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MYB-FL controls gain and loss of floral UV absorbance, a key trait affecting pollinator preference and reproductive isolation

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Abstract

Adaptations to new pollinators involve multiple floral traits, each requiring coordinated changes in multiple genes. Despite this genetic complexity, shifts in pollination syndromes have happened frequently during angiosperm evolution. Here we study the genetic basis of floral UV absorbance, a key trait for attracting nocturnal pollinators. In Petunia, mutations in a single gene, MYB-FL, explain two transitions in UV absorbance. A gain of UV absorbance in the transition from bee to moth pollination was determined by a cis-regulatory mutation, whereas a frameshift mutation caused subsequent loss of UV absorbance during the transition from moth to hummingbird pollination. The functional differences in MYB-FL provide insight into the process of speciation and clarify phylogenetic relationships between nascent species.

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Figure 1: The hawkmoth pollinator M. sexta prefers UV-absorbent to UV-reflective flowers.
Figure 2: A single major locus determines differences in UV absorption among P. inflata, P. axillaris and P. exserta.
Figure 3: FLS expression in floral buds from P. axillaris, P. inflata and P. exserta.
Figure 4: Transposon insertions in the MYB-FL gene reduce flavonol levels and increase anthocyanin levels.
Figure 5: MYB-FL shows differential expression potentially due to sequence differences in the promoter and second intron.
Figure 6: UV absorbance is associated with MYB-FL genotype in segregating populations.

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Acknowledgements

We thank C. Ball, J. Sekulovski, N. Signer and R. Zimmermann for expert care of our plants; M. Saxenhofer and A. Feller for help with genotyping and screening transposon populations; P. Morel and J. Zethof for help with the amplification and sequencing of dTph1 transposon-flanking sequences; A.L. Cazé for help with phenotyping hybrid progeny plants; and A.L. Segatto and C. Turchetto for field collections and DNA extraction. K. Snowden, A. Amrad, K. Hermann, H. Summers and S. Robinson provided constructive comments on manuscript drafts; A. Amrad, R. Koes, J. Stuurman and T. Gerats provided advice and suggestions throughout the project; and R. Köpfli assisted with figures. M.V. was financially supported by an ATIP-AVENIR (CNRS) and ANR-BLANC grant. L.F. was financed by Science without Borders, CNPq. The remaining authors were financed by the National Centre of Competence in Research “Plant Survival” and grants from the Swiss National Science Foundation and the University of Bern.

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Conceptualization: H.S., U.K., L.F. and C.K. Methodology: H.S., M.M., U.K., A.D., M.V. and C.K. Software: M.M. Formal analysis: H.S., M.M. and U.K. Performed all experiments: H.S., M.M., U.K., A.D., K.E., T.M., S.M. and M.V. Resources: M.V., L.F. and C.K. Writing: H.S., M.M., S.M., M.V. and C.K. Visualization: H.S. Funding acquisition: C.K.

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Correspondence to Cris Kuhlemeier.

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Integrated supplementary information

Supplementary Figure 1 UV-absorbance differs significantly between P. axillaris Rio Arapey (Arapey) and P. axillaris S7 (S7) but floral traits important for pollinator attraction do not differ substantially.

(a) UV-absorbance of the corolla limb. (b) Visible absorbance of the corolla limb. (c) Nectar volume. Note that the difference between Arapey and S7 does not affect the time spent feeding Fig. 1e). (d) Quantity of scent (methylbenzoate) emission. Scent had high variability within S7 but this did not affect the overall preference of M. sexta for Arapey flowers (Fig. 1c,d). (e) Surface area of the corolla limb. Bars show mean ± s.d.; for Rio Arapey samples, (a,b,e) n = 5, (c) n = 4, (d) n = 6; for S7 samples, (a-d) n = 4, (e) n = 5. Statistical tests were carried out using a student’s t test and bars with different letters are significantly different (p < 0.05).

Supplementary Figure 2 Protein sequence alignment of flavonol synthase in P. inflata, P. exserta and P. axillaris, and developmental series of floral buds used for gene expression analyses.

(a) Flavonol synthase (FLS) is a member of the 2-oxoglutarate iron-dependent oxygenase (2-ODD) proteins43. The predicted protein sequence of FLS shows no differences between P. axillaris and P. exserta, and only two residue changes between P. inflata and P. axillaris or P. exserta. Neither residue corresponds to conserved and/or functionally important residues, making differences in enzymatic activity unlikely. Dark grey boxes indicate residues that are conserved between 2-ODD proteins from different plant species87. His34, Asp36 and His290 are required for iron binding, and Arg300 and Ser302 bind the 2-oxoglutarate substrate88. (b,c) Developmental stages of floral buds of P. axillaris (b, top), P. exserta (b, bottom) and P. inflata (c). Numbers define the stages of development, as used for quantitative RT-PCR and RNA-seq experiments. For P. axillaris and P. exserta, stages are separated by approximately 1 d each. For P. inflata, stages 2-4 are separated by 1 to 2 d each, and stages 4-8 are separated by approximately 1 d each. Scale bars, 2 cm.

Supplementary Figure 3 Appearance of various somatic and germline mutants in UV and visible light, including a revertant sector, and details of associated MYB-FL alleles.

(a-d) Four examples of sectors obtained from P. axillaris x W138 F1 (a,c) and IL2-1Pax x W138 F1 (b,d) individuals. Each example is shown in UV light (left) and visible light (right), with UV and visible light absorbance measurements below. (e) A flower from the germline mutant, A1-95 (genotype: MYB-FLPax-dTph4/MYB-FLW138) shown in UV (left) and visible (right) light. (f) The germline mutant, A1-95, was crossed with P. exserta to test for genetic complementation (Fig. 4e) and one of the progeny from this cross that is heterozygous for MYB-FLPax-dTph4 and MYB-FLPex is shown in UV (left) and visible (right) light. This individual has a primarily UV-reflective corolla limb but a UV-absorbent sector is present caused by excision of the dTph4 transposon from the MYB-FLPax-dTph4 allele. (g) DNA was extracted from the sector and the surrounding corolla tissue of the flower shown in f, and the alleles of MYB-FL that were derived from P. axillaris were sequenced from each of these different tissues and are represented in the schematic. The UV-reflective surrounding tissue showed two alleles: allele 1 contained the dTph4 transposon and allele 2 contained a 5 bp footprint left by excision of this transposon (which ultimately causes a frameshift resulting in 21 non-homologous amino acid residues before a stop codon). In the UV-absorbent sector tissue, an allele with a 6 bp footprint due to dTph4 excision was found. This results in the addition of two amino acid residues (phenylalanine and lysine), but the protein sequence remains otherwise intact, allowing the MYB-FLPax-dTph4 allele to return to a functional state after excision of the dTph4 transposon.

Supplementary Figure 4 Protein sequence alignment of MYB111 from Arabidopsis and MYB-FL from P. inflata, P. exserta and P. axillaris.

The N terminus of R2R3-MYBs is highly conserved, with two repeats (R2 and R3; green) which each contain three α helices (yellow) and are involved in DNA-binding, whereas the C terminus is typically less conserved89. The motif that defines subgroup 7 of the R2R3-MYBs in Arabidopsis (AtMYB11, AtMYB12 and AtMYB111; [K/R][R/x][R/K]xGRT[S/x][R/G]xx[M/x]K) is present in Petunia sequences (light blue) with two mismatches (dark blue)90,91. Residues that were conserved in the N-terminus in over 90% of the R2R3-MYBs from Arabidopsis that were analysed are shown in purple90. Only one was not conserved in the Petunia sequences (grey). The positioning of the first two introns (black triangles) is the most common intron pattern in R2R3-MYBs92 and is conserved in the Arabidopsis MYB111 and Petunia MYB-FL sequences. The presence of a third intron (red triangle) is present only in MYB-FL but conserved in all three alleles. The numbers inside the triangles indicate the phase of the introns. Alignment was made using Geneious (Biomatters) with manual modifications.

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Sheehan, H., Moser, M., Klahre, U. et al. MYB-FL controls gain and loss of floral UV absorbance, a key trait affecting pollinator preference and reproductive isolation. Nat Genet 48, 159–166 (2016). https://doi.org/10.1038/ng.3462

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