More than the (human) eye can see: the regulation of floral UV absorbance

Beverley Glover

Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, UK. bjg26@cam.ac.uk

Flowers come in many different colours, and those colours can be arranged in a wide variety of patterns. Colour itself arises from a combination of differential reflection of light of different wavelengths, combined with the ability of an animal to perceive that differential reflection. If light across the human visible spectrum (blue to red) is all reflected equally, we perceive the reflecting object as white (no colour). If light of a shorter wavelength, in the ultraviolet (UV) range, is differentially reflected then we will still perceive the object as white, but an animal with UV-sensitive photoreceptors in its eyes will perceive the object as coloured. Most insects and many other animals have UV-sensitive vision. I was once asked in a popular science talk whether it’s possible that there is a planet out there on which colour doesn’t exist, and everything is black or white. To which the answer is, there are many planets out there on which no organism has the ability to perceive differential light reflection, but I’m sure that on all planets on which light falls, there are materials which reflect different wavelengths to different extents. Whether that means those planets have colour, if nothing can see it, is a question for a philosopher to tackle!

Bev blog on UV light

Arctotis fastuosa photographed under visible (left) and UV (right) light. The human visible patterning resulting from differential anthocyanin pigmentation is very different from the insect visible patterning that results from UV absorbance by the inner ring of ray florets but UV reflectance by the outer ring.

The colour of flowers is widely considered to be part of the signalling between plants and animal pollinators, with the brightly coloured petals acting as part of a ‘sensory billboard’ (Raguso, 2004) to attract animals to the floral rewards (usually nectar or pollen). It is clear that different colours are visible to different animals, and so particular colours or colour patterns can be interpreted as part of an evolutionary trend in response to attracting particular groups of pollinators. The extent to which specific flower colours do attract specific pollinators is a source of great debate in the literature, with some authors arguing that, like a human-attracting billboard, any colour that stands out against the green vegetation will be as attractive as any other.
While there are clearly some very specific cases of colour (and scent) patterns combining to attract very specific pollinators, such as in the sexually deceptive orchids like Ophrys speculum , it is likely that most flower colours are signalling more broadly.

Ophrys speculum has a highly zygomorphic flower, with inner tepals and both whorls of reproductive organs affected

One good example of specific colours signalling to specific pollinators is found in the genus Petunia, where bee-pollinated flowers such as P. inflata are purple and reflect UV, moth-pollinated P. axillaris flowers are white and absorb UV, and hummingbird-pollinated P. exserta flowers are red and reflect UV. The evolutionary transitions between these different pollination syndromes must have involved switches in regulation of pigment production, to cause both human-visible and human-invisible colour changes. Previous studies have uncovered some of the mechanisms underpinning the human-visible shifts between purple and white flowers, which are the result of changes in the regulation of anthocyanin production. In Petunia this evolutionary transition is caused by changes to the activity of a MYB transcription factor which regulates the genes encoding the enzymes of anthocyanin biosynthesis (Hoballah et al., 2007; Quattrocchio et al., 1999). Interestingly, in various other phylogenetically unrelated examples of evolutionary transitions in anthocyanin content, MYB genes have also been implicated (eg. Yuan et al., 2013).

Recent work by Sheehan et al. (2016) has tackled the much more difficult problem of how the human-invisible UV absorbance has changed between different Petunia species. UV absorbance is the result of flavonol production – these compounds are produced by a biosynthetic pathway which branches off the main anthocyanin pathway using dihydroflavonols as substrate. Flavonols appear white to the human eye, but they absorb UV, and so to a moth with UV-sensitive vision flavonol-pigmented flowers will reflect longer wavelengths more strongly that shorter ones, and appear blue-green. Sheehan and colleagues first used accessions of P. axillaris differing in flavonol production to confirm that hawkmoths preferentially visit UV-absorbing flowers over UV-reflecting ones. They then used QTL mapping with an F2 population derived from a cross between P. exserta and P. axillaris to identify a major locus explaining 79% of the variation in flavonol levels between the 2 species. In combination with previous reports of a flavonol QTL on chromosome II when comparing P. integrifolia (UV reflecting) and P. axillaris (UV absorbing), these data led the authors to investigate the gene encoding flavonol synthase (FLS), the enzyme that catalyses the production of flavonols from dihydroflavonols. FLS had been previously mapped to chromosome II. They found that the FLS locus itself could not explain the differences between species – there were no differences in coding sequence, and although the gene was differentially expressed in different parent species, when alleles from different parents were combined in a single heterozygous plant the different alleles did not show differential expression. Sheehan and colleagues concluded that changes to a regulator of FLS was the likely explanation for the evolutionary transitions. They therefore used a transposon mutagenesis screen to search for candidates, crossing a UV-reflective line of P. hybrida with active transposons to UV-absorbing P. axillaris. The F1 generation is heterozygous for UV absorption, which is a dominant trait. Flowers were screened for UV reflecting sectors where the transposon had disrupted the target gene in the P. axillaris chromosome. This work was made much easier by the surprising realisation that UV reflecting sectors were also a deeper pink in the human-visible spectrum. Identification of the gene disrupted in these sectors revealed the key regulator to be another MYB transcription factor, named MYB-FL, which is linked to and regulates the expression of FLS. Following this molecular genetic detective work Sheehan and colleagues were able to trace the evolution of UV absorbance between their 3 Petunia species, finding that MYB-FL is not expressed in bee-pollinated P. inflata. Expression is activated in moth-pollinated P. axillaris, inducing UV absorbance, by mutation of the MYB-FL promoter region. This expression is maintained in hummingbird-pollinated P. exserta but UV absorbance is lost due to a single base pair deletion in the coding region, causing a frameshift and an early stop codon.
This dissection of the molecular basis for shifts in UV absorption is a powerful example of the use of molecular genetic tools to tackle evolutionary questions even outside the range of what is usually visible to humans. However, the interesting twist in the tale is the discovery that UV reflecting sectors were also brighter pink to the human eye. We cannot see flavonols, so the explanation proposed by Sheehan et al. is that the FLS enzyme is in competition with dihydroflavonol reductase (DFR) for dihydroflavonol substrates. As FLS activity reduced with the inactivation of MYB-FL, then so DFR could channel more dihydroflavonol into the anthocyanin pathway. This hypothesis remains to be tested, but it is certainly logical and, if proven correct, will generate a need to re-think our ideas about the independence of different pigment classes and the ability of different aspects of flower colour to evolve independently.

References

Hoballah M,  Gubitz T, Stuurman J, Broger L,  Barone M,  Mandel T, Dell’Olivo A, Arnold M, and Kuhlemeier C. 2007. Single gene-mediated shift in pollinator attraction in Petunia. Plant Cell 19, 779-790.

Quattrocchio F,  Wing J, van der Woude K, Souer E, de Vetten N, Mol J, and  Koes R. 1999. Molecular analysis of the anthocyanin2 gene of petunia and its role in the evolution of flower colour. Plant Cell 11, 1433–1444.

Raguso RA. 2004. Flowers as sensory billboards: progress towards an integrated understanding of floral advertisement. Current Opinion in Plant Biology 7, 434-440.

Sheehan H, Moser M, Klahre U, Esfeld K, Dell’Olivo A, Mandel T, Metzger S, Vandenbussche M, Freitas L, and Kuhlemeier C. 2016. MYB-FL controls gain and loss of floral UV absorbance, a key trait affecting pollinator preference and reproductive isolation. Nature Genetics 48, 159-166.

Yuan Y, Sagawa J, Young R, Christensen B, and Bradshaw HD Jr. 2013. Genetic dissection of a major anthocyanin QTL contributing to pollinator-mediated reproductive isolation between sister species of Mimulus. Genetics 194, 255-263.

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Flowering Newsletter published by the Journal of Experimental Botany
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