Ultraviolet radiation and the snow alga Chlamydomonas nivalis (Bauer) Wille[para]

Photochemistry and Photobiology, Jun 2003 by Gorton, Holly L, Vogelmann, Thomas C

Cells for microscopy were taken from melted snow collected in the field. Cells were cleaned using a series of nylon mesh screens (14), collected by centrifugation (3600 g, 2 min) and then further purified with a sucrose step gradient using 0%, 20%, 40%, 60%, 80% and 100% saturated sucrose. After layering cells on the gradient and centrifugation for 5 min, cells were collected from all interfaces except the 80%/100% interface, which accumulated dirt as well as cells. Cells were then washed thoroughly on nylon mesh with deionized water. In some experiments, cells were broken using a mortar and pestle to release cytoplasmic compounds and were washed by centrifugation so we could examine the cell walls. In other experiments, we plasmolyzed cells in 50% saturated sucrose before microscopy.

Image analysis. To find percent transmittance (%T) for cells at each wavelength, we divided each image by a background image taken with the same wavelength and for the same exposure time but of a region of the sample with no cells. Stray light or poor optical quality of the lenses caused some fogging of the images, especially at the shortest wavelengths. Therefore, images were corrected on the basis of the assumptions that transmittance in the blue was negligible (14) and that transmittance in regions without cells should be 100%.

Extraction of screening compounds. We ground cells in aqueous acidified methanol (79:20:1 methanol-water-HCl) with a mortar and pestle, clarified the extract by centrifugation and measured absorption spectra using a Hewlett-Packard 8453 diode-array spectrophotometer. For comparison, we also measured an absorption spectrum of pure astaxanthin (Sigma-Aldrich Corporation, St. Louis, MO) in chloroform.

Wall isolation and spectral analysis. Filtered cells were washed by centrifugation, twice with acetone and twice with diethyl ether. They were ground with diethyl ether and liquid nitrogen using a mortar and pestle, the ether was evaporated and cells were washed in acetone. These grinding and washing steps were repeated at least three times, until the supernatant was no longer colored. Ninety-seven percent of the cells were broken by this treatment. Walls were resuspended in water and stored frozen for 10 days before analysis. Transmission spectra of a paste of cell wall material between two quartz windows were measured using a UV-VIS integrating sphere (14).

RESULTS

UV penetration through snow

Surface solar radiation decreased markedly as wavelength decreased below 400 nm, dropping to levels in the range of instrument noise at about 300 nm. The same pattern was evident in up to 8 cm of snow, but we could not detect measurable UV below this depth. The UV signal disappeared into noise at longer wavelengths with increasing depths. For example, whereas reliable data were obtained for wavelengths down to 300 nm at the snow surface, data for even part of the UV-B range were reliable only to 2 cm and for UV-A only to 8 cm. %T for sunlight at different depths in the snow was calculated by dividing a spectrum at each depth by the surface spectrum (Fig. 1). The transmittance spectra in Fig. 1 are truncated in the UV-B, reflecting the limitations of the instrument. All show a gradual decrease in transmittance with decreasing wavelength.