RNAi Modulation of Chlorogenic Acid and Lignin Deposition in Nicotiana tabacum and Insufficient Compensatory Metabolic Cross-Talk.

Chlorogenic acid (CGA) and guaiacyl/syringyl (G/S) lignin formation involves hydroxycinnamoyl ester intermediacy, the latter formed via hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferase (HCT) and hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase (HQT) activities. HQT and HCT RNAi silencing of a commercial tobacco (Nicotiana tabacum) K326 line was examined herein. NtHQT gene silencing gave relatively normal plant phenotypes, with CGA levels reduced (down to 1% of wild type) with no effects on lignin. RNAi NtHCT silencing had markedly adverse phenotypes (e.g., stunted, multiple stems, delayed flowering, with senescence delayed by several months). Lignin contents were partially lowered, with a small increase in cleavable p-hydroxyphenyl (H) monomers; those plants had no detectable CGA level differences relative to wild type. In vitro NtHCT kinetic parameters revealed preferential p-coumaroyl CoA and shikimate esterification, as compared to other structurally related potential acyl group donors and acceptors. In the presence of coenzyme A, NtHCT catalyzed the reverse reaction. Site-directed mutagenesis of NtHCT (His153Ala) abolished enzymatic activity. NtHQT, by comparison, catalyzed preferential conversion of p-coumaroyl CoA and quinic acid to form p-coumaroyl quinate, the presumed CGA precursor. In sum, metabolic pathways to CGA and lignins appear to be fully independent, and previous conflicting reports of substrate versatilities and metabolic cross-talk are resolved.

Insights into lignin primary structure and deconstruction from Arabidopsis thaliana COMT (caffeic acid O-methyl transferase) mutant Atomt1.

The Arabidopsis mutant Atomt1 lignin differs from native lignin in wild type plants, in terms of sinapyl (S) alcohol-derived substructures in fiber cell walls being substituted by 5-hydroxyconiferyl alcohol (5OHG)-derived moieties. During programmed lignin assembly, these engender formation of benzodioxane substructures due to intramolecular cyclization of their quinone methides that are transiently formed following 8-O-4' radical-radical coupling. Thioacidolytic cleavage of the 8-O-4' inter-unit linkages in the Atomt1 mutant, relative to the wild type, indicated that cleavable sinapyl (S) and coniferyl (G) alcohol-derived monomeric moieties were stoichiometrically reduced by a circa 2 : 1 ratio. Additionally, lignin degradative analysis resulted in release of a 5OHG-5OHG-G trimer from the Atomt1 mutant, which then underwent further cleavage. Significantly, the trimeric moiety released provides new insight into lignin primary structure: during polymer assembly, the first 5OHG moiety is linked via a C8-O-X inter-unit linkage, whereas subsequent addition of monomers apparently involves sequential addition of 5OHG and G moieties to the growing chain in a 2 : 1 overall stoichiometry. This quantification data thus provides further insight into how inter-unit linkage frequencies in native lignins are apparently conserved (or near conserved) during assembly in both instances, as well as providing additional impetus to resolve how the overall question of lignin macromolecular assembly is controlled in terms of both type of monomer addition and primary sequence.

Plant cell walls are enfeebled when attempting to preserve native lignin configuration with poly-p-hydroxycinnamaldehydes: evolutionary implications.

The lignin deficient double mutant of cinnamyl alcohol dehydrogenase (CAD, cad-4, cad-5 or cad-c, cad-d) in Arabidopsis thaliana [Sibout, R., Eudes, A., Mouille, G., Pollet, B., Lapierre, C., Jouanin, L., Séguin, A., 2005. Cinnamyl alcohol dehydrogenase-C and -D are the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis. Plant Cell 17, 2059-2076], was comprehensively examined for effects on disruption of native lignin macromolecular configuration; the two genes encode the catalytically most active CAD's for monolignol/lignin formation [Kim, S.-J., Kim, M.-R., Bedgar, D.L., Moinuddin, S.G.A., Cardenas, C.L., Davin, L.B., Kang, C., Lewis, N.G., 2004. Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis. Proc. Natl. Acad. Sci., USA 101, 1455-1460]. The inflorescence stems of the double mutant presented a prostrate phenotype with dynamic modulus properties greatly reduced relative to that of the wild type (WT) line due to severe reductions in macromolecular lignin content. Interestingly, initially the overall pattern of phenolic deposition in the mutant was apparently very similar to WT, indicative of comparable assembly processes attempting to be duplicated. However, shortly into the stage involving (monomer cleavable) 8-O-4' linkage formation, deposition was aborted. At this final stage, the double mutant had retained a very limited ability to biosynthesize monolignols as evidenced by cleavage and release of ca. 4% of the monolignol-derived moieties relative to the lignin of the WT line. In addition, while small amounts of cleavable p-hydroxycinnamaldehyde-derived moieties were released, the overall frequency of (monomer cleavable) 8-O-4' inter-unit linkages closely approximated that of WT for the equivalent level of lignin deposition, in spite of the differences in monomer composition. Additionally, 8-5' linked inter-unit structures were clearly evident, albeit as fully aromatized phenylcoumaran-like substructures. The data are interpreted as a small amount of p-hydroxycinnamaldehydes being utilized in highly restricted attempts to preserve native lignin configuration, i.e. through very limited monomer degeneracy during template polymerization which would otherwise afford lignins proper in the cell wall from their precursor monolignols. The defects introduced (e.g. in the vascular integrity) provide important insight as to why p-hydroxycinnamaldehydes never evolved as lignin precursors in the 350,000 or so extant vascular plant species. It is yet unknown at present, however, as to what levels of lignin reduction can be attained in order to maintain the requisite properties for successful agronomic/forestry cultivation. Nor is it known to what extent, if any, such deleterious modulations potentially compromise plant defenses. Finally, prior to investigating lignin primary structure proper, it is essential to initially define the fundamental characteristics of the biopolymer(s) being formed, such as inter-unit frequency and lignin content, in order to design approaches to determine overall sequences of linkages.

Reassessment of effects on lignification and vascular development in the irx4 Arabidopsis mutant.

The Arabidopsis thaliana irregular xylem4 (irx4) cinnamoyl-CoA reductase 1 (CCR1) mutant was reassessed for its purported exclusive rate-limiting or key effects on lignification. Analyses of gross growth characteristics and stem cross-section anatomy, from seedling emergence to senescence, revealed that stunted irx4 mutant lines were developmentally delayed, which in turn indirectly but predictably led to modest reductions (ca. 10-15%) in overall lignin amounts. Such developmental changes are not generally observed in suppression of other monolignol pathway forming enzymes (e.g., 4-coumarate CoA ligase) even when accompanied by significant reductions in lignin amounts. With the greatly arrested development of the irx4 mutant, formation of the lignin-derived syringyl moieties was also predictably delayed (by about 1-2 weeks), although at maturation the final guaiacyl:syringyl ratios were essentially identical to wild-type. No evidence was obtained for so-called abnormal lignin precursors being incorporated into the lignin, as shown by solid-state 13C NMR spectroscopic analysis in contrast to a claim to the contrary [Jones, L., Ennos, A.R., Turner, S.R., 2001. Cloning and characterization of irregular xylem4 (irx4): a severely lignin-deficient mutant of Arabidopsis. Plant J. 26, 205-216]. A previous claim of an "abnormal" lignin present in stunted CCR downregulated tobacco was also not substantiated, with only trace differences being noted in the presumed cell-wall constituent levels. More importantly, a linear correlation between total lignin amounts and lignin-derived fragmentation products was observed at all stages of Arabidopsis growth/development in both wild-type and irx4 mutant lines, regardless of lignin content, i.e., in harmony with an exquisitely controlled and predictable macromolecular assembly process. Recombinant CCR1 displayed fairly broad substrate versatility for all phenylpropanoid CoA substrates, with both feruloyl and 5-hydroxyferuloyl CoA being the best substrates. Taken together, these data indicate that other CCR isoforms are apparently capable of generating monolignol-derived lignified elements in irx4 when CCR1 is impaired, i.e., indicative of a functionally redundant CCR metabolic network operative in Arabidopsis. Other dwarfed phenotypes have also been observed following downregulation/disruption of unrelated metabolic processes but which also involve CoA ester metabolism, i.e., with hydroxymethylglutaryl CoA reductases in Arabidopsis and a bacterial enoyl CoA hydratase/lyase overexpressed in tobacco. Although the reasons for dwarfing in each case are unknown, a common mechanism for the various pleiotropic effects is proposed through perturbation of CoASH pool levels. Finally, this study demonstrates the need for progressive analyses over the lifespan of an organism, rather than at a single time point which cannot reveal the progressive developmental changes occurring.

Characterization of lignin derived from water-only and dilute acid flowthrough pretreatment of poplar wood at elevated temperatures.

BACKGROUND: Flowthrough pretreatment of biomass is a critical step in lignin valorization via conversion of lignin derivatives to high-value products, a function vital to the economic efficiency of biorefinery plants. Comprehensive understanding of lignin behaviors and solubilization chemistry in aqueous pretreatment such as water-only and dilute acid flowthrough pretreatment is of fundamental importance to achieve the goal of providing flexible platform for lignin utilization. RESULTS: In this study, the effects of flowthrough pretreatment conditions on lignin separation from poplar wood were reported as well as the characteristics of three sub-sets of lignin produced from the pretreatment, including residual lignin in pretreated solid residues (ReL), recovered insoluble lignin in pretreated liquid (RISL), and recovered soluble lignin in pretreatment liquid (RSL). Both the water-only and 0.05 % (w/w) sulfuric acid pretreatments were performed at temperatures from 160 to 270 °C on poplar wood in a flowthrough reactor system for 2-10 min. Results showed that water-only flowthrough pretreatment primarily removed syringyl (S units). Increased temperature and/or the addition of sulfuric acid enhanced the removal of guaiacyl (G units) compared to water-only pretreatments at lower temperatures, resulting in nearly complete removal of lignin from the biomass. Results also suggested that more RISL was recovered than ReL and RSL in both dilute acid and water-only flowthrough pretreatments at elevated temperatures. NMR spectra of the RISL revealed significant ß-O-4 cleavage, α-ß deoxygenation to form cinnamyl-like end groups, and slight ß-5 repolymerization in both water-only and dilute acid flowthrough pretreatments. CONCLUSIONS: Elevated temperature and/or dilute acid greatly enhanced lignin removal to almost 100 % by improving G unit removal besides S unit removal in flowthrough system. Only mild lignin structural modification was caused by flowthrough pretreatment. A lignin transformation pathway was proposed to explain the complexity of the lignin structural changes during hot water and dilute acid flowthrough pretreatment.Graphical abstractLignin transformations in water-only and dilute acid flowthrough pretreatment at elevated temperatures.

Controlling porosity in lignin-derived nanoporous carbon for supercapacitor applications.

Low-cost renewable lignin has been used as a precursor to produce porous carbons. However, to date, it has not been easy to obtain high surface area porous carbon without activation processes or templating agents. Here, we demonstrate that low molecular weight lignin yields highly porous carbon with more graphitization through direct carbonization without additional activation processes or templating agents. We found that molecular weight and oxygen consumption during carbonization are critical factors to obtain high surface area, graphitized porous carbons. This highly porous carbon from low-cost renewable lignin sources is a good candidate for supercapacitor electrode materials.

Microwave-induced enzyme-catalyzed chemoselective reduction of organic azides.

A lipase enzyme, suspended in organic media along with organic azides and irradiated under microwaves, enhances the reaction rate over thermal heating and affords the corresponding amines in high yields. The present biocatalytic method employing lipase is a significant development with remarkable regio- and chemoselectivity under microwave irradiation in organic media with excellent yields for the reduction of azide functionality.

Tetramethylammonium hydroxide (TMAH) thermochemolysis for probing in situ softwood lignin modification in each gut segment of the termite.

Termites are highly effective in lignocellulose degradation; however, the process of lignin deconstruction along the alimentary canal is not well understood. In this study, the wood metabolites in each gut segment were tentatively analyzed using pyrolysis-gas chromatography-mass spectrometry in the presence of tetramethylammonium hydroxide. Collectively, the significant differences in the pyrolysate distribution among each sample established (1) conservation of the major ß-O-4' bonds of lignin during termite digestion, although a selective lignin substructure modification was observed across the whole gut; (2) initiation of lignin-polysaccharide dissociation, aliphatic oxidation/carboxylation, phenolic dehydroxylation in the foregut, and linkage modification of the 5-5', ß-5', and ß-1' substructures; (3) the continuation of foregut reactions into the midgut with further phenolic carboxylation/demethoxylation/carbonylation; and (4) phenolic/aliphatic esterifications in the hindgut. Overall, elucidation of the stepwise lignin unlocking mechanism in termites provides a valuable insight for understanding plant cell wall structure and its recalcitrance.

Advanced biorefinery in lower termite-effect of combined pretreatment during the chewing process.

BACKGROUND: Currently the major barrier in biomass utilization is the lack of an effective pretreatment of plant cell wall so that the carbohydrates can subsequently be hydrolyzed into sugars for fermentation into fuel or chemical molecules. Termites are highly effective in degrading lignocellulosics and thus can be used as model biological systems for studying plant cell wall degradation. RESULTS: We discovered a combination of specific structural and compositional modification of the lignin framework and partial degradation of carbohydrates that occurs in softwood with physical chewing by the termite, Coptotermes formosanus, which are critical for efficient cell wall digestion. Comparative studies on the termite-chewed and native (control) softwood tissues at the same size were conducted with the aid of advanced analytical techniques such as pyrolysis gas chromatography mass spectrometry, attenuated total reflectance Fourier transform infrared spectroscopy and thermogravimetry. The results strongly suggest a significant increase in the softwood cellulose enzymatic digestibility after termite chewing, accompanied with utilization of holocellulosic counterparts and an increase in the hydrolysable capacity of lignin collectively. In other words, the termite mechanical chewing process combines with specific biological pretreatment on the lignin counterpart in the plant cell wall, resulting in increased enzymatic cellulose digestibility in vitro. The specific lignin unlocking mechanism at this chewing stage comprises mainly of the cleavage of specific bonds from the lignin network and the modification and redistribution of functional groups in the resulting chewed plant tissue, which better expose the carbohydrate within the plant cell wall. Moreover, cleavage of the bond between the holocellulosic network and lignin molecule during the chewing process results in much better exposure of the biomass carbohydrate. CONCLUSION: Collectively, these data indicate the participation of lignin-related enzyme(s) or polypeptide(s) and/or esterase(s), along with involvement of cellulases and hemicellulases in the chewing process of C. formosanus, resulting in an efficient pretreatment of biomass through a combination of mechanical and enzymatic processes. This pretreatment could be mimicked for industrial biomass conversion.

Structural and thermal characterization of wheat straw pretreated with aqueous ammonia soaking.

Production of renewable fuels and chemicals from lignocellulosic feedstocks requires an efficient pretreatment technology to allow ready access of polysaccharides for cellulolytic enzymes during saccharification. The effect of pretreatment on wheat straw through a low-temperature and low-pressure soaking aqueous ammonia (SAA) process was investigated in this study using Fourier transform infrared (FTIR), pyrolysis-gas chromatography/mass spectroscopy (Py-GC/MS), solid and liquid state nuclear magnetic resonance (NMR), and thermogravimetry/differential thermogravimetry (TG/DTG) to demonstrate the changes in lignin, hemicellulose, and cellulose structure. After treatment of 60 mesh wheat straw particles for 60 h with 28-30% ammonium hydroxide (1:10 solid/liquid) at 50 °C, sugar recovery increased from 14% (untreated) to 67% (SAA treated). The FTIR study revealed a substantial decrease in absorbance of lignin peaks. Solid and liquid state NMR showed minimal lignin structural changes with significant compositional changes. Activation energy of control and pretreated wheat straw was calculated according to the Friedman and ASTM methods and found to be decreased for SAA-treated wheat straw, from 259 to 223 kJ/mol. The SAA treatment was shown to remove significant amounts of lignin without strongly affecting lignin functional groups or structure.

In situ lignocellulosic unlocking mechanism for carbohydrate hydrolysis in termites: crucial lignin modification.

BACKGROUND: Termites are highly effective at degrading lignocelluloses, and thus can be used as a model for studying plant cell-wall degradation in biological systems. However, the process of lignin deconstruction and/or degradation in termites is still not well understood. METHODS: We investigated the associated structural modification caused by termites in the lignin biomolecular assembly in softwood tissues crucial for cell-wall degradation. We conducted comparative studies on the termite-digested (i.e. termite feces) and native (control) softwood tissues with the aid of advanced analytical techniques: 13C crosspolarization magic angle spinning and nuclear magnetic resonance (CP-MAS-NMR) spectroscopy, flash pyrolysis with gas chromatography mass spectrometry (Py-GC/MS), and Py-GC-MS in the presence of tetramethylammonium hydroxide (Py-TMAH)-GC/MS. RESULTS: The 13C CP/MAS NMR spectroscopic analysis revealed an increased level of guaiacyl-derived (G unit) polymeric framework in the termite-digested softwood (feces), while providing specific evidence of cellulose degradation. The Py-GC/MS data were in agreement with the 13C CP/MAS NMR spectroscopic studies, thus indicating dehydroxylation and modification of selective intermonomer side-chain linkages in the lignin in the termite feces. Moreover, Py-TMAH-GC/MS analysis showed significant differences in the product distribution between control and termite feces. This strongly suggests that the structural modification in lignin could be associated with the formation of additional condensed interunit linkages. CONCLUSION: Collectively, these data further establish: 1) that the major ß-O-4' (ß-aryl ether) was conserved, albeit with substructure degeneracy, and 2) that the nature of the resulting polymer in termite feces retained most of its original aromatic moieties (G unit-derived). Overall, these results provide insight into lignin-unlocking mechanisms for understanding plant cell-wall deconstruction, which could be useful in development of new enzymatic pretreatment processes mimicking the termite system for biochemical conversion of lignocellulosic biomass to fuels and chemicals.

Probing native lignin macromolecular configuration in Arabidopsis thaliana in specific cell wall types: further insights into limited substrate degeneracy and assembly of the lignins of ref8, fah 1-2 and C4H::F5H lines.

The interest in renewable, plant-derived, bioenergy/biofuels has resulted in a renaissance of plant cell-wall/lignin research. Herein, effects of modulating lignin monomeric compositions in a single plant species, Arabidopsis, are described. The earliest stage of putative "AcBr/Klason lignin" deposition was apparently unaffected by modulating p-coumarate 3-hydroxylase or ferulate 5-hydroxylase activities. This finding helps account for the inability of many other studies to fully suppress the reported putative levels of lignin deposition through monolignol biosynthesis manipulation, and also underscores limitations in frequently used lignin analytical protocols. The overall putative lignin content was greatly reduced (circa 62%) in a plant line harboring an H-(p-hydroxyphenyl) enriched lignin phenotype. This slightly increased H-monomer deposition level apparently occurred in cell-wall domains normally harboring guaiacyl (G) and/or syringyl (S) lignin moieties. For G- and S-enriched lignin phenotypes, the overall lignification process appeared analogous to wild type, with only xylem fiber and interfascicular fiber cells forming the S-enriched lignins. Laser microscope dissection of vascular bundles and interfascicular fibers, followed by pyrolysis GC/MS, supported these findings. Some cell types, presumably metaxylem and possibly protoxylem, also afforded small amounts of benzodioxane (sub)structures due to limited substrate degeneracy (i.e. utilizing 5-hydroxyconiferyl alcohol rather than sinapyl alcohol). For all plant lines studied, the 8-O-4' inter-unit frequency of cleavable H, G and/or S monomers was essentially invariant of monomeric composition for a given (putative) lignin content. These data again underscore the need for determination of lignin primary structures and identification of all proteins/enzymes involved in control of lignin polymer assembly/macromolecular configuration.

Phenylalanine biosynthesis in Arabidopsis thaliana. Identification and characterization of arogenate dehydratases.

There is much uncertainty as to whether plants use arogenate, phenylpyruvate, or both as obligatory intermediates in Phe biosynthesis, an essential dietary amino acid for humans. This is because both prephenate and arogenate have been reported to undergo decarboxylative dehydration in plants via the action of either arogenate (ADT) or prephenate (PDT) dehydratases; however, neither enzyme(s) nor encoding gene(s) have been isolated and/or functionally characterized. An in silico data mining approach was thus undertaken to attempt to identify the dehydratase(s) involved in Phe formation in Arabidopsis, based on sequence similarity of PDT-like and ACT-like domains in bacteria. This data mining approach suggested that there are six PDT-like homologues in Arabidopsis, whose phylogenetic analyses separated them into three distinct subgroups. All six genes were cloned and subsequently established to be expressed in all tissues examined. Each was then expressed as a Nus fusion recombinant protein in Escherichia coli, with their substrate specificities measured in vitro. Three of the resulting recombinant proteins, encoded by ADT1 (At1g11790), ADT2 (At3g07630), and ADT6 (At1g08250), more efficiently utilized arogenate than prephenate, whereas the remaining three, ADT3 (At2g27820), ADT4 (At3g44720), and ADT5 (At5g22630) essentially only employed arogenate. ADT1, ADT2, and ADT6 had k(cat)/Km values of 1050, 7650, and 1560 M(-1) S(-1) for arogenate versus 38, 240, and 16 M(-1) S(-1) for prephenate, respectively. By contrast, the remaining three, ADT3, ADT4, and ADT5, had k(cat)/Km values of 1140, 490, and 620 M(-1) S(-1), with prephenate not serving as a substrate unless excess recombinant protein (>150 microg/assay) was used. All six genes, and their corresponding proteins, are thus provisionally classified as arogenate dehydratases and designated ADT1-ADT6.

The Arabidopsis cinnamoyl CoA reductase irx4 mutant has a delayed but coherent (normal) program of lignification.

Previous studies have indicated that the Arabidopsis thalianairregular xylem 4 (irx4) mutant is severely lignin-deficient, forming abnormal lignin from aberrant monomers. Studies of lignin structure in dwarfed cinnamoyl CoA reductase (CCR)-downregulated tobacco were also previously reported to incorporate feruloyl tyramine derivatives. The lignin in the Arabidopsis irx4 mutant was re-investigated at 6 weeks and at maturation (9 weeks). Application of (1)H, (13)C, 2D Heteronuclear Multiple Quantum Coherence and 2D Heteronuclear Multiple Bond Coherence spectroscopic analyses to the lignin-enriched isolates from both Arabidopsis wild-type (Ler) and the CCR-irx4 mutant at both developmental stages revealed that only typical guaiacyl/syringyl lignins were formed. For the irx4 mutant, the syringyl content at 6 weeks growth was lower, in accordance with a delayed but coherent program of lignification. At maturation, however, the syringyl/guaiacyl ratio of the irx4 mutant approached that of wild-type. There was no evidence for feruloyl tyramines, or homologues thereof, accumulating as a chemical signature in lignins resulting from CCR mutation. Nor were there any noticeable increases in other phenolic components, such as hydroxycinnamic acids. These findings were further confirmed by application of thioacidolysis, alkaline nitrobenzene oxidation and acetyl bromide analyses. Moreover, in the case of CCR downregulation in tobacco, there were no NMR spectroscopic correlations that demonstrated feruloyl tyramines being incorporated into the lignin biopolymers. This study thus found no evidence that abnormal lignin formation occurs when CCR activity is modulated.