Journl of Integrtive Agriulture 216, 15(2): 295 38 Aville online t www.sienediret.om SieneDiret RESEARCH ARTICLE Effets of hilling tolerne indued y spermidine pretretment on ntioxidtive tivity, endogenous hormones nd ultrstruture of indi-jponi hyrid rie seedlings ZENG Yn-hu 1, 2, ZAHNG Yu-ping 2, XIANG Jing 2, WU Hui 2, CHEN Hui-zhe 2, ZHANG Yi-ki 2, ZHU De-feng 1, 2 1 College of Agronomy, Nnjing Agriulturl University, Nnjing 2195, P.R.Chin 2 Stte Key Lortory of Rie Biology, Chin Ntionl Rie Reserh Institute, Hngzhou 316, P.R.Chin Astrt Spermidine (Spd) is known to e involved in the regultion of plnt responses to hilling stress nd ountert the dverse effet of stress onditions. Antioxidnt tivities, endogenous hormones nd ultrstruture hnge under hilling stress were investigted in indi-jponi hyrid rie seedlings. 12-d-old seedlings were sujeted to exogenous Spd (1 mmol L 1 ) nd then hilling stress (6 C, 4 d) ws indued, followed y susequent reovery (25 C, 4 d). Results showed tht mlondildehyde (MDA) nd proline ontent were enhned signifintly, wheres shoot fresh nd dry weights deresed during hilling stress nd fter reovery; hlorophyll ontent of hilling-stressed seedlings inresed slightly ut delined fter reovery; dditionlly, totl solule sugr, surose, frutose nd strh ontents inresed signifintly during hilling stress, nd only solule sugr nd frutose ontents were oserved in inrese fter reovery; hilling stress-indued inreses in superoxide dismutse (SOD), peroxidse (POD) nd tlse (CAT) tivities, ut delined fter reovery, nd the level of sorte peroxidse ws lower during hilling stress nd fter reovery; however, endogenous indole-3-eti id (IAA), zetin rioside (ZR), gierelli id (GA 3 ), nd sisi id (ABA) levels were indued deresed ompred with Spd pretretment. The mirosopi nlysis reveled tht hilling stress-indued destrution of the hloroplst envelope during hilling stress nd inresed the numer of plstoglouli long with errtions in thylkoid memrnes fter reovery. In ontrst, exogenous Spd proteted rie seedlings from hilling-indued injuries in terms of lower mlondildehyde, proline nd rohydrtes umultion oupled with inresed endogenous hormones metolism. After reovery, Spd pretretment hilling-exposed seedlings showed higher tivities of ntioxidnt enzymes nd norml physiologil funtion of hloroplsts. These results suggest tht Spd ould promote effetively hilling tolerne whih might e lrgely ttriutle to the integrity of ell struture nd norml metolism of endogenous hormones in indi-jponi hyrid rie seedlings. Keywords: polymines, hilling stress, ntioxidtive tivity, endogenous hormones, ultrstruture, indi-jponi hyrid rie (Oryz stiv L.) Reeived 6 Ferury, 215 Aepted 13 April, 215 Correspondene ZHU De-feng, Tel/Fx: +86-571-6337373, E-mil: nrie@qq.om; ZAHNG Yu-ping, Tel/Fx: +86-571- 63371376, E-mil: nrrizyp@163.om 216, CAAS. Pulished y Elsevier Ltd. This is n open ess rtile under the CC BY-NC-ND liense (http:// retiveommons.org/lienses/y-n-nd/4./). doi: 1.116/S295-3119(15)6151-6 1. Introdution Low temperture is limiting environmentl ftor on plnts tht n use signifint growth nd yield redutions in mny gronomi rops, inluding rie (Boyer 1982). Gen-
296 ZENG Yn-hu et l. Journl of Integrtive Agriulture 216, 15(2): 295 38 erlly, the men lethl temperture for rie is ~4.7 C, nd when the mient temperture goes elow 5 1 C, the plnt seedlings growth nd development is impeded (Snhez et l. 214). During hilling stress, ellulr memrnes n e ttked y free rdils, resulting in memrne lipid peroxidtion nd umulting mlondildehyde (MDA) nd proline (Di et l. 212). The genertion of retive oxygen speies (ROS) hs een ssoited with oxidses in plsm memrne nd the eletron trnsport of hloroplst (Lloi et l. 24) nd will ffet memrnes of ell ultrstruture (Tylor nd Crig 1971). Plnts exhiit severl ntioxidnt enzymes suh s superoxide dismutse (SOD), peroxidse (POD), tlse (CAT), nd sorte peroxidse (APX), whih hve een reported to e orrelted with inresed stress tolerne, engged in protetion of the ellulr memrnes nd llevition of ROS effets (Xu et l. 211; Mostof et l. 214). Jouve et l. (24) reported tht rohydrtes re mjor tegory of omptile solutes tht inlude hexoses (mostly frutose nd gluose), dishrides (surose), sugr lohols, ll of whih umulte during stress. In previous studies, rohydrtes, suh s sugrs (gluose, frutose, surose nd frutns) nd strh, were reported to umulte in plnts under slt stress (Prid et l. 22), drought stress (Sheorn nd Sini 1996) nd hilling stress (Morsy et l. 27), yet dt on polymines pplition is limited under hilling stress of rie (Morsy et l. 27). Chilling ondition uses rpid hnges in memrne struture of plnt leves (Xu et l. 28), resulting from losure of stomt indued y osmoti stress. Moreover, endogenous hormones, suh s ytokinins (ZR), sisi id (ABA), gierellin (GA 3 ), nd indole-3-eti id (IAA), regulte the growth nd development of plnt leves y ffeting ell division, elongtion nd differentition within pil meristems (Luoml et l. 25), re vitlly sensitive to mient environment lthough they re muh little onentrtion in vivo of plnt. When plnts were enountered to hilling stress, it ould djust the hnges of mient environment in the regultion of the ontent of endogenous hormones (Wng et l. 26). Polymines (PAs), inluding putresine, spermidine (Spd) nd spermine, re smll liphti, low moleulr weight polytions nitrogenous ompounds tht re uiquitous in prokryoti nd eukryoti orgnisms nd prtiipte in the regultion of physiologil nd developmentl proesses. Pretretment with PAs signifintly enhned the levels of ntioxidnt pity nd llevited osmoti dmge used y memrne lipid peroxidtion (Mostof et l. 214). Menwhile, PAs medited in ABA induing in stomt losure y proteting ell struture to void swollen hloroplst (Konstrntinos et l. 21). It hs een demonstrted tht exogenously pplied PAs n rpidly enter the intt hloroplst (He et l. 22) nd ply role in proteting the photosyntheti pprtus from dverse effets of environmentl stresses. There is lose reltionship etween PA nd hormones in the regultion of plnt growth (Liu et l. 25). The funtion of PAs nd hormones (CTK) ws superposition, euse of hloroplst eing rih in PAs, ut when it ws lked, it might ffet the hloroplst struture nd funtion (Shu et l. 212). In reent studies, exogenous Spd pplitions suessfully llevited vrious ioti stresses, nd proteted ell struture suh s slinity (He et l. 28; Shu et l. 212), drought (Kuis 28), het (Mostof et l. 214), nd hilling (Ymmoto et l. 212). Plnts re sujet to hnges in temperture, oth during hnges in seson nd more rpidly within individul dys. indi-jponi hyrid rie hd een widely pplied in the field prtie owing to the powerful heterosis nd super-high yield potentil, ut it is severely ffeted y errnt hnges in temperture (Li et l. 1997). This flutution in temperture might ffet the physiology of rie plnts nd n use severe dmge, espeilly during the seedling stge. Mostof et l. (214) reported tht folir pplition of Spd enhned het tolerne during het stress nd fter reovery of rie. However, the effet of Spd pplition on rie seedlings during hilling stress nd fter reovery is unler. Moreover, exogenous Spd pplitions were seldom tested s wy to llevite hilling stress, prtiulrly in oxidtive dmge nd rohydrte umultion, endogenous hormones nd ultrstruture of hloroplsts in indi-jponi hyrid rie (Ymmoto et l. 212). We hypothesize tht low temperture my use rohydrte, endogenous hormones nd ultrstruture hnges in leves of rie nd higher tivities of ntioxidnts with Spd pplition my llevite those hnges nd protet ell memrnes from lipid peroxidtion. Therefore, in this study, we exmined the effet of hilling stress with or without pplition of exogenous Spd on the rohydrte ontent, endogenous hormones, ultrstruture of hloroplsts, nd the tivity of the ntioxidnt system in rie plnts. The results will e helpful to understnding the roles of Spd pplitions on enhning hilling tolerne in indi-jponi hyrid rie. 2. Results 2.1. Plnt growth In the present study, the plnt height, shoot fresh weight (FW), shoot dry weight (DW), nd reltive wter ontent deresed signifintly during hilling stress nd fter reovery in the non-spd-pretreted hilling-stressed seedlings (6 C) s ompred with the ontrol, while the Spd pretretment seedlings showed signifintly lower redutions in these
ZENG Yn-hu et l. Journl of Integrtive Agriulture 216, 15(2): 295 38 297 indies during hilling stress nd they did not inrese during the reovery period (Tle 1). In the ontrol (Spd pretretment non-hilling stressed) seedlings, there ws slight inrese in the ove indies exept plnt height. Similrly, the reltive growth rte (RGR) deresed signifintly in the hilling stress fter reovery. However, the Spd pretretment signifintly inresed the RGR fter reovery. The pplition of 1 mmol L 1 exogenous Spd gretly llevited the hilling-medited growth redution in the rie seedlings. 2.2. Chl ontent The hilling stress-indued seedlings showed signifint inreses in the totl Chl ontent nd Chl during hilling stress, while Spd pretretment seedlings showed signifint redutions (Tle 2), nd the trends in CK nd CK+Spd were similr to tht. In ontrst, the Chl ontent nd Chl /Chl vlue deresed in hilling stress-indued seedlings during hilling stress nd inresed to high levels in the Spd pretretment hilling-stressed seedlings. There ws similr trend mong ll the tretments fter reovery nd hilling stress, ut no differene ws found in Chl. 2.3. Crohydrte nd strh onentrtions As shown in Fig. 1, the solule sugr, surose, frutose, nd strh ontents in leves of rie seedlings inresed signifintly during hilling stress s ompred with the ontrol. They re ~86% (Fig. 1-A), 42% (Fig. 1-B), 272% (Fig. 1-C), nd 16% (Fig. 1-D) higher, respetively, thn the ontrol, ut the signifintly inresing trend during hilling reovery ws only found in totl solule sugr nd frutose, nd no signifint differene ws found in surose nd strh. The Spd pretretment signifintly deresed the totl solule sugr, surose nd frutose ontents during hilling stress nd sustined signifint inrese exept surose fter reovery. There ws little signifint differene in strh, lthough it ws similr to tht during hilling stress nd fter reovery. 2.4. MDA nd proline ontent Under the non-spd-pretreted hilling-stress, the MDA level inresed signifintly during hilling stress nd fter reovery in the leves of rie seedlings (Fig. 2-A), while Tle 1 Effet of exogenous spermidine (Spd) on hilling-indued hnges in the growth prmeters of rie seedlings Tretments 1) Plnt height (m) Shoot fresh weight (FW, mg seedling 1 ) Shoot dry weight (DW, mg seedling 1 ) Reltive wter ontent (%) RGR 2) (mg g 1 d 1 ) 4 d L CK 21.73±2.48 151.22±14.24 22.26±2.9 85.28±.19 CK+Spd 21.9±1.57 162.22±3.15 23.12±3.16 85.77±1.71 T 17.35±.57 94.78±6.83 18.3±1.7 8.97±1.21 T+Spd 19.32±.35 123.67±12.55 21.31±1.98 82.76±.16 4 d R CK 27.29±.46 186.±6.93 26.19±1.32 85.92±.21 44.95±.32 CK+Spd 26.97±.27 21.67±16.37 27.78±2.13 86.22±.16 52.35±9.4 T 22.48±.74 116.67±4.98 2.76±1.36 82.22±.68 3.85±6.54 T+Spd 22.26±.97 164.78±21.89 25.92±3.68 84.29±.22 41.67±6.25 1) 4 d L, low temperture tretment for four onseutive dys; 4 d R, norml temperture reovery for four onseutive dys. CK, ontrol plnts under 25 C; CK+Spd, plnts under 25 C with Spd folir sprying; T, plnts under 6 C; T+Spd, plnts under 6 C with Spd folir sprying. The sme s elow. 2) RGR, reltive growth rte. Dt re represented s mens±sd from three seprte experiments s 3 replites. Different letters indite signifint differenes mong vrious tretments in the sme period y Dunn s multiple rnge test t P<.5. The sme s elow. Tle 2 Effet of exogenous Spd on hilling-indued hnges in the hlorophyll (Chl) ontent in rie seedlings Tretments Totl Chl (mg g 1 FW) Chl (mg g 1 FW) Chl (mg g 1 FW) Chl /Chl 4 d L CK 3.35±.4 1.43±.2 1.92±.5.75±.3 CK+Spd 3.5±.9 1.38±.3 2.13±.12.65±.5 T 3.72±.15 1.31±.4 2.41±.19.55±.6 T+Spd 3.16±.13 1.49±.4 1.67±.17.9±.11 4 d R CK 3.11±.27 1.53 ±.6 1.59 ±.33 1. ±.27 CK+Spd 3.14±.11 1.52±.3 1.61 ±.14.95 ±.1 T 2.88±.14 1.59±.1 1.29 ±.14 1.24 ±.15 T+Spd 2.99±.5 1.56±.1 1.42 ±.6 1.1 ±.5
298 ZENG Yn-hu et l. Journl of Integrtive Agriulture 216, 15(2): 295 38 A Totl solule sugr (mg g 1 DW) B Surose ontent (mg g 1 DW) C Frutose (mg g 1 DW) CK CK+Spd T T+Spd 4 35 3 25 2 15 1 5 5 4 3 2 1 4 3 2 1 D 16 Strh (mg g 1 DW) 12 8 4 Fig. 1 Effets of exogenous spermidine (Spd) on hillingindued hnges in solule sugr (A), surose (B), frutose (C), nd strh (D) in the leves of rie seedlings. Vertil rs represent SD of the men (n=3). Different letters indite signifint differenes mong vrious tretments in the sme period t P<.5, ording to Dunn s multiple rnge test. 4 d L, low temperture tretment for four onseutive dys; 4 d R, norml temperture reovery for four onseutive dys. CK, ontrol plnts under 25 C; CK+Spd, plnts under 25 C with Spd folir sprying; T, plnts under 6 C; T+Spd, plnts under 6 C with Spd folir sprying. The sme s elow. MDA (nmol g 1 FW) Pro (ug g 1 FW) 2 16 12 8 4 5 4 3 2 1 CK CK+Spd T T+Spd d Fig. 2 Effets of exogenous Spd on hilling-indued hnges in mlondildehyde (MDA) (A) nd proline (Pro) (B) ontents in the leves of rie seedlings. the MDA level deresed signifintly in the Spd pretretment seedlings during hilling stress nd did not inrese fter the reovery period ompred with hilling-stressed seedlings. The MDA level of hilling-stressed seedlings inresed signifintly during hilling stress nd fter reovery s ompred with the ontrol. Similr to MDA, the proline level inresed signifintly during hilling stress nd remined t this high level fter reovery (Fig. 2-B). The exogenous Spd pplition deresed signifintly the proline level during hilling stress nd styed t similr level fter reovery. Spd pplition lso deresed signifintly the proline level under the ontrol seedlings, nd mintined the trend fter reovery ut no signifint differene. 2.5. Antioxidnt enzyme tivities The tretments showed signifint effets on the four ntioxidnt enzymes (Fig. 3). After exposure to hilling stress for 4 d, SOD tivity inresed signifintly; however, fter reovery signifintly deresing differenes were found (Fig. 3-A). Spd pretretment seedlings hd signifintly inresed SOD tivities during hilling stress nd fter reovery. Chilling stress reted different effets on CAT tivity. CAT tivity during hilling stress ws signifintly
ZENG Yn-hu et l. Journl of Integrtive Agriulture 216, 15(2): 295 38 299 higher thn the ontrol nd it lso inresed fter reovery (Fig. 3-B). In ontrst, CAT tivity inresed signifintly in the Spd pretretment seedlings irrespetive of hilling stress or not. A slight inrese ws found in the POD tivity of hilling-stressed seedlings during hilling stress (Fig. 3-C), nd fter reovery POD tivity ws signifintly lower thn the ontrol plnts, ut POD tivity inresed signifintly under the Spd pretretment. APX tivity showed signifint derese during hilling stress nd mintined signifint deresing trend fter reovery ompred with the ontrol (Fig. 3-D). However, the exogenous Spd pplition enhned the APX tivity during hilling stress nd it did not derese fter reovery ompred with tht in the hilling-stressed seedlings, ut signifint deline ws oserved in ontrst to the ontrol. 2.6. Endogenous hormones Endogenous hormone ontents of seedlings were pprently ffeted y the tretments (Fig. 4). ABA ontent, exposure to hilling stress, inresed signifintly during hilling stress nd fter reovery ompred with ontrol (Fig. 4-A), moreover, ABA ontent ws high signifintly in Spd pretretment. Spd-tretment enhned signifintly (P<.5) IAA ontent during hilling stress, ut it did not derese during reovery (Fig. 4-B). Similrly, ytokinin ontent (ZR) ws deresed signifintly y hilling stress tretment during hilling stress nd fter reovery ompred with ontrol (Fig. 4-C), wheres the exogenous Spd pplition enhned the ZR ontent during hilling stress nd ws still mintined the higher level fter reovery. In ontrst, GA 3 ontent redued signifintly during hilling stress (Fig. 4-D) while it remined the sme level fter reovery. However, Spd pretretment signifintly inresed the ontent of GA 3 during hilling stress nd it did inrese during reovery. A SOD (U g 1 FW) B CAT (U min 1 g 1 FW) C POD (U min 1 g 1 FW) 39 325 26 195 13 65 3 25 2 15 1 5 6 5 4 3 2 1 CK CK+Spd T T+Spd 2.7. Chnges in ell ultrstruture D 9 Under ontrol ondition, the hloroplsts showed thylkoids with grn during hilling stress (Fig. 5) nd fter reovery (Fig. 6). The ultrstruture of the hloroplsts seemed to show no differene etween the ontrol plnts nd plnts treted with Spd. However, low temperture used remrkle struturl hnges during hilling stress nd fter reovery. Speifilly, some hloroplsts were swollen, nd internl lmelle of the stroml thylkoids were dmged to indue prtly grnl thylkoid lyers to loosen during hilling stress. Furthermore, the hloroplst memrnes were dmged nd eme indistint, ut Spd pretretment enhned reovery of the hloroplst shpe in tht the stroml lmelle seemed to form formlly the grn ultrstruture nd the hloroplsts were oserved higher distint memrnes. APX (U min 1 g 1 FW) 75 6 45 3 15 Fig. 3 Effet of exogenous Spd on hilling-indued hnges in superoxide dismutse (SOD) (A), tlse (CAT) (B), peroxidse (POD) (C), nd sorte peroxidse (APX) (D) tivities in the leves of rie seedlings.
3 ZENG Yn-hu et l. Journl of Integrtive Agriulture 216, 15(2): 295 38 A ABA (ng g 1 FW) 2 16 12 8 4 B 1 IAA (ng g 1 FW) C ZR (ng g 1 FW) D GA 3 (ng g 1 FW) 8 6 4 2 15 12 9 6 3 12 9 6 3 CK CK+Spd T T+Spd d Fig. 4 Effet of exogenous Spd on hilling-indued hnges in sisi id (ABA) (A), indole-3-eti id (IAA) (B), ytokinins (ZR) (C), nd gierellin (GA 3 ) (D) ontents in the leves of rie seedlings. d Moreover, mny riosoml prtiles were relesed fter reovery under hilling stress (Figs. 5 nd 6) 2.8. Phenotypi performne Generlly, phenotypi performne ws ffeted, long with the morphologil hrteristis, when the plnt seedlings were sujeted to hilling stress (Fig. 7). Phonotypil oservtions showed tht the Spd pretretment hilling-stressed seedlings hd etter visul performne of leves (less dehydrtion nd frizzle) ompred with the seedlings sujeted to the hilling stress without Spd pretretment (Fig. 7-A nd B). After reovery signifint deline, inluding wilting, yellowing nd mrginl urning in the lef pex, ws oserved (Fig. 7-C nd D). 3. Disussion Chilling stress leds to dverse effets on rop growth nd development, resulting in the redution of grin yield (Ymmoto et l. 212). Growth nd morphologil nlyses re widely used tools for hrterizing plnt growth. In this study, the results showed tht hilling exposure signifintly deresed the plnt height, oveground fresh nd dry iomss umultion nd reltive wter ontent during hilling stress nd fter reovery, resulting in lower RGR of the rie seedlings fter reovery (Tle 1). However, the exogenous 1 mmol L 1 Spd folir spry pretretment on rie leves signifintly llevited the growth inhiition used y hilling stress nd inresed the oveground iomss umultion. Our findings is orroorted those of Tin et l. (212) who stted tht folir spry with Spd effetively enhned the tolerne of the plnts to high-temperture stress. Higher plnt heights nd greter plnt iomsses re enefiil for mintining the dvntges of plnt growth nd physiologil tivity. This might e euse of the involvement of Spd in the regultion of plnt growth nd development under hilling stress (Ymmoto et l. 212). Chloroplsts re the mjor soure of retive oxygen speies (ROS) under oth stressed nd non-stressed onditions (Nkno nd Asd 1981). A low temperture tretment dereses the hlorophyll ontent y inhiiting protohlorophyllide oxidoredutse nd Chl synthesis (Liu et l. 212). However, in our study, hilling stress slightly inresed the totl Chl ontent y signifintly inresing Chl during hilling stress s ompred with the ontrol, while in Spd pretretment seedlings the totl Chl ontent delined signifintly (Tle 2) whih is not onsistent with the findings of Mostof et l. (214) under the het stress. This ould e possile sried tht the hilling-stressed rie seedlings my e exessively dehydrted, resulting to lower level of reltive wter ontent, nd then leding to higher
31 ZENG Yn-hu et l. Journl of Integrtive Agriulture 216, 15(2): 295 38 CK CK 2 μm CK+Spd CK 1 μm.2 μm CK+Spd CK+Spd GL SL 1 μm 2 μm T.2 μm T SG T SG P 2 μm T+Spd ER 1 μm T+Spd T+Spd.2 μm GL SL P SG 2 μm Mesophyll ells 1 μm.2 μm Chloroplsts Thylkoids Fig. 5 Chnges in ultrstruture of mesophyll ells, hloroplsts nd thylkoids of rie seedlings during hilling stress. The seond fully expnded leves, numered sipetlly, were smpled for ultrmirosopi oservtion during hilling stress nd fter reovery, respetively. SL, strom lmell; GL, grn lmelle; SG, strh grin; P, plstogloulus; ER, endoplsmi retiulum. The sme s elow. green sttus thn the seedlings under non-hilling stress tretment (Fig. 7-A nd B). Therefore, the performne of withered ut mintining green sttus ould e ppered in the hilling-stressed rie seedlings. After reovery, the hilling-stressed seedlings performne showed irreversile trends suggesting tht the photosyntheti pprtus were irreprly injury nd unle to synthesize Chl (Fig. 7-C nd D) nd the Chl ontent deresed ompred with tht of the Spd hilling-stressed tretment. Solule rohydrtes, s n osmosis sustne, re importnt ftors relted to hilling dpttion in plnts, nd it ws reently proposed tht sugrs n t s rel ROS svengers in plnts, espeilly when it ws presented t higher onentrtions (Keunen et l. 213). In our experiment, hilling stress signifintly inresed the totl solule sugr, surose, frutose, nd strh in the hilling-stressed leves of rie seedlings ompred with the ontrol, whih indited serious osmosis of memrne dmge due to eletrolyte lekge, nd fter hilling reovery this signifint inrese ws mintined only in totl solule sugr nd frutose. The most undnt solule sugr tht umultes is usully surose during the exposure of plnts to hilling temperture (Plonen et l. 2), yet frutose my ply importnt roles during hilling stress (Purvis nd Grierson 1982). The highest inrese ws found in frutose, followed y totl solule sugr. The results were in greement with
32 ZENG Yn-hu et l. Journl of Integrtive Agriulture 216, 15(2): 295 38 CK CK CK SL.2 μm 1 μm 2 μm CK+Spd CK+Spd CK+Spd SL GL P T SG 1 μm 2 μm T.2 μm T SG 1 μm 2 μm T+Spd T+Spd T+Spd.2 μm SG P.2 μm 2 μm 1 μm Mesophyll ells Chloroplsts Thylkoids Fig. 6 Chnges in ultrstruture of mesophyll ells, hloroplsts nd thylkoids of rie seedlings fter reovery. A B C D Fig. 7 Phenotypi pperne of hilling-stressed (B nd D) nd Spd pretretment hilling-stressed seedlings (A nd C). The photogrphs were tken 4 d fter tretment (left) nd 4 d fter reovery (right) from hilling stress. previous reports in ge (Sski et l. 21), ridopsis (Klotke et l. 24) nd olive (Gulen et l. 29) under old stress. The totl solule sugr, surose nd frutose deresed signifintly in Spd pretretment hilling-stressed seedlings during hilling stress, the reson might e tht Spd pplition ould llevite osmosis of memrne
ZENG Yn-hu et l. Journl of Integrtive Agriulture 216, 15(2): 295 38 33 dmge. However, inresing trends were oserved fter reovery supporting the well-estlished role of this sugr s n osmoprotetnt tht stilizes ellulr memrnes nd mintins turgor (Jouve et l. 24), whih indited tht Spd pplition llevited the osmoti injury used y hilling stress, nd mintined ertin osmoti pressure to sustin norml physiology tivity nd metolism. Certinly, the result of Spd pplition ws lso s tht in the ontrol with non stress. MDA is the ultimte produt of memrne peroxidtion, nd its ontent is relted to the dmge extent of ROS (Di et l. 212). Our results showed tht indued hilling inreses the MDA levels, whih still elevted trend fter reovery. This phenomenon showed high memrne dmge in the hilling-stressed seedlings nd therefore resulted in poor symptoms of stress, suh s hnges in growth prmeters nd Chl ontent (Tles 1 nd 2). In ontrst, Spd pretretment hilling-exposed rie seedlings mintined low level of MDA suggesting wek lipid peroxidtion (Fig. 2-A), onurring with Mostof et l. (214). Similr to MDA, the proline ontent in rie leves signifintly inresed due to the hilling stress, whih might e n dptive response to lose wter through muh serious lipid peroxidtion, nd the exogenous Spd redued hilling-indued proline umultion (Fig. 2-B). These results support the evidene tht exogenous PAs pplied deresed proline nd MDA level (Xu et l. 211; Mostof et l. 214) nd it is onsistent with the oservtion tht memrnes ultrstruture in leves of hilling stress were seriously ffeted y low temperture. Chilling stress ould result in hnges in the tivities of ntioxidnt enzymes, whih my ffet the physiologil trits of plnts nd djust stress onditions y reduing dmge used y ROS (Qiu et l. 25; Guo et l. 26). In previous study, Guo et l. (26) reported tht the tolerne to hilling in rie ws ssoited with the enhned pity of the ntioxidtive system under hilling onditions. Our results showed tht rie seedlings ould redue ROS y enhning the tivities of SOD, POD nd CAT during hilling stress (Fig. 3-A-C), whih ws in greement with the findings of Kumr et l. (211) nd Xu et l. (211). However, the trend ws inversed fter reovery from hilling stress owing to high oxidtive stress suh s MDA prodution in rie seedlings. In ontrst, Spd pretretment seedlings sustined high SOD, CAT nd POD tivities even fter reovery, whih might indite redued levels of MDA nd proline. The APX tivity of rie seedlings ws signifintly deresed during hilling stress nd fter reovery; nevertheless, Spd pretretment hilling-stressed plnts mintined higher APX tivities ompred with those under just the hilling-stress tretment. These results were orroorted y the findings of Mostof et l. (214) who suggested tht Spd pretretment enhnes het tolerne in rie seedlings y inresing the tivities of ntioxidnt enzymes. In the sme wy, most of ntioxidnt enzymes were high in Spd pretretment seedlings of the ontrol. Therefore, Spd pplition mintined plnt norml growth through improving old tolerne in relted to the higher ntioxidnt pity ompnying with lower oxidtive stress under hilling stress. Plnt hormones ply n importnt role in regulting plnt growth nd development to response for mient environment stress (Sevillno et l. 29). An inrese in ABA levels ws reported to relte to inresed hilling tolerne (Wng et l. 28), whih ould orrelte with induing stomt losure, reduing the trnspirtion rte nd mintining ell wter levels, nd inresing the expression of old-indued genes (Wng et l. 28). The results showed tht Spd enhned signifintly hilling tolerne y rising ABA ontent during hilling stress, nd then did shrp derese fter reovery (Fig. 4-A) whih grees with the findings in whet (Wng et l. 29). In ontrst, higher ABA ontent ws sustined even fter reovery in hilling-stressed seedlings inditing tht the struture of lef ws seriously dmge nd the physiologil tivities were irreversile (Fig. 3). Another notle differene etween hilling stress nd ontrol ws in their endogenous IAA, ZR nd GA 3 levels (Fig. 4-B-D). There deresed signifintly the IAA, ZR nd GA 3 levels during hilling stress nd fter reovery ompred with the ontrol whih might ontriute to higher oxidtive stress (Fig. 2) nd trigger osmoti mtters umultion (Fig. 1). Wng et l. (26) reported tht inresing the IAA nd ZR levels might e fesile for lleviting hilling in plum fruit. Similrly, inresing the IAA nd ZR levels in Spd pretretment during hilling stress nd fter reovery might e enefiil to inrese seedlings hilling tolerne whih sustined integrity struture of ell. Pusittigul et l. (212) reported tht derese in GA 3 onentrtion ws ssoited with plnt resistne to hilling stress. This suggests tht inrese hilling tolerne in rie my e relted to derese endogenous GA 3 levels during hilling stress nd it did not inrese fter reovery (Fig. 4-D), ut Spd pretretment sustined higher GA 3 levels during hilling stress nd even fter reovery suggesting tht the synthesis of GA 3 ws ffeted slightly y hilling stress. Under stress of low temperture, the memrnes of individul stroml thylkoids re losed together, while the hloroplst s whole egins to swell (Tylor nd Crig 1971). Under hilling stress, the hloroplsts lso swelled in leves, nd some thylkoids were swollen so tht mny vesiles were formed y lmell (Fig. 5). The sme results hve een oserved in uumer leves under hilling stress (Xu et l. 28). Compred with hilling stress tretment, hloroplsts of Spd pretretment ws not signifintly during hilling stress (Fig. 5). This result suggests tht Spd
34 ZENG Yn-hu et l. Journl of Integrtive Agriulture 216, 15(2): 295 38 might protet hloroplst memrnes to void e dmged through the tivted ehvior of ntioxidnt system (Fig. 3) s oserved y redued the level of MDA in Spd pretretment seedlings under hilling ondition. However, few studies on struture of ell were reported fter reovery from hilling stress. In this study, hloroplsts were isolted from the plsm memrne nd the numer of osmiophili plstoglouli ws inresed under hilling stress fter reovery, whih possily due to rekdown of thylkoid memrne degrdtion. It hs een indited tht the inresed mss of osmiophili plstoglouli in thylkoid memrnes is generlly ttriuted to lipid peroxidtion-medited destrution of the memrnes (Zhng et l. 21). Chnges in the lipid omposition of hloroplsts re responsile for ltered thylkoid memrne strutures, suh s disrupted hloroplst envelopes nd swollen thylkoids (Ymne et l. 23). Moreover, other orgnelles suh s endoplsmi retiulum hd ruptures under hilling stress, while tht of Spd pplition kept the si integrity. Chloroplst development derived from proplstid is minly ontrolled y ytokinin (ZR) indued with Spd (Polnská et l. 27), while SOD nd APX re key enzymes for svenging ROS of hloroplst, whih ould mintin redox equilirium of hloroplst (Myoug et l. 28). This might e responsile for higher endogenous hormones, higher SOD tivity nd APX nd lower lipid peroxidtion (Figs. 2 4). 4. Conlusion Chilling-indued oxidtive stress in rie seedlings ws evident through higher levels of MDA nd proline oupled with n inrese on rohydrte umultion, Chl ontent, nd lower plnt height, FW, DW, nd RGR. Chilling-exposed seedlings lso displyed inhiition of the ntioxidtive system nd poor indution of the endogenous hormones, prtiulrly dmge effet on struture of ell during hilling stress, ut the pperne ws oserved fter reovery of hilling stress owing to oxidtive urden nd ineffiient non-enzymti nd enzymti ntioxidnts s well s of endogenous hormones. However, exogenous Spd exhiited enefiil role through sustined nd simultneous tivtion of the ntioxidtive systems nd physiologil funtion of hloroplsts nd endogenous hormones metolism whih rendered rie plnts more tolernt to hilling-indued oxidtive nd osmoti dmge. The novel spet of the present work is tht exogenous Spd modultes the rohydrte level whih might e involved in the simultneous indution of the ntioxidnt defense systems nd endogenous hormones metolism nd therey enhne hilling-stress tolerne in rie seedlings. However, these results me only from lortory prties, nd further studies should e needed to lrify in the field onditions, to otin sustinle rop prodution under extreme hilling onditions. 5. Mterils nd methods 5.1. Plnt mterils, growth onditions nd tretments indi-jponi hyrid rie (Oryz stiv L. v. Yongyou 15, hill-sensitivity) seeds were surfe sterilized with H 2 O 2 solution for 2 min, wshed with distilled wter nd imied for 48 h. The seeds were sown on plsti nets floting on distilled wter in plsti pltes pked with towels nd kept in the drk t (32±.5) C for germintion. After 48 h, uniformly germinted seeds were trnsferred to seeding try overed in mtrix ontining roseite, hrol, silver snd, ontrolled relese fertilizer, miroelements nd other mixture mterils. The seeding try (length width height: 33. m 21.5 m 4. m) ws divided into two equl setions y lpord inserted in the mtrix soil. The trysown seeds were pled in greenhouse to grow. Twelve dys fter sowing, when two fully expended leves nd one tender lef hd emerged on the seedlings, one hlf of the seedlings in the plsti try were sujeted to folir spry pplition of Spd (Sigm-Aldrih Chemil Co., St. Louis, MO, USA) every fternoon from 17. to 18. h for four ontinuous dys. The result of preliminry experiments indited tht the effetive onentrtion of Spd ws 1. mmol L 1 to llevite hilling injuries (dte not shown). Tween-2 (.5%, v/v; Hijiehem, Zio, Chin) ws used s surftnt. The other hlf of the seedlings were sujeted to folir spry pplition of distilled wter s the ontrol. Eh hlf of the plsti try ontined ~3 5 rie seedlings, nd 2 ml of 1 mmol L 1 Spd or distilled wter ws spryed on eh hlf seedlings. After 4 d of pretretment, oth Spd pretretment nd ontrol seedlings were exposed to 6 C (dy/night: 8 C/4 C) for 4 d, followed y reovery t 25 C (dy/night: 27 C/23 C) for 4 d. The intensity of the hilling stress ws determined on the sis of preliminry experiment where 6 C used visile injury. The seedlings were grown under n illumintion inutor (photon density: 2 µmol m 2 s 1, reltive humidity (RH): 7 8%). Control nd Spd pretretment non-stressed seedlings were kept t 25 C throughout the experimentl period. The oveground rie seedlings were hrvested fter 4 d of hilling stress nd fter 4 d of reovery to determine the weight, Chl, rohydrte, nd vrious iohemil prmeters. This experiment inluded four tretments: ontrol, ontrol dditive Spd (pretreted with Spd), 6 C (hilling stress), nd 6 C dditive Spd (Spd pretretment followed y hilling stress). Eh tretment ws replited three times
ZENG Yn-hu et l. Journl of Integrtive Agriulture 216, 15(2): 295 38 35 under the sme experimentl onditions. 5.2. Determintion of plnt iomss nd hlorophyll (Chl) ontent Thirty rie seedlings were hrvested for eh replite. Plnt height ws mesured y ruler with n ury of 1 mm from stem-sheth to the pil point. Before determining the hrvested oveground fresh weight, the seedlings were wshed using deionized wter. The smples were weighed immeditely nd then oven-dried t 15 C for 15 min followed y 75 C for 72 h, until they rehed onstnt weight, nd then they were weighed for dry shoot iomss. The dry plnt tissues were ground into powder for rohydrte nlysis. An dditionl ~1 2 g fresh lef tissue of eh smple ws immerged in liquid nitrogen for 1 min nd then stored t 8 C for the iohemil nlysis. The reltively wter ontent ws determined s the weight of fresh smples minus dried smples, divided y the smples fresh weight. The reltive growth rte (RGR) ws mesured ording to the method of Hunt (23). The totl Chl ontent ws mesured ording to the method of Arnon (1949). Fresh leves (.2 g) were slied nd dded to 1 ml 95% ethnol in the drk t 25 C for 24 h. The superntnts were olleted nd the sorne vlues were reorded t 665 nd 649 nm. 5.3. Estimtion of MDA nd proline ontent The MDA ontent ws determined using the thiorituri id retion following the method of Heth nd Pker (1968), it ws lulted from the differene in sorne vlues t 45, 532 nd 6 nm using n extintion oeffiient of 155 mmol L 1 m 1. The proline ontent ws determined ording to the method of Btes et l. (1973), nd it ws lulted in sorne vlues t 52 nm using the stndrd urve of L-proline. 5.4. Antioxidnt enzyme extrtion nd enzymti tivity ssys Antioxidnt enzyme tivities were determined using ~.3 g smples homogenized in 8 ml of extrtion solution ontining 5 mmol L 1 N-phosphte uffer (ph 7.8),.2 mmol L 1 EDTA (ethylene dimine tetreti id) nd 1% insolule polyvinyl pyrrolidone with hilled mortr nd pestle. The homogente ws entrifuged t 12 g for 2 min nd the superntnt ws olleted to nlyze enzyme tivity nd protein ontent. The entire extrtion proedure ws rried out t 4 C. All spetrophotometri nlyses were onduted using spetrophotometer (DU-8, Bekmn Coulter, In., Fullerton, USA). SOD tivity ws estimted y mesuring its ility to inhiit the photohemil redution of nitrolue tetrzolium (NBT) y 5% (Mdhv et l. 2). The photo-redution of NBT ws mesured t 56 nm, nd SOD tivity ws expressed s U g 1 FW. CAT tivity ws mesured ording to the method of Ckmk nd Mrshner (1992) y determining the derese in sorne t 24 nm due to the deline of H 2 O 2 extintion. The enzyme tivity ws lulted in terms of 1 mol of H 2 O 2 g 1 FW min 1 t (25±2) C. POD tivity ws determined with guiol y the method of Nikel nd Cunninghm (1969). The enzyme tivity ws lulted in terms of 1 mol of guiol oxidized g 1 FW min 1 t (25±2) C. APX tivity ws mesured ording to Nkno nd Asd (1981) y monitoring the derese in sorte oxidtion t 29 nm s sori id ws oxidized. 5.5. Estimtion of rohydrte nd strh ontents Dried smples of ~5 mg were weighed into 15-mL entrifuge tue; 1 ml of 8% ethnol (v/v) ws dded nd gently shken for 3 min. The homogentes were wshed with ethnol, entrifuged t 4 g t 4 C for 1 min, nd the superntnt ws olleted. This extrtion ws repeted three dditionl times to ompletely remove the residul gluose from the mteril. A totl of 1 mg tive ron ws dded to the superntnt to sor the Chl for 3 min t 8 C nd diluted with wter to 25 ml, filtering the superntnt. Then, the totl solule sugr, surose nd frutose were determined for the filtered liquid, nd the level of strh ws determined in the residue. The rohydrte ontent ws expressed s mg g 1 DW ording to the method of Yoshid et l. (1976). Totl solule sugr The retion mixture of the totl solule sugr, onsisting of 1 ml extrtion liquid nd 5 ml.2% nthrnone sulfuri id solution (g/v: 2/1, 72% sulfuri id), ws pled in wter th for 15 min t 9 C. The retion solution ws tken out, rpidly ooled nd developed for 1 min. The sorne ws reorded t 62 nm using spetrophotometer (DU-8, Bekmn Coulter, In., Fullerton, USA). Surose determintion The retion mixture, onsisting of 1 ml extrtion liquid nd 5 µl 12 N NOH, ws pled in wter th for 5 min t 9 C, nd then rpidly ooled. A totl of 5 ml.2% nthrnone sulfuri id solution (g/v: 2 /1, 72% sulfuri id) ws dded for lending nd the mixture ws pled in wter th for 1 min t 8 C. The retion solution ws developed for 1 min. The sorne ws reorded t 62 nm under the sme onditions s ove. Frutose determintion The retion mixture, onsisting of 1 ml extrtion liquid nd 5 ml.2% nthrnone sulfuri
36 ZENG Yn-hu et l. Journl of Integrtive Agriulture 216, 15(2): 295 38 id solution (g/v: 2/1, 72% sulfuri id), ws wrmed t 4 C for 1 min, nd then the sorne ws reorded t 62 nm under the sme onditions s desried ove. Strh determintion The dry residue ws left in the entrifuge tue in n oven t ~5 6 C for strh extrtion. A totl of 2 ml of distilled wter ws dded to the entrifuge tue ontining the dried residue nd pled in oiling wter th for 15 min nd stirred osionlly. The tue ws llowed to ool. Then, 2 ml 9.2 N HClO 4 ws dded, stirred onstntly for 15 min, nd then entrifuged 1 min efore dding 2 ml of distilled wter. The superntnt ws olleted nd 2 ml 4.6 N HClO 4 ws dded to the residue. Then 5 ml of distilled wter ws dded nd the smples were entrifuged 1 min efore dding 2 ml of distilled wter. This ws repeted twie nd the superntnts were omined. The extrted strh ws mesured in the sme mnner s the solule sugr. respetively. The hormone frtions were dried in freeze dryer (Lono, Englnd), nd dissolved in 1 ml phosphte uffer sline (PBS) ontining.1% (v/v) Tween 2 nd.1% (w/v) geltin (ph 7.5) for the nlysis y n enzyme-linked immunosorent ssy (ELISA). The mouse monolonl ntigens nd ntiodies ginst ZR, GA 3, IAA, nd ABA nd the immunogloulin G-horserdish peroxidse (IgG-HRP) used in the ELISA were mnuftured y the Phytohormones Reserh Institute, Chin Agriulturl University. The quntifition of ZR, GA 3, IAA, nd ABA ws performed y ELISA s previously desried y Yng et l. (21). The perentge reovery of IAA, ZR, ABA, nd GA 3 in lef ws (9±3.7), (86±1.5), (98±5.2), nd (85±4.1)%, respetively. The speifiity of the monolonl ntiody nd other possile nonspeifi immunoretive interferene ws heked previously nd proved relile (Yng et l. 21). 5.6. Mirosopi nlysis 5.8. Sttistil nlysis The ultrstruture of hloroplsts ws nlyzed using trnsmission eletron mirosope (Crl Zeiss, Göttingen, Germny). The seond fully expnded leves, numered sipetlly, were ut into piees of pproximtely 1 mm 2. The ut leves were then immersed in solution ontining 3% glutrldehyde nd 1% formldehyde in.1 mol L 1 phosphte uffer (ph 7.4) for 2 h (primry fixtion), then immersed in 2% osmi id in the sme uffer for 2 h (seond fixtion). After dehydrtion in etone nd emedding in Durupn ACM (Fluk), the resulting leves were ut to otin ultrthin setions, stined with urnium ette nd led itrte in series nd exmined using HITACHI H-765 trnsmission eletron mirosope (Crl Zeiss, Göttingen, Germny) t n elerting voltge of 8 kv. 5.7. Hormone extrtion nd purifition nd quntifition Approximtely.5 g FW of grin ws smpled, nd eh endogenous hormone onentrtion ws mesured. The methods for extrtion nd purifition of ZR, GA 3, IAA, nd ABA were modified from those desried y Yng et l. (21). The smples were ground in mortr (on ie) with 5 ml of 8% (v/v) methnol s the extrtion medium, whih ontined 1 mmol L 1 utylted hydroxytoluene (BHT) s n ntioxidnt. The methnol extrts were inuted t 4 C for 4 h nd entrifuged t 1 g for 15 min t the sme temperture. The superntnts were pssed through Chromosep C 18 olumns (C 18 Sep-Prk Crtridge, Wters Corp., Millford, MA, USA). The olumns were prewshed with 1 ml of 1% methnol nd 5 ml of 8% methnol, The dt were sujeted to one-wy nlysis of vrine nd the men differenes were ompred y Dunn s multiple rnge test using SAS ver. 9.1 (SAS Institute, Cry, NC, USA). Mens were presented with stndrd devitions to indite the vrition of three replites for eh tretment. The differenes etween mens were determined using t-tests (P<.5). Aknowledgements This work ws supported y grnts from the the Ermrked Fund for Modern Agro-industry Tehnology Reserh System of Chin (CARS-1-9B), the Nturl Siene Foundtion of Zhejing Provine, Chin (Y13C1313) nd the Speil Fund for Bsi Sientifi Reserh Business of Centrl Puli Reserh Tnstitutes, Chinese Ademy of Agriulturl Sienes (212RG4-2). Referenes Arnon D I. 1949. Copper enzymes in isolted hloroplsts. Polyphenoloxidse in Bet vulgris. Plnt Physiology, 24, 1 15. Btes L S, Wldren R P, Tere I D. 1973. Rpid determintion of free proline for wter-stress studies. Plnt nd Soil, 39, 25 27. Boyer J S. 1982. Plnt produtivity nd environment. Siene, 218, 443 448. Ckmk I, Mrshner H. 1992. Mgnesium defiieny nd high light intensity enhne tivities of superoxide dismutse, sorte peroxidse, nd glutthione redutse in en leves. Plnt Physiology, 98, 1222 1227.
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