Short Title: High irradiance and NH + 4 stress. Correspondence: Jose F. Moran

Transcription

1 Short Title: High irrdince nd NH 4 stress Correspondence: Jose F. Morn Institute of gri-iotechnology Public University of Nvrre- CSIC- Government of Nvrre, Cmpus de rrosdí s/n, E Pmplon, Nvrre, Spin Phone: Fx: Emil: jose.morn@unvrr.es 11 12

2 High irrdince induces photoprotective mechnisms nd positive effect on NH 4 stress in Pisum stivum Idoi riz, Rquel Estebn b, Jose Igncio Grcí-Plzol b José Mrí ecerril b, Pedro Mrí pricio-tejo, Jose Fernndo Morn * Institute of groiotechnology, Idb-CSIC-Public University of Nvrre-Government of Nvrre, Cmpus de rrosdí s/n, E Pmplon, Nvrr, Spin. b Deprtment of Plnt iology nd Ecology, Fculty of Science nd Technology, University of sque Country (UPV-EHU), pdo. 644; E ilbo, Vizcy, Spin.

3 Summry Photosynthesis provides plnt metbolism with reduced crbon (C) but is lso the min source of oxidtive stress in plnts. Likewise, high doses of NH 4 s sole N source hve been reported to be toxic for most plnts, resulting in reduced plnt growth nd restricting C vilbility. The combintion of high photosynthetic photon flux densities (PPFD) nd NH 4 nutrition my provide higher C vilbility but could lso hve detrimentl effect on the plnts, therefore the objective of this study is to evlute whether NH 4 induces photooxidtive stress tht is excerbted under high light conditions. Pe plnts (Pisum stivum cv Sugr Snp) were grown hydroponiclly with NH 4 (0.5, 2.5, 5 nd 10 mm) under high (750 µmol photons m -2 s -1 ) or low PPFD conditions (350 µmol photons m -2 s -1 ). High PPFD contributes to higher tolernce to mmonium by pe plnts, s it originted higher biomss content due to higher photosynthetic rtes. However, deficit of N (0.5 nd 2.5 mm NH 4 ) under high PPFD conditions cused n nti-oxidnt response, s indicted by incresed photoprotective pigment nd chloroplstic superoxide dismutse contents. Plnts grown with higher doses of N nd high PPFD showed less need for photoprotection. n increse in the specific lef weight (SLW) rtio ws observed ssocited not only with high PPFDs but lso with the highest NH 4 dose. Overll, these results demonstrte tht, despite the ctivtion of some photoprotective responses t high PPFD, there were no photoinhibitory symptoms nd positive effect on NH 4 toxicity, thus suggesting tht the hrmful effects of NH 4 re not directly relted to the genertion of photooxidtive stress. Keywords: mmonium stress, chlorophyll, crotene, superoxide dismutse, violxnthin cycle. bbrevitions: : ntherxnthin; Chl: chlorophyll; HI: high irrdince; LI: low irrdince; PPFD: photosynthetic photon flux density; ROS: rective oxygen species; SLW: specific lef weight; SOD: superoxide dismutse; S.E.: stndrd error; V: violxnthin; Z: Zexnthin 51

4 Introduction In order to ttin the gol of sustinble griculture it is necessry to mintin n equilibrium between the best possible yield nd product qulity whilst ensuring miniml environmentl impct. Two of the most importnt environmentl problems resulting from griculturl prctices tht rely on nitrte fertilizers re wter pollution due to nitrte leching nd the increse in globl wrming due to tmospheric N-contining gs emissions. The use of stbilized-nh 4 -bsed fertilizers is promising strtegy to void this pollution s they re formulted with specific compounds such s nitrifiction inhibitors, which dely the nitrifiction of mmonium, thus mintining soil N s NH 4 for longer period of time. lso It is known tht NH 4 nutrition, s sole N source, cn be toxic to mny plnts, lthough brod degree of tolernce to NH 4 stress cn be chieved in plnts within certin NH 4 concentrtion rnges (Domínguez-Vldivi et l., 2008). NH 4 is direct precursor of nucleic cids, proteins nd other orgnic molecules, s well s product of their ctbolism. It is the preferred N source when NH 4 NO 3 is vilble, nd it - is well known tht combined-n nutrition, including NO 3 nd NH 4, generlly gurntees higher crop yields thn NO - 3 or NH 4 s the sole N source. Trditionl plnt breeding hs, however, been performed under nitric or combined nutrition, therefore commercil vrieties re not usully dpted to NH 4 -bsed nutrition. C-skeleton vilbility, which depends on photosynthetic ctivity nd is therefore directly linked to light intensity, my ffect NH 4 tolernce. Photosynthesis is lso the min source of oxidtive stress in plnt tissues, which is ssocited with negtive effects on plnt growth (Mittler, 2002). To counterct the genertion of rective oxygen species (ROS), chloroplsts possess highly efficient photoprotection defence systems tht operte by two min mechnisms. The first of these prevents ROS production by enhncing the dissiption of excess excittion energy. This process depends on the presence of zexnthin (Z), which is formed by light-induced de-epoxidtion of violxnthin (V) vi the intermedite ntherxnthin () in the so-clled violxnthin cycle, or xnthophyll, cycle (Demmig-dms & dms, 1992). The second line of defense is the detoxifiction of ROS by other ntioxidnt mechnisms, including superoxide dismutses (SOD) s the first enzymtic protective brrier, nd/or through system of smll molecules [e.g. α-tocopherol (Munné-osch, 2005)], which del with ROS produced in photosynthetic membrnes. ll these mechnisms cn be triggered in response to the genertion of ROS cused by n environmentl stress, such s high rdition. Most of the previous studies regrding the effect of light intensity nd its interction with N nutrition hve focused on studying the effect of light-induced stress (Zhu et l., 2000)

5 rther thn its effect on N nutrition, nd hve obtined widely differing results s regrds its effect on NH 4 tolernce (Mglhes & Wilcox, 1983 nd b; Zhu et l., 2000). It hs been shown recently tht NH 4 nutrition, despite hving negtive effect on plnt growth, does not produce sufficient oxidtive stress to explin such toxic effect in pe plnts (Domínguez- Vldivi et l., 2008). N-deficiency, however, leds to reduced plnt growth nd photosynthetic bility, which hs been considered to produce photoinhibition (Kto et l., 2002). Finlly, most studies on NH 4 tolernce re crried out t reltively low photosynthetic photon flux density (PPFD) compred to usul light irrdinces outside the growth chmber (Zhu et l., 2000; Domínguez-Vldivi et l., 2008). The objective of this work ws therefore to study the effect of two PPFDs (350 nd 750 µmol photons m -2 s -1 ), t incresing doses of NH 4 s sole N source, on the overll growth of the plnt nd the photoprotection mechnisms in pe leves Mterils nd methods Plnt mteril nd growth conditions. Pe seeds (cv. sugr snp) were surfcesterilized ccording to Lbhilili et l. (1995). They were then germinted t 26 ºC in drkness for 96 h, in perlite:vermiculite (1:2) nd dmpened with deionized wter, prior to plcement in growth chmber (22/18 ºC dy/night, 70 % RH, 150 µmol photons m -2 s -1 of light intensity nd 14 h light/10 h drk photoperiod). One-week-old seedlings were trnsferred into tnks in groups of eight (8 L volume) nd grown for three weeks in controlled-environment chmbers under different conditions of NH 4 dose nd light (two irrdinces, see below). The hydroponic vessels contined erted (0.4 L ir min -1 L -1 ) nd N-free modified Rigud nd Puppo (1975) solution. The solution ws buffered with CCO 3 (5 mm) to ph 7 7.5, K 2 HPO 4 ws dded insted of KH 2 PO 4 nd NH 4 ws supplied s (NH 4 ) 2 SO 4 during the tretment period, s the only N source, t different concentrtions (0.5; 2.5; 5 nd 10 mm). mmonium sulphte ws chosen since sulphte is known to hve little effect on the uptke of other ions (Mengel nd Kirkby, 1987). The nutrient solution ws exchnged weekly. Two photosynthetic photon flux densities (PPFD) were pplied during these three weeks: high irrdince (HI) of 750 µmol photons m -2 s -1 nd low irrdince (LI) of 350 µmol photons m -2 s -1. LI tretment ws chieved by shding (50% of the incident light) with shde cloth. Plnts were cultured in growth chmber t 22/18ºC (dy/night) temperture, 60/70% reltive humidity nd 14 h light/10 h drk photoperiod. Plnts were hrvested by seprting the shoot nd root of ech plnt. The dry weight of severl pe plnts ws obtined fter weighing nd keeping them in n oven t ºC for 72 h. Smples were collected, frozen in liquid N 2 nd stored t -80 ºC for further nlysis of the remining fresh pe plnts.

6 Physiologicl prmeters: CO 2 ssimiltion ws determined using portble IRG (LI- 6200, Li-Cor, Lincoln, NE, US) t LI nd HI. Mesurements were performed with the lst fully expnded lef nd with vrious different plnts in ech tretment. The folir re ws obtined by using LICOR LI-3000 re mesurer (Li-Cor). Specific lef weight rtio (SLW) ws clculted s the rtio lef dry weight to its surfce, nd it ws expressed in g DW m -2. Fluorescence mesurements were performed with portble modulted light fluorimeter (OS 5-FL, Optisciences, Tyngsboro, U.S..). The mximl photochemicl efficiency of the PSII ws clculted using the rtio F v /F m, where F v = F m F o (Genty et l., 1989). This ws clculted from initil (F o ) nd mximum fluorescence (F m ) mesured in vivo on the lst fully expnded lef pre-cclimtised to the drk for pproximtely 40 minutes. F m ws estimted by pplying light sturting flsh with n intensity of 8000 µmol photons m -2 s -1. Pigment nd ntioxidnt content: Lipophillic ntioxidnts nd photosynthetic pigments were extrcted in cetone (100%) nd nlysed by reversed phse HPLC (Grcí-Plzol & ecerril 1999) following the modifictions of Grcí-Plzol & ecerril (2001). Photosynthetic pigments were mesured with PD detector (PD996, Wters). Retention times nd conversion fctors for crotenoids were s described by Grcí-Plzol & ecerril (1999; 2001). Rubisco content: Frozen leves (0.2 g) were homogenized in mortr with liquid N 2 in phosphte buffer 0.1 M, ph 7. Smples were centrifuged t 20,000 g nd 4 ºC for 20 min, then 5 µg of protein from the superntnts ws loded onto SDS-PGE gel (12.5% p/v), nd stined with Gel-Code lue Stin regent (Pierce iotechnology, Inc., Rockford, US). The totl protein ws clculted ccording to rdford (1976). To estimte the Rubisco content, densitometry nlysis ws conducted using the progrmme Qunt 1 in GelDoc 2000 (io- Rd), tking 100% s the Rubisco content in the smples treted with 0.5 mm NH 4 nd LI. Superoxide Dismutse ctivity (SOD, EC ). Frozen leves (0.2 g pprox.) were homogenized in mortr with liquid N 2 nd potssium phosphte buffer 50 mm ph 7.8, mnnitol 300 mm, MgCl 2 20 mm nd EDTN 2 2 mm (1: 10 w/v). Smples were centrifuged t 20,000 g nd 4 ºC for 20 min. Superntnts (30 µg of protein) were electrophoresed on PGE-ntive gels (15%) nd SOD ctivity ws detected in gel (euchmp nd Fridovich, 1971; Lrrinzr et l., 2008). Isoenzyme identity ws ssigned ccording to Gómez et l. (2004). Sttisticl nlysis. The SPSS progrmme (v.15.0) ws used. Sttisticl clcultions were performed using unifctoril MNOVS (fctor: dose of NH 4 ), including s dependent vribles ll those tht could present reltionship between themselves (e.g. photosynthetic pigments nd ntioxidnt pigments). The Levene test ws used to determine vrince

7 homogeneity. The LSD nd Dunnett T3 post-hoc sttisticl tests were pplied to determine the homogeneity nd non-homogeneity of vrinces cses, respectively. Student s t test ws performed to compre the irrdinces for ech NH 4 dose, tking into ccount whether it complied with the principle of homoscedsticity upon pplying the Levene test. ll sttisticl nlyses were conducted t significnce level of 5% (P 0.05) Results Plnt growth under HI resulted in the highest biomss (shoot nd root; dry weight), with the highest vlue being obtined for 2.5 mm NH 4 (Fig. 1 nd ), nd highest photosynthesis rte (Fig. 2 ), with mximum rte for 5 mm NH 4. However, importnt growth restrictions were found t lowest nd highest NH 4 doses under HI, nd hence mximl differences in plnt growth between HI nd LI re seen t intermedite NH 4 concentrtions (Fig. 1 nd ). Plnts grown under LI did not show notble vrition between NH 4 tretments in terms of biomss or photosynthesis, with the highest vlues being obtined t n NH 4 dose of 2.5 mm. No mjor chnges were detected in lef re between irrdinces or mong NH 4 concentrtions (Fig. 1 C). However, the different PPFDs hd n importnt effect on the specific lef weight index (SLW; Fig. 1 D). Thus, the plnts exposed to HI were thicker nd showed higher SLW vlues thn those exposed to LI. On the other hnd, the SLW rtio tended to increse with externl NH 4 concentrtion under LI (Fig. 1 D). The F v /F m rtio ws independent of PPFD nd NH 4 dose pplied (Fig. 2 ). lthough both HI nd LI tretments showed similr PSII photochemicl efficiency (Fig. 2 ), pe plnts cultured under HI showed fr higher photosynthetic rte thn those exposed to LI (Fig. 2 ). The content of chlorophyll nd b, which re the min components in the photosynthetic ntenn, did not vry between HI nd LI (Fig. 3 ), lthough the chlorophyll content incresed notbly (threefold) on going from 0.5 to 5 mm NH 4 dose under HI. similr, lthough slightly lower (twofold), increse ws lso detected under LI. The Chl /b rtio remined constnt in LI t ll NH 4 doses tested, wheres it incresed with NH 4 dose under HI (Fig. 3 ). The totl crotenoid content bsed on Chl ws higher for HI thn for LI tretment (Fig. 4 ), with this increse being more evident t 0.5 mm NH 4. Pe plnts grown t 0.5 mm NH 4 showed the highest totl crotenoid content under HI, wheres the crotenoid content t 2.5, 5 nd 10 mm NH 4 ws essentilly the sme. The totl crotenoid content in plnts exposed to LI remined constnt regrdless of NH 4 dose (Fig. 4 ) (Sttistic not shown). The proportion for ech individul crotenoid bsed on Chl differed with

8 light tretment nd N pplied, with the lrgest differences being found for the extreme NH 4 tretments (0.5 nd 10 mm NH 4 ; Fig. 4 ). The content of other nti-oxidnt molecules such s α-tocopherol vried considerbly for ll mesurements (LI nd HI in ll NH 4 doses). However, the effect of different PPFDs ws only significnt t 2.5 mm NH 4, where α-tocopherol content ws fr higher in plnts exposed to HI thn those cultivted t LI (Fig. 4 ). Xnthophyll cycle pigment content (violxnthin-v, nterxnthin- nd zexnthin-z) ws lso higher in plnts exposed to HI (Fig. 5). Furthermore, HI chnged the proportion of V cycle components, incresing the deepoxidted forms (Z) with respect to those observed t LI t NH 4 doses of 0.5 nd 2.5 mm (Fig. 5). V cycle component content incresed with NH 4 dose under LI (totl cke re), wheres it tended to decrese with NH 4 dose t HI; the highest totl V content ws observed t 0.5 mm NH 4 (Fig. 5, totl cke res). Ribulose 1,5-bisphosphte crboxylse/oxygense (Rubisco) content from lef extrcts ws visulized in coomsie blue-stined SDS gel by loding equivlent mounts of totl protein in ech line (Fig 6 ). Rubisco content decresed t high NH 4 concentrtions (pprox. 33% for 5 mm NH 4 nd 28% for 10 mm NH 4, under both LI or HI, with respect to 0.5 mm NH 4 ). The highest Rubisco content ws observed t 2.5 mm NH 4 nd HI tretment (34% higher compred thn tht for 0.5 mm NH 4 under LI; Fig. 6 nd 1). Rubisco percentge vlues, either bsed on ChI or bsed on lef re, tended to increse with NH 4 dose for both irrdinces (Fig. 6 2 nd 3, respectively). Reltive Rubisco content per µmol of Chl or per unit of lef re showed higher vlues under HI, with the exception of 0.5 mm NH 4, (Fig. 6 2 nd 3 respectively). The ntive gel SOD ctivity test showed the different SOD isoenzymes present in pe leves. The bnds observed on the gels were identified on the bsis of the bnd pttern obtined previously for pe (Gómez et l., 2004). t lest four bnds, which corresponded, by order of migrtion, to MnSOD, FeSOD nd cytosolic nd chloroplstic CuZnSOD isoenzymes, were detected. SODs presented greter ctivity under HI thn under LI. However, irrespective of PPFD pplied, the highest CuZnSOD isoenzyme (cytosolic nd plstidil) ctivity ws observed t 0.5 mm NH 4, with even greter intensity in the chloroplstic isoform (CuZnSOD 2). FeSOD nd CuZnSOD ctivity decresed slightly t high NH 4 concentrtions (5 nd 10 mm) with respect to low NH 4 doses (0.5 nd 2.5 mm), wheres MnSOD ctivity (isoenzyme typiclly mitochondril) remined unchnged for ll tretments (Fig. 7). 224

9 Discussion We hve confirmed tht NH 4 nutrition t high concentrtions (5 nd 10 mm) hs negtive effect on biomss ccumultion in pe plnts (Fig. 1 nd ), s reported previously (Domínguez-Vldivi et l., 2008; Cruz et l., submitted). However, high PPFD (HI) ws found to hve positive effect on plnt growth nd photosynthetic rte t intermedite NH 4 concentrtions (Fig. 1 nd nd 2, respectively), where NH 4 stress is prtly llevited. This finding is in contrst to previous reports, which found tht the negtive effect of NH 4 nutrition on plnt growth is ccentuted under HI conditions in severl species (Zhu et l., 2000; Guo et l., 2007). However, numerous studies hve shown tht photosynthetic rte nd photosynthetic electronic trnsport responses to PPFD vry with species, N dose nd light intensity pplied throughout the culture period (Gstl & Lemire, 2002; Kto et l., 2002). The greter totl biomss (shoot nd root DW) found under HI compred to LI (except 0.5 mm of NH 4 ; Fig. 1 nd ) my well be due to the greter photosynthetic bility developed during the pe plnts dpttion to HI, s noted by Kto et l. (2002), which trnsltes into greter photosynthetic rte (Fig. 2 ). Indeed, the higher SLW observed in plnts grown under HI (except t 10 mm NH 4 ; Fig. 1 C) is ssocited with thickening of the leves under HI conditions. Thicker leves (Fig. 1 D), higher net photosynthesis (Fig. 2 ) nd higher Chl /b rtios t 5 nd 10 mm NH 4 under HI (Fig. 3 ), re common fetures of HI-cclimtised leves nd those exposed to sunlight. Interestingly, similr SLW levels were obtined t 10 mm NH 4 by plnts under both PPFDs, thus indicting n effect of high NH 4 dose on the folir thickness increse (Fig. 1 D). In line with observtions by Demmig-dms et l. (1995), the reltive lutein nd neoxnthin contents were lower in HI plnts thn in LI plnts (Fig. 4 ), wheres V cycle component (Fig. 4 nd Fig. 5) nd β-crotene levels (Fig. 4 ) incresed under HI. s documented in the literture (Verhoeven et l., 1997; Kto et l., 2002), the ppliction of low N doses (0.5 mm) leds to photoinhibitory effect tht leds to plnt biomss reduction irrespective of the light intensity supplied (Fig. 1 nd ). Furthermore, N-deficiency hs remrkble effect on other prmeters, such s lower photosynthesis rte (Fig. 2 ) nd lower ChI b content (Fig. 3 ), which is probbly relted to lower folir N content. The 0.5 mm NH 4 dose lso showed high vlues for prmeters relted to high photoprotective demnd s result of N-deficiency nd HI exposure. The crotenoid content (Fig. 4 ), especilly xnthophyll cycle components (Fig. 4 nd 5), nd CuZnSOD ctivity (Fig. 7) were fr higher t 0.5 mm NH 4 thn t other N doses. Moreover, plnts grown t 0.5 mm NH 4 nd HI lso presented higher Rubisco content thn those t 5 nd 10 mm NH 4

10 (Fig. 6 nd 1). However, despite showing higher photosynthetic rte (Fig. 2 ) thn those cultivted under LI, these plnts did not show higher growth bsed on dry weight (Fig. 1 nd ). This behviour is likely relted to the higher totl crotenoid nd V content (Fig. 4 nd 5, respectively) nd to the higher de-epoxidtion index ( Z / V) observed t 0.5 mm NH 4 nd HI (Fig. 5). ll these results reflect high degree of photoprotection nd dissiption of non-photochemicl energy (Müller et l., 2001) cused by N-deficiency, which could be explined by the risk of photo-oxidtive dmge being worsened by excessive light ppliction. Previous studies on Pisum stivum hve found observed tht 2.5 mm NH 4 is n inflection point in the response of lrge number of prmeters to externl N (Domínguez- Vldivi et l., 2008; Cruz et l., submitted for publiction). This my suggest tht this dose represents trnsition point in the metbolic response of the plnt to the externl NH 4 concentrtion, which my involve compromise between the N requirement for photosynthesis nd photoprotection nd NH 4 toxicity. Some of the ntioxidnt system indictors, such s α-toc/chl (Fig. 4 ) (Iturbe-Ormetxe et l., 1995) nd the de-epoxidtion index ( Z / V) (Fig. 5 ), lso presented significntly higher vlues here compred to other tretments. The higher Rubisco content observed t 2.5 mm NH 4 for both irrdinces (Fig. 6 ) is remrkble effect which seems to be relted to the inflection point in pe metbolism t this dose. Thus, n NH 4 dose of 2.5 mm nd HI gve the highest expression of Rubisco (28% more thn 2.5 mm NH 4 nd LI; Fig. 6 nd 1), which mtches the highest biomss pek (Fig. 1 nd ). Wrren et l. (2000) hve exmined severl ntive ustrlin plnts under different N nutrition conditions nd found widely differing Rubisco contents. However, the mount of Rubisco increses with N dose pplied, irrespective of its source (NO - 3, NH 4 NO 3 or NH 4 ) in most species (Mkino et l., 1997; ungrd et l., 1997; elstegui-mcdm, 2004; Fig. 6 2 nd 3). Guo et l. (2007) hve found tht Rubisco vlues follow different trends depending on how they re expressed (Rubisco protein -1, Rubisco Chl -1 or Rubisco cm -2 ; Fig. 6 ). The higher Rubisco content bsed on totl protein t low NH 4 doses (0.5 nd 2.5 mm; Fig 6 nd 6 1) my contribute to greter photorespirtion rtes, s reported by Murok et l. (2000), nd n increse in photorespirtory bility hs been found to be one of the photoprotective non-photochemicl mechnisms in leves exposed to excess light (Murok et l., 2000). SODs ctlyze the dismuttion of superoxide rdicls, whose production my be importnt in chloroplsts through Mehler s rection (sd, 1999). This flux of electrons into the oxygen my increse under conditions of stress, lthough it lso occurs under physiologicl photosynthetic conditions (Foyer et l., 1994). FeSODs re chloroplstic

11 isoenzymes in pe (Morn et l., 2003) nd hve often been ssocited with protection ginst light rdition (Kliebstein et l., 1998; Kmink et l., 1999), which could explin their higher ctivity under HI conditions (Fig. 7). However, the mjor isoenzymes detected correspond to cytosolic nd plstidil CuZnSOD isoforms (1 nd 2 respectively), which show higher induction under HI nd t low N doses (i.e. 0.5 nd 2.5 mm NH 4 ; Fig. 7). This induction is relted to the greter de-epoxidtion index seen (Fig. 5 ) nd the greter photoprotective bility, s shown by the higher V/crotenoid rtio (Fig. 4). These results therefore highlight the high level of photoprotection nd greter nti-oxidnt potentil in pe leves under such conditions (Grcí-Plzol et l., 2004). Despite showing greter growth (Fig. 1 nd ), plnts grown with n NH 4 dose of 2.5 mm under HI tretment could lso be N-deficient, s is the cse with 0.5 mm NH 4. oth tretments showed higher levels of photoprotection nd energy dissiption, which is usully proportionl to the level of light stress to which leves re exposed (Grcí-Plzol et l., 2008b). Furthermore, the ChI /b rtio, which is good indictor of plnt s cclimtiztion to light intensity (sun/shde), showed lower vlues t HI nd low N doses (0.5 nd 2.5 mm NH 4 ) thn t 5 nd 10 mm NH 4 (Fig. 3 ), which lso points to n N-deficiency in those tretments. The rtio of Chl to Chl b typiclly increses with irrdince once plnt becomes cclimtised to light environment (Demmig-dms nd dms 1992; Demmig- dms 1998; Thyer nd jorkmn 1990). The vlues reported herein correspond to those typiclly found for shded leves but re consistent with the trend of higher Chl /b rtio t higher irrdinces. Despite light regultion of Chl /b, n dditionl positive NH 4 effect ws observed, thus suggesting n N-deficiency in the 0.5 nd 2.5 NH 4 tretments. Despite the ctivtion of photoprotection systems in pe plnts treted with high NH 4 doses (5 nd 10 mm), s confirmed by the vrition in crotenoid pigments nd V cycle components under HI, not only were no photoinhibition symptoms observed, but there ws beneficil effect of the HI plnts dpttion in terms of NH 4 toxicity in comprison to LI. Furthermore, the ctivtion of other ntioxidnt mechnisms, such s detoxifiction of ROS production (i.e. SOD), is not required t these N doses under HI Conclusions lthough higher crotenoid nd V contents re induced by HI, this does not hve negtive effect on the overll development of pe plnts (cv. sugr-snp) grown in the presence of NH 4. Indeed, greter photosynthetic rte nd improved plnt growth re observed. However, N-deficiency triggers n increse in the elimintion of ROS, which becomes incresingly evident under HI conditions. HI-exposed plnts cultivted t high NH 4

12 doses show lower photoprotective demnd thn those cultivted t low NH 4 doses, possibly becuse they suffer greter energy drin in photosynthesis. cknowledgements. This work ws supported by the Spnish MICIIN (grnt nos. GL CO2-01 nd GL CO2-01 [to P..-T.] nd GL /GR [to J.F.M.]), by the Government of Nvrr (Res 57/2007 to J.F.M.), nd FU of MEC nd EHU-GV IT ) of the sque Government to JM nd JIG-P. I ws supported by doctorl Fellowship from the Public University of Nvrre, Spin References sd K. The wter-wter cycle in chloroplsts: scvenging of ctive oxygens nd dissiption of excess photons. nnu Rev Plnt Physiol Plnt Mol iol 1999; 50: euchmp CO, Fridovich I. Superoxide dismutse: Improved ssys nd n ssy pplicble to crylmide gels. nl iochem 1971;44: elstegui-mcdm X. Inhibidores de l nitrificción: fitotoxicidd e inducción de l nutrición monicl. Ph. D. Thesis. Universidd del Pís Vsco, Spin, rdford MM. rpid nd sensitive method for the quntittive determintion of microgrm quntities of protein utilizing the principle of protein-dye binding. nl iochem 1976;72: ungrd R, McNeil D, Morton JD. Effects of nitrogen on the photosynthetic pprtus of Clemtis vitlb grown t severl irrdinces. ust J Plnt Physiol 1997;24: Cruz C, Domínguez-Vldivi MD, pricio-tejo PM, Lmsfus C, io MF, Mrtins- Loução, M, Morn JF. Pe (Pisum stivum L) vrieties respond to mmonium nutrition with distinct strtegies nd obtin distinct degrees of tolernce. Environ Exp ot; Submitted. Demmig-dms, dms WW III. Photoprotection nd other responses of plnts to high light stress. Rev Plnt Physiol Mol iol 1992;43: Demmig-dms, dms WW III. Crotenoid composition in sun nd shde leves of plnts with different life forms. Plnt, Cell nd Environment 1992;15: Demmig-dms, dms WW III, Logn, Verhoeven S. Xnthophyll cycledependent energy dissiption nd flexible PSII efficiency in plnts cclimted to light stress. ust J Plnt Physiol 1995;22:

13 Demmig-dms. Survey of therml energy dissiption nd pigment composition in sun nd shde leves. Plnt Cell Physiology 1998;39: Domínguez-Vldivi MD, pricio-tejo PM, Lmsfus C, Cruz C, Mrtins-Loução M, Morn JF. Nitrogen nutrition nd ntioxidnt metbolism in mmonium-tolernt nd - sensitive plnts. Physiol Plnt 2008;132: Foyer CH, Descourvières P, Kunert KJ. Protection ginst oxygen rdicls: n importnt defence mechnism studied in trnsgenic plnts. Plnt Cell Environ 1994;17: Grcí-Plzol JI, ecerril JM. rpid HPLC method to mesure lipophilic ntioxidnts in stressed plnts: simultneous determintion of crotenoids nd tocopherols. Phytochem nl 1999;10: Grcí-Plzol JI, ecerril JM. Sesonl chnges in photosynthetic pigments nd ntioxidnts in beech (Fgus sylvtic) in Mediterrnen climte: implictions for tree decline dignosis. ust J Plnt Physiol 2001;28: Grcí-Plzol JI, ecerril JM, Hernández, Niinemets Ü, Kollist H. cclimtion of ntioxidnt pools to the light environment in nturl forest cnopy. New Phytol 2004;163:87 97 Grcí-Plzol JI, Estebn R, Hormetxe K, ecerril JM. Sesonl reversibility of cclimtion to irrdince in leves of common box (uxus sempervirens L.) in deciduous forest. Flor 2008b;203: Gstl F, Lemire G. N uptke nd distribution in crops: n gronomicl nd ecophysiologicl perspective. J Exp ot 2001;53: Genty, rintis JM, ker NR. The reltionship between the qumtum yield of photosynthetic electron trnsport nd quenching of chlorophyll fluorescence. iochem. iophys. ct 1989; Gómez JM, Jiménez, Olmos E, Sevill F. Loction nd effect of long-term NCl stress on superoxide dismutse nd scorbte peroxidse isoenzymes of pe (Pisum stivum cv. Puget) chloroplsts. J Exp ot 2004;55: Guo SW, Zhou Y, Go Y-X, Li Y, Shen Q-R New insights into the nitrogen form effect on photosynthesis nd photoprotection. Pedosphere 2007;17: Iturbe-Ormetxe I, Morn JF, rrese-igor C, Gogorcen Y, Klucs RV, ecn M ctivted oxygen nd ntioxidnt defences in iron-deficient pe plnts. Plnt Cell Environ 1995;18:

14 Kmink H, Morit S, Tokumoto M, Yokoym H, Msumur T, Tnk K. Moleculr cloning nd chrcteriztion of cdn for n iron-superoxide dismutse in rice (Oryz stiv L.). ios iotechnol iochem 1999;63: Kto MC, Hikosk K, Hirose T. Photoinctivtion nd recovery of photosystem II in Chenopodium lbum leves grown t different levels of irrdince nd nitrogen-vilbility. Funct Plnt iol 2002;29: Kliebenstein DJ, Monde R, Lst RL. Superoxide dismutse in rbidopsis: n eclectic enzyme fmily with disprte regultion nd protein locliztion. Plnt Physiol 1998;118: Lbhilili M, Joudrier P, Gultier M. Chrcteriztion of cdn encoding Triticum durum dehydrins nd their expression ptterns in cultivrs tht differ in drought tolernce. Plnt Sci 1995;112: Lrrinzr E, Urrte E, uzmendi I, riz I, rrese-igor C, González EM, Morn JF. Use of Recombinnt Iron-Superoxide Dismutse s Mrker of Nitrtive Stress. Methods Enzymol 2008;437: Mglhes JR, Wilcox GE. Tomto growth nd minerl composition s influenced by nitrogen form nd light intensity. J Plnt Nutr 1983;6: Mglhes JR, Wilcox GE. Tomto growth nd nutrient uptke ptterns s influenced by nitrogen form nd light intensity. J Plnt Nutr 1983b;6: Mkino, Sto T, Nkno H, Me T. Lef photosynthesis, plnt growth nd nitrogen lloction in rice under different irrdinces. Plnt 1997;203: Mittler R. Oxidtive stress, ntioxidnts nd stress tolernce. Trends Plnt Sci 2002;7: Mengel nd Kirkby, Sulphur in physiology. p In Principles of Plnt Nutrition. Eds. K Mengel nd E Kirkby Interntionl Potsh Institute. Worblufen-ern, Switzerlnd Morn JF, Jmes EK, Rubio MC, Srth G, Klucs RV, ecn M. Functionl chrcteriztion nd expression of cytosolic iron-superoxide dismutse from cowpe root nodules. Plnt Physiol 2003;133: Müller P, Li XP, Niyogi KK. Non-photochemicl quenching. response to excess light energy. Plnt Physiology 2001;125: Munné-osch S. The role of lph-tocopherol in plnt stress tolernce. J Plnt Physiol 2005;162:

15 Murok H, Tng Y, Tershim I, Koizumi H, Wshitni I. Contributions of diffusionl limittion, photoinhibition nd photorespirtion to middy depression of photosynthesis in risem heterophyllum in nturl high light. Plnt Cell Environ 2000;23: Rigud J, Puppo. Indole-3-cetic ctbolism by soyben bcteroids. J Gen Microbiol 1975;88: Thyer SS, jorkmn O. Lef xnthophyll content nd composition in sun nd shde determined by HPLC. Photosynthesis Reserch 1990;23: Verhoeven S, Demmig-dms, dms III WW. Enhnced employment of the xnthophyll cycle nd therml energy dissiption in spinch exposed to high light nd N stress. Plnt Physiol 1997;113: Wrren CR, dms M, Chen ZL. Is photosynthesis relted to concentrtions of nitrogen nd Rubisco in leves of ustrlin ntive plnts? ust J Plnt Physiol 2000;27: Zhu Z, Gerendás J, endixen R, Schinner K, Tbrizi H, Sttelmcher, Hnsen U. Different tolernce to light stress in NO - 3 nd NH 4 -grown Phseolus vulgris L. Plnt iol 2000;2:

16 Legends to figures Fig. 1. Effect of NH 4 doses on growth prmeters: () shoot dry weight (g), () root dry weight (g), (C) totl lef re (cm 2 ) nd (D) SLW (g DW m -2 ) in pe plnts cv. sugr-snp grown on LI ( ) or HI ( ). Dt represent verge vlues ± S.E. ( nd : n=14; C nd D: n=3). Letters show significnt differences (P 0.05) mong NH 4 doses for HI (,, C nd D) nd LI (, b, c nd d). The sterisk (*) denotes significnt differences (P 0.05) for ech NH 4 concentrtion between HI nd LI Fig. 2. Effect of NH 4 doses on physiologicl prmeters: () Photosynthesis (µmol CO -2 s 2 m -1 ), () Mximum photochemicl efficiency of PSII (F v/f m) in pe plnts cv. sugr-snp grown on incresing doses of NH 4 (0.5; 2.5; 5 nd 10 mm) nd dpted to LI ( ) or HI ( ). Dt represent verge vlues ± S.E. (: n=5-8; : n=8). Letters show significnt differences (P 0.05) mong NH 4 doses for HI (,, C nd D) nd LI (, b, c nd d). The sterisk (*) denotes significnt differences (P 0.05) for ech NH 4 concentrtion between HI nd LI Fig. 3. Effect of NH 4 doses on physiologicl prmeters: () totl Chl content (µmol Chl (b) m -2 ), () Chl /b rtio, in pe plnts cv. sugr-snp grown on incresing doses of NH 4 (0.5; 2.5; 5 nd 10 mm) nd dpted to LI ( ) or HI ( ). Dt represent verge vlues ± S. E. (n=4-5). Letters show significnt differences (P 0.05) mong NH 4 doses for HI (,, C nd D) nd LI (, b, c nd d). The sterisk (*) denotes significnt differences (P 0.05) for ech NH 4 concentrtion between HI nd LI Fig. 4. Crotenoids nd α-tocopherol reltive contents in pe leves cv sugr-snp, grown with incresing doses of NH 4 (0.5; 2.5; 5 nd 10 mm) nd dpted to low nd high light intensities: () content of crotenoids. LI (left) nd HI (right), respectively. Violxnthin ntherxnthin zexnthin (V), neoxnthin (N), lutein (L) nd β- crotene (β-crot) re expressed s individul percentge (reltive res of the brs) to the totl crotenoids content (mmol Crot mol -1 Chl (b)); line with errors brs. Sttistics of individul crotenoids not shown. NOV nlysis for the different NH 4 doses (P 0.05) nd verges comprison of independent smples for the irrdince fctor (with two levels; P 0.05) were crried out. () Content of α-tocopherol (α-toc; mmol α-toc mol -1 Chl ( b)). Dt represent verge vlues ± S. E. (n=5). Letters show significnt differences (P 0.05) mong NH 4 doses for HI (,, C nd D) nd LI (, b, c nd d). The sterisk (*) denotes significnt differences (P 0.05) for ech NH 4 concentrtion between HI nd LI Fig. 5. Reltive contents of V cycle crotenoids cycle in pe leves cv sugr-snp, grown on incresing doses of NH 4 (0.5; 2.5; 5 nd 10 mm): () contents of V cycle crotenoids: violxnthin (V) ntherxnthin (), nd zexnthin (Z) dpted to LI (left) nd HI (right), respectively. Vlues re expressed in individul crotenoid percentge (reltive brs res) to the totl V cycle crotenoids content (mmol Crot mol -1 Chl ( b); line with errors brs). Dt represent verge vlues (n=3-5). NOV nlysis for the different NH 4 doses (P 0.05) nd verges comprison of independent smples for the irrdince fctor (with two levels; P 0.05) were crried out. () Effect of NH 4 doses on the ( Z)/ VZ index. Dt represent verge vlues ± S. E. (n=3-5). Letters show significnt differences (P 0.05) mong NH 4 doses for HI (,, C nd D) nd LI (, b, c nd d). The sterisk (*) denotes significnt differences (P 0.05) for ech NH 4 concentrtion between HI nd LI. 483

17 Fig. 6. Rubisco contents in pe plnts cv. sugr-snp grown on incresing doses of NH 4 (0.5; 2.5; 5 nd 10 mm) nd dpted to LI ( ) or HI ( ): () Rubisco contents in SDS-PGE stined with Coomssie blue; 5 µg of protein were loded into ech line. () Reltive Rubisco contents respect to reference tretment, 0,5 mm NH 4 nd LI: (1) reltive percentge of Rubisco, protein (5 µg)-bsed (2) reltive percentge of Rubisco Chl (b) (µmol)-bsed, nd (3 ) reltive percentge of Rubisco, lef surfce (cm 2 )-bsed Fig. 7. Totl superoxide dismutse (SOD) ctivity in gel in pe plnts cv. sugr-snp grown on incresing doses of NH 4 (0.5; 2.5; 5 nd 10 mm) nd dpted to LI ( ) or HI ( ), s indicted in mterils nd methods. 30 µg of protein were loded into ech line. MnSOD, FeSOD, CuZnSOD-1(cytosolic) nd CuZnSOD-2 (plstidil).

18 Shoot DW (g) Root DW (g) C b bc c * * * * * () C b () (C) b b (D) b c * * * * b verge Lef re (cm 2 ) SLW (g DW m -2 ) Concentrtion of NH 4 (mm) LI HI Fig.1

19 Photosynthesis Rte (µmol CO 2 m -2 s -1 ) F v / F m b b b () * * * b () b Concentrtion of NH 4 (mm) LI HI Fig.2

20 µmol Chl (b) m -2 Chl /b (mol mol -1 ) * b C C b b () * * () Concentrtion of NH 4 (mm) LI HI Fig.3

21 Low Irrdince (LI) High Irrdince (HI) () mmol crotenoids mol -1 Chl (b) * * * 19 % 17 % 16 % 16 % 46 % 47 % 44 % 45 % 21 % 21 % 26 % 23 % 14 % 15 % 14 % 16 % mmol crotenoids mol -1 Chl (b) % 19 % 13 % 19 % 38 % 42 % 45 % 39 % 31 % 27 % 29 % 30 % 11 % 12 % 13 % 12 % Concentrtion of NH 4 (mm) Concentrtion of NH 4 (mm) N VZ L β Crot Totl Crot mmol α Toc mol -1 Chl (b) * () Concentrtion of NH 4 (mm) LI HI Fig.4

22 Low Irrdince (LI) High Irrdince (HI) () mmol V cycle crotenoids mol -1 Chl (b) * * * 9 % 4 % 1 % 8 % 8 % 3 % 88 % 96 % 83 % 93 % % 6 % mmol V cycle crotenoids mol -1 Chl (b) % 8 % 2 % 13 % 16 % 6 % 14 % 17 % 82 % 67 % 92 % 73 % Concentrtion of NH 4 (mm) Concentrtion of NH 4 (mm) V Z Totl V cycle 0.45 * * () [Z] / VZ b b b Concentrtion of NH 4 (mm) Fig.5

23 () Rubisco % (D.O. units x 100 x 5 µg -1 prot) Concentrtion of NH 4 (mm) 1 Rubisco % (D.O. units x 100 x µmol -1 Chl b) Concentrtion of NH 4 (mm) 2 Rubisco % (D.O. units x 100 x cm -2 ) Concentrtion of NH 4 (mm) () LI HI LI HI LI HI Fig.6

24 Fig.7