Introduction

Continued stratospheric ozone depletion and the resultant increase in ultraviolet-B radiation (UV-B) raises a concern for a potential decrease in crop yields and impacts on agricultural and natural ecosystems. Although the implementation of regulations that minimize inputs of chlorofluorocarbons into the stratosphere is resulting in recovery of the ozone layer, there is still uncertainly about the stability of future ozone levels (WMO, 2003). For example, the link between global warming and ozone depletion is not fully understood and warrants further investigation.

Warming trends in the Pacific Ocean affect the strength of the vortex at the South Pole allowing the photochemical reactions that deplete ozone to persist longer in the spring (Kerr, 1995). As CO2 levels increase and the troposphere warms, the associated cooling of the stratosphere leads to increased transport of water vapor to the stratosphere (Kirk-Davidoff et al., 1999). This water vapor is the basis for the formation of the polar stratospheric clouds (PSCs) that provide a surface on which ozone destruction takes place. Persistence of PSCs into the Antarctic spring due to stratospheric cooling may lead to enhanced rates of catalytic ozone destruction. This may be further exaggerated by atmospheric denitrification that reduces the effectiveness of nitric acid to combine with free chlorine and prevent ozone destruction (Salawich et al., 2002). Thirty-five percent of the ozone lost in the Arctic winter of 1994 -1995 was attributable to stratospheric denitrification (Waibel et al., 1999). Concerns such as these suggest that the possible implications of continued ozone depletion and concomitant increases in solar UV-B radiation (between 290 nm and 315 nm) need further evaluation.

Although it represents only a fraction of the total solar electromagnetic spectrum, UV-B may exert substantial photobiological effects when absorbed by important macromolecules such as proteins and nucleic acids (Giese, 1964). The highly energetic photons of UV-B radiation may reduce photosynthesis, alter stomatal development and functioning, damage proteins and membranes, and induce DNA lesions if absorbed in sufficient quantity (e.g., Caldwell et al., 2003; Sullivan, 2005). However, sensitivity to UV-B varies greatly among plant species. Some plants are quite tolerant to high fluences of UV-B (Sullivan et al., 1992; Ziska et al., 1992), while others are sensitive to present ambient fluences (Bogenrieder and Klein, 1982; Krizek et al., 1998). This variation in response to UV-B makes it difficult to generalize about UV-B effects, but overall meta-analysis of UV studies through the end of the Century suggested that major reductions in growth or biomass were rare in realistic field studies (Searles et al., 2001). This infers the importance of UV-B throughout the evolution of land plants that has led to well-developed UV protection mechanisms in many plant species (Beggs et al., 1986).

One of the protective responses that have generally been considered adaptive is the accumulation of epidermal UV-screening compounds that serve as UV filters. The accumulation of phenolics, and flavonoids in particular, has been frequently reported in response to UV-B radiation (e.g., Robberecht and Caldwell, 1978; Caldwell et al., 1983; Sullivan and Teramura, 1989; Tevini et al., 1991). In fact, Searles et al. (2001) found through a meta-analysis of 103 published articles that the most common response of plants to UV-B is an increase in UV-screening compounds. Flavonoids and other phenolics, especially hydroxycinnamic acids (Sheahan, 1996), absorb strongly in the UV-B range. The accumulation of these compounds in the epidermis has been shown to reduce UV-B radiation transmittance and hypothesized to protect sensitive targets (Robberecht and Caldwell, 1978; Robberecht and Caldwell, 1983; Beggs et al. 1986; and many others). The accumulation of these compounds is dependent upon a number of factors, including both visible and UV fluence (Mohr and Drumm-Herrel, 1983; Wellman, 1983) and many other environmental factors (McClure, 1986). The mechanistic basis of light or UV-induced accumulation lies at the gene level as several key enzymes in the flavonoid biosynthetic pathway, such as, phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS), are induced by UV radiation (Chappell and Hahlbrock, 1984; Beerhues et al., 1988; Liu and McClure, 1995; Schnitzler et al., 1997). There is little doubt that phenolics accumulate in leaf tissue in response to UV-B radiation, and that they are important in conferring photoprotection to "target" molecules that may be damaged by UV-B radiation.

One of the best methods utilized to date to evaluate the screening effectiveness of phenolics has been to actually measure UV-B penetration inside a leaf and compare this to expected values based on soluble phenolic levels. The research by Day et al. (1994), and other related studies (see Vogelmann, 1994, and references therein), have shown that the penetration of UV-B through the epidermis is quite variable and may be related to plant life form and growth habit. However, a rather poor correlation (r = 0.21) was found between epidermal transmittance of UV-B at 300 nm and absorbance of soluble phenolics (Day, 1993). Also, Day et al. (1994) measured epidermal transmittance of both UV-B at 300 nm and total

UV-B weighted with one of several weighting functions. They found that the expected extinction rates based on absorbance values across a wide range of species within a life-form group followed the theoretical expectation in only 1 of 42 species. In other words, "Beer-Lambert" type extinction was rarely observed, based on concentrations of soluble UV-absorbing compounds alone. The presence of bound phenolics in the epidermis that are not extracted, but alter epidermal transmittance, could contribute to this relationship.

Even when the concentration or screening ability of soluble and bound phenolics are considered, these parameters do not always correlate well with UV tolerance (e.g., Barnes et al., 1987; Dillenburg et al., 1994; Sullivan et al., 1996), and simple correlations may not exist between the apparent concentrations of soluble UV-absorbing compounds and UV-sensitivity. Barnes et al. (1987) suggested that some species adapted to high ambient UV-B fluxes were inherently tolerant to UV-B radiation, but we do not yet have a complete understanding of all tolerance mechanisms.

In addition to their role as sunscreens, phenolics are involved in numerous aspects of plant growth and development, such as serving as antioxidants (Sheahan, 1996), involvement with auxin-mediated responses (Stafford, 1991), cell wall extension (Dale, 1988; Liu et al., 1995), and lignification (Raven, 1977). Phenolic concentrations and composition also have important ecological implications for decomposition rates and nutrient cycling, as well as for plant-insect and plant-pathogen interactions. Therefore, the physiological and ecological roles of phenolics in plants, and the role that solar UV radiation plays in regulating the composition and concentration of them, needs further evaluation. For example, the specific dose response of accumulation of protective compounds, the action spectrum for this response, and the contribution of environmental factors other than UV-B to this response, have not been clearly resolved in field studies.

Soybean has been intensively studied for UV-B responses for the past quarter century and its production of primary flavonols in response to UV-B radiation has frequently been reported (Murali and Teramura, 1986; Sinclair et al., 1990; Mazza et al., 2000). Therefore, since its chemistry and response to UV-B are rather well-known, soybean is a good candidate for a model system to evaluate the protective role of phenolics, flavonoids in particular, in response to UV radiation. This chapter assesses the protective role of flavonoids in protecting soybean DNA from damage due to exposure to high levels of UV-B radiation in the field. DNA damage, primarily cyclobutane pyrimidine dimers (CPDs) caused by UV-B exposure in plants, has been studied in a variety of plant systems, including soybean (Sutherland et al., 1996; Mazza et al., 2000; Bennett et al., 2001; and others). In addition, it is known that DNA damage may accumulate in some species. However, it is not clear whether the absence of flavonoids as screening compounds would alter the diurnal progression of CPD formation.

In this study, we utilized soybean lines that differ in flavonoid quantity and composition in order to evaluate the protective role of flavonoids against UV-B

damage. These cultivars included: (1) the Clark cultivar, a moderately tolerant cultivar that produces primarily the flavonols quercetin and kaempferol for putative UV-screening; and (2) an isoline of Clark, Clark-magenta (Buzzell et al., 1977) that produces essentially no flavonols, but accumulates cinnamic acids in response to UV radiation. Two of three goals of this research were to determine if: (1) CPDs accumulate over the course of the day; and (2) the absence of flavonol glycosides lead to the formation of more photoproducts in the DNA.

A third goal was to compare two methods of CPD analysis. One method previously utilized to evaluate CPDs uses a gel electrophoresis and imaging system to quantify dimers, while many researchers employ an alternate method using a western blotting procedure, in which immobilized DNA is reacted with monoclonal antibodies specific to CPD DNA damage (Mori et al., 1991; Stapleton et al., 1993; Stapleton and Walbot, 1994; Mazza et al., 2000). As previous research has rarely used both methods of dimer quantification, a comparison of these two methods will lend perspective and increase the interpretive power of past and future studies that may utilize one method or another.

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