第73回千葉医学会総会記事

Microarray data of genes with lower expression levels (<0.74, p < 0.05) in 35S:ROXY19 plants as compared to control plants and their response to TIBA in the Col-0 background. The table contains the gene identity (AGI), description, the mean expression values of four independent biological replicates of the genotypes Col-0 and 35S:ROXY19#8, the ratios (FC, fold changes, log2) of the transcript levels in the transgenic line with respect to Col-0 and the corresponding p-values, the ratios between Col-0 treated with 0.1 % DMSO and Col-0 treated with 0.1 mM TIBA/0.1 % DMSO and the corresponding p-values. Since the microarray analysis of the TIBA-treated plants was performed with the Affimetrix ATH1 gene chip, the list contains 301 and not 321 genes. Genes that are not induced by TIBA are shown in light grey. FC, fold changes. For TIBA induction, plants were grown for six to seven weeks on steamed soil (Archut, Fruhstorfer Erde, T25, Str1fein) in growth chambers with light intensity at 37 to 45 μmol photons m−2 s−1 at 22 °C and 60 % humidity. Eight plants were sprayed with either 0.1 mM TIBA/0.1 % DMSO or with 0.1 % DMSO and leaves were harvested after eight h. The experiment was repeated three times. (XLSX 205 kb

Additional file 2: Table S2. of Ectopically expressed glutaredoxin ROXY19 negatively regulates the detoxification pathway in Arabidopsis thaliana

Microarray data of genes with lower expression levels (FC < 0.74, p < 0.05) in 35S:ROXY19 plants as compared to control plants. The table contains the gene identity (AGI), description, the mean expression values of four independent biological replicates of the genotypes Col-0, 35S:GRXC2, 35S:ROXY19 SSMS , 35S:ROXY19#8 and 35S:ROXY19#12, and the ratios (FC, fold changes, log2) of the transcript levels in the transgenic lines with respect to Col-0 and the corresponding p-values. (XLSX 305 kb

Proposed mechanism of RES/cyclopentenone modification at cysteine residues of proteins.

<p>The α,ß-unsaturated carbonyl group of RES acts as an electrophile and modifies the thiol group of proteins <i>via</i> Michael addition. In a first reversible step, a non-covalent adduct of a protein with a RES may occur thereby facilitating the subsequent irreversible reaction of an exposed thiolate with the α,ß-unsaturated carbonyl group of the RES leading to covalent conjugate formation. Changes of the target protein conformation by non-covalent and covalent ligand binding may lead to target activation or inhibition.</p

qPCR primer sequences and accession numbers.

<p>qPCR primer sequences and accession numbers.</p

TGA2 modification by PGA₁-biotin <i>in vitro</i>.

<p>Recombinant His-tagged TGA2 was incubated with 75 μM PGA₁-biotin for 2 h. Proteins were separated on SDS-PAGE and transferred to a PVDF membrane. Biotinylated proteins were visualized by NeutrAvidin-HRP. <b>(A)</b> Stability of modification under different pH conditions tested at ambient temperature. <b>(B)</b> Incubation of TGA2 with PGA₁-biotin in sodium phosphate, pH 7.5 at different temperatures. His-TGA2 has a predicted molecular weight of 42.3 kDa. The addition of six histidine residues may alter the migration slightly, resulting in a larger than expected protein band of ~50 kDa (Figs 2 and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195398#pone.0195398.g003" target="_blank">3</a>). Modification by PGA₁-biotin would only add 0.6 kDa if a single site was modified. Immunoblot analysis with an αTGA2 antibody [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195398#pone.0195398.ref022" target="_blank">22</a>] confirmed the identity of His-TGA2 at 50 kDa (data not shown).</p

Induction of oxylipin-responsive genes by RES in wild type and TGA mutant lines.

<p>Wild type and different mutant seedlings (14 d) were incubated without (black bars) or with (white bars) 75 μM PGA1 (<b>A</b>) or 75 μM OPDA (<b>B</b>) for 4 h. The following lines were tested: <i>tga2/5/6</i> (<i>tga2 tga5 tga 6</i> triple knock-out line), TGA2 (native TGA2 overexpression in the background of <i>tga2/5/6</i>, TGA2 C186S: Overexpression of mutant TGA2 C186S in the background of <i>tga2/5/6</i>. RNA was isolated and qPCR analysis was performed. Expression of <i>TolB</i>, <i>GST25</i> and <i>CYP81D11</i> is relative to 10,000 molecules <i>Actin2/8</i>. Data show means ± SD, n = 5. Similar results were obtained in two additional experiments for PGA₁ and another experiment for OPDA treatment with different TGA2 and TGA2_C186S lines.</p

Cysteine specific modification of proteins by RES.

<p>Proteins were extracted from leaves and incubated without (control, Con) or with the cysteine reactive reagents N-ethylmaleimide (NEM), iodoacetamide (IAM), S-methyl methanethiosulfonate (MMTS) for 1 h prior to RES lipid modification. Protein samples were separated by SDS-PAGE. (<b>A</b>) After modification with PGA₁-biotin and SDS-PAGE, proteins (90 μg) were transferred to a PVDF membrane. Biotinylated proteins were visualized by NeutrAvidin-HRP (upper panel) followed by protein staining using Coomassie blue (loading control, bottom panel). (<b>B</b>) After modification by [1-¹⁴C]OPDA (500000 cpm) for 4 h, the SDS–PAGE gel (loaded with 25 μg protein per lane) was analyzed by autoradiography (left panel) and Coomassie staining (right panel).</p

Influence of <i>Verticillium longisporum</i> VL43 infection on protein content of whole leaf extracts, in apoplastic washing fluid and on the number of protein spots after 2-D electrophoresis.

<p>Plants were analyzed 25 days post infection. Protein spots were determined by Proteomweaver software in silver stained gels (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031435#pone-0031435-g001" target="_blank">Fig. 1</a>). Data indicate means (N ± SE).</p

Proteins significantly affected in the apoplast of <i>Verticillilum longisporum</i> VL43-infected compared with that of mock-infected <i>Arabidopsis thaliana</i> leaves.

<p>*Factor: protein abundance in samples of VL-treated plants/protein abundance in mock-inoculated plants,</p><p>**92% homology to PRX At437520.</p><p>Only those spots were analyzed that were reproducibly observed in two independent experiments. In each experiment three biological replicates were analyzed. For further details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031435#pone-0031435-t004" target="_blank">Table 4</a>. Spot identification numbers refer to those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031435#pone-0031435-g001" target="_blank">Figure 1</a>. Peptides are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031435#pone.0031435.s003" target="_blank">Table S1</a>.</p

Effect of cysteine blocking reagents on TGA2 modification by PGA₁-biotin.

<p>Recombinant TGA2 was incubated with the cysteine reactive reagents N-ethylmaleimide (NEM), iodoacetamide (IAM) or S-methyl methanethiosulfonate (MMTS) prior to PGA₁-biotin modification. Protein samples (30 μg) were separated on SDS-PAGE and transferred to a PVDF membrane. Biotinylated proteins were visualized by NeutrAvidin-HRP, followed by protein staining with Coomassie (loading control, lower panel).</p