Nitrate reductase (NR) and glutamine synthetase (GS) can be used as indicators of nitrogen status in eucalyptus clones

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1 AJCS 9(6): (215) ISSN: Nitrte reductse (NR) nd glutmine synthetse (GS) cn e used s indictors of nitrogen sttus in euclyptus clones Eric Victor de Oliveir Ferreir 1*, Roerto Ferreir Novis 2, Flávi Aprecid dos Sntos 3, Cleerson Rieiro 4, Nirm Félix Brros 3 1 Deprtmento de Ciêncis Florestis, Escol Superior de Agricultur "Luiz de Queiroz", Universidde de São Pulo, Brzil 2 Itituto de Ciêncis Agráris, Cmpus de Rio Prní, Universidde Federl de Viços, Brzil 3 Deprtmento de Solos, Cmpus de Viços, Universidde Federl de Viços, Brzil 4 Deprtmento de Biologi Vegetl, Cmpus de Viços, Universidde Federl de Viços, Brzil *Corresponding uthor: ericsolos@yhoo.com.r Astrct Nitrte reductse (NR) (EC ) ctivity nd glutmine synthetse (GS) (EC ) ctivity, hve een proposed s more seitive indictors of nitrogen (N) sttus in plnts thn totl N content. The im of this study ws to ssess the ctivities of NR nd GS s indictors of the sttus of N in euclyptus clones grown under different conditio of N supply. For this purpose, experiments were crried out in oth greenhouse nd field conditio. In the greenhouse, the experiment ws conducted in nutrient solution nd coisted of 2 6 fctoril rrngement, with two euclyptus clones (VM1 nd I144) nd six ppliction rtes of N (,.74, 2.93, 4.39, 5.85, nd 8 mmol L 1 of NH 4 NO 3 ). The field experiment ws composed of fctoril rrngement, with two regio (Pompeu nd João Pinheiro, MG Stte, Brzil), two smpling periods (dry seson nd riny seson), nd two euclyptus clones (VM1 nd I144). We evluted the totl N nd totl protein contents nd enzymtic ctivities of NR nd GS in the leves. In the greenhouse, the clone I144 took up less N, inducing less protein synthesis; however, it reduced nd ssimilted more N (greter NR nd GS ctivity), which resulted in greter dry mtter production compred to VM1. Enzyme ctivity ws negtively ffected y greter N supply (r=.58 for NR nd r=.5 for GS), proly through n inhiitory effect from oth NO 3 (sustrte of NR) nd NH 4 + (sustrte of GS) t high concentrtio. In the greenhouse, the ctivity of these enzymes lso showed negtive correltion with the lef content of totl N (r=.79 for NR nd r=.64 for GS) of the euclyptus clones. Keywords: Enzyme ctivity; forest nutrition; lef dignosis; N metolism; totl N. Arevitio: ATP_denosine triphosphte; AsnA_sprgine synthetse; C_cron; CBH_circumference t rest height (1.3 m); FAD_ flvin denine dinucleotide; g_grvity force; GHA_FeLglutmylγhydroxmte; GS_glutmine synthetse; I144 nd VM1_euclyptus clones; m..p._months fter plnting; min_minute; Moco_molydenum cofctor; N_nitrogen; NH 4 + _mmonium, NiR_nitrite reductse; NO 2 _ nitrite; NO 3 _nitrte; NR_Nitrte reductse; PMSF_phenylmethylsulfonyl fluoride; PVPP_polyvinylpyrrolidone; r_person correltion coefficient; S_south; W_west. Introduction Lef content of totl nitrogen (N) hs trditionlly een used to estimte the sttus of N in plnts; however, there re limits to its use s nutritionl indictor, such s low respoe to high N supply in trees (Alv et l., 1998), s well s its ieitivity to sesonl demnd in use of the nutrient (Chpin et l., 1982). It hs een oserved tht totl N content in the lef tissues lso does not differentite etween respoive nd unrespoive sites for Euclyptus gloulus nd E. grndis (Perdomo et l., 27). Thus, enzymtic ctivities hve een proposed s more seitive indictors for the sttus of N in plnts. In the 197s, it hd lredy een suggested tht nutritionl deficiencies in plnts could e dignosed through mesurement of enzymtic ctivity (Br Akiv, 1971), nd this ws lso oserved in lter studies (Tvdgiridze nd Putkrdze, 1991; Lvres Jr et l., 21). One of the dvntges of the metolic dignosis is its high seitivity since smll vrition in nutrient content leds to high vrition in the metolite content (Mrtinez et l., 1999). Nevertheless, these uthors emphsize tht there re no stndrds or universlly ccepted methods of determintion, which indictes the relevnce nd necessity of discovering novel protocol. Nitrte reductse (NR) is cited s n importnt iochemicl mrker for N deficiency (Srivstv nd Singh, 25), nd mesurements of its ctivity re used s tool for evlution of the nutritionl sttus of N in tropicl grsses (Lvres Jr nd Monteiro, 26). The ctivity of NR in the recently expnded lef lde hd positive nd significnt correltion with totl N content in the lef nd yield of momç grss (Pnicum mximum Jcq.) (Lvres Jr et l., 21). In ddition, NR is n enzyme with gret potentil for improving wter qulity of environments polluted with NO 3, through converting it to NO 2 (Cmpell nd Cmpell, 1998). Normlly NR ctivity is high in the leves (Souz nd Fernndes, 26); however, ccording to Cmpell (1999), some plnts hve little or no ctivity of this enzyme in the leves, ut they hve greter ctivity in the roots. Activity of NR in the leves ws greter (8 %) thn in the roots of E. cmphor nd E. ovt 561

2 (Grnger et l., 1994). This enzyme exhiits n inductive chrcter; n increse in its ctivity is seen fter supplying NO 3 to plnts, nd decrese in its ctivity t low light levels (Souz nd Fernndes, 26). Souz nd Fernndes (26) cite glutmine synthetse (GS) s the min enzyme in ssimiltion of NH + 4 y plnts. The ctivity of GS my vry under different conditio of N (Mglhães et l., 1995). The ctivity of GS in shoots of Ctsetum fimritum plnts ws positively correlted with solule protein contents (Mjerowicz nd Keruy, 22). There is positive correltion etween grin yield nd GS ctivity (Gllis nd Hirel, 24). In corn nd whet, GS ctivity is representtive of the N sttus of the plnt (Hirel et l., 27). In light of the ove, the im of this study ws to evlute the enzymtic ctivities of NR nd GS s indictors of N sttus s sustitution for totl N content in the lef in euclyptus clones grown under different conditio of N supply. Although enzymtic ctivities were lredy proposed in the pst century s more seitive indictors for N sttus in plnts, the ctivities of NR nd GS hve still not een ssessed for this purpose in euclyptus clones. Results nd Discussion Greenhouse experiment There ws n increse in lef protein contents of the clone VM1 with the increse in the ppliction rtes of N in solution (Fig 1), reching pek t the intermedite rtes, ccording to the qudrtic model (Fig 1). There ws low correltion (r=.38) etween protein contents nd the ppliction rtes of N in solution for the two clones (Tle 1). In spite of different models (qudrtic nd squre root), the protein contents hd tendency similr to totl N contents in the lef (Fig 1) for the clone VM1 (r=.7), nd lso to dry mtter production of plnts (Fig 1e). As prt of the N tken up is incorported into the plnt s mino cids, nd the synthesized protei promote lef growth with n increse in its supply, incresing the re of photosynthesis (Dechen nd Nchtigll, 27), there is greter dry mtter production of the plnt. For clone I144, there ws no difference etween the lef contents of protein, which were less thn those of clone VM1 (Fig 1), corroorting with its lower totl N contents in the lef (Fig 1). An increse in NR ctivity is generlly seen with the increse in N supply in nitric form, with the increse in totl N content in the lef, or with the increse in yield of different plnt species, s in Momç grss (Lvres Jr et l., 21), mnioc (Cruz et l., 24), coffee (DMtt et l., 1999; Reis et l., 27), nd her species (Grnger et l., 1994), nd even in E. grndis, E. regn, nd E. olíqu (Adms nd Attiwill, 1982; Cldeir et l., 1994). In contrst, NR ctivity in the leves of oth clones ssessed ws greter in the sence of N in solution nd decresed with the increse in the rtes pplied of this nutrient, ccording to the squre root model (Fig 1c). Thus, there ws n inverse correltion etween NR ctivity nd the N supplied (r =.49) nd the totl N content in the lef (r =.7) (Tle 1). Kumr nd Singh (22) lso oserved greter NR ctivity with low ccumultion of N in corn hyrids during the months of June to Septemer. The NR ctivity is regulted y vrious environmentl nd intrcellulr fctors, such s light, concentrtion of N compounds, CO 2, Mo, Fe, plnt hormones, nd C metolites (Le nd Leegood, 1995). Light hs n effect which my e direct, ctivting the enzyme, or indirect, y photosynthesis, supplying energy for ssimiltion of NO 3 (Smirnoff et l., 1984). Moreover, ccording to the lst uthors mentioned, the ctivity of this enzyme is lso ffected y the concentrtion of NH + 4. Both the synthesis nd the ctivity of NR re induced y the presence of the sustrte (Adms nd Attiwill, 1982; Somers et l., 1983); therefore, reduction in uptke of NO 3 would led to reduction in the ctivity of this enzyme. Nevertheless, mnioc leves deficient in N, in which lef contents of NO 3 were not detectle, showed NR ctivity (Cruz et l., 24). According to Cmpell (1999), the NO 3 newly tken up nd trported to plnt shoot ppers to e more determinnt fctor for induction of NR ctivity thn the NO 3 stored in the vcuole, since the first phse of reduction of NO 3 occurs in the cytoplsm. Rpid trport of NO 3 to outside of the cytoplsm expli the declines in NR ctivity when the externl supply of this nion is reduced, even when the totl content of NO 3 in the plnt is high (Fernndes nd Souz, 26). The influx of NO 3 to the cytosol performs more relevnt role in induction of NR ctivity thn the NO 3 stored in the vcuole (Queiroz et l., 1993). A decrese ws oserved in NR ctivity in pech plm (Bctris gsipes) seedlings t high concentrtio of NO 3 in the externl medium, indicting negtive effect from excess of sustrte on enzyme ctivity (Oliveir et l., 25). The ddition of higher concentrtio of NO 3 lso reduced NR ctivity in lge (Chow et l., 27). In reltion to NH + 4, high contents my hve n inhiitory effect on NR ctivity (DeluFilho, 1994). This uthor oserved tht in the fternoon, there ws low NR ctivity in the roots of the ruer tree nd ttriuted this fct to possile retroinhiition of the enzyme, cused y + the ccumultion of NH 4 in this orgn. The high + concentrtion of NH 4 in the roots results from low trloction of this ction to plnt shoot, rought out y decrese in trpirtion flux, rising from greter stomtl closing of the plnts. Thus, the increse in the concentrtion of NH + 4 in nutrient solution led to greter lef content of NH + 4 (Ferreir, 213), with reduction in NR ctivity. This ecomes relevnt especilly for euclyptus species, which, in generl, hve preferentil uptke of NNH + 4 (Brros nd Novis, 1996) nd high contents of this ction in their tissues (Ferreir, 213). In ruer tree roots the NH + 4 hd strong repressive effect on NR ctivity, even in plnts tht were grown t ner optiml NO 3 ppliction rtes (6 mmol L 1 of NO 3 nd 2 mmol L 1 of NH + 4 ) (Lemos et l., 1999). According to Redinugh nd Cmpell (1993), this repression of NR ctivity in the roots my e due to the ccumultion of NH + 4 or of other N compounds tht would e inhiiting the synthesis or the ctivity of this enzyme (Lewis et l., 1982). The enzyme NR is high moleculr weight flvoprotein formed of two identicl suunits, with three FAD groups, heme nd molydenum cofctor (Moco) (Chow et l., 27). Thus, the high Mo contents seen in the leves of the clones grown under the highest ppliction rtes of N (Ferreir, 213) my lso hve hd negtive effect on enzyme ctivity, even knowing tht the Mo deficiency reduces its ctivity (Smirnoff et l., 1984). Potssium lso plys n importnt role in ctivtion of the enzymes of N ssimiltion when NH + 4 is t toxic levels in plnt tissues (Souz nd Fernndes, 26). Another fctor tht my lso hve hd n effect, lthough to lesser degree, on greter NR ctivity t the lower rtes of N ppliction is tht the NR ctivity ws expressed s function of protein contents. Tht is ecuse, t lest for the clone VM1, there were lower protein contents in the plnts grown t the lower ppliction rtes of N (Fig 1) s result of the lower totl N contents in the lef (Fig 1), which mde for n increse in the rtio, thus contriuting to the greter vlue of its ctivity. Greter NR ctivity t lower ppliction rtes of N could indicte tht plnts with lower lef contents of totl N would 562

3 Tle 1. Person correltion coefficients (r) of N supply (rtes) nd totl N content in the lef with totl protein, nitrte reductse (NR) ctivity nd glutmine synthetse (GS) ctivity in euclyptus clones grown in nutrient solution. Protein NR GS Clone Rte of N Totl N Rte of N Totl N Rte of N Totl N r VM I Generl* * Person correltion coefficient (r) coidering the vlues of oth clones Clone VM1 Clone I = *x.29*x 2 R 2 =.88 = 8.52 y = 8,52 () 5 4 = *x+24.15**x.5 R 2 =.98 = *x+21.95*x.5 R 2 =.96 () Totl Protein (µg µl 1 ) n.s. Clone (p<.5) Rte (p<.5) Cl x R (p<.5) Totl N (g kg 1 ) Clone (p<.5) Rte (p<.5) Cl x R (p>.5) NR Activity (µmol NO 2 h 1 mg 1 of protein) = x.46*x.5 R 2 =.94 = x.2 x.5 R 2 = (c) Clone (p<.5).2 Rte (p<.5) Cl x R (p>.5) GS Activity (µmol GHA h 1 mg 1 of protein) =.18.4*x+.4*x 2 R 2 =.93 =.15 y.2 (d) Clone (p<.5).6 Rte (p<.5) Cl x R (p>.5) = *x+2.41*x.5 R 2 =.92 =5.95.2*x+22.18*x.5 R 2 =.96 (e) Totl Dry Mtter (g/plnt) Clone (p<.5) Rte (p<.5) Cl x R (p>.5) Rte of N (mmol L 1 of NH 4 NO 3 ) Fig 1. Totl protein (), totl N (), nitrte reductse (NR) ctivity (c) nd glutmine synthetse (GS) ctivity (d) in the leves, nd totl dry mtter (e) of young euclyptus clones under N rtes in nutrient solution., º, * nd ** indicte not significnt, significnt t 1, 5 nd 1 %, y the, t 5 % proility. Cl (clone) nd R (rte) 563

4 Totl Protein (µg µl 1 ) p<.1 () Clyey soil Sndy soil () Dry seson p<.1 Riny seson (c) p< NR Activity (µmol NO2 h1 mg1 of protein) GS Activity (µmol GHA h1 mg1 of protein) VM1 I144 VM1 I144 VM1 I144 Clone (d) (g) Dry Dry p<.5 Dry Seson Fig 2. Lef contents of totl protein compring euclyptus clones (), regio of different soils y ech smpling seson () or different seso y ech soil (c), lef nitrte reductse (NR) ctivity compring euclyptus clones (d), regio of different soils y ech smpling seson (e) or different seso y ech soil (f) nd lef glutmine synthetse (GS) ctivity compring euclyptus clones (g), regio of different soils y ech smpling seson (h) or different seso y ech soil (i). No significnt t 1 % y the (p>.1). Letters different in ech set of two colum re different t 5 % y Tukey s test. Brs t the top of colum represent stndrd error (SE). Riny p<.1 Riny Riny (e) (h) Clyey p<.5 Clyey p<.1 Clyey Soil Sndy p<.1 Sndy p<.1 Sndy (f) (i) exhiit greter efficiency of this enzyme, indicting competion in this condition. This rgument is strengthened upon compring the NR ctivity of the two clones with I144 eing tht which showed the lowest totl N contents in the lef nd, in contrst, greter ctivities of this enzyme nd greter dry mtter production (Fig 1, 1c, 1e). Tht wy, NR ctivity showed n inverse respoe oth to N in the solution (ppliction rtes) nd the totl contents of this nutrient in the lef (Tle 1). For GS ctivity of the clone VM1, greter vlues were lso oserved t lower N ppliction rtes, vlues decresing with n increse in the rtes, following the qudrtic model (Fig 1d).. This could e explined y greter supply of NH + 4 s sustrte for GS s coequence of greter reduction of N under these conditio (greter NR ctivity). Nevertheless, it should e noted tht NH + 4 in plnts is not formed only through reduction of NO 3 ut lso y other processes, such s lignin iosynthesis, ctolism of mino cids, rekdown of protei in senescent tissue, nd photorespirtion (Mrschner, 212), s well s y direct uptke y plnts. The mount of + NH 4 produced during decroxyltion of glycine for production of serine under photorespirtory conditio is up to ten times greter thn the mount produced during reduction of NO 3 (Le nd Blckwell, 1992). Low GS ctivity results from regultory effect tht cts under conditio of low supply of C nd, or, N to the plnt, keeping the enzyme in its inctive form, s n lterntive for coervtion of ATP (DeluFilho, 1994). Nitrte hd positive effect on GS ctivity on ruer leves (DeluFilho, 1994) nd on corn cultivrs (Purcino, 1992). Nevertheless, + the increse in concentrtion of NH 4 in the nutrient solution contriuted to repressing GS ctivity in the roots of the ruer tree (Lemos et l., 1999). The lef contents of NH + 4 of the euclyptus clones of the present study hd liner increse with the increse in N ppliction rtes (Ferreir, 213). Thus, with the increse in N ppliction rtes, the high lef contents of NH + 4 my hve negtively ffected GS ctivity, suggesting n inhiitory effect on it. For the clone I 144, there ws no difference in GS ctivity, s there ws likewise no difference for protein contents (Fig 1, 1d).. The GS enzyme ctlyzes the conversion of glutmte into glutmine through the use of NH + 4, ATP nd divlent ction (Miflin et l., 1981), thus promoting synthesis of the mino cid. In contrst, this clone generlly showed greter GS ctivities in reltion to the clone VM1, which ws not reflected in greter protein contents. It is noteworthy tht GS represents just 1 to 2 % of totl solule protei in org cple of ssimilting NH + 4 (Hungri et l., 1992). Thus, greter GS ctivities do not necessrily men greter protein 564

5 contents, s seen in comprison of the two clones, since there re innumerle other enzymes nd fctors involved in protein synthesis in plnts. As in NR ctivity, n inverse correltion ws oserved etween GS ctivity nd the N supplied in solution (r =.34) nd the totl N contents in the lef (.48) (Tle 1). Field experiment For clones grown in the field, greter lef content of totl protein ws oserved in clone VM1 (Fig 2), like tht oserved in the experiment in the greenhouse (Fig 1). Lower lef contents of totl protein were oserved under field conditio, in which there were older plnts (pproximtely 17 months of ge) in comprison to plnts grown in the greenhouse (Fig 1, 2). When compring plnts from regio of sndy soil nd clyey soil in ech period of ssessment (dry nd riny seson), lef protein contents did not differ (Fig 2), despite the clyey soil hving doule the totl N content (Ferreir, 213). It should e noted tht, y itself, the presence of greter N content in the soil will not lwys men greter uptke of the nutrient, nd protein synthesis y plnts, since these processes re dependent on vrious fctors, such s vilility of C, energy, enzyme ctivity, nd even climte conditio like temperture nd rin. In oth clyey soil nd sndy soil, the riny seson contriuted to greter contents of lef protein (Fig 2c).. Greter decomposition of orgnic mtter in the soil through greter microil ctivity s result of greter moisture in the soils in this seson from greter rinfll my hve fvored greter vilility nd uptke of N y plnts (Ferreir, 213), contriuting to greter protein synthesis. The NR ctivity seen in euclyptus clones grown in the field (men vlue of.9 µmol of NO 2 h 1 mg 1 of protein, Fig 2d, 2e, 2f) is coidered moderte (Cmpos, 29). The NR ctivity is drsticlly ffected y high cidity conditio (Towsend, 197), nd this my e n indiction of lower NR ctivities in euclyptus since this species, in generl, is ssocited with cid soils (GmRodrigues et l., 25), where, moreover, the nitrifiction process is reduced (Moreir nd Siqueir, 22). The clones did not differ in regrd to NR lef ctivity in the field (Fig 2d).. In this cse, NR ctivity ws not seitive to detect differences in N sttus mong the clones since they differed in totl N contents in the lef (Fig 1). However, this fct could e dvntgeous since it would llow the use of n indictor of N sttus for the species, regrdless of the clone ssessed. The difficulty in ppliction of enzymtic methods results from the fct tht vrition in the ctivity of the determined enzyme is ffected y fctors other thn simply the nutrient under study (Mrtinez et l., 1999). The NR ctivity ws greter in plnts grown in region of sndy soil in the riny seson in reltion to plnts in clyey soil, there eing no differences in the dry seson (Fig 2e), in contrst with the totl N content in the soil, which ws greter in the clyey soil (Ferreir, 213). This fct once more indictes, just s oserved in the greenhouse (Fig 1c), greter enzyme ctivity in plnts grown in environments of lower N vilility. Also leding to this conclusion, greter NR ctivity ws likewise oserved in smpling undertken in the dry seson for the plnts from oth soils (Fig 2f), in which, once more, the totl N contents of the soil were lower (Ferreir, 213). NR ctivity vried widely oth mong plnt species nd mong smpling periods, nd herceous species generlly exhiited greter ctivity thn woody species (Grnger et l., 1994). The NR ctivity vried ccording to the ge of the coffee plnt (Crelli et l., 199) nd there ws reduction in its ctivity, in oth roots nd leves, with incresed ge of pech plm (Bctris gsipes) (Oliveir et l., 25). Thus, NR ctivity ssessed in the second smpling (riny seson) my lso hve een reduced y greter ge of the plnts. The NR ctivity my exhiit sesonl fluctutio in respoe to internl nd environmentl fctors (Beevers nd Hgemn, 1969), s in the sence of ctivity of this enzyme seen in coffee leves long with drop in temperture (elow 12.5 ºC) recorded in the winter (DMtt et l., 1999). The NR ctivity is lso strongly ffected y wter vilility in the soil (Oliveir et l., 25). Csrino (29) oserved greter NR ctivity in tree species in rinier months nd suggested tht the reduction in enzyme ctivity in the dry seson is due to low vilility of NO 3 in solution since soil moisture is lower. Decrese in trpirtion cuses lower influx of NO 3, which my ffect NR ctivity (Plhk, 23). NR synthesis my e inhiited even with rief period of negtive wter lnce (Hsio, 1973). Reis et l. (27) oserved positive respoe of NR ctivity to N ppliction from Ferury to Mrch nd ttriuted this to greter rinfll in this period, since its ctivity my lso e regulted y soil moisture. Better soil moisture sttus, in ddition to other fctors, stimultes the protein phosphtse, which dephosphoryltes vrious residues of serine in the NR protein, promoting the ctivtion of NR (Tiz nd Zeiger, 24). Results suggested strong reltiohip etween reduction of NR ctivity nd of wter potentil in the leves, which my e relted to greter trpirtion in the period of greter light inteity (Oliveir et l., 25). Greter lef NR ctivity in coffee plnts in Jnury ws ttriuted to the greter light inteity which occurs in this seson since the dys re longer, incresing the photosynthetic rte of this species (Reis et l., 27), with prt of the energy production eing used to ctivte NR (Queiroz et l., 1993). Even with ll these coidertio in fvor of greter NR ctivities in periods of rin nd with greter light inteity, lef ctivity of this enzyme in the clones ws, on the contrry, higher in the dry seson, nd thus without known justifiction for such fct. Perhps the lower lef contents of protein seen in plnts in the dry seson (Fig 2c) rised the vlues of NR ctivity since the results were expressed sed on protein contents nd the redings of enzymtic ctivity were similr. Moreover, s oserved for NR ctivity, there were no differences for GS ctivity mong the clones (Fig 2g). The plnts from sndy soil, where there ws lower content of totl N in the soil nd in the plnt (Ferreir, 213), exhiited greter GS ctivity in the dry seson, there eing no differences in the riny seson (Fig 2h). According to Invers et l. (24), some studies show tht GS ctivity correltes positively with N vilility (Pregnll et l., 1987; Kremer et l., 1997), ut others report tht its ctivity does not follow the conditio of externl vilility of N nd is, ove ll, controlled y internl requirements (Finnemn nd Schjoerring, 1998; Thompson nd Vliel, 1999). Invers et l. (24) only found n increse in in vivo GS ctivity in Posidoni ocenic plnts fertilized in the period of gretest demnd for N (mximum lengthening of the lef). In trgenic Brssic npus L., the effect of gene expression of sprgine synthetse (AsnA, E.C ) of Escherichi coli on the growth of these plnts my e more pronounced under conditio limiting GS ctivity (Seiffert et l., 24). Upon compring the GS ctivity etween the two smpling periods, greter vlues re seen in the dry seson for oth the clyey soil nd sndy soil regio (Fig 2i). Thus, in generl wy, similr respoe is oserved etween the NR nd GS ctivities, in certin wy indicting the effect of one upon the nother in the ssimiltion pthwys of N y the plnts since the product of NR (NO 2 ) is converted into NH 4 + y the 565

6 ction of nitrite reductse (NiR) nd tht, in the finl nlysis, will e the sustrte for incorportion of N in glutmine y GS. Low concentrtio of GS were correlted with low cpcity for reduction of lef NO 3 (Stewrt et l., 199). In short, generlly nd coequently, the ctivity of these two enzymes ssessed exhiited similr tendencies in regrd to the effect of different conditio of N supply to euclyptus clones. Mterils nd Methods Description of study sites One experiment ws conducted in greenhouse nd nother in the field. In the first, the tril ws undertken in the Soil Deprtment of the Federl University of Viços, ViçosMG ( S, W), Brzil, over three months (July to Septemer 211). For the field experiment, commercil euclyptus res in the municiplities of Pompeu nd João Pinheiro (stte of Mi Geris, Brzil) were used, elonging to the compny Vllourec & Mnnesmnn Tues. The soils of the Pompeu region (18º53' S, 45º2' W) nd João Pinheiro region (17º3' S, 46º7' W) contined men vlues of 4.85 nd 4.79 of ph (in wter), 2.25 nd 1.14 dg kg 1 of orgnic mtter (WlkleyBlck), 5.55 nd 6.37 mg dm 3 of P, nd 28 nd 1 mg dm 3 of K (Mehlich 1), nd 74 nd 18 % cly (Ruiz, 25) in the 1 m depth lyer, respectively. The soil of the Pompeu region (clyey) contined.8 dg kg 1 of totl N (Kjeldhl method), while in the region of João Pinheiro, the soil hd.4 dg kg 1 of totl N in the lyer indicted. Pompeu nd João Pinheiro hve soils clssified, respectively, s Ltossolo VermelhoAmrelo nd Neossolo Qurtzrênico y the Brzilin Soil Clssifiction System (Emrp, 213). Tretments nd experimentl procedure The experiment conducted in the greenhouse coisted of 2 6 fctoril rrngement, with two euclyptus clones (VM 1 nd I144) nd six ppliction rtes of N (,.74, 2.93, 4.39, 5.85, nd 8 mmol L 1 of NH 4 NO 3 ), with five replictio, in rndomized lock design. The clone VM 1 is hyrid of E. urophyll x E. cmlduleis nd I144 is n E. urophyll. Seedlings in tues t n ge of round 5 dys were used for oth clones. The nutrient solution of Clrk (1975), dpted y Loctelli et l. (1984), ws used to mintin the NNH + 4 /NNO 3 rtio t 1, with doule the concentrtion of P (Cldeir et l., 1994). The nutrient solution ws kept permnently erted nd its ph ws djusted dily to 5.5 (±.5) with solutio (.1 mol L 1 ) of NOH or H 2 SO 4, nd it ws chnged weekly. The first month of crrying out this experiment represented the cclimtion phse of the seedlings in the nutrient solution, where the plnts remined in collective trys (11 L) nd the concentrtion of the solution ws grdully incresed y ech seven dys (25, 5, 75 nd 1 % of the originl concentrtion). With the pssing of this period, homogeneous plnts for height nd vigor were selected, nd two of these plnts were trplnted in ech pot (6 L), thus representing n experimentl unit, where they remined for two months with the ppliction of the tretments. At the end of the experimentl period, leves were collected from the middle third of ech plnt; they were weighed nd then wrpped in luminum foil nd plced in liquid N until eing stored in freezer (8 C) in the lortory for nlyses of protein nd enzymtic ctivity (NR nd GS). At this sme time, the plnts were hrvested (seprting them into roots, stem, rnches nd leves), wshed in deionized wter, nd dried in n ir circultion oven (6 C/72 h) for ssessment of dry mtter. The leves were then ground in Wiley mill for lter determintion of totl N contents. For the experiment in the field, fctoril rrngement with five replictes ws used in completely rndomized design, coisting of two regio (Pompeu clyey soil nd João Pinheiro sndy soil), two smpling periods (dry nd riny seson), nd two euclyptus clones (VM1 nd I144). Smpling from the dry seson ws crried out in Septemer 211 nd smpling from the riny seson in Ferury 212; oth were undertken t the end of ech seson. The euclyptus clones were pproximtely 17 months of ge in oth regio t the time of the first smpling (Septemer 211). The useful re of the plot ws composed of 3 plnts (five rows with six plnts ech) in 225 m 2 ccording to the plnt spcing of 3 x 2.5 m (1,333 plnts h 1 ). In Pompeu, fertiliztio of NPK coisted of 36 kg h 1 (1271, t plnting), 24 kg h 1 (2321, seven months fter plnting m..p.) nd 35 kg h 1 (2321, 18 m..p.). For the João Pinheiro plots, 3 kg h 1 (1271, t plnting), 25 kg h 1 (2323, seven m..p.) nd the sme quntity nd formul used in Pompeu t 18 m..p. were pplied. The res lso received 2.5 (Pompeu) nd 2. (João Pinheiro) t h 1 of lime,.8 t h 1 of gypsum, nd lef fertiliztion of 9 L h 1 of mmonium orte (1.22 kg h 1 of B, eril ppliction). For the Pompeu region, 4 kg h 1 of B in the form of ulexite (1 % B) ws lso pplied in the soil. The circumferences t rest height (CBH 1.3 m) of ll the trees in ech plot (3 plnts) were mesured, clculting the men vlue nd stndrd devition, nd choosing five representtive trees within this intervl. Of these, one tree ws used for lef smpling. Leves were collected in the mount of 6/plnt in the middle prt of the cnopy, from the middle to the tip of the rnches (only completely developed leves) from two to three rnches t different sides of the plnt, from 8:.m. to 12: noon. For the smpling of Ferury 212, nother tree ws chosen for collection of the leves in ech plot since there my hve een interference from the previous smpling (Septemer 211) ecuse the plnts hd mny dmged rnches. The procedure for storge of leves for nlysis of protein nd enzymtic ctivity nd for drying nd grinding for nlysis of totl N contents ws the sme used for mteril from the greenhouse. Lortory nlyses Totl N contents in the lefwere nlyzed y the Kjeldhl method (Bremner, 1996) fter minerliztion with sulfuric cid nd heting of the smples. For nlyses of totl protein nd enzymtic ctivities in vitro of NR (EC ) nd GS (EC ), extrcts were otined ccording to Cmri et l. (1989) nd with modifictio (1 mmol L 1 phenylmethylsulfonyl fluoridepmsf) nd.2 g of polyvinylpyrrolidonepvpp) in order to optimize the ctivity these enzymes in euclyptus leves. The crude extrcts were filtered in four lyers of guze nd centrifuged (15, g t 4 C for 15 min.), using the superntnt for the respective determintio. The quntifiction of totl protein ws performed ccording to Brdford (1976). For ssessment of NR ctivity (Rdin, 1974; Cmri et l., 1989) nd GS ctivity (Elliott, 1953) some modifictio (volume nd concentrtion of the rection medium) were used, descried in detil in Ferreir (213). The enzymtic ctivity of NR ws expressed in µmol of NO 2 h 1 mg 1 of protein nd GS ctivity ws expressed in µmol of GHA h 1 mg 1 of protein. 566

7 Throughout ll the procedures, the smples were kept in ice nd protected from light up to the time of redings. Sttisticl nlyses The results were sujected to nlysis of vrince (), nd when the effect of the sources of vrition nd, or, interction etween them in the respoe vriles ws significnt (p.5), regression equtio were fitted (greenhouse experiment). Correltion nlyses were lso mde (Person coefficient r) of the protein contents nd NR nd GS ctivity, with the N supply (ppliction rtes) nd the lef content of totl N. To verify the effects of the tretments on the vriles in the field experiment, the ws dopted with 1 % significnce (p.1) nd me were compred y Tukey s test. The SAS (24) sttisticl progrm ws used for tht purpose, nd the Sigmplot softwre for creting figures. Conclusion In the greenhouse, the clone I144 took up less N, inducing less protein synthesis; however, it reduced nd ssimilted more this nutrient (greter ctivities of the enzymes NR nd GS), which ws converted into greter dry mtter production compred to VM1; The NR nd GS ctivities were negtively ffected y greter supply of N, proly through n inhiitory effect oth from NO 3 (sustrte of NR) nd from NH + 4 (sustrte of GS) t high concentrtio; Both in the greenhouse nd in the field, the gretest NR nd GS ctivities were ssocited with lower vilility of N in the growing medium of the plnts. The ctivity of these enzymes lso exhiited negtive correltion with the totl N content in the lef of the euclyptus clones grown in the greenhouse. Acknowledgments This reserch pper is prt of the PhD thesis of the first uthor nd it ws finncilly supported y the Ntionl Council for Scientific nd Technologicl Development CNPq (project funding no /2116 nd PhD Fellowship). 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