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.