R3-MYB repressor Mybr97 is a candidate gene associated with the Anthocyanin3 locus and enhanced anthocyanin accumulation in maize.

KEY MESSAGE: Anthocyanin3 inhibits the anthocyanin and monolignol pathways in maize. Transposon-tagging, RNA-sequencing, and GST-pulldown assays determine Anthocyanin3 may be R3-MYB repressor gene Mybr97. Anthocyanins are colorful molecules receiving recent attention due to their numerous health benefits and applications as natural colorants and nutraceuticals. Purple corn is being investigated as a more economical source of anthocyanins. Anthocyanin3 (A3) is a known recessive intensifier of anthocyanin pigmentation in maize. In this study, anthocyanin content was elevated 100-fold in recessive a3 plants. Two approaches were used to discover candidates involved with the a3 intense purple plant phenotype. First, a large-scale transposon-tagging population was created with a Dissociation (Ds) insertion in the nearby Anthocyanin1 gene. A de novo a3-m1::Ds mutant was generated, and the transposon insertion was found to be located in the promoter of Mybr97, which has homology to R3-MYB repressor CAPRICE in Arabidopsis. Second, a bulked segregant RNA-sequencing population found expression differences between pools of green A3 plants and purple a3 plants. All characterized anthocyanin biosynthetic genes were upregulated in a3 plants along with several genes of the monolignol pathway. Mybr97 was highly downregulated in a3 plants, suggesting its role as a negative regulator of the anthocyanin pathway. Photosynthesis-related gene expression was reduced in a3 plants through an unknown mechanism. Numerous transcription factors and biosynthetic genes were also upregulated and need further investigation. Mybr97 may inhibit anthocyanin synthesis by associating with basic helix-loop helix transcription factors like Booster1. Overall, Mybr97 is the most likely candidate gene for the A3 locus. A3 has a profound effect on the maize plant and has many favorable implications for crop protection, human health, and natural colorant production.

Functional Characterization of an Anthocyanin Dimalonyltransferase in Maize.

Anthocyanins are pigments with appealing hues that are currently being used as sources of natural colorants. The interaction of acylation on the stability of anthocyanin molecules has long been known. Maize is an abundant source of malonylglucoside and dimalonylglucoside anthocyanins. The enzyme Aat1 is an anthocyanin acyltransferase known to synthesize the majority of acylated anthocyanins in maize. In this paper, we characterize the substrate specificity and reaction kinetics of Aat1. It was found that Aat1 has anthocyanin 3-O-glucoside dimalonyltransferase activity and is only the second enzyme of this type characterized to this date. Our results indicate that Aat1 can utilize malonyl-CoA; succinyl-CoA and every anthocyanin 3-O-glucoside tested. Results of this study provide insight into the structure-function relations of dimalonyltransferases and give a unique insight into the activity of monocot anthocyanin acyltransferases.

Prospects for economical natural colorants: insights from maize.

KEY MESSAGE: Anthocyanin pigments from maize offer a natural yet economical alternative to artificial dyes. Breeding for optimal colorant production requires understanding and integrating all facets of anthocyanin chemistry and genetics research. Replacing artificial dyes with natural colorants is becoming increasingly popular in foods and beverages. However, natural colorants are often expensive, have lower stability, and reduced variability in hue. Purple corn is rich in anthocyanins and offers a scalable and affordable alternative to synthetic dyes ranging in color from orange to reddish-purple. This diversity is attributable to differences in anthocyanin composition and concentration. Here we review the chemistry, biosynthesis, and genetics of purple corn and outline key factors associated with the feasibility of producing an economical source of natural colorants. Anthocyanin compositional modifications including acylation, methylation, and polymerization with flavan-3-ols can influence color stability and hue, yet there is more to learn regarding the genetic factors responsible for these modifications. Activators and repressors of anthocyanin biosynthesis structural genes as well as factors controlling trafficking and storage largely control anthocyanin yield. Further knowledge of these mechanisms will allow breeders to apply molecular strategies that accelerate the production of purple corn hybrids to meet growing demands for natural colorants.

Increasing aleurone layer number and pericarp yield for elevated nutrient content in maize.

The bran is a nutritive fraction of the maize (Zea mays L.) kernel containing micronutrients, quality protein, and antioxidants beneficial for human health. Bran consists of two major components: aleurone and pericarp. Increasing this nutritive fraction would therefore have implications on biofortification of maize. Since quantification of these two layers is difficult, the goals of this study were to develop efficient techniques for analyzing these layers and to develop molecular markers for pericarp and aleurone yield. Two populations with various characteristics were genotyped using genotyping-by-sequencing. The first was a yellow corn population with contrasting pericarp thicknesses. The second was a blue corn population segregating for Intensifier1 alleles. Both populations segregated for the multiple aleurone layer (MAL) trait that is known to increase aleurone yield. In this study, it was found that MALs are mostly determined by a locus on chromosome 8, but several minor loci are also involved. The inheritance of MALs was complex and seemingly more additive than dominant. In the blue corn population, anthocyanin content increased 20 to 30% with the addition of MALs demonstrating its effectiveness at increasing aleurone yield. Elemental analysis was performed on MAL lines and indicated a role of MALs in increasing iron content in the grain. Iron content was increased 17.5% in the MAL lines over the single aleurone layer lines and 35.5% over the recurrent parent, Mo17. Zinc content was increased 15.5% in the MAL lines compared to the recurrent parent. QTL analyses are presented in this study on many pericarp, aleurone, and grain quality traits. Molecular markers were also tested for the MAL locus on chromosome 8, and candidate genes are discussed. Results of this study may assist plant breeders enhancing anthocyanin content and other beneficial phytonutrients in maize.

Assessing the diversity of anthocyanin composition in various tissues of purple corn (Zea mays L.).

Anthocyanins are natural pigments used in various foods, beverages, textiles, and nutraceuticals. Anthocyanins in the grain of purple corn (Zea mays L., Poaceae) have been a focus of many studies, but not much is known about anthocyanins in other maize tissues. In this study, purple corn variety Apache Red Cob was crossed to genetic stock 320 N, which is recessive for anthocyanin 3. The result was intense anthocyanin production in portions of the plant not normally pigmented. Anthocyanin extracts from anthers, cob glumes, husks, kernels, leaf sheaths, seedlings, silks, and tassels were assessed using UHPLC. A previously undescribed pigment produced in anthers was determined by NMR to be anthocyanidin 3-6″-phenylacetylglucoside. Multivariate analysis classified maize anthocyanins into 8 major compositional profiles. Results of this study show that maize produces anthocyanins abundantly in non-grain portions of the plant and that maize anthocyanin extracts have numerous applications due to the diversity in pigment profiles and hues.

Discovery of Anthocyanin Acyltransferase1 (AAT1) in Maize Using Genotyping-by-Sequencing (GBS).

The reduced acylation phenotype describes the inability of certain accessions of maize (Zea mays [L]) to produce significant amounts of acylated anthocyanins, which are typically the most abundant pigments. Acylated anthocyanins are important for their association with stability and are therefore important for the various industries using anthocyanins as natural colorants to replace synthetic dyes. Many anthocyanin acyltransferases have been characterized in other species; however, no anthocyanin acyltransferases have been characterized in maize. Therefore, a mapping population was developed from a cross between mutant stock 707G and wild-type acylation line B73 to identify the locus associated with the reduced acylation trait. High-performance liquid chromatography was used to assay the pigment content and composition of 129 F2 lines generated in the mapping population. Recessive alleles of Colorless1, Colored1, and the reduced acylation mutant all decreased anthocyanin content while Intensifier1 increased anthocyanin content in aleurone tissue. The association of increased proportions of acylation with increased anthocyanin content indicates acylation may be important for increasing the stability of anthocyanins in vivo Genotyping-by-sequencing was used to create SNP markers to map the reduced acylation locus. In the QTL analysis, a segment of Chromosome 1 containing transferase family protein GRMZM2G387394 was found to be significant. A UniformMu Mu transposon knockout of GRMZM2G387394 demonstrated this gene has anthocyanidin malonyltransferase activity and will therefore be named Anthocyanin Acyltransferase1 (AAT1). AAT1 is the first anthocyanin acyltransferase characterized in a monocot species and will increase our knowledge of all acyltransferase family members.

Comparison of chemical, color stability, and phenolic composition from pericarp of nine colored corn unique varieties in a beverage model.

The objective was to compare the chemical stability and color of nine unique anthocyanin-rich colored corn varieties named/coded as V1, V2, V3… V9. Extracts were added to a beverage model and stored at 4 °C, 22°C, or 32°C for 12weeks. After 12 weeks of storage at 32°C, variety V6 [high condensed form (CF), high cyanidin-3-O-glucoside (C3G)] had the longest anthocyanin half-life, based on the quantification by HPLC. V3 [high pelargonidin (Pg), high acylated form (C3-mal)] and V5 (high CF, high C3G, high C3-mal) had the most favorable hue. V5 and V6 had some of the smallest changes in color over time. These findings suggest that an abundance of condensed forms with C3G in corn extracts could contribute to the improved stability. Beverage storage parameters also influenced color parameters; low temperatures and low pH enhanced color and anthocyanin stability. The most promising corn varieties for future experiments are V3, V5, and V6 based on color retention.

Survey of Anthocyanin Composition and Concentration in Diverse Maize Germplasms.

Increasing consumer demand for natural ingredients in foods and beverages justifies investigations into more economic sources of natural colorants. In this study, 398 genetically diverse pigmented accessions of maize were analyzed using HPLC to characterize the diversity of anthocyanin composition and concentration in maize germplasm. One hundred and sixty-seven accessions were identified that could produce anthocyanins in the kernel pericarp or aleurone and were classified into compositional categories. Anthocyanin content was highest in pericarp-pigmented accessions with flavanol-anthocyanin condensed forms, similar to the Andean Maíz Morado landraces. A selected subset of accessions exhibited high broad-sense heritability estimates for anthocyanin production, indicating this trait can be manipulated through breeding. This study represents the most comprehensive screening of pigmented maize lines to date and will provide information to plant breeders looking to develop anthocyanin-rich maize hybrids as an economic source of natural colorants in foods and beverages.