Ferritin 2 domain-containing protein found in lacquer tree (<i>Toxicodendron vernicifluum</i>) sap has negative effects on laccase and peroxidase reactions

<p>Lacquer tree sap, a raw material of traditional paints in East Asia, is hardened through laccase-catalyzed oxidation and the following polymerization of phenolic compound urushiol. In the sap’s water-insoluble fraction, we found two plantacyanins and a ferritin 2 domain-containing protein (TvFe2D, a homolog of <i>Arabidopsis</i> AT1G47980 and AT3G62730). The recombinant TvFe2D protein suppressed the accumulation of laccase-catalyzed oxidation products of a model substrate syringaldazine without decreasing oxygen consumption, the second substrate of laccase. The suppression was also observed when another substrate guaiacol or another oxidizing enzyme peroxidase was used. The functional domain of the suppression was the C-terminal half, downstream of the ferritin 2 domain. The results suggest that this protein may be involved in regulating the sap polymerization/hardening. We also discuss the possibility that homologous proteins of TvFe2D in other plants might be involved in the laccase- or peroxidase-mediated polymerization of phenolic compounds, such as lignin and flavonoids.</p> <p>Sap exuded from the wound site of lacquer tree. The ferritin 2 domain protein in it is a novel candidate regulating urushiol polymerization/hardening.</p

Metabolite profiling of the fermentation process of "yamahai-ginjo-shikomi" Japanese sake

<div><p>Sake is a traditional Japanese alcoholic beverage prepared by multiple parallel fermentation of rice. The fermentation process of “yamahai-ginjo-shikomi” sake is mainly performed by three microbes, <i>Aspergillus oryzae</i>, <i>Saccharomyces cerevisiae</i>, and <i>Lactobacilli</i>; the levels of various metabolites fluctuate during the fermentation of sake. For evaluation of the fermentation process, we monitored the concentration of moderate-sized molecules (m/z: 200–1000) dynamically changed during the fermentation process of “yamahai-ginjo-shikomi” Japanese sake. This analysis revealed that six compounds were the main factors with characteristic differences in the fermentation process. Among the six compounds, four were leucine- or isoleucine-containing peptides and the remaining two were predicted to be small molecules. Quantification of these compounds revealed that their quantities changed during the month of fermentation process. Our metabolomic approach revealed the dynamic changes observed in moderate-sized molecules during the fermentation process of sake, and the factors found in this analysis will be candidate molecules that indicate the progress of “yamahai-ginjo-shikomi” sake fermentation.</p></div

PC scores with Top 20 PC1 score.

<p>PC scores with Top 20 PC1 score.</p

PCA analysis of metabolic profiles.

<p>(A) Score plot of the fermentation process. Metabolic profiles of samples at six time points were plotted using two analytical replicates from the same sample. (B) Loading scatter plot. Factors having values of PC1 from the top to the 20^th at each plot are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190040#pone.0190040.t001" target="_blank">Table 1</a>.</p

Validation of peptide leucine or isoleucine compositions.

<p>(A) Chromatograms of sake samples with eight variations of synthetic [Leu/Ile]–[Leu/Ile]–[Leu/Ile] peptides. Extracted ion chromatograms (XIC) of m/z = 358.26–358.28 are presented. (B) Chromatograms of sake samples with two variations of synthetic Phe–Pro–[Leu/Ile] peptides. XIC of m/z = 376.22–376.23 are shown. Dashed lines with arrowheads indicate retention times of the main peaks of XIC for two samples. [Leu/Ile] indicates leucine or isoleucine residues that could not be determined by MS.</p

Quantification of peptides and associated compounds.

<p>(A) Quantification of Leu–Leu–Leu (left) and Phe–Pro–Leu by LC-TQMS. (B) Relative quantification of four factors (ID 1, 2, 6, and 7) that were identified by metabolic profiling using nanoLC-MS.</p