Amerin-Eursin J. Agri. & Environ. Si., 15 (9): 1860-1867, 2015 ISSN 1818-6769 IDOSI Pulitions, 2015 DOI: 10.5829/idosi.ejes.2015.15.9.95303 Effet of High Levels of Ammonium or Nitrte on Growth nd Nitrogen Metolism in Roots nd Leves of Sorghum (Sorghum sudngrss) Plnts Redoune EL Omri nd Mohmed Nhiri Lortoire De Biohimie et Génétique Moléulire, Fulté Des Sienes et Tehniques, Université Adelmlek Essâdi, Tnger Prinipl BP 416, Moroo Astrt: Sorghum (Sorghum sudngrss) plnts were treted with 5, 20 nd 50 mm of Nitrogen (Nitrte or Ammonium). Growth prmeters (length, dry weight nd fresh weight), iohemistry prmeters (proteins, mino ids nd hlorophyll ontent) nd tivities of enzymes involved in nitrogen metolism suh s: Nitrte redutse (NR), Glutmine synthetse (GS) nd glutmte dehydrogense (GDH) were studied in the leves nd roots fter 20 dys of N exposure. Results showed tht sorghum plnts exhiited enhned iomss prodution under high levels of inorgni nitrogen supply. Biohemistry prmeters inresed with inresing nitrogen onentrtion in roots nd leves espeilly in mmonium-treted plnts. Exogenous nitrte ut not mmonium indued signifint differene in the leves nd roots nitrte redutse tivity when ompred with ontrol plnts (0.5 mm nitrte). While Glutmine synthetse 1 tivity nd protein umulted in roots t inresing onentrtion of either nitrte or mmonium, prtiulrly in mmonium-treted plnts, glutmine synthetse 2 protein ws more umulted in leves of nitrte-treted plnts. Glutmte dehydrogense tivity ws enhned in oth orgns y inresing N soure in the ulture medium this inrese is more pronouned in presene of mmonium. This dt showed tht sorghum plnts exhiited higher dptive potentil under exessive onentrtion of mmonium y ility to detoxify this ion vi nitrogen ssimiltion in roots. Key words: Glutmine synthetse Nitrte redutse nd Glutmte dehydrogense INTRODUCTION ording to the plnt speies, whih is generlly relted to the physiologil dpttions of plnts to nturl Most plnt speies n utilize nitrogen soures eosystems [4]. - s NH 4 or NO 3. Although inorgni nitrogen is Nitrte is tken up y roots moves in prt, vi the predominntly ville to plnts s nitrte in most soils, vsulr undle, to leves for redution. In leves, nitrte in ertin soil nd in hydroponi ultures, mmonium n is redued to mmonium y ytosoli nitrte redutse e the mjority nitrogen ion [1]. High onentrtions of (NR; EC 1.6.6.1) nd then y plstidil ferredoxin-nitrite mmonium nitrogen in the soil or in nutrient solution my redutse (Fd-NiR, EC 1.6.6.4) [5]. Ammonium originted led to umultion of toxi mounts of mmonium ions from diret sorption, NR tivity, photorespirtion, in the plnts [2, 3]. Depending on the nitrogen soure, the dinitrogen fixtion or demintion of nitrogenous response of plnt speies or ultivrs vries widely. For ompounds, is ssimilted y glutmine synthetse exmple, free mmonium n e potentilly use (ytosoli GS1 nd plstidil GS2, EC 6.3.1.2) nd derese in net photosynthesis nd therefore in the then y glutmte synthse (Fd- GOGAT, EC 1.4.7.1; growth of the plnts [2]. Nitrte ions n umulte in NADH-GOGAT, EC 1.4.1.14) in vegettive orgns [6]. the vuoles; thus most plnt speies n tolerte high Alterntively, mitohondril NADH-glutmte nitrte onentrtions without ny sign of toxiity. dehydrogense (NADH-GDH, EC 1.4.1.2) n inorporte However, preferene for nitrte or mmonium vries high levels of mmonium into glutmte under stress [7]. Corresponding Author: Redoune EL Omri, Lortoire De Biohimie et Génétique Moléulire, Fulté Des Sienes et Tehniques, Université Adelmlek Essâdi, Tnger Prinipl BP 416, Moroo. Fx: 21239393953. 1860
Am-Eurs. J. Agri. & Environ. Si., 15 (9): 1860-1867, 2015 Sorghum is n importnt stple food rop in developing wter to remove residul vermiulite. All experiments nd ountries nd ws rnked the seventh most importnt enzyme preprtions were performed with freshly rop worldwide in terms of hrvested re. We hve used hrvested plnts. For free mino ids (AA) estimtion Sorghum sudngrss s experimentl model, whih is ws determined in dried leves or roots. extensively used s rop for their fvourle hrteristis suh s ility to ssimilte nd stok Growth Prmeters: After 20 dys of N tretment, 10 exess of nitrogen, quik growth, nutritionl qulity nd plnts from eh group were divided into seprte leves tolerne to het nd drought. Intensive growth of nd roots frtions. Fresh weights of leves nd roots S. sudngrss hyrids in short periods of wter shortge were weighed nd lengths were mesured. The smples (summer) requires the supply of high levels of inorgni were then dried in oven t 70 C for 72 h nd dry weights nitrogen tht is provided usully in the form of nitrte. were determined. In previous work, we hve reported tht S. sudngrss hyrids exhiited enhned iomss prodution under Estimtion of Protein nd Free Amino Aids: Protein ws high levels of inorgni nitrogen supply s well s estimted y the method of Brdford et l. [10] using BSA inresed pity for mmonium ssimiltion in roots y s stndrd. umultion of GS1 polypeptide with low-ffinity [8]. Free mino ids (AA) were mesured in roots nd In this pper, we study in roots nd leves of leves extrts with ninhydrin regent ording to sorghum plnts, the influene of high level of nitrogen Mgné nd Lrher [11]. A stndrd urve ws prepred (mmonium or nitrte) on (i) tivities of other enzymes with glyine. involved in nitrogen metolism suh s: Nitrte redutse (NR), Glutmine synthetse (GS) nd Glutmte Estimtion of Chlorophyll: The extrtion of lef dehydrogense (GDH) (ii) onentrtions of free mino hlorophyll ws performed with 80% etone. The ids, proteins nd hlorophyll. hlorophyll, hlorophyll nd totl hlorophyll Therefore, the im of this study ws to nlyze the quntities were lulted ording to the method of prtitioning of N ssimiltion etween roots nd leves Arnon [12]. whih ould help to eluidte the mehnism of mmonium tolerne in S. sudngrss plnts. Extrtion nd Essy of Nitrte Redutse: Leves or roots were homogenized in hilled mortr nd pestle with MATERIALS AND METHODS 100 mm potssium phosphte uffer (ph 7.4) ontining 7.5 mm ysteine, 1 mm EDTA, 1 mm PMSF nd 1.5% Plnt Mteril nd Growth: Sorghum seeds (Sorghum (w/v) sein. The homogente ws entrifuged t 30, 000g sudngrss) were sterilized with 5% of NOCl for 15 for 15 min t 4 C. Nitrte redutse tivity (NRA) ws minutes nd wshed thoroughly with sterile wter nd determined ording to the method desried y Roin germinted on filter pper (Whtmnn pper) in petri [13]. dish soked in distilled wter for 4 dys under drk ondition t 26 C nd plnted in pot filled with Extrtion nd Assy of Glutmine Synthetse: Leves or vermiulite, then grown in growth hmer. The roots were extrted in old mortr nd pestle with environmentl onditions in the growth hmer were grinding medium ontining 50 mm Tris HCl ph 8.0, 5 70% humidity, 25 C nd light intensity of 1000 Lux with mm MgSO 2, 12 mm Glutmte, 2 mm EDTA, 10% (v/v) 14 h photoperiod. The plnts reeived (twie week glyerol, 0.1% (v/v) 2-merptoethnol. The homogente (100 ml/pot)) nutrient solution of 0.5 mm KNO 3, 0.375 ws entrifuged t 13 000g for 30 min t 4 C. Glutmine mm KH2PO 4, 0.125 mm K2HPO 4, 0.375 mm MgSO 4, 0.1 synthetse tivity ws mesured using the trnsferse mm NCl, 1.25 mm CSO 4, 10 mg/l Fe-ethylene-dimine ssy s desried y Shpiro nd Stdtmn [14]. tetrette (Fe-EDTA) nd mironutrients [9], ph 6.4 (±0.1). For the treted ultures, the nutrient solution ws Extrtion nd Assy of Glutmte Dehydrogense: supplemented with different onentrtions of KNO 3 or Leves or roots were extrted in old mortr nd pestle (NH 4) 2SO 4(5, 20, 50 mm). with grinding medium ontining 50 mm Tris-HCl ph 8.2, Plnts were hrvested 20 dys fter inititing N 5 mm 2-merptoethnol, 1 mm CC1 2 nd 5% PVP. tretments, then immeditely sorted into leves nd roots. The homogente ws entrifuged t 13000g for 20 min t Roots were quikly nd gently wshed with deionized 4 C. The mintion retion ws mesured t 30 C in 1861
Am-Eurs. J. Agri. & Environ. Si., 15 (9): 1860-1867, 2015 100 mm Tris-HCl, ph 8.2, ontining 100 mm NH4Cl, 10 Amino Aids: The presene of mmonium s sole mm 2-oxoglutrte, 0.16 mm NADH nd 4 mm CC1 2. nitrogen soure leds to signifint inrese in ontent The 2-oxoglutrte-dependent oxidtion of NADH of free mino ids in leves nd roots, this inrese is followed t 340 nm s desried y Loyol-vrgs nd De importnt tht the mmonium onentrtion is high Jimenez [15]. (Tle 2). Nitrte lso indues n inrese in free mino ids in oth orgns. However, this inrese is very SDS PAGE nd Western Blot Anlysis: Crude enzymti modest ompred to tht indued y mmonium (Tle 2). extrts were seprted on 12.5% SDS polyrylmide In present study, the mino id onentrtion gels, the resolved polypeptides were eletrotrnsferred (Tle 2) ws higher in the roots thn in the leves in onto nitroellulose memrnes nd the presene of GS presene of high nitrogen onentrtions, prtiulrly the polypeptides immunoreveled with speifi ntiodies. mmonium. This umultion is relted to the rpid Immunolelling ws rried out essentilly s desried ssimiltion of mmonium sored, without whih the y Cánovs et l. [16] using the ntiserum rised ginst umultion of NH 4 ions my reh toxi levels [19]. reominnt pine GS [17]. Susequent detetion of This requires keto ids espeilly 2-oxoglutrte nd immunoomplexes ws rried out y peroxidise ssy. oxloette for synthesis of glutmique id nd sprtique id, s well s their mides; glutmine nd Sttistil Anlysis: The dt shown men vlues ±S.E. sprgine. Our results shown tht phosphoenolpyruvte Results were sujeted to one-wy nlysis of vrine roxylse (PEPC) leds to n inrese prodution of with lest signifint differene (LSD) test etween itri id yle ron skeletons needed for mmonium mens using Sttgrphis 5.0. Levels of signifine ssimiltion y npleroti fixtion of CO 2 (dt not were represented y t P<0.05, t P<0.01 nd t shown). Brneix nd Cusin [20] reported tht the P<0.001. mmoni-grown plnts often hve higher free mino ids onentrtion in their tissues thn nitrte fed plnts. RESULTS AND DISCUSSION Proteins: The presene of different onentrtions of Growth Prmeters: Sorghum plnts were grown for 20 mmonium in nutrient medium leds to signifint dys under low nd high levels of inorgni nitrogen inrese of the protein ontent in the leves nd the roots supply (Tle 1). Similr iomss prodution ws found when ompred with ontrol plnts; eing pproximtely t moderte level of nitrte (5 mm) when ompred with 2 fold in 20 mm nd 3 fold in 50 mm (Tle 2). Wheres ontrol plnts (0.5mM nitrte) (Tle 1). However, the nitrte did not ffet the protein ontent tht from enhned iomss (dry weight, fresh weight nd height) 20 mm in the leves, ut in the roots indue signifint were oserved t high levels of nitrte (20 nd 50 mm). inrese in protein ontent t different onentrtions The utilistion of mmonium s sole N soure in the used ompred to the ontrol plnts (Tle 2). nutrient solution led to enhned vlues of dry mtter Our results shown tht the protein ontent ws prodution, fresh weight nd height when ompred with higher in plnts treted with high levels of mmonium those oserved in ontrol plnts (Tle 1). Mximl espeilly in leves (Tle 2). This shows tht prt of iomss umultion ws oserved t 5 mm of mino ids synthesized in the roots, trnsported to mmonium supply (2 fold of DW nd 2.6 fold of FW). leves vi xylem nd used for protein synthesis neessry Prdoxilly, plnts growing under extremely high level for tissue development in prtiulr ; enzymes. of mmonium (50 mm) exhiited similr iomss prodution thn ontrol plnts (Tle 1). Chlorophyll: In this study, hlorophyll ontent inresed Generlly most plnt speies show redued in leves sorghum plnts treted with different growth, smller leves nd stunted root system when onentrtions of oth nitrogen soures with remrkle exposed to high nitrogen onentrtions nd in severe inrese in presene of mmonium (Tle 3). Smolov et l. ses this leds to the deth of the plnt [18]. The ility [21] hve shown tht the presene of mmonium in the of S. sudngrss hyrids to growth nd yield enhned ulture medium of Hetero- nd Mixotrophi Glyine mx levels of plnt iomss under high levels of either nitrte indues sustntil inrese in hlorophyll moleules or mmonium nutrition nd did not show pprent signs involved in protein-pigment omplexes of hloroplsts. of toxiity is well orrelted with the inresed GS tivity Smolov nd Semenov [22] suggested tht mmonium nd the speifi regultion of GS1 isoforms in roots [8]. exerts n indiret influene on the pigment synthesis in 1862
Am-Eurs. J. Agri. & Environ. Si., 15 (9): 1860-1867, 2015 Tle 1: Effet of nitrogen supply (nitrte or mmonium) on length, fresh weight (FW) nd dry weight (DW) in sorghum plnts Nitrte (mm) Ammonium (mm) --------------------------------------------------------- ------------------------------------------------------------ Control 5 20 50 5 20 50 Height (m) 48±7 59±5 64±3 69±2 71±4 66±3 62±2 FW (mg) 265±31 260±42 476±13 518±18 693±23 456±15 308±40 DW(mg) 60±3 55±8 85±6 93±5 121±18 81±5 67±4 Eh vlue represents the men of ten leves or roots with S.D., nd indite signifine t P<0.05, P<0.01 nd P<0.001 respetively when ompred to ontrol -1-1 Tle 2: Effet of nitrogen (mmonium or nitrte) supply on protein ontent (mg g FW) nd mino id ontent (AA) ( mol g DW) in leves nd roots of sorghum plnts Nitrte (mm) Ammonium (mm) ------------------------------------------------------------------ ------------------------------------------------------------------- Control 5 20 50 5 20 50 Roots Protein 4.5±0.3 6.7±0.2 6.9±0.4 7.1±0.6 7.4±0.3 8±0.2 12±0.6 AA 0.43±0.04 0.5±0.06 0.56±0.08 0.78±0.09 0.65±0.04 1.02±0.12 1.41±0.16 Leves Protein 12.9±0.4 13.8±0.6 16.7±0.3 17.6±0.4 21±0.4 26.7±0.3 33±1 AA 0.38±0.05 0.44±0.06 0.41±0.01 0.51±0.08 0.42±0.01 0.63±0.08 0.8±0.09 Eh vlue represents the men of three or four independent oservtions with S.D., nd indite signifine t P<0.05, P<0.01 nd P<0.001 respetively when ompred to ontrol. Tle 3: Effet of nitrogen (mmonium or nitrte) supply on totl hlorophyll (mg/gfw), hlorophyll (mg/gfw) nd hlorophyll (mg/gfw) in leves sorghum plnts Nitrte (mm) Ammonium (mm) ------------------------------------------------------------------ ------------------------------------------------------------------- Control 5 20 50 5 20 50 Chl () 1.6±0.1 2±0.5 2.4±0.45 2.6±0.4 2.5±0.2 3.6±0.2 3±0.1 Chl () 0.47±0.09 0.92±0.07 1±0.06 0.86±0.8 0.62±0.5 0.91±0.02 0.92±0.7 Chl (tot) 2.35±0.3 2.92±0.4 3.4±0.4 3.6±0.3 3.6±0.4 4.8±0.2 4±0.1 Eh vlue represents the men of three or four independent oservtions with S.D., nd indite signifine t P<0.05, P<0.01 nd P<0.001 respetively when ompred to ontrol the Cells of Soyen Mixotrophi Cllus, for exmple, due 50 mm), nitrte redutse tivity inresed signifintly to the tivted synthesis of free mino ids triggering in oth orgns. the hin of hlorophyll iosynthesis or due to the Our study shows tht in sorghum sudngrss retion of dditionl sites for pigment inorportion into hyrids, nitrte ssimiltion is distriuted etween roots the newly formed memrnes euse of elerted nd leves in the presene of high levels of nitrte. synthesis of protein omponents. R nd Terry [23] These results re in greement with those otined y reported tht higher hlorophyll ontent in mmonium-fed Andrews (1986) [26] who found tht in Riinus ommunis, Bet vulgris L plnt my even result in higher net Oryz stiv nd Solnum tuerosum, the shoot is the photosyntheti rtes s ompred to nitrte-fed plnts. mjor site of nitrte ssimiltion when given 1 molxm 3 of nitrte nd the prtitioning of nitrte ssimiltion etween Nitrte Redutse: In most plnts, nitrte ssimiltion root nd shoot remins onstnt s externl nitrte tkes ple predominntly in leves. In ordne, our onentrtion is inresed. However, the presene of results shown tht NR tivity (Fig. 1) ws higher in mmonium in the feeding medium hs not ffeted the leves thn in roots of sorghum plnts. An elevted NRA of leves or roots. Oks et l. [27] reported tht nitrte redution in leves ompred to roots ws relted mize root NRA ws depressed y mmonium t feeding to the higher NR protein ontents [24] nd suffiient medium ph of 7.5, ut stimulted y mmonium t ph 5.8. vilility of light nd reduing power [25]. Figure 1 Thus, feeding medium ph ould e n importnt ftor in shows tht nitrte redutse tivity ppers unffeted determining the effet of nutrient mmonium on NRA. The y the presene of mmonium s nitrogen soure. feeding medium ph in this experiment ws mintined t However, in the presene of high levels of nitrte (20 nd the 6.4. 1863
Am-Eurs. J. Agri. & Environ. Si., 15 (9): 1860-1867, 2015 Fig. 1: Nitrte redutse tivity (NR) in the leves nd the roots of sorghum sudngrss hyrids grown under inresed levels of nitrte or mmonium supply ( : Ammonium, : Nitrte). Eh vlue represents the men of three or four independent oservtions with S.D., nd indite signifine t P<0.05, P<0.01 nd P<0.001 respetively when ompred to ontrol (0.5 mm KNO ) 3 Fig. 2: GS tivity nd GS protein undne in the leves nd the roots of sorghum sudngrss hyrids grown under inresed levels of nitrte or mmonium supply (mmonium:, Nitrte: ). Eh vlue represents the men of three or four independent oservtions with S.D., nd indite signifine t P<0.05, P<0.01 nd P<0.001 respetively when ompred to ontrol (0.5 mm KNO 3). Control smple orresponds to plnts grown under low nitrogen (nitrte 0.5 mm). The size of the GS polypeptide (40 kd) is indited on the left. The sme mount of protein ws loded per lne in the gels Glutmine Synthetse: In the present study, we hve The inresed GS tivity in roots in presene of found tht high levels of nitrte or mmonium led to mmonium nutrition ws due to umultion of GS1 umultion of GS tivity in sorghum roots. However, polypeptide with low-ffinity who would provide this effet ws more pronouned with mmonium (Fig. 2). sustined glutmine iosynthesis t high levels of Aumultion of GS tivity in sorghum roots in presene mmonium supply nd my represent t the sme time n of nitrte might e explined y n indiret effet of effiient system of mmonium detoxifition [8]. nitrte fter its redution to mmonium in roots. In ft, we The inrese of lef GS tivity oserved in presene hve deteted inresed levels of nitrte redutse of nitrte or mmonium is due to n umultion of tivity in the roots of nitrte-fed sorghum plnts (Fig. 1). hloroplsti GS (GS2), prtiulrly in the presene of 1864
Am-Eurs. J. Agri. & Environ. Si., 15 (9): 1860-1867, 2015 Fig. 3: Glutmte dehydrogense tivity (GDH) in the leves nd the roots of sorghum sudngrss hyrids grown under inresed levels of nitrte or mmonium supply ( : Ammonium, : Nitrte). Eh vlue represents the men of three or four independent oservtions with S.D., nd indite signifine t P<0.05, P<0.01 nd P<0.001 respetively when ompred to ontrol (0.5 mm KNO ) 3 nitrte (Fig. 2). Confliting results re reported on the mmonium-fed plnts (Fig. 3). In previous study; effet of NH 4 on GS hloroplsti; in mjority of plnt Ls et l. [1] hve een found tht GDH tivity speies, mmonium does not seem to hve ny effet on inresed in pe plnts (mmonium-tolernt speies) when hloroplsti GS tivity ut Mäk [28] ws reported tht swithed from nitrte to mmonium nd this inrese ws mmonium nutrition stimulted GS2 tivity in rley orrelted with n inrese in the mino id ontent, leves. Brehlin et l. [29] suggested tht the NH 4 ws showing tht the minting tivity of GDH my hve rpidly ssimilted y root GS1 nd presumly lso y role in the mmonium detoxifition. Cmmerts nd lef GS2. Wllsgrove et l. [30] onfirm the ssumption Jos [34] hve shown tht GDH tivity inrese in tht, esides its role in ressimiltion of photorespirtory roots of Aridopsis thlin in response to inresed NH 4, lef GS2 my funtion in the ssimiltion of primry free mmonium ontents nd suggest tht high GDH NH 4. tivity my e relted to high inorportion of The reltive inrese in GS2 protein nd Fd-GOGAT mmonium into mino ids to prevent toxiity. (dt not shown) in the presene of high onentrtions of nitrte, let us ssume tht prt of nitrte is CONCLUSION trnsported from the roots to the leves vi the xylem indues its redution to nitrite nd therefore inresing The results showed tht growth, proteins, mino the ssimiltion of mmonium in hloroplst. Our results ids, pigment levels nd tivities of enzymes involved re in greement with the work of Hykw et l. (1990) in nitrogen metolism suh s; glutmine synthetse [31] who hve een found tht NO 3 inresed the (GS) nd glutmte dehydrogense (GDH), were severl undne of GS2 nd Fd-GOGAT protein in ultured fond higher in leves nd roots of sorghum plnts under rie ells. Toin nd Ymy (2001) [32] hve oserved high nitrogen levels espeilly in presene of mmonium. tht 15 mm of nitrte indues n inrese in the ontent Thus, Sorghum (S. sudngrss) plnts exhiited of GS2 in rley leves s ompred to plnts ontrol higher dptive potentil under exessive onentrtion (0.1 mm NO 3 ). of mmonium y ility to detoxify this ion vi nitrogen ssimiltion in roots. Glutmte Dehydrogense: Even though the GS/GOGAT pthwy is the mjor route in higher plnts [33], they re REFERENCES le to use other lterntive routes suh s those tlyzed y GDH, whih tlyzes the reversile 1. Ls, B., S. Frehill, P.M. Apriio-Tejo nd mintion of 2-oxoglutrte to yield glutmte. Our C. Lmsfus, 2002. Role of glutmte dehydrogense experiment shows tht GDH tivity ws grdully nd phosphoenolpyruvte roxylse tivity in inresed in oth orgns with inresing externl mmonium nutrition tolerne in roots. Plnt Physiol. nitrogen soure (mmonium or nitrte) prtiulrly in Biohem., 40: 969-976. 1865
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