Molecular Elucidation of Two Novel Seed Specific Flavonoid Glycosyltransferases in Soybean

Flavonoids are specialized plant secondary metabolites that are mainly present as glycoconjugates and function as attractants to pollinators and symbionts, UV protectants, allelochemicals, and have antimicrobial and antiherbivore activity for plant health. Because of the heterogeneity of UDP-glycosyltransferases (UGTs) for glycosylation in plants, their function in flavonoid glycosylation remains largely unknown in soybean and other legumes, particularly that of the UGT92 genes. Here, we identified 152 putative UGT92 genes across 48 plant species and elucidated their mode of duplication, expansion/deletion pattern, alignment, phylogenetic analysis, and genome-wide distribution. Two novel UGT-encoding genes Glyma14g04790 (UGT92G1) and Glyma15g03670 (UGT92G2) were isolated from soybean and their heterologous expression was optimized in Escherichia. coli. Both genes exhibited catalytic activity toward quercetin, kaempferol, and myricetin, with UDPglucose as the sugar donor and were characterized as flavanol-specific UGTs. High expression of both UGTs was observed in adaxial and abaxial parenchyma, suspensor cells, and adaxial and abaxial epidermis cells during seed development, suggesting that they are seed-specific flavanol glycosyltransferases in soybean. Co-expression analysis of UGT92 genes with their first and second neighborhood genes provided a basis for their network elucidation in soybean. We provide valuable information on the role of UGT92 in seed development via the glycosylation of multiple flavanols and the potential metabolic engineering of flavonoid compounds in both plants and E. coli.     

Molecular Characterization and Phylogenetic Analysis of Two Novel Regio-specific Flavonoid Prenyltransferases from Morus alba and Cudrania tricuspidata

Prenylated flavonoids are attractive specialized metabolites with a wide range of biological activities and are distributed in several plant families. The prenylation catalyzed by prenyltransferases represents a Friedel-Crafts alkylation of the flavonoid skeleton in the biosynthesis of natural prenylated flavonoids and contributes to the structural diversity and biological activities of these compounds. To date, all identified plant flavonoid prenyltransferases (FPTs) have been identified in Leguminosae. In the present study two new FPTs, Morus alba isoliquiritigenin 3′-dimethylallyltransferase (MaIDT) and Cudrania tricuspidata isoliquiritigenin 3′-dimethylallyltransferase (CtIDT), were identified from moraceous plants M. alba and C. tricuspidata, respectively. MaIDT and CtIDT shared low levels of homology with the leguminous FPTs. MaIDT and CtIDT are predicted to be membrane-bound proteins with predicted transit peptides, seven transmembrane regions, and conserved functional domains that are similar to other homogentisate prenyltransferases. Recombinant MaIDT and CtIDT were able to regioselectively introduce dimethylallyl diphosphate into the A ring of three flavonoids with different skeleton types (chalcones, isoflavones, and flavones). Phylogenetic analysis revealed that MaIDT and CtIDT are distantly related to their homologs in Leguminosae, which suggests that FPTs in Moraceae and Leguminosae might have evolved independently. MaIDT and CtIDT represent the first two non-Leguminosae FPTs to be identified in plants and could thus lead to the identification of additional evolutionarily varied FPTs in other non-Leguminosae plants and could elucidate the biosyntheses of prenylated flavonoids in various plants. Furthermore, MaIDT and CtIDT might be used for regiospecific prenylation of flavonoids to produce bioactive compounds for potential therapeutic applications due to their high efficiency and catalytic promiscuity.     

Dynamics of Seed Protein Biosynthesis in Two Soybean Genotypes Differing in Drought Susceptibility

The dynamics of seed storage protein biosynthesis was studied under field conditions during two vegetative seasons. Two soybean (Glycine max L. Merr.) genotypes were examined: BOSA (drought tolerant) and L 121 (drought susceptible). Seed samples were taken from plants at three stages of seed maturation (50 and 70 d after flowering, and at full maturity). The earlier synthesis of the -subunit of the 7S protein occurred in the drought susceptible cultivar. We have not found such differences in the synthesis of the - and -subunits of the 7S protein. Our results did not confirm significant genotypic differences in protein composition of the mature seeds between the cultivars studied, but have pointed out to the differences in the dynamics of protein biosynthesis during seed maturation and desiccation.     

Elucidation of Molecular Identity of the W3 Locus and Its Implication in Determination of Flower Colors in Soybean

The wide range of flower colors in soybean is controlled by six independent loci (W1, W2, W3, W4, Wm, and Wp). Among these loci, mutations in the W3 locus under the w4 allelic background (i.e., w3w4) produce near-white flowers, while the W3w4 genotype produces purple throat flowers. Although a gene encoding dihydroflavonol 4-reductase, DFR1, has been known to be closely associated with the W3 locus, its molecular identity has not yet been characterized. In the present study, we aimed to determine whether DFR1 is responsible for allelic variations in the W3 locus. On the basis of the sequence of a DFR probe, Glyma.14G072700 was identified as a candidate gene for DFR1, and nucleotide sequences of Glyma.14G072700 from cultivars with previously validated genotypes for the W3 locus were determined. As a result, a number of nucleotide polymorphisms, mainly single-base substitutions, between both coding and 5′-upstream region sequences of the W3 and w3 alleles were identified. Among them, an indel of 311-bp in the 5′-upstream region was noteworthy, since the Glyma.14G072700 in all the w3 alleles examined contained the indel, whereas that in all the W3 alleles did not; the former was barely expressed, but the latter was well expressed. These results suggest that Glyma.14G072700 is likely to correspond to DFR1 for the W3 locus and that its expression patterns may lead to allelic color phenotypes of W3 and w3 alleles under the w4 allelic background.     

The Elucidation of the Structure of Thermotoga maritima Peptidoglycan Reveals Two Novel Types of Cross-link

Thermotoga maritima is a Gram-negative, hyperthermophilic bacterium whose peptidoglycan contains comparable amounts of l- and d-lysine. We have determined the fine structure of this cell-wall polymer. The muropeptides resulting from the digestion of peptidoglycan by mutanolysin were separated by high-performance liquid chromatography and identified by amino acid analysis after acid hydrolysis, dinitrophenylation, enzymatic determination of the configuration of the chiral amino acids, and mass spectrometry. The high-performance liquid chromatography profile contained four main peaks, two monomers, and two dimers, plus a few minor peaks corresponding to anhydro forms. The first monomer was the d-lysine-containing disaccharide-tripeptide in which the d-Glu-d-Lys bond had the unusual γ→ϵ arrangement (GlcNAc-MurNAc-l-Ala-γ-d-Glu-ϵ-d-Lys). The second monomer was the conventional disaccharide-tetrapeptide (GlcNAc-MurNAc-l-Ala-γ-d-Glu-l-Lys-d-Ala). The first dimer contained a disaccharide-l-Ala as the acyl donor cross-linked to the α-amine of d-Lys in a tripeptide acceptor stem with the sequence of the first monomer. In the second dimer, donor and acceptor stems with the sequences of the second and first monomers, respectively, were connected by a d-Ala⁴-α-d-Lys³ cross-link. The cross-linking index was 10 with an average chain length of 30 disaccharide units. The structure of the peptidoglycan of T. maritima revealed for the first time the key role of d-Lys in peptidoglycan synthesis, both as a surrogate of l-Lys or meso-diaminopimelic acid at the third position of peptide stems and in the formation of novel cross-links of the l-Ala¹(α→α)d-Lys³ and d-Ala⁴(α→α)d-Lys³ types.Peptidoglycan (or murein) is a giant macromolecule whose main function is the protection of the cytoplasmic membrane against the internal osmotic pressure. It is composed of alternating residues of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc)² cross-linked by short peptides (1). The composition of the peptide stem in nascent peptidoglycan is l-Ala¹-γ-d-Glu²-X³-d-Ala⁴-d-Ala⁵, where X is most often meso-diaminopimelic acid (meso-A₂pm) or l-lysine in Gram-negative and Gram-positive species, respectively (2, 3). In the mature macromolecule, the last d-Ala residue is removed. Cross-linking of the glycan chains generally occurs between the carboxyl group of d-Ala at position 4 of a donor peptide stem and the side-chain amino group of the diamino acid at position 3 of an acceptor peptide stem (4→3 cross-links). Cross-linking is either direct or through a short peptide bridge such as pentaglycine in Staphylococcus aureus (2, 3). The enzymes for the formation of the 4→3 cross-links are active-site serine dd- transpeptidases that belong to the penicillin-binding protein (PBP) family and are the essential targets of β-lactam antibiotics in pathogenic bacteria (4). Catalysis involves the cleavage of the d-Ala⁴-d-Ala⁵ bond of a donor peptide stem and the formation of an amide bond between the carboxyl of d-Ala⁴ and the side chain amine at the third position of an acceptor stem. Transpeptidases of the ld specificity are active-site cysteine enzymes that were shown to act as surrogates of the PBPs in mutants of Enterococcus faecium resistant to β-lactam antibiotics (5). They cleave the X³-d-Ala⁴ bond of a donor stem peptide to form 3→3 cross-links. This alternate mode of cross-linking is usually marginal, although it has recently been shown to predominate in non-replicative “dormant” forms of Mycobacterium tuberculosis (6).Thermotoga maritima is a Gram-negative, extremely thermophilic bacterium isolated from geothermally heated sea floors by Huber et al. (7). A morphological characteristic is the presence of an outer sheath-like envelope called “toga.” Although the organism has received considerable attention for its biotechnological potential, studies about its peptidoglycan are scarce (8–11), and in particular the fine structure of the macromolecule is still unknown. In their initial work, Huber et al. (7) showed that the composition of its peptidoglycan was unusual for a Gram-negative species, because it contained both isomers of lysine and no A₂pm. Recently, we purified and studied the properties of T. maritima MurE (12); this enzyme is responsible for the addition of the amino acid residue at position 3 of the peptide stem (13, 14). We demonstrated that T. maritima MurE added in vitro l- and d-Lys to UDP-MurNAc-l-Ala-d-Glu. Although l-Lys was added in the usual way, yielding the conventional nucleotide UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys containing a d-Glu(γ→α)l-Lys amide bond, the d-isomer was added in an “upside-down” manner, yielding the novel nucleotide UDP-MurNAc-l-Ala-d-Glu(γ→ϵ)d-Lys. We also showed that the d-Lys-containing nucleotide was not a substrate for T. maritima MurF, the subsequent enzyme in the biosynthetic pathway, whereas this ligase catalyzed the addition of dipeptide d-Ala-d-Ala to the l-Lys-containing tripeptide, yielding the conventional UDP-MurNAc-pentapeptide (12).However, both the l-Lys-containing UDP-MurNAc-pentapeptide and d-Lys-containing UDP-MurNAc-tripeptide were used as substrates by T. maritima MraY with comparable efficiencies in vitro (12). This observation implies that the unusual d-Lys-containing peptide stems are likely to be translocated to the periplasmic face of the cytoplasmic membrane and to participate in peptidoglycan polymerization. Therefore, we have determined here the fine structure of T. maritima peptidoglycan and we have shown that l-Lys- and d-Lys-containing peptide stems are both present in the polymer, the latter being involved in the formation of two novel types of peptidoglycan cross-link.     

A Genomewide Scan Identifies Two Novel Loci Involved in Specific Language Impairment

Approximately 4% of English-speaking children are affected by specific language impairment (SLI), a disorder in the development of language skills despite adequate opportunity and normal intelligence. Several studies have indicated the importance of genetic factors in SLI; a positive family history confers an increased risk of development, and concordance in monozygotic twins consistently exceeds that in dizygotic twins. However, like many behavioral traits, SLI is assumed to be genetically complex, with several loci contributing to the overall risk. We have compiled 98 families drawn from epidemiological and clinical populations, all with probands whose standard language scores fall 1.5 SD below the mean for their age. Systematic genomewide quantitative-trait–locus analysis of three language-related measures (i.e., the Clinical Evaluation of Language Fundamentals–Revised [CELF-R] receptive and expressive scales and the nonword repetition [NWR] test) yielded two regions, one on chromosome 16 and one on 19, that both had maximum LOD scores of 3.55. Simulations suggest that, of these two multipoint results, the NWR linkage to chromosome 16q is the most significant, with empirical P values reaching 10⁻⁵, under both Haseman-Elston (HE) analysis (LOD score 3.55; P=.00003) and variance-components (VC) analysis (LOD score 2.57; P=.00008). Single-point analyses provided further support for involvement of this locus, with three markers, under the peak of linkage, yielding LOD scores >1.9. The 19q locus was linked to the CELF-R expressive-language score and exceeds the threshold for suggestive linkage under all types of analysis performed—multipoint HE analysis (LOD score 3.55; empirical P=.00004) and VC (LOD score 2.84; empirical P=.00027) and single-point HE analysis (LOD score 2.49) and VC (LOD score 2.22). Furthermore, both the clinical and epidemiological samples showed independent evidence of linkage on both chromosome 16q and chromosome 19q, indicating that these may represent universally important loci in SLI and, thus, general risk factors for language impairment.     

Identification of Putative Rhamnogalacturonan-II Specific Glycosyltransferases in Arabidopsis Using a Combination of Bioinformatics Approaches

Rhamnogalacturonan-II (RG-II) is a complex plant cell wall polysaccharide that is composed of an α(1,4)-linked homogalacturonan backbone substituted with four side chains. It exists in the cell wall in the form of a dimer that is cross-linked by a borate di-ester. Despite its highly complex structure, RG-II is evolutionarily conserved in the plant kingdom suggesting that this polymer has fundamental functions in the primary wall organisation. In this study, we have set up a bioinformatics strategy aimed at identifying putative glycosyltransferases (GTs) involved in RG-II biosynthesis. This strategy is based on the selection of candidate genes encoding type II membrane proteins that are tightly coexpressed in both rice and Arabidopsis with previously characterised genes encoding enzymes involved in the synthesis of RG-II and exhibiting an up-regulation upon isoxaben treatment. This study results in the final selection of 26 putative Arabidopsis GTs, including 10 sequences already classified in the CAZy database. Among these CAZy sequences, the screening protocol allowed the selection of α-galacturonosyltransferases involved in the synthesis of α4-GalA oligogalacturonides present in both homogalacturonans and RG-II, and two sialyltransferase-like sequences previously proposed to be involved in the transfer of Kdo and/or Dha on the pectic backbone of RG-II. In addition, 16 non-CAZy GT sequences were retrieved in the present study. Four of them exhibited a GT-A fold. The remaining sequences harbored a GT-B like fold and a fucosyltransferase signature. Based on homologies with glycosyltransferases of known functions, putative roles in the RG-II biosynthesis are proposed for some GT candidates.     

Erratum to: Methods for Molecular Evolution of Polymerases

Russian Journal of Bioorganic Chemistry - erratum     

Functional Characterization of a Glucosyltransferase Specific to Flavonoid 7-O-Glucosides from Withania somnifera

Flavonoids are a large class of phenylpropanoid-derived secondary metabolites, which are usually glycosylated by UDP-glycosyltransferases with one or more sugar groups. Here, we report the cloning and biochemical characterization of a flavonoid glycosyltransferase gene from Withania somnifera (WsGT), which is an important medicinal plant used in Ayurvedic formulations. Using PCR primers, designed for a highly conserved region of previously reported glycosyltransferases, we were able to isolate the corresponding fragment of the WsGT gene. Rapid amplification of cDNA ends (RACE) was then employed to isolate full-length cDNA, which had an open reading frame of 1,371 bp that encode for 456 amino acids. Phylogenetic analysis indicated that WsGT was similar to that of family 1 GT-B glycosyltransferase. Biochemical analysis revealed that WsGT interacts with UDP-glucose and was capable of regiospecifically glycosylating flavonoid-7-ols, such as apigenin, naringenin, luteolin, diadzein and genistein. Expression profiling studies showed that WsGT was highly expressed in young and mature leaves of W. somnifera. Furthermore, exposure to salicylic acid enhanced the expression of WsGT in the leaves and heat shock treatment resulted in decreased expression of WsGT after an initial increase. This may suggest the role of WsGT in response to abiotic/biotic stresses.     

Elucidation of the Flavonoid Catabolism Pathway in Pseudomonas putida PML2 by Comparative Metabolic Profiling

Flavonoids are 15-carbon plant secondary metabolites exuded in the rhizosphere that hosts several flavonoid-degrading bacteria. We studied flavonoid catabolism in a plant growth-promoting rhizobacterial strain of Pseudomonas by using a combination of biochemical and genetic approaches. Transposants carrying mini-Tn5gfp insertions were screened for flavonoid auxotrophy, and these mutant strains were found to be unable to grow in the flavonols naringenin and quercetin, while their growth in glycerol was comparable to that of the parental strain. In order to understand flavonoid catabolism, culture supernatants, whole-cell fractions, cell lysate, and cell debris of the wild-type and mutant strains were analyzed. Intermediates that accumulated intracellularly and those secreted in the medium were identified by a combination of reversed-phase high-pressure liquid chromatography and electrospray ionization-mass spectrometry. Structures of four key intermediates were confirmed by one-dimensional nuclear magnetic resonance spectroscopy. Comparative metabolic profiling of the compounds in the wild-type and mutant strains allowed us to understand the degradation events and to identify six metabolic intermediates. The first step in the pathway involves 3,3′-didehydroxylation, followed by hydrolysis and cleavage of the C-ring, leading via subsequent oxidations to the formation of protocatechuate. This is the first report on quercetin dehydroxylation in aerobic conditions leading to naringenin accumulation.     

The Susceptibility of Soybean Seed Lipids to Artificially-Enhanced Atmospheric Oxidation

Priestley, D. A., Werner, B. G. and Leopold, A. C. 1985. Thesusceptibility of soybean seed lipids to artificially-enhancedatmospheric oxidation.—J. exp. Bot. 36: 1653–1659. As a model system for studying possible oxidation changes insoybeans with ageing, whole soybean seeds, ground soybeans orsoybean oil were exposed to a heated oxygen atmosphere (105°C)for periods of up to 6 d. With the exception of polar lipidsof the embryonic axis, seed lipids were highly resistant tooxidative degradation provided seed structure was maintainedintact; however, the non-lipid fraction of the seed rapidlybecame discoloured. Polar lipids of ground seed material, andboth total and polar lipids in isolated oil, were less stableto oxidation than similar lipids within whole seeds. These resultsindicate that seed organization protects the lipid componentsfrom atmospheric autoxidation. Key words: Soybean, seed lipids, oxidation     

Molecular Cloning of Cyanobacterial Pteridine Glycosyltransferases That Catalyze the Transfer of either Glucose or Xylose to Tetrahydrobiopterin

Here, we report cloning of cyanobacterial genes encoding pteridine glycosyltransferases that catalyze glucosyl or xylosyl transfer from UDP-sugars to tetrahydrobiopterin. The genes were cloned by PCR amplification from genomic DNA which was isolated from culture and environmental samples and overexpressed in Escherichia coli for an in vitro activity assay.Tetrahydrobiopterin (BH4) is well known among pteridine compounds as a cofactor for aromatic amino acid hydroxylases and nitric oxide synthases in animals (19). Pteridine glycosides such as biopterin and 6-hydroxymethylpterin glycosides have been found in cyanobacteria and anaerobic photosynthetic bacteria (2, 4, 5, 8, 11, 13, 15, 17, 21). Although the function of these glycosides remains unknown, they are abundant and ubiquitous in cyanobacteria, implying some essential role (3, 6, 16-18, 21). There is a group of enzymes, named pteridine glycosyltransferases (PGTs), known to catalyze a variety of glycosyl transfers to pteridines. The first PGT isolated from the cyanobacterium Synechococcus sp. PCC 7942 was shown to catalyze a glucosyl transfer from UDP-glucose to BH4 and was therefore named UDP-glucose:BH4 glucosyltransferase (BGluT) (7). After cloning of the gene encoding BGluT (6), a PGT that catalyzes the transfer of glucuronic acid for cyanopterin synthesis was identified (12). In addition, there are many putative PGT homologs encoded in bacterial genomes, although their exact catalytic functions have not been determined. We recently found that BGluT is useful for the simultaneous detection of oxidized and reduced forms of BH4 in animal samples (14). Glycosyltransferases are also being studied intensively for applications in the design of novel pharmaceutical derivatives (1, 10). We were thus encouraged to find PGTs with new substrate specificities or enzymatic properties not only for study of protein structure and function but also for application in BH4 research. In this study, we succeeded in cloning four cyanobacterial genes encoding PGTs with either glucosyl- or xylosyltransferase activity, and here we report the results.PGT genes were cloned from Arthrospira platensis CY-007 (obtained through Hawaii Oceanic Institute sampling) and Arthrospira maxima CY-049 (UTEX 2342), which were cultured in the Korea Marine Microalgae Culture Center, and from environmental DNA sequences (designated UCNR-001 and UCNR-002) isolated from wild algal mats in the Nakdong River, South Korea. In order to amplify conserved internal sequences of the unknown PGT genes, degenerate PCR primers were designed from the nucleotide sequences of cyanobacterial PGT homologs using GeneFisher2 (9). A protein homology search with BGluT against the bacterial genome database in NCBI revealed more than a hundred PGT homologs. When a phylogenetic tree was constructed from the putative sequences, there was a separate group comprising cyanobacterial PGTs. Figure ​Figure11 shows the cyanobacterial cluster, in which members shared sequence identities of more than 34%. Because the degenerate primers designed from all of the cyanobacterial PGTs were too highly degenerate, the cluster was divided into four subgroups, as shown in Fig. ​Fig.1:1: this division allowed primers to be designed for each of the four subgroups. The PGTs in subgroup I were clearly distinguishable from the others, because they all originated from marine picocyanobacteria, which are abundant in the pelagic realm. Subgroup I could be divided further into two groups comprising PGTs from either Prochlorococcus species (CIA) or marine Synechococcus species (CIB). Subgroup II was also divided into two groups, CIIA, consisting mostly of PGTs from Synechococcus species, and CIIB, containing the other PGTs. Among the primers designed for each subgroup, those for the CIIA and CIIB subgroups successfully amplified DNA sequences of the expected sizes. The primer sequences were 5′-GTTCAGGAWTAGGAGGTGGAGT-3′ (CIIA-forward)/5′-CGCYTCAATWGCTACATTTCCA-3′ (CIIA-reverse) and 5′-ACGACTGGCTMYCGYTTTAYCTGA-3′ (CIIB-forward)/5′-GCYTCCACCCAYTTRGGGGTCA-3′ (CIIB-reverse). Based on the determined partial gene sequences, additional sets of primer pairs were designed for the inverse PCR method (20). The sequences were 5′-GATGAACTACAACAGGGTCTGCGTC-3′ (CY-007 forward)/5′-CGGCTTTTTAAGGCTTTTGCCATATTC-3′ (CY-007 reverse), 5′-GTCTGCGTGAATGTCGAGG-3′ (CY-047 forward)/5′-ATGACCTCGGCTGTGTAAG-3′ (CY-047 reverse), and 5′-CCTACAAAAAGAGCTAGGCGACTGTTTTG-3′ (UCNR forward)/5′-CCAAAGAAACGGAAGCCATGCTG-3′ (UCNR reverse). Total genomic DNA samples were partially digested with RsaI and then self-ligated to be used as templates for PCR amplification with the primer pairs. The amplified DNA sequences revealed the missing 5′- and 3′-end sequences of the genes.Open in a separate windowFIG. 1.Neighbor-joining phylogenetic tree of cyanobacterial PGT protein sequences, identified by NCBI accession numbers. Bootstrap values are presented at the nodes. The names of strains whose PGTs are characterized are in bold.The deduced protein sequences were multiply aligned with BGluT (Fig. ​(Fig.2).2). Amino acid identities for all sequences in pairwise comparisons are given as percentages in Fig. ​Fig.2.2. Recently, draft assemblies of the genome sequences of Arthrospira platensis strain Paraca and Arthrospira maxima CS-328 (UTEX 2342) were announced. The annotated PGT (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"EDZ91868","term_id":"209491491","term_text":"EDZ91868"}}EDZ91868) of Arthrospira maxima CS-328 was identical to the PGT of CY-049 at both the amino acid and nucleotide levels, proving that the two organisms originated from the same UTEX stock (UTEX 2342). On the other hand, the PGTs of Arthrospira platensis strains Paraca and CY-007 were different at nine individual nucleotides, resulting in seven amino acid differences. A phylogenetic analysis showed that CY-007 and CY-049 PGTs belonged to the CIIB subgroup and that UCNR-001 and UCNR-002 PGTs clustered in the CIIA subgroup (data not shown).Open in a separate windowFIG. 2.Alignment of multiple PGT sequences. Conserved sequences are shaded at four levels using GeneDoc software. At the end of the alignment, amino acid identities in percentages are given for all sequences in pairwise comparisons.In order to identify the catalytic function of the putative PGTs, the recombinant proteins were produced in Escherichia coli. The complete open reading frame (ORF) sequences were amplified by PCR from the genomic DNA samples, cloned into the pGEM-T vector, and subsequently cloned as NdeI/BamHI restriction fragments into pET-28b (for CY-007 and CY-049 sequences) or pET-15b (for UCNR-001 and UCNR-002 sequences). E. coli BL21(DE3)/pLysS transformants were induced with 0.05 to 0.2 mM isopropyl-β-d-thiogalactopyranoside and were cultured for 8 h at 22°C. The recombinant proteins were purified by chromatography on Ni-nitrilotriacetic acid gel according to the instructions of the manufacturer (Qiagen). The proteins were eluted with 250 mM imidazole, dialyzed against a mixture of 20 mM Tris-HCl (pH 7.5) and 30% (vol/vol) glycerol, and stored in aliquots at −70°C until use. Purification of the proteins was confirmed by electrophoresis on an SDS-polyacrylamide gel (Fig. ​(Fig.3A).3A). BGluT from a previous purification was used (6). Aliquots of PGT were assayed at 37°C for 10 min in a reaction mixture of 100 μl containing 50 mM sodium phosphate, pH 7.5, 10 mM MnCl₂, 0.2% ascorbic acid, 1 μM BH4 (Schircks Lab, Switzerland), and 100 μM UDP-glucose or UDP-xylose. The reaction mixture was combined with an equal volume of acidic iodine solution (2% KI and 1% I₂ in 1 N HCl) for 1 h in the dark. After centrifugation, the supernatant was mixed in a 10:1 volume ratio with 5% ascorbic acid and subjected to high-performance liquid chromatography (HPLC). HPLC was performed with a Gilson 321 pump equipped with an Inertsil ODS-3 column (150 by 2.3 mm; particle size, 5 μm [GL Science, Japan]) and a fluorescence detector (Shimadzu RF-10AXL). Pteridines were eluted with 10 mM potassium phosphate buffer (pH 6.0) at a flow rate of 1.2 ml/min and were monitored at excitation and emission wavelengths of 350 and 450 nm, respectively.Open in a separate windowFIG. 3.Analysis of purified recombinant PGTs on an SDS-12.5% polyacrylamide gel (A) and HPLC analysis of the enzymatic products (B).The enzymatic products of PGTs (Fig. ​(Fig.3)3) appeared only when enzymes were incubated with BH4 as a sugar acceptor and either UDP-glucose (for CY-007, UCNR-001, and UNCR-002 PGTs) or UDP-xylose (for CY-049 PGT) as a sugar donor. HPLC analysis of cultured CY-007 and CY-049 cells confirmed the presence of the corresponding biopterin glycosides (data not shown), supporting the conclusion that the PGTs exhibited genuine in vivo activities. This is the first report of a gene encoding a PGT that catalyzes xylosyl transfer to BH4. Although the data are not shown here, we found additional xylosyl transfer PGTs in Anabaena sp. PCC 7120, Gloeobacter violaceus PCC 7421, and Thermosynechococcus elongatus BP-1, whose genomic sequences were determined. The putative PGT genes (represented in Fig. ​Fig.1)1) were amplified by PCR from the genomic DNA, which was a kind gift from the Kazusa DNA Research Institute (http://genome.kazusa.or.jp/cyanobase/). The recombinant proteins for the in vitro activity assay were prepared by cloning the genes into pET-28b and overexpressing the proteins in E. coli according to the same procedures performed for the other PGTs. Interestingly, CY-007 and CY-049 PGTs exhibited different substrate specificities, although they share 93% protein sequence identity, and they also had higher specific activities than the other PGTs (Fig. ​(Fig.4).4). The three-dimensional structures of the proteins are currently being investigated to further understanding of the structural properties involved. Considering the cyanobacterial PGTs hitherto identified, there seems to be little correlation between their substrate preferences and phylogenetic classification. However, the CI group PGTs, which diverged early from the CII group PGTs, might have some distinctive features. Finally, the successful cloning of PGT genes from environmental DNA allows for potentially new PGTs to be isolated from cyanobacteria, which are abundant in nature.Open in a separate windowFIG. 4.Comparative analysis of PGT activities. The maximal activity (100%) corresponds to complete glycosylation of 1 μM BH4 in the reaction mixture, which contained 0.5 mM UDP-xylose for CY-049 PGT or 0.5 mM UDP-glucose for the other PGTs. The mixtures were incubated for 10 min with the indicated amounts of proteins.     

Seed Water Relations and the Regulation of the Duration of Seed Growth in Soybean

Soybean [Glycine max (L.) Merrill] seeds and cotyledons weregrown in an in vitro culture system to investigate the relationshipsbetween cell expansion (net water uptake by the seed) and drymatter accumulation. Seeds or cotyledons grown in a completenutrient medium containing 200 mol m^–3 sucrose continueddry matter accumulation for up to 16 d after in planta seedsreached physiological maturity (maximum seed dry weight). Seedor cotyledon water content increased throughout the cultureperiod and the water concentration remained above 600 g kg^–1fresh weight. These data indicate that the cessation of seeddry matter accumulation is controlled by the physiological environmentof the seed and is not a pre-determined seed characteristic.Adding 600 mol m^–3 mannitol to the medium caused a decreasein seed water content and concentration. Seeds in this mediumstopped accumulating dry matter at a water concentration ofapproximately 550 g kg^–1. The data suggest that dry matteraccumulation by soybean seeds can continue only as long as thereis a net uptake of water to drive cell expansion. In the absenceof a net water uptake, continued dry matter accumulation causesdesiccation which triggers maturation. Key words: Glycine max (L.) Merrill, solution culture, duration of seed growth, water content, dry matter accumulation