Precise pigment biosynthesis for flower color design in Brassica napus

Abstract

The flower color has drawn extensive attention in rapeseed breeding for its ornamental value. However, the color formation and precise design are still elusive. Here, we successfully introduced betalain biosynthesis pathway into rapeseed and achieved constitutive betalain production by overexpressing RUBY. The varying expression levels of RUBY and the flower colors of the receptor materials jointly determined the final color presentation. When RUBY was expressed in yellow-flowered rapeseed (cultivar R10), the flower color turned into different shades of orange. In white-flowered background (cultivar R2), RUBY created red flowers. However, RUBY overexpression led to dark-red leaves and decreased photosynthesis. To recover normal photosynthesis, we created orange flowers with green leaves using petal-specific XY355 promoter in yellow-flowered R10. We further verified that white flower is dominant to yellow and created green leaves with shining red flowers by crossing orange flower (XY355:RUBY expressed in yellow background) with white flower (R2). Given that the widespread carotenoid, betalain, and anthocyanin can produce the three major colors of yellow, red, and blue, respectively, we provide a promising approach for creating derivative colors in Brassica napus by employing bioengineering approaches to precisely regulate the pigment biosynthesis.

Acknowledgments

This work was supported by the Talented scientist project in Qinghai Province (2023-NK-145), Wuhan Knowledge Innovation Special Project (2023020201020401), National Natural Science Foundation of China (3240152299), Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2021-OCRI), and China Agriculture Research System of MOF and MARA (CARS-12).

Author contributions

Xinfa Wang and Hanzhong Wang conceived the project. Xiaoling Dun provided experimental materials R2 and R10. Jinwu Deng supervised the project and modified the paper. Yuhan Zheng, Rui Shi, and Wan Chen performed the experiments. Yuhan Zheng analyzed the data and wrote the paper. All authors contributed to the article and approved the submitted version.

Data availability

Supplementary information for this article can be found online.

Conflict of interest statement:

The authors declare that they have no competing interests.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Wakefield A. International statistics flowers and plants 2024. AIPH/Union Fleurs. 2025; 72:9-13

[2] Ye SH, Huang YY, Ma TT. et al. BnaABF3 and BnaMYB44 regulate the transcription of zeaxanthin epoxidase genes in carotenoid and abscisic acid biosynthesis. Plant Physiol. 2024; 195:2372-88

[3] Li Z, Heneen WK. Production and cytogenetics of intergeneric hybrids between the three cultivated brassica diploids and Orychophragmus violaceus. Theor Appl Genet. 1999; 99:694-704

[4] Zhao ZG, Hu TT, Ge XH. et al. Production and characteriza-tion of intergeneric somatic hybrids between Brassica napus and Orychophragmus violaceus and their backcrossing progenies. Plant Cell Rep. 2008; 27:1611-21

[5] Zhao CJ, Safdar LB, Xie ML. et al. Mutation of the PHYTOENE DESATURASE 3 gene causes yellowish-white petals in Brassica napus. Crop J. 2021; 9:1124-34

[6] FuWQ, Dz C, Pan Q. et al. Production of red-flowered oilseed rape via the ectopic expression of Orychophragmus violaceus OvPAP2. Plant Biotechnol J. 2018; 16:367-80

[7] Grotewold E. The genetics and biochemistry of floral pigments. Annu Rev Plant Biol. 2006; 57:761-80

[8] Nisar N, Li L, Lu S. et al. Carotenoid metabolism in plants. Mol Plant. 2015; 8:68-82

[9] Meng YY, Wang ZY, Wang YQ. et al. The MYB activator WHITE PETAL1 associates with MtTT8 and MtWD40-1 to regulate carotenoid-derived flower pigmentation in Medicago truncatula. Plant Cell. 2019; 31:2751-67

[10] Lu SW, Ye JL, Zhu KJ. et al. A fruit ripening-associated transcrip-tion factor CsMADS5 positively regulates carotenoid biosynthe-sis in citrus. JExp Bot. 2021; 72:3028-43

[11] Li HL, Yu KD, Amoo O. et al. Site-directed mutagenesis of the carotenoid isomerase gene BnaCRTISO alters the color of petals and leaves in Brassica napus L. front. Plant Sci. 2022; 13:801456

[12] Zhang B, Liu C, Wang YQ. et al. Disruption of a CAROTENOID CLEAVAGE DIOXYGENASE 4 gene converts flower colour from white to yellow in brassica species. New Phytol. 2015; 206:1513-26

[13] Jia LD, Wang JS, Wang R. et al. Comparative transcriptomic and metabolomic analyses of carotenoid biosynthesis reveal the basis of white petal color in Brassica napus. Planta. 2021; 253:8

[14] Gandía-Herrero F, García-Carmona F. Biosynthesis of betalains: yellow and violet plant pigments. Trends Plant Sci. 2013; 18:334-43

[15] Polturak G, Aharoni A. Advances and future directions in beta-lain metabolic engineering. New Phytol. 2019; 224:1472-8

[16] Chen JY, Xie FF, Cui YZ. et al. A chromosome-scale genome sequence of pitaya (Hylocereus undatus) provides novel insights into the genome evolution and regulation of betalain biosynthe-sis. Hortic Res. 2021; 8:164

[17] He YB, Zhang T, Sun H. et al. A reporter for noninvasively monitoring gene expression and plant transformation. Hortic Res. 2020; 7:152

[18] Chen K, Yu Y, Zhang ZJ. et al. Engineering a complex, multiple enzyme-mediated synthesis of natural plant pig-ments in the silkworm, Bombyx mori. Proc Natl Acad Sci USA. 2023; 120:e2306322120

[19] Saigo T, Wang T, Watanabe M. et al. Diversity of anthocyanin and proanthocyanin biosynthesis in land plants. Curr Opin Plant Biol. 2020; 55:93-9

[20] Liu Y, Tikunov Y, Schouten RE. et al. Anthocyanin biosynthe-sis and degradation mechanisms in Solanaceous vegetables: a review. Front Chem. 2018; 6:52

[21] He J, Giusti MM. Anthocyanins: natural colorants with health-promoting properties. Annu Rev Plant Biol. 2010; 1:163-87

[22] Fukui Y, Tanaka Y, Kusumi T. et al. A rationale for the shift in colour towards blue in transgenic carnation flowers expressing the flavonoid 3’,5’-hydroxylase gene. Phytochemistry. 2003; 63: 15-23

[23] Katsumoto Y, Fukuchi-Mizutani M, Fukui Y. et al. Engineering of the rose flavonoid biosynthetic pathway successfully generated blue-hued flowers accumulating delphinidin. Plant Cell Physiol. 2007; 48:1589-600

[24] Noda N, Aida R, Kishimoto S. et al. Genetic engineering of novel bluer-colored chrysanthemums produced by accumu-lation of Delphinidin-based anthocyanins. Plant Cell Physiol. 2013; 54:1684-95

[25] Li SJ, Li X, Wang XD. et al. Flavonoid synthesis-related genes determine the color of flower petals in Brassica napus L. Int J Mol Sci. 2023; 24:6472

[26] Ye SH, Hua SJ, Ma TT. et al. Genetic and multi-omics anal-yses reveal BnaA07.PAP2In-184-317 as the key gene conferring anthocyanin-based color in Brassica napus flowers. JExp Bot. 2022; 73:6630-45

[27] Di HM, Zhao YT, Zhou AL. et al. Integrated metabolome and transcriptome analysis revealed color formation in purple leaf mustard (Brassica juncea). Sci Hortic. 2024; 337:113526

[28] Kevan PG, Baker HG. Insects as flower visitors and pollinatorsl. Annu Rev Entomol. 1983; 28:407-53

[29] Shen YX, Rao YF, Ma MN. et al. Coordination among flower pigments, scents and pollinators in ornamental plants. Hortic Adv. 2024; 2:6

[30] Li XQ, Mitchell M, Rolland V. et al. ‘Pink cotton candy’—a new dye-free cotton. Plant Biotechnol J. 2023; 21:677-9

[31] Wang D, Zhong Y, Feng B. et al. The RUBY reporter enables effi-cient haploid identification in maize and tomato. Plant Biotechnol J. 2023; 21:1707-15

[32] Tanaka Y, Brugliera F. Flower colour and cytochromes P450†. Philos Trans R Soc Lond Ser B Biol Sci. 2013; 368:20120432

[33] Aida R, Yoshida K, Kondo T. et al. Copigmentation gives bluer flowers on transgenic torenia plants with the antisense dihydroflavonol-4-reductase gene. Plant Sci. 2000; 160:49-56

[34] Dyer AG, Jentsch A, Burd M. et al. Fragmentary blue: resolving the rarity paradox in flower colors. Front Plant Sci. 2021; 11: 618203

[35] Mao MX, Han GX, Zhao YL. et al. A review of phycocyanin: production, extraction, stability and food applications. Int J Biol Macromol. 2024; 280:135860

[36] Holton TA, Brugliera F, Lester DR. et al. Cloning and expression of cytochrome P450 genes controlling flower colour. Nature. 1993; 366:276-9

[37] Noda N, Yoshioka S, Kishimoto S. et al. Generation of blue chrysanthemums by anthocyanin B-ring hydroxylation and glucosylation and its coloration mechanism. Sci Adv. 2017; 3:e1602785

[38] Chen DZ, Liu Y, Fu WQ. et al. Progress on genetics and breeding of rapeseed (Brassica napus L.) with colored flowers (in Chi-nese with an English abstract). Chin J Oil Crops Sci. 2019; 41: 309-16

[39] Cui C, Zhang K, Chai L. et al. Unraveling the mechanism of flower color variation in Brassica napus by integrated metabolome and transcriptome analyses. Front Plant Sci. 2024; 15:1419508

[40] Pino MT, Skinner JS, Park EJ. et al. Use of a stress inducible promoter to drive ectopic AtCBF expression improves potato freezing tolerance while minimizing negative effects on tuber yield. Plant Biotechnol J. 2007; 5:591-604

[41] Seyfabadi J, Ramezanpour Z, Amini KZ. Protein, fatty acid, and pigment content of Chlorella vulgaris under different light regimes. J Appl Phycol. 2011; 23:721-6

[42] Azuma M, Morimoto R, Hirose M. et al. A petal-specific InMYB1 promoter from Japanese morning glory: a useful tool for molec-ular breeding of floricultural crops. Plant Biotechnol J. 2016; 14: 354-63

[43] Gu ZY, Zhu J, Hao Q. et al. A novel R2R3-MYB transcription factor contributes to petal blotch formation by regulating organ-specific expression of PsCHS in tree peony (Paeonia suffruticosa). Plant Cell Physiol. 2019; 60:599-611

[44] Fattorini R, Khojayori FN, Mellers G. et al. Complex petal spot for-mation in the beetle daisy (Gorteria diffusa) relies on spot-specific accumulation of malonylated anthocyanin regulated by paral-ogous GdMYBSG6 transcription factors. New Phytol. 2024; 243: 240-57

[45] Yuan Y, Li X, Yao X. et al. Mechanisms underlying the formation of complex color patterns on Nigella orientalis (Ranunculaceae) petals. New Phytol. 2023; 237:2450-66

[46] Arnon DI. Copper enzymes in isolated chloroplasts. Polyphe-noloxidase in Beta vulgaris. Plant Physiol. 1949; 24:1-15

[47] Wu YH, Li J, Li XY. et al. Copy number and zygosity determination of transgenic rapeseed by droplet digital PCR. Oil Crop Sci. 2017; 2: 84-94