ORIGINAL ARTICLE Manganese-induced oxidative stress, ultrastructural changes, and proteomics studies in rice plants Ritika Rajpoot 1 & Rajneesh Kumar Srivastava 1 & Anjana Rani 1 & Poonam Pandey 1 & R. S. Dubey 1 Received: 2 June 2020 /Accepted: 13 October 2020 # Springer-Verlag GmbH Austria, part of Springer Nature 2020 Abstract 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 O 2 ·- ,H 2 O 2 , 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 ap- pearance 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 pro- teins, 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 down- regulated 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. Keywords Manganese . Oxidative stress . Chloroplast . Mitochondria . Proteomics . Photosynthesis . Oryza sativa L Introduction Manganese (Mn) is abundantly present in the earth’s crust and in soil and it is available in different ionic forms. It is an essential element for growth and metab- olism of plants. Though Mn is present in the soil in different oxidation states, divalent Mn (Mn 2+ ) is more easily available to plants due to its greater solubility in the soil (Millaleo et al. 2010). Availability of Mn 2+ to plants depends on several parameters such as pH of the soil, redox potential of the rhizosphere, temperature, and moisture status (Husson 2013). Mn has multiple roles in plants. It participates in photolysis of water as a part of oxygen-evolving complex (OEC) associated with PS II, and it is involved in ATP synthesis, ribulose- bisphosphate carboxylase (RUBISCO) activity, biosyn- thesis of fatty acids, amino acids, and proteins, and also as cofactor of many enzymes (Millaleo et al. 2010). Besides, it also participates in biosynthesis of chloro- phylls, nitrate assimilation, and hormonal activation (Boojar and Goodarzi 2008; Millaleo et al. 2010). Solubility of Mn in soil is low at alkaline or neutral pH. At alkaline pH, it gets adsorbed onto soil particles and becomes unavailable to plant roots, whereas in acidic soils with pH lower than 5.0, solubility of Mn increases and it becomes readily available to plants (Millaleo et al. 2010). Therefore Handling Editor: Handling Editor: Bhumi Nath Tripathi * R. S. Dubey rsdubey_biochem@bhu.ac.in 1 Department of Biochemistry, Institute of Science, Banaras Hindu University, Varanasi 221005, India Protoplasma https://doi.org/10.1007/s00709-020-01575-0