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Effect of Mixed Amino Acids on Crop Growth Xing-Qun Liu 1 nd Kyu-Seung Lee 2* 1 School of Agriculture nd Food Science, Zhejing Agriculture nd Forestry University, Hngzhou, 2 Deprtment of Bio-Environmentl Chemistry, College of Agriculture nd Life Sciences, Chungnm Ntionl Univeristy, Tejon, 1 P.R. Chin 2 Kore 7 1. Introduction 1.1 Nitrogen uptke nd ssimiltion Among the minerl nutrient elements, nitrogen is kind of mcronutrient. Most plnt species re le to sor nd ssimilte nitrte ( ), mmonium (NH 4 +), ure nd mino cids s nitrogen sources. Most soils do not hve sufficient N in ville form to support desired production levels. Therefore, ddition of N from fertilizer is typiclly needed to mximize crop yields. Mny kinds of N fertilizers re used which contin vrying forms of N such s N, NH 4 + N nd ure. However, form of nitrogen is the predominnt form of N sored y plnts, regrdless of the source of pplied N (Breteler nd Luczk, 1982). This preference is due to severl utotrophic soil cteri, which rpidly oxidize NH 4 + to NO 2, nd then to in wrm, well erted soils. Even though is the most ville form of N to plnts, it cn e more redily lost from the root zone ecuse it is very moile nd esy to lech. This economiclly nd environmentlly undesirle process perpetutes lrge mount of the uncertinty ssocited with N fertilizer mngement (Pessrkli, 2002). In the soil solution, nitrte is crried towrds the root y ulk flow nd is sored into the epiderml nd corticl symplsm. Within the root symplsm, nitrte hs four ftes: (1) reduced to nitrite y the cytoplsmic enzyme nitrte reductse; (2) efflux ck cross the plsm memrne to the poplsm; (3) influx nd stored in the vcuole; or (4) trnsported to the xylem for long distnce trnsloction to the leves (Andrews, 1986; Ashley et l., 1975; Blck et l., 2002; Cooper nd Chrkson, 1989). Trnslocted from the xylem, nitrte enters the lef poplsm to rech lef mesophyll cells, where nitrte is gin sored nd either reduced to nitrite or stored in the vcuole. Nitrte trnslocted from the roots through the xylem is sored y mesophyll cell vi one of the nitrte proton symporters into the cytoplsm, reduced to nitrite y nitrte reductse (NR) in the cytoplsm, nd then reduced to mmonium y nitrite reductse (NiR) * Corresponding uthor

120 Agriculturl Science in the chloroplst, which is then incorported into mino cids y the glutmine synthetse glutmine 2 oxoglutrte midotrnsferse (GS/GOGAT) enzyme system, giving rise to glutmine (Gln) nd ultimtely other mino cids nd their metolites (Fig. 1; Tiz nd Zeiger, 2002). Therefore, NR, NiR nd GS constitute the first three enzymes of the nitrte ssimiltory pthwy. The NR ctivity is the limiting step of N conversion to mino cid synthesis (Cmpell, 1999). In most plnt species only proportion of the sored nitrte is ssimilted in the root, the reminder eing trnsported upwrds through the xylem for ssimiltion in the shoot where it is reduced nd incorported into mino cids (Forde, 2000). Fig. 1. The min process of nitrte ssimiltion. 1.2 Avilility of mino cids Trditionl models of nutrient cycling ssume tht orgnic N mtter must e decomposed y soil microorgnisms to relese inorgnic N, efore tht N ecomes ville for plnt uptke. But, there re growing evidences tht plnt cn sor orgnic N directly. Erlier studies of nutrient sorption demonstrted tht higher plnts could tke up mino cids (Virtnen nd Linkol, 1946). More recent studies of mino cid sorption hve further focused on the chrcteristics of the crrier systems nd other mechnistic spects of the uptke process nd wide rry of mino cid trnsporters hs een identified in severl different plnts species (Frommer et l., 1993; Montmt et l., 1999; Neelm et l., 1999). In the moist tundr of the rctic, inorgnic N supplied to plnts y minerliztion is not sufficient to meet their requirement of N due to low tempertures nd noxic soils. But these soils hve lrge stocks of wter extrctle free mino cids (Atkin, 1996). The studies of nitrogen cycling in rtic tundr hve indicted tht some non mycorrhizl plnt species, such s Eriophorum vgintum, could sor mino cids rpidly, ccounting for t lest 60% of totl the nitrogen sored (Chpin et l., 1993). Ectomycorrhizl species hve higher mino cid uptke thn non mycorrhizl species (Kiellnd, 1994). Amino cid uptke ws the generl ility found widely in plnts from orel forest (Näsholm et l., 1998; Persson nd Näsholm, 2001). 1.3 Amino cids nd nitrte uptke Plnts cn store high levels of nitrte, or they cn trnslocte it from tissue to tissue without deleterious effect. However, hzrdous effects my occur when livestocks nd humns

Effect of Mixed Amino Acids on Crop Growth 121 consume plnt mteril with rich nitrte, they my suffer from methemogloinemi or crcinom y converting nitrte to nitrite of nitrosmines. Some countries limit the nitrte content in plnt mteril sold for humn consumption. Severl uthors reported tht free mino cids could down regulte nitrte uptke nd nitrte content in plnt. It ws found tht exogenously supplied mino cids nd mides could decrese the uptke of nitrte y soyen (Muller nd Tourine, 1992); whet (Rodgers nd Brneix, 1993); mize (Ivshikin nd Sokolov, 1997; Pdgett nd Leonrd, 1993, 1996; Sivsnkr et l., 1997); rley (Aslm et l., 2001) Tle 1. Plnts pper to hve multiple mechnisms for regulting nitrte uptke in ddition to mino cids or N- sttus (Pdgett nd Leonrd, 1993). Work y Breteler nd Arnozis (1985) determined tht pretretment of dwrf en roots with mny different individul mino cids inhiited nitrte uptke to vrying degrees dependent upon prior exposure of the plnts to nitrogen nd the specific mino cid tretment. No significnt effect of mino cids on nitrte trnsport ws detected when oth nd mino cids were present in the thing solution, nd no correltion emerged etween inhiition of nitrte uptke nd inhiition of nitrte reductse reltive to specific mino cids. A more detiled study, presented y Muller nd Tourine (1992), demonstrted inhiition of uptke y 50% or greter y lnine, glutmine, sprgines, rginine, β lnine nd serine when soyen seedlings were pretreted for 18 h prior to exposure to. The mechnisms of inhiition y rginine nd lnine ppered to differ, however. Arginine stopped uptke immeditely upon introduction to the uptke solution, kineticlly similr to NH 4 + inhiition. The uthors suggested tht this my e the result of non-metolic response such s ltertion of memrne potentils. Inhiition y lnine ws slower to develop, suggesting metolic component to the regultion rther thn physicl or chemicl interference. Plnt mterils Soyen Whet Amino cid NO tretment 3 supplied uptke Remrks (mm) (%) 10 0.5 5 85 14 mino cids 100 0.5 40 120 (Muller, 1992) 1.0 0.3 50 105 3 dys N strvtion 1.0 0.3 89 106 Non strvtion (Rodger, 1993) Mize root 15 5.0 84 (Pdgett, 1993) 1.0 0.1 40 50 Brley root (Aslm, 2001) 1.0 10 70 Tle 1. Effect of mino cid on uptke in severl plnts The N sttus of the plnts could lso ffect the inhiitory effect of mino cids on nitrte uptke. Rodger nd Brneix (1993) hd supplied mino cids exogenously to N strved or non strved whet seedlings. Exogenously supplied mino cids nd mides hd no effect on the whet seedlings under well nourishment. However, some of the mino cids nd mides supplied seedlings strved of N for 3 dys inhiited up to 50% of the nitrte uptke rte.

122 Agriculturl Science Aslm et l. (2001) hd conducted study on differentil effect of mino cids (Glu, Asp, Gln nd Asn) on nitrte uptke nd reduction systems in rley roots. Similr results were oserved i.e. 50 60% inhiition in the uptke when the roots were supplied with 0.1 mm. However, no inhiition occurred t 10 mm. In contrst, Kim (2002) hd conducted study on effect of mixed mino cids on nitrte uptke in rice, pe, cucumer nd red pepper. The result showed tht the effect of mixed mino cids (MAA) on nitrte uptke in nutrient solution ws unffected in low MAA concentrtion nd ccelerted in high MAA concentrtion. The results indicted tht externl MAA could regulte nitrte uptke. 1.4 Amino cids nd enzyme regultion Nitrte reductse (NR) is sustrte inducile enzyme involved in the nitrte ssimiltion in higher plnt, nd the enzyme occupying control point in the pthwy of nitrte ssimiltion. Activity of the NR fluctutes widely in response to mny environmentl or physiologicl fctors, such s the presence of NH 4 + or mino cids in the growth medium. In studies of the possile regultion of NR ctivity y mino cids in higher plnts, the results hve often een conflicting. For exmple, Rdin (1975, 1977) hd shown tht the reduction of nitrte to nitrite in cotton roots is inhiited y specific mino cids. On the other hnd, Oks (1977) hd found using n in vitro ssy those mino cids results in enhnced levels of NR nd lso cuse only minor inhiitions in oth intct nd excised corn roots. Aslm et l. (2001) reported tht the mino cids prtilly inhiited (35%) the induction of nitrte reductse ctivity (NRA) in rley roots supplied with 0.1 mm, ut no inhiition occurred t 10 mm. He hs concluded tht the inhiition of induction of NRA y the mino cids is result of the lck of sustrte vilility due to inhiition of the uptke system t low supply. It hs een suggested tht glutmte inhiited NRA in roots, ut not in shoots (Ivshikin nd Sokolov, 1997). This inhiition seems e dependent on plnt mterils, ge of plnts, growth conditions, nitrte concentrtion, mino cid kinds, mino cids concentrtion nd other fctors. Effect of mino cids on the regultion of NR gene expression hs een studied t the moleculr level. Deng et l. (1991) reported tht the ddition of 5 mm glutmine to the nutrient solution of tocco plnts grown in 1 mm resulted in pronounced inhiition of NR mrna ccumultion in the roots. Vincentz et l. (1993) showed, under low light conditions (limiting photo synthetic conditions), the supply of glutmine or glutmte led to drop in the level of NR mrna, while glutmine nd glutmte were less efficient t decresing NiR mrna thn NR mrna levels. Li et l. (1995) lso demonstrted tht 5 mm glutmine dded together with resulted in reduced levels of NR mrna in oth root nd shoot of mize. Sivsnkr et l. (1997) oserved tht Gln nd sprgine (Asn) inhiited the induction of NR ctivity (NRA) in corn roots t n externl supply of 250 M nd 5 mm. They concluded tht inhiition ws not the result of ltered uptke, nd tissue nitrte ccumultion ws reduced t 250 M externl nitrte in the presence of 1mM Asn, ut not t 5mM Asn. In the studies of the possile regultion of NR ctivity y multiple mino cids in higher plnts, the conclusions re gin contrdictory. The inhiition on NR ctivity y glycine, sprgines, nd glutmine could e prtilly or wholly prevented y the presence of other

Effect of Mixed Amino Acids on Crop Growth 123 mino cids during the induction (Rdin, 1977). However when glutmine nd sprgine were included long with the corn mino cid mixture, the inhiition on the induction of NR in corn roots ws more severe (Oks et l., 1977). Chen nd Go (2002) hve pplied different mixture of glycine, isoleucine nd proline replcing nitrte of solution prtilly (20%) to Chinese cge nd lettuce in hydroponics. Amino cids enhnced the NR ctivity in Chinese cge, while it decresed in lettuce. 1.5 Influence on yield nd N ssimiltion L tryptophn, considered s physiologicl precursor of uxins in higher plnts, ws pplied to soil to evlute its influence on yield of severl crops. Kuchrski nd Nowk (1994) found tht L tryptophn did not ffect the yield of ove ground prt nd roots of field en. On the other hnd, positive effects on corn nd cge growth were reported (Srwr nd Frnkenerger, 1994; Chen et l., 1997). Amino cids were used to prtilly replce or folir spry in mny plnts. In most cse, the ppliction of mino cids led to decresed nitrte content nd incresed totl nitrogen content in lettuce, Chinese cge, onion, pkchoi or other lefy crops (Gunes et l., 1994, 1996; Chen nd Go, 2002; Wng et l., 2004). Some uthors suggested tht plnts proly preferred mino cids s sources of reduced nitrogen, nd nitrte uptke ws inhiited y mino cids. In fct, there ws little evidence or dt to support the conclusions. It hs not een distinguished tht incresed totl nitrogen cme of nitrte or mino cids. 1.6 Ojectives Regultion of induction of the uptke nd reduction systems y nitrogen metolites hs een ttriuted to feed ck inhiition (Pl ove Blng, 2002). It ws found tht nitrte uptke rte follows iphsic reltionship with externl nitrte concentrtion, suggesting the existence of t lest two different uptke systems (Cerezo et l., 2000). At high externl nitrte concentrtion (> 0.5 mm), low ffinity trnsport system (LATS), which shows liner kinetics, contriutes significntly to the uptke rte nd ppers to e constitutively expressed nd essentilly unregulted. At low externl concentrtions (< 0.5 mm), two high ffinity trnsport systems (HATS) operte, one of these eing constitutive wheres the other is induced y nitrte. The HATS for nitrte uptke is sensitive to metolic inhiitors nd ppers to e n ctive trnsport system (Dniel Vedele et l., 1998). Although the regultory effect of mino cids on nitrte uptke nd NR hs een exmined extensively, its effect on GS hs not een exmined in detil. Otherwise, lot of mino cids were investigted out their regultion on nitrte uptke nd ssimiltion, ut very little informtion hs een reported out effect of mixed mino cids (MAA). In fct, there re two possile resons for the increse of totl N content in the plnts: preference for mino cids s sources of reduced nitrogen nd regultion of mino cids on inorgnic nitrogen uptke nd ssimiltion. The solution experiments were crried out to investigte the regultion of the induction of uptke, NRA, NiRA nd GSA in rdish nd red pepper y pplying mixed mino

124 Agriculturl Science cids (MAA) under the conventionl fertiliztion. These two plnts were selected ecuse rdish is preferred crop nd red pepper is NH 4 + preferred crop. The mino cids used in this experiment were lnine (Al), β lnine (β Al), sprtic cid (Asp), sprgine (Asn), glutmic cid (Glu), glutmine (Gln) nd glycine (Gly). These mino cids were selected for the resons include: (1) their structurl role in proteins, (2) significnt effect on uptke which ws found in mny works, nd (3) considerle mounts in plnt phloem nd xylem (Cputo nd Brneix, 1997; Lohus et l., 1997; Peeters nd Vn Lere, 1994; Winter et l., 1992). In the frme of the studies on the effect of the mixed mino cids (MAA) on nitrte uptke nd ssimiltion, the pot experiments were focused on the role of MAA in process of uptke nd ssimiltion. In order to distinguish the origin of N in rdish, 15 N leled nitrte ws used. In order to develop n pproch for more efficient N fertilizer use nd to prevent environmentl pollution due to nitrte leching, the im of the study presented here, is to investigte the effect of mino cid fertilizer (AAF) on nitrte removl in high nitrte soils. 2. Hydroponic experiment of rdish 2.1 Mterils nd method Seeds of Ilsn rdish (Rphnus stivus) soked for 6 h llowed to germinte on pper towels were soked in wter in the drk. After 5 dys the seedlings were trnsferred to 50 ml plstic tues contining 10 ml inorgnic nutrient solution. The nutrient solution ws renewed every dy. The composition of the inorgnic nutrient solution is given in Tle 2. Iron (Fe-EDTA), oron (H 3 BO 3 ), mngnese (MnCl 2 4H 2 O), zinc (ZnSO 4 7H 2 O), copper (CuSO 4 5H 2 O) nd molydenum (H 2 MoO 4 H 2 O) were supplied to ll tretments t rtes of 40, 460, 90, 7.7, 3.2 nd 0.1 M, respectively. Seedlings were grown in growth chmer mintined t 25 C, 70 80% reltive humidity, with 14 h light/10 h drk cycle nd light intensity of 300 mol m 2 s 1. Chemicls K + NO 3 C 2+ H 2 PO 4 Mg 2+ SO 4 2 K 1.25 1.25 C( ) 2 2.50 1.25 KH 2 PO 4 0.25 0.25 MgSO 4 0.50 0.50 Totl 1.50 3.75 1.25 0.25 0.50 0.50 Tle 2. The min compositions of the nutrient solution for hydroponic experiment (mm) The mixed mino cids (MAA) solution contined 7 equl concentrtions of mino cids were s follows: lnine (Al), β lnine (β Al), sprtic cid (Asp), sprgine (Asn), glutmic cid (Glu), glutmine (Gln) nd glycine (Gly). After 10 dys, rdish seedlings were plced in 10 ml inorgnic nutrient solution contining 5.0 mm nd 0, 0.3 or 3.0 mm MAA, s indicted in Tle 3. The ph of the nutrient solutions were mintined etween 6.0 6.1 y dding 1.0 M KOH ppropritely. The nutrient solutions were renewed t 4, 8 nd 16 h, respectively. The choice of the levels of MAA nd renewed time of the nutrient solutions were ccording to the study of Kim (2002)

Effect of Mixed Amino Acids on Crop Growth 125 K + Al β Al Asp Asn Glu Gln Gly A0 5.25 5.0 A1 6.78 5.0 0.3 0.3 0.3 0.3 0.3 0.3 0.3 A2 13.10 5.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 Tle 3. The compositions of the tretment solutions for rdish in hydroponic experiment (mm) Plnts were hrvested 24 h fter tretment nd seprted into roots nd shoots for enzymes ssy nd N content nlysis. Net uptke rtes were determined y mount of disppered from the initilly treted solution. 2.2 Results nd discussion 2.2.1 Effect on uptke The MAA tretments showed different effect on nitrte uptke depending on the concentrtions (Fig. 2). The uptke in tretment A1 ws similr to tht of A0 fter 8 h exposure to. However, exposure for longer hours (16 or 24 h) to 0.3 mm MAA inhiited the uptke y 38% compred with A0. In contrst, the highest uptke ws found in tretment A2 tht showed 305% higher thn A0. - uptke ( mol g - FW) 300 A0 A1 A2 200 100 0 0 5 10 15 20 25 Time (h) Fig. 2. Effect of mixed mino cids on the nitrte uptke in rdish supplied with 5.0 mm. Vlues re mens ± SD (n=5). Severl uthors reported tht free mino cids could down regulte uptke. It ws found tht exogenously supplied mino cids nd mides could decrese the uptke of y soyen (Muller nd Tourine, 1992); whet (Rodgers nd Brneix, 1993); mize (Ivshikin nd Sokolov, 1997; Pdgett nd Leonrd, 1996; Sivsnkr et l., 1997); rley (Aslm et l., 2001). In this experiment, the effectiveness of the MAA tretments on uptke ws similr to ove references t low MAA tretment rte (0.3 mm MAA, Fig. 2). However, contrry result ws found t high MAA tretment rte (3.0 mm MAA, Fig. 2), in

126 Agriculturl Science which uptke ws 4 fold higher thn the control. This result ws similr to rice, pe, cucumer nd red pepper, which were treted with 5.0 mm MAA (Kim, 2002). The effect on nitrte uptke seems to respond to kinds nd concentrtion of mino cids. Muller nd Tourine (1992) hd exmined the effect of 14 different mino cids on nitrte uptke in soyen seedlings supplied with 0.5 mm nitrte. After 10 mm single mino cid pretretment, out hlf of the tested mino cids hd sustntil inhiitory effect on nitrte uptke, minly Al, Glu (lmost 100% inhiition), Asn nd Arg (out 80%), nd Asp, Al, Scr, nd Gln (from 70% to 48%). However, when supplied t 100 mm mino cid to the tip cut cotyledons, only eight of fourteen mino cids hd inhiitory effect, nd four mino cids hd enhnced nitrte uptke. 2.2.2 Effect on nd NO 2 ccumultion The ppliction of MAA incresed the concentrtions oth in shoots nd in roots regrdless of ppliction rtes (Tle 4), resulting in the highest concentrtion in A1 nd the lowest concentrtion in A0. The high concentrtion of in A2 ws ttriuted to the high NR ctivity (Fig. 3). Although A1 tretment showed the lowest uptke of (Fig. 2), the highest concentrtion of ws found y the reson of tht low NR ctivity in A1 (Fig. 3) led to locking of the reduction of to NO 2. With respect to the NO 2 vlues (Tle 4), in our experiments, the highest NO 2 concentrtions in oth shoots nd roots were found in the A2. In shoots, the lowest NO 2 concentrtion ws found in A1 nd the lowest in A0 in roots. NO 3 ( mol g 1 ) NO 2 (nmol g 1 ) Shoot Root Shoot Root A0 62.47 ± 4.06 16.30 ±1.88 6.76 ± 0.62 11.43 ± 1.67 c A1 67.73 ± 7.49 22.99 ±2.23 3.77 ± 0.34 c 17.14 ± 2.10 A2 63.37 ± 3.58 17.41 ±1.92 29.70 ± 2.78 30.39 ± 4.13 Dt re mens ± SD (n=5). Anlysis of vrince (ANOVA) ws employed followed y Duncn's new multi rnge test. Vlues with similr superscripts re not significntly different (P>0.05). Tle 4. Effect of mixed mino cids on nd NO 2 concentrtion in fresh weight of rdish t 24 h fter tretment Although mny uthors gree tht mino cid cn negtively regulte nitrte content in higher plnts (Chen nd Go, 2002; Gunes et l., 1994, 1996; Wng et l., 2004), the results in the present experiment do not support this interprettion. Both in shoots nd in roots, the MAA used in this study led to little increse of concentrtions (Tle 4). The contrdiction my reside in tretment method nd tretment period of mino cids. It ws demonstrted in other studies tht mino cid pretretment decresed ccumultion slightly, ut Gln nd Asn incresed the concentrtion in rley roots when they were used together with nitrte (Aslm et l., 2001). The reson of difference etween this experiment nd others is tht content of shoots includes portion of in xylem sp. Concentrtions of in xylem sp cn e quite high, especilly in plnts tht trnsport most of the tken up to the shoot for reduction (e.g., mize 10.5 mm, Oks, 1986; rley 27 to 34 mm, Lews et l., 1982).

Effect of Mixed Amino Acids on Crop Growth 127 As interim product of ssimiltion procedure, the concentrtion of NO 2 depended on the reduction rte of nitrte nd nitrite. The highest concentrtion of NO 2 found in A2 (Tle 4) ws due to high NR ctivity (Fig. 3), nd the lowest concentrtion of NO 2 in shoots in A1 (Tle 4) ws due to low NR ctivity (Fig. 3) too. However, low NiR ctivity (Fig. 4) led to locking of the reduction of NO 2 to NH 4 + in roots in A1, so tht concentrtion of NO 2 showed higher thn A0 (Tle 4). 2.2.3 Effect on NRA, NiRA nd GSA For ssimiltion, is reduced to NO 2 y ctlysis of NR. In this experiment, low concentrtion nd high concentrtion of MAA tretments led to different effects on NR ctivity (Fig. 3). Both in the shoots nd in the roots NR ctivities were inhiited slightly in A1. Significnt increses of NR ctivities were found in A2 tretment, with 75% in shoots nd 340% in roots respectively, reltive to A0. 1.2 g -1 FW h -1 ) 1.0 0.8 A0 A1 A2 NRA ( mol NO 2-0.6 0.4 0.2 0.0 Shoot Root Fig. 3. Effect of mixed mino cids on nitrte reductse ctivity in rdish t 24 h fter tretment. Vlues re mens ± SD (n=5). There re contrdictory results for the possile regultion of NR ctivity y mino cids for higher plnts. For exmple, Rdin (1975, 1977) hs shown tht the reduction of to NO 2 in cotton roots is inhiited y specific mino cids. On the other hnd, Oks et l. (1979) hve found tht mino cids inhiited minor levels of NR in oth intct nd excised corn roots using n in vitro ssy. Aslm et l., (2001) reported tht the mino cids prtilly inhiited the increse of NR ctivity in rley roots where most uptke ws fcilitted vi high ffinity trnsport system (HATS) ut hd little effect where low ffinity trnsport system (LATS) is opertive. It hs een suggested tht glutmte inhiited NR ctivity in roots, ut no inhiition in shoots (Ivshikin nd Sokolov, 1997). Sivsnkr et l. (1997) oserved tht Gln nd sprgine (Asn) inhiited the induction of NR ctivity in corn roots t oth 250 M nd 5 mm of externl supply. They concluded tht inhiition ws not the result of ltered uptke, nd tissue nitrte ccumultion ws reduced t 250 M externl nitrte in the presence of 1 mm Asn, ut not t 5 mm Asn.

128 Agriculturl Science In the studies of the possile regultion of NR ctivity y multiple mino cids in higher plnts, the conclusions re lso contrdictory. The inhiition on NR ctivity y glycine, sprgines, nd glutmine could e prtilly or wholly prevented y the presence of other mino cids during the induction (Rdin, 1977). However when glutmine nd sprgines were included long with the corn mino cid mixture, the inhiition on the induction of NR in corn roots ws more severe (Oks et l., 1979). Chen nd Go (2002) hve pplied different mixture of glycine, isoleucine nd proline to Chinese cge nd lettuce in hydroponic experiment. They found the mino cids tretment enhnced NR ctivity in Chinese cge, while decresing it slightly in lettuce. In this experiment, t 5.0 mm which is fcilitted y LATS, the presence of 0.3 mm MAA prtilly inhiited NR ctivity, s oserved in other works, wheres the 3.0 mm MAA incresed the NR ctivity more thn 4 times (Fig. 3). In ddition, the very high NO 2 content ws found in A2 (Tle 4). These results suggest tht high concentrtion MAA cn increse uptke y enhncing NR ctivity in rdish, especilly in roots. The next step in ssimiltion is the conversion of NO 2 to NH 4 + y the ction of NiR. Both enzymes, NR nd NiR, re induced y the sme fctors, nd therefore the response of NiR to the MAA tretments resemled tht of NR in roots, ut ws little different with tht of the NR in shoots (Fig. 3 nd Fig. 4). NiR ctivities in shoots nd roots in A1 were inhiited y 17% nd 52% respectively in reltion to A0. In A2, NiR ctivity ws inhiited y 15% in shoots nd enhnced 8 times in roots. In the present study, the decrese of NiR ctivity in shoots in A2 might e ttriuted to the low concentrtion of mino cids in shoots, too. The increse of NiR ctivity in roots in A2 ws due to the sme reson with NR. NiRA ( mol NO 2 - g - FW h -1 ) 0.30 0.25 0.20 0.15 0.10 0.05 0.00 Shoot A0 A1 A2 c Root Fig. 4. Effect of mixed mino cids on nitrite reductse ctivity in rdish t 24 h fter tretment. Vlues re mens ± SD (n=5). The principl NH 4 + pthwy is the glutmine synthetse (GS)/glutmte synthse (GOGAT) cycle. The ehvior of GS ctivities in shoots ws not ffected y MAA tretments (Fig. 5). However differences were found in roots etween tretments, showing 22% inhiition in A1 nd 17% increse in A2 in reltion to A0.

Effect of Mixed Amino Acids on Crop Growth 129 The NH 4 + originting in the plnt from reduction is incorported into n orgnic form primrily y the enzyme GS. In the present experiment, GS ctivity ws inhiited y 0.3 mm MAA tretment in rdish roots, wheres 3.0 mm of MAA tretment enhnced the ctivity (Fig. 5). It is lso striking tht effect of MAA on ssimiltion in the roots ws higher thn in the shoots, presumly ws more ville nd the MAA content ws higher in the roots. The results of the present experiment clerly indicted tht uptke nd ssimiltion ws regulted y MAA in rdish, especilly t high concentrtion of MAA tretment. In conclusion, the ppliction of high MAA rtes (principlly A2) could e the direct cuse of incresed ctivities of the three enzymes (NR, NiR nd GS) of the ssimiltory pthwy nd the uptke ws enhnced when supplied with LATS rnge of. GSA ( mol C 5 H 10 N 2 O 4 g -1 FW h -1 ) 50 40 30 20 10 0 Shoot A0 A1 A2 c Root Fig. 5. Effect of mixed mino cids on glutmine synthetse ctivity in rdish t 24 h fter tretment. Vlues re mens ± SD (n=5). 3. Hydroponic experiment of red pepper 3.1 Mterils nd methods Seeds of Chongok red pepper (Cpsicum nnuum) were sown in Ferury 2005. The seedlings were grown in individul pots filled with commercilized rtificil soil in n experimentl greenhouse for 35 dys nd then trnsferred to 50 ml plstic tues contining 20 ml inorgnic nutrient solution. The nutrient solution ws renewed every dy. The composition of the inorgnic nutrient solution nd the culturl condition were the sme with hydroponic experiment of rdish. The mixed mino cids (MAA) solution ws the sme with tht used in hydroponic experiment of rdish which contined 7 equl concentrtions of mino cids. At 7 dys fter trnsferring, red pepper seedlings were plced in inorgnic nutrient solution contining 1.0 mm nd 0, 0.3 or 3.0 mm MAA, s indicted in Tle 5. The ph of the nutrient solutions were mintined etween 6.0 6.1 y dding 1.0 M KOH ppropritely. The nutrient solutions were renewed t 4, 8, nd 16 h, respectively.

130 Agriculturl Science K + Al β Al Asp Asn Glu Gln Gly A0 10.25 10.0 A1 11.78 10.0 0.3 0.3 0.3 0.3 0.3 0.3 0.3 A2 18.10 10.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 Tle 5. The compositions of the tretment solutions for red pepper in hydroponic experiment (mm) Plnts were hrvested 24 h fter tretment nd seprted into roots nd leves for enzymes ssy nd N content nlysis. Net uptke rtes were determined y mount of disppered from the initilly treted solution. 3.2 Results nd discussion 3.2.1 Effect on uptke The MAA tretments showed different effect on nitrte uptke depending on the concentrtions (Fig. 6). Appliction of MAA t oth 0.3 mm nd 3.0 mm concentrtions incresed uptke in red pepper (P < 0.001) nd the highest uptke ws found in tretment A2 showing 7 fold increses over A0. - uptke ( mol g - FW) 100 80 60 40 20 0 A0 A1 A2 0 5 10 15 20 25 Time (h) Fig. 6. Effect of mixed mino cids on the nitrte uptke in red pepper supplied with 10.0 mm. Vlues re mens ± SD (n=5). 3.2.2 Effect on nd NO 2 ccumultion The highest concentrtion oth in the roots nd leves were found in tretment A0 (Tle 6), with respect to the lowest content found in tretment A1 in the leves (P < 0.05) nd A2 in the roots (P < 0.01). With respect to the NO 2 vlues (Tle 6), in this experiment, the highest NO 2 concentrtions in roots were found in the A2 nd the lowest in A0 (P < 0.001). In leves, the lowest NO 2 concentrtion ws found in A2 nd the lowest in A1 (P > 0.05).

Effect of Mixed Amino Acids on Crop Growth 131 NO 2 Lef Root Lef Root A0 9.12±0.58 6.20±0.23 0.036±0.003 0.596±0.032 c A1 7.54±0.34 3.99±0.36 0.046±0.006 1.164±0.046 A2 8.31±0.43 2.66±0.19 c 0.024±0.004 c 2.371±0.085 Vlues re mens ± SD (n=5). Anlysis of vrince (ANOVA) ws employed followed y Duncn's new multi rnge test. Vlues with similr superscripts re not significntly different (P>0.05). Tle 6. Effect of mixed mino cids on nd NO 2 concentrtion in fresh weight of red pepper t 24 h fter tretment ( mol g 1 ) 3.2.3 Effect on NRA, NiRA nd GSA For ssimiltion, is reduced to NO 2 y ctlysis of NR. In this experiment, MAA tretments led to different effects on NR ctivity in leves nd in roots (Fig. 7). In the roots, tretment A1 nd tretment A2 showed increses of 35% nd 212% respectively in reltion to A0 (P < 0.01). In contrst, NR ctivities were inhiited slightly in leves y MAA tretments, showing 8.2% in A1 nd 10.5% in A2, respectively (P > 0.05). The response of NiR to the MAA tretments resemled tht of NR in roots, ut ws different with tht of the NR in leves (Fig. 8). NiR ctivities in leves nd roots in A1 were incresed y 18% nd 60% respectively in reltion to A0 (leves: P < 0.05; roots: P < 0.01). In A2, NiR ctivities were the sme with A0 in leves nd enhnced 138% in roots (leves: P > 0.05; roots: P < 0.01). g -1 FW h -1 ) NRA ( mol NO 2-0.5 0.4 0.3 0.2 0.1 0.0 A0 A1 A2 Lef c Root Fig. 7. Effect of mixed mino cids on nitrte reductse ctivity in red pepper t 24 h fter tretment. Vlues re mens ± SD (n=5).

132 Agriculturl Science NiRA ( mol NO 2 - g - FW h -1 ) 0.6 0.5 0.4 0.3 0.2 0.1 0.0 Lef c A0 A1 A2 Root Fig. 8. Effect of mixed mino cids on nitrite reductse ctivity in red pepper t 24 h fter tretment. Vlues re mens ± SD (n=5). The principl NH 4 + pthwy is the glutmine synthetse (GS)/glutmte synthse (GOGAT) cycle. The ehvior of GS ctivities in leves ws incresed y 16% in A1 ut not ffected in A2 (Fig. 9; P > 0.05). However, slight inhiitions were found in roots, showing 7% in A1 nd 17% in A2 in reltion to A0 (P < 0.05). GSA ( mol C 5 H 10 N 2 O 4 g -1 FW h -1 ) 30 25 20 15 10 5 0 Lef Root Fig. 9. Effect of mixed mino cids on glutmine synthetse ctivity in red pepper t 24 h fter tretment. Vlues re mens ± SD (n=5). The first step in nitrte ssimiltion is the reduction of to NO 2 y NR, the min nd most limiting step, in ddition to eing the most prone to regultion (Sivsnkr et l., 1997; Ruiz et l., 1999). The next step in ssimiltion is the conversion of the NO 2 to NH 4 + y the ction of NiR. Both enzymes, NR nd NiR, re induced y the sme fctors (Oks, 1994). In our experiment, t 10 mm which is fcilitted y LATS, the presence of MAA could increse the ctivities of NR nd NiR in roots (Fig. 7 nd Fig. 8). In ddition, the very high NO 2 content ws found in MAA tretments in roots (Tle 6). These results suggest tht MAA cn increse uptke y enhncing NR ctivity in roots of red pepper. It is A0 A1 A2

Effect of Mixed Amino Acids on Crop Growth 133 lso striking tht effect of MAA on ssimiltion in the roots ws higher thn in the leves, presumly ws more ville nd the MAA content ws higher in the roots. Ammonium ssimiltion in higher plnts ws long thought to egin with the synthesis of glutmte y glutmte dehydrogense (GDH). It is now elieved tht the mjor pthwy of NH 4 + ssimiltion is the GS-GOGAT pthwy, nd GDH generlly cts in deminting direction (Milflin nd Hsh, 2001). However, role in NH 4 + detoxifiction would explin the increse in GDH expression under conditions tht provoke high tissue NH 4 + levels (Lncien et l., 2000). Two possile effect wys of mino cids on N ssimiltion process hd een suggested: direct effect on mrna of NR (Deng et l., 1991; Li et l., 1995; Vincentz et l., 1993) nd feed ck inhiition on reduction systems (King et l., 1993; Ivshikin nd Sokolov, 1997; Sivsnkr et l., 1997). The hypothesis is tht these two effect wys cn collectively influence N ssimiltion in higher plnt. This might proly e the min reson for differentil effects on uptke oserved in different studies. In the present experiment, GS ctivity ws inhiited slightly y MAA tretments in roots, wheres irregulr results were otined in leves (Fig. 9). 3.2.4 Effect on mino cids nd proteins ccumultion With respect to the min products of ssimiltion, mino cids nd proteins (Tle 7), the plnts treted with MAA did not show increse in these compounds s eing supposed prt from mino cids in roots (P < 0.05). In contrst, the concentrtion of proteins in the roots (P < 0.05) nd leves (P > 0.05) decresed with the MAA rte. Amino cids in leves (P > 0.05) showed the sme tendency too. Amino cids re the uilding locks for proteins nd lso the products of their hydrolysis (Brneix nd Cusin, 1996). In the present experiment, mino cids concentrtions (Tle 7) were higher in the roots thn in leves. This is norml since the N ssimiltion occurs primrily in the roots thn in the leves. In roots, proteins concentrtions (Tle 7) were decresed y MAA tretment due to the possiility tht mino cids content hd effect on protein rekdown. Amino cids Proteins Lef Root Lef Root A0 0.93±0.03 2.35±0.12 5.03±0.27 1.92±0.12 A1 0.78±0.06 2.81±0.16 4.53±0.18 1.90±0.10 A2 0.67±0.03 3.05±0.08 4.35±0.24 1.45±0.11 Dt re mens ± SD (n=5). Anlysis of vrince (ANOVA) ws employed followed y Duncn's new multi rnge test. Vlues with similr superscripts re not significntly different (P>0.05). Tle 7. Effect of mixed mino cids on level of mino cids nd proteins in fresh weight of red pepper t 24 h fter tretment (mg g 1 ) In conclusion, the results of the present experiment clerly indicted tht uptke nd ssimiltion were regulted y MAA in red pepper. The ppliction of MAA rtes could e the direct cuse of incresed ctivities of the enzymes (NR nd NiR) of the ssimiltory pthwy nd the uptke ws enhnced when supplied with

134 Agriculturl Science LATS rnge of. In ddition, uptke y red pepper in unit weight plnt ws less thn tht of rdish due to the different preference on N form etween these two plnts. 4. Pot experiment of rdish with high soil 4.1 Mterils nd methods Commercilized rtificil soil (ph, 5.2; EC, 1240 ms m 1 ; N, 280 mg Kg 1 ; ville P 2 O 5, 1020 mg Kg 1 ) ws mixed with 15 N leled potssium nitrte (10 tom % 15 N) nd incuted t room temperture for 14 dys t 60% of their mximum wter holding cpcity. Finlly, the high nitrte soil (ph, 5.0; EC, 3230 ms m 1 ; N, 1906 mg Kg 1 ; ville P 2 O 5, 1060 mg Kg 1 ) ws otined nd used for this experiment. Seeds of rdish were sown into 100 ml pots filled with the incuted soil nd grown in glsshouse. The mixed mino cids (MAA) solution contined equl concentrtions of mino cids viz., lnine (Al), β lnine (β Al), sprtic cid (Asp), sprgines (Asn), glutmic cid (Glu), glutmine (Gln) nd glycine (Gly). From 17 or 24 dys fter sowing, seedlings of rdish were spryed with 0.2 or 0.5 mm MAA solution for 2 or 4 times, s indicted in Tle 8. The ph of the MAA solutions ws mintined etween 6.0 6.1 y dding 1.0 M KOH ppropritely. Composition of treted solutions (mm) Applied time K + Al β Al Asp Asn Glu Gln Gly DAS A0* A1 0.78 0.2 0.2 0.2 0.2 0.2 0.2 0.2 17, 20, 24, 27 A2 2.10 0.5 0.5 0.5 0.5 0.5 0.5 0.5 17, 20, 24, 27 A3 0.78 0.2 0.2 0.2 0.2 0.2 0.2 0.2 24, 27 * Sme mount of distilled wter spryed Tle 8. Composition of the treted solutions nd ppliction times for rdish in pot experiment Fresh leves were collected t 28 dys fter sowing to determine the content nd enzyme ctivities nd t 30 dys fter sowing to determine the, mino cids nd protein contents. Plnt shoots were hrvested t 30 dys fter sowing to determine crop yield nd N ssimiltion. After hrvest the soils were collected for chemicl nlysis. 4.2 Results nd discussion 4.2.1 Effect of MAA on enzyme ctivities Nitrte reductse is the first enzyme involved in the metolic route of ssimiltion in higher plnts. Significnt differences were found in the NR ctivity etween the tretments (P < 0.01) (Fig. 10). The highest ctivity ws ttined with A2, showing n increse of 30% compred with the ctivity ttined with A0. Tretment A1 nd A3 were less effective in incresing the ctivity of NR thn A2, with increse of 21% nd 7%, respectively.

Effect of Mixed Amino Acids on Crop Growth 135 NRA ( mol NO 2 - g -1 (FW) h -1 ) 3 2 1 0 A0 A1 A2 A3 Fig. 10. Effect of mixed mino cids on nitrte reductse ctivity of rdish leves 28 dy fter sowing in pot experiment with high soil. Vlues re mens ± SD (n=4). The next step in ssimiltion is the conversion of the NO 2 to NH 4 + y the ction of NiR. The MAA tretments showed different effects on NiR ctivity depending on the pplied concentrtions nd times of MAA (Fig. 11). The highest ctivity of NiR ws found in tretment A2, showing n increse of 7% compred with A0 (P < 0.1). However, the ctivity of NiR showed decrese of 11% in A1 (P < 0.05). NiRA ( mol NO 2 - g -1 (FW) h -1 ) 8 6 4 2 0 A0 A1 A2 A3 Fig. 11. Effect of mixed mino cids on nitrite reductse ctivity of rdish leves 28 dy fter sowing in pot experiment with high soil. Vlues re mens ± SD (n=4).

136 Agriculturl Science GSA ( mol C 5 H 10 N 2 O 4 g -1 (FW) h -1 ) 15 10 5 0 A0 A1 A2 A3 Fig. 12. Effect of mixed mino cids on glutmine synthetse ctivity of rdish leves 28 dy fter sowing in pot experiment with high soil. Vlues re mens ± SD (n=4). The response of GS to MAA tretments is showed in Fig. 12. The gretest ctivity ws oserved in tretment A2, with n increse of 7% over the reference tretment (P > 0.1). On the contrry, the lest ctivity of GS ws found in A1, with 12% decrese compred with A0 (P < 0.05). The results of ctivities of enzymes re similr to those of other reserch, which indicted tht tretment of MAA (Section 3.1) nd mino cid fertilizer (Section 3.7) could enhnce ctivity of NR in rdish when supplied with high rte. In the present experiment, the tretments of MAA led to different rtes of increse in NR ctivity nd lso ffect NiR nd GS ctivities depending on pplied rtes. Higher ctivities of three enzymes were found in A2 for the reson tht the positive effect on NR ws stronger thn the feed ck inhiition. However, decrese of NiR nd GS ws oserved in A1 due to the feed ck inhiition on reduction systems which ffected GS first. 4.2.2 Effect of MAA on N contents The dt in Tle 9 showed tht N contents of the plnts were ffected y using MAA. The content of rdish ws decresed y 24 38% y pplying MAA (P < 0.001) compred with the reference tretment. Amino cids Proteins Totl N (mg g 1 FW) (mg g 1 DW) A0 2.91 ± 0.10 9.05 ± 0.58 5.79 ± 0.59 44.9 ± 1.9 A1 2.93 ± 0.07 9.57 ± 0.46 3.85 ± 0.44 c 38.4 ± 1.3 A2 3.03 ± 0.07 9.77 ± 0.54 3.57 ± 0.45 c 41.9 ± 1.8 A3 2.99 ± 0.09 9.19 ± 0.69 4.38 ± 0.18 40.2 ± 1.7 Dt re mens ± SD (n=4). Anlysis of vrince (ANOVA) ws employed followed y Duncn's new multi rnge test. Vlues with similr superscripts re not significntly different (P>0.05). Tle 9. Effect of mixed mino cids on nitrogen contents of rdish leves 30 dy fter sowing in pot experiment with high soil

Effect of Mixed Amino Acids on Crop Growth 137 With respect to the min products of ssimiltion, mino cids nd proteins (Tle 9), the plnts treted with MAA showed little increse of these compounds (P > 0.05) nd the highest contents were found in A2. The totl N content of the plnts ws ffected significntly y using MAA (P < 0.01). of A1, A2 nd A3 showed to decrese the totl N content to 14%, 7% nd 10% compred with the control, respectively. The result of content grees with the interprettion tht mino cid cn negtively regulte nitrte content in higher plnts (Chen nd Go, 2002; Gunes et l., 1994, 1996; Wng et l., 2004). In the present experiment, surged vlue of content ws lso found t 24 h fter MAA treting (the dt were not shown). This ws proly due to the different response of individul plnt to the complex mechnism of MAA in ssimiltion process in short period. However, 3 dys fter MAA ppliction, regulr result of content in shoots of rdish ws found. The predominnce of mino cids nd proteins were ttriuted to high ctivities of min enzymes of ssimiltion nd the direct uptke of mino cids from MAA. The result of totl N content ws opposite from tht of field experiment in which totl N content ws incresed y pplying mino cid fertilizer. These contrdictory results were due to different stge of mino cids tretment. Possily, young plnts my lck complete functionl system for uptke nd ssimiltion (Pessrkli, 2002). Wng et l. (2004) reported tht ppliction of mino cids in utumn could increse totl N in pkchoi ut no significnt effect ws oserved when treted in summer. 4.2.3 Effect of MAA on rdish yield nd N utiliztion The plnt production in terms of fresh weight ws found to e significntly higher (P < 0.05) in tretment A1 nd A2, with increses of 13% nd 12% compred with the control, respectively (Tle 10). The response of production in dry weight to MAA tretments ws more sensitive thn tht of fresh weight (Tle 10), with significnt influences in MAA ppliction (P < 0.01). The highest yield in dry weight ws found in A2, with n increse of 44% in reltion to A0. The results of N utiliztion (Tle 10) were similr to dry yield descried ove, gin registering the highest vlue in A2, with n increse of 34% compred with A0 (P < 0.01). Furthermore, significnt effects were lso oserved in A1 nd A3, with increse of 27% nd 13% respectively, reltive to A0 (P < 0.01). Fresh weight Dry weight N utiliztion (g/plnt) (mg/plnt) A0 13.32 ± 0.71 0.86 ± 0.10 37.60 ± 2.87 c A1 14.99 ± 1.01 1.22 ± 0.13 47.60 ± 4.11 A2 14.86 ± 0.57 1.23 ± 0.11 50.72 ± 2.53 A3 13.01 ± 0.71 1.06 ± 0.07 42.43 ± 3.67 c Dt re mens ± SD (n=4). Anlysis of vrince (ANOVA) ws employed followed y Duncn's new multi rnge test. Vlues with similr superscripts re not significntly different (P>0.05). Tle 10. Effect of mixed mino cids on rdish yield nd nitrogen utiliztion 30 dy fter sowing in pot experiment with high soil

138 Agriculturl Science For responses of growth, the ppliction of MAA showed enhnced effects oviously. These results re in greement with those oserved y Chen et l. (1997), who reported tht ppliction of mino cids led to positive effects on Chinese cge growth. Among the tretments of MAA, the growth responses were incresed y incresing the ppliction rte of MAA. The increses of yield were due to the positive djusting of MAA on growth of plnts, thus contriuting to the increses of N utiliztion (Tle 10) even though the totl N content ws decresed in MAA tretments (Tle 9). 4.2.4 Recovered fertilizer nitrogen It hs ecome evident tht mino cids re principl source of nitrogen for certin plnts, such s mycorrihizl, hethlnd species (Red, 1993), non mycorrihizl plnts from rctic nd lpine ecosystems (Chpin et l., 1993; Kiellnd, 1994) nd orel forest plnts (Näsholm et l., 1998; Persson nd Näsholm, 2001). These systems re similr in tht N minerliztion rtes re hevily constrined y climte, nd plnt N demnds cnnot e met through the uptke of inorgnic ions (R et l., 1999). Bsed on these reserches, the mino cids were used to prtilly replce in hydroponic experiment or spry to leves in mny plnts. In most cse, the ppliction of mino cids led to the decrese of nitrte content nd totl nitrogen content in lettuce, Chinese cge, onion, pkchoi or other lefy crops (Chen nd Go, 2002; Gunes et l., 1994, 1996; Wng et l., 2004). It hd een suggested tht plnts proly preferred mino cids s sources of reduced nitrogen, nd nitrte uptke ws inhiited y mino cids. In this study, the high NdfF ws found in MAA tretments (Tle 11), indicting tht pplied MAA did not ct s source of nitrogen for plnts. On the contrry, plnts hd tken up more N from soil due to the regultion of MAA on uptke nd ssimiltion. The results for the possile regultion of uptke nd ssimiltion y mino cids for higher plnts re contrdictory. Mny uthors greed tht mino cids cn down regulte the uptke nd ssimiltion in higher plnts (Aslm et l., 2001; Ivshikin nd Sokolov, 1997; Oks et l., 1979; Rdin, 1975, 1977; Sivsnkr et l., 1997). But Aslm et l. (2001) reported tht inhiition did not occur when the concentrtion of in the externl solutions hd een incresed to 10 mm. This result is consistent with the other reserch, which indicted tht rdish treted with mixed mino cids contining 5.0 mm in growth medium show significntly incresed the uptke. In this experiment, the positive effect on uptke y pplying MAA ws due to very high content in soil (1906 mg Kg 1 ). NdfF QNdfF NdfFRec (%) (mg/plnt) (%) A0 65.9 ± 1.5 24.8 ± 0.7 d 33.0 ± 1.3 d A1 68.6 ± 2.2 32.7 ± 1.1 43.6 ± 2.0 A2 71.6 ± 0.9 36.3 ± 0.8 48.4 ± 1.9 A3 67.2 ± 2.1 28.5 ± 1.0 c 38.1 ± 2.1 NdfF; the percentge of N derived from fertilizer, QNdfF ; the quntity of N derived from fertilizer, NdfFRec ; the fertilizer-n recovery Dt re mens ± SD (n=4). Anlysis of vrince (ANOVA) ws employed followed y Duncn's new multi rnge test. Vlues with similr superscripts re not significntly different (P>0.05). Tle 11. Nitrogen derived from fertilizer in the rdish shoots

Effect of Mixed Amino Acids on Crop Growth 139 4.2.5 Effect of MAA on chemicl properties of soil The chemicl properties of soil t the end of experiment re showed in Tle 12. The plnting of rdish ffected these chemicl properties of soil clerly. However, there were no differences in ph of soil mong tretments plnted with rdish. On the other hnd, either plnting tretment or MAA tretment showed effect on nitrte in soil. Compred with the non plnting tretment, the tretments of plnting showed decrese of 65~81% nd 35~47% of nitrte nd ville P t 30 dys fter sowing, respectively. The different rtes of decrese were due to the different growth rtes led y MAA tretment. ph EC* Aville P 2 O 5 N (1:5) (ms m 1 ) (mg Kg 1 ) NP 5.0 1985 599.7 1008.6 A0 5.7 981 390.6 339.4 A1 5.7 902 344.5 298.5 A2 5.7 674 316.5 192.0 A3 5.7 894 356.0 355.0 * The soil used in these experiments ws commercilized rtificil soil with lower soil density (out 0.4 g cm 3 ) nd higher wter holding cpcity. Since determintion of soil chemicl properties is sed on dry weight, the determined vlues of EC nd NO3 N re quite high reltive to ordinry soil. However, these re not very higher in soil solution. Tle 12. Chemicl properties of soil t the end of pot experiment for rdish with high soil In conclusion, the results of the present experiment suggest tht ppliction of MAA cn ffect ctivities of three enzymes of N ssimiltion (NR, NiR nd GS). However, the exct reson for this oservtion is unknown nd further investigtion is necessry. Furthermore, the ppliction of MAA cn enhnce growth, N utiliztion, nd concentrtions of proteins nd mino cids, nd reduce the content in plnt shoots. Considerle increse of N uptke from soil ws indicted y the incresed 15 N recovery y pplying MAA compred with the control. These results suggest tht the min role of MAA on nitrte uptke nd ssimiltion might e reltion with the regultion of uptke nd ssimiltion, ut not s sources of reduced nitrogen. 5. Pot experiment of rdish with low soil 5.1 Mterils nd methods Commercilized rtificil soil (ph, 5.2; EC, 1240 ms m 1 ; N, 280 mg Kg 1 ; ville P 2 O 5, 1020 mg Kg 1 ) ws used for this experiment. Plnt culture nd MAA tretment were the sme with tht of pot experiment of rdish with high soil. The smpling nd nlysis of plnt nd soil lso were ccording to procedures dopted for rdish with high soil.

140 Agriculturl Science 5.2 Results nd discussion 5.2.1 Effect of MAA on enzyme ctivities Significnt differences were found in the NR ctivity mong the tretments (P < 0.01) (Fig. 13). The NR ctivity ws inhiited y folir ppliction of MAA in this experiment, contrry to tht of rdish in which ws plnted in high nitrte soil. The lowest ctivity ws ttined with A2, showing decrese of 28% compred with the ctivity ttined in tretment A0. Tretment A1 nd A3 were less effective in decresing the ctivity of NR thn A2, with decreses of 8% nd 17%, respectively. NRA ( mol NO 2 - g -1 (FW) h -1 ) 0.8 0.6 0.4 0.2 0.0 A0 A1 A2 A3 Fig. 13. Effect of mixed mino cids on nitrte reductse ctivity of rdish leves 28 dy fter sowing in pot experiment with low soil. Vlues re mens ± SD (n=4). The response of ctivity of NiR to the MAA ppliction resemled tht of NR (Fig. 14). The lowest ctivity of NiR ws found in tretment A2, showing 40% decrese compred with the control tretment (P < 0.001). Tretment A1 nd A3 lso showed 23% nd 32% decrese in reltion to A0, respectively. NiRA ( mol NO 2 - g -1 (FW) h -1 ) 4 3 2 1 0 c A0 A1 A2 A3 c Fig. 14. Effect of mixed mino cids on nitrite reductse ctivity of rdish leves 28 dy fter sowing in pot experiment with low soil. Vlues re mens ± SD (n=4).

Effect of Mixed Amino Acids on Crop Growth 141 GSA ( mol C 5 H 10 N 2 O 4 g -1 (FW) h -1 ) 20 15 10 5 0 A0 A1 A2 A3 Fig. 15. Effect of mixed mino cids on glutmine synthetse ctivity of rdish leves 28 dy fter sowing in pot experiment with low soil. Vlues re mens ± SD (n=4). With respect to enzyme ctivity of GS (Fig. 15), the ppliction of MAA led to significnt decrese in the ctivity in leves of rdish in this experiment, the lowest ctivity eing recorded in tretment A1, with decrese of 27% in reltion to the highest ctivity, found in the reference tretment A0 (P > 0.01). Tretment A2 nd A3 lso showed decreses of 22% nd 26%, respectively. Like some of the N trnsporters, NR is induced y its own sustrte,, nd this induction is fst, occurring within severl minutes, nd requires very low concentrtions (< 10 μm) (Crw ford, 1995; Sueyoshi et l., 1995). is the primry fctor, lthough other fctors lso influence the regultion of reduction nd ssimiltion, including the end products of ssimiltion such s mino cids. NiR nd NR re similrly trnscriptionlly regulted for the reson of tht NiR is strongly induced y the sme fctor,, proly to prevent the ccumultion of toxic NO 2 (Wng et l., 2000). The ctivities of NR nd NiR were much lower thn tht of rdish which ws plnted in high soil due to the poor in the soil used in the present experiment (Tle 15). In the present experiment, the ctivities of three enzymes decresed when treted with MAA. These results re in greement with other reserches which indicted tht downstrem N ssimiltion products such s mino cids cn feed ck to regulte uptke nd reduction (Deng et l., 1991; Sivsnker et l., 1997; Vincentz et l., 1993). However, the effects of MAA on ctivities of enzymes re opposite to other experiments of ours, which indicted tht tretment of MAA nd mino cid fertilizer could enhnce ctivity of NR in rdish when supplied with high rte of. The contrdictory results re due to the different levels of the soils. 5.2.2 Effect of MAA on N contents The dt in Tle 13 showed tht N contents of the plnts were not ffected significntly y using MAA. The highest concentrtions of ll N forms were oserved in tretment A2 (P > 0.05). These results differed from rdish which ws plnted in high soil. The different

142 Agriculturl Science effects of MAA on N contents of rdish in high soil nd low soil re in greement with the supposition tht mino cids hve different effect on uptke nd ssimiltion. Amino cids Proteins Totl N (mg g 1 FW) (mg g 1 DW) A0 1.89 ± 0.07 7.35 ± 0.40 1.30 ± 0.06 20.5 ± 1.5 A1 1.87 ± 0.11 7.60 ± 0.37 1.32 ± 0.16 20.3 ± 2.4 A2 1.77 ± 0.12 7.92 ± 0.46 1.35 ± 0.08 21.0 ± 0.7 A3 1.97 ± 0.12 7.06 ± 0.50 1.34 ± 0.08 20.0 ± 0.8 Dt re mens ± SD (n=4). Anlysis of vrince (ANOVA) ws employed followed y Duncn's new multi rnge test. Vlues with similr superscripts re not significntly different (P>0.05). Tle 13. Effect of mixed mino cids on nitrogen contents of rdish leves 30 dy fter sowing in pot experiment with low soil 5.2.3 Effect of MAA on rdish yield nd N utiliztion The plnt production in fresh weight ws found to e higher (P < 0.01) in tretment of A2, with n increse of 9% compred with the control tretment (Tle 14). The response of production in dry weight to MAA tretments ws not s sensitive s tht in fresh weight (Tle 14), only with slight influences. The results of N utiliztion (Tle 14) were similr to dry yield, registering the highest vlue in A1, with n increse of 15% compred with A0 (P < 0.01). Fresh weight Dry weight N utiliztion (g/plnt) (mg/plnt) A0 8.38 ± 0.40 0.84 ± 0.05 17.06 ± 0.85 A1 8.57 ± 0.34 0.88 ± 0.09 19.71 ± 0.58 A2 9.83 ± 0.32 0.89 ± 0.05 18.52 ± 0.51 A3 8.44 ± 0.26 0.85 ± 0.03 18.88 ± 0.42 Dt re mens ± SD (n=4). Anlysis of vrince (ANOVA) ws employed followed y Duncn's new multi rnge test. Vlues with similr superscripts re not significntly different (P>0.05). Tle 14. Effect of mixed mino cids on rdish yield nd nitrogen utiliztion 30 dy fter sowing in pot experiment with low soil 5.2.4 Effect of MAA on chemicl properties of soil The chemicl properties of soil t the end of experiment were showed in Tle 15. The plnting of rdish ffected these chemicl properties of soil clerly. However, there were no differences in ph of soil mong tretments plnted with rdish. On the other hnd, either plnting tretment or MAA tretment showed effect on soil nitrte reduction. Compred with the non plnting tretment, the tretments of plnting showed decrese of 86~88% for nitrte nd decrese of 56~70% for ville P t 30 dys fter sowing, respectively. The different rtes of decrese were due to the different growth rtes resulted from y MAA tretment. And the EC decresed ccordingly.

Effect of Mixed Amino Acids on Crop Growth 143 ph EC Aville P 2 O 5 N (1:5) (ms m 1 ) (mg Kg 1 ) NP 5.4 1485 878 214.7 A0 5.7 482 384 68.2 A1 5.7 515 303 62.2 A2 5.7 349 264 70.3 A3 5.7 503 277 68.6 Tle 15. Chemicl properties of soil t the end of pot experiment for rdish with low soil The commercilized rtificil soil used in this experiment ws with lower soil density (out 0.4 g cm 3 ) nd higher wter holding cpcity. Although the contents of 62.2~70.3 mg Kg 1 re not low in ordinry soil, ville for plnts is very poor in soil solution in this experiment. This might e the prole reson, tht effects of MAA on N ssimiltion in the present experiment were different from tht of rdish which ws plnted in high soil. Whether in our experiments or in other reserches, different effects of mino cids on reduction nd ssimiltion were oserved (Aslm et l., 2001). In conclusion, the results of the present experiment suggest tht ppliction of MAA cn decrese ctivities of three enzymes of N ssimiltion (NR, NiR nd GS). However, except N utiliztion, the ppliction of MAA did not hve significnt effects on growth, nd concentrtions of proteins, mino cids, totl N nd content in plnt shoots. The difference in the results were found in oth the present experiment nd pot experiment which rdish ws plnted in high soil my e due to different levels of content in soil solution. The hypothesis tht effect of mino cids on uptke, reduction nd ssimiltion depends on concentrtion of ws justified. 6. Field experiment of rdish 6.1 Mterils nd methods The study ws conducted in summer of 2005 t the experimentl frm of the Chungnm Ntionl University, Dejeon, Kore. The verge chemicl properties of the soil of the field re descried in Tle 16. The fertilizer mixture ws uniformly rodcsted onto the soil surfce nd incorported efore ridging. The seeds of rdish were sown t the end of My 2005 nd rrnged in completely rndomized lock design, with three replictions. The plots were 5 m 2 m consisting of 2 rows. At 15 nd 22 dys fter sowing, AAF ws pplied 2 times to plots y sprying to leves fter diluting 500, 1000 nd 2000 times y wter, respectively. The min chemicl contents of the AAF nd ppliction quntities re shown in Tle 17. Soils ph EC Orgnic Aville Totl mtter P 2 O 5 N N (1:5) (ms m 1 ) (g Kg 1 ) (mg Kg 1 ) (g Kg 1 ) (mg Kg 1 ) Before fertiliztion 6.0 122 15.6 170 0.81 80.2 After fertiliztion 6.0 191 15.8 279 0.87 191.2 Tle 16. Chemicl properties of soils used in field experiment of rdish

144 Agriculturl Science Fresh leves were collected t 23 dys fter sowing to determine the, mino cids nd protein contents nd enzyme ctivities. The plots were hrvested t 35 dys fter sowing to determine crop yield nd N ssimiltion. The topsoil smples (0 20 cm) were collected t 25 nd 35 dys fter sowing for chemicl nlysis. In order to compre the different AAF tretments for their N uptke, net N uptke ws estimted y lncing N utiliztion nd N input y pplying AAF thus: NN NU NAAF. (1) where N N is the net N uptke y plnt; N U is the totl N utiliztion t hrvest; N AAF is N input y pplying AAF. It ws ssumed tht N would hve een either tken up y the plnts or lost from the soil plnt system. In our experiment, leching ws the min wy of N loss. Furthermore, N loss ttriutle to soil erosion nd runoff ws considered for our site with 2~5% slope. Since these losses my e influenced y protecting of the plnts from the rin, the vegettion cover ws oserved t 25 nd 35 dys fter sowing. Clssifiction (%) NP* A0 A1 A2 A3 (mg m 2 ) AAF ppliction 750 1500 3000 Essentil mino cid 2.22 16.7 33.3 66.6 Totl mino cid 5.14 38.6 77.2 154.4 Totl N 3.80 28.5 57.0 114.0 Solule P 3.12 23.4 46.8 93.6 Solule K 4.97 37.3 74.6 149.2 Solule B 0.13 0.98 1.95 3.90 * NP: No-plnting Tle 17. Amino cid fertilizer pplied to rdish in the field experiment 6.2 Results nd discussion 6.2.1 Effect of AAF on enzyme ctivities Nitrte reductse is the first enzyme involved in the metolic route of ssimiltion in higher plnts. Significnt differences were found in the NR ctivity etween the tretments (P < 0.01) (Fig. 16). The highest ctivity ws otined with A1, showing n increse of 16% in reltion to the ctivity otined with A0. A2 ws less effective in incresing the ctivity of NR thn A1; wheres no increse of NRA occurred in A3, even treted with fourfold AAF thn A1. The next step in ssimiltion is the conversion of the NO 2 to NH 4 + y the ction of NiR. The AAF tretments showed different effect on NiR ctivity depending on the pplied rte of AAF (Fig. 17). The highest ctivity of NiR ws found in tretment A1, showing n increse of 4% compred with A0 (P < 0.05). However, the ctivities of NiR were inhiited y 12 nd 13% in A2 nd A3, respectively.

Effect of Mixed Amino Acids on Crop Growth 145 NRA ( mol NO 2 - g -1 (FW) h -1 ) 1.6 1.2 0.8 0.4 0.0 A0 A1 A2 A3 Fig. 16. Effect of mino cid fertilizer on nitrte reductse ctivity in leves of rdish 23 dy fter sowing. Vlues re mens ± SD (n=3). NiRA ( mol NO 2 - g -1 (FW) h -1 ) 4.0 3.2 2.4 1.6 0.8 0.0 A0 A1 A2 A3 Fig. 17. Effect of mino cid fertilizer on nitrite reductse ctivity in leves of rdish 23 dy fter sowing. Vlues re mens ± SD (n=3). The reversile mintion of 2 oxoglutrte to glutmic cid vi GDH hs long een considered s mjor route of NH 4 + ssimiltion (Srivstv nd Singh, 1987). However the discovery of the enzyme GS GOGAT system ltered this point of view, nd the incorportion of NH 4 + to glutmine vi GS nd susequently into glutmic cid y GOGAT is now widely ccepted s the min route of NH 4 + ssimiltion (Oks, 1994). The response of GS (Fig. 18) to AAF tretments ws similr to tht of the NiR (Fig. 17). The gretest ctivity ws reched in tretment A1, with n increse of 20% over the reference tretment (P < 0.001). On the contrry, the ctivity of GS ws the lowest in A3, with decline of 11% compred with A0 (P < 0.05).

146 Agriculturl Science GSA ( mol C 5 H 10 N 2 O 4 g -1 (FW) h -1 ) 40 30 20 10 0 A0 A1 A2 A3 Fig. 18. Effect of mino cid fertilizer on glutmine synthetse ctivity in leves of rdish 23 dy fter sowing. Vlues re mens ± SD (n=3). The reduction of to NO 2 y NR, is the min nd most limiting step, in ddition to eing the most prone to regultion (Sivsnkr et l., 1997; Ruiz et l., 1999). The synthesis of this enzyme is induced y nitrte (Oks, 1994), ut lthough its ctivity is known to e repressed y mient mmonium, there re evidences tht this enzyme cn e regulted y certin mino cids. The results for the possile regultion of NR ctivity y mino cids for higher plnts re contrdictory. Mny uthors gree with tht mino cids cn inhiit the ctivity of NR in higher plnts (Rdin, 1975, 1977; Oks et l., 1979; Ivshikin nd Sokolov, 1997; Sivsnkr et l., 1997; Aslm et l., 2001). But Aslm et l. (2001) reported tht inhiition did not occur when the concentrtion of in the externl solutions hd een incresed to 10 mm. This result is consistent with the other reserch, which indictes tht rdish treted with mixed mino cids contining 5.0 mm in growth medium showed significnt increse of NR ctivity (Liu et l., 2005). The effect of mino cids on NR ctivity seems to e depended on plnt mterils, ge of plnts, growth conditions, nitrte concentrtion, kinds of mino cids, mino cids concentrtion nd other fctors. In this experiment, the positive effect on NR ctivity y pplying AAF ws due to high content in soil. In the present experiment, the tretments of AAF led to different levels of increse of NR ctivity nd inhiition on GS ctivity depending on pplied rtes. The high ctivities of three enzymes were found in A1 due to the positive effect of AAF on process of ssimiltion. However, inhiition on NiR nd GS ws oserved in A2 nd A3 for the reson tht high rtes of AAF ppliction hd high feed ck inhiition on reduction systems which ffected GS first. This is proly the min reson why different effects on the enzymes were oserved in this study.

Effect of Mixed Amino Acids on Crop Growth 147 6.2.2 Effect of AAF on iomss nd utiliztion of N nd P The plnt iomss production in fresh weight ws found to e significntly higher (P < 0.01) in the AAF tretments (men iomss in fresh weight of A1, A2 nd A3 re 5.056, 4.738 nd 4.653 Kg m 2, respectively) compred with the control (men iomss in fresh weight is 4.026 Kg m 2 ) (Tle 18). Among AAF tretments, the tretment with low concentrtion of AAF (A1) hd higher (P > 0.05) iomss production thn the tretment with high concentrtion of AAF (A3). The response of iomss production in dry weight to AAF tretments resemled tht in fresh weight (Tle 28), with significnt influence y pplying AAF (P < 0.01). The highest iomss production in dry weight ws found in A1, with n increse of 17% in reltion to A0. Fresh weigh Dry weight N utiliztion P utiliztion A0 4026 ± 227 c 345.7 ± 14.2 c 9.33 ± 0.87 c 2.35 ± 0.09 A1 5056 ± 213 404.4 ± 11.6 14.48 ± 0.89 2.87 ± 0.11 A2 4738 ± 183 394.8 ± 12.1 13.04 ± 0.53 2.68 ± 0.12 A3 4653 ± 189 382.0 ± 14.5 12.83 ± 0.67 2.64 ± 0.07 Vlues re mens ± SD (n=3). Anlysis of vrince (ANOVA) ws employed followed y Duncn's new multi rnge test. Vlues with similr superscripts re not significntly different (P>0.05) Tle 18. Effect of mino cid fertilizer on rdish yield nd utiliztion of nitrogen nd phosphorus 35 dy fter sowing (g m 2 ) The result of N utiliztion (Tle 18) ws similr to iomss production s descried ove, gin registering the highest vlue in A1 (14.48 ± 0.89 g m 2 ), with n increse of 55% compred with A0 (9.33 ± 0.87 g m 2 ) (P < 0.01). Furthermore, significnt effects were oserved in A2 nd A3 too, with increses of 40% nd 37% respectively, in reltion to A0 (P < 0.01). Even though P content ws not influenced y the ppliction of AAF (Tle 20), P utiliztion incresed in AAF tretments due to the increse of iomss production (Tle 18). The oserved result of vegettion cover nd clculted vlues of net N uptke re showed in Tle 19. The tretments of AAF showed higher vegettion cover thn the control. Besides the N input y pplying of AAF, the tretments of AAF showed significnt increse of 36~55%net N uptke compred with the control. Gunes et l. (1996) suggested tht plnts proly preferred mino cids s sources of reduced nitrogen, ut they did not distinguish origin of the N contents in the plnts. In our experiment, the increse of N uptke is out 200 times (Tle 19) more thn N supplied y pplying AAF, indicting ppliction of AAF could enhnce the ility of uptke nd ssimiltion of inorgnic N y plnts. Vegettion cover (%) Net N uptke (g m 2 ) 25 DAS 35 DAS 35 DAS A0 63 ± 3 c 91 ± 2 9.33 ± 0.87 A1 85 ± 5 100 ± 0 14.45 ± 0.89 A2 79 ± 6 100 ± 0 12.98 ± 0.53 A3 76 ± 3 100 ± 0 12.72 ± 0.67 Vlues re mens ± SD (n=3). Anlysis of vrince (ANOVA) ws employed followed y Duncn's new multi rnge test. Vlues with similr superscripts re not significntly different (P>0.05) Tle 19. Net nitrogen uptke nd vegettion cover of rdish

148 Agriculturl Science These results re in greement with those oserved y Chen et l. (1997), who reported tht ppliction of mino cids led to positive effects on cge growth. However, mong the tretments of AAF, the growth responses were decresed y incresing the ppliction rte of AAF. This my proly e relted to the feed ck inhiition of high rte ppliction of mino cids. 6.2.3 Effect of AAF on contents of N nd P The dt in Tle 20 showed tht N contents of the plnts were ffected y using mino cid fertilizer. The content of rdish ws decresed y ppliction of AAF (P < 0.05) compred with the reference tretment. Among the tretments, A1 gve the est result in reducing the nitrte to 1.16 mg g 1 (FW), with decrese of 24% in reltion to the highest content found in A0. This result grees with the interprettion tht mino cid cn negtively regulte nitrte content in higher plnts (Gunes et l., 1994, 1996; Chen nd Go, 2002; Wng et l., 2004). But this interprettion ws not supported in ll cses. It ws oserved tht the mixed mino cids incresed content slightly in rdish when the plnts growing in nutrient solution. The contrdiction my reside in mino cids tretment method. It ws demonstrted in other studies tht mino cid pretretment decresed ccumultion slightly, ut Gln nd Asn led to concentrtion increse in rley roots when they were used together with nitrte (Aslm et l., 2001). With respect to the min products of ssimiltion, mino cids nd proteins (Tle 20), the plnts in tretment A1 gve the highest contents of these compounds (P < 0.01). In the A1 tretment, high ctivities of min enzymes of ssimiltion could explin the predominnce of these nitrogenous compounds in rdish. Under tretments of A2 nd A3, the increses of mino cids nd proteins derived from the direct uptke of mino cids from AAF. Amino cids Proteins Totl-N Totl-P (mg g 1 FW) (mg g 1 DW) A0 1.53 ± 0.11 1.29 ± 0.02 1.28 ± 0.08 c 27.2 ± 1.6 c 6.8 ± 0.3 A1 1.16 ± 0.17 1.38 ± 0.02 1.98 ± 0.16 35.9 ± 1.6 7.1 ± 0.4 A2 1.32 ± 0.20 1.33 ± 0.02 1.74 ± 0.06 33.0 ± 0.9 6.8 ± 0.4 A3 1.48 ± 0.08 1.32 ± 0.04 1.70 ± 0.09 31.2 ± 1.2 c 6.9 ± 0.5 Vlues re mens ± SD (n=3). Anlysis of vrince (ANOVA) ws employed followed y Duncn's new multi rnge test. Vlues with similr superscripts re not significntly different (P>0.05) Tle 20. Effect of mino cid fertilizer on contents of nitrogen nd phosphorus in rdish 23 dy fter sowing The totl N content of the plnts ws lso ffected significntly y the use of AAF (P < 0.01). of A1, A2 nd A3 showed to increse the totl N to 32%, 21% nd 15% reltive to the control, respectively. These increses were due to the positive djusting of AAF on uptke nd ssimiltion of N, nd ttriuting to the increses of N utiliztion nd net N uptke. The P content of rdish ws not ffected significntly y the ppliction of AAF (Tle 20).

Effect of Mixed Amino Acids on Crop Growth 149 6.2.4 Effect of AAF on chemicl properties of soil The chemicl properties of soil in middle growth period nd t the end of experiment were showed in Tle 21 nd Tle 22. The plnting of rdish ffected totl N of soil clerly, except t 35 dys fter sowing, with fll of 10% compred with non plnting tretment. However, there were no differences in totl N of soil mong tretments plnted with rdish. On the other hnd, either plnting tretment or AAF tretment showed effect on nitrte in soil. Orgnic Aville Totl ph EC mtter P 2 O 5 N N (1:5) (ms m 1 ) (g Kg 1 ) (mg Kg 1 ) (g Kg 1 ) (mg Kg 1 ) NP 6.3 81 15.4 267 0.70 75.3 A0 6.3 57 15.2 297 0.67 52.5 A1 6.4 55 15.6 285 0.67 55.7 A2 6.5 65 15.1 310 0.67 58.9 A3 6.4 54 15.3 305 0.66 60.0 Tle 21. Chemicl properties of soil in the middle of growth period (25 dy fter sowing) for rdish In the soil of non plnting, nitrte ws decresed y leching nd runoff y rin. Compred with the non plnting tretment, the tretments of plnting showed 20~30% decrese t 25 dys fter sowing nd 23~42% decrese t 35 dys fter sowing in the nitrte content of soil. Although with the lowest net N uptke, the lowest concentrtion of nitrte in soil ws found in A0 tretment oth t two smpling times. This ws due to the fct tht the vegettion covers of AAF tretments were higher thn tretment of A0, nd could effectively prevent nitrte of soil from leching or runoff. The plnting tretments showed lower vlues of EC thn non plnting tretment, ut ll were in the rnge of generl soil. There were no significnt differences mong ll tretments in ph nd orgnic mtter of soil. Moreover, very smll differences were oserved in ville P due to different growth rte of the plnts. ph EC Orgnic mtter Aville P 2 O 5 Totl N N (1:5) (ms m 1 ) (g Kg 1 ) (mg Kg 1 ) (g Kg 1 ) (mg Kg 1 ) Before experiment 6.0 191 15.8 279 0.87 191.2 NP 6.4 99 15.9 308 0.70 94.3 A0 6.4 41 16.0 283 0.63 55.0 A1 6.4 42 15.8 263 0.63 65.9 A2 6.4 49 15.2 270 0.63 65.0 A3 6.4 45 15.3 272 0.63 73.2 Tle 22. Chemicl properties of soil t the end of field experiment (35 dy fter sowing) for rdish

150 Agriculturl Science removl (0~20cm) Removl rte y plnt Removl rte y leching (g m 2 ) (%) NP 25.2 100.0 A0 35.4 26.3 73.7 A1 32.6 44.4 55.6 A2 32.8 39.7 60.3 A3 30.7 41.8 58.2 Tle 23. Effect of mino cid fertilizer on nitrte removl from the soil The dt of removl re showed in Tle 23. Even though the highest removl ws found in tretment A0, the most removed ws leched (73.7%) nd would led to pollution for groundwter. The ppliction of AAF cn enhnce removl rte y plnting, nd void N losses through leching nd runoff due to increses of N utiliztion (Tle 18) nd vegettion cover (Tle 19). In conclusion, the results of the present experiment suggest tht ppliction of mino cid fertilizer cn ffect ctivities of three enzymes of N ssimiltion (NR, NiR nd GS) nd increse the growth nd N ssimiltion in rdish. However, the exct reson for this oservtion is not known nd requires further investigtion. The plnting of rdish proves very effective for nitrte removl in soil y its fst growth nd very high iomss production (345.7~404.4 g DW m 2 ) nd N utiliztion (9.33~14.48 g m 2 ) in short time (only 35 dys in our experiment). Furthermore, the ppliction of mino cid fertilizer cn enhnce iomss production, N utiliztion, nd concentrtions of proteins nd mino cids, nd it cn reduce N losses through leching nd runoff. 7. Conclusions By conducting these experiments, severl findings were otined: (1) increse of uptke y ppliction of MAA, (2) different effect of MAA dictted y N sttus, (3) efficient removl y ppliction of AAF, nd (4) true role of MAA in process of uptke nd ssimiltion. Both for rdish nd red pepper, the ppliction of MAA led to significnt increse of uptke nd ctivities of the three enzymes (NR, NiR nd GS) of the ssimiltory pthwy in solution experiment. These results re different from other reserches which inhiition ws oserved in most cse. This difference ws cused y two min resons: (1) tht effect of MAA ws different to single mino cid, nd (2) comprtive high level ws supplied in these experiments. In pot experiments, responses to pplied MAA were ffected y plnt species nd level in soil. For rdish, ppliction of MAA led to increses of ctivities of three enzymes, growth, N utiliztion, nd concentrtions of proteins, nd decrese of content in plnt shoots, when the plnts were plnted in high soil. However, in the cse tht rdish ws plnted in low soil, ctivities of the enzymes were decresed y using MAA, nd growth, nd concentrtions of proteins, mino cids, totl N nd content were not ffected. These phenomen indicte tht the effect of MAA is dependent on level.

Effect of Mixed Amino Acids on Crop Growth 151 With respect to red pepper which ws plnted in high soil, folir MAA sprys incresed ctivities of the three enzymes, while reduced content, concentrtions of proteins nd mino cids, totl N nd N utiliztion. Prtilly different results were found in red pepper which ws plnted in low soil, including decresed ctivities of NiR nd GS nd incresed of content in plnt shoots y the ppliction of MAA. The reson for these differences is the sme to tht of rdish. In field experiment of rdish, the folir sprys of AAF incresed removl rte y plnting, nd void N losses through leching nd runoff due to increses of N utiliztion nd vegettion cover. In ddition, the ppliction of AAF enhnced ctivities of three enzymes, iomss production, nd concentrtions of proteins nd mino cids, reduced content in plnt shoots. Similrly, for red pepper, the use of AAF led to increse of N utiliztion. However, decrese of totl N content in red pepper plnts ws found in AAF tretments. These results of 15 N leled experiments nd field experiments suggest tht the min role of mino cids on nitrte uptke nd ssimiltion might e reltion with the regultion of uptke nd ssimiltion, ut not s sources of reduced nitrogen. In pot experiments, it ws indicted tht the N utiliztion of plnts ws depended on soil uptke which ws regulted y ppliction of MAA. In field experiment of rdish, the increse of N utiliztion is out 200 times more thn N supplied y pplying AAF, indicting ppliction of AAF could enhnce the ility of uptke nd ssimiltion of inorgnic N y plnts. Finlly, the effect of mino cids on uptke nd ssimiltion ws lso influenced y stge of plnt growth. For lef rdish, response of enzymes ctivity nd yield ws not ffected y the stge of growth, while N ccumultion (totl N content) ws more sensitive to pplied mino cids in vegettive stge thn tht of young stge. With regrd to red pepper, effects of mino cids on enzymes ctivity nd N content in different growth stge were quite similr, while growth (dry iomss) showed to e incresed significntly in vegettive stge. A etter understnding of effect of mino cid on process of uptke nd ssimiltion will undoutedly help in developing n pproch to improve the mngement of fertilizer nitrogen nd to prevent N loss through leching or runoff. In the further study, more detiled reserches should e crried out to investigte the precise mnner y which MAA influences uptke nd ssimiltion. The reserches will focus on the effect of MAA on NR gene expression nd reltion etween GDH nd GS. 8. References Andrews, M. 1986. The prtitioning of nitrte ssimiltion etween root nd shoot of higher plnts. Plnt Cell Environ. 9, 511 519. Ashley, D. A., W. A. Jckson, nd R. Volk. 1975. Nitrte uptke nd ssimiltion y whet seedlings during initil exposure to nitrte. Plnt Physiol. 55, 1102 1106. Aslm, M, R. L. Trvis, nd D. W. Rins. 1996. Evidence for sustrte induction of nitrte efflux system in rley roots. Plnt Physiol. 112, 1167 1175. Aslm, M., R. L. Trvis, nd D. W. Rins. 2001. Differentil effect of mino cids on nitrte uptke nd reduction systems in rley roots. Plnt Sci. 160, 219 228.

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Agriculturl Science Edited y Dr. Godwin Aflkpui ISBN 978-953-51-0567-1 Hrd cover, 252 pges Pulisher InTech Pulished online 27, April, 2012 Pulished in print edition April, 2012 This ook covers key res in griculturl science, nmely crop improvement, production, response to wter, nutrients, nd temperture, crop protection, griculture nd humn helth, nd niml nutrition. The contriutions y the uthors include mnipultion of the vriles nd genetic resources of inheritnce of quntittive genes, crop rottion, soil wter nd nitrogen, nd effect of temperture on flowering. The rest re protecting crops ginst insect pests nd diseses, linking griculture lndscpe to recretion y humns, nd smll ruminnt nutrition. This ook is vlule ddition to the existing knowledge nd is especilly intended for university students nd ll professionls in the field of griculture. How to reference In order to correctly reference this scholrly work, feel free to copy nd pste the following: Xing-Qun Liu nd Kyu-Seung Lee (2012). Effect of Mixed Amino Acids on Crop Growth, Agriculturl Science, Dr. Godwin Aflkpui (Ed.), ISBN: 978-953-51-0567-1, InTech, Aville from: http:///ooks/griculturl-science/effect-of-mixed-mino-cids-on-crop-growth InTech Europe University Cmpus STeP Ri Slvk Krutzek 83/A 51000 Rijek, Croti Phone: +385 (51) 770 447 Fx: +385 (51) 686 166 InTech Chin Unit 405, Office Block, Hotel Equtoril Shnghi No.65, Yn An Rod (West), Shnghi, 200040, Chin Phone: +86-21-62489820 Fx: +86-21-62489821