Visual Sensor Array for Multiple Aromatic Amines via Specific Ascorbic Acid Oxidase Mimic Triggered Schiff-Base Chemistry.

Redox nanozymes have exhibited various applications in recognizing environmental pollutants but not aromatic amines (a type of typical pollutant). Herein, with Cu2+ as a node and tryptophan (Trp) as a linker, Cu-Trp as a specific ascorbic acid oxidase mimic was synthesized, which could catalyze ascorbic acid (AA) oxidation to dehydroascorbic acid (DHAA). Alternatively, with other natural amino acids as linkers to synthesize Cu-based nanozymes, such catalytic performances are also observed. The as-produced DHAA could react with o-phenylenediamine (OPD) and its derivatives (2,3-naphthalene diamine (NDA), 4-nitro-o-phenylenediamine (4-NO2-OPD), 4-fluoro-o-phenylenediamine (4-F-OPD), 4-chloro-o-phenylenediamine(4-Cl-OPD), and 4-bromo-o-phenylenediamine(4-Br-OPD)) to form a Schiff base and emit fluorescence. Based on the results, with Cu-Trp + AA and Cu-Arg (with arginine (Arg) as a linker) + AA as two sensing channels and extracted red, green, and blue (RGB) values from emitted fluorescence as read-out signals, a visual sensor array was constructed to efficiently distinguish OPD, NDA, 4-NO2-OPD, 4-F-OPD, 4-Cl-OPD, and 4-Br-OPD as low as 10 µM. Such detecting performance was further confirmed through discriminating binary, ternary, quinary, and senary mixtures with various concentration ratios, recognizing 18 unknown samples, and even quantitatively analyzing single aromatic amine. Finally, the discriminating ability was further validated in environmental waters, providing an efficient assay for large-scale scanning levels of multiple aromatic amines.

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.

Impressive promiscuous biomimetic models of ascorbate, amine, and catechol oxidases.

Copper metalloenzymes ascorbate oxidase (AOase), amine oxidase (AmOase), and catechol oxidase (COase) possess copper(II) sites of coordination, which are trimeric, homodimeric, and dimeric, respectively. Two newly mononuclear copper(II) complexes, namely, [Cu(L)(bpy)](ClO4) (1) and [Cu(L)(phen)](ClO4) (2) where HL = Schiff base, have been synthesized. UV-visible, EPR and single-crystal X-ray diffraction examinations were used to validate the geometry in solution and solid state. For complex 1, the metal exhibits a coordination sphere between square pyramidal and trigonal bipyramidal geometry (τ, 0.49). A positive CuII/I redox potential indicates a stable switching between CuII and CuI redox states. Despite the monomeric origin, both homogeneous catalysts (1 or 2) in MeOH were found to favor three distinct chemical transformations, namely, ascorbic acid (H2A) to dehydroascorbic acid (DA), benzylamine (Ph-CH2-NH2) to benzaldehyde (Ph-CHO), and 3,5-di-tert-butylcatechol (3,5-DTBC) to 3,5-di-tert-butylquinone (3,5-DTBQ) [kcat: AOase, 9.6 (1) or 2.0 × 106 h-1(2); AmOase, 13.4 (1) or 9.4 × 106 h-1 (2); COase, 2.0 (1) or 1.9 × 103 h-1 (2)]. They exhibit higher levels of AOase activity as indicated by their kcat values compared to the AOase enzyme. The kcat values for COase activity in buffer solution [5.93 (1) or 2.95 × 105 h-1 (2)] are one order lower than those of the enzymes. This is because of the labile nature of the coordinated donor, the flexibility of the ligand, the simplicity of the catalyst-substrate interaction, and the positive CuII/I redox potential. Interestingly, more efficient catalysis is promoted by 1 and 2 concerning that of other mono- and dicopper(II) complexes.

A two-dimensional iron-doped carbon-based nanoenzyme with catalase-like activity for the detection of alkaline phosphatase and ascorbate oxidase.

Herein, we successfully synthesized two-dimensional iron-doped carbon-based nanosheets (Fe-N800 CS) with catalase-like activity through doping Fe into Zn MOF and introducing graphitic C3N4 (g-C3N4). The interaction of the Fe-N800 CS with hydrogen peroxide could generated abundant reactive oxygen species (ROS) and further oxidize o-Phenylenediamine (OPD) to 2,3-diaminophenazine (DAP) which has constant fluorescence at 560 nm. Ascorbic acid (AA) could be generated via the hydrolysis reaction between alkaline phosphatase (ALP) and ascorbic acid 2-phosphate (AAP). AA can be oxidized to dehy-droascorbic acid (DHA) by ROS, and then combined with OPD to generate 3-(1,2-dihydroxyethyl)furo[3,4b]-quinoxaline (QXD) with fluorescence at 440 nm, which could increase as the concentration of AA enhanced. DHA could also be generated through oxidation of AA by ascorbate oxidase (AAO). Thus, by monitoring the fluorescence ratio (I560/I440), a ratiometric fluorescence biosensing platform for ALP and AAO was established with the linear ranges in 0.2-10 U/L and 1-60 U/L, respectively. The limit of detection for ALP and AAO were 0.12 U/L and 0.59 U/L. Furthermore, the biosensing platform was successfully applied for the detection of ALP and AAO activity in human serum samples. This work provides a potential tool for future biomedical diagnostics.

Revisiting the role of ascorbate oxidase in plant systems.

Ascorbic acid (AsA) plays an indispensable role in plants, serving as both an antioxidant and a master regulator of the cellular redox balance. Ascorbate oxidase (AO) is a blue copper oxidase that is responsible for the oxidation of AsA with the concomitant production of water. For many decades, AO was erroneously postulated as an enzyme without any obvious advantage, as it decreases the AsA pool size and thus is expected to weaken plant stress resistance. It was only a decade ago that this perspective shifted towards the fundamental role of AO in orchestrating both AsA and oxygen levels by influencing the overall redox balance in the extracellular matrix. Consistent with its localization in the apoplast, AO is involved in cell expansion, division, resource allocation, and overall plant yield. An increasing number of transgenic studies has demonstrated that AO can also facilitate communication between the surrounding environment and the cell, as its gene expression is highly responsive to factors such as hormonal signaling, oxidative stress, and mechanical injury. This review aims to describe the multiple functions of AO in plant growth, development, and stress resilience, and explore any additional roles the enzyme might have in fruits during the course of ripening.

A facile fluorescence Eu MOF sensor for ascorbic acid and ascorbate oxidase detection.

In this work, a facile fluorescence Eu3+-based metal-organic framework (Eu MOF) sensor for ascorbic acid (AA) and ascorbate oxidase (AAO) detection was developed. The fluorescence of the Eu MOF could be effectively quenched by Ce3+ but not by Ce4+ at an appropriate concentration, and thus, when the reductant AA was added into the solution containing Ce4+, Ce4+ was chemically reduced to Ce3+, which induced the decreased fluorescence signal of the Eu MOF. However, when AAO was introduced, AA was effectively oxidized to dehydroascorbic acid (DHAA) under the catalysis of AAO, and thus, Ce4+ could not be reduced, resulting in the fluorescence restoration of the Eu MOF. Hence, the concentration of AA and AAO could be determined by the fluorescence decrease and restoration of the Eu MOF. The fluorescent platform showed high sensitivity with a limit of detection of 0.32 µM for AA and 1.18 U L-1 for AAO, respectively. Moreover, the proposed method was successfully applied for AA and AAO determination in real samples, indicating great potential for biomedical application in complex matrices.

Silencing of a Nicotiana benthamiana ascorbate oxidase gene reveals its involvement in resistance against cucumber mosaic virus.

MAIN CONCLUSION: Silencing of an ascorbate oxidase (AO) gene in N. benthamiana enhanced disease severity from cucumber mosaic virus (CMV), showing higher accumulation and expansion of the spreading area of CMV. A Nicotiana benthamiana ascorbate oxidase (NbAO) gene was found to be induced upon cucumber mosaic virus (CMV) infection. Virus-induced gene silencing (VIGS) was employed to elucidate the function of AO in N. benthamiana. The tobacco rattle virus (TRV)-mediated VIGS resulted in an efficient silencing of the NbAO gene, i.e., 97.5% and 78.8% in relative quantification as compared to the control groups (TRV::eGFP- and the mock-inoculated plants), respectively. In addition, AO enzymatic activity decreased in the TRV::NtAO-silenced plants as compared to control. TRV::NtAO-mediated NbAO silencing induced a greater reduction in plant height by 15.2% upon CMV infection. CMV titer at 3 dpi was increased in the systemic leaves of NbAO-silenced plants (a 35-fold change difference as compared to the TRV::eGFP-treated group). Interestingly, CMV and TRV titers vary in different parts of systemically infected N. benthamiana leaves. In TRV::eGFP-treated plants, CMV accumulated only at the top half of the leaf, whereas the bottom half of the leaf was "occupied" by TRV. In contrast, in the NbAO-silenced plants, CMV accumulated in both the top and the bottom half of the leaf, suggesting that the silencing of the NbAO gene resulted in the expansion of the spreading area of CMV. Our data suggest that the AO gene might function as a resistant factor against CMV infection in N. benthamiana.

Enzyme-Regulated In Situ Formation of Copper Hexacyanoferrate Nanoparticles with Oxidase-Mimetic Behaviour for Colorimetric Detection of Ascorbate Oxidase.

In this study, a copper hexacyanoferrate nanoparticle with excellent oxidase-mimetic behaviour has been synthesized through a simple precipitation method. The synthesized copper hexacyanoferrate nanoparticle has intrinsic oxidase-like activity, which can catalyze the chromogenic reaction of 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) through an O2•- reactive oxygen-species-participated process. On the other hand, K3[Fe(CN)6] can be reduced by ascorbic acid (AA) to produce K4[Fe(CN)6], thereby inhibiting the formation of the copper hexacyanoferrate nanoparticles. Furthermore, ascorbate oxidase (AAO) can catalyze the oxidation of AA to produce dehydroascorbic acid, which cannot reduce K3[Fe(CN)6]. Thus, a system for an AAO-regulated in situ formation of copper hexacyanoferrate nanoparticles was constructed by coupling a prepared copper hexacyanoferrate nanozyme with AA for the detection of AAO activity. This colorimetric sensing assay shows high sensitivity and selectivity for the detection of AAO activity (the limit of detection is 0.52 U/L) with a linear range of 1.1-35.7 U/L. Finally, the developed method was applied to detect the activity of AAO in normal human serum with a satisfactory sample spiked recovery (87.4-108.8%). In short, this study provides a good strategy for the construction of nanozyme-based multi-enzyme cascade-signal amplification assay.

Ascorbate oxidase-like nanozyme with high specificity for inhibition of cancer cell proliferation and online electrochemical DOPAC monitoring.

Despite the extensive investigation of the nanozymes exhibit their favorable performance compared to natural enzymes, nevertheless, the highly specific nanozyme still needs to be developed so that it can meet the requirements of exploring the mechanism as well as administration of related diseases and selective monitoring in biological system. In this study, self-assembled glutathione-Cu/Cu2O nanoparticles (GSH-Cu/Cu2O NPs) that exhibits specific ascorbic acid (AA) oxidase-like catalytic activity were constructed for AA-activated and H2O2-reinforced cancer cell proliferation inhibition and selective neurochemical monitoring. Cu/Cu2O NPs demonstrates effective AA oxidase-like activity and no common characteristics of other redox mimic enzymes often present in nanozyme. In particular, we found that the AA oxidase-like activity of GSH-Cu/Cu2O nanozyme was significantly improved by about 40% by improving the activation ability toward oxygen. The synthesized nanozyme can induce the generation of active oxygen by accelerating the oxidation of AA, which effectively suppresses the proliferation of cancer cells. We constructed an online electrochemical system (OECS) though loading nanozyme with enhanced ascorbate oxidase activity into a microreactor and setting it in the upstream of the detector. This GSH-Cu/Cu2O NPs-integrated microreactor can completely eliminate AA interference of the physical level toward 3,4-dihydroxy phenylacetic acid (DOPAC) electrochemical measurement, and the nanozyme-based OECS is able to continuously capture DOPAC alteration in rat brain acidosis model. Our findings may inspire rational design of nanozymes with high specificity as well as nanozyme-based selectivity solution for in vivo detection and show promising opportunities for their involvement in neurochemistry investigation.

Multicopper oxidases GbAO and GbSKS are involved in the Verticillium dahliae resistance in Gossypium barbadense.

Ascorbate oxidase (AO) and skewed5 (SKU5)-similar (SKS) proteins belong to the multicopper oxidase (MCO) family and play important roles in plants in response to environmental stress via modulation of oxidoreduction homeostasis. Currently, reports on the response of Gossypium barbadense MCO to Verticillium wilt (VW) caused by Verticillium dahliae are still limited. Herein, RNA sequencing of two G. barbadense cultivars of VW-resistant XH21 and VW-susceptible XH7 under V. dahliae treatment, combined with physiological and genetic analysis, was performed to analyze the function and mechanism of multicopper oxidases GbAO and GbSKS involved in V. dahliae resistance. The identified differentially expressed genes are mainly involved in the regulation of oxidoreduction reaction, and extracellular components and signaling. Interestingly, ascorbate oxidase family members were discovered as the most significantly upregulated genes after V. dahliae treatment, including GbAO3A/D, GbSKS3A/D, and GbSKS16A/D. H2O2 and Asc contents, especially reductive Asc in both XH21 and XH7, were shown to be increased. Silenced expression of respective GbAO3A/D, GbSKS3A/D, and GbSKS16A/D in virus-induced gene silencing (VIGS) cotton plants significantly decreased the resistance to V. dahliae, coupled with the reduced contents of pectin and lignin. Our results indicate that AO might be involved in cotton VW resistance via the regulation of cell wall components.

Characteristic of the Ascorbate Oxidase Gene Family in Beta vulgaris and Analysis of the Role of AAO in Response to Salinity and Drought in Beet.

Ascorbate oxidase, which is known to play a key role in regulating the redox state in the apoplast, cell wall metabolism, cell expansion and abiotic stress response in plants, oxidizes apo-plastic ascorbic acid (AA) to dehydroascorbic acid (DHA). However, there is little information about the AAO genes and their functions in beets under abiotic stress. The term salt or drought stress refers to the treatment of plants with slow and gradual salinity/drought. Contrastingly, salt shock consists of exposing plants to high salt levels instantaneously and drought shock occurs under fast drought progression. In the present work, we have subjected plants to salinity or drought treatments to elicit either stress or shock and carried out a genome-wide analysis of ascorbate oxidase (AAO) genes in sugar beet (B. vulgaris cv. Huzar) and its halophytic ancestor (B. maritima). Here, conserved domain analyses showed the existence of twelve BvAAO gene family members in the genome of sugar beet. The BvAAO_1-12 genes are located on chromosomes 4, 5, 6, 8 and 9. The phylogenetic tree exhibited the close relationships between BvAAO_1-12 and AAO genes of Spinacia oleracea and Chenopodium quinoa. In both beet genotypes, downregulation of AAO gene expression with the duration of salt stress or drought treatment was observed. This correlated with a decrease in AAO enzyme activity under defined experimental setup. Under salinity, the key downregulated gene was BvAAO_10 in Beta maritima and under drought the BvAAO_3 gene in both beets. This phenomenon may be involved in determining the high tolerance of beet to salinity and drought.

Carboxylated chitosan enabled platinum nanozyme with improved stability and ascorbate oxidase-like activity for a fluorometric acid phosphatase sensor.

Chitosan modification has attracted considerable interest in the nanozyme field last decade. As a chitosan derivative, carboxylated chitosan (CC) has been less explored. Herein, PtNPs with an average size of approximately 3.3 nm and zeta potential of -44.8 ± 0.3 mV (n = 3) have been prepared by using CC as the surface modification (CC-PtNPs). We have carried out an in-depth investigation of CC-PtNPs, including the characterization, colloidal stability, and ascorbate oxidase-like activity. Due to the contribution of carboxylated chitosan, CC-PtNPs present improved colloidal stability and ascorbate oxidase-like activity compared to chitosan-modified Pt nanozyme. Inspired by these results, a fluorometric acid phosphatase sensor was proposed based on the improved performance of CC-PtNPs. This sensor exhibits excellent sensitivity and selectivity towards acid phosphatase in the linear range of 0.25-18 U/L with a low limit of detection (1.31 × 10-3 U/L). The concentration of acid phosphatase in human semen samples has been successfully measured.

Co-evolved plant and blast fungus ascorbate oxidases orchestrate the redox state of host apoplast to modulate rice immunity.

Apoplastic ascorbate oxidases (AOs) play a critical role in reactive oxygen species (ROS)-mediated innate host immunity by regulating the apoplast redox state. To date, little is known about how apoplastic effectors of the rice blast fungus Magnaporthe oryzae modulate the apoplast redox state of rice to subvert plant immunity. In this study, we demonstrated that M. oryzae MoAo1 is an AO that plays a role in virulence by modulating the apoplast redox status of rice cells. We showed that MoAo1 inhibits the activity of rice OsAO3 and OsAO4, which also regulate the apoplast redox status and plant immunity. In addition, we found that MoAo1, OsAO3, and OsAO4 all exhibit polymorphic variations whose varied interactions orchestrate pathogen virulence and rice immunity. Taken together, our results reveal a critical role for extracellular redox enzymes during rice blast infection and shed light on the importance of the apoplast redox state and its regulation in plant-pathogen interactions.

Ascorbate oxidase enabling glucometer readout for portable detection of hydrogen peroxide.

A rapid, portable, and cost-effective method using personal glucose meter (PGM) for quantitative analysis of hydrogen peroxide (H2O2) was established based on ascorbate oxidase (AAO)-catalyzed reaction for the first time. Ascorbic acid (AA) can rapidly reduce ferricyanide (K3[Fe(CN)6]) to ferrocyanide (K4[Fe(CN)6]) in the glucose test strip and transfer electron to the electrode to generating a PGM detectable signal. Thus, the concentration of AA can be directly determined by the PGM as simple as measuring the blood glucose. On the other hand, AAO can catalyze the reduction of H2O2 and produce an enzyme-peroxide complex, which decreases the yields of dehydroascorbic acid formed by the oxidation of AA, resulting in the increase in PGM detectable signal of residual ascorbic acid (re-AA). Therefore, the concentration of H2O2 is proportional to the concentration of re-AA. After optimization of the experimental conditions, the developed method can be used to detect H2O2 at linear range of 2.5-5 × 103 µM with the quantification limit of 2.5 µM. In addition, the satisfactory spiked recoveries (95.3-108.9 %) of real samples (i.e., tap water, contact lens solution, medical hydrogen peroxide, and normal human serum) confirm its feasibility for practical applications. In short, this study provides a feasible PGM-based method for H2O2 detection with simple operations.

Modulation of inner filter effect between persistent luminescent particles and 2, 3-diaminophenazine for ratiometric fluorescent assay of ascorbic acid and ascorbate oxidase activity.

Ascorbate oxidase (AAO) and ascorbic acid (AA) play an important role in delaying lives senescence and metabolism. In this study, a sensitive ratiometric fluorescence sensing system based on the inner filter effect (IFE) between persistent luminescent particles (PLPs) and 2, 3-diaminophenazine (DAP), was designed for the detection of AA and AAO activity. Wherein, PLPs emit blue fluorescence at 475 nm with an excitation wavelength of 370 nm. CoOOH nanosheets with oxidase-like activity can oxidize o-phenylenediamine (OPD) to produce 2, 3-diaminophenazine (DAP) with orange fluorescence at 558 nm. The generated DAP quenched the fluorescence of PLPs by an inner filter effect (IFE). When AA was introduced to the system, CoOOH nanosheets were destroyed and reduced to Co2+, thereby inhibiting the oxidization of OPD and effectively preserving the blue fluorescence of PLPs at 475 nm. Besides, AAO can catalyse AA to produce the oxided dehydroascorbic acid (DHA). The dissipative AA can recover orange fluorescence of DAP with weakening the blue fluorescence of PLPs. Therefore, a sensitive ratio fluorescence sensing strategy was established by using PLPs as the reference signal and DAP as a reported signal for the detection of AA and AAO activity. Under optimal conditions, the obtained linear ranges were 1-45 µM and 1-20 mU/mL, and detection limits were 0.2 µM and 0.25 mU/mL, respectively. Finally, this proposed ratiometric fluorescent analytical strategy was used to detect AA in real samples (lemon, orange, tomato), which exhibited satisfactory results comparing with commercial kit.

A sensitive photothermometric biosensor based on redox reaction-controlled nanoprobe conversion from Prussian blue to Prussian white.

As a new low-cost photothermal nanoprobe, Prussian blue nanoparticles (PB NPs) have been demonstrated to have more potential in photothermometric-based point-of-care testing (POCT) application. However, most of the existing PB NP-based photothermometric sensors were constructed mainly relying on in situ generation of PB NPs or their combination with antigens and antibodies, therefore usually suffering from the inherent defects like complicated preparation and cumbersome surface process as well as high-cost modification. To break this limitation of PB NP-based photothermometric POCT, we proposed an ingenious redox reaction-controlled nanoprobe conversion strategy and successfully applied to photothermometric detection of ascorbate oxidase (AAO). In this design, the heat of PB NP photothermal system under 808-nm laser irradiation dramatically decreased with the addition of AA, due to a unique AA-induced Prussian blue to Prussian white (PB-to-PW) conversion. Upon AAO addition, the heat of reaction system increased because of the enzymatic catalytic reaction between AAO and AA, which led to a significant reduction of AA and resultantly inhibited PB-to-PW conversion. Such target-mediated nanoprobe conversion resulted in an obvious temperature change that could be easily detected by a common thermometer and exhibited good linear ranges from 0.25 to 14 mU/mL with a detection limit as low as 0.21 mU/mL for POCT analysis of AAO. This facile, convenient, and portable photothermometric sensing platform provides an innovative route for the design of PB NP nanoprobe-based photothermometric detection methods. A sensitive photothermometric AAO sensor based on a redox reaction-controlled nanoprobe conversion strategy from Prussian blue to Prussian white.

The miR528-AO Module Confers Enhanced Salt Tolerance in Rice by Modulating the Ascorbic Acid and Abscisic Acid Metabolism and ROS Scavenging.

The monocot lineage-specific miR528 was previously established as a multistress regulator. However, it remains largely unclear how miR528 participates in response to salinity stress in rice. Here, we show that miR528 positively regulates rice salt tolerance by down-regulating a gene encoding l-ascorbate oxidase (AO), thereby bolstering up the AO-mediated abscisic acid (ABA) synthesis and ROS scavenging. Overexpression of miR528 caused a substantial increase in ascorbic acid (AsA) and ABA contents but a significant reduction in ROS accumulation, resulting in the enhanced salt tolerance of rice plants. Conversely, knockdown of miR528 or overexpression of AO stimulated the expression of the AO gene, hence lowering the level of AsA, a critical antioxidant that promotes the ABA content but reduces the ROS level, and then compromising rice tolerance to salinity. Together, the findings reveal a novel mechanism of the miR528-AO module-mediated salt tolerance by modulating the processes of AsA and ABA metabolism as well as ROS detoxification, which adds a new regulatory role to the miR528-AO stress defense pathway in rice.

Elevating Ascorbate in Arabidopsis Stimulates the Production of Abscisic Acid, Phaseic Acid, and to a Lesser Extent Auxin (IAA) and Jasmonates, Resulting in Increased Expression of DHAR1 and Multiple Transcription Factors Associated with Abiotic Stress Tolerance.

Gene expression and phytohormone contents were measured in response to elevating ascorbate in the absence of other confounding stimuli such as high light and abiotic stresses. Young Arabidopsis plants were treated with 25 mM solutions of l-galactose pathway intermediates l-galactose (l-gal) or l-galactono-1,4-lactone (l-galL), as well as L-ascorbic acid (AsA), with 25 mM glucose used as control. Feeding increased rosette AsA 2- to 4-fold but there was little change in AsA biosynthetic gene transcripts. Of the ascorbate recycling genes, only Dehydroascorbate reductase 1 expression was increased. Some known regulatory genes displayed increased expression and included ANAC019, ANAC072, ATHB12, ZAT10 and ZAT12. Investigation of the ANAC019/ANAC072/ATHB12 gene regulatory network revealed a high proportion of ABA regulated genes. Measurement of a subset of jasmonate, ABA, auxin (IAA) and salicylic acid compounds revealed consistent increases in ABA (up to 4.2-fold) and phaseic acid (PA; up to 5-fold), and less consistently certain jasmonates, IAA, but no change in salicylic acid levels. Increased ABA is likely due to increased transcripts for the ABA biosynthetic gene NCED3. There were also smaller increases in transcripts for transcription factors ATHB7, ERD1, and ABF3. These results provide insights into how increasing AsA content can mediate increased abiotic stress tolerance.

Effects of exogenous ascorbic acid on the mycelia growth and primordia formation of Pleurotus ostreatus.

Primordia formation is the first and most critical step in the development of fruiting bodies of edible fungi. In this study, the effects of exogenous ascorbic acid (ASA) on the Pleurotus ostreatus mycelia growth and primordia formation were researched and the results showed that the growth rate of P. ostreatus mycelia was accelerated and the time of primordia formation was advanced. The protein content and ascorbate oxidase (AAO) activity analysis showed that with the increase of ASA concentration, the protein content of mycelia first decreased and then increased, and in a certain concentration range, exogenous ASA could significantly promote the activity of AAO. Further expression analysis of the development regulating genes (Pofst3 and Pofst4) as well as blue light receptor coding genes (PoWC-1 and PoWC-2) showed the expression levels of those four genes all changed after the exogenous ASA addition, which indicated that the expression changes of PoWC-1 and PoWC-2, two key genes in the light morphogenesis, might affect the expression levels of development regulating genes Pofst3 and Pofst4, so as to lead to the formation of primordia in advance.

Photoelectrochemical detection of human epidermal growth factor receptor 2 (HER2) based on Co3O4-ascorbic acid oxidase as multiple signal amplifier.

A sensitive photoelectrochemical (PEC) sensor based on hexagonal carbon nitride tubes (HCNT) as photoactive material was prepared for the detection of human epidermal growth factor receptor 2 (HER2). Magnetic Fe3O4 nanospheres (MNs) modified with anti-HER2 antibodies were employed for highly efficient capture of HER2 from serum sample, and Co3O4 nanoparticles (Co3O4 NPs) modified with ascorbic acid oxidase (AAO) as well as HER2 aptamer were used for signal amplification. When the aptamer-Co3O4-AAO probe was captured onto the electrode surface through the specific binding of the aptamer with HER2, the photocurrent intensity decreased. This was because Co3O4 NPs competed with HCNT for consumption of the excitation energy. As a consequence AAO catalyzed the oxidation of the electron donor (AA), and the aptamer-Co3O4-AAO probe increased the steric hindrance at the electrode surface, leading to significant photocurrent intensity decrease, thus realizing multiple signal amplification. Based on this signal amplification strategy, at 0 V (vs Ag/AgCl), the PEC sensor shows a wide linear response ranging from 1 pg mL-1 to 1 ng mL-1 with a low detection limit of 0.026 pg mL-1 for HER2. Importantly, the prepared PEC sensor was applied for detection of HER2 in human serum samples with recoveries between 98.8 and 101%. Sensitive photoelectrochemical sensor based on Co3O4 nanoparticles modified with ascorbic acid oxidase for signal amplification is reported.