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Functional analysis of tobacco LIM protein Ntlim1 involved in lignin biosynthesis.

The AC-rich motif, Pal-box, is an important cis-acting element for gene expression involved in phenylpropanoid biosynthesis. A cDNA clone (Ntlim1) encoding a Pal-box binding protein was isolated by Southwestern screening. The deduced amino acid sequence is highly similar to the members of the LIM protein family that contain a zinc finger motif. Moreover, Ntlim1 had a specific DNA binding ability and transiently activated the transcription of a beta-glucuronidase reporter gene driven by the Pal-box sequence in tobacco protoplasts. The transgenic tobacco plants with antisense Ntlim1 showed low levels of transcripts from some key phenylpropanoid pathway genes such as phenylalanine ammonia-lyase, hydroxycinnamate CoA ligase and cinnamyl alcohol dehydrogenase. Furthermore, a 27% reduction of lignin content was observed in the transgenic tobacco with antisense Ntlim1.

Amino Acid Sequence↗

High free-methionine and decreased lignin content result from a mutation in the Arabidopsis S-adenosyl-L-methionine synthetase 3 gene.

As an approach to understand the regulation of methionine (Met) metabolism, Arabidopsis Met over-accumulating mutants were isolated based on their resistance to selection by ethionine. One mutant, mto3, accumulated remarkably high levels of free Met - more than 200-fold that observed for wild type - yet showed little or no difference in the concentrations of other protein amino-acids, such as aspartate, threonine and lysine. Mutant plants did not show any visible growth differences compared with wild type, except a slight delay in germination. Genetic analysis indicated that the mto3 phenotype was caused by a single, recessive mutation. Positional cloning of this gene revealed that it was a novel S-adenosylmethionine synthetase, SAMS3. A point mutation resulting in a single amino-acid change in the ATP binding domain of SAMS3 was determined to be responsible for the mto3 phenotype. SAMS3 gene expression and total SAMS protein were not changed in mto3; however, both total SAMS activity and S-adenosylmethionine (SAM) concentration were decreased in mto3 compared with wild type. Lignin, a major metabolic sink for SAM, was decreased by 22% in mto3 compared with wild type, presumably due to the reduced supply of SAM. These results suggest that SAMS3 has a different function(s) in one carbon metabolism relative to the other members of the SAMS gene family.

Arabidopsis↗

Lignin-carbohydrate complexes in forages: structure and consequences in the ruminal degradation of cell-wall carbohydrates.

Lignin-carbohydrate complexes (LCCs) are recognised as key structures in forage degradability. Apart from ester bonds involving phenolic acids, which seem to play a major role in grasses, little is known about the other types of linkages that must exist but have proved difficult to demonstrate. The chemical nature of possible LCC linkages is presented and the various mechanisms through which LCCs in the cell-wall architecture may interfere with carbohydrate utilisation by rumen microorganisms are discussed.

Animal Feed↗

Cinnamyl-alcohol dehydrogenase, a molecular marker specific for lignin synthesis: cDNA cloning and mRNA induction by fungal elicitor.

Cinnamyl-alcohol dehydrogenase (CAD; EC 1.1.1.195) catalyzes the final step in a branch of phenylpropanoid synthesis specific for production of lignin monomers. We have isolated a full-length cDNA clone encoding CAD, as a molecular marker specific for lignification, by immunoscreening a lambda gt11 library containing cDNAs complementary to mRNA from elicitor-treated cell cultures of bean (Phaseolus vulgaris L.). The clone comprises a single long open reading frame of 1767 base pairs, 31 base pairs of 5' leader, and 152 base pairs of 3' untranslated sequence. The deduced 65-kDa CAD polypeptide has several features that are strongly conserved in alcohol dehydrogenases. Addition of fungal elicitor to cell cultures stimulates CAD transcription, which leads to a remarkably rapid, but transient, accumulation of CAD mRNA, with no detectable lag and maximal levels after 1.5 hr. Southern blot analysis of bean genomic DNA indicates that elicitor-induced CAD is encoded by a single gene. The regulatory significance of the rapid activation of this CAD gene and the possible existence of a second, divergent CAD gene involved in lignification during xylogenesis are discussed.

Alcohol Oxidoreductases↗

Cloning and characterization of cDNA encoding glyoxal oxidase, a H2O2-producing enzyme from the lignin-degrading basidiomycete Phanerochaete chrysosporium.

Glyoxal oxidase is produced by ligninolytic cultures of the white-rot fungus Phanerochaete chrysosporium and is a source of the extracellular H2O2 that is required by ligninolytic peroxidases. We report here the cloning and characterization of glx-1c cDNA, which encodes glyoxal oxidase. The deduced mature protein has 537 amino acids, a molecular size of 57 kDa, and a pI of 5.1. Five potential N-glycosylation sites are present. The predicted N-terminal sequence is identical to the experimentally determined sequence of purified enzyme and is preceded by a leader peptide of 22 amino acids. The sequence of glx-1c lacks significant homology with known sequences. Specific comparisons were made between the glx-1c translated sequence and that of galactose oxidase from Dactylium dendroides because of previously observed catalytic similarities of the enzyme. Although no significant homology is observed, in both cases extensive beta-sheet regions are predicted from the primary sequences. Glyoxal oxidase activity correlates with transcript levels and is also coordinate with the lignin peroxidases in nutrient nitrogen-starved cultures.

Alcohol Oxidoreductases↗

Coniferyl aldehyde 5-hydroxylation and methylation direct syringyl lignin biosynthesis in angiosperms.

A central question in lignin biosynthesis is how guaiacyl intermediates are hydroxylated and methylated to the syringyl monolignol in angiosperms. To address this question, we cloned cDNAs encoding a cytochrome P450 monooxygenase (LsM88) and a caffeate O-methyltransferase (COMT) from sweetgum (Liquidambar styraciflua) xylem. Mass spectrometry-based functional analysis of LsM88 in yeast identified it as coniferyl aldehyde 5-hydroxylase (CAld5H). COMT expressed in Escherichia coli methylated 5-hydroxyconiferyl aldehyde to sinapyl aldehyde. Together, CAld5H and COMT converted coniferyl aldehyde to sinapyl aldehyde, suggesting a CAld5H/COMT-mediated pathway from guaiacyl to syringyl monolignol biosynthesis via coniferyl aldehyde that contrasts with the generally accepted route to sinapate via ferulate. Although the CAld5H/COMT enzyme system can mediate the biosynthesis of syringyl monolignol intermediates through either route, k(cat)/K(m) of CAld5H for coniferyl aldehyde was approximately 140 times greater than that for ferulate. More significantly, when coniferyl aldehyde and ferulate were present together, coniferyl aldehyde was a noncompetitive inhibitor (K(i) = 0.59 microM) of ferulate 5-hydroxylation, thereby eliminating the entire reaction sequence from ferulate to sinapate. In contrast, ferulate had no effect on coniferyl aldehyde 5-hydroxylation. 5-Hydroxylation also could not be detected for feruloyl-CoA or coniferyl alcohol. Therefore, in the presence of coniferyl aldehyde, ferulate 5-hydroxylation does not occur, and the syringyl monolignol can be synthesized only from coniferyl aldehyde. Endogenous coniferyl, 5-hydroxyconiferyl, and sinapyl aldehydes were detected, consistent with in vivo operation of the CAld5H/COMT pathway from coniferyl to sinapyl aldehydes via 5-hydroxyconiferyl aldehyde for syringyl monolignol biosynthesis.

Cloning, Molecular↗

Remediation of phenol, lignin and paper effluents by advanced oxidative processes.

The tremendous environmental impact of pulping and bleaching effluents and the relatively low efficiency of the current biological remediation processes represent one of the most important problems of the paper industry. In this work the efficiency of heterogeneous and homogeneous advanced oxidative processes was evaluated toward the degradation of model substrates (phenol and lignin) and the remediation of paper effluents. Best results were found by application of the UV-H2O2 system, with almost total discoloration of both pulping and bleaching effluents and typical COD removal higher than 60%, at reaction times of 120 min. In view of the reported results, and mainly on account of the simplicity of the UV-H2O2 system, shows good potential for the advanced process to remediation of recalcitrant effluents like those studied in this present work.

Environmental Pollution↗

Influence of molasses lignin-hemicellulose fractions in rat nutrition.

Previous reports have shown that fractionation of the non-sugar, non-dialyzable components of cane molasses yielded a fraction designated as a black phenolic-carbohydrate complex. Incorporation of 0.03% of this complex into diets fed weanling male rats significantly increased the growth rate above that of rats fed the basal diet alone. This study was conducted to determine the chemical nature and growth stimulating action of the black phenol-carbohydrate complex. Alkaline cleavage under nitrogen yielded a mixture of phenols and a carbohydrate fraction which was recovered by precipitation at pH 6 in four volumes of ethanol. The alkaline cleaved, free hemicellulose was non-diayzable and stimulated growth when incorporated into rat diets at the 0.03% levels. Acid hydrolysis of the complex yielded an insoluble product identified as lignin and found to represent 18% to 20% of the entire complex. The chemical nature of this ligninhemicellulose fraction and the previously reported growth-enhancing acid resistant hemicellulose fraction isolated from various plant sources were found to be similar.

Animal Nutritional Physiological Phenomena↗

Effect of guar gum, lignin and pectin on proteolytic enzyme levels in the gastrointestinal tract of the rat: a time-based study.

The effects of dietary pectin (P), guar gum (G) and lignin (L) on stomach emptying and potential levels of pepsin, trypsin and chymotrypsin during a 2-h period after force-feeding were investigated in growing rats. All of the fibers delayed stomach emptying by 21-26 min. Total potential pepsin activity over 2 h decreased for P (57%), G (44%) and L (20%). In the intestine, total potential trypsin activity over 2 h increased for L (16%) but decreased for P (21%). Total potential chymotrypsin activity over 2 h increased for L (54%) and G (39%). Sixteen to 21% of the variability in intestinal activity over time was statistically attributable to variation in the weight of intestinal contents. The results indicate that fiber components altered proteolytic enzyme levels in the gastrointestinal tract, but the decreased protein utilization previously observed with these fibers is probably not due to reduced levels of intestinal proteases.

Administration, Oral↗

Regulation of enzymes involved in lignin biosynthesis: induction of O-methyltransferase mRNAs during the hypersensitive reaction of tobacco to tobacco mosaic virus.

The mRNAs encoding orthodiphenol-O-methyltransferases (OMTs; EC 2.1.1.6), which are involved in the biosynthesis of lignin precursors, are highly induced in tobacco leaves during the hypersensitive reaction to tobacco mosaic virus (TMV). OMT messengers were fractionated on a sucrose gradient and translated in vitro. Protein A-Sepharose columns adsorbed with specific antisera raised against purified OMTs were used to select translation products, and the translatable activity of OMT mRNA was measured at different stages of infection. Oligonucleotides derived from peptide sequences of purified OMT I were used to prime polymerase chain reactions; total RNA was used as template to allow the isolation of an OMT I clone. RNA blots, hybridized with the OMT I probe, revealed a unique messenger of 1.7 kb. The kinetics of accumulation of OMT I mRNAs during the hypersensitive reaction to TMV parallels the kinetics of translation and suggests that an increase in mRNA controls the increase in the rate of enzyme synthesis. In healthy plants, RNA blot hybridization showed that the steady-state level of OMT I mRNA is very high in vascular tissue compared to the level measured in leaves.

Amino Acid Sequence↗

Identification of a phenolic 3-O-methyltransferase in the lignin-degrading fungus Phanerochaete chrysosporium.

A methyltransferase enzyme catalysing the 3-O-methylation of isovanillic acid (3-hydroxy-4-methoxybenzoic acid) by S-adenosylmethionine (SAM) was identified in Phanerochaete chrysosporium and purified. Gel filtration indicated an M(r) of 71,000 and SDS-PAGE showed that the enzyme was composed of two subunits of M(r) approximately 36,000. Substrate utilization studies demonstrated that the enzyme was highly specific, displaying an exclusive preference for the methylation of the 3-hydroxyl group of several substituted benzoic acids. 3-Hydroxybenzoic acids with a methoxyl or hydroxyl substituent in the 2 or 4 position were the best substrates with isovanillic and 3,4-dihydroxybenzoic acids showing the highest rates of methylation. The 3-O-methyltransferase enzyme was induced later in the growth cycle than the 4-O-methyltransferase previously isolated from this fungus, which is believed to have a role in the 4-O-methylation of lignin degradation products. However the function of this meta-specific enzyme, the first phenolic 3-O-methyltransferase isolated from a fungus, remains unclear. The combined activities of the 3- and 4-O-methyltransferase enzymes satisfactorily account for the pattern of SAM-dependent methylating activity shown by whole mycelia to phenolic substrates.

Basidiomycota↗

Cloning and molecular characterization of the basic peroxidase isoenzyme from Zinnia elegans, an enzyme involved in lignin biosynthesis.

The major basic peroxidase from Zinnia elegans (ZePrx) suspension cell cultures was purified and cloned, and its properties and organ expression were characterized. The ZePrx was composed of two isoforms with a M(r) (determined by matrix-assisted laser-desorption ionization time of flight) of 34,700 (ZePrx34.70) and a M(r) of 33,440 (ZePrx33.44). Both isoforms showed absorption maxima at 403 (Soret band), 500, and 640 nm, suggesting that both are high-spin ferric secretory class III peroxidases. M(r) differences between them were due to the glycan moieties, and were confirmed from the total similarity of the N-terminal sequences (LSTTFYDTT) and by the 99.9% similarity of the tryptic fragment fingerprints obtained by reverse-phase nano-liquid chromatography. Four full-length cDNAs coding for these peroxidases were cloned. They only differ in the 5'-untranslated region. These differences probably indicate different ways in mRNA transport, stability, and regulation. According to the k(cat) and apparent K(m)(RH) values shown by both peroxidases for the three monolignols, sinapyl alcohol was the best substrate, the endwise polymerization of sinapyl alcohol by both ZePrxs yielding highly polymerized lignins with polymerization degrees > or =87. Western blots using anti-ZePrx34.70 IgGs showed that ZePrx33.44 was expressed in tracheary elements, roots, and hypocotyls, while ZePrx34.70 was only expressed in roots and young hypocotyls. None of the ZePrx isoforms was significantly expressed in either leaves or cotyledons. A neighbor-joining tree constructed for the four full-length cDNAs suggests that the four putative paralogous genes encoding the four cDNAs result from duplication of a previously duplicated ancestral gene, as may be deduced from the conserved nature and conserved position of the introns.

Amino Acid Sequence↗

Differential expression of two O-methyltransferases in lignin biosynthesis in Zinnia elegans.

Caffeic acid 3-O-methyltransferase (CAOMT) and caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT) are involved in different methylation pathways in lignin biosynthesis. We previously showed that only the CCoAOMT was markedly induced during lignification in in vitro differentiating tracheary elements (TEs) of Zinnia elegans. To further examine the expression patterns of CAOMT in lignification, we isolated a cDNA clone for Zinnia CAOMT. RNA gel blot analysis showed that the expression of the CAOMT gene did not correlate well with lignification during in vitro TE differentiation from Zinnia-isolated mesophyll cells. Tissue-print hybridization showed that, in the young internodes, the CAOMT mRNA signal was much more evident in phloem fibers than in xylem, whereas the CCoAOMT mRNA signal was predominantly present in differentiating xylem regions. In the older internode, both the CAOMT and CCoAOMT mRNAs markedly accumulated in phloem fibers and differentiating xylem regions. Immunocytochemical localization showed that the CAOMT protein staining was much more evident in phloem fibers and xylem fibers than in xylem TEs. These results indicate that the expression of these two O-methyltransferases is differentially regulated during lignification in different cell types in Zinnia. We suggest that all the intermediates in the CAOMT-mediated methylation pathway might become substrates for the CCoAOMT-mediated methylation pathway after coenzyme A ligation when these two pathways occur in the same lignifying cell types.

Cells, Cultured↗

Reduced phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities and lignin synthesis in wheat grown under low pressure sodium lamps.

Wheat (Triticum aestivum L. cv Fremont) grown in hydroponic culture under 24-hour continuous irradiation at 560 to 580 micromoles per square meter per second from either metalhalide (MH), high pressure sodium (HPS), or low pressure sodium (LPS) lamps reached maturity in 70 days. Grain yields were similar under all three lamps, although LPS-grown plants lodged at maturity. Phenylalanine ammonia-lyase (PAL) and a tyrosine ammonia lyase (TAL) with lesser activity were detected in all extracts of leaf, inflorescence, and stem. Ammonia-lyase activities increased with age of the plant, and plants grown under the LPS lamp displayed PAL and TAL activities lower than wheat cultured under MH and HPS radiation. Greenhouse solar-grown wheat had the highest PAL and TAL activities. Lignin content of LPS-grown wheat was also significantly reduced from that of plants grown under MH or HPS lamps or in the greenhouse, showing a correlation with the reduced PAL and TAL activities. Ratios of far red-absorbing phytochrome to total phytochrome were similar for all three lamps, but the data do not yet warrant a conclusion about specific wavelengths missing from the LPS lamps that might have induced PAL and TAL activities in plants under the other lamps.

Ammonia-Lyases↗

Mutation of a chitinase-like gene causes ectopic deposition of lignin, aberrant cell shapes, and overproduction of ethylene.

Chitinase-like proteins have long been proposed to play roles in normal plant growth and development, but no mutations in chitinase-like genes have been obtained previously to support this hypothesis. In this study, we have shown that the gene responsible for the elp1 mutation in Arabidopsis encodes a chitinase-like protein (AtCTL1). Mutation of this chitinase-like gene caused ectopic deposition of lignin and aberrant shapes of cells with incomplete cell walls in the pith of inflorescence stems. The AtCTL1 gene was expressed in all organs during normal plant growth and development, but it was not induced by wounding, salicylic acid, pectin fragments, or ethylene. Consistent with its ubiquitous expression pattern, mutation of the AtCTL1 gene affected many aspects of plant growth and development, including exaggerated hook curvature, reduced length and increased diameter of hypocotyls in dark-grown seedlings, and reduced root length and increased number of root hairs in light-grown seedlings. The mutant phenotypes could be rescued partially by ethylene inhibitors, and ethylene production in the mutant was significantly greater than in the wild type. Together, these results suggest that AtCTL1, a chitinase-like gene, is essential for normal plant growth and development in Arabidopsis.

Amino Acid Sequence↗

Chalcone epoxide intermediates in the syntheses of lignin-related phenylcoumarans.

Compounds (2R*,3S*)-1-(3,4-dimethoxyphenyl)-3-{3-methoxy-2-[(2R*)-tetrahydropyran-2-yloxy]phenyl}-2,3-epoxy-1-propanone, C23H26O7, (I), and trans-1-(3,4-dimethoxyphenyl)-3-[3-methoxy-2-(methoxymethoxy)phenyl]-2,3-epoxy-1-propanone, C20H22O7, (II), were obtained on epoxidation of chalcones. The stereochemistries of (I) and (II) were elucidated. In both compounds, the substituents on the oxirane ring are trans-oriented. Compound (I) was obtained together with a diastereometric form that differs from (I) with respect to the configuration of the asymmetric C atom in the tetrahydropyran group. The geometries of the substituted oxirane rings of (I) and (II) are very similar. The hydrogen-bonding patterns, mediated via weak C-H...O interactions, differ considerably. The crystal structures of (I) and (II) are compared with those of related chalcone epoxides. The conversion of (I) and (II) into lignin-related phenylcoumarans is discussed.

Benzofurans↗

Electrophoretic karyotyping of the lignin-degrading basidiomycete Phanerochaete chrysosporium.

Electrophoretic karyotyping of the two most widely studied strains of Phanerochaete chrysosporium, BKMF-1767 and ME-446, has been determined using transverse alternating field electrophoresis. The genomic DNA of BKMF-1767 was resolved into 10 chromosomes ranging in size from 1.8-5.0 Mb, amounting to a total genome size of about 29 Mb. The genomic DNA of strain ME-446, on the other hand, was resolved into 11 chromosomes, amounting to a total genome size of about 32 Mb. Lignin peroxidase genes have been localized to five chromosomes in strain BKMF-1767 and to four chromosomes in strain ME-446.

Basidiomycota↗

Global transcript profiling of primary stems from Arabidopsis thaliana identifies candidate genes for missing links in lignin biosynthesis and transcriptional regulators of fiber differentiation.

Different stages of vascular and interfascicular fiber differentiation can be identified along the axis of bolting stems in Arabidopsis. To gain insights into the metabolic, developmental, and regulatory events that control this pattern, we applied global transcript profiling employing an Arabidopsis full-genome longmer microarray. More than 5000 genes were differentially expressed, among which more than 3000 changed more than twofold, and were placed into eight expression clusters based on polynomial regression models. Within these, 182 upregulated transcription factors represent candidate regulators of fiber development. A subset of these candidates has been associated with fiber development and/or secondary wall formation and lignification in the literature, making them targets for functional studies and comparative genomic analyses with woody plants. Analysis of differentially expressed phenylpropanoid genes identified a set known to be involved in lignin biosynthesis. These were used to anchor co-expression analyses that allowed us to identify candidate genes encoding proteins involved in monolignol transport and monolignol dehydrogenation and polymerization. Similar analyses revealed candidate genes encoding enzymes that catalyze missing links in the shikimate pathway, namely arogenate dehydrogenase and prephenate aminotransferase.

Arabidopsis↗