Microsatellite genotyping of the PLP (A) and BLP (B) lines.

<p>Purple and black colors mark introgressed fragments in the PLP and BLP lines, respectively.</p

Regulation of the Flavonoid Biosynthesis Pathway Genes in Purple and Black Grains of <i>Hordeum vulgare</i>

<div><p>Barley grain at maturity can have yellow, purple, blue, and black pigmentations which are suggested to play a protective role under stress conditions. The first three types of the colors are caused by phenolic compounds flavonoids; the last one is caused by phytomelanins, oxidized and polymerized phenolic compounds. Although the genetic basis of the flavonoid biosynthesis pathway in barley has been thoroughly studied, there is no data yet on its regulation in purple and black barley grains. In the current study, genetic model of <i>Hordeum vulgare</i> ‘Bowman’ near-isogenic lines (NILs) was used to investigate the regulation of the flavonoid biosynthesis in white, purple, and black barley grains. Microsatellite genotyping revealed donor segments in the purple- and black-grained lines on chromosomes 2H (in region of the <i>Ant2</i> gene determining purple color of grains) and 1H (in region of the <i>Blp</i> gene determining black lemma and pericarp), respectively. The isolated dominant <i>Ant2</i> allele of the purple-grained line has high level of sequence similarity with the recessive Bowman’s <i>ant2</i> in coding region, whereas an insertion of 179 bp was detected in promoter region of <i>ant2</i>. This structural divergence between <i>Ant2</i> and <i>ant2</i> alleles may underlie their different expression in grain pericarp: Bowman’s <i>Ant2</i> is not transcribed, whereas it was up-regulated in the purple-grained line with coordinately co-expressed flavonoid biosynthesis structural genes (<i>Chs</i>, <i>Chi</i>, <i>F3h</i>, <i>F3’h</i>, <i>Dfr</i>, <i>Ans</i>). This led to total anthocyain content increase in purple-grained line identified by ultra-performance liquid chromatography (HPLC). Collectively, these results proved the regulatory function of the <i>Ant2</i> gene in anthocyanin biosynthesis in barley grain pericarp. In the black-grained line, the specific transcriptional regulation of the flavonoid biosynthesis pathway genes was not detected, suggesting that flavonoid pigments are not involved in development of black lemma and pericarp trait.</p></div

Expression of the flavonoid biosynthesis structural genes in grains of the barley NILs having different coloration of lemma and pericarp.

<p>The data are presented as mean value ± standard error. *—differences are statistically significant between NILs and Bowman at <i>p</i>≤ 0.05 (<i>U</i>-test).</p

Expression of the <i>Ant2</i> gene in lemma and pericarp of NILs differing by the coloration.

<p>The data are presented as mean value ± standard error. *—differences are statistically significant between NILs and Bowman at <i>p</i> ≤ 0.05 (<i>U</i>-test).</p

Anthocyanin profiles of Bowman (A), PLP (B) and BLP (C) genotypes.

<p>Seed extracts were prepared using acidified aqueous methanol as described in the materials and methods section. Extracts were separated by UPLC and compound elution was monitored by photodiode array (PDA) detection followed by MS analysis. The chromatograms were obtained by extracting the PDA data at 515 nm. X-axis represents time (min) and Y-axis represents absorbance in milliabsorbance units (mAU).</p

Flavonoid biosynthetic pathway in plants.

<p>The enzymes are: CHS (chalcone synthase); CHI (chalcone-flavanone isomerase); F3H (flavanone 3-hydroxylase); FLS (flavonol synthase); FNS (flavone synthase); F3’H (flavonoid 3’-hydroxylase); F3’5’H (flavonoid 3’,5’-hydroxylase); DFR (dihydroflavonol 4-reductase); ANS (anthocyanidin synthase); GT (glycosyltransferase); MT (methyltransferase), RT (rhamnosyltransferase); and LAR (leucoanthocyanidin reductase).</p