Identification of the pr1 gene product completes the anthocyanin biosynthesis pathway of maize

Abstract

In maize, mutations in the pr1 locus lead to the accumulation of pelargonidin (red) rather than cyanidin (purple) pigments in aleurone cells where the anthocyanin biosynthetic pathway is active. We characterized pr1 mutation and isolated a putative F3'H encoding gene (Zmf3'h1) and showed by segregation analysis that the red kernel phenotype is linked to this gene. Genetic mapping using SNP markers confirms its position on chromosome 5L. Furthermore, genetic complementation experiments using a CaMV 35S::ZmF3'H1 promoter-gene construct established that the encoded protein product was sufficient to perform a 3'-hydroxylation reaction. The Zmf3'h1-specific transcripts were detected in floral and vegetative tissues of Pr1 plants and were absent in pr1. Four pr1 alleles were characterized: two carry a 24 TA dinucleotide repeat insertion in the 5'-upstream promoter region, a third has a 17-bp deletion near the TATA box, and a fourth contains a Ds insertion in exon1. Genetic and transcription assays demonstrated that the pr1 gene is under the regulatory control of anthocyanin transcription factors red1 and colorless1. The cloning and characterization of pr1 completes the molecular identification of all genes encoding structural enzymes of the anthocyanin pathway of maize.

Figure 1.—

Biosynthesis and accumulation of anthocyanins in maize. (A) Phenylpropanoid biosynthetic pathway leading to the production of anthocyanins. Genes (enzymes) in the pathway are: pal (PAL), phenylalanine-ammonia lyase; c4h (C4H), cinnamic acid hydroxylase; 4cl (4CL), 4-coumaryl:CoA ligase; c2 (CHS), chalcone synthase; chi1 (CHI), chalcone isomerase; f3h (F3H), flavanone 3-hydroxylase; pr1 (F3′H), flavonoid 3′-hydroxylase (* = based on enzyme activity assay); a1 (DFR), dihydroflavanone reductase; a2 (AS), anthocyanidin synthase; bz1 (UFGT), UDP-glucose flavonoid 3-O-glucosyltransferase; and bz2 (GST), glutathione S-transferase. (B) Close-up photos show red and purple aleurones in kernels of pr1 and Pr1 ears, respectively.

Figure 2.—

Multiple sequence alignments of deduced amino acid sequences of F3′H from maize and other plant species using the ClustalX program. Lightly shaded areas are F3′H-specific sequences and darkly shaded regions are conserved domains found in the CYP450 proteins. Other regions shown are: hydrophobic anchor, proline-rich region, and heme-binding site (HBS).

Figure 3.—

Characterization of Zmf3′h1, isolation of pr1 insertion, and deletion alleles. (A) A segment of λ-clone shows Zmf3′h1 and its 5′- and 3′-flanking sequences. FR and F387 are the probes used to screen the genomic library. Shaded boxes represent two exons that are joined by a bent line, which corresponds to the single intron of the Zmf3′h1 gene. Enlarged 5′-flanking region shows the location of 24 dinucleotide repeats and a deletion near the TATA box in mutant pr1 alleles. Positions of TA repeats and a 17-bp deletion are marked with respect to transcription start site (shown as bent arrow and marked +1). The position of the Ds insertion in exon 1 is shown and allele's name is indicated. Small arrows below the map illustrate the orientation and position of the PCR primers. (B) Self-pollinated ear of a plant with the genotype pr1::Ds/pr1-ref. Note the purple revertant kernel (arrow), indicating a potential excision event of Ds, which would result in the restoration of Pr1 gene expression.

Figure 4.—

Mutant pr1 alleles do not accumulate Zmf3′h1 transcripts in aleurone tissues. Expression of Zmf3′h1, c2, bz2, and c1 in aleurones was detected by RT–PCR from three pr1/pr1 mutant alleles and Pr1/Pr1 wild-type plants. Purple aleurones from W22 were used as positive control and α-tubulin1 was used as a loading control.

Figure 5.—

pr1 aleurones produce pelargonidin, while Pr1 aleurones produce cyanidin. (A) TLC of extracts from Pr1 or pr1 kernels. Samples shown in each lane are: 1, control C1 for cyanidin; 2, control C2 for pelargonidin; 3–5, Pr1; and 6–8, pr1. (B) HPLC analysis of silk methanolic extracts from sibling plants carrying pr1 or Pr1. Extracts were analyzed at wavelength 230 nm for DHK and DHQ. pr1 shows single peak representing DHK, while Pr1 has two peaks representing both compounds; thus DHK is converted into DHQ through the action of F3′H (Figure 1).

Figure 6.—

Complementation of tt7 mutant with Zmf3′h1 resulted in accumulation of pigments in seedling and seed coat. Seedling and seed phenotypes are shown for wild-type Landsberg erecta, tt7 mutant, and 35S::ZmF3′H1-complemented tt7.

Figure 7.—

Mutant c1 and r1 plants carrying Pr1 do not accumulate pr1 transcripts. Expression of pr1 and other anthocyanin genes in aleurones were analyzed by RT–PCR in c1-MGS131036, c1-MGS14633, r1-MGS167054, and r1-MGS14638 alleles. Purple aleurones from W22 were used as positive control for anthocyanin genes’ expression, while actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as loading controls.