Proteomic Identification Of Redox Sensitive Proteins And Functional Characterization Of Redox Protein Fumarase In Brassica Napus L.

Disruption of redox metabolism under environmental stresses results in enhanced production of intracellular reactive oxygen species?ROS?,which may lead to posttranslational modifications?PTMs?in structure and molecular function of responsive proteins.Redox PTMs are important mediators of cellular signaling and regulation and several proteomic approaches have been attempted to quantify them under various stresses in many crops.Salt stress brings dynamic changes in plant proteome by triggering enhanced production of reactive oxygen species,ultimately leading to oxidative modifications within the cell,which can be quantified through various proteomic approaches.We aimed to generate large-scale redox proteomics data in response to short-term salt stress in B.napus by analyzing reversible cysteine modification.For this purpose,we employed iodoTMT approach to analyze the redox proteome of B.napus seedlings subjected to 200 m M salt stress for four hours.This approach involves iodoTMT labelling of cysteine residues,HPLC fractionation,affinity enrichment and LC-MS/MS analysis followed by computational analysis.Using this approach,we identified 2010 peptides in1017 proteins,of which 1809 sites in 909 proteins had oxidative modification.These oxidization modified proteins were involved in regulation of various metabolic,molecular and cellular processes.Proteins involved in photosynthesis and nitrogen metabolism had reduced expression,while those mediating cysteine and methionine metabolism,arginine biosynthesis,carbon fixation and regulation of protein processing had increased abundance.We were able to locate position of modified cysteines within protein,which can help in elucidating the mechanism of salt stress response by plant at molecular level,as cysteine residues are main target of post translational modifications induced by stress.This technique also detected less abundant,acidic,basic and even hydrophobic proteins.Two oxidatively modified proteins,fructose 1-6,bisphosphatase and phosphoglycerate kinase were selected from the dataset and their in vitro activity under oxidative stress was assayed to validate the findings of proteomics study.The identified proteins are potential candidates for future functional characterization studies aimed to obtain exquisitely detailed information about the molecular mechanism of plants under salt stress.In addition,mitochondrial fumarase,a key enzyme of TCA cycle,has been identified in previous redox proteomic studies.We intended to check the role of fumarase in plant growth,metabolism and stress response.Transgenic lines with fumarase overexpression were developed in both Arabidopsis and B.napus.Our results confirmed the redox regulation of fumarase expression,as H₂O₂content was significantly higher in OE lines.Analysis of photosynthetic parameters in B.napus showed improved photosynthesis in terms of more chlorophyll content,stomatal conductance,carbon dioxide assimilation and electron transport rate.Over expression?OE?lines also performed well under both short term and long term salt stress,where PSII quantum yield and carbon assimilation was higher,while MDA and H₂O₂ contents were lesser than WT in saline environment.In order to observe corresponding lipid remodeling under salt stress,physiological levels of galactolipids?MGDG and DGDG?and phospholipids?PC,PE,PG,PI,PS,PA,DAG and TAG?were measured under both control and long term salt stress conditions.No significant change was observed in levels of MGDG,DGDG and phospholipid species under control conditions,but PC,PS,PG and PA had significantly altered levels under varying salt concentrations highlighting the influence of redox regulation over phospholipid species.Growth performance was also checked in pot experiment under control and sequential salt stress.Salt stress reduced rate of flowering in both arabidopsis and B.napus.Also,antioxidant activities?SOD and CAT?were higher in OE lines as compared to WT under both conditions,but fumarase activity was arrested at flowering stage.Under field conditions,overexpressing fumarase had early flowering and more biomass than wild type?WT?,resulting from increased number of effective branches and lesser nonproductive silique,while enhanced oil content was coupled with higher levels of desirable fatty acids.From all these findings,it can be concluded that overexpression of mitochondrial fumarase improves photosynthesis and aerial growth and plays a vital role in growth regulation and salt stress response in B.napus.Our current findings highlighted the potential role of fumarase in increasing crop productivity under both normal and saline environment.We suggest that these transgenic lines should be incorporated in future breeding programs intended for such purpose.