Formation Of Lignans Research Articles

Characterization of Peroxidase and Laccase Gene Families and In Silico Identification of Potential Genes Involved in Upstream Steps of Lignan Formation in Sesame.

Peroxidases and laccases are oxidative enzymes involved in physiological processes in plants, covering responses to biotic and abiotic stress as well as biosynthesis of health-promoting specialized metabolites. Although they are thought to be involved in the biosynthesis of (+)-pinoresinol, a comprehensive investigation of this class of enzymes has not yet been conducted in the emerging oil crop sesame and no information is available regarding the potential (+)-pinoresinol synthase genes in this crop. In the present study, we conducted a pan-genome-wide identification of peroxidase and laccase genes coupled with transcriptome profiling of diverse sesame varieties. A total of 83 and 48 genes have been identified as coding for sesame peroxidase and laccase genes, respectively. Based on their protein domain and Arabidopsis thaliana genes used as baits, the genes were classified into nine and seven groups of peroxidase and laccase genes, respectively. The expression of the genes was evaluated using dynamic transcriptome sequencing data from six sesame varieties, including one elite cultivar, white vs black seed varieties, and high vs low oil content varieties. Two peroxidase genes (SiPOD52 and SiPOD63) and two laccase genes (SiLAC1 and SiLAC39), well conserved within the sesame pan-genome and exhibiting consistent expression patterns within sesame varieties matching the kinetic of (+)-pinoresinol accumulation in seeds, were identified as potential (+)-pinoresinol synthase genes. Cis-acting elements of the candidate genes revealed their potential involvement in development, hormonal signaling, and response to light and other abiotic triggers. Transcription factor enrichment analysis of promoter regions showed the predominance of MYB binding sequences. The findings from this study pave the way for lignans-oriented engineering of sesame with wide potential applications in food, health and medicinal domains.

Genome-Wide Identification and Characterization of DIR Genes in Medicago truncatula.

Dirigent proteins (DIRs) are critically involved in the formation of lignans, a diverse and widely distributed class of secondary plant metabolites exhibiting interesting pharmacological activities and implicated in natural plant defense. However, no detailed information is available about DIR gene family in Medicago truncatula. In this study, a total of 45 DIR genes were identified in M. truncatula. DIR proteins have variability in sequence. Most MtDIR genes have no intron. All MtDIR proteins contain single dirigent domain. A large number of MtDIR genes were expanded via gene duplication, and 37 MtDIR genes were duplicated in tandem. Digital expression data showed that 40% MtDIR genes had a higher expression level in the root. Analysis of RNA-seq and microarray data indicated that more than 30% MtDIR genes were responsive to biotic and/or abiotic treatments. This study will facilitate further studies on DIR family and provide useful clues for functional validation ofDIR genes in higher plants.

Knockdown of PCBER1, a gene of neolignan biosynthesis, resulted in increased poplar growth

Poplar trees displayed an increased plant height due to the transgenic knockdown of PCBER1, a gene of lignan biosynthesis. The wood composition was slightly altered in both overexpression and knockdown lines. The gene PHENYLCOUMARAN BENZYLIC ETHER REDUCTASE1 (PCBER1) is well known as an important gene in the synthesis of lignans, a group of diverse phenylpropanoid derivatives. They are widely distributed in the plant kingdom and may have a role in both plant defense and growth regulation. To analyze its role in biomass formation and wood composition in poplar, both overexpression and knockdown approaches have been performed. Transgenic lines were analyzed on genetic and phenotypic levels, and partly in regard to their biomass composition. While the PCBER1 overexpression approach remained unremarkable concerning the plant height, biomass composition of obtained transgenic lines was modified. They had a significantly increased amount of ethanol extractives. The PCBER1 knockdown resulted in significantly deviating plants; after 17months of greenhouse cultivation, transgenic plants were up to 38% higher compared to non-transgenic wild type. Most examined transgenic lines did not reveal a significantly enhanced stem diameter after three vegetation periods in the greenhouse. Significant changes were not obtained with regard to the three major wood components, lignin, cellulose and hemicelluloses. As a slight but not significant reduction in ethanol extractives was detected, the hypothesis arises that the lignan content could be influenced. Lignans become important in the pharmaceutical industry and clinical studies concerning cancer and other diseases, thus further investigations on lignan formation in poplar and its connection to biomass formation seem promising.

Differential expression of SofDIR16 and SofCAD genes in smut resistant and susceptible sugarcane cultivars in response to Sporisorium scitamineum

Proteomic profiling of the stalk of a smut resistant and a susceptible sugarcane cultivars revealed the presence of dirigent and dirigent-like proteins in abundance in the pool of high molecular mass (HMMG) and mid-molecular mass (MMMG) glycoproteins, produced as part of the defensive response to the fungal smut pathogen. Quantitative RT-PCR analysis showed that expression levels of SofDIR16 (sugarcane dirigent16) and SofCAD (sugarcane cinnamyl alcohol dehydrogenase) were higher in the smut resistant My 55-14 cultivar than in the sensitive B 42231 cultivar prior to infection. Inoculation with fungal sporidia or water decreased the level of SofCAD transcripts in My 55-14, indicating that regulation of SofCAD expression does not take part of the specific response to smut infection. In contrast, SofDIR16 expression was almost nullified in My 55-14 after inoculation with fungal sporidia, but not after water injection. It is proposed that the decreased expression of dirigent proteins induces the formation of lignans, which are involved in the defense response of the smut resistant My 55-14 cultivar.

Natural hypolignification is associated with extensive oligolignol accumulation in flax stems.

Flax (Linum usitatissimum) stems contain cells showing contrasting cell wall structure: lignified in inner stem xylem tissue and hypolignified in outer stem bast fibers. We hypothesized that stem hypolignification should be associated with extensive phenolic accumulation and used metabolomics and transcriptomics to characterize these two tissues. (1)H nuclear magnetic resonance clearly distinguished inner and outer stem tissues and identified different primary and secondary metabolites, including coniferin and p-coumaryl alcohol glucoside. Ultrahigh-performance liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry aromatic profiling (lignomics) identified 81 phenolic compounds, of which 65 were identified, to our knowledge, for the first time in flax and 11 for the first time in higher plants. Both aglycone forms and glycosides of monolignols, lignin oligomers, and (neo)lignans were identified in both inner and outer stem tissues, with a preponderance of glycosides in the hypolignified outer stem, indicating the existence of a complex monolignol metabolism. The presence of coniferin-containing secondary metabolites suggested that coniferyl alcohol, in addition to being used in lignin and (neo)lignan formation, was also utilized in a third, partially uncharacterized metabolic pathway. Hypolignification of bast fibers in outer stem tissues was correlated with the low transcript abundance of monolignol biosynthetic genes, laccase genes, and certain peroxidase genes, suggesting that flax hypolignification is transcriptionally regulated. Transcripts of the key lignan genes Pinoresinol-Lariciresinol Reductase and Phenylcoumaran Benzylic Ether Reductase were also highly abundant in flax inner stem tissues. Expression profiling allowed the identification of NAC (NAM, ATAF1/2, CUC2) and MYB transcription factors that are likely involved in regulating both monolignol production and polymerization as well as (neo)lignan production.

Formation of lignans in plant cell culture of Schisandra chinensis

The fruit of Schisandra chinensis, a woody liana, has been used for centuries in traditional Chinese medicine. The fruit is prescribed for the treatment of hepatitis in China. The active principles are lignans with unusual structures derived from dibenzo[a,c]cyclooctadiene. These lignans have been shown to possess a broad range of biological activities. Recently, it has been found that lignan gamma-schizandin strongly inhibits P-glycoprotein, the overexpression of which is the most frequent cause for cancer multidrug resistance. S. chinensis cultures derived from immature zygotic embryos were developed on Murashige and Skoog and VW5 medium. Embryogenic cultures were established on the medium containing thidiazuron, 2,4-dichlorophenoxyacetic acid and benzylaminopurine. An analytical method based on HPLC-UV was used for the determination of six lignans in plant cell cultures. Our previous results showed that the concentration of lignans in plant cell cultures was mostly lower than that in fruit and leaves. We also detected the lignans in liquid culture media, but the amount of lignans inside the cells was substantially higher. In order to release the lignans stored within the cells, we explored the cultivation of five cell lines with neutral polymeric resin, Amberlite XAD2. We found that the addition of Amberlite XAD2 not only notably enlarged portions of extracellular lignans, but also greatly enhanced the production of lignan deoxyschizandrin, whereas the formation of other lignans was increased only moderately (less than 5fold). Importantly, Amberlite XAD-2 did not reduce the growth of cell cultures. Deoxyschizandrin adsorbed on the polymeric resin can be easily recovered and purified without destruction of the cells.

Dirigent Proteins in Conifer Defense: Gene Discovery, Phylogeny, and Differential Wound- and Insect-induced Expression of a Family of DIR and DIR-like Genes in Spruce (Picea spp.)

The outer stem tissues of conifers provide a durable constitutive and inducible defense barrier consisting of suberized or lignified periderm, sclereids, a network of terpenoid-filled resin ducts, and phenolic phloem parenchyma cells. Microarray gene expression profiling of Sitka spruce (Picea sitchensis) bark attacked by stem-boring weevils (Pissodes strobi) or through mechanical wounding demonstrated significant accumulation of transcripts resembling dirigent protein (DIR) genes. To investigate this gene family and its spatial and temporal patterns of expression in conifer defense, we isolated cDNAs representing 19 unique DIR and DIR-like genes from Sitka spruce, white spruce (P. glauca), and interior spruce (P. glauca x engelmannii). Sequence alignments also identified a large number of DIR-like proteins in other plant species, which share several conserved protein motifs with known DIR proteins. Phylogenetic analysis of 72 DIR and DIR-like proteins suggests five distinct subfamilies, DIR-a and four DIR-like subfamilies (DIR-b, DIR-c, DIR-d and DIR-e). Previously characterized members of the DIR-a subfamily direct stereoselective phenolic coupling reactions in the formation of lignans and possibly lignins. The spruce genes identified here are members of the DIR-a and DIR-b subfamilies. Using gene-specific quantitative real-time PCR we measured constitutive expression for six DIR-a genes and three DIR-like genes in different stem tissues, green shoot tips, and roots of Sitka spruce. DIR-like genes revealed ubiquitous high expression in all tissues. In contrast, the six DIR-a genes showed a gradient of transcript abundance in stem tissues with highest levels in the outer cortex and lowest levels in the inner xylem. Gene-specific transcript profiling of six DIR-a genes confirmed rapid and strong accumulation (up to 500-fold) in wound- and weevil-induced stem bark and xylem. These findings suggest a role for spruce DIR genes in constitutive and induced phenolic defense mechanisms against stem-boring insects.

Determination of lignans in human plasma by liquid chromatography with coulometric electrode array detection

The presence of mammalian lignans, mainly enterolactone, in human plasma has been related to lower incidence of certain cancers and cardiovascular disease. The plant lignans secoisolariciresinol and matairesinol have been reported to be precursors of mammalian lignans, but recently other plant lignans relatively abundant in the diet have also been identified as precursors. To evaluate the importance and contribution of these new dietary precursors to the mammalian lignan formation in vivo, metabolic studies in human subjects must be carried out. For this purpose a method based on high-performance liquid chromatography using coulometric electrode array detection for the simultaneous determination of nine plant lignans and two mammalian lignans in human plasma was developed, validated, and shown to fulfill the reliability criteria.

Kinetic study of coniferyl alcohol radical binding to the (+)-pinoresinol forming dirigent protein.

An essential step in lignan and lignin formation in planta is one electron oxidation of (E)-coniferyl alcohol (CA) to generate the radical intermediate (CA(*)), which can then undergo directed radical-radical couplings in vivo. For lignan formation in vitro and in vivo, stereoselective coupling of CA(*) only occurs to afford (+)-pinoresinol in the additional presence of (+)-pinoresinol forming dirigent protein (DP). Presented herein is a kinetic and thermodynamic study which reveals the central mechanistic details of the coupling process involved in DP-mediated coupling. DP activity was maximal between pH 4.25 and pH 6.0, with activity being maintained at temperatures below 33 degrees C. Equilibrium binding assays revealed that coniferyl alcohol was only weakly bound to the DP, with a K(D) of 370 +/- 65 microM. On the other hand, the enantiomeric excess of (+)-pinoresinol formed was dependent on both DP concentration and rate of CA oxidation and, thus, on apparent steady-state [CA(*)]. The data obtained could best be explained using a kinetic model where radical-radical coupling via DP competes with that occurring in open solution. Using this model, an apparent K(M) of about 10 nM was estimated from the saturation behavior of (+)-pinoresinol formation with respect to apparent steady-state [CA(*)]. These data strongly suggest that CA(*), rather than CA, is the substrate for DP, in agreement with earlier predictions. A mechanism of directed radical-radical coupling, where two coniferyl alcohol radical substrates are bound per protein dimer, is proposed.

Mammalian Lignan Formation in Rats Fed a Wheat Bran Diet

The dietary origin of lignan phytoestrogens is still poorly understood more than 20 years after their discovery in human urine. Their level in urine has been associated with the consumption of dietary fiber. This paper reports the study of the excretion of enterolactone, assayed by a time-resolved fluoroimmunoassay, in rats fed a diet supplemented with 15% wheat bran, one of the main sources of fiber in Western countries. Enterolactone excretion regularly increased during the two weeks of the diet to reach a value of 45 nmol/day. The level of excretion also increased upon preadaptation to ferulic acid, structurally related to secoisolariciresinol, an established precursor of enterolactone in flaxseeds, and decreased upon preadaptation to potato starch rich in fiber. These results show that the formation of lignans from wheat bran is influenced by the diet, possibly because of an adaptation of the colonic microflora.

Stereochemical diversity in lignan biosynthesis of Arctium lappa L.

The stereochemistry of lignan biosynthesis in Arctium lappa L. is regulated organ-specifically. (+)-Secoisolariciresinol [81% enantiomeric excess (e.e.)] was isolated from A. lappa petioles. In sharp contrast, lignans whose predominant enantiomers have the opposite absolute configuration to that of (+)-secoisolariciresinol [i.e., (-)-matairesinol (>99% e.e.), (-)-arctigenin (>99% e.e.), and (-)-secoisolariciresinol (65% e.e.)] were isolated from seeds of the species. The stereochemical diversity of secoisolariciresinol was demonstrated with enzyme preparations from A. lappa petioles and seeds. Thus, a petiole enzyme preparation catalyzed the formation of (+)-pinoresinol (33% e.e.), (+)-lariciresinol (30% e.e.), and (+)-secoisolariciresinol (20% e.e.) from achiral coniferyl alcohol in the presence of NADPH and H202, whereas that from ripening seeds catalyzed the formation of (-)-pinoresinol (22% e.e.), (-)-lariciresinol (>99% e.e.), and (-)-secoisolariciresinol (38% e.e.) under the same conditions. In addition, the ripening seed enzyme preparation mediated the selective formation of the optically pure (>99% e.e.) (-)-enantiomer of matairesinol from racemic (+/-)-secoisolariciresinols in the presence of NADP. These results indicate that the stereochemical mechanism for lignan biosynthesis in A. lappa varies with organs, suggesting that multiple lignan-synthesizing isozymes are involved in the stereochemical control of lignan formation in A. lappa.

Biosynthesis of (+)-syringaresinol inLiriodendron tulipifera I: feeding experiments withl-[U-14C]phenylalanine and [8-14C]sinapyl alcohol

To clarify the biosynthesis of syringyl lignans and lignan formation by stereoselective coupling of monolignols, formation of (+)-syringaresinol and (+)-pinoresinol inLiriodendron tulipifera were investigated by means of feeding experiments. Following individual administration ofl-[U-14C]phenylalanine and [8-14C]sinapyl alcohol to excised shoots ofL. tulipifera and their subsequent metabolism for 3h, free [14C] lignans and [14C] lignan glucosides were extracted from both of the stems and leaves with methanol and divided into an ether fraction and an aqueous one, respectively. The glucosides were hydrolyzed by a combination of cellulase andΒ-glucosidase to liberate [14C]lignans as aglycones.l-[U-14C]Phenylalanine was incorporated into free (+)-[14C]syringaresinol and its glucosides; the (+)-[14C]syringaresinols in the stems and leaves had 52% enantiomeric excess (% e.e.) and 42% e.e., respectively; and the (+)-[14C]syringaresinol aglycones from the glucosides in the stems and leaves had 20% e.e. and 22% e.e., respectively. Furthermore, [8-14C]sinapyl alcohol was incorporated into (+)-[14C]syringaresinol and its glucosides in the stems. These results suggest that the (+)-enantiomer of syringaresinol was enantioselectively formed from two molecules of sinapyl alcohol inL. tulipifera followed by transformation into the (+)-syringaresinol glucosides, accompanying the formation of racemic syringaresinol by nonselective coupling and the subsequent transformation of the racemate into their glucosides.l-[U-14C]Phenylalanine was incorporated also into free (+)-[14C]pinoresinol and its glucosides with 12%–42% e.e.

Stable isotope-labeling studies on the oxidative coupling of caffeic acid via o-quinone.

The formation of ortho-quinone from ortho-diphenol is a key step in its dimerization. An NMR analysis of the oxidation of 3,4-dihydroxycinnamic acid (caffeic acid) by NaIO4 revealed the formation of 3-(3',4'-dioxo-1',5'-cyclohexadienyl) propenoic acid (o-quinone) prior to the formation of furofuran-type lignan 4,8-exo-bis (3,4-dihydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane-2,6-dione. Both electrolytic and enzymatic oxidation of caffeic acid also generated o-quinone. The yields of o-quinone from caffeic acid were quantified by NMR and HPLC analyses. A stable isotope-labeling study of the formation of lignans directly proved the random radical coupling of semiquinone radicals formed from a set of caffeic acid and o-quinone.

Metabolic Activities of the Gut Microflora in Relation to Cancer

The human large intestine is host to a diverse range of bacteria with numbers reaching 10 11 /ml of faecal material. This population plays a significant role in colonic metabolism and health. Undigested dietary substrates and endogenous residues are metabolised by the gut flora. Some of the products of this metabolism have been associated with carcinogenic processes such as tumour promotion (ammonia, secondary bile acids), mutagenesis (fecapenaenes) and carcinogenesis (N-nitroso compounds). Bacterial enzymes involved with carcinogen formation include β -glucuronidase, β -glycosidase, azoreductase, nitroreductase, nitrate reductase, the conversion of pre-carcinogen 2 amino-3-methyl-7H-imidazo[4,5-f]quinoline(IQ) to 7-hydroxy-2-amino-3,6-dihydro-3-methyl-7H-imidazo[4,5-f]quinoline-7-one(7OHIQ). Protective effects of gut bacterial metabolism include carcinogen binding, detoxification of methylmercury and formation of lignans and isoflavones. Diet is known to play a role in gut microflora metabolism and cancer development. Studies have shown that gut bacterial enzymes are affected by diet and bacterial metabolism of dietary protein releases toxic products including ammonia, phenols and cresols. The interrelationships between diet, gut microflora metabolism and effects on the host are complicated however they may be important in the understanding of epidemiological associations between exogenous factors and colorectal cancer risk. Keywords: microflora metabolism, colon cancer, toxic end-products, detoxification.

Metabolic Activities of the Gut Microflora in Relation to Cancer

The human large intestine is host to a diverse range of bacteria with numbers reaching 10 11 /ml of faecal material. This population plays a significant role in colonic metabolism and health. Undigested dietary substrates and endogenous residues are metabolised by the gut flora. Some of the products of this metabolism have been associated with carcinogenic processes such as tumour promotion (ammonia, secondary bile acids), mutagenesis (fecapenaenes) and carcinogenesis ( N -nitroso compounds). Bacterial enzymes involved with carcinogen formation include &#103 -glucuronidase, &#103 -glycosidase, azoreductase, nitroreductase, nitrate reductase, the conversion of pre-carcinogen 2 amino-3-methyl-7H-imidazo[4,5-f]quinoline (IQ) to 7-hydroxy-2-amino-3,6-dihydro-3-methyl-7H-imidazo[4,5-f]quinoline-7-one (7OHIQ). Protective effects of gut bacterial metabolism include carcinogen binding, detoxification of methylmercury and formation of lignans and isoflavones. Diet is known to play a role in gut microflora metabolism and cancer development. Studies have shown that gut bacterial enzymes are affected by diet and bacterial metabolism of dietary protein releases toxic products including ammonia, phenols and cresols. The interrelationships between diet, gut microflora metabolism and effects on the host are complicated however they may be important in the understanding of epidemiological associations between exogenous factors and colorectal cancer risk.

Regiochemical control of monolignol radical coupling: A new paradigm for lignin and lignan biosynthesis

Although the lignins and lignans, both monolignol-derived coupling products, account for nearly 30% of the organic carbon circulating in the biosphere, the biosynthetic mechanism of their formation has been poorly understood. The prevailing view has been that lignins and lignans are produced by random free-radical polymerization and coupling, respectively. This view is challenged, mechanistically, by the recent discovery of dirigent proteins that precisely determine both the regiochemical and stereoselective outcome of monolignol radical coupling. To understand further the regulation and control of monolignol coupling, leading to both lignan and lignin formation, we sought to clone the first genes encoding dirigent proteins from several species. The encoding genes, described here, have no sequence homology with any other protein of known function. When expressed in a heterologous system, the recombinant protein was able to confer strict regiochemical and stereochemical control on monolignol free-radical coupling. The expression in plants of dirigent proteins and proposed dirigent protein arrays in developing xylem and in other lignified tissues indicates roles for these proteins in both lignan formation and lignification. The first understanding of regiochemical and stereochemical control of monolignol coupling in lignan biosynthesis has been established via the participation of a new class of dirigent proteins. Immunological studies have also implicated the involvement of potential corresponding arrays of dirigent protein sites in controlling lignin biopolymer assembly.

Furanofuran lignan metabolism as a function of seed maturation in sesamum indicum: methylenedioxy bridge formation

Sesamum indicum seeds accumulate the antioxidant lignans, (+)-sesamin and (+)-sesamolin. It was established that seeds of 8-week old S. indicum plants catalyzed distinct lignan transformations (methylenedioxy bridge formation in piperitol and sesamin, as well as oxygen insertion in sesamolin) depending upon the stage of seed maturity, i.e. lignan formation was developmentally regulated. The most mature seeds at the eight week developmental stage efficiently converted (+)-[3,3′-O14CH3] pinoresinol into (+)-piperitol and (+)-sesamin, whereas younger seeds had a higher conversion into (+)-sesamolin; the corresponding (−)-[3,3′-O14CH3] pinoresinol antipode was not metabolized. Microsomal preparations obtained from S. indicum seeds catalyzed methylenedioxy bridge formation via an O2\\NADPH\\cytochrome P-450 dependent transformation of (+)-pinoresinol into (+)-piperitol. Preliminary characterization of the enzyme established its cytochrome P-450 dependence.

Stereochemical Difference in Secoisolariciresinol Formation between Cell-free Extracts from Petioles and from Ripening Seeds of Arctium lappa L.

Cell-free extracts from ripening seeds of Arctium lappa L. catalyzed the enantioselective formation of (-)-pinoresinol, (-)-lariciresinol and (-)-secoisolariciresinol from achiral coniferyl alcohol in the presence of NADPH and H2O2. The enantioselectivity of the lignan formation was opposite to that of the (+)-secoisolariciresinol formation catalyzed by cell-free extracts from petioles of the same plant species.

Jatrodien, a lignan from stems of Jatropha gossypifolia

A novel lignan, jatrodien, representing and intermediate in Haworth's biosynthetic scheme for the formation of lignans, has been isolated from the stems of Jatropha gossypifolia. The structure of the compound was derived from its spectroscopic data. Synthesis of the compound, starting from piperonal has been achieved.

Formation of Lignans in Suspension Cultures otLinum album

Formation of Lignans in Suspension Cultures ot<i>Linum album</i>