Plant Science 151 (2000) 9–18

Repression of cystathionine g-synthase in Arabidopsis thalianaproduces partial methionine auxotrophy and developmental

abnormalities

Jungsup Kim, Thomas Leustek *Biotechnology Center for Agriculture and the En6ironment, Rutgers Uni6ersity, Cook College, 59 Dudley Road, New Brunswick,

NJ 08901-8520, USA

Received 2 July 1999; received in revised form 1 September 1999; accepted 1 September 1999

Abstract

Cystathionine g-synthase (CGS), a key enzyme in methionine biosynthesis, was repressed in transgenic Arabidopsis thaliana byantisense expression of CGS RNA. CGS activity was reduced by 5–9-fold in the antisense plants resulting in severe growthstunting, morphological abnormalities and an inability to flower. Feeding the plants methionine (Met) or Met metabolites reversedthe morphological effects of CGS repression. There was little change in the content of free Met and S-methylmethionine despitethe need for exogenously applied Met for growth. The overall amino acid content was significantly increased. The CGS antisensetransgene is inherited as a single recessive locus. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Antisense repression; Arabidopsis thaliana ; Cystathionine g-synthase; Methionine biosynthesis

www.elsevier.com/locate/plantsci

1. Introduction

The sulfur containing amino acid methionine(Met) is a fundamental metabolite in plant cells. Itis both a protein constituent and the precursor ofS-adenosyl-L-methionine (SAM) the primary bio-logical methyl-group donor. Met is of equal im-portance to animals that lack the ability tosynthesize this amino acid and must obtain it fromtheir diet or from enteric bacteria. The nutritionalvalue of some crops, legumes in particular, is

limited by low Met content [1]. Despite its bio-chemical and agronomic importance the regula-tion of Met synthesis in higher plants is not wellunderstood.

Met is a 4-carbon amino acid synthesized fromindependently derived components (Fig. 1). Thesulfur atom is derived from Cys. The carbon skele-ton is derived from Asp as are the amino acids Lysand Thr. The immediate precursor of both Metand Thr is O-phosphohomoserine (OPH). Thefirst Met-specific reaction is catalyzed by cys-tathionine g-synthase (CGS) which condenses Cysand OPH to form cystathionine. Next, cystathion-ine b-lyase carries out b-cleavage to form homo-cysteine (Hcy). Met is produced by transmethyl-ation of homocysteine. Aside from its incorpora-tion into proteins Met is the precursor of S-methylmethionine (SMM), a compound that is anintermediate in the synthesis of dimethylsulfonio-propionate (DMSP) in some angiosperms [2,3],and SAM, which is synthesized by Met adenosyl-transferase (SAM synthetase). The Met pathway

Abbre6iations: ACC, 1-aminocyclopropane-1-carboxylic acid; CGS,cystathionine g-synthase; DMS, dimethylsulfide; DMSP, dimethylsul-foniopropionate; Hcy, homocysteine; Kan®, kanamycin resistant;MTA, 5-methylthioadenosine; MTHB, 4-methylthio-2-hydroxy bu-tyric acid; MTOB, 4-methylthio-2-oxobutanoic acid; OPH, O-phos-phohomoserine; PITC, phenylisothiocyanate; SAH, S-adenosyl-L-homocysteine; SAM, S-adenosyl-L-methionine; SAT, serine acetyl-transferase; SMM, S-methylmethionine; TS, threonine synthase.

* Corresponding author. Tel.: +1-732-9320312, ext. 326; fax: +1-732-9328165, ext. 326.

E-mail address: leustek@aesop.rutgers.edu (T. Leustek)

0168-9452/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.

PII: S 0 1 6 8 -9452 (99 )00188 -0

J. Kim, T. Leustek / Plant Science 151 (2000) 9–1810

enzymes are distributed between plastids and thecytosol [4]. Plastids contain all the Asp-familyenzymes, the complete Cys pathway, CGS andcystathionine b-lyase. Met synthase and SAM syn-thetase are exclusively cytosolic as are the enzymesfor SMM synthesis [5].

Met synthesis is regulated at multiple levels. Ageneral mechanism for control of the Asp-familyamino acids centers on feedback inhibition of Aspkinase (AK), the first enzyme of the Asp pathway,by Lys, Thr, and SAM [6]. Combined treatmentwith Lys and Thr is herbicidal because they re-press the Asp pathway blocking the synthesis ofthe carbon skeleton and causing Met starvation[7]. A Met-specific control mechanism centers onthe competition between CGS and Thr synthase(TS) for their common substrate OPH. TS activityis stimulated by SAM and it has a much higheraffinity for OPH than does CGS. Thus, it has beenproposed that CGS may compete poorly for OPHwhen Met (hence SAM) is abundant [8,9]. Bycontrast, when Met is limiting and TS less activeCGS has a greater ability to compete for OPH.There is also evidence that when Met is limitingCGS expression is induced. For example, com-bined treatment with Thr and Lys causes CGSactivity to increase, whereas Met treatment causesit to decrease [10,11].

With the recent cloning of the CGS cDNA fromArabidopsis thaliana [12] it became possible tostudy its function in transgenic plants. Repressionof CGS activity was found to limit the ability ofA. thaliana to grow autonomously without exoge-nous application of Met. The CGS-repressedplants show an abnormal morphology that is in-herited as a recessive trait.

2. Materials and methods

2.1. Preparation of antibodies against recombinantCGS

Recombinant A. thaliana CGS was synthesizedas an S-TAG and hexa-His fusion protein ex-pressed from vector pET30c (Novagen). A 1.4 kbpXhoI fragment from the CGS cDNA [12] (Gen-Bank Accession Number U43709) was cloned intopET30c to produce the pET-CGS construct whichwas used to transform Escherichia coli strainBL21(DE3)pLysS (Novagen). Transformants wereselected on LB medium with 40 mg/ml chloram-phenicol and 30 mg/ml kanamycin. The culturewas grown in liquid LB medium with the antibi-otics at 37°C until an OD at 600 nm of approxi-mately 0.3 was achieved. Then 2.0 mM IPTG wasadded and the culture incubated further for 5 h at30°C. The recombinant enzyme, purified by Ni-affinity chromatography, as described by the pETprotocol from Novagen, showed a characteristicabsorption peak at 415 nm associated with pyri-doxal phosphate enzymes. The ratio between ab-sorbency maxima at 282 and 415 nm was 3.88,identical to native CGS from spinach [13]. CGSactivity was measured as described by Ravanel etal. [13] using O-succinylhomoserine. The pure en-zyme showed a specific activity of approximately2.1 mmol cystathionine formed/min/mg protein at24°C, pH 7.5. Although plant CGS uses OPH asthe physiological substrate it can also use a varietyof homoserine esters including O-succinylhomos-erine [13] which is commercially available fromSigma.

A New Zealand White rabbit was immunizedsubcutaneously with purified recombinant CGSprotein (1 mg) in Freund’s Complete Adjuvant.The rabbit was boosted with 1 mg CGS in salinesolution at intervals of 1 month. Serum sampleswere taken 7 days after boost immunization. The

Fig. 1. Met metabolism and regulation in plants. The path-ways for Met synthesis and metabolism are shown along withtheir subcellular compartmentation. Enzymes thought to beinvolved in regulation of Met synthesis are shown in boldlettering.

J. Kim, T. Leustek / Plant Science 151 (2000) 9–18 11

sample taken after the third boost was used di-rectly on immunoblots.

2.2. Construction of transgenic plants

Antisense repression was used to reduce CGSactivity in A. thaliana. A 1.1 kbp SalI fragmentfrom the 5% end of the CGS cDNA [12] was clonedinto pFF19 [14] placing the CGS gene in theantisense orientation with respect to the 35S pro-moter. The expression cassette was subcloned intothe transformation vector pBI101 (Clonetech) asan approximately 2.0 kbp HindIII–EcoRI frag-ment to produce the CGS[− ] construct. TheCGS[− ] construct was used to transformAgrobacterium tumefaciens strain pGV2260, andthen A. thaliana (C24) by vacuum infiltration [15].KanR plants were selected on medium with 50mg/ml kanamycin.

2.3. Analysis of CGS[− ] plants

All soil grown plants were raised in a 23°Cgrowth chamber with a light intensity of 100 mE/m2 per second and a photoperiod of 14 h lightfollowed 10 h of darkness. The plants were wa-tered with one-quarter strength Peters™ water-sol-uble (20:20:20) fertilizer (Grace-Sierra, Milpitas,CA) prepared in distilled water. Plants requiringexogenously supplied Met for growth in soil were,in addition, watered daily in the root zone with 1ml 0.2 mM Met. The nutritional requirements ofthe CGS[− ] plants were tested with axenicallygrown plants raised on agar solidified MS mediumsupplemented as described in the figure or tablelegends. The plants were incubated in a 21°Cgrowth chamber with a light intensity of 90 mE/m2/s, 13 h light/11 h dark period. Transgenicplants were confirmed to carry the CGS[− ] con-struct by a PCR method [16] using a 35S promoterprimer (5%-TATCTCCACTGACGTAAGGGAT-GA-3%) and a CGS specific primer (5%-ATGGC-ATCTGGGATGTGTGC-3%) and by genomicDNA blotting using the CGS cDNA as a probe[17].

The content of the CGS protein was determinedby immunoblotting carried out as described inWang et al. [18]. Soluble protein extracts wereprepared from the entire shoot of 40-day-oldplants. Total protein (40 mg) was analyzed bySDS-PAGE in a gel containing 10% (w/v) acry-

lamide. The protein concentration was measuredusing the Bradford dye-binding assay with BSA asa standard (BioRad). Antisera against CGS or A.thaliana serine acetyltransferase (SAT) [19] wereused at a dilution of 1:2000. The SAT antibodyserved to control for protein loading. A secondaryantibody was horseradish peroxidase-linked goatanti-rabbit diluted 1:8000 and immune complexeswere detected with the Renaissance™ Kit (DupontNEN).

Soluble amino acids were measured in the shootof 19- and 40-day-old CGS[− ] plants and mto1plants [20] (provided by Dr Satoshi Naito, Hok-kaido University). Amino acids were analyzed byHPLC after alkylation with phenylisothiocyanate(PITC) [21]. Plant tissues were extracted and theamino acids purified by chromatography on AG50W-8 [20]. After binding to the ion exchangeresin and then washing, the column was elutedwith 2 N NH4OH. The eluate was dried by evapo-ration under a stream of nitrogen and the residuedissolved in a solution of 7:1:1:1 (v/v/v/v)ethanol–water–triethanolamine–PITC. Met andThr were specifically measured. Their recoverieswere estimated by comparing the amount of eachamino acid from a typical tissue sample with thatin a spiked tissue sample. The recoveries were, Thrapproximately 81% and Met approximately 88%.Total amino acid content was calculated as thesum of all peak areas from a chromatographdivided by the fresh weight of the plant sample.

SMM was measured as dimethylsulfide (DMS)released from fresh plant samples after alkalinetreatment at 90°C. The assay was developed foralgal samples [22] and was optimized here for A.thaliana. A similar method has been reported forvascular plant samples [23]. Tissue samples rang-ing from 25 to 150 mg fresh weight were combinedwith 400 ml 1 M NaOH in a 2 ml gas tight vialfitted with double-faced PTFE/silicone septa (SU-PELCO, Bellefonte, PA). The vials were heatedfor 3 h at 90°C in a heating block. After cooling toroom temperature, 200 ml of the gas phase wasremoved using a gas tight syringe and injected intoa Shimadzu GC-17A gas chromatograph with aPoraplot Q, 10 m×0.53 mm fused silica capillarycolumn (Chrompack, The Netherlands). DMS wasdetected by flame ionization. The chromatographwas resolved with a mixture of hydrogen, nitrogen,and air at 60, 70, and 50 kPa, respectively. Thetemperatures of the column, injector, and detector

J. Kim, T. Leustek / Plant Science 151 (2000) 9–1812

Fig. 2. Release of DMS from dimethyl sulfonium compoundsand A. thaliana. DMSP (100 nmol) ( ), SMM (100 nmol)(�, �) or various amounts of A. thaliana tissue () wereadded to a vial containing 400 ml 1 M NaOH. (A) Incubationwas for the specified time at 23°C ( , �) or 90°C (�, ).(B) Incubation was for 3 h at 90°C ().

The SMM assay is extremely simple and givesreliable measurements. Release of DMS fromSMM requires strongly alkaline conditions andincubation at 90°C for about 2 h (Fig. 2A) [22].GC analysis of the volatilized product from pureSMM subjected to these conditions shows a singlepeak of DMS with a retention time of 2.5 min. A.thaliana leaf samples produce four volatile prod-ucts. The peak that migrates with DMS is pro-duced at a rate identical to that from pure SMM(Fig. 2A). Moreover, the DMS peak increasesselectively when more sample is added (Fig. 2B) orpure SMM is added with the leaf sample. Thethree additional peaks have retention times of0.77, 0.95 and 2.2 min. They are released from theleaf sample only after alkaline incubation at 90°C,but they appear more rapidly than does SMM-derived DMS or the peak area does not correlatewith the amount of tissue added to the assay.Therefore, they are probably unrelated to SMM.

3. Results

Six KanR plants were isolated from a transfor-mation with the CGS[− ] vector. All were confi-rmed to carry the CGS[− ] transgene constructusing a tissue PCR method (not shown). Four ofthe transformants, analyzed by genomic DNAblotting with the CGS cDNA as a probe showed apattern of hybridization indicative of having arisenthrough independent single integration events. Thetransformants were analyzed with HindIII orBamHI, enzymes that produce, respectively, eitherapproximately 5 or approximately 15 kbp hy-bridizing fragments in wild type A. thaliana. Theendogenous CGS fragment was observed in eachof the transformants, but they showed in addition,hybridization to a second fragment of variablelength, depending upon the transformant, corre-sponding to the transgene construct (Fig. 3).

Analysis of the T2 revealed that without Metfeeding all the transgenic plants (KanR plants)appeared normal up to 10 days after germination.However, after transfer to soil one-third developedsevere growth stunting and where unable to repro-duce while the other two thirds developed nor-mally and produced viable progeny. Abnormaland normal siblings are shown in Fig. 4A, com-pare plant A1 with A2. In addition to growthstunting the abnormal plants developed a clusterof apical shoots and the oldest leaves, which ap-

Fig. 3. Southern blot analysis of CGS[− ] lines. Total DNAwas digested with HindIII (lanes 1–5) or BamHI (lanes6–10). The plant samples were: 415-5 (lanes 1 and 7), 415-7(lanes 2 and 8), 415-11 (lanes 3 and 9), 415-16 (lanes 4 and10), untransformed A. thaliana ecotype C24 (lanes 5 and 6).The blot was probed with a 1.1 kbp fragment from the 5% endof the CGS1 cDNA. Faint bands are highlighted with aster-isks (*). The approximate 15 kbp fragment corresponding tothe endogenous CGS gene was probably not detected due toinefficient transfer from the gel to the membrane. All the largefragments in this experiment show weak hybridization for thesame reason.

were 120, 160, and 160°C, respectively. DMS hada retention time of 2.5 min and it was quantitatedby preparing a standard curve with pure SMM(Sigma) or DMSP (Research Plus, Bayonne, NJ).DMSP is a sulfonium compound that is less stablethan SMM, decaying to DMS under alkaline con-ditions at room temperature.

J. Kim, T. Leustek / Plant Science 151 (2000) 9–18 13

Fig. 4. Morphology of plants with repressed levels of CGS. (A), siblings from CGS[− ] line 415-7 (T2 generation) showingabnormal (A1) and normal (A2) morphology. (B), siblings of the plants in (A) grown for 51 days without Met feeding. The plantsat the top (B2) are normal while those at the bottom (B1) are of the abnormal type. (C), the same plants shown in (A) werephotographed 16 days after initiating the feeding of a 0.2 mM solution of Met. The plants were grown for 35 days at the timeMet feeding was initiated. (D), Wild-type A. thaliana 25 days after being watered with a 1 mM solution of propargylglycine.Fig. 5. Suppression of the abnormal morphology in CGS[− ] plants grown with nutritional supplements. Homozygous plantsfrom abnormal CGS[− ] line 415-5 (T3 generation) were grown axenically for 30 days on unsupplemented agar medium (A) orwith 0.2 mM Met (B). The photographs show a typical result that occurs with all the CGS[− ] lines. Similar changes inappearance were used to determine whether other nutrient supplements suppress the abnormal phenotype, reported in Table 2.

J. Kim, T. Leustek / Plant Science 151 (2000) 9–1814

peared normal early on, became thickened, curledand in some of the CGS[− ] lines accumulated ared-brown pigment in the petioles. The changes inmorphology became clearly evident within 40 daysafter germination. The abnormal plants were un-able to produce flowers and eventually died with-out reproducing (Fig. 4B, observe plants labeledB1). However, when watered with a solution ofMet their growth was restored sufficiently to allow

them to flower and to set viable seed. Plant A1was photographed before Met-feeding (Fig. 4A).The stimulation of growth is evident in the sameplant 16 days after initiating Met-feeding (Fig. 4C,plant C1). Visible signs of growth became evidentwithin 48 h after initiating Met-feeding. The pro-liferation of apical shoots observed in abnormalCGS[− ] plants became clearly evident after Metwas fed and each of the shoots developed into anindependent inflorescence (Fig. 4C, plant C1).

The progenies derived from abnormal CGS[− ]plants were all KanR and developed the abnormalphenotype. By contrast, the morphologically nor-mal KanR plants produced KanR progeny at aratio of approximately 3:1. The KanR plants segre-gated into normal and abnormal phenotypes at aratio of 2:1 (Table 1). The segregation resultsindicate that the antisense transgene behaves as asingle recessive locus that produces abnormalitywhen in the homozygous state rather than thehemizygous condition. Similar results were ob-tained with all six independently isolated CGS[− ]transgenic lines. The abnormal morphology hasbeen stable over six generations.

The developmental progression of plants fromabnormal homozygous CGS[− ] lines was studiedby recording the time of emergence of new leaves.Homozygous CGS[− ] plants grew like wild-typeand hemizygous siblings until 24–28 days aftergermination after which new leaves stoppedemerging and the single apical shoot proliferatedinto a mass of apical shoots. The growth of thehomozygous CGS[− ] plants arrested prior toconversion to reproductive growth.

Testing a variety of sulfur compounds for theability to suppress the abnormal phenotype pin-pointed the lesion in the CGS[− ] lines. This ex-periment was carried out on agar medium underaxenic conditions in order to eliminate the possi-bility of interference from or metabolism of thesupplements by microorganisms. After growth onunsupplemented agar medium the abnormal phe-notype was clearly visible after 30 days (Fig. 5A).The phenotype differs slightly from plants grownon soil in that the plants become chlorotic and theapical shoot remains very small (Fig. 5A). Thephenotype is suppressed by Met feeding (Fig. 5,compare A and B) similar to the results with soilgrown plants. Cystathionine and Hcy also sup-pressed the abnormal phenotype but neither Cysor glutathione, a Cys-containing metabolite, wereable to restore the normal phenotype (Table 2).

Table 1Inheritance of the abnormal phenotype in CGS[−] plantsa

KanR PhenotypeLine Abnormalfrequency

AbnormalNormal

41 24415-4b 17 0.41

0.26415-5b 246993169 0415-5c 169 1.00

442 299415-7b 143 0.32179 0415-7c 179 1.00

0.344383126415-11b

137 0415-11c 137 1.00

65 45 20415-16b 0.31

65 48 17415-18b 0.26

a Segregation analysis was performed with self-pollinatedCGS[−] plants in the T3 generation. KanR plants wereselected, transferred to soil, and the plants scored for mor-phology after 45 days. The total number of KanR and thenumber segregating into normal and abnormal plants aregiven.

b Seeds were from morphologically normal KanR plants.c Seeds were from abnormal KanR plants.

Table 2Chemical supplements suppress the abnormal morphology ofCGS[−] plantsa

Chemical Appearance

BD- or L-MetCystathionine B

BD,L-homocysteineAL-CysAGlutathione

5-Methylthioadenosine (MTA) BB4-Methylthio-2-oxobutanoic acid (MTOB)

4-Methylthio-2-hydroxy butyric acid B(MTHB)

a CGS[−] plants were germinated and axenically grown for45 days on M-S agar medium with the indicated chemicalsupplement at 0.2 mM. The ability of the chemical to sup-press the abnormal phenotype was scored. A and B refers tothe plant appearance as shown in Fig. 5A and B.

J. Kim, T. Leustek / Plant Science 151 (2000) 9–18 15

Table 3CGS activity in A. thalianaa

Specific activity (nmol/min per mg)Line

A. thaliana 0.33090.060

0.04090.010415-50.03690.009415-7

415-11 0.07390.011

a CGS enzyme activity was measured in the shoot of 40-day-old plants grown in soil. The transgenic lines analyzedwere homozygous T3 plants. The averages9S.D. of threeindependent experiments are shown.

2) that plants can convert to Met without the aidof CGS (see Fig. 1). MTA is an intermediate in theYang cycle that functions in recycling of Met fromSAM [4]. MTHB and MTOB are hydroxy and oxoacids that can be converted to Met via ubiquitousamino acid transaminases and dehydrogenases [3].Yet another indication that low CGS activity mayaccount for the growth abnormalities of CGS[− ]plants is that treatment of wild type A. thalianawith the CGS inhibitor, propargylglycine (PAG)[10] produces similar growth abnormalities (Fig.4D).

CGS activity is repressed in abnormal CGS[− ]lines (Table 3). Compared with wild-type, theCGS[− ] lines 415-5, 415-7 and 415-11 had 5–9-fold lower CGS activity and a comparably loweramount of CGS protein as measured by im-munoblotting (Fig. 6). Analysis of the amino acidcontent in the transgenic lines revealed that thelevel of free Met and SMM is similar to wild typeat 19 and 40 days after germination (Table 4). Thelevel of Thr and total amino acids was increased3–5-fold in 40-day-old but not in 19-day-oldplants. Therefore, the development of abnormalityis correlated with the increase in total amino acids.However, Thr is not specifically increased. Bycomparison the mto1 mutant of A. thaliana shows19–fold higher Met and 14-fold higher SMM in19-day-old plants but no higher level of totalamino acids. By 40 days there was no greater Metthan in the wild type. Similar results with mto1, anethionine-resistant mutant, which accumulatesMet in the early vegetative stage, have been previ-ously published [20].

4. Discussion

Gene families encode many amino acid biosyn-thetic enzymes in plants. CGS is unusual in that itexists as a single copy gene in A. thaliana [12].Being a single copy gene there was a greaterlikelihood that its expression could be effectivelyrepressed using an antisense RNA method. In-deed, all of the six independently isolated trans-genic A. thaliana lines transformed with theCGS[− ] construct showed pronounced affects ongrowth and a 5–9-fold reduction in the level ofCGS and CGS enzyme activity. Application ofMet or a diverse collection of Met metabolites tothe plants reversed the abnormal phenotype. How-

Fig. 6. Immunoblot of CGS[− ] plants. CGS (A) and SAT(B) protein was measured in wild type A. thaliana (C24), lanes1 and 2; and in abnormal CGS[− ] plants from line 415-5,lanes 3 and 4; or 415-7, lanes 5 and 6. Lane 7 contains 1 ngof pure recombinant CGS. A lower level of CGS protein wasdetected in CGS[− ] line 415-11 (not shown). The faint bandmigrating above CGS is not consistently observed on blots,therefore it is not thought to be related to CGS. Immunoreac-tion with SAT antibodies was used as a protein loadingcontrol.

The sulfur compounds differ in that cystathionineand Hcy can be converted to Met without theaction of CGS whereas Cys, the CGS substrate,cannot. The results indicate that the abnormalphenotype is caused by an inability to synthesizecystathionine, as expected from the design of theCGS[− ] construct. The abnormal phenotype isalso suppressed by a range of compounds (Table

J. Kim, T. Leustek / Plant Science 151 (2000) 9–1816

ever, Cys and glutathione, two sulfur-containingmetabolites that must be acted upon by CGS inorder to be converted to Met, were unable toreverse the abnormalities. Both Cys and glu-tathione produce physiological affects when sup-plied to A. thaliana [24], therefore, it is unlikelythat the antisense plants were unable to importthese compounds. Further evidence implicatingthe reduction of CGS activity as being responsiblefor the growth abnormalities is that application ofthe CGS inhibitor PAG to wild-type A. thalianaproduces similar growth defects. Just as with theCGS[− ] plants the morphological changes associ-ated with PAG treatment could be reversed byexogenous application of Met [25]. Nearly identi-cal morphological and developmental abnormali-ties are associated with repression of SAMsynthetase in A. thaliana [26], which is accompa-nied by the massive accumulation of free Met inthe leaves [27]. Thus, it is unlikely that the pheno-type of CGS[− ] plants is due solely to a defi-ciency of Met for protein synthesis. A deficiency inSAM may also be partly responsible. SAM isnecessary for a wide range of processes includingthe methylation of DNA and lignin precursors,ethylene production and polyamine synthesis. In-terestingly, some of the abnormalities observed inthe CGS[− ] lines, such as proliferation of theapical shoot (loss of apical dominance) and aninability to progress into the reproductive growthphase have been observed in A. thaliana plants inwhich DNA methylation was inhibited [28]. Al-though the red-brown pigment produced byCGS[− ] plants could be anthocyanin the localiza-

tion near the vascular bundles is reminiscent of thebrown midrib mutant of maize (bm3) which accu-mulates unmethylated lignin precursors [29].Taken together the experimental results and simi-larities with other transgenic and mutant plantmodels suggest that the CGS[− ] plants are unableto produce sufficient Met for growth beyond 24–28 days after germination.

Although the CGS[− ] plants require exogenousapplication of Met to complete their life cycle thelevel of free Met and its metabolite SMM werefound to be similar in wild type and the transgenicantisense lines. The expectation was that Met andSMM levels would be lower in the CGS antisenseplants. To date, this counterintuitive result has notbeen satisfactorily explained, although it is un-likely to be the result of faulty analytical methods.The Met and SMM assay procedures were testedby analyzing the mto1 mutant of A. thaliana that isknown to accumulate Met in young plants but notin older plants [20]. Our measurements were con-sistent with and confirmed the original study.Thus, it is more likely that the free Met and SMMmeasurements accurately reflect the condition inCGS[− ] plants. There are several potential expla-nations for why the level of free Met and SMMare similar in wild type and CGS[− ] plants. Al-though standard and widely accepted methodswere employed for measurement of Met, perhapsthe analytical results must be more critically inter-preted. For example, it is widely assumed that themeasured level of an amino acid in plants repre-sents the metabolically active pool. However, theresults with CGS[− ] plants could be explained by

Table 4Metabolite content in A. thalianaa

Age Metabolite Amino acid content (pmol/mg FW)

WT 415-5 415-7 mto1

Met 209219 Days 2295 1492 38794510.192.1SMM 1389124.191.43.991.9

Thr 410919 413934 422957 485954Totalb 1.190.2 1.190.1 1.290.2 1.290.2

Met 209840 Days 3599 4596 2997SMM 11.391.7 10.192.2 22.992.3 71.8919.7

452962 23639515Thr 31819791 4819191.290.2 4.590.3 6.091.1Totalb 1.390.2

a Soluble amino acids were measured in the shoot of 19 and 40-day-old plants. The transgenic lines analyzed were homozygousT3 generation. The averages9S.D. of three independent experiments are shown.

b Total amino acid content is expressed as total peak area of all amino acids divided by the fresh weight of plant tissue.

J. Kim, T. Leustek / Plant Science 151 (2000) 9–18 17

the existence of a metabolically inactive pool thatis not in equilibrium with the metabolically activepool. Another trivial explanation relates to thevery small size of the free Met pool in plantscompared with other free amino acids and withthe bound Met in proteins. Thus, the analyticalresults could easily have been skewed by relativelyminor amounts of protein hydrolysis during sam-ple processing.

Young antisense plants appear to be unaffectedby the CGS[− ] transgene whereas growth abnor-malities appear abruptly 24–28 days after germi-nation. This pattern suggests that young antisenseplants produce sufficient Met for growth. How-ever, at a critical stage in development the rate ofMet synthesis becomes insufficient. It is notewor-thy that the transition to abnormal growth occursat precisely the time when wild type A. thalianaand hemizygous antisense plants begin the transi-tion to reproductive growth. Perhaps a new andgreater demand for Met develops at this time withwhich the CGS[− ] plants are unable to cope.After the development of abnormality the anti-sense plants show an overall increase in aminoacids, indicating that general amino acidmetabolism is effected by the CGS[− ] transgene.Little is known about the global regulation ofamino acids in plants although a similar generalincrease in amino acids was observed in transgenicplants repressed for the branched chain aminoacid enzyme acetolactate synthase (ALS) [30]. Likethe CGS[− ] plants, the ALS antisense plantsshow severe growth retardation and a wide rangeof morphological deformations. Therefore, it ap-pears that disruption of single amino acids canhave pleiotropic effects on the homeostasis of allamino acids.

A decrease in CGS activity of as little as 5-foldwas correlated with gross changes in developmentand morphology observed in the CGS[− ] line415-11. This result is an indication that themetabolic flux coefficient for CGS is quite high asis usually associated with a metabolic regulationpoint [31]. By comparison, a 7-fold reduction inALS, a known control point in the branched chainamino acid pathway, produced severe growthstunting [30]. The early studies on Met biosynthe-sis in Lemna were the first to indicate that CGS islikely a key rate limiting step in Met biosynthesis[10,25]. The results with the CGS[− ] plants are inagreement with this hypothesis and indicate that

similar processes operate for Met synthesis in A.thaliana, a more complex vascular plant. Hofgenet al. [30] proposed that antisense RNA experi-ments may be useful for the identification of po-tential enzyme targets for herbicide discovery, andthey were able to demonstrate the concept bycreating antisense plants with repressed ALS, aknown target for the commercial sulfonylureaclass of herbicides. The results reported here sug-gest that CGS may be a good potential target forherbicide discovery.

The analysis of CGS[− ] lines raises anotherpotentially significant finding. Up to now a dogmaof the sulfur metabolism field has been that re-duced sulfur is transported throughout plants asglutathione [32]. However, application of Met orMet-derived metabolites to the roots of CGS[− ]plants reverses the growth abnormalities in theplant shoot even when measures were taken toprevent Met from contacting the leaves. From thiswe can conclude that A. thaliana is capable ofvascular transport of Met or a Met metabolite.The compound cannot be glutathione or Cys sinceboth would require CGS for conversion to Metonce transported to the shoot. This result suggeststhat Met or one of its metabolites has the poten-tial to play a heretofore unsuspected role in thetransport of reduced sulfur. One candidatemolecule is SMM, which has recently been shownto be phloem transported in vascular plants [33].

Acknowledgements

This work was supported by the National Sci-ence Foundation (Grant cMCB-9728661). Wewish to thank Dr Satoshi Naito for providingmto1 seed and Dr Andrew Hanson for manyhelpful discussions.

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