Suppressed Methionine g-Lyase Expression CausesHyperaccumulation of S-Methylmethionine inSoybean Seeds1[OPEN]

Takuya Teshima,a Naohiro Yamada,b Yuko Yokota,c Takashi Sayama,c,2 Kenji Inagaki,d Takao Koeduka,a

Masayoshi Uefune,e Masao Ishimoto,c and Kenji Matsuia,3,4

aDivision of Agricultural Sciences, Graduate School of Sciences and Technology for Innovation, YamaguchiUniversity, Yamaguchi 753–8515, JapanbNagano Vegetable and Ornamental Crops Experiment Station, Shiojiri, Nagano 399–6461, JapancInstitute of Crop Science, National Agriculture and Food Research Organization, Tsukuba, Ibaraki 305–8518,JapandDepartment of Biofunctional Chemistry, Graduate School of Environmental and Life Science, OkayamaUniversity, Okayama700–8530, JapaneDepartment of Agrobiological Resources, Faculty of Agriculture, Meijo University, Nagoya, Aichi 468–8502,Japan

ORCID IDs: 0000-0002-0786-3242 (T.K.); 0000-0002-4875-5176 (K.M.)

Several soybean (Glycine max) germplasms, such as Nishiyamahitashi 98-5 (NH), have an intense seaweed-like flavor aftercooking because of their high seed S-methylmethionine (SMM) content. In this study, we compared the amounts of aminoacids in the phloem sap, leaves, pods, and seeds between NH and the common soybean cultivar Fukuyutaka. This revealed acomparably higher SMM content alongside a higher free Met content in NH seeds, suggesting that the SMM-hyperaccumulationphenotype of NH soybean was related to Met metabolism in seeds. To investigate the molecular mechanism behind SMMhyperaccumulation, we examined the phenotype-associated gene locus in NH plants. Analyses of the quantitative trait loci insegregated offspring of the cross between NH and the common soybean cultivar Williams 82 indicated that one locus onchromosome 10 explains 71.4% of SMM hyperaccumulation. Subsequent fine-mapping revealed that a transposon insertioninto the intron of a gene, Glyma.10g172700, is associated with the SMM-hyperaccumulation phenotype. The Glyma.10g172700-encoded recombinant protein showed Met-g-lyase (MGL) activity in vitro, and the transposon-insertion mutation in NHefficiently suppressed Glyma.10g172700 expression in developing seeds. Exogenous administration of Met to sections ofdeveloping soybean seeds resulted in transient increases in Met levels, followed by continuous increases in SMMconcentrations, which was likely caused by Met methyltransferase activity in the seeds. Accordingly, we propose that theSMM-hyperaccumulation phenotype is caused by suppressed MGL expression in developing soybean seeds, resulting intransient accumulation of Met, which is converted into SMM to avoid the harmful effects caused by excess free Met.

In the 2017/2018 market year, 346.2 million tons ofsoybean (Glycine max) were produced (Food and Ag-riculture Organization Corporate Statistical Database[FAOSTAT]; http://www.fao.org/faostat/en/#home).This large amount of soybean production was drivenby the high oil and protein contents of the plant. Soy-bean meal is widely used as animal feed and has highprotein contents and a well-balanced amino acid pro-file. However, its nutritional value for monogastricanimals could be improved by increasing its Cys andMet contents. Accordingly, the regulatory mechanismsrelating to Met and Cys biosynthesis in soybean havebeen investigated extensively (Hesse and Hoefgen,2003; Galili et al., 2016; Krishnan and Jez, 2018; Amiret al., 2019). In the central region of Japan, specificsoybean seeds have been cultivated for their seaweedodor, which is strongly related to dimethyl sulfide.Dimethyl sulfide is formed spontaneously fromS-methylmethionine (SMM) during heating of the seeds

for cooking (Morisaki et al., 2014). In a representative cul-tivar, Nishiyamahitashi 98-5 (NH), the SMM level in theseeds is more than 100-fold higher than in common soy-bean cultivars, such as Fukuyutaka (FY; Morisaki et al.,2014). Because SMM is a direct product of Met metabo-lismbymethioninemethyltransferase (MMT), it is assumedthat the cultivars with higher SMM content are likely tometabolizeMet and its derivatives viamechanisms that aredistinctive from that in ordinary soybean cultivars.Met is a biosynthetic member of the Asp family

(Fig. 1; Galili et al., 2016; Amir et al., 2019). Cystathio-nine g-synthase (CGS) performs the crucial regulatorystep in Met biosynthesis and determines the rate of Metproduction from O-phosphohomoserine (Galili et al.,2016). Free Met is used to form proteins, but a portionof Met is converted to SAM by a SAM synthetase. An-other portion of free Met accepts a methyl group fromSAM through MMT and is converted into SMM. Ho-mocysteine methyltransferase (HMT) converts SMM

Plant Physiology�, July 2020, Vol. 183, pp. 943–956, www.plantphysiol.org � 2020 American Society of Plant Biologists. All Rights Reserved. 943 www.plantphysiol.orgon September 30, 2020 - Published by Downloaded from

Copyright © 2020 American Society of Plant Biologists. All rights reserved.

back to Met. MMT and HMT constitute the SMM cycle,which seems to operate throughout plant tissues, in-cluding reproductive tissues of various plant species(Ranocha et al., 2001). In several flowering plants, SMMis produced in the leaves by MMT and is transportedthrough the phloem toward the reproductive organs,where it is reconverted to Met by HMT (Bourgis et al.,1999; Cohen et al., 2017c). With developing seeds ofMedicago truncatula, SMM is converted back to Met viaHMT in seed coats, and Met released into the seedapoplast is taken up by seeds (Gallardo et al., 2007).Both the in situ formation of Met through Asp familyenzymes and the biosynthesis of SMM in the leavesfollowing phloem transport likely regulate Met con-tents simultaneously in the seeds (Cohen et al., 2017c;Amir et al., 2019). Met catabolism also controls Metlevels in plant tissues. Met g-lyase (MGL) is a pyridoxalphosphate (PLP)-dependent enzyme that metabolizesMet into 2-ketobutyric acid, methanethiol, and ammo-nia (Sato and Nozaki, 2009). 2-Ketobutyric acid can bemetabolized to form Ile even though this pathway isauxiliary to the major Ile biosynthetic pathway throughThr deaminase (Joshi and Jander, 2009).

Based on accumulating studies of Met regulation inplant tissues, numerous attempts to improveMet levelsin crops have been made through genetic engineeringparticularly of the Asp family pathway (Hacham et al.,2008; Hanafy et al., 2013; Song et al., 2013; Cohen et al.,2014; Kumar and Jander, 2017; Amir et al., 2019). Ac-celeration of SMM transport from nonseed tissues toseeds was also attempted to increaseMet levels in seeds(Lee et al., 2008; Cohen et al., 2017c). As such, attemptsto increase seed Met levels are becoming more suc-cessful but are sometimes disturbed by abnormal phe-notypes of the plants (Krishnan and Jez, 2018; Amiret al., 2019). Severe growth retardation was observedin potato (Solanum tuberosum) plants overexpressing thefeedback-insensitive CGS to form more Met and b-zeinto store Met (Dancs et al., 2008). Tobacco (Nicotiana

tabacum) plants overexpressing CGS and with elevatedfree Met levels also had increased sensitivity to oxida-tive stress (Hacham et al., 2017). In addition, Arabi-dopsis (Arabidopsis thaliana) seeds overexpressing amutant CGS accumulated Met to 2.5-fold higher levels,and these conditions were associated with increasedexpression of stress-related transcripts (Cohen et al.,2014; Cohen and Amir, 2017). These studies suggestthatMet levels are tightly controlled in plant tissues andthat excessive free Met is deleterious to plant health.Yet, the mechanisms by which Met levels are regulatedin some tissues remain poorly understood.

The soybean SMM-hyperaccumulation phenotype,like that of NH, was assumed to be attributable to agenotype related to Met metabolism in seeds. We inves-tigated the genotype of SMM-hyperaccumulating soy-bean plants and identified the gene that is responsible forSMMaccumulation in soybean. Through analyses of genefunction and NH phenotypes, we propose a mechanismunderlying SMM hyperaccumulation.

RESULTS

SMM and Free Met Levels

The quantities of SMMand freeMetwere determinedin the leaves, pods, and seeds of two soybean cultivars,FY and NH, at the flowering stage, immature greenseed stage, mature green seed stage, and in dry seeds(Fig. 2). The seed coats were removed from the otherparts of seeds (i.e. cotyledons, plumules, and radicles)before analyses to avoid mixing tissues of maternal gen-otype (i.e. seed coats) and those of offspring genotypes(cotyledons, plumules, and radicles). The contents ofSMM and free Met were low in the leaves, and no sig-nificant differences were identified between FY and

Figure 1. Schematic representation of Met metabolism in plants. Thepathways mentioned in this study are highlighted. Solid arrows repre-sent onemetabolic step,whereas dashed arrows represent severalmetabolicsteps. The enzymenames are underlined. The canonical Asp family pathwayis shown with gray background. The SMM cycle is shown with stripedbackground. HomoCys, Homocysteine; 2KB, 2-ketobutyric acid; 5MeTHF,5-methyltetrahydrofolate; MS, Met synthase; O-PhosphohomoSer,O-phosphohomoserine; SAM, S-adenosyl-Met.

1This work was supported by the Japan Society for the Promotionof Science (grant nos. 16H03283 and 19H02887), the Tojuro IijimaFoundation for Food Science and Technology, and the Fuji Founda-tion for Protein Research (to K.M.).

2Present address: Western Region Agricultural Research Center,National Agriculture and Food Research Organization, 6-12-1 Nishi-fukatsu, Fukuyama, Hiroshima 721–8514, Japan.

3Author for contact: matsui@yamaguchi-u.ac.jp.4Senior authorThe author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Kenji Matsui (matsui@yamaguchi-u.ac.jp).

K.M. conceived this project, designed experiments, and wrote thearticle; T.T. analyzed metabolites and characterized recombinant en-zyme; N.Y. performed cross-breeding and harvested seeds; M.I., T.S.,and Y.Y. performed genotyping and positional cloning; K.I. providedenzymes; T.K. mined database and constructed phylogenetic trees;M.U. performed statistical analyses.

[OPEN]Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.20.00254

944 Plant Physiol. Vol. 183, 2020

Teshima et al.

www.plantphysiol.orgon September 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.

NH. The SMM level in the pods seemed to be a littlehigher than that found in the leaves, but a statisticallysignificant difference in its level between FY and NHwas hardly detected. The free Met level in the NH podsat the immature green stage was significantly lowerthan that of FY, but the level in the FY pods lowered atthe mature green stage to the level found in the NHpods. The levels of SMM in theNH seedswere eightfoldand 15.6-fold higher at the immature and mature greenstage, respectively, than those found in the FY seeds. Asubstantial amount of SMM was detected in dry NHseeds, whereas it was under the detection limit in thedry FY seeds. The levels of free Met in the NH seedswere significantly higher at the mature green stage andin dry seeds than those found in the FY seeds.The SMM level in the phloem exudate collected

through a cut petiole of NH at the seed-developing stagewas significantly higher than that found for FY, whereasthe level of free Met was lower than that found for FY(Table 1). The levels of SMM and free Met in phloem

exudate collected using the same procedure throughArabidopsis petioles at the seed-filling stage were morethan 93-fold higher than those found for NH and FY.The free amino acid contents of mature seeds were

mostly similar between FY and NH, yet the His, Met,Phe, Thr, and homoserine contents were significantlyhigher in NH than in FY seeds (Fig. 3). Homocysteinewas under the detection level (4.2 mg g21) in both NHand FY. The total protein contents, protein profiles ex-amined using Coomassie Brilliant Blue staining afterSDS-PAGE (Supplemental Fig. S1), and total aminoacid contents showed no significant differences be-tween the FY and NH seeds (Supplemental Table S1).

Positional Cloning of the Gene Responsible forHyperaccumulation of SMM

The contents of SMM and free Met were determinedwith four F1 seeds after reciprocal crossing of the FY

Figure 2. SMMandMet contents in soybean plants. SMM (top row) andMet (bottom row) contents in the leaves (A), pods (B), andseeds (without seed coats; C) harvested at the flowering stage (F), the immature green seed stage (IG2; corresponding to stage 2 inFig. 8), and the mature green seed stage (MG5; corresponding to stage 5 in Fig. 8) are shown as means 6 SE of four replicates.Significant differences were identified using a two-way ANOVA after Box-Cox transformation and Fisher’s LSD test (P , 0.05).Different lowercase letters indicate significant differences between the developing stages (P, 0.05, Tukey’s honestly significantdifference [HSD] test after two-way ANOVA). Different uppercase 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; and NS, 0.05, P (simple main-effecttest after two-way ANOVA). n.d., Not detected.

Table 1. Levels of SMM and Met in soybean and Arabidopsis phloem exudates

To all the values of Met, 1 was added before Box-Cox transformation to avoid values of 0.

Plant Cultivar/Ecotype SMM Met

nmol g21 leaf DWSoybean NH 3.40 6 1.06a 0.063 6 0.032b

Soybean FY 0.63 6 0.23 0.203 6 0.033Arabidopsis Wassilewskija-0 990 6 150 18.8 6 5.2

aStudent’s t test after Box-Cox transformation (between soybean NH and FY), P 50.0163. bStudent’s t test after Box-Cox transformation (between soybean NH and FY), P 5 0.0292.

Plant Physiol. Vol. 183, 2020 945

Met g-Lyase Suppression Leads to High SMM

www.plantphysiol.orgon September 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.

and NH cultivars. The hyperaccumulation of SMMwasonly evident in self-pollinatedNH seeds, and thematernaland paternal genotypes did not play a significant role inthe accumulation of SMM in seeds (Fig. 4). Moreover, F2seeds of the FY 3 NH cross were segregated into highSMM:low SMM at a ratio of 3:17 with a consistent segre-gation ratio of 1:3 (x2 test, P5 0.3; Supplemental Fig. S2A).These data indicate that hyperaccumulation of SMM isessentially regulated by a single recessive allele. In order toidentify the gene responsible for hyperaccumulation ofSMM, we cross-bred NH to the Williams 82 (WI) cultivar.WI was used because the SMM contents inWI seeds wereas low as those in FY seeds (see below) and also becausethe reference genome sequence was produced with WI(Schmutz et al., 2010). A total of 156 F5 recombinant inbredlines (RILs) were generated from the cross between NHand WI, and SMM levels in their mature seeds were de-termined (Supplemental Fig. S2B). Quantitative trait locus(QTL) analyses with molecular markers indicated that anallele near a simple sequence repeat marker (Satt477) onchromosome 10 explained 71.4% of the phenotypic varia-tion (Fig. 5A).No otherQTLwith a significant influence onthe hyperaccumulation of SMM was detected. Fine-mappingof the allele responsible for the hyperaccumulationof SMM in F6 and F7 residual heterozygous linesnarrowed down this region to a 12-kb sequence onchromosome 10 (Fig. 5B). Furthermore, examinationof the Phytozome soybean genome sequence database(https://phytozome.jgi.doe.gov/pz/portal.html) revealedonly one open reading frame (ORF) ofGlyma.10g172700 inthis region. Glyma.10g172700 comprises two exons flank-ing a single intron. Finally, sequence analyses of Gly-ma.10g172700 in NH (GenBank accession no. MK887190)indicated that a copia-type retrotransposon (AB370254; Liuet al., 2008) was inserted into the intron.

Hyperaccumulation of SMM Correlates Well withTransposon Insertion in the Glyma.10g172700 Gene

We collected local soybean cultivars in Nagano Pre-fecture, Japan (Supplemental Fig. S3). These were

previously shown to have differing SMM levels(Morisaki et al., 2014). We extracted genomic DNA andthen amplified theGlyma.10g172700 gene using primersfor 59 and 39 termini of its deduced ORF. In normalsoybean cultivars, such as FY and WI, the resultingDNA fragment was 2,885 bp, as expected from genomesequences in the Phytozome database. The Gly-ma.10g172700 gene length was the same in five of the 10local cultivars as that detected in normal cultivars, but itwas longer in the other five cultivars and was similar inlength to that amplified from NH (approximately 9 kb;Fig. 6A). Therefore, it was expected that these latter fivesoybean cultivars carry an inserted transposon in theGlyma.10g172700 gene of the same size as the inserted

Figure 3. Free amino acids in seeds. Quantities offree amino acids in dry matured seeds of FY (whitebars) and NH (gray bars) are shown as mg g21 mature dry seeds. The data are shown as means6 SE of three replicates. Significant differenceswere identified using Student’s t test after Box-Coxtransformation (**, P , 0.01; *, 0.01 , P , 0.05;and n.s., 0.05 , P). HomoCys, Homocysteine;HomoSer, homoserine; n.d., not detected.

Figure 4. Inheritance of hyperaccumulation of SMM. Concentrations ofSMM in F1 progeny that were generated by reciprocal crossing of FY(MGL1) and NH (mgl1) soybean strains are shown as means 6 SE ofthree replicates. Significant differences between plant lines in SMMwere identified using Tukey’s HSD tests after Box-Cox transformation(P , 0.05).

946 Plant Physiol. Vol. 183, 2020

Teshima et al.

www.plantphysiol.orgon September 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.

transposon inNH.Determinations of SMM levels in seedsof these cultivars showed higher SMM levels in cultivarsharboring the Glyma.10g172700 gene with the transposoninsertion than in those without the transposon insertion(Fig. 6B). Free Met levels were higher in some seedshaving the transposon insertion in Glyma.10g172700 thanin those without the insertion, but the correlation was notalways evident. Reverse transcription quantitative PCR(RT-qPCR) analyses of RNA extracted from maturingseeds showed that the lower levels of transcript derivedfrom Glyma.10g172700 were detected with cultivars thathad the transposon insertion in Glyma.10g172700(Fig. 6C). The product sizes obtained with RT-PCR withNH and FY seeds with excess amplification cycles usingprimers for the 59 and 39 termini of GmMGL1 ORF werethe same (Supplemental Fig. S4), and no sign of alterna-tive splicing was detected.

Glyma.10g172700 Encodes a Functional Met g-Lyase

The ORF of Glyma.10g172700 encodes a protein of48,069 D, yet the deduced protein sequence had no

predictable targeting signal in TargetP analyses(http://www.cbs.dtu.dk/services/TargetP/), sug-gesting a cytosolic location of the protein. The Gly-ma.10g172700 gene was tentatively assigned as a geneencoding an MGL, and the deduced protein sequencehad 77.2% and 78.6% identities with Arabidopsis MGL(Q9SGU9, AEE34271.1) andmelon (Cucumis melo) MGL(M1NFB7, NP_001315378.1), respectively (SupplementalFig. S5; Rébeillé et al., 2006; Gonda et al., 2013). Moreover,the protein sequence has a motif (Ser-237-Xaa-Xaa-Lys-240) that is conserved in PLP enzymes of the g sub-family and associates with the cofactor PLP(Supplemental Fig. S5; Martel et al., 1987; Sato andNozaki, 2009). Tyr-142, Asp-216, and Arg-410 resi-dues are also involved in substrate binding and catal-ysis at appropriate positions (Goyer et al., 2007), andGly-144 is conserved as in Arabidopsis and melonMGLs that retain restricted substrate specificity forL-Met (Supplemental Fig. S5; Gonda et al., 2013).Glyma.10g172700 cDNA was cloned using RNA

that was extracted from developing WI seeds. Weexpressed the recombinant protein as an N-terminalHis-tagged protein and purified it using Ni21-affinity

Figure 5. Map-based cloning of the allele responsible for the hyperaccumulation of SMM in soybean. A, QTL regions detected inchromosome 10 of soybean. cM, Centimorgan; LOD, log of the odds. B, Graphical genotypes of 26 F6 and F7 residual heter-ozygous lines determinedwith markers are shown. The amounts of SMM in the respective lines are shown in the bar graph on theright. After delimiting the region, only one ORF (Glyma.10g172700) was identified.Glyma.10g172700was tentatively assignedas the gene encoding MGL and comprises two exons flanking one intron. The DNA sequencing of genes from NH andWI strainsshowed a copia-type retrotransposon inserted into the intron of the gene in the NH strain only.

Plant Physiol. Vol. 183, 2020 947

Met g-Lyase Suppression Leads to High SMM

www.plantphysiol.orgon September 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.

chromatography (Fig. 7A). Subsequently, L-Metreacted with the recombinant protein in the presenceof PLP, and the products were converted into their 3-methyl-2-benzothiazolinone hydrazone derivatives.This derivatization resulted in increased absorptionat 320 nm (Fig. 7B), suggesting the formation of analiphatic carbonyl compound (Esaki and Soda, 1987;Inoue et al., 1995). To confirm its structure, the reactionproduct that was extracted using ethyl acetate was reac-ted with N,O-bis(trimethylsilyl)trifluoroacetamide and

was then analyzed using gas chromatography-massspectrometry (GC-MS). A peak at the retention timeof 9.5 min was assigned as trimethylsilylated 2-ketobutyric acid by comparing its MS profile andretention time with that prepared from a standardcompound (Fig. 7, C and D). Accordingly, we con-cluded that Glyma.10g172700 encodes MGL that cat-alyzes g-elimination of L-Met. We denoted the geneGmMGL1. This reaction had optimal activity at pH 7and followedMichaelis-Menten kinetics, withKm andVmax values of 7.72 mM and 0.55 mmol mg21 min21,respectively (Supplemental Fig. S6).

Comparison of Met Metabolism Genes in FY and NH

BLAST searches forGmMGL1 indicated that the soybeangenome encodes theMGL-like genes Glyma.02g087900 andGlyma.13g001200 (hereafter referred to as GmMGL2 andGmMGL3, respectively). These genes encode proteins withthe amino acid signatures that are conserved among theMGLs described above (Supplemental Fig. S5). Among thethree GmMGLs, GmMGL2 and GmMGL3 showedhigher sequence similarity than the other combinations.The phylogenetic analyses with MGL and MGL-like se-quences found in several plant species indicated thatGmMGL1 is located in a clade different from the one thatGmMGL2 and GmMGL3 belong to (Supplemental Fig.S7; Supplemental Table S2).

The RT-qPCR analyses of the FY seeds showed thatGmMGL1mRNA expression was enhanced at the earlystage of seed maturation (from stages 1 to 2) andremained constant thereafter until the mature greenstage (stage 5; Fig. 8A). However, GmMGL1 expressionwas considerably lower in NH seeds than in FY seedsthroughout seed development and differed little be-tween developmental stages. GmMGL2 and GmMGL3expression levels were transiently induced during stage3, but only in NH seeds, and they were not significantlydifferent between FY and NH cultivars at the otherstages. The MGL activity in crude protein extractsprepared from developing seeds (at stage 4) of NH (5.866 0.81 nmol h21 g21) was significantly lower than thatdetected in FY seeds (12.1 6 2.28 nmol h21 g21; P ,0.05, Student’s t test, n 5 4). GmMGL1 expression wassignificantly lower in the leaves, stems, and roots of NHplants than in the leaves, stems, and roots of FY plantletsat the leaf-expansion stage before flowering (Fig. 8B).Among these, the transcript levels of GmMGL2 andGmMGL3 were highest in the leaves and did not differsignificantly between the soybean cultivars.

Because the genes of Met metabolism are regulatedcoordinately (Liao et al., 2012), we examined the effectsof GmMGL1 suppression on the expression of CGS,which catalyzes a key regulatory step of the Met bio-synthetic pathway (Hesse and Hoefgen, 2003), and ofMMT and HMT, which are directly involved in theformation and decomposition of SMM (Fig. 1; Cohenet al., 2017c). In SoyBase BLAST searches using AtCGS(At3g01120),AtMMT (At5g49810), andAtHMT1 (At3g25900)

Figure 6. Hyperaccumulation of SMM correlates with the insertion of atransposon into the GmMGL1 gene. A, Sizes of DNA fragments thatwere amplified with primers for the full-length coding sequence ofGlyma.10g172700. M, Molecular mass marker (l/HindIII digests); 1,Shinano-Kurakake; 2, Nishiyamahitashi 94-5; 3, Sinanomachi Arase-bara 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. B, Quantities of SMM (top, blackbars) andMet (bottom,white bars) in drymatured seeds of each soybeanline. Data are shown as means 6 SE of three replicates. C, Expressionlevels of Glyma.10g172700 in seeds at the green mature stage (stage 5in Fig. 8). The soybean 20S proteasome subunit (Glyma.06g078500)was used as an internal control in RT-qPCR analyses. Transcript levelsrelative to the internal control are shown asmultiples of the lowest valueof 1. Data are presented as means 6 SE (n 5 3). Significant differencesindicated with different lowercase letters were identified using Tukey’sHSD tests after Box-Cox transformation (P , 0.05).

948 Plant Physiol. Vol. 183, 2020

Teshima et al.

www.plantphysiol.orgon September 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.

as queries, two CGS homologs (Glyma.18g261600and Glyma.09g235400; referred to as GmCGS1 andGmCGS2, respectively), two MMT homologs (Gly-ma.12g163700 and Glyma.16g000200; GmMMT1 andGmMMT2, respectively), and three HMT homologs(Glyma.08g261200,Glyma.19g158800, andGlyma.20g148900;GmHMT1, GmHMT2, and GmHMT3, respectively) wereidentified. RT-qPCR analyses of mRNA expression fromsoybean seeds at different developing stages revealedno significant differences between FY and NH, exceptfor GmCGS1/2, GmHMT2, and GmHMT3 at stage1 (Supplemental Fig. S7).

Administration of Met Causes Accumulation of SMM inDeveloping Seeds

Suppression of GmMGL1 expression in developingsoybean seeds might lead to the accumulation of Met,

which would otherwise be catabolized to ammonia,methanethiol, and 2-ketobutyric acid. One of the alter-native fates of free Met is the formation of SMM via theactivity of MMT (Fig. 1), which is likely to occur indeveloping soybean seeds because of the substantialexpression levels of GmMMT1 and GmMMT2(Supplemental Fig. S8). To examine whether MMTs areactive in developing soybean seeds, we conducted aMet-feeding experiment. We fed free Met solution ontoslices of immature green soybean seeds of the FY andNH cultivars and determined SMM and Met contentsusing liquid chromatography with tandem mass spec-trometry (LC-MS/MS; Fig. 9). Inclusion of 1 or 5 mM

Met in the solution covering the cut surfaces of the FYseeds yielded incremental increases in the SMM con-tents, and after 24 h of treatment, the SMM levels in-creased up to 37.9 and 135 mg g21 for the 1 and 5 mM

Met solutions, respectively. The SMM levels of the NHseeds also showed similar incremental increases, but in

Figure 7. Glyma.10g172700 encodes a functional MGL. A, SDS-PAGE analysis of the recombinant soybean MGL1 protein withan N-terminal His tag. Lanes 1 and 4, Molecular mass markers; 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 of3-methyl-2-benzothiazolinone hydrazone (MBTH) derivatives of the product formed during metabolism of Met by recombinantGmMGL1. C, Chromatograms of trimethylsilylated products of recombinant GmMGL1 and Met from retention times of 8.25 to10.75 min. The solid line represents chromatograms from experiments with the recombinant enzyme, and the broken line showsthose without enzyme (negative control). The peak specifically found with the product formed with recombinant enzyme isshown with the arrow (peak A). D, Mass spectra obtainedwith authentic trimethylsilylated 2-ketobutyric acid (top) andwith peakA (bottom).

Plant Physiol. Vol. 183, 2020 949

Met g-Lyase Suppression Leads to High SMM

www.plantphysiol.orgon September 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.

a more prominent manner, and after 24 h, the SMMlevels increased up to 214 and 316 mg g21 for the 1 and5 mM Met solutions, respectively. The SMM level in theNH seeds treated only with water also significantlyincreased to 80 mg g21 after 24 h. No significant dif-ference in the Met levels was observed for FY and NHseeds treated with 1 mM Met in comparison with thelevels in seeds treatedwith water except those after 24 hwith NH seeds; however, following feeding with 5 mM

Met solution, the Met levels increased significantly inboth FY and NH, with more prominent increases inNH. The highest Met level for FY was 9.71 mg g21 at 8 hand for NH was 27.2 mg g21 at 24 h.

To confirm substrate-product relationships, weperformed feeding experiments using [13C]Met (C5,99 atom %) with NH seeds. Subsequent LC-MS/MSanalyses of SMM in the extract confirmed that it waspredominantly formed from [13C]Met, as indicatedby mass-to-charge ratio (m/z) values of 169.1 and106.1 that were generated from 13C5-labeled SMM(Fig. 9B).

DISCUSSION

Using a molecular genetic approach to locate the al-lele responsible for hyperaccumulation of SMM, wefound that a transposon insertion into the intron ofGmMGL1 is strongly associated with SMM hyper-accumulation in soybean seeds. Expression of theGmMGL1 gene and, accordingly, MGL activity in seedswere suppressed due to the transposon insertion. Un-der these conditions, Met catabolism would be low inseeds, leading toMet accumulation. Because excessMetlevels have been associatedwith various adverse effects

in plant tissues, we hypothesized that surplus Met insoybean seeds with MGL deficiencies was converted tothe better-tolerated compound SMM by MMT activity(Fig. 10). In line with this hypothesis, Met-feeding ex-periments showed that surplus Met was efficientlyconverted into SMM in green mature soybean seedsand that the conversionwasmore prominentwithMGLdeficiency.

The Transposon Insertion Suppresses the Expressionof GmMGL1

The insertion of a transposon into the intron ofGmMGL1 strongly suppressed its mRNA expression,whereas processing of the corresponding precursormRNA through splicing at the inherent positions waslittle affected by the intronic insertion. Intronic insertionof transposons generally has minimal impacts on geneexpression levels or splicing events (Hirsch andSpringer, 2017). For example, insertion of the retro-transposon Ty1-copia, which is approximately 5,000 bpin length, had little impact on transcription with flax(Linum usitatissimum; Galindo-González et al., 2016).Hence, the present marked repressive effects of trans-poson insertion into the intron of GmMGL1 are unique.Alternatively, in a previous study of soybeans, trans-posons in or near a gene were related to increasedCHG/CHH methylation and, consequently, lower ex-pression levels (Kim et al., 2015). Hence, epigeneticregulatory mechanisms likely play roles in the presentrepression of GmMGL1. As such, analyses of DNAmethylation should be one of the next priority researchareas to reveal more details about the mechanisms ofthis type of gene suppression.

Figure 8. Expression ofGmMGL1,GmMGL2, andGmMGL3 in soybean plants. Expression ofGmMGL1, GmMGL2, and GmMGL3 is shown indeveloping seeds (A) and in the leaves, stems, androots (B) of FYand NH soybean cultivars. Sizes ofNH and FY seeds collected for RNA extraction(stages 1–5) are shown in the photograph in A. Thesoybean 20S proteasome subunit (Glyma.06g078500)was used as an internal control in RT-qPCRanalyses. Transcript levels relative to the inter-nal control are shown as multiples of the lowestvalue of 1. Data are presented as means 6 SE

(n 5 3). Significant differences were identifiedusing a two-way ANOVA after Box-Cox trans-formation. Triple asterisks above the symbols inA indicate significant differences between plantlines in each developing stage (P , 0.001,simple main-effect test after two-way ANOVA).Double asterisks indicate significant differencesbetween plant lines in B (MGL1; P , 0.05,simple main-effect test after two-way ANOVA).Different lowercase letters above columns in-dicate significant differences between organs inB (MGL2 andMGL3; P , 0.05, Tukey’s HSD testafter two-way ANOVA).

950 Plant Physiol. Vol. 183, 2020

Teshima et al.

www.plantphysiol.orgon September 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.

Suppression of GmMGL1 Accounts forSMM Hyperaccumulation

In plant tissues, Met levels are tightly regulatedthrough biosynthesis and catabolism (Fig. 1). Higherfree Met contents (in addition to SMM contents) in NHseeds than in FY seeds prompted us to assume thatGmMGL1 participated in controlling free Met levels inseeds. If this is the case, in the absence of substantialMGL activity, as foundwith NH, freeMet levels shouldincrease, and surplusMet could be converted into SMMby the MMT activity in seeds. This scenario showed nofundamental inconsistency with the results obtained inthis study about the Met metabolism of NH. Thefunction of MGL to adjust free Met levels has beendemonstrated with Arabidopsis, in which knockout ofthe AtMGL gene increased free Met contents in leaves,flowers, and seeds (Goyer et al., 2007; Joshi and Jander,2009). Notably, the Arabidopsis knockout mutant con-tained 4.5-fold higher SMM contents in leaves than itsparental wild type; therefore, it is presumed that con-version of surplusMet to SMM is common among plants.In support of this hypothesis, SMM accumulation has

been reported in multiple transgenic plants with highfree Met levels (Kim et al., 2002; Hacham et al., 2008,2017). Taken together, it is suggested that GmMGL1was involved in controlling free Met levels in devel-oping soybean seeds. SMMhyperaccumulation is likelyto be a consequence of suppressed GmMGL1 and asubsequent fail-safe system employingMMT activity toavoid the adverse effect of excess Met.One of the MGL products, 2-ketobutyric acid, is

partly converted to Ile in Arabidopsis, especially underdrought stress (Rébeillé et al., 2006; Joshi and Jander,2009). However, we found no significant difference ineither free or total Ile content between NH and FYseeds, suggesting that GmMGL1 accounted little for Ileformation in developing seeds. On the contrary, dryNH seeds had higher free Thr, Phe, His, and homo-serine levels in addition to increased free Met and SMMlevels. Therefore, the MGL deficiency is likely to causepleiotropic effects on the metabolism of other aminoacids. Accumulation of free amino acids was often ob-served in several transgenic plants generated to en-hance Met levels (Hanafy et al., 2013; Cohen et al., 2014;Huang et al., 2014; Hacham et al., 2017). Accordingly, it

Figure 9. Absorption and conversion of exogenously supplied Met to SMM in a section of a developing soybean seed. A, SMMandMet contents after treating with 0 (white circles), 1 (gray squares), and 5 (black triangles) mMMet. Data are shown as means6SE (n 5 3). Significant differences were identified using a two-way ANOVA after Box-Cox transformation. Different letters abovethe symbols indicate significant differences between Met concentrations in each hour (P , 0.05, Tukey’s HSD tests after simplemain-effect tests). B, Top, Mass spectrum of SMM extracted from the seed sections treated with 5 mM [13C5]Met for 24 h. Thereaction of MMT from [13C5]Met is shown in the inset. The positions of 13C in Met and SMM are shown with asterisks. Bottom,Mass spectrum of nonlabeled SMM. Tentative assignments of molecular and fragment ions are also shown.

Plant Physiol. Vol. 183, 2020 951

Met g-Lyase Suppression Leads to High SMM

www.plantphysiol.orgon September 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.

is suggested that the adverse effect of surplus Met inNH induced a stress response that led to higher Thr,Phe, His, and homoserine levels.

Limited Significance of Phloem Transport of SMM forSMM Hyperaccumulation

It has been reported for several plant species, in-cludingArabidopsis andwheat (Triticum aestivum), thatSMM formed in vegetative tissues is transported toseeds through the phloem (Bourgis et al., 1999; Leeet al., 2008; Frank et al., 2015; Cohen et al., 2017b).Therefore, it was assumed that the phloem trans-portation of SMM formed in vegetative tissues to seedscould also be accountable for the hyperaccumulation ofSMM in NH seeds. The level of SMM in the phloemexudate collected from leaves of NH was higher thanthat found for FY plants. Therefore, transportation ofSMM through the phloem toward the seeds is likely tobe at least partly accountable for the hyper-accumulation of SMM in NH seeds. The SMM levels inthe pods of both NH and FY showed a tendency todecrease during maturation; thus, the transportation ofSMM from the pods to the seeds should also be takeninto consideration. However, it was remarkable that theSMM levels found in the phloem exudate of NH plantswere 291-fold lower than that in Arabidopsis phloemexudate. Concordant with the fact that amino acidlevels in soybean phloem exudate were eightfold lowerthan those in Arabidopsis and wheat (Bourgis et al.,1999), our observation of low levels of SMM in soy-bean phloem exudate prompted us to consider that thecontribution of phloem transport of SMM toward seedsfor SMM hyperaccumulation is not negligible but is

limited. Furthermore, the results of the reciprocalcrossing of NH and FY indicated that maternal aswell as paternal genotypes did not play a substantialrole in determining the seed phenotype of SMMhyperaccumulation, which was caused only when thegenotype of the seeds was homozygous for mgl1, andthus, the involvement of vegetative organs in SMMhyperaccumulation in the seeds of NH is likelylimited.

The extensively lower levels of SMM and Met in thesoybean phloem, compared with levels in the Arabi-dopsis phloem, are noteworthy because the levels offree and total amino acids including Met are more than10-fold higher in soybean seeds than in Arabidopsisseeds (Cohen et al., 2017a). Source-sink transport ofamino acids from vegetative organs to seeds, and in situsynthesis of amino acids in seeds, might be accountablein different ways for the accumulation of amino acids inseeds in these two plant species.

Exogenous Met Is Converted into SMM

The addition ofMet at 1 mM onto developing seeds ofFY had only a slight effect on Met or SMM concentra-tions, probably because Met, supplied exogenously,was appropriately catabolized in part by intrinsicGmMGL1 activity in FY seeds. This Met-catabolizingsystem was, however, overwhelmed by treatmentswith 5 mM Met solution, as indicated by transientincreases in free Met in developing FY seeds, fol-lowed by a significant increase in the amount ofSMM. The accumulation of Met and SMM seemed tobe further emphasized for NH. This result indicatedthat both NH and FY seeds exhibited enoughMMT toconvert Met supplied exogenously into SMM. Theexaggerated responses of the accumulation of Metand SMM in NH seeds could be explained by thelower activity of GmMGL1 in the seeds and the sur-plus Met left behind in the seed tissues being con-verted into SMM byMMT. SMM has been consideredto be a tentative storage form of Met to avoid exces-sive Met concentrations (Mudd and Datko, 1990). Inagreement, the SMM concentrations in soybean seedstreated with 5 mM Met solution were much higherthan the Met concentrations, suggesting that SMM isa safer storage form of Met. In summary, SMMhyperaccumulation was caused exclusively by thesuppression of GmMGL1 that regulates free Metlevels in developing soybean seeds. MMT activity indeveloping soybean seeds should be sufficient toconvert surplus Met into SMM, irrespective of MGLactivity (Fig. 10).

The data presented here indicate that the geneticsuppression of MGL in soybean seeds affects Met me-tabolism, favors the hyperaccumulation of SMM, andprovides further insights into the regulatory mecha-nisms of Met metabolism. This knowledge should betaken into consideration when attempting to modifyMet metabolism in soybean seeds.

Figure 10. A proposed mechanism of hyperaccumulation of SMM insoybean seeds with low MGL activity. A, When the MGL activity issufficient, as in normal soybean seeds, the level of free Met is properlycontrolled. B, When MGL activity is suppressed by transposon inser-tion, as in NH soybean seeds, surplus Met left behind is converted intoSMM, which seems to account for the hyperaccumulation of SMM.HomoCys, Homocysteine; 2KB, 2-ketobutyric acid; SAM, S-adenosyl-Met.

952 Plant Physiol. Vol. 183, 2020

Teshima et al.

www.plantphysiol.orgon September 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.

MATERIALS AND METHODS

Plant Materials

Seeds of the soybean (Glycinemax) cultivarsNH, FY, andWIwere grown andharvested during 2016 in an experimental field at the Nagano Vegetable andOrnamental Crops Experiment Station, Shiojiri City (E 137°579, N 36°069; an-nual mean temperature, 11°C). For the quantification of SMM levels in leaves,pods, and seeds, NH and FY plants were grown and harvested in 2019 in anexperimental field at the Yoshida campus of Yamaguchi University, YamaguchiCity (E 131°479, N 34°159; annual mean temperature, 15°C). To prepare samplesat a similar developmental stage, each organ was collected at slightly differentdates because FY showed a little early-growth phenotype when compared withNH. For collecting seeds for the RT-qPCR analyses and Met-feeding experi-ments, plants were grown and harvested in 2017 and 2019 in an experimentalfield at the Yoshida campus of Yamaguchi University. For extraction of RNAfrom leaves, stems, and roots, plantlets (NH and FY) were germinated withvermiculite and were transplanted to the hydroponic culture system (Kurodaand Ikenaga, 2015) with a 12-h-light (at 27°C)/12-h-dark (22°C) cycle. NH andFY plants were cultured under these conditions for 40 and 34 d, respectively.Seeds of the cultivars Shinano-Kurakake, Nishiyamahitashi 94-5, SinanomachiArasebara Kurakake, Togakushi Morozawa Ganimame, Usuda Zairai, ShinanoMidori, Nishiyamahitashi 94-1, Tousanhitashi 94-1, Ogawa Zairai 5, and ChinoZairai 4 were harvested in 2012 and 2015 from the experimental field at theNagano Vegetable and Ornamental Crops Experiment Station or in 2019 fromthe experimental field at the Yoshida campus of Yamaguchi University.

Determination of SMM Contents

Seed coatswere carefully removed and soybean seeds containing hypocotylswere then powdered using a multibeads shocker (PM2000; Yasui Kikai)equipped with stainless metal cones (MC-0316S; Yasui Kikai) and operated at2,500 rpm for two 30-s periods with a 10-s interval. Powder samples of 20 mgwere then mixed with 1 mL of distilled water containing 50 mg mL21 L-Met-S-methyl d6 sulfonium chloride (d6-SMM; Toronto Research Chemicals) andwere then placed in awater bath sonicator (US-2; SND) for 10min. The resultingsuspensions were centrifuged at 15,000 rpm (20,000g) for 10 min at 4°C. Sub-sequently, 100-mL aliquots were added to Strata C18-E (100 mg mL21; Phe-nomenex), and SMM was eluted twice with 0.5-mL aliquots of distilled water.Elutes were cleared using an Ekicrodisc 3 (0.45 mm, 3 mm; Pall).

LC-MS/MS analyses were performed using an AB Sciex 3200 Q-TRAP LC-MS/MS system equipped with a Prominence UFLC (Shimadzu) in multiplereaction monitoring (MRM) mode with positive electrospray ionization (ESI).Chromatography was conducted using a Discovery HS F5 column (15 cm3 2.1mm, 3 mm; Supelco), and HPLC and MS analyses were performed using pre-viously described conditions (Morisaki et al., 2014). To quantify 13C-labeledSMM formation from [13C]Met (Cambridge Isotope Laboratories), MS analy-ses were performed in the enhanced production mode with m/z 169.2 (for la-beled SMM) or 164.2 (for nonlabeled SMM) using positive ESI with a capillaryvoltage of 4,500 V, an arbitrary source temperature, a curtain gas of 10 (arbitraryunits), ion source gases 1 and 2 of 16 and 0 (arbitrary units), respectively, adeclustering potential of 26 V, and an entrance potential of 2.5 V. The level offree Met was also analyzed using the same LC-MS/MS condition but withdifferentMRM transitions. The detailed parameters for LC-MS/MS analysis areshown in Supplemental Table S3.

To collect phloemexudate, fully expanded leaves ofNHandFYplants at theirseed-filling stage were detached with the base of the petiole under a solution of20 mM EDTA (pH 7), immersed into 0.2 mL of the same solution in 0.5-mLmicrotubes, and placed in humid chambers in the dark at 25°C (Urquhart andJoy, 1981). After 5 h, the EDTA solution was collected and used for the LC-MS/MS analyses as described above to estimate the concentrations of SMM and freeMet. As a comparison, fully expanded rosette leaves of Arabidopsis (Arabidopsisthaliana; ecotype Wassilewskija-0) were used at its seed-filling stage.

Determination of Amino Acid and Protein Contents

Soluble amino acids were extracted from dry seed flour (20 mg) as describedby Hanafy et al. (2013). Flour samples were suspended in 240 mL of 3% (w/v)sulfosalicylic acid and were suspended with vigorous shaking for 30 min. Aftercentrifugation at 12,000g for 10 min at 25°C, precipitates were extracted twomore times as described above. Combined supernatants were then filtered andanalyzed using LC-MS/MS with an ESI interface (Tomita et al., 2016) as

detailed above. An Intrada amino acid column (1003 3 mm internal diameter,3 mm; Imtakt) was used with a column temperature of 40°C. Mobile phaseswere applied at 0.4 mL min21 and comprised solvents A (acetonitrile:formicacid at 100:0.3 [v/v]) and B (0.1 M acetonitrile:ammonium formate at 20:80 [v/v]) at 15% B for 10 min, followed by a linear increase from 15% B to 60% B over15 min, then from 60% B to 100% B over 5 min, and then 100% B for 10 min. Theinjection volume was 4 mL. The MS system was operated in MRM mode usingpositive ESI with a capillary voltage of 3,000 V, a source temperature at 550°C, acurtain gas of 35 (arbitrary units), ion source gases 1 and 2 of 80 and 60 (arbi-trary units), respectively, a declustering potential of 16 V, and an entrancepotential of 5 V. MRM transitions of the precursor to product ions used for thequantification and collision energy are summarized in Supplemental Table S3.Quantification was done using calibration curves constructed using amino acidmixture standard solution (Wako Pure Chemicals) supplemented with homo-serine and homocysteine (Wako Pure Chemicals).

Proteins were extracted from 20-mg samples of soybean flour using 500-mLaliquots of 10 mM Tris HCl (pH 8) containing 2.5% (w/v) SDS and 10 mM 2-mercaptoethanol. Proteins were extracted for 10 min with vigorous vor-texing at the highest speed using a Micro Tube Mixer (MT-360; Tomy Seiko)and subsequent treatment with a water bath sonicator (120 W, 38 kHz; US-2; SND) for 10 min at 25°C. A clear protein solution was obtained aftercentrifugation at 20,000g for 20 min at 4°C. Protein contents were deter-mined using Protein Assay (Bio-Rad). After the separation of proteins withSDS-PAGE, each protein band was quantified using ImageJ 1.48v (http://imagej.nih.gov/ij).

Total amino acids were determined according to the method employed byIshimoto et al. (2009). In brief, 10mg of soybean seed flourwas hydrolyzedwith1 mL of 6 N HCl at 100°C for 22 h under argon. The dried hydrolysate wasdissolved in 0.02 N HCl and served to LC-MS/MS analysis, as describedabove. To analyze total Cys and Met, the flour was oxidized with 1 mL ofperformic acid at 0°C for 16 h to give cysteic acid and Met sulfone prior toacid hydrolysis.

Analyses of QTLs for SMM Contents

RILs, including 155F5 lines,weredeveloped fromasingle seeddescendant ofthe cross between NH and WI. Total DNA extraction and linkage map con-struction by simple sequence repeat markers (WSGP version 2) were performedas described previously (Fujii et al., 2018). SMM contents in each RIL werequantified using bulked F6 seeds that were derived from the F5 individual.Because SMM contents varied widely among RILs, QTL analysis was con-ducted with the common logarithm value for contents.

QTL analyses were performed using composite interval mapping, asimplemented inQTLCartographer 2.5 software (Wang et al., 2005). The genomewas scanned at 1-cM intervals. One thousand permutation tests were con-ducted to determine the threshold value of the limit of detection score.

Mapping of Responsible Genes

F6 and F7 progeny of parent individuals with hetero genotypes in thechromosomal region that corresponded with QTLs were used for fine geneticmapping of responsible genes. Initial QTL analyses indicated a region of around486 kb ranging from Satt477 and Satt592 on chromosome 10. A population of F7progeny was then used to delimit the locus using marker genotyping and SMMquantification. Genemappingwas performed using the BARCSOYSSRmarkers(BSSR) described by Song et al. (2010). The tail sequence CACGACGTTGTAAAACGACwas added to the 59 end of the reverse primer, and oligonucleotidesthat were complementary to tail sequences were fluorescently labeled with 6-FAM, VIC, NED, and PET (Thermo Fisher Scientific) before addition to PCRsolutions. PCRs and PCR fragment length analyses were conducted followingFujii et al. (2018). Only one ORF (i.e. Glyma.10g172700) was identified in theregion after the second round of fine-mapping.

cDNA Cloning and Expression of Recombinant Proteins

Total RNA was extracted from developing seeds of WI using RNeasy PlantMini Kits (Qiagen) according to the manufacturer’s instructions. DNA wasdegraded using DNA-free Kits (Ambion, Thermo Fisher Scientific), and cDNAwas synthesized using SuperScript VILO cDNA Synthesis Kits (Invitrogen).Subsequently, Glyma.10g172700 (GmMGL1) cDNA was PCR amplified usingprimers for 59 and 39 ends of the translation initiation site (Supplemental Table

Plant Physiol. Vol. 183, 2020 953

Met g-Lyase Suppression Leads to High SMM

www.plantphysiol.orgon September 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.

S4). The resulting PCR products were cloned into pGEM T-easy vectors(Promega) for sequencing. PCR products were then subcloned into the EcoRI-XhoI site of the pET24a vector (Merck), and the resulting plasmid was trans-fected into Escherichia coli Rosetta2 (DE3) pLysS cells (Merck). Cells were sub-sequently grown in Luria broth supplemented with kanamycin (50 mg mL21)and chloramphenicol (30 mg mL21) at 37°C to an OD of 0.6 to 0.8 at 600 nm.After chilling the cultures on ice for 15 min, isopropyl b-D-1-thio-galactosylpyranoside was added to a concentration of 1mM, and cells were thencultured at 30°C for 16 h.

Cells from 50-mL cultures were recovered by centrifugation at 4,000g for20 min at 4°C and were resuspended in 5 mL of 100 mM potassium phosphatebuffer (pH 7.5) containing 0.01% (w/v) DTT and 1.3 mM PLP. After the additionof 5-mL aliquots of 100 mM phenylmethane sulfonyl fluoride and 50 mg mL2

1 lysozyme, suspensions were kept on ice for 15 min, and cells were then dis-rupted using a tip-type ultrasonic disruptor (UD-211; Tomy Seiko). After cen-trifugation at 12,000g for 10 min, supernatants were directly applied to acolumn (2 mL) of Ni-NTA agarose (Nacalai Tesque) that had been equili-brated with 100 mM potassium phosphate buffer (pH 7.5) containing 0.01%(w/v) DTT and 10 mM PLP. The column was then washed with 10 mL of thesame buffer containing 10 mM imidazole, and His-tagged recombinantproteins were eluted with 10 mL of the same buffer containing 250 mM

imidazole. Active fractions were finally combined and desalted using a PD-10 column (GE Healthcare).

Enzyme Assays

MGL enzyme assays were performed according to previous reports (Esakiand Soda, 1987; Takakura et al., 2004). Given volumes of purified enzyme so-lutionweremixedwith 100mMpotassiumphosphate buffer (pH 7.5) containing10 mM PLP and 40 mM L-Met and were incubated at 30°C with gentle shaking.Aliquots of 2 mLwere taken every 2min andwere added to 100mL of 50% (w/v)TCA. After centrifugation at 20,000g for 8 min at 25°C, 0.8-mL supernatantswere mixed with 1.6-mL aliquots of 1 M sodium acetate buffer (pH 5) and0.6-mL aliquots of 0.1% (w/v) MBTH. Reaction tubes were then tightly closedand incubated at 50°C for 40 min. The MGL product 2-ketobutyric acid wasquantified according to A278, which is derived from the MBTH derivative of2-ketobutyric acid. The absorbance at 0 min was subtracted from later mea-surements, and a calibration curvewas generated using authentic 2-ketobutyricacid (Sigma-Aldrich). The structure of 2-ketobutyric acid was confirmed usingGC-MS after converting the acid into a trimethylsilylated product (Gonda et al.,2013). After incubating the recombinant enzyme with L-Met and PLP overnightin a total volume of 3 mL, reactions were terminated by adding 40-mL aliquotsof 6 N HCl. Products were then extracted in 2 mL of ethyl acetate, and extractswere washed once with 1 mL of water prior to removing the solvent under astream of nitrogen gas. After confirming complete dryness, extracts were in-cubated with 100-mL aliquots of anhydrous pyridine at 25°C for 90 min.Thereafter, 100-mL aliquots of O-bis(trimethylsilyl)trifluoroacetamide (TokyoChemical Industry) were added and incubated for 90 min. GC-MS wasperformed using a QP-5050 (Shimadzu) instrument equipped with a0.25-mm 3 30-m DB-5MS column (film thickness, 0.25 mm; Restek). Thecolumn temperature was programmed as follows: 50°C for 1 min, in-creasing by 5°C min21 to 120°C, then by 20°C min21 to 280°C, and thenmaintenance at 280°C for 1 min. The carrier gas (He) was delivered at a flowrate of 30 kPa. Injector and interface temperatures were 240°C and 300°C,respectively. The mass detector was operated in electron impact mode with anionization energy of 70 eV. Compounds were assigned by comparingMS profilesand retention times with those of trimethylsilyl-derivatized 2-ketobutyric acidthat was prepared separately.

TodetermineMGLactivity indeveloping soybean seeds, the seedsofNHandFY at seed developmental stage 4 (compare with the photograph in Fig. 8) werehomogenized with 4 volumes of 50 mM sodium phosphate buffer (pH 7.5)containing 5% (w/v) sorbitol, 10 mM DTT, 5 mM sodium metabisulfite, and2.5 mM PLP. After centrifugation at 20,000g for 20 min at 4°C, the cleared su-pernatant (0.5 mL) was mixed with 0.25 mL of 0.2 M Met in a buffer (50 mM

sodium phosphate, pH 7.5, containing 2.5 mM PLP) in a total volume of4.5 mL and incubated at 30°C with a shaking water bath for 17 h. After thereaction, the reaction mixture was acidified by adding 66.6 mL of 6 N HCl,and then the products were extracted with 4 mL of ethyl acetate. Afterwashing the ethyl acetate extract with 1 mL of saturated NaCl solution, theextract was used for derivatization with O-bis(trimethylsilyl)tri-fluoroacetamide as described above. The amount of 2-ketobutyric acid wasdetermined using GC-MS analysis and a calibration curve constructed with

authentic 2-ketobutyric acid. Molecular ion chromatograms with m/z 73and 115 were used for quantification.

Genomic PCR and RT-qPCR Analysis

GenomicDNAwas isolated according toHanafy et al. (2013). Total RNAwasisolated using the Qiagen RNeasy Plant Mini Kit according to the manufac-turer’s instruction. Total RNA (0.25 mg) was then reverse transcribed with2.5 mM aliquots of oligo(dT)15 primer (Invitrogen) and ReverTra Ace (derivedfromMoloney murine leukemia virus reverse transcriptase; Toyobo) accordingto the manufacturer’s instructions. RT-qPCR was performed with an Eco Real-Time PCR System (Illumina). cycle threshold (Ct) values for the genes of interestwere normalized to means of the reference gene for the 20S proteasome subunitb (Glyma.06g078500; Pereira Lima et al., 2017). Expression levels were calcu-lated as relative amounts using DDCt values. The lowest DDCt value in eachexperiment was set at 1.

Homologs of MGL, CGS, MMT, and HMT were searched using BLASTPanalysis on SoyBase (https://soybase.org/) with GmMGL1 (Glyma.10g17200),AtCGS (At3g01120), AtMMT (At5g49810), and AtHMT1 (At3g25900) asqueries, respectively. Primers for genomic PCR and RT-qPCR are shown inSupplemental Table S4.

Met Feeding

Pods harboring the seeds of developmental stage 2 (compare with thephotograph in Fig. 8) were collected and were gently removed with their seedcoats. Thin sections of 1-mm thickness were excised at the short axis using arazor blade, and they were immediately placed on a sheet of Parafilm (BemisFlexible Packaging) in a glass petri dish. The inner surface of the petri dish wascovered with a moistened paper towel. Fifty-microliter aliquots of 0, 1, or 5 mM

Met or [13C]Met in water were then placed on the surfaces of seed sections at11 AM, and the petri dish was immediately closed and incubated at 25°C for 0, 4,8, and 24 h under light/dark conditions of 14 h of light (8 AM–10 PM)/10 h of dark(10 PM–8 AM). To determine Met and SMM concentrations, sections were care-fully washed with water and were mixed with 1-mL aliquots of distilled watercontaining 1 mg mL21 d6-SMM. Sections were then homogenized in a mortar,and homogenates were placed in a water bath sonicator (US-2; SND) for 10 minto facilitate extraction of Met and SMM. Suspensions were centrifuged at15,000 rpm (20,000g) for 10 min at 4°C, and 100-mL aliquots were applied to aStrata C18-E (100 mg mL21) cartridge (Phenomenex). Met and SMM wereeluted twice with 0.5 mL of distilled water, and eluted solutions were clearedwith Ekicrodisc 3 (0.45 mm, 3 mm; Pall) prior to LC-MS/MS analyses asdescribed above.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL datalibraries under accession number MK887190 (Glyma.10g172700 in NH).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Protein contents and profile of soybean cultivarsused in this study.

Supplemental Figure S2. The inheritance pattern of SMM contents.

Supplemental Figure S3. Appearance of local soybean cultivars collectedin Nagano Prefecture, Japan (bar 5 3 cm).

Supplemental Figure S4. PCR with complementary and genomic DNA.

Supplemental Figure S5. Amino acid sequence alignments of MGLs fromsoybean, melon, Arabidopsis, potato, Pseudomonas putida, and Streptomy-ces avermitilis.

Supplemental Figure S6. Properties of recombinant GmMGL1.

Supplemental Figure S7. Phylogenetic analysis of GmMGL1 and its re-lated MGL-like proteins.

Supplemental Figure S8. Expression levels of GmCGS1/2, GmMMT1,GmMMT2, GmHMT1, GmHMT2, and GmHMT3 in developing FY andNH seeds from stages 1 to 5 (refer to Fig. 8).

954 Plant Physiol. Vol. 183, 2020

Teshima et al.

www.plantphysiol.orgon September 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.

Supplemental Table S1. Total amino acid contents in the dry seeds of theFY and NH soybean cultivars.

Supplemental Table S2. Protein sequences used to construct thephylogenetic tree.

Supplemental Table S3. MRM transitions and MS parameters used todetect amino acids.

Supplemental Table S4. Primers used in this study.

ACKNOWLEDGMENTS

We thank Shiori Yamanaka, Yuumi Miyazaki, and Mai Nanri for their helpconducting the experiments.

Received March 2, 2020; accepted April 16, 2020; published April 28, 2020.

LITERATURE CITED

Amir R, Cohen H, Hacham Y (2019) Revisiting the attempts to fortifymethionine content in plant seeds. J Exp Bot 70: 4105–4114

Bourgis F, Roje S, Nuccio ML, Fisher DB, Tarczynski MC, Li C,Herschbach C, Rennenberg H, Pimenta MJ, Shen TL, et al (1999)S-Methylmethionine plays a major role in phloem sulfur transport and issynthesized by a novel type of methyltransferase. Plant Cell 11:1485–1498

Cohen H, Amir R (2017) Dose-dependent effects of higher methioninelevels on the transcriptome and metabolome of transgenic Arabidopsisseeds. Plant Cell Rep 36: 719–730

Cohen H, Hacham Y, Matityahu I, Amir R (2017a) Elucidating the effect ofhigher expression level of cystahionine g-synthase on methionine con-tents in transgenic Arabidopsis, soybean and tobacco seeds. In LJ DeKok, M Hawkesford, S Haneklaus, and E Schnug, eds, Sulfur Metabo-lism in Higher Plants: Fundamental, Environmental and AgriculturalAspects. Springer, Cham, Switzerland, pp 39–48

Cohen H, Hacham Y, Panizel I, Rogachev I, Aharoni A, Amir R (2017b)Repression of CYSTATHIONINE g-SYNTHASE in seeds recruits theS-methylmethionine cycle. Plant Physiol 174: 1322–1333

Cohen H, Israeli H, Matityahu I, Amir R (2014) Seed-specific expression ofa feedback-insensitive form of CYSTATHIONINE-g-SYNTHASE inArabidopsis stimulates metabolic and transcriptomic responses associ-ated with desiccation stress. Plant Physiol 166: 1575–1592

Cohen H, Salmon A, Tietel Z, Hacham Y, Amir R (2017c) The relativecontribution of genes operating in the S-methylmethionine cycle tomethionine metabolism in Arabidopsis seeds. Plant Cell Rep 36: 731–743

Dancs G, Kondrák M, Bánfalvi Z (2008) The effects of enhanced methio-nine synthesis on amino acid and anthocyanin content of potato tubers.BMC Plant Biol 8: 65

Esaki N, Soda K (1987) L-Methionine g-lyase from Pseudomonas putida andAeromonas. Methods Enzymol 143: 459–465

Frank A, Cohen H, Hoffman D, Amir R (2015) Methionine andS-methylmethionine exhibit temporal and spatial accumulation patternsduring the Arabidopsis life cycle. Amino Acids 47: 497–510

Fujii K, Sayama T, Takagi K, Kosuge K, Okano K, Kaga A, Ishimoto M(2018) Identification and dissection of single seed weight QTLs byanalysis of seed yield components in soybean. Breed Sci 68: 177–187

Galili G, Amir R, Fernie AR (2016) The regulation of essential amino acidsynthesis and accumulation in plants. Annu Rev Plant Biol 67: 153–178

Galindo-González L, Mhiri C, Grandbastien MA, Deyholos MK (2016)Ty1-copia elements reveal diverse insertion sites linked to polymor-phisms among flax (Linum usitatissimum L.) accessions. BMC Genomics17: 1002

Gallardo K, Firnhaber C, Zuber H, Héricher D, Belghazi M, Henry C,Küster H, Thompson R (2007) A combined proteome and transcriptomeanalysis of developing Medicago truncatula seeds: Evidence for meta-bolic specialization of maternal and filial tissues. Mol Cell Proteomics 6:2165–2179

Gonda I, Lev S, Bar E, Sikron N, Portnoy V, Davidovich-Rikanati R,Burger J, Schaffer AA, Tadmor Y, Giovannonni JJ, et al (2013) Ca-tabolism of L-methionine in the formation of sulfur and other volatiles inmelon (Cucumis melo L.) fruit. Plant J 74: 458–472

Goyer A, Collakova E, Shachar-Hill Y, Hanson AD (2007) Functionalcharacterization of a methionine g-lyase in Arabidopsis and its impli-cation in an alternative to the reverse trans-sulfuration pathway. PlantCell Physiol 48: 232–242

Hacham Y, Matityahu I, Amir R (2017) Transgenic tobacco plants having ahigher level of methionine are more sensitive to oxidative stress. PhysiolPlant 160: 242–252

Hacham Y, Matityahu I, Schuster G, Amir R (2008) Overexpression ofmutated forms of aspartate kinase and cystathionine g-synthase in to-bacco leaves resulted in the high accumulation of methionine andthreonine. Plant J 54: 260–271

Hanafy MS, Rahman SM, Nakamoto Y, Fujiwara T, Naito S, Wakasa K,Ishimoto M (2013) Differential response of methionine metabolism intwo grain legumes, soybean and azuki bean, expressing a mutated formof Arabidopsis cystathionine g-synthase. J Plant Physiol 170: 338–345

Hesse H, Hoefgen R (2003) Molecular aspects of methionine biosynthesis.Trends Plant Sci 8: 259–262

Hirsch CD, Springer NM (2017) Transposable element influences on geneexpression in plants. Biochim Biophys Acta Gene Regul Mech 1860:157–165

Huang T, Joshi V, Jander G (2014) The catabolic enzyme methioninegamma-lyase limits methionine accumulation in potato tubers. PlantBiotechnol J 12: 883–893

Inoue H, Inagaki K, Sugimoto M, Esaki N, Soda K, Tanaka H (1995)Structural analysis of the L-methionine g-lyase gene from Pseudomonasputida. J Biochem 117: 1120–1125

Ishimoto M, Rahman SM, Hanafy MS, Khalafalla MM, El-Shemy HA,Nakamoto Y, Kita Y, Takanashi K, Matsuda F, Murano Y, et al (2009)Evaluation of amino acid content and nutritional quality of transgenicsoybean seeds with high-level tryptophan accumulation. Mol Breeding25: 313–326

Joshi V, Jander G (2009) Arabidopsis methionine g-lyase is regulated ac-cording to isoleucine biosynthesis needs but plays a subordinate role tothreonine deaminase. Plant Physiol 151: 367–378

Kim J, Lee M, Chalam R, Martin MN, Leustek T, Boerjan W (2002)Constitutive overexpression of cystathionine g-synthase in Arabidopsisleads to accumulation of soluble methionine and S-methylmethionine.Plant Physiol 128: 95–107

Kim KD, El Baidouri M, Abernathy B, Iwata-Otsubo A, Chavarro C,Gonzales M, Libault M, Grimwood J, Jackson SA (2015) A comparativeepigenomic analysis of polyploidy-derived genes in soybean and com-mon bean. Plant Physiol 168: 1433–1447

Krishnan HB, Jez JM (2018) The promise and limits for enhancing sulfur-containing amino acid content of soybean seed. Plant Sci 272: 14–21

Kumar P, Jander G (2017) Concurrent overexpression of Arabidopsisthaliana cystathionine g-synthase and silencing of endogenous methio-nine g-lyase enhance tuber methionine content in Solanum tuberosum.J Agric Food Chem 65: 2737–2742

Kuroda M, Ikenaga S (2015) Single-tube hydroponics as a novel idea forsmall-scale production of crop seed in a plant incubator. Biosci Bio-technol Biochem 79: 63–67

Lee M, Huang T, Toro-Ramos T, Fraga M, Last RL, Jander G (2008) Re-duced activity of Arabidopsis thaliana HMT2, a methionine biosyntheticenzyme, increases seed methionine content. Plant J 54: 310–320

Liao D, Pajak A, Karcz SR, Chapman BP, Sharpe AG, Austin RS, Datla R,Dhaubhadel S, Marsolais F (2012) Transcripts of sulphur metabolicgenes are co-ordinately regulated in developing seeds of common beanlacking phaseolin and major lectins. J Exp Bot 63: 6283–6295

Liu B, Kanazawa A, Matsumura H, Takahashi R, Harada K, Abe J (2008)Genetic redundancy in soybean photoresponses associated with dupli-cation of the phytochrome A gene. Genetics 180: 995–1007

Martel A, Bouthier de la Tour C, Le Goffic F (1987) Pyridoxal 59 phosphatebinding site of Escherichia coli beta cystathionase and cystathioninegamma synthase comparison of their sequences. Biochem Biophys ResCommun 147: 565–571

Morisaki A, Yamada N, Yamanaka S, Matsui K (2014) Dimethyl sulfide asa source of the seaweed-like aroma in cooked soybeans and correlationwith its precursor, S-methylmethionine (vitamin U). J Agric Food Chem62: 8289–8294

Mudd SH, Datko AH (1990) The S-methylmethionine cycle in Lemna pau-cicostata. Plant Physiol 93: 623–630

Plant Physiol. Vol. 183, 2020 955

Met g-Lyase Suppression Leads to High SMM

www.plantphysiol.orgon September 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.

Pereira Lima JJ, Buitink J, Lalanne D, Rossi RF, Pelletier S, da Silva EAA,Leprince O (2017) Molecular characterization of the acquisition of lon-gevity during seed maturation in soybean. PLoS ONE 12: e0180282

Ranocha P, McNeil SD, Ziemak MJ, Li C, Tarczynski MC, Hanson AD(2001) The S-methylmethionine cycle in angiosperms: Ubiquity, antiq-uity and activity. Plant J 25: 575–584

Rébeillé F, Jabrin S, Bligny R, Loizeau K, Gambonnet B, Van Wilder V,Douce R, Ravanel S (2006) Methionine catabolism in Arabidopsis cellsis initiated by a g-cleavage process and leads to S-methylcysteine andisoleucine syntheses. Proc Natl Acad Sci USA 103: 15687–15692

Sato D, Nozaki T (2009) Methionine gamma-lyase: The unique reactionmechanism, physiological roles, and therapeutic applications againstinfectious diseases and cancers. IUBMB Life 61: 1019–1028

Schmutz J, Cannon SB, Schlueter J, Ma J, Mitros T, Nelson W, Hyten DL,Song Q, Thelen JJ, Cheng J, et al (2010) Genome sequence of the pa-laeopolyploid soybean. Nature 463: 178–183

Song Q, Jia G, Zhu Y, Grant D, Nelson RT, Hwang EY, Hyten DL, CreganPB (2010) Abundance of SSR motifs and development of candidate

polymorphic SSR markers (BARCSOYSSR_1.0) in soybean. Crop Sci 50:1950–1960

Song S, Hou W, Godo I, Wu C, Yu Y, Matityahu I, Hacham Y, Sun S, HanT, Amir R (2013) Soybean seeds expressing feedback-insensitive cysta-thionine g-synthase exhibit a higher content of methionine. J Exp Bot 64:1917–1926

Takakura T, Mitsushima K, Yagi S, Inagaki K, Tanaka H, Esaki N, SodaK, Takimoto A (2004) Assay method for antitumor L-methionine g-ly-ase: Comprehensive kinetic analysis of the complex reaction withL-methionine. Anal Biochem 327: 233–240

Tomita R, Todoroki K, Maruoka H, Yoshida H, Fujioka T, Nakashima M,Yamaguchi M, Nohta H (2016) Amino acid metabolomics using LC-MS/MS: Assessment of cancer-cell resistance in a simulated tumor mi-croenvironment. Anal Sci 32: 893–900

Urquhart AA, Joy KW (1981) Use of phloem exudate technique in the studyof amino acid transport in pea plants. Plant Physiol 68: 750–754

Wang S, Basten CJ, Zeng ZB (2005) Windows QTL Cartographer 25. De-partment of Statistics, North Carolina State University, Raleigh, NC

956 Plant Physiol. Vol. 183, 2020

Teshima et al.

www.plantphysiol.orgon September 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.