Ultraperformance Liquid Chromatography Tandem Mass Spectrometry Determination of Cyanogenic Glucosides in <i>Trifolium</i> Species

Cyanogenic glucosides were analyzed by ultra-high-performance liquid chromatography combined with mass spectrometry in 88 <i>Trifolium</i> species grown at the same site. On the basis of the occurrence of cyanogenic glucosides and the linamarin/lotaustralin ratio species could be grouped into five clusters. Cluster C1 included 37 species, which did not contain cyanogens. Cluster C2 (22 species) included plants containing only lotaustralin. In clusters C3 (14 species), C4 (13 species), and C5 (2 species) both linamarin and lotaustralin were present but at different ratios. In C3 and C4 the linamarin/lotaustralin ratio was below 1, whereas in cluster C5 the ratio was much higher. Generally, the total content of cyanogens was below 500 μg/g dry matter. Only in <i>Trifolium repens</i> var. <i>biasoletti</i> and <i>Trifolium montanum</i> extremely high cyanogen concentrations were observed. There was no general rule of occurrence of cyanogens. Samples of the same species from different countries accumulated cyanogens or could be free of these compounds

Number of rye plants inoculated with VIGS-BSMV control vectors (BSMV:00) and vectors designed for TGS containing promoter fragments of <i>ScBx1</i> (BSMV:<i>pScBx1</i>).

<p>Number of rye plants inoculated with VIGS-BSMV control vectors (BSMV:00) and vectors designed for TGS containing promoter fragments of <i>ScBx1</i> (BSMV:<i>pScBx1</i>).</p

Three new triterpene saponins from roots of <i>Eryngium planum</i>

<div><p>Saponin composition of the roots of <i>Eryngium planum</i> L. was investigated. Triterpene saponins found in <i>E. planum</i> and also present in <i>Eryngium maritimum</i> were different from those described previously in <i>Eryngium campestre</i> L. Three primary saponins were isolated and their tentative identifications, based on the electrospray MS/MS fragmentation patterns, were subsequently confirmed by 1D and 2D NMR analyses. Their structures were established as 3-<i>O</i>-β-d-glucopyranosyl-(1 → 2)-β-d-glucuronopyranosyl-21-<i>O</i>-acetyl-22-<i>O</i>-angeloyl-R1-barrigenol (<b>1</b>) and 3-<i>O</i>-β-d-glucopyranosyl-(1 → 2)-β-d-glucuronopyranosyl-22-<i>O</i>-angeloyl-A1-barrigenol (<b>2</b>) and 3-<i>O</i>-β-d-glucopyranosyl-(1 → 2)-β-d-glucuronopyranosyl-22-<i>O</i>-angeloyl-R1-barrigenol (<b>3</b>). Concentrations of the newly identified compounds in aerial parts and roots of both species were estimated using the liquid chromatography–mass spectrometry method.</p></div

Identification and VIGS-based characterization of <i>Bx1</i> ortholog in rye (<i>Secale cereale</i> L.)

<div><p>The first step of the benzoxazinoid (BX) synthesis pathway is catalyzed by an enzyme with indole-3-glycerol phosphate lyase activity encoded by 3 genes, <i>Bx1</i>, <i>TSA</i> and <i>Igl</i>. A gene highly homologous to maize and wheat <i>Bx1</i> has been identified in rye. The goal of the study was to analyze the gene and to experimentally verify its role in the rye BX biosynthesis pathway as a rye ortholog of the <i>Bx1</i> gene. Expression of the gene showed peak values 3 days after imbibition (dai) and at 21 dai it was undetectable. Changes of the BX content in leaves were highly correlated with the expression pattern until 21 dai. In plants older than 21 dai despite the undetectable expression of the analyzed gene there was still low accumulation of BXs. Function of the gene was verified by correlating its native expression and virus-induced silencing with BX accumulation. <i>Barley stripe mosaic virus</i> (BSMV)-based vectors were used to induce transcriptional (TGS) and posttranscriptional (PTGS) silencing of the analyzed gene. Both strategies (PTGS and TGS) significantly reduced the transcript level of the analyzed gene, and this was highly correlated with lowered BX content. Inoculation with virus-based vectors specifically induced expression of the analyzed gene, indicating up-regulation by biotic stressors. This is the first report of using the BSMV-based system for functional analysis of rye gene. The findings prove that the analyzed gene is a rye ortholog of the <i>Bx1</i> gene. Its expression is developmentally regulated and is strongly induced by biotic stress. Stable accumulation of BXs in plants older than 21 dai associated with undetectable expression of <i>ScBx1</i> indicates that the function of the <i>ScBx1</i> in the BX biosynthesis is redundant with another gene. We anticipate that the unknown gene is a putative ortholog of the <i>Igl</i>, which still remains to be identified in rye.</p></div

Parameters of mass spectrometer analysis.

<p>Parameters of mass spectrometer analysis.</p

Timetable of VIGS experiment design.

<p>Inoculation of the first (I) leaf of 5 days old seedlings 0 dpi, collection of third (III) and fourth (IV) leaves with virus infection symptoms 14 dpi, collection of young leaves with virus infection symptoms 21 and 99 dpi.</p

Rye seedlings used for inoculation and symptoms of <i>PDS</i> silencing.

<p>Rye seedlings 5 days after imbibition used for inoculation with the BSMV-derived vectors (<b>A</b>). Rye seedlings 14 days post inoculation (<b>B</b>) and 21 dpi (<b>C</b>). Control plant leaf (<b>D</b>), symptoms of BSMV infection (<b>E</b>), symptoms of <i>PDS</i> silencing (<b>F</b>-<b>J</b>).</p

Schematic representation of gDNA (5029 bp) containing <i>ScBx1</i>.

<p>Indicated are: GC-islands and TATA-box motifs, start codon, exons, introns and the two gDNA fragments cloned to the VIGS-BSMV vector for transcriptional gene silencing (<i>pScBx1-fragment I</i> and <i>pScBx1-fragment II</i>). The sites recognized by methylation-sensitive restriction enzymes–<i>Mlu</i>I, <i>Hha</i>I and <i>Hpy</i>CH4IV—located within the cloned fragments of the <i>pScBx1</i> promoter region are indicated. The intron-exon structure of the <i>ScBx1</i> gene is based on [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171506#pone.0171506.ref023" target="_blank">23</a>].</p

Relative level of <i>ScBx1</i> transcript and amount of benzoxazinoids in rye.

<p>Relative level of <i>ScBx1</i> transcript (<b>A</b>) and total amount of DIBOA/DIBOA-Glc and total amount of tested benzoxazinoids (<b>B</b>) in hypocotyls/leaves of rye cv. Stach F1 collected 1, 2, 3, 4, 7, 14, 21 and 99 days after imbibition. The results represent the mean values and the standard deviations from three biological replicates. *—total amount of: HBOA, DIBOA, DIBOA-Glc, DIMBOA, DIMBOA-Glc and MBOA.</p

Normalized expression of <i>ScBx1</i> and normalized content of benzoxazinoids in TGS experiments.

<p>Normalized expression of <i>ScBx1</i> (<b>A</b>) and normalized content of benzoxazinoids (BXs) (<b>B</b>) in control plants (BSMV:00) compared to expression in experimental plants inoculated with BSMV:α,β_{(<i>pScBx1-fragment I</i>)},γ_{(<i>pScBx1-fragment II</i>)} (BSMV:<i>pScBx1</i>). Results are shown on a logarithmic scale. BSMV:00 –the mean value of <i>ScBx1</i> relative expression in plants inoculated with BSMV:α,β_(-),γ_(-) vector assumed as 1.00. BSMV:<i>pScBx1</i>—the mean value of <i>ScBx1</i> normalized expression in leaves inoculated with silencing vector. #17, #22, #29 –the <i>ScBx1</i> normalized expression in plants inoculated with silencing vector. Statistical significance (Student’s <i>t</i>-test): ** p < 0.005, * 0.005 < p < 0.05.</p