Image_2_Polyphenol oxidase genes in barley (Hordeum vulgare L.): functional activity with respect to black grain pigmentation.tif

Polyphenol oxidase (PPO) is an oxidoreductase. In damaged plant tissues, it catalyzes enzymatic browning by oxidizing o-diphenols to highly reactive o-quinones, which polymerize producing heterogeneous dark polymer melanin. In intact tissues, functions of PPO are not well understood. The aim of the study was to investigate the barley PPO gene family and to reveal the possible involvement of Ppo genes in melanization of barley grain, which is controlled by the Blp1 gene. Based on known barley Ppo genes on chromosome 2H (Ppo1 and Ppo2), two additional genes—Ppo3 and Ppo4—were found on chromosomes 3H and 4H, respectively. These genes have one and two exons, respectively, contain a conserved tyrosinase domain and are thought to be functional. Comparative transcriptional analyzes of the genes in samples of developing grains (combined hulls and pericarp tissues) were conducted in two barley lines differing by melanin pigmentation. The genes were found to be transcribed with increasing intensity (while grains mature) independently from the grain color, except for Ppo2, which is transcribed only in black-grained line i:BwBlp1 accumulating melanin in grains. Analysis of this gene’s expression in detached hulls and pericarps showed its elevated transcription in both tissues in comparison with yellow ones, while it was significantly higher in hulls than in pericarp. Segregation analysis in two F2 populations obtained based on barley genotypes carrying dominant Blp1 and recessive ppo1 (I) and dominant Blp1 and recessive ppo1 and ppo2 (II) was carried out. In population I, only two phenotypic classes corresponding to parental black and white ones were observed; the segregation ratio was 3 black to 1 white, corresponding to monogenic. In population II, aside from descendants with black and white grains, hybrids with a gray phenotype — light hulls and dark pericarp — were observed; the segregation ratio was 9 black to 3 gray to 4 white, corresponding to the epistatic interaction of two genes. Most hybrids with the gray phenotype carry dominant Blp1 and a homozygous recessive allele of Ppo2. Based on transcription and segregation assays one may conclude involvement of Ppo2 but not Ppo1 in melanin formation in barley hulls.</p

Table_1_Polyphenol oxidase genes in barley (Hordeum vulgare L.): functional activity with respect to black grain pigmentation.xlsx

Polyphenol oxidase (PPO) is an oxidoreductase. In damaged plant tissues, it catalyzes enzymatic browning by oxidizing o-diphenols to highly reactive o-quinones, which polymerize producing heterogeneous dark polymer melanin. In intact tissues, functions of PPO are not well understood. The aim of the study was to investigate the barley PPO gene family and to reveal the possible involvement of Ppo genes in melanization of barley grain, which is controlled by the Blp1 gene. Based on known barley Ppo genes on chromosome 2H (Ppo1 and Ppo2), two additional genes—Ppo3 and Ppo4—were found on chromosomes 3H and 4H, respectively. These genes have one and two exons, respectively, contain a conserved tyrosinase domain and are thought to be functional. Comparative transcriptional analyzes of the genes in samples of developing grains (combined hulls and pericarp tissues) were conducted in two barley lines differing by melanin pigmentation. The genes were found to be transcribed with increasing intensity (while grains mature) independently from the grain color, except for Ppo2, which is transcribed only in black-grained line i:BwBlp1 accumulating melanin in grains. Analysis of this gene’s expression in detached hulls and pericarps showed its elevated transcription in both tissues in comparison with yellow ones, while it was significantly higher in hulls than in pericarp. Segregation analysis in two F2 populations obtained based on barley genotypes carrying dominant Blp1 and recessive ppo1 (I) and dominant Blp1 and recessive ppo1 and ppo2 (II) was carried out. In population I, only two phenotypic classes corresponding to parental black and white ones were observed; the segregation ratio was 3 black to 1 white, corresponding to monogenic. In population II, aside from descendants with black and white grains, hybrids with a gray phenotype — light hulls and dark pericarp — were observed; the segregation ratio was 9 black to 3 gray to 4 white, corresponding to the epistatic interaction of two genes. Most hybrids with the gray phenotype carry dominant Blp1 and a homozygous recessive allele of Ppo2. Based on transcription and segregation assays one may conclude involvement of Ppo2 but not Ppo1 in melanin formation in barley hulls.</p

Image_4_Polyphenol oxidase genes in barley (Hordeum vulgare L.): functional activity with respect to black grain pigmentation.tif

Polyphenol oxidase (PPO) is an oxidoreductase. In damaged plant tissues, it catalyzes enzymatic browning by oxidizing o-diphenols to highly reactive o-quinones, which polymerize producing heterogeneous dark polymer melanin. In intact tissues, functions of PPO are not well understood. The aim of the study was to investigate the barley PPO gene family and to reveal the possible involvement of Ppo genes in melanization of barley grain, which is controlled by the Blp1 gene. Based on known barley Ppo genes on chromosome 2H (Ppo1 and Ppo2), two additional genes—Ppo3 and Ppo4—were found on chromosomes 3H and 4H, respectively. These genes have one and two exons, respectively, contain a conserved tyrosinase domain and are thought to be functional. Comparative transcriptional analyzes of the genes in samples of developing grains (combined hulls and pericarp tissues) were conducted in two barley lines differing by melanin pigmentation. The genes were found to be transcribed with increasing intensity (while grains mature) independently from the grain color, except for Ppo2, which is transcribed only in black-grained line i:BwBlp1 accumulating melanin in grains. Analysis of this gene’s expression in detached hulls and pericarps showed its elevated transcription in both tissues in comparison with yellow ones, while it was significantly higher in hulls than in pericarp. Segregation analysis in two F2 populations obtained based on barley genotypes carrying dominant Blp1 and recessive ppo1 (I) and dominant Blp1 and recessive ppo1 and ppo2 (II) was carried out. In population I, only two phenotypic classes corresponding to parental black and white ones were observed; the segregation ratio was 3 black to 1 white, corresponding to monogenic. In population II, aside from descendants with black and white grains, hybrids with a gray phenotype — light hulls and dark pericarp — were observed; the segregation ratio was 9 black to 3 gray to 4 white, corresponding to the epistatic interaction of two genes. Most hybrids with the gray phenotype carry dominant Blp1 and a homozygous recessive allele of Ppo2. Based on transcription and segregation assays one may conclude involvement of Ppo2 but not Ppo1 in melanin formation in barley hulls.</p

Table_2_Polyphenol oxidase genes in barley (Hordeum vulgare L.): functional activity with respect to black grain pigmentation.xlsx

Polyphenol oxidase (PPO) is an oxidoreductase. In damaged plant tissues, it catalyzes enzymatic browning by oxidizing o-diphenols to highly reactive o-quinones, which polymerize producing heterogeneous dark polymer melanin. In intact tissues, functions of PPO are not well understood. The aim of the study was to investigate the barley PPO gene family and to reveal the possible involvement of Ppo genes in melanization of barley grain, which is controlled by the Blp1 gene. Based on known barley Ppo genes on chromosome 2H (Ppo1 and Ppo2), two additional genes—Ppo3 and Ppo4—were found on chromosomes 3H and 4H, respectively. These genes have one and two exons, respectively, contain a conserved tyrosinase domain and are thought to be functional. Comparative transcriptional analyzes of the genes in samples of developing grains (combined hulls and pericarp tissues) were conducted in two barley lines differing by melanin pigmentation. The genes were found to be transcribed with increasing intensity (while grains mature) independently from the grain color, except for Ppo2, which is transcribed only in black-grained line i:BwBlp1 accumulating melanin in grains. Analysis of this gene’s expression in detached hulls and pericarps showed its elevated transcription in both tissues in comparison with yellow ones, while it was significantly higher in hulls than in pericarp. Segregation analysis in two F2 populations obtained based on barley genotypes carrying dominant Blp1 and recessive ppo1 (I) and dominant Blp1 and recessive ppo1 and ppo2 (II) was carried out. In population I, only two phenotypic classes corresponding to parental black and white ones were observed; the segregation ratio was 3 black to 1 white, corresponding to monogenic. In population II, aside from descendants with black and white grains, hybrids with a gray phenotype — light hulls and dark pericarp — were observed; the segregation ratio was 9 black to 3 gray to 4 white, corresponding to the epistatic interaction of two genes. Most hybrids with the gray phenotype carry dominant Blp1 and a homozygous recessive allele of Ppo2. Based on transcription and segregation assays one may conclude involvement of Ppo2 but not Ppo1 in melanin formation in barley hulls.</p

Image_1_Polyphenol oxidase genes in barley (Hordeum vulgare L.): functional activity with respect to black grain pigmentation.tif

Polyphenol oxidase (PPO) is an oxidoreductase. In damaged plant tissues, it catalyzes enzymatic browning by oxidizing o-diphenols to highly reactive o-quinones, which polymerize producing heterogeneous dark polymer melanin. In intact tissues, functions of PPO are not well understood. The aim of the study was to investigate the barley PPO gene family and to reveal the possible involvement of Ppo genes in melanization of barley grain, which is controlled by the Blp1 gene. Based on known barley Ppo genes on chromosome 2H (Ppo1 and Ppo2), two additional genes—Ppo3 and Ppo4—were found on chromosomes 3H and 4H, respectively. These genes have one and two exons, respectively, contain a conserved tyrosinase domain and are thought to be functional. Comparative transcriptional analyzes of the genes in samples of developing grains (combined hulls and pericarp tissues) were conducted in two barley lines differing by melanin pigmentation. The genes were found to be transcribed with increasing intensity (while grains mature) independently from the grain color, except for Ppo2, which is transcribed only in black-grained line i:BwBlp1 accumulating melanin in grains. Analysis of this gene’s expression in detached hulls and pericarps showed its elevated transcription in both tissues in comparison with yellow ones, while it was significantly higher in hulls than in pericarp. Segregation analysis in two F2 populations obtained based on barley genotypes carrying dominant Blp1 and recessive ppo1 (I) and dominant Blp1 and recessive ppo1 and ppo2 (II) was carried out. In population I, only two phenotypic classes corresponding to parental black and white ones were observed; the segregation ratio was 3 black to 1 white, corresponding to monogenic. In population II, aside from descendants with black and white grains, hybrids with a gray phenotype — light hulls and dark pericarp — were observed; the segregation ratio was 9 black to 3 gray to 4 white, corresponding to the epistatic interaction of two genes. Most hybrids with the gray phenotype carry dominant Blp1 and a homozygous recessive allele of Ppo2. Based on transcription and segregation assays one may conclude involvement of Ppo2 but not Ppo1 in melanin formation in barley hulls.</p

Image_3_Polyphenol oxidase genes in barley (Hordeum vulgare L.): functional activity with respect to black grain pigmentation.tif

Polyphenol oxidase (PPO) is an oxidoreductase. In damaged plant tissues, it catalyzes enzymatic browning by oxidizing o-diphenols to highly reactive o-quinones, which polymerize producing heterogeneous dark polymer melanin. In intact tissues, functions of PPO are not well understood. The aim of the study was to investigate the barley PPO gene family and to reveal the possible involvement of Ppo genes in melanization of barley grain, which is controlled by the Blp1 gene. Based on known barley Ppo genes on chromosome 2H (Ppo1 and Ppo2), two additional genes—Ppo3 and Ppo4—were found on chromosomes 3H and 4H, respectively. These genes have one and two exons, respectively, contain a conserved tyrosinase domain and are thought to be functional. Comparative transcriptional analyzes of the genes in samples of developing grains (combined hulls and pericarp tissues) were conducted in two barley lines differing by melanin pigmentation. The genes were found to be transcribed with increasing intensity (while grains mature) independently from the grain color, except for Ppo2, which is transcribed only in black-grained line i:BwBlp1 accumulating melanin in grains. Analysis of this gene’s expression in detached hulls and pericarps showed its elevated transcription in both tissues in comparison with yellow ones, while it was significantly higher in hulls than in pericarp. Segregation analysis in two F2 populations obtained based on barley genotypes carrying dominant Blp1 and recessive ppo1 (I) and dominant Blp1 and recessive ppo1 and ppo2 (II) was carried out. In population I, only two phenotypic classes corresponding to parental black and white ones were observed; the segregation ratio was 3 black to 1 white, corresponding to monogenic. In population II, aside from descendants with black and white grains, hybrids with a gray phenotype — light hulls and dark pericarp — were observed; the segregation ratio was 9 black to 3 gray to 4 white, corresponding to the epistatic interaction of two genes. Most hybrids with the gray phenotype carry dominant Blp1 and a homozygous recessive allele of Ppo2. Based on transcription and segregation assays one may conclude involvement of Ppo2 but not Ppo1 in melanin formation in barley hulls.</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

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

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

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