Agriculturl Advnces (214) 3(9) 244-248 ISSN 2251-782 doi: 1.14196/.v3i9.1626 Contents lists vilble t Sjournls Journl homepge: www.sjournls.com Originl rticle Study on germintion of sunflower seeds tht ffected by ROS S.M. Azimi, M. Shbn b, * Former M.Sc student of Agronomy, Islmic Azd University, Boroujerd, IRAN. b Young Reserchers nd Elite Club, Boroujed brnch, Islmic Azd University, Boroujerd, IRAN. *Corresponding uthor; Young Reserchers nd Elite Club, Boroujed brnch, Islmic Azd University, Boroujerd, IRAN. A R T I C L E I N F O A B S T R A C T Article history, Received 3 August 214 Accepted 29 August 214 Avilble online 27 September 214 Keywords, Germintion nd rective oxygen species This study investigted in order to evlution of reltionship between germintion nd produce of rective oxygen species in sunflower seeds. This study ws performed in two seprte experiments, ech in completely rndomized design with fctoril design with four replictions. In both experiments, uses from dormnt nd non dormnt seeds of sunflower. It lso pplies of tretments Methylviologen nd Cynide in dormnt seeds which re the producers of rective oxygen species. Finlly, germintion, the content of rective oxygen species such s hydrogen peroxide nd superoxide nion production were evluted s well. The results showed tht the min reson for relese of sunflower seeds dormncy is production of rective oxygen species is n cceptble level so tht seed germintion of dormnt seeds which ws treted with Methylviologen nd Cynide ws more thn dormnt control seeds nd ws similr to non dormnt seeds. Also, production of Hydrogen peroxide nd superoxide nion production of seeds treted with Methylviologen nd Cynide ws similr to non dormnt seeds nd they hd nerly doubled of dormnt seeds. 214 Sjournls. All rights reserved. 244
S.M. Azimi nd M. Shbn / Agriculturl Advnces (214) 3(9) 244-248 1. Introduction Plnting sunflower oil production nd consumption of nuts hs risen drmticlly (Khje poor, 24 nd Leubner-Metzger, 25). Sunflower is one indifferent towrds the dy but needs lot of light. Rtio is reltively resistnt to soil slinity. Forge legumes in crop rottion, usully fter the first weeding the crop is plnted with plnts tht hve common root disese such s pes, sugr beets nd pottoes, re not included in the frequency. With sunflower, soyben, cnol, cottonseed nd penut oil yer, which is the most importnt crop plnts, it hs long been n importnt prt of griculture is formed Estern countries (Alyree nd Shekri, 2). Frequently red in vrious sources tht the old rective oxygen species Rective Oxygen Species or ROS briefly toxic molecules tht dmge produced by stress conditions in plnts nd seed production is sign of oxidtive stress. But tody is proved tht the ctive oxygen species, despite the dmging effects they hve useful roles in the body re living things. Orgnelles such s chloroplsts, mitochondri nd oxidtive ctivity proxies zoom or ultr-high-speed electron flow re the mjor source of ROS in plnt cells (Gy et l, 1991; Ginnopolitis et l, 1977; Gill et l, 21 nd Grnt et l, 2). In seed physiology (ROS) re generlly regrded s toxic molecules re resulted in the ccumultion of cell dmge nd impired developmentl processes of germintion or sprouting. Crucil role of these compounds in the seeds of tody's ge hs been well estblished (McDonld, 1999 nd Moller, 22). It hs been recognized tht ROS expression of some genes nd signl trnsduction pthwys tht ffect showing tht the cells grow some strtegy to tke dvntge of the ROS s stimulting the biologicl signl nd to which the ppliction of genetic stress nswers to ctivte or control (Dlton, 1999) Recently, it hs been found tht plnts ctively produce ROS tht there my be mny different physiologicl processes such s biologicl stress response non biologicl defense ginst disese nd signl to control systemic formtion (Gill et l, 21 ). There is evidence tht suggests tht ROS ply key role in seed germintion nd suggested tht the cell wll loosening in the context of growing contributes to the (Liszky et l, 24 nd Luck, 1962). The im of this study ws to investigte the role of rective oxygen species in the regultion of dormncy nd germinting in sunflower seeds. 2. Mterils nd methods This reserch ws crried out in order to study on effect of rective oxygen species on sunflower seed germintion nd dormncy of Rekord cultivr using reserch budget of Young Reserchers Club, Islmic Azd University Borojerd brnch, Irn. Experiment 1, The experimentl design ws completely rndomized with four replictions. Sunflower seeds re hrvested t dormncy they need to fter-ripening. For pply the fter-ripening, dormncy bunch of seed them for two months nd were incubted t 6% reltive humidity dormncy them to be broken. The tretments consisted of non dormnt seed, dormnt seeds, dormnt seeds treted with methylviologeen (producing rective oxygen species) nd dormnt seeds treted with hydrogen cynide (gs producing rective oxygen species ) tretments. For germintion test 25 seed petri-dishes on filter pper 9 cm (Top of pper) in foer repliction. Seed germintion tests were conducted in the drk nd t temperture of 25 degrees Celsius. Seeds were checked twice dy for 1 dys nd the number of germinted seeds were recorded. Seed germintion, root exclusion bsed on the mount of 2 to 3 mm. Experiment 2, The experimentl design ws completely rndomized with four replictions. The tretments consisted of non dormnt seed, dormnt seeds, dormnt seeds treted with methylviologen (producing rective oxygen species) nd dormnt seeds treted with hydrogen cynide (gs producing rective oxygen species). To extrct hydrogen peroxide.2 g of seed smples with 3 ml tri chloro cetic cid,.1% nd the porcelin mortr homogenized nd extrcted t 15, rpm for 15 min t 4 C centrifuge nd the resulting extrct ws used to mesure hydrogen peroxide. Active mesurement ws lid out s Jizdwsk et l (21). Superoxide rdicl content using Elstner nd Hpel (1976) method ws mesured. In this method,.2 g fresh weight of sunflower seed xes in 4 ml of 5 mm sodium phosphte buffer with ph of 7.8 nd t temperture of 4 C ws solved. The resulting solution for 15 min nd centrifuged t 15, rpm superoxide nion ws used to estimte the resulting. To estimte superoxide nion ml of the extrct obtined t 25 C for 3 min in the presence of hydroxylmine hydrochloride mm sodium phosphte buffer 5 mm with ph of 7.8 dt nd incubted. Then.5 ml of it with 5/ ml of 17 mm sulfnilmide nd /5 ml of 2 - Nftil mine 7 mm t 25 C for 3 min nd incubted the smples were then centrifuged for 1 min t 13, rpm nd the bsorbnce of the 245
germintion percentge S.M. Azimi nd M. Shbn / Agriculturl Advnces (214) 3(9) 244-248 smple t 54 nm ws red. Stndrd curves were plotted with the use of sodium nitrte. The results in terms of micromoles per grm dry weight of seed smples were estimted (Orcz et l, 27). The sttisticl nlyses to determine the individul nd interctive effects of time cultivtion nd weeds control methods were conducted using JMP 5..1.2 (SAS Institute Inc., 22). Sttisticl significnce ws declred t P.5 nd P.1. Tretment effects from the two runs of experiments followed similr trend, nd thus the dt from the two independent runs were combined in the nlysis. 3. Results nd discussion Between non dormnt seeds nd seeds treted with methylviologen nd cynide these significnt differences were observed mong the three tretments, lthough the highest germintion tretment without dormncy nd 98% were chieved. After tretment without dormncy, seeds treted with cynide (96%) nd seeds treted with methylviologen (94%) hd the highest rte of germintion (Fig 1). Rective oxygen species cuse dormncy relese of sunflower seeds re such tht the production of rective oxygen species during germintion s new mechnism known to relese of dormncy nd sunflower seed germintion were introduced. Around the time of sunflower seeds in the presence of methylviologen.1mm seed dormncy efficiently remedied. These events cn occur while the incubtion of sunflower seeds with methylviologen cuse prticulr purpose in dormnt seeds nd crboniltion proteins occurs in them only (Orcz et l, 27). 12 1 8 6 4 2-2 5 1 15 Time(Dy) b c d Fig. 1. Germintion percentge in different tretments. A= dormnt seeds B= non-dormnt seeds C= dormnt seeds treted with Methylviologen D= dormnt seeds treted with Cynid Production of rctive oxygen species such s hydrogen peroxide during the occurrence of seed germintion cn be chnged. The results showed tht hydrogen peroxide produced fewer seeds thn dormnt seeds nd dormnt seeds treted with methylviologen nd cynid. Hydrogen peroxide production in non dormnt seeds bout 1.11 micromoles per grm dry weight of the seed, which is bout hlf the mount of seed mteril non dormnt (2.45 micromoles per grm dry weight of seed) nd between them in terms of There ws no significnt difference. Peroxide levels mesured in the seeds treted with methylviologen (2.4 micromoles per grm dry weight of seed) nd cynide (2.33 micromoles per grm dry weight of seeds), roughly the sme mount of hydrogen peroxide ws produced in the dormnt seeds. There ws no significnt difference between them in terms of the mount of hydrogen peroxide produced in the dormnt seeds were no significnt differences (Fig 2). Sunflower seeds nd embryonic xes within the cell cytoplsm, stining ws observed nd the mount of peroxide stins ws very higher in non dormnt seeds thn dormnt seeds (Orcz et l, 27). 246
Anion superoxide (Micromol/g) Hydrogen peroxide (Micromol/g) S.M. Azimi nd M. Shbn / Agriculturl Advnces (214) 3(9) 244-248 3 2.5 2 1.5 1 b.5 b c d tretments Fig. 2. H2O2 content in different tretments. A= dormnt seeds B= non-dormnt seeds C= dormnt seeds treted with Methylviologen D= dormnt seeds treted with Cynid Dormnt seeds thn non dormnt seeds nd seeds treted with the superoxide nion production of methylviologen nd cynide production of superoxide nion ws less, so tht the dormnt seeds (4.28 micromoles per grm dry weight of seed) ws bout hlf the rte of its production to other tretments There ws significnt difference of opinion between them. In non dormnt seed tretments nd seed treted with methylviologen nd cynide, this difference ws not significnt, lthough the highest rte of superoxide nion production rte (9.2 micromoles per grm dry weight of seed) of non dormnt seed (Fig 3). Formzn ccumultion in seed xes dormnt nd within cells is not visible, but the seeds tht indicte the presence of drk spots non dormnt of superoxide nion hs been observed (Orcz et l, 27). 1 9 8 7 6 5 4 3 2 1 b b b c d tretments Fig. 3. Superoxide nion content in different tretments. A= dormnt seeds B= non-dormnt seeds C= dormnt seeds treted with Methylviologen D= dormnt seeds treted with Cynid Acknowledgments This reserch ws funded nd supported by reserch budget (reserch project code= 92313) of Young Reserchers nd Elite Club, Boroujerd Brnch, Islmic Azd University, Boroujerd, Irn. Their contribution is grtefully cknowledged. Code. 247
S.M. Azimi nd M. Shbn / Agriculturl Advnces (214) 3(9) 244-248 References Dlton, T.P., Shertzer, H.G., Pug, A., 1999. Regultion of gene expression by rective oxygen, Annu. Rev. Phrmcol. Toxicol., 39, 67 11,. Elstner, E.F., Heupel, A., 1976. Inhibition of nitrite formtion from hydroxylmmoniumchloride, simple ssy for superoxide dismutse. Anl. Biochem., 7, 616 62. Gy, C., Corbineu, F., Coˆ me, D., 1991. Effects of temperture nd oxygen on seed germintion nd seedling growth in sunflower (Helinthus nnuus L.). Environ. Exp. Bot., 31, 193 2. Ginnopolitis. C.N., Ries S.K., 1977. Superoxide dismutses i. Occurrence in higher plnts. Plnt Physiol., 59, 39-314. Gill, S.S., Tutej, N., 21. Rective oxygen species nd ntioxidnt mchinery in biotic stress tolernce in crop plnts. Plnt Physiol. Biochem., 48, 99-93. Grnt, J.J., Loke, G.J., 2. Role of rective oxygen intermedites nd cognte redox signling in disese resistnce. Plnt Physiol., 124, 21 3. Heth, R.L., Pcker, L., 1968. Photoperoxidtion in isolted choloroplst.1. Kinetics nd stoichiometry of ftty cid peroxidtion. Arch. Biochem. Biophys., 12, 189-198. Khje poor, M.R., 24. Industril plnts. Jihd Isfhn Technol. Press., 564pp. Leubner-Metzger, G., 25. Bet-1,3-glucnse gene expression in low-hydrted seeds s mechnism for dormncy relese during tobcco fter-ripening. Plnt J., 41,133-45. Liszky, A., vn der Zlm, E., Schopfer, P., 24. Production of rective oxygen intermedites (O2.-, H2O2, nd. OH) by mize roots nd their role in wll loosening nd elongtion growth. Plnt Physiol., 136,3114-23. Luck, H., 1962. Methods of enzymtic nlysis. E.B. By Bergmeyer (1th edition). Verlg chem. weinheim., Pp,885-894. McDonld, M.B., Seed deteriortion, physiology, repir nd ssessment. 1999. Seed Sci. Technol., 27,177 237. Moller, I.M., 21. Plnt mitochondri nd oxidtive stress, electron trnsport, NADPH turnover, nd metbolism of rective oxygen species. Annu. Rev. Plnt Physiol. Plnt Molecul. Biol., 52, 561 591. Krystyn, O., El-Mrouf Bouteu, H., Frrnt, J.M., Cooper, K., Belghzi, M., Cludette J., Dominique J., Frnc, O., Corbineu., Christophe B., 27. ROS production nd protein oxidtion s novel mechnismfor seed dormncy llevition. Plnt J., 217, 1-14. 248