A Biological-chemical Test System to Investigate the Influence of Sulfonylurea Herbicides such as Metsulfuron-methyl on Nitrogen Metabolism in Soybean

Transcription

1 Europ.J.Hort.Sci., 69 (4). S , 4, ISSN Verlg Eugen Ulmer GmH & Co. Stuttgrt A Biologicl-chemicl Test System to Investigte the Influence of Sulfonylure Hericides such s Metsulfuron-methyl on Nitrogen Metolism in Soyen A.J. Keutgen 1), N. Keutgen 1), M. Ji 1), S. Mtsuhshi 1), C. Mizuniw 1), T. Ito 1), T. Fujimur 1), T. Sekine ) nd S. Hshimoto 1) ( 1) Deprtment of Rdition Reserch for Environment nd Resources nd ) Deprtment of Mterils Science Tkski Rdition Chemistry Reserch Estlishment, JAERI, Tkski, Jpn) Summry A simple nd relile method for the identifiction of residues of sulfonylure hericides in rottionl frming systems hs een developed using metsulfuron-methyl s test sustnce. Metsulfuron-methyl such s other sulfonylure hericides impirs the synthesis of rnched-chin mino cids nd, in consequence, protein synthesis. Thus, nitrogen consumption is reduced, leding to n ccumultion of mino cids nd reduction of nitrogen uptke. The iologicl-chemicl test system is sed on the determintion of sprtic cid, sprgine, glutmine, nd + y ion chromtogrphy nd is suitle for monitoring chnges of nitrogen metolism in soyen seedlings. Of the mesured free mino cids, glutmine nd sprtic cid represent key sustnces tht reflect chnges in nitrogen uptke, ssimiltion, trnsport, nd storge. Their concentrtions incresed due to metsulfuron-methyl 15 times in roots, stems, nd leves. The sprgine content is mesure of the nitrogen sttus of soyen. However, the sensitivity of sprgine content to metsulfuron-methyl depended on the nutritious sttus of the plnt. In cse the sprgine level ws lredy high, the hericide did not induce significnt increse in sprgine. The + concentrtion my lso dd vlule informtion in cse + is present in the root medium. With respect to the test system, the lower stem section of soyen seedlings represents the optiml plnt section for free mino cid nlyses, ecuse smples cn reproducily e tken, the concentrtion of the cell sp is, compred to roots, less influenced y the wter sttus, nd this section is, compred to leves, rther sensitive to sulfonylure hericides in the root medium. A reduction of lef trnspirtion rte is further effect of metsulfuron-methyl, so tht its mesurement my represent vlule dditionl prmeter to the descried iologicl-chemicl test system. Key words. sprgine sprtic cid glutmine ion chromtogrphy lef trnspirtion nitrte uptke PETIS [ 13 N] Zusmmenfssung Ein iologisch-chemisches Testsystem zur Untersuchung des Einflusses von Sulfonylure-Heriziden wie Metsulfuron-methyl uf den Stickstoffmetolismus von Soj. Eine einfche und verlässliche Methode für die Rückstndsnlyse von Sulfonylure-Heriziden im Fruchtfolgenu wurde entwickelt und m Beispiel von Metsulfuron-methyl getestet. Metsulfuron-methyl eeinträchtigt wie ndere Sulfonylure-Herizide die Synthese von verzweigten Aminosäuren und, ls Konsequenz, die Proteinsynthese. In Folge dessen wird der Stickstoffverruch reduziert, ws zu einer Akkumultion von Aminosäuren und zu einer Reduktion der Stickstoffufnhme führt. Ds iologisch-chemische Testsystem siert uf der Bestimmung von Asprginsäure, Asprgin, Glutmin und NH + 4 mittels Ionenchromtogrphie und ermöglicht, Veränderungen im Stickstoffmetolismus von Soj zu erfssen. Von den gemessenen freien Aminosäuren stellen Glutmin und Asprginsäure Schlüsselsustnzen dr, die Veränderungen in der Aufnhme, der Assimiltion, dem Trnsport und der Speicherung von Stickstoff nzeigen. Ihre Konzentrtionen in Wurzeln, Stängeln und Blättern steigen durch Metsulfuron-methyl um ds 15-fche n. Der Asprgingehlt ist ein Mß für den Stickstoffsttus von Soj. Die Empfindlichkeit der Asprginkonzentrtion gegenüer Metsulfuron-methyl hängt jedoch vom Ernährungszustnd der Pflnze. Im Flle eines schon erhöhten Asprginniveus verurschte ds Herizid keine signifiknte Steigerung des Asprgingehltes. Die NH + 4 -Konzentrtion knn eenflls wertvolle Hinweise geen, wenn NH + 4 im Wurzelrum vorhnden ist. Im Bezug uf ds Testsystem stellt der untere Stängelereich von Soj ds optimle Pflnzenteil für die Anlyse von freien Aminosäuren dr, weil Proen reproduzierr gezogen werden können, die Konzentrtion des Zellsftes im Vergleich zur Wurzel in einem geringeren Mß durch den Wssersttus eeinflusst wird, und weil der untere Stängelereich im Vergleich zu Blättern sehr empfindlich uf Sulfonylure-Herizide im Wurzelrum regiert. Eine Anhme der Bltttrnspirtionsrte ist ein weiterer Effekt von Metsulfuron-methyl, so dss deren Messung einen wertvollen zusätzlichen Prmeter für ds eschrieene iologisch-chemische Testsystem drstellt. Europ.J.Hort.Sci. 4/4

2 134 Keutgen et l.: A Biologicl-chemicl Test System to Investigte the Influence of Sulfonylure Hericides in Soyen Introduction The sulfonylure hericide metsulfuron-methyl (MS) intercts with cetohydroxycid synthse, which is lso known s cetolctte synthse. The enzyme ctlyses the first rection, tht is common to the iosynthesis of the rnched-chin mino cids vline, leucine, nd isoleucine (SOUTHAN nd COPELAND 1996). MS is rpidly sored vi the roots nd leves nd is distriuted throughout the entire plnt within few hours. Since the 198s, sulfonylure hericides hve een studied nd pplied in griculture of mny countries nd re mong the most effective weed killers with low ppliction rtes, e.g. 8 g/h (DROBNY 1984; ACKERSON nd DAVIES 1987). Unfortuntely, residues of these hericides, even t low levels, represent potentil risk to certin following crops such s soyen, mize, or sorghum due to their slow degrdtion in certin groecosystems (VEGA et l. 199). Nowdys, the use of sulfonylure-tolernt vrieties of these crops insted of trditionl vrieties hs promise to minimise this prolem (SALISBURY nd BEAN 1998). In soyen, sprgine (Asn) cts s shunt for nitrogen storge nd/or long distnce nitrogen trnsport. It is sid to reflect the nitrogen sttus of the plnt (BACANAMWO nd HARPER 1997) nd together with glutmine (Gln) is considered to represent potentil cndidte to regulte nitrogen uptke in non-nodulted soyen roots (RUFTY et l. 1993). Therefore, the present uthors intended to develop simple ion chromtogrphic method for the determintion of these key mino cids. The hypothesis ws tested, whether chnges of their concentrtions re le to reflect n influence of the sulfonylure hericide MS on nitrogen metolism. Soyen ws chosen s model plnt, ecuse it is sensitive to low levels of sulfonylure hericides in the root medium. The ion chromtogrphic method for the detection of Asn nd Gln ws developed y seprtely injection of vrious mino cids onto the system descried elow nd evlution of the system responses. Asprtic cid (Asp), Asn, Gln, α-lnine (α-al) s well s + could e determined. The contents of the first three of these mino cids in ddition to + nd were determined for roots, stems, nd leves of soyen exposed to MS. In the first experiment, soyen seedlings supplied with in the nutrient solution were treted with different concentrtions of MS in the root medium in order to evlute the sensitivity of the test system. In second experiment, only ws the nitrogen source to investigte the influence of the nutritious sttus on the response of the key mino cids in soyen. For the third experiment, MS ws supplied onto side leflet of trifolite to study, whether the ppliction of MS into the root medium or onto the lef influenced chnges of the key mino cid concentrtions. Moreover, we tried to investigte the spreding of MS effects in soyen trifolite from the MS-treted leflet to the non-treted ones. To distinguish etween effects of MS on nitrogen metolism nd nitrogen trnsloction, the short-lived 13 N rdioisotope ws used. The prtitioning of [ 13 N] within the trifolite ws chosen s n exmple nd its distriution ws followed using positron-emitting trcer imging system (PETIS). Mterils nd Methods Plnt mteril nd growth conditions Soyen seeds [Glycine mx (L.) Merr. cv. Enrei ] were surfce-sterilised y soking in 7 % ethnol solution (v/v) for one minute, then in.5 % sodium hypochlorite solution (w/v) for three minutes. After thorough rinsing in sterile deionised wter, seeds were germinted on deionised wter in the drk t 5 C. Five dys lter, the seedlings were trnsferred to 5 l plstic continers (1 seedlings per continer) filled with vermiculite nd supplied with sl nutrient solution contining either.45 mm K SO 4,.4 mm K HPO 4,.5 mm MgSO 4 x 7H O,.6 mm CCl, 1. mm,.1 mm Fe(III)-EDTA (nutrient solution 1, NS1) or.4 mm K HPO 4,.5 mm MgSO 4 x 7H O,.6 mm CCl, 1. mm K,.1 mm Fe(III)-EDTA (nutrient solution, NS). Noteworthy, in NS the phosphorus content ws y fctor of 1 smller thn in NS1. However, sed on the phosphorus concentrtion used y RUFTY et l. (1993) the phosphorus level in NS ws considered sufficient to void deficiency symptoms in cse the nutrient solution ws exchnged dily (cf. experiment ). The soyen seedlings were grown in controlled environment chmer under 16 h photoperiod, 6/4 C dy/night temperture regime, nd reltive humidity of out 7 %. Light intensity ws out 3 µmol m s 1 photosyntheticlly ctive rdition (PAR) t plnt height. For ll experiments, the plnts were neither incuted with Brdyrhizoium jponicum nor nodulted. For the first set of experiments with metsulfuron-methyl [MS, methyl -[[(4methoxy-6-methyl-1,3,5-trizin--yl)minocronyl]minosulfonyl]enzote, Wko Pure Chemicl Industries, Tokyo, Jpn], soyen seedlings remined in the plstic continers with vermiculite for three weeks. Experiments were conducted with the first trifolite lef. After removl of the cotyledons, plnts were individully trnsferred into plstic continers filled with 1 ml of nutrient solution NS1. The solution ws continuously erted nd exchnged every morning. After four-dy dpttion period to hydroculture conditions, 16 plnts were selected for uniformity nd four plnts ech received nutrient solution with.5, 5 or 5 pp MS or nutrient solution without MS s control. The nutrient solution ws not exchnged during the next four dys. After these four dys plnts were hrvested nd seprted into leves, stems, nd roots. Fresh mss of the smples ws determined, the smples were frozen t 5 C, nd stored for finl mino cid nd + nlyses. In the second experiment, thirty two-week old seedlings were trnsplnted to hydroponic culture with nutrient solution NS. These seedlings hd just expnded their primry leves. Three seedlings were situted in one 1 ml plstic continer. The nutrient solution ws continuously erted nd exchnged every morning. Cotyledons were removed nd soyen seedlings cclimtised for three dys efore the MS tretment. To hlf of the plnts MS ws supplied to 9 ml of modified nutrient solution NS without K so tht the MS concentrtion reched 5 pp fter ddition of further 1 ml K -solution (1 mm). K ws Europ.J.Hort.Sci. 4/4

3 Keutgen et l.: A Biologicl-chemicl Test System to Investigte the Influence of Sulfonylure Hericides in Soyen 135 dded four hours fter MS ppliction nd 1 ml smples of the nutrient solution were tken 1 min, 6 h nd 7h fter the ppliction to mesure uptke of soyen seedlings. The differences in the concentrtions t 1 min nd 6 h s well s t 1 min nd 7 h were clculted nd expressed in µmol h 1 g 1 fresh weight. The men of oth vlues ws clculted nd regrded s mesure of uptke. One set of control nd MS-treted plnts ws hrvested t the end of ech experimentl dy nd seprted into leves, stems, nd roots. After weighing, the smples were frozen t 5 C nd stored for finl mino cid nd nlyses. These trils were repeted nine times. Additionl mesurements on leves of these plnts were performed to study MS-effects on lef gs exchnge. For the third experiment, plnts were grown s for the first (NS1), ut 1 ppm MS ws supplied to side leflet of young, not completely expnded trifolite overnight. Dipping tissue pper in the MS solution nd ttching it onto the leflet surfces pplied the hericide. The leflet nd tissue were plced into plstic g. The high MS concentrtion ws chosen, ecuse preliminry experiments reveled tht lower concentrtions frequently did not ffect the trifolite, possily ecuse the hericide could not esily enter the lef (ut cf. DROBNY 1984). The remining leflets of the trifolite served s controls. Contemporneous to the MS ppliction, the second side leflet ws treted with deionised wter nd the middle leflet remined untreted. Some of the MS-treted nd untreted soyen seedlings were trnsferred to the rdioisotope lortory nd lelled with [ 13 N] for 3 min. Further seedlings were hrvested efore nd 13 dys fter MS ppliction, seprted into the three leflets of the trifolite nd the upper petiole, nd, fter fresh mss determintion, frozen t 5 C for finl mino cid nd nlyses. Additionl mesurements on single leflets of the trifolites were performed to investigte the effects of the MS ppliction on lef gs exchnge. Amino cid nd + nlyses The frozen plnt smples were cut into smll pieces nd trnsferred into ml Ultrfree-MC tues (the exclusion limit for gloulr protein = 1 x 1 4 D, Millipore, Milford, MA, USA). The smples were centrifuged for 6 min t 3 g t 4 C nd the mss of the plnt residue ws determined with lnce. The filtrte ws stored t 5 C. For the nlyses of + nd the mino cids sprtic cid (Asp), sprgine (Asn), α-lnine (α-al), nd glutmine (Gln) modified ion-chromtogrphic method ws chosen (BLACK- BURN 1968). The filtrte ws ppropritely diluted with deionised wter (ph 6.) nd injected onto precolumn (Shim-pck IC-GC1, Ø 4 mm x 1 mm, Shimdzu, Kyoto, Jpn) connected to seprtion column (Shim-pck IC-C1, Ø 5 mm x 15 mm, Shimdzu, Kyoto, Jpn) filled with ction exchnge resin of polystyrene-divinylenzene, which incorportes sulfonic cid se s functionl group. Free mino cids nd + were seprted on the column using 5 mm H (flow rte:.8 ml min 1 ) s moile phse. The column temperture ws set t 3 C nd conductivity detector ws used for mino cid nd + detection. The peks were recorded with desktop recorder (Ohkur Electric, Tokyo, Jpn). Anlysis of plnt content nd uptke The content of the soyen frctions nd the nutrient solutions were lso nlysed y ion chromtogrphy. The nlyticl system consisted of precolumn nd seprtion column (TSKgel, IC-Anion-PW, Ø 4.6 mm x 5 mm, Tosoh Corportion, Tokyo, Jpn) with TSK IC-Amino-A s moile phse (1. ml min 1 ). Column temperture ws set t 3 C nd conductivity detector ws used for detection. Visulistion of [ 13 N] y PETIS An queous solution of [ 13 N] ws produced ccording to the rection 16 O (p, α) 13 N y omrding wter with 1 µa of MeV H + -prticles from the TIARA AVF cyclotron. A ction exchnge column (Accell Plus CM, Wters, Eschorn, Germny) ws used to purify the [ 13 N] solution from trces of other lelled 13 N compounds (ISHIOKA et l. 1999). The PETIS (Positron-Emitting Trcer Imging System) consists of pir of lock detectors (5 x 5 cm ), which re composed of Bi 4 Ge 3 O 1 scintilltor rry coupled with position sensitive photomultiplier tue (PS-PMT, Hmmtsu R3941-, Hmmtsu Photonics Co., Hmkit, Shizuok, Jpn). They produce time-series of two-dimensionl imges with sptil resolution of mm. The position of trcer ctivity in the soyen trifolite ws determined y coincidence mesurements of ntiprllel γ-ry pirs produced from the nnihiltion of positrons emitted y the trcer (Fig. 1). The use of mm thick soring crylic plte plced out mm from the plnt surfce is necessry to quntify imges of lef tissues, ecuse nnihiltion of positrons does not provide n ccurte trcer position in the plnt if the positron escpes the tissue (SATO et l. 1999; KUME et l. 1999). At the dy of the trcer experiment, soyen petioles were cut under deionised wter nd the young trifolites were rrnged in front of the PETIS detectors. Imme- Fig. 1. Sketch of the experimentl set-up for the mesurement of the distriution of [ 13 N] rdioctivity in soyen trifolites with the Positron-Emitting Trcer Imging System (PETIS). Europ.J.Hort.Sci. 4/4

4 136 Keutgen et l.: A Biologicl-chemicl Test System to Investigte the Influence of Sulfonylure Hericides in Soyen ditely efore the experiment, the wter ws replced y the trcer solution. [ 13 N] (5 to 1 Mq/ml) ws supplied to soyen petioles for 3 min. During the trcer pplictions, dt were recorded continuously with smpling intervl of 15s. The rdioctivity tht ccumulted within the soyen trifolite is given in order to visulise [ 13 N] quntity nd distriution. Lef gs exchnge Net photosynthesis rte (A), trnspirtion rte (E), stomtl conductnce (g s ), nd intercellulr CO concentrtion (C i ) of single soyen leflets were mesured with the LI-64 portle photosynthesis system (LI-COR Inc., Lincoln, Nersk, USA). Gs exchnge prmeters were recorded t 5 µmol m s 1 PAR, t out 5 C lef temperture, nd out 6 % reltive humidity. The mient CO concentrtion ws out 35 ppm in the lef cuvette. Gs exchnge mesurements were conducted during the first six hours of the photoperiod. Dt nlysis The experimentl dt were nlysed with the SPSS-sttisticl pckge (SPSS, Chicgo, USA). Concentrtions of free mino cids, +,, uptke y roots nd lef gs exchnge mesurements were in most cses compred y t-tests. In some instnces n ANOVA ws performed, ut sometimes trnsformtion of dt ws necessry to stilise the vrinces. In cse of vrince homogeneity, Duncn tests were used. In cse the vrinces were not homogeneously distriuted, the non-prmetric Wilcoxon test ws used nd the 5 % proility level ws corrected ccording to Bonferroni. Results Amino cid nlysis The response of the nlyticl system ws evluted y seprte injection of vrious mino cids. Asprtic cid (Asp), serine (Ser), threonine (Thr), sprgine (Asn), glutmic cid (Glu), glutmine (Gln), cysteine (Cys), α-lnine (α-al), β-lnine (β-al), proline (Pro), vline (Vl), γ-minoutyric cid (γ-aba), methionine (Met), tyrosine (Tyr), isoleucine (Ile), nd leucine (Leu) could e detected, wheres glycine (Gly), histidine (His), rginine (Arg), phenyllnine (Phe), tryptophn (Trp), lysine (Lys), nd ornithine (Orn) could not. The system showed lso high sensitivity for N +, +, nd K +. Unfortuntely, some of the peks consisted of more thn one component. For soyen smples, the first detectle pek consisted of Asp (t R =.6 min). The second single pek contined four mino cids, viz. Asn, Glu, Ser, nd Thr (t R =3. min). This pek ws followed y peks for Gln (t R =3.3 min), α-al (t R =4. min), N + (t R =4.8 min), + (t R =6.3 min) nd K + (t R =7.3 min), nd severl rod peks for Met, Tyr, Ile, nd Leu (their t R vlues rnged etween 11. min nd 13.8 min). The concentrtion of Cys (t R =6.3 min) ws too low to e detected. The determintion of Vl nd γ-aba turned out to e impossile due to overlpping with the K + pek. The peks of N + nd + impired the detection of Pro nd β-al, respectively. To summrise, in soyen, relile determintions were mesured for Asp, Glu, α-al, nd + (Fig. ). The concentrtion of the key mino cid Asn could e identified y quntittive hydrolysis to Asp y dding.1 µl of 35 % HCl p.. to.4 µl of soyen filtrte (48 h incution t 8 C) in seprte run. The reltionship of pek height to mino cid nd + concentrtion ws liner. Judging from the slope of the regression lines, the ion chromtogrphic nlysis ws most sensitive to +, followed y Asn, α-al, Gln, nd Asp (Tle 1). To ssess the ccurcy of the ion chromtogrphic method, primry lef, stem, nd root smples of soyen were spiked with.,.5, nd 1. µmol g 1 of ech sustnce, frozen over night, centrifuged, nd nlysed. The results, which re summrised in Tle 1, indicte tht the recoveries were etween 8 % nd 13 %. Men vlues re 88 % for α-al, 91 % for Gln, 93 % for Asp, 98 % for Asn, nd 1 % for +, respectively. Pek height [cm] content [µmol g -1 ] Retention time [min] Asn, Glu, Ser, Thr Asp Gln Al N + + K Fig.. Ion chromtogrphy pttern of selected free mino cids nd further ctions in filtrte from stem section of soyen. root stem lef MS content in nutrient solution [pp] Fig contents per g fresh weight in leves, stems, nd roots of soyen plnts s ffected y different concentrtions of MS in the nutrient solution. Plnts received nutrient solution NS1 contining 1 mm. Vlues represent mens of four replictes. Europ.J.Hort.Sci. 4/4

5 Keutgen et l.: A Biologicl-chemicl Test System to Investigte the Influence of Sulfonylure Hericides in Soyen 137 Tle 1. Linerity of concentrtion responses (correltion coefficient r ) nd recoveries () of sprtic cid (Asp), sprgine (Asn), glutmine (Glu), α-al (Al), nd NH4 + in soyen primry leves, stems, nd roots y ion chromtogrphy. Stndrd Lef smple Stem smple Root smple slope [µmol ml 1 ] r Added [µmol g 1 ] found [µmol g 1 ] [µmol g 1 ] [%] CV (%) found [µmol g 1 ] [µmol g 1 ] [%] CV (%) found [µmol g 1 ] [µmol g 1 ] [%] CV (%) Asp Asn Gln Al NH Europ.J.Hort.Sci. 4/4

6 138 Keutgen et l.: A Biologicl-chemicl Test System to Investigte the Influence of Sulfonylure Hericides in Soyen Tle. Chnges of mino cid concentrtions per g fresh weight in soyen due to,.5, 5 nd 5 pp metsulfuron-methyl in the nutrient solution. MS (pp) Gln (µmol g 1 ) Asp (µmol g 1 ) Asn (µmol g 1 ) Lef.17 ±.6.96 ±..98 ± ± ± ± ± ± ± ± ± ± 1.89 Stem 1. ± ± ± ± ± ± ± ± ± ± ± ± 19. Root.16 ±.8.44 ± ± ± ± ± ± ± ± ± ± ± 4.38 Vlues represent mens ± stndrd devitions of four replictes. Mens of leves, stems nd roots within column were compred y Duncn or Wilcoxon tests. Different letters indicte significnt differences t the 5% level. Root ppliction of different MS concentrtions with s nitrogen source Soyen seedlings exposed to,.5, 5 or 5 pp MS in nutrient solution NS1 for four dys were chrcterised y significnt concentrtion chnges of Gln nd Asn in roots, stems, nd leves (Tle ). With respect to Asp, significnt hericide effect ws detectle for the stem. In roots, however, the effect ws distinct, ut not detectle for leves. The results clerly indicte tht concentrtion of MS s low s.5 pp ffected mino cid content. In cses of MS effect, however, sttisticlly significnt differences mong the three MS concentrtions could not e found. By contrst, + concentrtions rose in roots nd stems in mnner tht is typicl of sturtion curve (Fig. 3). Root ppliction of 5 pp MS with only K s nitrogen source This experiment ws performed to investigte the influence of nitrogen source on mino cid response nd lso the time dependency of mino cid concentrtion chnges due to MS. First effects of the tretment with 5 pp MS in the root medium were oserved two dys fter MS ppliction. content of roots rose significntly nd concentrtions of Gln nd Asp in tendency. The increse of these mino cids ecme significnt t the third dy. uptke from the nutrient solution ws significntly reduced three dys fter MS ppliction (Fig. 4). In the lower stem section, the concentrtion chnges of mino cids were similr to those in roots, with the exception tht MS effect ws lredy significnt two dys fter MS ppliction in cse of Gln. Differences in concentrtions of stems, however, could not e detected (Fig. 5). In the lef section, MS effect ws rely detectle. Only Asp content strted to rise three dys fter MS ppliction, which is one dy lter thn in the root nd lower stem section (Fig. 6). Lef gs exchnge ws significntly impired y the ppliction of 5 pp MS to the nutrient solution. Stomtl conductnce (g s ) nd trnspirtion rte (E) were significntly lower t the third nd fourth dy fter MS ppliction. Net photosynthesis rte (A), however, ws not ffected, which corresponded well with the reduction of lef intercellulr CO concentrtion (C i ) during the lst two dys of the experiment (Fig. 7). Lef ppliction of metsulfuron-methyl The ppliction of solution contining 1 ppm MS onto trifolite resulted in time-dependent response of Gln, Asp, Asn, nd concentrtions (Fig. 8). In the MS-treted leflet Gln, Asp, nd Asn concentrtions were somewht elevted y the end of the first dy fter MS ppliction, wheres the concentrtions of these mino cids rose with dely of one dy in the remining leflets of the hericide-treted trifolite nd lso the petiole. However, in the petiole the Asn concentrtion ws not ffected y the hericide tretment. concentrtions were distinctly reduced t the third dy fter the MS ppliction in ll leflets nd the petiole. The decrese ws most pronounced in the petiole nd middle leflet, ut lest in the MS-treted one (Fig. 8). Mesurements of lef gs exchnge indicted significnt reduction of trnspirtion rte three dys fter MS ppliction (dt not shown). Noteworthy, ll leflets of the treted trifolite responded similrly t Europ.J.Hort.Sci. 4/4

7 Keutgen et l.: A Biologicl-chemicl Test System to Investigte the Influence of Sulfonylure Hericides in Soyen 139 Asn [µmol g -1 ] Asp [µmol g -1 ] Gln [µmol g -1 ] NO3 - [µmol g -1 ] 3 - uptke NO -1 g -1 ] [µmol h Dys fter first MS ppliction the third dy of the experiment despite the different time response of mino cids. The hypothesis ws tested, whether the differences in leflet contents t the lst dy of the experiment, resulted from different rtes of uptke y the leflets (cf. Fig. 8). The experiment with [ 13 N] indicted tht comprle reltive mount of trcer, entering the view of the PETIS detector, ws trnslocted into ech leflet of trifolite efore nd during the three dys fter MS ppliction, which ws evident from the comprle distriution pttern of pixels in the leflets of the trifolites shown in ech of the Figures 9 nd 1. Nevertheless, the solute mount of trcer tht ws trnslocted into the trifolites ws different in these figures. Wheres the shpe of the trifolite ws well reflected y the pixels in Fig. 9, it ws hrdly visile in Fig. 1, where fewer rdioctivity entered the trifolite. In oth figures, the highest mount of rdioctivity ppered in the petiole, which ws situted in the lower right (Fig. 9) or lower left side (Fig. 1) of the imges. The comprison of trcer intensity in Fig. 9, which is representtive of dys efore nd during the first two dys of MS ppliction, nd of Fig. 1, which represents the sitution t the third dy fter MS ppliction, thus proved tht distinctly less [ 13 N] entered the entire trifolite t the lst dy of the experiment. This result corresponded well with the mesured reduction of leflet trnspirtion rtes. If follows tht the differences in leflet contents t the lst dy of the experiment cnnot e explined y differences in uptke y the leflets. Discussion In rottionl frming systems residues of sulfonylure hericides my represent potentil risk to sensitive crops. Therefore, relile methods re needed to detect low levels of sulfonylure hericides in the soil. Biossys, using whole plnt or cell tissues (BERGER 199; OLOFSDOTTER et l. 1994), re commonly used to detect pesticide residues, frequently in comintion with high-performnce liquid chromtogrphy nd gs chromtogrphy methods (HERSHBERGER nd BREN- NAN 1988; COTTERILL 199; GALLETTI et l. 1995). In some cses, in vivo AHAS ssys (SIMPSON et l. 1995) nd immunossys (KELLEY et l. 1985; JIA 1998) re ville. However, iologicl-chemicl test system sed on the ion chromtogrphic nlysis of the free key mino cids Gln nd Asn in sulfonylure hericide sensitive soyen seedlings my represent n esy to hndle, cost-effective, quick, nd relile lterntive to Fig. 4. uptke y roots s well s chnges of, glutmine (Gln), sprtic cid (Asp), nd sprgine (Asn) concentrtions per g fresh weight in roots fter ppliction of 5 pp MS to the nutrient solution contining 1 mm K s nitrogen source. Mens nd stndrd devitions re given for controls (o) nd MS-treted ( ) soyen seedlings. Men vlues of ech dy were compred y t-tests (P=.5). [µmol g -1 ] Asn [µmol g -1 ] Asp [µmol g -1 ] Gln [µmol g -1 ] Dys fter first MS ppliction Fig. 5. Chnges of, glutmine (Gln), sprtic cid (Asp), nd sprgine (Asn) concentrtions per g fresh weight in the lower stem section fter ppliction of 5 pp MS to the nutrient solution contining 1 mm K s nitrogen source. Mens nd stndrd devitions re given for controls (o) nd MS-treted ( ) soyen seedlings. Men vlues of ech dy were compred y t-tests (P=.5). Europ.J.Hort.Sci. 4/4

8 14 Keutgen et l.: A Biologicl-chemicl Test System to Investigte the Influence of Sulfonylure Hericides in Soyen [µmol g -1 ] Asn [µmol g -1 ] Asp [µmol g -1 ] Gln [µmol g -1 ] C i [ppm] A [µmol m - s -1 ] g s [mmol m - s -1 ] E [µmol m - s -1 ] Dys fter first MS ppliction Dys fter first MS ppliction Fig. 6. Chnges of, glutmine (Gln), sprtic cid (Asp), nd sprgine (Asn) concentrtions per g fresh weight in lefs fter ppliction of 5 pp MS to the nutrient solution contining 1 mm K s nitrogen source. Mens nd stndrd devitions re given for controls (o) nd MS-treted ( ) soyen seedlings. Men vlues of ech dy were compred y t-tests (P=.5). Concentrtion [µmol g -1 ] nd 1 3 Dys fter MS ppliction - Gln Asp Asn Fig. 8. Chnges of, glutmine (Gln), sprtic cid (Asp), nd sprgine (Asn) concentrtions per g fresh weight in soyen trifolite nd upper petiole fter ppliction of 1 ppm MS to single leflet overnight etween dys nd 1. A = MS-treted side leflet, B = H O-treted side leflet, C = untreted middle leflet, D = upper petiole. Sttistics nd stndrd devitions re not given, ecuse mens were clculted from two mesurements, only. nd nd A B C D Fig. 7. Chnges of trnspirtion rte (E), stomtl conductnce (g s ), net photosynthesis rte (A), nd lef intercellulr CO concentrtion (C i ) fter ppliction of 5 pp MS to the nutrient solution contining 1 mm K s nitrogen source. Mens nd stndrd devitions re given for controls (o) nd MS-treted ( ) soyen seedlings. Men vlues of ech dy were compred y t-tests (P=.5). test for residues in the soil, especilly in developing countries. Soyen seedlings exposed to different mounts of MS in the root medium in the first experiment responded quite sensitive with respect to mino cid concentrtion chnges. Contents of Gln, Asp, nd Asn rose significntly or distinctly with the exception of Asp in leves (Tle ). However, when only K ws the nitrogen source in the root medium, the response ws slightly different. Gln nd Asp rose significntly in roots nd stems, wheres Asn concentrtions did not chnge. In leves, only Asp content rose significntly. The ltter difference could e explined y the fct tht MS effect might not hve een estlished completely. For exmple, lter increse of Gln content in the second experiment cnnot e ruled out. Nevertheless, with respect to Asn levels especilly in roots nd stems this interprettion is not stisfying. In cse only ws the nitrogen source, it ws converted vi nitrte reductse nd nitrite reductse into +, which ws converted y glutmine synthse nd GOGAT to glutmic cid in plstids. Glutmic cid ws the first mino cid to pper in the cytosol nd ws the nitrogen donor for the conversion of oxlocette to Asp, the immedite precursor of Asn (MENGEL 1991). Thus, the increse of Asp clerly showed MS effect in the root nd stem section, ut not Asn levels. A comprison of the Asn concentrtions in roots, stems nd leves of control plnts indicted tht in the second experiment with only s nitrogen source Europ.J.Hort.Sci. 4/4

9 Keutgen et l.: A Biologicl-chemicl Test System to Investigte the Influence of Sulfonylure Hericides in Soyen 141 Fig. 9. PETIS imge of the [ 13 N] rdioctivity ccumulted during 3 min in n untreted soyen trifolite. The rdioctivity is expressed in reltion to the lrgest mount, which ws oserved in the petiole (lck pixels). The righter the pixel, the less is the reltive rdioctivity. Fig. 1. PETIS imge of the [ 13 N] rdioctivity ccumulted during 3 min in MS-treted soyen trifolite three dys fter MS ppliction. The rdioctivity is expressed in reltion to the lrgest mount, which ws oserved in the petiole (lck pixels). The righter the pixel, the less is the reltive rdioctivity. the Asn level ws distinctly higher (Figs 46), eing lredy t the level found in the MS-treted plnts of the first experiment (Tle ). As Asn is considered to reflect the nitrogen sttus of soyen (BACANAMWO nd HARPER 1997), the differences my simply indicte tht the nitrogen supply ws somewht limited in the first experiment, when the nutrient solution ws not replced for four dys, wheres it ws exchnged every dy in the second. Alterntively, we my ssume tht in the second experiment the soyen seedlings consumed less nitrogen. Another explntion could e the lower phosphorus level in the nutrient solution NS, which might hve cused n increse of Asn content in the second experiment due to phosphorus deficiency (RUFTY et l. 1993). To summrise, the nutritious sttus of the plnt influenced the chnges of Asn concentrtions in response to the hericide tretment in soyen. The loction of the MS ppliction, for instnce onto soyen trifolite in the third experiment, lso ffected the response of the mino cid concentrtions. Gln, Asp, nd Asn contents incresed distinctly in leves, wheres in the petiole only concentrtions of Gln nd Asp rose. A comprison of the first nd third experiment, when the supplied nutrient solutions were similr ut the loctions of MS ppliction different, reveled tht Asp content of leves rose significntly in the third ut not in the first experiment. An explntion of this result is yet difficult. Nevertheless, in the second experiment the increse of Asp content due to MS ws the only detectle response for leves. Hence, its increse in the third experiment seems relile. With respect to petioles, it hs to e considered tht the min trnsport forms of nitrogen sored y non-nodulted roots re nd Asn. Both re trnsported to the leves vi the xylem (DELHON et l. 1995; SATO et l. 1999). The in spite of MS tretment unchnged Asn content in the petiole my thus indicte tht Asn trnsloction hd not een ffected considerly y MS during the experiment. Consequently, the results of the third experiment showed tht the loction of the MS ppliction lso influenced the concentrtion responses of Asn. Although nutrient supply nd loction of MS ppliction ffect mino cid concentrtion chnges, the descried ion chromtogrphic method for the determintion of key mino cids within soyen seedlings my e used s iologicl-chemicl test system to identify the presence of sulfonylure hericides such s MS in the soil in rottionl frming systems. In this test system, the hericide enters the plnt vi the root; hence the effect of the loction of hericide ppliction my e neglected. According to the present results, the optiml plnt section for mino cid nlyses represents the lower stem section of soyen seedlings, ecuse here significnt effects were first detectle (cf. Figs 46). Moreover, stem smples cn reproducily e tken nd the concentrtion of the cell sp is, when compred to roots, less influenced y the wter sttus. Independent from the nitrogen source Gln nd Asp contents rose significntly in stems. In cse of the presence of + in the root medium, n increse of its concentrtion in stem smples my lso e detectle (Fig. 3). The mino cid Asn represents less suitle indictor, ecuse its concentrtion is oviously influenced y the nutritious sttus of the plnt. Asp is to e used insted of Asn, which is dvntgeous, ecuse the second ion chromtogrphic nlysis to determine Asn y quntittive hydrolysis to Asp cn e omitted. The descried test system hs een investigted only for the sulfonylure-sensitive soyen cv. Enrei, which ws not incuted with Brdyrhizoium jponicum. In consequence, the influence of the occurence of Brdyrhizoium in the soil on the results of the test system is uncler. It hs een proved tht in soyen the mjor trnsport forms of nitrogen ssimilted y root nodules re ureides, llntoic cid, nd llntoin, wheres the min trnsport forms of nitrogen - Europ.J.Hort.Sci. 4/4

10 14 Keutgen et l.: A Biologicl-chemicl Test System to Investigte the Influence of Sulfonylure Hericides in Soyen sored from roots re nitrte nd sprgine (SATO et l. 1999). In consequence, differences in responses of Gln, Asp, nd + contents to sulfonylures in the soil my e expected when Brdyrhizoium estlished on soyen roots. However, the ppernce of Brdyrhizoium cn esily een checked y looking for nodules on the root system. The nitrogen sttus of plnt should lso influence its nitrogen ssimiltion. ent models derived from moleculr iologicl studies on Aridopsis thlin (L.) propose tht the trnscript undnces for nd + trnsporters re regulted y Gln, wheres root nd + contents my influence trnsporter ctivity (GLASS et l. 1, ). This model corresponds well with the results of the second experiment with K s nitrogen source. uptke ws reduced three dys fter MS ppliction, one dy fter Gln content strted to rise nd content of MS-exposed roots ws elevted (Fig. 4). A regultion of uptke y Asn cn e ruled out, ecuse its concentrtion did not chnge significntly. The present results indicte tht uptke ws lso impired y MS due to its influence on root Gln level nd ssimiltion (GODON et l. 1996)., which hs not een ssimilted in the roots, is trnslocted in the xylem nd my rech the leves, where it is ssimilted into mino cids. In the third experiment, when MS ws pplied onto leflet, concentrtions decresed in the soyen trifolite three dys fter MS ppliction, with the highest content remining in the MS-treted leflet (Fig. 8). The [ 13 N] trcer experiment showed tht the import of into the leflets did not differ within single trifolite (Fig. 1). It follows tht the different concentrtions in the leflets of trifolite cnnot e explined y different rtes of import, ut y more impired ssimiltion in the MS-treted leflet. This response could e expected, ecuse the MS effect ws detectle in the MS-treted leflet one dy erlier (Fig. 8). However, the low leflet [ 13 N] levels t the lst dy of the experiment lso indicted tht MS impired trnsport (cf. Figs 91), proly due to n impirment of lef trnspirtion (E). This effect of sulfonylure hericides, which hs yet rrely een focussed in literture (MASHHADI et l. 1986), ws proved y gs exchnge mesurements (cf. Fig. 7) nd my represent n dditionl prmeter tht could esily e checked in the mentioned iologicl-chemicl test system for the identifiction of sulfonylure hericide residues in the soil in rottionl frming systems. Noteworthy, the reduction of E is not closely relted to the mino cid metolism in the leflets, s the differences in nitrogen metolism in the time response of single leflets were not reflected in the modifiction of E nd stomtl conductnce (g s ). According to LEONHARDT et l (1997), sulfonylure hericides cn modulte the stomtl movements y their effect on gurd cell potssium chnnels. For exmple, glienclmide nd tolutmide resulted in n increse of g s. Thus, the immedite effect of these sulfonylure hericides differed from the oserved reduction of g s. Presumly, the oserved nrrowing of stomtl perture did not represent n immedite MS effect. Future investigtions of chnges in the scisic cid content my help to understnd the oserved response. Acknowledgements This reserch ws enled y the Science nd Technology Agency, Jpn, tht supported Ann nd Norert Keutgen during their sty in Jpn. N. Keutgen ws moreover grnted n Alexnder von Humoldt-fellowship, Germny, nd Minghong Ji ws supported y the STA Scientist Exchnge Progrm fellowship of the Science nd Technology Agency. References ACKERSON, R.C. nd L.A. DAVIS 1987: Metsulfuron methyl new hericide for use in rice nd plnttion crops. Proceedings, 11 th Asin Pcific Weed Science Society Conference, Asin Pcific Weed Science Society, Tipei, Tiwn, pp BACANAMWO, M. nd J.E. HARPER 1997: The feedck mechnism of nitrte inhiition of nitrogense ctivity in soyen my involve sprgine nd/or products of its metolism. Physiologi Plntrum 1, BERGER, B. 1993: Biossy for the determintion of the degrdtion of metsulfuron-methyl in soil. Gesunde Pflnzen 45, BLACKBURN, S. 1968: The chromtogrphic seprtion of mino cids. In: BLACKBURN, S. (ed.): Amino cid determintion. Methods nd techniques. Mrcel Dekker, Inc., New York, USA, pp COTTERILL, E.G. 199: Determintion of the sulfonylure hericides chlorsulfuron nd metsulfuron-methyl in soil, wter nd plnt mteril y gs chromtogrphy of their pentfluorenzyl derivtes. Pestic. Sci. 34, DELHON, P., A. GOJON, P. TILLARD nd L. PASSAMA 1995: Diurnl regultion of uptke in soyen plnts. I. Chnges in influx, efflux, nd N utiliztion in the plnt during the dy/night cycle. Journl of Experimentl Botny 46, DROBNY, H.G. 1984: DPX-T 6376 new hericide for cerels. Zeitschrift für Pflnzenkrnkheiten und Pflnzenschutz, Sonderheft 1, GALLETTI, G.C., A. BONETTI nd G. DINELLI 1995: High-performnce liquid chromtogrphic determintion of sulfonylures in soil nd wter. J. Chromtogr. 69, 737. GLASS, A.D.M., D.T. BRITTO, B.N. KAISER, H.J. KRO- NZUCKER, A. KUMAR, M. OKAMOTO, S.R. RAWAT, M.Y. SIDDIQI, S.M. SILIM, J.J. VIDMAR nd D. ZHUO 1: Nitrogen trnsport in plnts, with n emphsis on the regultion of fluxes to mtch plnt demnd. Journl of Plnt Nutrition nd Soil Science 164, GLASS, A.D.M., D.T. BRITTO, B.N. KAISER, J.R. KING- HORN, H.J. KRONZUCKER, A. KUMAR, M. OKAMOTO, S. RAWAT, M.Y. SIDDIQI, S.E. UNKLES nd J.J. VIDMAR : The regultion of nitrte nd mmonium trnsport systems in plnts. Journl of Experimentl Botny 53, GODON, C., A. KRAPP, M.-T. LEYDECKER, F. DA- NILE-VEDELE nd M. CABOCHE 1996: Methylmmonium-resistnt mutnts of Nicotin plumginifoli re ffected in nitrte trnsport. Moleculr nd Generl Genetics 5, Europ.J.Hort.Sci. 4/4

11 Keutgen et l.: A Biologicl-chemicl Test System to Investigte the Influence of Sulfonylure Hericides in Soyen 143 HERSHBERGER, L.W. nd D.E. BRENNAN 1988: Metsulfuron methyl. Anl. Methods Pestic. Plnt Growth Regul. 16, ISHIOKA, N.S., H. MATSUOKA, S. WATANABE, A. OSA, M. KOIZUMI, T. KUME, S. MATSUHASHI, T. FUJIMURA, A. TSUJI, H. UCHIDA nd T. SEKINE 1999: Production of positron emitters nd ppliction of their lelled compounds to plnt studies. Journl of Rdionlyticl nd Nucler Chemistry 39, JIA, M.H. 1998: Development of immunossy for sulfonylure hericide in Chin. Proceedings of n interntionl workshop on seeking griculturl products free of pesticide residue, Fe. 1719, 1998, Austrlin Center of Interntionl Agriculturl Reserch, Yogykrt, Indonesi, pp KELLEY, M.M., E.W. ZAHNOW, W.C. PETERSEN nd S.T. TOY 1985: Chlorsulfuron determintion in soil extrcts y enzyme immunossy. J. Agric. Food Chem. 33, KUME, T. 1999: Appliction of positron emitting trcer imging system (PETIS) for plnt reserch. Rdioisotopes 48, LEONHARDT, N., E. MARIN, A. VAVASSEUR nd C. FOR- ESTIER 1997: Evidence for the existence of sulfonylure-receptor-like protein in plnts: Modultion of stomtl movements nd gurd cell potssium chnnels y sulfonylures nd potssium chnnel openers. Proceedings of the Ntionl Acdemy of Science of the United Sttes of Americ 94, MASHHADI, H., B. BUGBEE nd J.O. EVANS 1986: The use of direct photosynthetic mesurement in hericide reserch. Proceedings of the Western Society of Weed Science 1986, 445. MENGEL, K. 1991: Ernährung und Stoffwechsel der Pflnze. 7. üerreitete Auflge, Gustv Fischer Verlg Jen, Germny, pp OLOFSDOTTER, M., A. OLESEN, S.B. ANDERSEN nd J.C. STREIBIG 1994: A comprison of hericide iossys in cell cultures nd whole plnts. Weed Reserch 34, RUFTY, T.W.Jr., D.W. ISRAEL, R.J. VOLK, J. QIU nd T. SA 1993: Phosphte regultion of nitrte ssimiltion in soyen. Journl of Experimentl Botny 44, SALISBURY, C.D. nd B.W. BEAN 1998: Tolernce of imidzolinone resistnt corn nd sulfonylure tolernt soyen to sulfonylure hericide residues fter whet. In: Prepring for the New Millenium. 51 st Annul Meeting Jnury 68, 1998, Birminghm, Alm. Southern Weed Science Society (USA) 51, p.19. SATO, T., N. OHTAKE, T. OHYAMA, N.S. ISHIOKA, S. WA- TANABE, A. OSA, T. SEKINE, H. UCHIDA, A. TSUJI, S. MATSUHASHI, T. ITO nd T. KUME 1999: Anlysis of nitrte sorption nd trnsport in non-nodulted nd nodulted soyen plnts with 13 nd 15. Rdioisotopes 48, SIMPSON, D.M., E.W. STOLLER nd L.M. WAX 1995: An in vivo cetolctte synthse ssy. Weed Technol. 9, 17. SOUTHAN, M.D. nd L. COPELAND 1996: Physicl nd kinetic properties of cetohydroxycid synthse from whet leves. Physiologi Plntrum 98, VEGA, D., J. BASTIDE nd C. POULAIN 199: Chemicl or microil degrdtion of sulfonylures in the soil. II. Metsulfuron methyl. Weed Reserch 3, eived Octoer 8, 3 / Accepted My 6, 4 Addresses of uthors: A. J. Keutgen (corresponding uthor), Institut für Agrikulturchemie (Agriculturl Chemistry), Crl-Sprengel-Weg 1, D 3775 Göttingen, Germny, Tel. 551/395565, Fx 551/39557, Emil: AKeutge@gwdg.de, N. Keutgen, M. Ji, S. Mtsuhshi, C. Mizuniw, T. Ito, T. Fujimur nd S. Hshimoto, Deprtment of Rdition Reserch for Environment nd Resources, Tkski Rdition Chemistry Reserch Estlishment, JAERI, Tkski, Gunm 3719, Jpn nd T. Sekine, Deprtment of Mterils Science, Tkski Rdition Chemistry Reserch Estlishment, JAERI, Tkski, Gunm 3719, Jpn Europ.J.Hort.Sci. 4/4