Measurements of UVB Absorbing Compounds and Chlorophyll Concentrations

Extraction of leaf UV-B absorbing-compounds, mainly flavonoids and related phenolic compounds, was based on the method by Gorton and Vogelmann (1996). Two 10-mm-diameter leaf discs (the total leaf area was 1.57 cm ) were placed in a 1.5 mL microfuge tube and ground to a fine powder in liquid nitrogen using a Teflon pestle. One mL of acidified methanol (methanol:H2O:HCl 79:20:1 v/v) was added to the microfuge tube and homogenized well. The extracts were stored frozen (- 80°C) for up to one week, then clarified by centrifugation (Micro 12, National Labnet Company, Inc.). Forty ^l of the supernatant were transferred to a quartz cuvette and diluted in 3 mL of extraction medium (resulting in a volume dilution factor of 76). Spectra of the extracts were obtained from 200 nm - 820 nm with a computer-controlled, split-beam, dual-detector UV/Visible spectrophotometer (Genesis 2 Spectronic, Inc.) at 1 nm increments. The absorbance spectra of the extract were then standardized for the leaf area and volume to obtain the absorbance values on a leaf area basis (A/cm2). In order to calculate the UV-B absorbing compound concentration for each sample, we decided to include the entire UV-B wavelength region by calculating the cumulative absorbance values from 280 nm - 320 nm for each sample. This avoided the arbitrary selection of the absorbance at a single wavelength, e.g., at 300 nm, 310 nm, or 330 nm, which has been used in numerous studies. Our recent report also indicates that the values of UV-B absorbing compounds measured at 280 nm, 300 nm, and 310 nm were not always in agreement (R ranged from 0.76 - 0.93) due to their wavelength specific nature (Qi et al., 2002). Therefore, the cumulative absorbance from

280 nm - 320 nm at 1 nm intervals was used, then standardized for leaf area and volume, and expressed as total absorbance (A280 nm-320 nm/cm ) to represent the total UV-B absorbing compound content or concentrations per unit of leaf area. Leaf total chlorophyll content was measured with a Minolta Chlorophyll Meter (Spad-502, Spectrum Technologies Inc, IL). The values were then converted to the true chlorophyll content based on the method developed by Yadava (1986).

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