Comparative Proteomic Analysis of Puccinellia tenuiflora Leaves under Na 2 CO 3 Stress

Transcription

1 Int. J. Mol. Sci. 213, 14, ; doi:1.339/ijms Article OPEN ACCESS Interntionl Journl of Moleculr Sciences ISSN Comprtive Proteomic Anlysis of Puccinelli tenuiflor Leves under N 2 CO 3 Stress Junjun Yu 1,2, Sixue Chen 3, Ti Wng 4, Guorong Sun 5 nd Shojun Di 1, * College of Life nd Environmentl Sciences, Shnghi Norml University, Shnghi 2234, Chin; E-Mil: yujun8186@gmil.com Snqun Medicl College, Xinxing Medicl University, Xinxing 4533, He nn, Chin Deprtment of Biology, Genetics Institute, Plnt Moleculr nd Cellulr Biology Progrm, Ginesville, FL 3261, USA; E-Mil: schen@ufl.edu Institute of Botny, Chinese Acdemy of Sciences, Beijing 193, Chin; E-Mil: twng@ics.c.cn Binzhou Polytechnic College, Binzhou 25663, Shndong, Chin; E-Mil: grsun@live.cn * Author to whom correspondence should e ddressed; E-Mil: dishojun@hotmil.com; Tel./Fx: Received: 17 Octoer 212; in revised form: 31 Decemer 212 / Accepted: 6 Jnury 213 / Pulished: 15 Jnury 213 Astrct: Soil slt-lkliniztion is widespred environmentl stress tht limits crop growth nd griculturl productivity. The influence of soil lkliztion cused y N 2 CO 3 on plnts is more severe thn tht of soil sliniztion. Plnts hve evolved some unique mechnisms to cope with lkli stress; however, the plnt lkline-responsive signling nd moleculr pthwys re still unknown. In the present study, N 2 CO 3 responsive chrcteristics in leves from 5-dy-old seedlings of hlophyte Puccinelli tenuiflor were investigted using physiologicl nd proteomic pproches. Comprtive proteomics reveled 43 differentilly expressed proteins in P. tenuiflor leves in response to N 2 CO 3 tretment for seven dys. These proteins were minly involved in photosynthesis, stress nd defense, crohydrte/energy metolism, protein metolism, signling, memrne nd trnsport. By integrting the chnges of photosynthesis, ion contents, nd stress-relted enzyme ctivities, some unique N 2 CO 3 responsive mechnisms hve een discovered in P. tenuiflor. This study provides new moleculr informtion towrd improving the lkli tolernce of cerels. Keywords: proteomics; hlophyte; Puccinelli tenuiflor; N 2 CO 3 response

2 Int. J. Mol. Sci. 213, Arevition: 6PGDH, 6-phosphogluconte dehydrogense; AAIR, cetohydroxy cid isomeroreductse; AIPLP, luminum-induced protein-like protein; ALD, ldolse; AST, sprtte minotrnsferse; ATP se CF1, ATP synthse CF1 lph suunit; ATPse 1, ATP synthse lph suunit; CA, cronic nhydrse; CAT, ctlse; CPS, counts per second; DREPP, developmentlly regulted plsm memrne polypeptide; FBA, fructose-isphosphte ldolse; FRKs, fructokinses; FTR, ferredoxin-thioredoxin reductse; Fv/Fm, mximum quntum efficiency of PSII photochemistry; Fv /Fm, PSII mximum efficiency; GAPDH, glycerldehyde-3-phosphte dehydrogense; GLO, glyoxlse; GLP, germin-like protein 1; Gs, Stomtl conductnce; IDs, identities; IF4A, eukryotic initition fctor 4A; M3, memrne-ssocited 3 kd protein; MDA, mlondildehyde; MDH, mlte dehydrogense; MS, methionine synthse; PDI, protein disulfide-isomerse; PGK, phosphoglycerte kinse; Pn, photosynthetic rte; POD, peroxidse; PPIse, peptidyl-prolyl cis-trns isomerse; PRK, phosphoriulokinse; Protesome α3, protesome lph type 3; PSII, photosystem II; qnp, non-photochemicl quenching coefficient; ROS, rective oxygen species; RP, Riosoml protein S1-like RNA-inding domin; RRM, RNA recognition motif; Ruisco LSU, Ruisco lrge suunit; Ruisco SSU, Ruisco smll suunit; SOD, superoxide dismutse; TIL, temperture stress-induced lipoclin; TIM, triosephosphte isomerse; Tr, trnspirtion rte; V-ATPse, vcuolr-type H + -ATPse. 1. Introduction Slt-lkli soil is one of the mjor iotic constrints limiting crop distriution nd yield worldwide [1 3]. Plnt slinity tolernce hs een extensively studied, ut the understnding of plnt lklinity tolernce is lcking. The lkli slt ffects plnt growth nd development through sodium toxicity nd high ph, which re more likely to cuse serious dmge thn neutrl slt to the plnt [4]. The high soil ph (minly ttriuted to cronte slts, e.g., N 2 CO 3 nd NHCO 3 ) could directly ffect nutrient uptke, orgnic cid lnce, nd ion homeostsis, especilly the ph stility t cellulr nd whole plnt levels [4,5]. Menwhile, some hlophyte species cn nturlly survive in high lkline soil, nd hve evolved vrious regultory nd metolic mechnisms for lkli tolernce. Alkli grss (Puccinelli tenuiflor) is n lkli tolernt hlophyte species tht cn survive in highly lkline soil (e.g., ph 1). Thus, it is considered s n outstnding psture for soil improvement. Although some slt tolernt mechnisms in P. tenuiflor hve een studied efore [6], few studies were focused on the specific moleculr mechnisms underlying lkli tolernce. To cope with sline or lkline stress, P. tenuiflor hs developed vrious strtegies, such s ion lnce [7 9], osmotic djustment [9 11], nd rective oxygen species (ROS) scvenging [12]. Previous studies hve reveled tht P. tenuiflor cn remrkly ccumulte citric cid in leves nd roots when exposed to lkline stress. This is different from slt stress under which citric cid levels remin unchnged [9,11]. The ccumultion of citric cid in P. tenuiflor my ply n importnt role in ph djustments used to cope with lkline stress [9,11]. Besides, severl ion slt-responsive genes encoding ntiporters/chnnel proteins in P. tenuiflor hve een isolted nd trnsformed into yest, rice nd Aridopsis to test their iologicl functions. These genes include PutPMP3-1/2 [13], PutHKT2;1 [14], PutAKT1 [15], KPutB1 [16], PutCAX1 [17], nd PtNHA1 [18]. The specific functions of these genes hve een summrized in our previous rticle [6]. Furthermore, some cndidte slt/lkli-responsive

3 Int. J. Mol. Sci. 213, genes/proteins in P. tenuiflor hve een found using high-throughput trnscriptomic nd proteomic pproches. A cdna lirry ws constructed for P. tenuiflor under 45 mm NHCO 3 stress for 48 h. It contined totl of 95 differentilly regulted trnscripts [19]. Our previous comprtive proteomic nlysis reveled 93 unique NCl-responsive proteins in P. tenuiflor leves [6]. These studies hve provided importnt informtion for understnding slt-tolernce mechnisms nd cndidte gene functions. However, the lkli responsive moleculr processes remin elusive. In the present study, we nlyzed the chrcteristics of P. tenuiflor leves in response to N 2 CO 3 using physiologicl nd comprtive proteomic pproches. By integrting the chnges of photosynthesis, ROS scvenging enzymes ctivities, ion contents, nd lkli-responsive proteins, some unique mechnisms of P. tenuiflor in response to N 2 CO 3 hve een reveled, leding to etter understnding of the underlying moleculr mechnisms of lkli tolernce in cerels. 2. Results 2.1. Effects of N 2 CO 3 Stress on the Growth nd Photosynthesis of P. tenuiflor To evlute the effects of lkline stress on the growth of P. tenuiflor, shoot length, lef fresh weight, dry weight, nd wter content were determined (Figure 1). The shoot length declined grdully with the increse in N 2 CO 3 concentrtion (Figure 1A). The fresh weight nd wter content decresed when seedlings were treted with 95 mm N 2 CO 3 (Figure 1B,D). Figure 1. Biomss of P. tenuiflor seedlings grown under N 2 CO 3 conditions. (A) shoot length of seedlings; (B) fresh weight of leves; (C) dry weight of leves; (D) wter content in leves. The vlues were determined fter plnts were treted with mm, 38 mm, nd 95 mm N 2 CO 3 for seven dys, nd were presented s mens ± SE (n = 9). The different smll letters indicte significnt differences (p <.5). mm N 2 CO 3 38 mm N 2 CO 3 95 mm N 2 CO 3 shoot length (cm) dry weight (g) A c C fresh weight (g) wter content (g g -1 FW) B D The photosynthesis indexes of P. tenuiflor under N 2 CO 3 tretment were nlyzed. After seven dys of 38 mm nd 95 mm N 2 CO 3 tretments, the P. tenuiflor seedlings did not show ovious dmge to lef morphology (dt not shown), implying the high cpcity of P. tenuiflor seedlings to tolerte N 2 CO 3. However, photosynthesis ws ffected y N 2 CO 3 stress. Stomtl conductnce (Gs) (Figure 2A), photosynthetic rte (Pn) (Figure 2B), nd trnspirtion rte (Tr) (Figure 2C) exhiited little chnges under 38 mm N 2 CO 3 tretment, ut showed mrked decreses under 95 mm N 2 CO 3. In

4 Int. J. Mol. Sci. 213, ddition, chlorophyll fluorescence prmeters were monitored to determine the performnce of photosystem II (PSII) photochemistry. The mximum quntum efficiency of PSII photochemistry (Fv/Fm) (Figure 2D) nd the PSII mximum efficiency (Fv /Fm ) (Figure 2E) were not significntly ltered under 38 mm N 2 CO 3, ut were reduced remrkly under 95 mm N 2 CO 3. The non-photochemicl quenching coefficient (qnp) (Figure 2F) remined constnt under 38 mm nd then incresed under 95 mm N 2 CO 3 tretment. Figure 2. Photosynthetic chrcteristics (A, B, C) nd chlorophyll fluorescence prmeters (D, E, F) of P. tenuiflor leves under N 2 CO 3 tretment. (A) stomt conductnce (Gs); (B) photosynthesis rte (Pn); (C) trnspirtion rte (Tr); (D) Fv/Fm; (E) Fv /Fm ; (F) qnp. The vlues were determined fter plnts were treted with mm, 38 mm, nd 95 mm N 2 CO 3 for seven dys, nd were presented s mens ± SE (n = 9). The different smll letters indicte significnt differences (p <.5). mm N 2CO 3 38 mm N 2CO 3 95 mm N 2CO 3 Gs (mol H 2O m -2 s -1 ) Fv/Fm A D Pn (μmol CO 2 m -2 s -1 ) Fv'/Fm' B E Tr qnp (mmol H2O m -2 s -1 ) C F Chnges to Lef Osmotic Potentil, Plsm Memrne Integrity nd Antioxidnt Enzyme Activities Lef osmotic potentil showed significnt decrese under N 2 CO 3 tretments (Figure 3A), indicting the seedlings suffered from osmotic stress. The electrolyte lekge rtio (Figure 3B) nd mlondildehyde (MDA) contents (Figure 3C) were incresed significntly under N 2 CO 3. This indictes tht the plsm memrne integrity ws dmged y N 2 CO 3 tretment, proly resulting from ROS generted under high ph nd ion stress conditions. The ctivities of representtive ntioxidtive enzymes were ltered with different ptterns under N 2 CO 3 stress. The superoxide dismutse (SOD) ctivity decresed under N 2 CO 3 stress (Figure 3D), ut the peroxidse (POD) ctivity incresed under 38 mm N 2 CO 3 (Figure 3E), nd the ctlse (CAT) ctivity incresed oviously under oth N 2 CO 3 concentrtions (Figure 3F) Ion Content Chnges in Leves under N 2 CO 3 Stress Ion homeostsis is importnt in plnt response to slt stress. Lef N content incresed with increses in N 2 CO 3 concentrtion (Figure 4A). Lef K contents lso incresed under 95 mm N 2 CO 3 tretment (Figure 4B). This led to declined K/N rtios under the N 2 CO 3 tretment (Figure 4C). Furthermore, N nd K contents on the lef surfce incresed grdully with the increses in N 2 CO 3 concentrtion, nd K/N rtio ws reduced on the lef surfce (Figure 4D,E,F). Moreover, s n importnt signling

5 Int. J. Mol. Sci. 213, messenger, clcium contents in different sucellulr comprtments of epiderml cells nd mesophyll cells were ffected y N 2 CO 3. In epiderml cells, clcium content in the cell wll nd cytoplsm incresed grdully with the increses in N 2 CO 3 concentrtion, ut reduced in the vcuoles under 38 mm N 2 CO 3 tretment (Figure 4G). Similrly, in mesophyll cells, clcium contents in the cell wll nd cytoplsm incresed under 95 mm N 2 CO 3 tretments, while the clcium content in vcuoles reduced shrply in the N 2 CO 3 treted smples (Figure 4H). Figure 3. Chnges of some ntioxidnt-relted indexes in leves of P. tenuiflor under N 2 CO 3 tretment. (A) osmotic potentil; (B) electrolyte lekge rtio; (C) MDA contents; (D) SOD ctivity; (E) POD ctivity; (F) CAT ctivity. The vlues were determined fter plnts were treted with mm, 38 mm, nd 95 mm N 2 CO 3 for seven dys, nd were presented s mens ± SE (n = 6). The smll letters indicte significnt difference (p <.5). osmotic potentil inside leves ( 1 5 P) SOD ctivity in leves (unit min -1 mg -1 protein) c mm N 2CO 3 A D reltive electric conductnce in leves (%) POD ctivity in leves (unit min -1 mg -1 protein ) mm N 2CO 3 95 mm N 2CO 3 c B E MDA content in leves (μmol g -1 FW) CAT ctivity in leves (unit min -1 mg -1 protein ) c c C F Figure 4. Chnges in ionic contents in leves of P. tenuiflor under N 2 CO 3 stress. (A) N content in leves; (B) K content in leves; (C) K/N rtio in leves; (D) N content outside leves; (E) K content outside leves; (F) K/N rtio outside leves; (G) C content in epiderml cells; (H) C content in mesophyll cells. The vlues were determined fter plnts were treted with mm, 38 mm, nd 95 mm N 2 CO 3 for seven dys, nd were presented s mens ± SE (n = 6). The different letters indicte significnt differences (p <.5). CPS, counts per second. mm N 2CO 3 38 mm N 2CO 3 95 mm N 2CO 3 N content in leves(mmol/l) N content outside leves(cps) A c D c K content in leves(mmol/l) K content outside leves(cps) B E c K/N rtio in leves K/N rtio outside leves C c F C content in epiderml cell (CPS) C content in mesophyll cell (CPS) G c c H c c cell wll cytoplsm vcuole

6 Int. J. Mol. Sci. 213, Identifiction nd Functionl Ctegoriztion of N 2 CO 3 Responsive Proteins The lkli-responsive protein profiles were otined using 2-DE nlysis of lef smples under mm, 38 mm, nd 95 mm N 2 CO 3 tretments (Figure 5, Supplementl Figure S1). Approximtely 1 Coomssie Brillint Blue-stined protein spots were detected on the pi 4 7 gels. A totl of 43 protein spots were identified using LC ESI Q-TOF MS/MS nd Mscot dtse serching (Figure 5, Tle 1). Bsed on Gene Ontology, BLAST lignments, nd informtion from literture, the protein identities (IDs) were clssified into 1 functionl ctegories including photosynthesis, stress nd defense, memrne nd trnsport, crohydrte nd energy metolism, mino cid metolism, trnscription, protein synthesis, protein folding nd trnsporting, protein degrdtion, nd signling (Tle 1). Among these ctegories, crohydrte nd energy metolism (41%), trnscription nd protein metolism (27%), photosynthesis (14%), s well s stress nd defense (9%), were over-represented. Figure 5. 2-DE profiling of proteins extrcted from P. tenuiflor leves treted with different concentrtions of N 2 CO 3. Gels A, B, nd C re protein smples from leves treted with, 38, nd 95 mm N 2 CO 3 for seven dys, respectively. Moleculr weight (MW) in kilodltons (kd) nd pi of proteins re indicted on the right nd top of gel A nd B, respectively. The 43 gel spots with protein IDs were mrked with spot numers. Detiled informtion cn e found in Tle 1.

7 Int. J. Mol. Sci. 213, Tle 1. Proteins nd their reltive chnges in leves from P.tenuiflor under N 2 CO 3 tretment. Spot No. Protein nme Plnt species c gi Numer d Thr. MW (D)/pI e Exp. MW (D)/pI f Sco g Photosynthesis (6) Cov (%) h QM i, 38, 95 mm N 2 CO 3 V% ± SE j 182 cronic nhydrse, chloroplst precursor (CA) Hordeum vulgre ,736/8.93 3,29/ Ruisco lrge suunit (Ruisco LSU) Tristchy leucothrix ,294/ ,412/ Ruisco lrge suunit (Ruisco LSU) 68 Ruisco smll suunit (Ruisco SSU) Oronche coerulescens Aven sterilis susp. ludovicin ,41/ ,47/ ,3/ / phosphoriulokinse (PRK), chloroplst precursor ferredoxin-thioredoxin reductse, vrile chin (FTR) Stress nd defense (4) Oryz stiv ,486 53,645/ Ze mys ,937/ / temperture stress-induced lipoclin (TIL) Triticum estivum ,89/5.5 19,791/ glyoxlse I (GLO) O. stiv (jponic cultivr-group) ,861/ ,631/ germin-like protein 1 (GLP) O. stiv ,17/6.1 22,973/ luminum-induced protein-like protein (AIPLP) Setri itlic ,4/6.5 39,899/ Memrne nd trnsport (3) 133 Os1g233,contining pfm5558 DREPP plsm memrne polypeptide domin (DREPP) O.stiv (jponic cultivr-group) ,788/4.92 4,22/

8 Int. J. Mol. Sci. 213, Tle 1. Cont. Spot No. Protein nme Plnt species c gi Numer d Thr. MW (D)/pI e Exp. MW (D)/pI f Sco g Cov (%) h QM i, 38, 95 mm N 2 CO 3 V% ± SE j 125 Vcuolr-type H + -ATPse (V-ATPse) H. vulgre ,86/ ,714/ memrne-ssocited 3 kd protein, chloroplst precursor (M3) Crohydrte nd energy metolism (18) Pisum stivum ,79/9.3 39,41/ Os8g1131, contining cd1167 fructokinses (FRKs) domin O. stiv (jponic cultivr-group) ,893/5.2 49,892/ puttive fructose-isphosphte ldolse (FBA) Phleum prtense ,28/ ,664/ cytoplsmic ldolse (ALD) O. stiv ,151/ ,317/ triosephosphte isomerse, cytosolic (TIM) Secle cerele ,138/ ,657/ Os3g1293, contining pfm44 Gp_dh_N (GAPDH) domin O. stiv (jponic cultivr-group) ,537/ ,685/ phosphoglycerte kinse (PGK) Vitis vinifer ,51/ ,21/ enolse2 Z. mys ,418/5.7 66,697/ mlte dehydrogense, mitochondril precursor (MDH) Citrullus lntus vr. lntus ,46/9.68 5,177/ puttive cytosolic 6-phosphogluconte dehydrogense (6PGDH) Z. mys ,24/ ,17/ ATP synthse lph (ATPse 1) T. estivum ,557/5.7 64,698/

9 Int. J. Mol. Sci. 213, Tle 1. Cont. Spot No. Protein nme Plnt species c gi Numer d Thr. MW (D)/pI e Exp. MW (D)/pI f Sco g Cov (%) h QM i, 38, 95 mm N 2 CO 3 V% ± SE j 123 ATP synthse lph suunit (ATPse 1) T. estivum ,557/5.7 64,82/ ATP synthse lph suunit (ATPse 1) Elymus siiricus ,549/6.3 66,615/ ATP synthse CF1 lph suunit (ATP se CF1) Agrostis stolonifer ,491/ ,44/ mitochondril ATP synthse precursor (ATPse 1) T. estivum ,9/ ,228/ Os12g231, contining two AAA ATPse fmily protein domin O. stiv (jponic cultivr-group) ,68/ ,28/ hypotheticl protein OsI_36614, contining two AAA ATPse fmily protein domin O. stiv (indic cultivr-group) ,826/ ,75/ unnmed protein product, contining cd9 the AAA, ATPses domin Vitis vinifer ,95/5.8 78,76/ hypotheticl protein OsI_23646, contining AAA, ATPses domin O. stiv (indic cultivr-group) ,173/ ,936/ Amino cid metolism (2) 349 sprtte minotrnsferse (AST) O. stiv ,16/5.9 58,376/ methionine synthse (MS) Trnscription relted (1) cp31bhv, contining cd59 RNA recognition 121 motif (RRM) domin Protein synthesis (2) 333 eukryotic initition fctor 4A (IF4A) H. vulgre susp. vulgre H. vulgre susp. vulgre O. stiv (jponic cultivr-group) ,794/ ,241/ ,662/ ,167/ ,187/ ,45/

10 Int. J. Mol. Sci. 213, Tle 1. Cont. Spot No. Protein nme Plnt species c gi Numer d Thr. MW (D)/pI e Exp. MW (D)/pI f Sco g Cov (%) h QM i, 38, 95 mm N 2 CO 3 V% ± SE j 415 Os7g168, contining cd164 Riosoml protein S1-like RNA-inding domin(rp) O. stiv (jponic cultivr-group) ,23/ ,993/ Protein folding nd trnsporting (3) 255 peptidyl-prolyl cis-trns isomerse, chloroplst precursor (PPIse) Glycine mx ,841/ ,18/ protein disulfide-isomerse precursor (PDI) Nicotin tcum ,82/ ,4/ puttive SecA Protein degrdtion (3) O. stiv (jponic cultivr-group) ,899/5.78 8,931/ Os1g8111, contining cd3751 protesome lph type 3 (Protesome α3) domin O. stiv (jponic cultivr-group) ,56/ ,44/ unknown, contining cd3751 protesome lph type 3 (Protesome α3) domin H. vulgre ,448/ ,21/ Os5g5737, contining pfm7991 cetohydroxy cid isomeroreductse, ctlytic domin (AAIR) O. stiv (jponic cultivr-group) ,68/6.1 68,899/ Signling (1) 334 plnt dhesion molecule PAM1 Aridopsis thlin ,36/8.8 12,776/ Assigned spot numer s indicted in Figure 5; The nme nd functionl ctegories of the proteins identified using LC ESI Q-TOF MS/MS; c The plnt species tht the peptides mtched to; d Dtse ccession numers from NCBInr; e, f Theoreticl (e) nd experimentl (f) mss (kd) nd pi of identified proteins. Experimentl vlues were clculted using Imge Mster 2D Pltinum Softwre. Theoreticl vlues were retrieved from the protein dtse; g The mino cid sequence coverge for the identified proteins; h The Mscot score otined fter serching ginst the NCBInr dtse; i The numer of unique peptides identified for ech protein; j The men vlues of protein spot volumes reltive to totl volume of ll the spots. Three N 2 CO 3 tretments ( mm, 38 mm, 95 mm) were performed. Error rs indicte ± stndrd error (SE).

11 Int. J. Mol. Sci. 213, Figure 6. Hierrchicl clustering nlysis of the expression profiles of the identified 43 proteins. The three columns represent different tretments, i.e., mm, 38 mm, nd 95 mm. The rows represent individul proteins. The protein cluster is on the left, nd the tretment cluster is on the top. The incresed nd decresed protein spots were indicted in red nd green, respectively. The intensities of the colors increse with the increse of expression differences, s shown in the r on the top. The protein spot numers re listed on the right, nd the letters efore the spot numers represent vrious functionl ctegories of the proteins. A, photosynthesis; B, stress nd defense; C, memrne nd trnsport; D, crohydrte nd energy metolism; E, mino cid metolism; F, trnscription relted; G, protein synthesis; H, protein folding nd trnsporting; I, protein degrdtion; J, signling (mm) I-1 Cluster I Cluster II II Protein Clustering nd the Dynmics of Protein Networks One importnt gol of system iology is to understnd the interdependence of proteins nd their expression profiles in certin tissue or other iologicl smples [6,2]. An effective method to determine the regultory mechnisms for protein interctions is the ppliction of hierrchicl clustering lgorithms used in DNA microrry experiments. With this method, the proteins ppering on the sme

12 Int. J. Mol. Sci. 213, rnches re ssumed to e involved in relted moleculr functions [6,2]. Thus, to nlyze the expression chrcteristics of proteins involved in ech functionl ctegory, we performed hierrchicl clustering nlysis of the 43 IDs, which reveled two min clusters. Cluster I included 32 lkli-induced IDs nd cluster II contined 11 lkli-reduced IDs (Figure 6). The numer of lkli-induced proteins ws oviously lrger thn tht of reduced proteins. The nlysis of protein functionl ctegories showed heterogeneous distriution etween the two clusters (Figure 6). For exmple, most of the IDs involved in crohydrte nd energy metolism were grouped in cluster I, wheres photosynthesis-relted proteins were minly in cluster II (Figure 6). This suggests tht switch of iologicl processes occurred in the course of lkline tretment. Interestingly, the clustering result supports the previous notion tht 38 mm is the turning point concentrtion of N 2 CO 3 for P. tenuiflor [12,21,22], ecuse there were 28 out of 43 protein IDs whose protein undnces chnged. Among them, 22 proteins (su-cluster I-1) reched the mximum levels, nd six proteins (sucluster II-1) got to the minimum levels under 38 mm N 2 CO 3 tretment (Figure 6). Such protein ptterns correlted well with our forementioned physiologicl results, e.g., the photosynthetic cpility (e.g., Pn, nd qp) nd ntioxidnt-relted indexes. 3. Discussion 3.1. Photosynthesis Is Inhiited y N 2 CO 3 The effects of slinity nd lklinity on plnt growth nd development vry mongst plnts. For glycophytes, slt stress generlly reduces plnt growth nd development, ut for most hlophytes, moderte slt ccumultion promotes the plnt growth [23]. However, the less-tolernt dicotyledonous hlophytes nd monocotyledons hlophytes, especilly grsses, grow etter in non-slinized conditions [23,24]. Our results showed tht the growth of P. tenuiflor, monocot hlophyte grss, ws not oviously ffected y low N 2 CO 3 concentrtion, ut ws inhiited y higher N 2 CO 3 concentrtion (Figure 1). This correltes well with the iomss chnges of P. tenuiflor under NCl stress [6]. In this study, Gs, Pn, nd Tr of P. tenuiflor seedlings exhiited little chnges t 38 mm N 2 CO 3, ut decresed significntly t 95 mm N 2 CO 3 (Figure 2A,B,C), indicting photosynthesis ws reduced t the higher N 2 CO 3 concentrtion. Moreover, Fv/Fm nd Fv'/Fm' were stle t 38 mm N 2 CO 3, ut reduced t 95 mm N 2 CO 3 (Figure 2D,E). This implied tht the efficiency of PSII photochemistry ws not ffected y the low N 2 CO 3 concentrtion, ut inhiited y the high N 2 CO 3 concentrtion. The reduced Fv/Fm of seedlings under 95 mm N 2 CO 3 implied the occurrence of photoinhiition in P. tenuiflor under the higher concentrtion of N 2 CO 3. The decrese of Fv/Fm ws usully ccompnied y increse of therml dissiption, which ws evluted y nonphotochemicl quenching of Chl fluorescence (qnp) [25]. Here qnp ws mintined lmost constnt t 38 mm N 2 CO 3, ut incresed significntly t 95 mm N 2 CO 3 (Figure 2F), which corresponded with the chnge of Fv/Fm. This result implied tht therml dissiption remined stedy t 38 mm N 2 CO 3, ut incresed t 95 mm N 2 CO 3. Previous studies hve found tht slt nd lkli stresses ffected photosynthetic cron fixtion [1,26]. Our present proteomics dt reveled tht some of the enzymes in Clvin cycle were reduced y N 2 CO 3 stress, which implied tht the decrese in Pn ws due to the less efficient cron fixtion under N 2 CO 3 stress. These enzymes included cronic nhydrse (CA), RuBisCO, phosphoriulokinse (PRK), nd ferredoxin-thioredoxin reductse (FTR). CA cn help increse the

13 Int. J. Mol. Sci. 213, concentrtion of CO 2 within the chloroplst in order to increse the croxyltion rte of RuBisCO [27]. The chnge tendency of CA is similr to those of Gs, Pn, nd Tr in P. tenuiflor. These results indicte tht the decrese of Pn is minly resulted from the declined cron fixtion. RuBisCO ctlyzes the first mjor step of cron fixtion in C3 plnts, nd PRK is the key enzyme tht functions in phosphorylting RuP into riulose-1,5-isphosphte (RuBP) in the Clvin cycle [28]. FTR is n iron-sulfur enzyme, which links light to enzyme regultion in oxygenic photosynthesis, ctlyzing the ctivtion of fructose 1,6-isphosphtse [29]. The decline of these enzymes in the Clvin cycle under N 2 CO 3 tretment could led to the decrese of cron fixtion Antioxidnt Mechnisms in Leves to Cope with N 2 CO 3 Slt nd lkli stresses enhnce the production of ROS, resulting in vrious ROS-ssocited perturtions in the seedlings [2]. Chloroplsts re key intrcellulr ROS genertors. In chloroplsts, the production of O 2 is minly determined y the lnce etween sorption nd utiliztion of light energy [25]. The energy consumption for CO 2 ssimiltion suppressed y N 2 CO 3 stress led to ROS imlnce, which would cuse oxidtive dmge to enzymes nd thus the photosynthetic pprtus [2,25,3]. In the present study, the significntly incresed electrolyte lekge rtio nd MDA contents indicte tht plsm memrne ws dmged y lipid peroxidtion under N 2 CO 3 stress [3]. Our results hve reveled severl mechnisms of light energy lnce nd ntioxidtion used to cope with N 2 CO 3 stress in P. tenuiflor seedlings. Slt ccumultion on the lef surfce would prevent excessive light sorption in N 2 CO 3 stressed plnts. The contents of N nd K on the surfce of P. tenuiflor leves incresed grdully with incresing N 2 CO 3 concentrtions (Figure 4A,B,D,E), which suggested tht P. tenuiflor possesses the ility to secrete slts under N 2 CO 3 stress. Other ions such s C, Mg, nd Si hve lso een found to increse in concentrtion on the P. tenuiflor lef surfce with the incresing N 2 CO 3 concentrtions [1]. The ccumultion of slts on lef surfces developed greter surfce reflectnce, contriuting to reduce light sorption [31]. At the sme time, therml dissiption ws enhnced to remove excess energy t high N 2 CO 3 concentrtion. The increse of qnp demonstrtes tht the therml dissiption ws enhnced to protect the photochemicl pprtus under N 2 CO 3 stress (Figure 2F). In ddition, the enhncement of the xnthophyll cycle would increse the thylkoid ph, which is helpful for induced therml dissiption [25]. The increse of temperture-induced lipoclin (TIL) in our proteomics results supported this notion. Lipoclin ws found to e key enzyme in the xnthophyll cycle responsile for protection ginst photo-oxidtive dmge [32]. It ws lso found to e incresed in Solnum lycopersicum under slt stress [33]. The ROS scvenging system ws ctivted in seedlings to cope with N 2 CO 3 stress. The ctivities of POD (Figure 3E) nd CAT (Figure 3F), the two enzymes involved in the removl of H 2 O 2, were incresed under N 2 CO 3 tretments, especilly under 38 mm N 2 CO 3. However, SOD ctivity ws reduced drmticlly under N 2 CO 3 stress (Figure 3D). SOD is n enzyme for dismuttion of O 2 to produce H 2 O 2. Thus, in P. tenuiflor seedlings under N 2 CO 3, H 2 O 2 might e minly produced in peroxisome from oxidtion of glycolte during photorespirtion rther thn from dismuttion of O 2 [6]. In ddition, our proteomics dt showed tht other ntioxidnt nd detoxifiction mechnisms were enhnced in seedlings to cope with N 2 CO 3 stress. Glyoxlse (GLO) ws found to e induced in seedlings with incresing concentrtions of N 2 CO 3. GLO is memer of the glyoxlse system tht

14 Int. J. Mol. Sci. 213, crries out the detoxifiction of methylglyoxl nd other rective ldehydes produced in plnt metolism. Previous studies hve documented tht slinity stress induced high ccumultion of methylglyoxl, potent cytotoxin in vrious plnt species [34]. In trnsgenic tocco plnts, overexpression of glyoxlse I cn tolerte n increse in methylglyoxl nd mintin high levels of reduced glutthione under slinity stress [35]. Our results implied tht GLO might e n importnt cndidte for conferring high lkli tolernce in P. tenuiflor. Furthermore, germin-like protein (GLP) ws lso induced under N 2 CO 3 stress. Germin ws first detected in germinting whet seeds, ut its homologs hve now ecome uiquitous in the plnt kingdom nd hve vrious functions, not only during emryogenesis, ut lso in iotic or iotic stress conditions [36]. The incresed GLPs were detected in Nicotin tcum lef poplst [37] nd Aridopsis thlin roots [38] fter exposure to slt stress. Whet germin hs een shown to disply oxlte oxidse ctivity; this ctivity is shred mong most plnts [36]. Germin-like oxlte oxidse is involved in degrding the oxlic cid, highly toxic chemicl, through production of H 2 O 2 [36]. Thus, the N 2 CO 3 incresed GLP might provide n explntion for the decrese of oxlic cids in shoots nd roots of P. tenuiflor [9]. Additionlly, n luminum-induced protein-like protein (AIPLP) ws induced under 95 mm N 2 CO 3 tretment. Previous studies hve shown tht the AIPLP ws not specific to luminum stress, ut lso involved in other metl, wounding [39], nd drought stresses [4]. This protein might lso contriute to the tolernce to N 2 CO 3 stress in P. tenuiflor when t high concentrtion Ion Homeostsis nd Trnsport under N 2 CO 3 Stress The intrcellulr ion homeostsis is fundmentl to living cells. Under slinity conditions, high poplstic levels of N + would lter the queous nd ionic thermodynmic equilirium, resulting in hyperosmotic stress, ionic imlnce, nd toxicity [26]. Thus, proper regultion of ion flux is necessry for cells to keep the concentrtions of toxic ions low nd to ccumulte essentil ions. Our results implied tht P. tenuiflor developed some protective mechnisms to reestlish cellulr ion homeostsis through selective slt ccumultion or exclusion, in vivo comprtmentliztion, nd C 2+ signling. Mintining high cytosolic K + /N + rtio is one of the most importnt mechnisms for plnt slt tolernce [8]. In our study, the N content in leves incresed remrkly in ll lkline tretments. However, the K content did not increse under 38 mm N 2 CO 3, ut incresed slightly under 95 mm N 2 CO 3 (Figure 4A,B). This led to the decresed intrcellulr K/N rtio in P. tenuiflor leves (Figure 4C), lthough some N ions were secreted to the lef surfce (Figure 4D). It is ovious tht the ion homeostsis in P. tenuiflor leves ws ffected under N 2 CO 3 stress. In previous studies, P. tenuiflor hd lower net N + uptke rtes thn whet (less thn 5% under 15 mm NCl) [8], which indictes tht P. tenuiflor hs greter cpcity thn whet to restrict unidirectionl N + influx to mintin low net N + uptke[9]. Besides, the incresed contents of K nd N on the leves surfce with the increse of externl N 2 CO 3 concentrtions (Figure 4D,E) support the hypothesis tht P. tenuiflor leves could exude slts through stomt or together with wx secretion [1]. Ion comprtmentliztion in different tissues cn fcilitte their metolic functions [2]. The slt-inducile N + /H + ntiporter is in chrge of N removl from the cytoplsm or comprtmentliztion in the vcuoles [2]. The vcuolr N + /H + ntiporters were induced y NHCO 3 in P. tenuiflor, suggesting its key role in ph regultion under lkline conditions [41]. Vcuolr-type N + /H + ntiporter

15 Int. J. Mol. Sci. 213, ws minly driven y the proton grdient cross the vcuolr memrne generted y vcuolr type H + -ATPses (V-ATPses) [2,42]. The V-ATPse is indispensle for plnt growth under norml conditions due to its roles in energizing secondry trnsport, mintining solute homeostsis, nd fcilitting vesicle fusion. Under stress conditions (e.g., slinity, drought, cold, cid, noxi, nd hevy metls), the survivl of the cells depends strongly on mintining or djusting the ctivities of the V-ATPses [2,42]. In the present study, suunit of V-ATPse ws induced under N 2 CO 3 stress. The corresponding increse of N content in vcuoles under N 2 CO 3 stress ws much higher thn in cytoplsm of epiderml cells nd mesophyll cells [43]. These findings suggest tht the N 2 CO 3 -induced V-ATPse ws required to energize the tonoplst for ion uptke into the vcuoles. Importntly, clcium content ws chnged with diverse cellulr structures (e.g., cell wll, cytoplsm, nd vcuole) in epiderml cells nd mesophyll cells in leves of P. tenuiflor when exposed to N 2 CO 3 stress (Figure 4G,H). Clcium is principl signling molecule for slinity tolernce [2,3]. High slinity leds to incresed cytosolic C 2+, which initites the stress signl trnsduction pthwys [3]. In this study, the clcium content in cytoplsm of epiderml cells (Figure 4G) nd mesophyll cells (Figure 4H) incresed significntly under 95 mm N 2 CO 3. In contrst, the clcium content in vcuoles of epidermis cells (Figure 4G) nd mesophyll cells (Figure 4H) decresed. This indictes tht the incresed cytosolic C 2+ might e trnsported from the poplst nd intrcellulr comprtments [2]. In ddition, our proteomics dt reveled tht developmentlly regulted plsm memrne polypeptide (DREPP PM)-like protein incresed under N 2 CO 3 stress. DREPP-like protein contins possile Glu-rich site t the C terminus responsile for clcium inding. DREPP-like protein hs een found to e incresed temporrily in rice under cold cclimtion [44] nd slt stress [45]. This result suggests tht DREPP-like protein my e ssocited with the C 2+ signl trnsduction pthwy in the seedlings of P. tenuiflor under N 2 CO 3 stress Enhncement of Energy Supply nd Other Specilized Metolism In this study, nine protein IDs were crohydrte metolism-relted enzymes, nd nine were involved in energy production (Tle 1). Among them, seven IDs (representing six unique proteins) were enzymes in glycolysis, including fructokinses (FRK), fructose-isphosphte ldolse (FBA), triosephosphte isomerse (TIM), glycerldehyde 3-phosphte dehydrogense (GAPDH), phosphoglycerte kinse (PGK), nd enolse. They were ll induced under N 2 CO 3 stress. In fct, glycolysis shred series of reversile rections with gluconeogenesis. Interestingly, except for FRK, the remining five enzymes ctlyzing the reversile rections were shred etween glycolysis nd gluconeogenesis. FRK, n enzyme tht irreversily ctlyzes the trnsfer of phosphte group from ATP to fructose in glycolysis, is the most importnt gtewy in the control of sugr influx into glycolysis. Thus, the increse of FRK under N 2 CO 3 long with the other five enzymes would contriute to glucose rekdown for energy genertion to cope with N 2 CO 3 stress. In ddition, mlte dehydrogense (n enzyme in citric cid cycle) nd cytosolic 6-phosphogluconte dehydrogense (n enzyme involved in pentose phosphte pthwy) were induced y N 2 CO 3 stress. Moreover, five homologs of ATP synthses nd four proteins contining the AAA ATPse fmily protein domin were induced with similr ptterns to forementioned crohydrte metolism-relted enzymes in leves under N 2 CO 3 stress. This implies tht the enhncement of the metolism provides ATP for plnt

16 Int. J. Mol. Sci. 213, dpttion to N 2 CO 3 stress. Similr results hve lso een found in slt-stressed Slicorni europe [2] nd P. tenuiflor [6]. Two mino cid metolism-relted proteins, sprtte minotrnsferse (AST) nd methionine synthse (MS), incresed in levels under N 2 CO 3 tretment (Tle 1). AST ctlyzes the interconversion of sprtte nd α-ketoglutrte to oxlocette nd glutmte, nd plys n importnt role in nitrogen ssimiltion nd iosynthesis of mino cids [2]. MS is n enzyme tht ctlyzes the finl step in the regenertion of methionine from homocysteine [46]. L-methionine is the sustrte for the synthesis of S-denosyl-L-methionine (AdoMet), which is the mjor methyl-group donor for numerous trnsmethyltions importnt in plnts secondry metolism. Lignin iosynthesis hs een suggested to represent mjor sink for AdoMet consumption in vsculr plnts. Thus, the increse of MS in N 2 CO 3 -stressed P. tenuiflor could reflect n enhnced demnd of AdoMet in the synthesis of lignin. In ddition, AST nd MS hve een thought to e relevnt to cell wll lignifiction [2], nd previous study hs reported the incresed deposition of lignin in the vsculr tissues of plnts under slinity stress [46]. Therefore, the increse of these two proteins might imply tht N 2 CO 3 tretment cn increse the vessel development in P. tenuiflor. The enhnced cell wll lignifiction incresed the mechnicl rigidity of cell wll, strengthening the vsculr tissue nd susequently permitting wter to e conducted through the xylem under negtive pressure without collpse of the vessels. This structurl ltertion my enhnce the cell-to-cell pthwy for wter trnsport, imprt greter selectivity nd reduced ion uptke, nd compenste for diminished ulk flow of wter nd solutes long the poplstic pthwy [6,2,46]. Additionlly, numer of proteins involved in trnscription, protein synthesis, protein folding nd trnsport, s well s protein degrdtion showed N 2 CO 3 response (Tle 1). Among them, two N 2 CO 3 -induced proteins were involved in protein folding. One ws protein disulfide-isomerse (PDI), memer of thioredoxin superfmily inserting disulfide onds into folding proteins. PDI is specilized to ccommodte the structurl fetures of the memrne nd secreted proteins. The other ws puttive SecA, the moile suunit of n integrl memrne trnsporter in Escherichi coli, consuming ATP during the insertion nd deinsertion phses of its ctlytic cycle while guiding preprotein segments cross the memrne [47]. While the exct function of SecA in plnts exposed to klinity stress is not known, its function is likely to ffect memrne protein dynmics in P. tenuiflor under lkline stress. 4. Experimentl Section 4.1. Plnt Cultivtion nd Tretment Seeds of Puccinelli tenuiflor (Turcz.) scrin. et Merr. were sowed on perlite nd cultured in controlled environment chmer under white fluorescent light (3 µm m 2 s 1 ; 13 h light/11 h drk) t 25 C nd out 75% reltive humidity for 5 dys, s previously descried [6,12]. The plnts were divided into three groups. In order to correlte with numer of our previous studies tht used,.4%, 1.% N 2 CO 3 tretments, these plnts were treted with mm, 38 mm nd 95 mm N 2 CO 3, respectively. Three iologicl replictes were grown nd treted with different concentrtions. To mintin stle N concentrtions in Hoglnd medium, we chnged the culture medium dily nd monitored the solution ion content nd the osmotic potentil using SevenMulti Neutrl meter (Mettler Toledo, London, UK) nd vpor pressure osmometer (552, Wescor Inc., Logn, UT, USA),

17 Int. J. Mol. Sci. 213, respectively. Seven dys lter, the treted nd untreted leves were hrvested nd used fresh or immeditely frozen in liquid nitrogen nd stored t 8 C for physiologicl nd proteomic nlysis. Shoot length nd fresh weight were mesured right fter hrvesting. Dry weight ws determined fter dehydrtion t 6 C until constnt weight ws mintined. Lef wter content ws estimted s the difference etween the fresh weight nd dry weight divided y the fresh weight [6] Photosynthesis nd Chlorophyll Fluorescence Anlysis Net photosynthetic rte (Pn), stomtl conductnce (Gs), nd trnspirtion rte (Tr) were determined t 1: m using portle photosynthesis system LI-COR 64 (LI-COR Inc., Lincoln, NE, USA). The induction kinetics of chlorophyll fluorescence ws recorded t room temperture using pulse modultion chlorophyll fluorometer (FMS-2, Hnstech, King s Lynn, UK). After drk dpttion for 3 min, the initil fluorescence yield (Fo) in wek modulted light (.12 μmol photons m 1 s 1, 6 Hz), nd mximum fluorescence yield (Fm) emitted during sturting light pulse (5 μmol photons m 2 s 1,.7 s) were mesured. Vrile fluorescence (Fv) ws clculted y the formul: Fv = Fm Fo. Then Fm' ws mesured using n irrdince of 4 μmol photons m 2 s 1 s the ctinic light nd 5 μmol photons m 2 s 1 s the sturting flshes. Fo' ws mesured using n irrdince of fr-red light (1.67 μmol photons m 2 s 1, 3 s). The vrile fluorescence (Fv') ws lso clculted y Fv' = Fm' Fo'. Mximl photochemicl efficiency of PSII (Fv/Fm), photochemicl efficiency of PSII in the light (Fv'/Fm'), non-photochemicl quenching coefficient (qnp; 1 (Fm' Fo')/(Fm Fo)), could e cquired directly from the instrument [12] Anlysis of Osmotic Potentil, Electrolyte Lekge, MDA Content, nd Antioxidnt Enzymes Osmotic potentil ws determined y vpor pressure osmometer (552, Wescor Inc., Logn, UT, USA). The MDA content ws determined using the thiorituric cid (TBA) rection s descried y Wng et l. [48]. The electrolyte lekge rtio ws otined ccording to Akrm et l. [49]. The three ntioxidnt enzymes (SOD, POD, nd CAT) were extrcted nd detected ccording to the method of Yu et l. [6] Determintion of Ion Content For mesuring the reltive contents of K nd N on the lef surfce, fresh leves (1 2 cm) were cemented to smple plte, then oserved nd nlyzed with XL-3 surrounding scnning electron microscope (The Netherlnds Phillips Compny, Eindhoven, Netherlnds) nd Kevex energy petrometer (Thermo Compny, Schumurg, IL, USA). The working voltge ws 2 kv, nd the time difference for the mesurement of smples ws 6 s. The reltive contents of elements were represented s CPS (counts per second), which were computed y sutrcting the ckground vlue from pek vlues of vried elements. The smple preprtion nd microscopic nlysis for C determintion ws done ccording to Qi et l. [5]. The reltive C contents in diverse cellulr structures (including cell wll, cytoplsm, vcuole) were detected y trnsmission electron microscope (TEM)-X ry micronlysis of 1 µm sections fter freeze-drying nd wter-free emedding in plstic. The leves (2 mm 2 mm) were plced

18 Int. J. Mol. Sci. 213, in luminum mesh oxes, nd then frozen quickly in the pre-cooling isopentne nd propne mixture (the volume rtio is 1:3). Once frozen, the mteril ws plced in freeze-dryer for removing ll of the wter. Dry mterils were then loded into the T-type vcuum permele tue, nd infiltrted with diethyl ether in vcuum t 27 C for 24 h. After eing infiltrted with phenylethylene-utyl methcrylte under norml temperture, the mterils were trnsferred into cpsules for polymeriztion. The emedding mterils were cut into 1 μm thickness sections on n ultrmicrotome (Reichert Ultrcut E, Vienn, Austri). For X-ry micronlysis, dry sections were trnsferred to folding copper grids, coted with cron nd stored over silic gel. Sections were nlyzed in Hitchi 8 trnsmission electron microscope (fitted with n energy-dispersive X-ry nlyzer EDAX 91). The ccelerting voltge ws 15 kv, the ngle etween smple nd proe ws 33, nd the counting time ws 6 s. The reltive C contents in diverse cellulr structures (including cell wll, cytoplsm, vcuole) were lso represented y CPS. For mesuring the concentrtions of K + nd N + in the seedling leves,.5 g fresh leves of ech tretment were cut to 2 cm, nd put into smll eker, then immersed in 3 ml deionized wter nd shken for 3 min. The leves were tken out nd put into nother smll eker with out 3 ml deionized wter, then cooked for 15 min in the utoclve with high temperture (121 C) in order to kill the cells nd relese ll the ions. Finlly, fter the temperture of the solution ws cooled to room temperture, the leves were filtered from the solution, nd the extrcted solution ws dded to the volume of 5 ml. We used the SevenMulti tester to determine the concentrtions of N + nd K Protein Smple Preprtion, 2DE, nd Imge Anlysis The proteins from leves under different tretment conditions were extrcted ccording to the method of Wng et l. [48]. Protein smples were prepred independently from three different tches of plnts. Protein concentrtion ws determined using Qunt-kit ccording to mnufcture s instructions (GE Helthcre, Pisctwy, NJ, USA). The protein smples were seprted nd visulized using 2DE pproches ccording to Yu et l. [6]. Protein smples were seprted on 24 cm IPG strips (ph 4 7 liner grdient) through isoelectric focusing (IEF) in the first dimension, followed y 12.5% SDS-PAGE gels in the second dimension. Aout 1.6 mg protein ws loded per gel. Gels were stined y Coomssie Brillint Blue (CBB). Three iologicl replictes for ech smple nd three technicl replictes for ech iologicl replicte were run on 2D gels. Gel imge cquisition nd nlysis were conducted s previously descried [48]. For imge cquisition, the gels were scnned using n ImgeScnner III (GE Helthcre) t resolution of 3 dpi nd 16-it gryscle pixel depth. The imges were nlyzed with ImgeMster 2D softwre (version 5.) (GE Helthcre, Pisctwy, NJ, USA).The verge vol% vlues were clculted from three technicl replictes to represent the finl vol% vlues of ech iologicl replicte. Spots with more thn 1.5-fold chnge mong the tretments nd p vlue smller thn.5 were considered to e differentilly expressed Protein Identifiction nd Dtse Serching The differentilly expressed spots were excised from the gels nd digested with trypsin [51]. MS/MS spectr were cquired on LC ESI Q-TOF MS/MS (AB Sciex, Frminghm, MA, USA) ccording to the method of Yu et l. [6]. The MS/MS spectr were serched ginst the NCBInr protein dtses [52]

19 Int. J. Mol. Sci. 213, using Mscot softwre (Mtrix Sciences, London, UK). The txonomic ctegory ws green plnts. The serching criteri were ccording to Yu et l. [6] Protein Clssifiction nd Hierrchicl Cluster Anlysis The identified proteins were serched ginst the NCBI dtse [52] nd UniProt dtse [53] to determine if their functions were known. Comined with the result of BLAST lignments, these proteins were clssified into different ctegories sed on iochemicl functions. Self-orgnizing tree lgorithm hierrchicl clustering of the expression profiles ws performed on the log trnsformed fold chnge vlues of protein spots [2,54] Sttisticl Anlysis All results were presented s mens ± stndrd error (SE) of t lest three replictes. Dt were nlyzed y one-wy ANOVA using the sttisticl softwre SPSS 17. (SPSS Inc., Chicgo, IL, USA). The tretment men vlues were compred y post hoc lest significnt difference (LSD) test. A p vlue less thn.5 ws considered sttisticlly significnt. 5. Conclusion Current proteomics llows detiled inspection of individul proteins on lrge scle nd their dynmic chnges underlying different lkline-responsive cellulr processes. In the present study, we discovered severl N 2 CO 3 -induced enzymes, such s xnthophyll cycle-relted TIL, ROS scvenging enzymes (e.g., GLO nd GLP), enzymes involved in crohydrte metolism, nd mino cid metolism-relted proteins (e.g., AST nd MS). A numer of proteins involved in cron ssimiltion, trnscription, s well s proteins synthesis nd fte were found to e reduced under N 2 CO 3 tretment. In ddition, severl proteins were proposed to e involved in cross-tolernce (e.g., AIPLP nd DREPP-like protein). More thn hlf of the N 2 CO 3 -responsive proteins reched the mximum or minimum undnces under 38 mm N 2 CO 3 tretment. This correltes to the performnce of plnt growth nd physiologicl indexes (e.g., photosynthetic cpility nd ntioxidnt enzyme ctivities), which supports the previous notion tht 38 mm N 2 CO 3 is the turning point concentrtion for P. tenuiflor. Bsed on the integrtion of proteomics nd physiologicl results, our study reveled severl N 2 CO 3 -responsive mechnisms in P. tenuiflor, including declined photosynthesis (e.g., light sorption nd cron ssimiltion) nd ctivtion of multiple ntioxidnt mechnisms (e.g., slt ion exclusion nd comprtmentliztion; POD pthwy, CAT pthwy, nd glyoxlse system), s well s enhnced energy supply nd other specilized metolisms. All these findings provide useful moleculr informtion towrd improving lkli tolernce of cerels. Acknowledgments The project ws supported y grnts from Ntionl Nturl Science Foundtion of Chin (No , ), Fundmentl Reserch Funds for the Centrl Universities (No. DL11EA1), Chin, nd Funds for Distinguished Young Scientists of Heilongjing Province, Chin (No. JC2111) to S.D.

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