Masoudi-Sadaghiani Farhad 1, Abdollahi Mandoulakani Babak 2, 3 *, Zardoshti Mohammad Reza 2, Rasouli-Sadaghiani Mir Hassan 4 and Tavakoli Afshin 5

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1 AJCS 5(1):55-60 (2011) ISSN: Response of proline, solule sugrs, photosynthetic pigments nd ntioxidnt enzymes in potto (Solnum tuerosum L.) to different irrigtion regimes in greenhouse condition Msoudi-Sdghini Frhd 1, Adollhi Mndoulkni Bk 2, 3 *, Zrdoshti Mohmmd Rez 2, Rsouli-Sdghini Mir Hssn 4 nd Tvkoli Afshin 5 1 Fculty of Agriculture, Urmi University, Urmi, Irn 2 Deprtment of Agronomy, Fculty of Agriculture, Urmi University, Urmi, Irn 3 Deprtment of Agriculturl Biotechnology, Institute of Biotechnology, Urmi University, Urmi, Irn 4 Deprtment of Soil Science, Fculty of Agriculture, Urmi University, Urmi, Irn 5 Deprtment of Agronomy, Fculty of Agriculture, Znjn University, Znjn, Irn * Corresponding uthor:.dollhi@urmi.c.ir, delk2000@yhoo.com Astrct To evlute the response of proline nd solule sugrs content, chlorophyll (CHL), chlorophyll (CHL), totl chlorophyll (TC), ctlse (CAT) nd scorte peroxidse (APX) ctivity in potto (Solnum tuerosum L.) leves to different irrigtion regimes t two growth stges, greenhouse fctoril experiment ws conducted in completely rndomized design (CRD) with three replictions. The fctors consisted of four different irrigtion regimes s 100% (I 0 ), 80% (I 1 ), 60% (I 2 ) nd 40% (I 3 ) of field cpcity nd growth stges: 50% emergence to 50% flowering (GS 1 ) nd 50% flowering to physiologicl mturity (GS 2 ). Fresh lef tissues were used to determine proline nd solule sugrs content, CHL, CHL, TC nd CAT nd APX ctivity. According to the results, irrigtion regimes hd significnt effect on proline content, solule sugrs nd ctlse (CAT) ctivity, ut no significnt differences were detected mong irrigtion regimes for CHL, CHL, TC nd APX ctivity. Limited irrigtion incresed proline concentrtion nd totl solule sugrs in leves. None of studied trits were ffected y growth stges. Interction etween irrigtion regimes nd growth stges ws not significnt for ll studied trits. Result lso indicted tht the highest proline content (4.9µmol.g -1 FW ), totl solule sugrs (55.9 mg.g -1 FW) nd CAT ctivity (12.7 µmol H 2 O 2 min -1 g -1 FW) were relted to irrigtion t 40% of field cpcity (I 3 ). It ws concluded tht proline nd solule sugr levels were incresed in potto leves under deficit irrigtion regimes. Key words: Ascorte peroxidse ctivity; ctlse ctivity; growth stges; osmoregultion; wter deficit Arevitions: CAT-ctlse; APX-scorte peroxidse; CHL-chlorophyll CHL-chlorophyll ; TC-totl chlorophyll; θ-grvimetric wter content Introduction Recent pulictions (FAO, 2004) hve shown the importnce of the potto (Solnum tuerosum, L.) s glol food crop, rnking fourth mong others. Wter deficit nd slt stresses re glol issues to ensure survivl of griculturl crops nd sustinle food production (Jleel et l., 2007). Drought is the most importnt limiting fctor for crop production nd it is ecoming n incresingly severe prolem in mny regions of the world (Pssiour, 2007). Mny investigtions hve demonstrted tht potto is reltively sensitive to wter deficit stress (Open nd Porter, 1999; Porter et l., 1999; Feiro et l., 2001). Reltive wter content (RWC), lef wter potentil, stomtl resistnce, trnspirtion rte nd lef nd cnopy temperture re importnt chrcteristics tht influence plnt wter reltions (Siddique et l., 2001). Under severe trnspirtion or wter deficit, potto RWC is lower thn tht of mny other crops (Loon, 1981). This might e one of the resons for susceptiility of potto to wter deficit. The vlues of RWC for irrigted pottoes is %, while for those who non-irrigted is 76-87% (Loon, 1981).Thornton (2002) nd Shock (2004) found tht potto re very sensitive to wter deficit stress in ll growth stges, especilly tuer formtion. Photosynthetic pigments re importnt to plnts minly for hrvesting light nd production of reducing power such s ATP nd NADPH. Both the chlorophyll (CHL) nd (CHL) re prone to soil drying dmges. However, crotenoids hve dditionl roles nd prtilly help the plnts to withstnd dversries of drought (Frooq et l., 2009). Drought stress induced chnges in the rtio of CHL nd CHL nd crotenoids (Frooq et l., 2009). The chlorophyll content decresed to significnt level t higher wter deficits in sunflower (Kini et l., 2008) nd Vccinium myrtillus (Thkokorpi et l., 2007). Rective oxygen species (ROS) re prtilly reduced forms of tmospheric oxygen. ROS typiclly result from the excittion of O 2 to form singlet oxygen (O 2 ) or 55

2 Tle 1. Vrince nlysis for proline, Chlorophyll (CHL), Chlorophyll (CHL) totl chlorophyll (TC), solule sugrs content nd ctlse (CAT) nd scorte peroxidse (APX) ctivity s effected y different irrigtion regimes nd growth stges Source of vrition df Proline Chlorophyll Chlorophyll Totl Solule CAT APX chlorophyll sugrs Irrigtion regime (I) ** ** ** 0.77 Growth stge (Gs) I Gs Error **, Significnt t P<0.01 from the trnsfer of one, two or three electrons to O 2, nd genertion of superoxide (O 2 ), hydrogen peroxide (H 2 O 2 ) or hydroxyl (OH) rdicls, respectively (Shigeok et l., 2002). The cells re normlly protected ginst ROS y the opertion of n ntioxidnt defense system, comprised of enzymtic (superoxide dismutse, ctlse, glutthione reductse, scorte peroxidse) nd nonenzymtic (scorte, α- tocopherol, crotenoids, glutthione) components (Shigeok et l., 2002). ROS production nd consequently ntioxidnt enzymes ctivities is further enhnced when plnts exposed to vrious iotic stresses, such s drought (Ruio et l., 2002), slinity (Broetto et l., 2002) nd low nd high temperture (Pstori nd Foyer 2002). Osmotic djustment is mechnism to mintin wter reltions under osmotic stress. It involves the ccumultion of rnge of osmoticlly ctive molecules/ions including solule sugrs, sugr lcohols, proline, glycineetine, orgnic cids, clcium, potssium, chloride ions nd etc. Under wter deficit nd s result of solute ccumultion, the osmotic potentil of the cell is lowered, which ttrcts wter into the cell nd helps with the mintennce of turgor. Potto responds to drought nd slt stresses y ccumulting proline which functions s n osmoprotector, osmoregultor nd ROS scvenger (Benvides et l., 2000). Regrding to involvement of the ove mentioned mechnisms in wter deficit stress, n investigtion ws designted to study the effects of different irrigtion regimes during two growth stges on totl chlorophyll (TC), CHL, CHL, proline nd totl solule sugrs content nd ntioxidnt enzymes ctivity (CAT nd APX) in potto leves. Mterils nd methods Certified seeds of S. tuerosum L. cv. Mrfun with uniform size (35 50mm) were used s experimentl mteril during 2008, from April to August t the reserch greenhouse of Urmi University, Irn (37 32' N, 45 5' E; ltitude, 1320 m). Potto tuers were plnted in pots (25 cm dimeter nd 40 cm height) filled with 24 kg cly lom soil with ulk density of 1.51 gcm - 3. The soil moisture content ws mesured grvimetriclly on every lternte dy immeditely efore ech irrigtion. Soil moisture contents (% θ, weight sed) t field cpcity (FC) nd permnent wilting point were 17% nd 3.0%, respectively. Until 30 dys fter plnting, pots were wtered eqully then, irrigtion tretments were pplied s: 100% of field cpcity (FC) (I 0 ), 80% of field cpcity (0.8 FC) (I 1 ), 60% of field cpcity (0.6 FC) (I 2 ), nd 40% of field cpcity (0.4 FC) (I 3 ). Pots were weighed dily nd wter dded to ech pot s lost weight of pots. Growth stges consisted of 50% emergence to 50% flowering (GS 1 ) nd 50% flowering to physiologicl mturity (GS 2 ). Fertilizers were pplied t rtes of 150 N, 30 P, 220 K kg/h ccording to the soil test nlysis. All tretments were repeted three times in experimentl pots in fctoril form sed on complete rndomized design. Greenhouses were kept s close s possile to the externl ir temperture (22-25ºC dy nd 19ºC night). The tuers were plnted on 21 April, 2008 nd emerged out three weeks lter. They were hrvested on 6 August, Pigments extrction nd estimtion The extrction of chlorophyll nd nd TC ws crried out ccording to Gross (1991). At the end of ech growth stges (GS 1 nd GS 2 ), the fresh tissue of young nd expnded leves collected nd freeze t -80ºC then, the leves (0.25 g) were homogenized with 80% cetone. The opticl density (O.D.) of the extrcted chlorophyll ws mesured t 645 nd 663 nm y using spectrophotometer PD-303. TC, CHL nd CHL were clculted y the following formule (Gross, 1991). CHL = ( OD663) ( OD645) CHL = ( OD645) ( OD663) TC = ( OD645) + ( OD663) Enzyme extrction nd ssy One-tenth g of fresh folir tissue (uppermost leves tken t the end of two growth stges) ws nlyzed for enzymtic ssys. Ctlse ctivity (µmol H 2 O 2 min -1 g -1 FW) ws ssyed y mesuring the initil rte of hydrogen peroxide disppernce (Chnce nd Mehly, 1959). The rection mixture contined 2.5 ml of 50 mm potssium phosphte uffer (ph 7.4), 0.1 ml of 1% hydrogen peroxide nd 50 µl of enzyme extrct. The homogente ws centrifuged t g for 15 min t 4 ºC nd the superntnt ws immeditely used for the enzyme ssy. The decrese in hydrogen peroxide ws followed s decline in opticl density t 240 nm nd the ctivity ws clculted using the extinction coefficient of 36 mm cm 1 for hydrogen peroxide. Ascorte peroxidse (µmolg -1 FW min -1 ) ctivity ws determined s descried y Asd (2001). The rection mixture contined 2.5 ml of 50 mm potssium phosphte uffer (ph 7.0), 0.1 mm EDTA, 0.5 mm scorte, 0.2 ml of 1% hydrogen peroxide nd 0.1 ml enzyme extrct. The homogente ws centrifuged t g for 15 min t 4 ºC nd the superntnt ws used to mesure enzyme ctivity. The hydrogen peroxide-dependent oxidtion of scorte ws 56

3 followed y monitoring the decrese in sornce t 290 nm, using the extinction coefficient of 2.8 mm cm -1. Determintion of proline nd solule sugrs content To determine the proline content, 0.5 g of dry leves ws homogenized with 5 ml of 95% ethnol. Aove phse of filtrte ws seprted nd its sediments were wshed y 5 ml of 70% ethnol for two times nd its ove phse dded to the previous over comprtment. The mixture ws centrifuged t 3500 g for 10 min t 4ºC nd the superntnt ws recovered nd lcoholic extrct kept in refrigertor t 4ºC (Pquin nd Lechsseur, 1979). One ml of lcoholic extrct ws diluted with 10 ml of distilled wter nd 5 ml of ninhydrin (0.125 g ninhydrin, 2 ml of 6 mm NH 3 PO 4, 3 ml of glcil cetic cid) nd 5 ml of glcil cetic cid dded then mixture plced in oiling wter th for 45 min t 100ºC. The rection ws stopped y plcing the test tues in cold wter. The smples were rigorously mixed with 10 ml enzene. The light sorption of enzene phse ws estimted t 515 nm using PD-303 model spectrophotometer. The proline concentrtion ws determined using stndrd curve. Free proline content ws expressed s µmol g -1 DW of leves (Irigoyen et l., 1992). To mesuring the content of solule sugrs, 0.5 g of dry leves ws homogenized with 5 ml of 95% ethnol. One-tenth ml of lcoholic extrct preserved in refrigertor mixed with 3 ml nthrone (150 mg nthrone, 100 ml of 72% sulphuric cid,w/w). The smples plced in oiling wter th for 10 minutes. The light sorption of the smples ws estimted t 625 nm using PD-303 model spectrophotometer. Contents of solule sugr were determined using glucose stndrd nd expressed s mg g -1 DW of leves. Sttisticl nlysis All experimentl dt reported were verges of three replictes nd the SE of the mens ws determined. Anlysis of vrince nd Duncn s multiple rnge test were performed using SPSS softwre version Results nd Discussion As indicted y nlysis of vrince, irrigtion regimes hd significnt effects on proline nd solule sugrs content nd CAT ctivity (P < 0.01), while no significnt differences were detected for chlorophyll content nd APX ctivity mong irrigtion regimes. Also, no considerle ltertion ws found in chlorophyll content t two growth stges. Interction effects etween the irrigtion regimes nd growth stges were not significnt for ll mesured prmeters (Tle.1). Proline content Sttisticl nlysis showed tht proline content ws highly ffected (P< 0.01) y irrigtion regimes, ut not ffected y growth stges nd the interction etween growth stges nd irrigtion regimes (Tle.1). The mximum proline content (4.94 µmol g -1 FW) otined in the most restrictive irrigtion regime I 3 (0.4 FC) (Fig.1). In generl, proline content of leves incresed with the decline in irrigtion wter, suggesting tht the production of proline is proly common response of potto under drought conditions. Proline (µmol.g -1 DW) c c 100% 80% 60% 40% Irrigtion tretments (% FC) Fig 1. Effect of irrigtion regimes on proline content in the leves of S. tuerosum c.v. Mrfun. Vlues re the mens ± SE of three replictes. Mens with different letters re significntly different (P<0.05). Shoot smples were tken nd mesured t the end of two growth stges. The role of proline in dpttion nd survivl of plnts under drought stress reported y Wtne et l (2000) nd Sruhn et l (2006). Osmotic djustment through the ccumultion of cellulr solutes, such s proline, hs een suggested s one of the possile mens for overcoming osmotic stress cused y the loss of wter (Cllero et l., 2005). Proline is non-protein mino cid formed in most tissues sujected to wter stress nd together with solule sugrs is redily metolized following recovery from drought (Singh et l., 2000). Proline lso serves s sink for energy to regulte redox potentils, hydroxyl rdicl scvenger (Shrm nd Dietz, 2006), solute tht protects mcromolecules ginst denturtion nd s mens for reducing cidity in the cell (Kishor et l., 2005). Loto et l (2008) reported tht the ccumultion of proline nd free mino cids in soyen (Glycine mx cv. Smi) leves were incresed under wter deficit 67 nd 388.1%, respectively. Teixeir nd Pereir (2006) indicted tht proline content significntly incresed in ll potto orgns in response to the stress conditions. This increse ws more remrkle in roots nd tuers thn in the leves. High levels of proline enle the plnt to mintin low wter potentils cusing the ccumultion of comptile osmolytes tht llows dditionl wter to e tken up from the environment, thus uffering the immedite effect of wter deficit within the orgnism (Mous nd Adel- Aziz, 2008). Solule sugrs content Solule sugrs content in leves significntly incresed under stress condition. The effect of growth stges nd the interction of growth stges y irrigtion regimes were not significnt for this trit (Tle.1). The highest content of these osmolytes (55.99 mg g -1 DW) ws found in leves under irrigtion regime 0.4 FC (I 3 ) (P< 0.01). Solule sugrs content in irrigtion regime I 3 were 1.1, 2.5 nd 5.1 times more thn those of I 2, I 1 nd I 0 irrigtion regimes, respectively. The mount of solule sugrs decresed with the decline of irrigtion wter (Fig.2). Irrigtion regimes I 0 nd I 1 were significntly different while no difference ws oserved etween I 2 nd I 3. The ccumultion of sugrs in response to drought is quite well documented (Iznloo et l., 2008, Wtne et l., 2000). Solule sugrs my function s typicl osmoprotectnt, stilizing cellulr memrnes nd mintining turgor pressure. Gene ontology 57

4 ttriutes such s proline nd solule sugr ccumultions were highly enriched in the drought-up-regulted genes, suggesting tht those metolic pthwys re importnt in responses to drought stress. Indeed, the importnce of mny of these pthwys to drought tolernce hs een empiriclly supported y trnsgenic experiments (Umezv et l., 2006). Chlorophyll content CHL, CHL nd TC in different irrigtion regimes were not significntly ffected. The effect of growth stges nd the interctive effect of irrigtion regimes y growth stges on photosynthetic pigments were not significnt s well (Tle. 1). Otined results regrding to chlorophyll content in the current study re similr to those otined y Sltmrsh et l (2006). Ynqiong et l (2007) found tht the content of chlorophyll, free proline nd solule sugrs increse under drought stress. Mnirnnn et l (2007) detected depression in CHL nd nd TC in Helinthus nnuus L. under wter stress. In this study the chlorophyll content remined unchnged under drought nd this is similr to wht ws recorded y Jensen (1985) in onions. According to Poljkoff nd Gle (1975), the ility to synthesize more chlorophyll under wter stress is good criterion for the species tolernt to drought. Teixeir nd Pereir (2006) reported tht chlorophyll content of potto leves incresed significntly in response to drought (5.6%). The decrese in chlorophyll content oserved in the leves collected from slt-stressed plnts (Soussi et l., 1998) indicting tht slt stress is more severe to potto plnts thn drought, where the chlorophyll content slightly incresed, proly ecuse of some wter turgor loss (Bussis nd Heineke, 1998). Khosrvifr et l (2008) reported tht in potto cultivr Agri, the mximum content of chlorophyll ws 72.3% nd its minimum content ws 37.6% during the irrigtion fter 175 mm nd 35 mm evportion from Clss A pn, respectively. Protective enzymes ctivities Ctlse ctivity incresed with the decrese of irrigtion wter (P< 0.01) ut, scorte peroxidse ctivity remins unchnged (Tle.1). Ctlse ctivity incresed in irrigtion regimes 0.6 nd 0.4 FC compred to FC nd 0.8 FC. The I 2 nd I 3 irrigtion regimes hd 56% nd 65% higher CAT ctivity thn the I 0 tretment (Fig.3). Drought, like other environmentl stresses induces oxidtive stress. To e le to endure oxidtive dmge under unfvourle conditions, plnts possess oth nonenzymtic ntioxidnts such s crotenoid, flvonoids, α- tocopherol, scoric cid nd glutthione, nd enzymtic ntioxidnts such s CAT nd peroxidse (POX) (Smirnoff, 1993; Munné-Bosch nd Alegre, 2000). These enzymtic nd nonenzymtic ntioxidnts hve een reported to ccumulte under vrious environmentl stresses (Acr et l., 2001), while comprtively higher ctivity of ntioxidnts hve een shown in tolernt cultivrs thn those in sensitive ones (Reddy et l., 2004), indicting tht higher ntioxidnt enzyme ctivity hs role in imprting tolernce ginst environmentl stress. Mechnisms tht reduce oxidtive injury my ply secondry role during drought tolernce. CAT is only present in peroxisomes nd it is indispensle for ROS detoxifiction Solule sugrs (mg.g -1 DW) c 100% 80% 60% 40% Irrigtion tretments (% FC) Fig 2. Effect of irrigtion regimes on solule sugrs content in the leves of S. tuerosum c.v. Mrfun. Vlues re the mens ± SE of three replictes. Mens with different letters re significntly different (P<0.05). Shoot smples were tken nd mesured t the end of two growth stges. CAT ctivity (µmol H 2 O2.min -1 ) % 80% 60% 40% Irrigtion tretments (% FC) Fig 3. Ctlse ctivity sujected to different irrigtion regimes in the leves of potto. Vlues re the mens ± SE of three replictes. Mens with different letters re significntly different (P<0.05). Shoot smples were tken nd mesured t the end of two growth stges. during stress when high levels of ROS re produced. The lnce etween SOD, APX or CAT ctivities in cells is crucil for determining the stedy-stte level of superoxide rdicls nd H 2 O 2 (Mittler, 2002). Incresed CAT ctivity under wter stress hs een reported y Agrwl nd Pndey (2003) nd D et l., (2005). Benevides et l (2000) reported the enzymes responsile for hydrogen peroxide detoxifiction such s scorte peroxidse nd ctlse in S. tuerosum. However, they suggested tht scorte peroxidse ws likely to e more importnt thn ctlse in the ROS detoxifiction. Since hydrogen peroxide ws lso involved in peroxidse-medited oxidtive polymeriztion, which results in cell wll strengthening, the ctivtion of peroxidse my hve protective role. However under iotic stress cusing hydrogen peroxide ccumultion, this my e one of the fctors tht results in the inctivtion of ctlse (Velikov et l., 2000). It is suggested tht the higher concentrtions of ctlse nd scorte peroxidse might hve removed the O2 rdicls nd its product H 2 O 2 induced y wter stress (Mous nd Adel- Aziz, 2008). Under the conditions of the present study, it is concluded tht the mount of the irrigtion wter influenced the 58

5 proline content, solule sugrs nd CAT ctivity. Mximum proline content ws recorded in the leves of potto grown under most restrictive irrigtion regime (I 3 ). Solule sugrs content ws highest in I 3 tretment s well. Also high CAT ctivity ws otined y ppliction of I 3 tretment. We re expnding the current investigtion to different potto cultivrs in greenhouse nd field conditions to precisely study the ove mentioned trits nd other importnt trits involved in drought tolernce in potto. Acknowledgment Authors grtefully thnked to Urmi University for finncil support of the work. References Acr O, Türkn I, Özdemir F (2001) Superoxide dismutse nd peroxidse ctivities in drought sensitive nd resistnt rley (Hordeum vulgre L.) vrieties. Act Physiol Plnt 3: Agrwl S, Pndey V (2003) Stimultion of stress-relted ntioxidtive enzymes in comting oxidtive stress in Cssi seedlings. Indin J Plnt Physiol 8: Asd K (2001) Ascorte peroxidse; hydroxide scvenging enzyme in plnts. 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