Ten rice peroxidases redundantly respond to multiple stresses including infection with rice blast fungus

Class III plant peroxidases are believed to function in diverse physiological processes including disease resistance and wound response, but predicted low substrate specificities and the presence of 70 or more isoforms have made it difficult to define a specific physiological function(s) for each gene. To select pathogen-responsive POX genes, we analyzed the expression profiles of 22 rice POX genes after infection with rice blast fungus. The expression of 10 POX genes among the 22 genes was induced after fungal inoculation in both compatible and incompatible hosts. Seven of the 10 POX genes were expressed at higher levels in the incompatible host than in the compatible host 6-24 h after inoculation by which time no fungus-induced lesions have appeared. Organ-specific expression and stress-induced expression by wounding and treatment with probenazole, an agrichemical against blast fungus, jasmonic acid, salicylic acid and 1-aminocyclopropane-1-carboxylate, a precursor of ethylene, indicated that rice POXs have individual characteristics and can be classified into several types. A comparison of the amino acid sequences of POXs showed that multiple isoforms with a high sequence similarity respond to stress in different or similar ways. Such redundant responses of POX genes may guarantee POX activities that are necessary for self-defense in plant tissues against environmental stresses including pathogen infection.     

SUPPRESSING A PEROXIDASE GENE REDUCES SURVIVAL IN THE WHEAT APHID Sitobion avenae

Peroxidases (POXs) make up a large superfamily of enzymes that act in a wide range of biological mechanisms, including maintaining appropriate redox balances within cells, among other actions. In this study, we cloned a sequence that encodes a POX protein, SaPOX, from wheat aphids, Sitobion avenae. Amino acid sequence alignment showed the SaPOX sequence was conserved with POXs from other insect species. SaPOX mRNA accumulations were present in all nymphal and adult stages, at higher levels during the first and second instar, and lower during later stages in the life cycle. Ingestion of dsRNA specific to POX led to reduced SaPOX mRNA accumulation. Sitobion avenae nymphs continuously exposed to dietary dsPOX via an artificial diet led to reduced survival rate and ecdysis index. We infer that POX is important to maintain the growth and development of S. avenae.     

Isolation of Tobacco Isoperoxidases Accumulated in Cell-Suspension Culture Medium and Characterization of Activities Related to Cell Wall Metabolism

All of the most important guaiacol-type peroxidase (POX) isoforms accumulated in the culture medium of BY-2 tobacco (Nicotiana tabacum L. cv Bright Yellow 2) cells have been isolated. Five basic and two acidic isoforms were found. The four major isoforms (B2, B3, P1, and P2), all strongly basic, have been purified to homogeneity and partially sequenced. B2 and B3 are new isoforms showing high homology to only one POX isolated so far. Amino acid sequencing and specific activities indicated that basic isoPOXs constitute two pairs of strictly related isoforms (P1/P2 and B2/B3). Their specific activities measured in the presence of different substrates, as monolignols and NAD(P)H, indicated possible specialized functions in cell wall metabolism. Only P-type POXs were able to oxidize indoleacetic acid. Variations in pH could play a regulatory role by changing the relative contribution of different isoforms to total POX activity. Apart from cell culture medium, polyclonal antibodies obtained against P1 and P2 detected P1 in roots and in lower parts of stems. Immunocytochemical labeling indicated that P-type POXs were expressed in stem phloem and in phloem and epidermal cells of roots.     

Peroxidase genes differentially respond to auxin during the formation of adventitious roots in soybean hypocotyl

In our investigation, auxins (IAA, IBA and NAA) effectively promoted rooting in soybean hypocotyls. The activity of anionic peroxidase (POX) (pI 3.7) and cationic POX (pI 8.5) was significantly suppressed by exogenous auxins on day 2 (the inductive phase). Some particular anionic POXs (pI 4.0 and pI 5.3) significantly increased in IBA-treated tissues as compared with the control when the incubation time was prolonged to day 3 and day 4 (the initiation phase). We sequenced 5′-flanking region of pI 8.5 and pI 5.3 POX genes using the PLACE and PlantCARE databases to identify several potential cis-regulatory elements. The pI 8.5 POX gene promoter contained two sites that were homologous to sequences commonly found in auxin response elements; motifs ARF/AuxRE and CATATGGMSAUR. During the inductive phase, the activity of pI 8.5 POX was significantly suppressed by the exogenously applied auxins. The pI 8.5 POX gene promoter contained both ARF/AuxRE and CATATGGMSAUR motifs that responded to auxins earlier than the pI 5.3 POX gene. Hence, the pI 8.5 POX gene might belong to primary auxin response genes. The pI 5.3 POX gene, which responded to auxins a day or two later, contained only ARF/AuxRE motif. Moreover, unlike pI 8.5 and pI 3.7 POXs that were suppressed by auxins, the pI 5.3 POX was induced or enhanced by the applied auxins, especially IBA. The pI 5.3 POX might generate H₂O₂ which caused the auxin-induced growth at the initiation phase during the formation of adventitious root in soybean hypocotyls.     

Biodepollution of wastewater containing phenolic compounds from leather industry by plant peroxidases

This study deals with the use of peroxidases (POXs) from Allium sativum, Ipomoea batatas, Raphanus sativus and Sorghum bicolor to catalyze the degradation of free phenolic compounds as well as phenolic compounds contained in wastewater from leather industry. Secretory plant POXs were able to catalyze the oxidation of gallic acid, ferulic acid, 4-hydroxybenzoic acid, pyrogallol and 1,4-tyrosol prepared in ethanol 2% (v:v). Efficiency of peroxidase catalysis depends strongly on the chemical nature of phenolic substrates and on the botanical source of the enzymes. It appeared that POX from Raphanus sativus had the highest efficiency. Results show that POXs can also remove phenolic compounds present in industrial wastewater such as leather industry. Removal of phenolic compounds in wastewater from leather industry by POX was significantly enhanced by polyethylene glycol.     

Sites and regulation of polyamine catabolism in the tobacco plant. Correlations with cell division/expansion, cell cycle progression, and vascular development

We previously gave a picture of the homeostatic characteristics of polyamine (PA) biosynthesis and conjugation in tobacco (Nicotiana tabacum) plant organs during development. In this work, we present the sites and regulation of PA catabolism related to cell division/expansion, cell cycle progression, and vascular development in the tobacco plant. Diamine oxidase (DAO), PA oxidase (PAO), peroxidases (POXs), and putrescine N-methyltransferase expressions follow temporally and spatially discrete patterns in shoot apical cells, leaves (apical, peripheral, and central regions), acropetal and basipetal petiole regions, internodes, and young and old roots in developing plants. DAO and PAO produce hydrogen peroxide, a plant signal molecule and substrate for POXs. Gene expression and immunohistochemistry analyses reveal that amine oxidases in developing tobacco tissues precede and overlap with nascent nuclear DNA and also with POXs and lignification. In mature and old tissues, flow cytometry indicates that amine oxidase and POX activities, as well as pao gene and PAO protein levels, coincide with G2 nuclear phase and endoreduplication. In young versus the older roots, amine oxidases and POX expression decrease with parallel inhibition of G2 advance and endoreduplication, whereas putrescine N-methyltransferase dramatically increases. In both hypergeous and hypogeous tissues, DAO and PAO expression occurs in cells destined to undergo lignification, suggesting a different in situ localization. DNA synthesis early in development and the advance in cell cycle/endocycle are temporally and spatially related to PA catabolism and vascular development.     

A large family of class III plant peroxidases

Class III plant peroxidase (POX), a plant-specific oxidoreductase, is one of the many types of peroxidases that are widely distributed in animals, plants and microorganisms. POXs exist as isoenzymes in individual plant species, and each isoenzyme has variable amino acid sequences and shows diverse expression profiles, suggesting their involvement in various physiological processes. Indeed, studies have provided evidence that POXs participate in lignification, suberization, auxin catabolism, wound healing and defense against pathogen infection. Little, however, is known about the signal transduction for inducing expression of the pox genes. Recent studies have provided information on the regulatory mechanisms of wound- and pathogen-induced expression of some pox genes. These studies suggest that pox genes are induced via different signal transduction pathways from those of other known defense-related genes.     

Roles of the reactive oxygen species-generating peroxidase reactions in plant defense and growth induction

Extracellularly secreted plant peroxidases (POXs) are considered to catalyze the generation of reactive oxygen species (ROS) coupled to oxidation of plant hormone indole-3-acetic acid (IAA) and defense-related compounds salicylic acid (SA), aromatic monoamines (AMAs) and chitooligosaccharides (COSs). This review article consists of two parts, which describe H(2)O(2)-dependent and H(2)O(2)-independent mechanisms for ROS generation, respectively. Recent studies have shown that plant POXs oxidize SA, AMAs and COSs in the presence of H(2)O(2) via a conventional POX cycle, yielding the corresponding radical species, such as SA free radicals. These radical species may react with oxygen, and superoxide (O(2)(.-)) is produced. Through the series of reactions 2 moles of O(2)(.-) can be formed from 1 moles of H(2)O(2), thus leading to oxidative burst. It has been revealed that the ROS induced by SA, AMAs and COSs triggers the increase in cytosolic Ca(2+) concentration. Actually POXs transduce the extracellular signals into the redox signals that eventually stimulate the intracellular Ca(2+) signaling required for induction of defense responses. On the other hand, IAA can react with oxygen and plant POXs in the absence of H(2)O(2), by forming the ternary complex enzyme-IAA-O(2), which readily dissociates into enzyme, IAA radicals and O(2)(.-). This article covers the recent reports showing that extracellularly produced hydroxy radicals derived from O(2)(.-) mediate the IAA-induced cell elongation. Here a novel model for IAA signaling pathway mediated by extracellular ROS produced by cell-wall POXs is proposed. In addition, possible controls of the IAA-POX reactions by a fungal alkaloid are discussed.     

Induction and expression of peroxidase in tomato is age-dependent

Peroxidases (POXs) are actively synthesised under stress conditions and induced by biotic/abiotic elicitors. Their age-regulated expression in plants has been a matter of investigation during recent times. The present study focuses on studying the expression of POX at different stages of growth and variation in its inducibility by aqueous extract of Azadirachta indica (neem) fruits. Tomato plants at 6, 8, 10 and 12 weeks were chosen for the study. The third nodal leaf of each plant was treated with neem fruit extract alone or in combination with Pseudomonas syringae pv. tomato. Sampling was performed at an interval of 24 h up to five days and after two weeks of treatment, from both the treated and distal untreated nodes of the plants. POX activity and its isoforms were analysed. The results demonstrated that POX expression and its inducibility by the biotic inducer (neem fruit extract) varies with age of the plant.     

Influence of plant secondary metabolites on in vitro oxidation of methyl ferulate with cell wall peroxidases from lupine apoplast

Ionically bound cell wall peroxidases (POXs) were liberated to intercellular washing fluids (IWFs) and isolated together with other proteins and metabolites present in the apoplast of white lupine (Lupinus albus L. var. Bac) root. After separation of proteins from low molecular weight compounds, activity of peroxidases was monitored in in vitro experiments. Oxidation of methyl ferulate with H2O2 was studied in multi-component mixtures of plant metabolites. Secondary metabolites identified in IWFs or other natural products playing important roles in different physiological processes were applied as modifiers of the dehydrodimerization process during oxidation reactions performed in vitro. These were isoflavones and their conjugates, lupanine representing quinolizidine alkaloids synthesized in lupine, or other natural products such as quercetin, ascorbic, and salicylic acid. The influence of these substances on the oxidation kinetics of methyl ferulate was monitored with liquid chromatography with ultraviolet detection (LC/UV), and identification of compounds was confirmed with the liquid chromatography/mass spectroscopy (LC/MS) system. On the basis of data collected, it was possible to reveal changes in the activities of cell wall POXs. Application of the LC system permitted us to monitor, independently, quantitative changes of two or more reaction products in the mixtures. In multi-component combinations, oxidation yields of methyl ferulate by POXs were modified depending on the actual composition of the reaction mixture. We conclude that various classes of plant secondary metabolites can modify the yield of methyl ferulate oxidation by hydrogen peroxide in the presence of POX, due to interactions with the enzyme's active site (genistein) or radical scavenging properties of metabolites present in the reaction mixture.     

Phytotoxic effects of β-pinene on early growth and associated biochemical changes in rice

β-Pinene, an oxygenated monoterpene, is one of the major monoterpenes emitted into the atmosphere from forest areas and trees. Besides, it is a principal component of essential oils of a number of aromatic plants, which are involved in a variety of ecological interactions, including allelopathy, in the natural environment. However, studies pertaining to phytotoxicity and biochemical effect(s) of β-pinene are largely lacking. We investigated the effect of β-pinene (0.02, 0.04, 0.08, 0.20, 0.40 and 0.80 mg/ml) in a dose- and time-dependent manner on early seedling growth, dry weight accumulation, photosynthetic pigments and changes in macromolecule (protein and carbohydrate) content and activities of enzymes—proteases, α- and β-amylases, polyphenol oxidases and peroxidases- in rice (Oryza sativa) after 3rd, 5th and 7th day of exposure. β-pinene (≥0.04 mg/ml) significantly reduced the root (by 13–87%) and coleoptile (by 5–80%) length of rice. Exposure to β-pinene reduced total chlorophyll content in rice coleoptiles suggesting a negative impact on photosynthesis. The content of macromolecules (proteins and carbohydrates) enhanced significantly in response to β-pinene, whereas the activities of hydrolyzing enzymes—proteases, α-amylases, and β-amylases—declined (by 30–85, 26–84, 27–74%, respectively) in β-pinene-exposed seedlings. In contrast, the activities of peroxidases (POX) and polyphenol oxidases (PPO) enhanced significantly (by 16–152 and 53–290%, respectively) in rice roots in response to β-pinene in a dose- and time-dependent manner. Increased activities of POX and PPO indicate their involvement in providing protection and/or conferring resistance against β-pinene-induced stress. The study concludes that β-pinene inhibits the early growth of rice by altering the plant biochemical status and enhancing activities of POXs and PPOs involved in general plant defense.     

Reactivation of model cholinesterases by oximes and intermediate phosphyloximes: a computational study

Phosphyloximes (POX) are generated upon the reactivation of organophosphorus (OP)-inhibited cholinesterases (ChEs) by pyridinium oximes. These POXs are known to be potent inhibitors of the ChEs following reactivation. However, they can also decompose to give an OP derivative and a cyano derivative of the oxime when a base abstracts the benzylic proton. Using density functional theory, thermodynamic properties were calculated for the reactivation and decomposition pathways of three different oximes (2-PAM, 3-PAM and 4-PAM) with six different OPs (cyclosarin, paraoxon, sarin, tabun, VR and VX). For reactivation purposes, 2-PAM is predicted to be more efficient than 3- and 4-PAM. Based on atomic charges and relative energies, 2-POXs were found to be more inclined towards the decomposition process.     

Differential effect of sorbitol and polyethylene glycol on antioxidant enzymes in rice leaves

Polyethylene glycol (PEG) and sorbitol (ST) have each been used inosmotically induced water stress studies in plants, however, these osmotica maynot have equivalent effects in plants. The present study was designed to examinewhether antioxidant enzyme responses in rice leaves are different for PEG and STof osmotic potential –1.5 MPa. As judged by relative watercontent, PEG treatment resulted in a higher degree of water stress in riceleaves than ST treatment. PEG treatment markedly increased lipid peroxidation,judged by malondialdehyde content, in rice leaves. However, ST treatment had noeffect on lipid peroxidation. An increase in peroxidase (POX), ascorbateperoxidase (APX) and glutathione reductase (GR) activities was observed in riceleaves treated with ST. PEG treatment had no effect on POX and APX activitiesand decreased GR activity in rice leaves. The decrease in superoxide dismutaseactivity induced by PEG was more pronounced than by ST. Cycloheximide blockedthe enhanced activities of POX, APX and GR by ST, indicating de novo synthesisof the enzymes. Results suggest that ST but not PEG treatment can up-regulateantioxidant system in rice leaves.     

Enhancement of Peroxidase-Dependent Oxidation of Sinapyl Alcohol by an Apoplastic Component, 4-Coumaric Acid Ester Isolated from Epicotyls of Vigna angularis L.

A water-soluble component that enhanced the peroxidase-dependent(POX-dependent) oxidation of sinapyl alcohol was isolated fromepicotyls of Vigna angularis. This compound was an ester of4-coumaric acid and a hexose, and it was found in both the apoplastand the symplast. The ester was oxidized by a basic POX isozyme(K_m, about 20 µM) and by an acidic POX isozyme (K_m, about40 µM) that had been partially purified from the apoplasticfraction of epicotyls of V. angularis. These POX isozymes oxidizedsinapyl alcohol at only a very low rate, but a 15-fold enhancementwas observed upon addition of the ester. The concentrationsof the ester required for the half-maximal enhancement weresimilar to the K_m values of the ester for its oxidation by therespective isozymes. The apoplastic concentration of the esterwas higher than 130 µM, suggesting that this ester mightact as a donor of electrons to the apoplastic POX isozymes insitu. Coniferyl alcohol also enhanced the POX-catalyzed oxidationof sinapyl alcohol. The concentrations of coniferyl alcoholrequired for half-maximal enhancement of the oxidation of sinapylalcohol were about 23 and 250 µM when reactions were catalyzedby the basic and acidic POXs, respectively. These values weresimilar to the K_m values of coniferyl alcohol for its oxidationby the respective isozymes. These results suggest that 4-coumaricacid ester and coniferyl alcohol, if it is present in the apoplast,can enhance the POX-dependent oxidation of sinapyl alcohol inthe apoplast of epicotyls of V. angularis. (Received July 1, 1996; Accepted February 5, 1997)     

Determination of Candida tropicalis acyl coenzyme A oxidase isozyme function by sequential gene disruption.

A recently developed transformation system has been used to facilitate the sequential disruption of the Candida tropicalis chromosomal POX4 and POX5 genes, encoding distinct isozymes of the acyl coenzyme A (acyl-CoA) oxidase which catalyzes the first reaction in the beta-oxidation pathway. The URA3-based transformation system was repeatedly regenerated by restoring the uracil requirement to transformed strains, either through selection for spontaneous mutations or by directed deletion within the URA 3 coding sequence, to permit sequential gene disruptions within a single strain of C. tropicalis. These gene disruptions revealed the diploid nature of this alkane- and fatty acid-utilizing yeast by showing that it contains two copies of each gene. A comparison of mutants in which both POX4 or both POX5 genes were disrupted revealed that the two isozymes were differentially regulated and displayed unique substrate profiles and kinetic properties. POX4 was constitutively expressed during growth on glucose and was strongly induced by either dodecane or methyl laurate and to a greater extent than POX5, which was induced primarily by dodecane. The POX4-encoded isozyme demonstrated a broad substrate spectrum in comparison with the narrow-spectrum, long-chain oxidase encoded by POX5. The absence of detectable acyl-CoA oxidase activity in the strain in which all POX4 and POX5 genes had been disrupted confirmed that all functional acyl-CoA oxidase genes had been inactivated. This strain cannot utilize alkanes or fatty acids for growth, indicating that the beta-oxidation pathway has been functionally blocked.     

Roles of Peroxinectin in PGE2-Mediated Cellular Immunity in Spodoptera exigua

Background

Prostaglandins (PGs) mediate insect immune responses to infections and invasions. Although the presence of PGs has been confirmed in several insect species, their biosynthesis in insects remains a conundrum because orthologs of the mammalian cyclooxygenases (COXs) have not been found in the known insect genomes. PG-mediated immune reactions have been documented in the beet armyworm, Spodoptera exigua. The purpose of this research is to identify the source of PGs in S. exigua.

Principal Findings

Peroxidases (POXs) are a sister group of COX genes. Ten putative POXs (SePOX-A ∼ SePOX-J) were expressed in S. exigua. Expressions of SePOX-F and -H were induced by bacterial challenge and expressed in the hemocytes and the fat body. RNAi of each POX was performed by hemocoelic injection of their specific double-stranded RNAs. dsPOX-F or, separately, dsPOX-H, but not the other eight dsRNA constructs, specifically suppressed hemocyte-spreading behavior and nodule formation; these two reactions were also inhibited by aspirin, a COX inhibitor. PGE₂, but not arachidonic acid, treatment rescued the immunosuppression. Sequence analysis indicated that both POX genes were clustered with peroxinectin (Pxt) and their cognate proteins shared some conserved domains corresponding to the Pxt of Drosophila melanogaster.

Conclusions

SePOX-F and -H are Pxt-like genes associated with PG biosynthesis in S. exigua.     

Expression of POX2 gene and disruption of POX3 genes in the industrial Yarrowia lipolytica on the γ-decalactone production

The yeast Yarrowia lipolytica growing on methyl ricinoleate can produce γ-decalactone, the worthy aroma compound, which can exhibit fruity and creamy sensorial notes, and recognized internationally as a safe food additive. Unfortunately, the yield is poor because of lactone degradation by enzyme Aox3 (POX3 gene encoded), which was responsible for continuation of oxidation after C(10) level and lactone reconsumption. In this paper, we chose the industrial Y. lipolytica (CGMCC accession number 2.1405), which is the diploid strain as the starting strain and constructed the recombinant strain Tp-12 by targeting the POX3 locus of the wild type, one copy of POX3 was deleted by CRF1+POX2 insertion. The other recombinant strain Tpp-11, which was a null mutant possessing multiple copies of POX2 and disrupted POX3 genes on two chromosomes, was constructed by inserting XPR2+hpt into the other copy of POX3 of Tp-12. The growth ability of the recombinants was changed after genetic modification in the fermentation medium. The production of γ-decalactone was increased, resulting from blocking β-oxidation at the C(10) Aox level and POX2 overexpression. The recombinant strain Tpp-11 was stable. Because there was no reconsumption of γ-decalactone, the mutant strain could be grown in continuous fermentation of methyl ricinoleate to produce γ-decalactone.     

Role of beta-oxidation enzymes in gamma-decalactone production by the yeast Yarrowia lipolytica.

Some microorganisms can transform methyl ricinoleate into gamma-decalactone, a valuable aroma compound, but yields of the bioconversion are low due to (i) incomplete conversion of ricinoleate (C(18)) to the C(10) precursor of gamma-decalactone, (ii) accumulation of other lactones (3-hydroxy-gamma-decalactone and 2- and 3-decen-4-olide), and (iii) gamma-decalactone reconsumption. We evaluated acyl coenzyme A (acyl-CoA) oxidase activity (encoded by the POX1 through POX5 genes) in Yarrowia lipolytica in lactone accumulation and gamma-decalactone reconsumption in POX mutants. Mutants with no acyl-CoA oxidase activity could not reconsume gamma-decalactone, and mutants with a disruption of pox3, which encodes the short-chain acyl-CoA oxidase, reconsumed it more slowly. 3-Hydroxy-gamma-decalactone accumulation during transformation of methyl ricinoleate suggests that, in wild-type strains, beta-oxidation is controlled by 3-hydroxyacyl-CoA dehydrogenase. In mutants with low acyl-CoA oxidase activity, however, the acyl-CoA oxidase controls the beta-oxidation flux. We also identified mutant strains that produced 26 times more gamma-decalactone than the wild-type parents.     

红光,远红光和植物激素对水稻幼苗生长及CAT,POX活性的影响

红光(R)和远红光(Fr)都抑制水稻胚芽的生长,但对胚芽鞘来说,红光抑制其生长,远红光表现出部分逆转效应。一定浓度单一生长素(IAA)促进水稻胚芽鞘的生长,而赤霉素(GA_3)与生长素作用相反。对于水稻的过氧化氢酶(CAT)、过氧化物酶(POX),红光促进两种酶的活性,远红光则表现出逆转效应。单一10~(-2)ppmIAA、10~(-2)ppmGA_3都促进其活性。照光时,在10~(-2)ppm IAA存在的条件下,红光表现为促进,远红光则表现为抑制;但在10~(-2)ppm GA_3存在的条件下,红光反而对两种酶的活性有抑制作用,远红光则表现为促进作用。     

Functional consequences of PRODH missense mutations

PRODH maps to 22q11 in the region deleted in the velocardiofacial syndrome/DiGeorge syndrome (VCFS/DGS) and encodes proline oxidase (POX), a mitochondrial inner-membrane enzyme that catalyzes the first step in the proline degradation pathway. At least 16 PRODH missense mutations have been identified in studies of type I hyperprolinemia (HPI) and schizophrenia, 10 of which are present at polymorphic frequencies. The functional consequences of these missense mutations have been inferred by evolutionary conservation, but none have been tested directly. Here, we report the effects of these mutations on POX activity. We find that four alleles (R185Q, L289M, A455S, and A472T) result in mild (<30%), six (Q19P, A167V, R185W, D426N, V427M, and R431H) in moderate (30%-70%), and five (P406L, L441P, R453C, T466M, and Q521E) in severe (>70%) reduction in POX activity, whereas one (Q521R) increases POX activity. The POX encoded by one severe allele (T466M) shows in vitro responsiveness to high cofactor (flavin adenine dinucleotide) concentrations. Although there is limited information on plasma proline levels in individuals of known PRODH genotype, extant data suggest that severe hyperprolinemia (>800 microM) occurs in individuals with large deletions and/or PRODH missense mutations with the most-severe effect on function (L441P and R453C), whereas modest hyperprolinemia (300-500 microM) is associated with PRODH alleles with a moderate reduction in activity. Interestingly, three of the four alleles associated with or found in schizophrenia (V427M, L441P, and R453C) resulted in severe reduction of POX activity and hyperprolinemia. These observations plus the high degree of polymorphism at the PRODH locus are consistent with the hypothesis that reduction in POX function is a risk factor for schizophrenia.