Identification and Characterization of Genes Involved in the Fruit Color Development of European Plum

in Journal of the American Society for Horticultural Science

European plum fruit (Prunus domestica) are normally blue-black to dark purple. However, some genotypes remain green/yellow after ripening. We hypothesized that in such genotypes anthocyanin biosynthesis is genetically disturbed. To examine this hypothesis, six european plum genotypes with diverse fruit colors were investigated for the expression pattern of several anthocyanin biosynthetic genes (ABGs)—e.g., phenylalanine ammonia-lyase, chalcone synthase (CHS), dihydroflavonol 4-reductase (DFR), anthocyanin synthase (ANS), and UDP-glucose:flavonoid 3-O-glucosyltransferase 1 and 2 (UFGT 1 and 2). Expression profiles indicated that ABGs, especially Pd-CHS and UFGT 2, were significantly downregulated in the green/yellow fruit compared with the dark-purple fruit. Furthermore, the quantification of total polyphenols and individual flavonoid compounds showed substantial differences between the off-colored and the purple genotype. To further examine the contribution of each of the ABGs in color development, the open reading frame (ORP) of Pd-CHS, Pd-DFR, Pd-ANS, and Pd-UFGT 2 was ectopically expressed in tobacco (Nicotiana tabacum). The characterization of transgenic plants showed that the petals of plants expressing Pd-CHS were darker in color and had higher anthocyanin content than control or even other transgenic types, suggesting the significant contribution of CHS in determining anthocyanin production levels and hence fruit coloration. The results of this study provides better understanding of color development in european plum, which can be rewarding in developing european plum cultivars with desired colors through classical or modern breeding tools.

Contributor Notes

This work was supported by grants from Ontario Ministry of Agriculture, Food and Rural Affairs (Canada) and Ontario Tender Fruit Producers Marketing board (OTFPMB).

We would like to thank K.S. Tiwari, C.M. Ajila, and Renu Chandrasekaran (University of Guelph, ON, Canada) for their technical support during the research work.

Current address: Department of Horticulture, Faculty of Agriculture, Damanhour University, Al-Gomhuria Street, P.O. Box 22516, Damanhour, Al-Behira, Egypt.

Current address: Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.

Corresponding author. E-mail: jsubrama@uoguelph.ca.

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    Novel genotypes of european plum examined in this study. Genotypes (A) purple (V982017), (B) greenish purple (V90271), (C) yellow 1 (V91058), (D) yellow 2 (V91057), (E) green 1 (V91048), and (F) green 2 (V95141).

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    Total polyphenol content expressed as gallic acid equivalents in different genotypes of european plum. The bars represent the mean ± se of three biological replicates (n = 10 fruit). Values marked with same letters are not significantly different at P < 0.05 by one-way analysis of variance followed by post hoc Tukey’s test.

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    Amount of individual (A) phenolic acids, (B) flavonoids, and (C) anthocyanin derivatives in different genotypes of european plum. Anthocyanin derivatives such as delphinidin, cyanidin, and pelargonidin are present in all genotypes but with varying concentrations. Delphinidin derivatives are present in very low amounts in green genotypes compared with yellow and purple genotypes. The bars represent the mean ± se of three biological replicates (n = 10 fruit). Values marked with same letters are not significantly different at P < 0.05 by one-way analysis of variance followed by post hoc Tukey’s test.

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    Gene expression analysis of (A) phenylalanine ammonia-lyase (PAL), (B) chalcone synthase (CHS), (C) dihydroflavonol reductase (DFR), (D) anthocyanin synthase (ANS), (E) UDP-glucose flavanoid 3-O glucosyl transferase (UFGT 1), and (F) UDP-glucose flavanoid 3-O glucosyl transferase (UFGT 2) in six genotypes of european plum. The expression of each gene was normalized to that of Pd-Actin and was calculated relative to the expression in the purple genotype (calibrator). The values are mean ± se of three biological replicates. Values marked with same letters are not significantly different at P < 0.05 by one-way analysis of variance followed by post hoc Tukey’s test.

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    Phenotypic characterization of control and transgenic tobacco flowers. (A) Flowers collected from five lines of transgenic plants expressing Prunus domestica chalcone synthase (Pd-CHS), P. domestica dihydroflavonol reductase (Pd-DFR), P. domestica anthocyanin synthase (Pd-ANS), and P. domestica UDP-glucose flavanoid 3-O-glucosyl transferase (Pd-UFGT) were evaluated based on corolla color intensity. (B) Schematic representation of anthocyanin biosynthetic pathway. All the enzymes involved in the pathway are in bold. The candidate genes examined in this study are boxed.

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    Anthocyanin content in flowers of wild-type and transgenic tobacco plants. (A) Anthocyanin absorption spectrum at 520 nm (optical density) for wild-type and transgenic tobacco plants. (B) Anthocyanin content present in flowers of transgenic plants expressing Prunus domestica chalcone synthase (Pd-CHS), P. domestica dihydroflavonol reductase (Pd-DFR), P. domestica anthocyanin synthase (Pd-ANS), and P. domestica UDP-glucose flavanoid 3-O-glucosyl transferase (Pd-UFGT) along with wild-type (WT) tobacco plants. The concentration of anthocyanin content is calculated according to the extinction coefficient of cyanidin 26,900 L·cm−1·mol−1. Values marked with same letters are not significantly different at P < 0.05 by one-way analysis of variance followed by post hoc Tukey’s test.

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