DIEL PERIODICITY OF NITRATE REDUCTASE ACTIVITY AND PROTEIN LEVELS IN THE MARINE DIATOM THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)

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Abstract
The diel variation and regulation of the enzyme nitrate reductase (NR) were examined in the diatom Thalassiosira weissflogii (Gru.) Fryxell et Hasle. NR was purified, and polyclonal antibodies were raised to a 98‐kD polypeptide. The antibodies cross‐reacted only with proteins from closely related diatom species, suggesting significant epitopic variation of this enzyme within algal divisions. Neither NR enzymatic activity nor protein was detected in cells grown with ammonium as the sole nitrogen source; the addition of ammonium to cells growing on nitrate decreased both protein levels and enzyme activity by 40% within 2 h. In cells grown on a 12:12 h LD cycle, NR activity and NR protein levels were highly correlated, with a peak at midday, a decrease toward the end of the photoperiod, and an increase in activity beginning near the end of the dark period. The addition of actinomycin D (an inhibitor of RNA synthesis) and cycloheximide (an inhibitor of protein synthesis) affected NR activity and NR protein levels identically, strongly suggesting that this nuclear‐encoded protein is regulated primarily at a transcriptional level. The diel pattern of NR protein and activity ceased immediately following transfer to continuous light, indicating that the periodicity is not directly controlled by a circadian rhythm. Time‐lagged cross‐correlation analysis revealed a 6‐h phased difference between the minimum enzyme activity or protein levels and the maximum cellular carbon pool. On the basis of the experimental results, we develop a model proposing that (1) NR activity is regulated primarily by transcriptional regulation of NR synthesis and that (2) the level of expression of the enzyme during a given day is correlated with the integrated pool of organic carbon accumulated during the preceding photoperiod.

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