Key words: salinity; growth; phenylalanine ammonia - lyase; phenols, flavonoids

Transcription

1 INFLUENCE OF SALT STRESS ON EX VITRO GROWTH AND ANTIOXIDATIVE RESPONSE OF TWO PAULOWNIA CLONES K. Mildinov*, K. Ivnov**, T. Georgiev*, M. Genev***, Y. Mrkovsk** * Biotree, 8 Iliensko shoes str., 1220 Sofi, Bulgri; ** Fculty of Biology, University of Sofi, 8 Drgn Tsnkov Blvd., Sofi 1164, Bulgri *** Istitute of Plnt Physiology nd Genetics, Bulgrin Acdemy of Sciences, Acd. Georgi Bonchev str., l. 21, Sofi 1113, Bulgri E-mil: pmmster2001@yhoo.com The morphologicl response of Pulowni tomentos x fortunei clone TF 01 nd Pulowni elongt x fortunei clone EF 02, grown in hydroponic t three levels of slinity, 50 mmol/l, 100 mmol/l, 200 mmol/l NCl ws compred. The content of mlondildehyde (MDA) ws enhnced with incresing of slt stress. Activity of phenyllnine mmoni - lyse (PAL), which prticipte in the control of phenolic metolism, ws incresed more in the leves of Pulowni elongt x fortunei. Under slt stress phenolic nd flvonoid contents were incresed in the leves of oth clones selected. The results were discussed from the view of chnges in synthesis of secondry metolites nd prticiption of seprte clsses of them in ntioxidtive response, chrcterizing different slt tolernce of Pulowni tomentos x fortunei nd Pulowni elongt x fortunei. Key words: slinity; growth; phenyllnine mmoni - lyse; phenols, flvonoids Section: Biology High slinity is the most widespred iotic stress nd constitutes the most stringent fctor in limiting plnt distriution nd productivity (Flowers et l., 1995:1). Slinity ffects plnt growth, metolism nd photosynthetic efficiency of crop plnts (Med nd Nkzw, 2008:2; Misr et l., 1997:3). Much effort is eing directed towrds the identifiction of physiologicl nd iochemicl processes tht re ffected y NCl in order to increse slt tolernce (Apse et l., 2002:4). Moleculr pproches to trget highly tolernt plnts re lso difficult s these plnts frequently survive the stress periods, eing in stte of suspended metolism. The study of plnt stress tolernce is suggested for understnding nd trnsfer of tolernce trits to sensitive crop plnts in future (Demmig-Adms et l., 2002:5). Pulowni is ntive from Chin. Pulowni tomentos hs een introduced into USA nd Europe s n ornmentl plnt nd is still widely used for this purpose. Trees introduced in Bulgri rech 12 m verge height nd 13.4 cm verge dimeter during 7 yers (Klmukov, 1995:6). Over the lst two decdes Pulowni species hs een extensively studied due to its ility to uptke nitrtes nd lnd contminnts, nmely hevy metls (Wng et l., 2010:7). This high-yielding tree cn e used for the production of energy, pper pulp nd wooden uilding mterils. The geneticlly tissue cultured Pulowni seedlings produced y The World Pulowni Institute (WPI) llow production of iofuels fter introducing of cultivrs without detrimentl impcts on food supply or the environment. Reserch on in vitro propgtion of P. elongt nd P. fortunei hs een reported (Bergmnn et l., 1997:8). Appliction of this technology for micropropgtion of tree species offers rpid mens of producing clonl plnting stock for fforesttion, woody iomss production nd it is effective wy to mintin the genetic gin (Prk nd Bong, 1992: 9). Plnts used in the current pper re propgted nd rooted ccording technology registered of Biotree Ltd., Bulgri. This lortory is lrgest producer nd supplier of geneticlly superior Pulowni tissue-cultures in vitro seedlings. The frmers preferred Pulowni tomentos x fortunei clone TF 01 due to fst development uniform regulr growth. Pulowni elongt x fortunei clone EF 02 is less rnchy for the purpose of wood mteril formtion. There is no informtion out its tolernce to slt stress nd possiilities to improvement of sline soil

2 utiliztion with these clones. The results derived from tissue cultures cn e used to predict the responses of plnts to environmentl contminnts nd to improve the design nd thus reduce the cost of susequent conventionl whole plnt experiments (Dorn, 2009:10). In this reserch, the effect of NCl on growth, ctivity of phenyllnine mmoni lyse, phenolic nd flvonoid contents in leves of Pulowni tomentos x fortunei clone TF 01 nd Pulowni elongt x fortunei clone EF 02, grown in hydroponic fter trnsplnt the explnts were compred so s to provide fundmentl se for vegettion restortion in contminted soils. Mterils nd methods Plnt mteril. Seeds nd in vivo explnts from the species P. tomentos, P. elongt nd their hyrids with P. fortunei re used for developing of in vitro multipliction protocol. For induction of shoots, explnts re cultured on Murshige nd Skoog (MS) nutrient medium included 2.5% (w/v) sucrose, 0.8% (w/v) gr nd vitmins. For multipliction of shoots MS medium ws used supplemented with µm 6-enzylminopurine (ВАР) nd µm indolilcetic cid (IAA). The medium included 3.0% sucrose (w/v), 0.8% gr nd vitmins. After multipliction the shoots were trnsferred to rooting medium sed on hlf strength sl slts MS medium, 2 % sucrose, 6 % gr nd vitmins supplemented with 4.92 µm indole-3- utyric cid (IBA) nd µm IAA. The ph of ll medi ws djusted to 5.7 using 0.1 N HCl nd 0.1 N NOH efore utoclving. All cultures were incuted under controlled conditions 16 h photoperiod, light intensity of 35 µmol m -2 s -1 nd 24/18±1ºC dy/night temperture. After three weeks of rooting, the shoots were rinsed with 1.5 ml L -1 Proplnt solution. Hydroponic experiment. The experiments were set s four tretments including control, ech tretment with 3 replictions. The uniform explnts were selected nd trnsplnted to polyethylene vessels contining 1.2 l of 1/4 Hellriegel solution (1898:11) with n ddition of A- Z microelements fter Hoglnd (ph 5.9) in growth chmer with 16-h photoperiod (PAR 100 µmol m -2 s -1 on the upper lef surfce, 25/23±1 ºC dy/night temperture, reltive humidity 60/70%). Ech vessel contined two explnts which represented one repliction. After 21 dys of cultivtion the plnts were trnsferred to 1/2 Hellriegel solution with the ddition of A-Z microelements (ph 5.9). The slt tretment ws pplied on the 48 th dy fter trnsplnting of explnts when the plnts hd dpted to the conditions of 1/2 Hellriegel nutrient solution nd 0 (control), 50, 100, nd 200 mm/l NCl ws dded. The solutions were erted every dy nd were chnged every 3 d to prevent depletion of nutrients nd NCl. Plnts were hrvested fter 10 d of tretment. Toxicity symptoms (e.g. discolortion, pigmenttion, yellowing nd stunting) were ssessed y eye through-out the experiment. At the end of the experiment the plnt smples were collected, wshed with tp wter nd rinsed with distilled wter efore eing seprted into lef, petiole, stem nd root nd fresh mss of ech plnt smple were mesured grvimetriclly. Dry mss of shoots nd roots were determined fter oven-drying (60 o C) for 2 dys until constnt weight ws otined. Lef re ws clculted using softwre progrm. Enzyme nd metolite ssys. For mesurements of phenyllnine mmoni-lyse (PAL), 0.2 g of FW of the third sl lef of plnts t ech slt tretment were ground with 0.05 g PVP-40 into fine powder with liquid nitrogen in 4 ml of ice-cold uffer contining 100 mm potssium phosphte (ph 7.2), 2 mm EDTA, 4 mm DTT. The suspensions were centrifuged (16 000g, 20 min, 4 o C) (Yun et l., 2002:12). Enzyme mesurement of PAL (EC ) ctivity ws mde: 0.1 ml superntnt ws mixed with 0.25 ml 20 mm phenyllnine (dissolved in 100 mм orte uffer, рн 8.8), then 2ml of the sme orte uffer nd 1 ml of distilled wter were dded. Control smples contined the superntnt nd uffer insted of phenyllnine. After 30, 60 nd 90 min of incution t 30ºC, sorption ws mesured t λ=290nm. Activity unit ws clculted s А = 0.01, equivlent to the production of 3.09 nmol

3 cinnmic cid (Yun et l., 2002:12). Protein content ws mesured with Folin regent (Lowry et l., 1951:13). For determintion of mlondildehyde (MDA), 0.3 g FW of the third sl lef of plnts t ech slt tretment were homogenized in mortr t 4 С with 3 ml 0.1 % trichlorocetic cid nd centrifuged for 20 min t rpm. 0.5 ml of the superntnt were mixed with 0.5 ml phosphte uffer ph 7.4 nd fter the ddition of 1 ml 0.5 % thiorituric cid dissolved in 20 % trichlorocetic cid, the smples were oiled for 30 min (Dhinds et l., 1981:14). After rpid cooling of the smples in n ice-th, sorption ws mesured t λ=532 nd 600nm using the extinction 3oefficient 155 mm -1 cm -1 (Heth nd Pcker 1968:15). For determintion of the phenols nd flvonoids, dry smples (100 mg) from third sl lef of plnts t ech slt tretment were ground nd exhustively extrcted with 96% (v/v) methnol. Totl phenolics were determined y the Folin & Cioclteu s colorimetric method (Pfeffer et l.,1998:16), modified y us s follows: n liquot of the extrct ws plced in test-tue nd distilled wter, 1:1 Folin & Cioclteu s regent nd 20% N 2 CO 3 were dded. The sortion ws mesured t λ=730 nm nd the totl phenolics were clculted y mens of clirtion curve of chlorogenic cid (in the rnge of 30µg/ml to 100 µg/ml) nd expressed s mg of chlorogenic cid equivlent per 1 g DW of the smple. Totl flvonoids content of the whole shoot smples of the plnt ws mesured using colorimetric ssy (Zhishen, 1999:17), modified s follows: Aliquots of the methnol extrct were plced in test-tue nd distilled wter, 5% NNO 2 nd 10% AlCl 3 were dded. After the ddition of 1N NOH nd distilled wter, the sorption t λ=510 nm ws mesured nd the concentrtion ws clculted y mens of clirtion curve of (+) ctechin (in the rnge of 2 µg/ml to 80 µg/ml). The totl flvonoids of the smples were expressed in mg of (+) ctechin equivlent per 1 g DW of the smple. All mesurements were performed in triplicte with three repetitions. Results nd disccusion Effect of slt stress on plnts growth. Seedlings growth is normlly limited y incresing concentrtion of NCl (Sreenivsulu et l., 2000:18). In our study, with incresing slinity levels, the root nd stem length, lef numer nd totl lef re in oth plnts were reduced (Tle 1). The root nd stem length of Pulowni tomentos x fortunei clone TF 01 ws reduced more thn tht of Pulowni elongt x fortunei clone EF 02. The vlues for stem length mesured t 200 mm/l NCl for Pulowni elongt x fortunei clone TF 01 were close to the control. The lef numer of Pulowni tomentos x fortunei clone EF 02 rose, ut totl lef re declined shrply with incresing slinity levels. Mximum reduction of totl lef re ws oserved t 200 mm/l NCl (80%).The lef numer of Pulowni elongt x fortunei clone ws the sme t 100 nd 200 mm/l NCl. With incresing slinity levels totl lef re ws reduced y 40%; 28% nd 36%, respectively, compred to control. Totl lef re showed the cpility of plnt in forming of photosynthetic surfce. The totl FW/DW per plnt ws incresed for Pulowni tomentos x fortunei y 21%; 13% nd 47%, respectively, ut ws decresed for Pulowni elongt x fortunei y 2%; 12% nd 5%, respectively, compred to control (dt not showed). The present results otined for Pulowni elongt x fortunei were in line with those of Prveen nd Frrukh (2009:19), who reported decline in fresh nd dry weights of shoots under high slinity stress. Effect of slt stress on plnts mlondildehyde. The incresed ccumultion of lipid peroxides is indictive of enhnced production of toxic oxygen (Mrkovsk et l., 2009:20). In this study, MDA concentrtion ws enhnced with incresing slinity levels, the mximum vlues ws oserved t 200 mm/l NCl (Tle 2). The results indicted tht slt stress produced more rective oxygen species, resulting in more incresed lipid peroxidtive products nd oxidtive stress in Pulowni tomentos x fortunei clone thn in Pulowni elongt x fortunei clone.

4 Effect of slt stress on plnts secondry metolism. PAL is involved in the control of phenolic metolism nd provides precursors for lignin iosynthesis (Hhlrock nd Scheel, 1989:21). ). It is known tht the level of PAL re ffected y ge, light, phytochrome, wonding, infection (Cmm nd Towers, 1973:22).Our results showed tht PAL ctivity in the leves of Pulowni elongt x fortunei ws three time higher compred to tht in the leves of Pulowni tomentos x fortunei (Fig. 1). PAL ctivity ws enhnced in oth plnts with incresing slinity levels. Flvonoids nd phenolic sustnces isolted from wide rnge of vsculr plnts, ct in plnts s ntioxidnts nd ntimicroils (Piett, 2000:23). Shukl et l. (2009:24) reported out significnt nd liner reltionship etween the ntioxidnt ctivity nd phenolic content, indicting tht phenolic compounds could e mjor contriutors to ntioxidnt ctivity. Our results showed tht PAL ctivity, phenolic nd flvonoid content rose with incresing slinity levels. The mximum vlues were oserved t 100 nd 200 mm/l NCl. There is some evidence of the induction of phenolic metolism in plnts s response to multiple stress (including slt stress) (Michlk, 2006:25). Phenolics including vrious flvonoids ply pivotl roles in soring free rdicls, quenching singlet oxygen, nd decomposing peroxides. Pulowni elongt x fortunei clone TF 01 is more tolernt to slt stress thn Pulowni tomentos x fortunei clone EF 02. It is chrcterized with smller reduction of root nd stem length, totl lef re nd higher PAL ctivity, totl phenolic nd flvonoid content in the leves. Tle 1. Men vlues ± SD (n = 5-6) of root nd stem length, lef numer nd totl lef re of Pulowni tomentos x fortunei clone TF 01 nd Pulowni elongt x fortunei clone EF 02, grown in hydroponic in response to slt stress Tretments Root length Stem length Lef numer Lef re [cm] [cm] [cm 2] Pulowni tomentosx fortunei Control 50 mm/l NCl 100 mm/l NCl 200 mm/l NCl Pulowni elongte x fortunei Control 50 mm/l NCl 100 mm/l NCl 200 mm/l NCl 28.25± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±1.7 8±0.6 9±1.5 11±1.5 10±0.8 9±1.1 8±1.2 8± ±16 126± 5 109±22 87± 4 502±39 300±40 360±23 321±67 Vlues with the sme letter re not significntly different when mens re seprted y Fisher`sLSD test (P<0.05). Tle 2. Men vlues ± SD (n = 5-6) of MDA content (in nm/gfw), determined in leves of Pulowni tomentos x fortunei clone TF 01 nd Pulowni elongt x fortunei clone EF 02, grown in hydroponic in response to slt stress Tretments Pulowni tomentos x fortunei Pulowni elongt x fortunei Control 0.017± ± mm/l NCl 0.298± ± mm/l NCl 0.281± ± mm/l NCl 0.396±0.031c 0.191±0.013c

5 Vlues with the sme letter re not significntly different when mens re seprted y Fisher`s LSD test (P<0.05). TF 01 EF 02 PAL [µm mg pr -1 min -1 ] c c Phenols [mg g -1 DW] c c Flvonoids [mg g -1 DW] 11,2 8,4 5,6 2,8 c Slts [mg L -1 ] Slts [mg L -1 ] 11,2 8,4 5,6 2,8 Figure 1. Chnges in the PAL ctivity, phenol nd flvonoid content, determined in leves of Pulowni tomentos x fortunei nd Pulowni elongt x fortunei clones, grown in hydroponic in response to the ddition of NCl The tolernce of Pulowni elongt x fortunei clone TF 01 for high-slinity environments mkes it possile cndidte for studying the moleculr mechnisms y which plnts respond to slinity stress. Chnges in the levels of some nonenzymtic ntioxidnts, such s phenolics nd flvonoids my estimte for their use s mrkers of slt tolernce in geneticlly diverse Pulowni clones.

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7 20. Mrkovsk, Y.K., Gorinov, N.I., Nedkovsk, M.P., Mitev, K Cdmium-induced oxidtive dmge nd ntioxidnt response in Brssic junce plnts. Biol. Plnt., 53, Hhlrock, K., Scheel, D Physiology nd moleculr iology of phenylpropnoid metolism. Ann. Rev. Plnt Phys. Plnt Mol. Biol., 40, Cmm, E.L., Towers, G.H.N Phenyllnine mmoni lyse. Phytochemistry, 12, Piett P.G Flvonoids s ntioxidnts, J. Nt. Products, 63, Shukl S., Meht A., Bjpi V.K., Shukl S In vitro ntioxidnt ctivity nd totl phenolic content of ethnolic lef extrct of Stevi reudin Bert, Food Chem. Toxicol., 47, Michlk, A Phenolic compounds nd their ntioxidnt ctivity in plnts growing under hevy metl stress. Pol. J. Environm. Stud., 15, Yulin Kostdinov Mrkovsk Associte Prefessor Fculty of Biology, University of Sofi, 8 Drgn Tsnkov Blvd., Sofi 1164, Bulgri Tel.: GSM: E-mil: pmmster2001@yhoo.com