Taiji Nomura

Molecular characterization and chromosomal localization of cytochrome P450 genes involved in the biosynthesis of cyclic hydroxamic acids in hexaploid wheat.

Abstract. The cyclic hydroxamic acids, 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), are defensive secondary metabolites found in gramineous plants including wheat, maize and rye. cDNAs for five cytochromes P450 (P450s) involved in DIBOA biosynthesis (CYP71C6, CYP71C7v2, CYP71C8v2, CYP71C9v1 and CYP71C9v2) were isolated from seedlings of hexaploid wheat [(Triticum aestivum L. cv. Chinese Spring (2n=6x=42, genomes AABBDD)] by RT-PCR and screening of a cDNA library. CYP71C9v1 and CYP71C9v2 are 97% identical to each other in amino acid and nucleotide sequences. The cloned P450 species showed 76–79% identity at the amino acid level to the corresponding maize P450 species CYP71C1–C4, which are also required for DIBOA biosynthesis. The wheat P450 cDNAs were heterologously expressed in the yeast (Saccharomyces cerevisiae) strain AH22. Microsome fractions from yeast cells expressing these P450 species catalyzed the same reactions as their maize orthologs. The chromosomes carrying the cyp71C6–C9v1 orthologs were identified by Southern hybridization using aneuploid lines of Chinese Spring wheat. The cyp71C9v1 orthologs were located on the chromosomes of wheat homoeologous group-4. The orthologs of the other P450 genes, cyp71C7v2, cyp71C6 and cyp71C8v2, were located on group-5 chromosomes. The same P450 genes were also present in the three ancestral diploid species of hexaploid wheat, T. monococcum (AA), Aegilops speltoides [BB (≈SS)] and Ae. squarrosa (DD).

Rearrangement of the genes for the biosynthesis of benzoxazinones in the evolution of Triticeae species

Gramineous plants, including the major agricultural crops wheat (Triticum aestivum L.), rye (Secale cereale L.) and maize (Zea mays L.), accumulate benzoxazinones (Bxs) as defensive compounds. Previously, we isolated cDNAs of the Bx biosynthetic genes in wheat, TaBx2–TaBx5, that encode the enzymes catalyzing the sequential hydroxylation of indole to Bxs. In this study we isolated a cDNA of TaBx1, which encodes the first enzyme of the Bx pathway of wheat. The level of identity (80%) in deduced amino-acid sequence between TaBx1 and the corresponding maize gene Bx1 was as high as those shown between TaBx2–TaBx5 and the corresponding maize genes Bx2–Bx5, respectively. Southern blot analysis using the TaBx1–TaBx5 cDNAs as probes was conducted with aneuploid lines of hexaploid wheat in order to determine sub-chromosomal locations of the five Bx biosynthetic genes in Triticeae species. In wheat, TaBx1 and TaBx2 co-existed in specific regions of chromosomes 4A, 4B and 4D, and TaBx3–TaBx5 were localized together in the distal regions of the short arms of chromosomes 5A, 5B and 5D. TaBx3 and TaBx5 were found to have duplicated loci in the long arm and the short arm of chromosome 5B, respectively. In rye, homoeoloci of TaBx1 and TaBx2 were located on chromosome 7R and those for TaBx3–TaBx5 were located on chromosome 5R. In barley, no Southern blot band was detected with any of the probes under the highly stringent hybridization conditions, suggesting that the non-Bx phenotype of barley is attributable to the loss of Bx biosynthetic genes.

Molecular and Structural Characterization of Hexameric β-d-Glucosidases in Wheat and Rye

The wheat (Triticum aestivum) and rye (Secale cereale) β-d-glucosidases hydrolyze hydroxamic acid-glucose conjugates, exist as different types of isozyme, and function as oligomers. In this study, three cDNAs encoding β-d-glucosidases (TaGlu1a, TaGlu1b, and TaGlu1c) were isolated from young wheat shoots. Although the TaGlu1s share very high sequence homology, the mRNA level of Taglu1c was much lower than the other two genes in 48- and 96-h-old wheat shoots. The expression ratio of each gene was different between two wheat cultivars. Recombinant TaGlu1b expressed in Escherichia coli was electrophoretically distinct fromTaGlu1a and TaGlu1c. Furthermore, coexpression of TaGlu1a and TaGlu1b gave seven bands on a native-PAGE gel, indicating the formation of both homo- and heterohexamers. One distinctive property of the wheat and rye glucosidases is that they function as hexamers but lose activity when dissociated into smaller oligomers or monomers. The crystal structure of hexameric TaGlu1b was determined at a resolution of 1.8 Å. The N-terminal region was located at the dimer-dimer interface and plays a crucial role in hexamer formation. Mutational analyses revealed that the aromatic side chain at position 378, which is located at the entrance to the catalytic center, plays an important role in substrate binding. Additionally, serine-464 and leucine-465 of TaGlu1a were shown to be critical in the relative specificity for DIMBOA-glucose (2-O-β-d-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one) over DIBOA-glucose (7-demethoxy-DIMBOA-glucose).

Dispersed Benzoxazinone Gene Cluster: Molecular Characterization and Chromosomal Localization of Glucosyltransferase and Glucosidase Genes in Wheat and Rye

Benzoxazinones (Bxs) are major defensive secondary metabolites in wheat (Triticum aestivum), rye (Secale cereale), and maize (Zea mays). Here, we identified full sets of homeologous and paralogous genes encoding Bx glucosyltransferase (GT) and Bx-glucoside glucosidase (Glu) in hexaploid wheat (2n = 6x = 42; AABBDD). Four GT loci (TaGTa–TaGTd) were mapped on chromosomes 7A, 7B (two loci), and 7D, whereas four glu1 loci (Taglu1a–Taglu1d) were on chromosomes 2A, 2B (two loci), and 2D. Transcript levels differed greatly among the four loci; B-genome loci of both TaGT and Taglu1 genes were preferentially transcribed. Catalytic properties of the enzyme encoded by each homeolog/paralog also differed despite high levels of identity among amino acid sequences. The predominant contribution of the B genome to GT and Glu reactions was revealed, as observed previously for the five Bx biosynthetic genes, TaBx1 to TaBx5, which are separately located on homeologous groups 4 and 5 chromosomes. In rye, where the ScBx1 to ScBx5 genes are dispersed to chromosomes 7R and 5R, ScGT and Scglu were located separately on chromosomes 4R and 2R, respectively. The dispersal of Bx-pathway loci to four distinct chromosomes in hexaploid wheat and rye suggests that the clustering of Bx-pathway genes, as found in maize, is not essential for coordinated transcription. On the other hand, barley (Hordeum vulgare) was found to lack the orthologous GT and glu loci like the Bx1 to Bx5 loci despite its close phylogenetic relationship with wheat and rye. These results contribute to our understanding of the evolutionary processes that the Bx-pathway loci have undergone in grasses.

A novel xylogenic suspension culture model for exploring lignification in Phyllostachys bamboo

BackgroundSome prominent cultured plant cell lines, such as the BY-2 cell line of tobacco (Nicotiana tabacum cv. ‘Bright Yellow 2’) and the T87 cell line of Arabidopsis (Arabidopsis thaliana L. Heynh., ecotype Columbia) are used as model plant cells. These suspension cell culture systems are highly applicable for investigating various aspects of plant cell biology. However, no such prominent cultured cell lines exist in bamboo species.ResultsWe standardized a novel xylogenic suspension culture model in order to unveil the process of lignification in living bamboo cells. Initial signs of lignin deposition were able to be observed by a positive phloroglucinol-HCl reaction at day 3 to 5 under lignification conditions (LG), i.e., modified half-strength Murashige and Skoog medium (m1/2MS) containing 10 μM 6-benzyladenine (BA) and 3% sucrose. Two types of xylogenic differentiation, both fiber-like elements (FLEs) with cell wall thickening and tracheary elements (TEs) with formation of perforations in the cell wall, were observed under these conditions. The suspension cells rapidly formed secondary cell wall components that were highly lignified, making up approximately 25% of the cells on a dry weight basis within 2 weeks. Detailed features involved in cell growth, differentiation and death during lignification were characterized by laser scanning microscopic imaging. Changes in transcript levels of xylogenesis-related genes were assessed by RT-PCR, which showed that the transcription of key genes like PAL1, C4H, CCoAOMT, and CCR was induced at day 4 under LG conditions. Furthermore, interunit linkage of lignins was compared between mature bamboo culms and xylogenic suspension cells by heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The presence of the most common interunit linkages, including β-aryl ether (β-O-4), phenylcoumaran (β-5) and resinol (β-β) structures was identified in the bamboo cultured cell lignin (BCCL) by HSQC NMR. In addition to these common features of lignin, several differences in lignin substructures were also found between the BCCL and the bamboo milled wood lignin (BMWL).ConclusionsOur xylogenic suspension culture model could be used for detailed characterization of physiological and molecular biological events in living bamboo cells.

A Novel Lactone-Forming Carboxylesterase: Molecular Identification of a Tuliposide A-Converting Enzyme in Tulip

Tuliposides, the glucose esters of 4-hydroxy-2-methylenebutanoate and 3,4-dihydroxy-2-methylenebutanoate, are major secondary metabolites in tulip (Tulipa gesneriana). Their lactonized aglycons, tulipalins, function as defensive chemicals due to their biological activities. We recently found that tuliposide-converting enzyme (TCE) purified from tulip bulbs catalyzed the conversion of tuliposides to tulipalins, but the possibility of the presence of several TCE isozymes was raised: TCE in tissues other than bulbs is different from bulb TCE. Here, to prove this hypothesis, TCE was purified from petals, which have the second highest TCE activity after bulbs. The purified enzyme, like the bulb enzyme, preferentially accepted tuliposides as substrates, with 6-tuliposide A the best substrate, which allowed naming the enzyme tuliposide A-converting enzyme (TCEA), but specific activity and molecular mass differed between the petal and bulb enzymes. After peptide sequencing, a novel cDNA (TgTCEA) encoding petal TCEA was isolated, and the functional characterization of the recombinant enzyme verified that TgTCEA catalyzes the conversion of 6-tuliposide A to tulipalin A. TgTCEA was transcribed in all tulip tissues but not in bulbs, indicating the presence of a bulb-specific TgTCEA, as suggested by the distinct enzymatic characters between the petal and bulb enzymes. Plastidial localization of TgTCEA enzyme was revealed, which allowed proposing a cytological mechanism of TgTCE-mediated tulipalin formation in the tulip defensive strategy. Site-directed mutagenesis of TgTCEA suggested that the oxyanion hole and catalytic triad characteristic of typical carboxylesterases are essential for the catalytic process of TgTCEA enzyme. To our knowledge, TgTCEA is the first identified member of the lactone-forming carboxylesterases, specifically catalyzing intramolecular transesterification.

Environmentally benign process for the preparation of antimicrobial α-methylene-β-hydroxy-γ-butyrolactone (tulipalin B) from tulip biomass

Tulipalin B (α-methylene-β-hydroxy-γ-butyrolactone, PaB) is an antimicrobial natural product occurring in tulip (Tulipa gesneriana). PaB is directly formed from the precursor glucose ester 6-tuliposide B (PosB) by endogenous Pos-converting enzyme (TCE). Despite the potential usefulness of antibacterial PaB in various industrial applications, lack of facile synthetic schemes hampers its practical use. Herein, we describe an environmentally benign and facile process for the preparation of PaB using tulip biomass materials based on one-step enzyme reaction catalyzed by TCE without the use of petroleum-derived solvents. By screening 115 tulip cultivars, we found three elite cultivars, which accumulated PosB almost exclusively in flower tissues. The flower extracts with aqueous ethanol were partially purified with activated charcoal and subjected to the enzyme reaction with reusable immobilized TCE prepared from bulb crude extracts. The reaction was completed in a few hours at room temperature, and PaB was purified with activated charcoal and ethanol in a batch-wise manner. Graphical Abstract Tulipalin B preparation from tulip biomass by one-step reaction with reusable immobilized enzyme and purification with activated charcoal and ethanol.

Molecular Diversity of Tuliposide A-Converting Enzyme in the Tulip

Tuliposide A-converting enzyme (TCEA) catalyzes the conversion of 6-tuliposide A to its lactonized aglycon, tulipalin A, in the tulip (Tulipa gesneriana). The TgTCEA gene, isolated previously from petals, was transcribed in all tulip tissues but not in the bulbs despite the presence of TCEA activity, which allowed prediction of the presence of a TgTCEA isozyme gene preferentially expressed in the bulbs. Here, the TgTCEA-b gene, the TgTCEA homolog, was identified in bulbs. TgTCEA-b polypeptides showed approximately 77% identity to the petal TgTCEA. Functional characterization of the recombinant enzyme verified that TgTCEA-b encoded the TCEA. Moreover, the TgTCEA-b was found to be localized to plastids, as found for the petal TgTCEA. Transcript analysis revealed that TgTCEA-b was functionally transcribed in the bulb scales, unlike the TgTCEA gene, whose transcripts were absent there. In contrast, TgTCEA-b transcripts were in the minority in other tissues where TgTCEA transcripts were dominant, indicating a tissue preference for the transcription of those isozyme genes.

Function and application of a non-ester-hydrolyzing carboxylesterase discovered in tulip

Plants have evolved secondary metabolite biosynthetic pathways of immense rich diversity. The genes encoding enzymes for secondary metabolite biosynthesis have evolved through gene duplication followed by neofunctionalization, thereby generating functional diversity. Emerging evidence demonstrates that some of those enzymes catalyze reactions entirely different from those usually catalyzed by other members of the same family; e.g. transacylation catalyzed by an enzyme similar to a hydrolytic enzyme. Tuliposide-converting enzyme (TCE), which we recently discovered from tulip, catalyzes the conversion of major defensive secondary metabolites, tuliposides, to antimicrobial tulipalins. The TCEs belong to the carboxylesterase family in the α/β-hydrolase fold superfamily, and specifically catalyze intramolecular transesterification, but not hydrolysis. This non-ester-hydrolyzing carboxylesterase is an example of an enzyme showing catalytic properties that are unpredictable from its primary structure. This review describes the biochemical and physiological aspects of tulipalin biogenesis, and the diverse functions of plant carboxylesterases in the α/β-hydrolase fold superfamily. Graphical abstract Both canonical ester-hydrolyzing and atypical non-ester hydrolyzing functions of carboxylesterases are required for secondary metabolism in plants.

Molecular diversity of tuliposide B-converting enzyme in tulip (Tulipa gesneriana): identification of the root-specific isozyme

6-Tuliposide B (PosB) is a glucose ester accumulated in tulip (Tulipa gesneriana) as a major secondary metabolite. PosB serves as the precursor of the antimicrobial lactone tulipalin B (PaB), which is formed by PosB-converting enzyme (TCEB). The gene TgTCEB1, encoding a TCEB, is transcribed in tulip pollen but scarcely transcribed in other tissues (e.g. roots) even though those tissues show high TCEB activity. This led to the prediction of the presence of a TCEB isozyme with distinct tissue specificity. Herein, we describe the identification of the TgTCEB-R gene from roots via native enzyme purification; this gene is a paralog of TgTCEB1. Recombinant enzyme characterization verified that TgTCEB-R encodes a TCEB. Moreover, TgTCEB-R was localized in tulip plastids, as found for pollen TgTCEB1. TgTCEB-R is transcribed almost exclusively in roots, indicating a tissue preference for the transcription of TCEB isozyme genes. Graphical Abstract Tissue-specific isozymes are involved in the conversion of 6-tuliposide B, a major secondary metabolite in tulip, to antibacterial tulipalin B.