Spatiotemporal Characteristics Determining the Multifaceted Nature of Reactive Oxygen, Nitrogen, and Sulfur Species in Relation to Proton Homeostasis.

Significance: Reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species (RSS) act as signaling molecules, regulating gene expression, enzyme activity, and physiological responses. However, excessive amounts of these molecular species can lead to deleterious effects, causing cellular damage and death. This dual nature of ROS, RNS, and RSS presents an intriguing conundrum that calls for a new paradigm. Recent Advances: Recent advancements in the study of photosynthesis have offered significant insights at the molecular level and with high temporal resolution into how the photosystem II oxygen-evolving complex manages to prevent harmful ROS production during the water-splitting process. These findings suggest that a dynamic spatiotemporal arrangement of redox reactions, coupled with strict regulation of proton transfer, is crucial for minimizing unnecessary ROS formation. Critical Issues: To better understand the multifaceted nature of these reactive molecular species in biology, it is worth considering a more holistic view that combines ecological and evolutionary perspectives on ROS, RNS, and RSS. By integrating spatiotemporal perspectives into global, cellular, and biochemical events, we discuss local pH or proton availability as a critical determinant associated with the generation and action of ROS, RNS, and RSS in biological systems. Future Directions: The concept of localized proton availability will not only help explain the multifaceted nature of these ubiquitous simple molecules in diverse systems but also provide a basis for new therapeutic strategies to manage and manipulate these reactive species in neural disorders, pathogenic diseases, and antiaging efforts.

Pleiotropic Functions of Nitric Oxide Produced by Ascorbate for the Prevention and Mitigation of COVID-19: A Revaluation of Pauling's Vitamin C Therapy.

Linus Pauling, who was awarded the Nobel Prize in Chemistry, suggested that a high dose of vitamin C (l-ascorbic acid) might work as a prevention or treatment for the common cold. Vitamin C therapy was tested in clinical trials, but clear evidence was not found at that time. Although Pauling's proposal has been strongly criticized for a long time, vitamin C therapy has continued to be tested as a treatment for a variety of diseases, including coronavirus infectious disease 2019 (COVID-19). The pathogen of COVID-19, SARS-CoV-2, belongs to the ß-coronavirus lineage, which includes human coronavirus, severe acute respiratory syndrome (SARS), and Middle East respiratory syndrome (MERS). This review intends to shed new light on vitamin C antiviral activity that may prevent SARS-CoV-2 infection through the chemical production of nitric oxide (NO). NO is a gaseous free radical that is largely produced by the enzyme NO synthase (NOS) in cells. NO produced by upper epidermal cells contributes to the inactivation of viruses and bacteria contained in air or aerosols. In addition to enzymatic production, NO can be generated by the chemical reduction of inorganic nitrite (NO2-), an alternative mechanism for NO production in living organisms. Dietary vitamin C, largely contained in fruits and vegetables, can reduce the nitrite in saliva to produce NO in the oral cavity when chewing foods. In the stomach, salivary nitrite can also be reduced to NO by vitamin C secreted from the epidermal cells of the stomach. The strong acidic pH of gastric juice facilitates the chemical reduction of salivary nitrite to produce NO. Vitamin C contributes in multiple ways to the host innate immune system as a first-line defense mechanism against pathogens. Highlighting chemical NO production by vitamin C, we suggest that controversies on the therapeutic effects of vitamin C in previous clinical trials may partly be due to less appreciation of the pleiotropic functions of vitamin C as a universal bioreductant.

Blood nitrate and nitrite modulating nitric oxide bioavailability: Potential therapeutic functions in COVID-19.

Most outcomes of COVID-19 are associated with dysfunction of the vascular system, particularly in the lung. Inhalation of nitric oxide (NO) gas is currently being investigated as a treatment for patients with moderate to severe COVID-19. In addition to the expected vasodilation effect, it has been also suggested that NO potentially prevents infection by SARS-CoV-2. Since NO is an unstable radical molecule that is easily oxidized by multiple mechanisms in the human body, it is practically difficult to control its concentration at lesions that need NO. Inorganic nitrate and/or nitrite are known as precursors of NO that can be produced through chemical as well enzymatic reduction. It appears that this NO synthase (NOS)-independent mechanism has been overlooked in the current developing of clinical treatments. Here, I suggest the missing link between nitrate and COVID-19 in terms of hypoxic NO generation.

d-Cysteine-Induced Rapid Root Abscission in the Water Fern Azolla Pinnata: Implications for the Linkage between d-Amino Acid and Reactive Sulfur Species (RSS) in Plant Environmental Responses.

Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) have been proposed as universal signaling molecules in plant stress responses. There are a growing number of studies suggesting that hydrogen sulfide (H2S) or Reactive Sulfur Species (RSS) are also involved in plant abiotic as well as biotic stress responses. However, it is still a matter of debate as to how plants utilize those RSS in their signaling cascades. Here, we demonstrate that d-cysteine is a novel candidate for bridging our gap in understanding. In the genus of the tiny water-floating fern Azolla, a rapid root abscission occurs in response to a wide variety of environmental stimuli as well as chemical inducers. We tested five H2S chemical donors, Na2S, GYY4137, 5a, 8l, and 8o, and found that 5a showed a significant abscission activity. Root abscission also occurred with the polysulfides Na2S2, Na2S3, and Na2S4. Rapid root abscission comparable to other known chemical inducers was observed in the presence of d-cysteine, whereas l-cysteine showed no effect. We suggest that d-cysteine is a physiologically relevant substrate to induce root abscission in the water fern Azolla.

Biological consilience of hydrogen sulfide and nitric oxide in plants: Gases of primordial earth linking plant, microbial and animal physiologies.

Hydrogen sulfide (H2S) is produced in the mammalian body through the enzymatic activities of cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST). A growing number of studies have revealed that biogenic H2S produced in tissues is involved in a variety of physiological responses in mammals including vasorelaxation and neurotransmission. It is now evident that mammals utilize H2S to regulate multiple signaling systems, echoing the research history of the gaseous signaling molecules nitric oxide (NO) and carbon monoxide (CO) that had previously only been recognized for their cytotoxicity. In the human diet, meats (mammals, birds and fishes) and vegetables (plants) containing cysteine and other sulfur compounds are the major dietary sources for endogenous production of H2S. Plants are primary producers in ecosystems on the earth and they synthesize organic sulfur compounds through the activity of sulfur assimilation. Although plant H2S-producing activities have been known for a long time, our knowledge of H2S biology in plant systems has not been updated to the extent of mammalian studies. Here we review recent progress on H2S studies, highlighting plants and bacteria. Scoping the future integration of H2S, NO and O2 biology, we discuss a possible linkage between physiology, ecology and evolutional biology of gas metabolisms that may reflect the historical changes of the Earth's atmospheric composition.

An Overview of Methods in Plant Nitric Oxide (NO) Research: Why Do We Always Need to Use Multiple Methods?

The free radical nitric oxide (NO) is a universal signaling molecule among living organisms. To investigate versatile functions of NO in plants it is essential to analyze biologically produced NO with an appropriate method. Owing to the uniqueness of NO, plant researchers may encounter difficulties in applying methods that have been developed for mammalian study. Based on our experience, we present here a practical guide to NO measurement fitted to plant biology.

Bimodal effect of hydrogen peroxide and oxidative events in nitrite-induced rapid root abscission by the water fern Azolla pinnata.

In the genus Azolla rapid abscission of roots from floating fronds occurs within minutes in response to a variety of stresses, including exposure to nitrite. We found that hydrogen peroxide, though itself not an inducer of root abscission, modulates nitrite-induced root abscission by Azolla pinnata in a dose-dependent manner, with 2 mM H2O2 significantly diminishing the responsiveness to 2 mM NaNO2, and 10 mM H2O2 slightly enhancing it. Hypoxia, which has been found in other plants to result in autogenic production of H2O2, dramatically stimulated root abscission of A. pinnata in response to nitrite, especially for plants previously cultivated in medium containing 5 mM KNO3 compared to plants cultivated under N2-fixing conditions without combined nitrogen. Plants, including Azolla, produce the small signaling molecule nitric oxide (NO) from nitrite using nitrate reductase. We found Azolla plants to display dose-dependent root abscission in response to the NO donor spermine NONOate. Treatment of plants with the thiol-modifying agents S-methyl methanethiosulfonate or glutathione inhibited the nitrite-induced root abscission response. Synchrotron radiation-based Fourier transform infrared spectromicroscopy revealed higher levels of carbonylation in the abscission zone of dropped roots, indicative of reaction products of polysaccharides with potent free radical oxidants. We hypothesize that metabolic products of nitrite and NO react with H2O2 in the apoplast leading to free-radical-mediated cleavage of structural polysaccharides and consequent rapid root abscission.

Controlled free radical attack in the apoplast: a hypothesis for roles of O, N and S species in regulatory and polysaccharide cleavage events during rapid abscission by Azolla.

Shedding of organs by abscission is a key terminal step in plant development and stress responses. Cell wall (CW) loosening at the abscission zone can occur through a combination chain breakage of apoplastic polysaccharides and tension release of cellulose microfibrils. Two distinctly regulated abscission cleavage events are amenable to study in small water ferns of the genus Azolla; one is a rapid abscission induced by environmental stimuli such as heat or chemicals, and the other is an ethylene-induced process occurring more slowly through the action of hydrolytic enzymes. Although free radicals are suggested to be involved in the induction of rapid root abscission, its mechanism is not fully understood. The apoplast contains peroxidases, metal-binding proteins and phenolic compounds that potentially generate free radicals from H2O2 to cleave polysaccharides in the CW and middle lamella. Effects of various thiol-reactive agents implicate the action of apoplastic peroxidases having accessible cysteine thiols in rapid abscission. The Ca(2+) dependency of rapid abscission may reflect the stabilization Ca(2+) confers to peroxidase structure and binding to pectin. To spur further investigation, we present a hypothetical model for small signaling molecules H2O2 and NO and their derivatives in regulating, via modification of putative protein thiols, free radical attack of apoplastic polysaccharides.

Polyamine-Induced Rapid Root Abscission in Azolla pinnata.

Floating ferns of the genus Azolla detach their roots under stress conditions, a unique adaptive response termed rapid root abscission. We found that Azolla pinnata plants exhibited dose-dependent rapid root abscission in response to the polyamines spermidine and spermine after a substantial time lag (>20 min). The duration of the time lag decreased in response to high pH and high temperature whereas high light intensity increased the time lag and markedly lowered the rate of abscission. The oxidation products of polyamines, 1,3-diaminopropane, ß-alanine and hydrogen peroxide all failed to initiate root abscission, and hydroxyethyl hydrazine, an inhibitor of polyamine oxidase, did not inhibit spermine-induced root abscission. Exposure of A. pinnata to the polyamines did not result in detectable release of NO and did not affect nitrite-dependent NO production. The finding of polyamine-induced rapid root abscission provides a facile assay for further study of the mode of action of polyamines in plant stress responses.

Nitric oxide-dependent hypotensive effects of wax gourd juice.

ETHNOPHARMACOLOGICAL RELEVANCE: The wax gourd (Benincasa hispida (Thunb) Cong.) is a long-season vegetable that has been used in traditional Chinese medicine to treat high blood pressure. However, precise details of its effect and the mechanism of action involved are still lacking. MATERIALS AND METHODS: Ten-fold-condensed wax gourd juice was used for the experiments. We measured (1) blood pressure of anesthetized normal Wistar rats in vivo, (2) isolated rat aortic contraction and relaxation, and (3) nitric oxide production from cultured porcine endothelial cells. The rats mentioned had not been treated with the investigational medicine. RESULTS: Intravenous injection of the juice produced a dose-dependent decrease in blood pressure. Treatment with the juice induced concentration-dependent relaxation of isolated rat aortic rings that had been precontracted with noradrenaline. The relaxation induced by the juice was strongly inhibited by treatment with the nitric oxide (NO) synthase inhibitor N(G)-nitro-l-arginine methyl ester hydrochloride (l-NAME) or endothelial denudation. Treatment with the juice produced NO from cultured porcine aortic endothelial cells. This NO production was significantly inhibited by l-NAME. CONCLUSIONS: The present findings suggest that wax gourd juice exerts a hypotensive effect via endothelium-dependent vasodilation. The main endothelium-derived relaxing factor involved might be NO.

Physiological characterization of the cold-shock-induced humoral factor for wing color-pattern changes in butterflies.

Butterfly wing color patterns can be modified by the application of temperature shock to pupae immediately after pupation, which has been attributed to a cold-shock-induced humoral factor called cold-shock hormone (CSH). Here, we physiologically characterized CSH and pharmacological action of tungstate, using a nymphalid butterfly Junonia orithya. We first showed that the precise patterns of modification were dependent on the time-point of the cold-shock treatment after pupation, and confirmed that the modification properties induced in a cold-shocked pupa were able to be transferred to another pupa in a parabiosis experiment. Cold-shock application after removal of the head and prothorax together still produced modified wings, excluding major involvement of the brain-retrocerebral neuroendocrine complex. Furthermore, tungstate injection induced modifications even in individuals whose head and prothorax were removed. Importantly, transplantation of tracheae isolated from cold-shocked pupae induced modifications in the recipient wings. We identified a chemical peak in hemolymph of the cold-shocked individuals using HPLC, which corresponded to dopamine, and demonstrated that dopamine and its related biogenic amines have ability to induce small color-pattern changes. Taken together, the present study suggests that CSH is likely to be secreted from trachea-associated endocrine cells upon cold-shock treatment and that tungstate may change color patterns via its direct action on wings.

Chemical induction of rapid and reversible plastid filamentation in Arabidopsis thaliana roots.

Plastids assume various morphologies depending on their developmental status, but the basis for developmentally regulated plastid morphogenesis is poorly understood. Chemical induction of alterations in plastid morphology would be a useful tool for studying this; however, no such chemicals have been identified. Here, we show that antimycin A, an effective respiratory inhibitor, can change plastid morphology rapidly and reversibly in Arabidopsis thaliana. In the root cortex, hypocotyls, cotyledon epidermis and true leaf epidermis, significant differences in mitochondrial morphology were not observed between antimycin-treated and untreated tissues. In contrast, antimycin caused extreme filamentation of plastids in the mature cortices of main roots. This phenomenon was specifically observed in the mature root cortex. Other mitochondrial respiratory inhibitors (rotenone and carbonyl cyanide m-chlorophenylhydrazone), hydrogen peroxide, S-nitroso-N-acetylpenicillamine [a nitric oxide (NO) donor] and 3-(3,4-dichlorophenyl)-1,1-dimethylurea did not mimic the phenomenon under the present study conditions. Antimycin-induced plastid filamentation was initiated within 5 min after the onset of chemical treatment and appeared to complete within 1 h. Plastid morphology was restored within 7 h after the washout of antimycin, suggesting that the filamentation was reversible. Co-applications of antimycin and cytoskeletal inhibitors (demecolcine or latrunculin B) or protein synthesis inhibitors (cycloheximide or chloramphenicol) still caused plastid filamentation. Antimycin A was also effective for plastid filamentation in the chloroplast division mutants atftsZ1-1 and atminE1. Salicylhydroxamic acid, an alternative oxidase inhibitor, was solely found to suppress the filamentation, implying the possibility that this phenomenon was partly mediated by an antimycin-activated alternative oxidase in the mitochondria.

Purification and molecular cloning of xylanases from the wood-feeding termite, Coptotermes formosanus Shiraki.

Coptotermes formosanus is one of the most destructive termites in the southern part of Japan as well as in the United States. Hemicellulose is a noncellulosic polysaccharide found in plant cell walls, and xylan is the major constituent of hemicellulose. Since hemicellulose prevents access of cellulolytic enzymes to cellulose, enzymatic hydrolysis of hemicellulose is beneficial for cellulose digestion. We purified three functional xylanases to homogeneity from C. formosanus for the first time. Elution profiles from the whole termite extract suggest that these three xylanases play major roles in xylan digestion in the gut of the termites. The corresponding cDNAs were successfully cloned based on the N-terminal amino acid sequences, encoding GHF11 xylanases. Reverse transcription-PCR using manipulated protozoan cells in the hindgut revealed that the corresponding genes were expressed in the symbiotic flagellate Holomastigotoides mirabile. These results suggest that the GHF11 xylanases that are produced by the symbiotic flagellates play a primary role in xylan degradation in C. formosanus.

Heat stress stimulates nitric oxide production in Symbiodinium microadriaticum: a possible linkage between nitric oxide and the coral bleaching phenomenon.

Nitric oxide (NO) is a gas displaying multiple physiological functions in plants, animals and bacteria. The enzymes nitrate reductase and NO synthase have been suggested to be involved in the production of NO in plants and algae, but the implication of those enzymes in NO production under physiological conditions remains obscure. Symbiodinium microadriaticum, commonly referred to as zooxanthellae, is a marine microalga commonly found in symbiotic association with a cnidarian host including reef-building corals. Here we demonstrate NO production in zooxanthellae upon supplementation of either sodium nitrite or L-arginine as a substrate. The nitrite-dependent NO production was detected electrochemically and confirmed by the application of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a specific NO scavenger. Cells stained with the diaminofluorescein, DAF-2 DA, an NO fluorescent probe, showed an increase in fluorescence intensity upon supplementation of both sodium nitrite and L-arginine. Microscopic observations of DAF-stained cells verified that NO was produced inside the cells. NO production in S. microadriaticum was found to increase upon exposure of cells to an acute heat stress which also caused a decline in the photosynthetic efficiency of PSII (F(v)/F(m)). This study provides substantial evidence to confirm that zooxanthellae can synthesize NO even when they are not in a symbiotic association with a coral host. The increase in NO production at high temperatures suggests that heat stress stimulates the microalgal NO production in a temperature-dependent manner. The implications of these findings are discussed in the light of the coral bleaching phenomenon which is associated with elevated sea surface temperature due to global warming.

Occurrence and recent long-distance dispersal of deep-sea hydrothermal vent shrimps.

Deep-sea hydrothermal vents and methane seeps are extreme environments that have a high concentration of hydrogen sulphide. However, abundant unique invertebrates including shrimps of the family Bresiliidae have been found in such environments. The bresiliid shrimps are believed to have radiated in the Miocene (less than 20 Myr); however, the period when and the mechanisms by which they dispersed across the hydrothermal vents and cold seeps in oceans worldwide have not been clarified. In the present study, we collected the deep-sea blind shrimp Alvinocaris longirostris from the hydrothermal vent site in the Okinawa Trough and carried out the first investigation of the 18S rRNA gene of a bresiliid shrimp. The phylogenetic analysis revealed that the bresiliid shrimp is situated at an intermediate lineage within the infraorder Caridea and shows monophyly with palaemonid shrimps, which live in shallow sea and freshwater. Furthermore, the mitochondrial cytochrome oxidase I (COI) gene sequences were analysed to determine the phylogenetic relationship with known bresiliid shrimps. A. longirostris of the Okinawa Trough had two haplotypes of the COI gene, one of which was identical to the Alvinocaris sp. of the cold seeps in Sagami Bay. These results indicate that a long-distance dispersal of A. longirostris occurred possibly within the last 100,000 years.

NO signal at the crossroads: polyamine-induced nitric oxide synthesis in plants?

Polyamines, such as spermine, spermidine and putrescine, are ubiquitous polycationic compounds that are produced by almost all living organisms, including plants, animals, fungi and bacteria. Polyamines are multifunctional and interact with polyanionic biomolecules such as DNA or protein. However, despite their potential significance, the polyamine-dependent signal transduction system has not been revealed yet. Ni Ni Tun and colleagues have recently reported a possible linkage between polyamine and nitric oxide (NO), another ubiquitous signalling molecule.

Isoprene emission from tropical trees in Okinawa Island, Japan.

This study surveyed isoprene emission from 42 indigenous and exotic tropical trees in subtropic Okinawa, Japan. Of the 42 trees studied, 4 emitted isoprene at a rate in excess of 20 microg g(-1)h(-1), and 28 showed the rates of 1-10 microg g(-1)h(-1). The remainder emitted less than 1 microg g(-1)h(-1). The majority of trees in this study may therefore fall within the lower emitting species. However, species in Moraceae that is indigenous in Okinawa emitted isoprene at relatively higher rates with an average of 14.2 microg g(-1)h(-1). The highest emission rate of 107.1 microg g(-1)h(-1) for Ficus virgata yielded the area basis rate of 47.4 nmol m(-2)s(-1), which is almost equivalent to the rate of high emitting species. Furthermore, a linear relationship between light intensity and isoprene emission was noted with Ficus virgata up to 1700 micromol m(-2)s(-1). These findings may show the potential importance of subtropical areas as sources of isoprene to the atmosphere.

Repair machinery of symbiotic photosynthesis as the primary target of heat stress for reef-building corals.

In a coral-algae symbiotic system, heat-dependent photoinhibition of photosystem II (PSII) leads to coral bleaching. When the reef-building coral Acropora digitifera was exposed to light, a moderate increase of temperature induced coral bleaching through photobleaching of algal pigments, but not through expulsion of symbiotic algae. Monitoring of PSII photoinhibition revealed that heat-dependent photoinhibition was ascribed to inhibition of the repair of photodamaged PSII, and heat susceptibility of the repair machinery varied among coral species. We conclude that the efficiency of the photosynthesis repair machinery determines the bleaching susceptibility of coral species under elevated seawater temperatures.

Enzymatic nitration of phytophenolics: evidence for peroxynitrite-independent nitration of plant secondary metabolites.

Peroxynitrite (ONOO(-)), a reactive nitrogen species, is capable of nitrating tyrosine residue of proteins. Here we show in vitro evidence that plant phenolic compounds can also be nitrated by an ONOO(-)-independent mechanism. In the presence of NaNO(2), H(2)O(2), and horseradish peroxidase (HRP), monophenolic p-coumaric acid (p-CA, 4-hydroxycinnamic acid) was nitrated to form 4-hydroxy-3-nitrocinnamic acid. The reaction was completely inhibited by KCN, an inhibitor for HRP. The antioxidant ascorbate suppressed p-CA nitration and its suppression time depended strongly on ascorbate concentration. We conclude that nitrogen dioxide radical (NO(2)(radical)), but not ONOO(-), produced by a guaiacol peroxidase is the intermediate for phytophenolic nitration.

Involvement of nitric oxide synthase in sucrose-enhanced hydrogen peroxide tolerance of Rhodococcus sp. strain APG1, a plant-colonizing bacterium.

Hydrogen peroxide (H2O2) tolerance of Rhodococcus sp. strain APG1, previously isolated from the aquatic fern Azolla pinnata, was examined in relation to nitric oxide (NO) production by cells cultured on a variety of C sources. Cells inoculated onto A. pinnata fronds established a surface-sterilant resistant density of 2-4x10(7) cells g(-1) without causing disease. Compared to cultures containing glucose, fructose, mannitol, or glycerol, those provided only with sucrose displayed, on a per C basis, substantially lower (<10%) growth yields and higher resistance to H2O2. NO, a positive regulator of catalase synthesis in bacteria, was produced in larger amounts in sucrose-grown cells as evidence by eightfold greater per cell accumulations in the medium of nitrite (NO2-), a stable oxidation product of NO. Addition to cells of L-arginine, the substrate for nitric oxide synthase (NOS), stimulated production of NO, detected both by fluorometric reaction with diaminofluorescein-FM diacetate (DAF-FM DA) and by increased levels of NO2- in the culture medium. These results suggest that sucrose may enhance H2O2 tolerance of Rhodococcus APG1 by increasing cellular NO producing capacity. We propose a regulatory role for NOS in promoting tolerance of Rhodococcus APG1 to oxidative stress in the phyllosphere.