Genetic factors involved with increased anthocyanin production in maize

Anthocyanins are the pigments present in nearly every plant species. These pigments have numerous roles in the plant and are associated with visual signaling and free-radicle scavenging. Anthocyanins have numerous human applications as well. The antioxidant properties of these molecules confer numerous health benefits. In addition, the orange to red to violet hues of anthocyanins makes them suitable as natural colorants. In particular, anthocyanins are capable of replacing FD&C Red No. 40, which is the most common synthetic dye on the market. Replacing synthetic dyes is important for human health and for sustainability. Interest in maize as a source of natural colorants is growing due to the abundance and diversity of pigment production and due to the economy of scale of maize production. In Chapter 1, a population with novel characteristics was developed to assess the diversity of anthocyanin composition in maize and to see the potential of anthocyanin production in tissues outside the grain. Results of this study show that maize is an abundant source of natural colorants, especially in non-grain portions of the plant, and the diversity in hues makes it suitable for many natural colorant applications. Additionally, a novel anthocyanin type was discovered in this population that broadens our knowledge of anthocyanin synthesis in maize. Chapter 2 is also focused on understanding in detail how anthocyanins are synthesized in maize. In this chapter, recombinant Anthocyanin acyltransferase1 (Aat1) was isolated to determine the specificity and reaction kinetics of this enzyme. Aat1 is one of only two characterized anthocyanin dimalonyltransferases in the plant kingdom. Integrating regulatory factors involved with increasing anthocyanin content in maize will assist in breeding for economical natural colorants. In all plants, the activation is stimulated by the core set of transcriptional regulators referred to as the MBW complex for the Myb, bHLH, and WD-repeat that must physically interact for anthocyanin-related genes to be transcribed. In maize, the MBW complex is composed of multi-allelic members that coordinate spatial and temporal patterning of anthocyanin synthesis in the plant. Aleurone pigment is activated by Myb Colored aleurone1, bHLH Colored1, and WD-repeat Pale aleurone color1. In mainly vegetative tissues, synthesis is regulated by Myb Purple plant1, bHLH Booster1 (B1), and a currently unknown WD-repeat protein. Additionally, there are two genes known to have a profound effect on anthocyanin synthesis. The first is Intensifier1, a classic negative regulator of anthocyanin synthesis in aleurone. The second is Anthocyanin3 (A3), which is a known negative regulator of pigmentation in tissues where B1 is most active. The gene or element involved with A3 was unknown prior to this work. In Chapter 3, two genetic approaches are utilized to determine the gene involved. The first was a large-scale transposon-tagging population utilizing a Dissociation element in the upstream Anthocyanin1 gene. The second was a bulk-segregant RNA-seq population segregating for the a3 phenotype comprised of pools of green and purple husk tissues. Together, these populations pinpointed the gene to R3-Myb-like gene Mybr97. Anthocyanin and phenolic content increased 100-fold in mutant husks. Transcriptomic analysis revealed that the entire phenylpropanoid pathway was upregulated in mutants and revealed novel regulators that may be involved with anthocyanin synthesis. An additional method for enhancing anthocyanin content is through the increased yield of anthocyanin-producing tissues in the grain. Pericarp and aleurone produce most of the anthocyanins in maize kernels. In Chapter 4, two populations with contrasting pericarp and aleurone yield were developed. Both populations utilized a unique trait referred to as the multiple aleurone layer (MAL) phenotype. Two populations were genotyped using genotyping-by-sequencing. It was found that the MAL trait is conferred mostly by a locus on chromosome 8 with several other minor loci modulating the number of aleurone layers. Anthocyanin content increased nearly 30 to 40% in MAL lines indicating an increase in aleurone yield. Genetic markers on Chromosome 8 were developed to make breeding for the MAL trait easier. Finally, the classic way to increase anthocyanin content is by utilizing genetic resources in maize and breeding for higher anthocyanin content. Chapter 5 discusses methods for breeding for higher grain yield and anthocyanin yield in temperate dent varieties. As a result of the breeding program, three near-isogenic purple corn lines in a temperate inbred background and multiple breeding populations were created. Methods for breeding purple corn and protocols for marker assisted selection are presented in this chapter. The results of this breeding program demonstrate that purple corn can match the Midwest USA average grain yield without a reduction in anthocyanin content. Altogether, maize has potential as an economical source of natural colorants and there is extensive genetic diversity of purple corn that can be utilized to make high anthocyanin-yielding varieties

Characterization of anthocyanin production in maize germplasm

Anthocyanins are the visually appealing red-orange to blue natural pigments present in most plant species. Because of their ease of aqueous extraction and rich color, anthocyanins make suitable natural replacements for synthetic dyes like FD&C Red 40. Increasing consumer demand for natural ingredients in foods and beverages has justified a search for more economic sources of natural colorants. Maize has been used a natural source of color for centuries. South American cultures in particular use purple corn varieties termed “Maize Morado” in foods and beverages. Maize has economic potential as a value-added source of natural colors. To characterize the diversity of anthocyanin production in maize germplasm, 398 diverse accessions of pigmented maize were analyzed with High Performance Liquid Chromatography (HPLC). To the best of our knowledge, this is the largest collection of pigmented maize investigated for anthocyanin diversity and composition. A subset of this collection was also used to test the repeatability of anthocyanin content and composition in several environments. Within the collection, 167 accessions could produce detectable amounts of anthocyanins. Clusters of accessions were created based on the abundance of pigments in the aleurone or pericarp layers and on compositional variations. These clusters were confirmed by principal component analysis and hierarchical clustering. Pericarp-pigmented accessions that could produce flavanol-anthocyanin dimers called “condensed forms” were the most important accessions in the survey in terms of total anthocyanin content. In the survey acylated anthocyanins were typically the most predominant pigments types, except in a few unique lines that only produce cyanidin 3-glucoside as their major pigment. The hypothesis tested was that this trait is due to partial loss of function anthocyanin acyltransferase. To characterize this phenotype, coined the “reduced acylation” trait, a mapping population was created from a mutant phenotype line crossed to B73, the reference genome. Genotyping-by-sequencing was used to generate single nucleotide polymorphisms that could be used to map the location of the trait in the maize genome. QTL analysis found one significant loci at the end of chromosome 1 which corresponds to a candidate anthocyanin acyltransferase GRMZM2G387394. A UniformMu Mu transposon knockout of this gene maintained the phenotype when crossed to reduced acylation mutants meaning the gene is responsible for a majority of the anthocyanin acyltransferase activity in maize. Overall, information provided here will assist plant breeders looking to develop anthocyanin-rich purple corn hybrids as a source of natural colorants for food and beverages

ATSeqMAF05.hmp.txt

Filtered and imputed reads from the F2 population of B73 x 707G. This population was developed for the purpose of discovering the position of the reduced acylation trait in the genome

ATSeqMAF05REF.txt

Genotype dataset from the F2 population of B73 x 707G coded as 1 = major allele, 0.5 = heterozygote, and 0 = minor allele

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

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

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 <i>Maı́z Morado</i> 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