Indian Journal of BiotechnologyVol 2, April 2003, pp 251-258

Salt stress Induced Changes on Enzyme Activities during DifferentDevelopmental Stages of Rice COryza sativa Linn.)

T S Swapna*Post Graduate Department of Botany, Sree Narayana College, University of Kerala, S N Puram, Alappuzha 688 582, India

Received 10 June 2002; accepted 73 August 2002

The effect of NaCI stress was studied in four rice varieties, 'Pokkali'- (salt tolerant variety), 'MI 48','Annapoorna' and 'Jyothi'- (salt sensitive varieties). The enzymes-esterase, isocitric dehydrogenase, superoxidedismutase, peroxidase and catalase were studied in different developmental stages such as embryo, 14-day-oldseedling, tillering and flowering stage, and in undifferentiated embryo derived callus, before and after giving NaCIstress (100 mM). Esterase activity was found to be higher in embryo stage and isocitric dehydrogenase activity washigher in callus, but the stress caused a reduction in activity of these two enzymes during other developmental stages.An increase in activity of superoxide dismutase and peroxidase was also observed during the different developmentalstages under stress. There was a fluctuation "incatalase activity under NaCI stress during the different developmentalstages in all the varieties. Variety specific and developmental stage specific variation was found in activity of all theenzymes studied and can be reflected in metabolic processes which, when well-defined can serve as markers for salttolerance.

Keywords: rice, salt tolerance, esterase, isocitric dehydrogenase, superoxide dismutase, peroxidase and catalase

IntroductionGenerally plants differ in their tolerance to salt

stress. Activities of several important enzymes havebeen found to be affected by salt stress in differentplants. The banding patterns of isozymes, particularlyof peroxidase (Edreva et al, 1989), acid phosphatase(Dubey & Sharma, 1989) and esterase(Gangopadhayay et al, 1995; Harsanein, 1999) werereported to show differences in response totemperature, salinity and water stresses. In case of saltstress the same changes will be effected by direct orindirect exposure to NaCl depending on the extent ofsalt accumulation in the cytoplasm. Enzymes likesuperoxide dismutase, catalase, peroxidase andglutathione reductase showed significantly higheractivities under NaCI stress and chilling stress (Gossetet al, 1994; Lee & Lee, 2000).

However, there is a scarcity for information on thechanges in various enzymes in varieties of ricediffering in salt tolerance during differentdevelopmental stages and in undifferentiated callustissue. Therefore, to understand the varietaldifferences in enzyme activity and to elucidate thepossible role of enzymes in salt tolerance, four ricevarieties namely 'Pokkali', 'MI 48', 'Annapooma'

*Tel: 91- 0478- 2864297; Fax: 91-0478 - 2865497E-mail: swapnats@yahoo.com

and 'Jyothi' were selected for this study. Enzymesesterase, isocitric dehydrogenase, superoxidedismutase, peroxidase and catalase were studiedduring different developmental stages of riceincluding, embryo, l-t-day-old seedling, tillering andflowering stages, and in undifferentiated callus,before and after giving NaCl stress. Inter varietaldifferences in salt tolerance will be reflected in theactivities of these enzymes and the same can be usedas a marker for salt tolerance during plant breedingprogrammes.

Materials and MethodsSeeds of all the four different rice vaneties were

collected from the germplasm reserve of KeralaAgricultural University. Seeds were soaked overnightin water and the embryos dissected out upon initiationof germination. Plants were raised on sand bed andtissue samples from shoot portions were collectedfrom these plants for enzyme analysis during differentdevelopmental stages. For preparation of embryoderived callus, dehusked and surface sterilized maturerice seeds were inoculated under aseptic conditions ona solid callus induction medium-MS medium(Murashige & Skoog, 1962) with 2 mg/l 2,4-0 and0.5 mg/l kinetin. Plants were separated and subjectedto salt stress by immersing the roots in NaCl(100 mM) solution for 48 hrs. In the case of calli

252 INDIAN J BIOTECHNOL, APRIL 2003

100 mM NaCl was added to the medium. Plant partsdissected for analysis were immediately transferred toice. Tissue samples were weighed and ground using achilled mortar and pestle with acid washed sand in abuffer containing 0.1 M Tris-HCl, pH 6.8, 0.25 Msucrose, 1% poly vinyl pyrrolidone, 0.1 % ascorbicacid, 0.1 % cysteine hydrochloride,1 mM EDT A and 1 mM MgCh at pH 6.8 (Pramanik etal, 1996), and centrifuged at 12,000 rpm for 20 min ina refrigerated centrifuge. The supernatant wascollected and used for enzyme assay.

Enzyme AssayEsterase activity was assayed by the method of

Huggins and Lapides (1947), using p-nitrophenylacetate as substrate. One unit of esterase activity wasdefined as the amount of enzyme liberating 1 millimolof p-nitrophenol in 20 min at 25°C and pH 7 inphosphate buffer when the substrate is at aconcentration of 0.666 micromol. The units wereexpressed per ml of the undiluted fluid. Catalase wasassayed by the method of Luck (1974), by measuringthe decomposition rate of hydrogen peroxide bycatalase and activity was expressed as units per litre.Isocitric dehydrogenase activity was estimated by themethod of Wolfson and Ashman (1957), andexpressed in terms of micromols of NADPH2 formedper litre of sample per hour at 25°C and expressed asunits per litre.

Superoxide dismutase was assayed by the methodof Marklund & Marklund (1974). A unit of superoxide dismutase was defined as the amount ofenzyme, which inhibits the pyrogallol autooxidationby 50% and was expressed in units per litre. Similarlyperoxidase activity was determined by the method ofPutter (1974), and expressed as the rate ofdehydrogenation of guaiacol. All the experimentswere conducted in duplicate and mean values arereported.

Results and DiscussionThe assay results indicate that the activity of

esterase, in different rice varieties, was higher inembryo stage as compared to other stages and showeda significant reduction in activity in the presence ofNaCl (Fig. 1). Generally there was a gradualreduction in esterase activity from embryo to tilleringstage, which increase in flowering stage, except in MI48 where lowest enzyme activity was found in 14-day-old seedling stage. Salt stress caused reduction inesterase activity in embryo stage but it increased in

tillering stage in all the varieties. Effect of stress onactivity varied in seedling and flowering stage andalso in callus. Alves et al (1994) reported similardecline in esterase activity in sweet potatoembryogenic callus and the same was used as amarker to distinguish between embryogenic and non-embryogenic callus.

In case of isocitric dehydrogenase results show thatin the absence of salt stress enzyme activity wassignificantly higher in callus as compared to thesamples from other developmental stages (Fig. 2).Salt stress induced reduction in isocitricdehydrogenase activity except in embryo stage of'Pokkali', 'Annapoorna' and 'Jyothi'.

Superoxide dismutase activity in 'Pokkali' wasdifferent from other rice varieties (Fig. 3). Superoxidedismutase activity was higher in embryo stage anddecreased in the 14-day-old seedling stage. However,it again increased in tillering stage. But in all the othervarieties superoxide dismutase activity was lower inembryo stage. Effect of salt stress on superoxidedismutase activity was evidently varietal specificduring different stages in development.

Peroxidase showed an increase in activity undersalt stress in all the four varieties during the differentdevelopmental stages (Fig. 4). In 'Pokkali', tilleringstage showed maximum catalase activity, whereas inall salt sensitive varieties, flowering stage showedhigher catalase activity (Fig. 5). Studies conducted oncallus cultures of salt susceptible and tolerantcultivars revealed that salinity level increased theactivities of different isozymes including peroxidase(Subhashini & Reddy, 1990). The increase in enzymeactivities can be taken as a protective measure againstthe salt stress induced cell damages.

Results obtained from the present study clearlyindicate that the salt tolerant rice variety 'Pokkali'showed major differences in activity of variousenzymes in the presence and absence of stress fromsalt sensitive varieties. This may be due to differencesin genotype resulting in synthesis of these enzymes.Based on this study and also from the informationavailable in the literature, it could be predicted thatthe characterization of biochemical and geneticmarkers for salt tolerance might aid breedingprogrammes for the development of salt tolerantplants by reducing the time needed for screening.Accuracy of genetic selection will be greatlyenhanced because plants are screened based onultimate gene product level and also due to thereduced involvement of environmental factors,

SWAPNA: SALT STRESS INDUCED CHANGES IN ENZYME ACTIVITIES IN RICE 253

Annapoorna

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Fig. I-Effect of NaCI treatment on esterase activity in rice varieties at different developmental stages and in callus.

254

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INDIAN J BIOTECHNOL, APRIL 2003

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Fig. 2-Effect of NaCI treatment on isocitric dehydrogenase activity in rice varieties at different developmental stages and in callus.

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SWAPNA: SALT STRESS INDUCED CHANGES IN ENZYME ACTIVITIES IN RICE 255

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256 INDIAN J BIOTECHNOL, APRIL 2003

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SWAPNA: SALT STRESS INDUCED CHANGES IN ENZYME ACTIVITIES IN RICE

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Fig. 5--Effect of NaCl treatment on catalase activity in rice varieties at different developmental stages and in callus.

258 INDIAN J BIOTECHNOL, APRIL 2003

affecting the qualitative traits. Biochemical selectioncriteria might prove the simplest and best method forbreeding salt tolerance.

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