Title Participants Abstract "H2O2-dependent oxidation of the transcription factor GmNTL1 promotes salt tolerance in soybean" "Wenxiao Zhang, Wenjiao Zhi, Hong Qiao, Jingjing Huang, Shuo Li, Qing Lu, Nan Wang, Qiang Li, Qian Zhou, Jiaqi Sun, Yuting Bai, Xiaojian Zheng, Mingyi Bai, Frank Van Breusegem, Fengning Xiang" "Reactive oxygen species (ROS) play an essential role in plant growth and responses to environmental stresses. Plant cells sense and transduce ROS signaling directly via hydrogen peroxide (H2O2)-mediated post translational modifications (PTMs) on protein cysteine residues. Here, we show that the H2O2-mediated cysteine oxidation of NAC WITH TRANS-MEMBRANE MOTIF1-LIKE 1 (GmNTL1) in soybean (Glycine max) during salt stress promotes its release from the endoplasmic reticulum (ER) membrane and translocation to the nucleus. We further show that an oxidative post-translational modification on GmNTL1 residue Cys-247 steers downstream amplification of ROS production by binding to and activating the promoters of RESPIRATORY BURST OXIDASE HOMOLOG B (GmRbohB) genes, thereby creating a feed-forward loop to fine-tune GmNTL1 activity. In addition, oxidation of GmNTL1 Cys-247 directly promotes the expression of CATION H+ EXCHANGER 1 (GmCHX1)/SALT TOLERANCE-ASSOCIATED GENE ON CHROMOSOME 3 (GmSALT3) and Na+/H+ Antiporter 1 (GmNHX1). Accordingly, transgenic overexpression of GmNTL1 in soybean increases the H2O2 levels and K+/Na+ ratio in the cell, promotes salt tolerance and increases yield under salt stress, while an RNA interference-mediated knockdown of GmNTL1 elicits the opposite effects. Our results reveal that the salt-induced oxidation of GmNTL1 promotes its relocation and transcriptional activity through an H2O2-mediated post-translational modification on cysteine that improves resilience of soybean against salt stress." "Quantitative measurements of biochemical and molecular markers of oxidative stress signaling and responses" "Graham Noctor, Mathias Cohen, Frank Van Breusegem, Amna M'Hamdi" "Increases in cellular oxidation are a part of most plant responses to challenging conditions and are commonly described as oxidative stress. While this phenomenon is closely related to the accumulation of reactive oxygen species, these latter compounds can be difficult to measure. Complementary measurements to assess cellular redox state are, therefore, very useful in studies of plant responses to stress. Here, we detail protocols for three complementary approaches that can be used to assess the intensity of oxidative stress. These involve quantification of marker transcripts, assays of the extractable activities of major antioxidative enzymes, and measurement of antioxidant buffers. We confirm experimentally that the data obtained by such approaches can provide reliable information on the intensity of oxidative stress." "Mutation of Arabidopsis SME1 and Sm core assembly improves oxidative stress resilience" "Valerie Van Ruyskensvelde, Takanori Maruta, Robin Pottie, Álvaro Daniel Fernández Fernández, Matthew A. Hannah, Frank Van Breusegem, Katrien Van Der Kelen" "Mechanisms controlling plant proteases and their substrates" "Álvaro Daniel Fernández Fernández, Simon Stael, Frank Van Breusegem" "Metabolite modification in oxidative stress responses : a case study of two defense hormones" "Caroline Lelarge-Trouverie, Mathias Cohen, Lug Trémulot, Frank Van Breusegem, Amna M'Hamdi, Graham Noctor" "Hydrogen peroxide in plants" "Amna M'Hamdi" "Oxidative stress responses in plants" "Frank Van Breusegem, Claire Remacle" "Cysteine thiol–based post-translational modification : what do we know about transcription factors?" "Heng Zhou, Jingjing Huang, Patrick Willems, Frank Van Breusegem, Yanjie Xie" "Reactive electrophilic species are ubiquitous in plant cells, where they contribute to specific redox-regulated signaling events. Redox signaling is known to modulate gene expression during diverse biological processes, including plant growth, development, and environmental stress responses. Emerging data demonstrates that transcription factors (TFs) are a main target of cysteine thiol-based oxidative post-translational modifications (OxiPTMs), which can alter their transcriptional activity and thereby convey redox information to the nucleus. Here, we review the significant progress that has been made in characterizing cysteine thiol-based OxiPTMs, their biochemical properties, and their functional effects on plant TFs. We discuss the underlying mechanism of redox regulation and its contribution to various physiological processes as well as still outstanding challenges in redox regulation of plant gene expression." "CysQuant : simultaneous quantification of cysteine oxidation and protein abundance using data dependent or independent acquisition mass spectrometry" "Jingjing Huang, An Staes, Francis Impens, Vadim Demichev, Frank Van Breusegem, Kris Gevaert, Patrick Willems" "Protein cysteinyl thiols are susceptible to reduction-oxidation reactions that can influence protein function. Accurate quantification of cysteine oxidation is therefore crucial for decoding protein redox regulation. Here, we present CysQuant, a novel approach for simultaneous quantification of cysteine oxidation degrees and protein abundancies. CysQuant involves light/heavy iodoacetamide isotopologues for differential labeling of reduced and reversibly oxidized cysteines analyzed by data-dependent acquisition (DDA) or data-independent acquisition mass spectrometry (DIA-MS). Using plexDIA with in silico predicted spectral libraries, we quantified an average of 18% cysteine oxidation in Arabidopsis thaliana by DIA-MS, including a subset of highly oxidized cysteines forming disulfide bridges in AlphaFold2 predicted structures. Applying CysQuant to Arabidopsis seedlings exposed to excessive light, we successfully quantified the well-established increased reduction of Calvin-Benson cycle enzymes and discovered yet uncharacterized redox-sensitive disulfides in chloroplastic enzymes. Overall, CysQuant is a highly versatile tool for assessing the cysteine modification status that can be widely applied across various mass spectrometry platforms and organisms." "Quantitative measurement of ascorbate and glutathione by spectrophotometry" "Graham Noctor, Amna M'Hamdi" "Ascorbate and glutathione are key chemical antioxidants present at relatively high concentrations in plant cells. They are also reducing cofactors for enzymes that process hydrogen peroxide in the ascorbate-glutathione pathway. Due to these two related biochemical functions, the compounds form an interface between reactive oxygen species and sensitive cellular components. Therefore, their status can provide reliable and direct information on cell redox state, signaling, and plant health. While several methods exist for quantification of ascorbate and glutathione, simple enzyme-dependent assays allow them to be measured easily and inexpensively in common extracts. This chapter describes a protocol to measure total contents, as well as the major oxidized and reduced forms, of both compounds in plant tissues."