Maize near-isogenic lines with enhanced flavonoids alleviated dextran sodium sulfate-induced murine colitis via modulation of the gut microbiota.

The rising incidence of inflammatory bowel disease (IBD) has necessitated the search for safe and effective novel therapeutic strategies. Dietary flavonoids exhibited antioxidant, antiproliferative, and anticarcinogenic activities in several model systems with proven abilities to reduce inflammation and oxidative stress, thus they could be promising therapeutic agents for IBD prevention/treatment. However, understanding the role of a specific class of compounds in foods that promote health is difficult because of the chemically complex food matrices. This study aimed to utilize four maize near-isogenic lines to determine the anti-colitis effects of specific classes of flavonoids, anthocyanins and/or phlobaphenes, in a whole-food matrix. Results showed that the intake of anthocyanin and phlobaphene-enriched maize diets effectively alleviated dextran sodium sulfate (DSS)-induced colitis in mice via reducing the intestinal permeability and restoring the barrier function. Anthocyanin diets were more effective in maintaining the crypt structure and muc2 protein levels and reducing inflammation. Bacterial communities of mice consuming diets enriched with anthocyanins and phlobaphenes were more similar to the healthy control compared to the DSS control group, suggesting the role of flavonoids in modulating the gut microbiota to retrieve intestinal homeostasis. Microbiota depletion rendered these compounds ineffective against colitis. Lower serum concentrations of several phenolic acids were detected in the microbiota-depleted mice, indicating that gut microbiota plays a role in flavonoid metabolism and bioavailability.

Inclusion of high-flavonoid corn in the diet of broiler chickens as a potential approach for the control of necrotic enteritis.

Avian necrotic enteritis (NE) is an infectious disease that impacts poultry worldwide causing economic losses. Discontinued use of antimicrobial growth promoters has been associated with high incidence of the disease, which has led to a necessity for finding new therapeutic alternatives. Flavonoids are polyphenolic compounds that have been studied for their health-promoting properties in animals and humans. This study presents a flavonoid-rich corn (PennHFD), as a potential alternative for ameliorating NE in broiler chickens. The effect of a diet formulated with PennHFD was compared to a diet based on commercially available corn in chickens subjected to a controlled challenge of NE based on a co-infection of Eimeria maxima and Clostridium perfringens. Birds fed on the PennHFD-based diet had lower incidence of intestinal lesions (P = 0.048), higher body weight gain (P < 0.01), lower feed conversion ratio (P < 0.01), and lower mortality rates (P = 0.023) compared to the control diet. Therefore, we concluded that the inclusion of the high-flavonoid PennHFD reduces the severity of an experimental challenge of NE in broiler chickens.

Raffinose Family Oligosaccharides: Friend or Foe for Human and Plant Health?

Raffinose family oligosaccharides (RFOs) are widespread across the plant kingdom, and their concentrations are related to the environment, genotype, and harvest time. RFOs are known to carry out many functions in plants and humans. In this paper, we provide a comprehensive review of RFOs, including their beneficial and anti-nutritional properties. RFOs are considered anti-nutritional factors since they cause flatulence in humans and animals. Flatulence is the single most important factor that deters consumption and utilization of legumes in human and animal diets. In plants, RFOs have been reported to impart tolerance to heat, drought, cold, salinity, and disease resistance besides regulating seed germination, vigor, and longevity. In humans, RFOs have beneficial effects in the large intestine and have shown prebiotic potential by promoting the growth of beneficial bacteria reducing pathogens and putrefactive bacteria present in the colon. In addition to their prebiotic potential, RFOs have many other biological functions in humans and animals, such as anti-allergic, anti-obesity, anti-diabetic, prevention of non-alcoholic fatty liver disease, and cryoprotection. The wide-ranging applications of RFOs make them useful in food, feed, cosmetics, health, pharmaceuticals, and plant stress tolerance; therefore, we review the composition and diversity of RFOs, describe the metabolism and genetics of RFOs, evaluate their role in plant and human health, with a primary focus in grain legumes.

Maize unstable factor for orange1 is essential for endosperm development and carbohydrate accumulation.

Maize (Zea mays L.) Ufo1-1 is a spontaneous dominant mutation of the unstable factor for orange1 (ufo1). We recently cloned ufo1, which is a Poaceae-specific gene highly expressed during seed development in maize. Here, we have characterized Ufo1-1 and a loss-of-function Ds insertion allele (ufo1-Dsg) to decipher the role of ufo1 in maize. We found that both ufo1 mutant alleles impact sugars and hormones, and have defects in the basal endosperm transfer layer (BETL) and adjacent cell types. The Ufo1-1 BETL had reduced cell elongation and cell wall ingrowth, resulting in cuboidal shaped transfer cells. In contrast, the ufo1-Dsg BETL cells showed a reduced overall size with abnormal wall ingrowth. Expression analysis identified the impact of ufo1 on several genes essential for BETL development. The overexpression of Ufo1-1 in various tissues leads to ectopic phenotypes, including abnormal cell organization and stomata subsidiary cell defects. Interestingly, pericarp and leaf transcriptomes also showed that as compared with wild type, Ufo1-1 had ectopic expression of endosperm development-specific genes. This study shows that Ufo1-1 impacts the expression patterns of a wide range of genes involved in various developmental processes.

Genome wide association mapping of epi-cuticular wax genes in Sorghum bicolor.

Sorghum accumulates epi-cuticular wax (EW) in leaves, sheaths, and culms. EW reduces the transpirational and nontranspirational (nonstomatal) water loss and protects the plant from severe drought stress in addition to imparting resistance against insect pests. Results presented here are from the analysis of EW content of 387 diverse sorghum accessions and its genome-wide association study (GWAS). EW content in sorghum leaves ranged from 0.1 to 29.7 mg cm-2 with a mean value of 5.1 mg cm-2. GWAS using 265,487 single nucleotide polymorphisms identified thirty-seven putative genes associated (P < 9.89E-06) with EW biosynthesis and transport in sorghum. Major EW biosynthetic genes identified included 3-Oxoacyl-[acyl-carrier-protein (ACP)] synthase III, an Ankyrin repeat protein, a bHLH-MYC, and an R2R3-MYB transcription factor. Genes involved in EW regulation or transport included an ABC transporter, a Lipid exporter ABCA1, a Multidrug resistance protein, Inositol 1, 3, 4-trisphosphate 5/6-kinase, and a Cytochrome P450. This GWA study thus demonstrates the potential for genetic manipulation of EW content in sorghum for better adaptation to biotic and abiotic stress.

Intestinal Mucosal Barrier Function Restoration in Mice by Maize Diet Containing Enriched Flavan-4-Ols.

Inflammatory bowel disease (IBD), a chronic intestinal inflammatory condition, awaits safe and effective preventive strategies. Naturally occurring flavonoid compounds are promising therapeutic candidates against IBD due to their great antioxidant potential and ability to reduce inflammation and improve immune signaling mediators in the gut. In this study, we utilized two maize near-isogenic lines flavan-4-ols-containing P1-rr (F+) and flavan-4-ols-lacking p1-ww (F-) to investigate the anti-inflammatory property of flavan-4-ols against carboxymethylcellulose (CMC)-induced low-grade colonic inflammation. C57BL/6 mice were exposed to either 1% CMC (w/v) or water for a total of 15 weeks. After week six, mice on CMC treatment were divided into four groups. One group continued on the control diet. The second and third groups were supplemented with F+ at 15% or 25% (w/w). The fourth group received diet supplemented with F- at 15%. Here we report that mice consuming F+(15) and F+(25) alleviated CMC-induced increase in epididymal fat-pad, colon histology score, pro-inflammatory cytokine interleukin 6 expression and intestinal permeability compared to mice fed with control diet and F-(15). F+(15) and F+(25) significantly enhanced mucus thickness in CMC exposed mice (p < 0.05). These data collectively demonstrated the protective effect of flavan-4-ol against colonic inflammation by restoring intestinal barrier function and provide a rationale to breed for flavan-4-ols enriched cultivars for better dietary benefits.

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 < 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.

Sorghum 3-Deoxyanthocyanidin Flavonoids Confer Resistance against Corn Leaf Aphid.

In this study we examined the role of sorghum flavonoids in providing resistance against corn leaf aphid (CLA) Rhopalosiphum maidis. In sorghum, accumulation of these flavonoids is regulated by a MYB transcription factor, yellow seed1 (y1). Functional y1 alleles accumulate 3-deoxyflavonoids (3-DFs) and 3-deoxyanthocyanidins (3-DAs) whereas null y1 alleles fail to accumulate these compounds. We found that significantly higher numbers of alate CLA adults colonized null y1 plants as compared to functional y1 plants. Controlled cage experiments and pairwise choice assays demonstrated that apterous aphids preferred to feed and reproduce on null y1 plants. These near-isogenic sorghum lines do not differ in their epicuticular wax content and were also devoid of any leaf trichomes. Significantly higher mortality of CLA was observed on artificial aphid diet supplemented with flavonoids obtained from functional y1 plants as compared to null y1 plants or the relevant controls. Our results demonstrate that the proximate mechanism underlying the deleterious effects on aphids is y1-regulated flavonoids which are important defense compounds against CLA.

The Dominant and Poorly Penetrant Phenotypes of Maize Unstable factor for orange1 Are Caused by DNA Methylation Changes at a Linked Transposon.

The maize (Zea mays) mutant Unstable factor for orange1 (Ufo1) has been implicated in the epigenetic modifications of pericarp color1 (p1), which regulates the production of the flavonoid pigments phlobaphenes. Here, we show that the ufo1 gene maps to a genetically recalcitrant region near the centromere of chromosome 10. Transcriptome analysis of Ufo1-1 mutant and wild-type plants identified a candidate gene in the mapping region using a comparative sequence-based approach. The candidate gene, GRMZM2G053177, is overexpressed by >45-fold in multiple tissues of Ufo1-1, explaining the dominance of Ufo1-1 and its phenotypes. In the mutant stock, GRMZM2G053177 has a unique transcript originating within a CACTA transposon inserted in its first intron, and it is missing the first four codons of the wild-type transcript. GRMZM2G053177 expression is regulated by the DNA methylation status of the CACTA transposon, explaining the incomplete penetrance and poor expressivity of Ufo1-1 Transgenic overexpression lines of GRMZM2G053177 (Ufo1-1) phenocopy the p1-induced pigmentation in coleoptiles, tassels, leaf sheaths, husks, pericarps, and cob glumes. Transcriptome analysis of Ufo1 versus wild-type tissues revealed changes in several pathways related to abiotic and biotic stress. Thus, this study addresses the enigma of Ufo1 identity in maize, which had gone unsolved for more than 50 years.

Overlapping RdDM and non-RdDM mechanisms work together to maintain somatic repression of a paramutagenic epiallele of maize pericarp color1.

Allelic variation at the Zea mays (maize) pericarp color1 (p1) gene has been attributed to epigenetic gene regulation. A p1 distal enhancer, 5.2 kb upstream of the transcriptional start site, has demonstrated variation in DNA methylation in different p1 alleles/epialleles. In addition, DNA methylation of sequences within the 3' end of intron 2 also plays a role in tissue-specific expression of p1 alleles. We show here a direct evidence for small RNAs' involvement in regulating p1 that has not been demonstrated previously. The role of mediator of paramutation1 (mop1) was tested in the maintenance of somatic silencing at distinct p1 alleles: the non-paramutagenic P1-wr allele and paramutagenic P1-rr' epiallele. The mop1-1 mutation gradually relieves the silenced phenotype after multiple generations of exposure; P1-wr;mop1-1 plants display a loss of 24-nt small RNAs and DNA methylation in the 3' end of the intron 2, a region close to a Stowaway transposon. In addition, a MULE sequence within the proximal promoter of P1-wr shows depletion of 24nt siRNAs in mop1-1 plants. Release of silencing was not correlated with small RNAs at the distal enhancer region of the P1-wr allele. We found that the somatic silencing of the paramutagenic P1-rr' is correlated with significantly reduced H3K9me2 in the distal enhancer of P1-rr'; mop1-1 plants, while symmetric DNA methylation is not significantly different. This study highlights that the epigenetic regulation of p1 alleles is controlled both via RdDM as well as non-RdDM mechanisms.

A sorghum MYB transcription factor induces 3-deoxyanthocyanidins and enhances resistance against leaf blights in maize.

Sorghum responds to the ingress of the fungal pathogen Colletotrichum sublineolum through the biosynthesis of 3-deoxyanthocyanidin phytoalexins at the site of primary infection. Biosynthesis of 3-deoxyanthocyanidins in sorghum requires a MYB transcription factor encoded by yellow seed1 (y1), an orthologue of the maize gene pericarp color1 (p1). Maize lines with a functional p1 and flavonoid structural genes do not produce foliar 3-deoxyanthocyanidins in response to fungal ingress. To perform a comparative metabolic analysis of sorghum and maize 3-deoxyanthocyanidin biosynthetic pathways, we developed transgenic maize lines expressing the sorghum y1 gene. In maize, the y1 transgene phenocopied p1-regulated pigment accumulation in the pericarp and cob glumes. LC-MS profiling of fungus-challenged Y1-maize leaves showed induction of 3-deoxyanthocyanidins, specifically luteolinidin. Y1-maize plants also induced constitutive and higher levels of flavonoids in leaves. In response to Colletotrichum graminicola, Y1-maize showed a resistance response.

Comparative proteomics analysis by DIGE and iTRAQ provides insight into the regulation of phenylpropanoids in maize.

The maize pericarp color1 (p1) gene encodes a Myb transcription factor that regulates the accumulation of 3-deoxyflavonoid pigments called phlobaphenes. The Unstable factor for orange1 (Ufo1) is a dominant epigenetic modifier of the p1 that results in ectopic pigmentation in pericarp. Presence of Ufo1-1 correlates with pleiotropic growth and developmental defects. To investigate the Ufo1-1-induced changes in the proteome, we conducted comparative proteomics analysis of P1-wr; Ufo1-1 pericarps using the 2-D DIGE and iTRAQ techniques. Most of the identified proteins were found to be involved in glycolysis, protein synthesis and modification, flavonoid and lignin biosynthesis and defense responses. Further, immunoblot analysis of internode protein extracts demonstrated that caffeoyl CoA O-methyltransferase (COMT) is post-transcriptionally down regulated in P1-wr; Ufo1-1 plants. Consistent with the down regulation of COMT, the concentrations of p-coumaric acid, syringaldehydes, and lignin are reduced in P1-wr; Ufo1-1 internodes. The reductions in these phenylpropanoids correlate with the bent stalk and stunted growth of P1-wr; Ufo1-1 plants. Finally, over-expression of the p1 in transgenic plants is also correlated with a lodging phenotype and reduced COMT expression. We conclude that ectopic expression of p1 can result in developmental defects that are correlated with altered regulation and synthesis of phenylpropanoid compounds including lignin. BIOLOGICAL SIGNIFICANCE: Transcription factors have specific expression patterns that ensure that the biochemical pathways under their control are active in relevant tissues. Plant breeders can select for alleles of transcription factors that produce desirable expression patterns to improve a plant's growth, development, and defense against insects and pathogens. The resulting de novo accumulation of metabolites in plant tissues in significant quantities could have beneficial and/or detrimental consequences. To understand this problem we investigated how the aberrant expression of a classically-studied transcription factor pericarp color1 (p1) which regulates phenylpropanoid metabolism, affects the maize proteome in pericarp tissue. We utilized a dominant mutant Unstable factor for orange 1-1 (Ufo1-1) which reduces the epigenetic suppression of p1 in various tissues throughout the maize plant. Our proteomic analysis shows how, in the presence of Ufo1-1, key enzymes of the glycolytic and shikimic acid pathways were modulated to produce substrates required for flavonoid synthesis. The finding that the presence of Ufo1-1 affected the expression levels of various enzymes in the lignin pathway was of particular interest. We show that lignin was reduced in Ufo1-1 plants expressing p1 and was associated with the post-transcriptional down regulation of CoA O-methyltransferase (COMT) enzyme. We further correlated the down-regulation of COMT with plant bending phenotype in Ufo1-1 plants expressing p1 and to a stalk lodging phenotype of transgenic p1 plants. This study demonstrates that although there can be adverse consequences to aberrantly overexpressing transcription factors, there might also be benefits such as being able to reduce lignin content for biofuel crops. However, more research will be required to understand the genetic and epigenetic regulation of transcription factors and how their expression can be optimized to obtain desired traits in preferred tissue types. This article is part of a Special Issue entitled: Translational Plant Proteomics.

Expression of flavonoid 3'-hydroxylase is controlled by P1, the regulator of 3-deoxyflavonoid biosynthesis in maize.

BACKGROUND: The maize (Zea mays) red aleurone1 (pr1) encodes a CYP450-dependent flavonoid 3'-hydroxylase (ZmF3'H1) required for the biosynthesis of purple and red anthocyanin pigments. We previously showed that Zmf3'h1 is regulated by C1 (Colorless1) and R1 (Red1) transcription factors. The current study demonstrates that, in addition to its role in anthocyanin biosynthesis, the Zmf3'h1 gene also participates in the biosynthesis of 3-deoxyflavonoids and phlobaphenes that accumulate in maize pericarps, cob glumes, and silks. Biosynthesis of 3-deoxyflavonoids is regulated by P1 (Pericarp color1) and is independent from the action of C1 and R1 transcription factors. RESULTS: In maize, apiforol and luteoforol are the precursors of condensed phlobaphenes. Maize lines with functional alleles of pr1 and p1 (Pr1;P1) accumulate luteoforol, while null pr1 lines with a functional or non-functional p1 allele (pr1;P1 or pr1;p1) accumulate apiforol. Apiforol lacks a hydroxyl group at the 3'-position of the flavylium B-ring, while luteoforol has this hydroxyl group. Our biochemical analysis of accumulated compounds in different pr1 genotypes showed that the pr1 encoded ZmF3'H1 has a role in the conversion of mono-hydroxylated to bi-hydroxylated compounds in the B-ring. Steady state RNA analyses demonstrated that Zmf3'h1 mRNA accumulation requires a functional p1 allele. Using a combination of EMSA and ChIP experiments, we established that the Zmf3'h1 gene is a direct target of P1. Highlighting the significance of the Zmf3'h1 gene for resistance against biotic stress, we also show here that the p1 controlled 3-deoxyanthocyanidin and C-glycosyl flavone (maysin) defence compounds accumulate at significantly higher levels in Pr1 silks as compared to pr1 silks. By virtue of increased maysin synthesis in Pr1 plants, corn ear worm larvae fed on Pr1; P1 silks showed slower growth as compared to pr1; P1 silks. CONCLUSIONS: Our results show that the Zmf3'h1 gene participates in the biosynthesis of phlobaphenes and agronomically important 3-deoxyflavonoid compounds under the regulatory control of P1.

Maize Unstable factor for orange1 is required for maintaining silencing associated with paramutation at the pericarp color1 and booster1 loci.

To understand the molecular mechanisms underlying paramutation, we examined the role of Unstable factor for orange1 (Ufo1) in maintaining paramutation at the maize pericarp color1 (p1) and booster1 (b1) loci. Genetic tests revealed that the Ufo1-1 mutation disrupted silencing associated with paramutation at both p1 and b1. The level of up regulation achieved at b1 was lower than that at p1, suggesting differences in the role Ufo1-1 plays at these loci. We characterized the interaction of Ufo1-1 with two silenced p1 epialleles, P1-rr' and P1-pr(TP), that were derived from a common P1-rr ancestor. Both alleles are phenotypically indistinguishable, but differ in their paramutagenic activity; P1-rr' is paramutagenic to P1-rr, while P1-pr(TP) is non-paramutagenic. Analysis of cytosine methylation revealed striking differences within an enhancer fragment that is required for paramutation; P1-rr' exhibited increased methylation at symmetric (CG and CHG) and asymmetric (CHH) sites, while P1-pr(TP) was methylated only at symmetric sites. Both silenced alleles had higher levels of dimethylation of lysine 9 on histone 3 (H3K9me2), an epigenetic mark of silent chromatin, in the enhancer region. Both epialleles were reactivated in the Ufo1-1 background; however, reactivation of P1-rr' was associated with dramatic loss of symmetric and asymmetric cytosine methylation in the enhancer, while methylation of up-regulated P1-pr(TP) was not affected. Interestingly, Ufo1-1-mediated reactivation of both alleles was accompanied with loss of H3K9me2 mark from the enhancer region. Therefore, while earlier studies have shown correlation between H3K9me2 and DNA methylation, our study shows that these two epigenetic marks are uncoupled in the Ufo1-1-reactivated p1 alleles. Furthermore, while CHH methylation at the enhancer region appears to be the major distinguishing mark between paramutagenic and non-paramutagenic p1 alleles, H3K9me2 mark appears to be important for maintaining epigenetic silencing.

Responses to low phosphorus in high and low foliar anthocyanin coleus (Solenostemon scutellarioides) and maize (Zea mays).

Foliar anthocyanin production is frequently induced by phosphorus deficiency, but the adaptive significance of increased anthocyanin production under P stress, if any, remains unknown. In this study we hypothesised that if anthocyanin expression is an adaptive response to mitigate the stress effects of P deficiency, genotypes with constitutive anthocyanin expression would have greater tolerance to P stress than low anthocyanin-producing genotypes. Four studies were conducted in greenhouse, outdoor chamber and field conditions to compare genetically similar maize and coleus plants with contrasting anthocyanin accumulation (i.e. 'red-leafed' vs 'green-leafed'). In low-P treatments, anthocyanin production did not consistently result in greater photosynthesis or biomass. In coleus, red-leafed phenotypes showed lower chlorophyll a/b ratios suggesting photoprotection by anthocyanins against degradation of light harvesting complex proteins. However, the opposite trend was observed in maize, where red-leafed phenotypes showed greater chlorophyll a/b ratios and lower qP (oxidation state of PSII). Based on results from the various treatments and growth conditions of this study, it could not be concluded that high foliar anthocyanin production confers a general functional advantage under low-P stress. More research comparing inducible vs constitutive production may help elucidate the role of anthocyanin biosynthesis in P deficiency responses.

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.

Tissue culture-induced novel epialleles of a Myb transcription factor encoded by pericarp color1 in maize.

Plants regenerated from tissue culture often display somaclonal variation, that is, somatic and often meiotically heritable phenotypic variation that can result from both genetic and epigenetic modifications. To better understand the molecular basis of somaclonal variation, we have characterized four unique tissue culture-derived epialleles of the pericarp color1 (p1) gene of maize (Zea mays L.). The progenitor p1 allele, P1-wr, is composed of multiple head-to-tail tandemly arranged copies of the complete gene unit and specifies brick-red phlobaphene pigmentation in the cob glumes. The novel epialleles identified in progeny plants regenerated from tissue culture showed partial to complete loss of p1 function indicated by pink or colorless cob glumes. Loss of pigmentation was correlated with nearly complete loss of p1 steady-state transcripts. DNA gel-blot analysis and genomic bisulfite sequencing showed that silencing of the epialleles was associated with hypermethylation of a region in the second intron of P1-wr. Presence of Unstable factor for orange1 (Ufo1), an unlinked epigenetic modifier of p1, restored the cob glume pigmentation in the silenced alleles, and such reactivation was accompanied by hypomethylation of the p1 sequence. This observation confirmed that silencing of the epialleles is indeed due to epigenetic modifications and that the p1 epialleles were capable of functioning in the presence of the correct trans-acting factors. While the low-copy regions of the genome generally undergo hypomethylation during tissue culture, our study shows that the tandemly repeated genes are also prone to hypermethylation and epigenetic silencing.

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.

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.

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 approximately 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.