Physiological function and regulation of ascorbate peroxidase isoforms.

Ascorbate peroxidase (APX) reduces H2O2 to H2O by utilizing ascorbate as a specific electron donor and constitutes the ascorbate-glutathione cycle in organelles of plants including chloroplasts, cytosol, mitochondria, and peroxisomes. It has been almost 40 years since APX was discovered as an important plant-specific H2O2-scavenging enzyme, during which time many research groups have conducted molecular physiological analyses. It is now clear that APX isoforms function not only just as antioxidant enzymes but also as important factors in intracellular redox regulation through the metabolism of reactive oxygen species. The function of APX isoforms is regulated at multiple steps, from the transcriptional level to post-translational modifications of enzymes, thereby allowing them to respond flexibly to ever-changing environmental factors and physiological phenomena such as cell growth and signal transduction. In this review, we summarize the physiological functions and regulation mechanisms of expression of each APX isoform.

Statistical evaluation of individual external exposure dose of outdoor worker and ambient dose rate at evacuation ordered zones after the Fukushima Daiichi Nuclear Power Station accident.

Following the accident at the Fukushima Daiichi Nuclear Power Station, evacuation orders were issued for the surrounding communities. In order to lift the evacuation order, it is necessary to determine individual external doses in the evacuated areas. The purpose of this study was to determine the quantitative relationship between individual external doses and ambient dose rates per hour as conversion coefficients. More specifically, individual external doses of Tokyo Electric Power Company Holdings employees in difficult-to-return zone were measured broadly over a long period (fiscal year 2020 to fiscal year 2022). To obtain highly accurate estimates, we used not only ambient dose rates based on airborne radiological monitoring data, but also Integrated dose rate map data that had been statistically corrected to correspond to local ambient dose rate gradients on the ground. As a result, the conversion coefficients based on the ambient dose rate map measured by airborne radiological monitoring were 0.42 for the Evacuation-Order Lifted Zones (ELZs), 0.37 for the Special Zones for Reconstruction and Rehabilitation (SZRRs), and 0.47 for the Difficult-to-Return Zones without SZRRs (DRZs). On the other hand, the conversion coefficients based on the Integrated dose rate map which is a highly accurate dose rate map based on statistical analysis of various types of monitoring that have been studied in government projects in recent years, were 0.78 for the ELZs, 0.72 for the SZRRs and 0.82 for the DRZs. Using these conversion coefficients, the individual external dose can be estimated from two representative ambient dose rate maps provided by the government.

Effect of radioactive cesium-rich microparticles on radioactive cesium concentration and distribution coefficient in rivers flowing through the watersheds with different contaminated condition in Fukushima.

Radioactive cesium-rich microparticles (CsMPs) derived from the Fukushima Daiichi Nnuclear Power Plant accident were detected from soils and river water around Fukushima Prefecture, Japan. Because CsMPs are insoluble and rich in radioactive cesium (RCs), they may cause the overestimation of solid-water distribution coefficient (Kd) for RCs in the water. Previous studies showed the proportion of RCs derived from CsMPs on RCs concentration in soils collected from areas with different contaminated levels. Because the proportion of RCs concentration derived CsMPs to the RCs concentration of soils in the less contaminated areas is higher than that in the highly contaminated areas, the effect of CsMPs on particulate RCs concentration in river water may be larger in the less contaminated areas. However, the difference in the effects of CsMPs on the particulate RCs concentration and Kd in river water flowing through watersheds with different contaminated levels has not been clarified. In this study, we investigated the effect of CsMPs on the particulate RCs concentration and Kd in two rivers, Takase River and Kami-Oguni River, flowing through the watersheds with different RCs contaminated levels in Fukushima Prefecture. CsMPs might enter rivers due to soil erosion because they were detected only in some samples collected from both rivers during flood events. CsMPs accounted for more than half of particulate RCs concentration in some water samples collected in the flood condition. In particular, the proportion of CsMPs in particulate RCs for the Kami-Oguni River was greater than that for the Takase River. However, when evaluating for the entire water sampling in the flood condition, a proportion of RCs concentration derived from CsMPs in the average RCs concentrations per unit mass of SS in both river waters collected in the flood condition was not large. CsMPs might temporarily increase the particulate RCs concentration and Kd in the flood event, but CsMPs did not significantly affect them when evaluated throughout the event.

Validation study of ambient dose equivalent conversion coefficients for radiocaesium distributed in the ground: lessons from the Fukushima Daiichi Nuclear Power Station accident.

Ambient dose equivalent conversion coefficients (ADCRCs) for converting a radiocaesium inventory to ambient dose equivalent rates (air dose rates) depend on the vertical distribution of radiocaesium in soil. To access the validity of ADCRCs, the air dose rate at 1 m above ground and the vertical distribution of radiocaesium in the soil around the Fukushima Daiichi Nuclear Power Station (FDNPS) present between 2011 and 2019 were measured in the current study. ADCRCs were calculated using air dose rates and three different parameters representing the vertical distribution of radiocaesium in soil: (1) relaxation mass depth (ß), (2) effective relaxation mass depth (ßeff) and (3) relaxation mass depth recommended by the International Commission on Radiation Units and Measurements before the FDNPS accident (ßICRU). When ADCRCs based on ß and ßeff were compared to those based on ß and ßICRU, a positive correlation was found. To confirm the applicability of the ADCRCs based on the three types of ß values, radiocaesium inventories were estimated using the air dose rates and ADCRCs, and the obtained results were compared to the radiocaesium inventory calculated using soil sample measurements. Good agreement was observed between the radiocaesium inventories estimated using the ADCRCs based on ß and ßeff and measured by investigating soil samples. By contrast, the radiocaesium inventory estimated using the ADCRCs based on ßICRU was overestimated compared with that measured by investigating soil samples. These findings support the applicability of ADCRCs based on ß and ßeff in the Fukushima region. Furthermore, the ßICRU result suggests that differences in soil characteristics between Japan and other countries should be considered for evaluating ADCRCs.

Transport and Redistribution of Radiocesium in Fukushima Fallout through Rivers.

The Fukushima Daiichi Nuclear Power Plant (FDNPP) accident released the most significant quantity of radiocesium into the environment since Chernobyl, and detailed measurements over the initial 5 years provide new insights into fluvial redistribution of radiocesium. We found that the high initial activity concentration of 137Cs-bearing suspended sediment in rivers was followed by a steep exponential decline (λ1) which extended to approximately 1 year after the accident, while the rate of initial decline in radiocesium activity concentration in water was an order of magnitude higher than rates measured after Chernobyl. Fluvial transport of 137Cs to the ocean from the Abukuma river totaled 12 TBq between June 2011 and August 2015 and almost all this radiocesium (96.5%) was transported in the particulate form. The primary sources of 137Cs were paddy fields, farmland, and urban areas [plaque-forming unit (PFU)], discharging 85% of the exported 137Cs from 38% of the watershed area. After 1 year, activity concentrations were lower and exhibited a more gradual secondary decline (λ2) which was associated with reduced radiocesium losses from PFU areas, while forest areas continue to represent more stable contaminant stores.

Biochemistry and Physiology of Vitamins in Euglena.

Euglena gracilis Z requires vitamins B1 and B12 for growth. It takes up and accumulates large amounts of these exogenous vitamins through energy-dependent active transport systems. Except for these essential vitamins, E. gracilis Z has the ability to synthesize all human vitamins. Euglena synthesizes high levels of antioxidant vitamins such as vitamins C and E, and, thus, are used as nutritional supplements for humans and domestic animals. Methods to effectively produce vitamins in Euglena have been investigated.Previous biochemical studies indicated that E. gracilis Z contains several vitamin-related novel synthetic enzymes and metabolic pathways which suggests that it is a highly suitable organism for elucidating the physiological functions of vitamins in comparative biochemistry and biological evolution. E. gracilis Z has an unusual biosynthetic pathway for vitamin C, a hybrid of the pathways found in animals and plants. This chapter presents up-to-date information on the biochemistry and physiological functions of vitamins in this organism.

Modulation of NADH Levels by Arabidopsis Nudix Hydrolases, AtNUDX6 and 7, and the Respective Proteins Themselves Play Distinct Roles in the Regulation of Various Cellular Responses Involved in Biotic/Abiotic Stresses.

Arabidopsis Nudix hydrolases, AtNUDX6 and 7, exhibit pyrophosphohydrolase activities toward NADH and contribute to the modulation of various defense responses, such as the poly(ADP-ribosyl)ation (PAR) reaction and salicylic acid (SA)-induced Nonexpresser of Pathogenesis-Related genes 1 (NPR1)-dependent defense pathway, against biotic and abiotic stresses. However, the mechanisms by which these enzymes regulate such cellular responses remain unclear. To clarify the functional role(s) of AtNUDX6 and 7 and NADH metabolism, we examined the effects of the transient expression of the active and inactive forms of AtNUDX6 and 7 under the control of an estrogen (ES)-inducible system on various stress responses. The transient expression of active AtNUDX6 and 7 proteins suppressed NADH levels and induced PAR activity, whereas that of their inactive forms did not, indicating the involvement of NADH metabolism in the regulation of the PAR reaction. A transcriptome analysis using KO-nudx6, KO-nudx7 and double KO-nudx6/7 plants, in which intracellular NADH levels increased, identified genes (NADH-responsive genes, NRGs) whose expression levels positively and negatively correlated with NADH levels. Many NRGs did not overlap with the genes whose expression was reported to be responsive to various types of oxidants and reductants, suggesting a novel role for intracellular NADH levels as a redox signaling cue. The active and inactive AtNUDX6 proteins induced the expression of thioredoxin-h5, the activator of NPR1 and SA-induced NPR1-dependent defense genes, while the active and inactive AtNUDX7 proteins suppressed the accumulation of SA and subsequent gene expression, indicating that AtNUDX6 and 7 proteins themselves play distinct roles in stress responses.

Time Dependence of the (137)Cs Concentration in Particles Discharged from Rice Paddies to Freshwater Bodies after the Fukushima Daiichi NPP Accident.

The concentration of particulate (137)Cs in paddy fields, which can be a major source of (137)Cs entering the water system, was studied following the Fukushima Daiichi Nuclear Power Plant accident. To parametrize the concentration and to estimate the time dependence, paddy fields covering various levels of (137)Cs deposition were investigated over the period 2011-2013 (n = 121). The particulate (137)Cs concentration (kBq kg-SS(-1)) showed a significant correlation with the initial surface deposition density (kBq m(-2)). This suggests that the entrainment coefficient (m(2) kg-SS(-1)), defined as the ratio between the particulate (137)Cs concentration and the initial surface deposition density, is an important parameter when modeling (137)Cs wash-off from paddy fields. The entrainment coefficient decreased with time following a double exponential function. The decrease rate constant of the entrainment coefficient was clearly higher than that reported for other land uses and for river water. The difference in the decrease rates of the entrainment coefficient suggests that paddy fields play a major role in radiocesium migration through the water system. An understanding of the decrease rate of the entrainment coefficient of paddy fields is therefore crucial to understand the migration of radiocesium in the water system.

Potential involvement of drought-induced Ran GTPase CLRan1 in root growth enhancement in a xerophyte wild watermelon.

Enhanced root growth is known as the survival strategy of plants under drought. Previous proteome analysis in drought-resistant wild watermelon has shown that Ran GTPase, an essential regulator of cell division and proliferation, was induced in the roots under drought. In this study, two cDNAs were isolated from wild watermelon, CLRan1 and CLRan2, which showed a high degree of structural similarity with those of other plant Ran GTPases. Quantitative RT-PCR and promoter-GUS assays suggested that CLRan1 was expressed mainly in the root apex and lateral root primordia, whereas CLRan2 was more broadly expressed in other part of the roots. Immunoblotting analysis confirmed that the abundance of CLRan proteins was elevated in the root apex region under drought stress. Transgenic Arabidopsis overexpressing CLRan1 showed enhanced primary root growth, and the growth was maintained under osmotic stress, indicating that CLRan1 functions as a positive factor for maintaining root growth under stress conditions.

A gain-of-function mutation of plastidic invertase alters nuclear gene expression with sucrose treatment partially via GENOMES UNCOUPLED1-mediated signaling.

Plastid gene expression (PGE) is one of the signals that regulate the expression of photosynthesis-associated nuclear genes (PhANGs) via GENOMES UNCOUPLED1 (GUN1)-dependent retrograde signaling. We recently isolated Arabidopsis sugar-inducible cotyledon yellow-192 (sicy-192), a gain-of-function mutant of plastidic invertase, and showed that following the treatment of this mutant with sucrose, the expression of PhANGs as well as PGE decreased, suggesting that the sicy-192 mutation activates a PGE-evoked and GUN1-mediated retrograde pathway. To clarify the relationship between the sicy-192 mutation, PGE, and GUN1-mediated pathway, plastid and nuclear gene expression in a double mutant of sicy-192 and gun1-101, a null mutant of GUN1 was studied. Plastid-encoded RNA polymerase (PEP)-dependent PGE was markedly suppressed in the sicy-192 mutant by the sucrose treatment, but the suppression as well as cotyledon yellow phenotype was not mitigated by GUN1 disruption. Microarray analysis revealed that the altered expression of nuclear genes such as PhANG in the sucrose-treated sicy-192 mutant was largely dependent on GUN1. The present findings demonstrated that the sicy-192 mutation alters nuclear gene expression with sucrose treatment via GUN1, which is possibly followed by inhibiting PEP-dependent PGE, providing a new insight into the role of plastid sugar metabolism in nuclear gene expression.

Identification and characterization of Arabidopsis AtNUDX9 as a GDP-d-mannose pyrophosphohydrolase: its involvement in root growth inhibition in response to ammonium.

GDP-d-mannose (GDP-d-Man) is an important intermediate in ascorbic acid (AsA) synthesis, cell wall synthesis, protein N-glycosylation, and glycosylphosphatidylinositol-anchoring in plants. Thus, the modulation of intracellular levels of GDP-d-Man could be important for maintaining various cellular processes. Here an Arabidopsis GDP-d-Man pyrophosphohydrolase, AtNUDX9 (AtNUDT9; At3g46200), which hydrolysed GDP-d-Man to GMP and mannose 1-phosphate, was identified. The K m and V max values for GDP-d-Man of AtNUDX9 were 376±24 µM and 1.61±0.15 µmol min(-1) mg(-1) protein, respectively. Among various tissues, the expression levels of AtNUDX9 and the total activity of GDP-d-Man pyrophosphohydrolase were the highest in the roots. The GDP-d-Man pyrophosphohydrolase activity was increased in the root of plants grown in the presence of ammonium. No difference was observed in the levels of AsA in the leaf and root tissues of the wild-type and knockout-nudx9 (KO-nudx9) plants, whereas a marked increase in N-glycoprotein levels and enhanced growth were detected in the roots of KO-nudx9 plants in the presence of ammonium. These results suggest that AtNUDX9 is involved in the regulation of GDP-d-Man levels affecting ammonium sensitivity via modulation of protein N-glycosylation in the roots.

Versatile physiological functions of the Nudix hydrolase family in Arabidopsis.

Nudix hydrolases are widely distributed in all kingdoms of life and have the potential to hydrolyze a wide range of organic pyrophosphates, including nucleoside di- and triphosphates, nucleotide coenzymes, nucleotide sugars, and RNA caps. However, except for E. coli MutT and its orthologs in other organisms that sanitize oxidized nucleotides to prevent DNA and RNA mutations, the functions of Nudix hydrolases had largely remained unclear until recently, because many members of this enzyme family exhibited broad substrate specificities. There is now increasing evidence to show that their functions extend into many aspects of the regulation of cellular responses. This review summarizes current knowledge on the molecular and enzymatic properties as well as physiological functions of Arabidopsis Nudix hydrolases. The information presented here may provide novel insights into the physiological roles of these enzymes in not only plant species, but also other organisms.

Ectopic expression of the human MutT-type Nudix hydrolase, hMTH1, confers enhanced tolerance to oxidative stress in arabidopsis.

Oxidized nucleotides produced by oxidative stress cause DNA mutations and the production of abnormal proteins. Thus, mammalian cells have developed multiple MutT-type Nudix hydrolases that exhibit pyrophosphohydrolase activity toward oxidized nucleotides in the cytosol, mitochondria and nucleus. On the other hand, AtNUDX1 is the only MutT-type Nudix hydrolase in the cytosol of Arabidopsis plants. To clarify the physiological significance of the defenses against oxidatively induced DNA damage in plant organelles, we analyzed the effects of the ectopic expression of the human MutT-type Nudix hydrolase, hMTH1, which was localized in the cytosol (cyt-hMTH1), chloroplasts (chl-hMTH1) and mitochondria (mit-hMTH1) of Arabidopsis cells, on tolerance to oxidative stress. Tolerance to oxidative stress caused by heating and paraquat (PQ) treatment was higher in the mit-hMTH1 and chl-hMTH1 plants than in the control and cyt-hMTH1 plants. The accumulation of H2O2 and the frequency of dead cells were lower in the mit-hMTH1 and chl-hMTH1 plants under stressful conditions. The poly(ADP-ribosyl)ation (PAR) reaction, which regulates repair systems for damaged DNA, was activated in the mit-hMTH1 and chl-hMTH1 plants under heat stress and PQ treatment. Furthermore, DNA fragmentation, which caused programmed cell death, was clearly suppressed in the mit-hMTH1 and chl-hMTH1 plants under heat stress. These results demonstrated that the ectopic expression of hMTH1 in the chloroplasts and mitochondria of Arabidopsis enhanced oxidative stress tolerance by activating the PAR reaction and suppressing programmed cell death.

Transient expression analysis revealed the importance of VTC2 expression level in light/dark regulation of ascorbate biosynthesis in Arabidopsis.

Ascorbate (AsA) is an important antioxidant and an enzyme cofactor involved in various metabolic pathways. In this study, we investigated the effects of estrogen (ES)-inducible transient expression of genes encoding enzymes involved in the d-mannose/l-galactose (d-Man/l-Gal) pathway for plant AsA biosynthesis on AsA levels under light and dark conditions. No significant difference was observed in AsA levels between Arabidopsis plants transiently expressing phosphomannose isomerase (PMI1), GDP-d-Man pyrophosphorylase (GMP/VTC1), GDP-Man-3',5'-epimerase (GME), and l-Gal 1-phosphate phosphatase (GPP/VTC4), but AsA levels in the plants transiently expressing GDP-l-Gal phosphorylase (GGP/VTC2) were 2.5-fold higher than those in control plants 7 d after ES treatment. The increase in AsA levels under continuous light conditions and the decrease in AsA levels under dark conditions were enhanced and suppressed, respectively, in the ES-treated plants. These results suggest that GGP/VTC2 acts as a rate-limiting step regulating AsA biosynthesis in response to light and dark conditions.

AtDREB2G is involved in the regulation of riboflavin biosynthesis in response to low-temperature stress and abscisic acid treatment in Arabidopsis thaliana.

Riboflavin (RF) serves as a precursor to flavin mononucleotide and flavin adenine dinucleotide, which are crucial cofactors in various metabolic processes. Strict regulation of cellular flavin homeostasis is imperative, yet information regarding the factors governing this regulation remains largely elusive. In this study, we first examined the impact of external flavin treatment on the Arabidopsis transcriptome to identify novel regulators of cellular flavin levels. Our analysis revealed alterations in the expression of 49 putative transcription factors. Subsequent reverse genetic screening highlighted a member of the dehydration-responsive element binding (DREB) family, AtDREB2G, as a potential regulator of cellular flavin levels. Knockout mutants of AtDREB2G (dreb2g) exhibited reduced flavin levels and decreased expression of RF biosynthetic genes compared to wild-type plants. Conversely, conditional overexpression of AtDREB2G led to an increase in the expression of RF biosynthetic genes and elevated flavin levels. In wild-type plants, exposure to low temperatures and abscisic acid treatment stimulated enhanced flavin levels and upregulated the expression of RF biosynthetic genes, concomitant with the induction of AtDREB2G. Notably, these responses were significantly attenuated in dreb2g mutants. Our findings establish AtDREB2G is involved in the positive regulation of flavin biosynthesis in Arabidopsis, particularly under conditions of low temperature and abscisic acid treatment.

H2O2-triggered retrograde signaling from chloroplasts to nucleus plays specific role in response to stress.

Recent findings have suggested that reactive oxygen species (ROS) are important signaling molecules for regulating plant responses to abiotic and biotic stress and that there exist source- and kind-specific pathways for ROS signaling. In plant cells, a major source of ROS is chloroplasts, in which thylakoid membrane-bound ascorbate peroxidase (tAPX) plays a role in the regulation of H(2)O(2) levels. Here, to clarify the signaling function of H(2)O(2) derived from the chloroplast, we created a conditional system for producing H(2)O(2) in the organelle by chemical-dependent tAPX silencing using estrogen-inducible RNAi. When the expression of tAPX was silenced in leaves, levels of oxidized protein in chloroplasts increased in the absence of stress. Microarray analysis revealed that tAPX silencing affects the expression of a large set of genes, some of which are involved in the response to chilling and pathogens. In response to tAPX silencing, the transcript levels of C-repeat/DRE binding factor (CBF1), a central regulator for cold acclimation, was suppressed, resulting in a high sensitivity of tAPX-silenced plants to cold. Furthermore, tAPX silencing enhanced the levels of salicylic acid (SA) and the response to SA. Interestingly, we found that tAPX silencing-responsive genes were up- or down-regulated by high light (HL) and that tAPX silencing had a negative effect on expression of ROS-responsive genes under HL, suggesting synergistic and antagonistic roles of chloroplastic H(2)O(2) in HL response. These findings provide a new insight into the role of H(2)O(2)-triggered retrograde signaling from chloroplasts in the response to stress in planta.

Cytosolic ascorbate peroxidase 1 protects organelles against oxidative stress by wounding- and jasmonate-induced H(2)O(2) in Arabidopsis plants.

BACKGROUND: Reactive oxygen species (ROS) are not only cytotoxic compounds leading to oxidative damage, but also signaling molecules for regulating plant responses to stress and hormones. Arabidopsis cytosolic ascorbate peroxidase 1 (APX1) is thought to be a central regulator for cellular ROS levels. However, it remains unclear whether APX1 is involved in plant tolerance to wounding and methyl jasmonate (MeJA) treatment, which are known to enhance ROS production. METHODS: We studied the effect of wounding and MeJA treatment on the levels of H(2)O(2) and oxidative damage in the Arabidopsis wild-type plants and knockout mutants lacking APX1 (KO-APX1). RESULTS: The KO-APX1 plants showed high sensitivity to wounding and MeJA treatment. In the leaves of wild-type plants, H(2)O(2) accumulated only in the vicinity of the wound, while in the leaves of the KO-APX1 plants it accumulated extensively from damaged to undamaged regions. During MeJA treatment, the levels of H(2)O(2) were much higher in the leaves of KO-APX1 plants. Oxidative damage in the chloroplasts and nucleus was also enhanced in the leaves of KO-APX1 plants. These findings suggest that APX1 protects organelles against oxidative stress by wounding and MeJA treatment. GENERAL SIGNIFICANCE: This is the first report demonstrating that H(2)O(2)-scavenging in the cytosol is essential for plant tolerance to wounding and MeJA treatment.

Reduction of furan derivatives by overexpressing NADH-dependent Adh1 improves ethanol fermentation using xylose as sole carbon source with Saccharomyces cerevisiae harboring XR-XDH pathway.

Several alcohol dehydrogenase (ADH)-related genes have been identified as enzymes for reducing levels of toxic compounds, such as, furfural and/or 5-hydroxymethylfurfural (5-HMF), in hydrolysates of pretreated lignocelluloses. To date, overexpression of these ADH genes in yeast cells have aided ethanol production from glucose or glucose/xylose mixture in the presence of furfural or 5-HMF. However, the effects of these ADH isozymes on ethanol production from xylose as a sole carbon source remain uncertain. We showed that overexpression of mutant NADH-dependent ADH1 derived from TMB3000 strain in the recombinant Saccharomyces cerevisiae, into which xylose reductase (XR) and xylitol dehydrogenase (XDH) pathway of Pichia stipitis has been introduced, improved ethanol production from xylose as a sole carbon source in the presence of 5-HMF. Enhanced furan-reducing activity is able to regenerate NAD(+) to relieve redox imbalance, resulting in increased ethanol yield arising from decreased xylitol accumulation. In addition, we found that overexpression of wild-type ADH1 prevented the more severe inhibitory effects of furfural in xylose fermentation as well as overexpression of TMB3000-derived mutant. After 120 h of fermentation, the recombinant strains overexpressing wild-type and mutant ADH1 completely consumed 50 g/L xylose in the presence of 40 mM furfural and most efficiently produced ethanol (15.70 g/L and 15.24 g/L) when compared with any other test conditions. This is the first report describing the improvement of ethanol production from xylose as the sole carbon source in the presence of furan derivatives with xylose-utilizing recombinant yeast strains via the overexpression of ADH-related genes.