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ThesisItem Open Access Biochemical changes in tomato (Solanum lycopersicum L.) under the influence of salicylic acid(CCSHAU, 2017) Naresh Kumar; Singal, H.R.The present investigation was aimed to study the effect of exogenous application of salicylic acid on two tomato varieties Hisar Arun (Short shelf life) and BSS-488 (Long shelf life). Tomato fruits of both varieties were harvested at mature green and turning stage and treated with the salicylic acid at different concentrations (0.5 mM, 0.75 mM, 1.0 mM, 1.25 mM and 1.5 mM). The fruit sample was analysed for various morpho-physiological and biochemical parameters at every 3rd day after salicylic acid treatment. The molecular parameters were analysed only at optimized concentration of salicylic acid (i.e. 0.75 mM) afterwards. The morpho-physiological and biochemical parameters like PLW, TSS, color change, lycopene, β-carotene, PG and cellulase increased progressively while the parameters like cellulose, hemicellulose, lignin and pectin decreased gradually throughout the storage period which leads to the ripening of tomato. The exogenous application of salicylic acid mainly at concentrations 0.75 mM improved the shelf life of tomato by delaying the change in the above mentioned morpho-physiological and biochemical parameters. Moreover, during ripening the parameters such as total sugars, reducing sugars, non-reducing sugars, ascorbic acid, ethylene, PPO and PME activity increased initially and declined thereafter with advanced ripening in control as well as in all treated fruits. However, the treated fruits at SA concentration 0.75 and 1.0 mM expressed the delay in the initial increase in above parameters. Similarly, the expression of ripening related genes (ACS and ACO) of tomatoes of both varieties at both maturation stages increased initially and decreased thereafter which is delayed by the exogenous application of salicylic acid. In addition, the quantitative analysis (via PD Quest) of 2-DE gels of tomato proteins demonstrated the overlapping gel coordinates between control and treated fruits. But the treated fruits expressed the differentially expressed protein spots that showed >1.5 fold expression change as compared to control. So it can be concluded that the treatment at 0.75 mM concentration of salicylic acid was most effective in enhancement of shelf life of tomato fruits.ThesisItem Open Access Transcriptome analysis for zinc and iron homeostasis and carbohydrate metabolism in rice (Oryza sativa L.)(CCSHAU, 2017) Sweety; Jain, VeenaBiochemical and molecular changes in root and shoot tissues of four rice varieties (HKR47, PUSA1121, HBC19 and PAU201) were studied in response to varied iron and zinc concentrations (T1 = 0.1 mM Fe(II)-EDTA+ 0.06 mM ZnSO4.7H2O, T2 = 0.1 mM Fe(II)-EDTA+ 0.12 mM ZnSO4.7H2O, T3 = 0.1 mM Fe(II)-EDTA+ 0.48 mM ZnSO4.7H2O, T4 = 0.5 mM Fe(II)-EDTA+ 0.06 mM ZnSO4.7H2O, T5 = 0.5 mM Fe(II)-EDTA+ 0.12 mM ZnSO4.7H2O, T6 = 0.5 mM Fe(II)-EDTA+ 0.48 mM ZnSO4.7H2O and T7 = 0.05 mM Fe(II)-EDTA+ 0.48 mM ZnSO4.7H2O) at vegetative and reproductive stages. Fe content in roots, shoots and grains was maximum at T4 treatment while contrarily Zn content was highest at T7 treatment in all the four varieties. Highest iron and zinc in dehusked grains was recorded in HBC19 at all the treatments. The maximum specific enzymes’ activity of glycolytic pathway (Hexokinase, phosphofructokinase, fructose-1,6 bisphosphate aldolase, pyruvate kinase), Hexose monophosphate pathway (Glucose 6 phosphate dehydrogenase and 6-phosphate gluconate dehydrogenase) and tricarboxylic acid cycle (isocitrate dehydrogenase and malate dehydrogenase) was observed at T7 while at T4 treatment activity of these enzymes reduced the most. Reducing sugar and total soluble sugar content increased at T1, T4, T5 and T6 treatments as compared to control (T2); maximum increase being at T4. Expression of genes involved in iron and zinc homeostasis [Nicotinamine synthase 1 (OsNAS1), Nicotinamine synthase 2 (OsNAS2), Nicotinamine synthase 3 (OsNAS3), Iron-regulated transporter 1 (OsIRT1) and Yellow stripe like 2 (OsYSL2), zinc-regulated transporter (ZRT), iron-regulated transporter (IRT)-like protein 1 (OsZIP1), OsZIP3,OsZIP4 and OsZIP8] were analyzed at T2, T4 and T7 treatments at vegetative stage; reduced expression of OsNAS1,OsNAS2, OsIRT1and OsYSL2 was observed at T4 treatment in all the four varieties as compared to the control (T2) in both the tissues. Conversely, these genes had enhanced expressions at T7 treatment. OsNAS3, OsZIP1, OsZIP3, OsZIP4 and OsZIP8 genes contrarily showed enhancement in relative expressions at T4 treatment and reduction at T7 treatment as compared to control (T2). Differential gene expression under low iron or low zinc conditions suggests that poor uptake of zinc and iron via roots and further translocation to shoots may not be the reason for low iron and zinc in grains. All the four rice varieties had better growth, grain yield and yield attributes at T7 (low iron and high zinc) treatment as compared to control (T2). Higher activities of carbohydrate metabolism’s enzymes, plant growth and productivity under high zinc environment clearly indicate the importance of Zn for plants.ThesisItem Open Access Biochemical and molecular analysis of postharvest chitosan treatments in guava (Psidium guajava L.) fruits(CCSHAU, 2017) Chawla, Shilpa; Jain, VeenaDuring present investigations, guava fruits of variety Hisar Surkha harvested at mature stage and were analyzed for various physico-chemical, biochemical and molecular changes during storage. Pre-treatment of fruits with chitosan and CaCl2 alone and in combination significantly delayed decline in physiological loss in weight, total soluble solids and maintenance of fruit firmness, delay in loss of acidity, phenolics, ascorbic acid and total antioxidant activity during storage. Cell wall components viz. hemicellulose, cellulose and pectin decreased progressively throughout the storage at room temperature and low temperature. The decrease in cell wall components were in co-ordination with enhanced activities of cell wall degrading enzymes viz. PME, PG and cellulase. The storage of fruits at low temperature showed slow increase in their activities at all stages of storage thereby retaining the integrity of membrane. Oxidative stress indicies viz. superoxide radicals, hydrogen peroxide, malondialdehyde content and LOX activity increased progressively during storage at both conditions but fruit stored at low temperature showed lower oxidative stress. The activities of ROS scavenging enzymes viz. SOD, CAT, APX, POX and PPO and the content of antioxidative metabolite, ascorbic acid increased initially for few days of storage followed by decline at later stages of storage at both the temperatures. However, β-carotene content decreased continuously throughout the storage period. Ethylene production and ACC oxidase activity increased initially and decreased at later stages of storage. Pre-treatment of fruits with chitosan and CaCl2 alone and in combination resulted in significant inhibition of cell wall degrading enzymes, activation of ROS scavenging enzymes and reduction in ethylene production during storage. Transcript profiling of PG and ACO genes revealed the expression at 301 bp and 320 bp respectively. Fruits treated with combination of chitosan and CaCl2 was the most effective in inhibiting the expression of PG and ACO genes. The treatment of CaCl2 (1.5%)+chitosan (1.5%) was most effective treatment in modulating biochemical and molecular changes in guava fruits and enhancing keeping quality of guava during storage. Hence fruits given these treatments and stored at low temperature retained their quality for longer duration of time.ThesisItem Open Access Biochemical studies on synthesis, hydrolysis, characterization and storage of pearl millet lipids(CCSHAU, 2017) Bajaj, Sonali; Chugh, L.K.The present investigation is the first ever attempt for studying synthesis, hydrolysis, physico-chemical characterization and storage stability of pearl millet lipids. A field experiment on high-lipids hybrid HHB 94 raised under irrigated and water deficit condition was carried out during kharif-2014 to correlate deposition of lipids and activity of acetyl CoA carboxylase (ACCase) in developing grains. Activity of ACCase could not be determined and correlated with higher amount of deposition of lipids in the grains under irrigated condition due to very high labile nature of the enzyme. For studying hydrolysis of lipids, flour of two R-lines (male parents) viz., fast lipids hydrolyzing (FLH) G73-107 and slow lipids hydrolyzing (SLH) HBL11and two A-Lines (female parents) viz., ICMA 97111 (FLH) and ICMA 95222 (SLH), F1 hybrids: ICMA 97111A × G73-107, ICMA 97111A × HBL 11, ICMA 95222 × HBL 11and ICMA 95222 × G73-107 along with that of one population WHC 901-445, seven check hybrids, two check varieties and thirty designated B-lines was stored at 37°C and RH 40-50% for 10 days. Lipids content, rate of hydrolysis of lipids determined as periodic increase of fat acidity (FA) in flour during storage, total hydrolysis of lipids measured as total buildup of FA on day 10, level of in vitro and in situ activities of lipolytic enzymes recorded periodically and changes manifested in the water soluble fraction of flour reflected as changes in pH were determined. Fresh flour (0 day storage) served as control. Crossing of either of SLH A-line (ICMA 95222) or R-line (HBL 11) with any of FLH A-line (ICMA 97111) or R-line (G73-107) did not result in development of F1 hybrids those were SLH. No correlation was found between lipids content and either total FA or rate of development of FA or activities of esterase and lipase of R-lines, A-lines, F1 hybrids, populations, check varieties, check hybrids, or designated B-lines. Strong correlation coefficients between rate of increase in FA and activities of lipolytic enzymes (r=0.855** for in vitro esterase, r=0.773** for in situ lipase and r=0.718** for in situ esterase) in stored flour of the designated B-lines indicated that development of FA is controlled by the level of activities of lipolytic enzymes and not by lipids content. No correlation was found between decrease in pH of water extract of flour and any of the other parameters associated with hydrolysis of lipids which indicated chemical changes taking place in water soluble fraction of flour of designated B-lines are independent of fat content and activities of lipolytic enzymes. To further investigate role of lipolytic enzymes in hydrolysis of lipids in pearl millet flour, one p-nitophenyl butyrate (p-NPB) dependent esterase was partially purified by ammonium sulphate precipitation (30-60% saturation), gel filtration through Sephadex G-75. Molecular weight of the purified enzyme was approx 60 kDa. The enzyme exhibited Michaelis-Menten kinetics for its substrates, p-NPB. The Km of purified enzyme was 0.65 mM. The purified preparation exhibited optimum activity at 45°C. The enzyme was stable up to 60°C for 20 min. Studies on stored flour showed that the activities of esterase (as well as lipase) did not change for 10 days of storage. This indicated in situ stability of lipolytic enzyme at 37°C. The purified preparation showed maximum activity at pH 8.2 and substantial activity at pH between 6 and 7 (which prevails in pearl millet flour) which explains presence of highly active esterase in situ. DTT and EDTA were potent inhibitors of the purified esterase followed by PMSF and ascorbic acid. Inhibition of the enzyme by ascorbic acid might have implications for developing processing technologies for improving shelf life of flour by arresting hydrolysis of lipids. The released variety HC 20 was found to be SLH because of low activities of lipolytic enzymes compared to the population WHC 901-445. Physico-chemical properties i.e. specific gravity, saponification value, unsaponifiable matter and refractive index of lipids extracted from bran and flour of both the genotypes were near identical. Total tocopherol content in bran and flour lipids of HC 20 was higher than that of WHC 901-445. In terms of hydrolysis and oxidative changes, lipids of HC 20 were comparatively more stable during storage compared to that of WHC 901-445.ThesisItem Open Access Exogenous spermine mediated biochemical changes in wheat (Triticum aestivum L. em Thell.) under salinity(CCSHAU, 2017) Midathala, Raghavendra; Madaan, ShashiThe present investigation was aimed to study the effect of spermine and salt stress on two tolerant (Kharchia 65 and KRL 210) and two susceptible wheat varieties (DBW 88, HD 3086). Salt stress was imposed before sowing by irrigating the pots with artificial saline water of 8 and 12 dSm-1. Spermine of 0.5 and 1.0 mM were sprayed over the plants at 21 and 90 days after sowing. The leaf samples were analysed for various physiological and biochemical parameters at 0, 5, 10 and 15 days after spermine treatment. The physiological parameters like RWC, osmotic potential, chlorophyll fluorescence and leaf membrane injury declined with increasing salinity levels and decrease was more in susceptible varieties than tolerant varieties. Compared to susceptible varieties, Kharchia 65 and KRL 210 had lower levels of H2O2, O2˙ˉ and MDA which can be ascribed to an elevated levels of antioxidant metabolites like glutathione, carotenoids and effective ROS scavenging enzymes such as SOD, APX, GR, Gpx. The exogenous spermine improved the plant water status, activities of the antioxidant enzymes, metabolites and was effective in triggering the protection against salt-induced cellular damages by scavenging H2O2, O2˙ˉ and MDA content. In addition, accumulation of elevated levels of proline in tolerant varieties may be due to the increased activity of proline biosynthesizing enzymes (P5CS, P5CR) and repressed proline oxidase enzyme. The exogenous spermine may be associated with the capacity to maintain high levels of Spm, Spd and lower level of Put which in turn may be due to stimulated DAO activity and suppressed PAO activity thus accumulating higher PAs like Spm & Spd under salt stress. Isoenzymic pattern revealed single form for DAO whereas two forms of PAO were visualized on native PAGE gels under salt stress and spermine treatment. Further, application of spermine improved grain quality, yield and prolong grain filling duration under salinity. The results demonstrated that exogenous application of 0.5mM spermine could partially alleviate the deleterious effects of salinity.ThesisItem Open Access Biochemical and molecular characterization of aerobic and lowland indica rice (Oryza sativa L.) varieties(CCSHAU, 2017) Dahiya, Anjali; Jain, SunitaIn the present investigation, biochemical and molecular characterization of aerobic (MAS25 and MAS26) and high yielding lowland (HBC19, HKR47, PUSA1121 and PAU201) indica rice varieties were carried out under aerobic and well watered conditions in the greenhouse to understand the mechanism(s) of water efficiency in rice. Data was recorded on various physio-morphological parameters and activities of key enzymes and metabolites of glycolytic, HMP, fermentative and proline metabolizing pathways in root and shoot tissues at early vegetative, late vegetative and reproductive stages. Relative expression of DRO1 and genes of proline metabolism using real time PCR were also observed. Aerobic compared to low-land rice varieties, invariably had higher root length, and root biomass under under aerobic conditions. Activities of the enzymes of glycolytic (hexokinase, phoshofructo kinase, fructose-1,6-bisphosphate aldolase, pyruvate kinase), HMP (glucose -6- phosphate dehydrogenase, 6-phospho gluconate dehydrogenase) and fermentative (alcohol dehydrogenase, lactate dehydrogenase and alanine aminotransferase) pathways in both the tissues declined under aerobic conditions but reduction was significantly lower in aerobic (MAS25 and MAS26) varieties. Rice cultivation under aerobic conditions also affected the levels of various metabolites. While ethanol, lactate and alanine contents decreased, levels of reducing sugar and total sugars increased in both the tissues of all the six rice varieties under aerobic conditions. Significant positive correlation was observed between per cent change in PFK1 activity and other glycolytic (HXK, FBP aldolase and PK) enzymes’ activities, between enzymes and metabolites of fermentative pathway in roots and shoots at all the stages. High basal levels of proline content and proline synthesizing enzymes, pyrroline-5-carboxylate synthetase (P5CS) and pyrroline-5-carboxylate reductase (P5CR) were observed in root as well as shoot tissues of aerobic rice genotypes in comparison to lowland rice varieties, however, per cent enhancement was more pronounced in lowland varieties under aerobic conditions. Contrarily, proline oxidase (ProDH), the proline degrading enzyme had lower activity in aerobically grown plants of all the six varieties. Relative expression of P5CS and P5CR genes increased while that of ProDH declined more in MAS26 under aerobic conditions as compared to HBC19 in both the tissues. A positive correlation was observed between proline content and both the enzymes involved in its biosynthesis while there was a negative correlation between proline content and proline oxidase activity. In roots, expression of DRO1 declined in HBC19 (57%) but increased by 41% in MAS26 under aerobic conditions. Higher expression of DRO1, P5CS and P5CR genes and levels of proline in aerobic compared to lowland indica rice varieties may be the potential reasons for the better growth and productivity in aerobic rice under less water conditions.ThesisItem Open Access Studies on post-harvest treatments of chitosan and selenium on membrane lipids and antioxidant status in guava (Psidium guajava L.) fruit(CCSHAU, 2017) Poonam; Jain, VeenaDuring present investigations, guava fruits of variety Hisar Surkha (shelf-life 2-3 days) were harvested at immature green, mature green, ripe and over-ripe stages of ripening during summer and winter seasons. The lipoxygenase activity increased progressively throughout ripening in both the seasons but fruits harvested during winter season showed lower activity as compared tofruits of summer season. The amount of total lipids, polar lipids, non-polar lipids, phospholipids and glycolipids increased upto ripe satge and declined thereafter at the over-ripe stage in both the seasons. The ratio of unsaturated to saturated fatty acids also declined during ripening but the decline waslower in winter fruits. Pre-treatment of fruits with chitosan and seleniumalone and in combination significantly delayed decline in physiological loss in weight, total soluble solids and more retention of firmness, acidity, sugars, phenolics, flavonoids and total antioxidant acivity during storage. However, the higher doses (0.04-0.05 ppm) of selenium showed negative effect on quality of guava fruits. The oxidative stress indicated by superoxide radicals, hydrogen peroxide content, melondialdehyde content and lipoxygenase activity, increased significantly during storage at both room temperature (20±2 0C) and low temperature (5±2 0C) but fruit stored at low temperature showed lower oxidative stress. The activities of reactive oxygen species scavenging enzymes viz.superoxide dismutase, catalase, glutathione reductase, glutathione peroxidaseand glucose 6-phosphate dehydrogenase decreased continuously while the activities of ascorbate peroxidase, peroxidase and polyphenol oxidase increased initially and invariably low at the later stages during storage at both temperatures. Antioxidative metabolites, ascorbic acid increased initially and declined at later stages while glutathione and β- carotene content decreased continuously throughout the storage period. However,decrease was less in all treated fruits except higher Se concentration (0.05 ppm) and storage at low temperature. During storage all classes of lipids decreased continuously and the ratio of unsaturated to saturated fatty acid also decreased but the fruits treated with chitosan along with Se stored at low temperatue retained high amount of all lipid classes and fatty acids.The treatment of chitosan (1.5%) and Se (0.02 ppm) was most effective at low temperature than at room temperature in delaying the lipid peroxidation and oxidative stress and maintaining the textural quality and fluidity of the membrane.