The effects of methyl jasmonate and salinity on germination and seedling growth in Ocimum basilicum L.

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1 749 The effects of methyl jsmonte nd slinity on germintion nd seedling growth in Ocimum bsilicum L. Shekoofeh Enteshri* nd Thereh Jfri Biology Deprtment, Pym Noor University, I.R. Irn Abstrct About 1 percen of lnds round the world under crops plnting re ffected by slinity. Therefore, comprehensive reserches hve been conducted to minimize the hrmful effects of slinity in griculture. The purpose of this study is exmintion of the Mmethyl jsmonte effect on reducing the effects of slinity in germintion nd vegettive stges of the bsil plnt.for the methyl jsmonte tretment, seeds were soked in methyl jsmonte (,/1 nd /1 µm) nd then seeds were plced on filter ppers wet using NCl (5, 1, 2 mm). Then, the morphologicl prmeters exmined. The results indicted tht high concentrtion of slinity cuse to decrese in germintion percentge nd Methyl jsmonte cuse increse in germintion percentge, nd this indicted the positive effect of methyl jsmonte on improvement of stress conditions in plnt. Furthermore, slinity increses the growth of root nd shoot, nd methyl jsmonte offsets this reduction. Keywords: slinity; methyl jsmonte; Ocimum bsilicum L. Enteshri, Sh. nd T. Jfri 'The effects of methyl jsmonte nd slinity on germintion nd seedling growth in Ocimum bsilicum L. stress'. Irnin Journl of Plnt Physiology 3 (3), Introduction About 1 percent of lnds round the world under crops plnting re ffected by slinity. Plnt growth is limited by unfvorble environmentl conditions nd slinity stress is discussed s one of the biggest problems in griculture round the world. (Kong et l., 25). Slinity cuses imblnce in ions of plnt cells, cretion of oxidtive stress nd in consequence, decrese in the growth of plnt (Kepczynski nd Bilech, 1994). Slinity inhibits wter uptke by plnt nd decreses soil wter *Corresponding uthor E-mil ddress: sh_enteshri@yhoo.com Received: November, 212 Accepted: Mrch, 213 potentil nd puts the plnts under drought stress too (Hgemeyer, 1996). Entrnce of ions from the membrne into the internl prts cuses blnce ltertion of plnt solution nd hormones, nd by this, the growth of root nd shoot re ffected (Lerner et l., 1999). The effect of slinity on lef number, lef re, nd dry weight of shoot nd root of colz plnt hs been reported. The growth of rootlet nd shootlet nd percentge of germintion decreses by slinity nd this reduction rises by incresing the level of slinity (Steppuhn et l, 21). The plnt cn rect to slinity by some chnges nd offsets in cell process nd morphologicl structure in order to decrese ion toxicities nd keep blnce between production nd destruction of oxygen rdicls (Kepczynski

2 75 Irnin Journl of Plnt Physiology, Vol (3), No (3) nd Bilech, 1994). These kind of morphologicl chnges in plnts djust pce nd mount of missed wter by plnt, indirectly increse wter potentil nd dilution of solute concentrtion in cell wter, nd offset dverse effects of slinity (Jin et l., 21). The first line of defense ginst excessive sodium entry into the plnt is cell plsm membrne of root. After ltering the ionic composition, wter nd solute get into the root center by symplst. Through the xylem, prenchyml cells of vsculr elements flow to trnsfer to the eril. Prenchyml cells re lso ble to trnsport slt to the phloem nd decrese slt concentrtion in xylem nd prevent it from getting into leves. Another physiologicl rection to slinity is xylem loding by str shped cells. These cells trnsfer K + nd N + ions to xylem which is controlled geneticlly (Gibberd et l., 22). Assignment of slt to the cell vcuole is nother physiologicl mechnism to reduce the level of cytoplsmic ions. If N + nd Cl - re ccumulted within the vcuole, K + nd soluble mteril must ccumulte in the cell cytoplsm to blnce the osmotic pressure of ions into the vcuole. Accumultion of N + nd Cl - in vcuoles cuses high osmotic potentil for wter bsorption nd cell turgor supply which is one of the effective fctors in the growth of plnt cell (Munns, 22). Jsmontes re one of the newest plnt growth regultors which cuse decrese in dmges due to environmentl stresses in plnt (Wng, 1999). Jsmonic methyl ester cid ws discovered first s mtter of frgment from oil of Jsmonium grndiflorum (Sembdner nd Prhtier, 1993), nd jsmonic cid ws derived from fungl culture in These compounds hve lso been observed in plnts e.g., in te, rosemry nd mint nd some fungi (Creelmn nd mullet, 1995). Pericrps, reproductive structure prticulrly ovrin nd the elongtion zone of root nd stem hve high mount of jsmonte. Jsmontes exist in 15 type including ferns, mosses nd fungi nd 26 plnt species hve been identified to contin these compounds (Creelmn nd Mullet, 1997). Jsmonic cid is produced s signling molecule in response to externl stimultors such s wounding, mechnicl force, pthogen ttck nd osmotic stress (Molin et l., 22). For exmple, t the wound site, cysteine with 18 mino cid trnsports by vessels to different prts of plnt nd by poly-peptides hydrolysis of the wound site cused induction of jsmonic cid. Genes responsible for the Jsmonte cid re expressed in picl prt of plnt. Increse in jsmonic cid controls compound biosynthesis such s proline nd putrescine in environmentl stresses nd induces the genes of these compounds in plnts. In the fungl pthogen ttck, jsmonte ctivities encode proteses, nd reduced the dmges. Furthermore, it djusts proteins of wll like PRP which my be necessry for brrier synthesis ginst infection (Go et l., 24). In soyben, t seedling stge, the level of jsmonte hs mostly been observed in hypocotyls, the region of cell division, root nd old leves. In mture leves of soyben, it is observed in mesophilic cells nd vsculr sheth surrounding the vein, nd the smll mount in epiderml cells. A smll mount of jsmonte ws observed in plisde prenchym cells, nd it hs been reported tht the low level of jsmonte in these cells is due to division of prenchym cells or low sensitivity of these cells to jsmonte (Creelmn, Mullet. 1997). In rice root under slinity stress, the mount of methyl jsmonte incresed nd hrmful effects of slinity on photosynthesis nd growth decresed (Wng et l., 21). Jsmonte is involved in physiologicl processes including seed germintion s plnt growth regultor. In some cses it ws reported tht jsmontes inhibited germintion of seeds without dormncy nd stimulted germintion of dormnt seeds. In pple, jsmonte stimultes germintion of dormnt embryo. In dormnt seeds, jsmonte my stimulte germintion of seed by decrese in sensitivity to bscisic cid. One reserch lso showed tht methyl jsmonte t the concentrtion of 1-3 M prevents from germintion of pigweed seeds (Kepczynski nd kepczynsk, 1999). On the other hnd, germintion in sunflower, whet nd ot ws inhibit by jsmonte (Nojvn nd Ishizw, 1998)

3 germintion percentge(%) men time need for germintion(dy) The effects of methyl jsmonte nd slinity on Ocimum bsilicum L. 751 The present work ws crried out to investigte the effect of different concentrtions of metyl jsmont nd NCl on germintion nd seedling growth of Ocimum bsilicum L. Mterils nd Methods Seeds of green bsil plnt were prepred from griculturl reserch center in Isfhn, Irn. After seprting helthy nd uniform seeds, 25 seeds were plced in Petri dishes contining filter pper nd for ech tretment 4 Petri dishes were considered s the 4 replictes. In order to give slinity tretment, sodium chloride solution with concentrtions of, 5, 1 nd 2 mm were dded to Petri dishes contining the seeds. For methyl jsmonte tretment, seeds were soked t concentrtions of,.1 nd.1 µm for 6 hours nd then were trnsferred to Petri dishes. Also for the combined tretments, fter soking the seeds t the mentioned methyl jsmonte concentrtions, they were trnsferred to Petri dishes nd irrigted by sodium chloride solution t the mentioned concentrtions. Then, the Petri dishes were plced in germintor (model KH RH 1, Irn) t the temperture of 16 C under light conditions 16:8. Germinted seeds in Petri dishes were counted fter 24, 36 nd 48 nd 6 nd 72 hours fter cultivtion. Percentge of germintion nd the required times for germintion were clculted using the following formuls (Torbi et l 212): Percentge of germintion = number of germinted seed / totl number of seeds 1 Averge time for germintion = N 1 T 1 + N 2 T 2 + N 3 T 3 / N where N 1, N 2, N n show number of germinted seeds in the first, second nd n th dy, respectively. T 1, T 2, T n show the count of first, second, n th dy, nd N shows the totl number of germinted seeds. The shootlet nd rootlet length nd wet nd dry weight were then mesured. Sttisticl nlysis ws done by SAS softwre with 4 replictes nd men vlues nd significnce were determined by Duncns multiple rnge test t 5% probbility level. Results Percentge of germintion Interction of effects of methyl jsmonte nd slinity on germintion percentge indicted tht compound tretment of methyl jsmonte nd slinity t concentrtion of 2 mm decresed this prmeter significntly (Fig. I. b) (P.5). Also, slinity levels hd no significnt effect on the verge time for germintion. The compound tretment of methyl jsmonte (.1 µm) nd slinity t concentrtion of 2 mm incresed this prmeter s compred with the control slinity (Fig. I. ) (P.5). Rootlet length b mj mj b Ncl concentrtion(mm) mj.1 mj b b b b b b b b b b bc Ncl concentrtion(mm) mj.1 mj.1 Fig. I. Interction of effects of methyl jsmonte nd slinity on the verge time for germintion nd percentge of germintion; verges with similr letters re in one ctegory nd re not significntly different t 5%.

4 fresh weight (gr) dry weight(gr) shootlet lenght(cm) rootlet lenght (cm) 752 Irnin Journl of Plnt Physiology, Vol (3), No (3) Rootlet length ws decresed by incresing level of slinity, so tht the lowest level of this prmeter ws observed t 2 mm of slinity s compred to control plnts. Use of Methyl jsmonte in tretment of.1 nd.1 µm nd slinity concentrtion of 2 mm cused significnt decrese in rootlet length (Fig. II. b) (P.5). Shootlet length Increse in slinity levels of 1 nd 2 mm cused significnt decrese in shootlet length. The shortest length of shootlet goes to slinity level of 2 mm. Methyl jsmonte t concentrtion of.1 µm nd slinity level of 1 mm cused significnt djustment in decrement of shootlet length cused by slinity (Fig. II. ) (P.5). Seedling fresh weight Slinity tretment t concentrtions of 1 nd 2 mm cused significnt decrese in fresh weight of seedlings. The compound tretment of methyl jsmonte nd slinity t slinity level of 2 mm indicted significnt increse in fresh weight s compred with slinity tretment without methyl jsmonte (Fig. III. ) (P.5). Seedling dry weight b b b b bc cd d de de e Ncl concentrtion(mm) mj mj.1 mj bc bc b b b bc c bc d e Ncl concentrtion(mm) b d mj mj.1 mj.1 Fig. II. Interction of effects of methyl jsmonte nd slinity on rootlet nd shootlet length; verges with similr letters re in one ctegory nd re not significntly different t l5% bc bcd b bcd b mj mj.1 bcd cd d bcd d e mj b b b b bc b b b cd cd d d mj mj.1 mj.1 Ncl concentrtion(mm) Ncl concentrtion(mm) Fig. III. Interction of effects of methyl jsmonte nd slinity on fresh nd dry weight of seedling; verges with similr letters re in one ctegory nd re not significntly different t level of 5%.

5 The effects of methyl jsmonte nd slinity on Ocimum bsilicum L. 753 Different slinity concentrtions cused significnt increse in dry weight of seedlings s compred to control plnts. Methyl jsmonte t concentrtion of.1 µm nd slinity levels of 5 nd 1 mm cused significnt decrese in this prmeter s compred to slinity level of 5 mm without methyl jsmonte (Fig. II. b) (P.5). Discussion The most importnt vegettive stge of plnt under slinity stress is the stge of germintion. High concentrtion of slt reduces the germintion percentge which my be probbly due to the incresed osmotic pressure or sodium ions within the seed. Sodium ion could be toxicity fctor resulting in reduced seed germintion (Ozkn et l., 25). Absorption of sodium nd chloride ions cuses impired wter uptke nd secretion of hormones, nd modifies the ctivity of enzymes, such s Mlte dehydrogense nd the glucose 6 phosphte dehydrogense enzymes which hve mjor role in grin growth. Plnts resistnt to slinity trnsport less sodium nd chloride to the leves nd re ble to seprte these ions in vcuoles to prevent their ccumultion in the cytoplsm or cell wll, nd prevent the ionic toxicity of them (Munns, 22). Furthermore, incresed slinity level cuses decrese in germintion percentge nd increse in verge time for germintion. Incresed resistnce to slt stress depends on the plnt species nd the growth stge. For exmple, rice is resistnt plnt to slinity t germintion stge but it is sensitive plnt to slinity t the erly stges of growth nd flowering. Sugr beet, mize, nd sunflower re sensitive to slinity t the germintion stge, but lter in the growth their sensitivity to stress becomes less. In colz types, germintion stge is sensitive to slinity nd cuses production of bnorml seedlings (Zheng et l., 21). Kv nd his collbortors (24) exposed cultivr colz nd trnsgenic colz to slinity stress, nd observed tht germintion increses in cultivr type by incresing slinity, but trnsgenic plnts re resistnt to slinity. In this study, high concentrtions of sodium chloride cused decrese in germintion percentge due to slowing wter bsorption by seeds. Toxicity of bsorbed sodium nd chloride by seed cuses modifiction in enzymes ctivity nd hormones secretion, nd cn decrese germintion percentge. Also methyl jsmonte in the study incresed germintion percentge t slinity level of 2 mm which shows its positive role in improving the conditions of stress in plnt. Although the effect of methyl jsmonte on the stimultion or inhibition of germintion depends on the concentrtion of methyl jsmonte, Jsik nd Klerk (26) indicted tht methyl jsmonte increses germintion percentge. Methyl jsmonte nd jsmonic cid hve inhibitory role in dormnt seeds nd stimultory role in seeds without dormnt period. It is reported tht this composition hs n effect on kind of ok (Quercus robur) nd methyl jsmonte concentrtion increses before drying nd loss of seed vibility. Incresed methyl jsmonte cused membrne peroxidtion nd dmge to the cell membrne (Creelmn nd Mullet, 1997). In eggplnt, sodium chloride concentrtion of higher thn 5 µm delyed germintion, but the finl germintion percentge did not fll, s germintion percentge significntly decresed only t high slinity (1-15 µm (Dnts et l., 25). By incresing slinity up to 1-15 mm, rootlet length ws reduced 36 nd 74 percent. Seedling ppernce lso delyed t high levels of slinity (Cheong nd Choi, 23). Seedling stge in eggplnt is more sensitive thn germintion stge. Similr results re observed in pepper (Chrtzoulkis nd Klpki 2). Reports hve shown tht ACC levels in treted cotyledons with jsmonte decresed. Thus, methyl jsmonte decresed ethylene synthesis by ffecting ACC nd ACO, cusing decrese in germintion. In pigweed (Kepczynski nd Bileck, 1994) nd sunflower (Corbineu et l., 1988), jsmonte decresed germintion. Acid jsmonte lso stimulted germintion in pple nd cused ctivtion of lkline lipse. Activtion of lipse stimultes fts nd supplies required sugrs for the germintion stge. Inhibition of lipooxygense ctivity, which is one

6 754 Irnin Journl of Plnt Physiology, Vol (3), No (3) of the key enzymes in biosynthesis pthwy of jsmontes, decreses germintion (Creelmn nd Mullet, 1997). Furthermore, by blocking other intermedites of biosynthesis pthwy of jsmontes, germintion would be inhibited. The growth of plnts is result of cell division nd irreversible increse in cell volume. Non-environmentl stresses ffect cell division nd decrese the growth. In this reserch, methyl jsmonte compensted decrese in germintion only t high slinity concentrtions. Slinity concentrtions of 5 nd 1 µm hd no significnt effect on percentge of germintion nd verge time for germintion. This ws probbly due to the rpid germintion of green bsil seeds. Slinity cuses ion imblnce in plnt cells, cusing oxidtive stress nd decrese in the growth of plnt (Kepczynski nd Bileck, 1994). Plnts through series of chnges or djustments in the cellulr process nd morphologicl structures cn respond to slinity stress in order to reduce ion toxicity nd mintin the blnce between production nd loss of oxygen rdicls (Kepczynski nd Bileck, 1994). Under slinity, osmotic pressure decreses nd decrese in turgescence is n importnt fctor in the inhibition of growth, s turgor pressure stops cell division nd cell elongtion. Entrnce of slt from root membrne to the inner prts cuses ltertion in the blnce of plnt solutions nd hormones, nd ffects the growth of roots nd shoots (Lerner et l., 1999). Reserches on colz plnt demonstrted tht following the slinity stress, chlorophyll concentrtion, root growth (Ky, et l., 21), shoot nd root dry weight (Steppuhn et l., 21) decresed in plnts. It is reported tht slinity decreses the growth of root nd shoot, nd this reduction will increse by incresing slinity. Decrese in length cn be due to decrese in turgor nd cell elongtion. Similr results hve been observed in slinity tretment of two sensitive nd resistnt types of rice (Kng et l., 25) nd cowpe (Dnts et l., 25). It is reported tht jsmonte prevents coleoptiles elongtion through inhibition of glucose formtion in polyscchrides of the two wlls (Creelmn nd Mullet, 1997). Obtined results conformed the decrese in the growth of shootlet by methyl jsmonte in seedling of rice (Yeh et l., 1995) in phrbitis nil nd pepper. In the experiment conducted on phrbitis nil, jsmonte tretment incresed root length. This ws ttributed to recretion of the root meristem (Mciejewsk nd Kopcewicz, 22). In this reserch, high levels of sodium chloride decresed shootlet nd rootlet length..1 µm of methyl jsmonte nd 1 mm of slinity decresed shootlet length while 2 mm of methyl jsmonte nd slinity decresed rootlet length. Thus, the stimultory effect on rootlet nd shootlet growth cn only be observed under sodium chloride inhibitory conditions..1 µm of methyl jsmonte cused significnt decrese in dry weight of seedlings t slinity levels of 5 nd 1 µm which might be due to inhibitory effect of methyl jsmonte on photosynthesis. On the other hnd, ll sodium chloride concentrtions in the study cused significnt increse in dry weight nd sodium chloride concentrtions of 1 nd 2 mm decresed fresh weight significntly. Therefore, we cn conclude tht high levels of sodium chloride due to chnges in wter potentil mke it difficult for wter bsorption while the mount of dry mtter in plnt increses. References Corbineu, F., R. M. Rudnicki. nd D. Come 'The effects of methyl jsmonte on sunflower (Helinthus nnuus L.) seed germintion nd seedling development'. Plnt Growth Regultion. 7: Chrtzoulkis, K. nd G. Klpki. 2. 'Response of two green house pepper hybrids to NCl slinity during different growth stges'. Scienti Horticulture. 86(3): Creelmn R. nd J. E. Mullet 'Jsmonic cid distribution nd ction in plnts: regultion during development nd response to biotic nd biotic stress'. Proceedings of the Ntionl Acdemy of Sciences. 92: Creelmn, R. nd J. E. Mullet ' Biosynthesis nd ction of jsmonte in plnt'. Annul Review of Plnt Physiology nd Plnt Moleculr Biology. 48:

7 The effects of methyl jsmonte nd slinity on Ocimum bsilicum L. 755 Dnts, B. F., S. Lucinde. nd A. Crlos. 25. Physiologicl response of cowpe seeds to slinity stress. Journl of Amricn Society Horticulturl Science.27(1): Go, X. P., X. F. Wng., Y. F. LU., L. Y. Zhng., Y. Y. Shen., Z. Ling. nd D.P Zhng,. 24. Jsmonic cid is involved in the wter-stressinduced betine ccumultion in per leves. Plnt Cell nd Environment. 27(4): Gibberd, M. R., N. C. Turner. nd R. storey. 22. Influence of sline irrigtion on growth, ion ccumultion nd prtitioning nd lef gs exchng of crrot (Ducus crot L.). Annuls of Botny. 9(6): Hgemeyer, J Slt. IN: Plnt Ecophsiology. (PRASAD, M. N. V.), Jhon wiley. ISBNO PP Jin, M., G. Mthur., S. Koul. nd N. B. Srin. 21. Ameliortive effects of prline on slt stress-induced lipid peroxidtion in cell lines of groundnut (Archis hypoge L). Plnt Cell Rep. 2: Kng, D. J., Y. J. Seo., J. D. Lee., R. Ishii., K. U. Lim., D. H. Shin., S., K. Prk., S. W. Jng. nd I. J. Lee. 25. Jsmonic cid differentilly ffects growth ion up tke nd bscisic cid concentrtion in slt-tolernt nd slt sensitve rice cultvrs. Journl of Agronomy nd Crop Science. 191: Kv, N. N. V., B. Fristensky. nd S. Srivstv. 24. Constitutive expression of PR1 protein enhnces the germintion of Brssic npus under sline condition. Plnt Cell Physiology. 45 (9): Ky, C., D. Higges. nd H. kirnk. 21. The effects of high slinity (NCl) nd supplementry phosphorus nd potssium on physiology nd nutrition development of spinch Bulge. Journl of Plnt Physiology. 27(3-4): Kepczynski, J. nd B. Bileck Stimultory effect of ethephon, ACC, gibberellin A 3 nd A 4+7 on germintion of methyl jsmonte inhibited Amrnthus cudtus L. seeds. Plnt Growth Regultion. 14: Kepczynski, J., B. Bileck. nd E. kepczynsk Ethylene biosynthesis in Amrnthus cudtus seeds in response to methyl jsmonte. Plnt Growth Regultion. 28: Kong, L., M. Wng. nd Bi. Dongling. 25. Selenium modultes the ctivities of ntioxidnt enzymes, osmotic homeostsis nd promotes the growth of sorrel seedling under slt stress. Plnt Growth Regultion. 45(2): Lerner, H. R., G. N. Amzlly., Y. Friedmn. nd P.Goloubinoff The response of plnts to slinity from turgor djustment to genome modifiction. Irsel Journl of Plnt Science. 42: Mciejewsk, B. nd J. kopcewicz. 22. Inhibitory effect of methyl jsmonte on flowering nd elongtion growth in phrbitis nil. Plnt Growth Regultion. 21: Molin, A., P. Bueno., M. C. Rodriguez-rosles., A. Belver., K. venem. nd J. Donire. 22. Involvement of endogenous slicylic cid content lipoxygense nd ntioxidnt enzyme ctivities in the response of tomto cell suspension cultures to NCl. New Physiologist. 156: Munns, R. 22. Comprtive physiology of slt nd wter stress. Plnt Cell nd Environment. 25: Nojvn-Asghri, M. nd K. Ishizw Inhibitory effects of Methyl jsmonte on the germintion nd ethylene production in cocklebur seeds. Journl of Plnt Growth Regultion. 17(1): Ozkn, S., H. Sivritepe., N. Eris. nd E. Turhn. 25. The effects of NCl pre-tretments on slt tolernce of melons growth under longterm slinity. Scienti Horticulture. 16: Torbi,F., A. Mjd. nd S. Enteshri Effect of exogenous silicon on germintion nd seedling estblishment in Borgo officinlis L. Journl of Medicinl Plnts Reserch. 6(1), pp Sembdner., G. nd B. prthier The biochemistry nd the physiologicl moleculr ction of jsmonte. Annul Review Plnt Physiology Plnt Moleculr Biology. 44; Steppuhn, H.K., M. Volkmr nd P.R. Miller. 21. Compring cnol, field pe, dry ben nd durum whet crops grown is sline medi. Crop Science. 41:

8 756 Irnin Journl of Plnt Physiology, Vol (3), No (3) Wng, S.Y Methyl Jsmonte reduces wter stress in strwberry. Journl of Plnt Growth Regultion. 18(3): Yeh, C. C., H.S. Tsy., J.H. Yeh., F.Y. TsiShih. nd C. H. Ko A comprtive study of the effects of methyl jsmonte nd bscisic cid on some rice physiologicl processes. Plnt Growth Regultion. 14: Zheng, H. X., J. N. Hundson, J.P. Willims nd E. Blumwld. 21. Engineering slt tolernce Brssic plnts: chrcteriztion of yield nd seed oil qulity in trnsgenic plnts with incresed vcuolr sodium ccumultion. PNAS.98(22):