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PublicationArticle Nickel-induced oxidative stress and the role of antioxidant defence in rice seedlings(2009) Ruchi Maheshwari; R.S. DubeySeedlings of rice (Oryza sativa L.) cv. Pant-12 grown in sand cultures containing 200 and 400 μM NiSO4, showed a decrease in length and fresh weight of roots and shoots. Nickel was readily taken up by rice seedlings and the concentration was higher in roots than shoots. Nickel-treated seedlings showed increased rates of superoxide anion (O2•-) production, elevated levels of H2O2 and thiobarbituric acid reactive substances (TBARS) demonstrating enhanced lipid peroxidation, and a decline in protein thiol levels indicative of increased protein oxidation compared to controls. With progressively higher Ni concentrations, non-protein thiol and ascorbate (AsA) increased, whereas the level of low-molecular-weight thiols (such as glutathione and hydroxyl-methyl glutathione), the ratio of these thiols to their corresponding disulphides, and the ratio of AsA to dehydroascorbic acid declined in the seedlings. Among the antioxidant enzymes studied, the activities of all isoforms of superoxide dismutase (Cu-Zn SOD, Mn SOD and Fe SOD), guaiacol peroxidases (GPX) and ascorbate peroxidase (APX) increased in Ni-treated seedlings, while no clear alteration in catalase activity was evident. Activity of the ascorbate-glutathione cycle enzymes monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR)-significantly increased in Ni-treated seedlings. However such increase was apparently insufficient to maintain the intracellular redox balance. Results suggest that Ni induces oxidative stress in rice plants, resulting in enhanced lipid peroxidation and decline in protein thiol levels, and that (hydroxyl-methyl) glutathione and AsA in conjunction with Cu-Zn SOD, GPX and APX are involved in stress response. © Springer Science+Business Media B.V. 2009.PublicationArticle Alleviation of chromium toxicity in rice seedling using Phyllanthus emblica aqueous extract in relation to metal uptake and modulation of antioxidative defense(Elsevier B.V., 2019) A.K. Pandey; A. Gautam; P. Pandey; R.S. DubeyChromium is a key metal pollutant of the environment. When present in high concentrations in soil it adversely affects plant growth and productivity. Phyllanthus emblica (P. emblica) aqueous extract contains many natural antioxidants, and it has been widely used in traditional medicine for the treatment of severe oxidative stress-related diseases and disorders. The present study was aimed to investigate the possible role of P. emblica aqueous extract in alleviating Cr toxicity effects in rice (Oryza sativa) seedlings in relation to growth attributes, Cr uptake, production of reactive oxygen species (ROS), lipid peroxidation, and activities of antioxidative enzymes. Rice seedlings were raised for 4–8 days in Yoshida nutrient medium in hydroponics containing either 100 μM Cr (K2Cr2O7) or Cr + P. emblica aqueous extract. Cr treatment to the seedlings caused decrease in root and shoots length and fresh biomass, decline in relative water content (RWC) and photosynthetic pigments and increase in root membrane permeability. Uptake of Cr was more in roots than in shoots. Increased production of ROS, increase in lipid peroxidation and increased activities of antioxidative enzymes superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX) and ascorbate peroxidase (APX) were observed in Cr-treated seedlings as compared to controls. With the addition of aqueous extract of P. emblica (5 mg ml− 1) in the Cr treatment medium, significant alleviation of Cr-induced decline in growth, biomass, photosynthetic pigments, and reduction in Cr-induced elevated activities of antioxidative enzymes in the seedlings was observed. Our results suggest that P. emblica aqueous extract considerably alleviates the effects of Cr in rice seedlings by suppressing Cr uptake, reducing oxidative stress and modulating activities of antioxidative enzymes. © 2018PublicationArticle Manganese-induced oxidative stress, ultrastructural changes, and proteomics studies in rice plants(Springer, 2021) Ritika Rajpoot; Rajneesh Kumar Srivastava; Anjana Rani; Poonam Pandey; R.S. DubeyManganese (Mn) is an essential element for plant growth but it becomes phytotoxic at higher concentrations. The effect of Mn-excess in hydroponics medium was examined on growth, oxidative stress, and ultrastructural changes in chloroplasts and mitochondria as well proteomic alterations in rice (Oryza sativa L.) seedlings. Seedlings grown with 1 mM and 2 mM Mn in nutrient medium for 8 days showed decline in length and fresh biomass, and decline in net photosynthetic rate, transpiration rate, and stomatal conductance. Shoots of the seedlings had higher Mn content than roots. Mn-treated seedlings showed increased production of O2·−, H2O2, and .OH, increased lipid peroxidation, increased carbonylation of proteins, and increased proteolytic activity compared to untreated seedlings. Mn-treated seedlings showed disorganization and swelling of chloroplasts with appearance of plastoglobuli in TEM images and deformity in shape of mitochondria. Using confocal microscopy depolarization of mitochondrial membrane was observed marked by green fluorescence of JC-1 dye monomers in Mn-treated roots. Proteomics studies from leaves of Mn-treated seedlings involving 2DE and PDQuest analysis showed differential expression of 23 proteins, among which MALDI-TOF/TOF mass spectrometry analysis revealed Mn-led downregulation of photosynthesis-related proteins, namely oxygen-evolving complex protein associated with PSII, PAP-3, enzyme involved in protein folding peptidyl-prolyl cis-trans isomerase (PPIase) and carbohydrate metabolizing enzymes hydrolase, fructose-bisphosphate aldolase, transketolase, and isocitrate dehydrogenase, whereas ATP-dependent Clp protease, peroxidase, and nucleic acid–binding proteins were downregulated due to Mn treatment. Results indicate that Mn-excess inhibits growth of rice plants with induction of oxidative stress, causing structural alterations in chloroplasts, mitochondria, inhibiting photosynthesis, and downregulating many photosynthesis and carbohydrate metabolism–related proteins. © 2020, Springer-Verlag GmbH Austria, part of Springer Nature.PublicationArticle Cadmium and lead interactive effects on oxidative stress and antioxidative responses in rice seedlings(Springer-Verlag Wien, 2014) Rajneesh Kumar Srivastava; Poonam Pandey; Ritika Rajpoot; Anjana Rani; R.S. DubeyInteractive effects of two heavy metal pollutants Cd and Pb in the growth medium were examined on their uptake, production of reactive oxygen species (ROS), induction of oxidative stress and antioxidative defence responses in Indica rice (Oryza sativa L.) seedlings. When rice seedlings in sand culture were exposed to 150 μM Cd (NO3)2 or 600 μM Pb (CH3COO)2 individually or in combination for 8-16 days, a significant reduction in root/shoot length, fresh weight, relative water content, photosynthetic pigments and increased production of ROS (O2̇- and H2O2) was observed. Both Cd and Pb were readily taken up by rice roots and localisation of absorbed metals was greater in roots than in shoots. When present together in the growth medium, uptake of both the metals Cd and Pb declined by 25-40 %. Scanning electron microscope (SEM) imaging of leaf stomata revealed that Pb caused more distortion in the shape of guard cells than Cd. Dithizone staining of roots showed localisation of absorbed Cd on root hairs and epidermal cells. Both Cd and Pb caused increased lipid peroxidation, protein carbonylation, decline in protein thiol and increase in non-protein thiol. The level of reduced forms of non-enzymic antioxidants glutathione (GSH) and ascorbate (AsA) and their redox ratios (GSH/AsA) declined, whereas the activities of antioxidative enzymes superoxide dismutase (SOD) and guaiacol peroxidase (GPX) increased in metal treated seedlings compared to controls. In-gel activity staining also revealed increased intensities of SOD and GPX isoforms with metal treatments. Catalase (CAT) activity increased during early days (8 days) of metal exposure and declined by 16 days. Results suggest that oxidative stress is an important component in expression of Cd and Pb toxicities in rice, though uptake of both metals gets reduced considerably when present together in the medium. © 2014 Springer-Verlag Wien.PublicationArticle Manganese-excess induces oxidative stress, lowers the pool of antioxidants and elevates activities of key antioxidative enzymes in rice seedlings(2011) Sarita Srivastava; R.S. DubeyManganese (Mn) is an essential element for plant growth but in excess, specially in acidic soils, it can become phytotoxic. In order to investigate whether oxidative stress is associated with the expression of Mn toxicity during early seedling establishment of rice plants, we examined the changes in the level of reactive oxygen species (ROS), oxidative stress induced an alteration in the level of non-enzymic antioxidants and activities of antioxidative enzymes in rice seedlings grown in sand cultures containing 3 and 6 mM MnCl2. Mn treatment inhibited growth of rice seedlings, the metal increasingly accumulated in roots and shoots and caused damage to membranes. Mn treated plants showed increased generation of superoxide anion (O2.-), elevated levels of H2O2 and thiobarbituric acid reactive substances (TBARS) and decline in protein thiol. The level of nonprotein thiol, however, increased due to Mn treatment. A decline in contents of reduced ascorbate (AsA) and glutathione (GSH) as well as decline in ratios of their reduced to oxidize forms was observed in Mn-treated seedlings. The activities of antioxidative enzymes superoxide dismutase (SOD) and its isoforms Mn SOD, Cu/Zn SOD, Fe SOD as well as guaiacol peroxidase (GPX) increased in the seedlings due to Mn treatment however, catalase (CAT) activity increased in 10 days old seedlings but it declined by 20 days under Mn treatment. The enzymes of Halliwell-Asada cycle, ascorbate peroxidase (APX) monodehydoascorbate reductase (MDHAR), dehyroascorbate reductase (DHAR) and glutathione reductase (GR) increased significantly in Mn treated seedlings over controls. Results suggest that in rice seedlings excess Mn induces oxidative stress, imbalances the levels of antioxidants and the antioxidative enzymes SOD, GPX, APX and GR appear to play an important role in scavenging ROS and withstanding oxidative stress induced by Mn. © 2010 Springer Science+Business Media B.V.PublicationArticle Differential responses of antioxidative defense system to prolonged salinity stress in salt-tolerant and salt-sensitive Indica rice (Oryza sativa L.) seedlings(Springer-Verlag Wien, 2013) Pallavi Mishra; Kumari Bhoomika; R.S. DubeyThe present investigation evaluated the ability of an antioxidative defense system in terms of the tolerance against salinity-induced oxidative stress and also explored a possible relationship between the status of the components of an antioxidative defense system and the salt tolerance in Indica rice (Oryza sativa L.) genotypes. When the seedlings of a salt-sensitive cultivar was grown in sand cultures containing different NaCl concentrations (7 and 14 dS m-1) for 5-20 days, a substantial increase was observed in the rate of superoxide anion (O2·-) production, elevated levels of H2O2 and thiobarbituric acid reactive substances (TBARS) which indicated an enhancement in lipid peroxidation. A declination in the level of thiol clearly indicated an increase in the protein oxidation as well as a decline in the reduced forms of ascorbate (AsA) and glutathione (GSH) and the ratios of their reduced to oxidized forms occurred in the salt-sensitive seedlings. Similar treatment caused a very little alteration or no change in the levels of these components in the seedlings of salt-tolerant cultivar. The activity of antioxidative enzymes superoxide dismutase (SOD), its isoform Cu/Zn-SOD and ascorbate peroxidase (APX) increased in both the cultivars against salinity. In salt-sensitive seedlings, the activity of the various enzymes, guaiacol peroxidase (GPX), catalase (CAT), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) increased at moderate salinity treatment of 7 dS m-1 NaCl while the activities of these enzymes declined with higher salinity level of 14 dS m-1 NaCl. However, a consistent increase was observed in the activities of these enzymes of salt-tolerant seedlings with an increase in the duration and the level of the salinity treatment. The results suggest that a higher status of antioxidants (AsA and GSH) and a coordinated higher activity of the enzymes (SOD, CAT, GPX, APX, and GR) can serve as the major determinants in the model for depicting salt tolerance in Indica rice seedlings. © 2011 Springer-Verlag.PublicationArticle Salicylic acid alleviates aluminum toxicity in rice seedlings better than magnesium and calcium by reducing aluminum uptake, suppressing oxidative damage and increasing antioxidative defense(2013) Poonam Pandey; Rajneesh Kumar Srivastava; R.S. DubeyAluminum toxicity is a major constraint to crop production in acid soils. The present study was undertaken to examine the comparative ameliorating effects of salicylic acid, Ca and Mg on Al toxicity in rice (Oryza sativa L.) seedlings grown in hydroponics. Al treatment (0.5 mM AlCl3) caused decrease in plant vigour, loss of root plasma membrane integrity, increased contents of O2•-, H2O2, lipid peroxidation, protein carbonyls and decline in the level of protein thiol. Al treatment caused significant changes in activity of antioxidative enzymes in rice seedlings. Exogenously added salicylic acid (60 μM), Ca (1 mM) and Mg (0.25 mM) significantly alleviated Al toxicity effects in the seedlings marked by restoration of growth, suppression ofAl uptake, restoration of root plasma membrane integrity and decline in O2•-, H 2O2, lipid peroxidation and protein carbonyl contents. Salicylic acid, Ca and Mg suppressed Al-induced increase in SOD, GPX and APX activities while it elevated Al-induced decline in CAT activity. By histochemical staining of O2•- using NBT and H 2O2 using DAB, it was further confirmed that added salicylic acid, Ca orMg decreasedAl-induced accumulation ofO2 •- and H2O2 in the leaf tissues. Results indicate that exogenously added salicylic acid, Ca or Mg alleviates Al toxicity in rice seedlings by suppressing Al uptake, restoring root membrane integrity, reducing ROS level and ROS induced oxidative damage and regulating the level of antioxidative enzyme activities. Further salicylic appears to be superior toMg and Ca in alleviating Al toxicity effects in rice plants. © Springer Science+Business Media New York 2013.PublicationArticle Involvement of oxidative stress and role of antioxidative defense system in growing rice seedlings exposed to toxic concentrations of aluminum(2007) Pallavi Sharma; R.S. DubeyWhen seedlings of rice (Oryza sativa L.) cultivar Pant-12 were raised in sand cultures containing 80 and 160 μM Al3+ in the medium for 5-20 days, a regular increase in Al3+ uptake with a concomitant decrease in the length of roots as well as shoots was observed. Al3+ treatment of 160 μM resulted in increased generation of superoxide anion (O 2 -) and hydrogen peroxide (H2O2), elevated amount of malondialdehyde, soluble protein and oxidized glutathione and decline in the concentrations of thiols (-SH) and ascorbic acid. Among antioxidative enzymes, activities of superoxide dismutase (SOD EC 1.15.1.1), guaiacol peroxidase (Guaiacol POX EC 1.11.1.7), ascorbate peroxidase (APX EC 1.11.1.11), monodehydroascorbate reductase (MDHAR EC 1.6.5.4), dehydroascorbate reductase (EC 1.8.5.1) and glutathione reductase (EC 1.6.4.2) increased significantly, whereas the activities of catalase (EC EC 1.11.1.6) and chloroplastic APX declined in 160 μM Al3+ stressed seedlings as compared to control seedlings. The results suggest that Al3+ toxicity is associated with induction of oxidative stress in rice plants and among antioxidative enzymes SOD, Guaiacol POX and cytosolic APX appear to serve as important components of an antioxidative defense mechanism under Al3+ toxicity. PAGE analysis confirmed the increased activity as well as appearance of new isoenzymes of APX in Al3+ stressed seedlings. Immunoblot analysis revealed that changes in the activities of APX are due to changes in the amounts of enzyme protein. Similar findings were obtained when the experiments were repeated using another popular rice cv. Malviya-36. © 2007 Springer-Verlag.PublicationArticle Water deficit and aluminum interactive effects on generation of reactive oxygen species and responses of antioxidative enzymes in the seedlings of two rice cultivars differing in stress tolerance(Springer Verlag, 2016) Poonam Pandey; Rajneesh Kumar Srivastava; Ritika Rajpoot; Anjana Rani; Akhilesh Kumar Pandey; R.S. DubeyAluminum (Al) is a major constraint to crop productivity in acid soils, whereas water deficit severely limits crop production in arid and semi-arid regions of the world. The objective of the present study was to examine the effects of both stresses, Al excess and water deficit, individually and in combination on the production of the reactive oxygen species (ROS) superoxide anion (O2˙−), hydrogen peroxide (H2O2), hydroxyl radical, and lipid peroxidation and the activity of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and guaiacol peroxidase (GPX) in the seedlings of two rice (Oryza sativa L.) cvs. Malviya-36 (sensitive to water deficit and Al) and Vandana (tolerant to water deficit and Al). When 15-day grown seedlings were exposed to water deficit (created with 15 % polyethylene glycol, PEG-6000) or Al (1 mM AlCl3) treatment or both treatments together for 24–72 h, the lengths and fresh weights of root/shoot declined in the seedlings of the sensitive cultivar, whereas in the tolerant seedlings, either little or insignificant decline in these parameters was observed due to the treatments. Biochemical determinations and histochemical studies revealed that under a similar level of water deficit, Al, or combined treatment, seedlings of sensitive cultivar showed a higher level of production of O2˙−, H2O2, hydroxyl radical, and lipid peroxides compared to the tolerant seedlings. Seedlings of tolerant cultivars, both in roots and shoots, had constitutively higher activity levels of antioxidative enzymes SOD, CAT, and GPX and showed a greater increase in activity under water deficit or Al treatment alone or in combination compared to the similarly treated seedlings of sensitive cultivar. Our results suggest that a lower constitutive level of ROS and a high antioxidative enzyme capacity are associated with tolerance to both water deficit and Al excess in rice seedlings. © 2015, Springer-Verlag Berlin Heidelberg.PublicationArticle Nitric oxide alleviates manganese toxicity by preventing oxidative stress in excised rice leaves(2012) Sarita Srivastava; R.S. DubeyIn the present study, we have investigated the effects of nitric oxide (NO) on alleviating manganese (Mn)-induced oxidative stress in rice leaves. Exogenous MnCl 2 treatment to excised rice leaves for 24 and 48 h resulted in increased production of H 2O 2 and lipid peroxides, decline in the levels of antioxidants, glutathione and ascorbic acid, and increased activities of antioxidative enzymes, superoxide dismutase, guaiacol peroxidase, catalase, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. Treatment of rice leaves with 100 μM sodium nitroprusside (SNP), a NO donor, was effective in reducing Mn-induced increased levels of H 2O 2, lipid peroxides and increased activities of antioxidative enzymes. The levels of reduced ascorbate and glutathione were considerably recovered due to SNP treatment. The effect of SNP was reversed by the addition of NO scavenger, 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (c-PTIO) suggesting that ameliorating effect of SNP is due to release of NO. The results indicate that MnCl 2 induces oxidative stress in excised rice leaves, lowers the levels of reduced ascorbate and glutathione, and elevates activities of the key antioxidative enzymes. NO appears to provide a protection to the rice leaves against Mn-induced oxidative stress and that exogenous NO application could be advantageous in combating the deleterious effects of Mn-toxicity in rice plants. © 2011 Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków.