Environmentt Conservation Jou urnal 15(1 &2) ISSN 0972-33099 (Print) 2278-5 5124 (Online) Abstracted annd Indexed
, 2014
Effects of Zn streess on antiioxidant enzyme acttivity in Leemna polyyrrhiza L. P and Krupa K Unad dkat Punita Parikh Received d: 23.11.201 13
Revised d: 02.04.20114
Accep pted: 28.04.22014
Abstract The effect of o deleterious concentration off zinc provided individually was investigated in order to asseess the effect off metal interaction in Lemna polyrrrhiza L. The prresent study alsso emphasizes on o the responsee of catalase and d guaiacol perooxidase enzymes un nder zinc stresss. Both antioxid dant enzymes exxhibited an increasing trend under u different treatment cond ditions but it was reverse at high hly toxic metall concentration.. The antioxidaant activities off enzymes, i.e of o catalase, asccorbate peroxidase,, guaiacol pero oxidase and their activity prooportions weree examined. Caatalase activitiees were substaantially increased in n a stress envirronment as com mpared to guaiaacol peroxidasee. Further, cataalase and showeed increased activities in a combined stress enviironment. Physsiological role of o these enzymees in stress toleerance mechaniism is discussed. The ns of Zn appearrs to induce oxxidative damagge as observed by the response off Lemna polyrrrhiza L to toxicc concentration increase an ntioxidant metab bolism.
Keywordss- Antioxidant, ascorbate peroxidase, p c catalase, enzyyme activity, glutathione g reeductase, guaaiacol peroxidasse, Lemna pollyrrhiza L. , stress
Introducction Environm mental stress factors like drougght, temperatuure, high salin nity and heavvy metals are the major coonstraints thaat limit plaant growth and productivity, by disturbing the inttracellular waater balance. Usually, U in fields fi or on agricultural a laand, unlike inn a laborato ory or evenn a greenhoouse environment, plants are a subjectedd to a maniffold array of stress factorr (Siddiqui et e al. 2008 and Nawaz ett al. 2010).H However, mosst of the studdies have beeen devoted to t assess thhe physiological response of plants in a single streess environm ment 1 and Shharma and Gills G like salinnity (Sinha 1991 1994, Kum mar and Kum mar 1996), droought (Shinozzaki and Yamaaguchi 2000) and heavymeetals (Hameedd et al. 2000 and Jetley et e al, 2004). Studies on the physiologgical respon nses of pllants under a combinatiion of such sttresses are reestricted to just a few reporrts (Wang ett al. 2003 annd Dudley and Shani 20003, Wang and d Huang 20004 and Jakabb et al. 2005) and even theey are not diirectly relatedd to the combiination of streess factors likke heavy metaal. Plants havve utilized vaarious mechannisms to com mbat with abiottic stresses. Among A them, stress tolerannce gene exprression, com mpatible soluttes, phenols and antioxidannt enzyme pro oduction are some s examples Author’s Address Departmentt of Botany, Facu ulty of Science, The Maharaja Sayajirao University U of Baroda, Vadodara Email:
[email protected]
Copyright byy ASEA All rights of reproduction r in an ny form reserved
(Jakab et al. 20055, Siddiqui and Khan 2011). 2 Antioxiidant enzymes such as supperoxide dism mutase (SOD) ascorbate peroxidase p (A APX), glutatthione reductase, guaiacol peroxidase and catalase are well-knnown defensee systems prooviding proteection against the hazardss of reactivee oxygen sppecies (ROS) in i different stressful condditions (Allen et al. 1997, Kwon K et al. 20002, Ahmed et e al. 2010). These T defensees may also a involvve water-sooluble antioxiddants such as a ascorbate, glutathionee and phenoliic compoundds and lipid-soluble moleecules such as carotenoidds and tocoppherols (Foyyer et al,19944; Hodges ett al . 1997 and Hodgess and Forney 2000; Pastorri et al, 20000). Oxidative stress is charracterized byy the syntheesis of hyddrogen peroxidde, which is detoxified byy CAT activvity in the perroxisomes annd by APX X in the cyytosol, mitochoondria and chloroplasts (Foyer et al. 1997 and Assada 1999). On O the other hand, antioxxidant molecules such ass glutathionee, ascorbatee and soluble phenolic coompounds aree able to diirectly scavengge reactive oxxygen radicalls and, in thee case of asccorbate and glutathione, they are also substrattes for the antioxidant a ennzymes APX X and GPX, respectively. r Therefore, itt seems likelyy that both asscorbate and glutathione might play a key role in buffering b oxidative stress in most eukarryotic systemss (Noctor annd Foyer 19998; Smirnoff 2000 and Sm mirnoff and Conklin andd Loewus 2001). 2
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Parrikh and Unadkatt
Activitiess of these antioxidant enzymes are frequentlyy observed in n a single stress environm ment but the reesponse and the t proportionn of the relattive activities of these enzzymes in a combined c heavy metal (leead) environ nment have seldom been reported. To make up p for this laack, the pressent study exxamines thee response of antioxiddant enzymes and the prroportions of their relattive activities in heavy metal m (zinc sulphate) strress environment.
Materiall and Metho ods Plant Maaterial: The test plants p Lemna a polyrrhiza L. L was colleccted from the pond at Haraani, Vadodaraa (Fig 1 and 2). They werre allowed to t acclimatizze for 15 daays. Plants weere washed thoroughly undder a running tap water andd were grown n and propagaated for 4 weeeks in quarterr strength Ho oagland’s soluution (Hoagland and Arnoon, 1950). Plants of saame size were w selected for fo the experim ment. In the pilot p scale
ment, the testt plants weree exposed to wide experim range of o the metal ioon concentrattions i.e.10, 20, 30, 40, 50, 60, 70, 80, 90 9 and 100 pppm. It was nooticed that the plants weere unable to t survive inn the concenttration range between 20-1100 ppm Zn ioons. In the subsequent s exxperiments itt was revealedd that the concentration moortality (LC500) of Zinc sullphate on expposed plants was 9 ppm m during 2400 hrs. Therefoore, the tracee element unnder study ZnSO Z 4 (Zn) weere supplied at a 1, 3, 5, 7 and a 9 ppm foor 3, 6 and 9 days. Nutriient solution devoid of trace elementt served as a control. Both the controol and the treaated solutionns were mainntained at pH H 5.5 using dilute d HCl or NaOH. Expeerimental plannts (in triplicattes) were placed p in nutrient n solution. Solutionns were repleenished everyy 3 days to prrevent depletioon of metalls and nutriients. After each experim mental period, harvested plants were washed w in runnning tap watter and rinseed with deioonized water. Extraction E annd estimation of CAT and APX was folllowed the meethod of Thim mmaiah, 1999.
Fig 1: Maap of Collectioon site Environmentt Conservationn Journal
Effects of Zn strress on antioxidan nt enzyme activityy
Enzyme Extraction E an nd Assay: Catalase Plants (1000 mg) from each e treatmennt were colleccted at 3, 6 andd 9 days interrvals and hom mogenized in 0.1 M sodiuum phosphaate buffer (pH 7.0) and centrifugeed at 1,000 g for 10 min att 4°C. One ml m of the superrnatant was added a to a reaction r mixtture containingg 1 ml of 0.1 1 M H2O2 andd 3 ml of 0.11 M sodium phosphate p bu uffer (pH 7.00). The reacttion was stoppped by adding g 10 ml of 2% % H2SO4 afteer 1 min incuubation at 20 0°C. The accidified reacttion mixture with w or without supernatant was titraated against 0.01 M KMnO O4 to determinne the quantityy of H2O2 utiliised by the enzyme. e The catalase activvity was exprressed in en nzyme units as µmol H2O2 destroyedd mg protein-1 min-1.
Results and Discu ussion In the current c researrch it was invvestigated that the activitiees of both catalase c and peroxidase were significcantly higher in i tolerant plaants in compaarison with thhe uncontaminnated ones (F Fig. 3 and Fig 4.) Greaterr activities of catalasse and Guuaicol peroxiddase indicateed that the toolerant plant were under oxidative o streess, a featuree often assocciated with metal m tolerancce (Van annd Clisters, 1990) Nashikkkar and Chaakrabarti, (19994) reportedd that both hiigher activityy of catalase and peroxidaase in crops grrown on heavvy metal polluuted soil.
Fig.3:Catalase Actiivity
Fig 2: Leemna polyrhhiiza
Guaiacol Peroxidase e treatmennt were colleccted Plants (1000 mg) from each at 2 weekk intervals an nd homogenizzed with mortar and pestlee in cold 0.1 M phosphate buffer (pH 6.1). 6 filtered and The hom mogenate was d centrifugedd at 12000 g for 10 min at a 4°C. The supernatant was w used for the peroxidase assay. Thhe assay mixtture containedd 0.1 M phosp phate buffer (pH 6.1), 4 mM m guaiacol, 3 mM H2O2 and 0.4 ml of o crude enzyyme T total reacttion volume was w 1.2 ml. The T extract. The rate of chhange in abso orbance (OD)) at 420 nm was w measured using a UV V-Spectrophootometer (Jassco, UVIDEC--650, Japan)). The leveels of enzyyme activity were w expressed d as µmol H2O2 destroyed mg protein-1 min-1.
Peroxidase activity Fig: 4:P
In the present inveestigation it was reportedd that activitiees of catallase enhanceed linearly with increaseed metal ioon concentration whereas the activityy of guaicol peeroxidase shoowed increasee with metal ion i concentraation only at a 3 day expposure period. Thereafter it decreased at 6 and 9 day exposurre period. Heavy H metals in soil, wateer and atmosphhere, where plants are living l are seeen to demonsstrate interacctions betweeen these heavy h metals and the plannts. On the other o hand, heavy h
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Paarikh and Unadkaat
metals shoow negative effects e on plaants by inhibitting growth, damaging th he structure, affecting the physiologgical and biochemical activities and b decreasingg the functio ons of the plaants. The effeects and bioavvailability off heavy mettals depends on many facctors includin ng environmeental conditiions such as pH, p species of o heavy metals, and orgaanic substancees in the mediia as well as fertilization and the indiviidual plant sp pecies. Plantss have their own o mechanism ms of resisstance againsst the negattive effects off heavy metall by combininng heavy metals with proteeins and deveeloping enzym mes and nucleic acids to detoxify d heav vy metal polllution. Thus, the effects heeavy metals on plants are revealedd in several asspects and thee plants show w many kindss of in resistancee mechanissms. The changes antioxidattive enzymee activities in response to heavy meetal stress aree known to be b dependentt on heavy metal m concen ntration (Shaah et al.20001). Activitiess of these enzymes might increase i in orrder to cope with w the oxidaative stress im mposed by heavy metals onn plants, as was w repeateddly found in our experimennts. Alternaatively, theey might be diminisheed if the toxic effeccts of higgher concentraation of heavy y metals were greater than can be toleraated and com mbated by the antioxiddant enzymes, as is the casse in the pressent experimeent, particularly catalase acctivity. On the otther hand, deccreased activiity of peroxiddase in 6 and 9 days Zn treeated plant inndicated that the plants weere under trem mendous heaavy metal streess. This mighht have resu ulted in the accumulation a n of ROS (reaactive oxygen n species). RO OS products are by reported to t cause dam mage to the biomolecules b peroxidatiion, electrop philic substitution reactiion, reduction of mem mberane lippids, proteins, chloroplasst pigments, enzymes, nuucleic acids etc. (Comba et e al. 1998, Becana et al. 2000 2 and Majeed et al.2010). It is evident that each of Zn can separatelyy cause signifficant reductioon in most off the recorded structural parameters p w with detrimenntal physiologgical consequences. It migght be concluded that expossure of L.poyrrhhiza L. to tooxic levels off Zn triggers a number of o closely inter relatedd s structural and functio onal events in the stresssed plants.
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