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Journal of Plant Physiology 170 (2013) 338– 345

Contents lists available at SciVerse ScienceDirect

Journal of Plant Physiology

jou rna l h om epa ge: www.elsev ier .com/ locate / jp lph

olecular biology

ifferential response of methionine metabolism in two grain legumes, soybeannd azuki bean, expressing a mutated form of Arabidopsis cystathionine-synthase

oemen S. Hanafya,1, Shaikh M. Rahmanb,2, Yumi Nakamotoa, Toru Fujiwarac, Satoshi Naitod,yo Wakasae, Masao Ishimotoa,f,∗

National Agricultural Research Center for Hokkaido Region, 1 Hitsujigaoka, Toyohira, Sapporo, Hokkaido 062-8555, JapanNational Agricultural Research Center for Western Region, 6-12-1 Nishifukatsu, Fukuyama, Hiroshima 721-8514, JapanGraduate School of Agricultural and Life Sciences, University of Tokyo, Yayoi 1-1-1, Bunkyo, Tokyo 113-8657, JapanGraduate School of Agriculture, Hokkaido University, Kita 9 Nishi 9, Kita, Sapporo 060-8589, JapanGenome Research Center, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya, Tokyo 156-8502, JapanNational Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan

r t i c l e i n f o

rticle history:eceived 14 June 2012eceived in revised form 19 October 2012ccepted 19 October 2012

eywords:zuki beanystathionine �-synthaseethionine biosynthesis

oybean

a b s t r a c t

Methionine (Met) is a sulfur-containing amino acid that is essential in mammals and whose low abun-dance limits the nutritional value of grain legumes. Cystathionine �-synthase (CGS) catalyzes the firstcommitted step of Met biosynthesis, and the stability of its mRNA is autoregulated by the cytosolicconcentration of S-adenosyl-l-methionine (SAM), a direct metabolite of Met. The mto1-1 mutant ofArabidopsis thaliana harbors a mutation in the AtCGS1 gene that renders the mRNA resistant to SAM-dependent degradation and therefore results in the accumulation of free Met to high levels in youngleaves. To manipulate Met biosynthesis in soybean and azuki bean, we introduced the AtCGS1 mto1-1gene into the two grain legumes under the control of a seed-specific glycinin gene promoter. Transgenicseeds of both species accumulated soluble Met to levels at least twice those apparent in control seeds.

ransgenic plant However, the increase in free Met did not result in an increase in total Met content of the transgenic seeds.In transgenic azuki bean seeds, the amount of cystathionine, the direct product of CGS, was markedlyincreased whereas the total content of Met was significantly decreased compared with control seeds.Similar changes were not detected in soybean. Our data suggest that the regulation of Met biosynthesisdiffers between soybean and azuki bean, and that the expression of AtCGS1 mto1-1 differentially affectsthe metabolic stability of sulfur amino acids and their metabolites in the two grain legumes.

ntroduction

The sulfur-containing amino acid methionine (Met) is limiting inrain legumes with regard to their nutritional value for humans and

Abbreviations: CaMV, cauliflower mosaic virus; CGS, cystathionine �-synthase;FP, green fluorescent protein; OPH, O-phosphohomoserine; PAGE, polyacrylamideel electrophoresis; PCR, polymerase chain reaction; RT, reverse transcription; SAM,-adenosyl-l-methionine; TS, threonine synthase.∗ Corresponding author at: National Institute of Agrobiological Sciences, 2-1-2annondai, Tsukuba, Ibaraki 305-8602, Japan. Tel.: +81 29 8387452; fax: +81 29387452.

E-mail address: ishimoto@affrc.go.jp (M. Ishimoto).1 Permanent address: Plant Biotechnology Department, National Research Cen-

er, Tahrir Str. Dokki, Cairo 12311, Egypt. Present address: Faculty of Science andumanities, Biology Department, Salman bin Abdulaziz University, 11942 Al-kharj,audi Arabia.2 Present address: Fukuoka Agricultural Research Center, Yoshiki 587, Chikushino,

ukuoka 818-8549, Japan.

176-1617/$ – see front matter © 2012 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.jplph.2012.10.018

© 2012 Elsevier GmbH. All rights reserved.

nonruminant livestock. Mammals, unlike plants, cannot synthesizeMet and therefore must obtain it from their diets. It is thereforedesirable to increase the level of Met in foods with a low Met con-tent such as soybean. Cystathionine �-synthase (CGS) catalyzes thefirst committed step in Met biosynthesis, and the stability of itsmRNA is autoregulated by S-adenosyl-l-methionine (SAM), a directmetabolite of Met (Chiba et al., 2003).

Met is synthesized from aspartate (Asp) via a pathway sharedin part with lysine (Lys) and threonine (Thr) (Hesse and Hoefgen,2003; Amir, 2008; Fig. 1). In higher plants, three consecutive reac-tions beginning with O-phosphohomoserine (OPH) and catalyzedby CGS, cystathionine �-lyase (CBL), and methionine synthase(MS) are specific for Met biosynthesis (Ravanel et al., 1998). OPHserves as a substrate for both Thr and Met biosynthesis (Datko

et al., 1974; Giovanelli, 1987), and the biosynthesis of Met in higherplants is thus affected by competition between CGS and threoninesynthase (TS) for their common substrate (Thompson et al., 1982)(Fig. 1). This branch point between CGS and TS is thought to be a

M.S. Hanafy et al. / Journal of Plant Ph

2,3-Dihyd rodipicolinate

Aspart ate

β-Aspartyl phosphate

Aspart ic β-semia ldehyd e

5 steps

Lysine

Homoserine

Cysta thionine

Homocyste ine

Methioni ne

Protein synt hesis

Cyste ineOPH

SAM

Threonine

AK

TS

CGS

MS

CBL

-

Fig. 1. Biosynthetic pathway of Met from Asp. Cystathionine �-synthase (CGS)catalyzes the first committed step in Met biosynthesis, with the stability of itsmRNA being reduced in response to overaccumulation of soluble S-adenosyl-l-methionine (SAM), a direct metabolite of Met. Abbreviations within ovals representenzymes responsible for catalysis of the indicated reactions: AK, aspartate kinase;TO

d2mtty

pa1CmEa(tarmorAspmm2rotio

10 shallow vertical cuts were made on the adaxial surface of

S, threonine synthase; CBL, cystathionine �-lyase; MS, methionine synthase. OPH,-phosphohomoserine.

eterminant of carbon flux into Met and Thr synthesis (Amir et al.,002; Galili and Höfgen, 2002; Hesse and Hoefgen, 2003). Theto2-1 (methionine overaccumulation 2-1) mutant of Arabidopsis

haliana, for example, which harbors a single-base mutation inhe TS gene, was found to show a 22-fold increase in Met levels inoung rosettes (Bartlem et al., 2000).

In addition to its role as a constituent of proteins, Met serves as arecursor of the primary methyl-group donor SAM, of polyamines,nd of the ripening hormone ethylene in plants (Azevedo et al.,997; Ravanel et al., 1998) (Fig. 1). The AtCGS1 gene, which encodesGS in A. thaliana, was identified by complementation of a CGSutant strain (metB) of Escherichia coli (Kim and Leustek, 1996).

xpression of AtCGS1 is subject to negative feedback regulationt the level of mRNA stability in response to Met applicationChiba et al., 1999; Suzuki et al., 2001). The mto1-1 mutant of A.haliana harbors a point mutation within the first exon of AtCGS1nd accumulates high levels of soluble (free) Met in leaves as aesult of a defect in the negative feedback regulation of AtCGS1RNA stability (Inaba et al., 1994; Chiba et al., 1999). More-

ver, antisense RNA-mediated repression of AtCGS1 expressionesulted in growth retardation and morphological abnormalities in. thaliana, presumably as a consequence of the inability to synthe-ize Met (Kim and Leustek, 2000). Conversely, transgenic A. thalianalants overexpressing AtCGS1 under the control of the cauliflowerosaic virus (CaMV) 35S RNA promoter manifested increased accu-ulation of Met at specific stages of development (Kim et al.,

002). Furthermore, the expression of AtCGS1 in transgenic alfalfaesulted in increased levels of Met, of S-methyl-l-methionine, andf protein-bound Met in the water-soluble protein fraction of up

o 32-fold, 19-fold, and 2.2-fold, respectively, compared with thosen wild-type plants (Avraham et al., 2005). However, constitutiveverexpression of the potato CGS gene in potato plants resulted

ysiology 170 (2013) 338– 345 339

in a substantial increase in enzyme activity but did not lead toincreased accumulation of Met (Kreft et al., 2003), suggesting eitherthat CGS in potato does not play a prominent role in regulation ofMet levels or that the extent of overexpression was not sufficient totrigger a change in Met metabolism. The CGS gene in potato doesnot appear to contain sufficient elements for posttranscriptionalregulation (Amir, 2008), possibly explaining the discrepancy withthe results obtained with plants overexpressing of AtCGS1.

Modification of Met biosynthesis in plants has important agro-nomical implications, given that the nutritional value of crops suchas grain legumes is limited by their low Met content (Müntz et al.,1998; Tabe and Higgins, 1998; de Lumen et al., 1999). We havenow introduced genomic DNA containing AtCGS1 from the mto1-1mutant into two important grain legumes, soybean and azuki bean,in an attempt to alter Met levels of the edible seeds. The mto1-1 allele corresponds to one of the most stringent mto1 mutations(Chiba et al., 1999; Ominato et al., 2002). Expression of the mutantAtCGS1 gene gave rise to different amino acid profiles in the twograin legumes.

Materials and methods

Construction of the transformation vector

The modified coding region for jellyfish green fluorescent pro-tein [sGFP(S65T), 0.8 kb] with a flanking CaMV 35S RNA promotersequence (P35S) and 3′ terminator sequence of the nopaline syn-thase gene (Tnos) was excised from pTH-2 (Chiu et al., 1996)and inserted between the EcoRI and PacI sites of pZH2 (Kurodaet al., 2010), which is a derivative of the binary vector pPZP202(Hajdukiewicz et al., 1994), thereby generating pZHG. The SalI sitebetween P35S and sGFP(S65T) as well as the NotI and PstI sitesbetween sGFP(S65T) and Tnos were digested and filled in, and themulticloning site from pBluescript II (Stratagene, La Jolla, CA) wasintroduced between the expression cassettes for the hygromycinphosphotransferase gene (hpt) and sGFP(S65T), thereby yieldingpZHG(SK). AtCGS1 genomic DNA from the A. thaliana mto1-1 mutantwas cloned as a 3.3-kb BamHI-XbaI fragment in the correspond-ing restriction enzyme sites of pZHG(SK). The promoter sequenceof the soybean glycinin A2B1a subunit gene (Pgy2) was obtainedas a HindIII-PstI fragment from pHVS (El-Shemy et al., 2004) andplaced upstream of AtCGS1 mto1-1 to confer seed-specific expres-sion (Fig. 2). The resultant construct, pTF519, was then immobilizedin Agrobacterium tumefaciens strain EHA105 (Hood et al., 1993).

Soybean transformation

Two soybean [Glycine max (L.) Merrill] varieties, Misuzudaizuand Bert, were selected because of their high capacity for regen-eration from cotyledonary node explants. Tissue culture andtransformation were performed as previously described (Zhanget al., 1999; Olhoft et al., 2003), with some modifications. Matureseeds were surface-sterilized by overnight exposure to chlorinegas (Di et al., 1996) and were allowed to germinate for 5 dayson B5 medium (Gamborg et al., 1968) containing 3% sucrose and0.8% agar (pH 5.8). Cotyledonary node explants were subjectedto transformation with the use of A. tumefaciens strain EHA105harboring pTF519 (Fig. 2). For infection, a horizontal slice wasmade through the hypocotyl region of germinated seeds, ∼3–5 mmbelow the cotyledons. The cotyledonary explants were separatedby a longitudinal cut and the embryo axes were removed. About

the cotyledonary node. Bacteria were grown overnight at 28 ◦Cwith shaking at 180 rpm in 2 mL of liquid YEP medium con-taining the appropriate antibiotics. The A. tumefaciens cells were

340 M.S. Hanafy et al. / Journal of Plant Physiology 170 (2013) 338– 345

Fig. 2. Schematic representation of the binary vector pTF519 used for soybean and azuki bean transformation. P35S, 35S mRNA promoter of CaMV; hpt, hygromycinp ne; Pm green

o

towima1ait0dlbm(itwvlrt2ortspnr1

A

[fs(wolTas

V

hwG4

hosphotransferase gene; Tnos, 3′ terminator sequence of the nopaline synthase geto1-1, AtCGS1 genomic DNA harboring the mto1-1 mutation; sGFP(S65T), modified

f the T-DNA region. The construct is not shown to scale.

hen cultured in 200 mL of liquid YEP medium containing antibi-tics until an optical density at 650 nm of 0.8 was achieved, afterhich the cells were harvested by centrifugation, resuspended

n liquid cocultivation medium [CCM: 0.1 × B5 salts, 1 × B5 vita-ins, 20 mM MES, 6-benzyl-aminopurine (1.67 mg/L), gibberellic

cid (GA3, 0.25 mg/L), 0.2 mM acetosyringone, 8.8 mM l-cysteine,.0 mM sodium thiosulfate, 1.0 mM dithiothreitol, pH 5.4], anddjusted to an optical density at 650 nm of 0.8. The explants werencubated in the A. tumefaciens suspension for 30 min and thenransferred to a petri dish containing solidified CCM containing.5% agar. Five explants were cultured (adaxial side down) perish for 5 days in the dark at 24 ◦C on sterilized filter paper

aid on the surface of the CCM. The explants were then washedriefly with liquid shoot induction medium [SIM: B5 salts, B5 vita-ins, 3 mM MES, 6-benzyl-aminopurine (1.67 mg/L), vancomycin

50 mg/L), ticarcillin (50 mg/L), cefotaxime (100 mg/L)], culturedn solidified SIM for 2 weeks, and transferred to solid SIM con-aining hygromycin B (5 mg/L) for another 2 weeks. The explantsere transferred to shoot elongation medium [SEM: MS salts, B5

itamins, 3 mM MES, GA3 (0.5 mg/L), asparagine (50 mg/L), pyrog-utamic acid (100 mg/L), indole-3-acetic acid (0.1 mg/L), zeatiniboside (1 mg/L), hygromycin B (10 mg/L), vancomycin (50 mg/L),icarcillin (50 mg/L), cefotaxime (100 mg/L)] and subcultured every

weeks. Cultures were maintained at 24 ◦C under cool white flu-rescent light with a 16-h photoperiod. Regenerated plants wereecovered by grafting the selected shoots onto young seedlings ofhe same cultivar. They were then transferred to pots containingoil and maintained under high humidity until acclimation. The R0lants and their progeny were grown in a greenhouse at day andight temperatures of 28◦ and 22 ◦C, respectively, under a natu-al light environment but with supplementary light according to a6-h-light, 8-h-dark regimen.

zuki bean transformation

The Agrobacterium-mediated transformation of azuki beanVigna angularis (Willd.) Ohwi & Ohashi cv. Beni-dainagon] was per-ormed as previously described (Hanafy et al., 2006). The transgenichoots were selected on medium supplemented with hygromycin B15 mg/L) and on the basis of expression of GFP. Regenerated shootsith more than two leaves were transferred to MS medium with-

ut growth regulators but containing hygromycin B (15 mg/L) andilacillin (500 mg/L) in order to induce a substantial root system.he resulting transgenic lines were transferred to soil and grownt 28◦/22 ◦C (day/night) under a natural light environment but withupplementary light according to a 16/8 h (light/dark) regimen.

isualization of GFP expression

The expression of GFP was observed in selected shoots on

ygromycin B-containing medium and in the regenerated plantsith the use of a stereomicroscope (Leica Microsystems, Wetzlar,ermany) equipped with a filter set for excitation between 455 and90 nm and emission above 515 nm.

gy2, promoter sequence of the soybean glycinin A2B1a subunit gene (gy2); AtCGS1fluorescent protein sGFP(S65T) gene; RB and LB, right and left borders, respectively,

PCR and DNA blot analysis

Total DNA was isolated from transgenic and nontransgenicplants as described (Murray and Thompson, 1980) and was sub-jected either to the polymerase chain reaction (PCR) or to DNAblot analysis. PCR analysis was performed to screen transformedplants and to evaluate the segregation of transgenes in progeny.The reaction mixture (10 �L) contained 10 ng of total DNA, 0.2 �Mof each deoxynucleoside triphosphate, 0.2 �M of each primer, and2.5 U of AmpliTaq Gold polymerase (Applied Biosystems, FosterCity, CA) in polymerase buffer. The amplification protocol com-prised an initial incubation for 9 min at 94 ◦C; 30 cycles of 1 minat 94 ◦C, 1 min at 57 ◦C, and 30 s at 72 ◦C; and a final incubation for7 min at 72 ◦C. Three primer sets were designed for amplification ofa 198-bp fragment of hpt (forward, 5′-CGCAAGGAATCGGTCAATAC-3′; reverse, 5′-ACATTGTTGGAGCCGAAATC-3′), a 294-bp fragmentof sGFP(S65T) (forward, 5′-TGAACCGCATCGAGCTGAAG-3′; reverse,5′-ATGTGATCGCGCTTCTCGTT-3′), and an ∼455-bp fragment ofAtCGS1 mto1-1 (forward, 5′-TTTCCTCCTAATTTCGTCCG-3′; reverse,5′-TCTTTCACCTGTGTAAAACC-3′).

DNA blot analysis was performed to confirm the stable inte-gration of transgenes. Total DNA (10 �g) was digested overnightwith HindIII, and the resulting fragments were fractionated by elec-trophoresis through a 1% agarose gel and then transferred to aHybond N+ membrane (GE Healthcare, Little Chalfont, UK). A frag-ment of AtCGS1 mto1-1 amplified with the same primer set as forPCR analysis was used as a hybridization probe. Labeling and detec-tion of the probe were performed with the use of an ECL kit (GEHealthcare).

Isolation of total RNA and RT-PCR analysis

Total RNA was isolated from developing seeds with the TRI-zol reagent (Invitrogen, Carlsbad, CA). Portions (2 �g) of the RNAwere incubated for 30 min at 37 ◦C with 3 U of RNase-free DNase(RQ1; Promega, Madison, WI) in 30 �l of a solution containing40 mM Tris–HCl (pH 8.0), 10 mM MgSO4, and 1 mM CaCl2. The RNAwas then subjected to phenol–chloroform extraction and ethanolprecipitation before reverse transcription (RT) with an oligo(dT)20primer and a ThermoScript RT-PCR kit (Invitrogen). The resultingcDNA was subjected to PCR with the same AtCGS1 mto1-1 primerset and conditions as those used for PCR analysis of genomic DNA.Soybean Actin1 cDNA (J01298) was amplified as an invariant controlwith a primer set and conditions described previously (Nishizawaand Ishimoto, 2009).

Separation and detection of storage proteins and CGS

Total seed proteins were extracted from soybean or azukibean seed flour by grinding either in phosphate-buffered saline

containing 1 mM EDTA, 20% glycerol, 2% 2-mercaptoethanol, and5 mM thiourea or in SDS sample buffer [50 mM Tris–HCl (pH8.0), 0.2% SDS, 5 M urea, 2% 2-mercaptoethanol], respectively,followed by centrifugation at 15,000 × g for 10 min at 4 ◦C. The

lant Physiology 170 (2013) 338– 345 341

posaeawaIcLrt

A

(crtstnLoahwT0ltr1tTe

S

slt(t

R

G

btofrtpdFcttt

Fig. 3. Detection of AtCGS1 mto1-1 in transgenic plants by DNA blot analysis.Total DNA (10 �g) isolated from transgenic or nontransgenic (Wt) plants wasdigested with HindIII and subjected to hybridization with a probe for AtCGS1 mto1-1.Transgenic soybean plants (M-5, B-2) in the T0 generation and the corresponding

M.S. Hanafy et al. / Journal of P

rotein concentration of the extract was determined with the usef a Protein Assay kit (Bio-Rad, Richmond, CA) and with bovineerum albumin as the standard. Samples containing an appropri-te amount of protein were subjected to SDS-polyacrylamide gellectrophoresis (PAGE) (Laemmli, 1970) on a gel containing 10.5%crylamide and 0.2% bis-acrylamide, which was then either stainedith Coomassie brilliant blue R-250 or subjected to immunoblot

nalysis with rabbit polyclonal antibodies to Arabidopsis CGS.mmune complexes were detected with horseradish peroxidase-onjugated goat antibodies to rabbit immunoglobulin G (Cappelaboratories, West Chester, PA) and a Vectastain kit (Vector Labo-atories, Burlingame, CA). Total extractable seed protein (2 �g) ofhe A. thaliana mto1-1 mutant was analyzed as a positive control.

nalysis of soluble and total amino acid content

Soluble amino acids were extracted from dry seed flour10–25 mg) by shaking for 60 min with 240 �L of 3% sulfosali-ylic acid followed by centrifugation at 12,000 × g for 10 min atoom temperature. The resulting pellet was extracted an additionalwo times with the same amount of extraction solution and withhaking for 60 min. The three supernatants were then combined, fil-ered, diluted if necessary, and assayed for free amino acids with theinhydrin method and an automated amino acid analyzer (model-8900; Hitachi High-Technologies, Tokyo, Japan). For the analysisf total amino acids, protein hydrolysis was performed according to

previously described method (Ishimoto et al., 2010) prior to nin-ydrin derivatization. In brief, seed flour (10 mg) was hydrolyzedith 1 mL of 6 M HCl at 110 ◦C for 22 h with nitrogen replacement.

he hydrolysate was dried under reduced pressure, dissolved in.02 M HCl, and then subjected to analysis with the amino acid ana-

yzer. Cysteine (Cys) and Met in the sample (10 mg) were convertedo their acid-stable forms (cysteic acid and methionine sulfone,espectively) by oxidation with 1 mL of performic acid at 0 ◦C for6 h. Oxidative products were dried under vacuum, dissolved in dis-illed water, and then lyophilized prior to standard acid hydrolysis.otal Trp content was measured as described previously (Ishimotot al., 2010).

tatistical analysis

Quantitative data are presented as means ± SD. The statisticalignificance of differences was evaluated by analysis of variance fol-owed by the Tukey–Kramer honestly significant difference (HSD)est for pairwise comparisons, as performed with JMP 9 softwareSAS Institute, Cary, NC). A P value of <0.05 was considered statis-ically significant.

esults

eneration of transgenic soybean and azuki bean plants

The AtCGS1 mto1-1 gene was inserted into a derivative of theinary vector pPZP202 (Fig. 2) and was then introduced intowo varieties of soybean (Misuzudaizu and Bert) and one varietyf azuki bean (Beni-dainagon) by Agrobacterium-mediated trans-ormation. Five, two, and four putative transgenic shoots wereegenerated from Misuzudaizu, Bert, and Beni-dainagon, respec-ively, on a selection medium containing hygromycin B. Theresence of transgenes in the regenerated plants was provisionallyemonstrated by PCR analysis of AtCGS1 mto1-1 (Supplementaryig. S1) as well as by visual assessment of the expression of the asso-

iated sGFP(S65T) gene. The stable integration of AtCGS1 mto1-1 inhese plants was confirmed by DNA blot analysis, which revealedhat the transgenic lines showed different integration patterns forhe transgene, which was present in one or two copies (Fig. 3). One

nontransgenic plants of the original varieties Misuzudaizu and Bert (A) as well astransgenic azuki bean lines (L-1, L-2, L-3) in the T2 generation and the correspondingnontransgenic original variety Beni-dainagon (B) were analyzed.

transgenic plant (M-5 and B-2) was finally recovered from eachof the two soybean varieties, and three independent transgenicplants (L-1, L-2, and L-3) were obtained from the azuki bean vari-ety. These transgenic plants were fertile and showed no obviousdeleterious effects of AtCGS1 mto1-1 on growth and developmentunder greenhouse conditions.

Expression of AtCGS1 mto1-1 in seeds of transgenic soybean andazuki bean plants

RT-PCR analysis revealed that the AtCGS1 mto1-1 transgene wasexpressed in the developing seeds of transgenic plants, whereasno transcript signal was detected in nontransgenic control (wildtype, or Wt) or nontransgenic sib seeds (Supplementary Fig. S2).Immunoblot analysis revealed that the protein encoded by AtCGS1mto1-1 was expressed and accumulated in the seeds of these trans-genic soybean and azuki bean lines, as judged from the presenceof an ∼53-kDa protein that reacted with polyclonal antibodies toArabidopsis CGS (Fig. 4). No such immunoreactivity was detectedin nontransgenic (Wt) seeds. These data indicated that the AtCGS1mto1-1 transgene was expressed in the two grain legumes and gaverise to high levels of the protein product in seeds.

Inheritance of AtCGS1 mto1-1 was verified in further progeny ofall the original transgenic plants by PCR analysis. Homozygous lineswere established from four of the five independent soybean andazuki bean lines, with L-1 being the exception. These homozygouslines were then subjected to further analysis.

Soluble and total amino acid profiles of transgenic soybean andazuki bean seeds

As a preliminary analysis, the amounts of several soluble (free)amino acids related to Asp or to Met metabolism (Fig. 1) were mea-sured in the T1 seeds of M-5, L-2, and L-3 (Fig. 5) after verificationof the presence of the AtCGS1 mto1-1 transgene by PCR analysis.

342 M.S. Hanafy et al. / Journal of Plant Physiology 170 (2013) 338– 345

Fig. 4. Detection of AtCGS1 mto1-1 protein in transgenic seed extracts by immunoblot analysis. (A) Protein extracts (100 �g) prepared from individual segregating T1 seedso e subjp rol. (Ba as in (

NdpslcTwIdaei

tsp

FsT5tast

f a primary transgenic soybean plant (M-5) or from a nontransgenic (Wt) seed werrotein extract (2 �g) of the A. thaliana mto1-1 mutant (At) was used a positive contnd L-3 or from a nontransgenic (Wt) seed were subjected to immunoblot analysis

ontransgenic soybean (Misuzudaizu) and azuki bean (Beni-ainagon), which grew under the same conditions as transgeniclants, accumulated only small amounts of soluble Met in theireeds: 40 and 2 ppm, respectively. Seeds of the transgenic soybeanine M-5 showed a significant increase (up to 8.7-fold) in free Metontent, however, compared with the nontransgenic control seeds.he soluble Met content of seeds of the transgenic azuki bean linesas increased up to 38.5-fold compared with nontransgenic seeds.

n addition to soluble Met, the concentration of cystathionine, theirect product of CGS action, was significantly increased up to 19-nd 23-fold in transgenic soybean and azuki bean, respectively. Thexpression of AtCGS1 mto1-1 thus resulted in a pronounced increasen free Met levels in seeds of the two grain legumes.

To confirm the stability of transgene effects and to characterize

hose on amino acid metabolism, we determined the profiles ofoluble (Table 1) and total (Table 2) amino acids in seeds of therogeny of the transgenic soybean and azuki bean lines. Expression

ig. 5. Amounts of amino acids related to Asp or to Met metabolism in transgeniceeds expressing AtCGS1 mto1-1. The amounts of the free (soluble) amino acids Met,hr, Lys, and cystathionine in T1 seeds obtained from primary transgenic plants (M-, L-2, L-3) and their corresponding nontransgenic controls were measured withhe use of an automated amino acid analyzer. Data are expressed as milligrams ofmino acid per gram of dry weight and are means ± SD from at least three individualeeds. Different letters indicate statistically significant (P < 0.05) differences amongransgenic and nontransgenic plants (Tukey–Kramer HSD test).

ected to immunoblot analysis with polyclonal antibodies to Arabidopsis CGS. A seed) Protein extracts (45 �g) prepared from T2 seeds of transgenic azuki bean lines L-2A). Arrows indicate the position of the AtCGS1 mto1-1 protein.

of AtCGS1 mto1-1 resulted in a significant increase in free Met levelsin seeds of the four homozygous transgenic lines compared withthe corresponding nontransgenic plants (Table 1). Seeds of thetwo transgenic soybean lines, B-2 and M-5, contained about twicethe amount of free Met present in nontransgenic seeds, whereasthere were no significant differences in the content of other aminoacids, including cystathionine. With regard to azuki bean, theMet concentration was significantly increased in seeds of the twotransgenic lines, L-2 and L-3, by a factor of almost 3–4 comparedwith nontransgenic seeds. The levels of other amino acids, includ-ing asparagine (Asn), arginine (Arg), Lys, phenylalanine (Phe),as well as cystathionine, were also significantly increased in thetransgenic azuki bean seeds, resulting in a significant increase intotal soluble amino acid content. In contrast, the concentration oftryptophan (Trp) was significantly decreased in the two transgeniclines.

We examined whether the enhanced production of soluble Metresulted in an increase in total Met content in the transgenicseeds. Rather, expression of AtCGS1 mto1-1 was associated witha decrease in the total content of Met and Cys in transgenic azukibean (Table 2). Finally, we examined the protein components ofthe transgenic soybean and azuki bean seeds by SDS-PAGE andstaining with Coomassie brilliant blue (Supplementary Fig. S3). Nosubstantial differences in the band pattern between the transgenicand nontransgenic seeds was apparent.

Discussion

Modification of Met biosynthesis has been studied intensivelywith the aim of increasing the level of this amino acid in vegeta-tive tissue and, to a lesser extent, in seeds, despite the nutritionalimportance of Met in seeds (Amir, 2010). We therefore decidedto evaluate the potential application of CGS to the adjustment ofMet content in the seeds of two grain legumes, soybean and azukibean. Overexpression of wild-type AtCGS1 increases free Met lev-els in vegetative tissue, including potato tubers (Di et al., 2003) andalfalfa leaves (Avraham et al., 2005). Expression of a mutant form ofAtCGS1 with a deletion of the regulatory sequence that encodes theNH2-terminal region of the protein resulted in increased accumu-lation of free Met in leaves of transgenic tobacco plants comparedwith those of plants expressing full-length AtCGS1 (Hacham et al.,2006). Furthermore, the A. thaliana mto1-1 mutant, which harbors apoint mutation within this regulatory region, manifests a high levelof free Met in leaves as a result of a defect in the negative feedback

regulation of mRNA stability (Inaba et al., 1994; Chiba et al., 1999).We thus decided to introduce the AtCGS1 mto1-1 mutant gene intothe two legumes, and we recovered transgenic plants expressingthis gene from both crops.

M.S. Hanafy et al. / Journal of Plant Physiology 170 (2013) 338– 345 343

Table 1Soluble amino acid composition of seeds from homozygous transgenic soybean and azuki bean lines expressing AtCGS1 mto1-1.

Amino acid Free amino acid content (mg/g DW)

Soybean Azuki bean

Bert Misuzudaizu Beni-dainagon

Transgenic Transgenic Transgenic

B-2 Control (Wt) M-5 Control (Wt) L-2 L-3 Control (Wt)

Gly 0.08 ± 0.03 0.05 ± 0.00 0.11 ± 0.01 0.09 ± 0.02 0.18 ± 0.03 0.18 ± 0.05 0.09 ± 0.00Ala 0.34 ± 0.08 0.27 ± 0.02 0.35 ± 0.09 0.34 ± 0.05 0.28 ± 0.16 0.25 ± 0.12 0.14 ± 0.06Val 0.06 ± 0.02 0.06 ± 0.01 0.07 ± 0.02 0.09 ± 0.03 0.26 ± 0.17 0.35 ± 0.26 0.10 ± 0.04Leu 0.08 ± 0.02 0.07 ± 0.01 0.10 ± 0.02 0.08 ± 0.01 0.08 ± 0.03 0.10 ± 0.07 0.05 ± 0.02Ile 0.15 ± 0.07 0.08 ± 0.01 0.21 ± 0.09 0.11 ± 0.04 0.25 ± 0.23 0.32 ± 0.31 0.08 ± 0.05Pro 0.38 ± 0.16 0.31 ± 0.06 0.26 ± 0.07 0.38 ± 0.07 0.15 ± 0.01 0.11 ± 0.02 0.06 ± 0.00Ser 0.04 ± 0.02 0.03 ± 0.00 0.06 ± 0.04 0.06 ± 0.01 0.15 ± 0.06 0.18 ± 0.10 0.10 ± 0.04Thr 0.03 ± 0.01 0.03 ± 0.00 0.03 ± 0.01 0.05 ± 0.01 0.08 ± 0.02 0.10 ± 0.04 0.07 ± 0.01Cys 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.66 ± 0.03 0.72 ± 0.14 0.76 ± 0.09Met 0.15 ± 0.05 0.07 ± 0.00 0.26 ± 0.07 0.11 ± 0.01 0.08 ± 0.04 0.12 ± 0.11 0.03 ± 0.00Asp 0.69 ± 0.20 0.83 ± 0.11 0.58 ± 0.28 0.98 ± 0.19 0.44 ± 0.02 0.45 ± 0.09 0.34 ± 0.04Asn 1.78 ± 2.27 0.66 ± 0.27 1.15 ± 0.74 2.76 ± 0.44 5.73 ± 4.81 4.10 ± 2.60 0.83 ± 0.38Arg 2.19 ± 2.20 1.25 ± 0.32 2.28 ± 0.42 4.93 ± 1.83 13.77 ± 3.81 12.20 ± 2.76 3.20 ± 1.14Lys 0.11 ± 0.06 0.07 ± 0.01 0.10 ± 0.03 0.10 ± 0.03 0.30 ± 0.06 0.31 ± 0.18 0.15 ± 0.04His 0.13 ± 0.12 0.05 ± 0.01 0.80 ± 1.21 0.17 ± 0.07 0.56 ± 0.24 0.53 ± 0.27 0.66 ± 0.51Phe 0.13 ± 0.07 0.09 ± 0.03 0.14 ± 0.04 0.10 ± 0.02 0.85 ± 0.38 0.86 ± 0.13 0.12 ± 0.03Tyr 0.13 ± 0.09 0.06 ± 0.01 0.07 ± 0.02 0.07 ± 0.01 0.09 ± 0.01 0.09 ± 0.00 0.07 ± 0.03Trp 0.28 ± 0.05 0.29 ± 0.04 0.27 ± 0.06 0.27 ± 0.03 0.24 ± 0.05 0.18 ± 0.06 0.58 ± 0.13Cystathionine 0.05 ± 0.00 0.04 ± 0.00 0.07 ± 0.01 0.05 ± 0.00 0.22 ± 0.17 0.17 ± 0.08 0.06 ± 0.02

B ic and

arAsMcHow(

TT

BA

Sum 7.55 ± 5.59 4.84 ± 0.93 7.58 ± 2.85

old letters indicate statistically significant differences (P < 0.05) between transgen

The soluble (free) Met levels in the seeds of transgenic soybeannd azuki bean lines were about twice and up to four times,espectively, those in the corresponding nontransgenic seeds.tCGS1 mto1-1 thus functioned in the two grain legumes andignificantly increased the free Met content of seeds. Both Thr andet are synthesized from the common substrate OPH, with the

onversion of OPH to Thr being catalyzed by TS (Amir et al., 2002;

esse and Hoefgen, 2003). Antisense RNA-mediated suppressionf TS expression resulted in a marked increase in Met abundance,ith no reduction in the amount of Thr, in potato leaves and tubers

Zeh et al., 2001), whereas overexpression of AtCGS1 resulted

able 2otal amino acid composition of seeds from homozygous transgenic soybean and azuki b

Amino acid Total amino acid content (mg/g DW)

Soybean

Bert Misuzudaizu

Transgenic Transgenic

B-2 Control (Wt) M-5

Gly 14.4 ± 0.2 14.3 ± 0.7 15.4 ± 0.4

Ala 14.3 ± 0.1 14.1 ± 0.5 15.8 ± 0.4

Val 14.7 ± 0.4 14.7 ± 1.0 16.2 ± 0.8

Leu 22.7 ± 0.7 22.5 ± 1.4 25.0 ± 1.4

Ile 14.0 ± 0.4 13.9 ± 1.0 15.6 ± 0.9

Pro 16.4 ± 0.4 16.2 ± 1.3 18.4 ± 0.9

Ser 15.1 ± 0.3 15.3 ± 0.6 16.5 ± 0.8

Thr 12.5 ± 0.1 12.6 ± 0.4 13.5 ± 0.4

Cys 5.9 ± 0.3 6.7 ± 0.3 6.3 ± 0.4

Met 6.6 ± 0.1 6.6 ± 0.5 6.4 ± 0.1

Asx 37.2 ± 0.4 36.8 ± 1.7 40.7 ± 2.1

Glx 52.7 ± 1.1 52.4 ± 3.4 60.8 ± 3.3

Arg 23.9 ± 0.8 23.4 ± 1.3 28.2 ± 1.8

Lys 21.2 ± 0.3 21.2 ± 0.9 23.0 ± 0.8

His 8.3 ± 0.1 8.3 ± 0.3 9.7 ± 1.7

Phe 14.8 ± 0.5 14.6 ± 1.0 17.0 ± 0.9

Tyr 10.8 ± 0.1 10.6 ± 0.4 11.3 ± 0.5

Trp 4.7 ± 0.1 5.3 ± 0.6 4.8 ± 0.2

Sum 310.3 ± 2.7 309.3 ± 16.7 344.7 ± 16.0

old letters indicate statistically significant differences (P < 0.05) between transgenic andsx, Asn + Asp; Glx, Gln + Glu.

11.56 ± 1.86 25.29 ± 9.36 22.72 ± 7.02 8.33 ± 1.78

nontransgenic control (Wt) seeds. Data are means ± SD for three individual seeds.

in a pronounced increase in Met but no change in Thr levels invegetative tissues of alfalfa and A. thaliana (Avraham et al., 2005;Hacham et al., 2006). Similarly, seeds of transgenic soybean andazuki bean expressing AtCGS1 mto1-1 in the present study showeda significant increase in free Met levels without a change in freeThr levels (Table 1). The amount of OPH as well as the activity ofTS may thus have little relation to the regulation of free Thr levels

in these legumes.

The presence and function of the AtCGS1 mto1-1 transgenein soybean and azuki bean plants were stably inherited by theirprogeny. The levels of free Met in homozygous transgenic seeds

ean lines expressing AtCGS1 mto1-1.

Azuki bean

Beni-dainagon

Transgenic

Control (Wt) L-2 L-3 Control (Wt)

16.1 ± 1.8 8.7 ± 1.6 8.4 ± 0.1 8.7 ± 1.516.4 ± 1.4 9.6 ± 1.9 9.2 ± 0.1 9.4 ± 1.616.9 ± 1.7 10.9 ± 2.1 10.6 ± 0.2 10.8 ± 1.825.9 ± 3.3 15.8 ± 3.2 15.2 ± 0.3 16.1 ± 2.916.2 ± 1.8 9.0 ± 1.6 8.7 ± 0.2 8.8 ± 1.619.2 ± 2.7 9.5 ± 1.6 9.0 ± 0.4 9.7 ± 1.916.7 ± 2.4 10.4 ± 2.4 9.8 ± 0.1 10.9 ± 1.913.9 ± 1.5 7.4 ± 1.6 7.0 ± 0.1 7.6 ± 1.3

6.6 ± 0.2 2.6 ± 0.3 2.4 ± 0.2 3.3 ± 0.16.9 ± 0.8 4.1 ± 0.7 3.9 ± 0.5 5.3 ± 0.1

43.9 ± 5.6 28.6 ± 9.0 26.3 ± 2.1 25.1 ± 4.762.9 ± 9.2 35.1 ± 7.6 34.4 ± 0.8 35.2 ± 6.531.1 ± 5.5 24.4 ± 6.6 22.8 ± 2.1 15.1 ± 2.523.7 ± 2.7 17.1 ± 3.5 16.3 ± 0.1 17.3 ± 3.1

9.2 ± 1.0 6.9 ± 1.4 6.6 ± 0.3 7.3 ± 1.417.8 ± 2.5 12.3 ± 2.3 11.7 ± 0.5 11.7 ± 1.911.6 ± 1.3 6.3 ± 1.2 5.7 ± 0.5 6.1 ± 0.8

5.2 ± 0.1 2.5 ± 0.4 2.2 ± 0.2 3.4 ± 0.2360.0 ± 45.2 221.2 ± 48.6 210.2 ± 4.3 212.0 ± 35.3

nontransgenic control (Wt) seeds. Data are means ± SD for three individual seeds.

3 lant P

wsicadMdgaattrbdi

MapsSmpapWppmtfp

foeaGss2hwMoCisrTtt

A

oatwfuw

44 M.S. Hanafy et al. / Journal of P

ere thus also significantly higher than those in nontransgeniceeds, although in the case of soybean they were lower than thosen seeds of the T1 generation (Fig. 5 and Table 1), possibly indi-ating instability of free amino acid composition during growth in

natural light environment. Unexpectedly, the increased abun-ance of soluble Met did not result in an increase in the totalet content of transgenic soybean seeds and even resulted in a

ecrease in the total content of Met and Cys in seeds of trans-enic azuki bean. In addition to free Met, the levels of other freemino acids, including Asn, Arg, Lys, Phe, as well as cystathionine,

direct product of CGS action, were also significantly increased inhe transgenic azuki bean seeds, whereas free and total Trp concen-rations were significantly decreased. These results indicate thategulation of the Met biosynthetic pathway differs between soy-ean and azuki bean, and that expression of AtCGS1 mto1-1 mayisturb the balanced biosynthesis of sulfur-containing amino acids

n azuki bean.Heterologous expression of AtCGS1 and the resultant enhanced

et accumulation have been accompanied by abnormalities, suchs growth retardation and loss of apical dominance, in tobacco andotato plants (Hacham et al., 2002; Dancs et al., 2008). The synthe-is of SAM from Met and ATP is catalyzed by SAM synthetase, andAM regulates Met biosynthesis by reducing the stability of CGSRNA (Chiba et al., 2003; Onouchi et al., 2008). SAM also acts as a

recursor to several key metabolites such as ethylene, polyamines,nd biotin (Roje, 2006). Overexpression of CGS may thus also affectlant metabolism through Met and its direct metabolite SAM.e did not detect any abnormal phenotypes in the transgenic

lants expressing AtCGS1 mto1-1 in the present study, however,robably because we used the gy2 promoter to control AtCGS1to1-1 expression in a seed-specific manner. Regulation of the

emporal and spatial pattern of CGS gene expression is thus aeasible strategy for preventing deleterious effects in transgeniclants.

Although heterologous expression of AtCGS1 mto1-1 increasedree Met levels in soybean and azuki bean seeds, it had no effectn total Met content. An increased supply of free Met was found tonhance the accumulation of exogenous sulfur-rich proteins suchs sunflower seed albumin and maize zein (Chiaiese et al., 2004;olan et al., 2005), although we detected no obvious difference ineed protein composition between transgenic and nontransgenicoybean or azuki bean plants. Met-rich proteins such as Brazil nutS albumin, sunflower seed albumin, and maize 15-kDa delta zeinave been expressed in transgenic legume seeds including soybean,ith such expression resulting in a 1.5- to 2.0-fold increase in totalet content (Tabe and Higgins, 1998; Krishnan, 2005). Introduction

f these Met-rich proteins in coordination with overexpression ofGS is thus a potential strategy for markedly increasing Met content

n grain legumes. Food processing or cooking can result in a loss ofoluble amino acids, but such losses can be mitigated by incorpo-ation of the amino acids into storage proteins (Dancs et al., 2008).argeting of the entire process from sulfur assimilation to biosyn-hesis and storage of Met may thus prove beneficial in approacheso increase total Met content of seeds.

cknowledgments

We thank Shigeru Utsumi (Kyoto University Graduate Schoolf Agriculture) for providing the glycinin gene promoter as wells Yasuo Niwa (University of Shizuoka School of Food and Nutri-ional Science) for providing the sGFP(S65T) gene. M.S.H. and S.M.R.

ere supported by postdoctoral fellowships from the Japan Society

or the Promotion of Science. This study was supported by CREST,nder the aegis of the Japan Science and Technology Agency, asell as in part by a grant from the Ministry of Agriculture, Forestry,

hysiology 170 (2013) 338– 345

and Fisheries of Japan (Genomics for Agricultural Innovation, GMZ-1004) and by the Fuji Foundation for Protein Research (Osaka,Japan).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jplph.2012.10.018.

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