Atmospheric nitrogen dioxide suppresses the activity of phytochrome interacting factor 4 to suppress hypocotyl elongation.

MAIN CONCLUSION: Ambient concentrations of atmospheric nitrogen dioxide (NO2) inhibit the binding of PIF4 to promoter regions of auxin pathway genes to suppress hypocotyl elongation in Arabidopsis. Ambient concentrations (10-50 ppb) of atmospheric nitrogen dioxide (NO2) positively regulate plant growth to the extent that organ size and shoot biomass can nearly double in various species, including Arabidopsis thaliana (Arabidopsis). However, the precise molecular mechanism underlying NO2-mediated processes in plants, and the involvement of specific molecules in these processes, remain unknown. We measured hypocotyl elongation and the transcript levels of PIF4, encoding a bHLH transcription factor, and its target genes in wild-type (WT) and various pif mutants grown in the presence or absence of 50 ppb NO2. Chromatin immunoprecipitation assays were performed to quantify binding of PIF4 to the promoter regions of its target genes. NO2 suppressed hypocotyl elongation in WT plants, but not in the pifq or pif4 mutants. NO2 suppressed the expression of target genes of PIF4, but did not affect the transcript level of the PIF4 gene itself or the level of PIF4 protein. NO2 inhibited the binding of PIF4 to the promoter regions of two of its target genes, SAUR46 and SAUR67. In conclusion, NO2 inhibits the binding of PIF4 to the promoter regions of genes involved in the auxin pathway to suppress hypocotyl elongation in Arabidopsis. Consequently, PIF4 emerges as a pivotal participant in this regulatory process. This study has further clarified the intricate regulatory mechanisms governing plant responses to environmental pollutants, thereby advancing our understanding of how plants adapt to changing atmospheric conditions.

Persistent methicillin-resistant Staphylococcus aureus bacteremia in an adult patient with Netherton's syndrome: A case report.

Netherton's syndrome, a rare congenital disorder, is clinically characterized by chronic dermatologic disorders such as ichthyosiform erythroderma and ichthyosis linearis circumflexa. Curable treatment is yet to be established, and corticosteroid ointment is required to maintain good dermatological condition. Because of the permanent skin barrier impairment, patients with Netherton's syndrome are considered to be vulnerable to cutaneous infections. However, its clinical characteristics are yet to be elucidated due to the limited number of reported cases. Herein, we describe the clinical course of a patient who developed persistent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia. A 19-year-old Japanese woman who had been diagnosed with Netherton's syndrome in her infancy and had been applying topical corticosteroid agents all over her body since her then, was referred to our hospital because of persistent MRSA bacteremia and secondary adrenal insufficiency. The patient was diagnosed with a central line-associated bloodstream infection and was appropriately treated with antibiotics and corticosteroid therapies. We assume that the damaged skin barrier due to the congenital dermatological disorder causes a disruption in the normal bacterial flora of the skin, leading to the invasion of harmful bacteria, such as S. aureus. In addition, internal (humoral immunodeficiency by decreased antibody against bacterial polysaccharide antigens) and external (prolonged and systemic use of corticosteroid ointment) factors bring about an immunodeficiency state in such patients. We highlight that in the absence of radical treatment, clinicians need to recognize that patients with Netherton's syndrome are vulnerable to bacterial infections owing to the mixture of immunosuppressive factors.

Prevalence of medical factors related to aging among older car drivers: a multicenter, cross-sectional, descriptive study.

AIM: An increasing number of older adults in Japan are at an increased risk of road traffic crashes. This study aimed to investigate the prevalence of potential underlying medical factors that increase the risk of road traffic crashes among older people. METHODS: This cross-sectional observational study was conducted in 11 medical institutions in Japan using self-administered questionnaires and physical examination from January to May 2021. The background and social data, data on the use of nursing care insurance, and clinical data suggestive of polypharmacy, sarcopenia, cognitive impairment, and frailty/oral frailty were obtained. The prevalence of these factors was compared between everyday and occasional drivers. RESULTS: Data of 127 patients were collected; their median (interquartile range) age was 73 (70-78) years. Of the total participants, 82 were men (64.6%) and 45 were women (35.4%). There were 77 everyday drivers and 50 occasional drivers. Of these, 121 (95.3%) had not applied for nursing care insurance, but the numbers of those who required help 1 and 2 were 1 (0.8%) and 3 (2.4%), respectively. Prevalence of medical factors was as follows: polypharmacy, 27.6%; sarcopenia, 8.7%; dementia, 16.4%; frailty, 15.0%; and oral frailty, 54.3%; it was not significantly different between every day and occasional drivers. Intention to return the car license was significantly higher among the occasional drivers (2.6% vs. 14.0%; odds ratio: 6.7, 95% confidence interval: 1.2-70.6, p = 0.024). CONCLUSION: We uncovered the prevalence of medical factors that can be associated with road traffic crashes among Japanese older people aged ≥ 65 years in our community.

Dual selective nitration in Arabidopsis: Almost exclusive nitration of PsbO and PsbP, and highly susceptible nitration of four non-PSII proteins, including peroxiredoxin II E.

Protein tyrosine nitration is a selective process, as revealed in studies of animals. However, evidence for selective protein nitration in plants is scarce. In this study, Arabidopsis plants were exposed to air with or without nitrogen dioxide at 40 ppm for 8 h in light. Proteins extracted from whole leaves or isolated chloroplasts were subjected to 2D PAGE followed by SYPRO Ruby staining and immunoblotting using an anti-3-nitrotyrosine antibody. We determined the relative intensity of a spot on an immunoblot (designated RISI), and relative intensity of the corresponding spot on SYPRO Ruby gel (designated RISS). Proteins that exhibited a high RISI value and/or a high RISI/RISS ratio were considered selectively nitrated. In whole leaf proteins from exposed plants, all immunopositive spots were identified as PsbO1, PsbO2 or PsbP1 by PMF. Thus, nitration was exclusive to PsbO and PsbP, extrinsic proteins of photosystem II (PSII). Their RISI/RISS ratio was ≤1.5. Non-exposed plants showed very faint nitration. In purified chloroplast proteins, PsbO and PsbP accounted for >80% of the total RISI values, while four non-PSII proteins, including peroxiredoxin II E, exhibited high RISI/RISS ratios (2.5∼6.6). Tyr(9) of PsbO1 was identified as a nitration site. Thus, nitration is selective for two PSII and four non-PSII proteins in Arabidopsis.

Nitrogen dioxide regulates organ growth by controlling cell proliferation and enlargement in Arabidopsis.

• To gain more insight into the physiological function of nitrogen dioxide (NO2), we investigated the effects of exogenous NO2 on growth in Arabidopsis thaliana. • Plants were grown in air without NO2 for 1 wk after sowing and then grown for 1-4 wk in air with (designated treated plants) or without (control plants) NO2. Plants were irrigated semiweekly with a nutrient solution containing 19.7 mM nitrate and 10.3 mM ammonium. • Five-week-old plants treated with 50 ppb NO2 showed a ≤ 2.8-fold increase in biomass relative to controls. Treated plants also showed early flowering. The magnitude of the effects of NO2 on leaf expansion, cell proliferation and enlargement was greater in developing than in maturing leaves. Leaf areas were 1.3-8.4 times larger on treated plants than corresponding leaves on control plants. The NO2-induced increase in leaf size was largely attributable to cell proliferation in developing leaves, but was attributable to both cell proliferation and enlargement in maturing leaves. The expression of different sets of genes for cell proliferation and/or enlargement was induced by NO2, but depended on the leaf developmental stage. • Collectively, these results indicated that NO2 regulates organ growth by controlling cell proliferation and enlargement.

Clinical characteristics of Campylobacter bacteremia: a multicenter retrospective study.

Campylobacter species are the pathogens of the intestinal tract, which infrequently cause bacteremia. To reveal the clinical characteristics of Campylobacter bacteremia, we performed a retrospective, multicenter study. Patients diagnosed with Campylobacter bacteremia in three general hospitals in western Japan between 2011 and 2021 were included in the study. Clinical, microbiological, and prognostic data of the patients were obtained from medical records. We stratified the cases into the gastroenteritis (GE) and fever predominant (FP) types by focusing on the presence of gastrointestinal symptoms. Thirty-nine patients (24 men and 15 women) were included, with a median age of 57 years and bimodal distribution between those in their 20 s and the elderly. The proportion of GE and FP types were 21 (53.8%) and 18 (46.2%), respectively. Comparing these two groups, there was no significant difference in patient backgrounds in terms of sex, age, and underlying diseases. Campylobacter jejuni was exclusively identified in the GE type (19 cases, 90.5%), although other species such as Campylobacter fetus and Campylobacter coli were isolated in the FP type as well. Patients with the FP type underwent intravenous antibiotic therapy more frequently (47.6% vs. 88.9%), and their treatment (median: 5 days vs. 13 days) and hospitalization (median: 7 days vs. 21 days) periods were significantly longer. None of the patients died during the hospitalization. In summary, we found that nearly half of the patients with Campylobacter bacteremia presented with fever as a predominant manifestation without gastroenteritis symptoms.

A case of focal nodular hyperplasia-like lesion presenting unusual signal intensity on the hepatobiliary phase of gadoxetic acid-enhanced magnetic resonance image.

Focal nodular hyperplasia (FNH) or FNH-like lesions of the liver are benign lesions that can be mostly diagnosed by hepatobiliary phase gadoxetic acid-enhanced magnetic resonance imaging (MRI). Accurate imaging diagnosis is based on the fact that most FNHs or FNH-like lesions show characteristic hyper- or isointensity on hepatobiliary phase images. We report a case of an FNH-like lesion in a 73-year-old woman that mimicked a malignant tumor. Dynamic contrast-enhanced computed tomography (CT) and MRI using gadoxetic-acid revealed an ill-defined nodule showing early enhancement in the arterial phase and gradual and prolonged enhancement in the portal and equilibrium/transitional phases. Hepatobiliary phase imaging revealed inhomogeneous hypointensity, accompanied by a slightly isointense area compared to the background liver. Angiography-assisted CT showed a portal perfusion defect of the nodule, inhomogeneous arterial blood supply in the early phase, and less internal enhancement in the late phase, accompanied by irregularly shaped peritumoral enhancement. No central stellate scar was identified in any of the images. Imaging findings could not exclude the possibility of hepatocellular carcinoma, but the nodule was pathologically diagnosed as an FNH-like lesion by partial hepatectomy. In the present case, an unusual inhomogeneous hypointensity on hepatobiliary phase imaging made it difficult to diagnose the FNH-like lesions.

The reductive reaction mechanism of tobacco nitrite reductase derived from a combination of crystal structures and ultraviolet-visible microspectroscopy.

Assimilatory nitrite reductase (aNiR) reduces nitrite to an ammonium ion and has siroheme and a [Fe(4)S(4)] cluster as prosthetic groups. A reaction mechanism for Nii3, an aNiR from tobacco, is proposed based on high resolution X-ray structures and UV-Vis (ultraviolet-visible) microspectroscopy of Nii3-ligand complexes. Analysis of UV-Vis spectral changes in Nii3 crystals with increasing X-ray exposure showed prosthetic group reductions. In Nii3-NO2(-) structures, X-ray irradiation enhanced the progress of the reduction reaction, and cleavage of the N-O bond was observed when X-ray doses were increased. Crystal structures of Nii3 with other bound ligands, such as Nii3-NO and Nii3-NH(2)OH, were also determined. Further, by combining information from these Nii3 ligand-bound structures, including that of Nii3-NO2(-), with UV-Vis microspectral data obtained using different X-ray doses, a reaction mechanism for aNiR was suggested. Cleavage of the two N-O bonds of nitrite was envisaged as a two-step process: first, the N-O bond close to Lys224 was cleaved, followed by cleavage of the N-O bond close to Arg109. X-ray structures also indicated that aNiR-catalyzed nitrite reduction proceeded without the need for conformation changes in active site residues. Geometrical changes in the ligand molecules and the placement of neighboring water molecules appeared to be important to the stability of the active site residue interactions (Arg109, Arg179, and Lys224) and the ligand molecule. These interactions may contribute to the efficiency of aNiR reduction reactions.

X-ray crystal structure of a mutant assimilatory nitrite reductase that shows sulfite reductase-like activity.

Assimilatory nitrite reductase (aNiR) reduces nitrite ions (NO(2)(-)) to ammonium ions (NH(4)(+)), whereas assimilatory sulfite reductase reduces sulfite (SO(3)(2-)) to hydrogen sulfide (HS(-)). Although aNiR can also reduce SO(3)(2-), its activity is much lower than when NO(2)(-) is reduced as the substrate. To increase the SO(3)(2-)-reduction activity of aNiR, we performed a N226K mutation of Nii3, a representative aNiR. The resulting Nii3-N226K variant could bind non-native targets, SO(3)(2-), and HCO(3)(-), in addition to its native target, i.e., NO(2)(-). We have determined the high-resolution structure of Nii3-N226K in its apo-state and in complex with SO(3)(2-), NO(2)(-), and HCO(3)(-). This analysis revealed conformational changes of Lys226 and the adjacent Lys224 upon binding of SO(3)(2-), but not NO(2)(-)In contrast, HCO(3)(-) binding induced a conformational change at Arg179. After replacing Asn226 with a positively charged Lys, aNiR showed affinity for several anions. A comparison of all ligand-bound structures for Nii3-N226K revealed that structural changes in the active site depend on the size of the substrate.

Trends in the incidence of syphilis in the middle-aged and older adults in Japan: A nationwide observational study, 2009-2019.

AIM: Sexually transmitted infections remain a neglected area of research in geriatrics. However, in the global aging societies, sexual health among the middle-aged and older adults is an emerging public concern. High-income countries are facing a resurgence of syphilis cases among young generations, but little is known about its prevalence in older populations. We aimed to investigate the national trend of syphilis cases in Japan. METHODS: This nationwide observational study used the publicly-available database (2009-2019) to calculate crude and age-adjusted incidence rates of syphilis per 100 000 population by age, sex and clinical stage. We collected data from patients aged ≥50 years and performed joinpoint regression analysis to estimate long-term trends and average annual percentage changes (AAPCs). RESULTS: The total number of patients with syphilis increased about 8-fold from 165 in 2009 to 1280 in 2019. AAPCs of crude incidence rates significantly increased in every age category; 33.2% in 50-59 years, 23.8% in 60-69 years and 20.9% in ≥70 years. Age-adjusted incidence rates have surged at AAPCs of 28.7% in men and 23.1% in women, reaching 4.09 in men and 0.71 in women in 2019. By clinical stage, marked increases were observed in primary (AAPCs, 42.3% in men and 41.6% in women) and secondary syphilis (AAPCs, 24.9% in men and 24.2% in women). CONCLUSIONS: An up-toward trend of syphilis among people aged ≥50 years was observed. The importance of sexual health among older people should be highlighted in this aging Japanese society. Geriatr Gerontol Int 2022; 22: 1019-1024.

Antimicrobial prescription practices for outpatients with uncomplicated cystitis in Japan.

To promote antimicrobial stewardship, we studied antimicrobial prescription rates for uncomplicated cystitis, a common outpatient disease requiring antibiotic treatment. This multicenter retrospective study was performed from January 1, 2018, to December 31, 2020, in Japan, targeting outpatients aged ≥ 20 years whose medical records revealed International Classification of Diseases (ICD-10) codes suggesting uncomplicated cystitis (N300). The data of 1445 patients were collected and that of 902 patients were analyzed. The overall median patient age was 71 years and a proportion of those aged less than 50 years was 18.8% with a female dominance (82.6%). Antimicrobials were prescribed for 884 patients (98.0%) and a total of 623 patients (69.1%) were treated with broad-spectrum drugs, including fluoroquinolones (36.0%), third-generation cephalosporins (29.9%) and faropenem (3.1%). A logistic regression model revealed that the broad-spectrum agents were significantly prescribed for the older patients, male patients, and those who visited internists. Recurrence was observed in 37 (4.1%) cases, and the multivariate analysis suggested any of age, sex, or antimicrobial types were not associated with the recurrence. Collectively, approximately two-thirds of antimicrobials prescribed for uncomplicated cystitis were broad-spectrum agents. The present data would be an indicator for antimicrobial prescriptions in uncomplicated cystitis in Japan.

Nitrogen Dioxide at Ambient Concentrations Induces Nitration and Degradation of PYR/PYL/RCAR Receptors to Stimulate Plant Growth: A Hypothetical Model.

Exposing Arabidopsis thaliana (Arabidopsis) seedlings fed with soil nitrogen to 10-50 ppb nitrogen dioxide (NO2) for several weeks stimulated the uptake of major elements, photosynthesis, and cellular metabolisms to more than double the biomass of shoot, total leaf area and contents of N, C P, K, S, Ca and Mg per shoot relative to non-exposed control seedlings. The 15N/14N ratio analysis by mass spectrometry revealed that N derived from NO2 (NO2-N) comprised < 5% of the total plant N, showing that the contribution of NO2-N as N source was minor. Moreover, histological analysis showed that leaf size and biomass were increased upon NO2 treatment, and that these increases were attributable to leaf age-dependent enhancement of cell proliferation and enlargement. Thus, NO2 may act as a plant growth signal rather than an N source. Exposure of Arabidopsis leaves to 40 ppm NO2 induced virtually exclusive nitration of PsbO and PsbP proteins (a high concentration of NO2 was used). The PMF analysis identified the ninth tyrosine residue of PsbO1 (9Tyr) as a nitration site. 9Tyr of PsbO1 was exclusively nitrated after incubation of the thylakoid membranes with a buffer containing NO2 and NO2- or a buffer containing NO2- alone. Nitration was catalyzed by illumination and repressed by photosystem II (PSII) electron transport inhibitors, and decreased oxygen evolution. Thus, protein tyrosine nitration altered (downregulated) the physiological function of cellular proteins of Arabidopsis leaves. This indicates that NO2-induced protein tyrosine nitration may stimulate plant growth. We hypothesized that atmospheric NO2 at ambient concentrations may induce tyrosine nitration of PYR/PYL/RCAR receptors in Arabidopsis leaves, followed by degradation of PYR/PYL/RCAR, upregulation of target of rapamycin (TOR) regulatory complexes, and stimulation of plant growth.

Nitrate, but not nitrite, derived from nitrogen dioxide accumulates in Arabidopsis leaves following exposure to 15N-labeled nitrogen dioxide.

It is known that when plant leaves are exposed to exogenously applied nitrogen dioxide (NO2), nitrogen derived from NO2 is reduced to amino acid nitrogen. However, whether this is the sole metabolic fate of exogenously applied NO2 is unclear. In this study, Arabidopsis leaves were exposed to 4 ppm 15N-labeled NO2 for 4 h in light, followed by capillary ion analysis and elemental analysis-mass spectrometry with an elemental analyzer connected directly to a mass spectrometer. We found that leaf cells exposed to 15N-labeled NO2 accumulated a large amount of 15N-labeled nitrate. Neither 15N-labeled nitrite nor endogenous nitrite was present in exposed leaves. It is likely that exogenously applied NO2 is first converted to nitrite, and that nitrite is oxidized to nitrate in Arabidopsis leaf cells. The complete disappearance of nitrite derived from exogenously applied NO2 and endogenous nitrite supports this mechanism.

Dual nitrogen species involved in foliar uptake of nitrogen dioxide in Arabidopsis thaliana.

Foliar uptake of nitrogen dioxide (NO2) is governed by its reactive absorption mechanism, by which NO2 molecules diffuse through cell wall layers and simultaneously react with apoplastic ascorbate to form nitrous acid, which freely diffuses across plasmalemma. However, whether free diffusion of nitrous acid is the sole mechanism of foliar uptake of NO2 remains unknown. The involvement of ammonia-inhibitable nitrite transporters in the foliar uptake of NO2, as reported in nitrite transport in Arabidopsis roots, is also unknown. In this study, we treated Arabidopsis thaliana leaves with methionine sulfoximine (MSX) to inhibit incorporation of ammonia into glutamate and exposed them to 4 ppm 15N-labeled NO2 for 4 h in light followed by quantification of total nitrogen, reduced nitrogen, and ammonia nitrogen derived from NO2 using mass spectrometry and capillary electrophoresis. The total nitrogen derived from NO2 in leaves without MSX treatment was 587.0 nmol NO2/g fresh weight, of which more than 65% was recovered as reduced nitrogen. In comparison, MSX treatment decreased the total nitrogen and reduced nitrogen derived from NO2 by half. Thus, half of the foliar uptake of NO2 is not attributable to passive diffusion of nitrous acid but to ammonia-inhibitable nitrite transport. Foliar uptake of NO2 is mediated by a dual mechanism in A. thaliana: nitrous acid-free diffusion and nitrite transporter-mediated transport.

Molecular characterization of atmospheric NO2-responsive germin-like proteins in azalea leaves.

Atmospheric nitrogen dioxide (NO(2)) is an environmental oxidant that is removed through direct uptake by foliage, but plant responses to this highly reactive gas are not well understood at the molecular level. From NO(2)-exposed leaves of a woody azalea (Rhododendron mucronatum), we cloned two cDNAs (RmGLP1 and RmGLP2) for germin-like proteins (GLPs), a group of ubiquitous plant proteins that have been implicated in various plant physiological and developmental processes. Quantitative analysis of mRNA expression, together with immunoblotting data, showed that foliar exposure to NO(2) caused a robust induction of these GLP-encoding genes. When produced in tobacco cell culture, recombinant RmGLP2 was secreted into the apoplast, where it exhibited superoxide dismutase activity. RmGLP1 and RmGLP2 represent the first examples of plant genes that are responsive to airborne NO(2). These enzymes might have a potential role in extracellular defense mechanisms through attenuation of interactions between reactive nitrogen and oxygen species.

A novel role for PsbO1 in photosynthetic electron transport as suggested by its light-triggered selective nitration in Arabidopsis thaliana.

Exposure of Arabidopsis leaves to nitrogen dioxide (NO2) results in the selective nitration of specific proteins, such as PsbO1. The 9th tyrosine residue (9Tyr) of PsbO1 has been identified as the nitration site. This nitration is triggered by light and inhibited by photosynthetic electron transport inhibitors. During protein nitration, tyrosyl and NO2 radicals are formed concurrently and combine rapidly to form 3-nitrotyrosine. A selective oxidation mechanism for 9Tyr of PsbO1 is required. We postulated that, similar to 161Tyr of D1, 9Tyr of PsbO1 is selectively photo-oxidized by photosynthetic electron transport in response to illumination to a tyrosyl radical. In corroboration, after reappraising our oxygen evolution analysis, the nitration of PsbO1 proved responsible for decreased oxygen evolution from the thylakoid membranes. NO2 is reportedly taken into cells as nitrous acid, which dissociates to form NO2-. NO2- may be oxidized into NO2 by the oxygen-evolving complex. Light may synchronize this reaction with tyrosyl radical formation. These findings suggest a novel role for PsbO1 in photosynthetic electron transport.

Selective nitration of PsbO1 inhibits oxygen evolution from isolated Arabidopsis thylakoid membranes.

Treatment of isolated Arabidopsis thaliana thylakoid membranes with nitrogen dioxide (NO2) induces selective nitration of the tyrosine residue at the ninth amino acid (9Tyr) of PsbO1. This selective nitration is triggered by light and is inhibited by photosynthetic electron transport inhibitors. Therefore, we postulated that, similar to 161Tyr of D1 (YZ), 9Tyr of PsbO1 is redox active and is selectively oxidized by photosynthetic electron transport in response to illumination to a tyrosyl radical that is highly susceptible to nitration. This tyrosyl radical may combine rapidly at diffusion-controlled rates with NO2 to form 3-nitrotyrosine. If this postulation is correct, the nitration of 9Tyr of PsbO1 should decrease oxygen evolution activity. We investigated the effects of PsbO1 nitration on oxygen evolution from isolated thylakoid membranes, and found that nitration decreased oxygen evolution to ≥ 0% of the control. Oxygen evolution and nitration were significantly negatively correlated. This finding is consistent with redox active properties of the 9Tyr gene of PsbO1, and suggests that PsbO1 9Tyr acts as an electron relay, such as YZ in the photosystem II oxygenic electron transport chain.

Selective nitration of PsbO1, PsbO2, and PsbP1 decreases PSII oxygen evolution and photochemical efficiency in intact leaves of Arabidopsis.

Exposure of intact Arabidopsis leaves to 40 ppm nitrogen dioxide (NO2) in light resulted almost exclusively in nitration of PsbO1, PsbO2, and PsbP1 of photosystem II (PSII), with minor nitration of four non-PS II proteins, including peroxiredoxin II E, as reported previously. Our previous findings that light-triggered selective nitration of PsbO1 decreased oxygen evolution and that inhibition of photoelectric electron transport inhibited nitration of PsbO1 implied that the nitratable tyrosine residue of PsbO1 is redox-active. However, whether the nitratable tyrosine residues of PsbO2 and PsbP1 are redox-active is unknown. In this study, we determined the oxygen evolution and maximal photochemical efficiency of PSII in intact Arabidopsis leaves following exposure to 40 ppm NO2 in light and found that these parameters were decreased to 60 and 70% of the non-exposed control, respectively. Because PsbO1, PsbO2, and PsbP1 accounted for > 80% of anti-3-nitrotyrosine antibody signal intensities, observed decreases in the oxygen evolution and maximal photochemical efficiency of PSII were mainly attributable to nitration of the tyrosine residues of these PSII proteins. Thus, it is postulated that nitratable tyrosine residues of PsbO2 and PsbP1 are redox-active, as in the case of PsbO1. A new hypothetical model is proposed.

Light-triggered selective nitration of PsbO1 in isolated Arabidopsis thylakoid membranes is inhibited by photosynthetic electron transport inhibitors.

PsbO1 is exclusively nitrated when isolated thylakoid membranes are incubated in a buffer bubbled with nitrogen dioxide (NO2) containing NO2 and nitrite. NO2 is the primary intermediate for this selective nitration. Isolated thylakoid membranes were incubated in NO2-bubbled buffer at 25°C in the light or dark. Protein analysis confirmed the selective nitration of PsbO1. Illumination was found to be essential in PsbO1 nitration. A nitration mechanism whereby nitratable tyrosine residues of PsbO1 are, prior to nitration, selectively photo-oxidized by photosynthetic electron transport to tyrosyl radicals to combine with NO2 to form 3-nitrotyrosine was hypothesized. We tested the electron transport inhibitors 3-(3,4-dichlorophenyl)-1,1- dimethylurea, sodium azide, and 1,5-diphenylcarbazide and found distinct inhibition of nitration of PsbO1. We also propose a possible nitration mechanism.

Differential abilities of nitrogen dioxide and nitrite to nitrate proteins in thylakoid membranes isolated from Arabidopsis leaves.

Exposure of Arabidopsis leaves to nitrogen dioxide (NO2) results in nitration of specific chloroplast proteins. To determine whether NO2 itself and/or nitrite derived from NO2 can nitrate proteins, Arabidopsis thylakoid membranes were isolated and treated with NO2-bubbled or potassium nitrite (KNO2) buffer, followed by protein extraction, electrophoresis, and immunoblotting using an anti-3-nitrotyrosine (NT) antibody. NO2 concentrations in the NO2-bubbled buffer were calculated by numerically solving NO2 dissociation kinetic equations. The two buffers were adjusted to have identical nitrite concentrations. Both treatments yielded an NT-immunopositive band that LC/MS identified as PSBO1. The difference in the band intensity between the 2 treatments was designated nitration by NO2. Both NO2 and nitrite mediated nitration of proteins, and the nitration ability per unit NO2 concentration was ∼100-fold greater than that of nitrite.