Non-invasive measurements of epidermal UV-A transmittance were made on intact mature leaves of plants at the time of harvest with a field-portable pulse amplitude modulation (PAM) chlorophyll fluorometer (UVA-PAM; Gademann Instruments, Würzburg, Germany). The UVA-PAM provides non-destructive estimates of epidermal UV-A transmittance through fluorescence yields of chlorophyll induced by UV-A (375 nm) and blue (470 nm) radiation pulses (Kolb and Pfündel, 2005). Fluorescence induced by UV-A (Fuv) from the mesophyll is compared to fluorescence induced by blue light (FBL). By standardizing the ratio FW/FBL to unity for an epidermis-free leaf (represented by a blue plastic standard, Heinz Walz GmbH, Effeltrich, Germany), absorbance of UV by the epidermis will proportionately reduce FUV with the resulting ratio equivalent to the transmittance for UV-A. Since UV-A transmittance is a dynamic plant characteristic responding to changing environmental factors (Barnes et al., 2008), these measurements were taken on plants that had been moved indoors.
The UVA-PAM measures the changes in transmittance associated with changes in UV-A absorbing pigments (375 nm peak). To determine the relationship between UV-A and UV-B absorbing compounds in Vicia faba, we measured epidermal transmittance and UV-absorbing compounds from additional plants grown in varying light environments in North Logan, Utah. Epidermal transmittance for UV-A was measured on mature intact leaves using the UVA-PAM. The leaves in this study had developed under different densities of shade cloth and under premium cellulose triacetate (0.13 mm thick, Liard Plastics, Salt Lake City, UT, USA) and Llumar (0.13 mm thick, part no. UVCLSRPS, cutoff near 390 nm; CP Films, Inc., Martinsville, VA, USA) to create unfiltered and UV-screened environments (Fig. 13.2), respectively, of different levels of PAR and UV. Subsequently, leaf disks (1 cm2) were collected from each leaf, dried at 60°C for 24 hours and weighed. Samples were ground, placed in an acidified methanol solution (5 ml of 70% methanol, 29% H2O, and 1% HCl) in the dark at 20 °C for 24 hours. Absorbances at 305 nm (approximate midpoint of UV-B spectrum) and 360 nm (midpoint of UV-A spectrum) of the extracts were measured with a scanning UV/visible spectrophotometer (Model DU640; Beckman Coulter, Inc., Fullerton, CA, USA).
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