Flavonoids represent a large class of secondary plant metabolites, of which anthocyanins are the most conspicuous class, dueto the wide range of colors resulting from their synthesis. Anthocyanins are important to many diverse functions within plants. Synthesis of anthocyanins in petals is undoubtedly intended to attract pollinators, whereas anthocyanin synthesis in seeds and fruits may aid in seed dispersal. Anthocyanins and other flavonoids can also be important as feeding deterrents and as protection against damage from UV irradiation. The existence of such a diverse range of functions and types of anthocyanins raises questions about how these compounds are synthesized and how their synthesis is regulated. The study of the genetics of anthocyanin synthesis began last century with Mendel’s work on flower color in peas. Since that time, there have been periods of intensive study into the genetics and biochemistry of pigment production in a number of different species. In the early studies, genetic loci were correlated with easily observable color changes. After the structures of anthocyanins and other flavonoids were determined, it was possible to correlate single genes with particular structural alterations of anthocyanins or with the presence or absence of particular flavonoids. Mutations in anthocyanin genes have been studied for many years because they are easily identified and because they generally have no deleterious effect on plant growth and development. In most cases, mutations affecting different steps of the anthocyanin biosynthesis pathway were isolated and characterized well before their function was identified or the corresponding gene was isolated. More recently, many genes involved in the biosynthesis of anthocyanin pigments have been isolated and characterized using recombinant DNA technologies. Three species have been particularly important for elucidating the anthocyanin biosynthetic pathway and for isolating genes controlling the biosynthesis of flavonoids: maize (Zea mays), snapdragon (Anfirrhinum majus), and petunia (Wtunia hybrida). Petunia has more recently become the organism of choice for isolating flavonoid biosynthetic genes and studying their interactions and regulation. At least 35 genes are known to affect flower color in petuniawiering and de Vlaming, 1984). Because this field of research has been reviewed fairly extensively in the past (Dooner et al., 1991; van Tunen and MOI, 1991; Gerats and Martin, 1992), in this review we concentrate on the more recent developments in gene isolation and characterization. A review of the genetics of flavonoid biosynthesis in other species was recently covered by Forkmann (1993). The characterization of genetically defined mutations has enabled the order of many reactions in anthocyanin synthesis and their modification to be elucidated. Some reactions have been postulated only on the basis of genetic studies and have not yet been demonstrated in vitro. Chemico-genetic studies have been very important in determining the enzymatic steps involved in anthocyanin biosynthesis and modification. The generation of transposon-tagged mutations and the subsequent cloning of the transposons provided a relatively straightforward means of isolating many genes from maize (Wienand et al., 1990) and snapdragon (Martin et al., 1991). However, a number of genes in the pathway have not been amenable to transposon tagging. Anthocyanin biosynthetic genes have been isolated using a range of methodologies, including protein purification, transposon tagging, differential screening, and polymerase chain reaction (PCR) amplification. Functions of isolated anthocyanin genes can be confirmed by restriction fragment length polymorphism (RFLP) mapping, complementation, or expression in heterologous systems. Reverse genetics has also been used recently to identify gene function; this requires a welldefined pathway to correlate phenotype with gene function. Once a gene has been isolated from one species, it is usually a straightforward task to isolate the homologous gene from other species by using the original clone as a molecular probe.

/pdf/genetics-and-biochemistry-of-anthocyanin-biosynthesis-41091xx7zv.pdf

Genetics and Biochemistry of Anthocyanin Biosynthesis

The relative organization of genes and repetitive DNAs in complex eukaryotic genomes is not well understood. Diagnostic sequencing indicated that a 280-kilobase region containing the maize Adh1-F and u22 genes is composed primarily of retrotransposons inserted within each other. Ten retroelement families were discovered, with reiteration frequencies ranging from 10 to 30,000 copies per haploid genome. These retrotransposons accounted for more than 60 percent of the Adh1-F region and at least 50 percent of the nuclear DNA of maize. These elements were largely intact and are dispersed throughout the gene-containing regions of the maize genome.

https://science.sciencemag.org/content/sci/274/5288/765.full.pdf

Nested Retrotransposons in the Intergenic Regions of the Maize Genome

Previous studies have suggested that the R locus of maize is responsible for determining the temporal and spatial pattern of anthocyanin pigmentation in the plant. In this report we demonstrate that three members of the R gene family, P, S, and Lc, encode homologous transcripts 2.5 kilobases in length. The structure of one R gene, Lc, was determined by sequencing cDNA and genomic clones. The putative Lc protein, deduced from the cDNA sequence, is composed of 610 amino acids and has homology to the helix-loop-helix DNA-binding/dimerization motif found in the L-myc gene product and other regulatory proteins. It also contains a large acidic domain that may be involved in transcriptional activation. Consistent with its proposed role as a transcriptional activator is our finding that a functional R gene is required for the accumulation of transcripts of at least two genes in the anthocyanin biosynthetic pathway. We discuss the possibility that the diverse patterns of anthocyanin pigmentation conditioned by different R genes reflect differences in the R gene promoters rather than their gene products.

https://www.pnas.org/content/pnas/86/18/7092.full.pdf

Lc, a member of the maize R gene family responsible for tissue-specific anthocyanin production, encodes a protein similar to transcriptional activators and contains the myc-homology region.

Recombinant inbred lines of maize have been developed for the rapid mapping of molecular probes to chromosomal location. Two recombinant inbred families have been constructed from F2 populations of T232 X CM37 and CO159 X Tx303. A genetic map based largely on isozymes and restriction fragment length polymorphisms has been produced that covers virtually the entire maize genome. In order to map a new gene, an investigator has only to determine its allelic distribution among the recombinant inbred lines and then compare it by computer with the distributions of all previously mapped loci. The availability of the recombinant inbreds and the associated data base constitute an efficient means of mapping new molecular markers in maize.

/pdf/gene-mapping-with-recombinant-inbreds-in-maize-1ublx7zrgi.pdf

Gene mapping with recombinant inbreds in maize

The Genetics of Corn

The a1 locus of Zea mays has been cloned using transposable elements as gene tags. The strategy was to make genomic libraries from maize stocks with a1 mutations induced either by En(Spm) or by Robertson's Mutator-system. These libraries were then screened with either Spm-I8 and En1, for the En-containing mutant, or with Mu1 for the Mu-induced mutation. There are many En and Mu1 hybridizing sequences present in the maize genome, however, by a process of cross-screening of the positives from the two libraries and by molecular analysis of the En-positive clones it was possible to identify clones in both libraries carrying all or part of the a1 gene.

Molecular cloning of the a1 locus of Zea mays using the transposable elements En and Mu1

A newly isolated, unstable mutation wx-844::En-1 of Zea mays was proven to be caused by the insertion of the autonomous transposable element En into the Waxy (Wx) gene. Molecular analysis revealed that En-1 is 8.4 kb long, has a 13-bp long perfect inverted repeat at its termini and generates a 3-bp target site duplication. En-1 is integrated into an intron located approximately in the middle of the transcribed region of the Wx gene. Structural evidence is presented indicating that a receptor component (Inhibitor) can arise by internal deletion of an autonomous En element.

Genetic and molecular analysis of the Enhancer (En) transposable element system of Zea mays.

Fourteen recombinant clones from Zea mays were studied with regard to their composition of unique and repetitive sequences. Southern hybridization experiments were used to classify restriction fragments of the clones into a unique, middle or highly repetitive class of reiteration frequency. All three classes were often found on the same genomic clone. Crosshybridization studies between clones showed that a given repeat might be present on several clones, and thus four families of highly repetitive elements were established. Heteroduplex analysis was used to show the arrangement and size of repeats common between several clones. A short interspersion pattern of unique, middle and highly repetitive DNA was found. The dispersed repetitive elements were 300-1300 bp in length. Analysis of the pattern produced by a given repeat in genomic Southern experiments suggests that some small dispersed repeats may also exist as part of a larger repeating unit elsewhere in the genome.

/pdf/repetitive-sequences-and-their-organization-on-genomic-2q27cca5j4.pdf

Repetitive sequences and their organization on genomic clones of Zea mays.

The Corn Insert, Cin1, is shown to be a heterogenous family of dispersed repetitive elements in the genome of Zea mays. Fourteen different recombinant clones were isolated from the maize Line C genome using the original Cin1-001 insert as a hybridization probe. These clones were characterized by heteroduplex analysis and restriction enzyme mapping as to the position and extent of the Cin1 region(s). The Cin1 related sequences are shown to vary in size up to approximately 700 bp.

Isolde Bertram

Papers

Molecular cloning of the a1 locus of Zea mays using the transposable elements En and Mu1

Genetic and molecular analysis of the Enhancer (En) transposable element system of Zea mays.

Repetitive sequences and their organization on genomic clones of Zea mays.

Cin1, a family of dispersed repetitive elements in Zea mays