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A Malus crabapple chalcone synthase gene, McCHS, regulates red petal color and flavonoid biosynthesis.

Tai D, Tian J, Zhang J, Song T, Yao Y - PLoS ONE (2014)

Bottom Line: The results showed McCHS have their special expression patterns in each tested cultivars, and is responsible for the red coloration and color variation in crabapple petals, especially for color fade process in 'Radiant'.Furthermore, tobacco plants constitutively expressing McCHS displayed a higher anthocyanins accumulation and a deeper red petal color compared with control untransformed lines.We conclude that the endogenous McCHS gene is a critical factor in the regulation of anthocyanin biosynthesis during petal coloration in Malus crabapple.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Science and Technology, Beijing University of Agriculture, Beijing, China; College of Horticulture, Shanxi Agricultural University, Taigu, Shanxi, China.

ABSTRACT
Chalcone synthase is a key and often rate-limiting enzyme in the biosynthesis of anthocyanin pigments that accumulate in plant organs such as flowers and fruits, but the relationship between CHS expression and the petal coloration level in different cultivars is still unclear. In this study, three typical crabapple cultivars were chosen based on different petal colors and coloration patterns. The two extreme color cultivars, 'Royalty' and 'Flame', have dark red and white petals respectively, while the intermediate cultivar 'Radiant' has pink petals. We detected the flavoniods accumulation and the expression levels of McCHS during petals expansion process in different cultivars. The results showed McCHS have their special expression patterns in each tested cultivars, and is responsible for the red coloration and color variation in crabapple petals, especially for color fade process in 'Radiant'. Furthermore, tobacco plants constitutively expressing McCHS displayed a higher anthocyanins accumulation and a deeper red petal color compared with control untransformed lines. Moreover, the expression levels of several anthocyanin biosynthetic genes were higher in the transgenic McCHS overexpressing tobacco lines than in the control plants. A close relationship was observed between the expression of McCHS and the transcription factors McMYB4 and McMYB5 during petals development in different crabapple cultivars, suggesting that the expression of McCHS was regulated by these transcription factors. We conclude that the endogenous McCHS gene is a critical factor in the regulation of anthocyanin biosynthesis during petal coloration in Malus crabapple.

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Sequence characteristics of McCHS and relationships with other CHS proteins.(A) Protein sequence alignment of McCHS and other known anthocyanin biosynthetic proteins from other plant species. Identical residues are shown in black, conserved residues in dark grey and similar residues in light grey. (B) Phylogenetic relationships between McCHS and CHS sequences from other species involved in anthocyanin biosynthesis. Phylogenetic and molecular evolutionary analysis was conducted using MEGA version 5.10 (using the minimum evolution phylogeny test and 1000 bootstrap replicates). (C) Relative expression profile of McCHS at different developmental stages of the petals of three Malus crabapple cultivars: ‘Royalty’, ‘Radiant’ and ‘Flame’. Five stages were tested in this study: (I) 6 days before full bloom; (II) 3 days before full bloom; (III) 1 day before full bloom; (IV) full bloom; and (V) 3 days after full bloom. Error bars represent the standard error of the mean ± SE of three replicate reactions. All real-time PCR reactions were normalized using the Ct value corresponding to a Malus crabapple 18S ribosomal RNA gene (DQ341382). The GenBank accession numbers of the proteins are as follows: McCHS (Malus domestica; FJ599763), MdCHS (Malus domestica; AB074485), MdCHS-1 (Malus domestica; DQ026297), MdCHS-320 (Malus domestica; EU872158), PbCHS (Pyrus bretschneideri; KF148032), MdCHS-46 (Malus domestica; EU872156), MtCHS (Malus toringoides; HQ853494), MdCHS-2 (Malus toringoides; JQ582624), MdCHS-23 (Malus toringoides; EU872155), SaCHS (Sorbus aucuparia; DQ286037), PpCHS (Prunus persica; HM543568), FrCHS-2 (Fragaria ananassa; AB201756), RiPKS-5 (Rubus idaeus; EF694718).
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pone-0110570-g003: Sequence characteristics of McCHS and relationships with other CHS proteins.(A) Protein sequence alignment of McCHS and other known anthocyanin biosynthetic proteins from other plant species. Identical residues are shown in black, conserved residues in dark grey and similar residues in light grey. (B) Phylogenetic relationships between McCHS and CHS sequences from other species involved in anthocyanin biosynthesis. Phylogenetic and molecular evolutionary analysis was conducted using MEGA version 5.10 (using the minimum evolution phylogeny test and 1000 bootstrap replicates). (C) Relative expression profile of McCHS at different developmental stages of the petals of three Malus crabapple cultivars: ‘Royalty’, ‘Radiant’ and ‘Flame’. Five stages were tested in this study: (I) 6 days before full bloom; (II) 3 days before full bloom; (III) 1 day before full bloom; (IV) full bloom; and (V) 3 days after full bloom. Error bars represent the standard error of the mean ± SE of three replicate reactions. All real-time PCR reactions were normalized using the Ct value corresponding to a Malus crabapple 18S ribosomal RNA gene (DQ341382). The GenBank accession numbers of the proteins are as follows: McCHS (Malus domestica; FJ599763), MdCHS (Malus domestica; AB074485), MdCHS-1 (Malus domestica; DQ026297), MdCHS-320 (Malus domestica; EU872158), PbCHS (Pyrus bretschneideri; KF148032), MdCHS-46 (Malus domestica; EU872156), MtCHS (Malus toringoides; HQ853494), MdCHS-2 (Malus toringoides; JQ582624), MdCHS-23 (Malus toringoides; EU872155), SaCHS (Sorbus aucuparia; DQ286037), PpCHS (Prunus persica; HM543568), FrCHS-2 (Fragaria ananassa; AB201756), RiPKS-5 (Rubus idaeus; EF694718).

Mentions: Based on homology with Malus × domestica sequences and related sequences in the Genome Database for Rosaceae (http://www.rosaceae.org), a gene sequence for the full length CHS (McCHS) was amplified from total RNA of Malus crabapple ‘Royalty’ leaves by RT-PCR and RACE. Specifically, the McCHS cDNA (accession no. FJ599763) is 1,529 bp in length and is predicted to encode a protein of 389 amino acids with a high level of homology to with CHS genes from a range of species (Figure 3A), including Malus domestica (MdCHS2; 99% identity), Malus domestica (MdCHS1; 99% identity), Malus domestica (MdCHS320; 99% identity), Pyrus bretschneideri (PbCHS; 98% identity), Malus domestica (MdCHS46; 94% identity) and so on. It appears that chalcone synthase protein sequences have undergone little sequence diversification in the species examined and that they are highly similar in length in different plant species. Other than the putative CHS gene sequences, there are also related sequences that are more divergent from characterized CHS genes and although these genes show high sequence similarity to a Fragaria ananassa CHS (FrCHS; AB201756), e.g. Rubus idaeus (RiPKS-5; EF694718; 97% identity), their functions have yet to be determined. Collectively they are described as polyketide synthase (PKS) genes, a more general description for the CHS and CHS-Like gene family (Figure 3B).


A Malus crabapple chalcone synthase gene, McCHS, regulates red petal color and flavonoid biosynthesis.

Tai D, Tian J, Zhang J, Song T, Yao Y - PLoS ONE (2014)

Sequence characteristics of McCHS and relationships with other CHS proteins.(A) Protein sequence alignment of McCHS and other known anthocyanin biosynthetic proteins from other plant species. Identical residues are shown in black, conserved residues in dark grey and similar residues in light grey. (B) Phylogenetic relationships between McCHS and CHS sequences from other species involved in anthocyanin biosynthesis. Phylogenetic and molecular evolutionary analysis was conducted using MEGA version 5.10 (using the minimum evolution phylogeny test and 1000 bootstrap replicates). (C) Relative expression profile of McCHS at different developmental stages of the petals of three Malus crabapple cultivars: ‘Royalty’, ‘Radiant’ and ‘Flame’. Five stages were tested in this study: (I) 6 days before full bloom; (II) 3 days before full bloom; (III) 1 day before full bloom; (IV) full bloom; and (V) 3 days after full bloom. Error bars represent the standard error of the mean ± SE of three replicate reactions. All real-time PCR reactions were normalized using the Ct value corresponding to a Malus crabapple 18S ribosomal RNA gene (DQ341382). The GenBank accession numbers of the proteins are as follows: McCHS (Malus domestica; FJ599763), MdCHS (Malus domestica; AB074485), MdCHS-1 (Malus domestica; DQ026297), MdCHS-320 (Malus domestica; EU872158), PbCHS (Pyrus bretschneideri; KF148032), MdCHS-46 (Malus domestica; EU872156), MtCHS (Malus toringoides; HQ853494), MdCHS-2 (Malus toringoides; JQ582624), MdCHS-23 (Malus toringoides; EU872155), SaCHS (Sorbus aucuparia; DQ286037), PpCHS (Prunus persica; HM543568), FrCHS-2 (Fragaria ananassa; AB201756), RiPKS-5 (Rubus idaeus; EF694718).
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pone-0110570-g003: Sequence characteristics of McCHS and relationships with other CHS proteins.(A) Protein sequence alignment of McCHS and other known anthocyanin biosynthetic proteins from other plant species. Identical residues are shown in black, conserved residues in dark grey and similar residues in light grey. (B) Phylogenetic relationships between McCHS and CHS sequences from other species involved in anthocyanin biosynthesis. Phylogenetic and molecular evolutionary analysis was conducted using MEGA version 5.10 (using the minimum evolution phylogeny test and 1000 bootstrap replicates). (C) Relative expression profile of McCHS at different developmental stages of the petals of three Malus crabapple cultivars: ‘Royalty’, ‘Radiant’ and ‘Flame’. Five stages were tested in this study: (I) 6 days before full bloom; (II) 3 days before full bloom; (III) 1 day before full bloom; (IV) full bloom; and (V) 3 days after full bloom. Error bars represent the standard error of the mean ± SE of three replicate reactions. All real-time PCR reactions were normalized using the Ct value corresponding to a Malus crabapple 18S ribosomal RNA gene (DQ341382). The GenBank accession numbers of the proteins are as follows: McCHS (Malus domestica; FJ599763), MdCHS (Malus domestica; AB074485), MdCHS-1 (Malus domestica; DQ026297), MdCHS-320 (Malus domestica; EU872158), PbCHS (Pyrus bretschneideri; KF148032), MdCHS-46 (Malus domestica; EU872156), MtCHS (Malus toringoides; HQ853494), MdCHS-2 (Malus toringoides; JQ582624), MdCHS-23 (Malus toringoides; EU872155), SaCHS (Sorbus aucuparia; DQ286037), PpCHS (Prunus persica; HM543568), FrCHS-2 (Fragaria ananassa; AB201756), RiPKS-5 (Rubus idaeus; EF694718).
Mentions: Based on homology with Malus × domestica sequences and related sequences in the Genome Database for Rosaceae (http://www.rosaceae.org), a gene sequence for the full length CHS (McCHS) was amplified from total RNA of Malus crabapple ‘Royalty’ leaves by RT-PCR and RACE. Specifically, the McCHS cDNA (accession no. FJ599763) is 1,529 bp in length and is predicted to encode a protein of 389 amino acids with a high level of homology to with CHS genes from a range of species (Figure 3A), including Malus domestica (MdCHS2; 99% identity), Malus domestica (MdCHS1; 99% identity), Malus domestica (MdCHS320; 99% identity), Pyrus bretschneideri (PbCHS; 98% identity), Malus domestica (MdCHS46; 94% identity) and so on. It appears that chalcone synthase protein sequences have undergone little sequence diversification in the species examined and that they are highly similar in length in different plant species. Other than the putative CHS gene sequences, there are also related sequences that are more divergent from characterized CHS genes and although these genes show high sequence similarity to a Fragaria ananassa CHS (FrCHS; AB201756), e.g. Rubus idaeus (RiPKS-5; EF694718; 97% identity), their functions have yet to be determined. Collectively they are described as polyketide synthase (PKS) genes, a more general description for the CHS and CHS-Like gene family (Figure 3B).

Bottom Line: The results showed McCHS have their special expression patterns in each tested cultivars, and is responsible for the red coloration and color variation in crabapple petals, especially for color fade process in 'Radiant'.Furthermore, tobacco plants constitutively expressing McCHS displayed a higher anthocyanins accumulation and a deeper red petal color compared with control untransformed lines.We conclude that the endogenous McCHS gene is a critical factor in the regulation of anthocyanin biosynthesis during petal coloration in Malus crabapple.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Science and Technology, Beijing University of Agriculture, Beijing, China; College of Horticulture, Shanxi Agricultural University, Taigu, Shanxi, China.

ABSTRACT
Chalcone synthase is a key and often rate-limiting enzyme in the biosynthesis of anthocyanin pigments that accumulate in plant organs such as flowers and fruits, but the relationship between CHS expression and the petal coloration level in different cultivars is still unclear. In this study, three typical crabapple cultivars were chosen based on different petal colors and coloration patterns. The two extreme color cultivars, 'Royalty' and 'Flame', have dark red and white petals respectively, while the intermediate cultivar 'Radiant' has pink petals. We detected the flavoniods accumulation and the expression levels of McCHS during petals expansion process in different cultivars. The results showed McCHS have their special expression patterns in each tested cultivars, and is responsible for the red coloration and color variation in crabapple petals, especially for color fade process in 'Radiant'. Furthermore, tobacco plants constitutively expressing McCHS displayed a higher anthocyanins accumulation and a deeper red petal color compared with control untransformed lines. Moreover, the expression levels of several anthocyanin biosynthetic genes were higher in the transgenic McCHS overexpressing tobacco lines than in the control plants. A close relationship was observed between the expression of McCHS and the transcription factors McMYB4 and McMYB5 during petals development in different crabapple cultivars, suggesting that the expression of McCHS was regulated by these transcription factors. We conclude that the endogenous McCHS gene is a critical factor in the regulation of anthocyanin biosynthesis during petal coloration in Malus crabapple.

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