The evolution of fruit colour in plants is intriguing. Citrus fruit has repeatedly gained or lost the ability to synthesize anthocyanins. Chinese box orange, a primitive citrus, can accumulate anthocyanins both in its fruits and its leaves. Wild citrus can accumulate anthocyanins in its leaves. In contrast, most cultivated citrus have lost the ability to accumulate anthocyanins. We characterized a novel MYB regulatory gene, Ruby2, which is adjacent to Ruby1, a known anthocyanin activator of citrus. Different Ruby2 alleles can have opposite effects on the regulation of anthocyanin biosynthesis. AbRuby2Full encodes an anthocyanin activator that mainly functions in the pigmented leaves of Chinese box orange. CgRuby2Short was identified in purple pummelo and encodes an anthocyanin repressor. CgRuby2Short has lost the ability to activate anthocyanin biosynthesis. However, it retains the ability to interact with the same partner, CgbHLH1, as CgRuby1, thus acting as a passive competitor in the regulatory complex. Further investigation in different citrus species indicated that the Ruby2–Ruby1 cluster exhibits subfunctionalization among primitive, wild and cultivated citrus. Our study elucidates the regulatory mechanism and evolutionary history of the Ruby2–Ruby1 cluster in citrus, which are unique and different from that found in Arabidopsis, grape or petunia.
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We thank C.Y. Kang (Huazhong Agricultural University) for kindly providing the strawberry fruits for transient expression assay, L.Z. Xiong and W. Zong (Huazhong Agricultural University) for providing plasmids pM999-35 and 35S:Ghd7–CFP and A. Allan (The New Zealand Institute for Plant and Food Research Limited, Auckland, New Zealand) for providing the vectors for Dual Luciferase Assay. This project was supported by the National Natural Science Foundation of China (31872052, 31572105 and 31330066 to Q.X. and X.X.D.), the fundamental research funds for the central universities (2662015PY109 and 2662018PY008 to Q.X.) and Huazhong Agricultural University Scientific & Technological Self-innovation Foundation.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Figures 1–16 and Supplementary Tables 2–6.
Gene expression profiling in the fruit peel was compared between the purple pummelo (PP) and normal pummelo (NP).
List of oligonucleotide sequences used in this study.
Nucleotide sequence of Ruby2 alleles in different accessions of the genus Citrus.
Re-sequencing of Ruby1 promoter in different accessions of the genus Citrus.
SNPs from the 11 accessions in the regions of single-copy genes that are conserved among Citrinae genomes.
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Huang, D., Wang, X., Tang, Z. et al. Subfunctionalization of the Ruby2–Ruby1 gene cluster during the domestication of citrus. Nature Plants 4, 930–941 (2018). https://doi.org/10.1038/s41477-018-0287-6
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