Journal of Integrative Agriculture 2019, 18(2): Available online at ScienceDirect

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1 Journl of Integrtive Agriculture 219, 18(2): Aville online t ScienceDirect RESEARCH ARTICLE Effects of potssium deficiency on photosynthesis, chloroplst ultrstructure, ROS, nd ntioxidnt ctivities in mize (Ze mys L.) DU Qi 1, ZHAO Xin-hu 1, XIA Le 2, JIANG Chun-ji 1, WANG Xio-gung 1, HAN Yi 1, WANG Jing 1, YU Hi-qiu 1 1 College of Agronomy, Shenyng Agriculturl University, Shenyng 11866, P.R.Chin 2 Bureu of Lioning Provincil Seed Mngement, Shenyng 1134, P.R.Chin Astrct Potssium (K) deficiency significntly decreses photosynthesis due to lef chlorosis induced y ccumultion of rective oxygen species (ROS). But, the physiologicl mechnism for djusting ntioxidtive defense system to protect lef function in mize (Ze mys L.) is unknown. In the present study, four mize inred lines (K-tolernt, nd 99; K-sensitive, D937 nd 835) were used to nlyze lef photosynthesis, ntomicl structure, chloroplst ultrstructure, ROS, nd ntioxidnt ctivities. The results showed tht the chlorophyll content, net photosynthetic rte (P n ), stomtl conductnce (G s ), photochemicl quenching (q P ), nd electron trnsport rte of PSII (ETR) in nd 99 were higher thn those in D937 nd 835 under K deficiency tretment. Prmeters of lef ntomicl structure in D937 tht were significntly chnged under K deficiency tretment include smller thickness of lef, lower epidermis cells, nd vsculr undle re, wheres the vsculr undle re, xylem vessel numer, nd re in were significntly lrger or higher. D937 lso hd seriously dmged chloroplsts nd PSII rection centers long with incresed superoxide nion (- ) nd hydrogen peroxide (H 2 ). Activities of ntioxidnts, like superoxide dismutse (SOD), ctlse (CAT), nd scorte peroxidse (APX), were significntly stimulted in resulting in lower levels of - nd H 2. These results indicted tht the K-tolernt mize promoted ntioxidnt enzyme ctivities to mintin ROS homeostsis nd suffered less oxidtive dmge on the photosynthetic pprtus, therey mintining regulr photosynthesis under K deficiency stress. Keywords: potssium deficiency, mize, photosynthesis, chloroplst ultrstructure, ROS nd ntioxidnt 1. Introduction Potssium (K) is n essentil element for plnt growth nd Received 5 Decemer, 217 Accepted 11 Ferury, 218 DU Qi, E-mil: duqi56@126.com; Correspondence YU Hiqiu, Tel: , Fx: , E-mil: hiqiuyu@163.com 219 CAAS. Pulished y Elsevier Ltd. This is n open ccess rticle under the CC BY-NC-ND license ( cretivecommons.org/licenses/y-nc-nd/4./) doi: 1.116/S (18) development, ecuse it is involved in photosynthesis, enzyme ctivtion, electricl neutrliztion, trnsport of metolites, osmoregultion, nd control of turgor pressure in living plnt cells (Wng nd Wu 217). However, compred with nitrogen (N) nd phosphorus (P), K is typiclly pplied t lower level during fertiliztion. An ongoing downwrd trend of negtive K lnce of out 6 kg h 1 yr 1 ppered in intensive griculturl production res strting in the lte 199s, nd currently, more thn 75% of soil in Chin is deficient in ville K (Hu et l. 216). Mize (Ze mys L.) is n importnt grin ecuse it is n importnt source of feed, iofuel, nd forge crop worldwide. A series of prolems hve emerged due to

2 396 DU Qi et l. Journl of Integrtive Agriculture 219, 18(2): K deficiency in mize production systems, such s lef chlorosis nd reduced photosynthesis, cusing susequent inhiition of growth nd development (Lu et l. 216). Photosynthetic ctivity is known to decrese under K deficiency. Erly chlorophyll degrdtion is very noticele sed on rown scorching nd curling of lef tips nd mrgins s well s chlorosis (yellowing) under K deficiency (Tin et l. 28; Römheld nd Kirky 21). Trnsport of wter nd nutrients were found to e restricted y dmged ntomicl structures from roots to shoots (Mttiello et l. 215; Sun et l. 215), nd the process of photosynthetic cron dioxide uptke ws impeded under K deficiency (Bednrz et l. 1998). However, chnges in morphology nd structure of chloroplsts were significntly relted to chlorophyll content nd photosynthetic cpcity (Tin et l. 28). Under iotic stress, drmticlly low chlorophyll content, poor chloroplst ultrstructure, nd restricted scchrine trnsloction inhiited the photosynthetic rte in plnt leves (Zho et l. 21). Additionlly, Ckmk (25) concluded tht lef chlorosis cused y K-deficiency ws relted to oxidtive degrdtion of chlorophyll y excess production of rective oxygen species (ROS), response to K deficiency. Stle morphology nd structure of chloroplsts is n dvntge contriuting to tolernce in plnts under K deficiency stress (Ji et l. 27; Sho et l. 216). Run et l. (215) lso suggested low-k tolernt whet could djust development of ntomicl structures to dpt to K-strvtion through gene expression. However, comprehensive understnding of how chnges in ntomicl structure nd chloroplst ultrstructure ffect photosynthesis is lcking under K deficiency in mize. Under nutrient deficiency stress, oxidtive stress occurs when the equilirium etween the production of ROS nd ntioxidnt defense is disrupted (Shin et l. 25). ROS is toxic to plnt cells t high concentrtions nd thus excess oxidnts must e scvenged to void deleterious effects (del Río 215). Disturnce in electron trnsport during photosynthesis nd respirtion is mjor source of norml levels of ROS (Apel nd Hirt 24). Evidence indictes tht K deprivtion stimultes ethylene production nd results in further increses in ROS production (Shin nd Schchtmn 24; Kim et l. 21). Excessive ccumultion of ROS will inevitly distur physiologicl function in cells under long-term K deficiency. Plnts possess efficient systems to scvenge ROS, which provides protection from destructive oxidtive rections. Antioxidtive enzymes, such s superoxide dismutse (SOD) (Li C L et l. 211), ctlse (CAT), peroxidse (POX), or scorte peroxidse (APX) (Mittler et l. 24; Qi et l. 217), re regrded s the most importnt defense mechnisms for ROS. Enzymes tht re ssocited with ntioxidnts, including scoric cid nd glutthione, re highly efficient for detoxifying - nd H 2 in cells (You nd Chn 215; Veronic et l. 217). Studies hve lso suggested tht stronger photosynthetic ility is linked to higher ntioxidnt enzymes under K deficiency (Ji et l. 27; Chen et l. 28). Understnding the underlying physiologicl mechnisms in K-tolernt genotypes during photosynthesis is eneficil for selecting K-tolernt prents in trditionl reeding progrm. In our previous study, we found tht lef senescence of nd 99, K-tolernt inred lines, were significntly slower thn tht in D937 nd 835, K-sensitive inred lines. In ddition, the photosynthetic rte in ws higher thn tht in D937 under low K stress t lter growth stge, which prolonged the functionl period of leves (Wng et l. 212). However, we did not identify the cuse or cuses of internl physiologicl chnges in leves under K deficiency etween different mize genotypes. Therefore, the ims of this study were to: (1) determine wht regultes photosynthesis; (2) explin the vritions in ntomicl structure nd chloroplst ultrstructure; nd (3) identify the reltionship of ROS nd ntioxidnt ctivities etween different tolernt genotype mize lines under K deficiency stress. 2. Mterils nd methods 2.1. Plnt mterils nd tretments Two groups of mize inred lines, (K-tolernt) nd D937 (K-sensitive), 99 (K-tolernt) nd 835 (K-sensitive), were compred from more thn 2 lines cultivted for over 1 yers in K-deficient field (ville K ws less thn 55 mg kg 1 ) (Co et l. 27), in which K fertilizer hs not een supplied since 2. The study ws conducted in the long-term K fertilizer nchor pool (41 82 N, E) of Shenyng Agriculturl University in Lioning Province, Chin, on 5 My 216. The originl K-deficient soil ws otined from Liozhong County in Lioning Province. The plough lyer (2 cm) nutrient compositions were g kg 1 orgnic mtter, 5.4 mg kg 1 ville K, 98.5 mg kg 1 lkli-hydrolysle N, 15.3 mg kg 1 ville P, nd ph 7.3. Plnts were fertilized with 15 kg h 1 ure (N, 46%) nd 15 kg h 1 phosphorus dimine fertilizer (P 2 O 5, 46%) s se mnure efore sowing, nd 315 kg h 1 ure ws replenished for nitrogen supply t the ooting stge. According to K content in soil, sulfuric cid potssium fertilizer (K 2 O, 5%) ws regulted the ville K content to 13 mg kg 1 in controls, while none pplied in treted pools. Four rows of ech inred line were rrnged on ech experiment plot, with spcing of.5 m etween rows,.3 m in row, nd row lengths of 3.5 m.

3 DU Qi et l. Journl of Integrtive Agriculture 219, 18(2): Pigment content determintion Chlorophyll ws extrcted ccording to Li X T et l. (211). A totl of.5 g of fresh leves ws cut into smll pieces nd extrcted using 8% cetone t 4 C for 48 h until they were lnched. The sornce of the superntnt ws recorded t 645 nd 663 nm using UV-spectrophotometer (UV-18; Hitchi, Jpn) Lef ntomicl structure Ech 5 mm 5 mm lef ws cut in formlin-ceto-lcohol (FAA) solution contining 38% formldehyde:glcil cetic cid:7% lcohol (1:1:18, v/v) for 24 h t 4 C to retin the nturl lef structure ccording to Mttiello et l. (215), with some modifictions. The fixed lef pieces were dehydrted in scending series of ethnol (7, 85, 95, nd 1%), then clered in xylene nd emedded in prffin with the Epon 812 resin (Sigm, St. Louis, MO, USA), nd finlly cut into 1 mm sections using n Ultr-Thin Semiutomtic Microtome (Ultrcut-UC7; Leic, Germny). Sections were dewxed nd dyed in 1% sfrnine O wter solution nd.5% Fst Green solution (95% lcohol dissolved), then oserved nd photogrphed t 1 using microscope (ZEISS Axio Scope A1, Germny). The thickness of lef nd upper nd lower epidermis cells, the vsculr undle (Krnz) re, nd xylem vessel numer nd re, were rndomly determined nd nlyzed y ZEN Blue Lite Softwre (n=25) Chloroplst morphology nd ultrstructure To void structurl differences in prts of leves, pieces of leves (1 mm 2 mm) from the mrgin of the middle eye leves were cut into sodium phosphte uffer (PBS,.1 mol L 1, ph 6.8), contining 2.5% glutrldehyde (v/v) nd 2% prformldehyde. Air ws removed from the ottle in order to sok leves completely in uffer solution ccording to Chen et l. (24), with some modifictions. The pieces were fixed t 4 C for 24 h, followed y three rinses of 15 min ech with PBS, nd post-fixed in 1% osmic cid (OsO 4 ) for 1.5 h. The fixed smples were dehydrted for 15 min in n scending series of ethnol dilutions with 5, 7, 8, 9, nd 1% (three times). Then they were wshed with tert-butnol-ethnol solution (1:1, v/v) nd tert-butnol for 15 min, nd finlly emedded in Epon 812 resin (Sigm, USA). The sections were cut with n LKB-V ultrmicrotome (LKB, Sweden) nd stined with urnium cette-led citrte efore eing exmined under trnsmission electron microscope (HT77; Hitchi, Jpn) t 1 kv Gs exchnge nd chlorophyll fluorescence mesurement Gs exchnge prmeters were mesured using CIRAS-2 Portle Open-flow Gs Exchnge System with n internl red/white LED light source (PP Systems, USA). The lef chmer ws set t n irflow rte of 1 ml min 1, 9% reltive humidity, C concentrtion of (39±5) μmol mol 1, nd photon flux density (PFD) of 1 5 μmol m 2 s 1. Five eye leves were used to mesure net photosynthetic rte (P n ), trnspirtion rte (T r ), stomtl conductnce (G s ), nd intercellulr C concentrtion (C i ). Chlorophyll fluorescence prmeters including miniml fluorescence (F o ), the mximl fluorescence (F m ), the mximum quntum efficiency of PSII photochemistry (F v /F m ), ctul photochemicl efficiency of PSII (ΦPSII), photochemicl quenching (q P ), electron trnsport rte of PSII (ETR), nd non-photochemicl quenching (NPQ, NPQ= F m /F m 1) were mesured with n FMS-2 pulse modulted fluorometer (Hnstech, UK) on the sme leves dpted for 2 min in totl drkness Oxidnt production nd lipid peroxidtion - production ws mesured y monitoring nitrite formtion from hydroxylmine ccording to Tin et l. (23). A totl of.5 g leves were homogenized in 65 mmol L 1 K-phosphte uffer (ph 7.8) nd then centrifuged t 5 g for 1 min t 4 C. The superntnts (1 ml) nd hydroxylmine hydrochloride (1 ml) were incuted t 25 C for 1 h, then mixed in 1 ml of 58 mmol L 1 p-minoenzene sulfonic cid nd 1 ml of 7 mmol L 1 -nphthylmine solution t 25 C for 2 min. An identicl volume of chloroform ws replenished into rective solution to remove the pigment. The intensity of superntnt ws mesured t 53 nm using UV-spectrophotometer (UV-18; Hitchi, Jpn). - concentrtion ws clculted using liner clirtion curve prepred with known concentrtion of NN. The H 2 level ws detected using method sed on Tsi et l. (24). H 2 ws extrcted y homogenizing with 5 ml of 5 mmol L 1 phosphte uffer (ph 6.8) including 1 mmol L 1 hydroxylmine hydrochloride. The homogente ws centrifuged t 6 g for 25 min t 4 C. To determine the H 2 level, 3 ml of extrcted solution ws mixed with 1 ml of.1% titnium chloride in 2% (v/v) H 2 SO 4, nd then the mixture ws centrifuged t 6 g for 15 min. The intensity of superntnt solution (yellow color) ws mesured t 41 nm. H 2 level ws clculted with known concentrtion nd expressed s micromole per grm of fresh weight. Mlondildehyde (MDA) content ws determined with thiorituric cid s descried in Mir et l. (215). A totl

4 398 DU Qi et l. Journl of Integrtive Agriculture 219, 18(2): of.5 g lef tissues were homogenized with 5 ml of 5% trichlorocetic cid (TCA) nd centrifuged for 2 min t 5 g. The superntnt solution (1.5 ml) ws mixed with solution contining 2.5 ml of 5% TCA nd.5% thiorituric cid, then heted for 3 min t 1 C, nd susequently plced in n ice th for cooling. The rection mixture ws centrifuged t 5 g for 1 min, nd the resulting superntnt ws used for determintion of MDA content y recording the sornce t 532 nd 6 nm Enzyme extrction nd ssys To extrct ntioxidnt enzymes,.5 g of lef tissue ws homogenized in precooled 5 mmol L 1 phosphte uffer (ph 7.8) with.1 mmol L 1 EDTA nd 2% (w/v) insolule polyvinyl pyrrolidone. Then the homogente ws centrifuged t 1 g for 2 min t 4 C. The superntnt solution ws used for enzyme ssys, including SOD, CAT, POX, nd APX (Chen et l. 215). SOD ctivity ws mesured s descried y Neto et l. (26). The rection mixture (3 ml) contined 75 mmol NBT, 2 mmol rioflvin, 13 mmol methionine, 1 mmol EDTA, 5 mmol L 1 sodium phosphte uffer (ph 7.8), nd 5 ml enzyme solution. The rection mixture ws illuminted t 4 lx for 2 min, while the control group ws kept in drkness. The SOD ctivity ws recorded t 56 nm nd defined s the mount of SOD required to produce 5% inhiition of reduction of nitroluetetrzolium (NBT). POX nd CAT ctivities were mesured following Tin et l. (23). The rection mixture (3 ml) consisted of.25% (v/v) guicol nd 3 mmol L 1 hydrogen peroxide in 1 mmol L 1 sodium phosphte uffer (ph 6.). 1 U of POX ctivity ws defined s the increse in sornce of.1 min 1 t 47 nm g 1 FW. CAT ctivity ws detected in 3-mL 5 mmol L 1 sodium phosphte uffer (ph 7.) contining 3 mmol L 1 H 2. 1 U ws defined s the decrese in sornce of.1 min 1 t 24 nm g 1 FW. APX ctivity ws mesured ccording to Akcy et l. (21). The rection mixture (3 ml) consisted of 5 mmol L 1 sodium phosphte uffer (ph 7.),.25 mmol L 1 scorte, 1. mmol L 1 H 2, nd 1 ml of the enzyme extrct. 1 U of enzyme ctivity ws defined s decrese in sornce of.1 min 1 t 29 nm Sttisticl nlysis The experimentl design ws completely rndomized lock design with three replictes (n=3) for ech tretment. All dt were sujected to n nlysis of vrince in SPSS18. (SPSS Inc., Chicgo, IL, USA), nd the significnce of difference etween men vlues were compred using the lest significnt difference (LSD) test t.5 level of proility. All tles nd figures were mde using Excel Results 3.1. Chlorophyll content Under K deficiency, Chl, Chl (+), nd Chl / in nd 99 were slightly different thn controls, wheres these vlues in D937 nd 835 were significntly decresed t tsseling nd flowering stges compred to controls (Tle 1). Chl nd Chl (+) of D937 were significntly decresed y nd 9.3% in comprison to the control, nd 14.65%, respectively, in 835. Moreover, Chl/ ws significntly decresed y 8.85 nd 16.31% respectively due to the decresed Chl in D937 nd 835. Chl ws stle under K deficiency stress. Chlorophyll prmeters of nd 99 were ll significntly higher thn those in D937 nd 835 under K deficiency tretment Lef ntomicl structure The lef ntomicl structure in nd D937 were clerly visile in Fig. 1, including upper epidermis cells, lower epidermis cells, vsculr undles nd xylem vessels. Compred with the controls, the thickness of lef ws significntly decresed y 5.21% in D937 under K deficiency tretment, ut ws not significntly different in (Tle 2). Both vrieties were decresed in upper epidermis thickness nd mesophyll cells, with significnt decreses of nd 7.5%, respectively, in D937. There ws no Tle 1 Chlorophyll content in , D937, 99, nd 835 mize inred lines grown under control nd potssium deficient conditions Prmeter +K K +K K +K K +K K Chl (mg g 1 ) 2.96±.8 c 3.5± ±.17 cd 2.51±.13 e 3.1± ±.12 c 3.2± ±.15 de Chl (mg g 1 ).82±.2.79±.2.73±.4.72±.5.82±.5.8±.5.83±.6.82±.4 Chl (+) (mg g 1 ) 3.77± ± ±.21 cd 3.23±.17 e 3.83± ±.15 c 4.4± ±.15 de Chl / 3.62± ± ±.4 3.5±.9 c 3.71± ± ± ±.25 c Vlues re expressed s men±se with three replictes. Within row, vlues followed y different letters re significntly different (P<.5), determined y LSD.

5 399 DU Qi et l. Journl of Integrtive Agriculture 219, 18(2): A B UC Bc Ve Kr St 5 µm Kr St 5 µm D Bc UC LC Kr St LC C ET Kr Ve 5 µm Ve 5 µm Fig. 1 Lef ntomicl structure in nd D937 grown under control nd potssium deficient conditions. A D, the picture of lef ntomicl structure of K, K, D937+K, nd D937-K t 1, respectively. UC, upper epidermis cells; LC, lower epidermis cells; Kr, Krnz; Ve, vessel; St, stom; ET, epiderml tomentum; Bc, ulliform cells. Tle 2 Lef ntomicl structure in nd D937 grown under control nd potssium deficient conditions Prmeter Thickness of lef (µm) Upper epidermis cells (µm) Lower epidermis cells (µm) Mesophyll cells (µm) Distnce etween undle (µm) Krnz re (µm2) Xylem vessel numer Xylem vessel re (µm2) K K ± ± ± ± ±.81 c 16.5±.58 c 15.64± ± ± ± ± ± ± ± ± ±17.98 D937 +K K 145.8± ± ± ± ± ± ± ± ± ± ±196.2 c ± c 1.68± ± ±7.81 c 121.5±27.87 c Vlues re expressed s men±se with 25 replictes. Within row, vlues followed y different letters re significntly different (P<.5), determined y LSD. difference in distnce etween undles. The Krnz re nd xylem vessel re were not significntly different from controls under K deficiency lthough they were slightly decresed y 5.54 nd 12.9%, respectively, in , nd y 8.9 nd 13.57%, respectively, in D937. The vsculr undle re, nd xylem vessel numer nd re in were significntly higher thn those in D937 under K deficiency Chloroplst morphology nd ultrstructure Chloroplst morphology Norml chloroplst morphology nd structure in mesophyll cells (MCs) re necessry for ensuring norml lef photosynthesis of mize. In this study, chloroplst from oth lines ws irregulr, nd chloroplst numer in MCs ws reduced ccordingly under K deficiency (Tle 3). Compred with controls, the numer of chloroplsts in MCs ws decresed y 8.91 nd 2.61% in nd D937, respectively. In ddition, edem ws evident in some chloroplsts under K deficiency in nd D937. The externl surfces of chloroplsts were trnsformed from long nd ovl to ellipticl or lmost circulr. Accordingly, the vlues of length/width (L/W) of chloroplsts were significntly decresed y 15.42% in , nd y 32.64% in D937 under K deficiency due to the decresed length nd incresed width of chloroplsts. Compred with the controls, the length in D937 ws significntly decresed y 13.41% nd the width ws significntly expnded y 28.22%, while in the vlues were significntly decresed y 4.2 nd 13.4%, respectively. Chloroplst ultrstructure The ultrstructure of chloroplsts ws clerly visile in controls, with grn nd strom lmelle emedded in stroml mtrix nd ounded y doule-memrne envelope for oth genotypes (Fig. 2-A, B, G, nd H). However, pronounced difference in nd D937 ws oserved under K deficiency tretments. The externl envelope nd oundry, nd the thylkoid systems were well-preserved in (Fig. 2-D nd E), lthough plstoglouli were incresed nd swollen under K deficiency. In contrst, the ultrstructure of D937

6 4 DU Qi et l. Journl of Integrtive Agriculture 219, 18(2): Tle 3 Chloroplst morphology nd ultrstructure in nd D937 grown under control nd potssium deficient conditions Tretment K K D937+K D937 K Chloroplst size1) L (µm) W (µm) 43.14± ±1.45 c 41.32± ± ± ±2.59 c 37.27±2.24 c 28.67±2.5 L/W 2.14± ± ± ±.1 c Chloroplst numer per mesophyll cell 5.5± ± ± ±.69 c Grn numer per chloroplst 54.6± ± ± ±3.65 c Lmelle numer per grn 25± ± ± ±1.5 c 1) L, length; W, width. Vlues re expressed s men±se with 25 replictes. Within row, vlues followed y different letters re significntly different (P<.5), determined y LSD. A B 1 μm D C E 1 μm G F H 1 μm J I K 1 μm L Fig. 2 Chloroplst morphology nd ultrstructure in nd D937 grown under control nd potssium deficient conditions. A, the complete picture of chloroplst of control ( 4 ). B nd C, ultrstructure of chloroplst ( 15 ) nd mitochondri ( 2 ), respectively, of control D, E, nd F, the complete picture of chloroplsts ( 4, 15 ) nd mitochondri ( 2 ), respectively, of under tretment. G, H, nd I, chloroplst ( 4, 15 ) nd mitochondri ( 2 ), respectively, of control D937. J, K, nd L, chloroplst ( 4, 15 ) nd mitochondri ( 2 ), respectively, of D937 under tretment. Pi, pismodesm; Mi, mitochondri; CW, cell wll; PM, plsm memrne; CM, chloroplst memrne; MM, mitochondril memrne; GL, grn lmell; SL, stroml lmelle; PI, plstoglouli; Ch, chloroplst. Broken line, oundry. ws seriously destroyed under K deficiency stress. The chloroplst ws irregulr, with sustntilly gthered nd swollen plstoglouli, nd the lmelle structure ws loose (Fig. 2-J nd K). In these seriously dmged cells, the chloroplst envelopes were roken, the structure of lmelle ws not norml, nd most of them were dispersed in the cytoplsm for the K-sensitive genotype, with loose nd vcuolted structure. The numers of grn nd grn

7 DU Qi et l. Journl of Integrtive Agriculture 219, 18(2): lmelle were significntly reduced in D937 y 3.4 nd 33.49%, respectively, nd y 1.3 nd 9.32%, respectively, in (Tle 3). Mitochondri in nd D937 were decresed in size compred to the controls (Fig. 2-F nd L). The memrne structure of mitochondri in D937 ws lurry, nd ws more seriously dmged thn tht in Gs exchnge prmeters Vrieties of gs exchnge prmeters of four inred lines were ffected y K deficiency stress (Tle 4). P n, G s, nd T r were decresed nd C i ws incresed t the tsseling nd flowering stges compred to the controls. In , P n, G s, nd T r slightly decresed y 4.5, 8.76, nd 1.74%, respectively, nd C i incresed y 9.45% t the tsseling nd flowering stges. In D937, P n, G s, nd T r significntly decresed y 13.3, 2., nd 28.8%, respectively, nd C i significntly incresed y 19.9%. Compred with the controls, there ws no significnt difference in P n, G s, nd T r in 99 treted with K deficiency stress, while in 835 significntly decresed y 17.73, 35., nd 31.9%. The C i ws slightly incresed in 99, nd significntly incresed y 24.97% in Chlorophyll fluorescence chrcteristics Compred with the controls, prmeters including F m, F v /F m, ΦPSII, q P, nd ETR, were more seriously decresed in D937 nd 835 under K deficiency stress. The prmeters significntly decresed y 11.53, 17.5, 14.29, 35.94, nd 32.81% in D937, respectively, while F o nd NPQ incresed y 1.25 nd 3%, respectively (Tle 5). And the first five prmeters in 835 were significntly decresed y 14.7, 28.25, 25.24, 23.13, nd 25.24%, respectively, nd the lst two were incresed y 6.29 nd 24.88%, respectively. In nd 99, the first five prmeters were slightly decresed under K deficiency stress, nd the NPQ significntly incresed y 18.6 nd 34.52%, respectively ROS nd MDA As shown in Fig. 3, -, H 2, nd MDA in nd D937 were incresed under K deficiency stress. Compred with the controls, there ws no significnt difference in - content in treted with K deficiency stress, while in D937, - content significntly incresed y 18.79% (Fig. 3-A). The H 2 content of nd D937 ws significntly incresed y 2.41 nd 26.96%, respectively, t the tsseling nd flowering stges in comprison to the control (Fig. 3-B). Prllel to vrition in - nd H 2, the MDA content in the lef of nd D937 ws lso incresed y K stress (Fig. 3-C). Compred with the controls, the MDA in D937 ws rpidly incresed under K deficiency tretment, nd slightly incresed in Tle 4 Gs exchnge prmeters in , D937, 99, nd 835 grown under control nd potssium deficient conditions Prmeter 1) +K K +K K +K K +K K T r (mmol m 2 s 1 ) 5.82±.36 cd 5.19±.36 de 6.33± ±.34 e 6.6±.5 c 5.53±.19 cd 6.99± ±.45 e P n (μmol m 2 s 1 ) 31.13±1.53 c 29.73±2.2 c 33.78± ±1.19 c 33.33± ±2.5 c 35.25± ±1.41 c G s (mmol m 2 s 1 ).37±.2.34±.2.36±.4.29±.1 c.39±.3.35±.3.4±.4.26±.2 c C i (μmol mol 1 ) 218±12.27 c 249±17.91 c 236±14.9 c 289± ±8.76 e 195±12.26 de 183±11.84 de 229±8.83 c 1) T r, trnspirtion rte; P n, photosynthetic rte; G s, stomtl conductnce; C i, intercellulr C concentrtion. Vlues re expressed s men±se with five replictes. Within row, vlues followed y different letters re significntly different (P<.5), determined y LSD. Tle 5 Chlorophyll fluorescence in , D937, 99, nd 835 grown under control nd potssium deficient conditions Prmeter 1) +K K +K K +K K +K K F o 19±4.24 c 116±5.72 c 117±7.93 c 129± ±6.1 c 115±7.7 c 117±6.23 c 132±5.2 F m 643±32.68 cd 6±36.4 d 694± ±29.22 cd 66±36.37 c 63±3.67 cd 71± ±37.44 cd F v /F m.81±.1.76±.6.8±.2.66±.4 c.81±.1.77±.4.81±.1.58±.9 d ΦPSII.31±.1 c.29±.1 cd.35±.1.3±.2 c.33±.2.31±.2 c.35±.2.26±.1 d q P.62±.1 c.56±.4 cd.64±.2.41±.1 e.73±.3.69±.2.76±.3.59±.3 cd NPQ 1.72±.4 d 2.4±.11 c 1.2±.9 e 1.56±.11 d 1.77±.18 d 2.38± ±.9 c 2.67±.18 ETR 169±11.96 c 158±12.88 cd 191± ±26.38 c 183± ±26.5 c 192± ±9.73 d 1) F o, the miniml fluorescence; F m, the mximl flurescence; F v /F m, the mximum quntum efficiency of PSII photochemistry; ΦPSII, the ctul photochemicl efficiency of PSII; q P, photochemicl quenching; NPQ, non-photochemicl quenching; ETR, electron trnsport rte of PSII. Vlues re expressed s men±se with five replictes. Within row, vlues followed y different letters re significntly different (P<.5), determined y LSD.

8 42 DU Qi et l. Journl of Integrtive Agriculture 219, 18(2): Antioxidnt enzyme ctivity Lef ntioxidnt enzyme ctivity ws stimulted in oth lines over long-term exposure to K deficiency stress t tsseling nd flowering stges (Fig. 4). Compred with the controls, SOD ctivity ws significntly elevted y15.51% in ; SOD ctivity ws not significntly different etween D937 nd control (Fig. 4-A). POX ctivity ws significntly incresed in nd D937 t tsseling nd flowering stges, y nd 32.18%, respectively, compred to the controls (Fig. 4-B). A significnt increse of 69.48% in CAT ctivity ws oserved in the lef of under K deficiency stress, ut there ws no difference in D937 (Fig. 4-C). APX ctivity incresed in nd D937 in response to K deficiency. And it ws significntly incresed in y 3.31%, nd y 12.53% in D937 (Fig. 4-D). 4. Discussion 4.1. Photosynthetic prmeters Photosynthesis, the sis of plnt growth, development +K K A content (nmol g 1 FW) B H 2 content (μmol g 1 FW) c c C MDA (nmol g 1 FW) Fig. 3 Accumultion of rective oxygen systems (ROS) nd mlondildehyde (MDA) in nd D937 grown under control nd potssium deficient conditions. A C re differences of superoxide nion (- ), hydrogen peroxide (H 2 ), nd MDA contents, respectively in oth inred lines t the tsseling nd flowering stges. The rs re the stndrd errors of the men; the letters represent significnt differences t the P<.5 level. +K K A SOD ctivity (U g 1 FW) B POX ctivity (U g 1 min 1 FW) c C CAT ctivity (U g 1 min 1 FW) D APX ctivity (U g 1 min 1 FW) Fig. 4 Antioxidnt enzyme ctivity in nd D937 grown under control nd potssium deficient conditions. A D re differences in superoxide dismutse (SOD), peroxidse (POX), ctlse (CAT), nd scorte peroxidse (APX) ctivities, respectively, in oth inred lines t the tsseling nd flowering stges. The rs re the stndrd errors of the men; the letters represent significnt differences t the P<.5 level.

9 DU Qi et l. Journl of Integrtive Agriculture 219, 18(2): nd yield, includes light hrvesting, PSII photochemistry, nd C ssimiltion. Previous studies hve shown tht nitrogen, sulfur, nd iron deficiencies could directly lock the synthesis of protein complexes in photosynthetic rections (Klji et l. 214). Lef P n decresed under K deficiency stress in soyen, cotton, nd mize, resulting in suppressed electron trnsfer energy, photosynthetic enzyme ctivity, nd nitrogen metolism enzyme ctivity (Qu et l. 211; Wng et l. 212, 215; Hu et l. 217). Chlorophyll is the min photosynthetic pigment in plnts. Chlorophyll synthesis is stimulted y K ppliction, ut stopped y incresing ethylene nd scisic cid under K deficiency (Lwnson et l. 1977). In the present study, decreses in P n nd G s, nd increses in C i were more severe under K deficiency stress in D937 nd 835 thn in nd 99. However, K-tolernt mize, nd 99, hd higher P n nd G s thn D937 nd 835 under K deficiency. In ddition, K deficiency reduced chlorophyll in D937 nd 835, especilly Chl, ut did not ffect chlorophyll in K-tolernt nd 99. A similr result ws reported in cotton (Hu et l. 216). These dt indicte tht K-tolernt mize, nd 99, could mintin norml photosynthesis with higher chlorophyll y djusting the stomt conductnce to void increses in the intercellulr C concentrtion. Chlorophyll fluorescence prmeters could provide precise informtion out the stte of the photosynthetic pprtus nd especilly of PSII (Sun et l. 215). F v /F m is mjor indictor of photoinhiition or injury in the PSII complexes (Bednrz et l. 1998; Li X T et l. 211). q P is used to mesure the utiliztion rte of energy sored y ntenn pigment of PSII for photosynthesis, nd lso reflects the opening stte of the PSII rection center (Bilger nd Björkmn 199). In the present study, the F v /F m, ΦPSII, q P, nd ETR were significntly decresed in D937 nd 835, ut there were miniml chnges in nd 99. The decresed F v /F m in D937 nd 835 under K deficiency ws due to the reduction of F m, which indicted dmge to the rection centers of PSII, hindering electron trnsport. The decresed ETR directly indicted the disorgnized electron trnsport chin, which resulted in free rdicls, nd dmge of chloroplst memrnes nd chnges in chloroplst structure 4.2. Lef ntomicl structure nd chloroplst structure Norml lef ntomicl structure nd chloroplst structure re prticulrly eneficil for photosynthesis (Li et l. 217). Dmged ntomicl structure could influence the trnsport of wter nd nutrients from roots to shoots, nd eventully impede the process of photosynthesis under nutrient deficiency (Mttiello et l. 215; Sun et l. 215). However, low-k-tolernt whet could djust its ntomicl structure development to llevite negtive effects of K-strvtion (Run et l. 215). In this study, lef thickness, mesophyll cells, lower epidermis cells, nd Krnz re were significntly decresed in D937 under K deficiency stress. Although the Krnz re nd xylem vessel re were lso decresed y K deficiency, hd significntly higher re thn D937, nd thus retined higher trnsport cpcity for wter nd nutrients from roots to shoots (Mttiello et l. 215). In ddition, morphologicl nd structurl chnges in chloroplsts were importnt for the decrese of chlorophyll content nd photosynthetic cpcity (Sho et l. 214). Dysfunction of chloroplsts ws cused y degrded envelope, irregulr chloroplst morphology nd decresed grn nd thylkoid lmelle due to iotic stress (Xu et l. 28; Ren et l. 216). Zho et l. (21) indicted tht the function of chloroplsts ws ffected in cotton leves under K deficiency stress, resulting in n increse in strch grins nd plstoglouli nd decresed grn in chloroplsts. In this study, chloroplsts in leves hd more welldefined memrnes nd thylkoids with smller numers of plstoglouli, wheres chloroplsts in D937 were destroyed with deliquescent nd fuzzy memrnes nd thylkoids. As result, the photosynthetic process ws decresed nd chlorophyll content nd chlorophyll fluorescence prmeters were reduced, eventully resulting in the degrdtion of lef photosynthetic cpcity ROS nd ntioxidnt ctivities ROS ws rpidly generted when crops were exposed to iotic stress, especilly in chloroplsts (Xu et l. 26; Ahnger nd Agrwl 217). ROS led to disordered regulr cell structure nd function y dmging nucleic cids, oxidizing proteins, nd cusing lipid peroxidtion (Foyer nd Noctor 25). Long-time K deficiency resulted in severe memrne lipid peroxidtion due to high ROS production nd ccumultion in tomto (Hernndez et l. 212). In this study, - nd H 2 were incresed y K deficiency, with more ccumultion in D937 thn in MDA contents indicted more severe memrne lipid peroxidtion in D937 thn tht in under K deficiency. By scvenging the norml ROS, enzymtic ntioxidnt systems were considered s primry regultors for scvenging nd eliminting ROS in the plnt cells. First, SOD could specilly ctlyze the conversion of - into H 2, cting s the first line of defense ginst the potentil toxicity of superoxide rdicls. Then, other ntioxidnt enzymes ctlyzed the susequent rekdown of H 2 to wter (H 2 O) nd oxygen ( ) (Grrtt et l. 22). Higher SOD enzyme ctivity in slt-tolernt rice ws importnt for eliminting ROS s slt concentrtion incresed (Chwl et l. 213). In our other

10 44 DU Qi et l. Journl of Integrtive Agriculture 219, 18(2): study, we found higher SOD enzyme ctivity nd lower MDA in roots of under K deficiency stress (Zho et l. 216). CAT is principl enzyme for H 2 scvenging under slt nd P deficiency (Neto et l. 26; Chen et l. 215). In the present study, K deficiency stimulted ntioxidnt ctivity in nd D937. The incresed ctivity of SOD in the K-tolernt line, , plyed essentil roles in scvenging or eliminting - under K deficiency. Also, CAT nd APX in were higher thn those in the K-sensitive line, D937, under K deficiency. H 2 concentrtion in nd D937 leves ws considerly higher thn tht in the controls with incresed CAT, POX, nd APX ctivities. However, higher CAT nd APX ctivities nd lower MDA ws oserved in , lthough POX ctivity ws incresed in oth lines. This result suggested tht CAT nd APX enzymes plyed mjor role in scvenging H 2 in leves under K deficiency stress. These results indicted tht K-tolernt mize, , ws le to effectively stimulte ctivity of SOD enzymes nd coordinte expression of CAT, POX, nd APX, lleviting memrne lipid peroxidtion under K deficiency stress. 5. Conclusion Under K deficiency stress, the K-tolernt mize, nd 99, mintined higher chlorophyll content, nd hd stle morphology nd chloroplst structure, ll of which led to higher photosynthetic ility compred to D937 nd 835. In contrst, the rection centers of PSII nd electron trnsfer in D937 nd 835 were inhiited under K deficiency stress, which resulted in severe memrne lipid peroxidtion due to ccumultion of - nd H 2. Compred with K-sensitive mize, ntioxidnt enzyme ctivity in K-tolernt mize ws incresed, which promoted scvenging of ROS under K deficiency stress nd llevited oxidtive stress, llowing norml photosynthesis. Acknowledgements This work ws supported y the Ntionl Nturl Science Foundtion of Chin ( nd ) nd the Ntionl Key Technology R&D Progrm of Chin (213BAD7B3). References Ahnger M A, Agrwl R M Potssium up-regultes ntioxidnt metolism nd llevites growth inhiition under wter nd osmotic stress in whet (Triticum estivum L.). Protoplsm, 254, Akcy U C, Ercn O, Kvs M, Yildiz L, Yilmz C, Oktem H A, Yucel M. 21. Drought-induced oxidtive dmge nd ntioxidnt responses in penut (Archis hypoge L.) seedlings. Plnt Growth Regultion, 61, Apel K, Hirt H. 24. Rective oxygen species: Metolism, oxidtive stress, nd signl trnsduction. Annul Review of Plnt Biology, 55, Bednrz C W, Oosterhuis D M, Evns R D Lef photosynthesis nd cron isotope discrimintion of cotton in response to potssium deficiency. Environmentl & Experimentl Botny, 39, Bilger W, Björkmn O Role of xnthophyll cycle in photoprotection elucidted y mesurements of light-induced sornce chnges, fluorescence nd photosynthesis in leves of Heder cnriensis. Photosynthesis Reserch, 25, Ckmk I. 25. The role of potssium in lleviting detrimentl effects of iotic stresses in plnts. Journl of Plnt Nutrition nd Soil Science, 168, Co M J, Yu H Q, Yn H K, Jing C J. 27. Difference in tolernce to potssium deficiency etween two mize inred lines. Plnt Production Science, 1, Chwl S, Jin S, Jin V Slinity induced oxidtive stress nd ntioxidnt system in slt-tolernt nd sltsensitive cultivrs of rice (Oryz stiv L.). Journl of Plnt Biochemistry & Biotechnology, 22, Chen P, Mitsui T, Frmer D B, GolovchenkoJ, Gordon R G, Brnton D. 24. Atomic lyer deposition to fine-tune the surfce properties nd dimeters of fricted nnopores. Nno Letters, 4, Chen S, Zho H, Ding G, Xu F Genotypic differences in ntioxidnt response to phosphorus deficiency in Brssic npus. Plnt nd Soil, 391, Chen W, Yng X, He Z, Feng Y, Hu F. 28. Differentil chnges in photosynthetic cpcity, 77 K chlorophyll fluorescence nd chloroplst ultrstructure etween Zn-efficient nd Zn-inefficient rice genotypes (Oryz stiv) under low zinc stress. Physiologi Plntrum, 132, Foyer C H, Noctor G. 25. Redox homeostsis nd ntioxidnt signling: A metolic interfce etween stress perception nd physiologicl responses. The Plnt Cell, 17, Grrtt L, Jngoudr B, Lowe K, Anthony P, Power J, Dvey M. 22. Slinity tolernce nd ntioxidnt sttus in cotton cultures. Free Rdicl Biology & Medicine, 33, Hernndez M, Fernndez-Grci N, Grci-Grm J, Ruio- Asensio J S, Ruio F, Olmos E Potssium strvtion induces oxidtive stress in Solnum lycopersicum L. roots. Journl of Plnt Physiology, 169, Hu W, Coomer T D, Lok D A, Oosterhuis D M, Zhou Z Potssium deficiency ffects the cron-nitrogen lnce in cotton leves. Plnt Physiology & Biochemistry, 115, 48. Hu W, Lv X, Yng J, Chen B, Zho W, Meng Y, Wng Y, Zhou Z, Oosterhuis D M Effects of potssium deficiency on ntioxidnt metolism relted to lef senescence in cotton (Gossypium hirsutum L.). Field Crops Reserch, 191, Ji Y, Yng X, Islm E, Feng Y. 27. Effects of potssium

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