Cloning of the gene encoding avermectin B 5-O-methyltransferase in avermectin-producing Streptomyces avermitilis

Complementation of a mutant lacking avermectin B 5-O-methyltransferase (AveD) of Streptomyces avermitilis, which catalyses the methylation of the hydroxyl group at the C5 position of avermectin B compounds, revealed that the gene encoding AveD is in a 1.25-kb SalI–EcoNI fragment in the left region of the gene cluster for avermectin biosynthesis. The nucleotide sequence of this fragment predicted a 283-aa gene product homologous to several methyltransferases requiring S-adenosyl-l-methionine as a cofactor. After cloning of the aveD region from mutant not producing AveD, the complementation experiments were performed using a pair of hybrid fragments (AveD⁺/AveD⁻ and AveD⁻/AveD⁺). They suggest that the mutation(s) is in the N-terminus of AveD. SSCP analysis of amplified DNA of the aveD region derived from both wild type and mutant strains supports the results of the complementation experiments. Sequence analysis of the aveD region of the mutant strain revealed that a point mutation is within ORF, being Thr23→Ile substitution. This mutation causes the inactivation of O-methyltransferase activity of AveD.     

Modification of lignin biosynthesis in transgenic Nicotiana through expression of an antisense O-methyltransferase gene from Populus

An aspen lignin-specific O-methyltransferase (bi-OMT; S-adenosyl-l-methionine: caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase, EC 2.1.1.68) antisense sequence in the form of a synthetic gene containing the cauliflower mosaic virus 35S gene sequences for enhancer elements, promoter and terminator was stably integrated into the tobacco genome and inherited in transgenic plants with a normal phenotype. Leaves and stems of the transgenes expressed the antisense RNA and the endogenous tobacco bi-OMT mRNA was suppressed in the stems. Bi-OMT activity of stems was decreased by an average of 29% in the four transgenic plants analyzed. Chemical analysis of woody tissue of stems for lignin building units indicated a reduced content of syringyl units in most of the transgenic plants, which corresponds well with the reduced activity of bi-OMT. Transgenic plants with a suppressed level of syringyl units and a level of guaiacyl units similar to control plants were presumed to have lignins of distinctly different structure than control plants. We concluded that regulation of the level of bi-OMT expression by an antisense mechanism could be a useful tool for genetically engineering plants with modified lignin without altering normal growth and development.Abbreviations OMT O-methyltransferase - bi-OMT bispecific O-methyltransferase - CAD cinnamyl alcohol dehydrogenase - Ptomt1 Populus tremuloides bi-OMT cDNA clone     

Structure and expression of the lignin O-methyltransferase gene from Zea mays L.

The isolation and characterization of cDNA and homologous genomic clones encoding the lignin O-methyltransferase (OMT) from maize is reported. The cDNA clone has been isolated by differential screening of maize root cDNA library. Southern analysis indicates that a single gene codes for this protein. The genomic sequence contains a single 916 bp intron. The deduced protein sequence from DNA shares significant homology with the recently reported lignin-bispecific caffeic acid/5-hydroxyferulic OMTs from alfalfa and aspen. It also shares homology with OMTs from bovine pineal glands and a purple non-sulfur photosynthetic bacterium. The mRNA of this gene is present at different levels in distinct organs of the plant with the highest accumulation detected in the elongation zone of roots. Bacterial extracts from clones containing the maize OMT cDNA show an activity in methylation of caffeic acid to ferulic acid comparable to that existing in the plant extracts. These results indicate that the described gene encodes the caffeic acid 3-O-methyltransferase (COMT) involved in the lignin biosynthesis of maize.     

<Emphasis Type="Italic">SpnH</Emphasis> from <Emphasis Type="Italic">Saccharopolyspora spinosa</Emphasis> encodes a rhamnosyl 4′-<Emphasis Type="Italic">O</Emphasis>-methyltransferase for biosynthesis of the insecticidal macrolide,spinosyn A

Deoxysugar, 2′, 3′, 4′-tri-O-methylrhamnose is an essential structural component of spinosyn A and D, which are the active ingredients of the commercial insect control agent, Spinosad. The spnH gene, which was previously assigned as a rhamnose O-methyltransferase based on gene sequence homology, was cloned from the wild-type Saccharopolyspora spinosa and from a spinosyn K-producing mutant that was defective in the 4′-O-methylation of 2′, 3′-tri-O-methylrhamnose. DNA sequencing confirmed a mutation resulting in an amino acid substitution of G-165 to A-165 in the rhamnosyl 4′-O-methyltransferase of the mutant strain, and the subsequent sequence analysis showed that the mutation occurred in a highly conserved region of the translated amino acid sequence. Both spnH and the gene defective in 4′-O-methylation activity (spnH165A) were expressed heterologously in E. coli and were then purified to homogeneity using a His-tag affinity column. Substrate bioconversion studies showed that the enzyme encoded by spnH, but not spnH165A, could utilize spinosyn K as a substrate. When the wild-type spnH gene was transformed into the spinosyn K-producing mutant, spinosyn A production was restored. These results establish that the enzyme encoded by the spnH gene in wild-type S. spinosa is a rhamnosyl 4′-O-methyltransferase that is responsible for the final rhamnosyl methylation step in the biosynthesis of spinosyn A.     

Both caffeoyl Coenzyme A 3-<Emphasis Type="Italic">O</Emphasis>-methyltransferase 1 and caffeic acid <Emphasis Type="Italic">O</Emphasis>-methyltransferase 1 are involved in redundant functions for lignin,flavonoids and sinapoyl malate biosynthesis in Arabidopsis

Two methylation steps are necessary for the biosynthesis of monolignols, the lignin precursors. Caffeic acid O-methyltransferase (COMT) O-methylates at the C5 position of the phenolic ring. COMT is responsible for the biosynthesis of sinapyl alcohol, the precursor of syringyl lignin units. The O-methylation at the C3 position of the phenolic ring involves the Caffeoyl CoA 3-O-methyltransferase (CCoAOMT). The CCoAOMT 1 gene (At4g34050) is believed to encode the enzyme responsible for the first O-methylation in Arabidopsis thaliana. A CCoAOMT1 promoter-GUS fusion and immunolocalization experiments revealed that this gene is strongly and exclusively expressed in the vascular tissues of stems and roots. An Arabidopsis T-DNA null mutant named ccomt 1 was identified and characterised. The mutant stems are slightly smaller than wild-type stems in short-day growth conditions and has collapsed xylem elements. The lignin content of the stem is low and the S/G ratio is high mainly due to fewer G units. These results suggest that this O-methyltransferase is involved in G-unit biosynthesis but does not act alone to perform this step in monolignol biosynthesis. To determine which O-methyltransferase assists CCoAOMT 1, a comt 1 ccomt1 double mutant was generated and studied. The development of comt 1 ccomt1 is arrested at the plantlet stage in our growth conditions. Lignins of these plantlets are mainly composed of p-hydroxyphenyl units. Moreover, the double mutant does not synthesize sinapoyl malate, a soluble phenolic. These results suggest that CCoAOMT 1 and COMT 1 act together to methylate the C3 position of the phenolic ring of monolignols in Arabidopsis. In addition, they are both involved in the formation of sinapoyl malate and isorhamnetin.     

Immunolocalization of two ligninO-methyltransferases in stems of alfalfa (Medicago sativa L.)

Summary Caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT) catalyze parallel reactions that are believed to be involved in the biosynthesis of lignin monomers. Antisera specific for alfalfa (Medicago sativa L.) COMT or CCOMT were raised against the enzymes expressed inEscherichia coli, and were used for immunolocalization studies in lignifying alfalfa stem tissue. Both COMT and CCOMT were localized to xylem parenchyma cells, as assessed by light microscopy and immunocytochemistry. Electron microscopy revealed that both enzymes were located in the cytoplasm of xylem parenchyma cells, and to a lesser extent, in the cytoplasm of phloem cells. There was no significant difference in the localization pattern of COMT and CCOMT, suggesting that the two enzymes may be part of a metabolic grid leading to production of lignin monomers in lignifying tissue of mature alfalfa stem internodes.     

Organization of biosynthetic gene cluster for avermectin in Streptomyces avermitilis: analysis of enzymatic domains in four polyketide synthases

The analysis of the incorporation of ¹³C-labeled precursors into avermectins indicates that the avermectin aglycons are synthesized by head-to-tail condensation of various acyl groups, which is similar to the biosynthesis of other polyketides. Polyketide synthases (PKS) use the appropriate CoA ester as a primer and add acetate units from malonyl-CoA and propionate units from methylmalonyl-CoA to assemble the polyketides. Avermectin aglycons are formed by addition to the starter unit (2-methylbutyrate or isobutyrate) of 12 acyl condensations in the order P–A–A–A–A–P–P–A–P–A–P–A (P, propionyl; A, acetyl). Within the 90-kb gene cluster for avermectin biosynthesis, the central 65-kb segment was found to be required for aglycon biosynthesis by phenotypic analysis of strains containing deletion or insertion mutations in this region. A complete sequence analysis of the 65-kb segment indicated that this segment encodes avermectin PKS. The avermectin PKS genes are organized into two converging blocks of ORFs. From the results of sequencing analysis, a feature of the two regions, aveA1/aveA2 and avea3/aveA4, is that they encode four kinds of large multifunctional polypeptides containing 55 domains which possess putative fatty acid synthase-like activities. The avermectin PKS (AVES 1–4) appear to contain two, three, or four modules. AVES 1 and 2 contain two and four modules, respectively, whereas AVES 3 and AVES 4 each contains three modules. The 12 modules correspond to the 12 cycles required for synthesis of the avermectin aglycon. Journal of Industrial Microbiology & Biotechnology (2001) 27, 170–176. Received 21 September 1999/ Accepted in revised form 14 September 2000     

Improvement of in-rumen digestibility of alfalfa forage by genetic manipulation of lignin O-methyltransferases

Lignin inhibits forage digestibility by ruminant animals, and lignin levels and the proportion of dimethylated syringyl (S) lignin monomers increase with progressive maturity in stems of forage crops. We generated transgenic alfalfa (Medicago sativa L.) with reduced lignin content and altered lignin composition. Down-regulation of caffeic acid 3-O-methyltransferase (COMT) reduces lignin content, accompanied by near total loss of S lignin, whereas down-regulation of caffeoyl coenzyme A 3-O-methyltransferase (CCoAOMT) reduces lignin content without reduction in S lignin. These changes are not accompanied by altered ratios of cell wall polysaccharides. Analysis of rumen digestibility of alfalfa forage in fistulated steers revealed improved digestibility of forage from COMT down-regulated plants, but a greater improvement in digestibility following down-regulation of CCoAOMT. The results indicate that both lignin content and composition affect digestibility of alfalfa forage, and reveal a new strategy for forage quality improvement by genetic manipulation of CCoAOMT expression.     

Genetic engineering of Streptomyces bingchenggensis to produce milbemycins A3/A4 as main components and eliminate the biosynthesis of nanchangmycin

Milbemycins A3/A4 are important 16-membered macrolides which have been commercialized and widely used as pesticide and veterinary medicine. However, similar to other milbemycin producers, the production of milbemycins A3/A4 in Streptomyces bingchenggensis is usually accompanied with undesired by-products such as C5-O-methylmilbemycins B2/B3 (α-class) and β1/β2 (β-class) together with nanchangmycin. In order to obtain high yield milbemycins A3/A4-producing strains that produce milbemycins A3/A4 as main components, milD, a putative C5-O-methyltransferase gene of S. bingchenggensis, was biofunctionally investigated by heterologous expression in Escherichia coli. Enzymatic analysis indicated that MilD can catalyze both α-class (A3/A4) and β-class milbemycins (β11) into C5-O-methylmilbemycins B2/B3 and β1, respectively, suggesting little effect of furan ring formed between C6 and C8a on the C5-O-methylation catalyzed by MilD. Deletion of milD gene resulted in the elimination of C5-O-methylmilbemycins B2/B3 and β1/β2 together with an increased yield of milbemycins A3/A4 in disruption strain BCJ13. Further disruption of the gene nanLD encoding loading module of polyketide synthase responsible for the biosynthesis of nanchangmycin led to strain BCJ36 that abolished the production of nanchangmycin. Importantly, mutant strain BCJ36 (?milD?nanLD) produced milbemycins A3/A4 as main secondary metabolites with a yield of 2312?±?47 μg/ml, which was approximately 74 % higher than that of the initial strain S. bingchenggensis BC-109-6 (1326?±?37 μg/ml).     

基于比较代谢组学的理性优化方法提高阿维菌素产量

【目的】利用比较代谢组学的分析方法,研究不同发酵培养基中阿维链霉菌的胞内代谢差异,揭示合成阿维菌素的关键代谢物和代谢途径,再通过理性优化添加主要关键代谢物,提高阿维菌素产量。【方法】对M1和M2培养基中生长的菌体进行基于GC-MS的胞内代谢组学分析,通过理性添加强化前体代谢物,确定阿维菌素高产培养基。【结果】GC-MS共检测到232种物质,能够精确匹配70种胞内代谢物,通过PCA和PLS分析,最终确定了21种已知的胞内代谢物与阿维菌素的生物合成密切相关。其中乳酸、丙酮酸、琥珀酸、苏氨酸、异亮氨酸、缬氨酸和油脂类物质对阿维菌素的产量影响较为显著。通过单独或组合优化添加这些前体,阿维菌素的产量从5.36 g/L提高到了5.92 g/L,增加了10.4%。【结论】基于比较代谢组学分析的理性优化培养基的方法可有效提高阿维菌素的产量,并为提高当下生物基产品的产量提供了新思路。     

Identification of a gene cluster encoding meilingmycin biosynthesis among multiple polyketide synthase contigs isolated from<Emphasis Type="Italic"> Streptomyces nanchangensis</Emphasis> NS3226

A cluster encoding genes for the biosynthesis of meilingmycin, a macrolide antibiotic structurally similar to avermectin and milbemycin 11, was identified among seven uncharacterized polyketide synthase gene clusters isolated from Streptomyces nanchangensis NS3226 by hybridization with PCR products using primers derived from the sequences of aveE, aveF and a thioesterase domain of the avermectin biosynthetic gene cluster. Introduction of a 24.1-kb deletion by targeted gene replacement resulted in a loss of meilingmycin production, confirming that the gene cluster encodes biosynthesis of this important anthelminthic antibiotic compound. A sequenced 8.6-kb fragment had aveC and aveE homologues (meiC and meiE) linked together, as in the avermectin gene cluster, but the arrangement of aveF (meiF) and the thioesterase homologues differed. The results should pave the way to producing novel insecticidal compounds by generating hybrids between the two pathways.     

Limonoids from the fruits of Melia toosendan

Four (1–4) new and seven known limonoids were isolated from the EtOH extract of the fruits of Melia toosendan. The structures of the new compounds were established on the basis of spectroscopic methods to be 12-O-methyl-1-O-deacetylnimbolinin B (1), 12-O-methy-1-O-tigloyl-1-O-deacetylnimbolinin B (2), 12-O-ethylnimbolinin B (3), and 1-O-cinnamoyl-1-O-debenzoylohchinal (4). Additionally, two new tirucallane-type triterpenoids, named meliasenins S (5) and T (6), were obtained from the same fractions during purification of the limonoids.     

Reduced lignin in transgenic plants containing a caffeic acidO-methyltransferase antisense gene

Lignin is a major structural polymer of secondarily thickended plant vascular tissue and fibres, imparting mechanical strength to stems and trunks and hydrophobicity to conducting vessels. Constitutive expression of a lucerne caffeic acid 3-O-methyltransferase antisense RNA in transgenic tobacco leads to a significant reduction in lignin content, particularly in the younger parts of the stems, without apparent alterations in lignin monomer composition. These observations open up the possibility of genetically manipulating plants with reduced lignin for improved processing and biomass digestibility.     

Comparative macroarray analysis of morphine containing Papaver somniferum and eight morphine free Papaver species identifies an O-methyltransferase involved in benzylisoquinoline biosynthesis

Benzylisoquinoline alkaloids constitute a group of about 2,500 structures and are mainly produced by plants of the order Ranunculales. But only the opium poppy, Papaver somniferum, and Papaver setigerum are able to produce morphine. In this study, we started to investigate by gene expression analysis the molecular basis for this exceptional biosynthetic ability. A sequencing project from P. somniferum seedlings was initiated using a method based on the amplified fragment length polymorphism technique that resulted in 849 UniGenes. These cDNAs were analysed on macroarrays for differential expression between morphine-containing P. somniferum plants and eight other Papaver species, which accumulate other benzylisoquinolines instead of morphine. Three cDNAs showing increased expression in P. somniferum compared to all the other Papaver species were identified. Whereas two showed no significant homology to any known protein, one putatively encoded an O-methyltransferase. Analysis of substrate specificity of the heterologously expressed protein and mass spectrometric identification of the enzymatic products identified this protein as S-adenosyl-L-methionine:(R,S)-3-hydroxy-N-methylcoclaurine 4-O-methyltransferase (EC 2.1.1.116). Unlike other O-methyltransferases of different positional specificities implicated in benzylisoquinoline metabolism, the enzyme only accepted tetrahydroxylated tetrahydrobenzylisoquinolines as substrates; methylation was tolerated only at the 6-hydroxy position.     

Molecular cloning and functional expression of a stress-induced multifunctional O-methyltransferase with pinosylvin methyltransferase activity from Scots pine (Pinus sylvestris L.)

Formation of pinosylvin (PS) and pinosylvin 3-O-monomethyl ether (PSM), as well as the activities of stilbene synthase (STS) and S-adenosyl-l-methionine (SAM):pinosylvin O-methyltransferase (PMT), were induced strongly in needles of Scots pine seedlings upon ozone treatment, as well as in cell suspension cultures of Scots pine upon fungal elicitation. A SAM-dependent PMT protein was purified and partially characterised. A cDNA encoding PMT was isolated from an ozone-induced Scots pine cDNA library. Southern blot analysis of the genomic DNA suggested the presence of a gene family. The deduced protein sequence showed the typical highly conserved regions of O-methyltransferases (OMTs), and average identities of 20–56% to known OMTs. PMT expressed in Escherichia coli corresponded to that of purified PMT (40 kDa) from pine cell cultures. The recombinant enzyme catalysed the methylation of PS, caffeic acid, caffeoyl-CoA and quercetin. Several other substances, such as astringenin, resveratrol, 5-OH-ferulic acid, catechol and luteolin, were also methylated. Recombinant PMT thus had a relatively broad substrate specificity. Treatment of 7-year old Scots pine trees with ozone markedly increased the PMT mRNA level. Our results show that PMT represents a new SAM-dependent OMT for the methylation of stress-induced pinosylvin in Scots pine needles.     

Cloning and functional characterization of a caffeic acid O-methyltransferase from Trigonella foenum-graecum L

A cDNA encoding an O-methyltransferase (namely FGCOMT1) was identified from the medicinal plant Trigonella foenum-graecum L. The FGCOMT1 enzyme is a functional caffeic acid O-methyltransferase (COMT) and is localized in the cytosol. Kinetic analysis indicated that FGCOMT1 protein exhibited the highest catalyzing efficiency towards 5-hydroxy ferulic acid and caffeic acid as substrates, but did not possess the abilities to methylate either quercetin or tricetin in vitro. Furthermore, transformation of Arabidopsis loss-of-function Atomt1 mutant with a FGCOMT1 cDNA partially complements accumulation of sinapoyl derivatives but did not function to produce the major methylated flavonol isorhamnetin in seeds. The results from this study indicated that FGCOMT1 is a COMT with substrate preference to monomeric lignin precursors but is not involved in the flavonoid methylation in T. foenum-graecum L.     

Biotransformation of gallic acid by <Emphasis Type="Italic">Beauveria sulfurescens</Emphasis> ATCC 7159

Preparative-scale fermentation of gallic acid (3,4,5-trihydroxybenzoic acid) (1) with Beauveria sulfurescens ATCC 7159 gave two new glucosidated compounds, 4-(3,4-dihydroxy-6-hydroxymethyl-5-methoxy-tetrahydro-pyran-2-yloxy)-3-hydroxy-5-methoxy-benzoic acid (4), 3-hydroxy-4,5-dimethoxy-benzoic acid 3,4-dihydroxy-6-hydroxymethyl-5-methoxy-tetrahydro-pyran-2-yl ester (7), along with four known compounds, 3-O-methylgallic acid (2), 4-O-methylgallic acid (3), 3,4-O-dimethylgallic acid (5), and 3,5-O-dimethylgallic acid (6). The new metabolite genistein 7-O-β-D-4″-O-methyl-glucopyranoside (8) was also obtained as a byproduct due to the use of soybean meal in the fermentation medium. The structural elucidation of the metabolites was based primarily on 1D-, 2D-NMR, and HRFABMS analyses. Among these compounds, 2, 3, and 5 are metabolites of gallic acid in mammals. This result demonstrated that microbial culture parallels mammalian metabolism; therefore, B. sulfurescens might be a useful tool for generating mammalian metabolites of related analogs of gallic acid (1) for complete structural identification and for further use in investigating pharmacological and toxicological properties in this series of compounds. In addition, a GRE (glucocorticoid response element)-mediated luciferase reporter gene assay was used to initially screen for the biological activity of the 6 compounds, 2–6 and 8, along with 1 and its chemical O-methylated derivatives 9–13. Among the 12 compounds tested, 11–13 were found to be significant, but less active than the reference compounds of methylprednisolone and dexamethasone.     

An analysis of flavonoid compounds in leaves of Japonolirion (Petrosaviaceae)

Japonolirion, comprising Japonolirion osense Nakai, which occurs on serpentinite at two widely separated localities in Japan, has been considered as an isolated taxon, but more recently has been proved by molecular evidence to be a sister group to an achlorophyllous, mycoheterotrophic genus, Petrosavia. In an effort to research possible characters linking these groups, we analyzed the flavonoid compounds obtained from leaves of Japonolirion using UV spectra, mass spectrometry and ¹H and ¹³C nuclear magnetic resonance, and acid hydrolysis of the original glycosides as well as direct thin layer chromatography and high performance liquid chromatography comparisons with authentic specimens. As a result, we identified seven flavonoids, of which two were major components identified as 6-C-glucosylquercetin 3-O-glucoside and isoorientin. The remaining five were minor components identified as 6-C-glucosylkaempferol 3-O-glucoside, quercetin 3-O-glucoside, quercetin 3-O-arabinoside, vicenin-2 and orientin. Both 6-C-glucosylquercetin 3-O-glucoside and 6-C-glucosylkaempferol 3-O-glucoside were recorded for the first time in nature. Because of their restricted occurrence in angiosperms, both C-glycosylflavonols and 3-O-glycosides of C-glycosylflavonols may be significant chemical markers for assessing relationships of J. osense.     

The Lectin from the Crustacean Liocarcinus depurator Recognizes O-acetylsialic Acids

A lectin that recognized sialic acids and agglutinated mouse erythrocytes was purified from hemolymph of the crab Liocarcinus depurator. It consisted of 38-kDa subunits and had a pI about 6.0. The specificity of the lectin was assayed by hemagglutination inhibition. N-acetylneuraminic acid (Neu5Ac) was a good inhibitor and its N-acetyl group at C-5 was critical for lectin-ligand interaction. Substitution of the C-9 hydroxyl on Neu5Ac with an O-acetyl group (9-O-Ac-Neu5Ac) increased the inhibitory potency of this molecule. Furthermore, O-acetyl substitution of all the hydroxyl groups yielded even better inhibitors (2,4,7,8,9-O-Ac-Neu5Ac and its 1-O-methyl ester). Removal of the hydroxyl or O-acetyl group connected to C-2 reduced the potency of these inhibitors. The lectin agglutinated and stimulated human but not mouse lymphocytes. It was also inhibited by Escherichia coli (O111:B4) lipopolysaccharide and agglutinated specific gram-negative bacteria. In vitro labeling with [³⁵S]methionine indicated that the lectin was synthesized in hepatopangreas of L. depurator. Immunofluorescence showed that among hemocytes it localized mainly in the large-granule population.     

<Emphasis Type="Italic">Anthocyaninless1</Emphasis> gene of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> encodes a UDP-glucose:flavonoid-3-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase

We isolated several mutants of Arabidopsis thaliana (L.) Heynh. that accumulated less anthocyanin in the plant tissues, but had seeds with a brown color similar to the wild-type. These mutants were allelic with the anthocyaninless1 (anl1) mutant that has been mapped at 15.0 cM of chromosome 5. We performed fine mapping of the anl1 locus and determined that ANL1 is located between the nga106 marker and a marker corresponding to the MKP11 clone. About 70 genes are located between these two markers, including three UDP-glucose:flavonoid-3-O-glucosyltransferase-like genes and a glutathione transferase gene (TT19). A mutant of one of the glucosyltransferase genes (At5g17050) was unable to complement the anl1 phenotype, showing that the ANL1 gene encodes UDP-glucose:flavonoid-3-O-glucosyltransferase. ANL1 was expressed in all tissues examined, including rosette leaves, stems, flower buds and roots. ANL1 was not regulated by TTG1.