Although finely divided ball-milled whole cell walls do not completely dissolve in dimethylsulfoxide (DMSO), they readily swell producing a gel. Solution-state two-dimensional (2D) nuclear magnetic resonance (NMR) of this gel, produced directly in the NMR tube, provides an interpretable structural fingerprint of the polysaccharide and lignin components of the wall without actual solubilization, and without structural modification beyond that inflicted by the ball milling and ultrasonication steps. Since the cellulose is highly crystalline and difficult to swell, the component may be under-represented in the spectra. The method however provides a more rapid method for comparative structural evaluation of plant cell walls than is currently available. With the new potential for chemometric analysis using the 2D NMR fingerprint, this method may find application as a secondary screen for selecting biomass lines and for optimizing biomass processing and conversion efficiencies.

Poplar (Populus tremula 3 alba) lignins with exceedingly high syringyl monomer levels are produced by overexpression of the ferulate 5-hydroxylase (F5H) gene driven by a cinnamate 4-hydroxylase (C4H) promoter. Compositional data derived from both standard degradative methods and NMR analyses of the entire lignin component (as well as isolated lignin fraction) indicated that the C4H::F5H transgenic's lignin was comprised of as much as 97.5% syringyl units (derived from sinapyl alcohol), the remainder being guaiacyl units (derived from coniferyl alcohol); the syringyl level in the wild-type control was 68%. The resultant transgenic lignins are more linear and display a lower degree of polymerization. Although the crucial b-ether content is similar, the distribution of other interunit linkages in the lignin polymer is markedly different, with higher resinol (b-b) and spirodienone (b-1) contents, but with virtually no phenylcoumarans (b-5, which can only be formed from guaiacyl units). p-Hydroxybenzoates, acylating the g-positions of lignin side chains, were reduced by .50%, suggesting consequent impacts on related pathways. A model depicting the putative structure of the transgenic lignin resulting from the overexpression of F5H is presented. The altered structural features in the transgenic lignin polymer, as revealed here, support the contention that there are significant opportunities to improve biomass utilization by exploiting the malleability of plant lignification processes.

The ensemble of all phenolics for which the biosynthesis is coregulated with lignin biosynthesis, i.e., metabolites from the general phenylpropanoid, monolignol, and (neo)-lignan biosynthetic pathways and their derivatives, as well as the lignin oligomers, is coined the lignome. In lignifying tissues, the lignome comprises a significant portion of the metabolome. However, as is true for metabolomics in general, the structural elucidation of unknowns represents the biggest challenge in characterizing the lignome. To minimize the necessity to purify unknowns for NMR analysis, it would be desirable to be able to extract structural information from liquid chromatography-mass spectrometry data directly. However, mass spectral libraries for metabolomics are scarce, and no libraries exist for the lignome. Therefore, elucidating the gas-phase fragmentation behavior of the major bonding types encountered in lignome-associated molecules would considerably advance the systematic characterization of the lignome. By comparative MS n analysis of a series of molecules belonging to the -aryl ether, benzodioxane, phenylcoumaran, and resinol groups, we succeeded in annotating typical fragmentations for each of these bonding structures as well as fragmentations that enabled the identification of the aromatic units involved in each bonding structure. Consequently, this work lays the foundation for a detailed characterization of the lignome in different plant species, mutants, and transgenics and for the MS-based sequencing of lignin oligomers and (neo)lignans.Lignin is an aromatic heteropolymer that is mainly present in secondary-thickened plant cell walls where it provides the necessary strength and hydrophobicity for plants to grow in an upward direction and to enable the transport of water, nutrients, and photoassimilates. Lignin is mainly composed of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units derived from the combinatorial coupling of p-coumaryl, coniferyl, and sinapyl alcohols ( Figure 1A), 1 the so-called monolignols that are produced by the general phenylpropanoid and monolignol biosynthetic pathways. [2][3][4][5] Following oxidation by peroxidase and/or laccase, the resulting electron-delocalized monolignol radical has unpaired electron density at its 1-, 3-, O-4-, 5-, and 8-positions ( Figure 1B). As radical coupling at the 8-position is favored, coupling with another monolignol radical results in, after rearomatization, a mixture of dehydrodimers with 8-8′, 8-5′, and 8-O-4′ linkages ( Figure 1C).In addition to these major monomers, several other monomers have been identified in particular species or in plants with modified lignin biosynthesis, 1,3 such as 5-hydroxyconiferyl alcohol in caffeic acid O-methyltransferase (COMT) downregulated transgenic plants, 6,7 dihydroconiferyl alcohol in cinnamyl alcohol dehydrogenase (CAD) deficient loblolly pine, 8 acylated monolignols, such as sinapyl p-hydroxybenzoate in poplar, 9 and hydroxycinnamic acid or hydroxycinnamate esters, such as feruloyl tyramine in tobacco....

Decapentaplegic (Dpp), a Drosophila homologue of bone morphogenetic proteins, acts as a morphogen to regulate patterning along the anterior-posterior axis of the developing wing. Previous studies showed that Dally, a heparan sulfate proteoglycan, regulates both the distribution of Dpp morphogen and cellular responses to Dpp. However, the molecular mechanism by which Dally affects the Dpp morphogen gradient remains to be elucidated. Here, we characterized activity, stability, and gradient formation of a truncated form of Dpp (Dpp(Delta N)), which lacks a short domain at the N-terminus essential for its interaction with Dally. Dpp(Delta N) shows the same signaling activity and protein stability as wild-type Dpp in vitro but has a shorter half-life in vivo, suggesting that Dally stabilizes Dpp in the extracellular matrix. Furthermore, genetic interaction experiments revealed that Dally antagonizes the effect of Thickveins (Tkv; a Dpp type I receptor) on Dpp signaling. Given that Tkv can downregulate Dpp signaling by receptor-mediated endocytosis of Dpp, the ability of dally to antagonize tkv suggests that Dally inhibits this process. Based on these observations, we propose a model in which Dally regulates Dpp distribution and signaling by disrupting receptor-mediated internalization and degradation of the Dpp-receptor complex.

Down-regulation of the gene encoding 4-coumarate 3-hydroxylase (C3H) in alfalfa massively but predictably increased the proportion of p-hydroxyphenyl (P) units relative to the normally dominant guaiacyl (G) and syringyl (S) units. Stem levels of up to ϳ65% P (from wild-type levels of ϳ1%) resulting from down-regulation of C3H were measured by traditional degradative analyses as well as two-dimensional 13 C-1 H correlative NMR methods. Such levels put these transgenics well beyond the P:G:S compositional bounds of normal plants; p-hydroxyphenyl levels are reported to reach a maximum of 30% in gymnosperm severe compression wood zones but are limited to a few percent in dicots. NMR also revealed structural differences in the interunit linkage distribution that characterizes a lignin polymer. Lower levels of key ␤-aryl ether units were relatively augmented by higher levels of phenylcoumarans and resinols. The C3H-deficient alfalfa lignins were devoid of ␤-1 coupling products, highlighting the significant differences in the reaction course for p-coumaryl alcohol versus the two normally dominant monolignols, coniferyl and sinapyl alcohols. A larger range of dibenzodioxocin structures was evident in conjunction with an approximate doubling of their proportion. The nature of each of the structural units was revealed by long range 13 C-1 H correlation experiments. For example, although ␤-ethers resulted from the coupling of all three monolignols with the growing polymer, phenylcoumarans were formed almost solely from coupling reactions involving p-coumaryl alcohol; they resulted from both coniferyl and sinapyl alcohol in the wild-type plants. Such structural differences form a basis for explaining differences in digestibility and pulping performance of C3H-deficient plants.The effects on lignin structure of perturbing one crucial step in the monolignol biosynthetic pathway remain to be addressed. Genes encoding all of the enzymes in Fig. 1 have been identified, and the effects of perturbing (by down-and/or up-regulation in transgenic plants or via their knockouts in mutants) all but the p-coumarate 3-hydroxylase (C3H) 2 /hydroxycinnamoyl transferase (HCT) steps have been studied in some detail, as reviewed in Refs. 1-3. Down-regulation of some genes, particularly those early in the pathway, may limit the overall flux of metabolites into lignin. In other cases, the distribution of units resulting from the primary monomers (the three monolignols p-coumaryl 1a, coniferyl 1b, and sinapyl 1c alcohols, differing in their degree of methoxylation; Fig. 1) may be dramatically altered, sometimes far beyond the limits that have been observed in nature. In some intriguing cases, lignification appears to be able to accommodate phenolics (e.g. 5-hydroxyconiferyl alcohol) that are not normally considered to be lignin monomers when the biosynthesis of the normal monolignols is thwarted (1, 3, 4). Such studies are not only providing rich insights into the lignification process, but are also opening up opportunities for improving the util...

The enzyme hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (HCT) is involved in the production of methoxylated monolignols that are precursors to guaiacyl and syringyl lignin in angiosperm species. We identified and cloned a putative HCT gene from Pinus radiata, a coniferous gymnosperm that does not produce syringyl lignin. This gene was up-regulated during tracheary element (TE) formation in P. radiata cell cultures and showed 72.6% identity to the amino acid sequence of the Nicotiana tabacum HCT isolated earlier. RNAi-mediated silencing of the putative HCT gene had a strong impact on lignin content, monolignol composition, and interunit linkage distribution. AcBr assays revealed an up to 42% reduction in lignin content in TEs. Pyrolysis-GC/MS, thioacidolysis, and NMR detected substantial changes in lignin composition. Most notable was the rise of p-hydroxyphenyl units released by thioacidolysis, which increased from trace amounts in WT controls to up to 31% in transgenics. Two-dimensional 13 C-1 H correlative NMR confirmed the increase in p-hydroxyphenyl units in the transgenics and revealed structural differences, including an increase in resinols, a reduction in dibenzodioxocins, and the presence of glycerol end groups. The observed modifications in silenced transgenics validate the targeted gene as being associated with lignin biosynthesis in P. radiata and thus likely to encode HCT. This enzyme therefore represents the metabolic entry point leading to the biosynthesis of methoxylated phenylpropanoids in angiosperm species and coniferous gymnosperms such as P. radiata.lignin ͉ HCT ͉ tracheary elements T he global trend toward a biomaterials-based economy makes plant cell walls increasingly important as renewable resources for the production of biofuels and biocomposites. Lignin is the second most abundant terrestrial biopolymer after cellulose and a major structural component of cell walls in woodforming tissues (1). The content, composition, and structure of lignin all have considerable impact on the utilization of plantderived materials and have therefore been the subject of intensive research (1, 2). Lignins are heterogeneous cell wall polymers derived primarily from hydroxycinnamyl alcohols via combinatorial radical coupling reactions (3). Typically, they make up 20-30% of the cell wall material in woody tissue of both angiosperm and gymnosperm species. Lignin in coniferous gymnosperms such as Pinus radiata does not contain syringyl (S) components, which makes it different from lignin of many other vascular plants including angiosperms (4).We have developed a P. radiata callus culture system to better assign function to genes associated with cell wall-related processes such as lignification in conifers. These callus cultures can be transformed and subsequently induced to differentiate into tracheary elements (TEs), the main cellular components of wood in conifer species (5). The biochemical composition of cell wall polymers in differentiated TEs is similar to those produced in P. radiata wood...

As a central model for morphogen action during animal development, the bone morphogenetic protein 2/4 (BMP2/4)-like ligand Decapentaplegic (Dpp) is proposed to form a long-range signalling gradient that directs both growth and pattern formation during Drosophila wing disc development. While the patterning role of Dpp secreted from a stripe of cells along the anterior-posterior compartmental boundary is well established, the mechanism by which a Dpp gradient directs uniform cell proliferation remains controversial and poorly understood. Here, to determine the precise spatiotemporal requirements for Dpp during wing disc development, we use CRISPR-Cas9-mediated genome editing to generate a flippase recognition target (FRT)-dependent conditional null allele. By genetically removing Dpp from its endogenous stripe domain, we confirm the requirement of Dpp for the activation of a downstream phospho-Mothers against dpp (p-Mad) gradient and the regulation of the patterning targets spalt (sal), optomotor blind (omb; also known as bifid) and brinker (brk). Surprisingly, however, third-instar wing blade primordia devoid of compartmental dpp expression maintain relatively normal rates of cell proliferation and exhibit only mild defects in growth. These results indicate that during the latter half of larval development, the Dpp morphogen gradient emanating from the anterior-posterior compartment boundary is not directly required for wing disc growth.

The proportion of erythro- and threo-forms of β-O-4-structures in lignin was elucidated by ozonation analysis of 21 wood species, and the relationship to the syringyl and guaiacyl composition was investigated. For all hardwood species, the erythro-form of β-O-4-structures predominated, although the extent varied widely, depending on wood species. In contrast, the proportion and amount of erythro- and threo-forms were very similar in all softwood species. The proportion of the erythro-form was greater in species with a higher methoxyl content in the lignin (correlation coefficient, R2=0.83). The S/V ratio (molar ratio of syringaldehyde and syringic acid to that of vanillin and vanillic acid) obtained by nitrobenzene oxidation was also strongly correlated with the proportion of the erythro-form (R2=0.99). Accordingly, the syringyl/guaiacyl ratio is closely related to the erythro/threo ratio. This stereochemical characteristic of β-O-4-structures is discussed in relation to the process of lignin formation.