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
