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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

A Mutator transposon insertion is associated with ectopic expression of a tandemly repeated multicopy Myb gene pericarp color1 of maize.

The molecular basis of tissue-specific pigmentation of maize carrying a tandemly repeated multicopy allele of pericarp color1 (p1) was examined using Mutator (Mu) transposon-mediated mutagenesis. The P1-wr allele conditions a white or colorless pericarp and a red cob glumes phenotype. However, a Mu-insertion allele, designated as P1-wr-mum6, displayed an altered phenotype that was first noted as occasional red stripes on pericarp tissue. This gain-of-pericarp-pigmentation phenotype was heritable, yielding families that displayed variable penetrance and expressivity. In one fully penetrant family, deep red pericarp pigmentation was observed. Several reports on Mu suppressible alleles have shown that Mu transposons can affect gene expression by mechanisms that depend on transposase activity. Conversely, the P1-wr-mum6 phenotype is not affected by transposase activity. The increased pigmentation was associated with elevated mRNA expression of P1-wr-mum6 copy (or copies) that was uninterrupted by the transposons. Genomic bisulfite sequencing analysis showed that the elevated expression was associated with hypomethylation of a floral-specific enhancer that is approximately 4.7 kb upstream of the Mu1 insertion site and may be proximal to an adjacent repeated copy. We propose that the Mu1 insertion interferes with the DNA methylation and related chromatin packaging of P1-wr, thereby inducing expression from gene copy (or copies) that is otherwise suppressed.

Epigenetic modifications of distinct sequences of the p1 regulatory gene specify tissue-specific expression patterns in maize.

Tandemly repeated endogenous genes are common in plants, but their transcriptional regulation is not well characterized. In maize, the P1-wr allele of pericarp color1 is composed of multiple copies arranged in a head-to-tail fashion. P1-wr confers a white kernel pericarp and red cob glume pigment phenotype that is stably inherited over generations. To understand the molecular mechanisms that regulate tissue-specific expression of P1-wr, we have characterized P1-wr*, a spontaneous loss-of-function epimutation that shows a white kernel pericarp and white cob glume phenotype. As compared to its progenitor P1-wr, the P1-wr* is hypermethylated in exon 1 and intron 2 regions. In the presence of the epigenetic modifier Ufo1 (Unstable factor for orange1), P1-wr* plants exhibit a range of cob glume pigmentation whereas pericarps remain colorless. In these plants, the level of cob pigmentation directly correlates with the degree of DNA demethylation in the intron 2 region of p1. Further, genomic bisulfite sequencing indicates that a 168-bp region of intron 2 is significantly hypomethylated in both CG and CNG context in P1-wr* Ufo1 plants. Interestingly, P1-wr* Ufo1 plants did not show any methylation change in a distal enhancer region that has previously been implicated in Ufo1-induced gain of pericarp pigmentation of the P1-wr allele. These results suggest that distinct regulatory sequences in the P1-wr promoter and intron 2 regions can undergo independent epigenetic modifications to generate tissue-specific expression patterns.

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.

The maize unstable factor for orange1 is a dominant epigenetic modifier of a tissue specifically silent allele of pericarp color1.

We have characterized Unstable factor for orange1 (Ufo1), a dominant, allele-specific modifier of expression of the maize pericarp color1 (p1) gene. The p1 gene encodes an Myb-homologous transcriptional activator of genes required for biosynthesis of red phlobaphene pigments. The P1-wr allele specifies colorless kernel pericarp and red cobs, whereas Ufo1 modifies P1-wr expression to confer pigmentation in kernel pericarp, as well as vegetative tissues, which normally do not accumulate significant amounts of phlobaphene pigments. In the presence of Ufo1, P1-wr transcript levels and transcription rate are increased in kernel pericarp. The P1-wr allele contains approximately six p1 gene copies present in a hypermethylated and multicopy tandem array. In P1-wr Ufo1 plants, methylation of P1-wr DNA sequences is reduced, whereas the methylation state of other repetitive genomic sequences was not detectably affected. The phenotypes produced by the interaction of P1-wr and Ufo1 are unstable, exhibiting somatic mosaicism and variable penetrance. Moreover, the changes in P1-wr expression and methylation are not heritable: meiotic segregants that lack Ufo1 revert to the normal P1-wr expression and methylation patterns. These results demonstrate the existence of a class of modifiers of gene expression whose effects are associated with transient changes in DNA methylation of specific loci.

Ordered origin of the typical two- and three-repeat Myb genes.

Myb domain proteins contain a conserved DNA-binding domain composed of one to four conserved repeat motifs. In animals, Myb proteins are encoded by a small gene family and commonly contain three repeat motifs (R1R2R3); whereas, plant Myb proteins are encoded by a very large and diverse gene family in which a motif containing two repeats (R2R3) is the most common. In contrast to the conservation in the Myb domain, other regions of Myb proteins are highly variable. To explore the evolutionary origin of Myb genes, we cloned and sequenced Myb domains from maize and sorghum, and conducted a comprehensive phylogenetic analysis of Myb genes. The results indicate that the origins of individual Myb repeats are strikingly distinct, and that the R2 repeat has evolved more slowly than the R1 and R3 repeats. However, it is not clear which repeat is the most ancient one. The evidence also suggests that R2R3 and R1R2R3 Myb genes co-existed in eukaryotes before the divergence of plants and animals. Based on our results, we propose that R1R2R3 Myb genes were derived from R2R3 Myb genes by gain of the R1 repeat through an ancient intragenic duplication; this gain model is more parsimonious than the previous proposal that R2R3 Myb genes were derived from R1R2R3 Mybs by loss of the R1 repeat. A separate group of diverse non-typical Myb proteins exhibits a polyphyletic origin and a complex evolutionary pattern. Finally, a small group of ancient Myb paralogs prior to the amplification of current Myb genes is identified. Together, these results support a new model for the ordered evolution of Myb gene family.

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.

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.