GROWTH-CHARACTERISTICS OF PHYTOPLANKTON DETERMINED BY CELL-CYCLE PROTEINS - PCNA IMMUNOSTAINING OF DUNALIELLA-TERTIOLECTA (CHLOROPHYCEAE)

By immunohistochemistry and immunofluorescence methods, we observed that the analog of proliferating cell nuclear antigen (PCNA) in Dunaliella tertiolecta Butcher (Chlorophyceae) was exclusively located in the nucleus. Among positively stained cells, PCNA abundance varied, being highest in S-phase cells, lower in others, and undetectable in early G1- or late M-phase cells. In exponentially growing and partially synchronized cultures, the percentage of PCNA-stained cells (% PCNA-stained cells) oscillated in the photocycle (12:12 h LD). It increased during the light period and reached a peak (75%) before the onset of the dark period when the culture was mainly (71%) in the S phase of the cell cycle. The DNA synthesis inhibitor, hydroxyurea, depressed PCNA abundance, whereas no effect was detected for the mitosis inhibitor colchicine. We conclude that PCNA in D. tertiolecta is associated with the S phase of the cell cycle where it is accumulated and functioning. PCNA was used to characterize the growth pattern of cultures grown in different media, temperatures, and growth stages. The time lag between the PCNA-stained phase and the M phase was very short in a continuous culture grown in reduced f/2 medium at 22 degrees C and was considerably longer in the cultures grown in f/2 at 15 degrees C. When an exponentially growing culture grew older, % PCNA-stained cells decreased. In a late stationary culture where there was no net growth, a small number of cells were still cycling through the PCNA-stained phase and cell division. In the continuous culture grown at 22 degrees C, the duration of the PCNA-stained phase (T-s) was 13 h. Calculations with this T-s and % PCNA-stained cells yielded a growth rate of 0.77 d(-1), which was close to that obtained by cell counts (0.69 d(-1)). Taken together, the results suggest that PCNA is a useful indicator of growth status and a promising cell cycle marker for estimation of species-specific growth rate.