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.

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.

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.

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.

Facile Preparation of Homogeneous Copper Nanoclusters Exhibiting Excellent Tetraenzyme Mimetic Activities for Colorimetric Glutathione Sensing and Fluorimetric Ascorbic Acid Sensing.

Nanozymes are artificial enzymes, which can substitute traditional biological enzymes for multifield applications. However, to date, it remains challenging to search novel mimic enzymes or multienzyme mimics. Herein, a facile and green method for preparing monodisperse, homogeneous copper nanoclusters (Cu NCs) with smaller size was developed, which used cysteamine as a template and hydrazine hydrate as a reductant to reduce Cu2+. The as-prepared Cu NCs exhibited excellent tetraenzyme-like activities, including peroxidase (POD)-, catalase (CAT)-, superoxide dismutase (SOD)-, and ascorbic acid oxidase (AAO)-mimic activities. The mechanisms, kinetics, and catalytic performances of Cu NCs were systematically studied. Moreover, based on the POD-like activity of Cu NCs, sensitive and simple colorimetric sensing glutathione (GSH) was explored, with the low limit of detection of 0.89 µM GSH (S/N = 3). Additionally, a novel fluorimetric ascorbic acid (AA) sensor was developed with the linear range of 0.5-30 µM and limit of detection (LOD) of 0.144 µM, on the basis of the principle that AA is oxidized to dehydroascorbic acid (DHAA) specifically catalyzed by the AAO-like activity of Cu NCs, while DHAA can further react with o-phenylenediamine (OPDA) to generate a highly fluorescent quinoxaline (DFQ) derivative. The as-proposed colorimetric GSH sensor and the fluorimetric AA sensor were capable of detecting GSH and AA, respectively, in real samples accurately and reproducibly. Thus, the Cu NCs-based multienzyme mimic is a promising candidate for biocatalysis and biosensing.

A Fungal Ascorbate Oxidase with Unexpected Laccase Activity.

Ascorbate oxidases are an enzyme group that has not been explored to a large extent. So far, mainly ascorbate oxidases from plants and only a few from fungi have been described. Although ascorbate oxidases belong to the well-studied enzyme family of multi-copper oxidases, their function is still unclear. In this study, Af_AO1, an enzyme from the fungus Aspergillus flavus, was characterized. Sequence analyses and copper content determination demonstrated Af_AO1 to belong to the multi-copper oxidase family. Biochemical characterization and 3D-modeling revealed a similarity to ascorbate oxidases, but also to laccases. Af_AO1 had a 10-fold higher affinity to ascorbic acid (KM = 0.16 ± 0.03 mM) than to ABTS (KM = 1.89 ± 0.12 mM). Furthermore, the best fitting 3D-model was based on the ascorbate oxidase from Cucurbita pepo var. melopepo. The laccase-like activity of Af_AO1 on ABTS (Vmax = 11.56 ± 0.15 µM/min/mg) was, however, not negligible. On the other hand, other typical laccase substrates, such as syringaldezine and guaiacol, were not oxidized by Af_AO1. According to the biochemical and structural characterization, Af_AO1 was classified as ascorbate oxidase with unusual, laccase-like activity.

A fluorescence "off-on-off" sensing platform based on bimetallic gold/silver nanoclusters for ascorbate oxidase activity monitoring.

Herein, papain-protected bimetallic gold/silver nanoclusters (Au/Ag NCs) were successfully synthesized and applied for the detection of ascorbate oxidase (AAO). The doping of papain-protected Au nanoclusters with Ag enhanced the fluorescence intensity with an intense red fluorescence peak at 617 nm, and the red-emitting Au/Ag nanoclusters were further used to monitor the AAO activity. The fluorescence of Au/Ag NCs could be quenched by hydrogen peroxide (H2O2) due to the generation of hydroxyl radicals (˙OH) from the reaction of Ag/Au nanoclusters and H2O2. However, the addition of ascorbic acid (AA) effectively reacted with the free radicals and caused the fluorescence recovery of the Au/Ag NCs. Furthermore, AAO could catalyze the oxidation of AA to form dehydro-ascorbate (DHA). As a result, there was not enough AA to consume the hydroxyl radicals, which resulted in a decrease in the fluorescence of the papain-capped Au/Ag NCs. Therefore, the AAO activity can be monitored by measuring the fluorescence intensity of the red-emitting Au/Ag NCs. Moreover, the developed method for AAO detection displayed a good linear relationship from 5 to 80 mU mL-1 and the detection limit was 1.72 mU mL-1. Thus, a simple and selective method for the determination of the AAO activity was constructed and satisfactory results were obtained in real sample detection.

Ascorbate Oxidase Mimetic Activity of Copper(II) Oxide Nanoparticles.

Although oxidase mimetic nanozymes have been widely investigated, specific biological molecules have rarely been explored as substrates, particularly in the case of ascorbate oxidase (AAO) mimetic nanozymes. Herein, we demonstrate for the first time that copper(II) oxide nanoparticles (CuO NPs) catalyze the oxidation of ascorbic acid (AA) by dissolved O2 (as a green oxidant) to form dehydroascorbic acid (DHAA), thus functioning as a new kind of AAO mimic. Under neutral conditions, the Michaelis-Menten constant of CuO NPs (0.1302 mm) is similar to that of AAO (0.0840 mm). Furthermore, the robustness of CuO NPs is greater than that of AAO, thus making them suitable for applications under various conditions. As a demonstration, a fluorescence AA sensor based on the AAO mimetic activity of CuO NPs was developed. To obtain a fluorescent product, o-phenylenediamine (OPDA) was used to react with the DHAA produced by the oxidation of AA catalyzed by CuO NPs. The developed sensor was cost-effective and easy to fabricate and exhibited high selectivity/sensitivity with a wide linear range (1.25×10-6 to 1.125×10-4 m) and a low detection limit (3.2×10-8 m). The results are expected to aid in expanding the applicability of oxidase mimetic nanozymes in a variety of fields such as biology, medicine, and detection science.

Genome-Wide Identification and Expression Analysis of the Ascorbate Oxidase Gene Family in Gossypium hirsutum Reveals the Critical Role of GhAO1A in Delaying Dark-Induced Leaf Senescence.

Ascorbate oxidase (AO) plays important roles in plant growth and development. Previously, we reported a cotton AO gene that acts as a positive factor in cell growth. Investigations on Gossypium hirsutum AO (GhAO) family genes and their multiple functions are limited. The present study identified eight GhAO family genes and performed bioinformatic analyses. Expression analyses of the tissue specificity and developmental feature of GhAOs displayed their diverse expression patterns. Interestingly, GhAO1A demonstrated the most rapid significant increase in expression after 1 h of light recovery from the dark. Additionally, the transgenic ao1-1/GhAO1A Arabidopsis lines overexpressing GhAO1A in the Arabidopsis ao1-1 late-flowering mutant displayed a recovery to the normal phenotype of wild-type plants. Moreover, compared to the ao1-1 mutant, the ao1-1/GhAO1A transgenic Arabidopsis presented delayed leaf senescence that was induced by the dark, indicating increased sensitivity to hydrogen peroxide (H2O2) under normal conditions that might be caused by a reduction in ascorbic acid (AsA) and ascorbic acid/dehydroascorbate (AsA/DHA) ratio. The results suggested that GhAOs are functionally diverse in plant development and play a critical role in light responsiveness. Our study serves as a foundation for understanding the AO gene family in cotton and elucidating the regulatory mechanism of GhAO1A in delaying dark-induced leaf senescence.

Application of cyclic voltammetry to analyse uric acid and reducing agents in commercial milks.

Cyclic voltammetry (CV) and high performance liquid chromatography (HPLC), were compared to quantify uric acid and antioxidant reducing agents in 36 milk samples. The enzymes uricase and ascorbate oxidase were used to remove uric and ascorbic acids and showed that the peaks obtained by CV and HPLC did not contain contributions from other unknown compounds. The levels of uric acid obtained by CV compared well to those determined using HPLC, with only a few exceptions, and the average difference was around 6%. CV measurements were made using the main anodic peak seen at approximately 330 mV (Ag/AgCl), while a later oxidation peak at approximately 650 mV can be associated with further reducing agents present in milk. The electrochemical method was quicker to apply than HPLC that included a pre-treatment step, and provides an inexpensive and simple method for the reliable analysis of uric acid in milk.

Ascorbate and ferritin interactions: Consequences for iron release in vitro and in vivo and implications for inflammation.

This review discusses the chemical mechanisms of ascorbate-dependent reduction and solubilization of ferritin's ferric iron core and subsequent release of ferrous iron. The process is accelerated by low concentrations of Fe(II) that increase ferritin's intrinsic ascorbate oxidase activity, hence increasing the rate of ascorbate radical formation. These increased rates of ascorbate oxidation provide reducing equivalents (electrons) to ferritin's core and speed the core reduction rates with subsequent solubilization and release of Fe(II). Ascorbate-dependent solubilization of ferritin's iron core has consequences relating to the interpretation of 59Fe uptake sourced from 59Fe-lebelled holotransferrin into ferritin. Ascorbate-dependent reduction of the ferritin core iron solubility increases the size of ferritin's iron exchangeable pool and hence the rate and amount of exchange uptake of 59Fe into ferritin, whilst simultaneously increasing net iron release rate from ferritin. This may rationalize the inconsistency that ascorbate apparently stabilizes 59Fe ferritin and retards lysosomal ferritinolysis and whole cell 59Fe release, whilst paradoxically increasing the rate of net iron release from ferritin. This capacity of ascorbate and iron to synergise ferritin iron release has pathological significance, as it lowers the concentration at which ascorbate activates ferritin's iron release to within the physiological range (50-250 µM). These effects have relevance to inflammatory pathology and to the pro-oxidant effects of ascorbate in cancer therapy and cell death by ferroptosis.

A dual-model strategy for fluorometric determination of ascorbic acid and of ascorbic acid oxidase activity by using DNA-templated gold-silver nanoclusters.

Methods are described for the fluorometric and colorimetric determination of ascorbic acid (AA) and the activity of ascorbic acid oxidase (AA-Ox). The method for AA is based on AA-induced growth and aggregation of DNA-templated gold/silver nanoclusters (DNA-Au/Ag NC), which is accompanied by quenching of fluorescence emission at 605 nm upon 260 nm excitation and a visible color change of the solution from colorless transparent to yellow. The determination of the activity of AA-Ox is based on the finding that it catalyzes the oxidation of AA which results in the inhibition of growth and aggregation. AA can be determined with a 0.6 µmol·L-1 detection limit over the 5 to 150 µmol·L-1 concentration range. AA-Ox can be determined with a 0.0048 U·mL-1 detection limit over the 0.01 to 0.20 U·mL-1 range. Graphical abstract Schematic of a novel fluorometric and colorimetric platform for determination of ascorbic acid and ascorbic acid oxidase activity based on the use of DNA-templated gold-silver nanoclusters.

Nitrate- and nitric oxide-induced plant growth in pea seedlings is linked to antioxidative metabolism and the ABA/GA balance.

This study looks at the effects of potassium nitrate (KNO3) and sodium nitroprusside (SNP), a nitric oxide (NO)-donor, on the development, antioxidant defences and on the abscisic acid (ABA) and gibberellin (GA) levels in pea seedlings. Results show that 10 mM KNO3 and 50 µM SNP stimulate seedling fresh weight (FW), although this effect is not reverted by the action of 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a NO-scavenger. The KNO3 treatment increased peroxidase (POX) and ascorbate oxidase (AOX) activities. SNP, on the other hand, reduced monodehydroascorbate reductase (MDHAR) activity and produced a significant increase in superoxide dismutase (SOD), POX and AOX activities. The "KNO3 plus cPTIO" treatment increased ascorbate peroxidase (APX), MDHAR, glutathione reductase (GR) and SOD activities, but POX activity decreased in relation to the KNO3 treatment. The "SNP plus cPTIO" treatment increased APX and MDHAR activities, whereas a huge decrease in POX activity occurred. Both the KNO3 and the SNP treatments increased reduced ascorbate (ASC) concentrations, which reached control values in the presence of cPTIO. All treatments increased the dehydroascorbate (DHA) level in pea seedlings, leading to a decrease in the redox state of ascorbate. In the "KNO3 plus cPTIO" treatment, an increase in the redox state of ascorbate was observed. Glutathione contents, however, were higher in the presence of SNP than in the presence of KNO3. In addition, KNO3 produced an accumulation of oxidised glutathione (GSSG), especially in the presence of cPTIO, leading to a decrease in the redox state of glutathione. The effect of SNP on reduced glutathione (GSH) levels was reverted by cPTIO, suggesting that NO has a direct effect on GSH biosynthesis or turnover. Both the KNO3 and SNP treatments produced an increase in GA4 and a decrease in ABA concentrations, and this effect was reverted in the presence of the NO-scavenger. Globally, the results suggest a relationship between antioxidant metabolism and the ABA/GA balance during early seedling growth in pea. The results also suggest a role for KNO3 and NO in the modulation of GA4 and ABA levels and antioxidant metabolism in pea seedlings. Furthermore, this effect correlated with an increase in the biomass of the pea seedlings.

Drosophila multicopper oxidase 3 is a potential ferroxidase involved in iron homeostasis.

Multicopper oxidases (MCOs) are a specific group of enzymes that contain multiple copper centers through which different substrates are oxidized. Main members of MCO family include ferroxidases, ascorbate oxidases, and laccases. MCO type of ferroxidases is key to iron transport across the plasma membrane. In Drosophila, there are four potential multicopper oxidases, MCO1-4. No convincing evidence has been presented so far to indicate any of these, or even any insect multicopper oxidase, to be a ferroxidase. Here we show Drosophila MCO3 (dMCO3) is highly likely a bona fide ferroxidase. In vitro activity assay with insect-cell-expressed dMCO3 demonstrated it has potent ferroxidase activity. Meanwhile, the ascorbate oxidase and laccase activities of dMCO3 are much less significant. dMCO3 expression in vivo, albeit at low levels, appears mostly extracellular, reminiscent of mammalian ceruloplasmin in the serum. A null dMCO3 mutant, generated by CRISPR/Cas9 technology, showed disrupted iron homeostasis, evidenced by increased iron level and reduced metal importer Mvl expression. Notably, dMCO3-null flies phenotypically are largely normal at normal or iron stressed-conditions. We speculate the likely existence of a similar iron efflux apparatus as the mammalian ferroportin/ferroxidase in Drosophila. However, its importance to fly iron homeostasis is greatly minimized, which is instead dominated by another iron efflux avenue mediated by the ZIP13-ferritin axis along the ER/Golgi secretion pathway.

An Ascorbic Acid Oxidase-based Sensing Platform for Stereoselective Interaction with Ascorbic Acid and Isoascorbic Acid.

A simple enzyme-based nanohybrid material was fabricated via immobilizing ascorbic acid oxidase (AO) on the surface of flower-like electrodeposited gold nanoparticles (dpAu) and reduced graphene oxide (rGO) modified glassy carbon electrodes (GCEs). The composite material was used for stereoselective interaction with ascorbic acid (AA) and isoascorbic acid (IAA). Herein, AO was applied as a stereoselective selector, and the dpAu/rGO nanohybrid not only acted as a supporter for high loading of AO, but also served as the nanomaterial for signal amplification. The results showed obvious peak current differences between AA and IAA, indicating that this strategy could be employed to recognize AA and IAA. Under the optimum conditions, the sensor exhibited a good linear response to AA and IAA in a linear range of 1.0 × 10-4 - 5.0 × 10-3 M. This approach with the merits of simplicity and rapid response provided a promising perspective for identification of AA and IAA.

[Exogenous H2O2 regulated secondary metabolism of Scutellaria baicalensis and enhanced drug quality].

The increasing demand of Chinese materia medica could not be supplied by wild resource, and the cultivated medicinal materials become popular, which led to decreased quality of many medicinal materials due to the difference of the circumstance between the wild and the cultivated. How to improve quality becomes key points of Chinese medicine resource. The leaves of Scutellaria baicalensis were sprayed with H2O2, the activities of superoxide dismutase (SOD) and catalase (CAT) changed little, but there had been a marked decrease of peroxidase (POD) and ascorbic oxidase (APX), which showed that the antioxidase system declined. Meanwhile, H2O2, as enhanced the expression of phenylalnine ammonialyase (PAL) and ß-glucuronidase (GUS) as well as activity of PAL, promoted the biosynthesis and biotransformation of flavonoids. At the day 2 after treated, H2O2 of 0.004 µmol·L⁻¹ the contents of the baicalin and the wogonoside decreased slightly, but the contents of the baicalein and the wogonin increased significantly, the baicalein from 0.094% to 0.324%, the wogonin from 0.060% to 0.110%, i. e. increased 246% and 83.3%, respectively.

An in situ electron donor consumption strategy for photoelectrochemical biosensing of proteins based on ternary Bi2S3/Ag2S/TiO2 NT arrays.

An ascorbic acid oxidase (AAO)-ascorbic acid bioevent-based electron donor consumption mode was introduced into the PEC bioassay for the first time. Ternary hybrid bismuth sulfide/silver sulfide/TiO2 nanotube arrays as the photoelectrode coupled with AAO attached to SiO2 as a dual signal quenching strategy were employed for sensitivity enhancement.

Sparks fly between ascorbic acid and iron-based nanozymes: A study on Prussian blue nanoparticles.

Herein we reported Prussian blue nanoparticles (PBNPs) possess ascorbic acid oxidase (AAO)- and ascorbic acid peroxidase (APOD)-like activities, which suppressed the formation of harmful H2O2 and finally inhibited the anti-cancer efficiency of ascorbic acid (AA). This newly revealed correlation between iron and AA could provide new insight for the studies of nanozymes and free radical biology.

The redox state of the apoplast influences the acclimation of photosynthesis and leaf metabolism to changing irradiance.

The redox state of the apoplast is largely determined by ascorbate oxidase (AO) activity. The influence of AO activity on leaf acclimation to changing irradiance was explored in wild-type (WT) and transgenic tobacco (Nicotiana tobaccum) lines containing either high [pumpkin AO (PAO)] or low [tobacco AO (TAO)] AO activity at low [low light (LL); 250 µmol m-2  s-1 ] and high [high light (HL); 1600 µmol m-2  s-1 ] irradiance and following the transition from HL to LL. AO activities changed over the photoperiod, particularly in the PAO plants. AO activity had little effect on leaf ascorbate, which was significantly higher under HL than under LL. Apoplastic ascorbate/dehydroascorbate (DHA) ratios and threonate levels were modified by AO activity. Despite decreased levels of transcripts encoding ascorbate synthesis enzymes, leaf ascorbate increased over the first photoperiod following the transition from HL to LL, to much higher levels than LL-grown plants. Photosynthesis rates were significantly higher in the TAO leaves than in WT or PAO plants grown under HL but not under LL. Sub-sets of amino acids and fatty acids were lower in TAO and WT leaves than in the PAO plants under HL, and following the transition to LL. Light acclimation processes are therefore influenced by the apoplastic as well as chloroplastic redox state.