Kaempferol 3 - 0 - Galactoside , 7 - 0 - Rhamnoside is the Major Green Fluorescing Compound in the Epidermis of Vicia fabal

The vacuoles of lower epidermal strips from Vicia faba exhibit an intrinsic green fluorescence when incubated in alkaline buffers. Using an alkaline-induced absorbance change as a spectrophotometric assay, the major pigment responsible for this fluorescence was isolated and identified as the flavonoid: kaempferol 3-O-galactoside, 7-O-rhamnoside. The aqueous absorption maxima were 394 and 341 nanometers at pH 10.0 and 6.0, respectively, with a pKa of 8.3 and the fluorescence emission maximum was 494 nanometers at pH 10.0. The in vivo concentration was estimated to be between 3 and 10 micromolar. The absorption spectrum of this flavonoid is different from the action spectrum for stomatal opening indicating that this compound is not the photoreceptor pigment for the blue light response of Viciafaba guard cells. Plants control much of their water loss and gas exchange by regulation of their stomatal aperture through the swelling and shrinking of the two guard cells surrounding the stoma (4, 12). Guard cell size and hence, stomatal aperture, is influenced by several environmental factors, one ofwhich is light. Action spectra indicate the involvement of two distinct photoreceptor pigment systems in guard cell photophysiology; one requires high light intensities and is sensitive to both red and blue light (photosynthetic pigments), while the other is effective at much lower light intensities and is sensitive only to wavelengths less than 520 nm (2, 5, 9, 14, 15). Action spectra for this blue light response resembles those measured for other blue light-sensitive responses, with peaks at 380 and 450 nm (2, 9, 15), implying that a similar type of photoreceptor pigment may be involved. Experiments from the majority of other blue light-sensitive responses indicate that the chromophore is most likely a flavin (1 1, 13). Zeiger and Hepler (21, 22) discovered that the blue light-responsive guard cells of Allium exhibit an intrinsic green vacuolar fluorescence. More recently, epidermal strips from Viciafaba also have been reported to display the same type of intrinsic green fluorescence after incubation with alkaline buffers (19, 20). Based on the assumption that a flavin is involved in photoperception in guard cells and the knowledge that flavins are highly yellow-green fluorescent, it was suggested that this intrinsic fluorescence is mediated by a flavin and that this flavin is involved in the blue light response of guard cells (19, 20). ' Supported by the United States Department of Energy under Contract DE-AC02-76ER01338. 2 Present Address: Department of Botany, University of Wisconsin, 139 Birge Hall, 430 Lincoln Drive, Madison, WI 53706. 3Present Address: Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan, 48824. Using the ability of V. faba epidermal strips to exhibit green fluorescence after alkalinization as a spectrophotometric assay, we attempted to identify the pigment(s) responsible. The principal goal was whether this fluorescence is indeed flavin-mediated and possibly linked to guard cell photophysiology. MATERIALS AND METHODS V.faba (cv. Improved Long Pod, from Lagomarsine Seeds, Inc., Sacramento, CA) were grown in a peatlike soil mixture and watered once a week with Y2 Hoagland solution. Growth conditions consisted of a 16-h light period (light intensity was 40 w m-2) at 23°C. The relative humidity was 70%. Epidermal strips were obtained by peeling off the lower epidermis from young fully expanded leaves. Care was taken to avoid mesophyll contamination. Absorption spectra of a single leaf epidermal strip were measured using a vertical cuvette in conjunction with a single beam spectrophotometer similar to that described by Davis et al. (3). The epidermal strip (_ 100 mm2) was washed twice with distilled H20, placed at the bottom of the cuvette, and a 50 mm2 circular window placed on top of the sample. Absorption spectra were recorded with and without the addition of I drop of I N NH40H and a difference spectrum obtained by computer-assisted subtraction. Absorption spectra of crude extracts or partially purified compounds were measured using a Cary 15 spectrophotometer. Uncorrected fluorescence excitation and emission spectra were determined using an Aminco Bowman (Model H-8202) spectrophotofluorometer. Samples were made alkaline by the addition of several drops of I N NH40H or 0.5 M MeONa4 to the aqueous and methanol extracts, respectively. Isolation of fluorescing compounds was performed by two dimensional descending paper chromatography. Epidermal strips from approximately 20 leaves were immersed immediately after peeling in methanol. The strips were washed twice with methanol, the combined extracts dried in vacuo, redissolved in methanol, and stored at -20°C. This solution was spotted onto washed Whatmann No. 3 chromatographic paper (23 x 27 cm) and developed in the first dimension with t-butanol:acetic acid: H20 (3:1:1) (TBA) and in the second dimension with 15% acetic acid (7). Fluorescent spots, observed under UV light with and without exposure to NH3 vapors, were cut out of the chromatogram and eluted with methanol. The fluorescing compound was tentatively identified by its absorption spectra in several solvents (methanol + MeONa, methanol + AlCl3 + HCI, and methanol + sodium acetate i H3BO3) as described by Mabry et al. (7). The aglycone of the fluorescing compound, obtained after sugar hydrolysis, was identified by 4Abbreviations: MeONa, sodium methoxide; TBA, t-butanol:acetic acid:H20 (3:1:1).