Bicarbonate Induced Redox Proteome Changes in Arabidopsis Suspension Cells

Abstract

Climate change as a result of increasing atmospheric CO2 affects plant growth and productivity. CO2 is not only a carbon donor for photosynthesis but also an environmental signal that can perturb cellular redox homeostasis and lead to modifications of redox-sensitive proteins. Although redox regulation of protein functions has emerged as an important mechanism in several biological processes, protein redox modifications and how they function in plant CO2 response remain unclear. Here a new iodoTMTRAQ proteomics technology was employed to analyze changes in protein redox modifications in Arabidopsis thaliana suspension cells in response to bicarbonate (mimic of elevated CO2) in a time-course study. A total of 47 potential redox-regulated proteins were identified with functions in carbohydrate and energy metabolism, transport, ROS scavenging, cell structure modulation and protein turnover. This inventory of previously unknown redox responsive proteins in Arabidopsis bicarbonate responses lays a foundation for future research toward understanding the molecular mechanisms underlying plant CO2 responses.

Characterization of thiol‐based redox modifications of Brassica napusSNF1‐related protein kinase 2.6‐2C

“…MS‐based proteomics approaches have been used to identify redox‐regulated thiol modifications with different cysteine‐tagging techniques such as isotope‐coded affinity tagging (ICAT) , cysteine reactive tandem mass tagging (cysTMT) , and iodoacetyl tandem mass tagging (iodoTMT) . In spite of the rapid progress in discovering proteins with specific redox PTMs in response to biotic or abiotic stresses , only limited studies have characterized the biological functions of the redox PTMs .…”

“…Another two controls consisted of an IAM blocking step, a reducing step with either DTT or GSH and Grx, and an mBBr labeling step, for testing effects of reductants only. Excessive mBBr was removed by the dichloromethane extraction described above, and the samples were digested with a modified trypsin (1 : 1 w/w) (Promega, USA) at 37 °C for 12 h. The digestion was terminated by adding 0.1% formic acid, and the tryptic peptides were then lyophilized . The resulting samples were cleaned up by solid‐phase extraction with ZipTip (Millipore, USA) according to the manufacturer's instructions.…”

“…The peptides were dissolved in 0.1% formic acid and then subjected to LC‐MS/MS analysis. The scan range of the precursor ions on the Q Exactive Plus (Thermo Fisher Scientific Inc., USA) was set as 400–2000 m/z, and a top 20 data‐dependent acquisition method was used for MS/MS . The acquired MS/MS spectra were searched against a B. napus database (http://www.genoscope.cns.fr/brassicanapus/data/) with an additional sequence of the recombinant BnSnRK2.6‐2C using Mascot (v2.4, Matrix Science, UK).…”

S-Nitroso-Proteome Revealed in Stomatal Guard Cell Response to Flg22

“…To identify nitrosylated proteins and uncover their roles in flg22-triggered stomatal closure, we used a double-labeling strategy with iodoTMT and iTRAQ, which allows for simultaneous analysis of cysteine redox changes and total protein level change [38,39]. In this strategy, we conducted redox proteomic analysis of control and treatment samples at 15, 30, and 60 min time points.…”

NaCl-responsive ROS scavenging and energy supply in alkaligrass callus revealed from proteomic analysis

“…The activities of MDHAR, DHAR and GR were measured by detecting the absorbance changes at 340 nm as the oxidation of NADH, at 265 nm as the production of GSSG, and at 340 nm as the oxidation of NADPH, respectively. Their activities were expressed as the amount of NADH oxidized, GSSG produced, and NADPH oxidized per minute per milligram protein, respectively [80]. For all the enzyme activity assays, protein content was determined using the Bradford method [81].In addition, the contents of AsA, DHA, GSH, and GSSG were measured by recording the absorbance changes at 525 nm [82].…”