A major premise in evolutionary developmental biology is that regulatory changes, often involving cis-regulatory elements, are responsible for much morphological evolution. This premise is supported by recent investigations of animal development, but information is just beginning to accumulate regarding whether it also applies to the evolution of plant morphology1,2,3,4. Here, we identify the genetic differences between species in the genus Clarkia that are responsible for evolutionary change in an ecologically important element of floral colour patterns: spot position. The evolutionary shift in spot position was due to two simple genetic changes that resulted in the appearance of a transcription factor binding site mutation in the R2R3 Myb gene that changes spot formation. These genetic changes caused R2R3 Myb to be activated by a different transcription factor that is expressed in a different position in the petal. These results suggest that the regulatory rewiring paradigm is as applicable to plants as it is to animals, and support the hypothesis that cis-regulatory changes may often play a role in plant morphological evolution.
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Wray, G. A. The evolutionary significance of cis-regulatory mutations. Nat. Rev. Genet. 8, 206–216 (2007).
Carroll, S. B. Evo-devo and an expanding evolutionary synthesis: a genetic theory of morphological evolution. Cell 134, 25–36 (2008).
Wittkopp, P. J. & Kalay, G. Cis-regulatory elements: molecular mechanisms and evolutionary processes underlying divergence. Nat. Rev. Genet. 13, 59–69 (2011).
Martik, M. L., Lyons, D. C. & McClay, D. R. Developmental gene regulatory networks in sea urchins and what we can learn from them. F1000Research 5, 203 (2016).
Della Pina, S., Souer, E. & Koes, R. Arguments in the evo-devo debate: say it with flowers!. J. Exp. Botany 65, 2231–2242 (2014).
Specht, C. D. & Howarth, D. G. Adaptation in flower form: a comparative evodevo approach. New Phytol. 206, 74–90 (2015).
Arnaud, N. et al. The same regulatory point mutation changed seed-dispersal structures in evolution and domestication. Curr. Biol. 21, 1215–1219 (2011).
Doebley, J. & Lukens, L. transcriptional regulators and the evolution of plant form. Plant Cell. 10, 1075–1082 (1998).
Sobel, J. M. & Streisfeld, M. A. Flower color as a model system for studies in plant evo-devo. Front. Plant Sci. 4, 321 (2013).
Sicard, A. et al. Standing genetic variation in a tissue-specific enhancer underlies selfing-syndrome evolution in Capsella. Proc. Natl. Acad. Sci. USA 113, 13911–13916 (2016).
Kusters, E. et al. Changes in cis-regulatory elements of a key floral regulator are associated with divergence of inflorescence architectures. Development 142, 2822–2831 (2015).
Jones, K. N. Pollinator behavior and postpollination reproductive success in alternative floral phenotypes of Clarkia gracilis (Onagraceae). Int. J. Plant Sci. 157, 733–738 (1996).
Eckhart, V. M. et al. Frequency-dependent pollinator foraging in polymorphic Clarkia xantiana ssp. xantiana populations: Implications for flower-colour evolution and pollinator interactions. Oikos 112, 412–421 (2006).
Ellis, A. G. & Johnson, S. D. Floral mimicry enhances pollen export: the evolution of pollination by sexual deceit outside of the Orchidaceae. Am. Nat. 176, E143–E151 (2010).
Lewis, H. & Lewis, M. E. The genus Clarkia. Univ. Calif. Publ. Bot. 20, 241–392 (1955).
Gottlieb, L. D. & Ford, V. S. Genetic studies of the pattern of floral pigmentation in Clarkia gracilis. Heredity 60, 237–246 (1988).
Abdel-Hameed, F. & Snow, R. The origin of the allotetraploid Clarkia gracilis. Evolution 26, 74–83 (1972).
Martins, T. R., Jiang, P. & Rausher, M. How petals change their spots: cis-regulatory re-wiring in Clarkia (Onagraceae). New Phytol. 216, 510–518 (2017).
Martins, T. R. et al. Precise spatiotemporal regulation of the anthocyanin biosynthetic pathway leads to petal spot formation in Clarkia gracilis (Onagraceae). New Phytol. 197, 958–969 (2013).
Solovyev, V. V. & Salamov A. A. The Gene-Finder computer tools for analysis of human and model organisms genome sequences. In Proc. Fifth Int. Conf. Intelligent Systems for Molecular Biology (eds Rawling, C. et al.) 294-302 (AAAI Press, 1997).
Lescot, M. et al. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 30, 325–327 (2002).
Higo, K. et al. Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res. 27, 297–300 (1999).
Massari, M. E. & Murre, C. Helix-loop-helix proteins: regulators of transcription in eucaryotic organisms. Mol. Cell. Biol. 20, 429–440 (2000).
Bailey, P. C. et al. Update on the basic helix-loop-helix transcription factor gene family in Arabidopsis thaliana. Plant Cell. 15, 2497–2501 (2003).
Heim, M. A. et al. The basic helix-loop-helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity. Mol. Biol. Evol. 20, 735–747 (2003).
Ito, S. et al. Flowering bHLH transcriptional activators control expression of the photoperiodic flowering regulator CONSTANS. Arab. Proc. Natl. Acad. Sci. USA 109, 3582–3587 (2012).
Takahashi, Y. et al. Reconstitution of abscisic acid signaling from the receptor to DNA via bHLH transcription factors. Plant Physiol. 174, 815–822 (2017).
Nakata, M. et al. A bhlh-type transcription factor, aba-inducible bhlh-type transcription factor/ja-associated myc2-like1, acts as a repressor to negatively regulate jasmonate signaling in Arabidopsis. Plant Cell. 25, 1641–1656 (2013).
Zhu, H. F. et al. CPC, a single-repeat R3 MYB, is a negative regulator of anthocyanin biosynthesis in Arabidopsis. Mol. Plant 2, 790–802 (2009).
Wada, T. et al. Epidermal cell differentiation in Arabidopsis determined by a Myb homolog, CPC. Science 277, 1113–1116 (1997).
Wada, T. et al. Role of a positive regulator of root hair development, CAPRICE, in Arabidopsis root epidermal cell differentiation. Development 129, 5409–5419 (2002).
Ogata, K. et al. Comparison of the free and DNA-complexed forms of the DMA-binding domain from c-Myb. Nat. Struct. Mol. Biol. 2, 309–320 (1995).
Jin, H. & Cathie, M. Multifunctionality and diversity within the plant MYB-gene family. Plant Mol. Biol. 41.5, 577–585 (1999).
Gould, S. J. Wonderful Life: The Burgess Shale and the Nature of History 163–165 (W. W. Norton & Company, New York, 1990).
Blount, Z. D., Borland, C. Z. & Lenski, R. E. Historical contingency and the evolution of a key innovation in an experimental population of Escherichia coli. Proc. Natl. Acad. Sci. USA 105, 7889–7906 (2008).
Harms, M. J. & Thornton, J. W. Historical contingency and its biophysical basics in glucocorticoid receptor evolution. Nature 512, 203–207 (2014).
De Smet, R. & Van de Peer, Y. Redundancy and rewiring of genetic networks following genome-wide duplication events. Curr. Opin. Plant Biol. 15.2, 168–176 (2012).
Zhang, Z. L. et al. SCARECROW-LIKE3 promotes gibberellin signaling by antagonizing DELLA in Arabidopsis. Proc. Natl. Acad. Sci. USA 108, 2160–2165 (2011).
Wroblewski, T. et al. Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis. Plant Biotechnol. J. 3, 259–273 (2005).
Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using realtime quantitative PCR and the 2-ΔΔCT method. Methods 25, 402–408 (2001).
Javelle, M. et al. In situ hybridization for the precise localization of transcripts in plants. J. Vis. Exp. 57, 3328 (2011).
We thank T. Martins, S. Zebell, C. Wilson and R. Zentella for technical advice. We thank X. Dong and F. Nijhout for comments on the manuscript. Photographs are by M. Below, K. Morse, V. Smith and B. Breckling. This work was supported by a National Science Foundation grant to M.D.R.
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Jiang, P., Rausher, M. Two genetic changes in cis-regulatory elements caused evolution of petal spot position in Clarkia. Nature Plants 4, 14–22 (2018). https://doi.org/10.1038/s41477-017-0085-6
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