Turing’s reaction-diffusion mechanism explains dispersed petal spots in monkeyflowers

by Paula Elomaa
Department of Agricultural Sciences, Viikki Plant Science Centre, University of Helsinki, Finland
paula.elomaa@helsinki.fi

Colorful pigmentation patterns in plants – spots, blotches, stripes, or colored veins – are particularly fascinating. Anthocyanin pigments are synthesized via the well-known flavonoid pathway, and they form patterns that, especially in flowers, are associated with spatial developmental signals but may also be activated by environmental cues. Complex pigmentation patterns, including those not visible for human eye, have also evolved in response to specialized pollination strategies and contributed to speciation.

Studies in diverse model systems, including maize, petunia, snapdragon and Arabidopsis show that the anthocyanin regulators are highly conserved, and include MYB, bHLH, and WD40-repeat proteins functioning in a so-called MBW complex that activates the biosynthetic genes (Feller et al., 2011; Xu et al., 2015). In this complex, tissue specifically expressed R2R3-MYB proteins primarily define spatial pigmentation patterns. Anthocyanin regulation also involves repressor proteins. As shown in petunia, the EAR-domain containing R2R3-MYBs may function as a part of the DNA-binding MBW complex, but converts it from an activating into a repressing complex (Albert et al., 2014). Moreover, small R3-MYB proteins, with only a single MYB repeat, have been identified as repressors. They do not bind DNA but interact with bHLH activators, consequently sequestering them from the MBW complexes. R3-MYBs are suggested to provide feedback regulation that limits anthocyanin accumulation and affects pigmentation patterning (Albert et al., 2011, 2014).

The paper by Ding et al. (2020) focuses on anthocyanin spots in monkeyflowers (Mimulus) that dot the nectar guide of the ventral petal (Fig. 1A). These spots form in randomly dispersed patterns raising a question of how they emerge. Ding et al. (2020) refer to the classical reaction-diffusion (RD) model of patterning, originally proposed by Turing (1952) and Gierer and Meinhardt (1972). The model states that patterns in various biological systems arise autonomously through a network of reacting factors that combines a short-range positive feedback with a long-range negative feedback (reviewed in Kondo and Miura, 2010). In its most simple form, the model may include only two components; an activator that controls its own synthesis as well as the synthesis of a repressor that is able to diffuse or move to neighboring cells and, in turn, to inhibit the activator function. To test whether this model holds for spot formation in Mimulus, Ding et al. (2020) functionally validated the interactions of two MYB domain factors; a known, self-activating R2R3-type anthocyanin activator NECTAR GUIDE ANTHOCYANIN (NEGAN) and a newly identified R3-type inhibitor RED TONGUE (RTO).

The rto mutant with uniform red pigmentation in the nectar guide, was identified through EMS mutagenesis screen in M. lewisii. By independent mapping experiments, the authors identified the causal gene as an R3-MYB, and verified that mutations in the same gene were responsible for the naturally occurring rto phenotypes of M. guttatus found in Oregon and southern California (Fig. 1B). The repressor function of RTO was confirmed in transgenic Mimulus lines. Following the principle of the RD model, NEGAN, in a protein complex with bHLH and WD40 components, was shown to activate RTO expression in the nectar guides in both Mimulus species. On the other hand, analysis of the rto mutant, rto-like natural variants and RTO transgenic lines indicated that RTO in turn inhibits NEGAN. Repression was shown to occur through interaction with bHLH proteins sequestering them into inactive complexes.

Fig. 1. Dispersed anthocyanin spots in monkeyflowers. (A) Spots in Mimulus lewisii dot the nectar guide of the ventral petal. Photo courtesy of Jouko Rikkinen, Univ. of Helsinki, Finland. (B) Segregating variation at the RTO locus in a wild Mimulus guttatus population at the UC Davis McLaughlin Reserve. Uniform red pigmentation in nectar guide region is observed in rto mutants. Photo courtesy of Benjamin Blackman, Univ. of California, USA.

The RD model also assumes long-range movement of a repressor. Using fluorescent marker lines, Ding et al. (2020) showed that the RTO gene was transcribed in the pigmented spots but the RTO protein was localized in a broader domain indicating that it is moving from the source cell to the neighboring cells. Mobility of R3-MYBs has also previously been shown for the petunia MYBx (Albert et al., 2014). The observed regulatory interactions, and the movement of the repressor support a simple two-component RD model. The authors applied computer simulations to test patterning dynamics. Using diverse parameter values for degradation rate of the activator NEGAN as well as production and degradation rates of the inhibitor RTO, they were able to recapitulate the phenotypes observed  both in wild-type and transgenic lines. Finally, the authors showed that the rto alleles are retained in natural populations of M. guttatus. Using controlled laboratory experiments, they showed that naïve bumblebees preferred both the homozygote and heterozygote rto flowers over wild-type suggesting that the patterning impacts plant-pollinator interactions.

The paper by Ding et al. (2020) provides a real-life example of Turing’s mechanism operating in pattern formation in plants. The presented experimental data combined with computer simulations elegantly demonstrates the extremely fine-tuned interaction dynamics between the activator and the repressor molecules leading to dispersed anthocyanin spot patterns. It will be interesting to see how widely the two-component RD model underlies pigmentation patterns observed in diverse plant lineages, and how it is linked with spatial regulation of pigmentation, upstream of MYBs.

References

Albert NW, Lewis DH, Zhang H, Schwinn KE, Jameson PE, Davies KM. 2011. Members of an R2R3-MYB transcription factor family in Petunia are developmentally and environmentally regulated to control complex floral and vegetative pigmentation patterning. The Plant Journal 65, 771-784. https://doi.org/10.1111/j.1365-313X.2010.04465.x

Albert NW, Davies KM, Lewis DH, Zhang H, Montefiori M, Brendolise C, Boase MR, Ngo H, Jameson PE, Schwinn KE. 2014. A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots. The Plant Cell 26, 962-980. https://doi.org/10.1105/tpc.113.122069

Ding B, Patterson EL, Holalu SV, Li J, Johnson GA, Stanley LE, Greenlee AB, Peng F, Bradshaw Jr. HD, Blinov ML, Blackman BK, Yuan Y-W. 2020. Two MYB proteins in a self-organizing activator-inhibitor system produce spotted pigmentation patterns. Current Biology 30, 1-13. https://doi.org/10.1016/j.cub.2019.12.067

Feller A, Machemer K, Braun EL, Grotewold E. 2011. Evolutionary and comparative analysis of MYB and bHLH transcription factors. The Plant Journal 66, 94-116. https://doi.org/10.1111/j.1365-313X.2010.04459.x

Gierer A, Meinhardt H. 1972. A theory of biological pattern formation. Kybernetik 12, 30-39. https://doi.org/10.1007/BF00289234

Kondo S, Miura T. 2010. Reaction-diffusion model as framework for understanding biological pattern formation. Science 329, 616-620. DOI: 10.1126/science.1179047

Turing AM. 1952. The chemical basis of morphogenesis. Philosophical Transactions of the Royal  Society of London. Series B, Biological Sciences 237, 37-72. https://doi.org/10.1098/rstb.1952.0012

Xu W, Dubos C, Lepinic L. 2015. Transcriptional control of flavonoid biosynthesis by MYB-bHLH-WDR complexes. Trends in Plant Science 20, 176-185.https://doi.org/10.1016/j.tplants.2014.12.001

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