Analysis of Phenolics

Leaf samples were collected on plants at predetermined times following the commencement of irradiation in order to verify their chemical composition and to assess their response to UV-B radiation. Leaf disks (1.13 cm ) were made on the first true leaf of the plants for studies on emerging plants and on the most recently mature leaf on mature plants. These were placed on ice and taken to the laboratory where they were placed in 20 ml high density polyethylene (HDPE) scintillation vials covered with 10 ml of slightly acidified aqueous methanol (MeOH:H2O:AcOOH, 50:50:1, v:v:v), tightly capped, and held in the dark at room temperature with gentle agitation (50 rpm) on a G10 Gyrotory Shaker (New Brunswick Scientific, Edison, NJ, USA) for 48 hrs. - 72 hrs. During extraction, vials were sealed with polyethylene lined caps; since it was found that foil lined caps would occasionally corrode and contaminate the extracts leading to the nonuniform formation of metal-flavonol complexes and bathochromic shifting of the absorbance spectra. The extract absorbance was determined from 260 nm - 760 nm at 1 nm intervals with a Shimadzu UV-1601 dual beam spectrophotometer (Shimadzu Scientific Corp., Columbia, MD, USA), and data were expressed as absorbance units per unit leaf area (A cm ). Since UV level did not lead to qualitative differences in spectra within a species or cultivar, absorbance values at 300 nm were used for comparative purposes between the treatment and controls.

In order to verify presence or absence of flavonoids in the Clark and Clark-magenta isoline of soybean, High Performance Liquid Chromatography (HPLC) separation and determination of flavonoids were conducted. Leaf disks were milled under liquid nitrogen in microcentrifuge tubes, hydrolyzed in 1 N H2SO4 in 1 ml 50% aqueous methanol at 80° for 30 minutes under nitrogen, brought to 5 ml, and filtered. Forty (L of the filtrate was directly injected onto a 250 x 4.6 mm column packed with 7 (r spherical C8 stationary phase with gradient elution by 20% solvent B (aqueous 1% orthophosphoric acid) in solvent A (acetonitrile in 1% orthophosphoric acid) increased to 60% B over 20 min at a flow rate of 1.5 ml min-1. Detection at 370 nm was accomplished using a Waters 490E programmable multiwavelength detector. Naringenin (5(L) in 50% aqueous methanol (1 mg ml-1) was coinjected at the beginning of each run as a standard.

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