Identification of Two Plastid Proteins in the Dinoflagellate <i>Alexandrium affine</i> That Are Substantially Down-Regulated by Nitrogen-Depletion

The formation of harmful algal blooms (HABs) by dinoflagellates has been correlated with the nitrogen load in coastal waters. Nitrogen is implicated as an important factor in the initiation and maintenance of phytoplankton blooms. To characterize the cellular response to nitrogen, 2DE was used to compare protein expressions from dinoflagellates grown under nitrogen depleted and nitrogen replete conditions. A total of 17 differentially expressed protein spots were found, nine of which showed a roughly 16-fold decrease in N-depleted conditions. Five of these nine spots were all identified as isoforms of the plastid Form II ribulose-1,5 bisphosphate carboxylase/oxygenase (Rubisco II), while an additional four protein spots with a molecular weight of 50 kDa were identified as isoforms of a novel protein named nitrogen-associated protein 50 (NAP50). NAP50 was located in the plastids as shown by the presence of an N-terminal plastid targeting leader sequence and by immunohistochemistry. Levels of both Rubisco II and NAP50 decrease sharply between 24 and 36 h following nitrogen depletion and the decrease can be blocked if the N source is replenished before degradation occurs. Both proteins are rapidly resynthesized if the nitrogen source is replenished after degradation has occurred. These results are a first step in the dissection of the behavior of the dinoflagellate proteome under nitrogen stress conditions and may provide new insights into the relationship between dinoflagellate blooms and the nitrogen budget.