Impact of salicylic acid on antioxidants, biomass and osmotic adjustments in Vigna unguiculata L. walp. during water deficit stress

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1 Vol. 12(33), pp , 14 August, 2013 DOI: /AJB ISSN Acdemic Journls Africn Journl of Biotechnology Full Length Reserch Pper Impct of slicylic cid on ntioxidnts, biomss nd osmotic djustments in Vign unguicult L. wlp. during wter deficit stress Umebese, C. E.* nd Bnkole, A. E. Deprtment of Botny, P.M.B 1029 UNILAG Post Office, University of Lgos, Akok-Yb, Lgos, Nigeri. Accepted 26 July, 2013 Ameliortive impct of slicylic cid (SA) on Vign unguicult L. (cowpe) cultivr IT93k during wter deficit stress ws investigted. Plnts were subjected to wter deficit stress (WD) for 7 dys t either the vegettive stge (DVS) or the reproductive stge (DRS) nd the lef wter potentils (ψ w ) of MP nd MP were obtined, respectively. Stress cused reductions in lmost ll prmeters studied. 3 nd 5 mm SA folir tretment cused increses of 27% in lef ψ w, 94% in chlorophyll content, 75% in plnt biomss, 7% in nitrte reductse ctivity nd 38% in proline content in DVS plnts while the impct ws much lower in DRS plnts. Stress stimulted lmost 3-fold increse in ntioxidnt vitmin B 12 in DVS plnts nd 40% increse in vitmin C in DRS plnts nd SA (3 mm) further incresed the ltter by 14%. Overll, SA hd protective influence on the wter potentil nd growth of stressed plnts ccompnied by n increse in production of osmolyte proline nd ntioxidnt vitmin C. Key words: Antioxidnts, growth, slicylic cid, wter stress. INTRODUCTION Wter deficit stress elicits mny different physiologicl responses in plnts. It cuses reduction in lef wter potentil, stomtl conductnce, nitrte reduction nd inhibits lef enlrgement while osmolytes such s totl soluble sugrs nd proline re incresed (Jfr et l., 2004; Adejre nd Umebese, 2007). Prolonged wter stress ffects virtully ll metbolic processes nd often results in severe reductions in plnt productivity nd deth of plnts. The effect of wter deficit vries with the vriety nd the growth stge of the plnts. All biotic stresses such s wter deficit nd slt stress cuse incresed production of rective oxygen species leding to oxidtive dmge. Almost ll orgnisms possess ntioxidnt defense nd repir systems tht hve evolved to protect them ginst oxidtive dmge. Mjority of the ntioxidnt ctivity is from flvonoids, isoflvone, flvones, nthocynin, ctechin, isoctechine, vitmins B, C, E nd β-crotene (Bronzetti et l. 2001; Amic et l., 2003). Others re enzymes such s, ctlse, peroxidse nd superoxide dismutse (Jleel et l., 2009). Slicylic cid cts s n endogenous signl molecule responsible for the induction of ntioxidnt responses tht protect plnts from dmge (Senrtn et l., 2000; Hyt et l., 2007, 2010). Slicylic cid cn stimulte flowering, increse flower life, inhibit seed germintion, promote ethylene synthesis (Singh nd Ush, 2003), enhnce photosynthetic rte nd growth rte (Khn et l., 2003). Slicylic cid hs *Corresponding uthor: E-mil: cumebese@gmil.com Abbrevitions: SA, Slicylic cid; NRA, nitrte reductse ctivity; DVS, vegettive stges; DRS, reproductive stges; HPLC, high performnce liquid chromtogrphy; ABA, bscisic cid; P5CS, D -pyroline-5-crboxylte synthetse; ASC, scorbte; SOD, superoxide dismutse; APX, scorbte peroxidse; GR, glutthione reductse; WD, wter deficit.

2 Umebese nd Bnkole 5201 been shown to produce protective effect in plnts under the ction of stress fctors of different biotic nture, such s, het, chilling, osmotic nd slt stress (Borsni et l., 2001; Jnd et l., 1999; Singh nd Ush, 2003; Wng nd Li, 2006). Slicylic cid (SA) is n endogenous growth regultor of phenolic nture, which prticiptes in the regultion of physiologicl processes in plnts such s growth, photosynthesis, nitrte metbolism, ethylene production, het production nd flowering (Hyt et l., 2010) nd lso provides protection ginst biotic nd biotic stresses such s slinity (Ky et l., 2002). An efficient N ssimiltion is sid to be fvoured by high rte of CO 2 ssimiltion (Lrsson et l., 1989). SA induced conservtion of wter in stressed plnts lso results in the protection of nitrte reductse ctivity (NRA) in SA treted stressed plnts. The present study is imed t evluting the protective effect of slicylic cid on the development of Vign unguicult L. (cowpe) seedlings during wter deficit stress, thereby considering the lef wter potentils, plnt biomss, lef chlorophyll content, nitrte reductse ctivity, proline concentrtions, vitmine B12 nd scorbte contents t both vegettive nd reproductive stges. MATERIALS AND METHODS Plnt mteril nd plnting procedure Seeds of V. unguicult L. cultivr IT93k were collected from the Interntionl Institute of Tropicl Agriculture (IITA) Ibdn, Oyo Stte. The experiment ws set up t the botnic grden of the University of Lgos, Akok Lgos. Seeds of cowpe were plnted 2-3 cm deep in 48 pots of 30.4 cm dimeter (4 tretments) ech filled with 2 kg grden topsoil mixed with 2 g NPK fertilizer. After three weeks, seedlings were thinned to 4 plnts per pot. Plnts were subjected to wter deficit for 7 dys t vegettive (21 st -27 th dys fter sowing) nd reproductive (45 th - 52 nd dys fter sowing) stges (DVS nd DRS plnts). At the beginning of the 7 dy stress period btch of 12 plnts ws treted with 3 mm SA s folir spry, the other btch of 12 plnts ws treted with 5 mm SA, the third btch which consist of yet nother 12 plnts ws subjected to wter deficit only while the fourth btch of 12 plnts served s the control (no stress tretment). All plnts were wtered dily prt from the period of stress tretment. The plnts were rrnged in rndomized complete block design with 3 replictes. The pots were kept t the green house with 12 h photoperiod nd reltive humidity of 60% during the dy nd 68% t night. Plnts were hrvested on the lst dy of stress tretment t ech growth stge, dried in n oven t 80 C for 3 dys nd the biomss tken. Determintion of lef wter potentil After subjecting plnts to 7 dys wter deficit, the degree of stress in the plnts ws mesured using the tissue weight- chnge method outlined by Hopkins nd Hüner (2004). 0.5 g lef from the different tretments ws plced in ech of grded series of moll concentrtion of sucrose in different test tubes. The leves were removed fter 1 h equilibrtion nd dried between filter ppers nd reweighed. Osmotic potentil (ψ s ) of ech sucrose solution ws clculted using the vn t Hoff eqution: ψ s = - CγRT Where, C is the moll concentrtion, γ is the ctivity coefficient ( vlue of 1 for neutrl solutes such s sucrose in dilute solution), R is the gs constnt ( kg MP mol -1 o K -1 ), T is the bsolute temperture ( o K = o C + 273). The chnge in weight ws clculted s percentge of the originl weight nd plotted ginst ψ s of sucrose solutions. The lef wter potentil (ψ w ) is estimted s equivlent to the ψ s of the solution in which there is no chnge in weight (the intercept on the bsciss). Determintion of nitrte reductse ctivity Nitrte reductse ctivity in the leves ws ssyed s described by McCshin (2000). 5 ml of the incubtion medium (comprising 100 ml 0.1 M phosphte buffer, ph 7.5, g KNO 3 nd 1 ml 4% propn-1-ol), ws used to incubte 0.5 g smple of leves in boiling tubes in 3 replictes for period of 1 h t room temperture of C. Then the rection ws stopped by dding 1 ml 1% sulphnilic cid, followed by 1 ml of 0.02 % nphthylethylenedimine dichloride. It ws then crefully shken nd left for 20 min for colour development. The blnk ws prepred by dding 1 ml substrte ssy solution, 1 ml 1% sulphnilic cid nd 1 ml npthylethylenedimine dichloride together in boiling tube. After full colour development, the bsorbnce ws mesured t 540 nm opticl density of the test solution, ws determined using Corning spectrophotometer model 258. A stndrd curve ws prepred using 0-10 μm nitrite ml -1. NRA is proportionl to the concentrtion of nitrite in the rection medium. Determintion of proline content Proline content of leves ws determined s described by Btes et l. (1973). 0.5 g of dried ground leves ws homogenized in 10 ml 3% queous sulfoslicylic cid nd the homogente filtered through filter pper. 2 ml cid ninhydrin nd 2 ml glcil cetic cid were dded to 2 ml of the filtrte in digestion tube nd plced in boiling wter bth for 90 min. The rection ws terminted in n ice bth. The rection mixture ws extrcted with 4 ml toluene nd gitted vigorously for 30 s. The chromophore contining toluene ws spirted from the queous phse nd the bsorbnce red t 520 nm using toluene s the blnk. The concentrtion ws determined from stndrd curve prepred from 1-10 mg l -1 of proline nd clculted on dry weight bsis. Estimtion of chlorophyll content The mount of chlorophyll nd b (mg g -1 fresh weight) were clculted using the formul of Mclchlm nd Zlik (1963). Determintion of the ntioxidnt vitmins content Antioxidnt vitmins cynocoblmin (vitmin B 12 ) nd scorbic cid (vitmin C) contents were determined s described by Vn (2006). Ten grms of the fresh leves from different plnts were mcerted using cermic mortr nd pestle. The extrct ws mixed thoroughly using n electric mixer nd centrifuged t 1000 g t 5 C. 1 ml liquot ws mde up to 10 ml with distilled wter for cynocoblmin (vitmin B 12 ) content while 1 ml of the extrct ws mde up to 100 ml with distilled wter for scorbic cid (vitmin C). After filtrtion ech ws injected into the high performnce liquid chromtogrphy (HPLC) t wvelength of 204 nm. The chromtogrphic conditions consist of the following: Column-zorbx SB

3 5202 Afr. J. Biotechnol. Tble 1. Lef wter potentil of cowpe subjected to wter deficit (WD) nd slicylic cid (SA) tretment t the vegettive nd reproductive stges of growth. Tretment Wter potentil (MP) vegettive stge Reproductive stge Control WD b b WD + 3 mm SA b b WD + 5 mm SA d c Superscripts with sme letters t ech growth stge re not significntly different t p<0.05 using the Duncn s multiple rnge tests. C18 (250 x 4.6 mm, 5 nm), thermostticlly regulted t 20 C; the mobile phse ws grdient composition of ctivities: cetonitrite (A) nd 25mM KH 2 PO 4 buffer (B) pumped t flow rte of 0.5 ml min -1. When the grdient elution of A is 8 nd 16% nd B is 92 nd 84% t 0 min nd 5 min, respectively, 20 μl of reference stndrds were injected to determine their respective retention time, nd grded concentrtion of mixed reference stndrds (vitmin C nd B 12 ) were injected. The pek res (MAU) were cquired nd integrted by enhnced integrtor for plotting of clibrtion curve nd concentrtions given s μg ml -1 nd clculted s mg g -1 fresh weight. All dt were nlyzed using Sttisticl Anlysis System (SAS) softwre. When ANOVA showed significnt tretment effects, Duncn s multiple rnge test ws pplied to compre the mens t p<0.05. RESULTS AND DISCUSSION Lef wter potentil of V. unguicult L. cultivr IT93k ws significntly reduced when plnts were subjected to 7 dys wter deficit stress t the vegettive nd reproductive stges (DVS nd DRS respectively) of growth, with the reproductive stges (DRS) showing greter decrese in wter potentil (Tble 1). This stge dependent response to wter deficit ws shown in n erlier study on soyben leding to greter increse in stomtl resistnce nd reduced stomtl cpcity t the reproductive stge (Adejre nd Umebese, 2007). This resulted in much reduction in growth nd yield of plnts (Figure 3). Folir ppliction of slicylic cid (SA) ws effective in rising the wter potentil of stressed plnts by 27% though the vlues were still lower thn the control. Wter deficit did not ffect the totl chlorophyll content of cowpe t both stges of growth (Figure 1). This ws lso observed by Vsilev nd Lliev (2011), when lucerne ws subjected to wter deficiency stress. However, the ppliction of 5 mm SA to DVS plnts significntly (p<0.05) enhnced the chlorophyll content over the control. This corrobortes the report by Rjsekrn nd Blke (1999) tht SA hs positive effect on photosynthesis. Nitrte reductse ctivity (NRA) ws significntly reduced by wter deficit (Figure 2). 5 mm SA rised the NRA level of vegettive stges (DVS) of plnts to tht of the control while 3 mm SA incresed the NRA of DRS plnts though it ws still lower thn tht of the control. There is strong positive correltion between NRA nd reltive wter content (Umebese nd Okeol, 1998). SA incresed NRA of stressed plnts nd this corresponded with the increse in the lef wter potentil. This grees with n erlier report tht SA increses the conservtion of wter in tomto nd mrnth during wter stress nd enhnces NRA (Umebese et l., 2009). Plnt biomss ws significntly reduced by wter deficit, corresponding to the low wter potentil induced by the 7 dys wter deficit (Figure 3). Energy requiring processes such s NRA nd growth re reduced by wter stress since CO 2 fixtion is limited, reducing the NADP + regenertion by the Clvin cycle leding to n over-reduced photosynthetic electron trnsport chin (Kruse, 1994). Though tretment with SA significntly incresed (p<0.05) plnt biomss, vlues were still lower thn the control. In n erlier study, plnt biomss ws enhnced by SA when tomto nd mrnth were subjected to wter stress (Umebese et l., 2009). Concentrtion of SA s low s 0.05 mm incresed the level of cell division within the picl meristem of seedling roots which cused n increse in plnt growth (Bezrukov et l., 2001). Proline ws significntly decresed in the DVS plnts while it ws significntly incresed in DRS plnts. Both concentrtions of SA enhnced proline production during wter stress t both stges of growth (Figure 4). The ccumultion of the osmolyte proline, in plnts is mechnism by which plnts resist wter stress nd develop nti-stress rection. Wter deficit induces ccumultion of proline in seedlings nd lso results in decline in metbolic ctivity of plnts cells, nd this is inevitbly reflected in inhibition of their growth (Bezrukov et l., 2001; Umebese et l., 2009). SA induced bscisic cid (ABA)-medited protective rections in plnts involves incresed production of proline by the expression of the gene encoding D -pyroline-5-crboxylte synthetse (P5CS) in proline biosynthesis. When plnts constntly produce high levels of proline, they re cpble of surviving drought (Deluney nd Verm, 1993). Thus, the incresed production of proline by SA in plnts subjected to wter deficit t the two growth stges my hve

4 Nitrte Reductse Activity (x10-1 mole Fresh Weight ) Umebese nd Bnkole 5203 Figure 1. Chlorophyll content of cowpe subjected to wter deficit (WD) nd slicylic cid (SA) tretment t vegettive nd reproductive stges of growth (Brs with sme letters t ech growth stge re not significntly different t P<0.05 using the Duncn's multiple rnge test) b b Control WD WD + 3mMSA WD + 5mMSA b c d Vegettive stte Reproductive stte Tretments Figure 2. Nitrte reductse ctivity of cowpe subjected to wter deficit (D) nd slicylic cid (SA) tretment t vegettive nd reproductive stges of growth (Brs with sme letters t ech growth stge re not significntly different t P<0.05 using the Duncn's multiple rnge test).

5 Proline content (x10-1 mg g -1 Dry Weight ) 5204 Afr. J. Biotechnol. Figure 3. Plnt biomss of cowpe subjected to wter deficit (D) nd slicylic cid (SA) tretment t vegettive nd reproductive stges of growth (Brs with sme letters t ech growth stge re not significntly different t P<0.05 using the Duncn's multiple rnge test). Control 16 WD WD 3m MSA 14 c WD 3m MSA 12 b c 10 b c Vegettive Stte Reproductive Stge Tretments Figure 4. Proline content of cowpe subjected to wter deficit (D) nd slicylic cid (SA) tretment t vegettive nd reproductive stges of growth (Brs with sme letters t ech growth stge re not significntly different t P<0.05 using the Duncn's multiple rnge test).

6 Umebese nd Bnkole 5205 Figure 5. Antioxidnt vitmin B 12 of cowpe subjected to wter deficit (D) nd slicylic cid (SA) tretment t vegettive nd reproductive stges of growth (Brs with sme letters t ech growth stge re not significntly different t P<0.05 using the Duncn's multiple rnge test). resulted in the observed enhncement of plnt biomss. The ntioxidnt vitmin B 12 ws significntly enhnced (p<0.05) by wter stress in DVS plnts but significntly reduced in DRS plnts (Figure 5). Vitmin C ws lso stimulted by wter deficit but this ws in DRS plnts (Figure 6). Thus, the stge t which plnts were subjected to wter deficit ffected the type of ntioxidnt induced; vitmin B 12 t the vegettive stge nd vitmin C t the reproductive stge. An increse in the level of ntioxidnts is believed to reflect n increse in the formtion of rective oxygen species (Foyer et l., 1997). All biotic stresses such s wter deficit stimulte the production of significnt quntities of rective oxygen species in sub-cellulr comprtments or orgnelles which re highly destructive to their proteins, membrne lipids nd nucleic cids (Mtsumur et l., 2002). The control of these oxyrdicls is chieved by ntioxidnt systems within the cells. Experiments show tht vitmin B is potent quencher of singlet oxygen with quenching rtes comprble to or greter thn those of vitmin C nd E, two of the most efficient biologicl ntioxidnts known (Bilski et l., 2000). Tretment with 3 mm SA resulted in significnt reduction in vitmin B content t both stges of growth of stressed plnts while 5 mm SA cused reduction only t the reproductive stge. Thus, SA hd retrding effect on vitmin B 12 in stressed plnts. Both concentrtions of SA cused significnt reduction in vitmin C content in DVS plnts but in DRS plnts, 3 mm SA stimulted further increse in this vitmin. This enhncement of vitmin C by 3 mm SA corresponds with the increse in NRA observed in stressed plnts subjected to this dose of SA. Vitmin C supplement hs been shown to increse NRA in white heded cbbge (Heród- Leszczyńsk nd Miedzobrodzk, 1993). This shows the meliortive ction of SA when cowpe is subjected to wter stress t the reproductive stge of development. The sensitivity of vitmin C to reversible oxidtion suggests its role in cellulr oxidtion-reduction rections (Verm nd Verm, 2007). Plnts must possess efficient ntioxidnt system to enble them to endure oxidtive dmge under unfvorble conditions such s high/low tempertures, wter deficit nd slinity (Sreenivsulu et l., 2000). Vrious ssocitions between wter stress nd endogenous levels of wter-soluble ntioxidnts hve been described (Zmn nd Ds, 1991), in which scorbte (ASC) is lso required for the re-conversion of SA to SA yielding monodehydroscorbte, since ASC is highly rective ginst phenoxyl rdicls generted by peroxidses during oxidtive stress (Kwno nd Muto, 2000). It cn be suggested tht the exogenous ppliction of SA is ccompnied by incresed rtes of ROS, which leds to n incresed lod on the scorbte-glutthione cycle leding to depletion of tasc. The results re supported

7 5206 Afr. J. Biotechnol. Figure 6. Antioxidnt Vitmin C of cowpe subjected to wter deficit (D) nd slicylic cid (SA) tretment t vegettive nd reproductive stges of growth (Brs with sme letters t ech growth stge re not significntly different t P<0.05 using the Duncn's multiple rnge test). by the observtion of Shi nd Zhu (2008), where SAtretment enhnced the ctivities of dehydroscorbte reductse. Mwnmwenge et l. (1999) explined tht when ROS increses, chin rections strt, in which superoxide dismutse (SOD) ctlyzes the dismuttion of O 2 rdicls to moleculr O 2 nd H 2 O 2. The H 2 O 2 is then detoxified in the scorbte-glutthione cycle which involves the oxidtion nd re-reduction of scorbte nd glutthione through the scorbte peroxidse (APX) nd glutthione reductse (GR) ction (Ouchi, et l., 1990; Psternk et l., 2005). In conclusion, cowpe is more sensitive to wter deficit t the reproductive stge thn the vegettive stge resulting in reductions in plnt biomss nd nitrte reductse ctivity. Tretment of stressed plnts with slicylic cid cused significnt improvement of those prmeters nd enhnced proline ccumultion, n ntistress rection. Plnts subjected to wter deficit t different stges of growth produced different types of ntioxidnts: vitmin B 12 in plnts stressed t the vegettive stge nd vitmin C in those stressed t the reproductive stge. Slicylic cid enhnced vitmin C content in the ltter which my hve cused the enhncement of nitrte reductse ctivity. REFERENCES Adejre FB, Umebese CE (2007). Stomtl resistnce to low wter potentil t different growth stges ffects plnt biomss in Glycine mx L. Am. J. Agric. Biol. Sci. 3: Amic D, Dvidovic D, Beslo D, Trinjstic N (2003). Structure-rdicl scvenging ctivity reltionships of flvonoids. Crot. Chem. Act 76: Btes LS, Wldren RP, Ter ID (1973). Rpid determintion of free proline for wter stress studies. Plnt Soil 39: Bezrukov M, Skhbutdinov VR, Ftkhutdinov RA, Kyldirov I, Shkirov F (2001). The role of hormonl chnges in protective ction of slicylic cid on growth of whet seedlings under wter deficits. Agrochemiy (Russ.) 2: Bilski P, Li MY, Ehrenshft M, Dub ME, Chignell CF (2000). Vitmin B 6 nd its derivtives re efficient singlet oxygen quencher nd potentil fungl ntioxidnts. Photochem. Photobiol. 71: Borsni O, Vlpuest V, Botell MA (2001). Evidence for role of slicylic cid in the oxidtive dmge generted by Ncl nd osmotic stress in Arbidopsis seedling. J. Plnt Physiol. 126: Bronzetti G, Cini M, Adrenoli E, Cltvuturo L, Pinunzio M, Croce CD (2001). Protective effects of vitmin nd selenium compounds in yest. Mutt. Res. 496: Deluney AJ, Verm DPS (1993). Proline biosynthesis nd osmoregultion in plnts. Plnt J. 4: Foyer CH, Lopez-Delgdo H, Dt JF, Scott IM (1997). Hydrogen peroxide nd glutthione-ssocited mechnisms of cclmtory stress tolernce nd signling. Physiol. Plnt 100: Hyt Q, Hyt S, Irfn M, Ahmd A (2010). Effect of exogenous slicylic cid under chnging environment: A review. Environ. Exp. Bot. 68: Hyt S, Ali B, Ahmd A (2007). Slicylic cid: biosynthesis, metbolism nd physiologicl role in plnts. In: Hyt S, Ahmd A., (Eds.), Slicylic cid A plnt hormone. Springer Verlg, Dordrecht, Boston, London. pp Heród-Leszczyńsk T, Miedzobrodzk A (1993). An ttempt to investigte the effect of mgnesium ions nd vitmin C on ctivity of nitrte reductse of white heded cbbge. Rocz Pnstw Zki Hig. 44: Hopkins WG, Hüner NPA (2004). Introduction to plnt physiology. John Wiley nd Sons, Inc. USA. pp Jfr MS, Nourmohmmdi G, Meleki A (2004). Effect of wter deficit

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