The presence of anthocyanins in flowers and fruits is frequently attributed to attracting pollinators and dispersers. In vegetative organs, anthocyanins and other non-pigmented flavonoids such as flavones and flavonols may serve protective functions against UV radiation, cold, heat, drought, salinity, pathogens, and herbivores; thus, these compounds are usually produced as a plastic response to such stressors. Although, the independent accumulation of anthocyanins in reproductive and vegetative tissues is commonly postulated due to differential regulation, the accumulation of flavonoids within and among populations has never been thoroughly compared. Here, we investigated the shore campion (Silene littorea, Caryophyllaceae) which exhibits variation in anthocyanin accumulation in its floral and vegetative tissues. We examined the in-situ accumulation of flavonoids in floral (petals and calyxes) and vegetative organs (leaves) from 18 populations representing the species' geographic distribution. Each organ exhibited considerable variability in the content of anthocyanins and other flavonoids both within and among populations. In all organs, anthocyanin and other flavonoids were correlated. At the plant level, the flavonoid content in petals, calyxes, and leaves was not correlated in most of the populations. However, at the population level, the mean amount of anthocyanins in all organs was positively correlated, which suggests that the variable environmental conditions of populations may play a role in anthocyanin accumulation. These results are unexpected because the anthocyanins are usually constitutive in petals, yet contingent to environmental conditions in calyxes and leaves. Anthocyanin variation in petals may influence pollinator attraction and subsequent plant reproduction, yet the amount of anthocyanins may be a direct response to environmental factors. In populations on the west coast, a general pattern of increasing accumulation of flavonoids toward southern latitudes was observed in calyxes and leaves. This pattern corresponds to a gradual increase of UV-B radiation and temperature, and a decrease of rainfall toward the south. However, populations along the southern coast exposed to similar climatic stressors showed highly variable flavonoid contents, implying that other factors may play a role in flavonoid accumulation.

BackgroundAnthocyanins are the primary source of colour in flowers and also accumulate in vegetative tissues, where they have multiple protective roles traditionally attributed to early compounds of the metabolic pathway (flavonols, flavones, etc.). Petal-specific loss of anthocyanins in petals allows plants to escape from the negative pleiotropic effects of flavonoid and anthocyanins loss in vegetative organs, where they perform a plethora of essential functions. Herein, we investigate the degree of pleiotropy at the biochemical scale in a pink-white flower colour polymorphism in the shore campion, Silene littorea. We report the frequencies of pink and white individuals across 21 populations and underlying biochemical profiles of three flower colour variants: anthocyanins present in all tissues (pink petals), petal-specific loss of anthocyanins (white petals), and loss of anthocyanins in all tissues (white petals).ResultsIndividuals lacking anthocyanins only in petals represent a stable polymorphism in two populations at the northern edge of the species range (mean frequency 8–21%). Whereas, individuals lacking anthocyanins in the whole plant were found across the species range, yet always at very low frequencies (< 1%). Biochemically, the flavonoids detected were anthocyanins and flavones; in pigmented individuals, concentrations of flavones were 14–56× higher than anthocyanins across tissues with differences of > 100× detected in leaves. Loss of anthocyanin pigmentation, either in petals or in the whole plant, does not influence the ability of these phenotypes to synthesize flavones, and this pattern was congruent among all sampled populations.ConclusionsWe found that all colour variants showed similar flavone profiles, either in petals or in the whole plant, and only the flower colour variant with anthocyanins in photosynthetic tissues persists as a stable flower colour polymorphism. These findings suggest that anthocyanins in photosynthetic tissues, not flavonoid intermediates, are the targets of non-pollinator mediated selection.

Anthocyanin pigments have become a model trait for evolutionary ecology as they often provide adaptive benefits for plants. Anthocyanins have been traditionally quantified biochemically or more recently using spectral reflectance. However, both methods require destructive sampling and can be labor intensive and challenging with small samples. Recent advances in digital photography and image processing make it the method of choice for measuring color in the wild. Here, we use digital images as a quick, noninvasive method to estimate relative anthocyanin concentrations in species exhibiting color variation. Using a consumer‐level digital camera and a free image processing toolbox, we extracted RGB values from digital images to generate color indices. We tested petals, stems, pedicels, and calyces of six species, which contain different types of anthocyanin pigments and exhibit different pigmentation patterns. Color indices were assessed by their correlation to biochemically determined anthocyanin concentrations. For comparison, we also calculated color indices from spectral reflectance and tested the correlation with anthocyanin concentration. Indices perform differently depending on the nature of the color variation. For both digital images and spectral reflectance, the most accurate estimates of anthocyanin concentration emerge from anthocyanin content‐chroma ratio, anthocyanin content‐chroma basic, and strength of green indices. Color indices derived from both digital images and spectral reflectance strongly correlate with biochemically determined anthocyanin concentration; however, the estimates from digital images performed better than spectral reflectance in terms of r 2 and normalized root‐mean‐square error. This was particularly noticeable in a species with striped petals, but in the case of striped calyces, both methods showed a comparable relationship with anthocyanin concentration. Using digital images brings new opportunities to accurately quantify the anthocyanin concentrations in both floral and vegetative tissues. This method is efficient, completely noninvasive, applicable to both uniform and patterned color, and works with samples of any size.

Flower color polymorphisms are widely used as model traits from genetics to ecology, yet determining the biochemical and molecular basis can be challenging. Anthocyanin-based flower color variations can be caused by at least 12 structural and three regulatory genes in the anthocyanin biosynthetic pathway (ABP). We use mRNA-Seq to simultaneously sequence and estimate expression of these candidate genes in nine samples of Silene littorea representing three color morphs (dark pink, light pink and white) across three developmental stages in hopes of identifying the cause of flower color variation. We identified 29 putative paralogs for the 15 candidate genes in the ABP. We assembled complete coding sequences for 16 structural loci and nine of ten regulatory loci. Among these 29 putative paralogs, we identified 622 SNPs, yet only nine synonymous SNPs in Ans had allele frequencies that differentiated pigmented petals (dark pink and light pink) from white petals. These Ans allele frequency differences were further investigated with an expanded sequencing survey of 38 individuals, yet no SNPs consistently differentiated the color morphs. We also found one locus, F3h1, with strong differential expression between pigmented and white samples (>42x). This may be caused by decreased expression of Myb1a in white petal buds. Myb1a in S. littorea is a regulatory locus closely related to Subgroup 7 Mybs known to regulate F3h and other loci in the first half of the ABP in model species. We then compare the mRNA-Seq results with petal biochemistry which revealed cyanidin as the primary anthocyanin and five flavonoid intermediates. Concentrations of three of the flavonoid intermediates were significantly lower in white petals than in pigmented petals (rutin, quercetin and isovitexin). The biochemistry results for rutin, quercetin, luteolin and apigenin are consistent with the transcriptome results suggesting a blockage at F3h, possibly caused by downregulation of Myb1a.

Plants respond to changes in ultraviolet (UV) radiation both morphologically and physiologically. Among the variety of plant UV-responses, the synthesis of UV-absorbing flavonoids constitutes an effective non-enzymatic mechanism to mitigate photoinhibitory and photooxidative damage caused by UV stress, either reducing the penetration of incident UV radiation or acting as quenchers of reactive oxygen species (ROS). In this study, we designed a UVexclusion experiment to investigate the effects of UV radiation in Silene littorea. We spectrophotometrically quantified concentrations of both anthocyanins and UV-absorbing phenolic compounds in petals, calyces, leaves and stems. Furthermore, we analyzed the UV effect on the photosynthetic activity in hours of maximum solar radiation and we tested the impact of UV radiation on male and female reproductive performance. We found that anthocyanin concentrations showed a significant decrease of about 20% with UV-exclusion in petals and stems, and a 30% decrease in calyces. The concentrations of UV-absorbing compounds under UV-exclusion decreased by approximately 25% in calyces and stems, and 12% in leaves. Photochemical efficiency of plants grown under UV decreased at maximum light stress, reaching an inhibition of 58% of photosynthetic activity, but their ability to recover after light-stress was not affected. In addition, exposure to UV radiation did not affect ovule production or seed set per flower, but decreased pollen production and total seed production per plant by 31% and 69%, respectively. Our results demonstrate that UV exposure produced opposing effects on the accumulation of plant phenolic compounds and reproduction. UV radiation increased the concentration of phenolic compounds, suggesting a photoprotective role of plant phenolics against UV light, yet overall reproduction was compromised.