Enhancement of Secondary Cell Wall Formation in Poplar Xylem Using a Self-Reinforced System of Secondary Cell Wall-Related Transcription Factors.

The secondary cell wall (SCW) in the xylem is one of the largest sink organs of carbon in woody plants, and is considered a promising sustainable bioresource for biofuels and biomaterials. To enhance SCW formation in poplar (Populus sp.) xylem, we developed a self-reinforced system of SCW-related transcription factors from Arabidopsis thaliana, involving VASCULAR-RELATED NAC-DOMAIN7 (VND7), SECONDARY WALL-ASSOCIATED NAC-DOMAIN PROTEIN 1/NAC SECONDARY WALL THICKENING-PROMOTING FACTOR3 (SND1/NST3), and MYB46. In this system, these transcription factors were fused with the transactivation domain VP16 and expressed under the control of the Populus trichocarpa CesA18 (PtCesA18) gene promoter, creating the chimeric genes PtCesA18pro::AtVND7:VP16, PtCesA18pro::AtSND1:VP16, and PtCesA18pro::AtMYB46:VP16. The PtCesA18 promoter is active in tissues generating SCWs, and can be regulated by AtVND7, AtSND1, and AtMYB46; thus, the expression levels of PtCesA18pro::AtVND7:VP16, PtCesA18pro::AtSND1:VP16, and PtCesA18pro::AtMYB46:VP16 are expected to be boosted in SCW-generating tissues. In the transgenic hybrid aspens (Populus tremula × tremuloides T89) expressing PtCesA18pro::AtSND1:VP16 or PtCesA18pro::AtMYB46:VP16 grown in sterile half-strength Murashige and Skoog growth medium, SCW thickening was significantly enhanced in the secondary xylem cells, while the PtCesA18pro::AtVND7:VP16 plants showed stunted xylem formation, possibly because of the enhanced programmed cell death (PCD) in the xylem regions. After acclimation, the transgenic plants were transferred from the sterile growth medium to pots of soil in the greenhouse, where only the PtCesA18pro::AtMYB46:VP16 aspens survived. A nuclear magnetic resonance footprinting cell wall analysis and enzymatic saccharification analysis demonstrated that PtCesA18pro::AtMYB46:VP16 influences cell wall properties such as the ratio of syringyl (S) and guaiacyl (G) units of lignin, the abundance of the lignin ß-aryl ether and resinol bonds, and hemicellulose acetylation levels. Together, these data indicate that we have created a self-reinforced system using SCW-related transcription factors to enhance SCW accumulation.

Overexpression of VviPGIP1 and NtCAD14 in Tobacco Screened Using Glycan Microarrays Reveals Cell Wall Reorganisation in the Absence of Fungal Infection.

The expression of Vitis vinifera polygalacturonase inhibiting protein 1 (VviPGIP1) in Nicotiana tabacum has been linked to modifications at the cell wall level. Previous investigations have shown an upregulation of the lignin biosynthesis pathway and reorganisation of arabinoxyloglucan composition. This suggests cell wall tightening occurs, which may be linked to defence priming responses. The present study used a screening approach to test four VviPGIP1 and four NtCAD14 overexpressing transgenic lines for cell wall alterations. Overexpressing the tobacco-derived cinnamyl alcohol dehydrogenase (NtCAD14) gene is known to increase lignin biosynthesis and deposition. These lines, particularly PGIP1 expressing plants, have been shown to lead to a decrease in susceptibility towards grey rot fungus Botrytis cinerea. In this study the aim was to investigate the cell wall modulations that occurred prior to infection, which should highlight potential priming phenomena and phenotypes. Leaf lignin composition and relative concentration of constituent monolignols were evaluated using pyrolysis gas chromatography. Significant concentrations of lignin were deposited in the stems but not the leaves of NtCAD14 overexpressing plants. Furthermore, no significant changes in monolignol composition were found between transgenic and wild type plants. The polysaccharide modifications were quantified using gas chromatography (GC-MS) of constituent monosaccharides. The major leaf polysaccharide and cell wall protein components were evaluated using comprehensive microarray polymer profiling (CoMPP). The most significant changes appeared at the polysaccharide and protein level. The pectin fraction of the transgenic lines had subtle variations in patterning for methylesterification epitopes for both VviPGIP1 and NtCAD14 transgenic lines versus wild type. Pectin esterification levels have been linked to pathogen defence in the past. The most marked changes occurred in glycoprotein abundance for both the VviPGIP1 and NtCAD14 lines. Epitopes for arabinogalactan proteins (AGPs) and extensins were notably altered in transgenic NtCAD14 tobacco.

ELIMÄKI Locus Is Required for Vertical Proprioceptive Response in Birch Trees.

Tree architecture has evolved to support a top-heavy above-ground biomass, but this integral feature poses a weight-induced challenge to trunk stability. Maintaining an upright stem is expected to require vertical proprioception through feedback between sensing stem weight and responding with radial growth. Despite its apparent importance, the principle by which plant stems respond to vertical loading forces remains largely unknown. Here, by manipulating the stem weight of downy birch (Betula pubescens) trees, we show that cambial development is modulated systemically along the stem. We carried out a genetic study on the underlying regulation by combining an accelerated birch flowering program with a recessive mutation at the ELIMÄKI locus (EKI), which causes a mechanically defective response to weight stimulus resulting in stem collapse after just 3 months. We observed delayed wood morphogenesis in eki compared with WT, along with a more mechanically elastic cambial zone and radial compression of xylem cell size, indicating that rapid tissue differentiation is critical for cambial growth under mechanical stress. Furthermore, the touch-induced mechanosensory pathway was transcriptionally misregulated in eki, indicating that the ELIMÄKI locus is required to integrate the weight-growth feedback regulation. By studying this birch mutant, we were able to dissect vertical proprioception from the gravitropic response associated with reaction wood formation. Our study provides evidence for both local and systemic responses to mechanical stimuli during secondary plant development.

Functional metabolomics as a tool to analyze Mediator function and structure in plants.

Mediator is a multiprotein transcriptional co-regulator complex composed of four modules; Head, Middle, Tail, and Kinase. It conveys signals from promoter-bound transcriptional regulators to RNA polymerase II and thus plays an essential role in eukaryotic gene regulation. We describe subunit localization and activities of Mediator in Arabidopsis through metabolome and transcriptome analyses from a set of Mediator mutants. Functional metabolomic analysis based on the metabolite profiles of Mediator mutants using multivariate statistical analysis and heat-map visualization shows that different subunit mutants display distinct metabolite profiles, which cluster according to the reported localization of the corresponding subunits in yeast. Based on these results, we suggest localization of previously unassigned plant Mediator subunits to specific modules. We also describe novel roles for individual subunits in development, and demonstrate changes in gene expression patterns and specific metabolite levels in med18 and med25, which can explain their phenotypes. We find that med18 displays levels of phytoalexins normally found in wild type plants only after exposure to pathogens. Our results indicate that different Mediator subunits are involved in specific signaling pathways that control developmental processes and tolerance to pathogen infections.

Sll1783, a monooxygenase associated with polysaccharide processing in the unicellular cyanobacterium Synechocystis PCC 6803.

Cyanobacteria play a pivotal role as the primary producer in many aquatic ecosystems. The knowledge on the interacting processes of cyanobacteria with its environment - abiotic and biotic factors - is still very limited. Many potential exocytoplasmic proteins in the model unicellular cyanobacterium Synechocystis PCC 6803 have unknown functions and their study is essential to improve our understanding of this photosynthetic organism and its potential for biotechnology use. Here we characterize a deletion mutant of Synechocystis PCC 6803, Δsll1783, a strain that showed a remarkably high light resistance which is related with its lower thylakoid membrane formation. Our results suggests Sll1783 to be involved in a mechanism of polysaccharide degradation and uptake and we hypothesize it might function as a sensor for cell density in cyanobacterial cultures.

High-throughput microanalysis of large lignocellulosic sample sets by pyrolysis-gas chromatography/mass spectrometry.

High-throughput analytical techniques to assess the chemistry of lignocellulosic plant material are crucial to plant cell-wall research. We have established an analytical platform for this purpose and demonstrated its usefulness with two applications. The system is based on analytical pyrolysis, coupled to gas chromatography/mass spectrometry - a technique particularly suited for analysis of lignocellulose. Automated multivariate-based data-processing methods are used to obtain results within a few hours after analysis, with an experimental batch of 500 analyzed samples. The usefulness of multivariate sample discrimination methods and hierarchical clustering of samples is demonstrated. We have analyzed an Arabidopsis mutant collection consisting of 300 samples representing 31 genotypes. The mutant collection is presented through cluster analysis, based on chemotypic difference, with respect to wild type. Further, we have analyzed 500 thin sections from five biological replicate trees to create a spatial highly resolved profile of the proportions of syringyl-, guaiacyl- and p-hydroxyphenyl lignin across phloem, developing and mature wood in aspen. The combination of biologically easy to interpret information, the low demand of sample amount and the flexibility in sample types amenable to analysis makes this technique a valuable extension to the range of established high-throughput biomaterial analytical platforms.

High-throughput characterization of sediment organic matter by pyrolysis-gas chromatography/mass spectrometry and multivariate curve resolution: A promising analytical tool in (paleo)limnology.

Molecular-level chemical information about organic matter (OM) in sediments helps to establish the sources of OM and the prevalent degradation/diagenetic processes, both essential for understanding the cycling of carbon (C) and of the elements associated with OM (toxic trace metals and nutrients) in lake ecosystems. Ideally, analytical methods for characterizing OM should allow high sample throughput, consume small amounts of sample and yield relevant chemical information, which are essential for multidisciplinary, high-temporal resolution and/or large spatial scale investigations. We have developed a high-throughput analytical method based on pyrolysis-gas chromatography/mass spectrometry and automated data processing to characterize sedimentary OM in sediments. Our method consumes 200 µg of freeze-dried and ground sediment sample. Pyrolysis was performed at 450°C, which was found to avoid degradation of specific biomarkers (e.g., lignin compounds, fresh carbohydrates/cellulose) compared to 650°C, which is in the range of temperatures commonly applied for environmental samples. The optimization was conducted using the top ten sediment samples of an annually resolved sediment record (containing 16-18% and 1.3-1.9% of total carbon and nitrogen, respectively). Several hundred pyrolytic compound peaks were detected of which over 200 were identified, which represent different classes of organic compounds (i.e., n-alkanes, n-alkenes, 2-ketones, carboxylic acids, carbohydrates, proteins, other N compounds, (methoxy)phenols, (poly)aromatics, chlorophyll and steroids/hopanoids). Technical reproducibility measured as relative standard deviation of the identified peaks in triplicate analyses was 5.5±4.3%, with 90% of the RSD values within 10% and 98% within 15%. Finally, a multivariate calibration model was calculated between the pyrolytic degradation compounds and the sediment depth (i.e., sediment age), which is a function of degradation processes and changes in OM source type. This allowed validation of the Py-GC/MS dataset against fundamental processes involved in OM cycling in aquatic ecosystems.

Expression of a fungal glucuronoyl esterase in Populus: effects on wood properties and saccharification efficiency.

The secondary walls of angiosperms contain large amounts of glucuronoxylan that is thought to be covalently linked to lignin via ester bonds between 4-O-methyl-α-D-glucuronic acid (4-O-Me-GlcA) moieties in glucuronoxylan and alcohol groups in lignin. This linkage is proposed to be hydrolysed by glucuronoyl esterases (GCEs) secreted by wood-degrading fungi. We report effects of overexpression of a GCE from the white-rot basidiomycete Phanerochaete carnosa, PcGCE, in hybrid aspen (Populus tremula L. x tremuloides Michx.) on the wood composition and the saccharification efficiency. The recombinant enzyme, which was targeted to the plant cell wall using the signal peptide from hybrid aspen cellulase PttCel9B3, was constitutively expressed resulting in the appearance of GCE activity in protein extracts from developing wood. Diffuse reflectance FT-IR spectroscopy and pyrolysis-GC/MS analyses showed significant alternation in wood chemistry of transgenic plants including an increase in lignin content and S/G ratio, and a decrease in carbohydrate content. Sequential wood extractions confirmed a massive (+43%) increase of Klason lignin, which was accompanied by a ca. 5% decrease in cellulose, and ca. 20% decrease in wood extractives. Analysis of the monosaccharide composition using methanolysis showed a reduction of 4-O-Me-GlcA content without a change in Xyl contents in transgenic lines, suggesting that the covalent links between 4-O-Me-GlcA moieties and lignin protect these moieties from degradation. Enzymatic saccharification without pretreatment resulted in significant decreases of the yields of Gal, Glc, Xyl and Man in transgenic lines, consistent with their increased recalcitrance caused by the increased lignin content. In contrast, the enzymatic saccharification after acid pretreatment resulted in Glc yields similar to wild-type despite of their lower cellulose content. These data indicate that whereas PcGCE expression in hybrid aspen increases lignin deposition, the inhibitory effects of lignin are efficiently removed during acid pretreatment, and the extent of wood cellulose conversion during hydrolysis after acid pretreatment is improved in the transgenic lines possible due to reduced cell wall cross-links between cell wall biopolymers by PcGCE.

Deficient sucrose synthase activity in developing wood does not specifically affect cellulose biosynthesis, but causes an overall decrease in cell wall polymers.

The biosynthesis of wood in aspen (Populus) depends on the metabolism of sucrose, which is the main transported form of carbon from source tissues. The largest fraction of the wood biomass is cellulose, which is synthesized from UDP-glucose. Sucrose synthase (SUS) has been proposed previously to interact directly with cellulose synthase complexes and specifically supply UDP-glucose for cellulose biosynthesis. To investigate the role of SUS in wood biosynthesis, we characterized transgenic lines of hybrid aspen with strongly reduced SUS activity in developing wood. No dramatic growth phenotypes in glasshouse-grown trees were observed, but chemical fingerprinting with pyrolysis-GC/MS, together with micromechanical analysis, showed notable changes in chemistry and ultrastructure of the wood in the transgenic lines. Wet chemical analysis showed that the dry weight percentage composition of wood polymers was not changed significantly. However, a decrease in wood density was observed and, consequently, the content of lignin, hemicellulose and cellulose was decreased per wood volume. The decrease in density was explained by a looser structure of fibre cell walls as shown by increased wall shrinkage on drying. The results show that SUS is not essential for cellulose biosynthesis, but plays a role in defining the total carbon incorporation to wood cell walls.

High resolution metabolite imaging in the hippocampus following neonatal exposure to the environmental toxin BMAA using ToF-SIMS.

The environmental neurotoxin ß-N-methylamino-L-alanine (BMAA) is suggested to be linked with neurodegenerative disease. In a rat model, neonatal exposure to BMAA induced selective uptake in the hippocampus and caused cell loss, mineralization and astrogliosis as well as learning and memory impairments in adulthood. Moreover, neonatal exposure resulted in increased protein ubiquitination in the cornus ammonis 1 (CA1) region of the adult hippocampus indicating that BMAA may induce protein aggregation. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) based imaging is a powerful technology for spatial profiling of small molecular weight compounds in biological tissues with high chemical specificity and high spatial resolution. The aim of this study was to characterize neurochemical changes in the hippocampus of six month-old rats treated neonatally (postnatal days 9-10) with BMAA. Multivariate data analysis of whole section ToF-SIMS scans was performed to delineate anatomical regions of interest based on their chemical distribution pattern. Further analysis of spectral data obtained from the outlined anatomical regions, including CA1 and dentate gyrus (DG) revealed BMAA-induced long-term changes. Increased levels of phospholipids and protein fragments in the histopathologically altered CA1 region as well as phosphate depletion in the DG were observed. Moreover, high resolution SIMS imaging revealed a specific localization of phosphatidylcholine lipids, protein signals and potassium in the histopathologically altered CA1. These findings demonstrate that ToF-SIMS based imaging is a powerful approach for probing biochemical changes in situ and might serve as promising technique for investigating neurotoxin-induced brain pathology.

A genome-wide screen for ethylene-induced ethylene response factors (ERFs) in hybrid aspen stem identifies ERF genes that modify stem growth and wood properties.

Ethylene Response Factors (ERFs) are a large family of transcription factors that mediate responses to ethylene. Ethylene affects many aspects of wood development and is involved in tension wood formation. Thus ERFs could be key players connecting ethylene action to wood development. We identified 170 gene models encoding ERFs in the Populus trichocarpa genome. The transcriptional responses of ERF genes to ethylene treatments were determined in stem tissues of hybrid aspen (Populus tremula × tremuloides) by qPCR. Selected ethylene-responsive ERFs were overexpressed in wood-forming tissues and characterized for growth and wood chemotypes by FT-IR. Fifty ERFs in Populus showed more than five-fold increased transcript accumulation in response to ethylene treatments. Twenty-six ERFs were selected for further analyses. A majority of these were induced during tension wood formation. Overexpression of ERFs 18, 21, 30, 85 and 139 in wood-forming tissues of hybrid aspen modified the wood chemotype. Moreover, overexpression of ERF139 caused a dwarf-phenotype with altered wood development, and overexpression of ERF18, 34 and 35 slightly increased stem diameter. We identified ethylene-induced ERFs that respond to tension wood formation, and modify wood formation when overexpressed. This provides support for their role in ethylene-mediated regulation of wood development.

Non-cell-autonomous postmortem lignification of tracheary elements in Zinnia elegans.

Postmortem lignification of xylem tracheary elements (TEs) has been debated for decades. Here, we provide evidence in Zinnia elegans TE cell cultures, using pharmacological inhibitors and in intact Z. elegans plants using Fourier transform infrared microspectroscopy, that TE lignification occurs postmortem (i.e., after TE programmed cell death). In situ RT-PCR verified expression of the lignin monomer biosynthetic cinnamoyl CoA reductase and cinnamyl alcohol dehydrogenase in not only the lignifying TEs but also in the unlignified non-TE cells of Z. elegans TE cell cultures and in living, parenchymatic xylem cells that surround TEs in stems. These cells were also shown to have the capacity to synthesize and transport lignin monomers and reactive oxygen species to the cell walls of dead TEs. Differential gene expression analysis in Z. elegans TE cell cultures and concomitant functional analysis in Arabidopsis thaliana resulted in identification of several genes that were expressed in the non-TE cells and that affected lignin chemistry on the basis of pyrolysis-gas chromatography/mass spectrometry analysis. These data suggest that living, parenchymatic xylem cells contribute to TE lignification in a non-cell-autonomous manner, thus enabling the postmortem lignification of TEs.

MYB103 is required for FERULATE-5-HYDROXYLASE expression and syringyl lignin biosynthesis in Arabidopsis stems.

The transcription factor MYB103 was previously identified as a member of the transcriptional network regulating secondary wall biosynthesis in xylem tissues of Arabidopsis, and was proposed to act on cellulose biosynthesis. It is a direct transcriptional target of the transcription factor SECONDARY WALL ASSOCIATED NAC DOMAIN PROTEIN 1 (SND1), and 35S-driven dominant repression or over-expression of MYB103 modifies secondary wall thickness. We identified two myb103 T-DNA insertion mutants and chemically characterized their lignocellulose by pyrolysis/GC/MS, 2D NMR, FT-IR microspectroscopy and wet chemistry. The mutants developed normally but exhibited a 70-75% decrease in syringyl (S) lignin. The level of guaiacyl (G) lignin was co-ordinately increased, so that total Klason lignin was not affected. The transcript abundance of FERULATE-5-HYDROXYLASE (F5H), the key gene in biosynthesis of S lignin, was strongly decreased in the myb103 mutants, and the metabolomes of the myb103 mutant and an F5H null mutant were very similar. Other than modification of the lignin S to G ratio, there were only very minor changes in the composition of secondary cell-wall polymers in the inflorescence stem. In conclusion, we demonstrate that F5H expression and hence biosynthesis of S lignin are dependent on MYB103.

Strategy for minimizing between-study variation of large-scale phenotypic experiments using multivariate analysis.

We have developed a multistep strategy that integrates data from several large-scale experiments that suffer from systematic between-experiment variation. This strategy removes such variation that would otherwise mask differences of interest. It was applied to the evaluation of wood chemical analysis of 736 hybrid aspen trees: wild-type controls and transgenic trees potentially involved in wood formation. The trees were grown in four different greenhouse experiments imposing significant variation between experiments. Pyrolysis coupled to gas chromatography/mass spectrometry (Py-GC/MS) was used as a high throughput-screening platform for fingerprinting of wood chemotype. Our proposed strategy includes quality control, outlier detection, gene specific classification, and consensus analysis. The orthogonal projections to latent structures discriminant analysis (OPLS-DA) method was used to generate the consensus chemotype profiles for each transgenic line. These were thereafter compiled to generate a global dataset. Multivariate analysis and cluster analysis techniques revealed a drastic reduction in between-experiment variation that enabled a global analysis of all transgenic lines from the four independent experiments. Information from in-depth analysis of specific transgenic lines and independent peak identification validated our proposed strategy.

Fructokinase is required for carbon partitioning to cellulose in aspen wood.

Sucrose is the main transported form of carbon in several plant species, including Populus species. Sucrose metabolism in developing wood has therefore a central role in carbon partitioning to stem biomass. Half of the sucrose-derived carbon is in the form of fructose, but metabolism of fructose has received little attention as a factor in carbon partitioning to walls of wood cells. We show that RNAi-mediated reduction of FRK2 activity in developing wood of hybrid aspen (Populus tremula × tremuloides) led to the accumulation of soluble neutral sugars and a decrease in hexose phosphates and UDP-glucose, indicating that carbon flux to cell-wall polysaccharide precursors is decreased. Reduced FRK2 activity also led to thinner fiber cell walls with a reduction in the proportion of cellulose. No pleiotropic effects on stem height or diameter were observed. The results establish a central role for FRK2 activity in carbon flux to wood cellulose.

Ultra-structural organisation of cell wall polymers in normal and tension wood of aspen revealed by polarisation FTIR microspectroscopy.

Polarisation Fourier transform infra-red (FTIR) microspectroscopy was used to characterize the organisation and orientation of wood polymers in normal wood and tension wood from hybrid aspen (Populus tremula × Populus tremuloides). It is shown that both xylan and lignin in normal wood are highly oriented in the fibre wall. Their orientation is parallel with the cellulose microfibrils and hence in the direction of the fibre axis. In tension wood a similar orientation of lignin was found. However, in tension wood absorption peaks normally assigned to xylan exhibited a 90° change in the orientation dependence of the vibrations as compared with normal wood. The molecular origin of these vibrations are not known, but they are abundant enough to mask the orientation dependence of the xylan signal from the S2 layer in tension wood and could possibly come from other pentose sugars present in, or associated with, the gelatinous layer of tension wood fibres.

Ultrastructure and mechanical properties of populus wood with reduced lignin content caused by transgenic down-regulation of cinnamate 4-hydroxylase.

Several key enzymes in lignin biosynthesis of Populus have been down-regulated by transgenic approaches to investigate their role in wood lignification and to explore their potential for lignin modification. Cinnamate 4-hydroxylase is an enzyme in the early phenylpropanoid pathway that has not yet been functionally analyzed in Populus . This study shows that down-regulation of cinnamate 4-hydroxylase reduced Klason lignin content by 30% with no significant change in syringyl to guaiacyl ratio. The lignin reduction resulted in ultrastructural differences of the wood and a 10% decrease in wood density. Mechanical properties investigated by tensile tests and dynamic mechanical analysis showed a decrease in stiffness, which could be explained by the lower density. The study demonstrates that a large modification in lignin content only has minor influences on tensile properties of wood in its axial direction and highlights the usefulness of wood modified beyond its natural variation by transgene technology in exploring the impact of wood biopolymer composition and ultrastructure on its material properties.

Analysis of the Arabidopsis IRX9/IRX9-L and IRX14/IRX14-L pairs of glycosyltransferase genes reveals critical contributions to biosynthesis of the hemicellulose glucuronoxylan.

The hemicellulose glucuronoxylan (GX) is a major component of plant secondary cell walls. However, our understanding of GX synthesis remains limited. Here, we identify and analyze two new genes from Arabidopsis (Arabidopsis thaliana), IRREGULAR XYLEM9-LIKE (IRX9-L) and IRX14-LIKE (IRX14-L) that encode glycosyltransferase family 43 members proposed to function during xylan backbone elongation. We place IRX9-L and IRX14-L in a genetic framework with six previously described glycosyltransferase genes (IRX9, IRX10, IRX10-L, IRX14, FRAGILE FIBER8 [FRA8], and FRA8 HOMOLOG [F8H]) and investigate their function in GX synthesis. Double-mutant analysis identifies IRX9-L and IRX14-L as functional homologs of IRX9 and IRX14, respectively. Characterization of irx9 irx10 irx14 fra8 and irx9-L irx10-L irx14-L f8h quadruple mutants allows definition of a set of genes comprising IRX9, IRX10, IRX14, and FRA8 that perform the main role in GX synthesis during vegetative development. The IRX9-L, IRX10-L, IRX14-L, and F8H genes are able to partially substitute for their respective homologs and normally perform a minor function. The irx14 irx14-L double mutant virtually lacks xylan, whereas irx9 irx9-L and fra8 f8h double mutants form lowered amounts of GX displaying a greatly reduced degree of backbone polymerization. Our findings reveal two distinct sets of four genes each differentially contributing to GX biosynthesis.

Identification of lignin and polysaccharide modifications in Populus wood by chemometric analysis of 2D NMR spectra from dissolved cell walls.

2D 13C-(1)H HSQC NMR spectroscopy of acetylated cell walls in solution gives a detailed fingerprint that can be used to assess the chemical composition of the complete wall without extensive degradation. We demonstrate how multivariate analysis of such spectra can be used to visualize cell wall changes between sample types as high-resolution 2D NMR loading spectra. Changes in composition and structure for both lignin and polysaccharides can subsequently be interpreted on a molecular level. The multivariate approach alleviates problems associated with peak picking of overlapping peaks, and it allows the deduction of the relative importance of each peak for sample discrimination. As a first proof of concept, we compare Populus tension wood to normal wood. All well established differences in cellulose, hemicellulose, and lignin compositions between these wood types were readily detected, confirming the reliability of the multivariate approach. In a second example, wood from transgenic Populus modified in their degree of pectin methylesterification was compared to that of wild-type trees. We show that differences in both lignin and polysaccharide composition that are difficult to detect with traditional spectral analysis and that could not be a priori predicted were revealed by the multivariate approach. 2D NMR of dissolved cell wall samples combined with multivariate analysis constitutes a novel approach in cell wall analysis and provides a new tool that will benefit cell wall research.