C1- and R-dependent expression of the maize Bz1 gene requires sequences with homology to mammalian myb and myc binding sites.

Tissue-specific expression of the maize anthocyanin Bronze-1 (Bz1) gene is controlled by the products of several regulatory genes. These include C1 or Pl and R or B that share homology to the myb proto-oncogenes and myc-like genes, respectively. Bz1 expression in embryo tissues is dependent on C1 and an R-sc allele of R. Transient expression from mutated and deleted versions of the Bz1 promoter fused to a luciferase reporter gene was measured in C1, Rscm2 embryos after gene transfer by microprojectiles. This analysis revealed that the sequences between -76 base pairs (bp) and -45 bp and a 9-bp AT-rich block between -88 bp and -80 bp were critical for Bz1 expression. The -76 bp to -45 bp region includes two short sequences that are homologous to the consensus binding sites of the myb- and myc-like proteins. Site-specific mutations of these "myb" and "myc" sequences reduced Bz1 expression to 10% and 1% of normal, respectively. Additionally, a trimer of a 38-bp oligonucleotide containing these myb and myc sites increased the expression of a cauliflower mosaic virus 35S minimal promoter by 26-fold. This enhancement was dependent on both C1 and R. Because the sites critical for Bz1 expression are homologous to the myb and myc consensus binding sequences and the C1 and R proteins share homology with the myb and myc products, respectively, we propose that C1 and R interact with the Bz1 promoter at these sites.     

The Purple leaf (Pl) locus of rice: the Pl(w) allele has a complex organization and includes two genes encoding basic helix-loop-helix proteins involved in anthocyanin biosynthesis

The Purple leaf (Pl) locus of rice (Oryza sativa L.) affects regulation of anthocyanin biosynthesis in various plant tissues. The tissue-specific patterns of anthocyanin pigmentation, together with the syntenic relationship, indicate that the rice Pl locus may play a role in the anthocyanin pathway similar to the maize R/B loci. We isolated two cDNAs showing significant identity to the basic helix-loop-helix (bHLH) proteins found in the maize R gene family. OSB1 appeared to be allelic to the previously isolated R homologue, Ra1, but showed a striking difference at the C-terminus because of a 2-bp deletion. Characterization of the corresponding genomic region revealed that the sequence identical to a 5'-portion of OSB2 existed approximately 10-kb downstream of the OSB1 coding region. OSB2 lacks a conserved C-terminal domain. Restriction fragment length polymorphism analyses using an F(2) population indicate that both genes co-segregate with the purple leaf phenotype. A transient complementation assay showed that the anthocyanin pathway is inducible by OSB1 or OSB2. These results suggest that the Pl(w) allele may be complex and composed of at least two genes encoding bHLH proteins.     

Genetic control of quercetin formation in the aleurone tissue of maize

The genes C, C2, R, A, A2, Bz, Bz2 and Pr are required for the formation of purple anthocyanin in the aleurone tissue of maize, and the recessive gene(s) result in non-purple (red, bronze and colorless). Aleurone extracts of recessive a and certain double recessive combinations were analyzed by paper chromatography, absorption spectra in the ultraviolet (UV) and infrared, mass spectra, and other analytical techniques. Homozygous recessive a tissue accumulates the flavonol, quercetin, while the double combinations a c and a r lack it, suggesting that dominant genes C and R are required for its formation and act prior to A in the synthesis of flavonols, as in the gene action sequence for anthocyanin synthesis. Dominant C-I inhibits the formation of quercetin, whereas Bz, Bz2 and In do not affect its formation. These results suggest a close biogenetic relationship between quercetin and cyanidin-3-glucoside and also independently confirm the position of A in both sequences.     

Elements of the maize A1 promoter required for transactivation by the anthocyanin B/C1 or phlobaphene P regulatory genes.

The extensive genetic and molecular characterization of the flavonoid pathway's structural and regulatory genes has provided some of the most detailed knowledge of gene interactions in plants. In maize flavonoid biosynthesis, the A1 gene is independently regulated in the anthocyanin and phlobaphene pathways. Anthocyanin production requires the expression of the C1 or PI and R or B regulatory genes, whereas phlobaphene production requires only the P regulatory gene. By deletion analysis of the A1 promoter, we show that the sequences between -123 and -88 are critical for activation by anthocyanin and phlobaphene regulatory genes. Linker-scanner mutations indicated that the -123 to -100 region is more important for transactivation by the P protein. The -98 to -88 region is more important for B/C1 transactivation and shows a strong homology with the region of the Bz1 anthocyanin structural gene promoter shown to be activated by B/C1 and not by P. We identified a 14-bp consensus sequence that is also present in the promoters of three other genes in the anthocyanin pathway, and we propose a model for how the flavonoid regulatory proteins interact with the promoters of the structural genes.     

Chalcone synthase mRNA and activity are reduced in yellow soybean seed coats with dominant I alleles.

The seed of all wild Glycine accessions have black or brown pigments because of the homozygous recessive i allele in combination with alleles at the R and T loci. In contrast, nearly all commercial soybean (Glycine max) varieties are yellow due to the presence of a dominant allele of the I locus (either I or i) that inhibits pigmentation in the seed coats. Spontaneous mutations to the recessive i allele occur in these varieties and result in pigmented seed coats. We have isolated a clone for a soybean dihydroflavonol reductase (DFR) gene using polymerase chain reaction. We examined expression of DFR and two other genes of the flavonoid pathway during soybean seed coat development in a series of near-isogenic isolines that vary in pigmentation as specified by combinations of alleles of the I, R, and T loci. The expression of phenylalanine ammonia-lyase and DFR mRNAs was similar in all of the gene combinations at each stage of seed coat development. In contrast, chalcone synthase (CHS) mRNA was barely detectable at all stages of development in seed coats that carry the dominant I allele that results in yellow seed coats. CHS activity in yellow seed coats (I) was also 7- to 10-fold less than in the pigmented seed coats that have the homozygous recessive i allele. It appears that the dominant I allele results in reduction of CHS mRNA, leading to reduction of CHS activity as the basis for inhibition of anthocyanin and proanthocyanin synthesis in soybean seed coats. A further connection between CHS and the I locus is indicated by the occurrence of multiple restriction site polymorphisms in genomic DNA blots of the CHS gene family in near-isogenic lines containing alleles of the I locus.     

A mutation in the pale aleurone color1 gene identifies a novel regulator of the maize anthocyanin pathway.

By screening for new seed color mutations, we have identified a new gene, pale aleurone color1 (pac1), which when mutated causes a reduction in anthocyanin pigmentation. The pac1 gene is not allelic to any known anthocyanin biosynthetic or regulatory gene. The pac1-ref allele is recessive, nonlethal, and only reduces pigment in kernels, not in vegetative tissues. Genetic and molecular evidence shows that the pac1-ref allele reduces pigmentation by reducing RNA levels of the biosynthetic genes in the pathway. The mutant does not reduce the RNA levels of either of the two regulatory genes, b and c1. Introduction of an anthocyanin structural gene promoter (a1) driving a reporter gene into maize aleurones shows that pac1-ref kernels have reduced expression resulting from the action of the a1 promoter. Introduction of the reporter gene with constructs that express the regulatory genes b and c1 or the phlobaphene pathway regulator p shows that this reduction in a1-driven expression occurs in both the presence and absence of these regulators. Our results imply that pac1 is required for either b/c1 or p activation of anthocyanin biosynthetic gene expression and that pac1 acts independently of these regulatory genes.     

Arabidopsis thaliana mutant that develops as a light-grown plant in the absence of light

The signal transduction pathways that lead to chloroplast biogenesis in plants are largely unknown. We describe here the identification and initial characterization of a novel genetic locus which fits the criteria of a regulatory gene located in a central pathway controlling light-mediated development. In the absence of light, these Arabidopsis thaliana mutants, designated det1 (de-etiolated 1), constitutively display many characteristics that are light-dependent in wild-type plants, including leaf and chloroplast development, anthocyanin accumulation, and accumulation of mRNAs for several light-regulated nuclear and chloroplast genes. The switch between dark and light growth modes thus appears to be a programmed step in a developmental pathway that is defined by det1. We suggest a model where the primary role of light on gene expression is mediated by the activation of leaf development. Further, the recessive nature of the det1 mutation implies that there is negative growth control on leaf development in dicotyledonous plants in the absence of light.     

Flavonoid biosynthesis-related genes in grape skin are differentially regulated by temperature and light conditions

Temperature and light are important environmental factors that affect flavonoid biosynthesis in grape berry skin. However, the interrelationships between temperature and light effects on flavonoid biosynthesis have not been fully elucidated at the molecular level. Here, we investigated the effects of temperature and light conditions on the biosynthesis of flavonoids (anthocyanins and flavonols) and the expression levels of related genes in an in vitro environmental experiment using detached grape berries. Sufficient anthocyanin accumulation in the grape skin was observed under a low temperature (15?°C) plus light treatment, whereas high temperature (35?°C) or dark treatment severely suppressed anthocyanin accumulation. This indicates that the accumulation of anthocyanins is dependent on both low temperature and light. qRT-PCR analysis showed that the responses of three MYB-related genes (VlMYBA1-3, VlMYBA1-2, and VlMYBA2) to temperature and light differed greatly even though the products of all three genes had the ability to regulate anthocyanin biosynthesis pathway genes. Furthermore, the expression levels of other MYB-related genes and many flavonoid biosynthesis pathway genes were regulated independently by temperature and light. We also found that temperature and light conditions affected the anthocyanin composition in the skin through the regulation of flavonoid biosynthesis pathway genes. Our results suggest that low temperature and light have a synergistic effect on the expression of genes in the flavonoid biosynthesis pathway. These findings provide new information about the relationships between environmental factors and flavonoid accumulation in grape berry skin.     

Modification of the B-ring during flavonoid synthesis in Petunia hybrida: Effect of the hydroxylation gene Hf1 on dihydroflavonol intermediates

The white flowering mutant W48 of Petunia hybrida is dominant for the hydroxylation gene Hf1 and homozygous recessive for the hydroxylation gene Ht1 and the anthocyanin gene An1. Flower buds of this mutant accumulate dihydrokaempferol-glucosides. Thus the effect of Hf1 being dominant is not the hydroxylation of the C15 skeleton, as is the case in mutants that are able to synthesize anthocyanins. This can be explained either by a feed-back inhibition of the hydroxylation by small amounts of dihydromyricetin (glucosides), or by a controlling effect of the gene An1 on the expression of Hf1. However, the white flowering mutant W58, which is homozygous recessive for the gene An6 and dominant for Hf1, accumulates dihydromyricetin (glucosides). This excludes a possible feed-back inhibition by dihydromyricetin and we conclude that An1 controls the expression of Hf1. Feeding of radioactive malonic acid to isolated flower limbs of mutants able to synthesize anthocyanins, leads to the incorporation of radioactivity into dihydrokaempferol (glucosides) and dihydroquercetin (glucosides). These results show that glucosylation of dihydroflavonols is a normal event in anthocyanin biosynthesis and is not induced by an inhibition of anthocyanin synthesis.