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Immunocytochemistry of <i>S. moellendorffii</i> stem (A), schematic representation of a section through a <i>S. moellendorffii</i> stem showing the location of the cortex (C), sclerenchyma (S), xylem (X), phloem (P), leaf traces (L) and specialized endodermal cells known as trabeculae (T) in the air space (A) separating the stele from the cortex.

<p>Indirect immunofluorescence microscopy reveals the location of homogalacturonan (HG, mAb JIM5) (B), β-(1,4)-galactan (mAb LM5) (C), (D) and (E), α-(1,5)-arabinan (mAb LM6) (F) and (G) and β-(1,4)-xylan (mAb LM10) (H). The images in (E) and (G) show counterstaining of the sections shown in (D) and (F) with Calcoflour White that reveals all β-glucan-containing cell walls. In contrast to most higher plants, β-(1,4)-galactan had a highly restricted location in <i>S. moellendorffii</i> stems and was located mainly in certain phloem cells (C) and only at the cell junctions of cortical cells (D) and (E). α-(1,5)-arabinan was widely distributed in cortical cell walls, but labelling was restricted to the middle portion of walls rather than throughout their whole width (F) and (G). HG is typically not abundant in the vascular tissues of higher plants but in <i>S. moellendorffii</i> HG was located predominantly in the walls of xylem and phloem cells and only present at low levels in other stem tissues. In contrast, labeling of β-(1,4)-xylan was strong in most cell walls apart from those in phloem tissue. Scale bars in (B) and (C)  = 140 μm, in (D) and (F)  = 20 μm, and in (H)  = 200 μm.</p

The A and C clades of GT77.

<p>The A clade holds the genes affected in the <i>reduced residual arabinose</i> (<i>rra</i>) mutants considered to be impaired in extensin arabinosylation. The arabidopsis member of the C-clade has recently also been implied in extensin arabinosylation based on analysis of the <i>xeg113</i>-mutant. Both clades have members from all four taxa. The full GT77 tree is <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035846#pone.0035846.s010" target="_blank">Fig. S10</a>.</p

Analysis of polysaccharides in the cell wall of <i>S. moellendorffii</i> using the microarray and antibody based CoMPP technique (Moller et al., 2007).

<p>Leaves, root and stems (A) and aerial part and roots (B). The heatmap, in which mean spot signals are correlated with colour intensity, shows the relative abundance of cell wall components as extracted using 1,2-diaminocyclohexanetetraacetic acid (CDTA) and NaOH. A low end cut-off value of 5 was used. The same amount of cell wall material (alcohol insoluble residue) was used for each sample. HG, homogalacturonan. XG, xyloglucan. AGP, arabinogalactan protein. This analysis indicates that <i>S. moellendorffii</i> has many cell wall polysaccharides in common with higher plants. It is of particular note that in contrast to most other higher plants, HG from <i>S. moellendorffii</i> was extracted more readily with NaOH than with CDTA and the occurrence of mixed linkage glucan. However, in common with higher plants but in contrast to <i>P. patens</i>, <i>S. moellendorffii</i> contained fucosylated XyG.</p

Phylogenetic tree of the A-clade of GT47, which includes the known XyG galactosyltransferase MUR3 and putative galactosyltransferases GT13 and GT18.

<p>The A-clade includes a mixed subclade with an overrepresentation of <i>P. patens</i> and <i>S. moellendorffii</i> sequences, a subclade containing At5g62220 with no <i>P. patens</i> members, and a subclade containing At5g41250 with no rice members, but is otherwise poorly resolved. Naming according to Li et al (2004): At5g62220 (GT18), At2g32740 (GT13) and At1g68470 (GT17). GT47 clade A members that are not included due to incomplete sequences include At4g13990, Pp156314 and Pp201625. Branching distal to † is not resolved.</p

Phylogenetic tree of GT2.

<p>The genomes of <i>P. patens</i> and <i>S. moellendorffii</i> include GT2 sequences with no orthologs among seed plants. Putative cellulose synthases from cyanobacteria and the red alga <i>Porphyra yezoensis</i> are the most similar sequences from within the green plant lineage. Other similar sequences include known cellulose synthases from oomycetes, the slime mold <i>Dictyostelium discoideum</i>, and two species of tunicates, e.g. sea squirts (animalia), and ascomycete sequences of unknown function.</p

Histogram showing the occurrence of GT-entries across families in the CAZy database and in DUF266-containing genes.

<p><i>A. thaliana</i>, white bars; <i>O. sativa</i>, grey bars; <i>P. patens,</i> hatched bars and <i>S. moellendorffii</i>, black bars. GT-numbers on the abscissa are shown in bold for families known or expected to be of particular cell wall relevance. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035846#pone.0035846.s017" target="_blank">Table S1</a> lists the JGI protein IDs for both <i>P. patens</i> and <i>S. moellendorffii</i> and gene names for the latter. Sequences are accessible via the JGI websites and at NCBI.</p

Sequential fractionation of aerial parts of <i>S. moellendorffii</i> analyzed with quantitative sugar composition analysis.

<p>The recalcitrance to extraction of homogalacturonan as observed in the CoMMP analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035846#pone-0035846-g001" target="_blank">Figure 1</a>) was confirmed. A general recalcitrance to extraction was observed and only in the cases of xylose and mannose could a majority of the sugars be extracted.</p

Confirmation that the epitope recognized by homogalacturonan, mixed linkage glucan and mannan specific antibodies are susceptible to enzymatic degradation by enzymes specific for the given polymer.

<p>CoMMP arrays were treated with PBS, buffer or increasing amount of enzyme to investigate if the epitope in question could be abolished. In all four cases were the epitope recognized by the antibody susceptible to enzymatic degradation, confirming that the epitope is found in the proposed polymer.</p