Nondestructive Measurement of Carotenoids in Plant Tissues by Fluorescence Quenching

other compounds found in healthy cell walls (Cochrane et al., 2000), as well as from a number of compounds Carotenoids, compounds valuable for their antioxidant properties that increase in concentration during host–pathogen inin humans and animals, are sometimes present in the bran layers of teractions (Pierce and Essenberg, 1987; Belefant-Miller rice (Oryza sativa L.). We developed a nondestructive technique to et al., 1994). screen individual rice kernels for the presence of carotenoids in their bran for genetic selection of the carotenoid-containing lines. Most Fluorescence spectroscopy is a highly sensitive techplant tissues are highly autofluorescent. Carotenoids have a high abnique, used particularly in chemical and physical studies sorptivity for visible light, which allows them to absorb or quench of fluorescent compounds that are often present in orthis natural fluorescence. Synchronous fluorescence spectroscopy proganic pollutants. In fluorescence spectroscopy, the samvides the specificity and sensitivity that allows detection of the changes ple is illuminated by ultraviolet or visible light (excitain fluorescence in a single rice kernel that occur when carotenoids tion light) and the fluorescent light then emitted by are present. The fluorescence quenching technique identified three the sample is measured. Synchronous fluorescence (or rice lines as having carotenoids present in the bran, and three other luminescence) provides an even higher degree of seleclines as being carotenoid-deficient. Fluorescence quenching was also tivity since both the excitation and emission wavelengths used to monitor changes in carotenoid levels in living plants and are scanned simultaneously and a signal is generated enabled the measurement of carotenoids within the different colors of a variegated leaf. only where the resulting emission and excitation spectra overlap (Vo-Dinh, 1982). By combining the separate characteristics of autofluorescent emission of visible light by plant tissues with the light-absorbing ability of carotB rice is rice that has been hulled but not milled enoids, we developed a rapid, nondestructive technique so that the bran layers around the endosperm reable to determine the presence of carotenoids in a single main. Brown rice is considered to be a healthful food rice kernel. This technique also has potential uses for source as it contains a number of nutrients not present studies of living, rare, or difficult to separate plant tissues. or present at low quantities in endosperm (white rice) alone. However, bran also contains a number of antinutritive factors, including phytic acid, trypsin inhibiMATERIALS AND METHODS tors, and hemagglutinin (Juliano, 1994). Rice bran also Synchronous Fluorescence Spectroscopy contains carotenoids (Sechi and Rossi-Manaresi, 1958; Saunders, 1990; Juliano and Bechtel, 1994), though not Synchronous fluorescence scans ( 10 nm) were performed on a Spex/JY Horiba Fluoromax 3 fluorometer (Jobin in every cultivar (Juliano and Bechtel, 1994). ConcurYvon, Inc., Edison, NJ) using slits at 2-nm (excitation) and rently with another study to develop and identify brown 2-nm (emission) bandwidths. The solid sample holder with rice with reduced levels of antinutritive factors (Rutger sample was adjusted to produce a maximum signal at ex et al., 2004), we endeavored to identify brown rice that 400 nm and em 410 nm. After manual optimization of also contained carotenoids. position, the scans were made from 400/410 to 650/660 nm, The color of rice bran can vary from light tan to very unless noted otherwise. We were able to screen and analyze yellow, brown, red, and even purple and nearly black. 20 to 30 samples per hour. It is impossible to visually discern a yellowness that may indicate the presence of carotenoids. Thus, the fluoresFluorescence Quenching by -Carotene cence spectroscopy technique provides a means for idenTo demonstrate the mechanism of fluorescence quenching, tifying brans containing carotenoids. Carotenoids, which synchronous fluorescence measurements ( 10) were include carotenes and xanthophylls, have a high molar made from 275/285 to 650/660 nm of a kernel of white (milled) absorptivity and, thus, absorb light efficiently. Conversely, rice spotted with 2 L of chloroform and a kernel of white plant tissues are highly autofluorescent; plant cells, when rice spotted with 2 L of 10 3 M -carotene dissolved in excited by light of the appropriate wavelength, are able chloroform. The scan of the rice spotted with -carotene was to fluoresce without the addition of dyes or other chemimathematically subtracted from the scan of the white, chloroform-spotted rice. The resulting curve shows the difference cals. Autofluorescence results from lignin, suberin, and in emitted light between the two spectra as a result of light absorption by -carotene. USDA-ARS, Dale Bumpers National Rice Research Center, Stuttgart, AR 72160-1090. Received 6 Oct. 2004. *Corresponding author Integrated Fluorescence Measurements ([email protected]). To ensure that we were detecting changes in light absorbance Published in Crop Sci. 45:1786–1789 (2005). in our sample measurements, and not just an altered backCrop Physiology & Metabolism Note ground, we measured the light emitted over a wavelength range doi:10.2135/cropsci2004.0592 © Crop Science Society of America 677 S. Segoe Rd., Madison, WI 53711 USA Abbreviations: NIFI, net integrated fluorescence intensity. 1786 Published online August 1, 2005