Down-regulation Of Caffeic Acid O-methyltransferase Research Articles

Development and field assessment of transgenic hybrid switchgrass for improved biofuel traits

Development of commercially relevant bioenergy switchgrass cultivars requires reducing recalcitrance for bioprocessing without compromising biomass yield. Low-lignin transgenic switchgrass has been produced via down-regulation of caffeic acid O-methyltransferase (COMT), a lignin biosynthetic enzyme, or by over-expression of the MYB4 transcription factor, a repressor of the lignin biosynthetic pathway. The aim of this study was to evaluate parents and selected hybrids obtained from COMT and MYB4 hybrid families under field conditions for agronomic performance and biomass quality. Plant height, width, number of tillers, dry weight, cell wall composition including lignin content, and sugar release were measured after the establishment year (2014) and the second growing season (2015). For COMT hybrids, biomass yield of the transgenic hybrids was similar to or greater than the wild-type parents selected for high biomass. Lignin content of COMT transgenic hybrids was reduced by 10%, S/G ratio decreased by 27%, and sugar release increased between 20% and 44% compared to their wild-type parents. These results indicate that hybridization of COMT with a high-yielding locally selected genotype resulted in both improved agronomic performance and enhanced biomass quality in the offspring. On the other hand, the MYB transgenic hybrid showed a 10% reduction in biomass yield compared with its wild-type parent in year 1, but not in year 2. The lignin S/G ratio was not reduced in MYB transgenic hybrids, nor was sugar release increased. These data indicate that the MYB transgene may not be suitable for an agronomic setting. More testing is needed of transgenic and wild-type, high-biomass selections for use as breeding parents. These results show that combining low-lignin transgenic switchgrass with a breeding and selection program for biomass yield will allow for the deployment of effective transgenes in high-yielding genetic backgrounds.

Analytical Py-GC/MS of Genetically Modified Poplar for the Increased Production of Bio-aromatics

Genetic engineering is a powerful tool to steer bio-oil composition towards the production of speciality chemicals such as guaiacols, syringols, phenols, and vanillin through well-defined biomass feedstocks. Our previous work demonstrated the effects of lignin biosynthesis gene modification on the pyrolysis vapour compositions obtained from wood derived from greenhouse-grown poplars. In this study, field-grown poplars downregulated in the genes encoding CINNAMYL ALCOHOL DEHYDROGENASE (CAD), CAFFEIC ACID O-METHYLTRANSFERASE (COMT) and CAFFEOYL-CoA O-METHYLTRANSFERASE (CCoAOMT), and their corresponding wild type were pyrolysed in a Py-GC/MS. This work aims at capturing the effects of downregulation of the three enzymes on bio-oil composition using principal component analysis (PCA). 3,5-methoxytoluene, vanillin, coniferyl alcohol, 4-vinyl guaiacol, syringol, syringaldehyde, and guaiacol are the determining factors in the PCA analysis that are the substantially affected by COMT, CAD and CCoAOMT enzyme downregulation. COMT and CAD downregulated transgenic lines proved to be statistically different from the wild type because of a substantial difference in S and G lignin units. The sCAD line lead to a significant drop (nearly 51%) in S-lignin derived compounds, while CCoAOMT downregulation affected the least (7–11%). Further, removal of extractives via pretreatment enhanced the statistical differences among the CAD transgenic lines and its wild type. On the other hand, COMT downregulation caused 2-fold reduction in S-derived compounds compared to G-derived compounds. This study manifests the applicability of PCA analysis in tracking the biological changes in biomass (poplar in this case) and their effects on pyrolysis-oil compositions.

Simultaneous Downregulation of MTHFR and COMT in Switchgrass Affects Plant Performance and Induces Lesion-Mimic Cell Death.

Switchgrass (Panicum virgatum) has been developed into a model lignocellulosic bioenergy crop. Downregulation of caffeic acid O-methyltransferase (COMT), a key enzyme in lignin biosynthesis, has been shown to alter lignification and increase biofuel yield in switchgrass. Methylenetetrahydrofolate reductase (MTHFR) mediates C1 metabolism and provides methyl units consumed by COMT. It was predicted that co-silencing of MTHFR and COMT would impact lignification even more than either of the single genes. However, our results showed that strong downregulation of MTHFR in a COMT-deficient background led to altered plant growth and development, but no significant change in lignin content or composition was found when compared with COMT plants. Another unexpected finding was that the double MTHFR/COMT downregulated plants showed a novel lesion-mimic leaf phenotype. Molecular analyses revealed that the lesion-mimic phenotype was caused by the synergistic effect of MTHFR and COMT genes, with MTHFR playing a predominant role. Microarray analysis showed significant induction of genes related to oxidative and defense responses. The results demonstrated the lack of additive effects of MTHFR and COMT on lignification. Furthermore, this research revealed an unexpected role of the two genes in the modulation of lesion-mimic cell death as well as their synergistic effects on agronomic performance.

Study of traits and recalcitrance reduction of field-grown COMT down-regulated switchgrass

BackgroundThe native recalcitrance of plants hinders the biomass conversion process using current biorefinery techniques. Down-regulation of the caffeic acid O-methyltransferase (COMT) gene in the lignin biosynthesis pathway of switchgrass reduced the thermochemical and biochemical conversion recalcitrance of biomass. Due to potential environmental influences on lignin biosynthesis and deposition, studying the consequences of physicochemical changes in field-grown plants without pretreatment is essential to evaluate the performance of lignin-altered plants. We determined the chemical composition, cellulose crystallinity and the degree of its polymerization, molecular weight of hemicellulose, and cellulose accessibility of cell walls in order to better understand the fundamental features of why biomass is recalcitrant to conversion without pretreatment. The most important is to investigate whether traits and features are stable in the dynamics of field environmental effects over multiple years.ResultsField-grown COMT down-regulated plants maintained both reduced cell wall recalcitrance and lignin content compared with the non-transgenic controls for at least 3 seasons. The transgenic switchgrass yielded 35–84% higher total sugar release (enzymatic digestibility or saccharification) from a 72-h enzymatic hydrolysis without pretreatment and also had a 25–32% increase in enzymatic sugar release after hydrothermal pretreatment. The COMT-silenced switchgrass lines had consistently lower lignin content, e.g., 12 and 14% reduction for year 2 and year 3 growing season, respectively, than the control plants. By contrast, the transgenic lines had 7–8% more xylan and galactan contents than the wild-type controls. Gel permeation chromatographic results revealed that the weight-average molecular weights of hemicellulose were 7–11% lower in the transgenic than in the control lines. In addition, we found that silencing of COMT in switchgrass led to 20–22% increased cellulose accessibility as measured by the Simons’ stain protocol. No significant changes were observed on the arabinan and glucan contents, cellulose crystallinity, and cellulose degree of polymerization between the transgenic and control plants. With the 2-year comparative analysis, both the control and transgenic lines had significant increases in lignin and glucan contents and hemicellulose molecular weight across the growing seasons.ConclusionsThe down-regulation of COMT in switchgrass resulting in a reduced lignin content and biomass recalcitrance is stable in a field-grown trial for at least three seasons. Among the determined affecting factors, the reduced biomass recalcitrance of the COMT-silenced switchgrass, grown in the field conditions for two and three seasons, was likely related to the decreased lignin content and increased biomass accessibility, whereas the cellulose crystallinity and degree of its polymerization and hemicellulose molecular weights did not contribute to the reduction of recalcitrance significantly. This finding suggests that lignin down-regulation in lignocellulosic feedstock confers improved saccharification that translates from greenhouse to field trial and that lignin content and biomass accessibility are two significant factors for developing a reduced recalcitrance feedstock by genetic modification.

Potential of genetically engineered hybrid poplar for pyrolytic production of bio-based phenolic compounds

Wild-type and two genetically engineered hybrid poplar lines were pyrolyzed in a micro-pyrolysis (Py-GC/MS) and a bench scale setup for fast and intermediate pyrolysis studies. Principal component analysis showed that the pyrolysis vapors obtained by micro-pyrolysis from wood of caffeic acid O-methyltransferase (COMT) and caffeoyl-CoA O-methyltransferase (CCoAOMT) down-regulated poplar trees differed significantly from the pyrolysis vapors obtained from non-transgenic control trees. Both fast micro-pyrolysis and intermediate pyrolysis of transgenic hybrid poplars showed that down-regulation of COMT can enhance the relative yield of guaiacyl lignin-derived products, while the relative yield of syringyl lignin-derived products was up to a factor 3 lower. This study indicates that lignin engineering via genetic modifications of genes involved in the phenylpropanoid and monolignol biosynthetic pathways can help to steer the pyrolytic production of guaiacyl and syringyl lignin-derived phenolic compounds such as guaiacol, 4-methylguaiacol, 4-ethylguaiacol, 4-vinylguaiacol, syringol, 4-vinylsyringol, and syringaldehyde present in the bio-oil.

Two-year field analysis of reduced recalcitrance transgenic switchgrass.

Switchgrass (Panicum virgatum L.) is a leading candidate for a dedicated lignocellulosic biofuel feedstock owing to its high biomass production, wide adaptation and low agronomic input requirements. Lignin in cell walls of switchgrass, and other lignocellulosic feedstocks, severely limits the accessibility of cell wall carbohydrates to enzymatic breakdown into fermentable sugars and subsequently biofuels. Low-lignin transgenic switchgrass plants produced by the down-regulation of caffeic acid O-methyltransferase (COMT), a lignin biosynthetic enzyme, were analysed in the field for two growing seasons. COMT transcript abundance, lignin content and the syringyl/guaiacyl lignin monomer ratio were consistently lower in the COMT-down-regulated plants throughout the duration of the field trial. In general, analyses with fully established plants harvested during the second growing season produced results that were similar to those observed in previous greenhouse studies with these plants. Sugar release was improved by up to 34% and ethanol yield by up to 28% in the transgenic lines relative to controls. Additionally, these results were obtained using senesced plant material harvested at the end of the growing season, compared with the young, green tissue that was used in the greenhouse experiments. Another important finding was that transgenic plants were not more susceptible to rust (Puccinia emaculata). The results of this study suggest that lignin down-regulation in switchgrass can confer real-world improvements in biofuel yield without negative consequences to biomass yield or disease susceptibility.

Phenolic profiling of caffeic acid O-methyltransferase-deficient poplar reveals novel benzodioxane oligolignols.

Caffeic acid O-methyltransferase (COMT) catalyzes preferentially the methylation of 5-hydroxyconiferaldehyde to sinapaldehyde in monolignol biosynthesis. Here, we have compared HPLC profiles of the methanol-soluble phenolics fraction of xylem tissue from COMT-deficient and control poplars (Populus spp.), using statistical analysis of the peak heights. COMT down-regulation results in significant concentration differences for 25 of the 91 analyzed peaks. Eight peaks were exclusively detected in COMT-deficient poplar, of which four could be purified for further identification using mass spectrometry/mass spectrometry, nuclear magnetic resonance, and spiking of synthesized reference compounds. These new compounds were derived from 5-hydroxyconiferyl alcohol or 5-hydroxyconiferaldehyde and were characterized by benzodioxane moieties, a structural type that is also increased in the lignins of COMT-deficient plants. One of these four benzodioxanes amounted to the most abundant oligolignol in the HPLC profile. Furthermore, all of the differentially accumulating oligolignols involving sinapyl units were either reduced in abundance or undetectable. The concentration levels of all identified oligolignols were in agreement with the relative supply of monolignols and with their chemical coupling propensities, which supports the random coupling hypothesis. Chiral HPLC analysis of the most abundant benzodioxane dimer revealed the presence of both enantiomers in equal amounts, indicating that they were formed by radical coupling reactions under simple chemical control rather than guided by dirigent proteins.

Transgenic down-regulation of caffeic acid O-methyltransferase (COMT) led to improved digestibility in tall fescue (Festuca arundinacea).

Dry matter digestibility is one of the most important characteristics of forage. The major constraint on ruminant digestion of forage cell walls is lignin. Sequences of cDNA encoding a key lignin biosynthetic enzyme, caffeic acid O-methyltransferase (COMT), was cloned from the widely grown monocot forage species tall fescue (Festuca arundinacea Schreb.). Enzymatic properties of recombinant COMT protein expressed in E. coli were determined using six substrates. The preferred substrates for tall fescue recombinant COMT were 5-hydroxyferulic acid and caffeoyl aldehyde. Transgenic tall fescue plants carrying either sense or antisense COMT gene constructs were obtained by microprojectile bombardment of single-genotype-derived embryogenic suspension cells. Consistent and closely related molecular and biochemical data demonstrated that two co-suppressed transgenic lines were down-regulated in their lignin biosynthesis. These COMT down-regulated transgenic tall fescue plants showed substantially reduced levels of transcripts, significantly reduced enzymatic activities, significantly decreased lignin content, apparently altered lignin composition and significantly increased (9.8-10.8%) digestibility.

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.

Substrate preferences of O-methyltransferases in alfalfa suggest new pathways for 3-O-methylation of monolignols.

Measurement of relative O-methyltransferase activities against all potential substrates in the monolignol pathway in developing alfalfa stem extracts revealed activities in the order: caffeoyl CoA > caffeoyl alcohol > 5-hydroxyferulic acid > caffeoyl aldehyde > 5-hydroxyconiferyl alcohol > 5-hydroxyferuloyl CoA > 5-hydroxyconiferaldehyde > caffeic acid. Maxima for all activities occurred in the seventh internode. In stem extracts from transgenic alfalfa with antisense downregulated caffeoyl CoA O-methyltransferase (CCoAOMT), activities with all substrates except for the two coenzyme A esters were unaffected. In contrast, downregulation of caffeic acid O-methyltransferase (COMT) reduced activities against the non-esterifed substrates in the order: 5-hydroxyconiferyl alcohol > 5-hydroxyferulic acid and caffeoyl alcohol > caffeoyl aldehyde > caffeic acid > 5-hydroxyconiferaldehyde. Recombinant COMT expressed in Escherichia coli exhibited the highest V(max)/K(m) values with 5-hydroxyconiferaldehyde and caffeoyl aldehyde, and the lowest with caffeic acid. These results indicate that COMT is unlikely to methylate caffeic acid during lignin biosynthesis in vivo, and provide enzymatic evidence for an alternative pathway to monolignols involving methylation of caffeoyl aldehyde and/or caffeoyl alcohol by COMT. The concept of independent pathways to guaiacyl and syringyl monolignols is discussed.