A new regime of heme-dependent aromatic oxygenase superfamily.

Two histidine-ligated heme-dependent monooxygenase proteins, TyrH and SfmD, have recently been found to resemble enzymes from the dioxygenase superfamily currently named after tryptophan 2,3-dioxygenase (TDO), that is, the TDO superfamily. These latest findings prompted us to revisit the structure and function of the superfamily. The enzymes in this superfamily share a similar core architecture and a histidine-ligated heme. Their primary functions are to promote O-atom transfer to an aromatic metabolite. TDO and indoleamine 2,3-dioxygenase (IDO), the founding members, promote dioxygenation through a two-step monooxygenation pathway. However, the new members of the superfamily, including PrnB, SfmD, TyrH, and MarE, expand its boundaries and mediate monooxygenation on a broader set of aromatic substrates. We found that the enlarged superfamily contains eight clades of proteins. Overall, this protein group is a more sizeable, structure-based, histidine-ligated heme-dependent, and functionally diverse superfamily for aromatics oxidation. The concept of TDO superfamily or heme-dependent dioxygenase superfamily is no longer appropriate for defining this growing superfamily. Hence, there is a pressing need to redefine it as a heme-dependent aromatic oxygenase (HDAO) superfamily. The revised concept puts HDAO in the context of thiol-ligated heme-based enzymes alongside cytochrome P450 and peroxygenase. It will update what we understand about the choice of heme axial ligand. Hemoproteins may not be as stringent about the type of axial ligand for oxygenation, although thiolate-ligated hemes (P450s and peroxygenases) more frequently catalyze oxygenation reactions. Histidine-ligated hemes found in HDAO enzymes can likewise mediate oxygenation when confronted with a proper substrate.

Evolutionary aspects and enzymology of metazoan carotenoid cleavage oxygenases.

The carotenoids are terpenoid fat-soluble pigments produced by plants, algae, and several bacteria and fungi. They are ubiquitous components of animal diets. Carotenoid cleavage oxygenase (CCO) superfamily members are involved in carotenoid metabolism and are present in all kingdoms of life. Throughout the animal kingdom, carotenoid oxygenases are widely distributed and they are completely absent only in two unicellular organisms, Monosiga and Leishmania. Mammals have three paralogs 15,15'-ß-carotene oxygenase (BCO1), 9',10'-ß-carotene oxygenase (BCO2) and RPE65. The first two enzymes are classical carotenoid oxygenases: they cleave carbon­carbon double bonds and incorporate two atoms of oxygen in the substrate at the site of cleavage. The third, RPE65, is an unusual family member, it is the retinoid isomerohydrolase in the visual cycle that converts all-trans-retinyl ester into 11-cis-retinol. Here we discuss evolutionary aspects of the carotenoid cleavage oxygenase superfamily and their enzymology to deduce what insight we can obtain from their evolutionary conservation.

Exploring the codon patterns between CCD and NCED genes among different plant species.

Plant carotenoid cleavage oxygenase (CCO) is an enzyme which catalyzes carotenoids to apocarotenoid products that are involved in several vital physiological functions. The CCO exists in two forms, namely, CCD (Carotenoid Cleavage Dioxygenase) and NCED (Nine-Cis Epoxycarotenoid Dioxygenase). This paper relates to a comparative study on CCD and NCED genes through phylogeny and codon usage analysis. The result of the phylogenetic analysis indicates a closer relationship between CCD and NCED subclass genes, while the RSCU values indicate a high preference for CUC codon in both CCD and NCED gene families. The mean ENc value of NCED genes was found to be 48.76, suggesting a higher codon bias compared to CCD genes. However, the ENc-GC3S plot suggests that both the gene families are under mutational pressure with variations according to their species-specific role. Similarly, the multivariate analysis also suggests that nucleotide mutation bias influences codon usage. Correlation analysis of Axis I and codon adaptation index values indicate a significant correlation between critical indices. Even though the prominence of the variations in codon usage between the two gene families, they are exerted towards the time-specific functional requirement for that plant species. This is evident from the cleaving roles of these enzymes against various carotenoids at different growth stages. The result of this investigation indicates that CCD and NCED genes are under mutational pressure. This codon bias study paves the way for increasing the production of apocarotenoids, which have a great significance in the industry.

Structural basis for methylphosphonate biosynthesis.

Methylphosphonate synthase (MPnS) produces methylphosphonate, a metabolic precursor to methane in the upper ocean. Here, we determine a 2.35-angstrom resolution structure of MPnS and discover that it has an unusual 2-histidine-1-glutamine iron-coordinating triad. We further solve the structure of a related enzyme, hydroxyethylphosphonate dioxygenase from Streptomyces albus (SaHEPD), and find that it displays the same motif. SaHEPD can be converted into an MPnS by mutation of glutamine-adjacent residues, identifying the molecular requirements for methylphosphonate synthesis. Using these sequence markers, we find numerous putative MPnSs in marine microbiomes and confirm that MPnS is present in the abundant Pelagibacter ubique. The ubiquity of MPnS-containing microbes supports the proposal that methylphosphonate is a source of methane in the upper, aerobic ocean, where phosphorus-starved microbes catabolize methylphosphonate for its phosphorus.

Phylogenetic analysis of the metazoan carotenoid oxygenase superfamily: a new ancestral gene assemblage of BCO-like (BCOL) proteins.

Here we describe a new family of carotenoid cleavage oxygenases (CCOs) in metazoans, the BCO2-like (BCOL) clade, which contains lancelet, nematode, and molluscan carotenoid oxygenase sequences. Phylogenetic analysis of CCOs in all kingdoms of life confirmed that the BCOL enzymes are an independent clade of ancient origin. One of the predicted lancelet BCOL proteins, cloned and analyzed for carotenoid cleavage activity in a bacterial carotenoid expression system, had activity similar to lancelet BCO2 proteins, although with a preference for cis isomers. Our docking predictions correlated well with the cis-favored activity. The extensive expansions of the new animal BCOL family in some species (e.g., lancelet) suggests that the carotenoid cleavage oxygenase superfamily has evolved in the "extremely high turnover" fashion: numerous losses and duplications of this family are likely to reflect complex regulation processes during development, and interactions with the environment. These findings also serve to provide a rationale for the evolution of the BCO-related outlier RPE65 retinol isomerase, an enzyme that does not utilize carotenoids as substrate or perform double-bond cleavage.

A novel methanotroph in the genus Methylomonas that contains a distinct clade of soluble methane monooxygenase.

Aerobic methane oxidation is a key process in the global carbon cycle that acts as a major sink of methane. In this study, we describe a novel methanotroph designated EMGL16-1 that was isolated from a freshwater lake using the floating filter culture technique. Based on a phylogenetic analysis of 16S rRNA gene sequences, the isolate was found to be closely related to the genus Methylomonas in the family Methylococcaceae of the class Gammaproteobacteria with 94.2-97.4% 16S rRNA gene similarity to Methylomonas type strains. Comparison of chemotaxonomic and physiological properties further suggested that strain EMGL16-1 was taxonomically distinct from other species in the genus Methylomonas. The isolate was versatile in utilizing nitrogen sources such as molecular nitrogen, nitrate, nitrite, urea, and ammonium. The genes coding for subunit of the particulate form methane monooxygenase (pmoA), soluble methane monooxygenase (mmoX), and methanol dehydrogenase (mxaF) were detected in strain EMGL16-1. Phylogenetic analysis of mmoX indicated that mmoX of strain EMGL16-1 is distinct from those of other strains in the genus Methylomonas. This isolate probably represents a novel species in the genus. Our study provides new insights into the diversity of species in the genus Methylomonas and their environmental adaptations.

Bioinformatics Analysis of Phylogeny and Transcription of TAA/YUC Auxin Biosynthetic Genes.

Auxin is a main plant growth hormone crucial in a multitude of developmental processes in plants. Auxin biosynthesis via the tryptophan aminotransferase of arabidopsis (TAA)/YUCCA (YUC) route involving tryptophan aminotransferases and YUC flavin-dependent monooxygenases that produce the auxin indole-3-acetic acid (IAA) from tryptophan is currently the most researched auxin biosynthetic pathway. Previous data showed that, in maize and arabidopsis, TAA/YUC-dependent auxin biosynthesis can be detected in endoplasmic reticulum (ER) microsomal fractions, and a subset of auxin biosynthetic proteins are localized to the ER, mainly due to transmembrane domains (TMD). The phylogeny presented here for TAA/TAR (tryptophan aminotransferase related) and YUC proteins analyses phylogenetic groups as well as transmembrane domains for ER-membrane localisation. In addition, RNAseq datasets are analysed for transcript abundance of YUC and TAA/TAR proteins in Arabidopsis thaliana. We show that ER membrane localisation for TAA/YUC proteins involved in auxin biosynthesis is already present early on in the evolution of mosses and club mosses. ER membrane anchored YUC proteins can mainly be found in roots, while cytosolic proteins are more abundant in the shoot. The distribution between the different phylogenetic classes in root and shoot may well originate from gene duplications, and the phylogenetic groups detected also overlap with the biological function.

A novel enzyme with antioxidant capacity produced by the ubiquitous skin colonizer Propionibacterium acnes.

The role of the skin microbiota in human health is poorly understood. Here, we identified and characterized a novel antioxidant enzyme produced by the skin microbiota, designated RoxP for radical oxygenase of Propionibacterium acnes. RoxP is uniquely produced by the predominant skin bacterium P. acnes, with no homologs in other bacteria; it is highly expressed and strongly secreted into culture supernatants. We show that RoxP binds heme, reduces free radicals, and can protect molecules from oxidation. Strikingly, RoxP is crucial for the survival of P. acnes in oxic conditions and for skin colonization of P. acnes ex vivo. Taken together, our study strongly suggests that RoxP facilitates P. acnes' survival on human skin, and is an important beneficial factor for the host-commensal interaction. Thus, RoxP is the first described skin microbiota-derived mutualistic factor that potentially can be exploited for human skin protection.

Cloning of an anthocyanidin synthase gene homolog from blackcurrant (Ribes nigrum L.) and its expression at different fruit stages.

Anthocyanidin synthase (ANS), a 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase, catalyzes the penultimate step in anthocyanin biosynthesis, from leucoanthocyanidins to anthocyanidins, the first colored compound in the anthocyanin pathway. In this study, a full-length, 1427-bp long cDNA named RnANS1, which is homologous to the anthocyanidin synthase gene, was cloned from blackcurrant using a homologous cloning strategy. RnANS1 is highly homologous to other plant ANS genes at both the nucleotide and amino acid sequence levels. The deduced protein contains domains conserved in the 2OG and Fe(II)-dependent oxygenase, and is phylogenetically closely related to Paeonia suffruticosa and Paeonia lactiflora. The expression of RnANS1 was upregulated during fruit maturation, and correlated with the accumulation of anthocyanins and soluble carbohydrates in the fruit. Further characterization of the structure and expression patterns of RnANS1 will clarify our understanding of anthocyanin biosynthesis in blackcurrant, and support the development of molecular approaches to manipulate anthocyanin production in this plant.

Temporal and spatial regulation of anthocyanin biosynthesis provide diverse flower colour intensities and patterning in Cymbidium orchid.

MAIN CONCLUSION: This study confirmed pigment profiles in different colour groups, isolated key anthocyanin biosynthetic genes and established a basis to examine the regulation of colour patterning in flowers of Cymbidium orchid. Cymbidium orchid (Cymbidium hybrida) has a range of flower colours, often classified into four colour groups; pink, white, yellow and green. In this study, the biochemical and molecular basis for the different colour types was investigated, and genes involved in flavonoid/anthocyanin synthesis were identified and characterised. Pigment analysis across selected cultivars confirmed cyanidin 3-O-rutinoside and peonidin 3-O-rutinoside as the major anthocyanins detected; the flavonols quercetin and kaempferol rutinoside and robinoside were also present in petal tissue. ß-carotene was the major carotenoid in the yellow cultivars, whilst pheophytins were the major chlorophyll pigments in the green cultivars. Anthocyanin pigments were important across all eight cultivars because anthocyanin accumulated in the flower labellum, even if not in the other petals/sepals. Genes encoding the flavonoid biosynthetic pathway enzymes chalcone synthase, flavonol synthase, flavonoid 3' hydroxylase (F3'H), dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) were isolated from petal tissue of a Cymbidium cultivar. Expression of these flavonoid genes was monitored across flower bud development in each cultivar, confirming that DFR and ANS were only expressed in tissues where anthocyanin accumulated. Phylogenetic analysis suggested a cytochrome P450 sequence as that of the Cymbidium F3'H, consistent with the accumulation of di-hydroxylated anthocyanins and flavonols in flower tissue. A separate polyketide synthase, identified as a bibenzyl synthase, was isolated from petal tissue but was not associated with pigment accumulation. Our analyses show the diversity in flower colour of Cymbidium orchid derives not from different individual pigments but from subtle variations in concentration and pattern of pigment accumulation.

A mechanistic study on SMOB-ADP1: an NADH:flavin oxidoreductase of the two-component styrene monooxygenase of Acinetobacter baylyi ADP1.

Two styrene monooxygenase types, StyA/StyB and StyA1/StyA2B, have been described each consisting of an epoxidase and a reductase. A gene fusion which led to the chimeric reductase StyA2B and the occurrence in different phyla are major differences. Identification of SMOA/SMOB-ADP1 of Acinetobacter baylyi ADP1 may enlighten the gene fusion event since phylogenetic analysis indicated both proteins to be more related to StyA2B than to StyA/StyB. SMOB-ADP1 is classified like StyB and StyA2B as HpaC-like reductase. Substrate affinity and turnover number of the homo-dimer SMOB-ADP1 were determined for NADH (24 µM, 64 s(-1)) and FAD (4.4 µM, 56 s(-1)). SMOB-ADP1 catalysis follows a random sequential mechanism, and FAD fluorescence is quenched upon binding to SMOB-ADP1 (K d = 1.8 µM), which clearly distinguishes that reductase from StyB of Pseudomonas. In summary, this study confirmes made assumptions and provides phylogenetic and biochemical data for the differentiation of styrene monooxygenase-related flavin reductases.

Drug oxygenation activities mediated by liver microsomal flavin-containing monooxygenases 1 and 3 in humans, monkeys, rats, and minipigs.

Liver microsomal flavin-containing monooxygenases (FMO, EC 1.14.13.8) 1 and 3 were functionally characterized in terms of expression levels and molecular catalytic capacities in human, cynomolgus monkey, rat, and minipig livers. Liver microsomal FMO3 in humans and monkeys and FMO1 and FMO3 in rats and minipigs could be determined immunochemically with commercially available anti-human FMO3 peptide antibodies or rat FMO1 peptide antibodies. With respect to FMO-dependent N-oxygenation of benzydamine and tozasertib and S-oxygenation of methimazole and sulindac sulfide activities, rat and minipig liver microsomes had high maximum velocity values (Vmax) and high catalytic efficiency (Vmax/Km, Michaelis constant) compared with those for human or monkey liver microsomes. Apparent Km values for recombinantly expressed rat FMO3-mediated N- and S-oxygenations were approximately 10-100-fold those of rat FMO1, although these enzymes had similar Vmax values. The mean catalytic efficiencies (Vmax/Km, 1.4 and 0.4 min(-1)µM(-1), respectively) of recombinant human and monkey FMO3 were higher than those of FMO1, whereas Vmax/Km values for rat and minipig FMO3 were low compared with those of FMO1. Minipig liver microsomal FMO1 efficiently catalyzed N- and S-oxygenation reactions; in addition, the minipig liver microsomal FMO1 concentration was higher than the levels in rats, humans, and monkeys. These results suggest that liver microsomal FMO1 could contribute to the relatively high FMO-mediated drug N- and S-oxygenation activities in rat and minipig liver microsomes and that lower expression of FMO1 in human and monkey livers could be a determinant factor for species differences in liver drug N- and S-oxygenation activities between experimental animals and humans.

Metagenomic analysis of oxygenases from activated sludge.

Oxygenases play a key role in degradation of the aromatic compounds in the wastewater. This study explores the oxygenase coding gene sequences from the metagenome of activated biomass. Based on these results, the catabolic capacity of the activated sludge was assessed towards degradation of naphthalene, anthracene, phenol, biphenyl and o-toluidine. Oxygenases found in this study were compared with oxygenases from three other metagenome datasets. Results demonstrate that despite different geographical locations and source, many genes coding for oxygenases were common between treatment plants. 1, 2 Homogentisate dioxygenase and phenylacetate CoA oxygenases were present in all four metagenomes. Metagenomics provides a vast amount of data that needs to be mined with specific targets to harness the potential of the microbial world.

Phylogenetic analysis of putative genes involved in the tryptophan-dependent pathway of auxin biosynthesis in rice.

Plant proteome databases were mined for a flavin monooxygenase (YUCCA), tryptophan decarboxylase (TDC), nitrilase (NIT), and aldehyde oxidase (AO) enzymes that could be involved in the tryptophan-dependent pathway of auxin biosynthesis. Phylogenetic trees for enzyme sequences obtained were constructed. The YUCCA and TDC trees showed that these enzymes were conserved across the plant kingdom and therefore could be involved in auxin synthesis. YUCCAs branched into two clades. Most experimentally studied YUCCAs were found in the first clade. The second clade which has representatives from only seed plants contained Arabidopsis sequences linked to embryonic development. Therefore, sequences in this clade were suggested to be evolved with seed development. Examination of TDC activity and expression had previously linked this enzyme to secondary products synthesis. However, the phylogenetic finding of a conserved TDC clade across land plants suggested its essential role in plant growth. Phylogenetic analysis of AOs showed that plants inherited one AO. Recent gene duplication was suggested as AO sequences from each species were similar to each other rather than to AO from other species. Taken together and based on the experimental support of the involvement of AO in abscisic synthesis, AO was excluded as an intermediate in IAA production. Phylogenetic tree for NIT showed that the first clade contained sequences from species across the plant kingdom whereas the second branch contained sequences from only Brassicaceae. Even though NIT4 orthologues were conserved in the second clade, their major role seems to be detoxification of hydrogen cyanide rather than producing IAA.

Diversifying selection on flavanone 3-hydroxylase and isoflavone synthase genes in cultivated soybean and its wild progenitors.

Soybean isoflavone synthase (IFS) and flavanone 3-hydroxylase (F3H) are two key enzymes catalyzing the biosynthesis of isoflavonoids and flavonoids, both of which play diverse roles in stress responses. However, little is known about the evolutionary pattern of these genes in cultivated soybean and its wild progenitors. Herein, we investigated the nucleotide polymorphisms in Isoflavone synthase (IFS1, IFS2) and Flavanone 3-hydroxylase (F3H2) genes from 33 soybean accessions, including 17 cultivars (Glycine max) and 16 their wild progenitors (Glycine soja). Our data showed that the target genes shared the levels of nucleotide polymorphism with three reference genes involved in plant-microbe interactions, but possessed a much higher nucleotide polymorphism than other reference genes. Moreover, no significant genetic differentiation was found between cultivated soybean and its wild relatives in three target genes, despite of considering bottleneck and founder effect during domestication. These results indicate that IFS and F3H genes could have experienced gene introgressions or diversifying selection events during domestication process. Especially, F3H2 gene appears to evolve under positive selection and enjoy a faster evolutionary rate than IFS1 and IFS2 genes.

Catalytic properties and reaction mechanism of the CrtO carotenoid ketolase from the cyanobacterium Synechocystis sp. PCC 6803.

CrtW and CrtO are two distinct non-homologous ß-carotene ketolases catalyzing the formation of echinenone and canthaxanthin. CrtO belongs to the CrtI family which comprises carotene desaturases and carotenoid oxidases. The CrtO protein from Synechocystis sp. PCC 6803 has been heterologously expressed, extracted and purified. Substrate specificity has been determined in vitro. The enzyme from Synechocystis is basically a mono ketolase. Nevertheless, small amounts of diketo canthaxanthin can be formed. The poor diketolation reaction could be explained by the low relative turnover numbers for the mono keto echinenone. Also other carotenoids with an unsubstituted ß-ionone ring were utilized with low conversion rates by CrtO regardless of the substitutions at the other end of the molecule. The CrtO ketolase was independent of oxygen and utilized an oxidized quinone as co-factor. In common to CrtI-type desaturases, the first catalytic step involved hydride transfer to the quinone. The stabilization reaction of the resulting carbo cation was a reaction with OH(-) forming a hydroxy group. Finally, the keto group resulted from two subsequent hydroxylations at the same C-atom and water elimination. This reaction mechanism was confirmed by in vitro conversion of the postulated hydroxy intermediates and by their enrichment and identification as trace intermediates during ketolation.

Phylogenetic analysis reveals the surprising diversity of an oxygenase class.

As metalloenzymes capable of transforming a broad range of substrates with high stereo- and regio-specificity, the multicomponent Rieske oxygenases (ROs) have been studied in bacterial systems for applications in bioremediation and industrial biocatalysis. These studies include genetic and biochemical investigations, determination of enzyme structure, phylogenetic analysis, and enzyme classification. Although RO terminal oxygenase components (RO-Os) share a conserved domain structure, their sequences are highly divergent and present significant challenges for identification and classification. Herein, we present the first global phylogenetic analysis of a broad range of RO-Os from diverse taxonomic groups. We employed objective, structure-based criteria to significantly reduce the inclusion of erroneously aligned sequences in the analysis. Our findings reveal that RO biochemical studies to date have been largely concentrated in an unexpectedly narrow portion of the RO-O sequence landscape. Additionally, our analysis demonstrates the existence two distinct groups of RO-O sequences. Finally, the sequence diversity recognized in this study necessitates a new RO-O classification scheme. We therefore propose a P450-like naming system. Our results reveal a diversity of sequence and potential catalytic functionality that has been wholly unappreciated in the RO literature. This study also demonstrates that many commonly used bioinformatic tools may not be sufficient to analyze the vast amount of data available in current databases. These findings facilitate the expanded exploration of RO catalytic capabilities in both biological and technological contexts and increase the potential for practical exploitation of their activities.

From lipid transport to oxygenation of aromatic compounds: evolution within the Bet v1-like superfamily.

In absence of significant sequence similarity, remote homology between proteins can be confused with analogy and in such a case, shared ancestry can be inferred in light of certain unique and common features. In the present study, to understand the evolutionary origin of catalytic domain of large subunit of ring-hydroxylating oxygenases (RHOs), belonging to the Bet v1-like superfamily, structure-based phylogenies have been derived from structural alignment of representative proteins of the superfamily. A careful inspection of the structural relatedness of RHOs with the rest of the families showed closest similarity between RHO catalytic domain and PA1206-like protein. In addition, phylogenetic relationship of the Rieske domain of the large subunit of RHOs with functionally and structurally similar proteins has also been elucidated so as to postulate the most possible events leading to the genesis of the large subunit of RHOs.

Identification of a novel self-sufficient styrene monooxygenase from Rhodococcus opacus 1CP.

Sequence analysis of a 9-kb genomic fragment of the actinobacterium Rhodococcus opacus 1CP led to identification of an open reading frame encoding a novel fusion protein, StyA2B, with a putative function in styrene metabolism via styrene oxide and phenylacetic acid. Gene cluster analysis indicated that the highly related fusion proteins of Nocardia farcinica IFM10152 and Arthrobacter aurescens TC1 are involved in a similar physiological process. Whereas 413 amino acids of the N terminus of StyA2B are highly similar to those of the oxygenases of two-component styrene monooxygenases (SMOs) from pseudomonads, the residual 160 amino acids of the C terminus show significant homology to the flavin reductases of these systems. Cloning and functional expression of His(10)-StyA2B revealed for the first time that the fusion protein does in fact catalyze two separate reactions. Strictly NADH-dependent reduction of flavins and highly enantioselective oxygenation of styrene to (S)-styrene oxide were shown. Inhibition studies and photometric analysis of recombinant StyA2B indicated the absence of tightly bound heme and flavin cofactors in this self-sufficient monooxygenase. StyA2B oxygenates a spectrum of aromatic compounds similar to those of two-component SMOs. However, the specific activities of the flavin-reducing and styrene-oxidizing functions of StyA2B are one to two orders of magnitude lower than those of StyA/StyB from Pseudomonas sp. strain VLB120.

Abscisic acid levels in tomato ovaries are regulated by LeNCED1 and SlCYP707A1.

Although the hormones, gibberellin and auxin, are known to play a role in the initiation of fruits, no such function has yet been demonstrated for abscisic acid (ABA). However, ABA signaling and ABA responses are high in tomato (Solanum lycopersicum L.) ovaries before pollination and decrease thereafter (Vriezen et al. in New Phytol 177:60-76, 2008). As a first step to understanding the role of ABA in ovary development and fruit set in tomato, we analyzed ABA content and the expression of genes involved in its metabolism in relation to pollination. We show that ABA levels are relatively high in mature ovaries and decrease directly after pollination, while an increase in the ABA metabolite dihydrophaseic acid was measured. An important regulator of ABA biosynthesis in tomato is 9-cis-epoxy-carotenoid dioxygenase (LeNCED1), whose mRNA level in ovaries is reduced after pollination. The increased catabolism is likely caused by strong induction of one of four newly identified putative (+)ABA 8'-hydroxylase genes. This gene was named SlCYP707A1 and is expressed specifically in ovules and placenta. Transgenic plants, overexpressing SlCYP707A1, have reduced ABA levels and exhibit ABA-deficient phenotypes suggesting that this gene encodes a functional ABA 8'-hydroxylase. Gibberellin and auxin application have different effects on the LeNCED1 and SlCYP707A1 gene expression. The crosstalk between auxins, gibberellins and ABA during fruit set is discussed.