An Integrative Approach to the Identification of Arabidopsis and Rice Genes Involved in Xylan and Secondary Wall Development

Xylans constitute the major non-cellulosic component of plant biomass. Xylan biosynthesis is particularly pronounced in cells with secondary walls, implying that the synthesis network consists of a set of highly expressed genes in such cells. To improve the understanding of xylan biosynthesis, we performed a comparative analysis of co-expression networks between Arabidopsis and rice as reference species with different wall types. Many co-expressed genes were represented by orthologs in both species, which implies common biological features, while some gene families were only found in one of the species, and therefore likely to be related to differences in their cell walls. To predict the subcellular location of the identified proteins, we developed a new method, PFANTOM (plant protein family information-based predictor for endomembrane), which was shown to perform better for proteins in the endomembrane system than other available prediction methods. Based on the combined approach of co-expression and predicted cellular localization, we propose a model for Arabidopsis and rice xylan synthesis in the Golgi apparatus and signaling from plasma membrane to nucleus for secondary cell wall differentiation. As an experimental validation of the model, we show that an Arabidopsis mutant in the PGSIP1 gene encoding one of the Golgi localized candidate proteins has a highly decreased content of glucuronic acid in secondary cell walls and substantially reduced xylan glucuronosyltransferase activity

Comparison of dilute acid and ionic liquid pretreatment of switchgrass: Biomass recalcitrance, delignification and enzymatic saccharification

The efficiency of two biomass pretreatment technologies, dilute acid hydrolysis and dissolution in an ionic liquid, are compared in terms of delignification, saccharification efficiency and saccharide yields with switchgrass serving as a model bioenergy crop. When subject to ionic liquid pretreatment (dissolution and precipitation of cellulose by anti-solvent) switchgrass exhibited reduced cellulose crystallinity, increased surface area, and decreased lignin content compared to dilute acid pretreatment. Pretreated material was characterized by powder X-ray diffraction, scanning electron microscopy, Fourier transform infrared spectroscopy, Raman spectroscopy and chemistry methods. Ionic liquid pretreatment enabled a significant enhancement in the rate of enzyme hydrolysis of the cellulose component of switchgrass, with a rate increase of 16.7-fold, and a glucan yield of 96.0% obtained in 24 h. These results indicate that ionic liquid pretreatment may offer unique advantages when compared to the dilute acid pretreatment process for switchgrass. However, the cost of the ionic liquid process must also be taken into consideration

Rapid qualitative analysis of carbohydrate/lignin ratio in plant cell walls of untreated biomass by confocal lambda scanning.

<p>Cell wall particles from senesced, dry, and milled leaf and stem biomass samples were used for lambda scanning with a confocal laser-scanning microscope. Three defined cell wall regions in each leaf and stem sample were manually selected for measurement of emission spectra. Green fluorescence emitted from ferulic acid, cellulose, and additional carbohydrates, red fluorescence indicative for high lignin content. Micrographs are representative for each sample after evaluating at least five independent replicates.</p

Principal components analysis of leaf and stem biomass after different pretreatment methods.

<p>Two-factor principal component analysis on the basis of six parameters defining efficiency of lignocellulosic ethanol production. Bd: <i>B. distachyon</i>, Mg: <i>M.</i> x <i>giganteus</i>, Ta: <i>T. aestivum</i>, Zm: <i>Z. mays</i>.</p

Cell wall composition of leaf and stem biomass.

<p>Milled leaf and stem samples from senesced, dry plants were used for the determination of the three major cell wall polymers (<b>A</b>) lignin, (<b>B</b>) cellulose, and (<b>C</b>) hemicelluloses. Values represent the relative amount of each polymer. *<i>P</i><0.05 by Tukey's test. Error bars represent SE, and <i>n</i> = 3.</p

Glucose and ethanol concentration during fermentation of leaf and stem biomass.

<p>Glucose and ethanol concentrations in supernatants of the leaf and stem fermentation broth were determined by HPLC using a refractive index detector. Samples taken at the start of fermentation with <i>S. cerevisiae</i> and after 48 h and 72 h. (<b>A</b>) Glucose concentration and (<b>B</b>) ethanol concentration of biomass autoclaved in diluted sulfuric acid (1.75% (v/v)); (<b>C</b>) glucose concentration and (<b>D</b>) ethanol concentration of biomass autoclaved in diluted sulfuric acid with subsequent enzymatic hydrolysis (Accellerase 1500 enzyme mixture). Letters a, b, and c indicate groups with <i>P</i><0.05 by Tukey's test. Error bars represent SE, and <i>n</i> = 3.</p

Phylogenetic relation of tested plant species.

<p>Phylogenetic tree based on the nucleotide alignment of the ITS2 region of ribosomal DNA of the C₄ grasses <i>Z. mays</i> (maize), <i>S. officinarum</i> (sugarcane), <i>S. bicolor</i> (sorghum), and <i>M.</i> x <i>giganteus</i>, the C₃ grasses <i>O. sativa</i> (rice), <i>T. aestivum</i> (wheat), <i>H. vulgare</i> (barley), <i>S. cereale</i> (rye), and <i>B. distachyon</i>, and the C₃ dicots <i>G. max</i> (soybean), <i>S. lycopersicum</i> (tomato), and <i>A. thaliana</i>, which were used as an outgroup. Bold: plant species used in this study, dotted line: plant species with a C₄ carbon fixation.</p

Relative non-cellulosic monosaccharide composition of leaf and stem biomass.

<p>Cell wall extracts from senesced, dry, and milled leaf and stem biomass samples were used. Non-cellulosic monosaccharide composition determined by HPAEC-PAD from (<b>A</b>) untreated biomass, (<b>B</b>) biomass autoclaved in diluted sulfuric acid (1.75% (v/v)), and (<b>C</b>) biomass autoclaved in diluted sulfuric acid with subsequent enzymatic hydrolysis (Accellerase 1500 enzyme mixture). Letters a, b, c, and d indicate groups with <i>P</i><0.05 by Tukey's test. Error bars represent SE, and <i>n</i> = 3. GalA: galacturonic acid, GlcA: glucuronic acid.</p