Two Regulatory Genes of the Maize Anthocyanin Pathway Are Homologous: Isolation of B Utilizing R Genomic Sequences

Abstract

Genetic studies in maize have identified several regulatory genes that control the tissue-specific synthesis of the purple anthocyanin pigments during development. Two such genes, R and B, exhibit extensive allelic diversity with respect to the tissue specificity and developmental timing of anthocyanin synthesis. Previous genetic studies demonstrated that certain B alleles can substitute for R function, and in these cases only one functional allele at either locus is required for pigment synthesis in the aleurone. In addition, biochemical studies have shown that both genes act on the same biosynthetic pathway, suggesting that the genes are functionally duplicate. In this report we describe DNA hybridization experiments that demonstrate that the functionally duplicate nature of B and R is reflected in DNA sequence similarity between the two genes. We took advantage of this homology and used the R genomic sequences to clone B. Two different strategies were pursued and two genomic clones isolated, a 2.5-kilobase BgIII fragment linked to the b allele in W23 inbred stocks and a 1.0-kilobase HindIII fragment linked to the B allele in CM37 stocks. Examination of several independent transposable element insertion mutations in B and revertant derivatives demonstrated that our clones recognize the functional B gene. Genomic clones representing the entire B-Peru allele were isolated, and a detailed restriction map was prepared. Using these clones we have identified a 2.2-kilobase mRNA in husks from plants containing either B-I or B-Peru alleles, but no B mRNA was detected in plants containing a b allele. The transcript is at least 100 times more abundant in strongly pigmented B-I husks than in weakly pigmented B-Peru husk tissue. Expression of functional B alleles in husk tissue correlates with the coordinate increase in mRNA levels of two structural genes of the pathway, A1 and Bz1, consistent with the postulated role of B as a regulatory gene.

Control of Pigment Biosynthesis Genes during Petal Development.

“…This multiple transcriptional activation is coordinated by two transcription factors, R and C7, which act together to induce all the committed biosynthetic genes (Dooner, 1983;Cone et al, 1986;Paz-Ares et al, 1986;Chandler et al, 1989;Ludwig et al, 1989). In flowers of dicots, by contrast, there does not appear to be a single induction mechanism for all of the biosynthetic genes; rather, there is evidence for discrete control of different biosynthetic genes (Almeida et al, 1989; (Jackson et al, 1992) shows that the genes encoding the first two steps of the pathway, chalcone synthase (CHS) and chalcone isomerase (CHI) (early biosynthetic genes [EBGs]) have different expression profiles than the genes encoding flavanone 3-hydroxylase (F3H), dihydroflavonol4-reductase (DFR), anthocyanidin synthase (AS), and UDP-glucose flavonol 3-O-glucosyl transferase (UFGT) (late biosynthetic genes [LBGs]).…”

The Birth of a Black Rice Gene and Its Local Spread by Introgression

“…The anthocyanin biosynthesis pathway has been elucidated in various plant species (Supplemental Figure 1B and Supplemental Table 1) (Dooner et al, 1991;Shih et al, 2008). Transcriptional regulation related to anthocyanin biosynthesis has also been extensively studied, particularly for the maize (Zea mays) R/B and C1/Pl gene families, and orthologs in other plant species, which encode basic helix-loop-helix (bHLH)-type and R2R3-Mybtype transcription factors, respectively (Styles et al, 1973;Paz-Ares et al, 1987;Chandler et al, 1989;Cone et al, 1993). These regulatory proteins act in a ternary complex, as MBW (MYBbHLH-WD40) complex transcription factors (Hichri et al, 2011;Petroni and Tonelli, 2011).…”

Identification of functional domains in the maize transcriptional activator C1: comparison of wild-type and dominant inhibitor proteins.

“…The B gene is homologous to the R gene (Chandler et al 1989), and both B and R encode proteins with homology to the helix-loop-helix subunit dimerization region of the myc family of proteins (Chandler et al 1989;Ludwig et al 1989;Perrot and Cone 1989). The Cl gene encodes a protein with amino-terminal homology to the DNA-binding domain of members of the myh oncogene family (Paz-Ares et al 1987).…”

“…The Rscm2 allele of R conditions anthocyanin pigmentation in the maize embryo (Styles et al 1973). r or b mutations have been found to affect the levels of the mRNAs and/or enzymes encoded by some of the anthocyanin structural genes examined (Dooner and Nelson 1979;Dooner 1983;Chandler et al 1989;Ludwig et al 1989). cl (colorless) mutants have low levels of the mRNAs and enzymes ^Corresponding author.…”