Zhan-Wu Gao 1, Ji-Tao Zhang 2, Zhuang Liu 3, Qing-Tao Xu 1, Xiu-Jun Li 2 and Chun-Sheng Mu 4* of Education, Changchun , China

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1 AJCS 6(6): (2012) ISSN: Comprtive effects of two lkli stresses, N 2 CO 3 nd NHCO 3 on cell ionic blnce, osmotic djustment, ph, photosynthetic pigments nd growth in ot (Aven stiv L.) Zhn-Wu Go 1, Ji-To Zhng 2, Zhung Liu 3, Qing-To Xu 1, Xiu-Jun Li 2 nd Chun-Sheng Mu 4* 1 Bicheng Norml University, Deprtment of Geogrphy, Bicheng , Chin 2 Northest Institute of Geogrphy nd Agroecology, Chinese Acdemy of Sciences, Chngchun , Chin 3 Bicheng City Acdemy of Agriculture Sciences, Ji Lin Province Reserch Institute of Sunflower, , Chin 4 Institute of Grsslnd Science, Northest Norml University, Key Lbortory of Vegettion Ecology of Ministry of Eduction, Chngchun , Chin *Corresponding uthor: mucs821@yhoo.com.cn Abstrct This study exmines the comprtive effects of NHCO 3 nd N 2 CO 3 on young ot (Aven stiv L.) plnts to elucidte the species physiologicl dptive mechnisms to lkli stress. Fctors considered re the intrcellulr influx nd efflux of ions, ionic blnce, osmotic djustment, ph homeostsis, photosynthetic pigments nd growth. Results show tht, N 2 CO 3 hd stronger effects thn NHCO 3, nd tht with incresing concentrtions of both stresses the plnt showed rising N + influxes into the shoot resulting in N + ion toxicity. This is tolerted by N + sequestrtion in the vcuole; the ccumultion minly of Cl -, SO 4 2- nd the synthesis of high concentrtions of orgnic nions to mintin vcuolr ionic blnce nd, lstly by the synthesis of proline in the cytoplsm to void dehydrtion. Moreover, N 2 CO 3 stress inhibits growth more strongly, compred to NHCO 3, becuse of the higher energy costs ssocited with N + exclusion nd comprtmentlistion,the syntheses of orgnic nions, the syntheses of proline in the cytoplsm, reduced photosynthetic cpcity nd incresed membrne permebility. Compred to the shoot, lthough the root hd similr response to both stresses, it showed higher tolernce becuse high N 2 CO 3 stresses (>48 mmol L -1 ) resulted in significnt increses in root tissue ph, but did not ffect the ph homeostsis of the shoot. Additionlly, while both stresses decresed root dry weight, they did not significntly ffect root extension growth. This indictes tht ot dopts n opportunistic guerrill strtegy by which it voids resource-poor ptches of soil (e.g. high lkli) while preferentilly exploiting more fvorble hbitts by mintining root extension. Keywords: lkli; ionic blnce; N 2 CO 3 nd NHCO 3 stresses; ot; shoot nd root growth. Abbrevitions: ELR, electrolyte lekge rte; OA, orgnic cid. Introduction Alkli stress hs been demonstrted in number of reports (Kwnbe nd Zhu, 1991; El-Smd nd Shddd, 1996; Cmpbell nd Nishio, 2000; Hrtung et l., 2002; Ro et l., 2008; Wng et l., 2011; M et l., 2011). Previous studies hve suggested tht lkli stress results minly from levels of the lkline slts NHCO 3 nd N 2 CO 3 (Shi nd Yin, 1993; Shi nd Sheng, 2005; Yng et l., 2010; Liu et l., 2010). In some res, soil lklistion is severe problem. For exmple, pproximtely 7% ( hm 2 ) of the cultivted lnd in northest Chin hs lkline soils with only few lkli-tolernt hlophytes being ble to survive there (Kwnbe nd Zhu, 1991). Alkli stress usully involves combintion of stresses, osmotic, ion-induced injury nd high ph (Munns, 2002; yng et l., 2008; Chen et l., 2011). The high-ph environment tht surrounds the roots cn gretly ffect the bsorption of ctions nd inorgnic nions nd cn lso disrupt the ionic blnce nd ph homeostsis of the tissues (Yng et l., 2007, 2008b; Guo et l., 2010). The results re decrese in photosynthesis, dmge to the membrne system nd finlly reduction in growth. Although the effects of vrious mixed lkli stresses hve been studied extensively in few plnts (Shi nd Yin, 1993; Hrtung et l., 2002; Shi nd Wng, 2005; Yn et l., 2006; Guo et l., 2010; Rdi et l., 2012), these studies do not differentite between the effects of NHCO 3 nd of N 2 CO 3 in isoltion. Although these studies provide better understnding of the dptive responses of plnts under mixed lkline stress, nlysis of the stresses cused by the single lkli hve not been dequtely reserched. Exmintion of the effects of NHCO 3 lone, or of N 2 CO 3 lone, would seem to be logicl strting point for resolving the demogrphic mechnisms underlying these complex responses. Therefore, stress effects ssocited with NHCO 3 on its own or with N 2 CO 3 on its own should be investigted s thoroughly s those of mixed lkli stress, especilly, in regrd to the responses of the mjor plnt orgns, the shoot nd root. The present study ws conducted in n experimentl re of Northest Norml University during the 2008 seson. We compre the seprte effects of lkli stresses, NHCO 3 stress nd N 2 CO 3 stress on wter potentil, photosynthetic pigments, ionic blnce nd tissue ph nd growth in the shoot nd root of young ot seedlings, to enhnce our understnding of the mechnisms of lkli stress dmge to plnts nd lso those by which they dpt to such lkli stress. Results Tissue ph nd ELR nlysis of shoot nd root Shoot tissue ph did not show significnt differences between NHCO 3 nd N 2 CO 3 stresses but root tissue ph nd electrolyte lekge rte (ELR) were significntly higher under N 2 CO 3 stress thn under NHCO 3 stress (Tble 1). The shoot 995

2 tissue ph under both stresses ws closely similr to the controls (Fig. 1A, B) suggesting tht these stress intensities did not ffect the boveground tissue ph. However, the root ph showed different behviour. While NHCO 3 stress did not ffect root ph with incresing stress, middle nd high N 2 CO 3 contents ( 72mmol L -1 ) did cuse significnt increses. Both stresses incresed the ELR but the extent of the increment under N 2 CO 3 stress ws much greter thn under NHCO 3 stress (Fig. 1C). All the plnts treted with 120 nd 144 mmol L -1 of N 2 CO 3 died so their ph vlues were not recorded. Growth indices nlysis of ot With incresing lkli stress, the shoot survivl rte, number of tillers per plnt, root length, plnt height, shoot dry weight nd root dry weight ll decresed (Fig. 2A-F), but the extents of the reductions were different. The effect on root length ws especilly slight with the effect of NHCO 3 being not significnt nd only the most concentrted N 2 CO 3 (144mmol L -1 ) cused significnt decrese (Fig. 2C, tble 1). Furthermore, the reductions under N 2 CO 3 stress were much greter thn those under NHCO 3 stress (tble 1). Photosynthetic pigments nlysis of leves All photosynthetic pigments were significntly lower under N 2 CO 3 thn under NHCO 3 (Tble 1). The effects of NHCO 3 stress on Chl, Chl b nd crotenoid were similr with ll three prmeters decresing but the extents of the reductions under N 2 CO 3 stress were greter thn those under NHCO 3. When N 2 CO 3 stress ws 120 mmoll -1 the plnts died nd the contents of Chl, Chlb nd Crotenoid were not recorded. The sme concentrtions of NHCO 3 did not result in plnt deth (Fig. 3). Ctions nlysis of shoot nd root Ech of the ctions showed significnt differences between the NHCO 3 nd N 2 CO 3 stresses (Tble 1). In both the shoots nd the roots, the concentrtion rtios of N + nd N + K + in the N 2 CO 3 stressed plnts compred to the NHCO 3 stressed plnts were significntly higher, while for K + nd C 2+ the concentrtion rtios were significntly lower (Tble 1). Both shoots nd roots showed similr trends for these ctions (Fig. 4A, B, C, D, G, H). With incresing stress, both the NHCO 3 nd N 2 CO 3 stresses resulted in incresed N + contents nd decresed K + contents, nd finlly in n incresed N + K + rtio (Fig. 4A, B, C, D, G, H). When NHCO 3 nd N 2 CO 3 stresses were compred, the extents not only of the increses in N + but lso of the reductions in K + were much greter under N 2 CO 3 stress thn under NHCO 3 stress (Fig. 4A, B, C, D, G, H). In both shoot nd root, lthough the C 2+ content rnge ws fr less thn for either N + or K +, it showed similr trend for decrese compred to K + with incresing lkli stress, lso, the extent of the C 2+ decrese for N 2 CO 3 stress ws greter thn for the NHCO 3 stress (Fig. 4E, F). Anions nlysis of shoot nd root All the nions showed significnt differences between the NHCO 3 nd N 2 CO 3 stresses (Tble 1). In both shoot nd root, compred to the NHCO 3 stress, the N 2 CO 3 stress showed significntly higher concentrtions of SO 2-4 nd orgnic cids, while the Cl -, NO nd H 2 PO 4 concentrtions were significntly lower (Tble 1). In both shoot nd root Cl - nd 2- SO 4 incresed with incresing stress intensity for both stressors but compred with the NHCO 3 stress, the extent of increse in Cl - in the N 2 CO 3 ws higher thn in SO 2-4 (Fig. 5A, B, C, D). Both NO nd H 2 PO 4 decresed with incresing stress but the reductions with N 2 CO 3 were much greter thn with NHCO 3 (Fig. 5E, F, G, H). With incresing stress, both low NHCO 3 ( 72mmolL -1 ) stresses nd ll the N 2 CO 3 stresses cused increses in orgnic cid. However, when the NHCO 3 stresses were higher thn 72 mmol L -1,OA showed decreses (Fig. 5I, J). Wter nd proline contents nlysis of shoot nd root In both shoot nd root, proline concentrtions were significntly higher under N 2 CO 3 stress thn under NHCO 3 stress while wter contents were significntly lower (Tble 1). Both NHCO 3 nd N 2 CO 3 stress decresed the wter contents of shoots nd roots (Fig.6A, B) but the extent of the reductions with incresing N 2 CO 3 stress were greter thn with incresing NHCO 3 stress. For shoot nd roots, the chnges in wter content were in opposite directions with proline being incresed by strong lkline stress (>120mmolL -1 in NHCO 3 nd 96 mmoll -1 in N 2 CO 3 ) (Fig. 6C, D). Discussion Tissue ph Regrdless of environmentl ph, to mintin norml metbolism it is importnt tht plnts cn stbilise their tissue ph (Yng et l., 2007). Our observtions tht shoot tissue ph ws similr to the control vlues under both lkli stresses nd tht stress intensity increses did not hve significnt ffects on tissue ph suggests tht ot is ble to mintin stble cell ph (Fig.1A, B). However, it is surprising tht when the stress level rose bove certin threshold vlues (48 mmol L -1 with N 2 CO 3 ; 72 mmol L -1 with NHCO 3 ), root ph ws ffected by incresing levels of stress. This my be explined s tht t bove 48 mmol L -1 in N 2 CO 3 (or 72 mmol L -1 in NHCO 3 ) the hrmful effects of high ph were opposed by ph djustments outside the roots (by extrusion of H +, OA, mino cids or CO 2 produced by root respirtion) with the intrcellulr environment being unffected. However, when the stress intensity exceeded the cpcity for root djustment (>48 mmol L -1 in N 2 CO 3 or >72 mmol L -1 in NHCO 3,), the result ws reduction in photosynthetic pigment content (Fig. 3), nd shrp increse in ELR (Fig. 1C). This suggests tht the stress my hve wekened the controls, leding to increses in ph. Menwhile, compred to the shoot, it ws found tht the effects of both lkli stresses on root growth (i.e. root length nd biomss) ws reduced, indicting tht the root hs higher tolernce of elevted ph. This deserves further investigtion. Growth indices Our results show tht the injurious effects of N 2 CO 3 on growth were greter thn those of NHCO 3 for the sme lkli content (Fig. 2), nd this is consistent with previous reports (Shi nd Yin, 1993; Yng et l., 2007). The injurious effects of lkli re commonly thought to be due to combintion of low wter potentil, ion toxicity, nd, in prticulr, to high-ph stress (Munns, 2002). Firstly, the greter lkli stress due to N 2 CO 3 compred with NHCO 3 leds to severer reductions in photosynthetic pigment content (Fig. 3) nd shrp increse in ELR (Fig. 1C). These results indicte tht high ph from N 2 CO 3 stress my dmge root cell structure nd function ffecting such processes s the bsorption of ions, dmging photosynthetic pigments nd membrne systems (e.g. increses in ELR). This my be why growth under NHCO 3 stress ws 996

3 Tble 1. Results of two-wy ANOVA of plnt chrcteristics by stress ctegory, nd by stress grdient nd their interction. Dependent vrible Stress ctegory Stress grdient Ctegory Grdient (A) ions nd orgnic cid shoot N + content *** *** *** root N + content *** *** *** shoot K + content *** *** *** root K + content *** *** *** shoot C 2+ content *** *** *** root C 2+ content *** *** *** shoot N + K + rtio *** *** *** root N + K + rtio *** *** *** shoot Cl - content *** *** *** root Cl - content, *** *** 7.554*** shoot SO 2-4 content *** *** *** root SO 2-4 content *** *** 5.714** shoot NO 3 content *** *** *** root NO 3 content *** *** *** shoot H 2 PO - 4 content ** *** *** root H 2 PO - 4 content *** *** 7.879*** shoot orgnic cid content *** *** *** root orgnic cid content *** *** *** (B) wter nd proline shoot wter content *** *** n.s. root wter content *** 4.594** n.s. shoot proline content ** *** 3.025* root proline content *** *** 5.750** (C) tissue ph, ELR, Chl, Cr shoot tissue ph n.s n.s n.s. root tissue ph *** *** *** electrolyte lekge rte (ELR) *** *** 4.010** chlorophyll (Chl) *** *** 8.851*** chlorophyll (Chl) b *** *** 8.783*** Crotenoid (Cr) *** *** *** (D) growth index survivl rte *** *** *** number of tillers per plnt *** *** 3.669** root length n.s n.s n.s. plnt height *** *** n.s. shoot dry weight *** *** n.s. root dry weight ** *** n.s. Note: Numbers represent F vlues: * P 0.1; ** P 0.01; *** P 0.001; n.s., no significnt. less thn under N 2 CO 3 stress. Next, to tolerte ion toxicity, plnts cn exclude N + depending on N + H + ntiport, synthesise orgnic nions to mintin ionic blnce or synthesise comptible solutes in the cytoplsm to prevent dehydrtion. All of these cost energy nd hence involve growth penlty. Thus, the higher N + toxicity under N 2 CO 3 stress thn under NHCO 3 likely results in higher energy costs, which my be nother reson for the reduced growth under NHCO 3 stress. Additionlly, it is interesting tht, compred to the control, root length growth did not chnge significntly for ny NHCO 3 concentrtions or for the low nd medium concentrtions of N 2 CO 3 (<144mmolL -1 ). However, root dry weight growth did decrese significntly under >96mmolL -1. It is suggested tht, under nturl conditions, ot ppers to use n opportunistic guerrill strtegy whereby resources re llocted in the direction of more fvourble microsite by mintining root extension rtes even though root dry weight is significntly decresed s result of dmge to the photosynthetic pigments nd the membrne systems (e.g. increses of ELR). By this strtegy, ot cn void resource-poor (e.g. high lkli ptches of soil) while preferentilly exploiting more fvorble hbitts. Ion toxicity nd ion imblnce Under high concentrtions of either lkli, the first effect on the plnt is likely to be in the root due to high concentrtions of N + in the soil. The N + enters the roots pssively vi voltge independent nonselective ction chnnels nd possibly vi other N + trnsporters such s some members of the high-ffinity K + trnsporter (HKT) fmily (Munns nd Tester, 2008), resulting in N + ion toxicity in shoot. The similr rdii of the hydrted ions of N + nd K + mkes them difficult to discriminte, nd this is the bsis of N + toxicity (Blumwld, 2000). Under both lkli stresses, N + competes with K + for uptke into the roots (Munns, 2002; Munns nd Tester, 2008). Compred to the sme stress concentrtions of NHCO 3 nd N 2 CO 3, the ltter hs the higher concentrtion of N + resulting in incresed N + ccumultion (ssocited with decresed K + ccumultion) (Fig. 4ABCD). Also, the incresed N + in the stressed plnt my induce decrese in N + exclusion, which normlly enbles the plnt to void or defer ion toxicity problems. Mny plnt species hve N + exclusion mechnism tht depends on N + H + ntiport, such s slt overly sensitive 1 997

4 ELR (%) Shoot tissue ph Root tissue ph 8.0 [A] NHCO3 N2CO3 [B] b c c b b b c c [C] d e 60 b c de e 40 b bc cd b Alklinity (mm) Fig 1. Effects of NHCO 3 nd N 2 CO 3 stress on (A) shoot tissue ph, (B) root tissue ph, (C) ELR (electrolyte lekge rte) in ot shoots. The 4-week-old ot seedlings were treted with NHCO 3 stress (ph ) nd N 2 CO 3 stress (ph ) for 9 dys. In ech column, the dt mrkers identified with the sme letters re not significntly different (P < 0.05) ccording to Duncn test. The error brs represent ± stndrd error (n = 3) of three replictes. Shoot represents boveground prt of plnt. Fig 2. Effects of NHCO 3 nd N 2 CO 3 stress on (A) survivl rte, (B) number of tillers per plnt, (C) root length, (D) plnt height, (E) shoot dry weight, (F) root dry weight in ot. The 4-week-old ot seedlings were treted with NHCO 3 stress (ph ) nd N 2 CO 3 stress (ph ) for 9 dys. In ech column, the dt mrkers identified with the sme letters re not significntly different (P < 0.05) ccording to Duncn test. The error brs represent ± stndrd error (n = 3) of three replictes. DW, dry weight; Shoot represents boveground prt of plnt. 998

5 (SOS1), which exchnges cytoplsmic N + with externl H + (Zhu, 2003; Munns nd Tester, 2008). The exchnge ctivity relies on the trnsmembrne proton grdient chieved by H + -ATPse (Zhu, 2003). Compred to NHCO 3, the sme concentrtion of N 2 CO 3 hs higher ph, nd its reltively lck of externl protons my weken the exchnge ctivity of the N + H + ntiport on the root plsm membrne (Munns nd Tester, 2008), possibly reducing the exclusion of N + from the rhizosphere nd enhncing plnt ccumultion of N + (Fig. 4). In ddition, compred to N + nd K +, other ions, especilly C 2+ cn lso be influenced by both lkli stresses ech of which exhibits decresing trends with incresing concentrtions. However, the extent of this effect is significntly higher in N 2 CO 3 thn in NHCO 3. This is firstly becuse, higher N + in the shoot is influenced by N 2 CO 3 replcement of more intr-cellulr C 2+ which results in substntil C 2+ loss (Fig. 4EF ); secondly, the high-ph environment of the roots with N 2 CO 3 cn cuse some ions, such s C 2+ to precipitte directly (Shi nd Zho, 1997); thirdly, C 2+ cn be influenced in the plnt by higher contents of orgnic cid (minly oxlic cid) (Fig. 5IJ) through the formtion of clcium oxlte (n insoluble crystl) from oxlic cid nd C 2+. In the root, the high concentrtion of N + cuses this ion to move in the symplst cross the endodermis where it is relesed from the stelr cells to the stelr poplst, from where it moves through the xylem in the trnspirtion strem, finlly reching the shoot where the N + toxicity occurs (Munns nd Tester, 2008). The min site of N + toxicity for most plnts is in the lef blde. Here, it cn be tolerted by ntomicl dpttions nd by intrcellulr prtitioning with the N + remining in the cell being sequestered in the vcuoles to void N + toxicity in the cytosol (Serrno nd Rodriguez-Nvrro, 2001; Munns, 2002; Zhu, 2003). Ion blnce When comprtmentlistion of N + in the vcuoles tkes plce, plnts usully ccumulte inorgnic nions, such s Cl -, nd SO4 2-, or they synthesise orgnic nions to mintin the ionic blnce (Yng et l., 2007). This fits with our study (Fig. 5) where the concentrtions of inorgnic nions under NHCO 3 stress were significntly lower thn those under N 2 CO 3 stress of the sme intensity. This suggests tht the reltively high ph cused by N 2 CO 3 stress my inhibit the uptke of nions such s NO 3 - nd H 2 PO 4 - (Fig. 5EFGH). Menwhile, Cl - nd SO 4 2- in reltively high concentrtions in both lklis mke higher contributions to the ion blnce. Additionlly, we find tht, with stress concentrtion increses in the plnt, OA is strongly incresed only with N 2 CO 3 wheres with NHCO 3 it is decresed. This suggests tht under N 2 CO 3 stress, OA is the dominnt ion in the mintennce of equilibrium. This my be n lkli resistnce mechnism involving two components, intrcellulr ph djustment nd extrcellulr ph djustment (lso possibly, ph djustment in the root-externl microenvironment). The OA synthesised might lso be trnsported to roots for ph regultion. The process of ph djustment my occur outside the root or in the root poplst, or both. Therefore, the type of cells involved in ph djustment my be epiderml, corticl or xylem prenchym cells. The mechnism of ph djustment my involve the exudtion of buffer compound, such s H +, OA, mino cids or even CO 2 produced by root respirtion, or other fctors not considered bove. Osmotic djustment When plnts comprtmentlise N + into the vcuoles to void N + toxicity in the cytosol (Serrno nd Rodriguez-Nvrro, 2001; Zhu, 2003) nd ccumulte inorgnic nions nd synthesise orgnic nions to mintin vcuolr ionic blnce (Yng et l., 2007), they my lso synthesise comptible low moleculr weight orgnic solutes (comptible solutes) in the cytoplsm to mintin osmotic equilibrium nd so prevent dehydrtion nd protect biomcromolecules (Prid nd Ds, 2005). The mino cid proline is one of the most widely distributed comptible solutes occurring in mny orgnisms from bcteri to higher plnts (Flowers et l., 1977). In mny hlophytes, proline occurs t sufficiently high concentrtions in their leves (over 40 mm on tissue wter bsis) to contribute significntly (over 0.1 MP) to cell osmotic potentil (Fricke, 2004). Thus, in our study it is cler tht N + concentrtion increses in the vcuoles (which lso ccumulte inorgnic nd orgnic nions to mintin ionic blnce) nd s lkli stress increses, proline concentrtion increses to prevent cytoplsmic dehydrtion (Fig. 6). Furthermore, for the sme intensity of the two lkli stresses the especilly high concentrtions of N + with N 2 CO 3 led to reltively high concentrtions of proline (Fig. 6). In prticulr, the induction of proline synthesis is lso relted to the high ph vlue ssocited with lklinity. The involvement of proline ccumultion in the dmge cused by lkli stress is nother spect tht requires further investigtion. Aprt from the bove-mentioned osmotic djustment which comes with n energy cost, quick nd energeticlly economicl wy of reducing cell wter potentil to void osmotic stress is to llow decreses in wter content (Fig. 6AB). In summry, the key fetures pertining to osmotic djustment re n bility to ccumulte N + nd orgnic cids in the vcuoles nd to ccumulte lrge mounts of proline in the cytoplsm. Mterils nd methods Plnt mterils Ot (Aven stiv L.) is n nnul, grminceous plnt which is very tolernt of drought, cold, sline nd lkli stress nd minerl deficiency. The ot cultivr No.2 biynmi (numbered species is B in Bicheng City Acdemy of Agriculture Sciences, Chin) ws used. This cultivr is chrcterised by erly mturtion, high slt nd lkli tolernce nd high disese resistnce (Wei et l., 2007). It hs high grin yield which rises to over 2300 kg/hm 2. Its protein nd ft contents re 16.6% nd 5.6%, respectively (Wei et l., 2007). The plnt cn mture in live culms nd provides both grin nd strw. Cultivtion Seeds were sown in 20-cm dimeter plstic pots contining 3 kg of wshed snd. The pots were wtered dily with sufficient Hoglnd nutrient solution. Ech pot contined 25 seedlings. All pots were plced outdoors nd protected from the rin. The experiment ws crried out in n experimentl re of the Northest Norml University during the 2008 growth seson. Design of the simulted lkline conditions Two lkline slts, NHCO 3 nd N 2 CO 3 were pplied seprtely to crete two stress groups. Within ech group, six concentrtions were used: 0, 48, 72, 96, 120 nd 144 mmol L -1. Tretments in the NHCO 3 stress group were lbeled A 1 A 6 nd those in the N 2 CO 3 stress group were lbeled B 1 B 6. A1 nd B1 were the controls. There were three replictes per tretment. 999

6 Fig 3. Effects of NHCO 3 nd N 2 CO 3 stress on the (A) chlorophyll (Chl), (B) chlorophyll (Chl) b in ot. The 4-week-old ot seedlings were treted with NHCO 3 stress (ph ) nd N 2 CO 3 stress (ph ) for 9 dys. In ech column, the dt mrkers identified with the sme letters re not significntly different (P < 0.05) ccording to Duncn test. The error brs represent ± stndrd error (n = 3) of three replictes. FW, fresh weight; Shoot represent boveground prt of plnt. Stress tretments The stress tretments were pplied when the seedlings were four weeks old. Thirty-six pots of uniform seedlings were divided rndomly into 12 sets of 3 pots. Two sets were used for the controls nd the remining 10 sets were used for the stress tretments giving three replicte pots per tretment. The treted pots were wtered dily between h with excess of nutrient solution tht contined the pproprite stress slts. The control plnts were wtered with nutrient solution t the sme time. The durtion of stress tretment ws nine dys. Physiologicl indices mesurements Mesurement of tissue ph To determine tissue ph, fresh shoots nd roots were wshed thoroughly three times with neutrl deionised wter, followed by surfce-drying with filter pper. They were then crushed nd the ph of the expressed sp mesured with digitl ph meter PHS-3C; Shnghi precision & scientific instruments, Shnghi, Chin. Mesurement of the electricl conductivity of the leves Membrne permebility is reflected in reltive electricl conductivity, which is defined s the rtio of the electricl conductivity of leves with intct membrnes to those with membrnes destroyed by boiling wter tretment. Electrolyte lekge rte (ELR) ws determined s described by Lutts et l., (1996). Mesurement of chlorophyll After 9 dys of stress tretment, fresh helthy leves were cut into smll segments to determine the concentrtions of chlorophylls nd crotenoid b ccording to Arnon (1949). Mesurement of growth indices All plnts were hrvested in the morning fter the finl tretment. The number of tillers per plnt ws recorded. The plnts were first wshed with tp wter nd then with distilled wter. For ech plnt, the roots nd shoots were seprted nd their fresh weights (FW) nd the lengths of their shoots nd the totl root length per plnt were determined. The smples were then oven-dried t 105 for 15 min before being vcuum-dried t 80 to constnt weight. The shoot nd root dry weights (DW) were recorded. The wter content (WC) of both prts ws clculted using the formul WC= (FW-DW) / FW. Survivl rte (SR) ws expressed using the formul: SR= n / N where n is the number of plnts surviving from the totl of N plnts. Mesurement of ions Dry smples (0.1 g) of shoot nd root were treted with 20 ml of deionised wter t 100 for 1 h nd the resultnt extrct ws used to determine the contents of inorgnic ions nd orgnic cids (OA).The contents of NO 3 -, Cl -, SO 4 2-, H 2 PO 4 - were determined by ion chromtogrphy using DX-300 ion chromtogrphic system with n AS 4 A-SC ion-exchnge column nd CDM-II electricl conductivity detector (mobile 1000

7 Fig 4. Effects of NHCO 3 nd N 2 CO 3 stress on (A) shoot N + content, (B) root N + content, (C) shoot K + content, (D) root K + content, (E) shoot C 2+ content, (F) root C 2+ content, (G) shoot N + K + rtio, (H) root N + K + rtio in ot. The 4-week-old ot seedlings were treted with NHCO 3 stress (ph ) nd N 2 CO 3 stress (ph ) for 9 dys. In ech column, the dt mrkers identified with the sme letters re not significntly different (P < 0.05) ccording to Duncn test. The error brs represent ± stndrd error (n = 3) of three replictes. DW, dry weight; Shoot represent boveground prt of plnt. phse: N 2 CO 3 NHCO 3 = mmol L -1 ; DIONEX, Sunnyvle, CA, USA). N 2 CO 3 NHCO3=1.7 / 1.8 mmol L -1. The levels of the orgnic cids were lso determined by ion chromtogrphy using n DX-300 ion chromtogrphic system with n ICE-AS6 ion-exclusion column, CDM-II electricl conductivity detector nd n AMMS-ICE II MicroMembrne suppressor (mobile phse: 0.4 mmol L -1 heptfluorobutyric cid; DIONEX). An tomic bsorption spectrophotometer (TAS-990; Purkinje Generl, Beijing, Chin) ws used to determine the levels of N +, K + nd C 2+. Mesurement of proline The dried smples were homogenised to determine free proline contents which ws ssyed using the cid-ninhydrin method (Zhu et l. 1983). Fig 5. Effects of NHCO 3 nd N 2 CO 3 stress on (A) shoot Cl - content, (B) root Cl - content, (C) shoot SO 4 2- content, (D) root SO 4 2- content, (E) shoot NO 3 content, (F) root NO 3 content, (G) shoot H 2 PO 4 - content, (H) root H 2 PO 4 - content, (I)shoot OA (orgnic cid) content, (J) root OA (orgnic cid) content in ot. The 4-week-old ot seedlings were treted with NHCO 3 stress (ph ) nd N 2 CO 3 stress (ph ) for 9 dys. In ech column, the dt mrkers identified with the sme letters re not significntly different (P < 0.05) ccording to Duncn test. The error brs represent ± stndrd error (n = 3) of three replictes. DW, dry weight; Shoot represent boveground prt of plnt. 1001

8 Fig 6. Effects of NHCO 3 nd N 2 CO 3 stress on the (A) shoot wter content, (B) root wter content, (C) shoot proline content, (D) root proline content in ot. The 4-week-old ot seedlings were treted with NHCO 3 stress (ph ) nd N 2 CO 3 stress (ph ) for 9 dys. In ech column, the dt mrkers identified with the sme letters re not significntly different (P < 0.05) ccording to Duncn test. The error brs represent ± stndrd error (n = 3) of three replictes. Shoot represent boveground prt of plnt. Sttisticl nlyses Sttisticl nlysis of the dt ws performed using the sttisticl progrm SPSS 13.0 (SPSS, Chicgo, USA). All dt re represented by n verge of three replictes nd their stndrd errors (SE). Dt were nlysed by one-wy nd two-wy ANOVA. The tretment men vlues for the sme orgn under either lkli stress were compred by post-hoc Duncn tests. The term significnt indictes differences t P < Acknowledgments Finncil support ws provided by collbortive grnt from the Ntionl Science Foundtion ( ), the Project Foundtion from the Eduction Ministry of Chin (106063), the Specilized Reserch Fund for the Doctorl Progrm of Higher Eduction ( ), nd the Ntionl TCM Min Project in 11th Five-Yer-Period (2008BADB3B09) nd the Science nd Technology Project of Jilin Province ( ). References Arnon DI (1949) Copper enzymes in isolted chloroplsts. Polyphenooxidse in Bet vulgris. Plnt Physiol. 24:1 15 Blumwld E (2000) Sodium trnsport nd slt tolernce in plnts. Curr Opin Cell Biol. 12: Cmpbell SA, Nishio JN (2000) Iron deficiency studies of sugr beet using n improved sodium bicrbonte-buffered hydroponics growth system. J Plnt Nutr. 23: Chen W, Feng C, Guo W, Shi D, nd Yng C (2011) Comprtive effects of osmotic-, slt- nd lkli stress on growth, photosynthesis, nd osmotic djustment of cotton plnts. Photosynthetic. 49: El-Smd HMA, Shddd MAK (1996) Comprtive effect of sodium crbonte, sodium sulphte, nd sodium chloride on the growth nd relted metbolic ctivities of pe plnts. J Plnt Nutr. 19: Flowers TJ, Troke PF, nd Yeo AR (1977) The mechnism of slt tolernce in hlophytes. Annu Rev Plnt Physiol. 28: Fricke W (2004) Rpid nd tissue-specific ccumultion of solutes in the growth zone of brley leves in response to slinity. Plnt. 219: Guo LQ, Shi DC nd Wng DL (2010) The key physiologicl response to lkli stress by the lkli-resistnt hlophyte puccinelli tenuiflor is the ccumultion of lrge quntities of orgnic cids nd into the rhyzosphere. J Agron Crop Sci. 196: Hrtung W, Leport L, Rtcliffe RG, Suter A, Dud R, nd Turner NC (2002) Abscisic cid concentrtion, root ph nd ntomy do not explin growth differences of chickpe (Cicer rietinum L.) nd lupin (Lupinus ngustifolius L.) on cid nd lkline soils. Plnt Soil. 240: Kwnbe S, nd Zhu TC (1991) Degenertion nd conservtion of Aneurolepidium chinense grsslnd in Northern Chin. J Jpn Grssl Sci. 37: Liu J, Guo WQ, nd Shi DC (2010) Seed germintion, seedling survivl, nd physiologicl response of sunflowers under sline nd lkline conditions. Photosynthetic. 48: Lutts S, Kiner JM, nd Bouhrmont J (1996) NCl-induced senescence in leves of rice (Oryz stiv L.) cultivrs differing in slinity resistnce. Ann Bot. 78: M Y, Guo LQ, Wng HX, Bi B, nd Shi DC (2011) Accumultion, distribution, nd physiologicl contribution of oxlic cid nd other solutes in n lkli-resistnt forge plnt, Kochi sieversin, during dpttion to sline nd lkline conditions. J Plnt Nutr Soil Sc. 174:

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