DNA Damage

An assessment of how plants differing in flavonoid composition and quantities are affected at the DNA level may provide key information regarding UV tolerance mechanisms and the importance of flavonoids in this process. No significant difference was noted between blocks on July 9th, and the data were combined for all fUrther analysis. Replicate blocks were also sampled on July 22, but only samples from block 1 were processed using the gel electrophoretic method. Samples taken from both blocks were processed with the monoclonal antibody method.

Statistically significant differences in DNA integrity were observed using both methods of analysis. In the gel method, a significant effect was found for cultivar (P = 0.0064), UV treatment (P = 0.0103), day (P = 0.002), and the interaction between UV treatment and cultivar (P = 0.0293). On the cloudy day, supplemental UV-B induced more DNA dimers in the Clark-magenta line than in the ambient UV-treatment, and more than in either treatment in the Clark wild type with a normal flavonoid complement (Fig. 15.3). The Clark-magenta line showed a trend of increasing dimers on the sunny day, but this was not statistically significant. In the CPD analysis, significant effects were cultivar (P = 0.0001), time of day (P = 0.015), and sunny vs. cloudy day (P = 0.0001). Also, when considering the diurnal changes in CPDs, the Clark-magenta line had increased CPD amounts at 13:00 h on the cloudy day (Fig. 15.4). Overall, the Clark line generally had less DNA damage than the Clark-magenta line, but the difference was only significant on the cloudy day (Fig. 15.5).

The level of dimers tended to increase early in the day and then subside in the afternoon. This suggests that at some points, the number of dimers formed over the course of the day exceeds the capacity of repair mechanisms. This short term accumulation of damage might be responsible for some of the deleterious responses of plants to increased levels of UV-B radiation.

Figure 15.3 The number of ESS (measure of DNA lesions formed) in soybean grown in the field under either ambient or ambient plus supplemental levels of UV-B radiation. Each bar is the mean of 5 replicate samples from 2 treatment replicates plus 1 SD. Different letters over a bar indicate a significant difference between the bars

Figure 15.3 The number of ESS (measure of DNA lesions formed) in soybean grown in the field under either ambient or ambient plus supplemental levels of UV-B radiation. Each bar is the mean of 5 replicate samples from 2 treatment replicates plus 1 SD. Different letters over a bar indicate a significant difference between the bars

Figure 15.4 Diurnal changes in the number of CPDs formed in 3 soybean lines grown under ambient or ambient plus supplemental levels of UV-B radiation. Each point is the mean of 5 samples collected each 2-hour period (see text for details on sampling protocols)

Figure 15.5 CPDs found in two soybean lines grown in the field under either ambient or ambient plus supplemental levels of UV-B. LSMeans of CPDs formed over the course of the day. UV-B treatments are combined since there was no statistical difference between treatments. Each bar is the mean of 5 samples collected each 2-hour period (see text for details on sampling protocols). Different letter over a bar indicates a statistically significant difference in the means at P = 0.05

Clark C-magenta C loudy day

Figure 15.5 CPDs found in two soybean lines grown in the field under either ambient or ambient plus supplemental levels of UV-B. LSMeans of CPDs formed over the course of the day. UV-B treatments are combined since there was no statistical difference between treatments. Each bar is the mean of 5 samples collected each 2-hour period (see text for details on sampling protocols). Different letter over a bar indicates a statistically significant difference in the means at P = 0.05

Damage to DNA by UV-B radiation is one factor that could lead to abnormal plant growth and development, altered biochemical pathways and metabolism, inability to reproduce successfully, and even death. Therefore, plants have developed a suite of photorepair and dark repair processes. Ultraviolet-screening mechanisms, such as that afforded by flavonoids, is another protective response. In this study, minimal DNA damage was observed in all cases, but there was some indication that the absence of flavonoids increased damage at certain times. It is also possible that screening by compounds other than flavonoids, per se (e.g., HCAs), may also be important. The minimal damage that occurred on the cloudy day in Clark-magenta did not persist at the end of the day (20:00 h), so we may assume that the repair of lesions was sufficient to mitigate the majority of damage.

Several studies have shown that the repair of UV-induced damage is mediated by photolyase in mature leaves (Dany et al., 2001). Since photolyase requires a photon of light, preferentially UV-A or blue light (Stapleton et al., 1997) to reverse the damaged DNA, this process must be completed before the energy from the sun dissipates late in the evening, as was the case in our samples. It was primarily on the cloudy day that lesions accumulated in the afternoon in Clark-magenta. The higher ratio of UV-B to PAR was such that minor increases in CPDs occurred periodically. Perhaps PAR levels were not sufficiently high enough to prevent these accumulations. Alternatively, it is possible that the absence of flavonoids, which are more efficient in the absorption of UV-A (Mabry et al., 1970) may lead to increased penetration of UV-A into the leaf mesophyll and therefore, lead to more effective repair systems.

These measurements quantify net dimer levels, and an increased rate of dimer formation might possibly have been countered by enhanced repair capability. Xu et al. (2008a) also noted that the rate of oxidant production appeared to be increased in these same soybean lines in ambient compared to reduced UV-B levels, but observed an increase in the antioxidant system in the magenta soybean line. It is well-known that the balance of repair mechanisms vs. those that provide protection is important (Jansen et al., 1998; Frohnmeyer and Staiger, 2003), and that an increase in UV-absorbing compounds is a common response to UV-B radiation (Searles et al. 2001). However, the results of this study do not allow us to quantify if it was protection (in the case of Clark) and repair (in the case of Clark-magenta) that maintained the low levels of CPDs. Clearly, the effects of UV-A on plant metabolism and the interactions between PAR, UV-A, and UV-B, is not fully understood. This is an area in which continued research may lead to a better understanding of how plants are affected by the various components of sunlight.

When dimer levels from this experiment were compared to maximum levels reached in a exposure response study (~120 CPD's/Mb, data not shown), it was clear that although there was a significant difference in dimers produced by the two cultivars, UV treatments, and irradiance levels, the difference was not biologically significant. Although Clark had fewer dimers on both days (3.46 + 0.91 and 5.20 + 1.67) as compared to Clark-magenta (4.39 ± 1.07 and 6.65 +1.66), the effects of the small size differences on the plants is probably minimal. This indicates that although flavonoids and phenolics do protect plants from UV-B exposure, in cases where they are not present, plants must provide protection to DNA integrity through other pathways. For example, additional repair enzymes may be produced in plants lacking phenolic protection.

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