Molecular and Biochemical Analysis of Chalcone Synthase from Freesia hybrid in flavonoid biosynthetic pathway

Abstract

Chalcone synthase (CHS) catalyzes the first committed step in the flavonoid biosynthetic pathway. In this study, the cDNA (FhCHS1) encoding CHS from Freesia hybrida was successfully isolated and analyzed. Multiple sequence alignments showed that both the conserved CHS active site residues and CHS signature sequence were found in the deduced amino acid sequence of FhCHS1. Meanwhile, crystallographic analysis revealed that protein structure of FhCHS1 is highly similar to that of alfalfa CHS2, and the biochemical analysis results indicated that it has an enzymatic role in naringenin biosynthesis. Moreover, quantitative real-time PCR was performed to detect the transcript levels of FhCHS1 in flowers and different tissues, and patterns of FhCHS1 expression in flowers showed significant correlation to the accumulation patterns of anthocyanin during flower development. To further characterize the functionality of FhCHS1, its ectopic expression in Arabidopsis thaliana tt4 mutants and Petunia hybrida was performed. The results showed that overexpression of FhCHS1 in tt4 mutants fully restored the pigmentation phenotype of the seed coats, cotyledons and hypocotyls, while transgenic petunia expressing FhCHS1 showed flower color alteration from white to pink. In summary, these results suggest that FhCHS1 plays an essential role in the biosynthesis of flavonoid in Freesia hybrida and may be used to modify the components of flavonoids in other plants.

Fig 1. Schematic diagram of the generalized structure of FhCHS1 gene.

A, Exons-intron architecture of FhCHS1. exons, intron, initiation codon (ATG) and terminator codon (TAA) are labeled. B, Nucleotide sequences surrounding splice sites in FhCHS1, AmCHS (Antirrhinum majus CHS, X03710) and PpCHS11 (Physcomitrella patens CHS, ABU87504). Amino acid residues and codons (underlined) split by introns are shown.

Fig 2. Sequence and crystal structural analysis of FhCHS1.

A, Comparison of amino acid sequences between FhCHS1, AtCHS (At5913930), PhCHSA (X14591) and MsCHS2 (L02902). Functionally important conserved residues are highlighted with a colored background: pink, the three conserved catalytic residues in all CHS; blue, two important residues determining the substrate specificity of CHS; yellow, the malonyl-CoA binding motif; red, highly conserved CHS signature sequence. B, Functional unit of CHS. The homo-dimer with one of the monomer highlighting secondary structure components: cyan for α-helix, magenta for β-strand, pink for loop. C, Structure comparison of the current CHS of freesia (blue, PDB ID 4WUM) and previous structure model CHS2 of alfalfa (yellow, PDB ID 1BI5), indicating strong structural conservation of the CHS protein.

Fig 3. Phylogenetic tree based on the amino acid sequences of plant CHSs.

The tree was constructed using the MEGA 5.1 and neighbor-joining method with 2000 bootstrap replicates. Freesia hybrida CHS is boxed. GenBank accession numbers used are: Nelumbo nucifera (NnCHS, FJ999628.1), Abelmoschus manihot (AmCHS, EU573212.1), Nicotiana tabacum (NtCHS, AF311783.1), petunia hybrida (PhCHSA, X14591; PhCHSB, X14592; PhCHSD, X14593; PhCHSF, X14594; PhCHSG, X14595; PhCHSJ, X14597), Saussurea medusa (SmCHS, DQ350888.1), Humulus lupulus (HlCHS, CAK19317.1), Anthurium andraeanum (AaCHS, DQ421809.1), Freesia hybrida (FhCHS1, JF732897.1), Iris germanica (IgCHS, AB219147.1), Camellia chekiangoleosa (CcCHS1, JN944573.1; CcCHS2, JN944574.1), Rosa chinensis (RcCHS, HQ423171.1), Melastoma malabathricum (MmCHS, KF234569.1), Rhododendron simsii (RsCHS, AJ413277.1), Camellia nitidissima (CnCHS, HQ269804.1).

Fig 4. Enzyme activity of freesia (Freesia hybrida) chalcone synthase (FhCHS1).

A, The sample of naringenin standard. B, The reaction products catalyzed by FhCHS1. C, Control.

Fig 5. FhCHS1 gene expression profiles in Freesia hybrida.

A, The phenotypes of different samples. 1–5, represent the flowers of different developmental stages; Pe, petals; St, stamens; Pi, pistils; Ca, calyxes; To, toruses; Sc, scapes; Le, leaves; Ro, roots. B, Expression profile of FhCHS1 in flowers at different developmental stages. C, Expression levels of FhCHS1 in different tissues. Data represent means ± SD of three biological replicates.

Fig 6. Complementation of the pigmentation of Arabidopsis tt4 mutant seedlings with FhCHS1 gene.

A, Phenotypes of wild-type, tt4 mutant, and transgenic Arabidopsis seeds and seedlings. B, Expressional analysis of the FhCHS1 gene by reverse transcription polymerase chain reaction in the wild-type, tt4 mutant and transgenic lines. C, Contents of anthocyanidins and flavonols in Arabidopsis seedlings. Data correspond to means of three biological replicates. Means with different letters within the same column are significantly different at the 0.01 level of probability. N/D, not detected.

Fig 7. Functional characterization of FhCHS1 gene following its overexpression in transgenic petunia lines.

A, Differences in color between wild-type and transgenic petunia flowers. B, Expressional analysis of the FhCHS1 gene by reverse transcription polymerase chain reaction in the wild-type and transgenic lines. C, Extracted solutions from flowers of wild-type and transgenic lines. D, Contents of anthocyanidins and flavonols in the wild-type and transgenic petunia flowers. Data correspond to means of three biological replicates. Means with different letters within the same column are significantly different at the 0.01 level of probability. N/D, not detected.