Does elevated atmospheric [CO 2 ] alter diurnal C uptake and the balance of C and N metabolites in growing and fully expanded soybean leaves?

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1 Journl of Experimentl Botny, Vol. 58, No. 3, pp , 7 doi:1.193/jx/erl33 Advne Aess pulition 8 Deemer, This pper is ville online free of ll ess hrges (see for further detils) RESEARCH PAPER Does elevted tmospheri [CO ] lter diurnl C uptke nd the lne of C nd N metolites in growing nd fully expnded soyen leves? Elizeth A. Ainsworth 1,,, Alistir Rogers 3,, Andrew D. B. Lekey, Lindsey E. Hedy 3, Yves Gion 5, Mrk Stitt 5 nd Ulrih Shurr 1 USDA/ARS Photosynthesis Reserh Unit nd Deprtment of Plnt Biology, University of Illinois, Urn, IL 181, USA Institute for Genomi Biology, University of Illinois, Urn, IL 181, USA 3 Deprtment of Environmentl Sienes, Brookhven Ntionl Lortory, Upton, NY , USA Deprtment of Crop Sienes, University of Illinois, Urn, IL 181, USA 5 Mx Plnk Institut für Molekulre Pflnzenphysiologie, D-17 Golm, Germny ICG-III, Juelih Reserh Center, D-55, Juelih, Germny Reeived 19 June ; Revised 8 Septemer ; Aepted 1 Otoer Astrt Inreses in growth t elevted [CO ] my e onstrined y plnt s ility to ssimilte the nutrients needed for new tissue in suffiient quntity to mth the inrese in ron fixtion nd/or the ility to trnsport those nutrients nd ron in suffiient quntity to growing orgns nd tissues. Anlysis of metolites provides n indition of shifts in ron nd nitrogen prtitioning due to rising tmospheri [CO ] nd n help identify where ottleneks in ron utiliztion our. In this study, the ron nd nitrogen lne ws investigted in growing nd fully expnded soyen leves exposed to elevted [CO ]in free ir CO enrihment experiment. Diurnl photosynthesis nd diurnl profiles of ron nd nitrogen metolites were mesured during two different rop growth stges. Diurnl ron gin ws inresed y. % in elevted [CO ] in fully expnded leves, whih led to signifint inreses in lef hexose, surose, nd strh ontents. However, there ws no detetle differene in nitrogen-rih mino ids nd ureides in mture leves. By ontrst to mture leves, developing leves hd high onentrtions of ureides nd mino ids reltive to low onentrtions of rohydrtes. Developing leves t elevted [CO ] hd smller pools of ureides ompred with developing leves t mient [CO ], whih suggests N ssimiltion in young leves ws improved y elevted [CO ]. This work shows tht elevted [CO ] lters the lne of ron nd nitrogen pools in oth mture nd growing soyen leves, whih ould hve downstrem impts on growth nd produtivity. Key words: Amino ids, elevted [CO ], FACE, Glyine mx, hexose, strh, surose, ureide. Introdution Atmospheri CO onentrtion ([CO ]) is now higher thn it hs een t ny time in the pst million yers nd ontinues to rise t n unpreedented rte (Prentie et l., 1). Inresed photosynthesis (A) t elevted [CO ] ommonly leds to inresed plnt growth. However, mximum exploittion of CO -rih tmosphere n only e hieved when plnt hs suffiient pity to use the inresed supply of ron (C) ville t elevted [CO ], nd this is often limited y the vilility of nitrogen (N) (Stitt nd Krpp, 1999; Oren et l., 1; Downloded from y guest on Deemer 18 To whom orrespondene should e ddressed. E-mil: inswort@uiu.edu Arevitions: A, net lef CO uptke rte (lmol m s 1 ); A#, dily integrl of CO fixtion (mmol C m d 1 ); AA, mino id; C, ron; i, interellulr [CO ]; DOY, dy of yer; FACE, free ir CO enrihment; g s, stomtl ondutne to H O vpour (mol m s 1 ); LMA, lef mss per unit re (g m ); N, nitrogen; PPFD, photosyntheti photon flux density; Pro, protein. ª The Author(s). This is n Open Aess rtile distriuted under the terms of the Cretive Commons Attriution Non-Commeril Liense ( whih permits unrestrited non-ommeril use, distriution, nd reprodution in ny medium, provided the originl work is properly ited.

2 58 Ainsworth et l. Hungte et l., 3; Luo et l., ). Investigtion of this mehnism hs minly een onduted in mture (fully expnded) leves (Mtt et l., 1; Ellsworth et l., ). However, the stimultion of produtivity y elevted [CO ] will depend on the metoli sttus of growing tissue. Growing leves re prtiulrly importnt sine greter growth t elevted [CO ] will led to lrger lef re nd possily ompound gins in produtivity. In ddition to its gronomi importne, soyen (Glyine mx L. Merr.) is n interesting speies to investigte in elevted [CO ] euse it hs n ssoition with N-fixing teri (Rhizoiee) tht inreses N vilility to the plnt. Soyen hs oth lrge sink pity (Wlsh et l., 1987) nd the ility to mth its N supply to C supply t elevted [CO ] (Rogers et l., ). Therefore, indeterminte soyens re expeted to espe the limittion of sink pity tht other speies experiene t elevted [CO ]. Determining the presene of n N limittion t elevted [CO ] is hllenge sine mny of the eosystem-level prmeters tht might provide evidene of N limittion re not very sensitive, nd mny yers or dedes my e required to detet signifint effets (Luo et l., ). Mesurements of internl metolite pools ould provide fr more sensitive inditor, providing dignosti metolites n e identified tht rise or fll in N-limited mteril. Understnding the soure sink lne of plnt is eqully importnt sine the regultory role of rohydrtes is well known. Of prtiulr relevne to elevted [CO ] is the role of rohydrtes in down-regulting photosyntheti pity (Long et l., ; Rogers nd Ainsworth, ). Therefore, there is inresing interest in determining whether metolite pools n e used s mesure of metoli sttes like the N or the C/N sttus of plnts (Stitt nd Krpp, 1999; Mtt et l., 1; Foyer et l., 3; Kruse et l., 3; Jeong et l., ). Metolite profiles my give n indition of shifts in C nd N prtitioning due to rising tmospheri [CO ] nd n help identify if ottleneks in C utiliztion our. There is evidene tht metolite lne in growing leves is impted y growth t elevted [CO ] (Geiger et l., 1998; Mtt et l., 1). Nielsen nd Stitt (1) rried out detiled nlysis of fluxes in fully expnded nd developing too leves. Wheres the former synthesize predominntly rohydrtes, the ltter mke smll mounts of rohydrte ut lrge mounts of mino ids nd lso inorporte newly fixed CO into protein nd ell wll mteril. There hve een very few reports of the lne of C nd N metolites in growing (sink) leves exposed to elevted [CO ], prtiulrly under field onditions. Dt re emerging tht orrelte hnges in lef growth t elevted [CO ] with ltered metolite pool sizes. Tylor et l. (3) elegntly showed tht the pttern of lef expnsion ws ltered in Populus3eurmerin leves exposed to elevted [CO ] in the field, nd hypothesized tht C sttus of the lef plys n importnt regultory role. Wlter et l. (5) similrly showed tht diel expnsion ptterns were pertured y elevted [CO ] in Populus deltoides; in this system, trnsient derese in gluose pools ompnied ltered lef expnsion ptterns (Wlter et l., 5). In young too leves, iosynthesis nd growth dominted metolism, wheres in mture leves, ssimiltion nd export dominted (Mslux et l., ). Growing leves therefore hve higher demnd for C reltive to N ompred with mture leves euse protein synthesis requires more C per N thn protein export (Foyer nd Notor, ). If C export is inresed in elevted [CO ] in mture leves, then the limittion of iosynthesis y C supply my e llevited in growing leves. In this study, lef C nd N metolite ontent in fully expnded nd growing soyen leves exposed to elevted [CO ] were investigted using free ir CO enrihment (FACE). These experiments were onduted twie during the growing seson to ssess further how the developmentl stge of the rop ltered C nd N metoli profiles. The im of the present study ws to identify metoli imlne whih might onstrin soyen exploittion of future high-co environment, y mesuring C nd N vilility in soure nd sink soyen leves grown under elevted [CO ] in the field. Diurnl ourses of gs exhnge of young nd mture leves were mesured to investigte hnges in C flux under elevted [CO ]. Lef rohydrte nd mino id pools were mesured throughout the diurnl ourse to investigte if elevted [CO ] ltered the rohydrte nd mino id profiles in developing nd mture leves. Previous work t this site suggested tht mture leves would hve higher rohydrte ontent, ut tht the N-fixing ility of soyen would not result in dilution of N metolites in the leves (Rogers et l., ). Mterils nd methods Experimentl site Soyens v. 93B15 (Pioneer Hi-Bred) were grown under mient nd elevted [CO ] t the SoyFACE fility, loted in Chmpign, IL, USA ( # N, 88 1# W, 8 m sl). The field ws plnted on 8 My. Mesurements were mde on 7 8 July [dy of yer (DOY) 189] when the rop ws in the vegettive (V) growth phse nd on 11 1 August (DOY 3) when the rop ws in the reprodutive 5 (R5) growth phse. The SoyFACE experimentl design ws rndomized omplete lok design with four loks. Eh lok ontined two -m-dimeter otgonl plots, one t urrent mient [CO ](. 375 lmol mol 1 ) nd one fumigted from sunrise to sunset to n elevted trget [CO ]of 55 lmol mol 1, using the FACE design of Migliett et l. (1). In, the tul elevted [CO ] verged ross the growing seson ws 55 lmol mol 1 nd 1 min verges of [CO ] in the fumigted plots were within % of the trget 93% of the time (T Mies, personl ommunition). The SoyFACE experimentl fility is desried in detil elsewhere (Ort et l., ). Downloded from y guest on Deemer 18

3 C nd N metolites in soyens grown t elevted [CO ] 581 Gs exhnge mesurements The diurnl ourse of gs exhnge of young ( 5 m long) nd the most reently fully expnded (1 13 m long) middle leflets ws mesured from dwn to dusk on 7 July nd 11 August. Both the young nd mture leves were loted t the top of the soyen nopy on oth smpling dtes, nd therefore were not shded. Four portle, open gs exhnge systems (Li-Cor ; Li-Cor, Linoln, NE, USA) were used simultneously t intervls of. h from erly morning to sunset. Three plnts were mesured in eh plot t eh time intervl. Mesurements of gs exhnge prmeters on ll plnts were mde t growth [CO ] nd t mient ir temperture nd photosyntheti photon flux density (PPFD). The gs exhnge systems were rotted etween loks, nd strted in different [CO ] tretments t eh time point to ensure tht mesurements were not ised y differenes in mirolimte over time or y different gs exhnge systems (Lekey et l., ; Rogers et l., ). For the overll omprison of A nd g s etween trifolites nd [CO ] tretments over the diurnl period, mixed model ws fitted to repeted mesures in time. Eh dy ws nlysed independently, nd lef ge nd [CO ] were onsidered fixed effets, while lok ws rndom effet. Sttistis were performed on plot mens using the MIXED proedure of SAS (SAS Institute, Cry, NC, USA). The totl dily CO uptke (A#) ws lulted y integrting under the re of the diurnl urve of photosynthesis. Lef CO ssimiltion rte (A) ws determined in response to hnges in the interellulr [CO ] ( i ) for developing nd fully expnded leves in June nd July, using portle infrred gs exhnge system (LI-). Leves were smpled predwn nd kept t low light prior to mesurement in order to void trnsient dereses in wter potentil, dereses in hloroplst inorgni phosphte onentrtion, or dereses in mximum photosystem II effiieny (Bernhi et l., 5). Photosynthesis ws initilly indued t growth [CO ], then the [CO ] entering the hmer ws redued stepwise to lower onentrtion of 5 lmol mol 1, nd then inresed stepwise to n upper onentrtion of 1 lmol mol 1. Lef temperture ws mintined t 5 C nd PPFD ws 15 lmol m s 1. Photosyntheti prmeters were lulted y fitting the equtions of Frquhr et l. (198) nd y mximum likelihood regression (Sigmplot, Jndel Sientifi, Erkrth, Germny). Lef metolite mesurements Diretly fter mking photosyntheti mesurements in the field, three lef diss from eh plot nd eh developmentl stge were smpled for iohemil nlysis. Smples for lef rohydrtes were tken t four time points: predwn, pproximtely solr noon, dusk, nd the following dwn. Smples for totl solule protein nd totl mino ids were tken t middy. Eh dis (. 1.8 m ) ws removed from the middle leflet while voiding the midri, wrpped in foil, plunged immeditely into liquid N nd stored t 8 C until nlysis. Five diss per plot were lso smpled t middy for ureide ontent nd C nd N elementl ontent. These smples were dried to onstnt mss prior to nlysis. Folir ontents of rohydrtes, protein, nd mino ids were extrted from ground lef tissue in 8% (v/v), uffered ( mm HEPES, ph 7.8) ethnol t 8 C. Four min inutions were needed to reover the solule metolites. Gluose, frutose, nd surose were determined using ontinuous enzymti sustrte ssy (Jones et l., 1977). For protein nd strh determintion, pellets of the ethnol extrtion were soluilized y heting to 95 C in.1 M NOH. Protein ontent ws determined using ommeril kit ( protein ssy kit; Piere, IL, USA) with BSA s stndrd. The NOH solution ws then idified to ph.9 nd strh ontent ws determined from gluose equivlents (Rogers et l., ). Totl mino id ontents were determined using fluorogeni-sed miroplte ssy (Bntn-Polk et l., 1). Individul mino ids were mesured from ethnol/wter extrts using high performne liquid hromtogrphy s in Geigenerger et l. (199). Lef N nd C ontent were determined y dry omustion with n elementl nlyser (PE Series II CHN nlyser; Perkin Elmer, CT, USA) nd ureide ontent ws ssyed using olorimetri ssy (Vdez nd Sinlir, ). For the omprison of metolites, repeted-mesures mixed model ANOVA ws performed with trifolite nd [CO ] onsidered s fixed effets nd lok s rndom effet. Results Elevted [CO ] inresed in situ rtes of photosynthesis when mesured in the field in fully expnded leves (Fig. 1, ), despite deresed stomtl ondutne (Fig. 1, d; Tle 1). The dily integrl of ron uptke (A#) ws % higher in mture leves grown under elevted [CO ] on DOY 189 nd 1% higher on DOY 3 (Fig. ). However, signifint stimultion of diurnl C uptke in developing leves ws not deteted on either dy of mesurement during vegettive growth (DOY 189) or reprodutive growth (DOY 3) (Figs 1,, ; Tle 1). Stomtl ondutne ws signifintly lower in young leves grown t elevted [CO ], nd interellulr [CO ] ws 1 15% higher in developing leves ompred with fully expnded leves in oth mient nd elevted [CO ] (Fig. 1e, f). The A/ i response urves (Fig. 3) showed tht fully expnded leves hd higher photosyntheti pity thn developing leves, ut there ws no effet of [CO ] tretment on the shpe of the A/ i response urve. Elevted [CO ] inresed lef rohydrte ontents in fully expnded leves, ut not in developing leves (Fig. ). Lef hexose, surose, nd strh ontents were ll signifintly inresed y growth t elevted [CO ] in fully expnded leves over the diel yle during the first time point (Fig. ; Tle 1). There ws ler diel pttern of rohydrte umultion during the dy nd use t night on DOY 189; however, the pttern ws not ler on DOY 3, when surose nd strh umulted in the leves, prtiulrly in mture, elevted [CO ]-grown leves (Fig. ). Developing leves hd signifintly lower lef mss per unit re (LMA) thn fully expnded soyen leves (Fig. 5, ), nd were in the exponentil phse of expnsion in oth mient nd elevted [CO ] (dt not shown). Elementl nlysis of lef C nd N reveled tht developing leves hd the sme N ontent s fully expnded leves erly in the growing seson (Fig. 5) nd slightly lower N ontent lter in the growing seson (Fig. 5d), when mesured on n re sis. However, during oth time points, developing leves hd lower C:N rtios (Fig. 5e, f) thn fully expnded leves in oth mient nd elevted [CO ]. As expeted from the photosynthesis mesurements, totl hlorophyll ontent ws signifintly Downloded from y guest on Deemer 18

4 58 Ainsworth et l. A (µmol m - s -1 ) 3 1 DOY 189 DOY A (µmol m - s -1 ) g s (mol m - s -1 ) i (µmol mol -1 ) T ir (ºC) lower in developing leves (Fig. 5g, h). Totl protein ontent ws not ffeted y [CO ] tretment or developmentl stge on DOY 189 (Fig. 5i), ut ws signifintly lower in developing leves on DOY 3 (Fig. 5j). Free mino id ontent ws higher in developing leves ompred with fully expnded leves (Tle ; Fig. 5k, l), nd ws signifintly higher in young developing leves grown t elevted [CO ] on DOY 189 (Tle ; Fig. 5k). Ureide levels, mesured s llntoin, were signifintly nd mrkedly higher in developing leves on oth dys (Fig. ). There ws no effet of elevted [CO ] e g d h Fig. 1. Diurnl ourse of photosynthesis (A;, ), stomtl ondutne (g s ;, d) nd interellulr [CO ]( i ; e, f) on 7 July (DOY 189) nd 11 August (DOY 3) in fully expnded (irles) nd developing (tringles) soyens exposed to mient [CO ] (open symols) nd elevted [CO ] (losed symols). Diurnl ourses of photosynthesis (A) nd stomtl ondutne (g s ) were mesured in situ t growth [CO ], nd t mient light nd temperture (g, h) onditions. Dt re lest squre mens 1 stndrd error of the differene of mens. The results of the sttistil nlysis of A nd g s re shown in Tle 1. Signifint differenes etween elevted nd mient mens in fully expnded leves within time points re mrked with sterisks: P <.5; P <.1. f on ureide levels in fully expnded leves ut, in developing leves, elevted [CO ] resulted in signifint redution in ureide ontent (Fig. ). Individul mino ids were mesured over the ourse of the dy on DOY 3. Elevted [CO ] did not signifintly ffet mino id ontent (Tle 3; Figs 7 9). Individul mino id ontent ws highly dependent upon the developmentl stge of the lef. Glu (Fig. 7), Gln (Fig. 7), Gly (Fig. 8), Asn (Fig. 9), nd Al (Fig. 9) were ll higher in developing leves thn fully expnded leves (Tle 3). The ontent of most mino g s (mol m - s -1 ) i (µmol mol -1 ) PPFD (µmol m - s -1 ) Downloded from y guest on Deemer 18

5 C nd N metolites in soyens grown t elevted [CO ] 583 Tle 1. Sttistil nlysis of diurnl photosynthesis (A), stomtl ondutne (g s ), interellulr [CO ]( i ), lef hexose, surose, nd strh ontent A mixed model ws fitted to repeted mesures in time for overll omprisons, with [CO ] tretment ([CO ]) nd lef developmentl ge (Age) onsidered fixed effets, nd time repeted mesure. Eh dy ws nlysed independently. Signifint results (F, P) from the ANOVA re shown in old. A g s i Hexose Surose Strh DOY 189 [CO ] 17.17, <.1., , <.1.,.3 8.3,. 1.,.19 Age , < , < , < , <.1 1.1, <.1 8.1, <.1 [CO ]3Age 3.87, ,.181.7,.1 11.,. 7.3, , <.1 Time 3.5, <.1 8.8, <.1 1.3, < , < , <.1.3, <.1 [CO ]3Time 1.37, , ,.89.,.85.33, ,.15 [CO ]3Age3Time 1.99, < , <.1.8,.73.,.878.,.3.,. DOY 3 [CO ].,.15.78, , < , <.1.9, , <.1 Age 3., < , < , <.1.3, , < , <.1 [CO ]3Age 5.1,..95,.9.3,.1 5.5,.3.,.3.7, <.1 Time 15.79, <.1 3.8, <.1 8.9, <.1.,. 7., < , <.1 [CO ]3Time.3,.9 1.5,.175., <.1 3.5, ,.18.3,.798 [CO ]3Age3Time 11.95,.1 1.1, ,.315.,..9, <.1.7, <.1 A' (mol CO m - d -1 ) A' (mol CO m - d -1 ) Fully Expnded ids lso vried over the ourse of the dy, with middy ontent highest for Glu (Fig. 7), Gln (Fig. 7), Ser (Fig. 8), nd Al (Fig. 9). The rtio of Glu:Gln ws highest for fully expnded leves exposed to elevted [CO ], nd 37 ppm 55 ppm Developing Fig.. Dily integrl of net CO ssimiltion on () DOY 189 nd () DOY 3. On oth dys, there ws signifint intertion etween CO nd lef ge (P <.5). Signifint differenes etween elevted nd mient mens in fully expnded leves within time points re mrked with letters (P <.5). A (µmol m - s -1 ) i (µmol mol -1 ) Fig. 3. Representtive A/ i responses of fully expnded (irles) nd developing (tringles) soyens exposed to mient [CO ] (open symols) nd elevted [CO ] (losed symols). V,mx ws estimted from points elow the inflexion nd J mx ws estimted from points ove the inflexion. more thn three times higher in fully expnded leves ompred with young leves (Fig. 7). When verged ross lef ges, the rtio of Gly:Ser ws lower for plnts grown in elevted [CO ] ompred with mient [CO ] (Fig. 8). Disussion An inrese in tmospheri [CO ] to levels predited for 5 used hnges in C flux nd C nd N metolites in fully expnded nd developing soyen leves. Elevted [CO ] inresed photosynthesis in fully expnded soyen leves, nd deresed stomtl ondutne in leves of oth ges (Fig. 1). Crohydrte ontent ws high in fully expnded leves nd signifintly inresed y elevted [CO ], while in developing leves rohydrte ontent ws low nd unffeted y [CO ] (Fig. 3). By ontrst, Downloded from y guest on Deemer 18

6 58 Ainsworth et l. 5 DOY 189 DOY 3 5 hexose (mmol m - ) surose (mmol m - ) strh (mmol m - ) mino id ontent ws low nd unffeted y [CO ]in fully expnded leves, while mino id ontent in developing leves ws high (Figs, 8). Ureide ontent ws lso lower in fully expnded leves thn in developing leves, nd ws signifintly redued y elevted [CO ] in developing leves (Fig. 5). These generl hnges were not ffeted y the developmentl stge of the rop, nor ws lef re index signifintly inresed t elevted [CO ] t ny point during the growing seson (O Dermody, personl ommunition). The vrition tht did our etween the dtes of smpling ws onsistent with previously oserved developmentl e 1 d f 1 Fig.. Hexose, surose, nd strh ontent of developing nd fully expnded soyen leves exposed to mient nd elevted [CO ]. Results of the sttistil nlysis of hexose, surose, nd strh ontent re shown in Tle 1. Symols nd error rs re s desried for Fig. 1. Signifint differenes etween elevted nd mient mens in fully expnded leves within time points re mrked with sterisks: P <.5; P <.1; P <.1. ptterns (e.g. ureide ontent from Rogers et l., ) nd the effets of generl vriility in growth onditions (e.g. photosynthesis in Bernhi et l., ). Responses of mture leves to elevted [CO ] The response of fully expnded soyen leves to elevted [CO ] resemled tht oserved in erlier studies (Rogers et l.,, ). Photosynthesis in mture leves ws very similr t oth time points, despite different rtes of stomtl ondutne (Fig. 1). This n e explined y vrition in roxyltion effiieny, whih hs een demonstrted to vry y up to % over the Downloded from y guest on Deemer 18

7 C nd N metolites in soyens grown t elevted [CO ] 585 LMA (g m - ) 5 3 DOY 189 DOY LMA (g m - ) 1 1 AA (mmol m - ) Pro (g m - ) Chl (µmol m - ) C:N N (g m - ) d 1 e f 1 8 g i j k l h AA (mmol m - ) Pro (g m - ) Chl (µmol m - ) C:N N (g m - ) Downloded from y guest on Deemer 18 Fully Expnded Developing Fully Expnded Developing Fig. 5. Men speifi lef weight (SLW) lef N, lef C:N, totl hlorophyll (Chl), protein (Pro), nd mino id (AA) ontent of growing (grey olumns) nd fully expnded (white olumns) soyens exposed to mient [CO ] (open olumns) nd elevted [CO ] (hthed olumns). Error rs show the stndrd error of the lest squred men. Signifint differenes etween mens (P <.5) within time point re indited with letters.

8 58 Ainsworth et l. Tle. Sttistil nlysis (F, P) of lef N, C, the C:N rtio, totl lef hlorophyll ontent, protein, mino id ontent, nd llntoin ontent shown in Figs nd 5 A mixed model ws fitted for overll omprisons, with [CO ] tretment ([CO ]) nd lef developmentl ge (Age) onsidered fixed effets. Eh dy ws nlysed independently. Signifint results from the ANOVA (P <.5) re shown in old. DOY 189 DOY 3 [CO ] Age [CO ]3Age [CO ] Age [CO ]3Age SLW.,.5 1., <.1.,..,.53 8., <.1.55,.9 N.,.9.9,.77.1,.3.11, , <.1.58,.8 C:N 7.,. 5., <.1.87, , , < ,.8 Chlorophyll 1.8,.1 15., <.1.,.9., ,.3.95,.11 Protein.9,.37.9,.77.5,.1.3, ,..17,.9 Amino id., ,. 3.3,.1.17,.9 1.7, <.1.7,. Allntoin.7, , <.1.,..1,. 5.31, < ,.1 Allntoin (µmol m - ) Allntoin (µmol m - ) Fully Expnded ourse of the growing seson in field-grown soyen (Bernhi et l., 5). Bsed on the instntneous inrese in [CO ] from 38 to 55 lmol mol 1, n pproximte 9% stimultion would e ntiipted in photosynthesis in mture leves (Fig. 3), whih is onsistent with the mesured.5% nd.9% differenes in middy photosynthesis mesured under field onditions Developing Fig.. Middy ontent of llntoin mesured on () DOY 189 nd () DOY 3 for plnts grown t mient [CO ] (open olumns) nd elevted [CO ] (hthed olumns), s in Fig.. On oth dys, there ws signifint intertion etween CO nd lef ge (P <.1). Allntoin ontent ws not ffeted y elevted [CO ] in fully expnded leves, ut ws signifintly lower in developing leves exposed to elevted [CO ]. Signifint differenes etween elevted nd mient mens in developing leves within time points re mrked with letters (P <.5). (Fig. 1). Totl lef C, hexoses, surose, nd strh were signifintly higher in fully expnded leves grown t elevted [CO ], ompred with fully expnded leves grown t mient [CO ] (Fig. ), s hs een ommonly reported in rops exposed to elevted [CO ] (reviewed y Drke et l., 1997; Ainsworth et l., ; Ainsworth nd Long, 5). These physiologil mrkers of lef photosynthesis nd rohydrte sttus orresponded with inresed ove-ground produtivity t elevted [CO ]in multiple yers t this field site (Rogers et l.,, ; Morgn et l., 5). The greter strh ontent nd LMA t elevted [CO ] is lso onsistent with mny previous studies in whih greter strh ontent t elevted [CO ] led to signifintly greter LMA (Peterson et l., 1999; Ainsworth nd Long, 5). All metolite dt re therefore presented here on lef re sis. By ontrst to lef C pools, lef N pools were lrgely unffeted y elevted [CO ], using n inrese in the C:N rtio of mture leves. Ureide ontent ws the sme in fully expnded leves grown t mient nd elevted [CO ] (Fig. ), nd pools of mino ids were lso lrgely unffeted (Figs 7 9). These results gree with prior results from SoyFACE, where ureide ontent in fully expnded leves ws signifintly ffeted y growth stge, ut not y [CO ] (Rogers et l., ). Other studies hve shown tht ureide onentrtion in soyen leves dereses t elevted [CO ], nd tht elevted [CO ] hnges the response of N fixtion to soil wter ontent (Serrj et l., 1998; Serrj nd Sinlir, 3). Crops t SoyFACE did not experiene wter stress t ny time during the growing seson in when the mesurements were tken (Lekey et l., ); therefore, ny potentil hnges in the response of N metolites to drought t elevted [CO ] were unlikely to our during this study. Responses of developing leves to elevted [CO ] How developing leves respond to elevted [CO ] hs reeived muh less ttention thn mture leves. Osorne et l. (1998) nd Adm et l. () reported tht Downloded from y guest on Deemer 18

9 Tle 3. Sttistil nlysis (F, P) of mino id ontent mesured on DOY 3 C nd N metolites in soyens grown t elevted [CO ] 587 A mixed model ws fitted to repeted mesures in time for overll omprisons, with [CO ] tretment ([CO ]) nd lef developmentl ge (Age) onsidered fixed effets, nd time repeted mesure. Signifint results (P <.5) from the ANOVA re shown in old. [CO ] Age [CO ]3Age Time [CO ]3Time [CO ]3Age3Time Glu 1.3, ,..58,.5 1.3, <.1.39,.8 3.,.3 Gln.3, , < ,. 19., <.1.3,. 1.5,.3 Glu:Gln 3.8,.9 5.5, < ,. 9.85, <.1.18,.8.7, <.1 Gly 1.5,. 11.9,. 1.8,.3 13., <.1.87,.3.,.77 Ser.9,.5 1.5,.31.1, , <.1.3, ,.3 Gly:Ser 3.11, , <.1 3., , <.1.51, ,. Asn., ,.8.9, , <.1.5, ,.1 Asp 1.8,.3 8.1, <.1.1, , <.1.11,.9 3.7,. Al.78,.58.5,. 1.87, , <.1., ,.1 limtion of photosyntheti pity in whet ourred in older, shded leves, ut not young, photosynthetilly mture flg leves. An inrese in diurnl photosynthesis ould not e deteted t elevted [CO ] in developing soyen leves (Figs 1, ); however, developing leves hd signifintly higher interellulr [CO ] ompred with fully expnded leves (Fig. 1e, f). From the A/ i response urve, n 18.% inrese in light-sturted photosynthesis would e ntiipted in developing leves (Fig. 3) t 5 C. In ft, on DOY 189, middy photosynthesis ws. % higher t elevted [CO ], ut this ws outside of the rnge of sttistil detetion in the gs exhnge mesurements. On DOY 3, photosynthesis ws not higher in developing leves t ny time during the dy (Fig. 1). Developing leves hd low levels of hlorophyll ompred with fully expnded leves, ut there ws no [CO ] effet on hlorophyll ontent (Fig. g, h). There ws striking derese in stomtl ondutne in young leves grown t elevted [CO ] (Fig. 1, d), whih my e mehnism y whih developing leves prioritize wter for expnsion over trnspirtion. Ureides re delivered to the lef in the trnspirtion strem nd the redued stomtl ondutne t elevted [CO ] my explin the deresed ureide ontent. However, it is felt tht this is unlikely euse stomtl ondutne ws mrkedly higher in fully expnded leves where ureide levels were low nd lso where there ws no effet of the redued stomtl ondutne on ureide ontent. Developing leves hd unique metolite ontents, inluding low levels of rohydrte nd high levels of ureides nd mino ids, nmely Glu, Gln, nd Asn. These three mino ids were previously identified s mrkers of young leves in quking spen (Jeong et l., ). The low LMA of developing leves versus mture leves mens tht these mrkers of juvenile sttus reflet n even greter hnge in ureide nd mino id onentrtion per unit dry mss with developmentl stge. Elevted [CO ] deresed ureide ontent (Fig. ), trnsiently inresed mino id ontent (Fig. k), nd hd no ovious effet on lef rohydrtes (Fig. ). Without n inrese in rohydrte ontent t elevted [CO ], there ws no hnge in LMA of developing leves. Implitions for whole plnt C nd N sttus t elevted [CO ] When verged ross lef ges nd three time points, the Gly:Ser rtio ws 15% lower in plnts grown under elevted [CO ]. A deresed Gly:Ser rtio is frequently seen in elevted [CO ] (Stitt nd Krpp, 1999; Mtt et l., 1; Rogers et l., ). Novitsky et l. () demonstrted tht with inresed photorespirtory flux, C flooded into glyolte, leding to Gly umultion nd n inrese in the Gly:Ser rtio. Novitsky et l. () lso reported negtive orreltion etween Asp nd photorespirtion. The present dt lso show higher Asp level in the fully expnded leves grown t elevted [CO ]; oth oservtions, long with higher interellulr [CO ], provide strong evidene tht there ws derese in photorespirtory flux in soyens growing t elevted [CO ]. Clultion of C export y mss lne (Rogers et l., ) provides rude estimte of C ville to developing sink tissue. Consistent with deresed photorespirtion nd inresed photosynthesis, there ws 15 9% more ron exported from fully expnded soure leves t elevted [CO ] thn t mient [CO ]. The djent developing trifolite leves re likely to e strong proximl sinks for this dditionl photosynthte (Frrr nd Willims, 1991; Frrr, 199), prtiulrly during vegettive growth (DOY 189), ut lso during the reprodutive phse (DOY 3) in this indeterminte ultivr. Fixed N requires C skeletons for ssimiltion nd further iosynthesis (Todd et l., ).Therefore,importedCouldeusedtofuel iosynthesis in the developing leves, possily explining why ureide levels were lower in developing leves grown t elevted [CO ] ompred with those grown t urrent [CO ]. Further experiments would e needed to determine if more C were in ft exported from fully expnded leves to developing leves under elevted [CO ]. Another possiility is tht long-distne signls relted to higher rohydrte sttus in elevted [CO ] drive Downloded from y guest on Deemer 18

10 588 Ainsworth et l. 8 3 Glutmte (µmol m - ) Glyine (µmol m - ) 1 Glutmine (µmol m - ) Glu:Gln Fig. 7. Diurnl mesurements of glutmte (), glutmine (), nd the rtio of glutmte to glutmine [Glu:Gln ()] mesured on 11 August. Symols nd error rs re s desried for Fig. 1. Serine (µmol m - ) Gly:Ser Fig. 8. Diurnl mesurements of glyine (), serine (), nd the Gly:Ser rtio () mesured on 11 August. Symols nd error rs re s desried for Fig. 1. Downloded from y guest on Deemer 18 inresed growth in developing leves; however, if growth were inresed, it ws not pprent y mesurements of lef re index. Long-distne signlling from mture to developing leves is one mehnism y whih stomtl development responds to elevted [CO ] (Lke et l., ), so it is possile tht similr signl stimultes growth. Sims et l. (1998) provided further evidene for the role of long-distne signls. They found tht the photosyntheti pity of soyen lef depended on the [CO ] surrounding the plnt, not the [CO ] surrounding the lef. Controlled experiments where mture lef is mintined t one [CO ] nd developing lef t different [CO ] would e needed to investigte further how growth is ltered y long-distne signlling. Wht do the hnges in C nd N metolites suggest for the C nd N lne of the soyen plnts? N ontent of the soyens ws signifintly higher during the erly reprodutive phse ompred with the vegettive phse in

11 Asprgine (µmol m - ) Asprtte (µmol m - ) Alnine (µmol m - ) Fig. 9. Diurnl mesurements of sprgine (), sprtte (), nd lnine () mesured on 11 August. Symols nd error rs re s desried for Fig. 1. oth mient nd elevted [CO ] (Fig. ). Mximum N ontent of mny rops is rehed t the erly reprodutive phse nd, with further development, ville N is depleted nd nodules senese in nnul legumes (Peoples nd Gifford, 1997). Ureide ontent in young leves hrvested during erly reprodutive growth ws nerly doule tht in young leves hrvested during vegettive growth (Fig. 5), onsistent with the well-hrterized hnges in N fixtion known to our during rop development C nd N metolites in soyens grown t elevted [CO ] 589 (Rithie et l., 1997). Sine there ws no evidene of redution in N ontent, nd soyen ove-ground iomss ws 3% nd % greter in elevted [CO ] t the pproximte times when the smpling for the present study ws mde (K MConnughy, personl ommunition), it suggests tht N fixtion per plnt inresed proportionlly (Rogers et l., ), i.e. elevted [CO ] inresed C ville for N fixtion nd/or enled plnts t elevted [CO ] to tke dvntge of fixed N pools y mking more C skeletons ville for N ssimiltion (Rogers et l., ). Interestingly, despite the inrese in ove-ground iomss t elevted [CO ], lef re index ws not inresed. Adpting soyens to llote resoures towrds inresed lef re my e n importnt strtegy to improve plnt performne in future environmentl onditions. Aknowledgements We thnk Steve Long, Orl Dermody, Kelly MConnughy, Tim Mies, Joe Cstro, Kelly Gillespie, Mj Christ, Ktie Ciodiol, nd Regin Feil. EAA ws supported y n Alexnder von Humoldt post-dotorl reserh fellowship. MS knowledges support from the Mx Plnk Soiety nd from the Europen Commission (Integrted Projet Grin legumes ). AR nd LEH were supported y the US Deprtment of Energy Offie of Siene ontrt No. DE-AC-98CH188 to Brookhven Ntionl Lortory nd Siene Undergrdute Lortory Internship to LEH. SoyFACE ws supported y the Illinois Counil for Food nd Agriulturl Reserh, Arher Dniels Midlnd Compny, the US Deprtment of Agriulturl, nd the Illinois Agriulturl Experiment Sttion. Referenes Adm NR, Wll GW, Kimll BA, et l.. Alimtion response of spring whet in free-ir CO enrihment (FACE) tmosphere with vrile soil nitrogen regimes. 1. Lef position nd phenology determine limtion response. Photosynthesis Reserh, Ainsworth EA, Dvey PA, Bernhi CJ, et l.. A metnlysis of elevted [CO ] effets on soyen (Glyine mx) physiology, growth nd yield. Glol Chnge Biology 8, Ainsworth EA, Long SP. 5. Wht hve we lerned from 15 yers of free ir CO enrihment (FACE)? A met-nlyti review of the responses of photosynthesis, nopy properties nd plnt prodution to rising CO. New Phytologist 15, Bntn-Polk T, Kssi M, Grnt KB. 1. A omprison of fluoresmine nd nphthlene-,3-diroxldehyde fluorogeni regents for miroplte-sed detetion of mino ids. Anlytil Biohemistry 97, Bernhi CJ, Lekey ADB, Hedy LE, et l.. Hourly nd sesonl vrition in photosynthesis nd stomtl ondutne of soyen grown t future CO nd ozone onentrtions for 3 yers under fully open-ir field onditions. Plnt, Cell nd Environment 9, Bernhi CJ, Morgn PB, Ort DR, Long SP. 5. The growth of soyen under free ir [CO ] enrihment (FACE) stimultes photosynthesis while deresing in vivo Ruiso pity. Plnt, 3. Downloded from y guest on Deemer 18

12 59 Ainsworth et l. Drke BG, GonzlezMeler MA, Long SP More effiient plnts: onsequene of rising tmospheri CO? Annul Review of Plnt Physiology nd Plnt Moleulr Biology 8, Ellsworth DS, Reih PB, Numerg ES, Koh GW, Kuiske ME, Smith SD.. Photosynthesis, roxyltion nd lef nitrogen responses of 1 speies to elevted pco ross four free-ir CO enrihment experiments in forest, grsslnd nd desert. Glol Chnge Biology 1, Frquhr GD, von Cemmerer S, Berry JA A iohemil model of photosyntheti CO fixtion in C 3 speies. Plnt 19, Frrr JF Sinks: integrl prts of whole plnt. Journl of Experimentl Botny 7, Frrr JF, Willims ML The effets of inresed tmospheri ron dioxide nd temperture on ron prtitioning, soure sink reltions nd respirtion. Plnt, Cell nd Environment 1, Foyer CH, Notor G.. Photosyntheti nitrogen ssimiltion: inter-pthwy ontrol nd signling. In: Foyer CH, Notor G, eds. Photosyntheti nitrogen ssimiltion nd ssoited ron nd respirtory metolism. The Netherlnds: Kluwer Ademi Pulishers, 1. Foyer CH, Prry M, Notor G. 3. Mrkers nd signls ssoited with nitrogen ssimiltion in higher plnts. Journl of Experimentl Botny 5, Geigenerger P, Lerhl J, Stitt M, Sonnewld U Phloemspeifi expression of pyrophosphtse inhiits long-distne trnsport of rohydrtes nd mino ids in too plnts. Plnt, Cell nd Environment 19, Geiger M, Wlh-Liu P, Engels C, Hrneker J, Shulze ED, Ludewig F, Sonnewld U, Sheile WR, Stitt M Enhned ron dioxide leds to modified diurnl rhythm of nitrte redutse tivity in older plnts, nd lrge stimultion of nitrte redutse tivity nd higher levels of mino ids in young too plnts. Plnt, Cell nd Environment 1, Hungte BA, Dukes JS, Shw MR, Luo Y, Field CB. 3. Nitrogen nd limte hnge. Siene 3, Jeong ML, Jing HY, Chen HS, Tsi CJ, Hrding SA.. Metoli profiling of the sink-to-soure trnsition in developing leves of quking spen. Plnt Physiology 13, Jones MGK, Outlw WH, Lowry OH Enzymi ssy of 1 7 to 1 1 moles of surose in plnt tissues. Plnt Physiology, Kruse J, Hetzger I, Hnsh RH, Mendel RR, Rennenerg H. 3. Elevted pco ffets C nd N metolism in wild type nd trnsgeni too exhiiting ltered C/N lne in metolite nlysis. Plnt Biology 5, Lke JA, Woodwrd FI, Quik WP.. Long-distne CO signlling in plnts. JournlofExperimentlBotny53, Lekey ADB, Bernhi CJ, Dohlemn FG, Ort DR, Long SP.. Will photosynthesis of mize (Ze mys) in the US Corn Belt inrese in future [CO ] rih tmospheres? An nlysis of diurnl ourses of CO uptke under free-ir onentrtion enrihment (FACE). Glol Chnge Biology 1, Lekey ADB, Urielrre M, Ainsworth EA, Nidu SL, Rogers A, Ort DR, Long SP.. Photosynthesis, produtivity nd yield of Ze mys re not ffeted y open-ir elevtion of CO onentrtion in the sene of drought. Plnt Physiology 1, Long SP, Ainsworth EA, Rogers A, Ort DR.. Rising tmospheri ron dioxide: plnts FACE the future. Annul Review of Plnt Biology 55, Luo Y, Su B, Currie WS, et l.. Progressive nitrogen limittion of eosystem responses to rising tmospheri ron dioxide. Biosiene 5, Mslux C, Vldier M-H, Brugiere N, Morot-Gudry J-F, Hirel B.. Chrteriztion in the soure/sink in too (Niotin tum L.) shoots in reltion to nitrogen mngement nd lef senesene. Plnt 11, Mtt P, Geiger M, Wlh-Liu P, Engels C, Krpp A, Stitt M. 1. The diurnl hnges of nitrogen metolism in leves of nitrte-replete too re driven y n imlne etween nitrte redutse tivity nd the rte of nitrte uptke nd mmonium nd glutmine metolism during the first prt of the light period. Plnt, Cell nd Environment, Migliett F, Peressotti A, Vri FP, Zldei A, de Angelis P, Srsi-Mugnozz G. 1. Free-ir CO enrihment (FACE) of poplr plnttion: the POPFACE fumigtion system. New Phytologist 15, 5 7. Morgn PB, Bollero GA, Nelson RL, Dohlemn FG, Long SP. 5. Smller thn predited inrese in oveground net primry prodution nd yield of field-grown soyen under fully open-ir [CO ] elevtion. Glol Chnge Biology 11, Nielsen TH, Stitt M. 1. Too trnsformnts with strongly deresed expression of pyrophosphte:frutose--phosphte expression in the se of their sink leves ontin muh higher levels of frutose-,-isphosphte ut exhiit no mjor hnges in fluxes. Plnt 1, Novitsky L, Trevnion SJ, Drisoll S, Foyer CH, Notor G.. How does photorespirtion modulte lef mino id ontents? A dul pproh through modelling nd metolite nlysis. Plnt, Cell nd Environment 5, Oren R, Ellsworth DS, Johnsen KH, et l. 1. Soil fertility limits ron sequestrtion y forest eosystems in CO - enrihed tmosphere. Nture 11, 9 7. Ort DR, Ainsworth EA, Alde M, et l.. SoyFACE: the effets nd intertions of elevted [CO ] nd [O 3 ] on soyen. In: Nöserger J, ed. Mnged eosystems nd CO : se studies, proesses nd perspetives. Berlin: Springer Verlg, Osorne CP, LRohe J, Gri RL, Kimll BA, Wll GW, Pinter PJ, LMorte RL, Hendrey GR, Long SP Does lef position within nopy ffet limtion of photosynthesis to elevted CO? Anlysis of whet rop under free-ir CO enrihment. Plnt Physiology 117, Peoples MB, Gifford RM Regultion of the trnsport of nitrogen nd ron in higher plnts. In: Dennis DT, Turpin DH, Lefevre DD, Lyzell DB, eds. Plnt metolism. Hrlow: Addison Wesley Longmn, Peterson AG, Bll JT, Luo Y, et l Quntifying the response of photosynthesis to hnges in lef nitrogen ontent nd lef mss per re in plnts grown under tmospheri CO enrihment. Plnt, Cell nd Environment, Prentie IC, Frquhr GD, Fshm MJR, Goulden ML, Heimnn M, Jrmillo VJ, Kheshgi HS, LeQuere C, Sholes RJ, Wlle DWR. 1. The ron yle nd tmospheri ron dioxide. In: Houghton JT, Ding Y, Griggs DJ, Noguer M, Vn der Linder PJ, Di X, Mskell K, Johnson CA, eds. Climte hnge 1: the sientifi sis. Contriutions of Working Group I to the Third Assessment Report of the Intergovernmentl Pnel on Climte Chnge. Cmridge: Cmridge University Press, Rithie SW, Hnwy JJ, Thompson HE, Benson GO How soyen plnt develops. Speil Report No. 53, Iow Stte University, Ames, IA. Rogers A, Ainsworth EA.. 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13 C nd N metolites in soyens grown t elevted [CO ] 591 Rogers A, Allen DJ, Dvey PA, et l.. Lef photosynthesis nd rohydrte dynmis of soyens grown throughout their life-yle under free-ir ron dioxide enrihment. Plnt, Cell nd Environment 7, Rogers A, Gion Y, Stitt M, Morgn PB, Bernhi CJ, Ort DR, Long SP.. Inresed ron vilility t elevted ron dioxide onentrtion improves N ssimiltion in legume. Plnt, Cell nd Environment 9, Serrj R, Sinlir TR. 3. Evidene tht ron dioxide enrihment llevites ureide-indued deline of nodule nitrogense tivity. Annls of Botny 91, Serrj R, Sinlir TR, Allen LH Soyen nodultion nd N fixtion response to drought under ron dioxide enrihment. Plnt, Cell nd Environment 1, Sims DA, Luo Y, Seemnn JR Importne of lef versus whole plnt CO environment for photosyntheti limtion. Plnt, Cell nd Environment 1, Stitt M, Krpp A The intertion etween elevted ron dioxide nd nitrogen nutrition: the physiologil nd moleulr kground. Plnt, Cell nd Environment, Tylor G, Triker PJ, Zhng FZ, Alston VJ, Migliett F, Kuzminsky E. 3. Sptil nd temporl effets of free-ir CO enrihment (POPFACE) on lef growth, ell expnsion, nd ell prodution in losed nopy of poplr. Plnt Physiology 131, Todd CD, Tipton PA, Blevins DG, Piedrs P, Pined M, Polo JC.. Updte on ureide degrdtion in legumes. Journl of Experimentl Botny 57, 5 1. Vdez V, Sinlir TR.. Ureide degrdtion pthwys in intt soyen leves. Journl of Experimentl Botny 51, Wlsh KB, Vessey JK, Lyzell DB Crohydrte supply nd N fixtion in soyen: the effet of vried dylength nd stem girdling. Plnt Physiology 85, Wlter A, Christ MM, Brron-Gfford GA, Grieve KA, Murthy R, Rsher U. 5. The effet of elevted CO on diel lef growth yle, lef rohydrte ontent nd nopy growth performne of Populus deltoides. Glol Chnge Biology 11, Downloded from y guest on Deemer 18