Purple-grained barley (Hordeum vulgare L.): marker-assisted development of NILs for investigating peculiarities of the anthocyanin biosynthesis regulatory network.

BACKGROUND: Anthocyanins are plants secondary metabolites important for plant adaptation to severe environments and potentially beneficial to human health. Purple colour of barley grain is caused by the pigments synthesized in pericarp. One or two genes determine the trait. One of them is Ant2 mapped on chromosome 2HL and is known to encode transcription factor (TF) with a bHLH domain. In plants, bHLH regulates anthocyanin biosynthesis together with TF harboring an R2R3-MYB domain. In wheat, the R2R3-MYBs responsible for purple colour of grain pericarp are encoded by the homoallelic series of the Pp-1 genes that were mapped on the short arms of chromosomes 7. In barley, in orthologous positions to wheat's Pp-1, the Ant1 gene determining red colour of leaf sheath has been mapped. In the current study, we tested whether Ant1 has pleiotropic effect not only on leaf sheath colour but also on pericarp pigmentation. RESULTS: А set of near isogenic lines (NILs) carrying different combinations of alleles at the Ant1 and Ant2 loci was created using markers-assisted backcrossing approach. The dominant alleles of both the Ant1 and Ant2 genes are required for anthocyanin accumulation in pericarp. A qRT-PCR analysis of the Ant genes in lemma and pericarp of the NILs revealed that some reciprocal interaction occurs between the genes. Expression of each of the two genes was up-regulated in purple-grained line with dominant alleles at the both loci. The lines carrying dominant allele either in the Ant1 or in the Ant2 locus were characterized by the decreased level of expression of the dominant gene and scant activity of the recessive one. The Ant1 and Ant2 expression was barely detected in uncolored line with recessive alleles at both loci. The anthocyanin biosynthesis structural genes were differently regulated: Chs, Chi, F3h, Dfr were transcribed in all lines independently on allelic state of the Ant1 and Ant2 genes, whereas F3'h and Ans were activated in presence on dominant alleles of the both regulatory genes. CONCLUSIONS: The R2R3-MYB-encoding counterpart (Ant1) of the regulatory Ant2 gene was determined for the first time. The dominant alleles of both of them are required for activation of anthocyanin synthesis in barley lemma and pericarp. The R2R3-MYB + bHLH complex activates the synthesis via affecting expression of the F3'h and Ans structural genes. In addition, positive regulatory loop between Ant1 and Ant2 was detected. Earlier the interaction between the anthocyanin biosynthesis regulatory genes has been revealed in dicot plant species only. Our data demonstrated that the regulatory mechanism is considered to be more common for plant kingdom than it has been reported so far.

Regulation of the Flavonoid Biosynthesis Pathway Genes in Purple and Black Grains of Hordeum vulgare.

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 Hordeum vulgare '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 Ant2 gene determining purple color of grains) and 1H (in region of the Blp gene determining black lemma and pericarp), respectively. The isolated dominant Ant2 allele of the purple-grained line has high level of sequence similarity with the recessive Bowman's ant2 in coding region, whereas an insertion of 179 bp was detected in promoter region of ant2. This structural divergence between Ant2 and ant2 alleles may underlie their different expression in grain pericarp: Bowman's Ant2 is not transcribed, whereas it was up-regulated in the purple-grained line with coordinately co-expressed flavonoid biosynthesis structural genes (Chs, Chi, F3h, F3'h, Dfr, Ans). 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 Ant2 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.