H2O2 Signaling Research Articles

Sodium bisulfite boosted exopolysaccharide production by Auxenochlorella protothecoides: Potential mechanisms harnessing H2O2 signaling and carbon reallocation.

Microalgal exopolysaccharides (EPS) possess significant functional benefits across various industrial sectors, but their commercial feasibility is constrained by inefficient synthesis and poorly understood synthesis mechanisms. This study found that 1.25 mmol/L sodium bisulfite promoted EPS accumulation to 224.78 mg/L by Auxenochlorella protothecoides. Inhibiting endogenous H2O2 blocked the sodium bisulfite-induced increase in EPS content and reversed the upregulation of EPS biosynthesis-related genes, underscoring the involvement of H2O2 signaling in microalgal EPS accumulation. Furthermore, omics analysis revealed a reconfiguration of metabolites in carbohydrates, proteins, and lipids, facilitating the reallocation of carbon flux. Specifically, sodium bisulfite reduced protein storage while enhancing carbohydrate anabolism for EPS precursors, redirecting carbon towards EPS accumulation. This process acted as a defense against oxidative stress, aided by increased lipid and starch production. Collectively, the findings boosted microalgal EPS production and elucidated the mechanisms involving cellular H2O2 signaling and carbon redistribution, providing theoretical support for large-scale microalgal EPS production.

Astrocyte-to-neuron H2O2 signalling supports long-term memory formation in Drosophila and is impaired in an Alzheimer's disease model.

Astrocytes help protect neurons from potential damage caused by reactive oxygen species (ROS). While ROS can also exert beneficial effects, it remains unknown how neuronal ROS signalling is activated during memory formation, and whether astrocytes play a role in this process. Here we discover an astrocyte-to-neuron H2O2 signalling cascade in Drosophila that is essential for long-term memory formation. Stimulation of astrocytes by acetylcholine induces an increase in intracellular calcium ions, which triggers the generation of extracellular superoxide (O2•-) by astrocytic NADPH oxidase. Astrocyte-secreted superoxide dismutase 3 (Sod3) converts O2•- to hydrogen peroxide (H2O2), which is imported into neurons of the olfactory memory centre, the mushroom body, as revealed by in vivo H2O2 imaging. Notably, Sod3 activity requires copper ions, which are supplied by neuronal amyloid precursor protein. We also find that human amyloid-β peptide, implicated in Alzheimer's disease, inhibits the nAChRα7 astrocytic cholinergic receptor and impairs memory formation by preventing H2O2 synthesis. These findings may have important implications for understanding the aetiology of Alzheimer's disease.

A bi-kinase module sensitizes and potentiates plant immune signaling.

Systemic signaling is an essential hallmark of multicellular life. Pathogen encounter occurs locally but triggers organ-scale and organismic immune responses. In plants, elicitor perception provokes systemically expanding Ca2+ and H2O2 signals conferring immunity. Here, we identify a Ca2+ sensing bi-kinase module as becoming super-activated through mutual phosphorylation and as imposing synergistically enhanced NADPH oxidase activation. A combined two-layer bi-kinase/substrate phospho-code allows for sensitized signaling initiation already by near-resting elevations of Ca2+ concentration. Subsequently, it facilitates further signal wave proliferation with minimal Ca2+ amplitude requirement, triggering protective defense responses throughout the plant. Our study reveals how plants build and perpetuate trans-cellular immune signal proliferation while avoiding disturbance of ongoing cellular signaling along the path of response dissemination.

Apoplastic pH is a chemical switch for extracellular H2O2 signaling in abscisic acid-mediated inhibition of cotyledon greening.

The apoplastic pH (pHApo) in plants is susceptible to environmental stimuli. However, the biological implications of pHApo variation have remained largely unknown. The universal stress phytohormone abscisic acid (ABA) as well as the major environmental stimuli drought and salinity were selected as representative cases to investigate how changes in pHApo relate to plant behaviors in Arabidopsis. Variations in pHApo negatively regulated the cotyledon greening inhibition to the universal stress hormone ABA or environmental stimuli through the action of extracellular hydrogen peroxide (eH2O2). Further studies revealed that an increase in pHApo diminishes the chemical reactivity of eH2O2, effectively functioning as an 'off' switch for its action in oxidizing thiols of plasma membrane proteins. Consequently, this suppresses the eH2O2-mediated cotyledon greening inhibition to environmental stimuli and ABA, alongside inhibiting the eH2O2-mediated intracellular Ca2+ signaling. Conversely, a decrease in pHApo serves as an 'on' switch for the action of eH2O2. In summary, the pHApo is a crucial messenger and chemical switch for eH2O2 in signal transduction, notwithstanding the apparent simplicity of the underlying mechanism. Our findings provide a novel fundamental biological insight into the significance of pH.

Insights of cellular and molecular changes in sugarcane response to oxidative signaling

Main conclusionSignificant changes in the proteome highlight essential metabolic adaptations for development and oxidative signaling induced by the treatment of young sugarcane plants with hydrogen peroxide. These adaptations suggest that hydrogen peroxide acts not only as a stressor but primarily as a signaling molecule, triggering specific metabolic pathways that regulate growth and plant resilience.Sugarcane is a crucial crop for sugar and ethanol production, often influenced by environmental signals. Hydrogen peroxide (H2O2) is increasingly recognized as an important signaling molecule that regulates plant development and adaptation. In this study, two-month-old sugarcane plants were treated with varying concentrations of H2O2 to investigate how this molecule acts as a signal at the cellular, biochemical, and proteomic levels. Antioxidant enzyme activity exhibited fluctuations, suggesting a dynamic response to oxidative signaling. Lipid peroxidation, observed through TBARs and scanning electron microscopy, highlighted early membrane modifications. Proteomic analysis (ProteomeXchange PXD048142) identified 2,699 proteins, with 155 showing significant expression changes in response to H2O2 signaling. Bioinformatics, including Principal Component Analysis, revealed distinct proteomic profiles in roots and leaves, indicating tissue-specific metabolic reprogramming. Functional annotation through Gene Ontology and KEGG pathway enrichment showed that oxidative signaling led to the repression of photosynthesis-related pathways in leaves, while promoting pathways related to protein processing, glycolysis, and carbon metabolism in roots. Additionally, bioinformatic tools identified proteins involved in amino acid metabolism, the TCA cycle, and carbohydrate metabolism as critical components of sugarcane's adaptive signaling response. The data suggest that sugarcane plants responded to oxidative signals by adjusting their metabolic networks, promoting sustained development and potential pathways for targeted plant breeding.

Redox modification of m6A demethylase SlALKBH2 in tomato regulates fruit ripening.

Hydrogen peroxide (H2O2) functions as a critical signalling molecule in controlling multiple biological processes. How H2O2 signalling integrates with other regulatory pathways such as epigenetic modification to coordinately regulate plant development remains elusive. Here we report that SlALKBH2, an m6A demethylase required for normal ripening of tomato fruit, is sensitive to oxidative modification by H2O2, which leads to the formation of homodimers mediated by intermolecular disulfide bonds, and Cys39 serves as a key site in this process. The oxidation of SlALKBH2 promotes protein stability and facilitates its function towards the target transcripts including the pivotal ripening gene SlDML2 encoding a DNA demethylase. Furthermore, we demonstrate that the thioredoxin reductase SlNTRC interacts with SlALKBH2 and catalyses its reduction, thereby modulating m6A levels and fruit ripening. Our study establishes a molecular link between H2O2 and m6A methylation and highlights the importance of redox regulation of m6A modifiers in controlling fruit ripening.

Extracellular ATP activates H2O2 signaling to mitigate cadmium toxicity by restricting Cd2+ entry and triggering the antioxidant system in Arabidopsis

Extracellular ATP activates H2O2 signaling to mitigate cadmium toxicity by restricting Cd2+ entry and triggering the antioxidant system in Arabidopsis

5-Aminolevulinic acid improves strawberry salt tolerance through a NO–H2O2 signaling circuit regulated by FaWRKY70 and FaWRKY40

5-Aminolevulinic acid improves strawberry salt tolerance through a NO–H2O2 signaling circuit regulated by FaWRKY70 and FaWRKY40

A Dual‐Mode Electrochemical Aptasensor for Ultrasensitive Detecting Acetamiprid Based on Hollow Porous Fe2O3 Cubes

AbstractAcetamiprid, a widely utilized pesticide, poses risks to human health due to the bioaccumulation of its residues. Detecting pesticide residues is crucial for effective pesticide management. To enhance the sensitivity and accuracy of pesticide detection, researchers have developed dual‐mode sensors that allow for mutual validation of the two signals. In this paper, a dual‐mode electrochemical sensor for detecting acetamiprid was constructed using H2O2 and methylene blue (MB) as probes. The electrochemical signal of MB was measured using square wave voltammetry (SWV) mode, followed by the detection of the reduction signal of H2O2 in amperometric i‐t curve (i‐t) mode. The two signals complemented each other, thereby reducing errors and enhancing measurement accuracy. Additionally, hollow porous Fe2O3 cubes were synthesized to catalyze the reduction of H2O2 to enhance mass transfer rates. The detection limit in SWV mode was 0.034 pM (S/N = 3), with a linear equation represented as ΔI = 5.39lgc‐33.80 (R2 = 0.99) within the concentration range of 0.1 pM to 0 .1µM. The detection limit in i‐t mode was 0.38 nM (S/N = 3), with a linear equation expressed as ΔI = ‐7.26lgc‐77.20 (R2 = 0.99) within the concentration range of 0.1 nM to 0.1 µM. This dual‐mode electrochemical sensor offers a promising approach for detecting pesticide residues.

Hydrogen Peroxide Is Involved in Methane-Alleviated Cadmium Toxicity in Alfalfa (Medicago sativa L.) Seedlings by Enhancing Cadmium Chelation onto Root Cell Walls.

Although previous studies have demonstrated that methane (CH4) can mitigate the toxicity of cadmium (Cd) in alfalfa seedlings, the CH4-rich water used in these studies may create hypoxic conditions, potentially influencing the experimental outcomes. Therefore, this study aimed to investigate whether CH4 can reduce Cd toxicity in alfalfa seedlings without the interference of hypoxia and to analyze its underlying mechanisms. Here, it was observed that supplementing oxygen with saturated CH4-rich water can significantly alleviate the inhibition of 75 μM CdCl2 on the growth of alfalfa (Medicago sativa L.) seedlings. Less Cd accumulation was also observed in both root and shoot parts, which could be explained by the CH4-altered cell wall components in alfalfa seedling roots, including covalent and ionic soluble pectin, and the degree of demethylation in pectin, thus enabling a higher proportion of Cd binding to the cell walls and reducing the entry of Cd into the cells. The above actions of CH4 were accompanied by an increase in hydrogen peroxide (H2O2) content and NADPH oxidase activity, which could be blocked by the addition of the NADPH oxidase inhibitor diphenylene iodonium (DPI). Taken together, these results implied that exogenously applied CH4 could alleviate Cd toxicity in alfalfa seedlings by enhancing Cd chelation onto the root cell walls, which might be closely associated with NADPH oxidase-dependent H2O2 signals. These findings could provide insight into the mechanism through which CH4 alleviates Cd toxicity in alfalfa plants.

Gamma-aminobutyric acid elicits H2O2 signalling and promotes wheat seed germination under combined salt and heat stress.

In the realm of wheat seed germination, abiotic stresses such as salinity and high temperature have been shown to hinder the process. These stresses can lead to the production of reactive oxygen species, which, within a certain concentration range, may actually facilitate seed germination. γ-aminobutyric acid (GABA), a non-protein amino acid, serves as a crucial signaling molecule in the promotion of seed germination. Nevertheless, the potential of GABA to regulate seed germination under the simultaneous stress of heat and salinity remains unexplored in current literature. This study employed observational methods to assess seed germination rate (GR), physiological methods to measure H2O2 content, and the activities of glutamate decarboxylase (GAD), NADPH oxidase (NOX), superoxide dismutase (SOD), and catalase (CAT). The levels of ABA and GABA were quantified using high-performance liquid chromatography technology. Furthermore, quantitative real-time PCR technology was utilized to analyze the expression levels of two genes encoding antioxidant enzymes, MnSOD and CAT. The findings indicated that combined stress (30 °C + 50 mM NaCl) decreased the GR of wheat seeds to about 21%, while treatment with 2 mM GABA increased the GR to about 48%. However, the stimulatory effect of GABA was mitigated by the presence of ABA, dimethylthiourea, and NOX inhibitor, but was strengthened by H2O2, antioxidant enzyme inhibitor, fluridone, and gibberellin. In comparison to the control group (20 °C + 0 mM NaCl), this combined stress led to elevated levels of ABA, reduced GAD and NOX activity, and a decrease in H2O2 and GABA content. Further investigation revealed that this combined stress significantly suppressed the activity of superoxide dismutase (SOD) and catalase (CAT), as well as downregulated the gene expression levels of MnSOD and CAT. However, the study demonstrates that exogenous GABA effectively reversed the inhibitory effects of combined stress on wheat seed germination. These findings suggest that GABA-induced NOX-mediated H2O2 signalling plays a crucial role in mitigating the adverse impact of combined stress on wheat seed germination. This research holds significant theoretical and practical implications for the regulation of crop seed germination by GABA under conditions of combined stress.

Sly-miR398b Mediates Mature Leaf Flattening by Orchestrating Auxin and H2O2 Signalling in Tomato.

Leaf flattening plays a pivotal role in optimizing light capture and enhancing photosynthesis efficiency. While extensive research has clarified the molecular mechanisms governing the initial stages of leaf flattening, understanding the maintenance of this process in mature leaves remains limited. Our investigation focused on sly-miR398b in tomatoes and revealed its crucial role in maintaining leaf flattening. In situ hybridization experiments indicated predominant expression of sly-miR398b in the abaxial side. Disrupting sly-miR398b using CRISPR/Cas9 relieved its suppression on target gene (Cu/Zn-SOD, SlCSD1), elevating SlCSD1 levels specifically on the abaxial side. Consequently, this asymmetrical expression of SlCSD1 increased hydrogen peroxide (H2O2) levels in the abaxial side, hindering auxin influx genes while promoting auxin efflux gene expression. This shift reduced auxin response gene expression in the abaxial side of mature leaves compared to the adaxial side, leading to leaf epinasty in sly-miR398b mutants. Exogenous H2O2 spraying induced leaf epinasty, downregulating SlGH3.5 and upregulating SlPIN3 and SlPIN4. Remarkably, spraying with 1-naphthalacetic acid (NAA) restored leaf flattening in sly-miR398b mutants. Our findings offer novel insights into mature leaf flattening maintenance via sly-miR398b's regulation of auxin and H2O2 signalling pathways.

Aquaporin CmPIP2;3 links H2O2 signal and antioxidation to modulate trehalose-induced cold tolerance in melon seedlings.

Cold stress severely restricts the growth and development of cold-sensitive crops. Trehalose (Tre), known as the "sugar of life", plays key roles in regulating plant cold tolerance by triggering antioxidation. However, the relevant regulatory mechanism remains unclear. Here, we confirmed that Tre triggers apoplastic hydrogen peroxide (H2O2) production and thus plays key roles in improving the cold tolerance of melon (Cucumis melo var. makuwa Makino) seedlings. Moreover, Tre treatment can promote the transport of apoplastic H2O2 to the cytoplasm. This physiological process may depend on aquaporins. Further studies showed that a Tre-responsive plasma membrane intrinsic protein 2;3 (CmPIP2;3) had strong H2O2 transport function and that silencing CmPIP2;3 significantly weakened apoplastic H2O2 transport and reduced the cold tolerance of melon seedlings. Yeast library and protein-DNA interaction technology were then used to screen 2 Tre-responsive transcription factors, abscisic acid-responsive element (ABRE)-binding factor 2 (CmABF2) and ABRE-binding factor 3 (CmABF3), which can bind to the ABRE motif of the CmPIP2;3 promoter and activate its expression. Silencing of CmABF2 and CmABF3 further dramatically increased the ratio of apoplastic H2O2/cytoplasm H2O2 and reduced the cold tolerance of melon seedlings. This study uncovered that Tre treatment induces CmABF2/3 to positively regulate CmPIP2;3 expression. CmPIP2;3 subsequently enhances the cold tolerance of melon seedlings by promoting the transport of apoplastic H2O2 into the cytoplasm for conducting redox signals and stimulating downstream antioxidation.

Hydrogen peroxide positively regulates ABA signaling via oxidative modification of the C2H2-type zinc finger protein ZFP36 in rice

The rice zinc finger protein ZFP36 serves as a pivotal regulator of the hydrogen peroxide (H2O2) signaling pathway in response to abscisic acid (ABA). Its role is crucial for integrating H2O2 signals with the plant defense mechanisms against water deficit and oxidative stress. However, it remains unclear whether ZFP36 directly modulates ABA-induced H2O2 signaling. This study explored the effects of oxidative post-translational modifications (OxiPTMs) on ZFP36 in rice, with an emphasis on the H2O2-induced oxidation through its cysteine (Cys) residues. We found that ZFP36 undergoes oxidative modification as a target of H2O2 in the presence of ABA, specifically at Cys32. Employing quantitative detection and fluorescence assays, we observed that ZFP36 oxidation enhances the expression and activity of genes encoding protective antioxidant enzymes. Moreover, our investigation into the thioredoxin (Trx) and glutaredoxin (Grx) families revealed that OsTrxh1 facilitates the reduction of oxidized ZFP36. Genetic evidence indicates that ZFP36 positively influences rice resilience to oxidative and water stress, while OsTrxh1 exerts an opposing effect. These insights reveal a distinctive pathway for plant cells to perceive ABA-induced H2O2 signaling, advance our comprehension of H2O2 signaling dynamics, and ABA-related plant responses, and lay a vital groundwork for enhancing crop stress tolerance.

Visualizing hydrogen peroxide and nitric oxide dynamics in endothelial cells using multispectral imaging under controlled oxygen conditions

The complex interplay between hydrogen peroxide (H2O2) and nitric oxide (NO) in endothelial cells presents challenges due to technical limitations in simultaneous measurement, hindering the elucidation of their direct relationship. Previous studies have yielded conflicting findings regarding the impact of H2O2 on NO production. To address this problem, we employed genetically encoded biosensors, HyPer7 for H2O2 and geNOps for NO, allowing simultaneous imaging in single endothelial cells. Optimization strategies were implemented to enhance biosensor performance, including camera binning, temperature regulation, and environmental adjustments to mimic physiological normoxia. Our results demonstrate that under ambient oxygen conditions, H2O2 exhibited no significant influence on NO production. Subsequent exploration under physiological normoxia (5 kPa O2) revealed distinct oxidative stress levels characterized by reduced basal HyPer7 signals, enhanced H2O2 scavenging kinetics, and altered responses to pharmacological treatment. Investigation of the relationship between H2O2 and NO under varying oxygen conditions revealed a lack of NO response to H2O2 under hyperoxia (18 kPa O2) but a modest NO response under physiological normoxia (5 kPa O2). Importantly, the NO response was attenuated by l-NAME, suggesting activation of eNOS by endogenous H2O2 generation upon auranofin treatment. Our study highlights the intricate interplay between H2O2 and NO within the endothelial EA.hy926 cell line, emphasizing the necessity for additional research within physiological contexts due to differential response observed under physiological normoxia (5 kPa O2). This further investigation is essential for a comprehensive understanding of the H2O2 and NO signaling considering the physiological effects of ambient O2 levels involved.

Appropriate Drought Training Induces Optimal Drought Tolerance by Inducing Stepwise H2O2 Homeostasis in Soybean.

Soybean is considered one of the most drought-sensitive crops, and ROS homeostasis can regulate drought tolerance in these plants. Understanding the mechanism of H2O2 homeostasis and its regulatory effect on drought stress is important for improving drought tolerance in soybean. We used different concentrations of polyethylene glycol (PEG) solutions to simulate the progression from weak drought stress (0.2%, 0.5%, and 1% PEG) to strong drought stress (5% PEG). We investigated the responses of the soybean plant phenotype, ROS level, injury severity, antioxidant system, etc., to different weak drought stresses and subsequent strong drought stresses. The results show that drought-treated plants accumulated H2O2 for signaling and exhibited drought tolerance under the following stronger drought stress, among which the 0.5% PEG treatment had the greatest effect. Under the optimal treatment, there was qualitatively describable H2O2 homeostasis, characterized by a consistent increasing amplitude in H2O2 content compared with CK. The H2O2 signal formed under the optimum treatment induced the capacity of the antioxidant system to remove excess H2O2 to form a primary H2O2 homeostasis. The primary H2O2 homeostasis further induced senior H2O2 homeostasis under the following strong drought and maximized the improvement of drought tolerance. These findings might suggest that gradual drought training could result in stepwise H2O2 homeostasis to continuously improve drought tolerance.

Interactions between the nitrate reductase 2 and catalase 1 fine-tune disease resistance in cassava.

Cassava is one of the most important tuber crops that is used for food, starch and bio-energy. However, cassava is susceptible to a number of diseases, especially cassava bacterial blight (CBB). Nitric oxide (NO) and hydrogen peroxide (H2O2) regulate plant growth and development, as well as stress responses. However, no direct relationships between the enzymes involved in the metabolic enzymes that produce and process these key signaling molecules has been demonstrated. Here, we provide evidence for the interaction between the nitrate reductase 2 (MeNR2) and catalase 1 (MeCAT1) proteins in vitro and in vivo, using yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays, respectively. MeNR2 is a positive regulator and MeCAT1 is a negative regulator of CBB resistance. MeNR2 was localized in the nucleus, cell membrane and peroxisome, while MeCAT1 was localized in the peroxisomes. The interactions between MeNR2 and MeCAT1 also had effects of their respective enzyme activities. Taken together, the data presented here suggested that there is coordination between H2O2 and NO signaling in cassava disease resistance, through the interactions between MeCAT1 and MeNR2.

Decoding early stress signaling waves in living plants using nanosensor multiplexing.

Increased exposure to environmental stresses due to climate change have adversely affected plant growth and productivity. Upon stress, plants activate a signaling cascade, involving multiple molecules like H2O2, and plant hormones such as salicylic acid (SA) leading to resistance or stress adaptation. However, the temporal ordering and composition of the resulting cascade remains largely unknown. In this study we developed a nanosensor for SA and multiplexed it with H2O2 nanosensor for simultaneous monitoring of stress-induced H2O2 and SA signals when Brassica rapa subsp. Chinensis (Pak choi) plants were subjected to distinct stress treatments, namely light, heat, pathogen stress and mechanical wounding. Nanosensors reported distinct dynamics and temporal wave characteristics of H2O2 and SA generation for each stress. Based on these temporal insights, we have formulated a biochemical kinetic model that suggests the early H2O2 waveform encodes information specific to each stress type. These results demonstrate that sensor multiplexing can reveal stress signaling mechanisms in plants, aiding in developing climate-resilient crops and pre-symptomatic stress diagnoses.

The critical role of CmCIPK1-CmRbohD1/D2 complexes in generating H2O2 signals for enhancing salt tolerance in pumpkins

Pumpkin is of great economic importance not only as food resources but also as the most widely used rootstock to graft cucurbit crops. Pumpkin rootstock improves salt tolerance of cucumber scion through respiratory burst oxidase homolog protein D (CmRbohD)-mediated reactive oxygen species (ROS) burst, which further enhances Na+ export and K+ uptake. RbohD activation requires calcium signaling. However, the underlying mechanism remains largely unknown. In this study, we discovered two RbohD members from pumpkin involved in the ROS burst at the early stage of salt stress. CmRbohD1 and CmRbohD2 were functionally redundant and double mutation significantly impaired salt tolerance in pumpkin. Overexpression of CmRbohD1 and CmRbohD2 mitigated salinity-induced damage and maintained a relatively low Na+ content and high K+ content. We screened the potential calcineurin B-like interacting protein kinases (CmCIPKs) which could bind with CmRbohD1 and CmRbohD2. Our results revealed that two RbohDs formed complexes specifically with CmCIPK1, thereby bursting ROS production. Overexpression of CmCIPK1 promoted the early ROS burst under salt stress condition and maintained a relatively balanced Na+/K+ homeostasis. Altogether, we proposed a CmCIPK1-CmRbohD1/D2 complex for pumpkin salt stress signaling transduction, which regulates the ROS burst and Na+/K+ homeostasis. Our findings offer unprecedented insights into the general mechanism of pumpkin salt tolerance.

Pyrus betulaefolia ERF3 interacts with HsfC1a to coordinately regulate aquaporin PIP1;4 and NCED4 for drought tolerance.

Environmental disasters like drought reduce agricultural output and plant growth. Redox management significantly affects plant stress responses. An earlier study found that PbPIP1;4 transports H2O2 and promotes H2O2 downstream cascade signaling to restore redox equilibrium. However, this regulatory mechanism requires additional investigation. In this search, the AP2 domain-containing transcription factor was isolated by screening Y1H from the wild pear (Pyrus betulaefolia) cDNA library, named PbERF3. The overexpression of PbERF3 in pear callus and Arabidopsis enhanced plant resistance to drought and re-established redox balance. The transcripts of the NCEDs gene were upregulated under drought stress. The drought stress-related abscisic acid (ABA) signaling pathway modulates PbERF3. PbERF3 silencing lowered drought tolerance. Furthermore, yeast 2-hybrid, luciferase, bimolecular fluorescence complementation, and co-immunoprecipitation assays verified that PbERF3 physically interacted with PbHsfC1a. The PbERF3-PbHsfC1a heterodimer coordinately bound to PbPIP1;4 and PbNCED4 promoter, therefore activating both the H2O2 and the ABA signaling pathway. This work revealed a novel PbERF3-PbHsfC1a-PbNCED4-PbPIP1;4 regulatory module, in which PbERF3 interacts with PbHsfC1a to trigger the expression of target genes. This module establishes an interaction between the H2O2 signaling component PbPIP1;4 and the ABA pathways component PbNCED4, enabling a response to drought.