Research progress on plant stress-associated protein (SAP) family: Master regulators to deal with environmental stresses.

Every year, unfavorable environmental factors significantly affect crop productivity and threaten food security. Plants are sessile; they cannot move to escape unfavorable environmental conditions, and therefore, they activate a variety of defense pathways. Among them are processes regulated by stress-associated proteins (SAPs). SAPs have a specific zinc finger domain (A20) at the N-terminus and either AN1 or C2H2 at the C-terminus. SAP proteins are involved in many biological processes and in response to various abiotic or biotic constraints. Most SAPs play a role in conferring transgenic stress resistance and are stress-inducible. The emerging field of SAPs in abiotic or biotic stress response regulation has attracted the attention of researchers. Although SAPs interact with various proteins to perform their functions, the exact mechanisms of these interactions remain incompletely understood. This review aims to provide a comprehensive understanding of SAPs, covering their diversity, structure, expression, and subcellular localization. SAPs play a pivotal role in enabling crosstalk between abiotic and biotic stress signaling pathways, making them essential for developing stress-tolerant crops without yield penalties. Collectively, understanding the complex regulation of SAPs in stress responses can contribute to enhancing tolerance against various environmental stresses through several techniques such as transgenesis, classical breeding, or gene editing.

The role of ascorbate redox turnover in iron toxicity tolerance.

Iron (Fe) toxicity is a major abiotic stress in lowland rice production. Breeding tolerant varieties has proven challenging due to the complex genetic architecture of Fe toxicity tolerance and the strong genotype-by-environment interactions. Additionally, conventional methods for phenotyping visible stress symptoms are often inaccurate, inconsistent, and lack reproducibility. In our previous work, we identified that ascorbate redox regulation, mediated by the activities of dehydroascorbate reductase (DHAR) and ascorbate oxidase (AO), contributed to high tolerance in an indica rice genotype across various environments. To explore whether this mechanism is common among other rice genotypes, we selected ten genotypes with contrasting stress symptoms under Fe-toxic conditions to examine the roles of DHAR and AO in regulating Fe toxicity tolerance. Additionally, we aimed to develop objective and accurate image-based phenotyping methods to replace the traditional leaf bronzing scoring method. Among the ten genotypes we tested, we found significant positive correlations between DHAR activity and stress symptoms in plants grown under both Fe toxicity and control conditions, suggesting a general link between ascorbate redox regulation and Fe toxicity tolerance. Using RGB signals from leaf images of plants exposed to 1000 mg/L Fe2+, we evaluated 36 different color indices to quantify stress symptoms. We identified the normalized green‒red difference index as most significant in quantifying stress symptoms under Fe toxicity conditions. Our findings suggest that DHAR activity could be potentially employed as a biomarker in the screening of rice germplasms and breeding tolerant cultivars to Fe toxicity.

FDX2, an iron-sulfur cluster assembly factor, is essential to prevent cellular senescence, apoptosis or ferroptosis of ovarian cancer cells.

Recent studies reveal that biosynthesis of iron-sulfur clusters (Fe-Ss) is essential for cell proliferation, including that of cancer cells. Nonetheless, it remains unclear how Fe-S biosynthesis functions in cell proliferation/survival. Here, we report that proper Fe-S biosynthesis is essential to prevent cellular senescence, apoptosis, or ferroptosis, depending on cell context. To assess these outcomes in cancer, we developed an ovarian cancer line with conditional KO of FDX2, a component of the core Fe-S assembly complex. FDX2 loss induced global downregulation of Fe-S-containing proteins and Fe2+ overload, resulting in DNA damage and p53 pathway activation, and driving the senescence program. p53 deficiency augmented DNA damage responses upon FDX2 loss, resulting in apoptosis rather than senescence. FDX2 loss also sensitized cells to ferroptosis, as evidenced by compromised redox homeostasis of membrane phospholipids. Our results suggest that p53 status and phospholipid homeostatic activity are critical determinants of diverse biological outcomes of Fe-S deficiency in cancer cells.

Transgenic Drosophila melanogaster Carrying a Human Full-Length DISC1 Construct (UAS-hflDISC1) Showing Effects on Social Interaction Networks.

Disrupted in Schizophrenia 1 (DISC1) is a scaffold protein implicated in major mental illnesses including schizophrenia, with a significant negative impact on social life. To investigate if DISC1 affects social interactions in Drosophila melanogaster, we created transgenic flies with second or third chromosome insertions of the human full-length DISC1 (hflDISC1) gene fused to a UAS promotor (UAS-hflDISC1). Initial characterization of the insertion lines showed unexpected endogenous expression of the DISC1 protein that led to various behavioral and neurochemical phenotypes. Social interaction network (SIN) analysis showed altered social dynamics and organizational structures. This was in agreement with the altered levels of the locomotor activity of individual flies monitored for 24 h. Together with a decreased ability to climb vertical surfaces, the observed phenotypes indicate altered motor functions that could be due to a change in the function of the motor neurons and/or central brain. The changes in social behavior and motor function suggest that the inserted hflDISC1 gene influences nervous system functioning that parallels symptoms of DISC1-related mental diseases in humans. Furthermore, neurochemical analyses of transgenic lines revealed increased levels of hydrogen peroxide and decreased levels of glutathione, indicating an impact of DISC1 on the dynamics of redox regulation, similar to that reported in transgenic mammals. Future studies are needed to address the localization of DISC1 expression and to address how the redox parameter changes correlate with the observed behavioral changes.

Participation of the stress-responsive CDSP32 thioredoxin in the modulation of chloroplast ATP-synthase activity in Solanum tuberosum.

Plant thioredoxins (TRXs) are involved in numerous metabolic and signalling pathways, such as light-dependent regulation of photosynthesis. The atypical TRX CDSP32, chloroplastic drought-induced stress protein of 32 kDa, includes two TRX-fold domains and participates in responses to oxidative stress as an electron donor to other thiol reductases. Here, we further characterised potato lines modified for CDSP32 expression to clarify the physiological roles of the TRX. Upon high salt treatments, modified lines displayed changes in the abundance and redox status of CDSP32 antioxidant partners, and exhibited sensitivity to combined saline-alkaline stress. In non-stressed plants overexpressing CDSP32, a lower abundance of photosystem II subunits and ATP-synthase γ subunit was noticed. The CDSP32 co-suppressed line showed altered chlorophyll a fluorescence induction and impaired regulation of the transthylakoid membrane potential during dark/light and light/dark transitions. These data, in agreement with the previously reported interaction between CDSP32 and ATP-synthase γ subunit, suggest that CDSP32 affects the redox regulation of ATP-synthase activity. Consistently, modelling of protein complex 3-D structure indicates that CDSP32 could constitute a suitable partner of ATP-synthase γ subunit. We discuss the roles of the TRX in the regulation of both photosynthetic activity and enzymatic antioxidant network in relation with environmental conditions.

Regulation of arsenate stress by nitric oxide and hydrogen sulfide in Oryza sativa seedlings: Implication of sulfur assimilation, glutathione biosynthesis, and the ascorbate-glutathione cycle and its genes.

Seed priming by nitric oxide (NO) and hydrogen sulphide (H2S) in combating against abiotic stress in plants is well documented. However, knowledge of fundamental mechanisms of their crosstalk is scrambled. Therefore, the reported study examined the probable role of NO and H2S in the mitigation of arsenate toxicity (As(V)) in rice seedlings and whether their potential signalling routes crossover. Results report that As(V) toxicity limited shoot and root length growth with more As accumulation in roots. As(V) further caused elevated reactive oxygen species levels, inhibited ascorbate-glutathione cycle enzymes and relative gene expression of its enzymes and thus, causing lipid and protein oxidation. These results correlate with reduced nitric oxide synthase-like and L-cysteine desulfhydrase activity along with endogenous NO and H2S. While, L-NAME or PAG augmented As(V) toxicity, and addition of SNP or NaHS effectively reversed their respective effects. Furthermore, SNP under PAG or NaHS under L-NAME were able to pacify As(V) stress, implicating that endogenous NO and H2S efficiently ameliorate As(V) toxicity but without their shared signaling in rice seedlings.

Proton gradient across the chloroplast thylakoid membrane governs the redox regulatory function of ATP synthase.

Chloroplast ATP synthase (CFoCF1) synthesizes ATP by using a proton electrochemical gradient across the thylakoid membrane, termed ΔµH+, as an energy source. This gradient is necessary not only for ATP synthesis but also for reductive activation of CFoCF1 by thioredoxin, using reducing equivalents produced by the photosynthetic electron transport chain. ΔµH+ comprises two thermodynamic components: pH differences across the membrane (ΔpH) and the transmembrane electrical potential (ΔΨ). In chloroplasts, the ratio of these two components in ΔµH+ is crucial for efficient solar energy utilization. However, the specific contribution of each component to the reductive activation of CFoCF1 remains unclear. In this study, an in vitro assay system for evaluating thioredoxin-mediated CFoCF1 reduction is established, allowing manipulation of ΔµH+ components in isolated thylakoid membranes using specific chemicals. Our biochemical analyses revealed that ΔpH formation is essential for thioredoxin-mediated CFoCF1 reduction on the thylakoid membrane, whereas ΔΨ formation is nonessential.

Exploring the unmapped cysteine redox proteoform landscape.

Cysteine redox proteoforms define the diverse molecular states that proteins with cysteine residues can adopt. A protein with one cysteine residue must adopt one of two binary proteoforms: reduced or oxidized. Their numbers scale: a protein with 10 cysteine residues must assume one of 1,024 proteoforms. Although they play pivotal biological roles, the vast cysteine redox proteoform landscape comprising vast numbers of theoretical proteoforms remains largely uncharted. Progress is hampered by a general underappreciation of cysteine redox proteoforms, their intricate complexity, and the formidable challenges that they pose to existing methods. The present review advances cysteine redox proteoform theory, scrutinizes methodological barriers, and elaborates innovative technologies for detecting unique residue-defined cysteine redox proteoforms. For example, chemistry-enabled hybrid approaches combining the strengths of top-down mass spectrometry (TD-MS) and bottom-up mass spectrometry (BU-MS) for systematically cataloguing cysteine redox proteoforms are delineated. These methods provide the technological means to map uncharted redox terrain. To unravel hidden redox regulatory mechanisms, discover new biomarkers, and pinpoint therapeutic targets by mining the theoretical cysteine redox proteoform space, a community-wide initiative termed the "Human Cysteine Redox Proteoform Project" is proposed. Exploring the cysteine redox proteoform landscape could transform current understanding of redox biology.

Ten "Cheat Codes" for Measuring Oxidative Stress in Humans.

Formidable and often seemingly insurmountable conceptual, technical, and methodological challenges hamper the measurement of oxidative stress in humans. For instance, fraught and flawed methods, such as the thiobarbituric acid reactive substances assay kits for lipid peroxidation, rate-limit progress. To advance translational redox research, we present ten comprehensive "cheat codes" for measuring oxidative stress in humans. The cheat codes include analytical approaches to assess reactive oxygen species, antioxidants, oxidative damage, and redox regulation. They provide essential conceptual, technical, and methodological information inclusive of curated "do" and "don't" guidelines. Given the biochemical complexity of oxidative stress, we present a research question-grounded decision tree guide for selecting the most appropriate cheat code(s) to implement in a prospective human experiment. Worked examples demonstrate the benefits of the decision tree-based cheat code selection tool. The ten cheat codes define an invaluable resource for measuring oxidative stress in humans.

Microbe-induced coordination of plant iron-sulfur metabolism enhances high-light-stress tolerance of Arabidopsis.

High-light stress strongly limits agricultural production in subtropical and tropical regions owing to photo-oxidative damage, decreased growth, and decreased yield. Here, we investigated whether beneficial microbes can protect plants under high-light stress. We found that Enterobacter sp. SA187 (SA187) supports the growth of Arabidopsis thaliana under high-light stress by reducing the accumulation of reactive oxygen species and maintaining photosynthesis. Under high-light stress, SA187 triggers dynamic changes in the expression of Arabidopsis genes related to fortified iron metabolism and redox regulation, thereby enhancing the antioxidative glutathione/glutaredoxin redox system of the plant. Genetic analysis showed that the enhancement of iron and sulfur metabolism by SA187 is coordinated by ethylene signaling. In summary, beneficial microbes could be an effective and inexpensive means of enhancing high-light-stress tolerance in plants.

Exploring the Redox and pH Dimension of Carbonic Anhydrases in Cancer: A Focus on Carbonic Anhydrase 3.

Significance: Both redox and pH are important regulatory processes that underpin cell physiological functions, in addition to influencing cancer cell development and tumor progression. The thioredoxin (Trx) and glutathione redox systems and the carbonic anhydrase (CA) proteins are considered key regulators of cellular redox and pH, respectively, with components of the Trx system and CAs regarded as cancer therapeutic targets. However, the redox and pH axis in cancer cells is an underexplored topic of research. Recent Advances: Structural studies of a CA family member, CA3, localized two of its five cysteine residues to the protein surface. Redox-regulated modifications to CA3 have been identified, including glutathionylation. CA3 has been shown to bind to other proteins, including B cell lymphoma-2-associated athanogene 3, and squalene epoxidase, which can modulate autophagy and proinflammatory signaling, respectively, in cancer cells. Critical Issues: CA3 has also been associated with epithelial-mesenchymal transition processes, which promote cancer cell metastasis, whereas CA3 overexpression activates the phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway, which upregulates cell growth and inhibits autophagy. It is not yet known if CA3 modulates cancer progression through its reported antioxidant functions. Future Directions: CA3 is one of the least studied CA isozymes. Further studies are required to assess the cellular antioxidant role of CA3 and its impact on cancer progression. Identification of other binding partners is also required, including whether CA3 binds to Trx in human cells. The development of specific CA3 inhibitors will facilitate these functional studies and allow CA3 to be investigated as a cancer therapeutic target.

Phylogenetic Profiling Analysis of the Phycobilisome Revealed a Novel State-Transition Regulator Gene in Synechocystis sp. PCC 6803.

Phycobilisomes play a crucial role in the light-harvesting mechanisms of cyanobacteria, red algae, and glaucophytes, but the molecular mechanism of their regulation is largely unknown. In the cyanobacterium, Synechocystis sp. PCC 6803, we identified a gene, slr0244, as a phycobilisome-related gene using phylogenetic profiling analysis, a method to predict gene function based on comparative genomics. To investigate the physiological function of the slr0244 gene, we characterize the slr0244 mutants spectroscopically. The disruption of the slr0244 gene impaired state transition, a process by which the distribution of light energy absorbed by the phycobilisomes between two photosystems was regulated in response to the changes in light conditions. The Slr0244 protein seems to act somewhere at or downstream of the sensing step of the redox state of the plastoquinone pool in the process of state transition. These findings, together with the past report of the interaction of this gene product with thioredoxin or glutaredoxin, suggest that the slr0244 gene is a novel state-transition regulator that integrates the redox signal of plastoquinone pools with that of photosystem I-reducing side. The protein has two USP (universal stress protein) motifs in tandem. The second motif has two conserved cysteine residues found in USPs of other cyanobacteria and land plants. These redox-type USPs with conserved cysteines may function as redox regulators in various photosynthetic organisms. Our study also showed the efficacy of the phylogenetic profiling analysis in predicting the function of cyanobacterial genes that have not been annotated so far.

Redox takes control.

A study of two enzymes in the brain reveals new insights into how redox reactions regulate the activity of protein kinases.

Mitochondrial Dihydrolipoamide Dehydrogenase (mtLPD1): Expression, Purification, Activity, and Redox Regulation.

Mitochondrial dihydrolipoamide dehydrogenase (mtLPD1) is a central enzyme in primary carbon metabolism, since its function is required to drive four multienzymes involved in photorespiration, the tricarboxylic acid (TCA) cycle, and the degradation of branched-chain amino acids. However, in illuminated, photosynthesizing tissue a vast amount of mtLPD1 is necessary for glycine decarboxylase (GDC), the key enzyme of photorespiration. In light of the shared role, the functional characterization of mtLPD1 is necessary to understand how the three pathways might interact under different environmental scenarios. This includes the determination of the biochemical properties and all potential regulatory mechanisms, respectively. With regards to the latter, regulation can occur through multiple levels including effector molecules, cofactor availability, or posttranslational modifications (PTM), which in turn decrease or increase the activity of each enzymatic reaction. Gaining a comprehensive overview on all these aspects would ultimately facilitate the interpretation of the metabolic interplay of the pathways within the whole subcellular network or even function as a proof of concept for genetic engineering approaches. Here, we describe the typical workflow how to clone, express, and purify plant mtLPD1 for biochemical characterization and how to analyze potential redox regulatory mechanisms in vitro and in planta.

Effect of Gentianella acuta (Michx.) Hulten against the arsenic-induced development hindrance of mouse oocytes.

The current study was designed to investigate the alleviative effect of Gentianella acuta (Michx.) Hulten (G. acuta) against the sodium arsenite (NaAsO2)-induced development hindrance of mouse oocytes. For this purpose, the in vitro maturation (IVM) of mouse cumulus-oocyte complexes (COCs) was conducted in the presence of NaAsO2 and G. acuta, followed by the assessments of IVM efficiency including oocyte maturation, spindle organization, chromosome alignment, cytoskeleton assembly, cortical granule (CGs) dynamics, redox regulation, epigenetic modification, DNA damage, and apoptosis. Subsequently, the alleviative effect of G. acuta intervention on the fertilization impairments of NaAsO2-exposed oocytes was confirmed by the assessment of in vitro fertilization (IVF). The results showed that the G. acuta intervention effectively ameliorated the decreased maturation potentials and fertilization deficiency of NaAsO2-exposed oocytes but also significantly inhibited the DNA damages, apoptosis, and altered H3K27me3 expression level in the NaAsO2-exposed oocytes. The effective effects of G. acuta intervention against redox dysregulation including mitochondrial dysfunctions, accumulated reactive oxygen species (ROS) generation, glutathione (GSH) deficiency, and decreased adenosine triphosphate (ATP) further confirmed that the ameliorative effects of G. acuta intervention against the development hindrance of mouse oocytes were positively related to the antioxidant capacity of G. acuta. Evidenced by these abovementioned results, the present study provided fundamental bases for the ameliorative effect of G. acuta intervention against the meiotic defects caused by the NaAsO2 exposure, benefiting the future application potentials of G. acuta intervention in these nutritional and therapeutic research for attenuating the outcomes of arseniasis.

Regulation of Glutathione S-Transferase Omega 1 Mediated by Cysteine Residues Sensing the Redox Environment.

Glutathione S-transferase omega 1 (GstO1) catalyzes deglutathionylation and plays an important role in the protein glutathionylation cycle in cells. GstO1 contains four conserved cysteine residues (C32, C90, C191, C236) found to be mutated in patients with associated diseases. In this study, we investigated the effects of cysteine mutations on the structure and function of GstO1 under different redox conditions. Wild-type GstO1 (WT) was highly sensitive to hydrogen peroxide (H2O2), which caused precipitation and denaturation at a physiological temperature. However, glutathione efficiently inhibited the H2O2-induced denaturation of GstO1. Cysteine mutants C32A and C236A exhibited redox-dependent stabilities and enzyme activities significantly different from those of WT. These results indicate that C32 and C236 play critical roles in GstO1 regulation by sensing redox environments and explain the pathological effect of cysteine mutations found in patients with associated diseases.

ALDH2 polymorphism and myocardial infarction: From alcohol metabolism to redox regulation.

Acute myocardial infarction (AMI) remains a leading cause of death worldwide. Increased formation of reactive oxygen species (ROS) during the early reperfusion phase is thought to trigger lipid peroxidation and disrupt redox homeostasis, leading to myocardial injury. Whilst the mitochondrial enzyme aldehyde dehydrogenase 2 (ALDH2) is chiefly recognised for its central role in ethanol metabolism, substantial experimental evidence suggests an additional cardioprotective role for ALDH2 independent of alcohol intake, which mitigates myocardial injury by detoxifying breakdown products of lipid peroxidation including the reactive aldehydes, malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE). Epidemiological evidence suggests that an ALDH2 mutant variant with reduced activity that is highly prevalent in the East Asian population increases AMI risk. Additional studies have uncovered a strong association between coronary heart disease and this ALDH2 mutant variant. It appears this enzyme polymorphism (in particular, in ALDH2*2/2 carriers) has the potential to have wide-ranging effects on thiol reactivity, redox tone and therefore numerous redox-related signaling processes, resilience of the heart to cope with lifestyle-related and environmental stressors, and the ability of the whole body to achieve redox balance. In this review, we summarize the journey of ALDH2 from a mitochondrial reductase linked to alcohol metabolism, via pre-clinical studies aimed at stimulating ALDH2 activity to reduce myocardial injury to clinical evidence for its protective role in the heart.

Redox Regulation of Phosphatase and Tensin Homolog by Bicarbonate and Hydrogen Peroxide: Implication of Peroxymonocarbonate in Cell Signaling.

Phosphatase and tensin homolog (PTEN) is a negative regulator of the phosphoinositide 3-kinases/protein kinase B (PI3K/AKT) signaling pathway. Notably, its active site contains a cysteine residue that is susceptible to oxidation by hydrogen peroxide (H2O2). This oxidation inhibits the phosphatase function of PTEN, critically contributing to the activation of the PI3K/AKT pathway. Upon the stimulation of cell surface receptors, the activity of NADPH oxidase (NOX) generates a transient amount of H2O2, serving as a mediator in this pathway by oxidizing PTEN. The mechanism underlying this oxidation, occurring despite the presence of highly efficient and abundant cellular oxidant-protecting and reducing systems, continues to pose a perplexing conundrum. Here, we demonstrate that the presence of bicarbonate (HCO3-) promoted the rate of H2O2-mediated PTEN oxidation, probably through the formation of peroxymonocarbonate (HCO4-), and consequently potentiated the phosphorylation of AKT. Acetazolamide (ATZ), a carbonic anhydrase (CA) inhibitor, was shown to diminish the oxidation of PTEN. Thus, CA can also be considered as a modulator in this context. In essence, our findings consolidate the crucial role of HCO3- in the redox regulation of PTEN by H2O2, leading to the presumption that HCO4- is a signaling molecule during cellular physiological processes.

A perspective on exogenous redox regulation mediated by transfused RBCs subject to the storage lesion.

Granted with a potent ability to interact with and tolerate oxidative stressors, RBCs scavenge most reactive oxygen and nitrogen species (RONS) generated in circulation. This essential non-canonical function, however, renders RBCs susceptible to damage when vascular RONS are generated in excess, making vascular redox imbalance a common etiology of anemia, and thus a common indication for transfusion. This accentuates the relevance of impairments in redox metabolism during hypothermic storage, as the exposure to chronic oxidative stressors upon transfusion could be exceedingly deleterious to stored RBCs. Herein, we review the prominent mechanisms of the hypothermic storage lesion that alter the ability of RBCs to scavenge exogenous RONS as well as the associated clinical relevance.

The redox-sensitive R-loop of the carbon control protein SbtB contributes to the regulation of the cyanobacterial CCM.

SbtB is a PII-like protein that regulates the carbon-concentrating mechanism (CCM) in cyanobacteria. SbtB proteins can bind many adenyl nucleotides and possess a characteristic C-terminal redox sensitive loop (R-loop) that forms a disulfide bridge in response to the diurnal state of the cell. SbtBs also possess an ATPase/ADPase activity that is modulated by the redox-state of the R-loop. To investigate the R-loop in the cyanobacterium Synechocystis sp. PCC 6803, site-specific mutants, unable to form the hairpin and permanently in the reduced state, and a R-loop truncation mutant, were characterized under different inorganic carbon (Ci) and light regimes. Growth under diurnal rhythm showed a role of the R-loop as sensor for acclimation to changing light conditions. The redox-state of the R-loop was found to impact the binding of the adenyl-nucleotides to SbtB, its membrane association and thereby the CCM regulation, while these phenotypes disappeared after truncation of the R-loop. Collectively, our data imply that the redox-sensitive R-loop provides an additional regulatory layer to SbtB, linking the CO2-related signaling activity of SbtB with the redox state of cells, mainly reporting the actual light conditions. This regulation not only coordinates CCM activity in the diurnal rhythm but also affects the primary carbon metabolism.