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, 127 (1), 46-57

Functional Conservation of Plant Secondary Metabolic Enzymes Revealed by Complementation of Arabidopsis Flavonoid Mutants With Maize Genes

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Functional Conservation of Plant Secondary Metabolic Enzymes Revealed by Complementation of Arabidopsis Flavonoid Mutants With Maize Genes

X Dong et al. Plant Physiol.

Abstract

Mutations in the transparent testa (tt) loci abolish pigment production in Arabidopsis seed coats. The TT4, TT5, and TT3 loci encode chalcone synthase, chalcone isomerase, and dihydroflavonol 4-reductase, respectively, which are essential for anthocyanin accumulation and may form a macromolecular complex. Here, we show that the products of the maize (Zea mays) C2, CHI1, and A1 genes complement Arabidopsis tt4, tt5, and tt3 mutants, restoring the ability of these mutants to accumulate pigments in seed coats and seedlings. Overexpression of the maize genes in wild-type Arabidopsis seedlings does not result in increased anthocyanin accumulation, suggesting that the steps catalyzed by these enzymes are not rate limiting in the conditions assayed. The expression of the maize A1 gene in the flavonoid 3' hydroxylase Arabidopsis tt7 mutant resulted in an increased accumulation of pelargonidin. We conclude that enzymes involved in secondary metabolism can be functionally exchangeable between plants separated by large evolutionary distances. This is in sharp contrast to the notion that the more relaxed selective constrains to which secondary metabolic pathways are subjected is responsible for the rapid divergence of the corresponding enzymes.

Figures

Figure 1
Figure 1
Flavonoid biosynthetic pathway. Only the enzymatic steps significant for the studies presented here are indicated, with the Arabidopsis and maize genes labeled. Dihydrokaempferol (DHK), kaempferol (K), dihydroquercetin (DHQ), quercetin (Q).
Figure 2
Figure 2
Complementation of the pigmentation of Arabidopsis tt4, tt5, and tt3 mutant seedlings and seed coats with the maize C2, CHI1, and A1 genes, respectively. A, Pigment accumulation of Landsberg erecta seedlings grown in low-nitrogen media and of Landsberg erecta seed coats. Complementation of the tt4 mutant (transformed with the empty pBIB121plasmid; left colorless seedling) by 35S::C2 (three independent transformation events; red seedlings); tt5 mutant (transformed with the empty pBIB121plasmid; left colorless seedling) by 35S::CHI1 (three independent transformation events; red seedlings); and tt3 mutant (transformed with the empty pBIB121 plasmid; left colorless seedling) by 35S::A1 (three independent transformation events; red seedlings). Transgenic T2 seeds are visualized under visible light. B, Northern analysis of total RNA obtained from wild-type (Landsberg erecta) and mutant (tt4, tt5, and tt3) seedlings grown in low-nitrogen media expressing C2 (Landsberg erecta and tt4), CHI1 (Landsberg erecta and tt5), and A1 (Landsberg erecta and tt3), or from 4-week-old plants (Landsberg erecta or tt7) expressing the maize A1 gene. A probe corresponding to the 18S rRNA was used as a loading control. pBI- corresponds to lines that carry the empty pBIB121 plasmid.
Figure 3
Figure 3
Anthocyanidin and flavonoid accumulation in wild type and transgenic Arabidopsis plants. A, Thin-layer chromatography (TLC) analysis of the anthocyanidins that accumulate in wild-type (Landsberg erecta) or mutant tt3, tt4, or tt5 Arabidopsis seedlings grown on low-nitrogen media expressing the maize C2, CHI, or A1 genes. BMS R&C1 corresponds to the anthocyanidins found in BMS cells expressing the R and C1 regulators of anthocyanin biosynthesis (Grotewold et al., 1998). The pelargonidin standard (P) was obtained from geranium (Pelargonium cv Salmon Mbl Mix) flowers, and the delphinidin (D) from lisianthus (Eustoma grandiflorum Grise variety Royal Violet) flowers (see “Materials and Methods”). B, HPLC analysis of flavonoids present in methanol extracts of equivalent amounts (wet weight) of leaves from 4-week-old wild-type (Landsberg erecta) or mutant Arabidopsis, in the presence or absence of the maize C2, CHI1, or A1 genes. The left column shows the accumulation of the non-hydrolized glycosides, and the right column shows the accumulation of hydrolized aglycones. The mobility of Q or K standards is indicated by arrows.
Figure 4
Figure 4
Expression of the maize flavonoid biosynthetic genes in wild-type Arabidopsis seedlings does not result in an increase of anthocyanidin accumulation. A, Wild-type (Landsberg erecta) Arabidopsis seedlings expressing the maize A1 (A1-5), C2 (C2-2), or CHI1 (CHI-1) genes show similar levels of pigment accumulation as compared with wild-type seedlings carrying the empty binary vector (pBI-1) when grown in low-nitrogen media. B, Comparison of cyanidin accumulation in seedlings of three lines of wild-type (Landsberg erecta) Arabidopsis expressing the maize genes. The error bars correspond to the sd of triplicate measurements for each line.
Figure 5
Figure 5
Accumulation of pelargonidin in F3′H mutant seedlings. A, TLC analysis of anthocyanidins extracted from tt7 mutant seedlings expressing the maize A1 gene (tt7 A1-1) or an empty vector (tt7 pBI-1). The conditions and standards are identical to Figure 3. B, Comparison of pelargonidin accumulation in seedlings of three tt7 lines (Table I) expressing the maize A1 gene (tt7 + 35S::A1) or the empty vector (tt7 + pBI121). The error bars correspond to the sd of triplicates for each line.
Figure 6
Figure 6
Comparison of structural models for the maize and Arabidopsis CHI enzymes. A, Alignment of the maize CHI1 with the Arabidopsis TT5-encoded CHI proteins. Identical residues are highlighted with a black background and conservative substitutions are boxed. The β1a, β1b, and β2β-strands suggested as providing a potential protein-protein interaction surface (Jez et al., 2000) are highlighted in gray. Residues that are clearly exposed to the solvent (defined here as having either more than 10 Å2 exposed or being more than 33% solvent accessible as the same residue in an unfolded peptide) are indicated with red dots over the amino acid. B, Space filling model of the Arabidopsis CHI protein with the substrate-binding site indicated with an arrow. Residues that are identical in the maize and Arabidopsis proteins are indicated in red. C, Space filling model of the maize CHI1 protein, view is identical to B. D, Space filling model of the Arabidopsis CHI protein showing the opposite face. E, Space filling model of the maize CHI1, view is identical to panel D. The N- and C-terminal regions of the proteins were not included in the models.

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