A novel lignin in poplar trees with a reduced caffeic acid/5-hydroxyferulic acid O-methyltransferase activity

Lignin is a polymeric constituent of the cell wall that needs to be removed during the paper making process. Bi-specific caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT) catalyses the O-methylation of caffeic acid and 5-hydroxyferulic acid to ferulic acid and sinapic acid, respectively. These compounds are intermediates in the biosynthesis of the lignin precursors. Therefore, COMTs are potential target enzymes for reducing the amount, or modifying the composition, of lignin in plants. Different antisense and sense constructs have been expressed of a gene encoding a COMT from poplar (Populus trichocarpa x P. deltoides) in a P. tremula x P. alba clone under the control of the cauliflower mosaic virus 35S promoter. From all analysed transformants, four lines transformed with an antisense construct had a reduced COMT activity. Two showed a 50% reduction of COMT activity, which altered only slightly the monomeric composition. In the two other transformants, the COMT activity was reduced by 95%. In the latter case, the syringyl/ guaiacyl ratio (S/G) was reduced by sixfold (due to a decrease of S and an increase of G), as analysed by thioacidolysis. A new component of lignin, the 5-hydroxyguaiacyl residue, was detected among the thioacidolysis products. Moreover, in contrast to the white/yellow colour of wild-type wood, the xylem of the transgenic lines with a 95% reduction of COMT activity was pale rose. A similar phenotype was observed in brown-midrib mutants of maize and sorghum, known for their altered lignification. Although the lignin composition was consistently modified, the lignin content of the transgenic poplars was similar to that of the controls.     

Biochemical characterization of a putative wheat caffeic acid O-methyltransferase

A wheat (Triticum aestivum L., near isogenic line of Hamlet) O-methyltransferase (OMT) was previously reported as a putative caffeic acid OMT (TaCOMT1), involved in lignin biosynthesis, based on its high sequence similarity with a number of graminaceous COMTs. The fact that the putative TaCOMT1 exhibits a significantly high sequence homology to another recently characterized wheat flavone-specific OMT (TaOMT2), and that molecular modeling studies indicated several conserved amino acid residues involved in substrate binding and catalysis of both proteins, prompted an investigation of its appropriate substrate specificity. We report here that TaCOMT1 exhibits highest preference for the flavone tricetin, and lowest activity with the lignin precursors, caffeic acid/5-hydroxyferulic acid as the methyl acceptor molecules, indicating that it is not involved in lignin biosynthesis. We recommend its reannotation to a flavone-specific TaOMT1 that is distinct from TaOMT2.     

Purification and properties of caffeic acid O-methyltransferase from alfalfa root nodules

An O-methyltransferase which catalyses the methylation of caffeic acid to ferulic acid using S-adenosyl-l-methionine as methyl donor has been isolated and purified ca 70-fold from root nodules of alfalfa. The enzyme also catalysed the methylation of 5-hydroxyferulic acid. Chromatography on 1,6-diaminohexane agarose (AH-Sepharose-4B) linked with S-adenosyl-l-homocysteine (SAH) gave 35% recovery of enzyme activity. The K_m values for caffeic acid and S-adenosyl-l-methionine were 58 and 4.1 μM, respectively. S-Adenosyl-l-homocysteine was a potent competitive inhibitor of S-adenosyl-l-methionine with a K_i of 0.44 μM. The MW of the enzyme was ca 103 000 determined by gel filtration chromatography.     

Characterization of bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase from aspen.

Bispecific O-methyltransferase (OMT, EC 2.1.1.68) which catalyses the meta-specific methylation of caffeic acid and 5-hydroxyferulic acid was purified to homogeneity from the developing secondary xylem of aspen (Populus tremuloides). The enzyme was purified by conventional techniques and affinity chromatography on S-adenosyl-L-homocysteine agarose using substrate elution. The enzyme has a M(r) of 40,000 as determined by SDS-PAGE. Amino acid sequences of three peptides produced from a proteolytic digest of bispecific OMT were obtained by automated Edman degradation. Polyclonal antibodies raised against the purified OMT reacted strongly to OMT in both purified and unpurified fractions in western blots. Addition of an equal concentration of anti-OMT IgG to crude extract protein inhibited OMT activity by more than 70%.     

Cloning and functional characterization of a caffeic acid O-methyltransferase from Trigonella foenum-graecum L

A cDNA encoding an O-methyltransferase (namely FGCOMT1) was identified from the medicinal plant Trigonella foenum-graecum L. The FGCOMT1 enzyme is a functional caffeic acid O-methyltransferase (COMT) and is localized in the cytosol. Kinetic analysis indicated that FGCOMT1 protein exhibited the highest catalyzing efficiency towards 5-hydroxy ferulic acid and caffeic acid as substrates, but did not possess the abilities to methylate either quercetin or tricetin in vitro. Furthermore, transformation of Arabidopsis loss-of-function Atomt1 mutant with a FGCOMT1 cDNA partially complements accumulation of sinapoyl derivatives but did not function to produce the major methylated flavonol isorhamnetin in seeds. The results from this study indicated that FGCOMT1 is a COMT with substrate preference to monomeric lignin precursors but is not involved in the flavonoid methylation in T. foenum-graecum L.     

Variation in lignin and cell wall digestibility in caffeic acid O-methyltransferase down-regulated maize half-sib progenies in field experiments

Lignin content and structure were examined in seven caffeic acid O-methyltransferase antisense (COMT-AS) maize progenies and their corresponding normal inbred lines in relation to cell wall digestibility. The seven parental inbreds were chosen for their highly divergent in vitro wall digestibility. Maize plants were grown under field conditions to determine (i) if the positive effect of COMT down-regulation on wall chemistry and digestibility was similar to that previously observed for COMT-AS maize grown in the greenhouse and (ii) to what extent the genetic background was a factor in determining the effect of the transgene. All␣COMT-AS progenies displayed a significant reduction in endogenous COMT activity (14–43% residual activity). In all but one genetic background (F4), the COMT-AS gene resulted in an expected increase in wall digestibility accompanied by changes in lignin composition. These effects varied greatly among parental lines, and independently of the inherent digestibility values in the corresponding non-transformed lines. Curiously, in the highly digestible F4 background, the typical decrease in syringyl (S) unit lignin and a higher frequency of 5-OH guaiacyl lignin resulting from the introduction of the COMT-AS transgene were not observed. Our results indicate that COMT down-regulation via an antisense strategy is an efficient tool for forage maize improvement in the field.     

Down-regulation of caffeic acid o-methyltransferase in maize revisited using a transgenic approach

Transgenic maize (Zea mays) plants were generated with a construct harboring a maize caffeic acid O-methyltransferase (COMT) cDNA in the antisense (AS) orientation under the control of the maize Adh1 (alcohol dehydrogenase) promoter. Adh1-driven beta-glucuronidase expression was localized in vascular tissues and lignifying sclerenchyma, indicating its suitability in transgenic experiments aimed at modifying lignin content and composition. One line of AS plants, COMT-AS, displayed a significant reduction in COMT activity (15%-30% residual activity) and barely detectable amounts of COMT protein as determined by western-blot analysis. In this line, transgenes were shown to be stably integrated in the genome and transmitted to the progeny. Biochemical analysis of COMT-AS showed: (a) a strong decrease in Klason lignin content at the flowering stage, (b) a decrease in syringyl units, (c) a lower p-coumaric acid content, and (d) the occurrence of unusual 5-OH guaiacyl units. These results are reminiscent of some characteristics already observed for the maize bm3 (brown-midrib3) mutant, as well as for COMT down-regulated dicots. However, as compared with bm3, COMT down-regulation in the COMT-AS line is less severe in that it is restricted to sclerenchyma cells. To our knowledge, this is the first time that an AS strategy has been applied to modify lignin biosynthesis in a grass species.     

Enzymatic transformation of caffeic acid with enhanced cyclooxygenase-2 inhibitory activity

Convenient enzymatic transformation of the phenylpropanoid caffeic acid (1) with polyphenol oxidase originating from pear afforded three new oxidized metabolites, caffeoxynic acid (2), caffeicinic acid (3), and isocaffeicinic acid (4), along with, 7,8-erythro-caffeicin (5) and phellinsin A (6). The structures of the three new caffeic acid oxidation products were elucidated on the basis of spectroscopic methods. The new products 2 and 3 exhibited significantly enhanced inhibitory activity against cyclooxygenase-2 (COX-2) when compared to parent caffeic acid.     

Nuclease-producing bacteria in soil cultivated with herbicide resistant transgenic white poplars

This study was carried out using soil cultivated, under greenhouse conditions, with transgenic white poplars expressing thebar gene for tolerance to the Basta® herbicide. The occurrence of extracellular nucleolytic activity was monitored in soil samples collected at four different times over a 26-month period. The fraction of nuclease producing bacteria (NPB) ranged from 62.5 to 100% of the total culturable bacterial population. The DNA-methyl green plate assay allowed to distinguish five groups of bacteria showing increasing levels of extracellular DNase activity. The NPB isolates were classified by 16S rDNA sequence analysis as members of theBacillus, Brevibacillus, Microbacterium, Pseudomonas andStenotrophomonas genera. For each genus, NPB isolates were cultured in liquid medium and the nucleolytic activity during different growth phases was monitored. Production of extracellular nucleases was observed only during the mid-exponential growth phase of theBrevibaccillus Microbacterium andStenotrophomonas isolates, while no activity was evidenced for isolates classified within theBacillus andPseudomonas genera.     

Structure-function analyses of a caffeic acid O-methyltransferase from perennial ryegrass reveal the molecular basis for substrate preference

Lignin forms from the polymerization of phenylpropanoid-derived building blocks (the monolignols), whose modification through hydroxylation and O-methylation modulates the chemical and physical properties of the lignin polymer. The enzyme caffeic acid O-methyltransferase (COMT) is central to lignin biosynthesis. It is often targeted in attempts to engineer the lignin composition of transgenic plants for improved forage digestibility, pulping efficiency, or utility in biofuel production. Despite intensive investigation, the structural determinants of the regiospecificity and substrate selectivity of COMT remain poorly defined. Reported here are x-ray crystallographic structures of perennial ryegrass (Lolium perenne) COMT (Lp OMT1) in open conformational state, apo- and holoenzyme forms and, most significantly, in a closed conformational state complexed with the products S-adenosyl-L-homocysteine and sinapaldehyde. The product-bound complex reveals the post-methyl-transfer organization of COMT's catalytic groups with reactant molecules and the fully formed phenolic-ligand binding site. The core scaffold of the phenolic ligand forges a hydrogen-bonding network involving the 4-hydroxy group that anchors the aromatic ring and thereby permits only metahydroxyl groups to be positioned for transmethylation. While distal from the site of transmethylation, the propanoid tail substituent governs the kinetic preference of ryegrass COMT for aldehydes over alcohols and acids due to a single hydrogen bond donor for the C9 oxygenated moiety dictating the preference for an aldehyde.     

Lignification and structural biomass production in tobacco with suppressed caffeic/5-hydroxy ferulic acid-O-methyl transferase activity under ambient and elevated CO concentrations

To investigate the relationship between growth, biomass partitioning and lignification we used tobacco (Nicotiana tabacum) in which O-methyl transferase (OMT) activity, an enzyme involved in the pathway of sinapyl alcohol formation for lignin synthesis, was suppressed by antisense transformation. To modulate growth, controls and transformed tobacco plants were grown under ambient (approximately 380 p.p.m) or elevated CO(2) (700 p.p.m), respectively. Lignin concentrations and composition were determined with spectrophotometric methods (thioglycolate and acetyl bromide) and Fourier transform infrared (FTIR) spectroscopy, respectively. A comparison of the thioglycolate and acetylbromide method suggested that the thioglycolate method was sensitive to changes in the syringyl/guaiacyl (S/G)-ratio in lignin and therefore not suitable for quantification in tissues with altered lignin composition. Growth under elevated CO(2) increased leaf and stem biomass of both genotypes by 40 and 20%, respectively, compared with ambient CO(2) and had no effect on root biomass. OMT suppression did not affect lignin concentrations in the leaves but caused a shift in biomass partitioning from the structural to the non-structural fraction. Elevated CO(2) caused a shift towards production of structural compounds resulting in decreased foliar lignin concentrations and indicating that the lignin/structural mass ratio is flexible in leaves. By contrast, the lignin concentrations of stems were unaffected by elevated CO(2) or OMT suppression and increased about three-fold from the apex to the base. Antisense-OMT plants produced more stem biomass than controls but showed no changes of the relative partitioning of biomass to the different pools. This indicates that the metabolic control of carbon fluxes to the production of structural versus non-structural fractions is tighter in stems than in leaves. FTIR spectroscopy indicated a relative increase in guaiacyl- as compared with syringyl-units in antisense-OMT tobacco, which was more pronounced under elevated as compared with ambient CO(2). Since there was no evidence for decreased lignin concentrations in the antisense-OMT plants but increased biomass formation we suggest that less methylated lignins are 'cheaper' in biosynthetic carbon and energy demand and, thus, may enable plants to allocate increased resources to growth.     

The brown midrib3 (bm3) mutation in maize occurs in the gene encoding caffeic acid O-methyltransferase.

The brown midrib mutations are among the earliest described in maize. Plants containing a brown midrib mutation exhibit a reddish brown pigmentation of the leaf midrib starting when there are four to six leaves. These mutations are known to alter lignin composition and digestibility of plants and therefore constitute prime candidates in the breeding of silage maize. Here, we show that two independent brown midrib3 (bm3) mutations have resulted from structural changes in the COMT gene, which encodes the enzyme O-methyltransferase (COMT; EC 2.1.1.6), involved in lignin biosynthesis. Our results indicate that the bm3-1 allele (the reference mutant allele) has arisen from an insertional event producing a COMT mRNA altered in both size and amount. By sequencing a COMT cDNA clone obtained from bm3-1 maize, a retrotransposon with homology to the B5 element has been found to be inserted near the junction of the 3' coding region of the COMT gene intron. The second bm3 allele, bm3-2, has resulted from a deletion of part of the COMT gene. These alterations of the COMT gene were confirmed by DNA gel blot and polymerase chain reaction amplification analyses. These results clearly demonstrate that mutations at the COMT gene give a brown midrib3 phenotype. Thus, the gene genetically recognized as bm3 is the same as the one coding for COMT.     

Antiproliferative activity and SARs of caffeic acid esters with mono-substituted phenylethanols moiety

A series of CAPE derivatives with mono-substituted phenylethanols moiety were synthesized and evaluated by MTT assay on growth of 4 human cancer cell lines (Hela, DU-145, MCF-7 and ECA-109). The substituent effects on the antiproliferative activity were systematically investigated for the first time. It was found that electron-donating and hydrophobic substituents at 2′-position of phenylethanol moiety could significantly enhance CAPE’s antiproliferative activity. 2′-Propoxyl derivative, as a novel caffeic acid ester, exhibited exquisite potency (IC₅₀ = 0.4 ± 0.02 & 0.6 ± 0.03 μM against Hela and DU-145 respectively).     

Reduced wood stiffness and strength, and altered stem form, in young antisense 4CL transgenic poplars with reduced lignin contents

? Reduced lignin content in perennial crops has been sought as a means to improve biomass processability for paper and biofuels production, but it is unclear how this could affect wood properties and tree form. ? Here, we studied a nontransgenic control and 14 transgenic events containing an antisense 4-coumarate:coenzyme A ligase (4CL) to discern the consequences of lignin reduction in poplar (Populus sp.). During the second year of growth, trees were grown either free-standing in a field trial or affixed to stakes in a glasshouse. ? Reductions in lignin of up to 40% gave comparable losses in wood strength and stiffness. This occurred despite the fact that low-lignin trees had a similar wood density and up to three-fold more tension wood. In free-standing and staked trees, the control line had twice the height for a given diameter as did low-lignin trees. Staked trees had twice the height for a given diameter as free-standing trees in the field, but did not differ in wood stiffness. ? Variation in tree morphogenesis appears to be governed by lignin x environment interactions mediated by stresses exerted on developing cells. Therefore our results underline the importance of field studies for assessing the performance of transgenic trees with modified wood properties.     

我国抗虫转基因杨树生态安全性研究进展

转基因树木与农作物相比, 人们更关注其长时间种植可能导致转基因扩散到周围野生近缘种。由于生长周期长, 转基因树木会增加转基因不稳定性, 对非靶标生物的影响, 靶标害虫对转基因植物产生抗性, 增加树木入侵性(杂草化), 以及由于基因漂移或基因逃逸对环境产生的负面影响或新的环境风险。过去十几年, 针对我国抗食叶害虫的两个商业化转Bt基因欧洲黑杨(Populus nigra)和转双抗虫基因741杨[P. alba× (P. davidiana + P. simonii) × P. tomentosa], 已开展了有关生态安全性方面的多项研究。本文围绕抗虫转基因树木生态安全性研究进展进行了综述。抗虫转基因杨树对节肢动物种群和群落结构产生了一定影响, 使昆虫的多样性提高, 但对土壤微生物区系未见明显影响。转基因欧洲黑杨通过花粉和种子发生的基因漂移几率很低。转基因杨树通过内生菌发生的水平转移可能会对环境造成的潜在危险也进行了评价。文章最后指出对抗虫转基因杨树农林复合生态系统开展生物安全研究的必要性。     

Purification and properties of S-adenosyl-L-methionine: caffeic acid O-methyltransferase from leaves of spinach beet (Beta vulgaris L).

1. An enzyme catalysing the methylation of caffeic acid to ferulic acid, using S-adenosyl-L-methionine as methyl donor, has been extracted from leaves of spinach beet and purified 75-fold to obtain a stable preparation. 2. The enzyme showed optimum activity at pH 6.5, and did not require the addition of Mg2+ for maximum activity. 3. It was most active with caffeic acid, but showed some activity with catechol, protocatechuic acid and 3,4-dihydroxybenzaldehyde. The Km for caffeic acid was 68 muM. 4. 4. The Km for S-adenosyl-L-methionine was 12.5 muM. S-Adenosyl-L-homocystein (Ki = 4.4 muM) was a competitive inhibitor of S-adenosyl-L-methionine. 5. The synthesis of S-adenosyl-L-homocysteine from adenosine and L-homocysteine and its consequent effect on caffeic acid methylation were demonstrated with a partially-purified preparation from spinach-beet leaves, which possessed both S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) and adenosine nucleosidase (EC 3.2.2.7) activities. This preparation was also able to catalyse the rapid breakdown of S-adenosyl-L-homocysteine to adenosine and adenine; the possible significance of this reaction in relieving the inhibition of caffeic acid methylation by S-adenosyl-L-homocystein is discussed.