Characterization of Maize Near-Isogenic Lines With Enhanced Flavonoid Expression to Be Used as Tools in Diet-Health Complexity

Increasing incidence of chronic diseases in the 21st century has emphasized the importance of developing crops with enhanced nutritional value. Plant-based diets are associated with reduced incidence of many chronic diseases. The growing population and increased food demand have prioritized the development of high-yielding commercial crop varieties at the expense of natural flavors as well as health-benefiting compounds including polyphenols. Flavonoids are a large subfamily of polyphenols abundant in the plant kingdom with known health-promoting effects, making them a promising trait to be re-introduced into elite lines. Given the vast array of flavonoids and the complexity of plant food metabolome interactions, it is difficult to identify with certainty the specific class(es) of flavonoids in the food matrix that are anti-inflammatory. To address this, we have developed four maize near-isogenic lines (NILs); a line that lacked both anthocyanins and phlobaphenes, a second NIL containing phlobaphenes, a third line had anthocyanins, and a fourth line that contained both anthocyanins and phlobaphenes. The phytochemical profiles and the antioxidant potential of the NILs were characterized. The accumulation of anthocyanins and phlobaphenes contributed significantly to antioxidant capacity compared to maize lines that lacked one or both of the compounds (p \u3c 0.05). Pilot study showed that intake of flavonoid-rich maize diets were able to alleviate experimental colitis in mice. These NILs offer novel materials combining anthocyanins and phlobaphenes and can be used as powerful tools to investigate the disease-preventive effects of specific flavonoid compound in diet/feeding experiments

Characterization of Maize Near-Isogenic Lines With Enhanced Flavonoid Expression to Be Used as Tools in Diet-Health Complexity

Increasing incidence of chronic diseases in the 21st century has emphasized the importance of developing crops with enhanced nutritional value. Plant-based diets are associated with reduced incidence of many chronic diseases. The growing population and increased food demand have prioritized the development of high-yielding commercial crop varieties at the expense of natural flavors as well as health-benefiting compounds including polyphenols. Flavonoids are a large subfamily of polyphenols abundant in the plant kingdom with known health-promoting effects, making them a promising trait to be re-introduced into elite lines. Given the vast array of flavonoids and the complexity of plant food metabolome interactions, it is difficult to identify with certainty the specific class(es) of flavonoids in the food matrix that are anti-inflammatory. To address this, we have developed four maize near-isogenic lines (NILs); a line that lacked both anthocyanins and phlobaphenes, a second NIL containing phlobaphenes, a third line had anthocyanins, and a fourth line that contained both anthocyanins and phlobaphenes. The phytochemical profiles and the antioxidant potential of the NILs were characterized. The accumulation of anthocyanins and phlobaphenes contributed significantly to antioxidant capacity compared to maize lines that lacked one or both of the compounds (p \u3c 0.05). Pilot study showed that intake of flavonoid-rich maize diets were able to alleviate experimental colitis in mice. These NILs offer novel materials combining anthocyanins and phlobaphenes and can be used as powerful tools to investigate the disease-preventive effects of specific flavonoid compound in diet/feeding experiments

Gene Structure Induced Epigenetic Modifications of pericarp color1 Alleles of Maize Result in Tissue-Specific Mosaicism

BACKGROUND: The pericarp color1 (p1) gene encodes for a myb-homologous protein that regulates the biosynthesis of brick-red flavonoid pigments called phlobahpenes. The pattern of pigmentation on the pericarp and cob glumes depends upon the allelic constitution at the p1 locus. p1 alleles have unique gene structure and copy number which have been proposed to influence the epigenetic regulation of tissue-specific gene expression. For example, the presence of tandem-repeats has been correlated with the suppression of pericarp pigmentation though a mechanism associated with increased DNA methylation. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we extensively characterize a p1 allele called P1-mosaic (P1-mm) that has mosaic pericarp and light pink or colorless cob glumes pigmentation. Relative to the P1-wr (white pericarp and red cob glumes), we show that the tandem repeats of P1-mm have a modified gene structure containing a reduced number of repeats. The P1-mm has reduced DNA methylation at a distal enhancer and elevated DNA methylation downstream of the transcription start site. CONCLUSIONS/SIGNIFICANCE: Mosaic gene expression occurs in many eukaryotes. Herein we use maize p1 gene as model system to provide further insight about the mechanisms that govern expression mosaicism. We suggest that the gene structure of P1-mm is modified in some of its tandem gene repeats. It is known that repeated genes are susceptible to chromatin-mediated regulation of gene expression. We discuss how the modification to the tandem repeats of P1-mm may have disrupted the epigenetic mechanisms that stably confer tissue-specific expression

Maize Flavan-4-ols and Anthocyanins Alleviated Dextran Sulfate Sodium-Induced Colitis in Mice via Intestinal Barrier Function Restoration

This study investigated the anti-inflammatory effects of anthocyanins and flavan-4-ols (precursor of phlobaphenes) within a whole-food matrix against dextran sulfate sodium (DSS)-induced colitis using four maize near-isogenic lines (NILs) that differ only in a single class of flavonoids. The four NILs are A (lacks anthocyanins and phlobaphenes), B (phlobaphenes +), C (anthocyanins +) and D (anthocyanins + and phlobaphenes +)

Expression of a putative \u3ci\u3eflavonoid 3\u27-hydroxylase\u3c/i\u3e in sorghum mesocotyls synthesizing 3-deoxyanthocyanidin phytoalexins

In sorghum, ingress of Cochliobolus heterostrophus stimulates the synthesis of 3-deoxyanthocyanidins that act as phytoalexins. Apigeninidin and luteolinidin are two major phytoalexins induced in the first 24 h after infection. In an attempt to understand genetic regulation of the biosynthesis of sorghum phytoalexins, we isolated a differentially expressed partial cDNA. Characterization and comparison showed that this cDNA sequence corresponds to a putative flavonoid 3’-hydroxylase. Full length sequence characterization allowed us to establish that the sorghum putative f3’h cDNA encodes a peptide of 517 amino acids that has domains conserved among cytochrome P450 proteins functioning in the flavonoid biosynthetic pathway. Heterologous expression of the putative f3’h cDNA in Escherichia coli yielded a membrane preparation that catalyzed the hydroxylation of naringenin. We show here that transcription of the flavonoid 3’-hydroxylase was coordinately regulated with that of chalcone synthase and dihydroflavonol reductase, and expression of these genes was induced within the first 24 h of fungal challenge. Synthesis of apigeninidin and luteolinidin followed the induced expression of the f3’h gene, implicating its role in fungal induced expression of sorghum phytolaexins

Identification of the Pr1 Gene Product Completes the Anthocyanin Biosynthesis Pathway of Maize

In maize, mutations in the pr1 locus lead to the accumulation of pelargonidin (red) rather than cyanidin (purple) pigments in aleurone cells where the anthocyanin biosynthetic pathway is active. We characterized pr1 mutation and isolated a putative F3′H encoding gene (Zmf3′h1) and showed by segregation analysis that the red kernel phenotype is linked to this gene. Genetic mapping using SNP markers confirms its position on chromosome 5L. Furthermore, genetic complementation experiments using a CaMV 35S::ZmF3′H1 promoter–gene construct established that the encoded protein product was sufficient to perform a 3′-hydroxylation reaction. The Zmf3′h1-specific transcripts were detected in floral and vegetative tissues of Pr1 plants and were absent in pr1. Four pr1 alleles were characterized: two carry a 24 TA dinucleotide repeat insertion in the 5′-upstream promoter region, a third has a 17-bp deletion near the TATA box, and a fourth contains a Ds insertion in exon1. Genetic and transcription assays demonstrated that the pr1 gene is under the regulatory control of anthocyanin transcription factors red1 and colorless1. The cloning and characterization of pr1 completes the molecular identification of all genes encoding structural enzymes of the anthocyanin pathway of maize

Progressive Loss of DNA Methylation Releases Epigenetic Gene Silencing From a Tandemly Repeated Maize Myb Gene

Maize pericarp color1 (p1) gene, which regulates phlobaphene biosynthesis in kernel pericarp and cob glumes, offers an excellent genetic system to study tissue-specific gene regulation. A multicopy p1 allele, P1-wr (white pericarp/red cob) is epigenetically regulated. Hypomethylation of P1-wr in the presence of Unstable factor for orange1 (Ufo1), leads to ectopic pigmentation of pericarp and other organs. The Ufo1-induced phenotypes show incomplete penetrance and poor expressivity: gain of pigmentation is observed only in a subset of plants carrying Ufo1 mutation, and the extent of pigmentation is highly variable. We show that Ufo1 induces progressive hypomethylation of P1-wr repeats over generations. After five generations of exposure to Ufo1, a 30–40% decrease in CG and CNG methylation was observed in an upstream enhancer and an intron region of P1-wr. Interestingly, such hypomethylation correlated with an increase in penetrance of the Ufo1-induced pigmentation phenotype from ∼27 to 61%. Expressivity of the Ufo1-induced phenotype also improved markedly as indicated by increased uniformity of pericarp pigmentation in the later generations. Furthermore, the poor expressivity of the Uo1 is associated with mosaic methylation patterns of the P1-wr upstream enhancer in individual cells and distinct P1-wr gene copies. Finally, comparison of methylation among different tissues indicated that Ufo1 induces rapid CG and CNG hypomethylation of P1-wr repeats during plant development. Together, these data indicate that the poor penetrance and expressivity of Ufo1-induced phenotypes is caused by mosaicism of methylation, and progressive mitotic hypomethylation leads to improved meiotic heritability of the mutant phenotype. In duplicated genomes like maize, loss of an epigenetic regulator may produce mosaic patterns due to redundancy of epigenetic regulators and their target sequences. We show here that multiple developmental cycles may be required for complete disruption of suppressed epigenetic states and appearance of heritable phenotypes

Flavonoid Phytoalexin-Dependent Resistance to Anthracnose Leaf Blight Requires a Functional yellow seed1 in Sorghum bicolor

In Sorghum bicolor, a group of phytoalexins are induced at the site of infection by Colletotrichum sublineolum, the anthracnose fungus. These compounds, classified as 3-deoxyanthocyanidins, have structural similarities to the precursors of phlobaphenes. Sorghum yellow seed1 (y1) encodes a MYB transcription factor that regulates phlobaphene biosynthesis. Using the candystripe1 transposon mutagenesis system in sorghum, we have isolated functional revertants as well as loss-of-function alleles of y1. These near-isogenic lines of sorghum show that, compared to functionally revertant alleles, loss of y1 lines do not accumulate phlobaphenes. Molecular characterization of two null y1 alleles shows a partial internal deletion in the y1 sequence. These null alleles, designated as y1-ww1 and y1-ww4, do not accumulate 3-deoxyanthocyanidins when challenged with the nonpathogenic fungus Cochliobolus heterostrophus. Further, as compared to the wild-type allele, both y1-ww1 and y1-ww4 show greater susceptibility to the pathogenic fungus C. sublineolum. In fungal-inoculated wild-type seedlings, y1 and its target flavonoid structural genes are coordinately expressed. However, in y1-ww1 and y1-ww4 seedlings where y1 is not expressed, steady-state transcripts of its target genes could not be detected. Cosegregation analysis showed that the functional y1 gene is genetically linked with resistance to C. sublineolum. Overall results demonstrate that the accumulation of sorghum 3-deoxyanthocyanidin phytoalexins and resistance to C. sublineolum in sorghum require a functional y1 gene