Supplementary Information
METHOD
Plant material and pigment analysis. Mirabilis jalapa plants were grown in Murcia
(Spain).Yellow flowers contained mainly dopamine-betaxanthin, 0.215 mg g-1 fresh
flower; tyramine-betaxanthin, 0.046 mg g-1; and methionine sulfoxide-betaxanthin,
0.021 mg g-1. Red flowers and areas contained betaxanthins with the same profile as
that obtained for the yellow ones (dopamine-betaxanthin, 0.126 mg g-1; tyraminebetaxanthin, 0.059 mg g-1; methionine sulfoxide-betaxanthin, 0.016 mg g-1), together
with the presence of betanin (0.442 mg g-1) and other betacyanins (iso-betanin, 0.019
mg g-1; betanidin, 0.010 mg g-1). Small violet areas in the patterned petals may contain
mainly betanin, as found for the violet flowers (betanin, 0.761 mg g-1; iso-betanin, 0.069
mg g-1; betanidin, 0.015 mg g-1; dopamine betaxanthin, 0.022 mg g-1). HPLC profiles
are shown in Fig. S2.
Fluorescence and absorbance spectroscopy. Fluorescence excitation and emission
spectra were recorded in water at 25ºC in a LS50B spectrofluorometer (PerkinElmer
Life and Analytical Sciences, Boston, MA, USA). Excitation was measured for
emission at λ = 510 nm, and emission measured for excitation at λ = 463 nm. For
absorbance spectra an Uvikon 940 spectrophotometer (Kontron Instruments, Zurich,
Switzerland) was used.
Photography. Fluorescence photographs were taken using an excitation filter which
limited the flash output between 360 nm and 480 nm. A yellow barrier filter blocked off
the reflected blue light under 490 nm, and transmitted only the wavelengths emitted by
the fluorophore. Filters were performed and supplied by Physical Sciences (Andover,
MA, USA).
Microscopy. Brightfield and fluorescence microscopy were performed in a Leica
DMRB microscope (Leica Microsystems, Wetzlar, Germany) with incident light beam.
The filtercube I3 (Leica Microsystems) was used (excitation: 450-490 nm).
Supplementary Reference
1. Mertens, M. L. & Kägi, J. H. R. Anal Biochem. 96, 448-455 (1979).
Suplementary Figure S1
IFE on the visible fluorescence of dopaxanthin mediated by the presence of increasing
concentrations of betanin. Dopaxanthin concentration was 1.9 µM. In the presence of an
additional absorbing substance, the reduction of the intensity of fluorescence, FIFE, can
be given by an expression similar to that described for absorbance by Lambert-Beer´s
law1:
FIFE  F0  e  k xc
(1)
where F0 is the fluorescence intensity measured in the absence of the absorber, c is the
absorber concentration, x is fixed by the position of the cell (x = 0.5 cm), and k relates
with the capacity of the absorber of attenuating the fluorescence. The exponential
expression given by equation (1) fits with the results shown in Fig. S1, which allowed
the determination of the k value (k = 150,000 M-1cm-1).
Suplementary Figure S2
HPLC profiles for the analysis of the pigment content of yellow (a), red (b), and violet
(c) shades of M. jalapa flowers. Elutions were followed at 480 nm. Main peaks
corresponded to the following betalains: methionine sulfoxide-betaxanthin (1),
dopamine-betaxanthin (2), tyramine-betaxanthin (3), betanin (4), iso-betanin (4´), and
betanidin (5). Full scales are A480 = 0.10 absorbance units for a and b and A480 = 0.15
absorbance units for c.