CRISPR/Cas9-mediated targeted mutation reveals a role for AN4 rather than DPL in regulating venation formation in the corolla tube of Petunia hybrida

Abstract

Venation is a common anthocyanin pattern displayed in flowers that confers important ornamental traits to plants. An anthocyanin-related R2R3-MYB transcription factor, DPL, has been proposed to regulate corolla tube venation in petunia plants. Here, however, we provide evidence redefining the role of DPL in petunia. A CRISPR/Cas9-mediated mutation of DPL resulted in the absence of the vein-associated anthocyanin pattern above the abaxial surface of the flower bud, but not corolla tube venation, thus indicating that DPL did not regulate the formation of corolla tube venation. Alternately, quantitative real-time PCR analysis demonstrated that the spatiotemporal expression pattern of another R2R3-MYB gene, AN4, coincided with the formation of corolla tube venation in petunia. Furthermore, overexpression of AN4 promoted anthocyanin accumulation by increasing the expression of anthocyanin biosynthesis genes. CRISPR/Cas9-mediated mutation of AN4 led to an absence of corolla tube venation, suggesting that this gene in fact determines this key plant trait. Taken together, the results presented here redefine the prime regulator of corolla tube venation, paving the way for further studies on the molecular mechanisms underlying the various venation patterns in petunia.

References

[1] Grotewold, E. The genetics and biochemistry of floral pigments. Annu. Rev. Plant Biol. 57, 761-780 (2006).

[2] Quattrocchio, F., Wing, J. F., Leppen, H., Mol, J. & Koes, R. E. Regulatory genes controlling anthocyanin pigmentation are functionally conserved among plant species and have distinct sets of target genes. Plant Cell 5, 1497-1512 (1993).

[3] Kubasek, W. L. et al. Regulation of flavonoid biosynthetic genes in germinating arabidopsis seedlings. Plant Cell 4, 1229-1236 (1992).

[4] Martin, C., Prescott, A., Mackay, S., Bartlett, J. & Vrijlandt, E. Control of anthocyanin biosynthesis in flowers of Antirrhinum majus. Plant J. 1, 37-49 (1991).

[5] Zhang, Y., Butelli, E. & Martin, C. Engineering anthocyanin biosynthesis in plants. Curr. Opin. Plant Biol. 19, 81-90 (2014).

[6] Lai, Y., Li, H. & Yamagishi, M. A review of target gene specificity of flavonoid R2R3-MYB transcription factors and a discussion of factors contributing to the target gene selectivity. Front. Biol. 8, 577-598 (2013).

[7] Petroni, K. & Tonelli, C. Recent advances on the regulation of anthocyanin synthesis in reproductive organs. Plant Sci. 181, 219-229 (2011).

[8] Gao, Y. et al. Tomato SlAN11 regulates flavonoid biosynthesis and seed dormancy by interaction with bHLH proteins but not with MYB proteins. Hortic. Res. 5, 27 (2018).

[9] Sun, B. et al. Purple foliage coloration in tea (Camellia sinensis L.) arises from activation of the R2R3-MYB transcription factor CsAN1. Sci. Rep. 6, 32534 (2016).

[10] Yamagishi, M . A novel R2R3-MYB transcription factor regulates light-mediated floral and vegetative anthocyanin pigmentation patterns in Lilium regale. Mol. Breeding 36, 3 (2016).

[11] Xu, Z. S., Yang, Q. Q., Feng, K., Yu, X. & Xiong, A. S. DcMYB113, a root-specific R2R3-MYB, conditions anthocyanin biosynthesis and modification in carrot. Plant Biotechnol. J. 18, 1585-1597 (2020).

[12] Davies, K. M., Albert, N. W. & Schwinn, K. E. From landing lights to mimicry: the molecular regulation of flower colouration and mechanisms for pigmentation patterning. Funct. Plant Biol. 39, 619-638 (2012).

[13] Shang, Y. J. et al. The molecular basis for venation patterning of pigmentation and its effect on pollinator attraction in flowers of Antirrhinum. N. Phytologist 189, 602-615 (2011).

[14] Schwinn, K. et al. A small family of MYB-regulatory genes controls floral pigmentation intensity and patterning in the genus Antirrhinum. Plant Cell 18, 831-851 (2006).

[15] Hsu, C. C., Chen, Y. Y., Tsai, W. C., Chen, W. H. & Chen, H. H. Three R2R3-MYB transcription factors regulate distinct floral pigmentation patterning in Phalaenopsis spp. Plant Physiol. 168, 175-191 (2015).

[16] Gu, Z. 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. 60, 599-611 (2019).

[17] Yamagishi, M., Shimoyamada, Y., Nakatsuka, T. & Masuda, K. Two R2R3-MYB genes, homologs of Petunia AN2, regulate anthocyanin biosyntheses in flower Tepals, tepal spots and leaves of asiatic hybrid lily. Plant Cell Physiol. 51, 463-474 (2010).

[18] Ding, B. et al. Two MYB proteins in a self-organizing activator-inhibitor system produce spotted pigmentation patterns. Curr. Biol. 30, 802-814 (2020).

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

[20] Wiering, H. d. V. P. In Monographs on Theoretical and Applied Genetics 9: Petunia (ed Sink, K. C.) 49-76 (Springer, 1984).

[21] Albert, N. W. et al. Members of an R2R3-MYB transcription factor family in Petunia are developmentally and environmentally regulated to control complex floral and vegetative pigmentation patterning. Plant J. 65, 771-784 (2011).

[22] Yuan, Y. W., Rebocho, A. B., Sagawa, J. M., Stanley, L. E. & Bradshaw, H. D. Competition between anthocyanin and flavonol biosynthesis produces spatial pattern variation of floral pigments between Mimulus species. Proc. Natl Acad. Sci. USA 113, 2448-2453 (2016).

[23] Zhang, H. et al. Identification and functional analysis of three new anthocyanin R2R3-MYB genes in Petunia. Plant Direct 3, e00114 (2019).

[24] Yamagishi, M., Toda, S. & Tasaki, K. The novel allele of the LhMYB12 gene is involved in splatter-type spot formation on the flower tepals of Asiatic hybrid lilies (Lilium spp.). N. Phytologist 201, 1009-1020 (2014).

[25] Janick, J. Plant Breeding Reviews, Vol. 30 (Wiley, 2008).

[26] Quattrocchio, F., Wing, J. F., Leppen, H. T. C., Mol, J. N. M. & Koes, R. E. Regulatory genes-controlling anthocyanin pigmentation are functionally conserved among plant-species and have distinct sets of target genes. Plant Cell 5, 1497-1512 (1993).

[27] Povero, G. Physiological and Genetic Control of Anthocyanin Pigmentation in Different Species. PhD thesis (2011).

[28] Albert, N. W. et al. Light-induced vegetative anthocyanin pigmentation in Petunia. J. Exp. Bot. 60, 2191-2202 (2009).

[29] Ai, T. N., Arun, M., Naing, A. H., Lim, S. H. & Kim, C. K. Combinatorial expression of transcription factor genes B-Peru and mPAP1 enhances anthocyanin accumulation in transgenic Petunia hybrid. Sci. Hortic. 200, 186-196 (2016).

[30] Schwinn, K. E. et al. MYB and bHLH transcription factor transgenes increase anthocyanin pigmentation in petunia and lisianthus plants, and the petunia phenotypes are strongly enhanced under field conditions. Front. Plant Sci. 5, 603 (2014).

[31] van Houwelingen, A., Souer, E., Mol, J. & Koes, R. Epigenetic interactions among three dTph1 transposons in two homologous chromosomes activate a new excision-repair mechanism in petunia. Plant Cell 11, 1319-1336 (1999).

[32] Koseki, M., Goto, K., Masuta, C. & Kanazawa, A. The star-type color pattern in Petunia hybrida ‘Red star’ flowers is induced by sequence-specific degradation of chalcone synthase RNA. Plant Cell Physiol. 46, 1879-1883 (2005).

[33] Saito, R. et al. Regulation of anthocyanin biosynthesis involved in the formation of marginal picotee petals in Petunia. Plant Sci. 170, 828-834 (2006).

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

[35] Quattrocchio, F. et al. Molecular analysis of the anthocyanin2 gene of petunia and its role in the evolution of flower color. Plant Cell 11, 1433-1444 (1999).

[36] Quattrocchio, F., Wing, J. F., van der Woude, K., Mol, J. N. M. & Koes, R. Analysis of bHLH and MYB domain proteins: species-specific regulatory differences are caused by divergent evolution of target anthocyanin genes. Plant J. 13, 475-488 (1998).

[37] Stracke, R. et al. Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling. Plant J. Cell Mol. Biol. 50, 660-677 (2007).

[38] Dubos, C. et al. MYB transcription factors in Arabidopsis. Trends Plant Sci. 15, 573-581 (2010).

[39] Ito, Y. et al. Re-evaluation of the rin mutation and the role of RIN in the induction of tomato ripening. Nat. Plants 3, 866-874 (2017).

[40] Dell’Aglio, E., Dalvit, I., Loubery, S. & Fitzpatrick, T. B. Clarification of the dispensability of PDX1.2 for Arabidopsis viability using CRISPR/Cas9. BMC Plant Biol. 19, 464 (2019).

[41] Yan, S. et al. Anthocyanin Fruit encodes an R2R3-MYB transcription factor, SlAN2-like, activating the transcription of SlMYBATV to fine-tune anthocyanin content in tomato fruit. N. Phytol. 225, 2048-2063 (2020).

[42] Guo, Y. L. et al. Undesired small RNAs originate from an artificial microRNA precursor in transgenic Petunia (Petunia hybrida). PLoS ONE 9, e98783 (2014).

[43] Xie, X. R. et al. CRISPR-GE: a convenient software toolkit for CRISPR-based genome editing. Mol. Plant 10, 1246-1249 (2017).

[44] Zhang, B., Yang, X., Yang, C. P., Li, M. Y. & Guo, Y. L. Exploiting the CRISPR/Cas9 system for targeted genome mutagenesis in Petunia. Sci. Rep. 6, 20315 (2016).

[45] Liu, W. Z. et al. DSDecode: a web-based tool for decoding of sequencing chromatograms for genotyping of targeted mutations. Mol. plant 8, 1431-1433 (2015).

[46] Mallona, I., Lischewski, S., Weiss, J., Hause, B. & Egea-Cortines, M. Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida. BMC Plant Biol. 10, 4 (2010).

[47] Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative C-T method. Nat. Protoc. 3, 1101-1108 (2008).

[48] Viola, I. L., Camoirano, A. & Gonzalez, D. H. Redox-dependent modulation of anthocyanin biosynthesis by the TCP transcription factor TCP15 during exposure to high light intensity conditions in Arabidopsis. Plant Physiol. 170, 74-85 (2016).