Thioredoxins (Trxs) are ubiquitous oxidoreductases that maintain cellular redox homeostasis through thiol-disulfide exchange reactions. Escherichia coli thioredoxin 2 (EcTrx2) possesses a unique N-terminal zinc-binding domain absent from the canonical thioredoxin EcTrx1, but the physiological significance of this domain has remained unclear. Here we show that EcTrx2 undergoes reversible, redox-dependent structural switching accompanied by a functional conversion under oxidative stress. Oxidative conditions promoted the formation of high-molecular-weight (HMW) oligomeric complexes, whereas reducing conditions favored low-molecular-weight (LMW) species. Increased surface hydrophobicity of oxidized EcTrx2 correlated with a marked enhancement of holdase chaperone activity and a concomitant reduction in disulfide reductase activity. Size-exclusion chromatography coupled with transmission electron microscopy further revealed that the HMW oligomers were the predominant chaperone-active species, whereas the LMW form primarily retained reductase activity. Deletion of the N-terminal zinc-binding domain abolished the redox-dependent structural transition and impaired chaperone activation, demonstrating that this domain is required for stress-responsive functional switching. These findings identify EcTrx2 as a redox-regulated molecular chaperone and provide a mechanistic basis for reversible structural and functional switching in a bacterial thioredoxin during oxidative stress.

The pervasive accumulation of elevated heavy metal concentrations in the environment exerts significant selective pressures, driving the progressive evolution of a comprehensive suite of adaptive strategies. These include both physiological tolerance and biochemical detoxification mechanisms. Our research identified a novel role of a rice-specific m-type thioredoxin (OsTrx-m) in conferring tolerance specifically to copper-induced heavy metal stress. Upon exposure to CuSO4, recombinant OsTrx-m underwent a rapid functional transformation from a disulfide reductase to a holdase chaperone, accompanied by a concurrent alteration in protein conformation from a monomeric form to oligomeric complexes. This transition was highly selective for Cu2+ and did not occur with other metal cations, such as Fe2+, Zn2+, Cd2+, and Ni2+. The Cu2+-induced structural and functional transitions of OsTrx-m were reversibly mitigated by adding EDTA, a metal chelator. Comparative analysis of OsTrx-m with Arabidopsis m-type thioredoxins (AtTrx-m1, -m2, and -m3) revealed that this Cu2+-responsive property is a distinctive characteristic of OsTrx-m. Consequently, ectopic expression of OsTrx-m, but not AtTrx-m1, in Arabidopsis (OsTrx-mOE/Col-0 and AtTrx-m1OE/Col-0, respectively) conferred robust tolerance to copper-induced toxicity, as evidenced by enhanced root growth and fresh weight.

To coordinate growth and acclimation, plants must accurately discriminate physiological reactive oxygen species (ROS) signals from those derived from environmental stress. Xing et al. (2026) resolve the 'specificity paradox' of ROS signaling by identifying Radical-induced Cell Death1 (RCD1) as a redox-responsive molecular sieve. Under basal conditions, RCD1 utilizes its intrinsically disordered regions to drive liquid-liquid phase separation (LLPS), forming nuclear condensates that preferentially entrap the transcription factor ASYMMETRIC LEAVES1 (AS1). Conversely, stress-induced ROS accumulation triggers the oxidation of a conserved cysteine triad (C371/379/392), provoking a phase transition that dismantles these assemblies. This structural reorganization triggers ZAT12-mediated antioxidant defenses, establishing redox-driven phase separation as a pivotal regulatory nexus for the rapid, spatiotemporal recalibration of plant physiological states.

EMR, an endoplasmic reticulum (ER)-associated degradation-mediating RING-finger E3 ligase initially identified as an ER-resident protein, was unexpectedly detected in the cytoplasm under ER stress conditions. This unanticipated subcellular distribution prompted us to explore the uncharacterized functions of cytoplasmic EMR. Using an integrated approach that combines bioinformatics, biochemical, and physiological analyses, we show that EMR also acts as a specific heat-responsive regulator. Heat exposure causes Zn2+ to dissociate from the RING-finger domain of EMR, triggering a conformational change from monomeric to oligomeric form driven by increased hydrophobicity. This structural reorganization, mediated by Zn2+ coordination, transforms EMR's function from an E3 ligase to a molecular chaperone. Importantly, this functional switch contributes significantly to plant thermotolerance, as EMR's chaperone activity effectively prevents heat-induced protein unfolding and aggregation of vital cellular proteins. Consequently, transgenic Arabidopsis lines overexpressing EMR in an EMR-null-mutant (emr) background, 35S:EMR, showed significantly improved thermotolerance compared to wild-type and emr plants. The critical role of EMR's chaperone activity in conferring heat-stress resistance was further substantiated by the observation that 35S:EMR(C/S) plants, in which EMR(C/S) primarily retains chaperone function, exhibited heat-stress tolerance comparable to that of 35S:EMR plants. Proteomic profiling after heat shock identified candidate client substrates of EMR's chaperone activity, which are mainly involved in critical cellular processes such as protein translation and energy metabolism, highlighting EMR's broad cytoprotective role. Taken together, these results demonstrate that the bifunctional properties of EMR are essential for environmental-stress recognition and adaptive signaling, thereby driving thermotolerance in plants.

Within plant cells, a pivotal cohort of thiol-based redoxins, encompassing thioredoxins, glutaredoxins, and peroxiredoxins and characterized by pKa values spanning the physiological range of 6.0 to 8.0, orchestrates cellular redox homeostasis through the mechanism of thiol-disulfide exchange. Their active site cysteine residues enable redox modulation of target proteins under physiological pHs. Electron transfer-mediating proteins constitute critical molecular conduits that orchestrate fundamental cellular redox signaling mechanisms. These molecular processes subsequently modulate intricate cellular regulatory pathways, encompassing comprehensive developmental programming, hormonal signaling cascades, and the sophisticated synchronization of circadian rhythmicity within biological systems. In this review, we elucidate the definition, classification, molecular mechanisms, and physiological functions of thiol-based redoxins. Furthermore, it incorporates recent advances to reveal newly characterized physiological roles, governing a broad spectrum of biological activities. The integrated analysis of the proteins highlights the essential contribution of these molecules to cellular and metabolic balance while simultaneously delineating a trajectory for future research focused on their application in augmenting plant stress tolerance and productivity.

Thioredoxins are ubiquitous thiol-disulfide oxidoreductases that maintain intracellular redox homeostasis. In addition to its conserved catalytic domain, Escherichia coli thioredoxin 2 (EcTrx2) possesses a unique N-terminal zinc-binding domain whose physiological function remains largely unknown. Here, we identify a previously unrecognized DNA-binding activity of EcTrx2 and demonstrate its role in protecting DNA during oxidative stress. Electrophoretic mobility shift assays showed that EcTrx2 bound plasmid DNA in a concentration-dependent and GST-tag-independent manner, whereas EcTrx1 exhibited no detectable DNA-binding activity. DNA binding was abolished by deletion of the N-terminal zinc-binding domain and was blocked by zinc occupancy, indicating that this unique domain is essential for DNA interaction. Consistent with these findings, EcTrx2 significantly protected plasmid DNA from DNase I digestion and hydroxyl radical-mediated oxidative damage in vitro. Furthermore, EcTrx2 enhanced bacterial tolerance to the DNA-damaging agents zeocin and diamide, supporting the physiological relevance of its DNA-binding activity. Our results reveal a DNA-binding role for EcTrx2 and identify its N-terminal zinc-binding domain as a key determinant of DNA binding and protection against oxidative DNA damage.

The intricate interplay between cellular circadian rhythms,primarily manifested in the chloroplast redox os-cillations-characterized by diel hyperoxidation/reduction cycles of 2-Cys peroxiredoxins-and the nu-clear transcription/translation feedback loop(TTFL)machinery within plant cells,demonstrates a remark-able temporal coherence.However,the molecular mechanisms underlying the integration of these circadian rhythms remain elusive.In this study,we reveal that the chloroplast redox protein,NADPH-dependent thioredoxin reductase type C(NTRC),modulates the integration of the chloroplast redox rhythms and nuclear circadian clocks by regulating intracellular levels of reactive oxygen species and su-crose.In NTRC-def icient ntrc mutants,the perturbed temporal dynamics of cytosolic metabolite pools sub-stantially attenuate the amplitude of CIRCADIAN CLOCK ASSOCIATED 1(CCA1)mRNA oscillation while maintaining its inherent periodicity.In contrast,these fluctuations extend the period and greatly reduced the amplitude of GIGANTEA(GI).In alignment with its regulatory role,the chloroplast redox rhythm and TTFL-driven nuclear oscillators are severely disrupted in ntrc plants.The impairements are rescued by NTRC expression but not by the expression of catalytically inactive NTRC(C/S)mutant,indicating that NTRC's redox activity is essential for synchronizing intracellular circadian rhythms.In return,the canonical nuclear clock component,TIMING OF CAB EXPRESSION 1(TOC1),regulates the diel chloroplast redox rhythm by controlling NTRC expression,as evidenced by the redox cycle of chloroplast 2-Cys peroxiredox-ins.This reciprocal regulation suggests a tight coupling between chloroplast redox rhythms and nuclear oscillators.Collectively,our study has identified NTRC as a key circadian modulator,elucidating the intri-cate connection between the metabolite-dependent chloroplast redox rhythm and the temporal dynamics of nuclear canonical clocks.

The endoplasmic reticulum (ER) is a cellular organelle responsible for protein synthesis and folding. When its protein folding capacity is exceeded, unfolded or misfolded proteins accumulate, causing ER stress and triggering the unfolded protein response (UPR) to restore ER proteostasis. Although UPR gene expression in plants follows a diel cycle, the mechanisms by which the circadian clock regulates these genes remain unclear. Here, we demonstrate that sensitivity to ER stress in root growth exhibits time-of-day phases and that the circadian clock regulates UPR target gene expression during ER stress. Notably, mutations in the core morning clock component CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) impair ER stress recovery. CCA1 forms a complex with the UPR modulator basic leucine zipper 28 (bZIP28) and acts as an upstream regulator of ER stress recovery. Upon ER stress, CCA1 is stabilized and associates with bZIP28 at the ER stress response element within the BiP3 promoter, enhancing the ER stress response. Thus, CCA1 and bZIP28 coordinate a time-dependent adaptive response to ER stress to maintain ER proteostasis. Our results suggest that the circadian clock primes the timing and levels of ER chaperone expression to enhance ER stress tolerance.

A recent study has demonstrated that NADPH-thioredoxin reductase type C (NTRC) plays a crucial role in coordinating the diel redox rhythms of the chloroplast with nuclear circadian oscillators, particularly by regulating the expression of CCA1, which is significantly reduced in ntrc mutants compared to wild-type (WT) Col-0 plants. However, the specific molecules responsible for transmitting NTRC-mediated alterations of metabolite signals to nuclear clock components remained elusive. To address this intriguing question, we investigated the changes in intracellular reactive oxygen species (ROS) and sucrose levels in NTRC-deficient, ntrc, mutant, and compared them with WT plants. Our study revealed a notable increase in ROS levels and a decrease in sucrose concentration in ntrc mutant plants compared to Col-0 controls, highlighting the distinct influence of NTRC deficiency on these metabolites. Through extensive bioinformatic analyses that elucidated the mechanistic role of pseudo-response regulators (PRRs) linking fluctuations in cytoplasmic metabolites to the nuclear clock components CCA1/LHY, we performed comparative transcriptional profiling of PRR expression patterns between ntrc mutant and the WT controls. The results indicate a substantial upregulation of PRR genes in ntrc mutants compared to Col-0 plants. Furthermore, in prr975 triple mutants, the expression of CCA1 could not be restored by treatment with DMTU and sucrose. These results support that PRRs act as transducers, conveying NTRC-dependent metabolic signals to the nuclear TTFL clock component, CCA1.

Phytohormone auxin plays a pivotal role in governing plant growth, development, and responses to abiotic stresses. YUCCA6 (YUC6), an auxin biosynthetic enzyme belonging to the flavin monooxygenase (FMO) subfamily, converts indole-3-pyruvic acid to indole-3-acetic acid. Our prior investigation uncovered that YUC6 also functions as a thiol-reductase and chaperone in a Cys85-dependent manner, resulting in conferred tolerance to nickel heavy metal stress and drought and delayed leaf senescence. Notably, the conserved co-factor binding sites (FAD and NADPH) in YUC6, shared with FMOs and thioredoxin reductase, prompted our exploration into their significance for holdase chaperone activity and oxidative stress tolerance in Arabidopsis. We demonstrate that YUC6 transcripts are upregulated in response to methyl viologen (MV)-induced oxidative stress, implicating YUC6 in oxidative stress response. Mutations in co-factor binding sites markedly diminish the chaperone activity of YUC6, and reduce the YUC6-mediated oxidative stress tolerance in Arabidopsis. Furthermore, YUC6 proteins exist as oligomeric states under native conditions, formed by disulfide-bond bridges. Oligomeric YUC6 displays enhanced chaperone activity compared to its monomeric YUC6. We found here that co-factor binding sites of YUC6 are necessary for its chaperone properties.

Arabidopsis Universal Stress Protein (USP; At3g53990) plays critical roles in acting as a stress shield and regulating the expression of the nuclear clock gene, CIRCADIAN CLOCK ASSOCIATED 1(CCA1). To elucidate the reciprocal regulatory interplay between USP and CCA1 proteins, this study employed in silico analysis of USP promoter, uncovering four putative CCA1-binding motifs, and subsequently validating CCA1's interaction with these elements. We investigated the role of CCA1 in regulating USP expression by generating CCA1-deficient mutants (cca1-1) harboring a PUSP:LUC reporter. These mutants exhibited a pronounced enhancement in USP amplitude, implicating CCA1 as a negative regulator of USP expression. Subsequently, we investigated the protein dynamics of USP, utilizing plants expressing HA-tagged-USP prepared in usp background (PUSP:USP-HA/usp). Immunoblotting unveiled rhythmic oscillations in USP abundance, prompting us to delineate the regulatory mechanisms governing USP's circadian rhythm. Treatment of plants with protein synthesis and proteasome inhibitors revealed that USP abundance decreases during the night through proteasome-mediated degradation. Leveraging the STRING protein-interaction databases, we identified the culprit behind USP degradation and validated PUB35 as the USP-specific E3-ligase. Our findings elucidate the intricate reciprocal interplay between the stress-responsive protein and the circadian clock machinery, illuminating the mechanistic underpinnings that govern the daily oscillations in USP abundance.

The Arabidopsis quiescin sulfhydryl oxidase 1 (QSOX1) thiol-based redox sensor has been identified as a negative regulator of plant immunity. Here, we have found that small molecular weight proteins of QSOX1 were converted to high molecular weight (HMW) complexes upon exposure to heat stress and that this was accompanied by a switch in QSOX1 function from a thiol-reductase to a molecular chaperone. Plant treatment with S-nitrosoglutathione (GSNO), which causes nitrosylation of cysteine residues (S-nitrosylation), but not with H2O2, induced HMW QSOX1 complexes. Thus, functional switching of QSOX1 is induced by GSNO treatment. Accordingly, simultaneous treatment of plants with heat shock and GSNO led to a significant increase in QSOX1 chaperone activity by increasing its oligomerization. Consequently, transgenic Arabidopsis overexpressing QSOX1 (QSOX1OE) showed strong resistance to heat shock, whereas qsox1 knockout plants exhibited high sensitivity to heat stress. Plant treatment with GSNO under heat stress conditions increased their resistance to heat shock. We conclude that S-nitrosylation allows the thiol-based redox sensor, QSOX1, to respond to various external stresses in multiple ways.

Plants have developed multilayered defense strategies to adapt and acclimate to the kaleidoscopic environmental changes that rapidly produce reactive oxygen species (ROS) and induce redox changes. Thiol-based redox sensors containing the redox-sensitive cysteine residues act as the central machinery in plant defense signaling. Here, we review recent research on thiol-based redox sensors in plants, which perceive the changes in intracellular H2O2 levels and activate specific downstream defense signaling. The review mainly focuses on the molecular mechanism of how the thiol sensors recognize internal/external stresses and respond to them by demonstrating several instances, such as cold-, drought-, salinity-, and pathogen-resistant signaling pathways. Also, we introduce another novel complex system of thiol-based redox sensors operating through the liquid-liquid phase separation.

Ubiquitous disulfide reductases, thioredoxins (Trxs), function in the redox balance of all living organisms. Although the roles of the rice (Oryza sativa) Trx m-type isoform (OsTrxm) in chloroplast development have been already published, biochemical and molecular functions of OsTrxm remain to be elucidated for decades. The OsTrxm and its two conserved active cysteine mutant (OsTrxm C95S/C98S, referred to as OsTrxmC/S) proteins in Arabidopsis thaliana were overexpressed to characterize in vivo roles of active cysteines of OsTrxm. Interestingly, the OsTrxm overexpressed variant plants were resistant to heat shock treatment. Especially OsTrxmC/S with higher molecular weight (HMW) complexes showed higher heat tolerance than OsTrxm with lower molecular weight (LMW) structure in Arabidopsis thaliana. To confirm the importance of active cysteines on structural changes under heat stress, OsTrxm and OsTrxmC/S proteins were bacterially expressed and isolated. This study found that two proteins have various structures ranging from LMW to HMW complexes and have potential functions as a disulfide reductase and a molecular chaperone, which has never been reported anywhere. The function of molecular chaperone predominated in the HMW complexes, whereas the disulfide reductase function was observed in LMW forms. These results suggest that the active cysteines of OsTrxm play a critical role in protein structural change as well as heat tolerance in plants.

Abstract The precise timing of flowering in adverse environments is critical for plants to secure reproductive success. We report a mechanism in Arabidopsis (Arabidopsis thaliana) controlling the time of flowering by which the S-acylation-dependent nuclear import of the protein SALT OVERLY SENSITIVE3/CALCINEURIN B-LIKE4 (SOS3/CBL4), a Ca2+-signaling intermediary in the plant response to salinity, results in the selective stabilization of the flowering time regulator GIGANTEA inside the nucleus under salt stress, while degradation of GIGANTEA in the cytosol releases the protein kinase SOS2 to achieve salt tolerance. S-acylation of SOS3 was critical for its nuclear localization and the promotion of flowering, but partly dispensable for salt tolerance. SOS3 interacted with the photoperiodic flowering components GIGANTEA and FLAVIN-BINDING, KELCH REPEAT, F-BOX1 and participated in the transcriptional complex that regulates CONSTANS to sustain the transcription of CO and FLOWERING LOCUS T under salinity. Thus, the SOS3 protein acts as a Ca2+- and S-acylation-dependent versatile regulator that fine-tunes flowering time in a saline environment through the shared spatial separation and selective stabilization of GIGANTEA, thereby connecting two signaling networks to co-regulate the stress response and the time of flowering.

The tomato (Solanum lycopersicum) is widely consumed globally and renowned for its health benefits, including the reduction of cardiovascular disease and prostate cancer risk. However, tomato production faces significant challenges, particularly due to various biotic stresses such as fungi, bacteria, and viruses. To address this challenges, we employed the CRISPR/Cas9 system to modify the tomato NUCLEOREDOXIN (SlNRX) genes (SlNRX1 and SlNRX2) belonging to the nucleocytoplasmic THIOREDOXIN subfamily. CRISPR/Cas9-mediated mutations in SlNRX1 (slnrx1) plants exhibited resistance against bacterial leaf pathogen Pseudomonas syringae pv. maculicola (Psm) ES4326, as well as the fungal pathogen Alternaria brassicicola. However, the slnrx2 plants did not display resistance. Notably, the slnrx1 demonstrated elevated levels of endogenous salicylic acid (SA) and reduced levels of jasmonic acid after Psm infection, in comparison to both wild-type (WT) and slnrx2 plants. Furthermore, transcriptional analysis revealed that genes involved in SA biosynthesis, such as ISOCHORISMATE SYNTHASE 1 (SlICS1) and ENHANCED DISEASE SUSCEPTIBILITY 5 (SlEDS5), were upregulated in slnrx1 compared to WT plants. In addition, a key regulator of systemic acquired resistance, PATHOGENESIS-RELATED 1 (PR1), exhibited increased expression in slnrx1 compared to WT. These findings suggest that SlNRX1 acts as a negative regulator of plant immunity, facilitating infection by the Psm pathogen through interference with the phytohormone SA signaling pathway. Thus, targeted mutagenesis of SlNRX1 is a promising genetic means to enhance biotic stress resistance in crop breeding.

The activities of cold-responsive C-repeat-binding transcription factors (CBFs) are tightly controlled as they not only induce cold tolerance but also regulate normal plant growth under temperate conditions 1 – 4 . Thioredoxin h2 (Trx-h2)—a cytosolic redox protein identified as an interacting partner of CBF1—is normally anchored to cytoplasmic endomembranes through myristoylation at the second glycine residue 5 , 6 . However, after exposure to cold conditions, the demyristoylated Trx-h2 is translocated to the nucleus, where it reduces the oxidized (inactive) CBF oligomers and monomers. The reduced (active) monomers activate cold-regulated gene expression. Thus, in contrast to the Arabidopsis trx-h2 (AT5G39950) null mutant, Trx-h2 overexpression lines are highly cold tolerant. Our findings reveal the mechanism by which cold-mediated redox changes induce the structural switching and functional activation of CBFs, therefore conferring plant cold tolerance.

Plants are exposed to various environmental stimuli, including abiotic and biotic stresses, during their life cycle. In Arabidopsis thaliana, the Universal Stress Protein (AtUSP) acts as a protein chaperone and RNA chaperone to protect plants from heat shock and cold shock, respectively. This study aimed to explore the role of AtUSP in plant growth and development. We conducted morphological analyses of wild-type (WT; Col-0), AtUSP overexpression (AtUSPOE), and atusp knockout mutant plants during their vegetative growth, and measured the total leaf number, leaf size, and root length. Compared with the WT, AtUSPOE plants displayed enhanced growth, whereas atusp plants displayed reduced growth of all examined organs. To investigate whether these growth phenotypes were caused by changes in cell expansion and proliferation, we analyzed the mature leaves of all genotypes at the cellular level. The enlarged leaves of AtUSPOE plants showed an increase in cell size, but not in cell number, indicating that AtUSP promotes cell expansion. Moreover, expression analysis of cell growth-related genes revealed that AtUSP is involved in cell expansion rather than cell proliferation. These findings suggest that AtUSP acts as a positive regulator of cell expansion, and provide novel insights into its role in plant growth and development.

In the original publication [...].