Xylose content in cell wall polysaccharides isolated from stems of 6-week-old mutant plants transformed with the different constructs.

<p>Cell walls were prepared from stems, hydrolyzed in TFA, and the xylose content determined by HPAEC. The nomenclature for the constructs used to transform the <i>irx9</i> and <i>irx14</i> plants is explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105014#pone-0105014-t001" target="_blank">Table 1</a>. The bars show average ± SD (n = 4). Averages that are not significantly different (ANOVA, Tukey's test, p>0.05) are indicated with the same letter.</p

Phylogenetic tree of GT43 proteins from selected species.

<p>A multiple alignment of protein sequences from Arabidopsis (At), rice (<i>Oryza sativa</i>, Os), poplar (<i>Populus trichocarpa</i>, Pt), spikemoss (<i>Selaginella moellendorffii</i>, Sm) and human (<i>Homo sapiens</i>, Sp) was used to generate a neighbor-joining tree using MEGA5. Bootstrap values are indicated and the scale bar shows evolutionary distances in units of the number of amino acid substitutions per site. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105014#s2" target="_blank">Materials and Methods</a> section for sequence IDs.</p

Representative photos of wild type, mutants and transformed plants.

<p>The nomenclature for the constructs used to transform the <i>irx9</i> and <i>irx14</i> plants is explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105014#pone-0105014-t001" target="_blank">Table 1</a>. Rosettes were from 4-week-old plants and mature plants were 6-week-old.</p

Stem height of 6-week-old mutant plants transformed with the different constructs.

<p>The nomenclature for the constructs used to transform the <i>irx9</i> and <i>irx14</i> plants is explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105014#pone-0105014-t001" target="_blank">Table 1</a>. The bars show average ± SD (n = 6). Averages that are not significantly different (ANOVA, Tukey's test, p>0.05) are indicated with the same letter.</p

Microcopy analysis of stems from 6-week-old plants.

<p>The LM10 anti-xylan monoclonal antibody was used for immunodetection of xylan in transverse stem sections. The nomenclature for the constructs used to transform the <i>irx9</i> and <i>irx14</i> plants is explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105014#pone-0105014-t001" target="_blank">Table 1</a>. Irregular xylem phenotype (indicated with arrows) was observed in the <i>irx9</i> and <i>irx14</i> mutants, as well as in the <i>irx14</i> plants transformed with the 35S:IRX14-1 construct. All the other transformants showed normal vessel phenotype.</p

Rosette diameter of 4-week-old mutant plants transformed with the different constructs.

<p>The nomenclature for the constructs used to transform the <i>irx9</i> and <i>irx14</i> plants is explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105014#pone-0105014-t001" target="_blank">Table 1</a>. The bars show average ± SD (n = 6). Averages that are not significantly different (ANOVA, Tukey's test, p>0.05) are indicated with the same letter.</p

Alphabetical list of the CWGTs that were screened.

<p>Alphabetical list of the CWGTs that were screened.</p

Plant cell wall glycosyltransferases: High-throughput recombinant expression screening and general requirements for these challenging enzymes

<div><p>Molecular characterization of plant cell wall glycosyltransferases is a critical step towards understanding the biosynthesis of the complex plant cell wall, and ultimately for efficient engineering of biofuel and agricultural crops. The majority of these enzymes have proven very difficult to obtain in the needed amount and purity for such molecular studies, and recombinant cell wall glycosyltransferase production efforts have largely failed. A daunting number of strategies can be employed to overcome this challenge, including optimization of DNA and protein sequences, choice of expression organism, expression conditions, co-expression partners, purification methods, and optimization of protein solubility and stability. Hence researchers are presented with thousands of potential conditions to test. Ultimately, the subset of conditions that will be sampled depends on practical considerations and prior knowledge of the enzyme(s) being studied. We have developed a rational approach to this process. We devise a pipeline comprising <i>in silico</i> selection of targets and construct design, and high-throughput expression screening, target enrichment, and hit identification. We have applied this pipeline to a test set of <i>Arabidopsis thaliana</i> cell wall glycosyltransferases known to be challenging to obtain in soluble form, as well as to a library of cell wall glycosyltransferases from other plants including agricultural and biofuel crops. The screening results suggest that recombinant cell wall glycosyltransferases in general have a very low soluble:insoluble ratio in lysates from heterologous expression cultures, and that co-expression of chaperones as well as lysis buffer optimization can increase this ratio. We have applied the identified preferred conditions to Reversibly Glycosylated Polypeptide 1 from <i>Arabidopsis thaliana</i>, and processed this enzyme to near-purity in unprecedented milligram amounts. The obtained preparation of Reversibly Glycosylated Polypeptide 1 has the expected arabinopyranose mutase and autoglycosylation activities.</p></div

Hits from the test library screening.

<p>Hits from the test library screening.</p

<i>Arabidopsis thaliana</i> RGP1 scale-up, purification and activity determinations.

<p>(A) Coomassie stained SDS-PAGE of the final chromatographic step yielding almost pure RGP1 (42 kDa). (B) Phosphate-release assay showing autoglycosylating or hydrolytic activity of RGP1 on UDP-glucose. (C) UDP-arabinose mutase activity of RGP1. High-pressure liquid chromatograms of authentic UDP-arabinopyranose (UDP-Ara<i>p</i>) and UDP-arabinofuranose (UDP-Ara<i>f</i>) standards (grey) overlaid with the chromatogram of the reaction mixture of UDP-Ara<i>f</i> with recombinant, purified RGP1 (black).</p