. Bar = 3 cm. 723 724 Supplemental Figure S4. Polymerase chain reactions with complementary and 725 genomic DNA. 726 727 Supplemental Figure S5. Amino acid sequence alignments of methionine γ-lyases 728 (MGLs) from soybean, melon, Arabidopsis, potato, Pseudomonas putida, and 729 Streptomyces avermitilis. 730 731 Supplemental Figure S6. Properties of recombinant GmMGL1. 732 733 Supplemental Figure S7. Phylogenetic analysis of GmMGL1 and its related MGL-like 734 proteins. 735 736 Supplemental Figure S8. Expression levels of GmCGS1/2, GmMMT1, GmMMT2, 737



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GmHMT1, GmHMT2, and GmHMT3 in developing Fukuyutaka (FY) and 738 Nishiyamahitashi 98-5 (NH) seeds from stages one to five (refer to Fig. 8). 739 740 Supplemental Table S1. Total amino acid contents in the dry seeds of the Fukuyutaka 741 (FY) and Nishiyamahitashi 98-5 (NH) soybean cultivars. 742 743 Supplemental Table S2. Multiple reaction monitoring (MRM) transitions and MS 744 parameters used to detect amino acids. 745 746 Supplemental Table S3. Primers used in this study. 747 748 Supplemental Table S4. Protein sequences used to construct the phylogenetic tree. 749 750 Acknowledgments: 751 The authors are thankful to Mss. Shiori Yamanaka, Yuumi Miyazaki, and Mai Nanri for 752 their help to conduct the experiments. 753 754 TABLES 755 756 Table 1. Level of SMM and Met in soybean and Arabidopsis phloem exudates. 757 Plant Cultivar/Ecotype SMM

(nmol g-1 leaf DW) Met (nmol g-1 leaf DW)

Soybean NH 3.40 ± 1.06* 0.063 ± 0.032†

Soybean FY 0.63 ± 0.23 0.203 ± 0.033

Arabidopsis Ws-0 990 ± 150 18.8± 5.2

*: t-test after Box-Cox transformation (between soybean NH and FY) P = 0.0163, †: 758 t-test after Box-Cox transformation (between soybean NH and FY) P = 0.0292. To all 759 the values of Met 1 was added before Box-Cox transformation to avoid the values of 0. 760 761 FIGURE LEGENDS 762 Figure 1. Schematic representation of Met metabolism in plants. The pathways 763

mentioned in this study are highlighted. Solid arrows represent one metabolic step, 764



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whereas dashed arrows represent several metabolic steps. The enzyme names are 765 underlined. The canonical Asp family pathway is shown with gray background. The 766 S-methylmethionine cycle is shown with striped background. Asp, aspartic acid; 767 CGS, cystathionine γ-synthase; Cys, cysteine; HMT, homocysteine 768 methyltransferase; HomoCys, homocysteine; Ile, isoleucine; 2KB, 2-ketobutyric 769 acid; Met, methionine; MGL, methionine γ-lyase; MMT, methionine 770 methyltransferase; MS, methionine synthase; 5MeTHF, 5-methyltetrahydrofolate; 771 O-PhosphohomoSer, O-phosphohomoserine; SAM, S-adenosylmethionine; SMM, 772 S-methylmethionine; Thr, threonine. 773

774 Figure 2. S-Methylmethionine and methionine contents in soybean plants. 775 S-Methylmethionine (SMM) (upper panels) and methionine (Met) (lower panels) 776 contents in the leaves (A), pods (B), and seeds (without seed coats) (C) harvested at the 777 flowering stage (F), the immature green seed stage (IG; corresponding to stage two in 778 Fig. 8), and the mature green seed stage (MG; corresponding to stage five in Fig. 8) are 779 shown as means ± standard errors (SE) of four replicates. Significant differences were 780 identified using a two-way analysis of variance (ANOVA) after Box-Cox 781 transformation and Fisher’s least significant difference test (LSD; P < 0.05). Different 782 lowercase letters indicate significant differences between the developing stages (P < 783 0.05, Tukey’s HSD test after two-way ANOVA). Different capital letters indicate 784 significant differences between all treatments (P < 0.05, Tukey’s HSD test after 785 two-way ANOVA). ***: P < 0.001, **: 0.001 < P < 0.01, NS: 0.05 < P (simple main 786 effect test after two-way ANOVA). 787 788 Figure 3. Free amino acids in seeds. Quantities of free amino acids in dry matured 789 seeds of Fukuyutaka (FY; white bar) and Nishiyamahitashi 98-5 (NH; gray bar) are 790 shown as µg per g of mature dry seeds. The data are shown as means ± SE of three 791 replicates. Significant differences were identified using Student’s t-test after Box-Cox 792 transformation (**: P < 0.01, *: 0.01 < P < 0.05, n.s.: 0.05 < P). HomoSer: homoserine, 793 HomoCys: homocysteine, n.d.: not detected. 794 795 Figure 4. Inheritance of hyperaccumulation of S-methylmethionine. Concentrations of 796 S-methylmethionine (SMM) in F1 progenies that were generated by reciprocal crossing 797



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of Fukuyutaka (FY) (MGL1) and Nishiyamahitashi 98-5 (NH) (mgl1) soybean strains; 798 data are shown as means ± SE of three replicates. Significant differences between plant 799 lines in SMM were identified using Tukey’s HSD tests after Box-Cox transformation (P 800 < 0.05). 801 802 Figure 5. Map-based cloning of the allele responsible for the hyperaccumulation of 803 S-methylmethionine in soybean. A. QTL-regions detected in chromosome 10 of 804 soybean. B. Graphical genotypes of 26 F6 and F7 residual heterozygous lines determined 805 with markers are shown (WI: Williams 82, NH: Nishiyamahitashi 98-5). The amounts 806 of S-methylmethionine (SMM) in the respective lines are shown in the bar graph on the 807 right. After delimiting the region, only one open reading frame (Glyma.10g172700) was 808 identified. Glyma.10g172700 was tentatively assigned as the gene encoding Met γ-lyase 809 and comprises two exons flanking one intron. The DNA sequencing of genes from NH 810 and WI strains showed a copia-type retrotransposon inserted into the intron of the gene 811 in the NH strain only. 812 813 Figure 6. Hyperaccumulation of S-methylmethionine correlates with the insertion of a 814 transposon into the GmMGL1 gene. A. Sizes of DNA fragments that were amplified 815 with primers for the full-length coding sequence of Glyma.10g172700; M: molecular 816 weight marker (l/Hind III digests), 1: Shinano-Kurakake, 2: Nishiyamahitashi 94-5, 3: 817 Sinanomachi Arasehara Kurakake, 4: Togakushi Morozawa Ganimame, 5: Usuda Zairai, 818 6: Shinano Midori, 7: Nishiyamahitashi 94-1, 8: Tousanhitashi 106, 9: Ogawa Zairai 5, 819 10: Chino Zairai 4. NH: Nishiyamahitashi 98-5, WI: Williams 82, FY: Fukuyutaka. B. 820 Quantities of S-methylmethionine (SMM; upper panel: black bars) and Met (lower 821 panel: white bars) in dry matured seeds of each soybean line. Data are shown as means 822 ± SE of three replicates. C. Expression levels of Glyma.10g172700 in seeds at the green 823 mature stage (stage five in Fig. 8). The Glycine max 20S proteasome subunit 824 (Glyma.06g078500) was used as an internal control in RT-qPCR analyses. Transcript 825 levels relative to the internal control are shown as multiples of the lowest value of 1. 826 Data are presented as means ± SE (n = 3). Significant differences indicated with 827 different lowercase letters were identified using Tukey’s HSD tests after Box-Cox 828 transformation (P < 0.05). 829 830



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Figure 7. Glyma.10g172700 encodes a functional MGL. A, Sodium dodecyl 831 sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the recombinant 832 soybean MGL1 protein with an N-terminal His-tag; lanes 1 and 4, molecular weight 833 marker; lane 2, E. coli lysate expressing recombinant GmMGL1 protein; lane 3, E. coli 834 lysate with the empty vector (a negative control); lane 5, purified recombinant 835 GmMGL1. B, Absorption spectra of 3-methyl-2-benzothiazolinone hydrazone (MBTH) 836 derivatives of the product formed during metabolism of Met by recombinant GmMGL1; 837 C, Chromatograms of trimethylsilylated products of recombinant GmMGL1 and Met 838 from retention times of 8.25–10.75 min; the solid line represents chromatograms from 839 experiments with the recombinant enzyme, and the broken line shows those without 840 enzyme (negative control). The peak specifically found with the product formed with 841 recombinant enzyme is shown with arrow (peak A). D, Mass spectra obtained with 842 authentic trimethylsilylated 2-ketobutyric acid (upper panel) and with peak A (lower 843 panel). 844 845 Figure 8. Expression of GmMGL1, GmMGL2, and GmMGL3 in soybean plants. 846 Expression of GmMGL1, GmMGL2, and GmMGL3 in developing seeds (A) and in the 847 leaves, stems, and roots (B) of Fukuyutaka (FY) and Nishiyamahitashi 98-5 (NH) 848 soybean cultivars. Sizes of NH and FY seeds collected for RNA extraction (stages one 849 to five) are shown in the upper panel. The Glycine max 20S proteasome subunit 850 (Glyma.06g078500) was used as an internal control in RT-qPCR analyses. Transcript 851 levels relative to the internal control are shown as multiples of the lowest value of 1. 852 Data are presented as means ± SE (n = 3). Significant differences were identified using 853 a two-way analysis of variance (ANOVA) after Box-Cox transformation. *** above the 854 symbols in A indicate significant differences between plant lines in each developing 855 stage (P < 0.001, simple main effect test after two-way ANOVA). ** indicate significant 856 differences between plant lines in B (MGL1) (P < 0.05, simple main effect test after 857 two-way ANOVA). Different lowercase letters above columns indicate significant 858 differences between organs in B (MGL2 and MGL3) (P < 0.05, Tukey’ HSD test after 859 two-way ANOVA ). 860 861 Figure 9. Absorption and conversion of exogenously supplied methionine to 862 S-methylmethionine in a section of a developing soybean seed. A. S-methylmethionine 863



28

(SMM) and methionine (Met) contents after treating with 0 (circle and white)-, 1 864 (square and gray), and 5 (triangle and black) mM Met. Data are shown as means ± SE 865 (n = 3). Significant differences were identified using a two-way analysis of variance 866 (ANOVA) after Box-Cox transformation. Different letters above the symbols indicate 867 significant differences between Met concentrations in each hour (P < 0.05, Tukey’ HSD 868 tests after simple main effect tests). B. Mass spectrum of SMM extracted from the seed 869 sections treated with 5 mM 13C5-Met for 24 h (upper panel). Reaction of MMT from 870 13C5-Met is shown as an inset. The positions of 13C in Met and SMM are shown with 871 asterisks. Mass spectrum of non-labelled SMM is shown (lower panel). Tentative 872 assignments of molecular and fragment ions are also shown. 873 874 Figure 10. A proposed mechanism of hyperaccumulation of S-methylmethionine in 875 soybean seeds with low MGL activity. (A) When the MGL activity is sufficient as in 876 normal soybean seeds, the level of free Met is properly controlled. (B) When MGL 877 activity is suppressed by transposon insertion as in NH soybean seeds, surplus Met left 878 behind is converted into SMM, which seems to account for hyperaccumulation of SMM. 879 Met, methionine; SMM, S-methylmethionine; MGL, methionine γ-lyase; MMT, 880 methionine methyltransferase; HMT, homocysteine methyltransferase; SAM, 881 S-adenosylmethionine; HomoCys, homocysteine; 2KB, 2-ketobutyric acid. 882 883



29

884



Figure 1. Schematic representation of Met metabolism in plants. The pathways mentioned in this study are highlighted. Solid

arrows represent one metabolic step, whereas dashed arrows represent several metabolic steps. The enzyme names are underlined.

The canonical Asp family pathway is shown with gray background. The S-methylmethionine cycle is shown with striped

background. Asp, aspartic acid; CGS, cystathionine g-synthase; Cys, cysteine; HMT, homocysteine methyltransferase; HomoCys,

homocysteine; Ile, isoleucine; 2KB, 2-ketobutyric acid; Met, methionine; MGL, methionine g-lyase; MMT, methionine

methyltransferase; MS, methionine synthase; 5MeTHF, 5-methyltetrahydrofolate; O-PhosphohomoSer, O-phosphohomoserine;

SAM, S-adenosylmethionine; SMM, S-methylmethionine; Thr, threonine.

Phloem

Asp

2KB

O-PhosphohomoSer

Cys

Cystathionine

CGS

HomoCys

Thr

Ile

2KB

Proteins

Met

SAM

MGL

Vegetative organs

HMT

MMT

MS 5MeTHF

SMM

Asp

2KB

O-PhosphohomoSer

Cys

Cystathionine

CGS

HomoCys

Thr

Ile

2KB

Proteins

Met

SAM

MGL

Developing seeds

HMT

MMT

MS 5MeTHF

SMM



0.0

4.0

8.0

12.0

16.0S

MM

(µ

g/g

)

0.0

0.2

0.4

0.6

0.8

1.0

Met

(µg/g

)

0.0

4.0

8.0

12.0

16.0

0.0

1.0

2.0

3.0

4.0

5.0

A

B B B

Leaves Pods

FY NH

a

a

b b

b

a

b b

F IG2 MG5 IG2 MG5

Figure 2. S-Methylmethionine and methionine contents in soybean plants. S-Methylmethionine (SMM) (upper panels) and methionine (Met) (lower

panels) contents in the leaves (A), pods (B), and seeds (without seed coats) (C) harvested at the flowering stage (F), the immature green seed stage (IG;

corresponding to stage two in Fig. 8), and the mature green seed stage (MG; corresponding to stage five in Fig. 8) are shown as means ± standard

errors (SE) of four replicates. Significant differences were identified using a two-way analysis of variance (ANOVA) after Box-Cox transformation

and Fisher’s least significant difference test (LSD; P < 0.05). Different lowercase letters indicate significant differences between the developing stages

(P < 0.05, Tukey’s HSD test after two-way ANOVA). Different capital letters indicate significant differences between all treatments (P < 0.05, Tukey’s

HSD test after two-way ANOVA). ***: P < 0.001, **: 0.001 < P < 0.01, NS: 0.05 < P (simple main effect test after two-way ANOVA).

A B

development P < 0.0001

plant line P = 0.2116

development x line P = 0.4262

Seeds

(without seed coat)

0

40

80

120

160

0.0

3.0

6.0

9.0

12.0

15.0

n.d.

n.d.

IG2 MG5 Dry

C

***

*** ***

*** ** NS

development P = 0.0046





development x line

P = 0.1490


plant line P < 0.0001

development x line P < 0.0001



development x line

P = 0.0002






Figure 3. Free amino acids in seeds. Quantities of free amino acids in dry matured seeds of Fukuyutaka (FY; white bar) and

Nishiyamahitashi 98-5 (NH; gray bar) are shown as µg per g of mature dry seeds. The data are shown as means ± SE of

three replicates. Significant differences were identified using Student’s t-test after Box-Cox transformation (**: P < 0.01, *:

0.01 < P < 0.05, n.s.: 0.05 < P). HomoSer: homoserine, HomoCys: homocysteine, n.d.: not detected.

n.d.

FY NH

Am

ount (µ

g g

-1)

0

500

1000

1500

2000

2500

3000

n.s.

n.s. n.s.

n.s.

n.s.

n.s.

n.s. n.s. n.s. n.s.

n.s. n.s. n.s. n.s.

*

*

*

** n.d. n.d.

*

Ala Arg Asn Asp Cys Gln Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val Homo

Ser Homo

Cys



NH

x NH

FY

x FY

FY (♀)

x NH (♂)

NH (♀)

x FY (♂)

Figure 4. Inheritance of hyperaccumulation of S-methylmethionine. Concentrations of S-

methylmethionine (SMM) in F1 progenies that were generated by reciprocal crossing of Fukuyutaka (FY)

(MGL1) and Nishiyamahitashi 98-5 (NH) (mgl1) soybean strains; data are shown as means ± SE of three

replicates. Significant differences between plant lines in SMM were identified using Tukey’s HSD tests

after Box-Cox transformation (P < 0.05).

mgl1

mgl1

MGL1

MGL1

mgl1/

mgl1

MGL1/

MGL1 F1 seed

Maternal

genotype

Paternal

genotype

MGL1

MGL1 mgl1

mgl1

mgl1/

MGL1

MGL1/

mgl1

0

200

400

600

800

SM

M (

µg g

-1)

a

b b b



Figure 5. Map-based cloning of the allele responsible for the hyperaccumulation of S-methylmethionine in soybean. A. QTL-regions detected in chromosome

10 of soybean. B. Graphical genotypes of 26 F6 and F7 residual heterozygous lines determined with markers are shown (WI: Williams 82, NH:

Nishiyamahitashi 98-5). The amounts of S-methylmethionine (SMM) in the respective lines are shown in the bar graph on the right. After delimiting the

region, only one open reading frame (Glyma.10g172700) was identified. Glyma.10g172700 was tentatively assigned as the gene encoding Met g-lyase and

comprises two exons flanking one intron. The DNA sequencing of genes from NH and WI strains showed a copia-type retrotransposon inserted into the intron

of the gene in the NH strain only.

Gm10

40 cM 2

4

6

0

LO

D s

core

Sa

tt477

BS

SR

10 1

156

BS

SR

10 1

174

BS

SR

10 1

176

BS

SR

10 1

177

BS

SR

10 1

179

BS

SR

10 1

181

BS

SR

10 1

182

BS

SR

10 1

189

BS

SR

10 1

194

BS

SR

10 1

261

0 200 400 600

SMM (µg g-1)

Marker

Lines

Glyma.10g172700

copia-type retrotransposon

Homozygous WI

Homozygous NH

Heterozygous

Sa

t_196

BS

SR

10_0190

WG

SP

10_0040

Sa

tt653

Sa

tt345

Sa

tt479

Sa

tt478

Sa

tt477

BS

SR

10_1261

Sa

tt592

Sa

t_038

Sa

tt243

CS

SR

141 A

B

12 kb

2.3 Mb



A

B

Figure 6. Hyperaccumulation of S-methylmethionine correlates with the

insertion of a transposon into the GmMGL1 gene. A. Sizes of DNA

fragments that were amplified with primers for the full-length coding

sequence of Glyma.10g172700; M: molecular weight marker (l/Hind III

digests), 1: Shinano-Kurakake, 2: Nishiyamahitashi 94-5, 3: Sinanomachi

Arasehara Kurakake, 4: Togakushi Morozawa Ganimame, 5: Usuda Zairai,

6: Shinano Midori, 7: Nishiyamahitashi 94-1, 8: Tousanhitashi 106, 9:

Ogawa Zairai 5, 10: Chino Zairai 4. NH: Nishiyamahitashi 98-5, WI:

Williams 82, FY: Fukuyutaka. B. Quantities of S-methylmethionine (SMM;

upper panel: black bars) and Met (lower panel: white bars) in dry matured

seeds of each soybean line. Data are shown as means ± SE of three

replicates. C. Expression levels of Glyma.10g172700 in seeds at the green

mature stage (stage five in Fig. 8). The Glycine max 20S proteasome subunit

(Glyma.06g078500) was used as an internal control in RT-qPCR analyses.

Transcript levels relative to the internal control are shown as multiples of the

lowest value of 1. Data are presented as means ± SE (n = 3). Significant

differences indicated with different lowercase letters were identified using

Tukey’s HSD tests after Box-Cox transformation (P < 0.05).

23.1 9.4 6.6 4.4

2.3 2.0

(kbp) M 1 2 3 4 5 6 7 8 9 10 NH WI FY

Am

oun

t (µ

g g

-1)

0.0

20.0

40.0

60.0SMM

0.0

5.0

10.0

15.0 Met

ab

a

ab ab b

c c c c c

a

c

ab

a

a a

ab bc bc

abc

d c

a

bc

e

c

0.0

10.0

20.0

30.0

Tra

nscript le

vel

(re

lative to G

mP

BA

1)

a ab

ab

ab ab

ab

abc

bcd

cd cd

cd cd d

C

1 2 3 4 5 6 7 8 9 10 NH WI FY

1 2 3 4 5 6 7 8 9 10 NH WI FY



1 2 3 5

150

100 80

60

50

40

30 25

4

60

50

40

30

(kDa) (kDa)

300 320 340 360 380 400

Wavelength (nm)

DA (0.05)

Detector

response

(1 x 104)

8.5 9.0 9.5 10.0 10.5

Retention time (min)

With recombinant

enzyme

Without recombinant enzyme

A

B

C

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200

0.00

0.25

0.50

0.75

1.00 73

115 75

45 101

57 85 130 102 159

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 0.00

0.25

0.50

0.75

1.00 73

45 115 75

101 59 85 130 159

55 121 192 143 147

(x10,000)

(x10,000)

Dete

cto

r re

spon

se

D

ete

cto

r re

spon

se

D

Figure 7. Glyma.10g172700 encodes a functional MGL. A, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

analysis of the recombinant soybean MGL1 protein with an N-terminal His-tag; lanes 1 and 4, molecular weight marker; lane 2, E. coli

lysate expressing recombinant GmMGL1 protein; lane 3, E. coli lysate with the empty vector (a negative control); lane 5, purified

recombinant GmMGL1. B, Absorption spectra of 3-methyl-2-benzothiazolinone hydrazone (MBTH) derivatives of the product formed

during metabolism of Met by recombinant GmMGL1; C, Chromatograms of trimethylsilylated products of recombinant GmMGL1 and Met

from retention times of 8.25–10.75 min; the solid line represents chromatograms from experiments with the recombinant enzyme, and the

broken line shows those without enzyme (negative control). The peak specifically found with the product formed with recombinant enzyme

is shown with arrow (peak A). D, Mass spectra obtained with authentic trimethylsilylated 2-ketobutyric acid (upper panel) and with peak A

(lower panel).

Peak A

49.0

(kDa)



Figure 8. Expression of GmMGL1, GmMGL2, and GmMGL3 in soybean plants. Expression of GmMGL1, GmMGL2, and GmMGL3 in developing seeds (A) and in the leaves,

stems, and roots (B) of Fukuyutaka (FY) and Nishiyamahitashi 98-5 (NH) soybean cultivars. Sizes of NH and FY seeds collected for RNA extraction (stages one to five) are

shown in the upper panel. The Glycine max 20S proteasome subunit (Glyma.06g078500) was used as an internal control in RT-qPCR analyses. Transcript levels relative to the

internal control are shown as multiples of the lowest value of 1. Data are presented as means ± SE (n = 3). Significant differences were identified using a two-way analysis of

variance (ANOVA) after Box-Cox transformation. *** above the symbols in A indicate significant differences between plant lines in each developing stage (P < 0.001, simple

main effect test after two-way ANOVA). ** indicate significant differences between plant lines in B (MGL1) (P < 0.05, simple main effect test after two-way ANOVA).

Different lowercase letters above columns indicate significant differences between organs in B (MGL2 and MGL3) (P < 0.05, Tukey’ HSD test after two-way ANOVA ).

0

10

20

30

40MGL1

0.0

10.0

20.0

1 2 3 4 5

MGL3

0.0

2.5

5.0

7.5

10.0

MGL2

***

Seed developing stage

NH

2 cm

FY

A

Tra

nscript le

ve

l (r

ela

tive to G

mP

BA

1)

FY NH

***

***

***

*** ***

***

0

100

200

300

MGL1

0

5

10

15

20

MGL2 a

a

bc bc b

c

0

40

80

120

160

MGL3 a

a

b b b

c

Organ

NH FY

Leaf Stem Root T

ranscript le

ve

l (r

ela

tive to G

mP

BA

1)

**

**

**

organ P < 0.0001

pant line P < 0.0001

organ x line P = 0.709

organ P < 0.0001

pant line P = 0.1046

organ x line P = 0.0272

organ P < 0.0001

pant line P = 0.0005

organ x line P < 0.0001










B

1 2 3 4 5 Seed developing stage



50 100 150 200 250 0.00

2.00e6

4.00e6

6.00e6

8.00e6

1.00e7

1.20e7

Inte

nsity (

cp

s)

106.1

169.1

m/z (Da)

50 100 150 200 250

m/z (Da)

0.00

2.00e6

4.00e6

6.00e6

8.00e6

1.00e7

1.20e7

102.1

164.2

Inte

nsity (

cp

s)

B

Figure 9. Absorption and conversion of exogenously supplied methionine to S-methylmethionine in a section of a developing soybean seed. A. S-

methylmethionine (SMM) and methionine (Met) contents after treating with 0 (circle and white)-, 1 (square and gray), and 5 (triangle and black) mM

Met. Data are shown as means ± SE (n = 3). Significant differences were identified using a two-way analysis of variance (ANOVA) after Box-Cox

transformation. Different letters above the symbols indicate significant differences between Met concentrations in each hour (P < 0.05, Tukey’ HSD

tests after simple main effect tests). B. Mass spectrum of SMM extracted from the seed sections treated with 5 mM 13C5-Met for 24 h (upper panel).

Reaction of MMT from 13C5-Met is shown as an inset. The positions of 13C in Met and SMM are shown with asterisks. Mass spectrum of non-labelled

SMM is shown (lower panel). Tentative assignments of molecular and fragment ions are also shown.

0

100

200

300

0 4 8 12 16 20 24

SM

M (μ

g g

-1)

Met (μ

g g

-1)

Time after treatment (h)

b

a

a

a

c

NH

b

b c

a b

a

a

b

FY

c

A

0

10

20

30

0 4 8 12 16 20 24

a

time P < 0.0001

Met conc P < 0.0001

time x Met conc P < 0.0001

time P = 0.7956

Met conc P < 0.0001

time x Met conc P = 0.0459

time P < 0.0001

Met conc P = 0.0003

time x Met conc P = 0.0159

time P = 0.0066

Met conc P < 0.0001

time x Met conc P = 0.0069 a

b

c

a

b

b

a

ab

b

a

a

b

a a

b

b b

b

a

MMT SAM

*

* *

* *

*

* *

* *

m/z 169.1

m/z 106.1

m/z 164.2

m/z 102.1



2KB/CH3SH/NH3 SMM Met MGL

HomoCys

HMT

MMT

SAM

2KB/CH3SH/NH3 SMM Met MGL

HomoCys

HMT

MMT

SAM

Transposon

insertion

Figure 10. A proposed mechanism of hyperaccumulation of S-methylmethionine in soybean seeds

with low MGL activity. (A) When the MGL activity is sufficient as in normal soybean seeds, the level

of free Met is properly controlled. (B) When MGL activity is suppressed by transposon insertion as in

NH soybean seeds, surplus Met left behind is converted into SMM, which seems to account for

hyperaccumulation of SMM. Met, methionine; SMM, S-methylmethionine; MGL, methionine g-lyase; MMT, methionine methyltransferase; HMT, homocysteine methyltransferase; SAM, S-

adenosylmethionine; HomoCys, homocysteine; 2KB, 2-ketobutyric acid.

A

B



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https://www.ncbi.nlm.nih.gov/pubmed?term=Amir R%2C Cohen H%2C Hacham Y %282019%29 Revisiting the attempts to fortify methionine content in plant seeds%2E J Exp Bot&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Amir&hl=en&c2coff=1

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https://www.ncbi.nlm.nih.gov/pubmed?term=Bourgis F%2C Roje S%2C Nuccio ML%2C Fisher DB%2C Tarczynski MC%2C Li C%2C Herschbach C%2C Rennenberg H%2C Pimenta MJ%2C Shen TL%2C Gage DA%2C Hanson AD %281999%29 S%2DMethylmethionine plays a major role in phloem sulfur transport and is synthesized by a novel type of methyltransferase%2E Plant Cell&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Cohen M%2C Israeli H%2C Matityahu I%2C Amir R %282014%29 Seed%2Dspecific expression of a feedback%2Dinsensitive form of CYSTATHIONINE%2D%2DSYNTHASE in Arabidopsis stimulates metabolic and transcriptomic responses associated with desiccation stress%2E Plant Physiol&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Hacham Y%2C Matiyahu I%2C Schuster G%2C Amir R %282008%29 Overexpression of mutated forms of aspartate kinase and cystathionine %2Dsynthase in tobacco leaves resulted in the high accumulation of methionine and threonine%2E Plant J&dopt=abstract

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Wang S, Basten CJ, Zeng ZB (2005) Windows QTL Cartographer 2.5. Department of Statistics, North Carolina State University, Raleigh,NC.



https://www.ncbi.nlm.nih.gov/pubmed?term=Urquhart AA%2C Joy KW %281981%29 Use of phloem exudate technique in the study of amino acid transport in pea plants%2E Plant Physiol&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Urquhart&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Use of phloem exudate technique in the study of amino acid transport in pea plants&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Use of phloem exudate technique in the study of amino acid transport in pea plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Urquhart&as_ylo=1981&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Wang S%2C Basten CJ%2C Zeng ZB %282005%29 Windows QTL Cartographer 2%2E5%2E Department of Statistics%2C North Carolina State University%2C Raleigh%2C NC&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Wang&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Windows QTL Cartographer 2.5.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Windows QTL Cartographer 2.5.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wang&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1


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7 8 9 10 11 Methionine γ-Lyase€¦ · 97 that in ordinary soybean cultivars. 98 Met is a biosynthetic member of the aspartate (Asp) family (Fig. 1) (Galili et 99 al., 2016, Amir