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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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Phenotypic analysis of McCHS overexpressing transgenic tobacco flowers and expression profiles of target genes.(A) Typical flower phenotypes of control lines (CK) and McCHS-ox-1 and McCHS-ox-3 tobacco plants overexpressing McCHS. (B) Petal color of control plants (CK) and McCHS-ox-1 and McCHS-ox-3. (C) Microscopic observation of the transgenic tobacco petals. (D) Content of total anthocyanin in petals of control lines (CK) and McCHS-ox-1 and McCHS-ox-3. (E) Relative expression profiles of endogenous anthocyanin biosynthesis genes in transgenic tobacco flowers. A spectrophotometric colorimeter was used to measure color changes in petals and HPLC was used to analyze the total anthocyanin content in the petals of transgenic tobacco. Real-time PCR was used to assess the expression of target genes (NtCHS, NtF3H, NtF3′H, NtDFR, NtANS, NtUFGT) in McCHS-ox tobacco plants. CK refers to wild type tobacco. All real time-PCR reactions were normalized using the Ct value corresponding to the NtActin gene (GQ339768). Error bars correspond to the standard error of the mean ± SE of three replicate reactions. Analysis of relative expression levels of control and McCHS-ox lines and different letters above the bars indicate significantly different values (P<0.05) calculated using one-way analysis of variance (ANOVA) followed by a Duncan′s multiple range test.
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pone-0110570-g007: Phenotypic analysis of McCHS overexpressing transgenic tobacco flowers and expression profiles of target genes.(A) Typical flower phenotypes of control lines (CK) and McCHS-ox-1 and McCHS-ox-3 tobacco plants overexpressing McCHS. (B) Petal color of control plants (CK) and McCHS-ox-1 and McCHS-ox-3. (C) Microscopic observation of the transgenic tobacco petals. (D) Content of total anthocyanin in petals of control lines (CK) and McCHS-ox-1 and McCHS-ox-3. (E) Relative expression profiles of endogenous anthocyanin biosynthesis genes in transgenic tobacco flowers. A spectrophotometric colorimeter was used to measure color changes in petals and HPLC was used to analyze the total anthocyanin content in the petals of transgenic tobacco. Real-time PCR was used to assess the expression of target genes (NtCHS, NtF3H, NtF3′H, NtDFR, NtANS, NtUFGT) in McCHS-ox tobacco plants. CK refers to wild type tobacco. All real time-PCR reactions were normalized using the Ct value corresponding to the NtActin gene (GQ339768). Error bars correspond to the standard error of the mean ± SE of three replicate reactions. Analysis of relative expression levels of control and McCHS-ox lines and different letters above the bars indicate significantly different values (P<0.05) calculated using one-way analysis of variance (ANOVA) followed by a Duncan′s multiple range test.

Mentions: To investigate the temporal and spatial expression of McCHS in plants, transgenic tobacco plants expressing McCHS under the control of a constitutive CaMV35S promoter were generated, and two independent T2 lines were characterized. Compared to wild type, tobacco plants transformed with 35S::McCHS developed a more intense pigmentation in the petals, especially in McCHS-ox-1 line (Figure 7A). The lightness value L*, hue value a*, hue value b* of the McCHS-overexpressing tobacco petals were also measured. The ‘H’ value (hue angle) decreased to one-third of that of control plant petals and the ‘a*’ values were 4.0 and 5.0-fold higher than in control plant petals, indicative of a stronger red color (Figure 7B). In addition, we confirmed by microscopy that more anthocyanins accumulated in the petal cells of the transgenic tobacco lines (Figure 7C).


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)

Phenotypic analysis of McCHS overexpressing transgenic tobacco flowers and expression profiles of target genes.(A) Typical flower phenotypes of control lines (CK) and McCHS-ox-1 and McCHS-ox-3 tobacco plants overexpressing McCHS. (B) Petal color of control plants (CK) and McCHS-ox-1 and McCHS-ox-3. (C) Microscopic observation of the transgenic tobacco petals. (D) Content of total anthocyanin in petals of control lines (CK) and McCHS-ox-1 and McCHS-ox-3. (E) Relative expression profiles of endogenous anthocyanin biosynthesis genes in transgenic tobacco flowers. A spectrophotometric colorimeter was used to measure color changes in petals and HPLC was used to analyze the total anthocyanin content in the petals of transgenic tobacco. Real-time PCR was used to assess the expression of target genes (NtCHS, NtF3H, NtF3′H, NtDFR, NtANS, NtUFGT) in McCHS-ox tobacco plants. CK refers to wild type tobacco. All real time-PCR reactions were normalized using the Ct value corresponding to the NtActin gene (GQ339768). Error bars correspond to the standard error of the mean ± SE of three replicate reactions. Analysis of relative expression levels of control and McCHS-ox lines and different letters above the bars indicate significantly different values (P<0.05) calculated using one-way analysis of variance (ANOVA) followed by a Duncan′s multiple range test.
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pone-0110570-g007: Phenotypic analysis of McCHS overexpressing transgenic tobacco flowers and expression profiles of target genes.(A) Typical flower phenotypes of control lines (CK) and McCHS-ox-1 and McCHS-ox-3 tobacco plants overexpressing McCHS. (B) Petal color of control plants (CK) and McCHS-ox-1 and McCHS-ox-3. (C) Microscopic observation of the transgenic tobacco petals. (D) Content of total anthocyanin in petals of control lines (CK) and McCHS-ox-1 and McCHS-ox-3. (E) Relative expression profiles of endogenous anthocyanin biosynthesis genes in transgenic tobacco flowers. A spectrophotometric colorimeter was used to measure color changes in petals and HPLC was used to analyze the total anthocyanin content in the petals of transgenic tobacco. Real-time PCR was used to assess the expression of target genes (NtCHS, NtF3H, NtF3′H, NtDFR, NtANS, NtUFGT) in McCHS-ox tobacco plants. CK refers to wild type tobacco. All real time-PCR reactions were normalized using the Ct value corresponding to the NtActin gene (GQ339768). Error bars correspond to the standard error of the mean ± SE of three replicate reactions. Analysis of relative expression levels of control and McCHS-ox lines and different letters above the bars indicate significantly different values (P<0.05) calculated using one-way analysis of variance (ANOVA) followed by a Duncan′s multiple range test.
Mentions: To investigate the temporal and spatial expression of McCHS in plants, transgenic tobacco plants expressing McCHS under the control of a constitutive CaMV35S promoter were generated, and two independent T2 lines were characterized. Compared to wild type, tobacco plants transformed with 35S::McCHS developed a more intense pigmentation in the petals, especially in McCHS-ox-1 line (Figure 7A). The lightness value L*, hue value a*, hue value b* of the McCHS-overexpressing tobacco petals were also measured. The ‘H’ value (hue angle) decreased to one-third of that of control plant petals and the ‘a*’ values were 4.0 and 5.0-fold higher than in control plant petals, indicative of a stronger red color (Figure 7B). In addition, we confirmed by microscopy that more anthocyanins accumulated in the petal cells of the transgenic tobacco lines (Figure 7C).

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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