Dark Protein Synthesis: Physiological Response to Nutrient Limitation of a Natural Phytoplankton Population

Abstract

Dark ¹⁴CO₂ incorporation into protein was determined from 24-hr incubations using size-fractionated natural phytoplankton populations from Kāne'ohe Bay, Hawai'i, enriched with either ammonium or ammonium plus phosphorus. Response to ammonium addition was maximum at an ammonium concentration of 3-4 μM. Dark ¹⁴CO₂ assimilation was suppressed by addition of both ammonium and phosphorus, but percentage incorporation into protein was not significantly different from addition of ammonium alone. About 75±1% of the ¹⁴C taken up by the cells was incorporated into either protein or low-molecular-weight intermediate compounds. Cells smaller than 10 μm showed little response to nutrient additions. However, cells in the 10- to 35-μ size fraction incorporated significantly more ¹⁴C into protein when nutrients were added. C:N ratios calculated from the percentage of ¹⁴C incorporated into protein were most variable temporally in the 10- to 35-μm size group and least variable in the picoplankton (0.2-2.0 μm). Nutrient limitation indices (NLIs) calculated from the quotient of C:N ratios in control and nutrient-enriched cultures were not significantly different for the picoplankton and 2- to 10-μm size fraction. The NLI for the 10- to 35-μm size fraction was significantly lower and implied a modest degree of nutrient limitation. The results suggest that cells smaller than 10 μm are growing at close to nutrient-saturated rates much of the time in Kāne'ohe Bay. However, larger cells appear to experience a significant degree of nutrient limitation at some times, particularly when chlorophyll a concentrations are less than about 1 mg m-³. Dark protein synthesis appears to be a useful modification of previous methods based on the dark uptake of ¹⁴ CO₂ for studying nutrient limitation.

Nitrogen-Limited Phytoplankton are known to take up ammonium rapidly in the dark as well as in the light (Goldman and Glibert 1983). In contrast to ammonium, nitrate uptake is light-dependent (MacIsaac and Dugdale 1972) for most phytoplankton, with the exception of some dinoflagellates (Dortch and Maske 1982).

When ammonium is taken up in the dark, it may be incorporated into amino acids using carbon also assimilated in the dark (Mortain- Bertrand et al. 1988). Dark ammonium uptake is accompanied by enhanced dark ¹⁴CO₂ assimilation by nitrogen-limited phytoplankton. The degree of enhancement of dark ¹⁴CO₂ assimilation may be a function of nitrogen limitation. This idea was suggested by Morris et al. (1971) and Yentsch et al. (1977) and later confirmed experimentally by Goldman and Dennett (1983). Glibert et al. (1985) and Cook et al. (1992, 1994) used this approach to study nutrient limitation of natural phytoplankton populations and symbiotic zooxanthellae, respectively. Ammonium enrichment in the dark led to significant CO₂ uptake by the marine diatom Chaetoceros simplex regardless of whether the cultures were preconditioned with oxidized or reduced nitrogen (Goldman and Dennett 1986). Ammonium enrichment in the dark led to significant CO₂ assimilation by the marine diatom Skeletonema costatum preconditioned with limited nitrogen (Granum and Myklestad 1999). The results to date indicate that the response of dark CO₂ fixation to ammonium enrichment is a useful semiquantitative tool for studying phytoplankton nitrogen limitation, in the sense that it can be used to distinguish between no, moderate, and severe nitrogen limitation. Dodds and Priscu (1991) used the same approach to study nitrogen limitation of chemoautotrophic ammonium-oxidizing bacteria in fresh water.

Protein synthesis is known to occur at night (Cuhel et al. 1984). However dark protein synthesis as opposed to ¹⁴CO₂ uptake has never been related to nitrogen limitation. Because protein synthesis is directly related to the growth rate of phytoplankton (Goldman 1980, DiTullio and Laws 1986, Taguchi and Laws 1987), in this study we extended the use of dark ¹⁴CO₂ assimilation to study dark protein synthesis in size-fractionated phytoplankton populations to explore the degree of nitrogen limitation of growth rate. The method focuses on the metabolic incorporation of ¹⁴CO₂ into protein rather than...