Flavones and flavone synthases

Within the secondary metabolite class of flavonoids which consist of more than 9000 known structures, flavones define one of the largest subgroups. Their natural distribution is demonstrated for almost all plant tissues. Various flavone aglyca and their O- or C-glycosides have been described in the literature. The diverse functions of flavones in plants as well as their various roles in the interaction with other organisms offer many potential applications, not only in plant breeding but also in ecology, agriculture and human nutrition and pharmacology. In this context, the antioxidative activity of flavones, their use in cancer prevention and treatment as well as the prevention of coronary heart disease should be emphasized. The therapeutic potential of flavones makes these compounds valuable targets for drug design, including recombinant DNA approaches. The biosynthesis of flavones in plants was found to be catalyzed by two completely different flavone synthase proteins (FNS), a unique feature within the flavonoids. The first, FNS I, a soluble dioxygenase, was only described for members of the Apiaceae family so far. The second, FNS II, a membrane bound cytochrome P450 enzyme, has been found in all other flavone accumulating tissues. This phenomenon is particularly of interest from the evolutionary point of view concerning the flavone biosynthesis and functions in plants. Recently, FNS I and FNS II genes have been cloned from a number of plant species. This now enables detailed biochemical and molecular characterizations and also the development of direct metabolic engineering strategies for modifications of flavone synthesis in plants to improve their nutritional and/or biopharmaceutical value.     

Evolution of flavone synthase I from parsley flavanone 3beta-hydroxylase by site-directed mutagenesis

Flavanone 3beta-hydroxylase (FHT) and flavone synthase I (FNS I) are 2-oxoglutarate-dependent dioxygenases with 80% sequence identity, which catalyze distinct reactions in flavonoid biosynthesis. However, FNS I has been reported exclusively from a few Apiaceae species, whereas FHTs are more abundant. Domain-swapping experiments joining the N terminus of parsley (Petroselinum crispum) FHT with the C terminus of parsley FNS I and vice versa revealed that the C-terminal portion is not essential for FNS I activity. Sequence alignments identified 26 amino acid substitutions conserved in FHT versus FNS I genes. Homology modeling, based on the related anthocyanidin synthase structure, assigned seven of these amino acids (FHT/FNS I, M106T, I115T, V116I, I131F, D195E, V200I, L215V, and K216R) to the active site. Accordingly, FHT was modified by site-directed mutagenesis, creating mutants encoding from one to seven substitutions, which were expressed in yeast (Saccharomyces cerevisiae) for FNS I and FHT assays. The exchange I131F in combination with either M106T and D195E or L215V and K216R replacements was sufficient to confer some FNS I side activity. Introduction of all seven FNS I substitutions into the FHT sequence, however, caused a nearly complete change in enzyme activity from FHT to FNS I. Both FHT and FNS I were proposed to initially withdraw the beta-face-configured hydrogen from carbon-3 of the naringenin substrate. Our results suggest that the 7-fold substitution affects the orientation of the substrate in the active-site pocket such that this is followed by syn-elimination of hydrogen from carbon-2 (FNS I reaction) rather than the rebound hydroxylation of carbon-3 (FHT reaction).     

Female nuptial signal advertises spawning readiness in a population of <Emphasis Type="Italic">Halichoeres margaritaceus</Emphasis> on the Great Barrier Reef,Australia

Females of some polygynous fish species display female nuptial signals (FNSs), advertising spawning readiness to potential mates. Halichoeres margaritaceus, a polygynous coral reef fish, reportedly display red belly color and a unique bobbing behavior as FNSs advertising spawning readiness. In this study, I examined whether FNSs were observed in an H. margaritaceus population in Australia. Females in this study displayed red belly color as an FNS, although at a lower intensity than that reported in a Japanese population. Contrary to observations made in the Japanese population, bobbing behavior performed by females in this study showed no temporal trend as spawning approached, indicating bobbing behavior did not function as an FNS in the Australian population. The role of local selective pressures on FNS intensity and lifetime reproductive benefits H. margaritaceus may potentially gain in utilizing FNSs are briefly discussed.     

Flavone synthases from Medicago truncatula are flavanone-2-hydroxylases and are important for nodulation

Flavones are important copigments found in the flowers of many higher plants and play a variety of roles in plant adaptation to stress. In Medicago species, flavones also act as signal molecules during symbiotic interaction with the diazotropic bacterium Sinorhizobium meliloti. They are the most potent nod gene inducers found in root exudates. However, flavone synthase II (FNS II), the key enzyme responsible for flavone biosynthesis, has not been characterized in Medicago species. We cloned two FNS II genes from Medicago truncatula using known FNS II sequences from other species and named them MtFNSII-1 and MtFNSII-2. Functional assays in yeast (Saccharomyces cerevisiae) suggested that the catalytic mechanisms of both cytochrome P450 monooxygenases were similar to the other known legume FNS II from licorice (Glycyrrhiza echinata). MtFNSII converted flavanones to 2-hydroxyflavanones instead of flavones whereas FNS II from the nonlegume Gerbera hybrida, converted flavanones to flavones directly. The two MtFNSII genes had distinct tissue-specific expression patterns. MtFNSII-1 was highly expressed in roots and seeds whereas MtFNSII-2 was highly expressed in flowers and siliques. In addition, MtFNSII-2 was inducible by S. meliloti and methyl jasmonate treatment, whereas MtFNSII-1 was not. Histochemical staining of transgenic hairy roots carrying the promoter-reporter constructs indicated that the MtFNSII-2 induction was tissue specific, mostly localized to vascular tissues and root hairs. RNA interference-mediated suppression of MtFNSII genes resulted in flavone depleted roots and led to significantly reduced nodulation when inoculated with S. meliloti. Our results provide genetic evidence supporting that flavones are important for nodulation in M. truncatula.     

Flavone synthase II (CYP93B16) from soybean (Glycine max L.)

Flavonoids are a very diverse group of plant secondary metabolites with a wide array of activities in plants, as well as in nutrition and health. All flavonoids are derived from a limited number of flavanone intermediates, which serve as substrates for a variety of enzyme activities, enabling the generation of diversity in flavonoid structures. Flavonoids can be characteristic metabolites, like isoflavonoids for legumes. Others, like flavones, occur in nearly all plants. Interestingly, there exist two fundamentally different enzymatic systems able to directly generate flavones from flavanones, flavone synthase (FNS) I and II. We describe an inducible flavone synthase activity from soybean (Glycine max) cell cultures, generating 7,4′-dihydroxyflavone (DHF), which we classified as FNS II. The corresponding full-length cDNA (CYP93B16) was isolated using known FNS II sequences from other plants. Functional expression in yeast allowed the detailed biochemical characterization of the catalytic activity of FNS II. A direct conversion of flavanones such as liquiritigenin, naringenin, and eriodictyol into the corresponding flavones DHF, apigenin and luteolin, respectively, was demonstrated. The enzymatic reaction of FNS II was stereoselective, favouring the (S)- over the (R)-enantiomer. Phylogenetic analyses of the subfamily of plant CYP93B enzymes indicate the evolution of a gene encoding a flavone synthase which originally catalyzed the direct conversion of flavanones into flavones, via early gene duplication into a less efficient enzyme with an altered catalytic mechanism. Ultimately, this allowed the evolution of the legume-specific isoflavonoid synthase activity.     

Functional characterization of a Plagiochasma appendiculatum flavone synthase I showing flavanone 2-hydroxylase activity

FNS I is a 2-oxoglutarate dependent dioxygenase (2-ODD) found mainly in species of the Apiaceae family. Here, an FNS I cDNA sequence was isolated from the liverwort Plagiochasma appendiculatum (Aytoniaceae) and characterized. The recombinant protein exhibited high FNS I activity catalyzing the conversion of naringenin to apigenin and 2-hydroxynaringenin. The critical residue for flavanone-2-hydroxylation activity was Tyr240, as identified from homology modeling and site-directed mutagenesis. The recombinant protein also showed some flavonol synthase activity, as it can convert dihydrokaempferol to kaempferol. When the Leu311 residue was mutated to Phe, the enzyme’s capacity to convert dihydrokaempferol to kaempferol was substantially increased. PaFNS I represents a 2-ODD in which a hydrophobic π-stacking interaction between the key residue and the naringenin A-ring determines 2-hydroxyflavanone formation.     

Flavonoid Production Is Effectively Regulated by RNAi Interference of Two Flavone Synthase Genes from <Emphasis Type="Italic">Glycine max</Emphasis>

Flavonoids are a group of secondary metabolites found in many higher plants. The multiple roles of their flavone subclass include protection against UV damage, regulation of auxin transport, and modulation of flower color. In soybean (Glycine max), flavone synthase II (FNS II) is the key enzyme responsible for flavone biosynthesis. Two FNS II genes from soybean cultivar Hefeng 47 were cloned according to basic local alignment search tool (BLAST) contexts using flavone synthase sequences reported in other species. These were named GmFNSII-1 and GmFNSII-2. Sequence alignments showed that the cDNA of GmFNSII-1 was identical to that of CYP93B16, whereas GmFNSII-2 was clearly distinct. Functional assays in yeast (Schizosaccharomyces pombe) suggested that these two enzymes could convert (2S)-naringenin into apigenin. The two GmFNSII genes had similar tissue-specific expression patterns, but GmFNSII-2 was significantly expressed in the roots after treatment with 0.4 M glucose. This demonstrates that the gene plays an important role in the response to defense signals in soybean. RNA interference-mediated suppression of those GmFNSII genes effectively regulated flavone and isoflavone production in hairy roots that arose from soybean cotyledons transformed with Agrobacterium rhizogenes (ATCC15834). Our study also highlights some of the challenges associated with metabolic engineering of plant natural products.     

Molecular evolution of flavonoid dioxygenases in the family Apiaceae

Plant species of the family Apiaceae are known to accumulate flavonoids mainly in the form of flavones and flavonols. Three 2-oxoglutarate-dependent dioxygenases, flavone synthase or flavanone 3 beta-hydroxylase and flavonol synthase are involved in the biosynthesis of these secondary metabolites. The corresponding genes were cloned recently from parsley (Petroselinum crispum) leaves. Flavone synthase I appears to be confined to the Apiaceae, and the unique occurrence as well as its high sequence similarity to flavanone 3beta-hydroxylase laid the basis for evolutionary studies. In order to examine the relationship of these two enzymes throughout the Apiaceae, RT-PCR based cloning and functional identification of flavone synthases I or flavanone 3beta-hydroxylases were accomplished from Ammi majus, Anethum graveolens, Apium graveolens, Pimpinella anisum, Conium maculatum and Daucus carota, yielding three additional synthase and three additional hydroxylase cDNAs. Molecular and phylogenetic analyses of these sequences were compatible with the phylogeny based on morphological characteristics and suggested that flavone synthase I most likely resulted from gene duplication of flavanone 3beta-hydroxylase, and functional diversification at some point during the development of the apiaceae subfamilies. Furthermore, the genomic sequences from Petroselinum crispum and Daucus carota revealed two introns in each of the synthases and a lack of introns in the hydroxylases. These results might be explained by intron losses from the hydroxylases occurring at a later stage of evolution.     

Molecular Basis for Chemical Evolution of Flavones to Flavonols and Anthocyanins in Land Plants

During the course of evolution of land plants, different classes of flavonoids, including flavonols and anthocyanins, sequentially emerged, facilitating adaptation to the harsh terrestrial environment. Flavanone 3β-hydroxylase (F3H), an enzyme functioning in flavonol and anthocyanin biosynthesis and a member of the 2-oxoglutarate-dependent dioxygenase (2-ODD) family, catalyzes the hydroxylation of (2S)-flavanones to dihydroflavonols, but its origin and evolution remain elusive. Here, we demonstrate that functional flavone synthase Is (FNS Is) are widely distributed in the primitive land plants liverworts and evolutionarily connected to seed plant F3Hs. We identified and characterized a set of 2-ODD enzymes from several liverwort species and plants in various evolutionary clades of the plant kingdom. The bifunctional enzyme FNS I/F2H emerged in liverworts, and FNS I/F3H evolved in Physcomitrium (Physcomitrella) patens and Selaginella moellendorffii, suggesting that they represent the functional transition forms between canonical FNS Is and F3Hs. The functional transition from FNS Is to F3Hs provides a molecular basis for the chemical evolution of flavones to flavonols and anthocyanins, which contributes to the acquisition of a broader spectrum of flavonoids in seed plants and facilitates their adaptation to the terrestrial ecosystem.

The success of land plants in the colonization of and adaptation to terrestrial ecosystems has been particularly attributed to the emergence and evolution of a unique metabolic capacity that synthesizes diverse specialized metabolites, including flavonoids, a highly polymorphic class of polyphenols (Weng and Chapple, 2010). The flavonoid metabolites have been classified into several subgroups, namely flavanones, dihydroflavonols, flavones, flavonols, flavan-3,4-diols, flavan-3-ols, and anthocyanins, based on their oxidation status and substitution patterns of the core skeleton (Winkel-Shirley, 2001; Martens et al., 2010). Along with the evolution of land plants, different classes of flavonoids emerged (Koes et al., 1994). The basal land plants liverworts produce chalcones, flavanones, and flavones; whereas lycophytes gained the ability to produce proanthocyanidins (Markham, 1984; Koes et al., 1994). Furthermore, both pteridophyta and gymnosperms, while dominated with flavone production, began to produce flavonols (Markham, 1984; Koes et al., 1994). Finally, flavonols and anthocyanins are well represented in angiosperms. Flavonols, which bear a 3-hydroxyl group in the core structure, have been exploited as effective photoprotectants against UV-B radiation (Solovchenko and Schmitz-Eiberger, 2003), as signal providers to symbionts (Hungria et al., 1991), as regulators of the transport of phytohormones (Peer and Murphy, 2007), and as determinants of conditional male fertility (Muhlemann et al., 2018). Anthocyanins, derived from dihydroflavonol, are important for sexual reproduction, acting as attractants for insect pollinators and for animal dispersers of seed (Shimada et al., 2005). It is obvious that a clear chemical evolution trace from chalcones, flavanones, and flavones to flavonols and anthocyanins, occurs across plant phyla. However, the molecular basis for such a chemical evolution remains mysterious.The biosynthesis of flavones and flavonols requires chemical conversion of a common precursor, (2S)-flavanone, and is catalyzed by flavone synthase I (FNS I) and flavanone 3β-hydroxylases (F3Hs), respectively. Both enzymes as well as flavonol synthase (FLS) and anthocyanidin synthase (ANS) belong to a larger enzyme family, the 2-oxoglutarate-dependent dioxygenases (2-ODDs; Farrow and Facchini, 2014). FNS I converts (2S)-flavanone to flavone via desaturation of carbon 2 and 3 of the heterologous ring of flavanone (Gebhardt et al., 2005, 2007), while F3H catalyzes the conversion of (2S)-flavanone to (2R,3R)-dihydroflavonol by hydroxylation of the C-3β position (Supplemental Fig. S1). Subsequently, FLS converts (2R,3R)-dihydroflavonols to their corresponding flavonols, and ANS catalyzes the nonpigmented leucoanthocyanidins (leucopelargonidin, leucocyanidin, and leucodelphinidin) to the pigmented anthocyanidins (pelargonidin, cyanidin, and delphinidin, respectively; Supplemental Fig. S1). These four classes of 2-ODD enzymes phylogenetically form two distinct subgroups, one consisting of F3H and FNS I and the other consisting of FLS and ANS. FNS I and F3H both use flavanone as substrate and exhibit, in general, a relatively narrow substrate specificity (Turnbull et al., 2000; Martens et al., 2003), whereas ANS and FLS display some degree of promiscuity in their substrate preferences and catalytic activities. For example, Arabidopsis (Arabidopsis thaliana) FLS1 is not only capable of converting dihydroflavonols to their corresponding flavonols but also mediates the oxidation of 2S-flavanone (naringenin) to both dihydrokaempferol enantiomers, an activity normally associated with F3H (Prescott et al., 2002). While F3Hs are ubiquitous in vascular plants, FNS Is appear to be confined to the Apiaceae family as well as a few non-Apiaceae species such as rice (Oryza sativa; Lee et al., 2008), maize (Zea mays), and Arabidopsis (Falcone Ferreyra et al., 2015). Prior to the discovery of FNS Is in those non-Apiaceae species, it was assumed that the gene encoding FNS I arose from duplication and mutation of F3H (Martens et al., 2001, 2003; Gebhardt et al., 2005, 2007). However, the FNS Is revealed in both Z. mays and Arabidopsis show very poor sequence similarity with those present in Apiaceae species, which suggests that the evolution of the FNS Is was not as clear-cut as was originally believed. It is likely that the evolution of FNS occurred several times independently. In several cereal crops, such as Z. mays, O. sativa, and wheat (Triticum aestivum), flavones are the major flavonoid substances, which protect the plants during pathogen attack and under biotic or abiotic stress conditions (Righini et al., 2019).Previously, we found that the liverwort Plagiochasma appendiculatum FNS I (which should change to PaFNS I/F2H, according to the function) converted flavanone to 2-hydroxyflavanone and flavone (Han et al., 2014). The dual FNS I and F2H activities of PaFNS I/F2H, together with the fact that its amino acid sequence shares a higher identity with F3Hs than with FNS Is, implicates an evolutionary connection between liverwort FNS Is and seed plant F3Hs. On the other hand, previous in silico analysis failed in identifying any F3H sequences in either the bryophyte Physcomitrium (Physcomitrella) patens or the lycophyte Selaginella moellendorffii, even though both species produce dihydroflavonol-derived metabolites. To identify when and how F3H emerged and evolved to produce a vast variety of flavonoid metabolites, we systematically identified FNS I and F3H homologous sequences from species of different phyla, including liverworts, P. patens, S. moellendorffii, gymnosperms, and angiosperms. Subsequent biochemical characterization revealed that the functionally promiscuous FNS Is widely emerged in the liverworts, which evolved into a dual-function enzyme with both FNS I and F3H activities in both P. patens and S. moellendorffii. Further evolution led to the emergence of F3H with a minor level of FNS I activity in gymnosperm species, while those generated by angiosperm species showed a more specific F3H activity.     

Molecular systematics of Apiaceae subfamily Apioideae: phylogenetic analyses of nuclear ribosomal DNA internal transcribed spacer and plastid RPO C1 intron sequences

Evolutionary relationships among representatives of Apiaceae (Umbelliferae) subfamily Apioideae have been inferred from phylogenetic analyses of nuclear ribosomal DNA internal transcribed spacer (ITS 1 and ITS 2) and plastid rpoC1 intron sequences. High levels of nucleotide sequence variation preclude the use of the ITS region for examining relationships across subfamilial boundaries in Apiaceae, whereas the rpoC1 intron is more suitably conserved for family-wide phylogenetic study but is too conserved for examining relationships among closely related taxa. In total, 126 ITS sequences from subfamily Apioideae and 100 rpoC1 intron sequences from Apiaceae (all three subfamilies) and outgroups Araliaceae and Pittosporaceae were examined. Phylogenies estimated using parsimony, neighbor-joining, and maximum likelihood methods reveal that: (1) Apiaceae subfamily Apioideae is monophyletic and is sister group to Apiaceae subfamily Saniculoideae; (2) Apiaceae subfamily Hydrocotyloideae is not monophyletic, with some members strongly allied to Araliaceae and others to Apioideae + Saniculoideae; and (3) Apiaceae subfamily Apioideae comprises several well-supported subclades, but none of these coincide with previously recognized tribal divisions based largely on morphological and anatomical characters of the fruit. Four major clades in Apioideae are provisionally recognized and provide the framework for future lower level phylogenetic analyses. A putative secondary structure model of the Daucus carota (carrot) rpoC1 group II intron is presented. Of its six major structural domains, domains II and III are the most, and domains V and VI the least, variable.     

Three 2-oxoglutarate-dependent dioxygenase activities of Equisetum arvense L. forming flavone and flavonol from (2S)-naringenin

Equisetum arvense L. (Equisetaceae-horsetail) accumulates various flavones and flavonols in infertile shoot. Enzyme assays conducted with crude extracts of the green tissue revealed chalcone synthase activity and also three further activities assigned to flavonoid biosynthesis and identified as flavone synthase I, flavanone 3β-hydroxylase and flavonol synthase. The latter three activities were characterized as soluble, 2-oxoglutarate-dependent dioxygenases by their typical cofactor requirements and peculiar inhibition. Notably, this is the first report of flavone synthase I which had been considered to be restricted solely to species of the Apiaceae from a distant plant taxon.     

Helospectin I and II evoke vasodilation in the intact peripheral microcirculation

Helospectin I and II, two closely related mammalian neuropeptides of the secretin/glucagons/vasoactive intestinal peptide (VIP) superfamily of peptides, are co-localized with VIP in nerve fibers surrounding vascular smooth muscle. However, the role if any, VIP receptors play in transducing the vasorelaxant effects of helospectin I and II in the intact peripheral microcirculation is uncertain. The purpose of this study was to determine whether helospectin I and II elicit vasodilation in the intact peripheral microcirculation and, if so, whether this response is mediated, in part, by VIP or pituitary adenylate cyclase activating peptide (PACAP) receptor engagement, and through local elaboration of cyclooxygenase products of arachidonic acid metabolism. Using intravital microscopy, we found that suffusion of helospectin I and II (each, 1.0 nmol) evoked potent vasodilation and of similar magnitude in the intact hamster cheek pouch microcirculation (P < 0.05). Suffusion of 0.1 nmol helospectin I and II had no significant effects on arteriolar diameter. Pretreatment with VIP(10-28), a VPAC1/VPAC2 receptor antagonist, or PACAP(6-38), a PAC1/VPAC2 receptor antagonist, had no significant effects on helospectin I- and II-induced responses. In addition, pretreatment with indomethacin had no significant effects on helospectin I- and II-induced vasodilation. Collectively, these data indicate that helospectin I and II evoke potent vasodilation in the intact peripheral microcirculation that is not transduced by VIP or PACAP receptors nor through cyclooxygenase products of arachidonic acid metabolism.     

Screening and heterologous expression of flavone synthase and flavonol synthase to catalyze hesperetin to diosmetin

Biotechnology Letters - In this study, 44 flavone synthases (FNS) and flavonol synthases (FLS) from different origins were collected. The instability index and conserved domain of the enzymes were...     

Neurabins recruit protein phosphatase-1 and inhibitor-2 to the actin cytoskeleton

Inhibitor-2 (I-2) bound protein phosphatase-1 (PP1) and several PP1-binding proteins from rat brain extracts, including the actin-binding proteins, neurabin I and neurabin II. Neurabins from rat brain lysates were sedimented by I-2 and its structural homologue, I-4. The central domain of both neurabins bound PP1 and I-2, and mutation of a conserved PP1-binding motif abolished neurabin binding to both proteins. Microcystin-LR, a PP1 inhibitor, also attenuated I-2 binding to neurabins. Immunoprecipitation of neurabin I established its association with PP1 and I-2 in HEK293T cells and suggested that PP1 mediated I-2 binding to neurabins. The C terminus of I-2, although not required for PP1 binding, facilitated PP1 recruitment by neurabins, which also targeted I-2 to polymerized F-actin. Mutations that attenuated PP1 binding to I-2 and neurabin I suggested distinct and overlapping sites for these two proteins on the PP1 catalytic subunit. Immunocytochemistry in epithelial cells and cultured hippocampal neurons showed that endogenous neurabin II and I-2 colocalized at actin-rich structures, consistent with the ability of neurabins to target the PP1.I-2 complex to actin cytoskeleton and regulate cell morphology.     

Protein-protein interactions as a proxy to monitor conformational changes and activation states of the tomato resistance protein I-2

Plant resistance proteins (R) are involved in pathogen recognition and subsequent initiation of defence responses. Their activity is regulated by inter- and intramolecular interactions. In a yeast two-hybrid screen two clones (I2I-1 and I2I-2) specifically interacting with I-2, a Fusarium oxysporum f. sp. lycopersici resistance protein of the CC-NB-LRR family, were identified. Sequence analysis revealed that I2I-1 belongs to the Formin gene family (SlFormin) whereas I2I-2 has homology to translin-associated protein X (SlTrax). SlFormin required only the N-terminal CC I-2 domain for binding, whereas SlTrax required both I-2 CC and part of the NB-ARC domain. Tomato plants stably silenced for these interactors were not compromised in I-2-mediated disease resistance. When extended or mutated forms of I-2 were used as baits, distinct and often opposite, interaction patterns with the two interactors were observed. These interaction patterns correlated with the proposed activation state of I-2 implying that active and inactive R proteins adopt distinct conformations. It is concluded that the yeast two hybrid system can be used as a proxy to monitor these different conformational states.     

Tissue Prx I in the protection against Fe-NTA and the reduction of nitroxyl radicals

Peroxiredoxin I (Prx I) is a key cytoplasmic peroxidase that reduces intracellular hydroperoxides in concert with thioredoxin. To study the role of tissue Prx I in protection from oxidative stress, we generated Prx I-/- mice by gene trapping. We then evaluated the acute-phase tissue damage caused by ferric-nitrilotriacetate (Fe-NTA). Increases in serum aspartate aminotransferase and alanine aminotransferase levels were significantly greater in Prx I-/- than wild-type mice, 4 and 12 h after the injection of Fe-NTA. Using real-time EPR imaging, we examined the reduction of the stable paramagnetic nitroxyl radical 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl in vivo, and found that the half-life of this spin probe in the liver and kidney was significantly prolonged in the Prx I-/- mice. These results demonstrate that Prx I-/- mice have less reducing activity and are more susceptible to the damage mediated by reactive oxygen species in vivo than wild-type mice.