Leaf Phenolics

The first sampling collection day (July 9) was a clear, sunny day, while the second sampling day (July 22) was primarily overcast and rainy. This provided an opportunity to observe the occurrence of DNA damage and the levels of UV-absorbing compounds under contrasting conditions of PAR and UV radiation.

The absorption spectrum in the UV region of the extracts from the Clark-soybean line was typical of a common flavonoid spectrum (e.g., Mabry et al.,

1970). However, the spectrum from the magenta isoline was suggestive of a lack of flavonoids. The absorption peak in the 280 nm - 290 nm range did not exhibit the classic bathochromic shift indicative of flavonoids (data not shown). It is likely that the UV absorbance in the magenta line was due to the presence of hydroxycinnamates (HCAs) which also strongly absorb in the UV-B waveband. Analysis with HPLC confirmed that the primary flavonoids accumulating in the Clark cultivar were the flavonols quercetin and kaempferol, with quercetin accounting for a large proportion of the total flavonols present (Fig. 15.1). Only minute traces of quercetin were observed in the extract from the Clark-magenta plants.

400 600 800 Time (seconds)

Figure 15.1 HPLC trace from methanol extracts of Clark and Clark-magenta soybean lines grown in the field under ambient levels of UV radiation. The primary flavonol in Clark was found to be quercetin, and only trace amounts of this compound were found in the magenta line

The absorbance at 300 nm of the methanol extracts varied between the two sampling days with higher absorbance values found on the sunny day compared to the cloudy day (P = 0.02). There was also a significant difference between the Clark and the Clark-magenta lines on the sunny, but not the cloudy, day. Overall, however, there was little response in these measurements to supplemental UV-B radiation (Fig. 15.2).

It has been suggested (e.g., Searles et al., 2001) that the most common response to increasing levels of UV-B radiation is the increase in UV-absorbing compounds. Several studies with soybean have found that this is a common response (Murali and Teramura, 1986; Sullivan and Teramura, 1990; Gitz et al., 2005). Since the levels were higher on the sunny compared to the cloudy day, the short-term (day-to-day) variations observed in this study may have been linked with ambient levels of UV-B, UV-A, and/or PAR. However, Cosio and McClure (1984) found

Figure 15.2 The absorbance at 300 nm of a methanol extract from 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 15.2 The absorbance at 300 nm of a methanol extract from 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 that activities of key enzymes in the flavonoid biosynthetic pathway were greatly reduced following the completion of leaf expansion. Only exposure to UV-C, not found in sunlight reaching the earth's surface, resulted in renewed enzyme activity. Therefore, it seems unlikely that new synthesis of flavonoids on the sunny day would have explained the differences. It is more likely to have been due to environmental conditions (self shading, ambient UV levels, etc.) present during the development of these particular leaves, which led to leaf-to-leaf variations in flavonoid levels. Sullivan et al. (2007) found that soybean responded to short-term changes in ambient levels of both UV-A and UV-B during the initial phases of leaf development and that the spectral sensitivity varied with the species and soybean cultivar. Ultraviolet-A also had a greater effect than did UV-B in soybean lines that were lacking flavonoids. Therefore, it is unclear just how flavonoid synthesis is regulated and further studies on the spectral and fluence response are needed before we have a complete understanding of this process. It is important that we understand the regulation of this process in order to fully understand how plants have adapted to UV-B radiation and how plants and ecosystems will respond to potential changes in solar UV radiation reaching the earth's surface.

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