Monoallelic and bi-allelic variants in NCDN cause neurodevelopmental delay, intellectual disability, and epilepsy.

Neurochondrin (NCDN) is a cytoplasmatic neural protein of importance for neural growth, glutamate receptor (mGluR) signaling, and synaptic plasticity. Conditional loss of Ncdn in mice neural tissue causes depressive-like behaviors, impaired spatial learning, and epileptic seizures. We report on NCDN missense variants in six affected individuals with variable degrees of developmental delay, intellectual disability (ID), and seizures. Three siblings were found homozygous for a NCDN missense variant, whereas another three unrelated individuals carried different de novo missense variants in NCDN. We assayed the missense variants for their capability to rescue impaired neurite formation in human neuroblastoma (SH-SY5Y) cells depleted of NCDN. Overexpression of wild-type NCDN rescued the neurite-phenotype in contrast to expression of NCDN containing the variants of affected individuals. Two missense variants, associated with severe neurodevelopmental features and epilepsy, were unable to restore mGluR5-induced ERK phosphorylation. Electrophysiological analysis of SH-SY5Y cells depleted of NCDN exhibited altered membrane potential and impaired action potentials at repolarization, suggesting NCDN to be required for normal biophysical properties. Using available transcriptome data from human fetal cortex, we show that NCDN is highly expressed in maturing excitatory neurons. In combination, our data provide evidence that bi-allelic and de novo variants in NCDN cause a clinically variable form of neurodevelopmental delay and epilepsy, highlighting a critical role for NCDN in human brain development.

Plant-insect communication in urban forests: Similarities of plant volatile compositions among tree species (host vs. non-host trees) for alder leaf beetle Agelastica coerulea.

Behavior of insects, such as pollination and grazing, is usually determined by biogenic volatile organic compounds (BVOCs). However, particularly in O3-polluted urban forests, the BVOCs-based plant-insect communication can be disrupted by the reaction of O3 with leaf-emitted BVOCs, such as between Japanese white birch (Betula platyphylla var. japonica) and a leaf beetle (Agelastica coerulea). To understand plant-insect communication in O3-polluted environments, it is necessary to identify chemical species of BVOCs that contribute to attractiveness toward insects but are diminished by elevated O3. In this study, we conducted olfactory response tests and gas chromatography mass spectrometry (GC-MS) analyses to clarify whether there is a similarity of BVOC components among Betulaceae host trees that can explain the attraction of the stenophagous insect A. coerulea. The olfactory response tests indicated that Betulaceae host trees attract A. coerulea via leaf-emitted BVOCs, while there was no preference of the leaf beetles to non-host trees (Sorbus commixta and Morus bombycis). However, GC-MS analyses indicated that the composition of BVOC blends considerably differed among Betulaceae host trees, although alders (Alnus hirsuta and A. japonica) had a similar composition of BVOC blend in each season (June and September) during which the adult leaf beetle is active. A distinct characteristic of the emission from B. platyphylla was that 2-carene and limonene, which are O3-reactive species, were emitted with a high monoterpene ratio irrespective of the season. Thus, these volatiles and the blend could be expected to lead the disrupted communication found between B. platyphylla and A. coerulea under elevated O3 in previous field studies. In addition, our results indicated that A. coerulea is attracted to more than one blend within Betulaceae host trees, suggesting that grazing damages can be affected by different host preferences and O3 reactivity with specific BVOCs in the field. BVOCs-based plant-insect interactions should be further studied in multi-species communities to better understand plant-insect communication in O3-polluted environments.

Efficient detection of copy-number variations using exome data: Batch- and sex-based analyses.

Many algorithms to detect copy number variations (CNVs) using exome sequencing (ES) data have been reported and evaluated on their sensitivity and specificity, reproducibility, and precision. However, operational optimization of such algorithms for a better performance has not been fully addressed. ES of 1199 samples including 763 patients with different disease profiles was performed. ES data were analyzed to detect CNVs by both the eXome Hidden Markov Model (XHMM) and modified Nord's method. To efficiently detect rare CNVs, we aimed to decrease sequencing biases by analyzing, at the same time, the data of all unrelated samples sequenced in the same flow cell as a batch, and to eliminate sex effects of X-linked CNVs by analyzing female and male sequences separately. We also applied several filtering steps for more efficient CNV selection. The average number of CNVs detected in one sample was <5. This optimization together with targeted CNV analysis by Nord's method identified pathogenic/likely pathogenic CNVs in 34 patients (4.5%, 34/763). In particular, among 142 patients with epilepsy, the current protocol detected clinically relevant CNVs in 19 (13.4%) patients, whereas the previous protocol identified them in only 14 (9.9%) patients. Thus, this batch-based XHMM analysis efficiently selected rare pathogenic CNVs in genetic diseases.

Identification of new biomarkers of pyridoxine-dependent epilepsy by GC/MS-based urine metabolomics.

α-Aminoadipic semialdehyde and its cyclic form (Δ1-piperideine-6-carboxylate) accumulate in patients with α-aminoadipic semialdehyde dehydrogenase (AASADH; antiquitin; ALDH7A1) deficiency. Δ1-Piperideine-6-carboxylate is known to react with pyridoxal 5'-phosphate (PLP) to form a Knoevenagel condensation product, resulting in pyridoxine-dependent epilepsy. Despite dramatic clinical improvement following pyridoxine supplementation, many patients still suffer some degree of intellectual disability due to delayed diagnosis. In order to expedite the diagnosis of patients with suspected AASADH deficiency and minimize the delay in treatment, we used gas chromatography-mass spectrometry-based metabolomics to search for potentially diagnostic biomarkers in urine from four patients with ALDH7A1 mutations, and identified Δ2-piperideine-6-carboxylate, 6-oxopipecolate, and pipecolate as candidate biomarkers. In a patient at postnatal day six, but before pyridoxine treatment, Δ2-piperideine-6-carboxylate and pipecolate were present at very high concentrations, indicating that these compounds may be good biomarkers for untreated AASADH deficiency patients. On the other hand, following pyridoxine/PLP treatment, 6-oxopipecolate was shown to be greatly elevated. We suggest that noninvasive urine metabolomics screening for Δ2-piperideine-6-carboxylate, 6-oxopipecolate, and pipecolate will be useful for prompt and reliable diagnosis of AASADH deficiency in patients within any age group. The most appropriate combination among Δ2-piperideine-6-carboxylate, 6-oxopipecolate, and pipecolate as biomarkers for AASADH deficiency patients appears to depend on the age of the patient and whether pyridoxine/PLP supplementation has been implemented. We anticipate that the present bioanalytical information will also be useful to researchers studying glutamate, proline, lysine and ornithine metabolism in mammals and other organisms.

Interactive effect of leaf age and ozone on mesophyll conductance in Siebold's beech.

Mesophyll conductance (Gm ) is one of the most important factors determining photosynthesis. Tropospheric ozone (O3 ) is known to accelerate leaf senescence and causes a decline of photosynthetic activity in leaves. However, the effects of age-related variation of O3 on Gm have not been well investigated, and we, therefore, analysed leaf gas exchange data in a free-air O3 exposure experiment on Siebold's beech with two levels (ambient and elevated O3 : 28 and 62 nmol mol-1 as daylight average, respectively). In addition, we examined whether O3 -induced changes on leaf morphology (leaf mass per area, leaf density and leaf thickness) may affect CO2 diffusion inside leaves. We found that O3 damaged the photosynthetic biochemistry progressively during the growing season. The Gm was associated with a reduced photosynthesis in O3 -fumigated Siebold's beech in August. The O3 -induced reduction of Gm was negatively correlated with leaf density, which was increased by elevated O3 , suggesting that the reduction of Gm was accompanied by changes in the physical structure of mesophyll cells. On the other hand, in October, the O3 -induced decrease of Gm was diminished because Gm decreased due to leaf senescence regardless of O3 treatment. The reduction of photosynthesis in senescent leaves after O3 exposure was mainly due to a decrease of maximum carboxylation rate (Vcmax ) and/or maximum electron transport rate (Jmax ) rather than diffusive limitations to CO2 transport such as Gm . A leaf age×O3 interaction of photosynthetic response will be a key for modelling photosynthesis in O3 -polluted environments.

Methylprednisolone pulse therapy in 31 patients with refractory epilepsy: A single-center retrospective analysis.

PURPOSE: We investigated the efficacy of methylprednisolone pulse therapy (MP) and responder characteristics in patients with refractory epilepsy. METHODS: We reviewed medical records of our center to identify patients with refractory epilepsy treated with MP other than continuous spikes and waves during slow sleep (CSWS), Landau-Kleffner syndrome (LKS), or Rasmussen's syndrome (RS) between 2004 and 2015. A course of MP consisted of intravenous methylprednisolone (30 mg/kg/day) on three consecutive days. Patients received multiple courses at intervals of four weeks. We examined seizure outcome, developmental outcome, antibodies to N-methyl-d-aspartate (NMDA)-type glutamate receptors (GluRs), cerebral spinal fluid (CSF)-albumin/serum-albumin ratio, and interictal electroencephalograms (EEGs). Responder to MP was defined as maintaining seizure reduction rate (SRR) ≥50% for three months after the first course of MP. RESULTS: Thirty-one consecutive patients treated with MP at our center were studied. Seizure types were focal onset impaired awareness seizure (FIAS) only (n = 23), FIAS with epileptic spasms (ES) (n = 7), and ES only (n = 1). Responder rate was 32.2% (10/31 patients), and seizure-free rate was 9.7% (3/31). Responders constituted 43.5% of patients without ES. No patient with ES was responder. Behavior and cognition also improved in 6 of 10 responders. History of seizure aggravation after inactivated vaccine before MP was found significantly higher rate in responder patients, comparing with nonresponder patients (p = 0.01). CONCLUSION: Methylprednisolone pulse therapy may be considered for possible treatment in patients with focal epilepsy with drug-resistant seizures without ES, and it may improve cognitive function and behavioral comorbidities.

A quantitative assessment of hormetic responses of plants to ozone.

Evaluations of ozone effects on vegetation across the globe over the last seven decades have mostly incorporated exposure levels that were multi-fold the preindustrial concentrations. As such, global risk assessments and derivation of critical levels for protecting plants and food supplies were based on extrapolation from high to low exposure levels. These were developed in an era when it was thought that stress biology is framed around a linear dose-response. However, it has recently emerged that stress biology commonly displays non-linear, hormetic processes. The current biological understanding highlights that the strategy of extrapolating from high to low exposure levels may lead to biased estimates. Here, we analyzed a diverse sample of published empirical data of approximately 500 stimulatory, hormetic-like dose-responses induced by ozone in plants. The median value of the maximum stimulatory responses induced by elevated ozone was 124%, and commonly <150%, of the background response (control), independently of species and response variable. The maximum stimulatory response to ozone was similar among types of response variables and major plant species. It was also similar among clades, between herbaceous and woody plants, between deciduous and evergreen trees, and between annual and perennial herbaceous plants. There were modest differences in the stimulatory response between genera and between families which may reflect different experimental designs and conditions among studies. The responses varied significantly upon type of exposure system, with open-top chambers (OTCs) underestimating the maximum stimulatory response compared to free-air ozone-concentration enrichment (FACE) systems. These findings suggest that plants show a generalized hormetic stimulation by ozone which is constrained within certain limits of biological plasticity, being highly generalizable, evolutionarily based, and maintained over ecological scales. They further highlight that non-linear responses should be taken into account when assessing the ozone effects on plants.

High doses of ethylenediurea (EDU) as soil drenches did not increase leaf N content or cause phytotoxicity in willow grown in fertile soil.

Ground-level ozone (O3) levels are nowadays elevated in wide regions of the Earth, causing significant effects on plants that finally lead to suppressed productivity and yield losses. Ethylenediurea (EDU) is a chemical compound which is widely used in research projects as phytoprotectant against O3 injury. The EDU mode of action remains still unclear, while there are indications that EDU may contribute to plants with nitrogen (N) when the soil is poor in N and the plants have relatively small leaf area. To reveal whether the N content of EDU acts as a fertilizer to plants when the soil is not poor in N and the plants have relatively large total plant leaf area, willow plants (Salix sachalinensis Fr. Schm) were exposed to low ambient O3 levels and treated ten times (9-day interval) with 200mL soil drench containing 0, 800 or 1600mg EDU L-1. Fertilizer was added to a nutrient-poor soil, and the plants had an average plant leaf area of 9.1m2 at the beginning of EDU treatments. Indications for EDU-induced hormesis in maximum electron transport rate (Jmax) and ratio of intercellular to ambient CO2 concentration (Ci:Ca) were observed at the end of the experiment. No other EDU-induced effects on leaf greenness and N content, maximum quantum yield of photosystem II (Fv/Fm), gas exchange, growth and matter production suggest that EDU did not act as N fertilizer and did not cause toxicity under these experimental conditions.

A meta-analysis of leaf nitrogen distribution within plant canopies.

BACKGROUND AND AIMS: Leaf nitrogen distribution in the plant canopy is an important determinant for canopy photosynthesis. Although the gradient of leaf nitrogen is formed along light gradients in the canopy, its quantitative variations among species and environmental responses remain unknown. Here, we conducted a global meta-analysis of leaf nitrogen distribution in plant canopies. METHODS: We collected data on the nitrogen distribution and environmental variables from 393 plant canopies (100, 241 and 52 canopies for wheat, other herbaceous and woody species, respectively). KEY RESULTS: The trends were clearly different between wheat and other species; the photosynthetic nitrogen distribution coefficient (Kb) was mainly determined by leaf area index (LAI) in wheat, whereas it was correlated with the light extinction coefficient (KL) and LAI in other species. Some other variables were also found to influence Kb We present the best equations for Kb as a function of environmental variables and canopy characteristics. As a more simple function, Kb = 0·5KL can be used for canopies of species other than wheat. Sensitivity analyses using a terrestrial carbon flux model showed that gross primary production tended to be more sensitive to the Kb value especially when nitrogen content of the uppermost leaf was fixed. CONCLUSION: Our results reveal that nitrogen distribution is mainly driven by the vertical light gradient but other factors such as LAI also have significant effects. Our equations contribute to an improvement in the projection of plant productivity and cycling of carbon and nitrogen in terrestrial ecosystems.

Mesophyll conductance in leaves of Japanese white birch (Betula platyphylla var. japonica) seedlings grown under elevated CO2 concentration and low N availability.

To test the hypothesis that mesophyll conductance (gm ) would be reduced by leaf starch accumulation in plants grown under elevated CO2 concentration [CO2 ], we investigated gm in seedlings of Japanese white birch grown under ambient and elevated [CO2 ] with an adequate and limited nitrogen supply using simultaneous gas exchange and chlorophyll fluorescence measurements. Both elevated [CO2 ] and limited nitrogen supply decreased area-based leaf N accompanied with a decrease in the maximum rate of Rubisco carboxylation (Vc,max ) on a CO2 concentration at chloroplast stroma (Cc ) basis. Conversely, only seedlings grown at elevated [CO2 ] under limited nitrogen supply had significantly higher leaf starch content with significantly lower gm among the treatment combinations. Based on a leaf anatomical analysis using microscopic photographs, however, there were no significant difference in the area of chloroplast surfaces facing intercellular space per unit leaf area among treatment combinations. Thicker cell walls were suggested in plants grown under limited N by increases in leaf mass per area subtracting non-structural carbohydrates. These results suggest that starch accumulation and/or thicker cell walls in the leaves grown at elevated [CO2 ] under limited N supply might hinder CO2 diffusion in chloroplasts and cell walls, which would be an additional cause of photosynthetic downregulation as well as a reduction in Rubisco activity related to the reduced leaf N under elevated [CO2 ].

Photosynthetic responses of Monarch birch seedlings to differing timings of free air ozone fumigation.

To study the effects of different periods of ozone (O3) fumigation on photosynthesis in leaves of the Monarch birch (Betula maximowicziana), we undertook free air O3 fumigation to Monarch birch seedlings at a concentration of 60 nmol mol(-1) during daytime. Plants were exposed to O3 at early, late or both periods in the growing season. The light-saturated net photosynthetic rate (A(sat)) in July and August was reduced by O3 exposure through a reduction in the maximum rate of carboxylation (V(c,max)). In early September, on the other hand, despite a reduction in V(c,max), A(sat) was not reduced by O3 due to a counteracting increase in the stomatal conductance. Through the experiment, there was no difference in sensitivity to O3 between maturing and matured leaves. We analyzed the relationship between A(sat), V(c,max) and accumulated stomatal O3 flux (AF(st)). Whereas V(c,max) decreased with increasing AF(st), the correlation between A(sat) and AF(st) was weak because the response of stomatal conductance to O3 was affected by season. We conclude photosynthetic response of Monarch birch to O3 exposure changes with season. This is due to the inconstant stomatal response to O3 but not due to the respose of biochemical assimilation capacity in chloroplasts.

Direct mapping of morphological distribution of syringyl and guaiacyl lignin in the xylem of maple by time-of-flight secondary ion mass spectrometry.

Lignin, one of the main structural polymer of plant cell walls, varies in amount and monomeric composition among tissue and cell types, as well as among plant species. However, few analytical methods are available that can conveniently and accurately determine the morphological distribution of lignin units at the cellular level. In this report, we used time-of-flight secondary ion mass spectrometry (TOF-SIMS) to directly map guaiacyl (G) and syringyl (S) lignin units in several successive growth rings of the maple xylem. TOF-SIMS imaging and a semiquantitative approach revealed clear difference in the annual distribution of lignins between the fiber and vessel. While the vessel walls were constantly G-rich with varied S/G ratios through a growth ring, the fibers showed fairly regular annual distribution of lignins in which the earlywood was S-rich with an almost constant S/G ratio and the latewood was G-rich resulting from a decrease of the S unit. The reliability of TOF-SIMS results was demonstrated by its high correlation with the results of thioacidolysis on radial distribution of the S/G ratio in several contiguous tree rings and also in the latewood and earlywood of each ring. These results indicate that TOF-SIMS allows direct visualization of lignin composition in plant tissues.

Model-based analysis of avoidance of ozone stress by stomatal closure in Siebold's beech (Fagus crenata).

BACKGROUND AND AIMS: Resistance of plants to ozone stress can be classified as either avoidance or tolerance. Avoidance of ozone stress may be explained by decreased stomatal conductance during ozone exposure because stomata are the principal interface for entry of ozone into plants. In this study, a coupled photosynthesis-stomatal model was modified to test whether the presence of ozone can induce avoidance of ozone stress by stomatal closure. METHODS: The response of Siebold's beech (Fagus crenata), a representative deciduous tree species, to ozone was studied in a free-air ozone exposure experiment in Japan. Photosynthesis and stomatal conductance were measured under ambient and elevated ozone. An optimization model of stomata involving water, CO2 and ozone flux was tested using the leaf gas exchange data. KEY RESULTS: The data suggest that there are two phases in the avoidance of ozone stress via stomatal closure for Siebold's beech: (1) in early summer ozone influx is efficiently limited by a reduction in stomatal conductance, without any clear effect on photosynthetic capacity; and (2) in late summer and autumn the efficiency of ozone stress avoidance was decreased because the decrease in stomatal conductance was small and accompanied by an ozone-induced decline of photosynthetic capacity. CONCLUSIONS: Ozone-induced stomatal closure in Siebold's beech during early summer reduces ozone influx and allows the maximum photosynthetic capacity to be reached, but is not sufficient in older leaves to protect the photosynthetic system.

Photosynthetic downregulation in leaves of the Japanese white birch grown under elevated CO(2) concentration does not change their temperature-dependent susceptibility to photoinhibition.

To determine the effects of elevated CO(2) concentration ([CO(2)]) on the temperature-dependent photosynthetic properties, we measured gas exchange and chlorophyll fluorescence at various leaf temperatures (15, 20, 25, 30, 35 and 40°C) in 1-year-old seedlings of the Japanese white birch (Betula platyphylla var. japonica), grown in a phytotron under natural daylight at two [CO(2)] levels (ambient: 400 µmol mol(-1) and elevated: 800 µmol mol(-1)) and limited N availability (90 mg N plant(-1)). Plants grown under elevated [CO(2)] exhibited photosynthetic downregulation, indicated by a decrease in the carboxylation capacity of Rubisco. At temperatures above 30°C, the net photosynthetic rates of elevated-CO(2)-grown plants exceeded those grown under ambient [CO(2)] when compared at their growth [CO(2)]. Electron transport rates were significantly lower in elevated-CO(2)-grown plants than ambient-CO(2)-grown ones at temperatures below 25°C. However, no significant difference was observed in the fraction of excess light energy [(1 - q(P))× F(v)'/F(m)'] between CO(2) treatments across the temperature range. The quantum yield of regulated non-photochemical energy loss was significantly higher in elevated-CO(2)-grown plants than ambient, when compared at their respective growth [CO(2)] below 25°C. These results suggest that elevated-CO(2)-induced downregulation might not exacerbate the temperature-dependent susceptibility to photoinhibition, because reduced energy consumption by electron transport was compensated for by increased thermal energy dissipation at low temperatures.

Mixed-power scaling of whole-plant respiration from seedlings to giant trees.

The scaling of respiratory metabolism with body mass is one of the most pervasive phenomena in biology. Using a single allometric equation to characterize empirical scaling relationships and to evaluate alternative hypotheses about mechanisms has been controversial. We developed a method to directly measure respiration of 271 whole plants, spanning nine orders of magnitude in body mass, from small seedlings to large trees, and from tropical to boreal ecosystems. Our measurements include the roots, which have often been ignored. Rather than a single power-law relationship, our data are fit by a biphasic, mixed-power function. The allometric exponent varies continuously from 1 in the smallest plants to 3/4 in larger saplings and trees. Therefore, our findings support the recent findings of Reich et al. [Reich PB, Tjoelker MG, Machado JL, Oleksyn J (2006) Universal scaling of respiratory metabolism, size, and nitrogen in plants. Nature 439:457-461] and West, Brown, and Enquist [West GB, Brown JH, Enquist BJ (1997) A general model for the origin of allometric scaling laws in biology. Science 276:122 -126.]. The transition from linear to 3/4-power scaling may indicate fundamental physical and physiological constraints on the allocation of plant biomass between photosynthetic and nonphotosynthetic organs over the course of ontogenetic plant growth.

Elevated ozone alters long-chain fatty acids in leaves of Japanese white birch saplings.

Long-chain fatty acids (LCFAs) in leaves have attracted attention as nutritious phytochemicals and olfactory signals that influence the behavior and growth of herbivorous insects. In recognition of the negative effects of increasing tropospheric ozone (O3) levels on plants, LCFAs can be altered through peroxidation by O3. However, how elevated O3 changes the amount and composition of LCFAs in field-grown plants is still unknown. We investigated palmitic, stearic, oleic, linoleic, linolenic LCFAs in the two leaf types (spring and summer) and two stages (early and late stage after expansion) of Japanese white birch (Betula platyphylla var. japonica) after a multi-year O3 exposure on the field. Summer leaves exhibited a distinct composition of LCFAs under elevated O3 at the early stage, whereas both stages of spring leaves did not exhibit significant changes in LCFAs composition by elevated O3. In the spring leaves, the amounts of saturated LCFAs significantly increased at the early stage, however, the amount of total, palmitic, and linoleic acids at the late stage were significantly decreased by elevated O3. Summer leaves had a lower amount of all LCFAs at both leaf stages. Regarding the early stage of summer leaves, the lower amount of LCFAs under elevated O3 was possibly due to O3-suppressed photosynthesis in the current spring leaves. Furthermore, the decrease ratio of spring leaves over time was significantly increased by elevated O3 in all LCFAs, whereas summer leaves did not exhibit such an effect. These findings suggest that further studies should be conducted to reveal the biological functions of LCFAs under elevated O3, considering the leaf type- and stage-dependent changes of LCFAs.

Ethylenediurea protects against ozone phytotoxicity not by adding nitrogen or controlling stomata in a stomata-unresponsive hybrid poplar.

Ozone (O3) pollution is a persistent environmental issue worldwide, which causes widespread damage to vegetation, deteriorating plant health and reducing plant productivity. Ethylenediurea (EDU) is a synthetic chemical that has been widely applied in scientific studies as a protectant against O3 phytotoxicities. Despite four decades of active research, the exact mechanisms to explain its mode of action remain unclear. Here, we aimed to reveal whether EDU's phytoprotective property is due to its control over stomatal regulation and/or its action as a nitrogen (N) fertilizer, utilizing stomatal-unresponsive plants of a hybrid poplar (Populus koreana × trichocarpa cv. Peace) grown in a free-air O3-concenctration enrichment (FACE) facility. Plants were treated with water (WAT), EDU (400 mg L-1), or EDU's constitutive amount of N every nine days, and exposed to ambient (AOZ) or elevated (EOZ) O3 during a growing season (June-September). EOZ led to extensive foliar injuries (but protected against rust disease), lower photosynthetic rate (A), impaired dynamics of responses of A to changes in light intensity, and smaller total plant leaf area. EDU protected against common phytotoxicities caused by EOZ without inducing stomatal closure, since stomatal conductance (gs) was generally unresponsive to the experimental treatments. EDU also modulated the dynamic response of A to light fluctuations under O3 stress. N addition acted as a fertilizer but did not satisfactorily protect plants against O3 phytotoxicities. The results suggest that EDU protects against O3 phytotoxicity not by adding N or controlling stomata, which provides a new insight into our understanding of the mode of action of EDU as a protectant against O3 phytotoxicity.