Subfunctionalization of the Ruby2Ruby1 gene cluster during the domestication of citrus

Abstract

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 Ruby2Ruby1 cluster exhibits subfunctionalization among primitive, wild and cultivated citrus. Our study elucidates the regulatory mechanism and evolutionary history of the Ruby2Ruby1 cluster in citrus, which are unique and different from that found in Arabidopsis, grape or petunia.

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Fig. 1: Phylogeny and fruit and leaf colours of primitive, wild and cultivated citrus.
Fig. 2: Identification of an active Ruby1 allele in a ‘purple pummelo’ germplasm.
Fig. 3: Expression pattern of CgRuby2Short and its function as an anthocyanin repressor.
Fig. 4: AbRuby2Full is a positive regulator of anthocyanin biosynthesis.
Fig. 5: Expression pattern of AbRuby2Full and the cytosine methylation level in the promoter region of AbRuby2 in pigmented and green leaves of Chinese box orange.
Fig. 6: CgRuby2Short competes with CgRuby1 via binding to the same bHLH partner.
Fig. 7: Selection signatures in the Ruby cluster according to comparative genomic analyses of Atalantia, Ichang papeda and pummelo populations.
Fig. 8: The evolutionary history of the Ruby2Ruby1 gene cluster in citrus.

Data availability

The RNA-seq data have been deposited in NCBI under accession codes SRR7631524 (PP1), SRR7631526 (PP2), SRR7631531 (PP3), SRR7631532 (NP1), SRR7631692 (NP2) and SRR7631800 (NP3).

References

  1. 1.

    Swingle, W. T. & Reece, P. C. in The Citrus Industry Vol. 1 (eds Reuther, W. et al) 190–430 (Univ. of California Press, Berkeley, 1967).

  2. 2.

    Butelli, E. et al. Retrotransposons control fruit-specific, cold-dependent accumulation of anthocyanins in blood oranges. Plant Cell 24, 1242–1255 (2012).

    CAS  Article  Google Scholar 

  3. 3.

    Butelli, E. et al. Changes in anthocyanin production during domestication of Citrus. Plant Physiol. 4, 2225–2242 (2017).

    Article  Google Scholar 

  4. 4.

    Winkel-Shirley, B. Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol. 126, 485–493 (2001).

    CAS  Article  Google Scholar 

  5. 5.

    Ramsay, N. A. & Glover, B. J. MYB-bHLH-WD40 protein complex and the evolution of cellular diversity. Trends. Plant. Sci. 10, 63–70 (2005).

    CAS  Article  Google Scholar 

  6. 6.

    Koes, R., Verweij, W. & Quattrocchio, F. Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. Trends. Plant. Sci. 10, 236–242 (2005).

    CAS  Article  Google Scholar 

  7. 7.

    Xu, W., Dubos, C. & Lepiniec, L. Transcriptional control of flavonoid biosynthesis by MYB-bHLH-WDR complexes. Trends. Plant. Sci. 20, 176–185 (2015).

    CAS  Article  Google Scholar 

  8. 8.

    Albert, N. W. et al. A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots. Plant Cell 26, 962–980 (2014).

    CAS  Article  Google Scholar 

  9. 9.

    Walker, A. R. et al. White grapes arose through the mutation of two similar and adjacent regulatory genes. Plant J. 49, 772–785 (2007).

    CAS  Article  Google Scholar 

  10. 10.

    Espley, R. V. et al. Multiple repeats of a promoter segment causes transcription factor autoregulation in red apples. Plant Cell 21, 168–183 (2009).

    CAS  Article  Google Scholar 

  11. 11.

    Wang, Z. et al. The methylation of the PcMYB10 promoter is associated with green-skinned sport in ‘Max Red Bartlett’ pear. Plant Physiol. 162, 885–896 (2013).

    CAS  Article  Google Scholar 

  12. 12.

    Uematsu, C. et al. Peace, a MYB-like transcription factor, regulates petal pigmentation in flowering peach ‘Genpei’ bearing variegated and fully pigmented flowers. J. Exp. Bot. 65, 1081–1094 (2014).

    CAS  Article  Google Scholar 

  13. 13.

    Telias, A. et al. Apple skin patterning is associated with differential expression of MYB10. BMC Plant Biol. 11, 93–107 (2011).

    CAS  Article  Google Scholar 

  14. 14.

    Xu, Q. et al. The draft genome of sweet orange (Citrus sinensis). Nat. Genet. 45, 59–66 (2012).

    Article  Google Scholar 

  15. 15.

    Wu, G. A. et al. Sequencing of diverse mandarin, pummelo and orange genomes reveals complex history of admixture during citrus domestication. Nat. Biotechnol. 32, 656–662 (2014).

    CAS  Article  Google Scholar 

  16. 16.

    Wu, G. A. et al. Genomics of the origin and evolution of Citrus. Nature 554, 311–316 (2018).

    CAS  Article  Google Scholar 

  17. 17.

    Hawkins, C., Caruana, J., Schiksnis, E. & Liu, Z. Genome-scale DNA variant analysis and functional validation of a SNP underlying yellow fruit color in wild strawberry. Sci. Rep. 6, 29017 (2016).

    CAS  Article  Google Scholar 

  18. 18.

    Wang, X. et al. Genomic analyses of primitive, wild and cultivated citrus provide insights into asexual reproduction. Nat. Genet. 49, 765–772 (2017).

    CAS  Article  Google Scholar 

  19. 19.

    Lin-Wang, K. et al. An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae. BMC Plant Biol. 10, 50–67 (2010).

    Article  Google Scholar 

  20. 20.

    Chen, J., Hu, Q., Zhang, Y., Lu, C. & Kuang, H. P-MITE: a database for plant miniature inverted-repeat transposable elements. Nucleic Acids Res. 42, D1176–D1181 (2014).

    CAS  Article  Google Scholar 

  21. 21.

    Zimmermann, I. M., Heim, M. A., Weisshaar, B. & Uhrig, J. F. Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R/B-like BHLH proteins. Plant J. 40, 22–34 (2004).

    CAS  Article  Google Scholar 

  22. 22.

    Hichri, I. et al. The basic helix-loop-helix transcription factor MYC1 is involved in the regulation of the flavonoid biosynthesis pathway in grapevine. Mol. Plant 3, 509–523 (2010).

    CAS  Article  Google Scholar 

  23. 23.

    Gabrielsen, O., Sentenac, A. & Fromageot, P. Specific DNA binding by c-Myb: evidence for a double helix-turn-helix-related motif. Science 253, 1140–1143 (1991).

    CAS  Article  Google Scholar 

  24. 24.

    Jia, L., Clegg, M. T. & Jiang, T. Evolutionary dynamics of the DNA-binding domains in putative R2R3-MYB genes identified from rice subspecies indica and japonica genomes. Plant Physiol. 134, 575–585 (2004).

    CAS  Article  Google Scholar 

  25. 25.

    Wright, S. Evolution and Genetics of Populations Vol. 4 (Univ. Chicago Press, Chicago, USA, 1978).

  26. 26.

    Aharoni, A. et al. The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco. Plant J. 28, 319–332 (2001).

    CAS  Article  Google Scholar 

  27. 27.

    Dubos, C. et al. MYBL2 is a new regulator of flavonoid biosynthesis in Arabidopsis thaliana. Plant J. 55, 940–953 (2008).

    CAS  Article  Google Scholar 

  28. 28.

    Zhu, H. F., Fitzsimmons, K., Khandelwal, A. & Kranz, R. G. CPC, a single-repeat R3 MYB, is a negative regulator of anthocyanin biosynthesis in Arabidopsis. Mol. Plant 2, 790–802 (2009).

    CAS  Article  Google Scholar 

  29. 29.

    Jun, J. H., Liu, C., Xiao, X. & Dixon, R. A. The transcriptional repressor MYB2 regulates both spatial and temporal patterns of proanthocyandin and anthocyanin pigmentation in Medicago truncatula. Plant Cell 27, 2860–2879 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Duarte, J. M. et al. Expression pattern shifts following duplication indicative of subfunctionalization and neofunctionalization in regulatory genes of Arabidopsis. Mol. Biol. Evol. 23, 469–478 (2006).

    CAS  Article  Google Scholar 

  31. 31.

    Zhang, H. et al. Transposon-derived small RNA is responsible for modified function of WRKY45 locus. Nat. Plants. 2, 16016 (2016).

    CAS  Article  Google Scholar 

  32. 32.

    Gonzalez, A., Zhao, M., Leavitt, J. M. & Lloyd, A. M. Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant J. 53, 814–827 (2008).

    CAS  Article  Google Scholar 

  33. 33.

    Bombarely, A. et al. Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida. Nat. Plants. 2, 16074 (2016).

    CAS  Article  Google Scholar 

  34. 34.

    Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313 (2014).

    CAS  Article  Google Scholar 

  35. 35.

    Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome. Biol. 14, R36 (2013).

    Article  Google Scholar 

  36. 36.

    Trapnell, C. et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010).

    CAS  Article  Google Scholar 

  37. 37.

    Hoffmann, T., Kalinowski, G. & Schwab, W. RNAi-induced silencing of gene expression in strawberry fruit (Fragaria x ananassa) by agroinfiltration: a rapid assay for gene function analysis. Plant J. 48, 818–826 (2006).

    CAS  Article  Google Scholar 

  38. 38.

    Gruntman, E. et al. Kismeth: analyzer of plant methylation states through bisulfite sequencing. BMC Bioinformatics 9, 1–14 (2008).

    Article  Google Scholar 

  39. 39.

    Liu, C. et al. Characterization of a citrus R2R3-MYB transcription factor that regulates the flavonol and hydroxycinnamic acid biosynthesis. Sci. Rep. 6, 25352 (2016).

    CAS  Article  Google Scholar 

  40. 40.

    Hellens, R. P. et al. Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods 1, 13 (2005).

    Article  Google Scholar 

  41. 41.

    Cao, H. et al. Comprehending crystalline β-carotene accumulation by comparing engineered cell models and the natural carotenoid-rich system of citrus. J. Exp. Bot. 63, 4403–4417 (2012).

    CAS  Article  Google Scholar 

  42. 42.

    Zhu, F. et al. An R2R3-MYB transcription factor represses the transformation of alpha- and beta-branch carotenoids by negatively regulating expression of CrBCH2 and CrNCED5 in flavedo of Citrus reticulate. New Phytol. 216, 178–192 (2017).

    CAS  Article  Google Scholar 

  43. 43.

    Jefferson, R. A., Kavanagh, T. A. & Bevan, M. W. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901–3907 (1987).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

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.

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Authors

Contributions

Q.X. conceived and coordinated this project. D.H designed the experiments. D.H., Z.Z.T., Y.T.X., X.L.J, Y.Y. and J.X.H, performed the experiments. X.X.D. and S.A.P found and collected the purple pummelo. D.H. and X.W. analysed the bioinformatic data. L.L. and E.B. were involved in the research design and the improvement of the manuscript. D.H. and Q.X. wrote the article.

Corresponding author

Correspondence to Qiang Xu.

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Supplementary information

Supplementary Information

Supplementary Figures 1–16 and Supplementary Tables 2–6.

Reporting Summary

Supplementary Table 1

Gene expression profiling in the fruit peel was compared between the purple pummelo (PP) and normal pummelo (NP).

Supplementary Table 7

List of oligonucleotide sequences used in this study.

Supplementary Data Set 1

Nucleotide sequence of Ruby2 alleles in different accessions of the genus Citrus.

Supplementary Data Set 2

Re-sequencing of Ruby1 promoter in different accessions of the genus Citrus.

Supplementary Data Set 3

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 Ruby2Ruby1 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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