Inter-specific variability in protein use by two vegetable crop species

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1 2010 rzilin soiety of plnt physiology DOI /S RESERCH RTICLE Inter-speifi vriility in protein use y two vegetle rop speies rtosz mzyk*, Mirosłw Golewski Deprtment of Plnt Cytology n Cytohemistry, Institute of Plnt Physiology, Cytology n Cytogeneti, University of Łóź, Łóź, Poln * Corresponing uthor: rtosz mzyk, Deprtment of Plnt Cytology n Cytohemistry, Institute of Plnt Physiology, Cytology n Cytogeneti, University of Łóź, Poln, e-mil: rtek_mzyk_79@o2.pl Reeive: 04 My 2010; epte: 27 Septemer 2010 strt It is now well-known tht plnts n uptke not only inorgni nitrogen ut lso orgni nitrogen ompouns, minly mino is. However, soil proteins re the min pool of mino is. oring to our erlier ppers, plnts n get ess to this soure of nitrogen using root-serete proteses, ut the level of proteolyti tivity of suh root-serete proteses is speies-speifi. Our im ws to ompre the use of protein s nitrogen soure y two vegetle rops hving high (llium porrum) or low (Ltu stiv) level of tivity of root-serete proteses. Seelings were ultivte on Murshige n Skoog meium (MS), MS meium without inorgni nitrogen, MS meium without inorgni nitrogen, ut with sein in onentrtion of 0.01%, 0.1% or 1%. Fresh weight of shoot of. porrum ws the highest for seelings growing on ulture meium with sein, ut shoots of L. stiv otine the highest weight growing on the ulture meium with inorgni nitrogen. llium porrum seelings otine 15-fol higher proteolyti tivity in the ulture meium thn L. stiv. Seelings of. porrum using suh high tivity of proteses serete y roots oul provie sustntil pool of mino is for intensive growth. The urrent stuies onute on. porrum n L. stiv suggest tht the effiieny of protein use in nitrogen nutrition y plnts is speies-speifi. Keywors: orgni nitrogen, plnt nitrogen nutrition, seretion of proteses Introution It is now well estlishe tht plnts n uptke not only inorgni nitrogen, ut lso orgni forms of this nutrient, minly mino is, even in the presene of miroil ompetitors (Lipson n Näsholm, 2001; Ge et l., 2009; Näsholm et l., 2009), n espeilly in onitions of high mino i onentrtions in the soil (Jones et l., 2005; Suheitl et l., 2009). The ility of plnts to uptke mino is ws proven in numerous experiments for mny plnt speies (i.e. Flkengren-Grerup et l., 2000; Persson n Näsholm, 2001; Xu et l., 2004; Hirner et l., 2006; Rentsh et l., 2007; Svennerstm et l., 2007). mino is re the mjor frtion of soil nitrogen; however, they re minly present in polymeri form s proteins (Kye n Hrt, 1997). Protein utiliztion therefore is ssoite with proteolysis riven y miroil proteses. Our erlier stuies showe, tht lso plnts n serete proteses y intt roots (Golewski n mzyk, 2007) n tht Tritium estivum roots n iretly utilize proteins in the ulture meium s soure of nitrogen without prior igestion y miroil proteses (mzyk et l., 2008). This phenomenon ws lso proven y Pungfoo-Lonhienne et l., (2008); their stuies were onute on two non-myorrhizl speies - riopsis thlin n Hke tities; itionlly these uthors showe tht riopsis thlin growth ws supporte etter y protein n low inorgni nitrogen thn protein or low inorgni nitrogen lone. Moreover, Pungfoo-Lonhienne

2 172 rtosz mzyk n Mirosłw Golewski et l. (2008) suggeste possile uptke of intt protein y plnt roots vi enoytosis. In our erlier pper we prove tht proteses serete y plnt roots n igest proteins (sein, ovine serum lumin) to low-moleulr-mss prouts (mzyk et l., 2009) whih n e tken up iretly y plnt roots (Rentsh et l., 2007; Tsy et l., 2007; Pungfoo-Lonhienne et l., 2009). However, the level of proteolyti tivity in the ulture meium of ifferent plnt speies vrie signifintly (Golewski n mzyk, 2007). lso the uptke of mino is the prout of proteolysis proess - iffers etween vrious plnt speies (Weigelt et l., 2003, 2005; Okmoto n Ok, 2004; Reeve et l., 2009). In this pper we were looking for linkge etween these ifferenes in orgni nitrogen uptke n the level of proteolyti tivity in the ulture meium. We were stuying if there re some ifferenes in the use of proteins y vrious plnt speies llium porrum v. rtek n Ltu stiv v. Ewelin. We hve hosen these speies euse of high isrepny etween their root-serete proteolyti tivities in the ulture meium in preliminry stuies. porrum h the highest n L. stiv h the lowest proteolyti tivity, ompring to other stuie speies (Golewski n mzyk, 2007). These plnt speies were ultivte in sterile onitions on Murshige n Skoog meium (MS; Murshige n Skoog, 1962), MS meium without inorgni nitrogen, MS meium without inorgni nitrogen, ut with sein (0.01%, 0.1% or 1%). Our hypotheses were tht (1). porrum seelings woul hve higher shoot weight reltive to L. stiv, growing on meium ontining protein (2) L. stiv woul e less effetive t growing on protein ompre to. porrum seelings (3) these vritions in protein use oul e reflete lso in the level of proteolyti tivities in the ulture meium. Mterils n Methos Plnt mteril n ultivtion: Sees of llium porrum L. v. rtek n Ltu stiv v. Ewelin were otine from ommeril plnt istriutor (Torsee, Poln). Plnts were ultivte in septi onitions s in our previous stuies (Golewski n mzyk, 2007). riefly, sees were surfe sterilize with 70% ethyl lohol n with 10% soium hypohlorite; n germinte on Petri ishes (7 ys) n lter seprtely in tues (for 2 weeks; whole experiment took 3 weeks), ontining 15 ml of utolve liqui meium: Murshige n Skoog meium (MS), MS meium without inorgni nitrogen, MS meium without inorgni nitrogen, ut with sein in onentrtion of 0.01%, 0.1% or 1%. MS meium without moifitions inlues 60 mm of inorgni nitrogen, MS meium without inorgni nitrogen ut with sein inlue: 1.18 mm, 11.8 mm n 118 mm of nitrogen for 0.01%, 0.1% n 1% sein onentrtion, respetively. The experiment ws performe on liqui meium inste of sterilize soil, euse soil ontins oth, inorgni n orgni soures of nitrogen, so it woul not e possile to otin very ontrolle onitions of nitrogen soures. The seelings were ultivte t 23±1 C ir temperture, 70% reltive humiity, n 16:8 h photoperio with 380 µmol m-2 s-1 light intensity t plnt height. Sterility in the ulture meium fter ultivtion n purifition ws verifie with miroiologil tests (Miroount omi; Shülke-Myr). Proteolyti tivity mesurements: The ulture meium ws purifie n onentrte using Vivpore 5 solvent sorption onentrtor (Sigm) with pore size 7500 MWCO. Proteolyti tivity ws etermine using metho y Tomrelli et l. (1949), in whih the time-epenent relese of zo yeouple trihloroeti i solule peptie frgments from the sustrte, zosein, ws monitore. riefly, 200 μl of prtilly purifie n onentrte ulture meium ws mixe with 100 μl 0.5% (w/v) zosein issolve in 0.9% NCl in 50 mm phosphte uffer (ph 6.8). fter 4 h inution, the retion ws stoppe y ing 200 μl 20% (w/v) trihloroeti i. fter entrifugtion (10000g, 5 min.), 150 μl of the resulting superntnt ws mixe with 50 μl 1 M NOH, n fter 30 minutes sorne t 440 nm ws re (Hithi U-2000 Spetrophotometer, Jpn). In the ontrol, trihloroeti i ws e to the ulture meium prior to ition of zosein. One unit of protese tivity ws efine s the mount of enzyme tht inrese the sorne y 0.1 t 440 nm per 1 h. ll regents were purhse from Sigm. Proteolyti tivities re presente per one squre millimeter of the root surfe, whih ws mesure with stereologil metho, s esrie y He (1966), on the sis of length of the root n root fresh weight. zosein metho ws hosen for this stuy euse it is nonspeifi protese sustrte, wht mens tht zosein

3 inter-speifi vriility in protein use y two vegetle rop speies 173 n e esily igeste y ifferent proteses. zosein is often use for mesuring the overll proteolyti tivity (Hno et l., 2008). Methos of mesurements of proteolyti tivity on the sis of zosein igestion re wiely use (i.e. Zhng et l., 2007; Rojs et l., 2009). Sttistis: Eh experiment onsiste of 6 replites (n=6). Proteolyti tivity mens were ompre mong nitrogen tretments n fresh weight mens were ompre mong nitrogen tretments using one-wy NOV, followe y Tukey s test. We use Sttisti (Sttsoft, In.). Results Fresh weight of roots n shoots of. porrum n L. stiv: Fresh shoot weight of. porrum ws the lowest (9.8 mg) on MS meium without ny soure of nitrogen n the highest shoot growth (32.8 mg) ws otine on MS meium in whih inorgni nitrogen ws reple y sein (0.1% n 1% onentrtions) (P<0.01) (Figure 1 ). Growth of. porrum shoot ws similr on stnr MS meium ompre to MS meium without inorgni nitrogen ut with 0.01% sein (P<0.99). Fresh shoot weight of L. stiv ws the lowest (11 mg) on MS meium without ny soure of nitrogen n the highest shoot growth (71.2 mg) ws otine on MS meium with inorgni nitrogen (Figure 1 ). The lowest vlues of fresh weight of roots of. porrum (2.7 mg) were otine for seelings growing on MS without nitrogen; the rest of meium vrints gve higher results (from 5 to 6.5 mg), ut similr to eh other (no sttistil ifferenes) (Figure 1 ). The lowest fresh weight of roots of L. stiv (2.6 mg) ws otine for seelings growing on MS without nitrogen, higher vlues were otine for seelings ultivte on stnr MS meium n MS with 0.01% sein (6 n 8.2 mg, respetively), ut the highest L. stiv root fresh weight ws otine for seelings growing on MS with 0.1% or 1% sein (10.5 n 11.2 mg, respeitvely) (Figure 1 ). Fresh weigth, mg 8 6 shoot root Fresh weigth, mg 3 1 shoot root 1 Figure 1. Fresh weight of L. stiv (1) n. porrum seelings (1) of seelings ultivte on ifferent mei: (1) Murshige n Skoog meium, (2) MS meium without inorgni nitrogen, (3) MS meium without inorgni nitrogen, ut with 0.01% sein, (4) MS meium without inorgni nitrogen, ut with 0.1% sein, (5) MS meium without inorgni nitrogen, ut with 1% sein. Vlues re mens of six replites. Error rs inite stnr error of the men. Sttistilly signifint ifferenes (P<0.05) re inite y ifferent letters. Comprison of the level of growth of. porrum with tht of L. stiv: To ompre the level of growth of. porrum n L. stiv we presente the results lso in the form of perent of growth in omprison with growth on stnr MS meium, whih ws trete s 100% of growth (Figure 2). Inter-speies omprison of shoot weight revele tht. porrum seelings otine signifintly higher level of growth on MS mei with sein in omprison with the level of growth of shoot of L. stiv (P<0.05). In the sme wy we lso presente root growth (Figure 2). In omprison with root fresh weight of. porrum, L. stiv roots otine higher weight growing on mei with sein (sttistilly signifint for mei with 0.1% n 1% sein).

4 174 rtosz mzyk n Mirosłw Golewski Shoot iomss, % of ontrol porrum L. stiv f e e Root iomss, % of ontrol porrum L. stiv Figure 2. Comprison of growth of Ltu stiv n llium porrum shoot (2) n root (2) on ifferent ulture mei: (1) MS meium without inorgni nitrogen, (2) MS meium without inorgni nitrogen, ut with 0.01% sein, (3) MS meium without inorgni nitrogen, ut with 0.1% sein, (4) MS meium without inorgni nitrogen, ut with 1% sein. Results re presente s perent of growth on stnr MS meium (growth on stnr MS meium ws trete s 100%). Vlues re mens of six replites. Error rs inite stnr error of the men. Sttistilly signifint ifferenes (P<0.05) re inite y ifferent letters. Proteolyti tivity in the ulture meium: llium porrum seelings otine 15-fol higher proteolyti tivity thn L. stiv (Figure 3). There were lwys sttistilly signifint ifferenes etween tivities otine for L. stiv n. porrum in eh ulture meium (not shown on figure). Ltu stiv n. porrum seelings showe higher proteolyti tivities growing on mei with sein, espeilly in higher onentrtions (for 1% sein onentrtion n U m 2 of proteolyti tivity, respetively), in omprison with mei with inorgni nitrogen (0.002 n U m 2 of proteolyti tivity, respetively); ut in the se of. porrum, tht inrese in tivity ws greter. Proteolyti tivity, U/m² L. stiv. porrum Figure 3. Proteolyti tivity in the ulture meium of seelings ultivte on ifferent mei: (1) Murshige n Skoog meium, (2) MS meium without inorgni nitrogen, (3) MS meium without inorgni nitrogen, ut with 0.01% sein, (4) MS meium without inorgni nitrogen, ut with 0.1% sein, (5) MS meium without inorgni nitrogen, ut with 1% sein. Vlues re mens of six replites. Error rs inite stnr error of the men. Sttistilly signifint ifferenes (P < 0.05) etween. porrum mei re inite y ifferent letters n signifint ifferenes n etween L. stiv mei re inite y pitls. There were lwys sttistilly signifint ifferenes etween tivities otine for L. stiv n. porrum in eh ulture meium (not shown on figure). Disussion There is inresing interest in eveloping eologilfrienly methos for plnt ultivtion. primry mens of ttining this gol is to erese the use of inorgni nitrogen fertilizers, whih ontriute to environmentl pollution (Hung et l., 2003; Wng et l., 2004). Plnt ssimiltion of orgni nitrogen (reviewe y Näsholm et l., 2009) provies mens for mintining high plnt yiel n reuing environmentl pollution. Plnt speies n iffer in the ility to use soil orgni nitrogen soures (i.e. Okmoto n Ok, 2004). oring to our erlier pper, plnts growing in the sme onitions n iffer in the level of proteolyti tivity in the ulture meium, i.e. llium porrum showe severl fol higher proteolyti tivity thn L. stiv (Golewski n mzyk, 2007). In this pper, we expne tht oservtion y the use of ifferent vrints on MS mei (with inorgni or with orgni nitrogen soure). oth plnt

5 inter-speifi vriility in protein use y two vegetle rop speies 175 speies (. porrum n L. stiv) were le to use sein s nitrogen soure n in the se of oth speies ition of protein to the ulture meium resulte in signifint inrese of proteolyti tivity. However,. porrum ws signifintly more effetive t growing on sein s nitrogen soure thn L. stiv; suh result ws unerline y omprison of shoot growth of these two speies s perent of growth (growth on stnr MS meium ws trete s 100%) (Figure 2). However, similr omprison of the level of root growth showe ifferent pttern - roots of L. stiv otine higher fresh weight thn roots of. porrum. Inrese growth of roots in omprison with shoots n point to nitrogen efiieny (Roinson n Rorison, 1988; mezine et l., 1995) in L. stiv seelings ultivte on meium with orgni nitrogen. Plnts hving higher proteolyti tivity in the ulture meium shoul show higher ffinity to orgni nitrogen soures in the form of mino is - prouts of proteolysis. This is in orne with pper y Mtsumoto et l. (1999) in whih uthors showe tht L. stiv prefers inorgni over orgni nitrogen soures, n with Termine et l. (1987) stuies, in whih. porrum plnts were growing omprly on inorgni n orgni nitrogen soures. However, one shoul rememer tht these stuies were onute in lortory onitions n in fiel onitions prt of orgni soil nitrogen oes not exist in n esy essile form, i.e. in omplexes with tnnins (ening n Re, 1996). Moreover, tnnins n not only rete omplexes with soil proteins sustrtes for proteolysis, ut tnnins n lso erese proteolyti tivity (He et l., 2006; mzyk et l., 2009). oring to our hypotheses,. porrum seelings otine higher shoot fresh weight on meium with sein thn L. stiv. Signifint vritions in proteolyti tivities in ifferent ulture mei (highest in. porrum thn in L. stiv, espeilly in the se of mei with sein) point to mehnism hien y these ifferenes in plnt growth - seelings of. porrum y using higher proteolyti tivities in the ulture meium thn L. stiv were strongly inresing the pool of essile orgni nitrogen. Stuies onute on. porrum n L. stiv suggest, tht lso other plnt speies n effetively use proteins in nitrogen nutrition; however effiieny of this strtegy is speies-speifi. Referenes mzyk, Golewski M, Smolner, Kitunen V (2009) Degrtion of proteins y enzymes exue y llium porrum roots - potentilly importnt strtegy for quiring orgni nitrogen y plnts. Plnt Physiol. iohem. 47: mzyk, Golewski M, Zimny J, Zimny (2008) Whet (Tritium estivum) seelings serete proteses from the roots n, fter protein ition, grow well on meium without inorgni nitrogen. 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