Fluorescence microscopy III Fluorescence correlation spectroscopy (FCS)
Botany: Floral fluorescence effect
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BRIEF COMMUNICATIONS NATURE|Vol 437|15 September 2005 334 Ross, J. F. Science 146, 1170–1172 (1964). 5. Harkness, D. D. Nature 240, 302–303 (1972). 6. Spalding, K. L., Bhardwaj, R. D., Buchholz, B. A., Druid, H. & Frisén, J. Cell 122, 133–143 (2005). 7. Nolla, C. M. J. Dent. Child. 27, 254–266 (1960). 8. Bolanos, M. V., Manrique, M. C., Bolanos, M. J. & Briones, M. T. Foren. Sci. Int. 110, 97–106 (2000). 9. Ritz-Timme, S. et al. Int. J. Legal Med. 113, 129–136 (2000). 10. Otlet, R. L., Walker, A. J., Fulker, M. J. & Collins, C. J. Envir. Radioact. 34, 91–101 (1997). Supplementary information accompanies this communication on Nature’s website. Competing financial interests: declared (see online version of the communication). doi:10.1038/437333a The fluorescence micrograph shows that fluo- rescence is inhibited in areas where betaxan- thins coexist with betanin (Fig. 2d) — the dark area corresponds to the orange area in Fig. 2c. Fluorescence can be an important signal in mate choice for budgerigars 6 and possibly in mantis shrimp 7 , and it may be that in flowers it attracts pollinators. The patterns arising from the internal light-filtering effect between beta- lain pigments described here could encourage bees 1 and bats 8 , which have visual receptors that are sensitive to green light and can detect bright targets better than dim ones 9 . Variation in light emission by flowers at visible wave- lengths also modifies their colour, which would enhance their visibility to pollinators 10 . Fernando Gandía-Herrero, Francisco García-Carmona, Josefa Escribano Departamento de Bioquímica y Biología Molecular A, Unidad Docente de Biología, Facultad de Veterinaria, Universidad de Murcia, 30100 Espinardo, Murcia, Spain e-mail: [email protected] 1. Gumbert, A. Behav. Ecol. Sociobiol. 48, 36–43 (2000). 2. Giurfa, M., Eichmann, B. & Menzel, R. Nature 382, 458–461 (1996). 3. Heiling, A. M., Herberstein, M. E. & Chittka, L. Nature 421, 334 (2003). 4. Gandía-Herrero, F., García-Carmona, F. & Escribano, J. J. Chromatogr. A 1078, 83–89 (2005). 5. Gandía-Herrero, F., Escribano, J. & García-Carmona, F. Plant Physiol. 138, 421–432 (2005). 6. Arnold, K. E., Owens, I. P. F. & Marshall, N. J. Science 295, 92 (2002). 7. Mazel, C. H., Cronin, T. W., Caldwell, R. L. & Marshall, N. J. Science 303, 51 (2004). 8. Winter, Y., Lopez, J. & von Helversen, O. Nature 425, 612–614 (2003). 9. De Ibarra, N. H., Vorobyev, M., Brandt, R. & Giurfa, M. J. Exp. Biol. 203, 3289–3298 (2000). 10. Vorobyev, M., Marshall, J., Osorio, D., De Ibarra, N. H. & Menzel, R. Color Res. Appl. 26 (suppl.), 214–217 (2001). Supplementary information accompanies this communication on Nature’s website. Competing financial interests: declared none. doi:10.1038/437334a BRIEF COMMUNICATIONS ARISING online ➧ www.nature.com/bca see Nature contents. The way flowers appear to insects is crucial for pollination 1–3 . Here we describe an internal light-filtering effect in the flowers of Mirabilis jalapa, in which the visible fluorescence emit- ted by one pigment, a yellow betaxanthin, is absorbed by another, a violet betacyanin, to create a contrasting fluorescent pattern on the flower’s petals. This finding opens up new pos- sibilities for pollinator perception as fluores- cence has not previously been considered as a potential signal in flowers. We investigated the spectra and distribution of the pigments in the multicoloured, strik- ingly patterned flowers of M. jalapa (Nyctagi- naceae), which open only in the late afternoon. This and related plants, such as Bougainvillea, Celosia, Gomphrena and Portulaca, contain pigments known as betalains. These comprise the yellow, fluorescent betaxanthins 4 and vio- let betacyanins, of which betanin (betanidin- O-ȋ-glucoside) is the most common. We extracted and purified the pigments of M. jalapa flowers and analysed them by high- performance liquid chromatography, as previ- ously described 5 . The analysis confirmed that the pigmentation pattern on the flowers was due to a mixture of betaxanthins and betanins. Measurement of the fluorescence-emission spectrum of dopaxanthin and the absorbance spectrum of betanin indicates that the light emitted by the fluorophore is strongly re- absorbed (Fig. 1). Addition of increasing con- centrations of betanin to the dopaxanthin solution reduced the intensity of its fluores- cence, until only 30% of the initial fluorescence was detectable at a ratio of 8.5:1. (For details and methods, see supplementary information.) This internal light-filtering effect between the two types of betalain plant pigment causes a fading of visible fluorescence on parts of the flower where both types are present; areas containing only betaxanthins appear yellow under white light because of a combination of fluorescence and reflectance of non-absorbed radiation (Fig. 2a). The effect can be demon- strated in a system designed to visualize green fluorescence, which filters the incident light to blue and causes betaxanthins in the flower to fluoresce by emitting green light (Fig. 2b). Detailed images of different zones of petal coloration were obtained by using light and flu- orescence microscopy. A brightfield image under white light shows some cells containing only betaxanthins (Fig. 2c, yellow), others with betacyanins (Fig. 2c, deep-red spots), and some with both pigments together (Fig. 2c, orange). BOTANY Floral fluorescence effect Figure 2 | Visible fluorescence in Mirabilis jalapa petals. a, b, Flower with areas of red or yellow coloration under white light (a); only the yellow areas emit green fluorescence when excited by blue light (b) (scale bar, 1.5 cm). c, d, Light micrographs of a section of a single red-and-yellow petal, showing brightfield (c) and fluorescent (d; excitation wavelength, 450–490 nm) images (scale bar, 500 Ȗm). Green fluorescence is due to betaxanthins; dark areas correspond to orange areas in c, where light emitted from the fluorescent pigment is absorbed by betanin. a b c d Wavelength (nm) 300 400 500 600 700 Fluorescence intensity Absorbance Dopaxanthin Betanin (R=glucose) 0.0 0.2 0.4 0.6 0 10 20 30 40 50 HO HO HO N O O N H O OH OH OH RO HO HO (+) N O O N H O OH Figure 1 | Spectra of dopaxanthin and betanin. Dopaxanthin is used as a model betaxanthin because of its structural (insets) and biochemical similarity to betacyanins. When excited by blue light, betaxanthins emit green fluorescence 4 . Fluorescence spectra (blue line, excitation spectrum; green line, emission spectrum) for natural dopaxanthin (6.0 ȖM, in water) are shown; violet line, absorbance spectrum of pure betanin (8.4 ȖM, in water). Note the overlap of the emission and absorbance spectra of the pigments. Nature Publishing Group ©2005
Transcript of Botany: Floral fluorescence effect
BRIEF COMMUNICATIONS NATURE|Vol 437|15 September 2005
334
Ross, J. F. Science 146, 1170–1172 (1964).5. Harkness, D. D. Nature 240, 302–303 (1972).6. Spalding, K. L., Bhardwaj, R. D., Buchholz, B. A., Druid, H. &
Frisén, J. Cell 122, 133–143 (2005).7. Nolla, C. M. J. Dent. Child. 27, 254–266 (1960).8. Bolanos, M. V., Manrique, M. C., Bolanos, M. J. &
Briones, M. T. Foren. Sci. Int. 110, 97–106 (2000).9. Ritz-Timme, S. et al. Int. J. Legal Med. 113, 129–136 (2000).
10. Otlet, R. L., Walker, A. J., Fulker, M. J. & Collins, C. J. Envir. Radioact. 34, 91–101 (1997).
Supplementary information accompanies thiscommunication on Nature’s website.Competing financial interests: declared (see online versionof the communication).doi:10.1038/437333a
The fluorescence micrograph shows that fluo-rescence is inhibited in areas where betaxan-thins coexist with betanin (Fig. 2d) — the darkarea corresponds to the orange area in Fig. 2c.
Fluorescence can be an important signal inmate choice for budgerigars6 and possibly inmantis shrimp7, and it may be that in flowers itattracts pollinators. The patterns arising fromthe internal light-filtering effect between beta-lain pigments described here could encouragebees1 and bats8, which have visual receptorsthat are sensitive to green light and can detectbright targets better than dim ones9. Variationin light emission by flowers at visible wave-lengths also modifies their colour, whichwould enhance their visibility to pollinators10. Fernando Gandía-Herrero, Francisco García-Carmona, Josefa EscribanoDepartamento de Bioquímica y BiologíaMolecular A, Unidad Docente de Biología,Facultad de Veterinaria, Universidad de Murcia,30100 Espinardo, Murcia, Spaine-mail: [email protected]
1. Gumbert, A. Behav. Ecol. Sociobiol. 48, 36–43 (2000).2. Giurfa, M., Eichmann, B. & Menzel, R. Nature 382,
458–461 (1996).3. Heiling, A. M., Herberstein, M. E. & Chittka, L. Nature 421,
334 (2003). 4. Gandía-Herrero, F., García-Carmona, F. & Escribano, J.
J. Chromatogr. A 1078, 83–89 (2005).5. Gandía-Herrero, F., Escribano, J. & García-Carmona, F.
Plant Physiol. 138, 421–432 (2005).6. Arnold, K. E., Owens, I. P. F. & Marshall, N. J. Science 295,
92 (2002).7. Mazel, C. H., Cronin, T. W., Caldwell, R. L. & Marshall, N. J.
Science 303, 51 (2004). 8. Winter, Y., Lopez, J. & von Helversen, O. Nature 425,
612–614 (2003).9. De Ibarra, N. H., Vorobyev, M., Brandt, R. & Giurfa, M.
J. Exp. Biol. 203, 3289–3298 (2000).10. Vorobyev, M., Marshall, J., Osorio, D., De Ibarra, N. H. &
Menzel, R. Color Res. Appl. 26 (suppl.), 214–217 (2001).
Supplementary information accompanies thiscommunication on Nature’s website.Competing financial interests: declared none.doi:10.1038/437334a
BRIEF COMMUNICATIONS ARISING online➧ www.nature.com/bca see Nature contents.
The way flowers appear to insects is crucial forpollination1–3. Here we describe an internallight-filtering effect in the flowers of Mirabilisjalapa, in which the visible fluorescence emit-ted by one pigment, a yellow betaxanthin, isabsorbed by another, a violet betacyanin, tocreate a contrasting fluorescent pattern on theflower’s petals. This finding opens up new pos-sibilities for pollinator perception as fluores-cence has not previously been considered as apotential signal in flowers.
We investigated the spectra and distributionof the pigments in the multicoloured, strik-ingly patterned flowers of M. jalapa (Nyctagi-naceae), which open only in the late afternoon.This and related plants, such as Bougainvillea,Celosia, Gomphrena and Portulaca, containpigments known as betalains. These comprisethe yellow, fluorescent betaxanthins4 and vio-let betacyanins, of which betanin (betanidin-O-�-glucoside) is the most common.
We extracted and purified the pigments ofM. jalapa flowers and analysed them by high-performance liquid chromatography, as previ-ously described5. The analysis confirmed thatthe pigmentation pattern on the flowers wasdue to a mixture of betaxanthins and betanins.Measurement of the fluorescence-emission
spectrum of dopaxanthin and the absorbancespectrum of betanin indicates that the lightemitted by the fluorophore is strongly re-absorbed (Fig. 1). Addition of increasing con-centrations of betanin to the dopaxanthinsolution reduced the intensity of its fluores-cence, until only 30% of the initial fluorescencewas detectable at a ratio of 8.5:1. (For detailsand methods, see supplementary information.)
This internal light-filtering effect betweenthe two types of betalain plant pigment causesa fading of visible fluorescence on parts of the flower where both types are present; areascontaining only betaxanthins appear yellowunder white light because of a combination offluorescence and reflectance of non-absorbedradiation (Fig. 2a). The effect can be demon-strated in a system designed to visualize greenfluorescence, which filters the incident light toblue and causes betaxanthins in the flower tofluoresce by emitting green light (Fig. 2b).
Detailed images of different zones of petalcoloration were obtained by using light and flu-orescence microscopy. A brightfield imageunder white light shows some cells containingonly betaxanthins (Fig. 2c, yellow), others withbetacyanins (Fig. 2c, deep-red spots), and somewith both pigments together (Fig. 2c, orange).
BOTANY
Floral fluorescence effect
Figure 2 | Visiblefluorescence inMirabilis jalapa petals.a, b, Flower with areasof red or yellowcoloration under whitelight (a); only theyellow areas emit greenfluorescence whenexcited by blue light (b)(scale bar, 1.5 cm).c, d, Light micrographsof a section of a singlered-and-yellow petal,showing brightfield (c)and fluorescent (d;excitation wavelength,450–490 nm) images(scale bar, 500 �m).Green fluorescence isdue to betaxanthins;dark areas correspondto orange areas in c,where light emittedfrom the fluorescentpigment is absorbed by betanin.
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Figure 1 | Spectra of dopaxanthin and betanin.Dopaxanthin is used as a model betaxanthinbecause of its structural (insets) and biochemicalsimilarity to betacyanins. When excited by bluelight, betaxanthins emit green fluorescence4.Fluorescence spectra (blue line, excitationspectrum; green line, emission spectrum) fornatural dopaxanthin (6.0 �M, in water) are shown;violet line, absorbance spectrum of pure betanin(8.4 �M, in water). Note the overlap of theemission and absorbance spectra of the pigments.
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