The factors affecting the evolution of the anthocyanin biosynthesis pathway genes in monocot and dicot plant species

Abstract Background The available data demonstrate that even in universal metabolic pathways, some species-specific regulatory features of structural genes are present. For instance, in the anthocyanin biosynthesis pathway (ABP), genes may be regulated by ABP-specific regulatory factors, and their expression levels may be strongly associated with anthocyanin pigmentation, or they may be expressed independently of pigmentation. A dataset of orthologous ABP genes (Chs, Chi, F3h, F3’h, Dfr, Ans) from monocot and dicot plant species that have distinct gene regulation patterns and different types of pollination was constructed to test whether these factors affect the evolution of the genes. Results Using a maximum likelihood approach, we demonstrated that although the whole set of the ABP genes is under purifying selection, with greater selection acting on the upstream genes than on the downstream genes, genes from distinct groups of plant species experienced different strengths of selective pressure. The selective pressure on the genes was higher in dicots than in monocots (F3h and further downstream genes) and in pollinator-dependent plants than in pollinator-independent species (Chi and further downstream genes), suggesting an important role of pollination type in the evolution of the anthocyanin biosynthesis gene network. Contrasting effects of the regulation patterns on evolution were detected for the F3h and Dfr genes, with greater selective pressure on the F3h gene in plant species where the gene expression was not strongly associated with pigmentation and greater selective pressure on Dfr in plant species where the gene expression was associated with pigmentation. Conclusions We demonstrated the effects of pollination type and patterns of regulation on the evolution of the ABP genes, but the evolution of some of the genes could not be explained in the framework of these factors, such as the weaker selective pressure acting on Chs in species that attract pollinators or the stronger selective pressure on F3h in plant species where the gene expression was not associated with pigmentation. The observations suggest that additional factors could affect the evolution of these genes. One such factor could be an effect of gene duplication with further division of functions among gene copies and relaxed selective pressure acting on them. Additional tests with an appropriate dataset combining data on duplicated gene sequences and their functions in the flavonoid biosynthesis pathway are required to test this hypothesis

Ant13 Encodes Regulatory Factor WD40 Controlling Anthocyanin and Proanthocyanidin Synthesis in Barley (Hordeum vulgare L.)

Flavonoid compounds like anthocyanins and proanthocyanidins are important plant secondary metabolites having wide biological activities for humans. In this study, the molecular function of the Ant13 locus, which is one of the key loci governing flavonoid synthesis in barley, was determined. It was found that Ant13 encodes a WD40-type regulatory protein, which is required for transcriptional activation of a set of structural genes encoding enzymes of flavonoid biosynthesis at the leaf sheath base (colored by anthocyanins) and in grains (which accumulate proanthocyanidins). Besides its role in flavonoid biosynthesis, pleiotropic effects of this gene in plant growth were revealed. The mutants deficient in the Ant13 locus showed similar germination rates but a decreased rate of root and shoot growth and yield-related parameters in comparison to the parental cultivars. This is the seventh Ant locus (among 30) for which molecular functions in flavonoid biosynthesis regulation have been determined

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

Abstract 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 <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

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

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

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

Evaluating the Effects of Grain of Isogenic Wheat Lines Differing in the Content of Anthocyanins in Mouse Models of Neurodegenerative Disorders

Functional foods enriched with plant polyphenols and anthocyanins in particular attract special attention due to multiple beneficial bioactive properties of the latter. We evaluated the effects of a grain diet rich in anthocyanins in a mouse model of Alzheimer&rsquo;s disease induced by amyloid-beta (A&beta;) and a transgenic mouse model of Parkinson&rsquo;s disease (PD) with overexpression of human alpha-synuclein. The mice were kept at a diet that consisted of the wheat grain of near isogenic lines differing in anthocyanin content for five&ndash;six months. The anthocyanin-rich diet was safe and possessed positive effects on cognitive function. Anthocyanins prevented deficits in working memory induced by A&beta; or a long-term grain mono-diet; they partially reversed episodic memory alterations. Both types of grain diets prolonged memory extinction and rescued its facilitation in the PD model. The dynamics of the extinction in the group fed with the anthocyanin-rich wheat was closer to that in a group of wild-type mice given standard chow. The anthocyanin-rich diet reduced alpha-synuclein accumulation and modulated microglial response in the brain of the transgenic mice including the elevated expression of arginase1 that marks M2 microglia. Thus, anthocyanin-rich wheat is suggested as a promising source of functional nutrition at the early stages of neurodegenerative disorders