Lorenz Gerber

Non-Cell-Autonomous Postmortem Lignification of Tracheary Elements in Zinnia elegans

Here, we show that lignification occurs after programmed cell death in xylem tracheary elements (TEs) of Zinnia elegans xylogenic cell cultures. Living, parenchymatic xylem cells surrounding the TEs synthesize and transport lignin monomers and reactive oxygen species to the cell walls of the dead TEs, thereby contributing to TE lignification in a non-cell-autonomous manner. 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.

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.

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.

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.

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-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.

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.

Ethylene signaling via Ethylene Response Factors (ERFs) modifies wood development in hybrid aspen

Background The phytohormone ethylene (ET) has the potential to regulate secondary growth of plants and wood formation in trees. Application of exogenous ethylene or its in planta precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), to wood forming tissues of hybrid aspen (Populus tremula x Populus tremuloides) enhances xylem growth [1]. In the same study it was demonstrated that stimulation of enhanced xylem formation (tension wood, TW) at the upper side of leaning stems is mediated by endogenous ET. The production of endogenous ET in TW forming tissues is further supported by the increase of ACC oxidase gene transcript and enzyme activity on the TW side [2]. The ET perception and signal transmission cascade in Arabidopsis has been linked to the transcriptional activation of Ethylene Response Factors (ERFs) [3,4]. As transcription factors, ERFs regulate the expression of various specific downstream target genes by binding to cis-elements in their promoters [5]. We hypothesize that ERFs participate in xylem development through ethylene signaling and that they are involved in ET responses during TW formation.

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.