Toshiaki Umezawa

Diversity in lignan biosynthesis

AbstractLignans are phenylpropanoid dimers, where the phenylpropane units are linked by the central carbon (C8) of their side chains. Ligans vary substantially in oxidation level, substitution pattern, and the chemical structure of their basic carbon framework. In addition to structural diversity, lignans show considerable diversity in terms of enantiomeric composition, biosynthesis, and phylogenetic distribution. In this review, these diversities are outlined and the phylogenetic distribution of plants producing 66 typical lignans is listed. The distribution is correlated with the putative biosynthetic pathways of the lignans and discussed from evolutionary aspects. Abbreviations: SIRD – Secoisolariciresinol dehydrogenase; PLR – pinoresinol lariciresinol reductase; DP – dirigent protein

Degradation mechanisms of phenolic β-1 lignin substructure model compounds by laccase of Coriolus versicolor

Phenolic beta-1 lignin substructure model compounds, 1-(3,5-dimethoxy-4-hydroxy-phenyl)-2-(3,5-dimethoxy-4-ethoxyphenyl)propa ne-1, 3-diol (I) and 1-(3,5-dimethoxy-4-ethoxyphenyl)-2-(3, 5-dimethoxy-4-hydroxyphenyl)propane-1,3-diol (II) were degraded by laccase of Coriolus versicolor. Substrate I was converted to 1-(3,5-dimethoxy-4-hydroxyphenyl)-2-(3,5-dimethoxy-4-ethoxyphenyl)-3- hydroxypropanone (III), 1-(3,5-dimethoxy-4-ethoxyphenyl)-2-hydroxyethanone (IV), syringaldehyde (V), 1-(3,5-dimethoxy-4-ethoxyphenyl)-3-hydroxypropanal (VI), 2,6-dimethoxy-p-hydroquinone (VII), and 2,6-dimethoxy-p-benzoquinone (VIII). Furthermore, incorporations of 18O of 18O2 into ethanone (IV) and 18O of H218O into hydroquinone (VII) and benzoquinone (VIII) were confirmed. Substrate II gave 1-(3,5-dimethoxy-4-hydroxyphenyl)ethane-1, 2-diol (IX), 1-(3,5-dimethoxy-4-hydroxyphenyl)-2-hydroxyethanone (X), and 3,5-dimethoxy-4-ethoxybenzaldehyde (XI). Also 18O of H218O was incorporated into glycol (IX) and ethanone (X). Based on the structures of the degradation products and the isotopic experiments, it was established that three types of reactions occurred via phenoxy radicals of substrates caused by laccase: (i) C alpha-C beta cleavage (between C1 and C2 carbons); (ii) alkyl-aryl cleavage (between C1 carbon and aryl group); and (iii) C alpha (C1) oxidation.

A rice fungal MAMP‐responsive MAPK cascade regulates metabolic flow to antimicrobial metabolite synthesis

Plants recognize potential microbial pathogens through microbial-associated molecular patterns (MAMPs) and activate a series of defense responses, including cell death and the production of reactive oxygen species (ROS) and diverse anti-microbial secondary metabolites. Mitogen-activated protein kinase (MAPK) cascades are known to play a pivotal role in mediating MAMP signals; however, the signaling pathway from a MAPK cascade to the activation of defense responses is poorly understood. Here, we found in rice that the chitin elicitor, a fungal MAMP, activates two rice MAPKs (OsMPK3 and OsMPK6) and one MAPK kinase (OsMKK4). OsMPK6 was essential for the chitin elicitor-induced biosynthesis of diterpenoid phytoalexins. Conditional expression of the active form of OsMKK4 (OsMKK4DD) induced extensive alterations in gene expression, which implied dynamic changes of metabolic flow from glycolysis to secondary metabolite biosynthesis while suppressing basic cellular activities such as translation and cell division. OsMKK4DD also induced various defense responses, such as cell death, biosynthesis of diterpenoid phytoalexins and lignin but not generation of extracellular ROS. OsMKK4DD-induced cell death and expression of diterpenoid phytoalexin pathway genes, but not that of phenylpropanoid pathway genes, were dependent on OsMPK6. Collectively, the OsMKK4–OsMPK6 cascade plays a crucial role in reprogramming plant metabolism during MAMP-triggered defense responses.

KaPPA-View. A Web-Based Analysis Tool for Integration of Transcript and Metabolite Data on Plant Metabolic Pathway Maps

The application of DNA array technology and chromatographic separation techniques coupled with mass spectrometry to transcriptomic and metabolomic analyses in plants has resulted in the generation of considerable quantitative data related to transcription and metabolism. The integration of “omic” data is one of the major concerns associated with research into identifying gene function. Thus, we developed a Web-based tool, KaPPA-View, for representing quantitative data for individual transcripts and/or metabolites on plant metabolic pathway maps. We prepared a set of comprehensive metabolic pathway maps for Arabidopsis (Arabidopsis thaliana) and depicted these graphically in Scalable Vector Graphics format. Individual transcripts assigned to a reaction are represented symbolically together with the symbols of the reaction and metabolites on metabolic pathway maps. Using quantitative values for transcripts and/or metabolites submitted by the user as Comma Separated Value-formatted text through the Internet, the KaPPA-View server inserts colored symbols corresponding to a defined metabolic process at that site on the maps and returns them to the users browser. The server also provides information on transcripts and metabolites in pop-up windows. To demonstrate the process, we describe the dataset obtained for transgenic plants that overexpress the PAP1 gene encoding a MYB transcription factor on metabolic pathway maps. The presentation of data in this manner is useful for viewing metabolic data in a way that facilitates the discussion of gene function.

Biosynthesis of lignans and norlignans

Lignans and norlignans constitute abundant classes of phenylpropanoids. Biosynthesis of these compounds has received widespread interest, mainly because they have various clinically important biological activities. In addition, lignans and norlignans are often biosynthesized and deposited in significant amounts in the heartwood region of trees as a metabolic event of heartwood formation, probably preventing heart rot by heart-rot fungi. Furthermore, biosynthetic reactions of lignans and norlignans involve unique stereochemical properties that are of great interest in terms of bioorganic chemistry and are expected to provide a model for biomimetic chemistry and its application. We outline the recent advances in the study of lignan and norlignan biosynthesis.

Red-brown color of lignified tissues of transgenic plants with antisense CAD gene: Wine-red lignin from coniferyl aldehyde

Coniferyl aldehyde was converted to a wine-red lignin polymer with peroxidase/H2O2. Color of the lignin was very similar to that of lignified tissues of transgenic tobacco plants with an antisense gene of cinnamyl alcohol dehydrogenase (CAD; Schuch, 1993). Experimental results suggested that the red-brown color of lignified tissues of the transgenic plants is ascribed to the extendedly conjugated coniferyl aldehyde groups of abnormal lignin derived from dehydrogenative polymerization of coniferyl aldehyde. The result also showed that lignin content of the transgenic plants does not decrease but the red-brown lignin predominant with coniferyl aldehyde groups is easily digested by Kraft cooking in paper mills. The red-brown wood is expected to be used for furnitures without staining.

The cinnamate/monolignol pathway

The cinnamate/monolignol pathway provides precursors for various phenylpropanoid compounds including lignins, lignans, neolignans, p-hydroxycinnamate esters, coumarins, suberins, flavonoids, stilbenes and so on. Therefore, the pathway plays the central role in plant secondary metabolism. During the last decade, significant advances have been made in understanding the major routes and transcriptional control mechanisms of the pathway. In this review, the major routes and the transcriptional control are outlined in relation to lignification.

Aromatic ring cleavage of 4,6‐di(tert‐butyl)guaiacol, a phenolic lignin model compound, by laccase of Coriolus versicolor

It was found that 2,4‐di(tert‐butyl)‐4‐(methoxycarbonylmethyl)‐2‐buten‐4‐olide (II) was formed as an aromatic ring cleavage product of a phenolic lignin model compound, 4,6‐di(tert‐butyl)guaiacol (I), by laccase of Coriolus versicolor. Based on isotopic experiments with 18O2 H2 18O, the mechanism of formation of II from I is discussed.

Characterization of Arabidopsis thaliana Pinoresinol Reductase, a New Type of Enzyme Involved in Lignan Biosynthesis

A lignan, lariciresinol, was isolated from Arabidopsis thaliana, the most widely used model plant in plant bioscience sectors, for the first time. In the A. thaliana genome database, there are two genes (At1g32100 and At4g13660) that are annotated as pinoresinol/lariciresinol reductase (PLR). The recombinant AtPLRs showed strict substrate preference toward pinoresinol but only weak or no activity toward lariciresinol, which is in sharp contrast to conventional PLRs of other plants that can reduce both pinoresinol and lariciresinol efficiently to lariciresinol and secoisolariciresinol, respectively. Therefore, we renamed AtPLRs as A. thaliana pinoresinol reductases (AtPrRs). The recombinant AtPrR2 encoded by At4g13660 reduced only (–)-pinoresinol to (–)-lariciresinol and not (+)-pinoresinol in the presence of NADPH. This enantiomeric selectivity accords with that of other PLRs of other plants so far reported, which can reduce one of the enantiomers selectively, whatever the preferential enantiomer. In sharp contrast, AtPrR1 encoded by At1g32100 reduced both (+)- and (–)-pinoresinols to (+)- and (–)-lariciresinols efficiently with comparative kcat/Km values. Analysis of lignans and spatiotemporal expression of AtPrR1 and AtPrR2 in their functionally deficient A. thaliana mutants and wild type indicated that both genes are involved in lariciresinol biosynthesis. In addition, the analysis of the enantiomeric compositions of lariciresinol isolated from the mutants and wild type showed that PrRs together with a dirigent protein(s) are involved in the enantiomeric control in lignan biosynthesis. Furthermore, it was demonstrated conclusively for the first time that differential expression of PrR isoforms that have distinct selectivities of substrate enantiomers can determine enantiomeric compositions of the product, lariciresinol.

Aromatic ring cleavage of β-O-4 lignin substructure model dimers by lignin peroxidase of Phanerochaete chrysosporium

Extracellular lignin peroxidase (ligninase) from Phanerochaete chrysosporium catalyzed aromatic ring cleavage of β‐O‐4 lignin substructure model dimers to give three esters of arylglycerol, cyclic carbonate, formate and methyl oxalate. H2O2, was required for the activity of the enzyme.