Cell, Vol. 62, 649-851,

September

7, 1990, Copyright

0 1990 by Cell Press

Maize R Gene Family: TissueSpecif ic Helix-Loop-Helix Proteins Steven R. Ludwig and Susan ft. Wessler Botany Department University of Georgia Athens, Georgia 30602

The red and purple pigments that can color virtually any tissue of the maize plant are called anthocyanins. The anthocyanin biosynthetic pathway has attracted geneticists since the turn of the century because the mutant phenotypes are viable and the range of expression that can be visually detected is extraordinary (reviewed in Coe et al., 1968). Long before Mendel, native Americans selected and propagated maize strains that displayed diverse patterns of pigmentation. Only in recent years have we learned that they were selecting natural variants among the R gene family of helix-loop-helix (HLH) proteins that control the temporal and spatial distribution of anthocyanins in the maize plant. The genetics of anthocynanin biosynthesis in maize has a long and rich history beginning with Mendel, who studied the segregation of dark-red kernels on the cob. Shortly after the rediscovery of Mendel’s laws, East and Emerson, two pioneers of maize genetics, communicated the value of the anthocyanin pathway as a genetic research tool to many of their students, including Beadle, Brink, Demerec, Hayes, Mangelsdorf, and McClintock. East and Hayes (1911) named the regulatory gene R, signifying red plant color, and Emerson (1921) subsequently identified multiple alleles of both R and a functionally duplicate locus, 8. To characterize the R alleles, Fogel (1946) quantified the level of anthocyanins from many strains displaying diverse phenotypes. He determined that the R alleles control organ, tissue, and cell-type specificity of anthocyanin pigmentation. Genetic studies have revealed that the overall pattern of pigmentation displayed by a maize plant may reflect the expression of either one or several R genes. For example, the standard R locus (see table), which pigments seed and plant tissues, is actually comprised of two independently mutating but tightly linked genes, Sand P(for Seed and Plant color; Stadler, 1951). In certain strains, additional members of the R gene family are located two map units distal to the standard R locus. Although these R genes probably arose following duplication of the standard R locus, their tissue-specific patterns of expression are distinct from those of the S and P genes. Chromosomal duplication followed by dispersal may explain the origin of the 6 genes, members of the R family located on chromosome 2. Unlike many of the R genes (on chromosome lo), the B genes condition deep-purple pigmentation of most photosynthetic tissues. The duplicate nature of R and 6 genes was manifested by the observation that the B-Rsru gene can substitute for a functional R gene in the aleurone layer of the kernel (Styles et al., 1973). An understanding of the complex interactions between structural and regulatory genes is a necessary prerequi-

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site to unraveling the diversity of R phenotypes. Biochemical and genetic studies have identified many of the genes in the anthocyanin pathway and provided the initial evidence that R genes encode regulatory proteins. R has been shown to regulate the levels of expression of three enzymes involved in anthocyanin biosynthesis: chalcone synthase encoded by C2, Al-encoded dihydroquercetin reductase, and UDP glucose:flavonoid 3-O-glucosyltransferase encoded by Bronze7 (Bzl) @eddy et al., 1987; Dooner, 1983; Dooner and Nelson, 1979). Additional enzymes in the pathway, including hydroxylases, methylases, glycosylases, and acylases, function to produce different anthocyanin pigments; whether R regulates the level of expression of these enzymes remains to be determined. Further evidence for the regulatory role of the R gene family and insight into its allelic diversity have come from its molecular characterization. An R gene, R-nj, was cloned (Dellaporta et al., 1988) using a transposon tagging protocol that exploits the propensity of the transposable element Activator(Ac) to transpose into nearby chromosomal sites (Greenblatt and Brink, 1962). Greenblatt utilized an existing translocation between chromosomes 10 and 1 to move the target (the R gene on chromosome 10) closer to the transposable element donor site (an AC on chromosome l), thus enhancing the likelihood of AC transposition into R. The phenotype of R-nj, which conditions pigmentation of the crown of the aleurone, facilitated screening of the large numbers of kernels required to identify the spotted phenotype associated with a tagged gene. With the subsequent cloning of the R-nj gene (Dellaporta et al., 1988) questions regarding allelic diversity and gene function could be addressed. The genetic evidence for a small gene family was confirmed by Southern blot analysis. The correlation of specific hybridizing bands with particular R genes was possible because of the exis-

Some Members

of the R Gene Family

R Gene

Chromosome Position

Standard P

R

Tissues Pigmented

1 OL-61 Anthers, coleoptile, first and second sheath Aleurone

s Lc

1 OL-83

Leaf midrib, ligule, auricle, glume, lemma, palea, pericarp

R-SC

1OL

Aleurone,

R-nj

1OL

Crown of aleurone, roots, coleoptile

B-I

2s49

Coleoptile, blade, sheath, seedling auricle, husk, culm, tassel branch

B-Peru

2s49

Aleurone, scutellum, coleoptile, blade, sheath, seedling auricle, husk, culm, tassel branch

coleoptile anthers,

Cdl 850

tence of isogenic lines containing defined R family members. For example, strains containing only the P or S component of standard R, isolated following unequal crossing over, were used to identify the P and S genes (Dellaporta et al., 1988). Similarly, isogenic lines with or without the Lc gene facilitated the cloning of this family member and the isolation of a full-length Lc cDNA (Ludwig et al., 1989). The Lc protein predicted by its cDNA sequence shares features with many eukaryotic regulatory proteins. The most striking is a HLH motif, making Lc the first plant protein shown to have this DNA binding and dimerization domain. The HLH motif is involved in the formation of homoand heterodimers with other HLH proteins (Murre et al., 1989). The HLH of the Lc protein has 14 of the 18 amino acids conserved in other HLH proteins from Drosophila and mammals (Benezra et al., 1990). The Lc protein also contains a large acidic domain that may be involved in transcriptional activation (Ptashne, 1988). The R genes S, B-Peru, and B-I have also been cloned and sequenced. The cDNA sequences of S and Lc are 95% identical, with most of the differences residing in the 5’ untranslated region (Perrot and Cone, 1989). The B-Peru and B-I genes were recently analyzed and also found to be highly homologous to Lc (Goff et al., 1990). Consistent with their proposed role as transcriptional activators, a functional R or 8 gene is required for the accumulation of transcripts from three genes in the anthocyanin pathway: C2, Al, and Bzl (Ludwig et al., 1989; Chandler et al., 1989). The high degree of sequence similarity in R proteins implies that phenotypic diversity results either from minor variations in R protein structure or differences in their patterns of expression. Support for the latter view came from particle bombardment studies in which R-containing constructs were introduced into maize tissues (Ludwig et al., 1990). Specifically, the Lc cDNA was fused to a constitutive promoter, CaMV35S, and introduced into different tissues of the maize plant. This chimeric gene induces cellautonomous pigmentation in tissues that are not normally pigmented by the Lc gene. Similar experiments with chimerit constructs derived from the B-I gene (Goff et al., 1990) support the contention that various R proteins are functionally equivalent and pigmentation can be induced in a novel tissue by changing the R promoter. Particle bombardment has also provided a powerful transient assay that permits the complementation of mutations and the analysis of complex gene interactions. Introduction of the structural gene 6~7 into tissues mutant for bzl function produced red cells only when the tissue contained a functional R gene. In addition, transcriptional activation of the 6~7 and A7 genes could be quantitated indirectly by measuring induction of a reporter gene, Iuciferase, fused to the 8~7 or A7 promoter (Klein et al., 1989). Luciferase activity was not detected in genetic backgrounds that lacked a functional R family member However, co-bombardment of Bzl-luciferase and CaMV-B-I cDNA restored high levels of luciferase activity. These data support and extend previous molecular and biochemical studies showing that Bzl- and Al-encoded enzymes and transcripts do not accumulate in the absence of R proteins.

Genetic, biochemical, and molecular data indicate that R proteins are not the only regulatory proteins required for expression of the structural genes in the pathway. In the absence of light, functional copies of either the C7 or the PI gene must also be present. The C7 gene has been cloned and found to encode a protein that has sequence similarity to myb proto-oncogenes (Paz-Ares et al., 1987). Particle bombardment experiments are being used to study possible interactions between the Cl and R proteins. Co-bombardment of both Cl and B-Peru constitutively expressed cDNA constructs was required to activate Bzl-luciferase constructs in tissue mutant for C7 and R function (Goff et al., 1990). Consistent with the requirement for both of these proteins is the identification of potential myb and HLH binding sites in the Bz7 promoter. Mutagenesis of the putative HLH binding site reduced Iuciferase activity 30-fold, whereas alteration of the myb binding sequence resulted in only a P-fold reduction (Goff et al., 1990). Although these experiments provide additional evidence for the regulatory role of Cl and R proteins, direct interactions between these proteins and the 8~7 promoter must be demonstrated before definitive conclusions can be drawn. With the preliminary molecular characterization of the R gene family and the availability of a transient assay, the tools are now available to exploit the genetic resources. These resources will be valuable in understanding how the R protein rrans-activates structural genes, such as 8~7, as well as determining how the R genes are themselves regulated. Unlike other previously characterized HLH proteins, many mutant R proteins are available for molecular analysis because the mutant phenotype is viable and easily detected. For example, Kermicle et al. (1989) have generated a collection of 43 mutant R-SC genes, each containing an independent insertion of the Dissociation (Ds) transposable element. Of the 43 Ds alleles, 24 represent insertions into the protein coding region. These &-containing genes have the potential to produce hundreds of new R alleles, since Ds transposition leads to insertions (resulting in the addition of amino acids) at the site of Ds excision. Derivatives with amino acid changes that alter the level of anthocyanins can then be selected for molecular analysis. In addition to R alleles that encode mutant proteins, there is a large collection of natural variants of R that differ in their temporal and spatial patterns of anthocyanin accumulation. The genetic and molecular data summarized above suggest that these patterns reflect R promoter diversity. The fact that anthocyanins can be synthesized in most cell types means that the R system is uniquely suited to detect tissue- and cell type-specific enhancers. In this regard, the R transcription unit is analogous to promoterless /acZ constructs (so-called enhancer traps) that have been transformed into mouse and Drosophila genomes to identify enhancers. Interaction between these constructs and DNA at the site of insertion results in tissue-specific patterns of P-galactosidase expression @ ‘Kane and Gehring, 1987; Allen et al., 1988). In conclusion, a small gene family of HLH proteins is responsible for the diverse patterns of pigmentation seen

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in the maize plant. The ability of R proteins to activate the anthocyanin pathway in most cell types contrasts with the tissue-specific effects of certain HLH proteins in animals. For example, the HLH protein MyoD can activate musclespecific gene expression in fibroblasts but not hepatocytes (Schafer et al., 1990). MyoD activity is modulated by the formation of heterodimers with other tissue-specific HLH proteins (Murre et al., 1989; Benezra et al., 1990). In contrast, no such tissue-specific factors are required to interact with the R protein to activate transcription of the anthocyanin structural genes, because virtually all tissues and cell types have the potential to be pigmented. Therefore, an alternative way to control the tissue-specific patterns of gene expression is by the expansion and divergence of a multigene family of HLH proteins. References Allen, N. D., Cran, D. G., Barton, S. C., Hettle, S., Reik, W., and Surani, M. A. (1988). Nature 333, 852-855. Benezra, R., Davis, R. L., Lockshon, H. (1990). Cell 67, 49-59.

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