Assessment of DNA Damage as a Tool to Measure UVB Tolerance in Soybean Lines Differing in Foliar Flavonoid Composition

1 2 2 Joseph H. Sullivan , Linda C. Pope, Betsy M. Sutherland , Paula V. Bennett ,

James E. Blum3, Ann E. Stapleton4, and Dennis C. Gitz III5

1 Department of Plant Science and Landscape Architecture, 2122 Plant Sciences Building University of Maryland, College Park, MD 20742, USA E-mail: [email protected] 2 Brookhaven National Laboratory, P.O. Box 5000, Upton, NY 11973-5000, USA E-mail: [email protected] 3 Department of Mathematics and Statistics University of North Carolina at Wilmington, Wilmington, NC 28403, USA E-mail: [email protected] 4 Department of Biology and Marine Biology University of North Carolina at Wilmington, Wilmington, NC 28403, USA E-mail: [email protected] 5USDA-ARS, 3810 4th Street, Lubbock, Texas 79415,USA

Abstract Damage to DNA, in the form of cyclobutane pyrimidine dimers (CPD), may occur in soybean (Glycine max (L.) Merr) plants when they are exposed to increasing levels of ultraviolet-B (UV-B) radiation. Flavonoids and other phenolics accumulate in the epidermal layer of leaves and may provide protection for sensitive tissues including DNA molecules. We evaluated the steady state levels of accumulated damage and the protection afforded by flavonoids in two soybean isolines: Clark producing high levels of flavonoids, and Clark-magenta producing extremely low flavonoid levels. Both cultivars were grown in the field under ambient and supplemental UV-B radiation. Leaf tissue was harvested in a diurnal sequence, and the samples were analyzed. Two methods of analysis were used in order to develop a common reference point between the two. In one method, DNA was isolated and treated with UV endonuclease, and the DNA fragments were separated using unidirectional pulsed field electrophoresis and quantified through electronic imaging. In the alternate method, a western blotting procedure, immobilized DNA was reacted with monoclonal antibodies specific to CPD DNA damage. Results were similar in both techniques and show lesion frequency to be low in both isolines. However significant differences were found between cultivars, UV treatments, time of day collected, and levels of PAR. The average level of dimers per megabase for the isoline Clark was ~4 (with or without supplemental UV), and for Clark-magenta, ~4 for samples with no supplemental UV and ~6 for those exposed to supplemental UV radiation. Diurnally, dimer levels were frequently higher in the Clark-magenta isoline, especially when exposed to supplemental UV-B. Both isolines appear to be either well-protected from DNA damage, or repair is efficient enough to minimize biologically significant accumulation of DNA damage. This suggests that protection mechanisms, other than flavonoids alone, contribute to maintenance of DNA integrity in soybean.

Keywords Glycine max, soybean, DNA damage, pyrimidine dimers, UV-B radiation, stratospheric ozone depletion

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