Manganese-induced oxidative stress, ultrastructural changes, and proteomics studies in rice plants.

Manganese (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.

Terminalia arjuna bark extract alleviates nickel toxicity by suppressing its uptake and modulating antioxidative defence in rice seedlings.

Terminalia arjuna (Ta) bark contains various natural antioxidants and has been used to protect animal cells against oxidative stress. In the present study, we have examined alleviating effects of Ta bark aqueous extract against Ni toxicity in rice (Oryza sativa L.). When rice seedlings were raised for 8 days in hydroponics in Yoshida nutrient medium containing 200 µM NiSO4, a decline in height, reduced biomass, increased Ni uptake, loss of root plasma membrane integrity, increase in the level of O2˙-, H2O2 and ˙OH, increased lipid peroxidation, decline in photosynthetic pigments, increase in the level of antioxidative enzymes superoxide dismutase, catalase and glutathione peroxidase and alterations in their isoenzyme profile patterns were observed. Transmission electron microscopy (TEM) showed damage to chloroplasts marked by disorganised enlarged starch granules and disrupted thylakoids under Ni toxicity. Exogenously adding Ta bark extract (3.2 mg ml-1) to the growth medium considerably alleviated Ni toxicity in the seedlings by reducing Ni uptake, suppressing generation of reactive oxygen species, reducing lipid peroxidation, restoring level of photosynthesis pigments and ultrastructure of chloroplasts, and restoring levels of antioxidative enzymes. Results suggest that Ta bark extract considerably alleviates Ni toxicity in rice seedlings by preventing Ni uptake and reducing oxidative stress in the seedlings.

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.

Aluminum (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.

Exogenous application of calcium and silica alleviates cadmium toxicity by suppressing oxidative damage in rice seedlings.

The present study was undertaken to examine the possible roles of calcium (Ca(2+)) and silica (Si) in protection against oxidative damage due to Cd(2+) toxicity in rice (Oryza sativa L.) seedlings grown in hydroponics. Rice seedlings raised for 12 days in hydroponics containing Cd(NO3)2 (75 µM) showed reduced growth; increase in the level of reactive oxygen species (ROS) (O2 (·-) and H2O2), thiobarbituric acid reactive substances (TBARSs) and protein carbonylation; and increase in the activity of antioxidant enzymes-superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (GPX) compared to untreated controls. Exogenously added Ca(2+) (2 mM) and Si (200 µM) significantly alleviated negative effect of Cd(2+) by restoration of growth of the seedlings, suppression of Cd(2+) uptake and restoration of root plasma membrane integrity. The levels of O2 (·-), H2O2, lipid peroxidation and protein carbonyls were much lower when Ca(2+) and Si were added in the growth medium along with Cd(2+) as compared to Cd-alone-treated seedlings. Ca(2+) and Si lowered Cd-induced increase in SOD, GPX and APX activities while they elevated Cd-induced decline in CAT activity. Using histochemical staining of O2 (·-) and H2O2 in leaf tissues, it was further confirmed that added Ca(2+) and Si suppressed Cd-induced accumulation of O2 (·-) and H2O2 in the leaves. The results suggest that exogenous application of Ca(2+) and Si appears to be advantageous for rice plants in alleviating Cd(2+) toxicity effects by reducing Cd(2+) uptake, decreasing ROS production and suppressing oxidative damage. The observations indicate that Ca(2+) and Si treatments can help in reducing Cd(2+) toxicity in rice plants.

Cadmium and lead interactive effects on oxidative stress and antioxidative responses in rice seedlings.

Interactive 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 (O2˙- 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.