Effect of Mg Deficiency on Antioxidant Enzymes Activities and Lipid Peroxidation

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1 Journl of Agriculturl Science Vol. 2, No. 3; Septemer 21 Effect of Mg Deficiency on Antioxidnt Enzymes Activities nd Lipid Peroxidtion Eztollhe Esfndiri (Corresponding uthor) Deprtment of Agronomy nd Plnt Breeding, Fculty of Agriculture University of Mrgheh, Mrgheh, Irn Tel: E-mil: Mjid Shokrpour Deprtment of Agronomy nd Plnt Breeding, Fculty of Agriculture University of Mohghegh Ardili, Ardil, Irn Simk Alvi-Ki Deprtment of Agronomy nd Plnt Breeding, Fculty of Agriculture University of Triz, Triz, Irn Astrct The importnce of the physiologicl role of nutrient elements in plnt ehvior concerning higher nd more stle yield, using the durum whet vriety P1252 s model plnt, ws exmined under hydroponic conditions. To investigte the effects of, the element ws eliminted from the medi solutions. Results showed tht Mg elimintion decresed the chlorophyll content. Lck of Mg only ffected guicol peroxidse (GPX) nd ctlse (CAT) ctivities significntly. The SOD/APX+GPX+CAT rtio s n index of ssessing the lnce etween H 2 O 2 -producing nd H 2 O 2 -scvenging enzymes incresed leding to the ccumultion of H 2 O 2 in cell. The elevtion of SOD/APX+GPX+CAT rtio nd H 2 O 2 ccumultion indictes the occurrence of oxidtive stress in leve cells under. Lck of mgnesium (Mg) resulted in considerle increse in other oxidtive stress indices, cell deth nd Mlondildehyde (MDA). The reson is the occurrence of Her-Weiz rection in sence of Mg nd production of hydroxyl rdicl, very dngerous rdicl, leding to incresing dmge of cell iomolecules nd their poptosis. Keywords:, Oxidtive stress, ROS Scvenging 1. Introduction Crops re often exposed to vrious types of stresses including deficiencies during their life time. Due to vrious roles of nutrient in plnt cells, their deficiency my led to metolic disorders. The reson is tht n element such s Mg, mcro-nutrient element, hold criticl role in ctivtion of some importnt enzymes such s ATPses, riulose 1, 5-isphosphte (RuBP) croxylse, RNA polymerse nd protein kinses (Ckmk nd Kirky, 28). Mg holds fundmentl role in phloem export of photosynthtes from photosynthetic orgns to roots. results in drmtic increses in crohydrtes ccumultion of source leves (Hermns et l., 24), leds to reduced trnsport nd ccumultion of crohydrtes, lters photosynthetic cron metolism nd restricts CO 2 fixtion. Impirment of the photosynthetic electron trnsport to CO 2 through photosynthetic memrnes my cuse n ccumultion of non-utilized electrons nd sored energy (Hermns nd Verruggen, 25). Moreover, decreses pore conductivity (Velikov et l., 2). Under such conditions, the electrons nd excited energy tht not used in photosynthetic CO 2 fixtions re chnneled to moleculr O 2, leding to produce rective oxygen species (ROS). ROS re regrded s one of the min sources of cell dmges under iotic nd iotic stresses (Mittler, 22). ROS re prtilly reduced forms of tmospheric oxygen which re produced in vitl processes such s photorespirtion, photosynthesis nd respirtion (Esfndiri et l., 27; Jimenez et l., 1998; Mittler, 22; Kornyeyev, et l., 23; Vidynthn et l., 23; Uchid et l., 22). For production of wter in these processes, four electrons re required to full reduction of oxygen. ROS typiclly result from the trnsfer of one, two nd three electrons to O 2 in order to formtion of superoxide (O 2 - ), hydrogen peroxide (H 2 O 2 ) nd hydroxyl rdicl (HO ), respectively (Mittler, 22). These species of oxygen re highly Pulished y Cndin Center of Science nd Eduction 131

2 Journl of Agriculturl Science Vol. 2, No. 3; Septemer 21 cytotoxic nd cn seriously rect with vitl iomolecules (i.e. lipids, proteins, nucleic cids, etc.) cusing lipid peroxidtion, protein denturing nd DNA muttion, respectively (Breusegem et l., 23; Quiles nd López, 24; Scndlios, 1993). Evidence suggests tht memrnes re one the primry sites of cells nd orgnelles injuries (Cndn nd Trhn, 23). This is ecuse ROS cn rect with unsturted ftty cids to cuse peroxidtion of essentil memrne lipids in plsmlemm or intrcellulr orgnelles (Krl et l., 23). Peroxidtion of plsmlemm leds to the lekge of cellulr contents, rpid desicction nd cell deth. Intrcellulr memrne dmge cn ffect respirtory ctivity in mitochondri, cusing pigment deteriortion nd loss of the cron fixing ility in chloroplsts (Scndlios, 1993). Nevertheless, plnts hve evolved vrious protective mechnisms to eliminte or reduce ROS, which re effective t different levels of stress-induced deteriortion (Bek nd Skinner, 23). Enzymtic ntioxidnt system is one of the protective mechnisms including superoxide dismutse (SOD: EC ) which cn e found in vrious cell comprtments nd ctlyses the disproportionte concentrtion of two O - 2 rdicls to H 2 O 2 nd O 2 (Scndlios, 1993). H 2 O 2 is eliminted y vrious ntioxidnt enzymes such s ctlses (CAT: EC ) (Kono nd Fridovich, 1983) nd peroxidses (POX: EC ) (Gr et l., 23) which convert H 2 O 2 to wter. Other enzymes tht re very importnt in ROS scvenging system nd function in scorte-glutthione cycle re glutthione reductse (GR: EC ), monodehydro scorte reductse (MDHAR: EC ) nd dehydroscorte reductse (DHAR: EC ) (Cndn nd Trhn, 23). Moreover, ROS re inevitle y-products of norml cell metolism (Mrtinez et l., 21). But under norml conditions production nd destruction of ROS re well regulted in cell metolism (Mittler, 22). When plnt is encountered with hrsh conditions, ROS production will overcome scvenging systems nd consequently oxidtive stress will dominte. Knowing the physiologicl significnce of nutrient elements in plnt is one of the criticl issues to improve the yield nd yield stility. Regrding the mgnesium key role in ctivting importnt enzymes such s RuBP-croxylse, ATPses, Mg cheltse nd lloction of photosynthetic compounds to shoot nd root the present study ws designed to ssess effects on plnt metolism considering some physiologicl prmeters. 2. Mterils nd Methods In order to study of effects on ctivity of ntioxidnt enzymes, oxidtive stress indexes (mlondildehyde contents (MDA) nd H 2 O 2 ) nd cell deth, the P1252 vriety of durum whet ws plnted in hydroponic wy under controlled conditions. Nutrients used during growth period included mcroelements (C(NO 3 ) 2, KNO 3, MgSO 4 nd KH 2 PO 4 with concentrtions of 3, 2.5, 1.5 nd.17 mm, respectively) nd microelements (FeSO 4, H 3 Bo 3, MnSO 4, ZnSO 4, CuSO 4 nd H 2 MoO 4 with concentrtions 5,23, 5,.4,.2 nd.1 µm, respectively). MgSO 4 ws eliminted from medi solution in without Mg tretment (Grieve nd Grttn, 1983). The seedlings were plnted on hlf strength nutrition solution till 2-3 leves stge, then fter they were nourished with full strength nutrient solution. The solutions were renewed twice week to mintin the lnce of nutrients concentrtion. ph of solutions were set round Temperture, dy length nd light density were 25±2ºC, 14 h nd 2 µm photon m -2 s -1, respectively. The seedlings were grown under these conditions for two months. Smpling ws mde from completely-expnded leves (zdoks scle, 18-19) nd the plnt mterils were immeditely trnsferred to liquid nitrogen nd mintined t - 2 C until the mesurement of vriles under study. To mesure the enzyme ctivity t lest 5 leves smples were used. Compring the two levels of Mg (control nd ) were done y t- student test. Mesurement of ntioxidnt enzymes ctivities nd other prmeters: Enzyme extrction: For SOD, CAT nd GR extrction, lef smples (.5 g) were homogenized in ice cold.1 M phosphte uffer (ph 7.5) contining.5 mm EDTA with pre-chilled pestle nd mortr. Ech homogente ws trnsferred to centrifuge tues nd ws centrifuged t 4 C in Beckmn refrigerted centrifuge for 15 min t 15, g. The superntnt ws used for enzyme ctivity ssy. For APX extrction, lef smples (.5 g) were homogenized in ice cold.1 M phosphte uffer (ph 7.5) contining.5 mm EDTA, 2mM scorte (AsA) nd 5% poly vinyl pyrrolidin (PVP) with pre-chilled pestle nd mortr. Other stges were similr to extrction of other enzymes (Esfndiri et l., 27). Enzyme ctivity ssy: SOD ctivity ws estimted y recording the decrese in sornce of superoxide-nitro lue tetrzolium complex y the enzyme (Sirm et l., 22). Aout 3 ml of rection mixture, contining.1 ml of 2 mm 132 ISSN E-ISSN

3 Journl of Agriculturl Science Vol. 2, No. 3; Septemer 21 methionine,.1 ml of 2.25 mm nitro-lue tetrzolium (NBT),.1 ml of 3 mm EDTA, 1.5 ml of 1 mm potssium phosphte uffer, 1 ml distilled wter nd.5 ml of enzyme extrction, were tken in test tues in duplicte from ech enzyme smple. Two tues without enzyme extrct were tken s control. The rection ws strted y dding.1 ml rioflvin (6 μm) nd plcing the tues elow light source of two 15 W florescent lmps for 15 min. Rection ws stopped y switching off the light nd covering the tues with lck cloth. Tues without enzyme developed mximl color. A non-irrdited complete rection mixture which did not develop color served s lnk. Asornce ws recorded t 56 nm nd one unit of enzyme ctivity ws tken s the quntity of enzyme which reduced the sornce reding of smples to 5% in comprison with tues lcking enzymes. CAT ctivity ws mesured ccording to Aei (1984). Aout 3 ml rection mixture contined 1.5 ml of 1 mm potssium phosphte uffer (ph 7),.5 ml of 75 mm H2O2,.5 ml enzyme extrct nd distilled wter to mke up the volume to 3 ml. Rection strted y dding H2O2 nd decrese in sornce ws recorded t 24 nm for 1 min. Enzyme ctivity ws computed y clculting the mount of H2O2 decomposed. APX ctivity ws mesured ccording to Yoshimur et l. (2) y monitoring the rte of scorte oxidtion t 29 nm (E=2.8 mm 1 cm -1 ). The rection mixture contined 25 mm phosphte uffer (ph= 7),.1 mm EDTA, 1 mm H2O2,.25 mm AsA nd the enzyme smple. No chnge in sorption ws found in the sence of AsA in the test medium. Protein content of smples ws determined y the method of Brdford (1976), ovine serum lumin used s stndrd. Peroxidtion product estimtion: Mlondildehyde (MDA) ws mesured y colorimetric method..5 g of lef smples were homogenized in 5 ml of distilled wter. An equl volume of.5% thiorituric cid (TBA) in 2% trichlorocetic cid solution ws dded nd the smple incuted t 95 C for 3 min. The rection stopped y putting the rection tues in n ice th. The smples were then centrifuged t 1 g for 3 min. The superntnt ws removed, sorption red t 532 nm, nd the mount of nonspecific sorption t 6 nm red nd sutrcted from this vlue. The mount of MDA present ws clculted from the extinction coefficient of 155 mm -1 cm -1 (Stewrt nd Bewley, 198). Determintion of H 2 O 2 content: Hydrogen peroxide levels were determined ccording to Sergive et l., (1997). Lef tissues (.5g) were homogenized in ice th with 5 ml.1% (w/v) TCA. The homogente ws centrifuged t 12 g for 15 min nd.5 ml of the superntnt ws dded to.5 ml 1 mm potssium phosphte uffer (ph=7.) nd 1 ml 1 M KI. The sorncy of superntnt ws red t 39 nm. The content of H2O2 ws given on stndrd curve. Determintion of cell deth: Aliquots consisting of four lef discs were removed from tretments nd sumerged in 1 ml of.25% Evns lue in 1 ml disposle plstic ekers nd incuted on pltform shker t 8 rpm for 2 min. the eker contents were poured into smll Buchner funnel nd the discs rinsed well with deionized wter until no more lue stin ws eluted. The discs were ground with using pestle nd the homogente diluted with.5 ml of deionozed wter. The tue ws cpped, vortexed nd centrifuged t 1 g for 5 min. A.8 ml liquot of the superntnt ws removed nd the opticl density determined spectrophotometriclly t 6 nm (Bker nd Mock, 1994). 3. Results nd discussion Results indicted tht chlorophyll content hs een ffected y (Fig. 1). GPX nd CAT ctivity hd significnt decrese compred to control under lck-of-mg conditions (Figs 2 nd 3). Whilst the other H 2 O 2 scvenger ctivity, APX, did not disply ny chnge compred to control (Fig. 4). Moreover, the ctivity of H 2 O 2 producer enzyme, SOD, did not significntly increse in the Mg-deficit condition (Fig. 5). Oserving the lnce of the enzyme which produces H 2 O 2 (SOD) nd its scvenging enzyme (APX, GPX nd CAT) showed tht lck of Mg my disrupt the lnce. Becuse of the decrese in the scvenger enzymes ctivity, the rtio hd significnt nd considerle growth (% Fig. 6). The results showed tht, H 2 O 2 ccumulted t in whet seedling under regime (Fig. 7). The lck of Mg incresed MDA content (Fig. 8) nd cell deth significntly compring with control (Fig. 9). The results indicted H 2 O 2 ccumultion under conditions ecuse, it is n essentil element hs criticl role in cell metolism. Fe-SOD nd Cu/Zn-SOD ctivity is inhiited y high levels of H 2 O 2 (Milone et l., 23). Moreover, H 2 O 2 inctivtes iphophtes enzymes in Clvin cycle which decreses CO 2 fixtion (Ymzki et l., 23). Regrding the Mg roles in ctivting RuBP-Cr, ATP-ses nd decreses of ROS-scvenger mechnisms under, ROS ccumulte in plnt cells nd oxidtive stress occurs. The lnce of the enzymes relted to ROS scvenging mechnisms nd non-ccumultion of ROS is one of the Pulished y Cndin Center of Science nd Eduction 133

4 Journl of Agriculturl Science Vol. 2, No. 3; Septemer 21 indices expressing non-occurrence of oxidtive stress. The results showed tht SOD/APX+GPX+CAT rtio incresed under. The rtio suggests tht production rte of H 2 O 2 y SOD is fster thn its reduction y the scvenging enzymes. However, H 2 O 2 is not produced just y SOD. The imlnce of H 2 O 2 producing nd scvenging would cuse this toxic metolite to ccumulte in cells. Totl-SOD ctivity did not vry significntly s result of the lck of Mg compred with control. Furthermore, MDA content nd cell deth ws enhnced t, too. These indexes indicted tht oxidtive stress occurred ecuse Mg is n exceptionl element in plnt cell metolism (Ckmk nd Kirky, 28). Therefore, the produced O.- 2 t cell metolism is higher thn increse in totl-sod ctivity under lck of Mg condition. As result, O.- 2 nd H 2 O 2 will produce highly toxic nd rective HO. rdicl y Her-Weiz rection (Edrev, 25). Tewri et l. (25) reported tht under Fe deficiency conditions, SOD, POX nd CAT ctivities were decresed.- nd in consequence O 2 nd H 2 O 2 ccumulted in plnt cells. In these situtions Her-Weiz rection does not run nd this leds to low production of hydroxyl rdicles nd hence minimum oxidtive stress. The results displyed tht the content of chlorophyll (Chl) decresed under condition. The sme result ws reported y Ckmk nd Mrschner (1992). The reson is tht Mg cheltse uses Mg-ATP to incorporte Mg into protoporphyrin XI structure nd so e trnsformed (Ckmk nd Mrschner, 1992, Ckmk nd Kirky, 28). Protoporphyrin XI is highly toxic metolite which is ccumulted under condition (Ckmk nd Kirky, 28). 4. Conclusions It cn e concluded tht Mg is key element for cell metolism nd the deficiency of it leds to inctivities in criticl metolic points such s CO 2 fixtion, crohydrte up-loding to phloem, ATP-ses ctivity. Increse in ROS production nd/or ccumultion of protoporphyrin XI re the direct results of Mg-deficiency. The increse in ROS production nd their ccumultion trgets ntioxidnt enzymes involved in defense mechnisms, deilitting these enzymes. All these fctors put together, leds to incresed cell dmge nd high occurrence of cell progrmmed deth. Acknowledgement The reported study ws supported nd finncilly cked y reserch chncellorship t Triz University. References Aei, H. (1984). Ctlse in vitro. Method of Enzymology, 15, Bker, C.J. nd Mock. N.M. (1994). An improved method for monitoring cell deth in cell suspension nd lef disk ssy using Evns lue. Plnt Cell Tissue Orgn Culture, 39, Bek, K.H. nd Skinner, D.Z. (23). Altertion of ntioxidnt enzyme gene expression during cold cclimtion of ner-isogenic whet lines. Plnt Science, 165, Brdford, M. M. (1976). A rpid nd sensitive method for the quntittion of microgrm quntities of protein utilizing the principle of protein-dye inding. Anlyticl Biochemistry, 72, Breusegem, F. V., Vrnov, E., Dt, J. F., nd Inze, D. (21). The role of ctive oxygen species in plnt signl trnsduction. Plnt Science, 161, Ckmk, I. nd Kirky. E. (28). Role of mgnesium in cron prtitioning nd lleviting photooxidtive dmge. Physiologi Plntrum, 133, Ckmk, I. nd Mrschner, H. (1992). Mgnesium deficiency nd high light intensity enhnce ctivities of superoxide dismutse, scorte peroxidse, nd glutthione reductse in en leves. Plnt Physiology, 98, Cndn, N. nd Trhn, L. (23). The correltion etween ntioxidnt enzyme ctivities nd lipid peroxidtion levels in Menth pulegium orgns grown in C 2+, Mg 2+, Cu 2+, Zn 2+ nd Mn 2+ stress conditions. Plnt Science, 163, Edrev, A. (25). Genertion nd scvenging of rective oxygen species in chloroplsts: A sumoleculr pproch. Agriculture, Ecosystems nd Environment, 16, Esfndiri, E. A., Shki, M. R., Mhoo, S. A., Alyri, H., nd Toorchi, M. (27). Wter stress, ntioxidnt enzyme ctivity nd lipid peroxidtion in whet seedling. Journl of Food, Agriculture & Environment, 5, Gr, L. D., Pinto, M. C. nd Tommsi, F. (23). The ntioxidnt systems vis-á-vis rective oxygen species during plnt-pthogen interction. Plnt Physiology nd Biochemistry, 41, ISSN E-ISSN

5 Journl of Agriculturl Science Vol. 2, No. 3; Septemer 21 Grieve, C. nd Grttn, R. (1983). Rpid ssy for determintion of wter solule quternry mmonium componds. Plnt nd siol, 7, Hermns, C., Johnson, G. N. Strsser, R. J. nd Verruggen, N. (24). Physiologicl chrcteriztion of mgnesium deficiency in sugr eet: cclimtion to low mgnesium differentilly ffects photosystems I nd II. Plnt, 22, Hermns, C. nd Verruggen, N. (25). Physiologicl chrcteriztion of in Aridopsis thlin. Journl of experimentl otny. 56: Jimenez, A., Hernndez, J. A., Pstori, G., Rio, L.A. D. nd Sevill, F. (1998). Role of the scorte-glutthione cycle of mitochondri nd peroxisomes in the senescence of pe leves. Plnt Physiology, 118, Krl, E., Yücel, M. nd Öktem, H.A. (23). Antioxidnt responses of tolernt nd sensitive rley cultivrs to oron toxicity. Plnt Science, 164, Kono, Y. nd Fridovich, I. (1983). Inhiition nd rectivtion of Mn- ctlse. The Journl of Biologicl Chemistry, 258, Kornyeyev, D., Logn, B. A., Allen, R. D. nd Holdg, A. S. (23). Effect of chloroplstic overproduction of scorte peroxidse on photosynthesis nd photoproduction in cotton leves sujected to low temperture photoinhiition. Plnt Science, 165, Mrtinez, C. A., Loureiro, M. E., Oliv, M. A. nd Mestri, M. (21). Differentil responses of superoxide dismutse in freezing resistnt Solnum tuerosum sujected to oxidtive nd wter stress. Plnt Science, 16, Milone, M. T., Sgherri, C., Clijsters, H., nd Nvri-Izzo, F. (23). Antioxidtive responses of whet treted with relistic concentrtion of cdmium. Environmentl nd Experimentl Botny, 5, Mittler, R. (22). Oxidtive stress, ntioxidnts nd stress tolernce. Trends in Plnt Science, 7, Quiles, M. J. nd López, N. I. (24). Photoinhiition of photosystems I nd II induced y exposure to high light intensity during ot plnt grown effects on the chloroplstic NADH dehydrogense complex. Plnt Science, 166, Sirm, R. K., Ro, K. V. nd Srivstv, G. C. (22). Differentil response of whet genotypes to long term slinity stress in reltion to oxidtive stress, ntioxidnt ctivity nd osmolyte concentrtion. Plnt Science, 163, Scndlios, J. G. (1993). Oxygen stress nd superoxide dismutse. Plnt Physiology, 11, Sergiv, I., V. nd Alexiev, E. Krnov. (1997). Effect of spermine, trzine nd comintion etween them on some endogenous protective systems nd stress mrkers in plnts. Compt. Rend. Acd. Bulg. Sci., 51: Stewrt, R. R. C. nd Bewley, J. D. (198). Lipid peroxidtion ssocited ging of soyen xes. Plnt Physiology, 65, Tewri, R. Kumr, P nd Shrm, P. (25). Signs of oxidtive stress in the chlorotic leves of iron strved plnts. Plnt Science, 169, Uchid, A., Jgendorf, A. T., Hiino, T., Tke, T. nd Tke, T. (22). Effects of hydrogen peroxide nd nitric oxide on oth slt nd het stress tolernce in rice. Plnt Science, 163, Vidynthn, H., Sivkumr, P., Chkrrsty, R. nd Thoms, G. (23). Scvenging of rective oxygen species in NCl-stressed rice (Oryz stiv L.)-differentil response in slt-tolernt nd sensitive vrieties. Plnt Science, 165, Velikov, V., Yordnov, I. nd Edrev, A. (2). Oxidtive stress nd some ntioxidnt systems in cid rin-treted en plnts. Protective role of exogenous polymins. Plnt Science, 151, Ymzki, J, A, Ohshi, Y, Hshimoto, E, Negishi, S, Kumgi, T, Kuo, T, Oikw, E, Mrut, Y, Kmimur. (23). Effects of high light nd low temperture during hrsh winter on needle photodmge of Aies mriesii growing t the forest limit on Mt. Norikur in Centrl Jpn. Plnt Science, 165, Yoshimur, K., Yute, Y., Ishikw, T., nd Shigeok, S. (2). Expression of spinch scorte peroxidse isoenzymes in response to oxidtive stresses. Plnt Physiology, 123, Pulished y Cndin Center of Science nd Eduction 135

6 Journl of Agriculturl Science Vol. 2, No. 3; Septemer 21 Chlorophyll content (mm.g -1 FW) GPX ctivity (Units.mg -1 Protein.min -1 ) Figure 1. The effect of on chlorophyll content. Different letters show significnt difference Figure 2. The effect of on GPX ctivity. Different letters show significnt difference CAT ctivity (Units.mg -1 Protein.min -1 ) APX ctivity (Units.mg -1 Protein.min -1 ) Figure 3. The effect of on CAT ctivity. Different letters show significnt difference Figuer 4. The effect of on APX ctivity. Different letters show significnt difference SOD ctivity (Units.mg -1 Protein) SOD/APX+CAT+ GPX rtio (%) Figure 5. The effect of on APX ctivity. Different letters show significnt difference Figure 6. The effect of on SOD/APX+CAT+GPX rtio. Different letters show significnt difference. H 2 O 2 content (mm.g -1 FW) MDA content (nm.g -1 FW) Figure 7. The effect of on H 2 O 2 content. Different letters show significnt difference Figure 8. The effect of on MDA content. Different letters show significnt difference Cell deth (%) Figure 9. The effect of on cell deth. Different letters show significnt difference 136 ISSN E-ISSN