Exogenous catechin increases antioxidant enzyme activity and promotes flooding tolerance in tomato (Solanum lycopersicum L.)

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1 Plnt Soil (2011) 344: DOI /s y REGULAR ARTICLE Exogenous tehin inreses ntioxidnt enzyme tivity nd promotes flooding tolerne in tomto (Solnum lyopersium L.) Jinn-Chin Yiu & Menq-Jiu Tseng & Chung-Wei Liu Reeived: 13 Otoer 2010 / Aepted: 18 Jnury 2011 / Pulished online: 20 Ferury 2011 # Springer Siene+Business Medi B.V Astrt We studied the extent to whih tehin pplied s soil drenh modifies the effets of soil wterlogging on plnt growth, the funtioning of the free rdil svenging system nd on oxidtive stress levels. Forty-dy-old tomto plnts (Solnum lyopersium L.) were treted with 0 nd 2mM tehin48hpriorto5d wterlogging followed y 4 d dringe period. Exogenous tehin inresed totl fresh nd dry weight of flooded plnts, redued memrne dmge, mintined hlorophyll onentrtions, promoted photosynthesis nd inresed ATP onentrtion in the leves, nd rised surose synthse nd lohol dehydrogense tivities in the roots. Ctehin pretretment lso redued hydrogen peroxide nd superoxide rdil onentrtion nd inresed vrious omponents of the ntioxidtive system in leves. Ctehin tretment ffeted superoxide dismutse nd tlse tivities in lose oordintion with sorte peroxidses nd glutthione redutse. Exogenous tehin n mrkedly redue the wterlogging injury Responsile Editor: Eri J.W. Visser. J.-C. Yiu (*) : C.-W. Liu Deprtment of Hortiulture, Ntionl Iln University, 1, Se. 1, Shen-Lung Rod, I-Ln 260, Tiwn e-mil: jyiu@niu.edu.tw M.-J. Tseng Deprtment of Hortiulture, Ntionl Chung Hsing University, Tihung 402, Tiwn in leves nd roots of tomto y enhning free rdil svenging system suffiiently to lower hydrogen peroxide nd superoxide onentrtions. Keywords Alohol dehydrogense. Antioxidnt enzyme. Pretretment. Reovery. Surose synthse. Wterlogging Arevitions ADH lohol dehydrogense APX sorte peroxidse C ontrol Ct tehin CAT tlse Chl hlorophyll GR glutthione redutse MSI memrne stility index Pn CO 2 ssimiltion rte ROS retive oxygen speies RWC reltive wter ontent SOD superoxide dismutse SuSy surose synthse W wterlogging Introdution Wterlogging is defined s the sturtion of the soil with wter round the roots (Anton et l. 2002). The negtive impt of wterlogging on plnt growth nd

2 214 Plnt Soil (2011) 344: development is lrgely the onsequene of the slow diffusion rtes of gses in wter s ompred with ir nd the reltively low soluility of oxygen in wter (Vrtpetin nd Jkson 1997). Oxygen is vitl for the entrl energy-providing pthwy of ells; the presene or sene of oxygen determining metoli tivity nd energy prodution. Redution of root respirtion is one of the erliest responses of plnts under noxi, regrdless of whether the plnts re flooding-tolernt or intolernt (Lio nd Lin 2001). Kuo nd Chen (1980) demonstrted tht the reltively greter tolerne of the tomto genotype L-123 to soil flooding depends, in prt, on its ility to trnsport oxygen internlly from the eril prt to the roots. Oxygen defiieny due to wterlogging or flooding loses stomt, redues internl CO 2 onentrtions in leves, nd therefore slows photosynthesis severely (Olivell et l. 2000). As onsequene, the photosyntheti eletron trnsport hin my eome over-redued, forming superoxide rdils (O 2 ) nd singlet oxygen speies in hloroplsts. Any retive oxygen speies (ROS) generted in exess due to flooding onditions ould unlne the ellulr redox systems in fvor of oxidized forms, resulting in oxidtive dmge to lipids, proteins nd nulei ids (Hlliwell nd Gutteridge 1989). Wterlogging loks the oxygen supply to the roots, thus inhiiting root respirtion, resulting in severe deline in the energy sttus of root ells tht ffets importnt metoli proesses of plnts. Plnts ret to n sene of oxygen y swithing from n oxidtive to solely sustrte-level phosphoryltion of ADP to ATP, the ltter retions predominntly involving glyolysis nd fermenttion. The overll yield of ATP produed during fermenttion is only two moleules of ATP per gluose moleule, ginst 38 moleules of ATP produed during oxidtive phosphoryltion (Rird et l. 2006). Surose synthse (SuSy), whih tlyzes the reversile onversion of surose nd UDP to UDPgluose nd frutose, plys ruil role in providing n dequte sugr supply during noxi stress (Rird et l. 2006). Meristem deth of mize seedlings ws signifintly llevited in the presene of gluose in n noxi inution medium, inditing tht n inrese in glyolyti flux lone ws suffiient to restore root tip viility (Rird et l. 1998). The genotype T44 (tolernt) of the mung en showed greter inrese in SuSy tivity thn the PB genotype (suseptile) under wterlogging onditions (Sirm et l. 2009). Continuous supply of fermentle sugrs to roots is onsidered to e ritil for long-term survivl under noxi or flooding. Therefore, the greter inrese in SuSy tivity under wterlogged onditions resulted in n inresed vilility of reduing sugrs, whih sustined their energy requirement. Root neroi metolism depends on loholi fermenttion for ATP prodution. Alohol dehydrogense (ADH) is n essentil fermenttive enzyme whih is supported y rpid inreses in the tivity of ADH in rie seedlings when oxygen supply ws severely deprived (Rivol et l. 1989). Mize nd rley mutnts tht lk the ADH1 gene re more sensitive to flooding injury thn re wild-type plnts (Roerts et l. 1989). Wng et l. (2009) found ADH tivity of Kentuky luegrss (Po prtensis L.) to inrese drmtilly within 3 dys of flooding ut signifintly deresed s root internl ertion inresed. Higher ADH tivity is often ssoited with neroi onditions in plnts. The suseptiility of plnts to environmentl stress, inluding wterlogging, hs een onsidered to e ssoited with ntioxidtive ility (Zheng et l. 2009). To ontrol the level of ROS nd to protet ells under stress onditions, plnt tissues ontin severl ROS svenging enzymes, e.g., superoxide dismutse (SOD; EC ), tlse (CAT; EC ), sorte peroxidses (APX; EC ), glutthione redutse (GR; EC ), enzymes tht detoxify lipid peroxidtion produts (glutthione S-trnsferses, phospholipid- hydroperoxide-glutthione peroxidse nd sorte peroxidse), nd network of lowmoleulr-mss ntioxidnts (sorte, glutthione, phenoli ompounds nd α-toopherol). Inreses in the tivities of ntioxidnt enzymes in response to vrious environmentl stresses hve lso een reported in vrious studies. The omprtively greter ntioxidnt enzyme tivities tht result in less oxidtive stress in pigeon pe genotype ICP 301 (tolernt) ould e one of the determining ftors of greter tolerne to flooding s ompred with Pus 207 (suseptile) (Kumuth et l. 2009). Hossin et l. (2009) demonstrted tht oordinted ntioxidnt tivity, involving inresed tivities of SOD nd CAT together with modultion of the sorte glutthione yle, llowed plnts to ope with flooding-indued oxidtive stress up to ertin point. Some polyphenols, espeilly those in seeds nd green leves inhiit oxidtive dmge through their

3 Plnt Soil (2011) 344: ntioxidnt tivity (Rie-Evns et l. 1996). Ctehins re the mjor polyphenoli ompounds in green te. These inlude ( )-epigllotehin gllte (EGCG), ( )-epigllotehin (EGC), ( )-epitehin gllte (ECG), nd ( )-epitehin (EC). Colletively, they ount for out 10% of te lef dry weight (Hr et l. 1995). Some phenoli ompounds n estlish oopertive redox intertions with the endogenous ntioxidnt sustnes, whih reinfore synergistilly the resistne of the system to suffer oxidtive dmge. The mehnisms of polyphenol ntioxidnt tivity inlude their ility to svenge rdils nd intert with intrellulr signl trnsdution pthwys (Nijveldt et l. 2001; Willims et l. 2004). Te tehins svenge O 2, hydroxyl rdils, nd oxidized opper-medited low-density lipoproteins more effetively thn lph-toopherol. Along with the protetive effet ginst oxidtive stress polyphenols might t s prooxidnts (Chen nd Chn 1996; Zhng et l. 1997; Lo et l. 2006). In mny Asin ountries, hevy typhoon rins often distur tomto prodution during the hot summer seson (Lin et l. 2004). The physiologil nd iohemil responses of tomto plnts to wterlogging stress hve een extensively investigted (Jkson et l. 2003). Furthermore, exogenous ntioxidnt pplition is onvenient nd effetive pproh for enhning the stress tolerne of rops nd eventully improving rop produtivity. Tretment with 10mM L-sori id efore re-exposure to ir fter vrying periods of noxi shows tht this ntioxidnt n improve growth nd survivl of hikpe seedling during the post-noxi reovery period (Crwford nd Wollenweer-Rtzer 1992). The present study is the first we know of tht ssesses the ility of tehin to meliorte the effets of wterlogging on plnt growth nd link its effets to hnges in ROS nd omponents of the ntioxidnt system. Mterils nd methods Plnt growth, wterlogging tretment nd smple olletion Tomto seeds (Solnum lyopersium L. v. Kom) were sown in smll pots (3 m tll nd 2 m dimeter) ontining pet moss, vermiulite nd perlite (1:2:2, v/v) nd trnsferred to growth hmer t 24 C under fluoresent light nd tungsten light (350 μmol m 2 s 1 ) with 16-h photoperiod. Seven dys fter germintion, seedlings were trnsplnted one plnt per pot to 8 m tll nd 6 m dimeter plsti pots filled with similr ompost. Plnts were grown in plsti greenhouse t Ntionl Iln University, Tiwn, under nturl sunlight nd photoperiod for 4 weeks efore eing sujeted to wterlogging stress. Plnts were wtered with hlf-strength Hoglnd solution every other dy to mintin optiml irrigtion. Middy wether onditions during the experimentl periods were hrterized y reltively ler sky (i.e., photosyntheti photon flux density >900 μmol m 2 s 1 ), mximum ir temperture of C nd 72% reltive humidity. Eh plnt ws treted with single 50-mL dose of n queous solution of 2mM tehin (. 0.1mmol) (C1788, Sigm-Aldrih Lorhemiklien Gmh, Seelze, Germny), pplied s soil drenh. In preliminry experiments, when ompring the effet of Ct onentrtion, 2mM Ct proved to e the most effetive dose. A lower onentrtion (1mM) ws slightly effetive, nd 4mM Ct effets were little different to those of 2mM Ct. Forty eight hours fter tehin tretment, the plnts were wterlogged for 5 d y pling pots in 65 m 35 m 12 m plsti ontiners filled with suffiient wter to rise levels to 2 m ove the soil surfe (Yiu et l. 2009). Tretments onsisted of ontrol (C), 1, 3, nd 5 d of wterlogging, nd reovery fter 4 d dringe. Control (unwterlogged) plnts remined wellwtered during the period of the experiment. More mesurements, plnts were removed from pots nd plnts (roots nd leves) were wshed thoroughly with wter nd dried in pper towel, oven dried t 70 C to onstnt dry weight (DW). Three replites were lloted to eh tretment, eh replite onsisting of 30 plnts. Physiologil prmeters nd enzyme tivities were mesure on sl 3 full lef of 2nd nd 3rd node from the top nd ll prts of the root. Oservtions were reorded on reltive wter ontent (RWC), memrne stility index (MSI), hlorophyll (Chl) ontent, photosynthesis (Pn), tivity of SuSy nd ADH, ATP, O 2,H 2 O 2 onentrtion, nd tivity of ntioxidnt enzymes. After eh tretment, leves or roots were hrvested, immeditely frozen in liquid nitrogen nd kept t 80 C until nlysis.

4 216 Plnt Soil (2011) 344: Physiologil mesurements Lef RWC ws estimted y reording the fresh weight of 0.5 g fresh lef smples efore nd fter floting on wter for 4 h, followed y drying in hot ir oven (75 C) until onstnt dry weight ws hieved (Wetherley 1950). RWC ¼ðfresh weight dry weightþ =ðfresh weight t full turgor dry weightþ 100%: MSI ws estimted y pling two sets of 200 mg of lef or root mteril in test tues ontining 10 ml of doule-distilled wter (Sirm et l. 1997). One set ws heted t 40 C for 30 min in wter th, nd the eletril ondutivity of the solution reorded using ondutivity meter (C 1 ). The seond set ws oiled t 100 C on oiling wter th for 10 min efore ondutivity (C 2 ) ws mesured s ove. MSI ws lulted s MSI ¼½1 ðc 1 =C 2 ÞŠ100 Chlorophyll onentrtion ws estimted y extrting 0.5 g of the lef mteril in 25 ml dimethyl sulfoxide (DMSO). Smples were heted t 60 C for 12 h, ooled to room temperture efore mesuring sorne t nd nm nd lulting onentrtion ording to Brnes et l. (1992). Lef CO 2 ssimiltion rte (Pn) ws mesured ording to Yiu et l. (2009) on eh of the seleted leves using infrred gs nlyzers uilt into lef uvette in n open-gs exhnge system (Li-Cor-6400; Li-Cor In., Linoln, NE, USA). Enzyme ssys on roots Surose synthse ssy of root tissue ws sed on the synthesis of surose from UDP-gluose nd frutose nd its estimtion y nthrone regent fter the removl of untreted frutose y heting t 100 C (Zeng et l. 1999). Extrtion uffer onsisted of 200 mm HEPES uffer, ph 7.5, 1mM DTT, 5mM MgCl 2, 1mM EDTA, 20mM sodium sorte, 1mM PMSF, nd 10% (w/v) polyvinylpolypyrrolidone. The ufferto-tissue rtio ws 10:1. One grm root ws ground to fine powder in liquid nitrogen in hilled mortr nd pestle followed y extrtion with hilled extrtion uffer. Extrt ws entrifuged t 14,000 g t 4 C for 10 min, the superntnt dilyzed t 4 C for 24 h ginst extrtion uffer diluted 1:40 nd hnged t lest three times during dilysis. Dilyzed extrt ws used s n enzyme. The retion mixture in 3 ml ontined 50mM HEPES NOH uffer (ph 7.5), 15 mm MgCl 2, 10mM frutose, 5mM UDP-gluose, 50 μl enzyme extrt nd wter to mke finl volume 3.0 ml. Assys were t 30 C for 30 min in shking wter th nd the retion terminted y the ddition of 1 ml of 30% KOH. Controls were terminted t 0 min. The un-reted frutose is removed y susequent heting t 100 C for 10 min. After ooling, eh ssy mixture ws inuted with 1 ml of 0.14% nthrone in H 2 SO 4 t 40 C for 20 min nd sorne reorded t 620 nm in UV visile spetrophotometer. Ativity ws expressed s μmol per mg protein per min. Totl protein ws quntified ording to Brdford (1976), with ovine serum lumin s the stndrd. Alohol dehydrogense (ADH) tivity in roots ws ssyed y the reverse retion i.e., oxidtion of ethnol y ADH with the help of NAD, resulting into the synthesis of etldehyde nd NADH (Chung nd Ferl 1999). The extrtion uffer onsisted of 50mM Tris HCl nd 15mM DTT, ph 8.0. Root tissue (0.5 g) ws first powdered with liquid nitrogen nd then homogenized with 5.0 ml of extrtion uffer. The extrt ws entrifuged t 12,000 g for 15 min t 4 C in refrigerted entrifuge nd n liquot of 50 μl tken for wter-solule protein onentrtion ssy. The 3 ml retion mixture ontined 50mM Tris uffer, 0.867mM NAD, 20% ethnol, 50 μl enzyme, nd wter to mke finl volume of 3 ml. Retion mixture exept NAD ws prepred in test tues nd eh smple used s lnk to djust to zero. NAD ws then dded to initite the retion. The resultnt inrese in sorne t 340 nm ws reorded for 1 min in UV visile spetrophotometer. Amount of NADH formed ws omputed y drwing stndrd urve of NADH t 340 nm, tivity eing expressed s nmol NADH formed per mg protein per min. Mesurement of ATP, O 2 nd H 2 O 2 onentrtion in leves Lef smples were prepred for ATP, O 2 nd H 2 O 2 nlyses y homogenizing 0.2 g of frozen lef. Intrellulr ATP ws mesured ording to Yiu et l. (2009) using n ATP Bioluminesene Detetion Kit (Promeg Corportion, Mdison, WI, USA). ATP

5 Plnt Soil (2011) 344: onentrtion ws lulted from n ATP stndrd urve nd expressed s nmol g 1 DW. Superoxide nion nd H 2 O 2 levels were determined ording to Yiu et l. (2009). Determintions were performed on 5 replites. Protein extrtion nd ntioxidnt enzyme ssy in leves Approximtely 1.0 g fresh weight (. 10 leves) were homogenized t 4 C in 1.5 ml of extrtion uffer [100mM potssium phosphte, ph 7.5, ontining 1 mm EDTA nd 1% PVP-40] with mortr nd pestle. The homogente ws then entrifuged t 25,000 g for 20 min nd the superntnt used s the rude extrt for SOD, CAT nd GR ssys. For the nlysis of APX, the extrtion uffer lso ontined 5mM sorte s desried y Ro et l. (1995). For the spetrophotometri ssy of SOD, extrts were pssed through Sephdex G-25 olumns t 4 C using 1 ml of 0.1 M sodium phosphte, ph 7.8. All smples were mde to 40% (v/v) with glyerol nd then stored t 20 C until enzyme nlysis. All spetrophotometri ssys were performed in Hithi U-2001 UV/Vis spetrophotometer (Hithi Ltd, Tokyo, Jpn). Totl SOD tivity ws mesured spetrophotometrilly ording to MCord nd Fridovih (1969). The retion ws performed t 25 C in 3-mL uvette of 50mM potssium phosphte, ph 7.8, ontining 0.1mM EDTA, 0.06mM ferriytohrome, 0.05mM xnthine, nd 10 8 M xnthine oxidse. One unit of SOD is defined s the mount of enzyme tht inhiited the rte of ferriytohrome redution y 50% t A 550. Totl CAT tivity ws determined spetrophotometrilly y following the deline in A 240 s H 2 O 2 (extintion oeffiient 40mM 1 m 1 ) ws tolized s desried y Beers nd Sizer (1952). The 2-mL retion mixture ontined 50mM phosphte uffer (ph 7.0), 10mM DTT, nd 5mM H 2 O 2. One unit of CAT onsumes 1 nmol of H 2 O 2 min 1 under the ssy onditions. Totl APX tivity ws mesured t 25 C ording to Nkno nd Asd y the derese in A 290 due to sorte oxidtion y H 2 O 2 (extintion oeffiient 2.8mM 1 m 1 )(Nknond Asd 1981). The 2-mL retion volume ontined 100mM potssium phosphte uffer (ph 7.0), 0.5 mm sorte, nd 0.2mM H 2 O 2. One unit of APX forms 1 μmol of sorte oxidized min 1 under the ssy onditions. Totl GR tivity ws determined y following the oxidtion of NADPH t 340 nm (extintion oeffiient 6.2mM 1 m 1 ) s modified y Foyer nd Hlliwell (1976). The 2-mL ssy mixture ontined 0.1M Tris uffer (ph 7.8), 2mM EDTA, 0.5mM GSSG nd 150mM NADPH. One unit of GR oxidizes 1 μmol of NADPH min 1 under the ssy onditions. Sttistil nlysis To lulte the signifine of vlues, mens were ompred y Dunn s Multiple Rnge tests t P<0.05, using PC SAS 8.2 (SAS Institute, Cry, NC, USA). All dt presented re mens±se. Results Physiologil prmeters Chlorosis nd lef senesene were enhned y soil flooding whih lso redued lef re nd rnhing s flooding progressed. This injury ws entirely eliminted y tehin tretment. Compred with the well-drined ontrol plnts, root length of flooded plnts given tehin inresed ompred to tht of plnts wterlogged without tehin (W) (Fig. 1). Tle 1 shows the time ourse of hnges in whole plnt iomss during wterlogging nd reovery. After 9 d under non-wterlogging onditions, the FW nd DW of the tomto roots nd leves inresed when plnts were treted with 2mM tehin (Ct) ut ws little different to tht of plnts not reeiving tehin. In wterlogged plnts, FW nd DW delined from dy 1 to dy 5 of flooding ut inresed gin during 4 d reovery; However, wterlogged plnts given tehin ttined FW nd DWs tht exeeded those of untreted ounterprts nd grew lrger tht non-wterlogged plnts. After 4 d of reovery, exogenous pplition of tehin under wterlogging onditions, gve whole plnt FW nd DW vlues tht were, respetively, 46.8% nd 61.6% greter thn those of wterlogged plnts not given tehin. Wterlogging redued lef RWC over 5 d nd ws lmost 45% elow ontrol vlues t the time plnts were

6 218 Plnt Soil (2011) 344: Fig. 1 Photogrph showing the effet of root-zone flooding on tehin nd non-tehin tomto (Solnum lyopersium L.) plnts. The right plnt ws shown 40-dy-old tomto plnts were exposed to wterlogging onditions lone without tehin pre-tretment; the left plnt ws shown 40-dy-old tomto plnts were supplied with tehin exogenously for 48 h nd then exposed to wterlogging stress for 5 dys. Photogrph ws mde fter 4 d of reovery. Brs=5 m drined. However this deline ws limited to 12% in plnts given tehin (Tle 2). Reovery of RWC during 4 d dringe ws lso lrger. The Memrne Stility Index (MSI) of oth leves nd roots deresed with the durtion of wterlogging to minimum on dy 5. The deline in wterlogged plnts ws 60.2% nd 59.1% in the leves nd roots, respetively. However, in flooded plnts given tehin the dereses in MSI were only 26.8% nd 25.4% in the leves nd roots, respetively (Tle 2). Reovery ws lso fster in the tehin-treted plnts. Totl hlorophyll onentrtion, deresed under wterlogging. Wterlogging lone for 5 d deresed hlorophyll y 47.6% lthough levels reovered slightly fter 4 d reovery under well-drined onditions. Applition of tehin to ontrol plnts ws without effet ut, in wterlogged plnts, the loss of hlorophyll ws sustntilly slowed with onentrtions delining y only 24.92% fter 5 d flooding nd y only 11.85% fter the 4 d reovery period (Tle 3). After 1 d, ssimiltion of CO 2 (Pn) deresed signifintly in wterlogged plnts (y 12.5%) in omprison with non-wterlogged ontrols (Tle 3) ut tehin tretment prevented sttistilly signifint loss in Pn. If wterlogging ws prolonged for 5 d wterlogging, Pn ws hlved in the sene of tehin ut redued y only fifth when tehin ws given. Thus, the presene of tehin, wterlogging inhiited Pn to muh smller extent. After 5 d reovery, Pn levels remined t t. 66% elow ontrol vlues in wterlogged plnts while in tehin-treted plnts Pn hd returned to ontrol levels. Enzyme tivities Surose synthse tivity in the roots ws inhiited y wterlogging for 1 5 d (Fig. 2). In ontrst, tivity levels were stimulted strongly y tehin tretment to wterlogged plnts with levels pproximtely doule those of well-drined ontrols. Ctehin given Tle 1 Effets of wterlogging nd tehin tretment on the fresh nd dry weight of the tomto leves nd roots Dys of wterlogging/ reovery Fresh weight (mg) Dry weight (mg) C Ct W Ct+W C Ct W Ct+W 0 631±35 d 628±31 d 625±34 619± ±1.8 d 31.9±1.6 d 31.3± ± ±40 d 665±35 d 610±39 636± ±2.1 d 34.6±1.8 d 31.1± ± ±53 738±48 554±24 685± ± ± ± ± ±57 866±46 493±30 727± ± ± ± ±2.8 4 dys fter reovery 1219± ±82 558±51 819± ± ± ± ±3.1 C: the group without tehin tretment or wterlogging; Ct: 40-dy-old tomto plnts were supplied with tehin lone; W: 40-dyold tomto plnts were exposed to wterlogging onditions lone without tehin pre-tretment; Ct + W: 40-dy-old tomto plnts were supplied with tehin exogenously for 48 h nd then exposed to wterlogging stress for 5 dys, followed y 4-dy reovery period Eh vlue represents the men ± SE (n=15) of 3 independent experiments. Different letters indite signifint differenes etween tretments (P<0.05).

7 Plnt Soil (2011) 344: Tle 2 Effets of wterlogging nd tehin tretment on the reltive wter ontent (RWC) in leves (%) nd the memrne stility index (MSI) in the leves nd roots of the tomto RWC (%) MSI (%) leves MSI (%) roots Dys of wterlogging/ reovery C Ct W Ct+W C Ct W Ct+W C Ct W Ct+W ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±2.3 e 71.32± ± ± ±2.1 e 60.86±3.1 d 81.08± ± ±2.4 d 60.21±2.9 4 dys fter reovery 81.29± ± ±2.9 d 79.27± ± ± ±3.1 d 71.98± ± ± ± ±3.5 C: the group without tehin tretment or wterlogging; Ct: 40-dy-old tomto plnts were supplied with tehin lone; W: 40-dy-old tomto plnts were exposed to wterlogging onditions lone without tehin pre-tretment; Ct+W: 40-dy-old tomto plnts were supplied with tehin exogenously for 48 h nd then exposed to wterlogging stress for 5 dys, followed y 4-dy reovery period Note: Eh vlue represents the men±se (n=15) of 3 independent experiments. Different letters indite signifint differenes etween tretments (P<0.05). to well-drined plnts hd no effet. During reovery, wterlogged plnts showed slight inrese in SuSy tivity, while no further inrese ws oserved in wterlogged plnts given tehin. Alohol dehydrogense tivity in the roots ws inresed y 1 5 d wterlogging (Fig. 2). This effet ws very muh lrger in the presene of tehin; tivity inresing to pproximtely 14.7 times prestress levels fter 5 d Ativity of ADH delined fter the termintion of tretment in oth the presene nd sene of tehin. ATP onentrtion nd free rdil prodution In the leves of flooded plnts, rpid derese in ATP onentrtion ws oserved fter 3 d or 5 d ompred to well-drined ontrols (Fig. 3). Although exogenous tehin did not signifintly lter the ATP onentrtion of ontrol plnts, it prevented sttistilly signifint loss of ATP in flooded plnts. After 4 d reovery, the enefit of tehin to ATP onentrtions in flooded plnts ws still very mrked. Flooding inresed oxidtive stress levels of the leves. A 1.9-fold inrese in O 2 (Fig. 2) nd 1.4- fold inrese in H 2 O 2 (Fig. 3) ws oserved on dy 5 for non-tehin treted plnts. However, exogenous tehin redued the umultion of O 2 nd H 2 O 2 y 44.5% nd 36.7% under flooding tretment, respetively. After 4d reovery oth O 2 nd H 2 O 2 onentrtions deresed lthough levels in wterlogged plnts not given tehin remined sustntilly ove those of ontrols nd of flooded plnts treted with tehin. Antioxidnt enzyme tivities Soil flooding modified oth enzymti tivities nd metolites of the sorte glutthione yle in the leves. Among the different ntioxidnt enzymes, SOD nd CAT exhiited erly responses, wheres APX nd GR exhiited lte responses (Fig. 4). SOD tivity signifintly ws inresed y 1 d of wterlogging ut levels fell to elow ontrol vlues therefter, inluding fter 4 dringe. Ctehin hnged this flooding response y rising SOD tivity to levels tht exeeded those of ontrol plnts. This effet persisted fter 4 d reovery. In wterlogged plnts, CAT followed similr trend to SOD. Although exogenous tehin hd no

8 220 Plnt Soil (2011) 344: Tle 3 Effets of wterlogging nd tehin tretment on Chl + nd Pn in the tomto leves Dys of wterlogging/ reovery Chl + (μg g 1 FW) Pn (μmol CO 2 m 2 s 1 ) C Ct W Ct+W C Ct W Ct+W ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±92 e 2136± ± ± ±0.3 e 11.05±0.5 4 dys fter reovery 2882± ± ±64 d 2508± ± ± ±0.5 d 12.85±0.4 C: the group without tehin tretment or wterlogging; Ct: 40-dy-old tomto plnts were supplied with tehin lone; W: 40-dy-old tomto plnts were exposed to wterlogging onditions lone without tehin pre-tretment; Ct+W: 40-dy-old tomto plnts were supplied with tehin exogenously for 48 h nd then exposed to wterlogging stress for 5 dys, followed y 4-dy reovery period Note: Eh vlue represents the men±se (n=15) of 3 independent experiments. Different letters indite signifint differenes etween tretments (P<0.05). Fig. 2 Effet of wterlogging nd tehin tretment on the tivity of SuSy () nd ADH () in root tissues. Letters ove rs indite sttistilly-signifint differene (P 0.05) ording to Dunn s multiple rnge test. Vertil rs show ± SE of men. C: the group without tehin tretment or wterlogging; Ct: 40-dy-old tomto plnts were supplied with tehin lone; W: 40-dy-old tomto plnts were exposed to wterlogging onditions lone without tehin pre-tretment; Ct+W: 40-dy-old tomto plnts were supplied with tehin exogenously for 48 h nd then exposed to wterlogging stress for 5 dys, followed y 4-dy reovery period reovery effet on non-wterlogged ontrols this tretment inresed CAT tivity y 53.7% in flooded plnts, nd up to twie the tivity found in wterlogged plnts without tehin (Fig. 4). These effets were slightly diminished fter 4 d reovery. APX tivity ws deresed y 3 nd 5 d wterlogging (Fig. 4), to 27.2% of ontrol plnts. The depression ws still evident fter 4 d reovery fter drining. Ctehin tretment to non-wterlogged plnts rised APX tivity y up to 20.1% on the 9th dy of the experiment. In flooded plnts, tehin oosted APX tivity fter 3 d nd 5 d wterlogging nd fter 4 dys reovery. The effet ws suffiient to give APX tivities losely similr to those of nonwterlogged ontrol plnts. Wterlogging for 5 d redued GR tivity y 22.6% ompred to ontrols nd the effet extended to the end of the 4 d reovery period (Fig. 4d). Ctehin tretment inresed GR tivity y 22.7% in non-wterlogged plnts with respet to ontrols. Ctehin tretment to flooded plnts ompletely restored the GR tivity to ontrol levels nd, t the end of the experiment, GR tivity ws 24.9% more thn in flooded plnts not treted with tehin. Disussion The ommeril prodution of tomtoes is mostly limited to the hot nd wet summer seson in Tiwn. Exess wter produes hypoxi soil onditions within few hours, nd prolonged wterlogging results in noxi. As result of 5 d of wterlogging, the tomto

9 Plnt Soil (2011) 344: plnts in our tests suffered severe losses in growth, FW, DW, nd MSI in oth the roots nd leves, nd RWC, hlorophyll nd Pn in the leves (Fig. 1, Tles 1, 2 nd 3) leding to stunted plnts (Zhng et Fig. 3 Physiologil nd iohemil responses of wterlogging stress-treted tomto leves. The ATP, O 2 nd H 2 O 2 onentrtions of ontrol (drined) nd wterlogging-treted plnt leves re shown in (), () nd (), respetively. Eh point represents the verge of three individul identil experiments (±SE). Letters ove rs indite sttistillysignifint differene (P 0.05). C: the group without tehin tretment or wterlogging; Ct: 40-dy-old tomto plnts were supplied with tehin lone; W: 40-dy-old tomto plnts were exposed to wterlogging onditions lone without tehin pretretment; Ct + W: 40-dy-old tomto plnts were supplied with tehin exogenously for 48 h nd then exposed to wterlogging stress for 5 dys, followed y 4-dy reovery period reovery l. 2007). However, the growth in FW nd DW of the wterlogged plnts (roots nd leves) enefited mrkedly from pre-tretment with tehin (Tle 1). Sirm et l. (2009) reported derese in RWC during flooding, whih further delined with the durtion of flooding stress. Eletrolyte lekge is n inditor of ell memrne stility nd is widely used to sreen to evlute plnt speies or ultivrs in terms of their tolerne to temperture nd wter stresses (Spr nd Anele 1991). The higher MSI in flooded plnts given tehin indite n enhned pility to mintin root ell memrne stility. This proly ontriuted signifintly to the etter wterlogging tolerne (Tle 2). Sirm et l. (2009) reportedtht4nd8dofwterlogginginthemung en lowered hlorophyll onentrtions ut lso noted rise following dringe, inditing prtil restortion of the photosyntheti mhinery one oxygen reentered the wet soil. The dt in Tle 3 demonstrte tht tehin enefitted the flooded plnts y mintining higher hlorophyll onentrtions nd resultnt higher rte of CO 2 ssimiltion (Pn). Crohydrte strvtion hs een shown to e one of the numerous resons for hypoxi/noxi-indued injury (Shluter nd Crwford 2001). SuSy is key enzyme responsile for the hydrolysis of surose to frutose nd gluose (UDP-gluose) under oxygen deprivtion (Noguhi 2004). Both gluose nd frutose (reduing sugrs) re sustrtes for the glyolyti pthwy, whih is the mjor soure of energy under hypoxi in the sene of oxidtive phosphoryltion (Greenwy nd Gis 2003). In this ontext, the greter nd inresing tivity of SuSy in flooded plnts given tehin plnts s ompred with those without tehin nd with non-wterlogged plnts under tehin suggest rohydrte-sed tolerne mehnism in tomto roots (Fig. 2). The essentility of SuSy in noxi tolerne hs een demonstrted in mize using doule-mutnt of the enzyme (Rird et l. 1998). Furthermore, Sirm et l. (2009) reported tht the greter inrese in the SuSy tivity under wterlogged onditions in the tolernt T44 genotype of the mung en resulted in n inresed vilility of reduing sugrs, thus helping to fulfill their energy requirement. ADH tivity is ritil for the reyling of NADH nd thus for the ontinution of the glyolyti pthwy (Johnson et l. 1994). Inreses in ADH tivity my redue the detrimentl effets of the umultion of toxi etldehyde or ltte

10 222 Plnt Soil (2011) 344: R Fig. 4 Effets of wterlogging nd tehin tretment on the tivities of SOD, CAT, APX nd GR in tomto leves. Eh vlue represents the men of three replites with SE. Letters ove rs indite sttistilly-signifint differene (P 0.05). C: the group without tehin tretment or wterlogging; Ct: 40-dy-old tomto plnts were supplied with tehin lone; W: 40-dy-old tomto plnts were exposed to wterlogging onditions lone without tehin pre-tretment; Ct + W: 40-dy-old tomto plnts were supplied with tehin exogenously for 48 h nd then exposed to wterlogging stress for 5 dys, followed y 4-dy reovery period under flooding onditions (Pert nd Alpi 1993) nd help mintin ATP prodution in the sene of O 2 (Dennis et l. 2000). The importne of ADH in flooding tolerne hs een investigted in study of mize mutnt defiient in one of its ADH genes, reovery therefore unle to produe funtionl ADH enzyme. As there ws no ounterlne to lti dehydrogense, nd the ph ontinued to deline to very low levels, this mutnt ws more sensitive to flooding injury thn the wild-type plnt, nd died fter 3 d of sumergene (Roerts et l. 1984). The tivity of ADH in the roots ws tomtoes ws signifintly inresed y tehin plnts nd espeilly so under wterlogging onditions nd during reovery fter drining the soil (Fig. 2). Reently, gene expression studies during 24 h wterlogging on SuSy nd ADH gene expression suggested tht the flooding suseptile PB genotype of mung en filed to express SuSy nd ADH mrna under wterlogged onditions (Sirm et l. 2009). Light-dependent eletron trnsported y hlorophyll n e onverted into stle hemil energy nd prompt ATP formtion vi ATPse in the hloroplsts. In whet, the suppression of the tivities of Mg 2+ - ATPse nd C 2+ -ATPse in the hloroplsts my hve depressed ATP synthesis under wterlogging stresses (Zheng et l. 2009). Thus, the suppression in ATP synthesis might hve dmged PSII, resulting in n exess of sored energy y hlorophyll, nd the formtion of ROS. Figure 3 shows tht the ATP onentrtion mrkedly deresed under flooding stress on dys 1, 3 nd 5 of flooding in omprison with non-stressed onditions. Remrkly, tehin pre-tretment signifintly enhned the ATP onentrtion in the leves under wterlogging stress. The role of tehin (Ct) in plnt defense to environmentl stress hs not een widely disussed, so the urte mode of their tion still remins to e explored. Bsed on the ove results, we propose tht Ct my funtion s stress-signling regultor. A very striking oservtion under wterlogging is the prodution of vrious ROS, espeilly O 2 nd H 2 O 2, leding to n inrese in lipid peroxidtion. This inrese in ROS during wterlogging is primrily

11 Plnt Soil (2011) 344: due to n inrese in DPI-sensitive NADPH oxidsedependent O 2 prodution (Kumuth et l. 2009). The wterlogging-indued inrese in O 2 (Fig. 3) nd H 2 O 2 (Fig. 3) levels in lef tissue ws redued in tehin-treted plnts. This suggests tht tehin is lso involved in the diret svenging of free rdils, therey reduing oxidtive stress. ROS in the leves indued y oxygen shortge t the roots ould lso serve s signl for the indution of ntioxidnt enzymes (Agrwl et l. 2005). The results oserved for the ntioxidtive enzymes SOD, CAT, APX nd GR in the leves in wterlogged plnts given tehin revel ontinuous inrese in ll four enzymes up to 1 d of wterlogging. In plnts wterlogged without tehin, n inrese in ntioxidtive enzymes (SOD nd CAT) ws notied only in 1-d wterlogged plnts, nd t susequent stges there ws deline in the tivity of ll ntioxidnt enzymes s ompred with well-drined plnts (Fig. 4). Enhnement of SOD tivity under wterlogged environments my e n inditor of O 2 prodution (Fig. 4). The inrese in SOD tivity in response to flooding plnts might e relted to the overprodution of O 2 nd ssoited with oxygen deprivtion t the roots euse of inlnes in energy input nd usge s photosynthesis is inhiited through wilting (low RWC see Tle 2) nd stomtl losure (Else et l. 2009). Hene, the grdul inrese in the H 2 O 2 onentrtion in ontinuously-flooded plnts my e prtilly responsile for the deline in SOD tivity (Figs. 3 nd 4). High levels of SOD should e followed y the svenging of H 2 O 2 tlyzed y APX nd CAT. The erly tivtion of SOD nd CAT enzymes oserved in ontinuously-flooded plnts ppered to e n tive nd effiient response of flooded plnts enrihed with tehin (Fig. 4, ). Tseng et l. (2007) demonstrted tht trnsgeni Chinese ge plnts over-expressing SOD nd CAT isoforms were le to tolerte slt stress etter thn the non-trnsgeni line. These results llow us to suggest the o-ordinted involvement of these two enzymes in mitigting oxidtive stress dmge. The exess O 2 generted under wterlogging stress ould dismutte into H 2 O 2 through the tion of SOD, whih is then metolized y the omponents of the sorte glutthione yle. Asd (1992) reported tht the APX found in orgnelles svenges H 2 O 2 produed y the orgnelles, wheres the funtion of ytosoli APX is proly to eliminte H 2 O 2 tht is produed in the ytosol or poplst nd tht diffused from orgnelles. APX nd CAT tivities inresed in the leves of flooded plnt treted with tehin ut were muh depressed y wterlogging without tehin (Fig. 4, ). This response indites strong H 2 O 2 svenging ility y leves of flooded plnts given tehin. The oserved orreltion etween the two enzyme tivities strongly supports the oordinted tion of APX nd CAT. Previous work y Zhng et l. (2007) showed tht in the erly stges of wterlogging, oth peroxidse nd CAT tivities signifintly inresed in tolernt rley ultivr Xuimi 3, while they deresed in the flooding sensitive ultivr Gerdner. It n e onluded tht rpid development of higher peroxidse nd CAT tivities under stress re trit of tolernt plnt speies or genotypes, enling plnts to protet themselves ginst oxidtive stress (Zhng et l. 2007). Therefore, n inrese in the tivity of H 2 O 2 -snvenging enzymes with n ompnying inrese in SOD tivity is ruil for n effetive defense ginst oxidtive stress (Gossett et l. 1994). GR helps mintin high rtios of GSH/GSSG, whih is required for the regenertion of sorte, n importnt ntioxidnt in plnt ells. GR tivity n effetively reyle GSH t the expense of NADPH. GR tivity in flooded tomtoes pre-treted with tehin ws omprle with non-flooded plnts (Fig. 4d). In flood-sensitive genotypes of itrus, n impirment of the ollortive tion of GR nd APX hs een shown to lower the ility for AOS detoxifition nd to rise the sensitivity to flooding (Aron et l. 2008). Overll, these results suggest tht the enhnement in the level of most omponents in the ntioxidtive system y tehin pre-tretment resulted in muh greter resiliene of tomto plnts to 5 d flooding stress. To the est of our knowledge, this study is the first to demonstrte tehin s n effetive ntioxidnt in tomto plnts sujeted to soil flooding. High levels of ROS ould e redued in the leves fter tehin tretment to the soil nd roots. Inresed flooding tolerne from tehin tretment ws ssoited with inresed ntioxidnt enzyme tivity nd, redution in lipid peroxidtion, inditing tht the ntioxidnt property of tehin involves protetion ginst memrne oxidtion. Therefore, we onlude tht exogenous tehin tretment hs the potentil to redue injury

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