H2O2 In Plants Research Articles

Rapid and In-Field Sensing of Hydrogen Peroxide in Plant by Hydrogel Microneedle Patch.

The rapidly changing climate is exacerbating the environmental stress that negatively impacts crop health and yield. Timely sensing of plant response to stress is beneficial to timely adjust planting conditions, promoting the healthy growth of plants, and improving plant productivity. Hydrogen peroxide (H2O2) is an importantmolecule of signal transduction in plants. However, the common methods for detecting H2O2in plants are associated with certain drawbacks, such as long extraction time, cumbersome steps, dependence on large instruments, and difficulty in realizing in-field sensing. Therefore, it is urgent to establish more efficient detection methods to realize the rapid detection of H2O2 content in plants. In this research, poly (methyl vinyl ether-alt-maleic acid) (PMVE/MA) hydrogel microneedle (MN) patch for rapid extraction of leaf sap are prepared, and the extraction mechanism of PEG-crosslinked PMVE/MA hydrogel MN patch isstudied. A method of rapid detection of H2O2 content in plants based on MN patch with optical detection technology is constructed. The hydrogel MN patch can be usedfor timely H2O2 analysis. This application enables new opportunities in plant engineering, and can be extended to the safety and healthmonitoring of other plants and animals.

Root endophyte-mediated alteration in plant H2O2 homeostasis regulates symbiosis outcome and reshapes the rhizosphere microbiota.

Endophytic symbioses between plants and fungi are a dominant feature of many terrestrial ecosystems, yet little is known about the signaling that defines these symbiotic associations. Hydrogen peroxide (H2O2) is recognized as a key signal mediating the plant adaptive response to both biotic and abiotic stresses. However, the role of H2O2 in plant-fungal symbiosis remains elusive. Using a combination of physiological analysis, plant and fungal deletion mutants, and comparative transcriptomics, we reported that various environmental conditions differentially affect the interaction between Arabidopsis and the root endophyte Phomopsis liquidambaris, and link this process to alterations in H2O2 levels and H2O2 fluxes across root tips. We found that enhanced H2O2 efflux leading to a moderate increase in H2O2 levels at the plant-fungal interface is required for maintaining plant-fungal symbiosis. Disturbance of plant H2O2 homeostasis compromises the symbiotic ability of plant roots. Moreover, the fungus-regulated H2O2 dynamics modulate the rhizosphere microbiome by selectively enriching for the phylum Cyanobacteria, with strong antioxidant defenses. Our results demonstrated that the regulation of H2O2 dynamics at the plant-fungal interface affects the symbiotic outcome in response to external conditions and highlight the importance of the root endophyte in reshaping the rhizosphere microbiota.

Physiological and phosphoproteomic analyses revealed that the NtPOD63 L knockout mutant enhances drought tolerance in tobacco

Drought is an important environmental factor affecting plant growth and development. Tobacco is a widely cultivated and economically important crop worldwide. Drought can decrease the yield and quality of tobacco. Peroxidase III can maintain the balance of peroxy free radicals and H2O2 in plants and participate in a variety of physiological processes, including lignin biosynthesis and auxin catabolism. In this study, we first found that a CRISPR/Cas9-mediated peroxidase 63-like (NtPOD63 L) knockout mutant had high drought tolerance, such as reduced stomatal apertures, increased lignin content and interfascicular fiber number, and induced ABA-related gene expression. Then, a quantitative phosphoproteomic analysis of tobacco mutant leaves was conducted after PEG treatment. Phosphoproteomic analysis revealed 609 phosphorylated proteins, among which 266 were upregulated and 343 were downregulated. KEGG pathway analysis indicated that the cluster with “Pyruvate metabolism” contained the most upregulated pathways. The cluster with “RNA degradation”, “Cysteine and methionine metabolism,” and “Tryptophan metabolism contained the three primary downregulated pathways. Sixty-seven kinases were found to change phosphorylation levels. Our study provides new information for understanding NtPOD63 L function in drought stress and describes the molecular regulation mechanisms of the tobacco response to drought at the posttranslational level.

Dynamic and fluctuating generation of hydrogen peroxide via photorespiratory metabolic channeling in plants.

The homeostasis of hydrogen peroxide (H2 O2 ), a key regulator of basic biological processes, is a result of the cooperation between its generation and scavenging. However, the mechanistic basis of this balance is not fully understood. We previously proposed that the interaction between glycolate oxidase (GLO) and catalase (CAT) may serve as a molecular switch that modulates H2 O2 levels in plants. In this study, we demonstrate that the GLO-CAT complex in plants exists in different states, which are dynamically interchangeable in response to various stimuli, typically salicylic acid (SA), at the whole-plant level. More crucially, changes in the state of the complex were found to be closely linked to peroxisomal H2 O2 fluctuations, which were independent of the membrane-associated NADPH oxidase. Furthermore, evidence suggested that H2 O2 channeling occurred even invitro when GLO and CAT worked cooperatively. These results demonstrate that dynamic changes in H2 O2 levels are physically created via photorespiratory metabolic channeling in plants, and that such H2 O2 fluctuations may serve as signals that are mechanistically involved in the known functions of photorespiratory H2 O2 . In addition, our study also revealed a new way for SA to communicate with H2 O2 in plants.

Metal-Organic Framework-Based Biosensor for Detecting Hydrogen Peroxide in Plants through Color-to-Thermal Signal Conversion.

Plant biotic or abiotic stresses, such as pathogens, mechanical damage, or high temperature, can increase intracellular H2O2 concentration, damaging proteins, lipids, and DNA. Most current H2O2 detection methods require the separation or grinding of plant samples, inducing plant stresses, and the process is complicated and time-consuming. This paper constructed a metal-organic framework (MOF)-based biosensor for real-time, remote, and in situ detection of exogenous/endogenous H2O2 in plant organs through color-to-thermal signal conversion. By simply spraying horseradish peroxidase, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and the precursor of zeolite imidazolate frameworks-8 (ZIF-8), ZIF-8 biosensors were formed in situ on a plant root, petiole, or leaf. This biosensor could report sub-micromolar H2O2 in plants since the oxidation products, ABTS• +, emitted heat when they absorbed energy from near-infrared (NIR) light. Due to the plant's low absorption in the NIR region, the ZIF-8 biosensor allowed for remote thermal sensing of H2O2 transport or biotic/abiotic stresses in plants with a high signal-to-noise ratio combining NIR laser and thermometer. Our biosensor can be used for the future development of plant sensors for monitoring plant signaling pathways and metabolism that are nondestructive, minimally invasive, and capable of real-time, in situ analysis.

Measurement of foliar H2O2 concentration can be an indicator of riparian vegetation management

Riparian vegetation is frequently exposed to abiotic stress, which generates reactive oxygen species (ROS) caused by strong differences in a river’s hydrological conditions. Among different ROS, hydrogen peroxide (H2O2) is relatively steady and can be measured appropriately. Thus, the quantification of plant H2O2 can be used as a stress indicator for riparian vegetation management. The current study examines the spatial distribution of plants by riparian vegetation communities across the elevation gradient of riparian zones through quantification of environmental stress using foliar H2O2 concentration. The trees Salix spp., Robinia pseudoacacia, Ailanthus altissima with Juglans mandshurica, and the herbs Phragmites australis, Phragmites japonica, and Miscanthus sacchariflorus were selected for this study. Leaf tissues were collected to analyze H2O2 concentration, meanwhile riparian soil was sampled to measure total nitrogen (TN), total phosphorus (TP), and moisture content. The H2O2 concentration of tree species increased with higher soil moisture content, which was negatively correlated for Salix and herb spp., in which H2O2 concentration always decreased with high soil moisture. In this study, we found a unique significant interaction between soil moisture content and H2O2 concentration, both positively or negatively correlated relationships, when compared with other parameters, such as TN or TP concentrations or TN: TP in riparian soil. The species-specific distribution zones can be explained by the H2O2 concentration in the plant for gravelly and sandy channels on a theoretical range of soil moisture. Each species’ H2O2 concentration was estimated through derived equations and is directly related to an elevation above the channel. The comparison with the observed distribution of plant elevations in the field indicated that all species showed a spatial distribution that acts as species-specific elevations where H2O2 concentrations stayed below 40 μmol/gFW. Hence, the present study suggests that foliar H2O2 concentration can be a useful benchmark for the distribution potentiality of riparian vegetation.

Simple Phenotypic Sensor for Visibly Tracking H2O2 Fluctuation to Detect Plant Health Status.

Hydrogen peroxide (H2O2), as a main component of reactive oxygen species (ROS), serves as a key signaling molecule relevant to plant stress response and health status. Many strategies have been developed for detecting or quantifying H2O2 concentration. However, reports on simply, visibly tracking H2O2 fluctuation in vivo are limited. Here, for visibly tracking the plant H2O2 wave, a green fluorescent phenotypic probe was designed by merging a H2O2-sensitive tertiary amine moiety with the core fluorophore tetraphenylethene skeleton. The green fluorescence emission is quenched up to 52% by H2O2 with good sensitivity, selectivity, and reversibility within the plant physiological range of 10-100 μM H2O2. In response to various abiotic stresses, including mechanical damage, high salt, strong light and drought, fluorescence fluctuations, response to H2O2 concentration alterations in vivo was visible to the naked eye under irradiation of commercially available UV light (365 nm) after simple injection of this H2O2 probe solution into seedling leaves. This phenotypic fluorescent H2O2 probe illustrates great potential as early sensors of plant health under stress without the aid of skillful operation and specialized equipment.

CONTROLE DE MILHO VOLUNTÁRIO EM FUNÇÃO DE PERÍODOS DE AUSÊNCIA LUMINOSA APÓS A APLICAÇÃO DE DIQUAT

ABSTRACT The action of photosystem I (PSI) inhibiting herbicides depends on light to cause oxidative stress in plants. However, their translocation is inhibited due to their rapid action in the presence of light. The aim was to evaluate the efficacy of the herbicide diquat for control of corn plants subjected to different periods of absence of light after application. Two experiments (field and greenhouse) were conducted, applying the herbicide diquat (200 g a.i. ha−1) to maize plants at stage V4. The plants were subjected to different periods of absence of light after diquat application: 0; 1; 2; 3; 4; 5 and 6 hours. A treatment without herbicide application was used as a control. The control (%) and biomass of corn plants were evaluated in both experiments, and photosynthetic activity and hydrogen peroxide (H2O2) accumulation in leaves were evaluated in the greenhouse experiment. The results showed that diquat needs at least 5 hours of absence of light after application to fully control corn plants. The plants recovered when using shorter periods (4 hours or less) of darkness, and injuries were restricted to points where the herbicide had contact with the plant. The ability of plants to recover was related to the higher photosynthetic activity and oxidative stress induction due to early light exposure. Thus, the lower production of H2O2 in plants kept in the dark for longer periods after herbicide application allows the translocation of the herbicide to meristems, which prevents regrowth of corn plants.

Effect of Photoconversion Coatings for Greenhouses on Electrical Signal-Induced Resistance to Heat Stress of Tomato Plants.

The use of photoconversion coatings is a promising approach to improving the quality of light when growing plants in greenhouses in low light conditions. In this work, we studied the effect of fluoropolymer coatings, which produce photoconversion of UV-A radiation and violet light into blue and red light, on the growth and resistance to heat stress of tomato plants (Solanum lycopersicum L.). The stimulating effect of the spectrum obtained as a result of photoconversion on plant growth and the activity of the photosynthesis process are shown. At the same time, the ability to withstand heat stress is reduced in plants grown under a photoconversion coating. Stress electrical signals, which normally increase resistance, in such plants have a much weaker protective effect on the photosynthetic apparatus. The observed effects are apparently explained by a decrease in the concentration of H2O2 in plants grown using photoconversion technologies, which leads to a shift in the development program towards increased productivity to the detriment of the protective function. Thus, when using photoconversion technologies in agricultural practice, it is necessary to pay increased attention to maintaining stable conditions during plant cultivation.

Disposable Stainless-Steel Wire-Based Electrochemical Microsensor for In Vivo Continuous Monitoring of Hydrogen Peroxide in Vein of Tomato Leaf.

As one of the pivotal signal molecules, hydrogen peroxide (H2O2) has been demonstrated to play important roles in many physiological processes of plants. Continuous monitoring of H2O2 in vivo could help understand its regulation mechanism more clearly. In this study, a disposable electrochemical microsensor for H2O2 was developed. This microsensor consists of three parts: low-cost stainless-steel wire with a diameter of 0.1 mm modified by gold nanoparticles (disposable working electrode), an untreated platinum wire with a diameter of 0.1 mm (counter electrode), and an Ag/AgCl wire with a diameter of 0.1 mm (reference electrode), respectively. The microsensor could detect H2O2 in levels from 10 to 1000 µM and exhibited excellent selectivity. On this basis, the dynamic change in H2O2 in the vein of tomato leaf under high salinity was continuously monitored in vivo. The results showed that the production of H2O2 could be induced by high salinity within two hours. This study suggests that the disposable electrochemical microsensor not only suits continuously detecting H2O2 in microscopic plant tissue in vivo but also reduces the damage to plants. Overall, our strategy will help to pave the foundation for further investigation of the generation, transportation, and elimination mechanism of H2O2 in plants.

Atomically Dispersed Cu Nanozyme with Intensive Ascorbate Peroxidase Mimic Activity Capable of Alleviating ROS-Mediated Oxidation Damage.

Ascorbate peroxidase (APX) as a crucial antioxidant enzyme has drawn attentions for its utilization in preventing cells from oxidative stress responses by efficiently scavenging H2O2 in plants. For eliminating the specific inactivation of natural APXs and regulating the catalytic activity, single‐atom nanozymes are considered as promising classes of alternatives with similar active sites and maximal atomic utilization efficiency to natural APXs. Herein, graphitic carbon nitride (g‐C3N4) anchored with isolated single copper atoms (Cu SAs/CN) is designed as an efficient nanozyme with intrinsic APX mimetic behavior. The engineered Cu SAs/CN exhibits comparable specific activity and kinetics to the natural APXs. Based on the density functional theory (DFT), Cu‐N4 moieties in the active center of Cu SAs/CN are determined to exert such favorable APX catalytic performance, in which the electron transfer between Cu and coordinated N atoms facilitates the activation and cleavage of the adsorbed H2O2 molecules and results in fast kinetics. The constructed Cu SAs/CN nanozyme with superior APX‐like performance and high biocompatibility can be applied for effectively protecting the H2O2‐treated cells against oxidative injury in vitro. These findings report the single‐atom nanozymes as a successful paradigm for guiding nanozymes to implement APX mimetic performance for reactive oxygen species‐related biotherapeutic.

Jasmonic Acid Impairs Arabidopsis Seedling Salt Stress Tolerance Through MYC2-Mediated Repression of CAT2 Expression.

High salinity causes ionic, osmotic, and oxidative stresses to plants, and the antioxidant enzyme Catalase2 (CAT2) plays a vital role in this process, while how CAT2 expression is regulated during plant response to high salinity remains elusive. Here, we report that phytohormone jasmonic acid (JA) impairs plant salt stress tolerance by repressing CAT2 expression in an MYC2-dependent manner. Exogenous JA application decreased plant salt stress tolerance while the jar1 mutant with reduced bioactive JA-Ile accumulation showed enhanced salt stress tolerance. JA enhanced salt-induced hydrogen peroxide (H2O2) accumulation, while treatment with H2O2-scavenger glutathione compromised such effects of JA on plant H2O2 accumulation and salt stress tolerance. In addition, JA repressed CAT2 expression in salt-stressed wild-type plant but not in myc2, a mutant of the master transcriptional factor MYC2 in JA signaling, therefore, the myc2 mutant exhibited increased salt stress tolerance. Further study showed that mutation of CAT2 largely reverted lower reactive oxygen species (ROS) accumulation, higher CAT activity, and enhanced salt stress tolerance of the myc2 mutant in myc2 cat2-1 double mutant, revealing that CAT2 functions downstream JA-MYC2 module in plant response to high salinity. Together, our study reveals that JA impairs Arabidopsis seedling salt stress tolerance through MYC2-mediated repression of CAT2 expression.

Nighttime Temperatures and Sunlight Intensities Interact to Influence Anthocyanin Biosynthesis and Photooxidative Sunburn in “Fuji” Apple

Light and low temperatures induce anthocyanin accumulation, but intense sunlight causes photooxidative sunburn. Nonetheless, there have been few studies of anthocyanin synthesis under different sunlight intensities and low nighttime temperatures. Here, low nighttime temperatures followed by low light intensity were associated with greater anthocyanin accumulation and the expression of anthocyanin biosynthesis genes in “Fuji” apple peel. UDP-glucose flavonoid-3-O-glucosyltransferase (UFGT) activity was positively associated with anthocyanin enrichment. Ascorbic acid can be used as an electron donor of APX to scavenge H2O2 in plants, which makes it play an important role in oxidative defense. Exogenous ascorbate altered the anthocyanin accumulation and reduced the occurrence of high light–induced photooxidative sunburn by removing hydrogen peroxide from the peel. Overall, low light intensity was beneficial for the accumulation of anthocyanin and did not cause photooxidative sunburn, whereas natural light had the opposite effect on the apple peel at low nighttime temperatures. This study provides an insight into the mechanisms by which low temperatures induce apple coloration and high light intensity causes photooxidative sunburn.

A Phytophthora sojae CRN effector mediates phosphorylation and degradation of plant aquaporin proteins to suppress host immune signaling.

Phytophthora genomes encode a myriad of Crinkler (CRN) effectors, some of which contain putative kinase domains. Little is known about the host targets of these kinase-domain-containing CRNs and their infection-promoting mechanisms. Here, we report the host target and functional mechanism of a conserved kinase CRN effector named CRN78 in a notorious oomycete pathogen, Phytophthora sojae. CRN78 promotes Phytophthora capsici infection in Nicotiana benthamiana and enhances P. sojae virulence on the host plant Glycine max by inhibiting plant H2O2 accumulation and immunity-related gene expression. Further investigation reveals that CRN78 interacts with PIP2-family aquaporin proteins including NbPIP2;2 from N. benthamiana and GmPIP2-13 from soybean on the plant plasma membrane, and membrane localization is necessary for virulence of CRN78. Next, CRN78 promotes phosphorylation of NbPIP2;2 or GmPIP2-13 using its kinase domain in vivo, leading to their subsequent protein degradation in a 26S-dependent pathway. Our data also demonstrates that NbPIP2;2 acts as a H2O2 transporter to positively regulate plant immunity and reactive oxygen species (ROS) accumulation. Phylogenetic analysis suggests that the phosphorylation sites of PIP2 proteins and the kinase domains of CRN78 homologs are highly conserved among higher plants and oomycete pathogens, respectively. Therefore, this study elucidates a conserved and novel pathway used by effector proteins to inhibit host cellular defenses by targeting and hijacking phosphorylation of plant aquaporin proteins.

Methionine Sulfoxide Reductase B Regulates the Activity of Ascorbate Peroxidase of Banana Fruit.

Ascorbate peroxidase (APX) is a key antioxidant enzyme that is involved in diverse developmental and physiological process and stress responses by scavenging H2O2 in plants. APX itself is also subjected to multiple posttranslational modifications (PTMs). However, redox-mediated PTM of APX in plants remains poorly understood. Here, we identified and confirmed that MaAPX1 interacts with methionine sulfoxide reductase B2 (MsrB2) in bananas. Ectopic overexpression of MaAPX1 delays the detached leaf senescence induced by darkness in Arabidopsis. Sulfoxidation of MaAPX1, i.e., methionine oxidation, leads to loss of the activity, which is repaired partially by MaMsrB2. Moreover, mimicking sulfoxidation by mutating Met36 to Gln also decreases its activity in vitro and in vivo, whereas substitution of Met36 with Val36 to mimic the blocking of sulfoxidation has little effect on APX activity. Spectral analysis showed that mimicking sulfoxidation of Met36 hinders the formation of compound I, the first intermediate between APX and H2O2. Our findings demonstrate that the redox state of methionine in MaAPX1 is critical to its activity, and MaMsrB2 can regulate the redox state and activity of MaAPX1. Our results revealed a novel post-translational redox modification of APX.

Proposed mechanism for regulation of H2 O2 -induced programmed cell death in plants by binding of cytochrome c to 14-3-3 proteins.

Programmed cell death (PCD) is crucial for development and homeostasis of all multicellular organisms. In human cells, the double role of extra-mitochondrial cytochrome c in triggering apoptosis and inhibiting survival pathways is well reported. In plants, however, the specific role of cytochrome c upon release from the mitochondria remains in part veiled yet death stimuli do trigger cytochrome c translocation as well. Here, we identify an Arabidopsis thaliana 14-3-3ι isoform as a cytosolic cytochrome c target and inhibitor of caspase-like activity. This finding establishes the 14-3-3ι protein as a relevant factor at the onset of plant H2 O2 -induced PCD. The in vivo and in vitro studies herein reported reveal that the interaction between cytochrome c and 14-3-3ι exhibits noticeable similarities with the complex formed by their human orthologues. Further analysis of the heterologous complexes between human and plant cytochrome c with plant 14-3-3ι and human 14-3-3ε isoforms corroborated common features. These results suggest that cytochrome c blocks p14-3-3ι so as to inhibit caspase-like proteases, which in turn promote cell death upon H2 O2 treatment. Besides establishing common biochemical features between human and plant PCD, this work sheds light onto the signaling networks of plant cell death.

Biocontrol Using Bacillus amyloliquefaciens PP19 Against Litchi Downy Blight Caused by Peronophythora litchii.

Bacillus amyloliquefaciens has been widely used in the agriculture, food, and medicine industries. Isolate PP19 was obtained from the litchi fruit carposphere and showed biocontrol efficacy against litchi downy blight (LDB) whether applied preharvest or postharvest. To further understand the underlying regulatory mechanisms, the genome of PP19 was sequenced and analyzed. The genome comprised a 3,847,565 bp circular chromosome containing 3990 protein-coding genes and 121 RNA genes. It has the smallest genome among 36 sequenced strains of B. amyloliquefaciens except for RD7-7. In whole genome phylogenetic analysis, PP19 was clustered into a group with known industrial applications, indicating that it may also produce high-yield metabolites that have yet to be identified. A large chromosome structural variation and large numbers of single nucleotide polymorphisms (SNPs) between PP19 (industrial strain) and UMAF6639 (plant-associated strain) were detected through comparative analysis, which may shed light on their functional differences. Preharvest treatment with PP19 enhanced resistance to LDB, by decreasing the plant H2O2 content and increasing the SOD activity. This is the first report of an industrial strain of B. amyloliquefaciens showing a plant-associated function and with major potential for the biocontrol of LDB.

The role analysis of APX gene family in the growth and developmental processes and in response to abiotic stresses in Arabidopsis thaliana

Oxidative stress caused by reactive oxygen species (ROS) is one of the major abiotic stresses in plants. Under adverse growth conditions, the incoordination of various metabolic processes in plant cells can result in increased hydrogen peroxide (H2O2), thus causing a variety of threats and injuries to plant cells. Ascorbate peroxidase (APX) is an important enzyme to remove H2O2 in plants. In Arabidopsis thaliana, there are eight APX gene family members, including APX1?APX6, sAPX and tAPX. In this study, we analyzed the expression patterns of the eight APX genes in the wild-type and apx mutant plants at different developmental stages and under different abiotic stress conditions. Meanwhile, the tolerance of each apx mutant to salt, drought and heat stresses was studied. qRT-PCR analysis showed that during development (from 4 to 8 weeks old), APX1 and APX2 exhibited the highest and lowest expression levels, respectively. In addition, the expression levels of APX4, sAPX and tAPX decreased during development, while the expression of APX6 increased with the maturity of the plants. Moreover, under different abiotic stress conditions, APX1, APX2 and APX6 were significantly induced by heat stress, sAPX actively responded to salt stress, and APX3 and APX5 exhibited obvious responses to salt, drought and heat stresses. Further tolerance analysis showed that the resistance of all apx mutants to salt and drought stresses was lower than that of the wild-type plant at both germination and maturity stages. At germination stage, all apx mutants were more sensitive to drought stress than to salt stress. At maturity stage, the apx1 and apx6 mutants were more sensitive to salt and drought stresses than the wild-type and other apx mutant plants. The physiological indexes indicated that the H2O2 content in all mutants, especially in the apx1, sapx and tapx, was significantly higher than that in the wild type 10 days after drought stress treatment, the malondialdehyde (MDA) content in all mutants was significantly higher than that in the wild type 5 days after salt stress treatment, while heat stress treatment for 2 h resulted in a significant increase in the contents of H2O2 and MDA in apx1, apx2 and apx6, especially in apx2. Taken together, our study revealed that all eight APX members of Arabidopsis participate in the growth and developmental processes and the abiotic stress responses, with some specific APXs playing a major role in a certain process.

The fluorescent protein sensor roGFP2‐Orp1 monitors in vivo H2O2 and thiol redox integration and elucidates intracellular H2O2 dynamics during elicitor‐induced oxidative burst in Arabidopsis

Hydrogen peroxide (H2 O2 ) is ubiquitous in cells and at the centre of developmental programmes and environmental responses. Its chemistry in cells makes H2 O2 notoriously hard to detect dynamically, specifically and at high resolution. Genetically encoded sensors overcome persistent shortcomings, but pH sensitivity, silencing of expression and a limited concept of sensor behaviour invivo have hampered any meaningful H2 O2 sensing in living plants. We established H2 O2 monitoring in the cytosol and the mitochondria of Arabidopsis with the fusion protein roGFP2-Orp1 using confocal microscopy and multiwell fluorimetry. We confirmed sensor oxidation by H2 O2 , show insensitivity to physiological pH changes, and demonstrated that glutathione dominates sensor reduction invivo. We showed the responsiveness of the sensor to exogenous H2 O2 , pharmacologically-induced H2 O2 release, and genetic interference with the antioxidant machinery in living Arabidopsis tissues. Monitoring intracellular H2 O2 dynamics in response to elicitor exposure reveals the late and prolonged impact of the oxidative burst in the cytosol that is modified in redox mutants. We provided a well defined toolkit for H2 O2 monitoring in planta and showed that intracellular H2 O2 measurements only carry meaning in the context of the endogenous thiol redox systems. This opens new possibilities to dissect plant H2 O2 dynamics and redox regulation, including intracellular NADPH oxidase-mediated ROS signalling.

Influence of exogenously applied nitric oxide on strawberry (Fragaria × ananassa) plants grown under iron deficiency and/or saline stress.

A study was carried out to assess the protective effects of exogenously applied nitric oxide (NO) in the form of its donor sodium nitroprusside (SNP) to strawberry seedlings (Fragaria × ananassa cv. Camarosa) grown under iron deficiency (ID), salinity stress or combination of both. The experimental design contained control, 0.1 mM FeSO4 (ID, Fe deficiency); 50 mM NaCl (S, Salinity) and ID + S. Plants were sprayed with 0.1 mM SNP or 0.1 mM sodium ferrocyanide, an analogue of SNP containing no NO. The deleterious effects of ID + S treatments on plant fresh and dry matters, total chlorophyll and chlorophyll fluorescence were more striking than those caused by the ID or S treatment alone. Furthermore, combination of salinity and iron stress exacerbated electrolyte leakage (EL) and the levels of malondialdehyde (MDA) and hydrogen peroxide (H2 O2 ) in plant leaves compared to those in plants grown with either of the single stresses. NO treatment effectively reduced EL, MDA and H2 O2 in plants grown under stress conditions applied singly or in combination. Salt stress alone and with ID reduced the superoxide dismutase (EC1.15.1.1) and catalase (EC 1.11.1.6) activities but increased that of POD (EC 1.17.1.7). Exogenously applied NO led to significant changes in antioxidant enzyme activities in either ID or S than those by ID+S. Overall, exogenously applied NO was more effective in mitigating the stress-induced adverse effects on the strawberry plants exposed to a single stress than those due to the combination of both stresses.