Enhancement of Salinity Tolerance during Rice Seed Germination by Presoaking with Hemoglobin

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1 Int. J. Mol. Sci. 2011, 12, ; doi: /ijms Article OPEN ACCESS Interntionl Journl of Moleculr Sciences ISSN Enhncement of Slinity Tolernce during Rice Seed Germintion y Presoking with Hemogloin Sheng Xu 1, Bing Hu 1, Ziyi He 1, Fei M 1, Jinfei Feng 1, Wenio Shen 1, * nd Jie Yng 2, * 1 2 Lortory Centre of Life Sciences, College of Life Sciences, Nnjing Agriculturl University, Nnjing , Chin; E-Mils: @nju.edu.cn (S.X.); njhuing@sin.com (B.H.); njheziyi@sin.com (Z.H.); @nju.edu.cn (F.M.); @nju.edu.cn (J.F.) Institute of Food Crops, Jingsu Acdemy of Agriculturl Sciences, Nnjing , Chin * Authors to whom correspondence should e ddressed; E-Mils: wshenh@nju.edu.cn (W.S.); yngjie1698@hotmil.com (J.Y.); Tel.: ; Fx: Received: 7 Decemer 2010; in revised form: 6 April 2011 / Accepted: 6 April 2011 / Pulished: 12 April 2011 Astrct: Slinity stress is n importnt environmentl constrint limiting the productivity of mny crops worldwide. In this report, experiments were conducted to investigte the effects of seed presoking y ovine hemogloin, n inducer of heme oxygense-1 (HO-1), on slinity tolernce in rice (Oryz stiv) plnts. The results showed tht different concentrtions of the hemogloin (0.01, 0.05, 0.2, 1.0, nd 5.0 g/l) differentilly llevited the inhiition of rice seed germintion nd therefter seedling shoot growth cused y 100 mm NCl stress, nd the responses of 1.0 g/l hemogloin ws the most ovious. Further nlyses showed tht ppliction of hemogloin not only incresed the HO-1 gene expression, ut lso differentilly induced ctlse (CAT), scorte peroxidse (APX), nd superoxide dismutse (SOD) ctivities or trnscripts, thus decresing the lipid peroxidtion in germinting rice seeds sujected to slt stress. Compred with non-hemogloin tretment, hemogloin presoking lso incresed the potssium (K) to sodium (N) rtio oth in the root nd shoot prts fter slinity stress. The effect is specific for HO-1 since the potent HO-1 inhiitor zinc protoporphyrin IX (ZnPPIX) locked the positive ctions of hemogloin on seed germintion nd seedling shoot growth. Overll, these results suggested tht hemogloin performs n dvntgeous role in enhncement of slinity tolernce during rice seed germintion.

2 Int. J. Mol. Sci. 2011, Keywords: heme oxygense-1; hemogloin; slinity tolernce; seed germintion; Oryz stiv 1. Introduction Seed germintion is complex process involving vrious physicl nd iochemicl cues such s wter, light nd phytohormones. It hs een widely reported tht seed germintion s well s seedling growth re sensitive to vrious iotic stresses. A growing ody of evidence lso showed tht the inhiitory effect of slt stress on seed germintion is llevited y phytohormones, including ethylene [1], cytokinin (CTK) [2]), gierellin cid (GA) [3], nd messenger molecules such s hydrogen peroxide (H 2 O 2 ) [4], nitric oxide (NO) [5], nd cron monoxide (CO) [6]. Menwhile, high slt concentrtions could cuse ion imlnce nd hyperosmotic stress in plnts. Additionlly, secondry stresses often occur, such s oxidtive stress due to the overproduction of rective oxygen species (ROS) in slt stressed plnts [7]. To quench ROS, plnt cells possess oth enzymtic nd non-enzymtic mechnisms for the scvenging of ROS overproduction. It hs een well estlished tht the coordintion ctivities of the multiple forms of ntioxidnt enzymes, such s superoxide dismutse (SOD), ctlse (CAT), scorte peroxidse (APX) in the different cell comprtments, chieve lnce etween the rte of formtion nd the scvenging of ROS, nd mintin ROS t suitle levels required for cell signling. Heme ctolism in wide rnge of species is medited y the heme oxygense fmily of enzymes (HOs; EC ). In nimls, HOs re locted within the endoplsmic reticulum, where they serve, in concert with NADPH cytochrome P450 reductse, to oxidize heme to iliverdin, free ferrous iron, nd CO. It is well known tht HOs exist in three mjor isoforms: HO-1, n inducile form, nd HO-2/3 elonging to constitutively expressed isozymes. Further results in plnts hve confirmed tht plstidic HO-1 in Aridopsis ws the potentil source of CO s well [8], nd its role in the prticiption in the iosynthetic pthwy leding to phytochrome chromophore formtion nd/or light signling were lso reported [9]. Recent reports confirmed tht HO could ct s strong ntioxidnt enzyme ginst vrious iotic stresses, s well s the inducer functioning in different developmentl processes, including dventitious nd lterl root development [10,11]. In nimls, oxidtive stress-incresed HO-1 protein hs een well estlished. Further mple oservtions demonstrted tht incresed endogenous HO-1 provided cellulr protection ginst oxidnt injury [12]. Hemogloin nd its ctlyzed products including hemin were le to induce the expression of HO-1 in cererl rteril smooth muscle during the 24-h period in time- nd dose-dependent mode [13], suggesting tht the induction of HO-1 in mmmlin cells is generl response to heme-contining compounds nd proly to oxidnt stress which results from the oxidtion of ferrous to ferric hemogloin [14]. Hemogloin ws le to induce the lung HO-1 expression, which plys n importnt role in the defense ginst I/R-induced lung injury [15]. In plnts, it ws demonstrted tht the supplementtion of culture medium with ovine hemogloin clerly promoted mitotic division of protoplsts in jponic rice vriety, Tipei 309. Menwhile, n increse

3 Int. J. Mol. Sci. 2011, in shoot regenertion from protoplst-derived tissues lso occurred [16]. However, whether hemogloin could e used in seed pretretment to induce slt tolernce is still unknown. It is well known tht etter understnding of mechnisms(s) tht enle plnts to dpt slt stress is necessry to mke the est use of sline soils. In this report, we investigted some physiologicl nd iochemicl events induced y ovine hemogloin presoking, including the llevition of seed germintion nd growth inhiition, lipid peroxidtion, re-estlishment of ironic unlnce, nd the up-regultion of ntioxidnt enzyme expression. The possile mechnisms of hemogloin involved in conferring slt tolernce were preliminrily discussed. 2. Results 2.1. The Inhiition of Seed Germintion nd Seedling Shoot Growth Were Allevited y the Pretretment of Hemogloin In comprison with the control tretment (H 2 O H 2 O), rice seed germintion nd seedling growth were inhiited significntly y 100 mm NCl slt stress tretment (H 2 O S, Tle 1). Further results showed tht the different concentrtions of hemogloin pretretment were le to reverse the negtive impct of NCl on seed germintion nd shoot growth inhiition, with mximl response t 1.0 g/l hemogloin (H1.0 S). For exmple, when compred to the NCl-tretment lone smple, the ddition of 1.0 g/l hemogloin resulted in the increment of 48.0% nd 32.9% in seed germintion nd shoot length, respectively. Menwhile, the slight negtive effects on root length ppered when the higher doses of hemogloin (0.2, 1.0, nd 5.0 g/l) were dopted. Further prllel experiments suggested tht, except the ggrvtion of root growth inhiition conferred y 5.0 g/l hemogloin, no significnt differences in seed germintion nd seedling growth were oserved in the hemogloin pretretment lone, compred to slt stressed smple. Tle 1. Effects of different concentrtions of hemogloin (H, g/l) pretretments for 24 h on the inhiition of rice seed germintion, nd seedling shoot nd root length cused y 100 mm NCl stress for nother 60 h. Tretment Germintion rte (%) Shoot length (cm) Root length (cm) H 2 O H 2 O ± ± ± 0.05 H0.01 H 2 O ± ± ± 0.25 H0.05 H 2 O ± ± ± 0.07 H0.2 H 2 O ± ± ± 0.04 H1.0 H 2 O ± ± ± 0.22 H5.0 H 2 O ± ± ± 0.20 H 2 O S ± 7.26 c 0.70 ± 0.05 c 2.55 ± 0.25 c H0.01 S ± 3.44 c 0.76 ± 0.10 c 2.80 ± 0.48 c H0.05 S ± 2.69 c 0.76 ± 0.10 c 2.57 ± 0.51 c H0.2 S ± ± ± 0.29 c H1.0 S ± ± ± 0.01 c H5.0 S ± ± ± 0.24 c Vlues re the mens ± SE of t lest three independent experiments. Different letters in the sme column indicte significnt differences (P < 0.05) ccording to Duncn s multiple test.

4 Int. J. Mol. Sci. 2011, To test the hypothesis tht HO-1 ws involved in ove hemogloin-induced responses, the potent HO-1 inhiitor ZnPPIX proven in oth nimls nd plnts [17,18] ws pplied. In our experiment, the ppliction of ZnPPIX prevented the llevition ctions of hemogloin on the inhiitions of seed germintion (especilly) nd shoots growth cused y slt stress (Tle 2). In contrst, only slight ut not significnt decrese could e oserved in response to the ddition of ZnPPIX together with NCl (H 2 O S + ZnPPIX) compred with NCl stressed lone smple (H 2 O S). The ove results strongly suggested the role of HO-1 in hemogloin-induced cytoprotective roles. Tle 2. Effects of hemogloin (H, 1.0 g/l) pretretment for 24 h on the inhiition of rice seed germintion, nd seedling shoot nd root length, cused y 100 mm NCl stress in the presence nd sence of the HO-1 inhiitor ZnPPIX (100 μm) for nother 60 h. Tretment Germintion rte (%) Shoot length (cm) Root length (cm) H 2 O H 2 O ± ± ± 0.14 H1.0 H 2 O ± ± ± 0.23 H 2 O ZnPPIX ± ± ± 0.10 H1.0 ZnPPIX ± ± ± 0.30 H 2 O S ± 6.59 d 0.70 ± 0.08 c 2.58 ± 0.33 H1.0 S ± 3.77 c 0.90 ± ± 0.10 H 2 O S + ZnPPIX ± 5.04 d 0.66 ± 0.07 c 2.14 ± 0.25 c H1.0 S + ZnPPIX ± 2.58 d 0.79 ± 0.09 c 1.95 ± 0.21 c Vlues re the mens ± SE of t lest three independent experiments. Different letters within columns indicte significnt differences (P < 0.05) ccording to Duncn s multiple test Lipid Peroxidtion nd ROS-Scvenging Enzyme Activities In susequent experiment, TBARS contents of rice germinting seeds sujected to 100 mm NCl with or without hemogloin pretretment were compred. It ws found tht in comprison with the control, TBARS content ws enhnced y out 35.0% upon NCl stress lone, ut less thn 13.1% in rice seeds exposed to hemogloin pretretment followed y the ddition of NCl (Figure 1). Additionlly, no significnt difference in TBARS ws oserved etween hemogloin pplied lone (H H 2 O) nd control smples (H 2 O H 2 O). It ws well known tht APX, CAT, nd SOD enzymes detoxify ROS in plnt tissues. Thus, their ctivities were determined s representtive enzymes responsile for ntioxidnt metolism. The results shown in Figure 1-d showed tht with respect to the control smples, the ctivities of ll ove three enzymes decresed significntly upon slinity stress. While, the response ptterns of the smples sujected to hemogloin pretretment were opposite. For exmple, APX, CAT, nd SOD ctivities incresed fter 60 h of slinity stress, eing 31.3%, 18.3%, nd 15.3%, respectively, higher thn tht in the smple under slinity stress lone. Similrly, the inducile responses were lso oserved when hemogloin ws preincuted lone in comprison with the control smple.

5 CAT ctivity ( mol min -1 mg -1 protein) SOD ctivity (U mg -1 protein) TBARS content ( mol g -1 FW) APX ctivity ( mol min -1 mg -1 protein) Int. J. Mol. Sci. 2011, Figure 1. Effects of hemogloin pretretment on the TBARS content (), APX (), CAT (c), nd SOD (d) ctivities in rice germinting seeds upon slt stress. Dry rice seeds were presoked in distilled wter or 1.0 g/l H for 1 d, nd then shifted to 100 mm NCl solution for nother 60 h. Smple with distilled wter tretment lone (H 2 O) ws used s the control. Dt re the mens ± SE of t lest three independent experiments. Brs denoted y different letters were significntly different t P < 0.05 ccording to Duncn s multiple comprison. () 24 () c (c) (d) c c d H 2 O H 2 O H H 2 O H 2 O S H S 10 H 2 O H 2 O H H 2 O H 2 O S H S 2.3. Antioxidnt Enzyme Trnscripts Semi-quntittive RT-PCR ws pplied to investigte the responses of ntioxidnt enzyme trnscripts upon different tretments. Time-course experiment showed tht the pretretment of hemogloin could significntly strengthen the inducile effect of slinity on the up-regultion of HO-1 trnscripts through 24 h of incution (Figure 2). Menwhile, in comprison with the NCl-free control, the mount of the CATA, capx nd Cu/Zn-SOD trnscripts in NCl-treted smple ws decresed. While, no significnt chnges were oserved in the trnscripts of Mn-SOD. Furthermore, pretretment with hemogloin led to significnt increses in ove trnscripts compred with smples without corresponding pretretment efore NCl tretment. We lso noticed tht ove chnges in mrna levels pproximtely coincided with the chnges of the corresponding CAT, APX, nd SOD ctivities (Figure 1 d).

6 Int. J. Mol. Sci. 2011, Figure 2. Effects of hemogloin pretretment on the trnscription levels of HO1, CATA, capx, Cu/Zn-SOD, nd Mn-SOD, in rice germinting seeds upon slt stress. Dry rice seeds were presoked in distilled wter or 1.0 g/l H for 1 d, nd then shifted to 100 mm NCl solution for nother 0, 6, 12, nd 24 h. Smple with distilled wter tretment lone (H 2 O) ws the control. The numers elow the nd indicte reltive undnce of corresponding trnscripts with respect to the dt t time zero (T0). HO1 CATA capx Cu/Zn-SOD Mn-SOD 18S rrna H 2 O H 2 O H 2 O S H H 2 O H S T T h Chnges of K/N Rtio The ionic lnce inside the plnt cell is closely relted to the plnt dpttion to slt stress, so we compred K/N rtio in oth the whole shoot nd root prts nd especilly the root tips from seedlings under slt stress. Figure 3 showed tht the inducile chnges of K/N rtio were oserved in shoot (prticulrly) nd root tissues of hemogloin pretretment smples fter NCl tretment for 36 h nd 60 h, suggesting tht hemogloin pretretment could enhnce slinity tolernce y re-estlishing the ion homeostsis. Furthermore, the X-ry density mps of the distriution of N nd K elements in root tips of rice seedlings t 60 h fter slinity tretment showed tht in comprison with these of slt-stressed lone smples, more K ut less N were oserved in the root tip sections of smple pretreted with hemogloin (Figure 4), thus resulting in the enhncement of the K/N rtio, especilly in corticl cells of root tips (Figure 4).

7 K/N rtio K/N rtio Int. J. Mol. Sci. 2011, Figure 3. Effects of hemogloin pretretment on K/N rtio in shoot nd root prts from rice seedlings under slt stress. Dry rice seeds were presoked in distilled wter or 1.0 g/l H for 1 d, nd then shifted to 100 mm NCl solution for nother 36 h nd 60 h; Dt re the mens ± SE of t lest three independent experiments. Brs denoted y different letters were significntly different t P < 0.05 ccording to the Duncn s multiple comprison H 2 O S H S Shoot Root Time fter Tretment (h)

8 K/N rtio Int. J. Mol. Sci. 2011, Figure 4. Effects of hemogloin pretretment on K nd N element distriution in root prts of rice seedlings under slt stress. Dry rice seeds were presoked in distilled wter or 1.0 g/l H for 1 d, nd then shifted to 100 mm NCl solution. X-ry microgrphy of root tips of rice seedlings treted for 60 h, the enrichment of K or N distriution shown with white dots (); IM, scnning electron microscope (SEM) imge of corresponding section; Scle r = 100 μm; K/N rtio detected in the cortex nd stele of root tips (). Dt re the mens ± SE of t lest three independent experiments. Brs denoted y different letters were significntly different t P < 0.05 ccording to the Duncn s multiple comprison. () H 2 O S IM N + distriution K + distriution H S () 2.0 Cortex Stele c c 0.5 H 2 O S H S 3. Discussion Exogenous use of vrious chemicls to reduce the dverse effects of iotic stresses hs gret implictions oth from theoreticl nd prcticl perspectives [19]. In our test, soking of rice seeds in incresed hemogloin levels (from 0.01 to 5.0 g/l) for 24 h could differentilly modulte seed germintion nd seedling shoot growth under slt stress in rice plnts (Tle 1), nd the responses of 1.0 g/l hemogloin ws the most ovious. To the est of our knowledge, ove oservtions re new. In fct, similr inducile responses conferred y ovine hemogloin hve een oserved in the mitotic division nd plnt regenertion from cultured rice protoplsts [16], nd the growth of cultured cotton cells [20]. Use of hemogloin in seed presoking is dvntgeous only when it supports the emergence nd estlishment of seedlings. Initil sorption nd lter scvenging of ROS in the rice seeds provided evidence out the occurrence of chnges minly relted to the protection ginst oxidtive dmge.

9 Int. J. Mol. Sci. 2011, Therefore, we investigted the effect of hemogloin produced chnges on the lipid peroxidtion nd the expression of ntioxidnt enzymes in slinized hemogloin-pretreted seedlings of rice nd compred with wter control nd slinized lone smples. Further results showed tht 1.0 g/l hemogloin ws le to counterct the increses in TBARS content induced y slinity in germinting rice seeds (Figure 1), nd this effect my e ttriuted to the ility of hemogloin to ctivte ntioxidnt enzymes, including SOD, CAT, nd APX (Figure 1 d). Trnscription levels of corresponding genes were lso up-regulted (Figure 2). These findings were consistent with those reported y Grrtt et l. [20], in which reserchers found SOD ctivity incresed linerly with the incresing concentrtions of Erythrogen, commercil ovine hemogloin, in cultured cotton cells. Menwhile, higher concentrtion of Erythrogen could ring out concomitnt increse in CAT ctivity to mximum of 62% more thn the control smple. Another importnt mechnism regrding slt stress in plnts is ttriuted to ionic phyto-toxicity cused y excess mounts of slt ions in plnts. Thus, re-estlishment of ion homeostsis is essentil for plnts to resist slt stress. Here, the ppliction of hemogloin presoking tretment ws le to induce the increment of K/N rtio in slt stressed seedling shoots nd roots or even root tips (Figure 3 nd Figure 4). Thus, ion homeostsis is re-estlished so s to dpt the plnt cell to slt stress. Expression of stress protein is n importnt dptive strtegy of environmentl stress tolernce. Normlly, they re highly wter solule nd het stle. Our previous report showed tht slinity stress could induce HO ctivity in whet seedling roots, nd the product of HO, CO ws le to enhnce slt tolernce y NO-medited mintennce of ion homeostsis nd up-regultion of ntioxidnt defense [21]. In this test, we lso noticed tht 1.0 g/l hemogloin pretretment followed y slinity stress cn effectively strengthen the up-regultion of HO-1 gene expression induced y slinity stress lone tretment (Figure 2). Similr inducile response of HO-1 driven y hemogloin ws discovered in nimls previously [13 15]. Comined with the fct tht the ddition of ZnPPIX notly prevented the inducile ction of hemogloin on rice seed germintion nd seedling shoot growth (Tle 2), it is speculted tht the up-regultion of HO-1 expression might contriute to the responses of hemogloin. Previously, we discovered tht CO, one of the product of HO-1, plyed n importnt role ginst slinity- or osmotic-induced oxidtive cell dmge y enhncing ntioxidnt system prmeters in whet [22,23] nd rice seedlings [6]. In whet lurone cells, the up-regultion of HO-1 expression delyed progrmmed cell deth (PCD) y the down-regultion of H 2 O 2 production [24]. In view of the fct tht hemogloin relesed from senescing erythrocytes hs een confirmed s the resource of CO synthesis s erly s in 1949 [25], it is interesting nd necessry in the future to investigte the specific role of HO-1/CO in the ove hemogloin-induced plnt physiologicl processes. 4. Experimentl Section 4.1. Chemicls Commercil ovine hemogloin, purchsed from Shnghi Boo Ltd., Chin, ws used s n HO-1 inducer. Zinc protoporphyrin IX (ZnPPIX), specific inhiitor of HO-1 [17,18], ws otined from Sigm (St Louis, MO, USA) nd used t 100 μm.

10 Int. J. Mol. Sci. 2011, Plnt Mteril nd Experimentl Design For the presoking experiments, rice (Oryz stiv L., Wuyujing 7) seeds, kindly supplied y Jingsu Acdemy of Agriculturl Sciences, Jingsu Province, Chin, were presoked in 0.01, 0.05, 0.2, 1.0, nd 5.0 g/l commercil ovine hemogloin or distilled wter s control (Con). All seeds were incuted in growth chmer (Shnghi Yiheng Technology Co., Ltd., Shnghi, Chin) t 30 C under drkness condition for 24 h. Then, seeds were plced in petri dishes contining filter pper wet with 6 ml of distilled wter (Con), or 100 mm NCl, 100 μm ZnPPIX, or its comintion tretment, nd lso incuted in similr growth chmer. After 60 h or t the indicted time point of slinity tretment, the smples were hrvested, growth prmeters were determined, nd corresponding mterils were frozen t 80 C for further nlysis Germintion nd Growth Anlysis Germintion tests were performed on three replictes of 150 seeds ech. There were 50 seeds in ech petri dish. Cumultive germintion rte (%) ws recorded t 60 h fter slinity tretment, nd seeds were considered to hve germinted when the emerging shoot ws pproximtely hlf the length of the seeds. Menwhile, the mesurements of root length nd shoot length were crried out fter different tretments TBARS Determintion Oxidtive dmge ws estimted y mesuring the concentrtion of TBARS s descried previously [26] Antioxidnt Enzyme Assys Frozen rice plnts (pproximtely 200 mg) were homogenized in 10 ml of 50 mm potssium phosphte uffer (ph 7.0) contining 1 mm EDTA nd 1% polyvinylpyrrolidone (PVP) for CAT, nd SOD ssy, or comintion with the ddition of 1 mm scoric cid (ASC) in the cse of APX ssy. The homogente ws centrifuged t 12,000 g (Avnti J-25, Beckmn) for 20 min t 4 C nd the superntnt ws deslted immeditely y Sephdex G-25 gel filtrtion to remove interfering mterils nd used s the crude enzyme extrct. APX ctivity ws mesured y monitoring the decrese in sornce t 290 nm s ASC ws oxidized (ε = 2.8 mm 1 cm 1 ) for t lest 1 min in 3 ml rection mixture, s descried y Nkno et l. [27]. CAT ctivity ws spectrophotometriclly mesured y monitoring the consumption of H 2 O 2 (ε = 39.4 mm 1 cm 1 ) t 240 nm for t lest 3 min [22]. Totl superoxide dismutse (SOD) ctivity ws mesured on the sis of its ility to reduce nitrolue tetrzolium (NBT) y superoxide nion generted y the rioflvin system under illumintion. One unit of SOD (U) ws defined s the mount of crude enzyme extrct required to inhiit the reduction rte of NBT y 50% [28]. Protein concentrtion ws determined y the method of [29], using ovine serum lumin (BSA) s stndrd.

11 Int. J. Mol. Sci. 2011, Semi-Quntittive RT-PCR Anlysis 100 mg fresh rice tissue ws homogenized y grinding with mortr nd pestle in liquid nitrogen, nd totl RNA ws isolted using Trizol regent (Invitrogen) ccording to the mnufcturer s instructions. DNA-free totl RNA (5 μg) from different smples were used for first-strnd cdna synthesis in 20-μL rection volume contining 2.5 units of vin myelolstosis virus reverse trnscriptse XL (TKR) nd 2.5 μm rndom primer. PCR ws performed using 2 μl of 20-fold dilution of the cdna, 10 pmol of ech oligonucleotide primer, nd 1 unit of Tq polymerse (TKR) in 25-μL rection volume. cdna ws mplified y PCR using the following primers: for rice HO-1 (ccession numer EU781632), forwrd (5 -AGGAATACTGGGTTGGAGAG-3 ) nd reverse (5 -GCTTAGGTGAATATGTGACGGA-3 ), mplifying 416-p frgment; for CATA (ccession numer AK061923), forwrd (5 -AGGCCAGACAATGTCAGATG-3 ) nd reverse (5 -GTGGCATTAATACGCCAGTA-3 ), mplifying 202-p frgment; for capx (ccession numer AK061841), forwrd (5 -CATTGCCCGTGGTACTCT-3 ) nd reverse (5 -TTTCATACCAACACATCT-3 ), mplifying 199-p frgment; for Cu/Zn-SOD (ccession numer L36320), forwrd (5 -AATGGTGAAGGCTGTTGTTGT-3 ) nd reverse (5 -TAGCCTTGAAGTCCGATGA-3 ), mplifying 461-p frgment; for Mn-SOD (ccession numer L34038), forwrd (5 -CCAGAAGCACCACGCCACCTAC-3 ) nd reverse (5 -CTCCCAGACATCAATTCCCAAC-3 ), mplifying 418-p frgment; nd for 18S rrna (ccession numer AK059783), forwrd (5 -TACCGTCCTAGTCTCAACCA-3 ) nd reverse (5 -AGAACATCTAAGGGCATCACA-3 ), mplifying 451-p frgment. To stndrdize the results, the reltive undnce of 18S rrna ws determined nd used s the internl reference. The cycle numers of the PCR rections were djusted for ech gene to otin cler nds in grose gels. Aliquots of the PCR rections were loded in 1.2% grose gels with ethidium romide. Specific mplifiction products of the expected size were oserved, nd their identities were confirmed y sequencing Determintion of Ion Contents N nd K contents of shoot nd root prts were mesured y n tomic emission spectrophotometer (TAS-986; Beijing Purkinje Generl Instrument Co., Ltd., Beijing, Chin) X-ry Micronlysis Element rtio mesurement ws exmined with scnning electron microscope (SEM) (Model S-3000N, Hitchi High-Technologies Corportion, Jpn) equipped with n energy-dispersive X-ry detector (EDX, Hori Inc. Kyoto, Jpn) s descried previously [30,31] with some modifictions. After eing plced verticlly in the hole of n luminum stu nd immersed quickly into liquid nitrogen for freeze-drying, the tender root tips were trnsferred quickly into vcuum evportor nd dried under the vcuum of Torri for t lest 12 h. The root tips were coted with fine lyer of pure evported cron nd then oserved. Accelertion voltge of 10 kv ws used. Bem current ws djusted to fixed (0.06 μa), nd the working distnce from the EDX detector ws 13.5 mm. For

12 Int. J. Mol. Sci. 2011, nlyses of reltive elementl levels within corticl nd stelr cells of root tips ccurtely, point nlysis of ech smple ws repeted t lest four times. The results were clculted y expressing the tomic numer for prticulr element in given point or region s percentge of the totl tomic numer for ll the elements mesured (K, N, clcium, mgnesium, phosphorus, sulfur, nd chlorine) in the root tips Sttisticl Anlysis All results re expressed s men vlues ± SE of t lest three independent experiments. For sttisticl nlysis, Duncn s multiple test ws used s pproprite, fter testing for dt normlity. A vlue of P < 0.05 ws considered significnt for men differences. 5. Conclusions Hemogloin ttenuted slinity-induced inhiition of rice seed germintion nd seedling shoot growth. It ws prtilly due to the induction of ntioxidnt metolisms nd reestlishment of ion homeostsis. Such responses my e of considerle vlue in the development of improved methods for crop protection ginst environmentl stresses. Interestingly, our results in rice seeds correspondingly demonstrted tht the effects of hemogloin functions in niml reserch, such s chieving cytoprotective function, lso work in plnts. Acknowledgements This work ws supported y the Progrm for New Century Excellent Tlents in University (Grnt No. NCET ), the Priority Acdemic Progrm Development of Jingsu Higher Eduction Institutions, the Eduction Deprtment of Jingsu (Grnt No ), the Technology Support Progrm in Jingsu Province, Chin (Grnt No. BE ), Ntionl Science & Technology Key Specific Projects for Trnsgenic Vriety Development (Grnt No. 2009ZX B, 019B), nd the Fundmentl Reserch Funds for the Centrl Universities (Grnt No. KYZ200905). References 1. Chng, C.; Wng, B.; Shi, L.; Li, Y.; Duo, L.; Zhng, W. Allevition of slt stress-induced inhiition of seed germintion in cucumer (Cucumis stivus L.) y ethylene nd glutmte. J. Plnt Physiol. 2010, 167, Khn, A.A.; Hung, X.L. Synergistic enhncement of ethylene production nd germintion with kinetin nd 1-minocyclopropne-1-croxylic cid in lettuce seeds exposed to slinity stress. Plnt Physiol. 1988, 87, Kur, S.; Gupt, A.K.; Kur, N. Gierellin A 3 reverses the effect of slt stress in chickpe (Cicer rietinum L.) seedlings y enhncing mylse ctivity nd moiliztion of strch in cotyledons. Plnt Growth Regul. 1998, 26, Çvusoglu, K.; Kr, K. Effects of hydrogen peroxide on the germintion nd erly seedling growth of rley under NCl nd high temperture stresses. EurAsi. J. BioSci. 2010, 4,

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