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M. Sc. Dissertations

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20093
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Subject: Biochemistry × End date: 2009 × Start date: 2009 ×
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    ThesisItemOpen Access
    Biochemical studies in wheat (Triticum aestivum L.) seedlings under heat stress
    (CCSHAU, 2009) Reena; Madan, Shashi
    The present investigation was carried out to investigate the effect of heat stress and revival on some antioxidative enzymes, metabolites, chlorophyll stability and polypeptide pattern in the leaves of Wheat seedlings of heat susceptible (WH-147 and HS-277) and heat tolerant (WH-1021 and HW-2045) genotypes. Seven days old seedlings grown at 25ºC were exposed to 40ºC for 6 h and these seedlings were brought to 25ºC. The observations were recorded in the leaves of control, stressed and revived seedlings on 2nd and 4th day of revival. Screening of thirty six genotypes for thermo-tolerance was done based on wilting of primary leaf and values of chlorophyll fluorescence. Chlorophyll stability (%) declined in tolerant and susceptible genotypes under heat stress. There was enhancement in the activities of antioxidative enzymes, viz. CAT, POX, GR and APX in the leaves of tolerant and susceptible genotypes under heat stress, however, higher per cent increase was observed in tolerant genotypes. Heat stress increased the SOD activity in tolerant genotypes but activity declined in susceptible genotypes. On revival, the activities of the CAT, POX and GR declined in comparison to stressed seedlings but remained higher as compared to control. Ascorbate peroxidase activity remained higher on 2nd day and 4th day of revival in all the genotypes. The higher accumulation of H2O2, ascorbic acid and malondialdehyde content was observed under heat stress in all the genotypes but their level was more in heat susceptible genotypes as compared to heat tolerant genotypes. SDS-PAGE of seedlings under stress conditions revealed the appearance of polypeptide bands of different molecular weight in tolerant and susceptible genotypes, and these polypeptides bands disappeared on revival of 2nd and 4th day. Based on genotypes screening and antioxidant enzyme activities, out of four genotypes HW-2045 was found to be the most tolerant and WH-147 as the most susceptible genotype.
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    ThesisItemOpen Access
    High temperature induced changes in oxidative stress, antioxidant system and polypeptide pattern in Indian mustard [Brassica juncea (L.) Czern & Coss.]
    (CCSHAU, 2009) Babita Rani; Dhawan, Kamal
    The present investigations were undertaken to study the changes in lipid peroxidation, antioxidative enzymes, metabolites and protein pattern in Brassica juncea seedling after subjecting to high temperature stress (45±0.5C) and on revival. Two tolerant genotypes viz. BPR-542-6 and NRCDR-02 and two susceptible genotypes viz. NPJ-119 and RGN-152 were screened at 45±0.5C on the basis of time taken to 50% seedling mortality. Lipoxygenase (LOX), malondialdehyde (MDA) and hydrogen peroxide (H2O2) which are indicators of cell membrane damage increased under heat stress in all the genotypes but increase was higher in susceptible genotypes. On recovery, LOX, MDA and H2O2 content decreased in all the genotypes. The activities of antioxidative enzymes viz . superoxide dismutase (SOD), peroxidase (POX), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) increased under high temperature stress . SOD and CAT started decreasing but activity of POX and GR still continued increasing in all the genotypes. But APX enzyme exhibited differential behaviour on revival which increased in tolerant genotypes but decreased in susceptible genotypes. Ascorbic acid and carotenoids increased under heat stress but during recovery, ascorbic acid continued increasing while carotenoids started decreasing in all the genotypes. A major band of 53.12 kDa and minor bands of 100 kDa, 89.12 kDa, 74.13 kDa, 46.76 kDa and 38.9 kDa in tolerant genotypes and protein bands of molecular weight 25.79 kDa and 30.7 kDa in susceptible genotypes appeared under high temperature stress which disappeared when the stress was relieved.
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    ThesisItemOpen Access
    Isolation, partial purification and characterization of sucrose synthase from thermotolerant wheat (Triticum aestivum L.)
    (CCSHAU, 2009) Ekta; Singal, H.R.
    Wheat grain is the dominant grain of world commerce and is the staple food of millions of people world wide. High temperature beyond 300C which is usually encountered during later part of grain filling period, affects grain yield (reduction by 20-50 per cent) and grain quality. Starch is the major storage carbohydrate in wheat grains. It is synthesized from sucrose which is the principal product of leaf photosynthesis and transported to the wheat grain. Sucrose synthase is the first enzyme and an important link in sucrose-starch conversion pathway. Keeping above in view, the present investigation was conducted to purify and characterize sucrose synthase from thermotolerant wheat. Sucrose synthase was purified to near homogeneity (as revealed by single band on Native-PAGE) from immature grains (21 days after anthesis) of thermotolerant wheat WH-1021 by using conventional protein purification techniques viz. ammonium sulphate fractionation, gel filtration through sephadex G-100 and DEAE-cellulose ion exchange chromatography. The enzyme was purified about 27 fold with approximately 37 per cent recovery. The molecular weight as determined by gel filtration and subunit molecular weight as determined by SDS-PAGE (single band) were found to be 269 KDa and 63 KDa respectively indicating that enzyme is a homotetramer. The purified enzyme exhibited optimum activity at 370C temperature and pH 6.5. It was thermostable upto 500C. The activity followed Michaelis-Menten kinetics with Km value of 14.28 mM and 1.18 mM for sucrose and UDP, respectively. Among the various nucleotides tested the enzyme was highly specific for UDP as substrate. The kinetic studies revealed that sucrose synthase catalysed the sucrose degradation by ping-pong mechanism. The enzyme activity was inhibited by Mn2+ (38.5 % inhibition) while NO3- stimulated (20.8% stimulation) the activity at 2 mM concentration. Amongst various metabolites tested NADP+ and G-6-P were found to be the potent inhibitors of purified sucrose synthase (inhibiting the enzyme activity by 16 and 34%), respectively. To summarize, higher thermostability of enzyme is suggestive of enzyme’s adaptation to high temperature stress.