Lysine enhances methionine content by modulating the expression of S-adenosylmethionine synthase

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1 The Plnt Journl (7) 5, doi:./j x x ine enhnes methionine ontent y modulting the expression of S-denosylmethionine synthse Yel Hhm,, Luhu Song, Gdi Shuster nd Rhel Amir,3, Lortory of Plnt Siene, Migl Glilee Tehnology Center, PO ox 83, Kiryt Shmon,, Isrel, Deprtment of Biology, Tehnion, Hif, 3, Isrel, nd 3 Tel-Hi College, Upper Glilee,, Isrel Reeived 4 Mrh 7; revised 3 April 7; epted 3 My 7. For orrespondene (fx ; e-mil rhel@migl.org.il). Present ddress: Deprtment of Biohemistry, University of Nevd t Reno, Reno, NV 89557, USA. Summry ine nd methionine re two essentil mino ids whose levels ffet the nutritionl qulity of erels nd legume plnts. Both mino ids re synthesized through the sprtte fmily iosynthesis pthwy. Within this fmily, lysine nd methionine re produed y two different rnhes, the lysine rnh nd the threonine methionine rnh, whih ompete for the sme ron/mino sustrte. To eluidte the reltionship etween these iosyntheti rnhes, we rossed two lines of trnsgeni too plnts: one tht overexpresses the feedk-insensitive teril enzyme dihydrodipiolinte synthse (DHPS) nd ontins signifintly higher level of lysine, nd seond tht overexpresses Aridopsis ystthionine -synthse (AtCGS), the first unique enzyme of methionine iosynthesis. Signifintly higher levels of methionine nd its metolite, S-methylmethionine (SMM), umulted in the newly produed plnts ompred with plnts overexpressing AtCGS lone, while the level of lysine remined the sme s in those overexpressing DHPS lone. The inresed levels of methionine nd SMM were orrelted with inreses in the mrna nd protein levels of AtCGS nd redued mrna level for the genes enoding S-dnosylmethionine (SAM) synthse, whih onverts methionine to SAM. Redution in SAMS expression level leds most proly to the redution of SAM found in plnts tht feed with lysine. As SAM is negtive regultor of CGS, this redution leds to higher expression of CGS nd onsequently to n inresed level of methionine. Eluidting the reltionship etween lysine nd methionine synthesis my led to new wys of produing trnsgeni rop plnts ontining inresed methionine nd lysine levels, thus improving their nutritionl qulity. Keywords: methionine metolism, sprtte fmily, ystthionine -synthse, dihydrodipiolinte synthse, S-denosylmethionine (SAM), SAM synthse. Introdution ine, methionine nd threonine re of gret nutritionl importne in niml feeds nd humn foods euse of the limited mounts of these essentil mino ids in seeds nd vegettive tissues of mny rop plnts (reently reviewed y Azevedo et l., 6; Glili et l., 5; Hesse nd Hoefgen, 3; Hesse et l., 4). These three essentil mino ids re synthesized in plnts from sprtte vi two rnhes of the sprtte fmily iosyntheti pthwy: the lysine rnh nd the threonine methionine isoleuine rnh (Figure ). The sprtte fmily iosynthesis pthwy in plnts is regulted y severl feedk inhiition loops. Moleulr nd iohemil studies hve shown tht the isozymes of sprtte kinse (AK), the enzyme tlyzing the first step in this pthwy, re feedk-inhiited y severl produts long the iosynthesis pthwys. Threonine negtively regultes the tivity of AK homoserine dehydrogense (Curien et l., 5), while lysine negtively regultes the tivity of the monofuntionl AK isozymes (Frnkrd et l., 997; Tng et l., 997). Moreover, in vitro studies hve demonstrted tht lysine feedk inhiition is synergistilly enhned y S-denosylmethionine (SAM), methionine metolite, ut SAM y itself does not ffet the tivity of AK (Rognes et l., 98). In ddition, lysine lso inhiits the tivity of the first enzyme in its own pthwy, 85 ª 7 The Authors Journl ompiltion ª 7 Blkwell Pulishing Ltd

2 ine ffets the expression level of SAMS nd methionine ontent 85 Glutmine Ethylene Glutmte ine Asprtte AK 3-ASA DHPS HSD Methionine SAMS SAM Biotin CGS SMM Polymines OPH Asprgine dihydrodipiolinte synthse (DHPS), nd threonine inhiits the tivity of homoserine dehydrogense (Figure ), whih regultes the flux towrds methionine nd threonine (Azevedo et l., 6; Glili, 995, ). Therefore, the reltive mount of preursor in the two rnhes of the sprtte fmily iosynthesis pthwy, 3-sprti semi-ldehyde, is regulted y the levels of threonine nd lysine (Figure ). Moleulr studies hve demonstrted tht the inhiition of DHPS y lysine is the mjor rte-limiting step of lysine iosynthesis in vivo (Glili, 995; Shul nd Glili, 99). Aordingly, overexpression of teril feedk-insensitive DHPS in vrious plnt speies resulted in lysine overprodution (Flo et l., 995; Krhi et l., 994; Mzur et l., 999; Perl et l., 99; Shul nd Glili, 99). While DHPS regultes the level of lysine, AK regultes the level of threonine. Overexpression of teril feedk-insensitive AK in trnsgeni plnts led to overprodution of threonine, while the level of methionine, whih diverges from this rnh, did not hnge signifintly (Krhi et l., 993; Shul nd Glili, 99). TS Methyl group donor Threonine TDH Isoleuine Seondry metolites Figure. Sheme of the sprtte fmily iosynthesis pthwy nd methionine metolism. Only some of the enzymes nd metolites re indited. 3-ASA, 3-sprti semi-ldehyde; OPH, O-phosphohomoserine; SAM, S-denosylmethionine; SMM, S-methylmethionine; SAMS, SAM synthse; AK, sprtte kinse; DHPS, dihyrodipiolinte synthse; HSD, homoserine dehydrogense; TS, threonine synthse; CGS, ystthionine -synthse; TDH, threonine dehydrtse. Dshed rrows with minus sign represent feedk inhiition loopsonthetivitiesof thekeyenzymesinthenetwork; thedshedrrowwith plus sign represent tivtion of the enzyme. The dotted rrow indites regultion of the SAMS expression level y lysine s proposed in this pper. Methionine synthesis is regulted y the level of its first speifi iosynthesis enzyme, ystthionine -synthse (CGS), nd y the level of threonine synthse (TS), the lst enzyme in the threonine iosynthesis pthwy (Amir et l., ; Avrhm nd Amir, 5; Chi et l., 999; Droux et l., ; Glili et l., 5; Hesse nd Hoefgen, 3; Hesse et l., 4; Rvnel et l., 998; Zeh et l., ). These two enzymes ompete for their ommon sustrte, O-phosphohomoserine (OPH; Figure ). In vitro iohemil studies reveled tht the ffinity of TS for OPH is stimulted y SAM (Curien et l., 998), suggesting tht methionine regultes its own synthesis through SAM. When the level of methionine, nd hene SAM, is high, TS tivity is inresed, using redued OPH vilility for methionine synthesis (Curien et l., 996, 998). In ddition, in Aridopsis, SAM lso negtively regultes the trnsript level of CGS (Chi et l., 999, 3; Kreft et l., 3; Onouhi et l., 5). Therefore SAM plys mjor role in the regultion of methionine nd threonine synthesis nd ffets methionine umultion in plnts. In ddition to these effets on the regultion of methionine synthesis, the level of methionine is lso regulted y the rte of its tolism to SAM nd to SAM metolites (Giovnelli et l., 985). A redution in the mount or tivity of SAM synthse (SAMS), the enzyme tht onverts methionine to SAM, resulted in signifintly higher level of methionine in these plnts (Boerjn et l., 994; Goto et l., ; Shen et l., ). In n ttempt to revel the ross-tlk etween metolites nd genes tht elong to the sprtte fmily, nd to explore the regultion of methionine metolism, we ddress here the following questions: (i) onsidering tht the lysine nd methionine iosynthesis pthwys re loted on different rnhes of the sprtte fmily iosynthesis pthwy, how does the methionine level hnge when the flux towrds lysine synthesis is signifintly enhned? nd (ii) n lysine nd methionine umulte to signifintly high level in the sme plnt tissue? The ltter is prtiulrly importnt issue from iotehnologil stndpoint. To this end, we rossed too plnts overexpressing teril DHPS nd those overexpressing Aridopsis CGS (AtCGS). The results dislosed new regultory point t whih lysine ffets methionine ontent y modulting the expression level of the genes enoding the enzyme SAMS. In ddition, we show tht oth methionine nd lysine n signifintly overumulte in the sme plnt tissue. Results Methionine level is enhned in too plnts overexpressing oth Aridopsis CGS (AtCGS) nd teril DHPS ompred with plnts overexpressing AtCGS only An inrese in lysine ontent hs een reported for plnts overexpressing the Esherihi oli DHPS enzyme (Krhi ª 7 The Authors Journl ompiltion ª 7 Blkwell Pulishing Ltd, The Plnt Journl, (7), 5, 85 86

3 85 Yel Hhm et l. et l., 994; Perl et l., 99; Shul nd Glili, 99). In these plnts, lysine umultion refleted the mount of the enzyme s well s its feedk-inhiition insensitivity, unlike its plnt ounterprt (Glili, 995). With regrd to methionine, we hve previously otined signifint umultion of this mino id, s well s its metolite S-methylmethionine (SMM), in trnsgeni too plnts overexpressing AtCGS (Hhm et l.,, 6). To study the effet of enhned flux towrds lysine synthesis on methionine metolism, we rossed homozygous too plnts overexpressing feedk-insensitive DHPS with homozygous trnsgeni too plnts overexpressing AtCGS to produe new trnsgeni line tht overexpresses oth the teril DHPS nd AtCGS (see Experimentl proedures). Two seprte rosses were performed, nd s the results otined from the progeny of these two rosses were very similr, the results presented elow desried only one set of rosses. For these rosses, we seleted trnsgeni plnts expressing AtCGS without morphologil phenotype (Hhm et l., 6). However, plnts overexpressing teril DHPS exhiited the typil norml morphologil phenotype, inluding mosi green olor in newly developed leves t the tip of the pex nd prtil loss of pil dominne (Shul nd Glili, 99). When grown together with plnts expressing the DHPS gene lone, the newly produed trnsgeni line, expressing oth the AtCGS nd DHPS genes, exhiited weker phenotype thn DHPS plnts (dt not shown), the reson for whih is not known. The lysine rnh ompetes with tht of methionine/ threonine for the ron/mino skeleton 3-sprti semildehyde, originlly derived from sprtte (Figure ). As CGS is loted downstrem in the threonine/methionine rnh, it ws expeted tht the level of methionine would e redued in the kground of overexpression of teril DHPS, whih enhnes the flux of 3-sprti semi-ldehyde towrds lysine synthesis (Figure ). Unexpetedly, however, methionine levels were signifintly elevted in plnts o-expressing oth trnsgenes ompred with those expressing AtCGS lone (Figure ). The level of SMM, the storge nd moile form of methionine, whih is usully orrelted with the solule methionine ontent (Hhm et l., ), ws lso signifintly inresed in these plnts (Figure ). Notly, the level of SMM lso inresed signifintly in plnts overexpressing the teril DHPS lone ompred with wild-type plnts. The level of lysine did not signifintly differ in CGS/DHPS plnts ompred with those expressing only DHPS (Figure ). The higher levels of lysine nd methionine in plnts expressing oth trnsgenes were ompnied y signifint redution in the level of sprtte, the preursor of the sprtte fmily pthwy, nd glutmine, whih ws redued pproximtely fivefold reltive to wild-type plnts (Tle ). The levels of other mino ids did not differ signifintly from those of the wild-type plnts, inluding the () mol % from totl mino ids + SMM () mol % from totl mino ids + SMM Methionine SMM d ine WT Methionine ine In leves DHPS In seeds WT DHPS CGS CGS DHPS/CGS DHPS/CGS Figure. Amino id ontents in seeds nd leves of trnsgeni plnts. () Methionine, S-methylmethionine (SMM) nd lysine ontents in leves of trnsgeni plnts nd in ontrol, wild-type (WT) too plnts. () Methionine nd lysine ontents in seeds of trnsgeni nd wild-type plnts. Extrts were otined from the leves of -week-old wild-type plnts, or from seeds of trnsgeni plnts overexpressing teril DHPS, Aridopsis CGS, or oth. The mounts of mino ids were lulted from totl mino ids s deteted y HPLC nd re given in mol% of the totl. The dt presented represent the men nd SD of six plnts per line. ANOVA ws used to determine sttistilly signifint differenes (P <.5) s identified y different letters. ª 7 The Authors Journl ompiltion ª 7 Blkwell Pulishing Ltd, The Plnt Journl, (7), 5, 85 86

4 ine ffets the expression level of SAMS nd methionine ontent 853 Tle Solule mino id ontents in too plnts expressing Aridopsis CGS (AtCGS), teril DHPS or oth trnsgenes Amino id (% mol) Wild-type AtCGS plnts DHPS plnts AtCGS/DHPS plnts Met SMM.5. d Asp Thr Glu Asn Ser Gln Gly His Arg Al Pro Tyr Vl Ile Leu Phe Trp of methionine nd SMM in plnts expressing oth trnsgenes, we exmined the expression of AtCGS y immunolot nd Northern lot nlysis. The mrna nd protein expression levels of AtCGS in these CGS/DHPS plnts were inresed 5.5-fold ompred with plnts expressing AtCGS lone (Figure 3). These results demonstrte tht overexpression of DHPS nd the inresed mount of lysine modulte the umultion of AtCGS mrna nd protein. This higher mount of AtCGS proly ounts for the highlevel umultion of methionine nd SMM in these plnts. ine pplition inreses the mount of CGS in Aridopsis plnts The results desried so fr suggest tht lysine enhnes the expression level of CGS. To sustntite this suggestion y () Immunolot Stining AtCGS Ruiso The mounts of mino ids were lulted from totl mino ids s deteted y HPLC nd re given in mol% of this totl. The totl mino id ontent did not differ signifintly etween wild-type nd trnsgeni lines. Six plnts of eh type were nlyzed nd the dt re presented s mens SE. ANOVA ws used to determine sttistilly signifint differenes (P <.5) s identified y different supersript letters. Intensity rtio 5..5 CGS CGS/DHPS level of threonine, whih ompetes with methionine for the sme ron/mino skeleton (Tle ). High levels of mino ids in leves n influene the levels of these mino ids in seeds. Therefore, the levels of lysine nd methionine were exmined in seeds of these trnsgeni plnts s well s in wild-type seeds. It ws found tht the methionine level does not differ signifintly etween seeds of trnsgeni nd wild-type plnts (Figure ). However, the lysine level ws found to inrese in plnts expressing DHPS lone, ut this elevtion ws not found in plnts expressing oth trnsgenes, AtCGS nd DHPS (Figure ). These findings suggest tht the mrked inrese in the levels of these mino ids in leves does not neessrily ffet their levels in seeds. A similr oservtion ws lso mde in trnsgeni rley plnts overexpressing the teril AK or DHPS genes, in whih the levels of lysine nd threonine signifintly inresed in leves ut not in seeds (Brinh-Pedersen et l., 996). CGS over-umultes in CGS/DHPS plnts In trnsgeni too plnts, methionine ontent is positively orrelted with the expression level of AtCGS (Hhm et l., 6). In n ttempt to ount for the elevted levels () RNA lot Stining Intensity rtio 4 CGS CGS/DHPS AtCGS 8S rrna Figure 3. AtCGS umultion in CGS/DHPS plnts. () Immunolot nlysis of totl solule proteins using ntiodies ginst the 3HA epitope tg. Coomssie lue stining of the Ruiso nd ws used to determine equl loding. () RNA lot nlysis of totl RNA isolted from leves hyridized with Aridopsis CGS DNA s proe. Methylene lue stining of 8S rrna ws used s n internl loding ontrol. Quntifitions of nd intensities (men nd SD) of six plnts nlyzed y Western lot nd Northern lot, respetively, using the BioImge Intelligent Quntifier (Quntity One, Bio Rd, Herules, CA, USA), re shown elow the lots. Rtios were lulted ompring the nd intensities in the Western nlyses nd Northern lots ginst those for the nds otined for Coomssie lue nd methylene lue stining, respetively. Sttistilly signifint hnges (P <.5, using Student s t-test) re identified y sterisks. ª 7 The Authors Journl ompiltion ª 7 Blkwell Pulishing Ltd, The Plnt Journl, (7), 5, 85 86

5 854 Yel Hhm et l. nother pproh, wild-type Aridopsis seedlings were irrigted with mm of lysine for 4 h, nd the umultion of AtCGS protein nd its trnsript ws ompred with tht in plnts treted with wter. The expression level of AtCGS trnsript s well s tht of the protein were signifintly inresed in plnts irrigted with lysine (Figure 4). In Aridopsis, the mount of CGS mrna is modulted y SAM in mehnism involving trnsltion rrest nd regultion of RNA degrdtion (Onouhi et l., 5). This type of modultion hs een noted in n in vitro oupled trnsription/trnsltion system sed on whetgerm extrt (Chi et l., 3; Kreft et l., 3; Onouhi et l., 5). To exmine whether lysine diretly modultes the mount of CGS trnsript y similr mehnism, we used the sme system here. Thus, DNA enoding AtCGS ws trnsried/trnslted in the presene of.5 nd mm lysine, nd in the presene of methionine s ontrol. The () Immunolot Stining () Intensity rtio Intensity rtio 5.5 RT-PCR 4 H O H O AtCGS Ruiso AtCGS Atin Figure 4. Effet of pplied lysine on AtCGS expression levels. () Western lot of CGS nlyses of -dy-old wild-type Aridopsis plnts irrigted for 4 h with mm lysine () ompred with ontrol plnts irrigted with distilled wter (H O). The lot represents one of five repets. Totl solule proteins were extrted from five groups, eh ontining plnts, nd the proteins (4 lg) ws seprted y SDS PAGE nd sujeted to immunolot nlysis using nti-serum ginst AtCGS. Coomssie lue stining of the Ruiso nd ws used to determine equl loding. () Semi-quntittive RT-PCR nlysis of the sme plnts, using speifi primers for the Aridopsis CGS trnsript. Amplifition of tin DNA ws used s n internl loding ontrol. The nd intensities (men nd SD) of the five groups of plnts nlyzed y Western lot nd RT-PCR, respetively, nd quntified using the BioImge Intelligent Quntifier (Quntity One, Bio Rd, Herules, CA, USA), re shown elow the lots. Rtios were lulted ompring the nd intensities for the Western nlyses nd RT-PCR ginst those of the Ruiso nd otined in the Coomssie lue stining nd tin, respetively. Sttistilly signifint hnges (P <.5, using Student s t-test) re identified y sterisks. mount of CGS ws nlyzed y immunolot ssy (Figure 5). A low onentrtion of 4 lm lysine or methionine ws pplied to the ontrol smple euse, s oserved previously, protein synthesis is not limited t tht onentrtion (Hhm et l., 6). While methionine used redution in the level of AtCGS, lysine did not ffet the AtCGS level (Figure 5). Therefore, unlike methionine/sam, lysine does not ffet CGS umultion in the in vitro trnsription/trnsltion system. These results suggest tht the mehnism y whih lysine enhnes the expression level of CGS is not medited through diret inding to the protein or its trnsript. Therefore, dditionl omponents suh s metolites nd/or proteins tht re not present in the in vitro trnsription/trnsltion system re likely to e involved. ine negtively ffets the mrna level of the four Aridopsis SAMS genes In Aridopsis, the trnsript nd protein mounts of CGS re modulted in n opposite mnner to those of SAMS, fmily of enzymes tht onvert methionine to SAM () Intensity rtio () Intensity rtio..4.7 In vitro trns./trnsl. ine effet C.5 Methionine effet AtCGS Comssie lue mm AtCGS Comssie lue C 5 mm Met Figure 5. CGS umultion in whetgerm in vitro trnsription/trnsltion system in the presene of different onentrtions of lysine () or methionine (), whih ws used s ontrol. Smples ontining lg of linerized plsmid DNA (pbluesript KS ) ) hroring DNA enoding the Aridopsis CGS were inuted in whet germ extrts ontining.4 (C, for ontrol),.5 or mm lysine (or or 5 mm methionine) for 9 min t 3 C. The mount of CGS protein ws deteted y immunolot ssy. Rtios were lulted ompring the nd intensities in the Western lot reltive to the nds otined in the Coomssie lue stining. ANOVA ws used to determine sttistilly signifint differenes (P <.5) s identified y different letters. ª 7 The Authors Journl ompiltion ª 7 Blkwell Pulishing Ltd, The Plnt Journl, (7), 5, 85 86

6 ine ffets the expression level of SAMS nd methionine ontent 855 (Figures 5 nd 7 in Kim et l., ). This negtive orreltion etween the expression of CGS nd SAMS might e expeted euse n inrese in the level of SAMS ould led to n inrese in the mount of the produt, SAM, whih is negtive regultor of CGS expression (Chi et l., 3; Onouhi et l., 5) (Figure ). If lysine regultes the expression of SAMS (trnsript nd/or protein), then it will lso indiretly ffet tht of CGS. To test this hypothesis, the protein level of SAMS ws nlyzed in Aridopsis seedlings irrigted with mm lysine. The results showed nerly.3-fold derese in the SAMS protein level ompred with plnts treted with wter (Figure 6). To test whether similr effet lso ours t the trnsript level, semi-quntittive RT-PCR ws performed with oligonuleotides derived from the four Aridopsis SAMS genes. The mrna mounts of the four SAMS genes were signifintly redued (Figure 6). Next, the trnsript levels of SAMS were mesured in too plnts expressing AtCGS/DHPS, where the level of CGS ws signifintly inresed. Using semi-quntittive PCR, we found, s expeted from the results otined in Aridopsis, tht the level of SAMS ws redued in these plnts ompred with plnts expressing only AtCGS (Figure 6). A similr redution in SAMS level ws lso otined when leves of trnsgeni too plnts overexpressing the AtCGS were infiltrted with mm lysine for 6 h (Figure 6d). Suh redution ws not oserved when the leves were infiltrted with wter. () Immunolot Stining Aridopsis feeding experiments Intensity rtio H O H O AtSAMS Ruiso () Aridopsis feeding experiments RT-PCR H O SAMS SAMS SAMS3 SAMS4 Atin Intensity rtio 3 H O SAMS SAMS SAMS3 SAMS4 () RT-PCR (d) Trnsgeni too lines overexpressing CGS or CGS/DHPS Intensity rtio RT-PCR Intensity rtio 4 CGS NtSAMS Atin CGS/DHPS Trnsgeni too lines overexpressing CGS Feeding experiments..5 H O NtSAMS Atin Figure 6. Effet of lysine on SAMS expression levels. () Effets of exogenously pplied lysine on SAMS protein levels in -dy-old wild-type Aridopsis plnts irrigted for 4 h with mm lysine (). As ontrol, plnts were irrigted with distilled wter (H O). Totl solule proteins (4 lg) were seprted y SDS PAGE nd sujeted to immunolot nlysis. Coomssie lue stining of the Ruiso nd ws used s n equl loding ontrol. Quntifition of the nd intensity (men nd SD) of the five groups of plnts nlyzed y Western lot ws performed using the BioImge Intelligent Quntifier (Quntity One, Bio Rd, Herules, CA, USA). Rtios were lulted ompring the nd intensities in the Western nlyses ginst tht of the Ruiso nd otined in Coomssie lue stining. Sttistilly signifint hnges (P <.5, using Student s t-test) re identified y sterisks. () Semi-quntittive RT-PCR nlysis of DNA from Aridopsis plnts tht were irrigted with mm lysine () for 4 h, using primers enling detetion of the four SAMS isozymes. () Semi-quntittive RT-PCR nlysis of too SAMS (NtSAMS) in plnts expressing CGS/DHPS nd those expressing AtCGS. (d) Semi-quntittive RT-PCR nlysis of too SAMS (NtSAMS) in trnsgeni too plnts overexpressing AtCGS infiltrted with distilled wter, or mm of lysine for 6 h. In () (d), mplifition of tin DNA ws used s n internl loding ontrol. The rtios etween nd intensities were lulted reltive to the tin nd. Quntifition of the nd intensities (men nd SD) of six plnts ws performed using the BioImge Intelligent Quntifier (Quntity One, Bio Rd, Herules, CA, USA). Sttistilly signifint hnges (P <.5, using Student s t test) re identified y sterisks. ª 7 The Authors Journl ompiltion ª 7 Blkwell Pulishing Ltd, The Plnt Journl, (7), 5, 85 86

7 856 Yel Hhm et l. Low expression of SAMS n ffet the SAM level. Therefore, the SAM level ws determined using LC-MS in trnsgeni too leves overexpressing AtCGS, whih were infiltrted with mm lysine, or with wter or mm methionine s ontrol, for 5 h. It ws found tht, while the SAM level lmost douled in leves treted with methionine, it ws signifintly redued in leves treted with lysine (out 3% in omprison to leves treted with wter) (Figure 7). The redution in SAM level in the presene of lysine ould explin the higher expression level of AtCGS in AtCGS/DHPS trnsgeni plnts nd in Aridopsis plnts irrigted with lysine (Figure 4). These results suggest tht lysine negtively modultes the expression level of SAMS. This modultion ould e performed either y diret inding of lysine to domin(s) found in the SAMS trnsript, or indiretly y its effet on nother metolite or gene expression omponent(s) tht, s onsequene, ffets SAMS. To distinguish etween these mehnisms nd test whether lysine ffets the expression level of SAMS y diret inding to the protein or trnsript, we used the in vitro oupled trnsription/ trnsltion system desried ove. DNAs enoding SAMS nd SAMS3 were hosen s representtives of this fmily nd were trnsried/trnslted in the presene of.5 nd mm lysine. The results showed redutions of 45% nd 7% in the protein mounts of SAMS in the presene of.5 nd mm of lysine, respetively (Figure 8). A similr ut less pronouned effet ws found for SAMS3. Tken together, the dt otined suggest tht lysine diretly modultes the trnsript level of SAMS, leding to redution in the level of SAM. Disussion In order to study the ross-tlk etween the methionine nd lysine iosynthesis pthwys, we produed trnsgeni too plnts overexpressing the two key enzymes SAM nmol/g FW 5 5 H O Met Figure 7. The SAM level in leves of trnsgeni plnts overexpressing AtCGS infiltrted with distilled wter, mm lysine or mm methionine for 5 h. The infiltrted spots were ut from the leves nd frozen in liquid nitrogen. The mount of SAM ws determined y LC-MS nd is expressed in ppm (prts per million). The dt presented represent the men nd SD for six lef spots per tretment. ANOVA ws used to determine sttistilly signifint differenes (P <.5) s identified y different letters. Intensity rtio 3 SAMS In vitro trns./trnsl. SAMS3 C.5 C.5 AtSAMS Comssi lue mm Figure 8. Effet of lysine onentrtions on umultion of the Aridopsis SAMS nd SAMS3 proteins in the whetgerm in vitro oupled trnsription/ trnsltion system. Smples ontining lg of linerized plsmid DNA (pbluesript KS ) ) hroring DNA oding for Aridopsis SAMS or SAMS3 were inuted in whetgerm extrts ontining.4 (C, for ontrol),.5 or mm lysine for 9 min t 3 C. The mount of SAMS protein ws deteted y immunolot ssy. The rtios etween nd intensities in the Western lot were lulted reltive to the nds otined in Coomssie lue stining. ANOVA ws used to determine sttistilly signifint differenes (P <.5) s identified y different letters. ontrolling lysine nd methionine iosynthesis pthwys, DHPS nd CGS, respetively. We used too plnts expressing the teril DHPS gene, whih, unlike the plnt enzymes, is feedk-insensitive to lysine (Shul nd Glili, 99). Those trnsgeni plnts tht exhiited signifintly higher levels of lysine were rossed with too plnts overexpressing the AtCGS gene. The ltter plnts ontin signifintly higher levels of methionine nd its metolite, SMM (Hhm et l.,, 6). As these key enzymes re loted in ompeting rnhes of the sprtte fmily iosynthesis pthwy, it ws expeted tht the level of methionine would e redued in plnts expressing DHPS/AtCGS ompred with those expressing AtCGS lone. However, unexpetedly, the levels of methionine nd its metolite, SMM, were signifintly inresed ompred with plnts expressing AtCGS lone. In these high-methionine plnts, the expression level of CGS ws inresed, while tht of SAMS ws redued. The redution of SAMS ffets the SAM level, whih ws signifintly redued in plnts treted with lysine. In vitro studies onduted using the whetgerm trnsription/trnsltion system demonstrted tht lysine did not inrese the CGS expression level diretly, ut rther redued the mount of SAMS (Figures 5 nd 7). The expression level of SAMS is negtively orrelted with the expression level of CGS, s found in Aridopsis (Kim et l., ), nd negtive orreltion my e expeted s SAMS produes SAM, nd SAM is negtive regultor of AtCGS expression (Chi et l., 3). Tking into ount these new results, we propose the following sheme for the ross-tlk etween the lysine nd ª 7 The Authors Journl ompiltion ª 7 Blkwell Pulishing Ltd, The Plnt Journl, (7), 5, 85 86

8 ine ffets the expression level of SAMS nd methionine ontent 857 methionine iosynthesis pthwys (Figure, dotted line): A high level of lysine rings out redution in the mount of the enzyme SAMS due to redution in the level of trnsripts enoding this enzyme. This leds to redution in the mount of SAM, whih negtively regultes the mount of CGS trnsript (Chi et l., 3; Onouhi et l., 5). As result, the expression of CGS is inresed, nd onsequently the level of methionine (Figure ). The methionine level n lso e enhned from redution of the flux towrds SAM nd its metolites (Giovnelli et l., 985; Rnoh et l., ). Tken together, this mehnism ensures fine lne etween the mounts of lysine nd methionine through n indiret iohemil ross-tlk mehnism involving regultion of the expression of SAMS. The proposed iohemil ross-tlk sheme lso explins our results showing tht the level of SMM is signifintly elevted in plnts overexpressing teril DHPS (Figure ). As the onentrtion of lysine inreses, the SAMS level is downregulted, enling higher prodution of SMM, even though the expression of CGS is not trnsgenilly inresed. Moreover, this model further explins the hnges in lysine nd methionine ontent otined previously in three sets of Aridopsis trnsgeni plnts: (i) n Aridopsis mutnt with knokout in lysine ketoglutrte redutse/ shropine dehydrogense tht expresses the himeri phseoline::dhps gene, (ii) Aridopsis seeds expressing phseoline::dhps together with phseoline::rnai of lysine ketoglutrte redutse/shropine dehydrogense, nd (iii) Aridopsis seeds expressing teril DHPS lone (Zhu nd Glili, 3, 4). In seeds of these types of trnsgeni plnts, in ddition to signifint 8-fold inrese in lysine ontent, the methionine level inresed signifintly up to 5- fold ompred with wild-type seeds (Zhu nd Glili, 3, 4). The positive orreltion etween high lysine nd methionine levels ws lso found in trnsgeni rley plnts onstitutively expressing teril DHPS. These plnts exhiited 4-fold inrese in free lysine nd n eightfold inrese in free methionine (Brinh-Pedersen et l., 996). These results demonstrte tht, in seeds, s in the vegettive tissues, higher level of lysine enhnes the prodution of methionine, or redues its tolism, s explined y the sheme proposed here. This new regultion trk within the sprtte fmily iosynthesis pthwy etween lysine nd SAMS suggests ontrol mehnism y whih the mounts of SAM nd lysine nnot e o-enhned in the sme ell. This is intriguing euse, s suggested previously on the sis of in vitro studies, the tivity of the first enzyme of this fmily, AK, is not only sensitive to feedk inhiition y lysine ut is lso synergistilly enhned y SAM (Rognes et l., 98). Although the results of the urrent study suggest tht the synergisti effet of these two metolites on AK tivity does not our in vivo, dditionl studies re required to eluidte this regultion of AK. The ross-tlk sheme etween lysine nd SAMS hs een determined y nlyzing trnsgeni plnts overexpressing teril DHPS nd AtCGS, y in vitro trnsription/trnsltion ssys, nd y lysine feeding experiments. However, when onsidering wild-type plnts, the question remin s to the onditions under whih lysine umultes to high levels nd thus modultes the methionine ontent nd its metolites. A reent report hs shown tht elevted lysine levels re otined in plnts exposed to ABA nd sugr strvtion, s exmples of hormonl nd metoli signls (Stepnsky et l., 5). Therefore, the high lysine ontent under suh stress onditions n ffet the level of SAM nd its metolites. In ddition, the levels of solule lysine re found to inrese in young leves nd t shoot pies where it is required for protein synthesis (G. Glili, Weizmnn Institute, Rehovot, Irel, personl ommunition). As methionine is generlly onverted rpidly to SAM nd its other metolites (Giovnelli et l., 985; Rnoh et l., ), suh n effet of lysine on the level of SAMS enzymes my redue the onversion rte of methionine to SAM nd inrese the level of solule methionine, whih is required together with lysine for protein synthesis. The inrese of lysine nd methionine in the too leves of CGS/DHPS plnts ws ssoited with redued levels of sprtte nd glutmine (Tle ). This finding is not surprising s sprtte is the preursor of the sprtte fmily iosynthesis pthwy leding to lysine, threonine nd methionine (Figure ), nd glutmine is the preursor of glutmte, whih, together with sprgine, is preursor of sprtte (Figure ) (Coruzzi nd Lst, ). The results otined in the urrent study suggest tht high flux towrds lysine nd methionine iosynthesis my trigger the onversion of glutmine, vi glutmte, to sprtte nd sprtte metolites. However, this regultion pprently does not tke ple in other plnts or tissues, s the levels of glutmine nd sprgine were not redued nd even inresed in Aridopsis seeds umulting higher levels of lysine nd methionine (Zhu nd Glili, 3, 4). Therefore, differenes in the level of sprtte fmily produts n ffet the level of mide mino ids s well s glutmte nd sprtte, the four mjor mino ids in plnts, ut the vilility of these mino ids is tissuedependent nd, most proly, plnt speies-dependent. The level of threonine ws not signifintly ltered in the CGS/DHPS plnts ompred with wild-type plnts (Tle ). This finding ws unexpeted for two min resons. First, ssuming tht the level of SAM is redued in CGS/DHPS plnts, this should lower the tivity of TS (Curien et l., 998) nd therefore led to lower level of threonine. Seond, the enhned flux of the ron/mino skeleton towrd lysine nd methionine synthesis should led to redution in threonine ontent. The ltter mehnism is supported y severl lines of evidene s explined elow. Plnts expressing only teril DHPS hve signifintly ª 7 The Authors Journl ompiltion ª 7 Blkwell Pulishing Ltd, The Plnt Journl, (7), 5, 85 86

9 858 Yel Hhm et l. lower level of threonine (Ben-Tzvi-Tzhori et l., 996; Zhu nd Glili, 3, 4) (Tle ). As these two mino ids (lysine nd threonine) ompete for the sprtte metolite, 3-sprti semi-ldehyde, s ommon sustrte in their iosynthesis pthwys, this redution in threonine level ws ntiipted (Glili, 995). In the CGS/DHPS plnts, lysine ontent ws the sme s in DHPS plnts, thus the threonine level ws expeted to e redued. Moreover, in these plnts, methionine, whose iosynthesis pthwy diverges from threonine rnh, ws lso signifintly elevted, mening tht more OPH ws hnneled towrds methionine synthesis. Therefore, due to the signifint elevtion in lysine nd methionine, signifint redution in threonine level in these plnts ws expeted. However, the min differene etween plnts expressing only DHPS nd those expressing oth genes ws the higher methionine ontent in the ltter. Previously, it ws oserved tht methionine enhned the expression level of Aridopsis TS, nd plnts irrigted with methionine demonstrted higher level of threonine (Avrhm nd Amir, 5). Therefore, one of the possile mehnisms for the non-redued threonine ontent in CGS/DHPS plnts is tht the expression level of too TS in these plnts is enhned y methionine, whih leds to enhned threonine synthesis. Alterntively, two other mehnisms ould explin this oservtion. First, it is possile tht the non-redued threonine level in the CGS/ DHPS plnts originted from redued flux towrds methionine synthesis nd its metolites, leving more OPH for threonine synthesis. If this is the se, it my e ssumed tht enhnement of methionine in these plnts stemmed not from elevtion in its synthesis ut rther from redution of its tolism to SAM. Seond, s the threonine level is regulted not only y the rte of its synthesis ut lso y the rte of its tolism (Joshi et l., 6), it my e ssumed tht, when the methionine nd lysine levels re inresed, the level of threonine tolism is redued due to lower expression of threonine ldolse nd/or threonine deminse. Indeed, n ltered expression level of oth of these enzymes leds to hnges in threonine ontent in Aridopsis (Joshi et l., 6). In either se, further studies re required to eluidte regultion of the threonine ontent in the different trnsgeni lines. From iotehnologil stndpoint, the results otined suggest novel pproh to improve the nutritionl qulity of rop plnts y inresing the mounts of the nutritionlly importnt mino ids lysine nd methionine. Our results demonstrte tht expression of feedk-insensitive DHPS nd AtCGS ould onstitute fesile pproh for inresing lysine nd methionine in rop plnts. Notly, in the CGS/DHPS trnsgeni plnts, the level of threonine, the third most importnt essentil mino id of the sprtte fmily, ws not redued. Applition of this pproh is importnt minly for erels nd legume grins, the min protein donor rops for humn nd domesti nimls, whih suffer from low lysine/methionine/threonine ontent in their proteins (Glili et l., 5). Experimentl proedures Plnt growth onditions The Aridopsis plnts were grown in ontrolled growth hmer (6 h light/8 h drk, t 3 C). The too plnts were grown in nother growth hmer under 4 h light nd h drk, t 7 3 C. Sttistil nlysis The dt otined from this study were nlyzed sttistilly using SPSS softwre dpted to Windows, version. Within this softwre, we used ANOVA nd Student s t-test progrms s indited. Genertion of trnsgeni plnts expressing DHPS/AtCGS Homozygous too plnts expressing teril feedk-insensitive DHPS (kindly donted y G. Glili) (Shul nd Glili, 99) were rossed with homozygous trnsgeni too plnts overexpressing full-length AtCGS (Hhm et l.,, 6). The expression level of oth trnsgenes ws verified y Western lot nlysis in the leves of -week-old heterozygous F plnts (not shown). Following self-pollintion, 3 F plnts were grown in the greenhouse. These F plnts were divided into three lsses: rriers of the AtCGS gene, rriers of teril DHPS, nd those expressing oth trnsgenes. Plnts of ll lsses were heterozygous. Non-trnsformed wild-type plnts were used s ontrol. Anlysis of mino ids Smples from -week-old too leves were ground in liquid nitrogen nd kept frozen. Free mino ids were extrted from smple of frozen leves, essentilly s desried y Bieleski nd Turner (996). Approximtely mg tissue ws homogenized y mortr nd pestle in the presene of 6 ll of wter:hloroform:methnol (3:5: v/v). Following entrifugtion ( min, top speed), the superntnt ws olleted nd the residue ws reextrted with 6 ll of the sme mixture. The two superntnts were omined, hloroform (3 ll) nd wter (45 ll) were dded, nd the resulting mixture ws re-entrifuged for 5 min t top-speed. The upper wter methnol phse ws olleted, dried, nd dissolved in ll wter. The onentrtion of free mino ids ws determined using O-phthlldehyde regent, followed y mesurement of the fluoresene t 335/447 nm. The mino id omposition ws determined y loding 66 nmol of the smples onto n RP-HPLC. We used Nov Pk C 8 olumn (4 lm, 3.9 mm 5 mm; Wters, Milford Msshusetts, USA) in Hewlett-Pkrd liquid hromtogrph 9 with n utomti injetion system (Hewlett-Pkrd Agilent Tehnologie, Wldronn, Germny). The.4 M sodium ette uffer, ph 6.4, ws repled with orresponding mmonium ette uffer (Mrti et l., 994). Immunolot nlysis Twenty-dy-old Aridopsis rosette leves or -week-old too leves were homogenized using mortr nd pestle in uffer ontining mm Tris HCl, ph 7.5, mm EDTA, mm DTT nd ª 7 The Authors Journl ompiltion ª 7 Blkwell Pulishing Ltd, The Plnt Journl, (7), 5, 85 86

10 ine ffets the expression level of SAMS nd methionine ontent 859 mm PMSF t 4 C. After min of entrifugtion (6 g t 4 C), the superntnt ws olleted. Protein smples ( 4 lg) were frtionted on % SDS PAGE (Lemmli, 97), nd trnsferred to PVDF memrne using Bio-Rd protein trns-lot pprtus ( The memrne ws loked overnight t 4 C in 5% v/v non-ft dried milk, nd then reted for h t room temperture with either ommeril nti-ha monolonl ntiodies (Rohe, Mnnheim, Germny), CGS rit ntiserum (Hhm et l., 6) or SAMS hiken ntiserum (kindly donted y J. Shroeder, Universitet Freiurg, Freiurg, Germny). After inution with the mthing nti-igg ntiody onjugted to horserdish peroxidse under the sme onditions, immunodetetion ws performed using n enhned hemiluminesene kit (Piere, Rokford, IL, USA) in ordne with the mnufturer s instrutions. RNA extrtion nd Northern nlysis Totl RNA ws extrted from frozen mteril using the Tri regent (Sigm-Aldrih; ording to the mnufturer s instrutions. RNA smples ( lg) were sujeted to eletrophoresis in % grose gel ontining. M formldehyde nd 5 mm MOPS, ph 7., nd trnsferred onto nitroellulose Hyond N memrne (Amershm; mershmiosienes.om/). The lots were hyridized for h t 65 C with proes leled with - 3 P dctp using Rediprime kit (Amershm). The proe ws SphI nd EoRI frgment of the Aridopsis CGS (Hhm et l., ). The mount of 8S rrna (used to determine equl loding) ws visulized y stining the memrne with methylene lue in.5 M sodium ette, ph 5.3. The intensity of the nds ws quntified using the BioImge Intelligent Quntifier (Quntity One, Bio Rd, Herules, CA, USA). Semi-quntittive RT-PCR RNA smples were treted with.4 U of DNse (Amion, Austin, USA) for 3 min. First-strnd DNA ws synthesized t 4 C with lg totl RNA s the templte, oligo(dt) primer (5 ng per retion) nd U of AMV reverse trnsriptse (Promeg; in ll retion mixtures, ording to the mnufturer s instrutions. Aridopsis CGS ws mplified using forwrd primer 5 -GTCCGTCAGCTGAGCATTAAAGCC nd reverse primer 5 -CACTGCAGCTTCAAGCCCTG. Aridopsis SAMS (ession numer M5577) ws mplified using forwrd primer 5 - GTCTCAAACCGTTCCTTC nd reverse primer 5 - TGTGGAATGAA- GACAGAAACA. Aridopsis SAMS (ession numer M337) ws mplified using forwrd primer 5 -GTCTCGTTTCCTTCTTCGCT nd reverse primer 5 -TATTGGTCCATGGTAGATGT. Aridopsis SAMS3 (ession numer NM 68) ws mplified using forwrd primer 5 -GTACTACTACTACCACCACA nd reverse primer 5 -CTGATTCTGGACAGAAGTG. Aridopsis SAMS4 (ession numer NM 943) ws mplified using forwrd primer 5 -TCTC- TACTTCCTTCAACAGA nd reverse primer 5 -TGGTATAATGATAA- GTTCCCA. Too SAMS (AY44558) ws mplified using forwrd primer 5 -ATTGGAGCTGGTGACCAAGGTC nd reverse primer 5 -CATCACGGCCAAAGTGACCATAAG. The gene enoding tin (ession numer AT5 G98), used s n internl ontrol to normlize smples of Aridopsis nd too, ws mplified using forwrd primer 5 -GTGACAATGGAACTGGAATG nd reverse primer 5 -AGACGAAGGATAGCATGAGG. The PCR retion ws rried out in totl volume of 5 ll with.5 U of Tq polymerse (Sigm-Aldrih, Sigm, St. Louis, MO, USA), 5 lm dntp, 5 nm primers nd ll of first-strnd DNA s the templte. The thermoyling onditions were: 95 C for min, then yles of 95 C for 3 se, 6 C for min nd 7 C for min, then 7 C for min. PCR produts ( ll) were loded on.5% TAE grose gel. The rtio of the nd intensity of trget genes versus tht of tin ws lulted using the BioImge Intelligent Quntifier. In vitro trnsription/trnsltion system The DNA form of Aridopsis CGS ws mplified s previously desried (Hhm et l., 6). Aridopsis SAMS DNA ws mplified using forwrd primer 5 -CATGGAGACTTTTCTATT- CACATCTGAG nd reverse primers 5 -ACCCGGGAGCTTGAGGTT- TGTCCCACTTGAG (ontins n SmI site). Aridopsis SAMS3 DNA ws mplified using forwrd primer 5 -CATGGAATCTTTTT- TGTTCACATCTG nd reverse primers 5 -ACCCGGGAGCTTGGAC- CTTGTTAGACTTG (ontins n SmI site). The DNA forms of Aridopsis CGS nd SAMS were ligted into pbluesript KS ) (Strtgene; ontining 3HA epitope tg vi SII nd SmI restrition sites, nd sujeted to further nlysis. In vitro trnsription nd trnsltion of CGS DNA or SAMS DNA were performed using the TNT oupled whetgerm system (Promeg). Retion mixtures (5 ll eh) were prepred utilizing n mino id mixture supplied y the mnufturer t finl onentrtion of 4 lm. Finl onentrtions of.5 nd mm lysine were djusted y dding pproprite quntities of lysine seprtely to eh retion mixture. As ontrol, the respetive DNAs were expressed in the presene of 4 lm lysine. Following inution t 3 C for 9 min, the retion ws stopped y freezing the smples in liquid nitrogen. CGS or SAMS protein levels were susequently determined y Western lot nlysis using nti-ha monolonl ntiodies. Feeding experiments Aridopsis seedlings were grown on Nitsh medi (Duhef, Biohemie B.V., Hrlem, Netherlnds) in ontrolled growth hmer (6 h photoperiod t 3 C) for dys. Feeding experiments were done y dding 5 llofmm lysine to eh plnt for 4 h. As ontrol, 5 ll of distilled wter ws dded. Twentydy-old plnts were ssemled into four groups of plnts eh, nd the smples were frozen in liquid nitrogen until use. Eh group ws sujeted to Western lot nd RT-PCR nlyses. Anlysis of SAM ontent using LC-MS The entire proedure ws performed t 4 C in the drk. Lef smples were ground in liquid nitrogen nd kept frozen t ) C. Then 5 ll of pre-ooled.5% TEA (triethnolmine) t ph 7. were dded to mg of ground plnt mteril. After 5 min of entrifugtion t g, the superntnt ws olleted nd filtered in lm syringe filter. The extrts olleted were nlyzed using Wters 79 HPLC system (Wters, Milford, USA) equipped with miromss triple qudruple Qutro-Ultim LC-MS instrument (Miro- Mss, Mnhester, UK). A smple volume of ll ws injeted into the HPLC using loop injetor t moile phse flow of.5 ml min ) into n eletron spry ioniztion (ESI) interfe in the positive (ESI+) mode. The moile phse ws.5% TEA t ph 7.. High seletivity of the ompound ws hieved using the multiple retion monitoring (MRM) method. The SAM mother ion (m/z 399.) ws frgmented using rgon t ollision energy of 7 ev (eletron volute). The frgmented ion (m/z 5) ws olleted for quntifition of the SAM. Conformtion ws hieved y spiking ª 7 The Authors Journl ompiltion ª 7 Blkwell Pulishing Ltd, The Plnt Journl, (7), 5, 85 86

11 86 Yel Hhm et l. the smples with pure SAM (Fluk, Sigm-Aldrih, Buhs, Switzerlnd), whih ws dded to the lef extrts. For quntifition, the pek res for the smples were ompred with stndrd urve pek re of SAM onentrtion rnging from prts per million (ppm) to 5 prt per illion (pp). The SAM level ws lulted s nmol g ) fresh weight (FW). Aknowledgements We thnk Gd Glili for providing the DHPS too line, Johim Shroeder for providing the ntiser ginst Aridopsis SAMS, Igl Br-Iln nd Gershon Melmn for their help with the SAM nlysis, nd Johnn Shller nd Psle Lht (University of Bern, Bern, Switzerlnd) for their invlule help with the mino id nlysis. This work ws supported y the Isrel Siene Foundtion (grnt numer 566/-) nd prtly y the Frme-Work progrm of the ommission of the Europen Communities (QLRT- -3). Y.H. ws supported y n Eshol fellowship from the Isrel Ministry of Siene. Referenes Amir, R., Hhm, Y. nd Glili, G. () Cystthionine -synthse nd threonine synthse operte in onert to regulte ron flow towrds methionine in plnts. Trends Plnt Si. 7, Avrhm, T. nd Amir, R. (5) Methionine nd threonine regulte the rnhing point of their iosynthesis pthwys nd thus ontrolling the level of eh other. Trnsgeni Res. 4, Azevedo, R.A., Lnien, M. nd Le, P.J. (6) The sprti id metoli pthwy, n exiting nd essentil pthwy in plnts. Amino Aids, 3, Ben-Tzvi-Tzhori, I., Perl, A. nd Glili, G. (996) ine nd threonine metolism re sujet to omplex ptterns of regultion in Aridopsis. Plnt Mol. Biol. 3, Bieleski, R.L. nd Turner, N.A. (996) Seprtion nd estimtion of mino-ids in rude plnt extrts y thin-lyer eletrophoresis nd hromtogrphy. Anl. Biohem. 7, Boerjn, W., Buw, G., Montgu, D. nd Inze, D. (994) Distint phenotypes generted y overexpression nd suppression of S-denosyl-L-methionine synthetse revel developmentl ptterns of gene silening in too. Plnt Cell, 6, Brinh-Pedersen, H., Glili, G., Knudsen, S. nd Holm, P.B. (996) Engineering of the sprtte fmily iosyntheti pthwy in rley (Hordeum vulgre L.) y trnsformtion with heterologous genes enoding feed-k-insensitive sprtte kinse nd dihydrodipiolinte synthse. Plnt Mol. Biol. 3, 6 6. Chi, Y., Ishikw, M., Kijim, F., Tyson, R.H., Kim, J., Ymmoto, A., Nmr, E., Leustek, T., Wllsgrove, R.M. nd Nito, S. (999) Evidene for utoregultion of ystthionine gmm-synthse mrna stility in Aridopsis. Siene, 86, Chi, Y., Skuri, R., Yoshino, M., Ominto, K., Ishikw, M., Onouhi, H. nd Nito, S. (3) S-denosyl-L-methionine is n effetor in the posttrnsriptionl utoregultion of the ystthionine gmm-synthse gene in Aridopsis. Pro. Ntl Ad. Si. USA,, 5 3. Coruzzi, G. nd Lst, T.R. () Amino ids. In Biohemistry nd Moleulr Biology of Plnts (Buhnn, B.B., Gruissem, W. nd Jones, R.L., eds). Rokville, MD: Amerin Soiety of Plnt Physiologists, pp Curien, G., Dums, R., Rvnel, S. nd Doue, R. (996) Chrteriztion of n Aridopsis thlin DNA enoding n S-senosylmethionine-sensitive threonine synthse from higher plnts. FEBS Lett. 39, Curien, G., Jo, D., Doue, R. nd Dums, R. (998) Allosteri tivtion of Aridopsis threonine synthse y S-denosylmethionine. Biohemistry, 37, 3 3. Curien, G., Rvnel, S., Roert, M. nd Dums, R. (5) Identifition of six novel llosteri effetors of Aridopsis thlin sprtte kinse homoserine dehydrogense isoforms. Physiologil ontext sets the speifiity. J. Biol. Chem. 8, Droux, M., Gkiere, B., Denis, L., Rvnel, S., Te, L., Lpprtient, A.G. nd Jo, D. () Methionine iosynthesis in plnts: iohemil nd regultory spets. In Sulfur Nutrition nd Sulfur Assimiltion in Higher Plnts (Brunold, C., ed.). Bern, Switzerlnd: Pul Hupt, pp Flo, S.C., Guid, T., Loke, M., Muvis, J., Sndres, C., Wrd, R.T. nd Weer, P. (995) Trnsgeni nol nd soyen seeds with inresed lysine. Bio/Tehnology, 3, Frnkrd, V., Vuterin, M. nd Jos, M. (997) Moleulr hrteriztion of n Aridopsis thlin DNA oding for monofuntionl sprtte kinse. Plnt Mol. Biol. 34, Glili, G. (995) Regultion of lysine nd threonine synthesis. Plnt Cell, 7, Glili, G. () New insights into the regultion nd funtionl signifine of lysine metolism in plnts. Annu. Rev. Plnt Physiol. Plnt Mol. Biol. 53, Glili, G., Amir, R., Hoefgen, R. nd Hesse, H. (5) Improving the levels of essentil mino ids nd sulfur metolites in plnts. Biol. Chem. 386, Giovnelli, J., Mudd, S.H. nd Dtko, A.H. (985) Quntittive nlysis of pthwys of methionine metolism nd their regultion in Lemn. Plnt Physiol. 78, Goto, D.B., Ogi, M., Kijim, F., Kumgi, T., Werven, F.V., Onouhi, H. nd Nito, S. () A single-nuleotide muttion in gene enoding S-denosylmethionine synthetse is ssoited with methionine over-umultion phenotype in Aridopsis thlin. Genes Genet. Syst. 77, Hhm, Y., Avrhm, T. nd Amir, R. () The N-terminl region of Aridopsis ystthionine gmm synthse plys n importnt role in methionine metolism. Plnt Physiol. 8, Hhm, Y., Shuster, G. nd Amir, R. (6) An in vivo internl deletion in the N-terminus region of Aridopsis ystthionine gmm-synthse results in GCS expression tht is insensitive to methionine. Plnt J. 45, Hesse, H. nd Hoefgen, R. (3) Moleulr spets of methionine iosynthesis. Trends Plnt Si. 8, Hesse, H., Kreft, O., Mimnn, S., Zeh, M. nd Hoefgen, R. (4) Current understnding of the regultion of methionine iosynthesis in plnts. J. Exp. Bot. 55, Joshi, V., Luengyer, K.M., Shuer, N., Fernie, A.R. nd Jnder, G. (6) Two Aridopsis threonine ldolses re nonredundnt nd ompete with threonine deminse for ommon sustrte pool. Plnt Cell, 8, Krhi, H., Shul, O. nd Glili, G. (993) Seed-speifi expression of teril desensitized sprtte kinse inreses the prodution of seed threonine nd methionine in trnsgeni too. Plnt J. 3, Krhi, H., Shul, O. nd Glili, G. (994) ine synthesis nd tolism re oordintely regulted during too seed development. Pro. Ntl Ad. Si. USA, 9, Kim, J., Lee, M., Chlm, R., Mrtin, M.N., Leustek, T. nd Boerjn, W. () Constitutive overexpression of ystthionine gmm-synthse in Aridopsis leds to umultion of solule methionine nd S-methylmethionine. Plnt Physiol. 8, ª 7 The Authors Journl ompiltion ª 7 Blkwell Pulishing Ltd, The Plnt Journl, (7), 5, 85 86